48 results on '"Katrin S. Lips"'
Search Results
2. Differences in expression of Wnt antagonist Dkk1 in healthy versus pathological bone samples
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Katrin S. Lips, N.D. Wijekoon, T El Khassawna, Christian Heiss, Ursula Sommer, Seemun Ray, Ulrich Thormann, and Volker Alt
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musculoskeletal diseases ,030203 arthritis & rheumatology ,0301 basic medicine ,Cell type ,Pathology ,medicine.medical_specialty ,Histology ,Cartilage ,Wnt signaling pathway ,Osteoarthritis ,Bone healing ,Biology ,medicine.disease ,Chondrogenesis ,Pathology and Forensic Medicine ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,DKK1 ,Bone cell ,medicine ,Cancer research - Abstract
Wnt/β-catenin signalling components was shown to affect bone cells function including chondrocytes.Secreted Dkk1, a potent osteogenesis inhibiting factor mediates bone loss in diseased bones by suppressing the biological actions of Wnt proteins. In addition, increased Dkk1 signalling inhibits chondrogenesis in new bone formation. Recent findings also show there exists a cross-talk between the chondrocytes and the cells of the osteoblast lineage, which are the most affected cell types in muskuloskeletal disorders. This study investigated whether spatial expression of Dkk1 is confined to only osteoblasts, osteocytes or chondrocytes. The second objective was to detect a difference in the Dkk1 expression pattern in healthy subjects when compared to pathological state. To elucidate the cell specificity of Dickkopf-1 (Dkk1) in healthy bones, samples from female Sprague-Dawley rats were tested against two different antibodies with the two most widely accepted visualization system (ABC and Envision). The findings show Dkk1 specificity predominantly for osteoblasts, chondrocytes and osteocytes depending upon the antibody used. In addition, Dkk1 expression was evaluated in different cells of human osteoarthritis (OA) and rheumatoid arthritis (OA) patients. Its overexpression in pathologic state also suggests the role of Dkk1 in bone formation. This is scientifically and clinically important in studying the effect of Dkk1 in bone healing and in designing treatments for patients with compromised bone status. Taking into consideration the paradigm that cartilage and subchondral bone behave as an interconnected functional unit, normalization of cell behaviour in one compartment may have benefits in both tissues.
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- 2016
3. Regulation of acetylcholine receptors during differentiation of bone mesenchymal stem cells harvested from human reaming debris
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Olga Dakischew, Katja Trinkaus, Sonja Hartmann, Katrin S. Lips, Christian Heiss, Andreas Zablotni, and Gabor Szalay
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Adult ,Male ,medicine.medical_specialty ,Immunology ,Osteoporosis ,Receptors, Nicotinic ,Biology ,Real-Time Polymerase Chain Reaction ,Chondrocyte ,Bone remodeling ,Young Adult ,Internal medicine ,Muscarinic acetylcholine receptor ,Adipocytes ,medicine ,Humans ,Immunology and Allergy ,RNA, Messenger ,Cells, Cultured ,Aged ,Aged, 80 and over ,Pharmacology ,Osteoblasts ,Reverse Transcriptase Polymerase Chain Reaction ,Mesenchymal stem cell ,Cell Differentiation ,Mesenchymal Stem Cells ,Osteoblast ,Middle Aged ,medicine.disease ,Receptors, Muscarinic ,RUNX2 ,Endocrinology ,medicine.anatomical_structure ,Nicotinic agonist ,Gene Expression Regulation ,Female - Abstract
Acetylcholine (ACh) is an important signaling molecule in non-neuronal systems where it is involved in regulation of viability, proliferation, differentiation and migration of mesenchymal stem cells (MSC) that are capable to differentiate into osteoblasts, chondrocytes and adipocytes. Patients with the systemic disease osteoporosis show altered MSC properties, reduced bone formation and mineral density followed by increased bone fragility and high fracture incidence. Here we asked whether nicotinic and muscarinic acetylcholine receptors (AChR) are expressed in osteoblasts, adipocytes and chondrocytes differentiated from bone MSC extracted from human reaming debris (RDMSC) that was harvested during surgery of long bone diaphyseal fractures. Using RT-PCR, AChR were detected in RDMSC, osteoblasts, chondrocytes and adipocytes of male and female bone-healthy and of female osteoporotic donors. An up-regulation in multiplicity and occurrence of AChR subtypes was found in female compared to male donors and in osteoblast of male donors compared to adipocytes. Real-time RT-PCR analysis resulted in a significant increase in relative expression of nAChR α9 in chondrocytes compared to adipocytes of healthy female donors. The nAChR subunit α10 was significantly up-regulated in osteoblasts of healthy compared to osteoporotic donors as well as the mAChR M5 that is additionally decreased in osteoporotic osteoblasts compared to MSC and chondrocytes of osteoporotic donors. In summary, the gene expression of AChR during differentiation of RDMSC and its regulation in cells of osteoporotic donors lead to the assumption that AChR signaling is involved in bone formation and might be utilized to stimulate bone remodeling processes.
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- 2015
4. Altered ultrastructure, density and cathepsin K expression in bone of female muscarinic acetylcholine receptor M3 knockout mice
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Katrin S. Lips, Imke Panzer, Sonja Hartmann, Vivien Kauschke, Christian Heiss, Mathias Kneffel, Lutz Dürselen, Marian Kampschulte, Fee Willscheid, and Frank Martin Mathies
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Male ,medicine.medical_specialty ,Cathepsin K ,Immunology ,Osteoporosis ,Bone and Bones ,Mice ,Sex Factors ,Bone Density ,Internal medicine ,Trabecular Pattern ,Muscarinic acetylcholine receptor ,medicine ,Animals ,Immunology and Allergy ,Acetylcholine receptor ,Mice, Knockout ,Receptor, Muscarinic M3 ,Pharmacology ,biology ,Chemistry ,Osteoblast ,medicine.disease ,Biomechanical Phenomena ,medicine.anatomical_structure ,Endocrinology ,Gene Expression Regulation ,RANKL ,Osteocyte ,biology.protein ,Female - Abstract
High frequency of osteoporosis is found in postmenopausal women where several molecular components were identified to be involved in bone loss that subsequently leads to an increased fracture risk. Bone loss has already been determined in male mice with gene deficiency of muscarinic acetylcholine receptor M3 (M3R-KO). Here we asked whether bone properties of female 16-week old M3R-KO present similarities to osteoporotic bone loss by means of biomechanical, radiological, electron microscopic, cell- and molecular biological methods. Reduced biomechanical strength of M3R-KO correlated with cortical thickness and decreased bone mineral density (BMD). Femur and vertebrae of M3R-KO demonstrated a declined trabecular bone volume, surface, and a higher trabecular pattern factor and structure model index (SMI) compared to wild type (WT) mice. In M3R-KO, the number of osteoclasts as well as the cathepsin K mRNA expression was increased. Osteoclasts of M3R-KO showed an estimated increase in cytoplasmic vesicles. Further, histomorphometrical analysis revealed up-regulation of alkaline phosphatase. Osteoblasts and osteocytes showed a swollen cytoplasm with an estimated increase in the amount of rough endoplasmatic reticulum and in case of osteocytes a reduced pericellular space. Thus, current results on bone properties of 16-week old female M3R-KO are related to postmenopausal osteoporotic phenotype. Stimulation and up-regulation of muscarinic acetylcholine receptor subtype M3 expression in osteoblasts might be a possible new option for prevention and therapy of osteoporotic fractures. Pharmacological interventions and the risk of side effects have to be determined in upcoming studies.
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- 2015
5. Effects of BDNF and PEC Nanoparticles on Osteocytes
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Katrin S. Lips, Vivien Kauschke, Christian Heiss, Martin Müller, Thomas Leonhard Loy, and David Vehlow
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proliferation ,osteocyte ,Pharmaceutical Science ,Bone healing ,Osteocytes ,Article ,vitality ,Cell Line ,Analytical Chemistry ,lcsh:QD241-441 ,polyelectrolyte complex nanoparticles ,Mice ,03 medical and health sciences ,0302 clinical medicine ,lcsh:Organic chemistry ,Neurotrophic factors ,Drug Discovery ,medicine ,Animals ,Physical and Theoretical Chemistry ,Cell Proliferation ,030304 developmental biology ,Brain-derived neurotrophic factor ,0303 health sciences ,Osteoblasts ,biology ,Chemistry ,Brain-Derived Neurotrophic Factor ,neurotrophin ,Organic Chemistry ,Mesenchymal stem cell ,dorsal root ganglia ,Cell Differentiation ,Osteoblast ,Polyelectrolytes ,Cell biology ,MLO-Y4 ,medicine.anatomical_structure ,nervous system ,Chemistry (miscellaneous) ,030220 oncology & carcinogenesis ,Osteocyte ,Drug delivery ,biology.protein ,Nanoparticles ,Molecular Medicine ,Neurotrophin - Abstract
Bone substitute materials loaded with mediators that stimulate fracture healing are demanded in the clinical treatment in trauma surgery and orthopedics. Brain-derived neurotrophic factor (BDNF) enhances the proliferation and differentiation of mesenchymal stem cells into osteoblast. To load the implants with BDNF, a drug delivery system that allows the release of BDNF under spatiotemporal control would improve functionality. Polyelectrolyte complex nanoparticles (PECNP) have been reported as a suitable drug delivery system. The suitability of PECNP in contact with osteocytes as the main cell type of bone is not known so far. Thus, we aimed to verify that BDNF and PECNP loaded with BDNF (PECNP+BDNF) as well as pure PECNP have no negative effects on osteocytes in vitro. Therefore, the murine osteocyte cell line MLO-Y4 was treated with BDNF and PECNP+BDNF. The effects on proliferation were analyzed by the BrdU test (n = 5). The results demonstrated a significant increase in proliferation 24 h after BDNF application, whereas PECNP+BDNF did not lead to significant changes. Thus, we conclude that BDNF is an appropriate mediator to stimulate osteocytes. Since the addition of PECNP did not affect the viability of osteocytes, we conclude that PECNP are a suitable drug delivery system for bone implants.
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- 2020
6. Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections
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Walid Mohamed, Eugen Domann, Katrin S. Lips, Cathrin Spröer, Jörg Overmann, Ursula Sommer, Julian Koettnitz, Boyke Bunk, Gopala Krishna Mannala, Torsten Hain, Volker Alt, and Christian Heiss
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0301 basic medicine ,Microbiology (medical) ,Staphylococcus aureus ,Histidine Kinase ,Virulence ,Biology ,Moths ,medicine.disease_cause ,Microbiology ,Bone and Bones ,Pathogenesis ,03 medical and health sciences ,Bacterial Proteins ,medicine ,Animals ,Humans ,Gene ,Peroxidase ,Comparative genomics ,Osteoblasts ,Whole Genome Sequencing ,Biofilm ,Quorum Sensing ,Osteomyelitis ,General Medicine ,Gene Expression Regulation, Bacterial ,Staphylococcal Infections ,medicine.disease ,Interspersed Repetitive Sequences ,Quorum sensing ,030104 developmental biology ,Infectious Diseases ,Bacteremia ,Biofilms ,Protein Kinases ,Genome, Bacterial - Abstract
Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of β-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections.
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- 2017
7. Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis
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Hans-Georg Morhenn, Katrin S. Lips, Reinhard Schnettler, Janet Beckmann, Jan Schubert, and Veronika Grau
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musculoskeletal diseases ,medicine.medical_specialty ,Pathology ,Histology ,Arthritis ,Cartilage metabolism ,Osteoarthritis ,Biology ,Pathology and Forensic Medicine ,Choline ,Non-neuronal cholinergic system ,Arthritis, Rheumatoid ,chemistry.chemical_compound ,Internal medicine ,medicine ,Humans ,Choline transporter-like proteins ,RNA, Messenger ,Rheumatoid arthritis ,Neurons ,Organic cation transporter ,Macrophages ,Synovial Membrane ,Endothelial Cells ,Membrane Transport Proteins ,Regular Article ,Cell Biology ,Fibroblasts ,medicine.disease ,Immunohistochemistry ,Acetylcholine ,Choline transporter ,medicine.anatomical_structure ,Endocrinology ,Cartilage ,chemistry ,Gene Expression Regulation ,Choline transport ,Synovial membrane ,medicine.drug ,Human - Abstract
Increasing evidence is showing that the non-neuronal cholinergic system plays an important role in the pathology of rheumatoid arthritis (RA). Choline transport into the cell is the rate-limiting step for the synthesis of acetylcholine (ACh), which can be released directly or in vesicles from the cell. However, in the human joint little is known about choline import or the release of ACh from the cell. Thus, we analyze the expression of members of the organic cation transporter (OCT), of the newly discovered choline transporter-like (CTL) family and of classical neuronal components such as the high-affinity choline transporter (CHT1) and the vesicular ACh transporter (VAChT) in the synovium and cartilage of the human hip joint from patients with osteoarthritis (OA) and RA. OCT1, OCT3 and OCTN1 and all members of the CTL family were expressed in synovial and cartilage samples. The expression of CTL1 and CTL2 was localized in synovial macrophages and fibroblasts. CHT1 mRNA expression was detectable only in the synovium, whereas VAChT was completely absent in all samples. Therefore, in the human joint, choline transport into the cell and the release of ACh seems to be mediated mainly by members of the OCT and CTL family. Expression of transporters appears not to be influenced by the pathological state, as no differences have been detected between joints from OA or RA patients. Importantly, however, all necessary components for choline import and the release of non-neuronal ACh are present in the human joint. Electronic supplementary material The online version of this article (doi:10.1007/s00441-014-2036-0) contains supplementary material, which is available to authorized users.
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- 2014
8. Nicotinic Acetylcholine Receptor α9 and α10 Subunits Are Expressed in the Brain of Mice
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Olena Lykhmus, Larysa P. Voytenko, Katrin S. Lips, Ivonne Bergen, Gabriela Krasteva-Christ, Douglas E. Vetter, Wolfgang Kummer, and Maryna Skok
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0301 basic medicine ,medicine.medical_specialty ,Cerebellum ,animal structures ,brain ,Thalamus ,Central nervous system ,RT-PCR ,Hippocampus ,Biology ,lcsh:RC321-571 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,sandwich ELISA ,Internal medicine ,medicine ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Original Research ,α7 ,Cell biology ,Nicotinic acetylcholine receptor ,α10 nicotinic acetylcholine receptors ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,nervous system ,α9 ,immunohistochemistry ,Medulla oblongata ,Alpha-4 beta-2 nicotinic receptor ,030217 neurology & neurosurgery ,Acetylcholine ,medicine.drug ,Neuroscience - Abstract
The a9 and a10 nicotinic acetylcholine receptor (nAChR) subunits are likely to be the evolutionary precursors to the entire cys-loop superfamily of ligand-gated ion channels, which includes acetylcholine, GABA, glycine, and serotonin ionotropic receptors. nAChRs containing a9 and a10 subunits are found in the inner ear, dorsal root ganglia and many non-excitable tissues, but their expression in the central nervous system has not been definitely demonstrated. Here we show the presence of both α9 and α10 nAChR subunits in the mouse brain by RT-PCR and immunochemical approaches with a range of nAChR subunit-selective antibodies, which selectivity was demonstrated in the brain preparations of a7-/-, a9-/- and a10-/- mice. The α9 and α10 RNA transcripts were found in medulla oblongata, cerebellum, midbrain, thalamus and putamen, somatosensory cortex, frontal cortex and hippocampus. High a9-selective signal in ELISA was observed in the frontal cortex, somatosensory cortex, medulla oblongata, thalamus-putamen and hippocampus and a10-selective signal was the highest in medulla oblongata and frontal cortex. The α9 and α10 proteins were found in the brain mitochondria, while their presence on the plasma membrane has not been definitely confirmed The a7-, a9- and a10-selective antibodies stained mainly neurons and hypertrophied astrocytes, but not microglia. The a9- and a10-positive cells formed ordered structures or zones in cerebellum and superior olive and were randomly distributed among α7-positive cells in the frontal cortex; they were found in CA1, CA3 and CA4, but not in CA2 region of the hippocampus. The a9 and α10 subunits were up-regulated in a7-/- mice and both α7 and a9 subunits were down-regulated in a10-/- mice. We conclude that a9 and a10 nAChR subunits are expressed in distinct neurons of the mouse brain and in the brain mitochondria and are compensatory up-regulated in the absence of α7 subunits.
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- 2017
9. Osteocyte Regulation of Receptor Activator of NF-κB Ligand/Osteoprotegerin in a Sheep Model of Osteoporosis
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Anja Schlagenhauf, Katrin S. Lips, Hans-Joachim Wilke, Wolfgang Böcker, Stefanie Kern, Fathi Hassan, Anita Ignatius, Sabine Stoetzel, Deeksha Malhan, Angela Rösen-Wolff, Christian Heiss, Felix Merboth, Dirk Rosenbaum, Felix Schulze, Natali Bauer, Judith Langenstein, Thaqif El Khassawna, Dirk Hose, Anja Secklinger, Diaa Eldin S. Daghma, Markus Rupp, Hematology, and Basic (bio-) Medical Sciences
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0301 basic medicine ,Bone density ,Osteoporosis ,Methylprednisolone/pharmacology ,surgery ,chemistry.chemical_compound ,0302 clinical medicine ,Bone Density ,Signal Transduction/drug effects ,Osteoporosis/metabolism ,Orthopedics and Sports Medicine ,Osteoprotegerin/metabolism ,biology ,hematology ,NF-kappa B ,RANK Ligand/metabolism ,medicine.anatomical_structure ,RANKL ,Osteocyte ,Female ,Spine/drug effects ,Signal Transduction ,musculoskeletal diseases ,medicine.medical_specialty ,Ovariectomy ,030209 endocrinology & metabolism ,Methylprednisolone ,Osteocytes ,Pathology and Forensic Medicine ,03 medical and health sciences ,Osteoprotegerin ,Internal medicine ,medicine ,Internal Medicine ,Animals ,Sheep ,business.industry ,RANK Ligand ,NF-κB ,Bone Density/drug effects ,medicine.disease ,Spine ,Disease Models, Animal ,030104 developmental biology ,Endocrinology ,chemistry ,NF-kappa B/metabolism ,biology.protein ,Sclerostin ,business ,Osteocytes/drug effects - Abstract
Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.
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- 2017
10. Intracellular proliferation of S. aureus in osteoblasts and effects of rifampicin and gentamicin on S. aureus intracellular proliferation and survival
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Katrin S. Lips, Walid Mohamed, Ursula Sommer, T Chakraborty, Reinhard Schnettler, Volker Alt, S Sethi, Ulrich Thormann, E Domann, and I Schütz
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intracellular proliferation ,Staphylococcus aureus ,lcsh:Diseases of the musculoskeletal system ,Cell ,lcsh:Surgery ,Biology ,gentamicin ,medicine.disease_cause ,rifampicin ,Microbiology ,Cell Line ,chemistry.chemical_compound ,medicine ,Humans ,Pathogen ,Cytochalasin D ,Cell Proliferation ,Cell growth ,osteoblasts ,lcsh:RD1-811 ,infection ,Anti-Bacterial Agents ,medicine.anatomical_structure ,chemistry ,Cell culture ,Gentamicin ,Gentamicins ,Rifampin ,lcsh:RC925-935 ,Intracellular ,medicine.drug - Abstract
Staphylococcus aureus is the most clinically relevant pathogen regarding implant-associated bone infection and its capability to invade osteoblasts is well known. The aim of this study was to investigate firstly whether S. aureus is not only able to invade but also to proliferate within osteoblasts, secondly to delineate the mechanism of invasion and thirdly to clarify whether rifampicin or gentamicin can inhibit intracellular proliferation and survival of S. aureus. The SAOS-2 osteoblast-like cell line and human primary osteoblasts were infected with S. aureus EDCC5055 and S. aureus Rosenbach 1884. Both S. aureus strains were able to invade efficiently and to proliferate within human osteoblasts. Immunofluorescence microscopy showed intracellular invasion of S. aureus and transmission electron microscopy images could demonstrate bacterial division as a sign of intracellular proliferation as well as cytosolic bacterial persistence. Cytochalasin D, the major actin depolymerisation agent, was able to significantly reduce S. aureus invasion, suggesting that invasion was enabled by promoting actin rearrangement at the cell surface. 7.5 μg/mL of rifampicin was able to inhibit bacterial survival in SAOS-2 cells with almost complete elimination of bacteria after 4 h. Gentamicin could also kill intracellular S. aureus in a dose-dependent manner, an effect that was significantly lower than that observed using rifampicin. In conclusion, S. aureus is not only able to invade but also to proliferate in osteoblasts. Invasion seems to be associated with actin rearrangement at the cell surface. Rifampicin is effective in intracellular eradication of S. aureus whereas gentamicin only poorly eliminates intracellularly replicating bacteria.
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- 2014
11. Bone formation induced by strontium modified calcium phosphate cement in critical-size metaphyseal fracture defects in ovariectomized rats
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Gudrun Schlewitz, Seemun Ray, Katrin S. Lips, Matthias Schumacher, Christian Heiss, Ursula Sommer, Michael Gelinsky, Jürgen Janek, Anja Henß, Marvin Hundgeburth, Gabor Szalay, Volker Alt, Tanja Rehling, Thaqif ElKhassawna, Reinhard Schnettler, Ulrich Thormann, and Marcus Rohnke
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Calcium Phosphates ,medicine.medical_specialty ,Materials science ,Endpoint Determination ,Ovariectomy ,medicine.medical_treatment ,Osteocalcin ,Biophysics ,chemistry.chemical_element ,Dentistry ,Biocompatible Materials ,Bioengineering ,Calcium ,Bone Morphogenetic Protein Receptors, Type II ,Osteotomy ,Rats, Sprague-Dawley ,Biomaterials ,Fractures, Bone ,Osteoprotegerin ,Osteogenesis ,Internal medicine ,medicine ,Animals ,Femur ,Bone ,Strontium ,biology ,business.industry ,Bone Cements ,Alkaline Phosphatase ,Immunohistochemistry ,Biomaterial ,Rats ,Fracture ,Endocrinology ,Calcium phosphate ,chemistry ,Mechanics of Materials ,Ceramics and Composites ,Ovariectomized rat ,biology.protein ,Alkaline phosphatase ,Female ,business - Abstract
The first objective was to investigate new bone formation in a critical-size metaphyseal defect in the femur of ovariectomized rats filled with a strontium modified calcium phosphate cement (SrCPC) compared to calcium phosphate cement (CPC) and empty defects. Second, detection of strontium release from the materials as well as calcium and collagen mass distribution in the fracture defect should be targeted by time of flight secondary ion mass spectrometry (TOF-SIMS). 45 female Sprague Dawley rats were randomly assigned to three different treatment groups: (1) SrCPC (n = 15), (2) CPC (n = 15), and (3) empty defect (n = 15). Bilateral ovariectomy was performed and three months after multi-deficient diet, the left femur of all animals underwent a 4 mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with SrCPC or CPC or was left empty. After 6 weeks, histomorphometric analysis showed a statistically significant increase in bone formation of SrCPC compared to CPC (p = 0.005) and the empty defect (p = 0.002) in the former fracture defect zone. Furthermore, there was a statistically significant higher bone formation at the tissue implant interface in the SrCPC group compared to the CPC group (p < 0.0001). These data were confirmed by immunohistochemistry revealing an increase in bone-morphogenic protein 2, osteocalcin and osteoprotegerin expression and a statistically significant higher gene expression of alkaline phosphatase, collagen10a1 and osteocalcin in the SrCPC group compared to CPC. TOF-SIMS analysis showed a high release of Sr from the SrCPC into the interface region in this area compared to CPC suggesting that improved bone formation is attributable to the released Sr from the SrCPC. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
- Published
- 2013
12. The Non-Neuronal Cholinergic System in Health and Disease
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Katrin S. Lips and Janet Beckmann
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Pharmacology ,Neurotransmitter transporter ,Polymorphism, Genetic ,General Medicine ,Biology ,Acetylcholine ,Nicotinic agonist ,Drug Design ,Vesicular acetylcholine transporter ,Muscarinic acetylcholine receptor M5 ,Disease Progression ,Muscarinic acetylcholine receptor M4 ,medicine ,Animals ,Humans ,Cholinergic ,Receptors, Cholinergic ,Molecular Targeted Therapy ,Receptor ,Neuroscience ,Autoantibodies ,medicine.drug - Abstract
Acetylcholine (ACh) is not only a neurotransmitter but is an ancient molecule that can be released by and act on non-neuronal cells. In these cells the system of ACh-synthesizing enzymes, transporters, receptors and degrading enzymes is termed the non-neuronal cholinergic system (NNCS). There is increasing evidence that the NNCS is dysregulated in various diseases and can have an influence on their pathology. However, for many organ systems not much is known about the expression and function of the NNCS. Thus, this review focusses on the role of the NNCS in different organ systems in health and disease. Dysregulation of ACh synthesis and release, mutations or polymorphisms in genes encoding NNCS components, and auto-antibodies against NNCS components are common factors influencing disease progression. Pharmacological agents targeting the NNCS are already successfully in clinical use for some disorders, indicating that interfering with this system is very promising and more research is needed to elucidate the role of the NNCS in different tissues and pathological states.
- Published
- 2013
13. Mitochondrial complex II is essential for hypoxia-induced pulmonary vasoconstriction of intra- but not of pre-acinar arteries
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Katrin S. Lips, Anna Goldenberg, Barbara Gries, José López-Barneo, Ralph T. Schermuly, Petra Faulhammer, José I. Piruat, Norbert Weissmann, Renate Paddenberg, Wolfgang Kummer, Uwe Pfeil, and Martina Tiefenbach
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Heterozygote ,medicine.medical_specialty ,Pathology ,Contraction (grammar) ,Physiology ,SDHB ,SDHA ,Blood Pressure ,Pulmonary Artery ,Biology ,Mice ,Physiology (medical) ,Internal medicine ,Hypoxic pulmonary vasoconstriction ,medicine ,Animals ,RNA, Messenger ,Hypoxia ,Lung ,Electron Transport Complex II ,Membrane Proteins ,Hypoxia (medical) ,Mice, Mutant Strains ,Succinate Dehydrogenase ,medicine.anatomical_structure ,Endocrinology ,Vasoconstriction ,Models, Animal ,SDHD ,medicine.symptom ,Cardiology and Cardiovascular Medicine - Abstract
Aims Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II ( SDHD +/−). Methods and results Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD +/− mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20–50 µm) intra-acinar and larger (51–100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD +/− mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD +/− than in wild-type mice. Conclusion SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.
- Published
- 2012
14. A new animal model for implant-related infected non-unions after intramedullary fixation of the tibia in rats with fluorescent in situ hybridization of bacteria in bone infection
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Volker, Alt, Katrin S, Lips, Christoph, Henkenbehrens, Dominik, Muhrer, Márcia Cristina, Oliveira Cavalcanti, Marcia, Cavalcanti-Garcia, Ursula, Sommer, Ulrich, Thormann, Gabor, Szalay, Christian, Heiss, Theodoros, Pavlidis, Eugen, Domann, and Reinhard, Schnettler
- Subjects
Staphylococcus aureus ,Pathology ,medicine.medical_specialty ,Prosthesis-Related Infections ,Histology ,Physiology ,Endocrinology, Diabetes and Metabolism ,Bone healing ,Biology ,law.invention ,Rats, Sprague-Dawley ,Bone Infection ,Intramedullary rod ,law ,medicine ,Animals ,Tibia ,In Situ Hybridization, Fluorescence ,Fixation (histology) ,Colony-forming unit ,X-Rays ,X-Ray Microtomography ,Bone Diseases, Infectious ,Fracture Fixation, Intramedullary ,Rats ,Tibial Fractures ,Disease Models, Animal ,Biofilms ,Fractures, Ununited ,Implant - Abstract
There is no adequate animal model to mimic the difficult clinical situation of infected non-union of the tibia after intramedullary stabilization. The purpose was to establish an animal model of implant-related infected non-unions of the tibia in rats . Furthermore, it was evaluated if detection of bacteria by fluorescent in situ hybridisation (FISH) technique is possible in bone infection. 17 rats were used in which osteotomy of the midshaft tibia was performed and stabilized with an intramedullary device. Two groups were tested: group 1: contamination of the osteotomy site with 10 4 colony forming units (CFUs) of Staphylococcus aureus (11 animals), group 2: no bacterial contamination (6 animals). The animals were sacrificed after 42 days and bone healing and infection were assessed clinically, by X-ray, micro-CT, and microbiological methods including FISH technique using EUB and STAPHY probes. Histology and scanning electron microscopy (SEM) for biofilm formation were performed. All animals of the control group showed uneventful bone healing after 6 weeks without any signs of local infections. 10 of 11 (90.9%) animals of group 1 with bacterial contamination exhibited infected non-union formation with positive clinical, radiological and microbiological infection signs of the tibia but without any systemic infection signs. FISH technique was able to identify bacteria in the infected bone. All intramedullary implants from the infected animals showed positive biofilm formation in SEM. This work presents the first animal model for the induction of intramedullary device-related infected non-union in the tibia and detection of bacteria by FISH technique in infected bone.
- Published
- 2011
15. Expression of non-neuronal cholinergic system in osteoblast-like cells and its involvement in osteogenesis
- Author
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Katja Trinkaus, Katrin S. Lips, Christian Heiss, Volker Alt, Brigitte Stigler, Olaf Kilian, Reinhard Schnettler, Maryam En-Nosse, and Sonja Hartmann
- Subjects
Histology ,Organic Cation Transport Proteins ,Bone Morphogenetic Protein 2 ,Receptors, Nicotinic ,Biology ,Cell Line ,Pathology and Forensic Medicine ,Mice ,chemistry.chemical_compound ,Osteogenesis ,Muscarinic acetylcholine receptor ,medicine ,Animals ,Humans ,Receptor ,Fracture Healing ,Osteoblasts ,Osteoblast ,Cell Biology ,Receptors, Muscarinic ,Acetylcholinesterase ,Choline acetyltransferase ,Acetylcholine ,Rats ,Up-Regulation ,Cell biology ,medicine.anatomical_structure ,Nicotinic agonist ,Carnitine Acyltransferases ,chemistry ,Biochemistry ,Butyrylcholinesterase ,Cholinergic ,Bone Remodeling ,medicine.drug - Abstract
Acetylcholine (ACh) is detected in a variety of non-neuronal cells where it acts as a para/autocrine signaling molecule controlling basic cell functions such as proliferation, differentation, and maintenance of cell-cell contacts. ACh-synthesizing enzymes include choline acetyltransferase and carnitine acetyltransferase (CarAT). ACh is released through vesicular exocytosis or directly from the cytoplasm via organic cation transporters (OCT). Extracellular ACh binds to nicotinic (nAChR) and muscarinic receptors (MR). Degradation of ACh is performed by acetylcholinesterase and butyrylcholinesterase (BChE). Here, we have determined whether these molecules are expressed in osteoblast-like cells, by means of reverse transcription polymerase chain reaction and immunohistochemistry, focusing on nAChR subunits alpha3 and alpha5. RNA for CarAT, OCT-1, M2R, M5R, nAChR subunits alpha3, alpha5, alpha9, alpha10, beta2, beta3, and BChE were detected in human (SAOS-2) and murine (MC3T3-E1) osteoblast-like cells. Other cholinergic components were only expressed species-specifically, e.g., M3R and nAChR subunit alpha7. Immunhistochemistry localized the nAChR subunits alpha3 and alpha5 in osteoblasts in vitro and in vivo where they were up-regulated after application of bone morphogenetic protein-2 (BMP-2) during fracture healing in a rat model. Thus, the cholinergic system of osteoblast-like cells might be regulated by BMP-2 during bone remodeling. Osteoblast-like cells express all necessary enzymes, transporters, and receptors for ACh synthesis and recycling.
- Published
- 2009
16. The epithelial cholinergic system of the airways
- Author
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Uwe Pfeil, Wolfgang Kummer, and Katrin S. Lips
- Subjects
medicine.medical_specialty ,Histology ,Airway epithelium ,Organic Cation Transport Proteins ,Cell ,Review ,Respiratory Mucosa ,Choline O-Acetyltransferase ,Paracrine signalling ,Brush cell ,Internal medicine ,medicine ,Animals ,Humans ,Molecular Biology ,Neuroendocrine cell ,Acetylcholine receptor ,Neurons ,Organic cation transport proteins ,biology ,Symporters ,Epithelial Cells ,Cell Biology ,Acetylcholine ,Cell biology ,Trachea ,Medical Laboratory Technology ,medicine.anatomical_structure ,Endocrinology ,biology.protein ,Respiratory epithelium ,Cholinergic ,medicine.drug - Abstract
Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types--ciliated, basal and secretory--are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.
- Published
- 2008
17. Immunohistochemical detection of nicotinic acetylcholine receptor subunits α9 and α10 in rat lung isografts and allografts
- Author
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Veronika Grau, Winfried Padberg, Katrin S. Lips, Simone Biallas, Wolfgang Kummer, Sigrid Wilker, and Sergei A. Grando
- Subjects
Pathology ,medicine.medical_specialty ,Time Factors ,medicine.medical_treatment ,Receptors, Nicotinic ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Macrophages, Alveolar ,Parenchyma ,medicine ,Animals ,Transplantation, Homologous ,Lung transplantation ,General Pharmacology, Toxicology and Pharmaceutics ,Receptor ,Lung ,Acetylcholine receptor ,General Medicine ,respiratory system ,Immunohistochemistry ,Rats ,Protein Subunits ,Transplantation, Isogeneic ,Nicotinic acetylcholine receptor ,surgical procedures, operative ,medicine.anatomical_structure ,Nicotinic agonist ,Rats, Inbred Lew ,Alveolar macrophage ,sense organs ,Lung Transplantation - Abstract
The success of clinical lung transplantation is poor in comparison to other solid organ transplants and novel therapeutic approaches are badly needed. In the view of the recent discovery of anti-inflammatory pathways mediated via nicotinic acetylcholine receptors, we investigated changes in this system in pulmonary isografts and allografts by immunohistochemistry. Lung transplantation was performed in the isogeneic Lewis to Lewis rat strain combination. For allogeneic transplantation Dark Agouti rats were used as donors. Nicotinic alpha9 and alpha10 acetylcholine receptor subunits were detected on alveolar macrophages as well as in the lung parenchyma of native and transplanted lungs. The expression of both receptor subunits was up-regulated in the parenchyma of day 4 allografts. These allografts were characterized by accumulations of alveolar macrophages strongly expressing the alpha9 and the alpha10 receptor subunit. Therapeutic application of nicotinic agonists might down-modulate pro-inflammatory functions of alveolar macrophages and protect pulmonary transplants.
- Published
- 2007
18. Polyspecific Organic Cation Transporters: Structure, Function, Physiological Roles, and Biopharmaceutical Implications
- Author
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Christopher Volk, Hermann Koepsell, and Katrin S. Lips
- Subjects
Models, Molecular ,Organic cation transport ,SLC47A1 ,Drug-Related Side Effects and Adverse Reactions ,Organic Cation Transport Proteins ,Protein Conformation ,Pharmaceutical Science ,Biopharmaceutics ,Structure-Activity Relationship ,chemistry.chemical_compound ,Cations ,Animals ,Humans ,Drug Interactions ,Pharmacokinetics ,Tissue Distribution ,Pharmacology (medical) ,Cloning, Molecular ,Transcellular ,Pharmacology ,Neurotransmitter Agents ,Binding Sites ,Polymorphism, Genetic ,Organic cation transport proteins ,biology ,Organic Chemistry ,Transporter ,Antiporters ,Pharmaceutical Preparations ,Biochemistry ,chemistry ,biology.protein ,Molecular Medicine ,Cholinergic ,Histamine ,Biotechnology - Abstract
The body is equipped with broad-specificity transporters for the excretion and distribution of endogeneous organic cations and for the uptake, elimination and distribution of cationic drugs, toxins and environmental waste products. This group of transporters consists of the electrogenic cation transporters OCT1-3 (SLC22A1-3), the cation and carnitine transporters OCTN1 (SLC22A4), OCTN2 (SLC22A5) and OCT6 (SLC22A16), and the proton/cation antiporters MATE1, MATE2-K and MATE2-B. The transporters show broadly overlapping sites of expression in many tissues such as small intestine, liver, kidney, heart, skeletal muscle, placenta, lung, brain, cells of the immune system, and tumors. In epithelial cells they may be located in the basolateral or luminal membranes. Transcellular cation movement in small intestine, kidney and liver is mediated by the combined action of electrogenic OCT-type uptake systems and MATE-type efflux transporters that operate as cation/proton antiporters. Recent data showed that OCT-type transporters participate in the regulation of extracellular concentrations of neurotransmitters in brain, mediate the release of acetylcholine in non-neuronal cholinergic reactions, and are critically involved in the regulation of histamine release from basophils. The recent identification of polymorphisms in human OCTs and OCTNs allows the identification of patients with an increased risk for adverse drug reactions. Transport studies with expressed OCTs will help to optimize pharmacokinetics during development of new drugs.
- Published
- 2007
19. Expression and distribution of cholinergic receptors in the human urothelium
- Author
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Katrin S. Lips, Ulrich Schwantes, Wolfgang Weidner, Sergei A. Grando, Thomas Bschleipfer, Wolfgang Kummer, and Konstantin Schukowski
- Subjects
medicine.medical_specialty ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Bladder Urothelium ,Internal medicine ,Muscarinic acetylcholine receptor ,medicine ,Humans ,Receptors, Cholinergic ,General Pharmacology, Toxicology and Pharmaceutics ,Urothelium ,Receptor ,Aged ,Acetylcholine receptor ,Receptor, Muscarinic M3 ,Receptor, Muscarinic M2 ,Receptor, Muscarinic M5 ,Receptor, Muscarinic M4 ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Receptor, Muscarinic M1 ,General Medicine ,Middle Aged ,Immunohistochemistry ,Receptors, Muscarinic ,Cell biology ,Protein Subunits ,Endocrinology ,Cholinergic ,Female ,Acetylcholine ,Ionotropic effect ,medicine.drug - Abstract
The bladder urothelium not only provides a diffusion barrier but it also serves a sensor function and releases signalling molecules that are considered to act in a paracrine and autocrine fashion, e.g. by acetylcholine. Its actions are conferred by two classes of receptors, i.e. G-protein-coupled muscarinic receptors (MR) and ionotropic nicotinic receptors (nAChR). In this study we set out to determine the expression and distribution of all MR subtypes (M1R-M5R) and nAChR alpha-subunits 7, 9 and 10 in the human urothelium by means of RT-PCR and immunohistochemistry, respectively. Real-time RT-PCR revealed a rank order of MR subtype expression of M2R>>M3R=M5R>M4R=M1R. Immunohistochemistry demonstrated differential distribution patterns with M1R being restricted to basal cells, M2R nearly exclusively found in umbrella cells, whereas M3R and M4R were homogenously distributed and M5R was seen in a decreasing gradient from luminal to basal. As for nAChR alpha-subunits, rank order of expression is alpha7>>alpha10>alpha9, and they were observed throughout the urothelium with a gradient decreasing from luminal to basal in intensity. In conclusion, the human urothelium carries multiple cholinergic receptor subtypes, with predominant expression of M2R, M3R and alpha7-nAChR. Their distribution as well as that of the less expressed subtypes is layer-specific in the urothelium. In view of the multiplicity of pathways to which different cholinergic receptor subtypes are coupled, we propose that this layer-specific distribution serves to stratify cholinergic regulation of human urothelial function.
- Published
- 2007
20. Administration of keratinocyte growth factor (KGF) modulates the pulmonary expression of nicotinic acetylcholine receptor subunits α7, α9 and α10
- Author
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Katrin S. Lips, Wolfgang Kummer, Veronika Grau, Winfried Padberg, Sigrid Wilker, Heinz Fehrenbach, Sergei A. Grando, and Petra Hartmann
- Subjects
medicine.medical_specialty ,Fibroblast Growth Factor 7 ,Blotting, Western ,Gene Expression ,Receptors, Nicotinic ,Pharmacology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Ganglion type nicotinic receptor ,Internal medicine ,Muscarinic acetylcholine receptor ,Muscarinic acetylcholine receptor M5 ,medicine ,Animals ,RNA, Messenger ,General Pharmacology, Toxicology and Pharmaceutics ,Lung ,Acetylcholine receptor ,Reverse Transcriptase Polymerase Chain Reaction ,Muscarinic acetylcholine receptor M3 ,Muscarinic acetylcholine receptor M2 ,General Medicine ,Immunohistochemistry ,Recombinant Proteins ,Rats ,Protein Subunits ,Nicotinic acetylcholine receptor ,Endocrinology ,Nicotinic agonist ,sense organs - Abstract
Administration of recombinant human keratinocyte growth factor (rHuKGF, Delta23N-KGF, palifermin) protects the lung against a variety of injurious stimuli. The exact mechanisms leading to lung protection are unknown. Alterations in the non-neuronal cholinergic system of the lung might be involved, as vital pulmonary functions are regulated by acetylcholine. Here, we investigated the effect of KGF on the expression of nicotinic acetylcholine receptor subunits alpha7, alpha9 and alpha10 in rat lungs. Adult rats were treated via intratracheal instillation with rHuKGF or with an equivalent volume of PBS. The expression of nicotinic acetylcholine receptor subunits was analyzed by real-time RT-PCR, immunoblotting and immunohistochemistry. Treatment with rHuKGF led to a decreased expression of nicotinic receptor subunit alpha7 in the total lung. In contrast, the expression of the receptor subunits alpha9 and alpha10 was up-regulated. In conclusion, nicotinic acetylcholine receptors are differentially regulated by KGF treatment in vivo, which might result in changes in the biological effects of acetylcholine.
- Published
- 2007
21. Down-regulation of the non-neuronal acetylcholine synthesis and release machinery in acute allergic airway inflammation of rat and mouse
- Author
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Reinhard Pabst, Katrin S. Lips, Thomas Tschernig, Wolfgang Kummer, Hermann Koepsell, Francesca Alessandrini, Veronika Grau, Tobias Stoeger, Rainer Viktor Haberberger, and Anke Lührmann
- Subjects
Ovalbumin ,Vesicular Acetylcholine Transport Proteins ,Blotting, Western ,Down-Regulation ,Stimulation ,Pharmacology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Choline O-Acetyltransferase ,Pathogenesis ,Mice ,chemistry.chemical_compound ,Rats, Inbred BN ,Vesicular acetylcholine transporter ,Macrophages, Alveolar ,Respiratory Hypersensitivity ,medicine ,Animals ,Choline ,RNA, Messenger ,General Pharmacology, Toxicology and Pharmaceutics ,Cation Transport Proteins ,Lung ,Inflammation ,Neurons ,Mice, Inbred BALB C ,Reverse Transcriptase Polymerase Chain Reaction ,Organic Cation Transporter 1 ,Membrane Transport Proteins ,Epithelial Cells ,General Medicine ,Allergens ,respiratory system ,Choline acetyltransferase ,Acetylcholine ,Rats ,Choline transporter ,chemistry ,Immunology ,Respiratory epithelium ,Female ,medicine.drug - Abstract
Acetylcholine (ACh), derived both from nerve fibres and from non-neuronal sources such as epithelial cells, is a major regulator of airway function. There is evidence that dysfunction of the neuronal cholinergic system is involved in the pathogenesis of asthma. Here, we asked whether the pulmonary non-neuronal ACh-synthesis and release machinery is altered in a rat and a mouse model of allergic airway disease. Animals were sensitized against ovalbumin, challenged by allergen inhalation, and sacrificed 24 or 48 h later. Targets of investigation were the high-affinity choline transporter-1 (CHT1), that mediates cellular uptake of choline, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the polyspecific organic cation transporters (OCT1-3), which are able to translocate choline and ACh across the plasma membrane. With cell-type specific distribution patterns, immunohistochemistry identified these proteins in airway epithelial cells and alveolar macrophages. Real-time RT-PCR revealed significant decreases in ChAT-, CHT1-, VAChT-, OCT-mRNA in the lung of sensitized and allergen challenged animals. These data were supported by immunohistochemistry, demonstrating reduced labeling intensity of airway epithelial cells. ChAT-, CHT1-, VAChT-, and OCT1-mRNA were also significantly reduced in cells recovered by bronchoalveolar lavage from sensitized and challenged rats. In conclusion, the pulmonary non-neuronal cholinergic system is down-regulated in acute allergic airway inflammation. In view of the role of ACh in maintenance of cell-cell-contacts, stimulation of fluid-secretion and of ciliary beat frequency, this down-regulation may contribute to epithelial shedding and ciliated cell dysfunction that occur in this pathological condition.
- Published
- 2007
22. Acetylcholine and Molecular Components of its Synthesis and Release Machinery in the Urothelium
- Author
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Ignaz Wessler, Ulrich Schwantes, Hermann Koepsell, Shirin Zarghooni, Julia Wunsch, Katrin S. Lips, Wolfgang Weidner, Konstantin Schukowski, Wolfgang Kummer, and Thomas Bschleipfer
- Subjects
Organic cation transport proteins ,biology ,business.industry ,Urology ,Anatomy ,Synaptic vesicle ,Molecular biology ,Choline acetyltransferase ,Acetylcholine ,Mice ,Vesicular acetylcholine transporter ,medicine ,biology.protein ,Animals ,Humans ,Cholinergic ,Urothelium ,business ,Cation transport ,medicine.drug - Abstract
Objectives Previous studies provided indirect evidence for urothelial synthesis and release of acetylcholine (ACh). We aimed to determine directly the ACh content in the urothelium and to characterize the molecular components of its synthesis and release machinery. Methods The study was performed on mouse bladder and abraded urothelium, and human mucosal bladder biopsies. ACh content was measured by high-performance liquid chromatography-electrochemical. Reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry served to investigate expression of ACh-synthesizing enzymes—choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT)—vesicular ACh transporter (VAChT), and polyspecific organic cation transporters (OCTs; isoforms 1–3). Transfected cells served to investigate whether the anticholinergic drug trospium chloride interferes with ACh-transporting OCTs. Results ACh is present in the urothelium in a nanomolar range per gram of wet weight. RT-PCR data support the presence of CarAT but not ChAT. VAChT, used by neurons to shuffle ACh into synaptic vesicles, is detected in subepithelial cholinergic nerve fibres, but not by RT-PCR or immunohistochemistry in the urothelium. OCT1 and OCT3 are expressed by the urothelium. The quarternary ammonium base trospium chloride inhibits cation transport by OCTs with a potency rank order of OCT2 (IC 50 =0.67±0.42μmol/l)>OCT1 (IC 50 =6.2±2.1μmol/l)>OCT3 (IC 50 =871±177μmol/l). Conclusions This study demonstrates a urothelial non-neuronal cholinergic system that differs widely from that of neurons with respect to molecular components of the ACh synthesis and release machinery. Consequently, these two systems might be differentially targeted by pharmacologic approaches.
- Published
- 2007
23. Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats
- Author
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Ulrich Thormann, Katrin S. Lips, Anita Ignatius, Christian Heiss, Alexander C. Langheinrich, Parameswari Govindarajan, Katharina Brodsky, David Weisweiler, Marcus Rohnke, Thaqif El Khassawna, Natali Bauer, Marian Kampschulte, Anja Henss, R. Müller, Reinhard Schnettler, Andreas Deutsch, Wolfgang Böcker, and Lutz Dürselen
- Subjects
Genetic Markers ,medicine.medical_specialty ,Integrins ,Histology ,Ovariectomy ,Osteoporosis ,Biology ,Bone resorption ,Bone remodeling ,Rats, Sprague-Dawley ,Internal medicine ,Bone cell ,medicine ,Animals ,Extracellular Signal-Regulated MAP Kinases ,Molecular Biology ,Extracellular Matrix Proteins ,Adipogenesis ,Malnutrition ,Osteoblast ,Cell Biology ,medicine.disease ,Extracellular Matrix ,Rats ,Medical Laboratory Technology ,Disease Models, Animal ,medicine.anatomical_structure ,Endocrinology ,Collagen catabolic process ,Bone Morphogenetic Proteins ,Female ,Bone marrow ,Collagen - Abstract
Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.
- Published
- 2015
24. Drug specificity and intestinal membrane localization of human organic cation transporters (OCT)
- Author
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Katrin S. Lips, Linda Metzner, Matthias Brandsch, Reinhard H.H. Neubert, Johanna Müller, and Hermann Koepsell
- Subjects
1-Methyl-4-phenylpyridinium ,Organic Cation Transport Proteins ,Brush border ,Molecular Sequence Data ,Transfection ,Biochemistry ,Intestinal absorption ,Cell membrane ,Immunolabeling ,medicine ,Humans ,Amino Acid Sequence ,Pharmacology ,Organic cation transport proteins ,biology ,Chemistry ,Cell Membrane ,Organic Cation Transporter 2 ,Biological Transport ,Intestines ,medicine.anatomical_structure ,Caco-2 ,Cell culture ,biology.protein ,Biophysics ,Caco-2 Cells ,Octamer Transcription Factor-1 - Abstract
This study was performed to investigate which human organic cation transporter, hOCT1, hOCT2 or hOCT3, participates with regard to cation specificity and membrane localization in the intestinal absorption of orally available cationic drugs. Inhibition of N-[methyl-3H]4-phenylpyridinium ([3H]MPP+) uptake by various compounds into Caco-2 cells and into cells (HEK-293 or CHO) that were stably transfected with hOCT1, hOCT2 or hOCT3 was compared. The uptake of [3H]MPP+ into Caco-2 cells was inhibited by atropine, butylscopolamine, clonidine, diphenhydramine, etilefrine, quinine and ranitidine with IC50 values between 6 microM and 4 mM. Transepithelial, apical to basal flux of [3H]MPP+ across Caco-2 cell monolayers was also strongly inhibited by these compounds. The inhibitory potency of the cationic drugs and prototypical organic cations at Caco-2 cells correlated well with the inhibitory potency measured at CHO-hOCT3 cells but much less with that at HEK-hOCT1 and -hOCT2 cells. This is functional evidence for the predominant role of hOCT3. Etilefrine and atropine were specifically transported into CHO cells by hOCT3. In Caco-2 cells, the mRNA of all three hOCT and the proteins hOCT2 and hOCT3 were detected. More importantly, immunocytochemical analyses of human jejunum revealed for the first time that hOCT3 is localized to the brush border membrane whereas hOCT1 immunolabeling was mainly observed at the lateral membranes of the enterocytes.
- Published
- 2005
25. Polyspecific Cation Transporters Mediate Luminal Release of Acetylcholine from Bronchial Epithelium
- Author
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Katrin S. Lips, Hermann Koepsell, Uwe Pfeil, Leander Ermert, Bernhard M. Schmitt, Christopher Volk, Petra Arndt, Dagmar Miska, and Wolfgang Kummer
- Subjects
Patch-Clamp Techniques ,Xenopus ,Clinical Biochemistry ,Xenopus Proteins ,Epithelium ,Xenopus laevis ,Cricetinae ,Protein Isoforms ,Budesonide ,Cation Transport Proteins ,Organic cation transport proteins ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Organic Cation Transporter 2 ,Immunohistochemistry ,Cell biology ,DNA-Binding Proteins ,Electrophysiology ,Trachea ,Protein Transport ,medicine.anatomical_structure ,Efflux ,Acetylcholine ,medicine.drug ,Pulmonary and Respiratory Medicine ,Nicotine ,medicine.medical_specialty ,Organic Cation Transport Proteins ,Bronchi ,CHO Cells ,Cell Line ,Inhibitory Concentration 50 ,Species Specificity ,Internal medicine ,medicine ,Animals ,Humans ,Patch clamp ,Glucocorticoids ,Molecular Biology ,DNA Primers ,Dose-Response Relationship, Drug ,Membrane Transport Proteins ,Biological Transport ,Transporter ,Cell Biology ,biology.organism_classification ,Asthma ,Rats ,Kinetics ,Endocrinology ,Microscopy, Fluorescence ,Oocytes ,biology.protein ,Cholinergic ,Catecholamine Plasma Membrane Transport Proteins ,Corticosterone ,Octamer Transcription Factor-1 ,Transcription Factors - Abstract
In airway epithelia, non-neuronal cholinergic regulations have been described; however, the route for acetylcholine (ACh) release has not been verified. To investigate whether organic cation transporters (OCTs) serve this function, we studied the expression of OCTs in airway epithelia and their capability to translocate ACh. Using immunohistochemistry in rats and humans, OCT1, OCT2, and OCT3 were localized to the luminal membrane of ciliated epithelial cells. In humans, OCT2 showed the strongest expression in the luminal membrane. We expressed the OCT isoforms in oocytes of Xenopus laevis and measured uptake and efflux of ACh. Tracer flux measurements showed that ACh is transported by OCT1 and OCT2 but not by OCT3. Two-electrode-voltage-clamp measurements revealed that OCT2 mediates electrogenic uptake and efflux of ACh. For ACh uptake by human OCT2, a K(M) value of approximately 0.15 mM was determined. At -50 mV, ACh efflux by human OCT2 was trans-inhibited by micromolar concentrations of the inhalational glucocorticoid budesonide, which is used in treatment of asthma (K(i) approximately 2.7 microM). The data show that OCT1 and OCT2 mediate luminal ACh release in human airways and suggest that ACh release is blocked after inhalation of budesonide.
- Published
- 2005
26. Expression of high affinity choline transporter during mouse development in vivo and its upregulation by NGF and BMP-4 in vitro
- Author
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Beata Madziar, Weronika Szczecinska, Uwe Pfeil, Katrin S. Lips, Jan Krzysztof Blusztajn, Victoria Zemelko, Brygida Berse, Ignacio Lopez-Coviella, Rafal Kaminski, and Katarzyna Kozar
- Subjects
Central Nervous System ,Vesicular Acetylcholine Transport Proteins ,Bone Morphogenetic Protein 4 ,Biology ,Choline O-Acetyltransferase ,Mice ,Phosphatidylinositol 3-Kinases ,chemistry.chemical_compound ,Developmental Neuroscience ,Vesicular acetylcholine transporter ,Nerve Growth Factor ,medicine ,Animals ,Choline ,RNA, Messenger ,Cholinergic neuron ,Cells, Cultured ,Phosphoinositide-3 Kinase Inhibitors ,Neurons ,Brain ,Gene Expression Regulation, Developmental ,Membrane Transport Proteins ,Immunohistochemistry ,Choline acetyltransferase ,Acetylcholine ,Up-Regulation ,Cell biology ,Choline transporter ,Nerve growth factor ,Animals, Newborn ,Cholinergic Fibers ,Spinal Cord ,chemistry ,Biochemistry ,Bone Morphogenetic Proteins ,Choline transport ,Developmental Biology ,medicine.drug - Abstract
An important feature of cholinergic neurons is high-affinity choline transport, which allows them to reuse choline for the synthesis of ACh needed to support cholinergic neurotransmission. The choline transporter, designated CHT, was recently cloned. We applied RT/PCR to monitor the expression of CHT in the developing mouse CNS from embryonic day 14 (E14) to postnatal day 30 (P30). We found that CHT was expressed early in development, predominantly in the regions containing cholinergic neurons. In the spinal cord, CHT mRNA was present at close to adult levels at the earliest time point examined (E14) and showed almost no changes after birth. In the striatum and the septum, CHT mRNA increased steadily during embryonic stages and leveled off after birth. Surprisingly, CHT mRNA expression was also detected in other brain regions, notably in the cerebellum, where it peaked on E19, and then rapidly declined during postnatal development. CHT protein was detected by Western blotting as a band of apparent molecular weight of 70 kDa. The accumulation of this protein during development lagged behind mRNA accumulation in all tissues. We also examined the effects of NGF and BMP-4, the potent inducers of choline acetyltransferase and vesicular acetylcholine transporter genes, on CHT expression. Both factors increased CHT mRNA accumulation in primary septal cultures. The effect of NGF was dependent on the PI3K signaling, as it was abolished by the PI3K inhibitor LY294002. This result indicates that some of the signals regulating other cholinergic-specific genes also control CHT expression.
- Published
- 2005
27. Developmental changes in the expression of nicotinic acetylcholine receptor ?-subunits in the rat heart
- Author
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Katrin S. Lips, Dörthe Brüggmann, Magdalena Chottova Dvorakova, Wolfgang Kummer, Jana Slavikova, and Jitka Kuncova
- Subjects
Histology ,Protein subunit ,Receptors, Nicotinic ,Biology ,Pathology and Forensic Medicine ,Pregnancy ,medicine ,Animals ,Myocyte ,Myocytes, Cardiac ,RNA, Messenger ,Rats, Wistar ,Fluorescent Antibody Technique, Indirect ,Receptor ,Acetylcholine receptor ,Neurons ,Messenger RNA ,Reverse Transcriptase Polymerase Chain Reaction ,Cryoelectron Microscopy ,Skeletal muscle ,Heart ,Cell Biology ,Fibroblasts ,Immunohistochemistry ,Molecular biology ,Rats ,Nicotinic acetylcholine receptor ,Nicotinic agonist ,medicine.anatomical_structure ,Animals, Newborn ,nervous system ,Female ,sense organs - Abstract
Neuronal nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels that consist of various subunits. During ontogeny, muscular and neuronal nAChR undergo changes in the distribution and subunit composition in skeletal muscle and brain, respectively. Here, we have investigated the occurrence of the ligand-binding alpha-subunits of neuronal nAChR by means of reverse transcription/polymerase chain reaction and immunohistochemistry in the rat heart during prenatal and postnatal development and after capsaicin-induced sensory denervation. mRNAs coding for the alpha4, alpha5, alpha7 and alpha10 subunits were detected throughout all developmental stages. Messenger coding for the alpha2 subunit was first detectable at developmental stage E20; alpha3 subunit mRNA was expressed throughout all prenatal developmental stages, whereas it was restricted postnatally to the atria. mRNA for alpha6 was observed at E14-P8 but was absent thereafter. At no developmental stage could an unequivocal signal for alpha9 nAChR subunit mRNA be obtained. The expression pattern was unchanged by capsaicin treatment. Immunohistochemistry demonstrated alpha7 subunits on cardiac neurons, fibroblasts and cardiomyocytes and alpha2/4 subunits on cardiomyocytes with a postnatal redistribution to intercalated discs, as shown by cryo-immunoelectron microscopy. Our results indicate an additional non-neuronal expression of nAChR subunits in the rat heart that, as in skeletal muscle, precedes functional innervation and then undergoes changes in its distribution on the surface of cells.
- Published
- 2004
28. Nicotinic acetylcholine receptor subtypes in nociceptive dorsal root ganglion neurons of the adult rat
- Author
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Rainer Viktor Haberberger, Katrin S. Lips, Michaela Kress, Nadia Bernardini, Wolfgang Kummer, and Petra Hartmann
- Subjects
Male ,Nicotine ,Central nervous system ,Pain ,Receptors, Nicotinic ,Biology ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Dorsal root ganglion ,Ganglia, Spinal ,medicine ,Animals ,RNA, Messenger ,Rats, Wistar ,Acetylcholine receptor ,Neurons ,Methyllycaconitine ,Endocrine and Autonomic Systems ,Rats ,Nicotinic acetylcholine receptor ,medicine.anatomical_structure ,Nicotinic agonist ,nervous system ,chemistry ,Female ,Neurology (clinical) ,Neuron ,Neuroscience ,Sensory nerve - Abstract
Stimulation of nicotinic acetylcholine receptors (nAChR) excites peripheral sensory nerve fibres, but also exert antinociceptive effects. The differences in these nAChR-mediated effects could be related to the expression of different nAChR subtypes located on nociceptive neurons. In the present study, we focused on the recently described alpha 10-nAChR subunit, and on alpha 4 and alpha 7 subunits, which are the most abundant subunits in the central nervous system. In nociceptive neurons from thoracic and lumbar dorsal root ganglia (DRG), nAChR subunits were found at transcriptional (RT-PCR), translational (immunohistochemistry) and functional levels. Cultured DRG neurons express mRNA for the subunits alpha 2-7 and alpha 10. The alpha-subunit proteins 4, 7 and 10 were colocalised in virtually all nociceptive neurons that were identified by immunoreactivity for the vanilloid receptor TRPV-1. These findings were corroborated by current recordings and calcium measurements, which revealed excitatory inward currents and calcium responses in capsaicin sensitive neurons.
- Published
- 2004
29. Expression of the High-Affinity Choline Transporter, CHT1, in the Neuronal and Non-neuronal Cholinergic System of Human and Rat Skin
- Author
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Uwe Pfeil, Katrin S. Lips, Wolfgang Kummer, and Rainer Viktor Haberberger
- Subjects
keratinocytes ,vasculature ,HaCaT cells ,Dermatology ,Biology ,Biochemistry ,chemistry.chemical_compound ,medicine ,Choline ,Molecular Biology ,sweat glands ,hair follicle ,integumentary system ,Transporter ,Cell Biology ,Choline acetyltransferase ,Cell biology ,Choline transporter ,HaCaT ,medicine.anatomical_structure ,chemistry ,Keratinocyte ,Rat Protein ,Acetylcholine ,medicine.drug - Abstract
Choline is an essential component in acetylcholine biosynthesis, and is involved in cell signaling. It is unable to permeate the cell membrane and requires a transporter to enter the cell. Neurons that synthesize acetylcholine take up choline by a recently cloned high-affinity choline transporter (choline transporter 1) that is Na+-dependent and can be blocked by hemicholinium-3. The aim of this study was to determine the expression and to analyze the distribution of choline transporter 1 in human and rat skin. The mRNA for choline transporter 1 was detected in rat and human skin and in the human keratinocyte cell line HaCaT. A polyclonal anti-serum was developed against the N-terminal region of the human and rat protein. In rat and human skin, choline transporter 1 immunoreactivity was present in nerve fibers. In addition, keratinocytes, HaCaT cells and cells of the internal root sheath of the hair follicle contained choline transporter 1 immunoreactivity. The labeling patterns of nonconfluent vs confluent cultured cells and the distribution of choline transporter 1 along the epidermal layer suggest an association of choline transporter 1 with keratinocyte differentiation. In conclusion, this study shows the presence of the high-affinity choline transporter choline transporter 1 in nerve fibers and epithelial cells in the human and rat skin supporting the pivotal role of this transporter in both the neuronal and non-neuronal cholinergic system of the skin.
- Published
- 2002
30. Expression of muscarinic acetylcholine receptors M3 and M5 in osteoporosis
- Author
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Christian Heiss, Katrin S. Lips, Vivien Kauschke, and Reinhard Schnettler
- Subjects
medicine.medical_specialty ,Osteoporosis ,chemistry.chemical_element ,Biology ,Calcium ,Pharmacology ,Bone resorption ,Rats, Sprague-Dawley ,Internal medicine ,Muscarinic acetylcholine receptor ,medicine ,Animals ,RNA, Messenger ,Receptor ,Bone ,Regulation of gene expression ,Receptor, Muscarinic M3 ,Messenger RNA ,Receptor, Muscarinic M5 ,Animal Study ,Non-Neuronal Cholinergic System ,General Medicine ,medicine.disease ,Endocrinology ,chemistry ,Gene Expression Regulation ,Cholinergic ,Rat ,Muscarinic Acetylcholine Receptors ,Female - Abstract
Background Cholinergic signaling via muscarinic acetylcholine receptors (mAChR) is known to influence various physiological functions. In bone, M3 mAChR and M5 mAChR were identified on the membrane of osteoblast-like cells. M3 mAChR seems to be particularly relevant for bone physiology, as signaling via this receptor was reported to increase bone formation and decrease bone resorption. Thus, in the present study we investigated the relative mRNA expression of M3 and M5 mAChR in bones of a rat osteoporosis model. Material and methods Osteoporosis was induced in Sprague-Dawley rats by bilateral ovariectomy and additional feeding of a diet deficient in calcium, vitamins C, D2, D3, and phosphorus, and free of soy and phytoestrogen. After a period of 3, 12, and 14 months, relative mRNA expression of M3 mAChR and M5 mAChR was analyzed in the 11th thoracic vertebra by real-time RT-PCR. Results Relative mRNA expression of M3 mAChR was significantly reduced in bones of osteoporotic rats compared to sham operated animals that served as controls. Further, M3 mAChR mRNA expression was significantly down-regulated when comparing 14-month osteoporotic rats to 3-month osteoporotic rats. Relative M5 mAChR mRNA was expressed to a lesser extent than M3 mAChR and did not show significant differences in mRNA expression level between the experimental groups. Conclusions M3 mAChR mRNA expression was reduced upon induction of osteoporosis and progression of disease was associated with further decrease of this receptor, indicating that M3 mAChR is involved in the development and regulation of osteoporosis.
- Published
- 2014
31. Cholinergic receptors in the murine oviduct: inventory and coupling to intracellular calcium concentration
- Author
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Katrin S. Lips, Wolfgang Kummer, Katharina Noreikat, Inés Ibañez-Tallon, Sabine Kölle, and Miriam Wolff
- Subjects
medicine.medical_specialty ,Transcription, Genetic ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Calcium in biology ,Mice ,Pregnancy ,Internal medicine ,Muscarinic acetylcholine receptor ,Mecamylamine ,medicine ,Animals ,Receptors, Cholinergic ,General Pharmacology, Toxicology and Pharmaceutics ,Fallopian Tubes ,Acetylcholine receptor ,Reverse Transcriptase Polymerase Chain Reaction ,Purinergic receptor ,General Medicine ,Epithelium ,Mice, Inbred C57BL ,Endocrinology ,medicine.anatomical_structure ,Oviduct ,Cholinergic ,Calcium ,Female ,medicine.drug - Abstract
Aims In the oviduct, muscarinic acetylcholine receptors (MR) are linked with motility regulation and nicotinic receptors (nAChR) with ectopic pregnancy. We here aimed to determine the repertoire of cholinergic receptor expression in the murine oviduct and their functional coupling to regulation of intracellular calcium concentration ([Ca 2+ ] i ). Main methods Cholinergic receptor transcripts were assessed by RT-PCR in oviductal segments (ampulla, isthmus, uterotubar junction) in all cyclic stages and pregnancy, and in laser-microdissected samples of epithelium and smooth muscle, nAChR subunit α3 distribution in tissue sections using an appropriate genetic reporter mouse strain. [Ca 2+ ] i responses were monitored in ciliated and non-ciliated oviductal cells isolated from wild-type and MR subtypes 1 and 3 gene deficient mice. Key findings Transcripts for all MR subtypes (M1–M5) are constantly expressed whereas there is some variability in nAChR expression from individual to individual. The qualitative expression pattern is independent from the hormonal status of the animal, except for nAChR α7, which is less present during pregnancy. The epithelium expresses M1, M3, nAChR α7 (data from laser-assisted microdissection) and nAChR α3 (ultrastructural investigation of reporter mice). MR dominate over nAChR in increasing [Ca 2+ ] i with being M3 the major, but not sole subtype driving this effect. The general nAChR inhibitor mecamylamine enhances muscarinic and purinergic responses. Significance In conclusion, the murine oviduct is endowed with a multiplicity of muscarinic and nicotinic receptors subtypes that, with respect to regulation of [Ca 2+ ] i , are inversely linked to each other. The major, but not sole, cholinergic receptor driving increase in [Ca 2+ ] i is M3.
- Published
- 2011
32. Does bladder outlet obstruction alter the non-neuronal cholinergic system of the human urothelium?
- Author
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Katrin S. Lips, Thomas Bschleipfer, Wolfgang Weidner, and Wolfgang Kummer
- Subjects
Male ,medicine.medical_specialty ,Organic Cation Transport Proteins ,Vesicular Acetylcholine Transport Proteins ,Fluorescent Antibody Technique ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Choline O-Acetyltransferase ,Bladder outlet obstruction ,chemistry.chemical_compound ,Internal medicine ,Vesicular acetylcholine transporter ,Muscarinic acetylcholine receptor ,medicine ,Choline ,Humans ,Receptors, Cholinergic ,General Pharmacology, Toxicology and Pharmaceutics ,Urothelium ,Aged ,Symporters ,Reverse Transcriptase Polymerase Chain Reaction ,General Medicine ,Middle Aged ,Choline acetyltransferase ,Urinary Bladder Neck Obstruction ,Nicotinic agonist ,Endocrinology ,chemistry ,Gene Expression Regulation ,RNA ,Acetylcholine ,medicine.drug - Abstract
Aims Alterations of the bladder sensory system are considered to contribute to detrusor overactivity (DO) when patients suffer from bladder outlet obstruction (BOO). The urothelium is one part of this sensory system and it harbors a non-neuronal cholinergic system (NNCS). We aimed to investigate if BOO causes alterations in the NNCS. Main methods Urothelial specimens were collected by endoscopy from six male controls and eight male patients suffering from BOO and DO. The samples were examined by immunofluorescence (IF) and real-time RT-PCR for high-affinity choline transporter-1 (CHT1), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), organic cation transporters OCT1–3, muscarinic receptor (mAChR) subtypes M1–M5 and nicotinic receptor (nAChR) subunits α7, α9 and α10. Key findings ChAT, VAChT and OCT2 are not present in the male urothelium. Real-time RT-PCR and IF detected all other investigated targets. Rank order of expression was M2 ≫ M3 = M5 > M4 = M1 for mAChR subtypes and α7 ≫ α10 > α9 for nAChR subunits. Statistical analysis of RT-PCR results did not detect significant differences between patients and controls. Only IF detected differences between both groups: α9-Immunolabeling was increased in all BOO/DO patients. Significance BOO does not induce considerable alterations of the human urothelial NNCS on mRNA level. Expression of mAChRs, CHT1, OCT1 and OCT3 is not significantly affected by BOO. Thus, transport mechanisms for choline and acetylcholine (ACh) stay unaltered. BOO increases immunolabeling of α9-nAChR but whether this sole finding contributes to the onset of DO seems questionable. Comparing the present results with our previous work, the urothelial NNCS does not differ between men and women.
- Published
- 2011
33. Presence of alpha7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective alpha-neurotoxins in histochemistry
- Author
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Katrin S. Lips, Elena V. Kryukova, Irina V. Shelukhina, Victor I. Tsetlin, and Wolfgang Kummer
- Subjects
Genotype ,alpha7 Nicotinic Acetylcholine Receptor ,Neurotoxins ,Biology ,In Vitro Techniques ,Receptors, Nicotinic ,Torpedo ,complex mixtures ,Biochemistry ,Iodine Radioisotopes ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Mice ,Radioligand Assay ,0302 clinical medicine ,Dorsal root ganglion ,Ganglia, Spinal ,medicine ,Neurotoxin ,Animals ,Axon ,Cobra Neurotoxin Proteins ,Muscle, Skeletal ,030304 developmental biology ,Acetylcholine receptor ,Mice, Knockout ,Neurons ,0303 health sciences ,Histocytochemistry ,Reverse Transcriptase Polymerase Chain Reaction ,Bungarotoxins ,3. Good health ,Cell biology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Nicotinic agonist ,Spectrometry, Fluorescence ,nervous system ,Knockout mouse ,Female ,sense organs ,Neuron ,Neuroscience ,030217 neurology & neurosurgery ,Acetylcholine ,medicine.drug - Abstract
In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.
- Published
- 2009
34. Pivotal Advance: Up-regulation of acetylcholine synthesis and paracrine cholinergic signaling in intravascular transplant leukocytes during rejection of rat renal allografts
- Author
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Zbigniew Mikulski, Uwe Pfeil, Petra Hartmann, Wolfgang Kummer, Anna Zakrzewicz, Veronika Grau, Winfried Padberg, Katrin S. Lips, Ignaz Wessler, Sigrid Wilker, and Andreas Hecker
- Subjects
Graft Rejection ,Pathology ,medicine.medical_specialty ,Isograft ,Immunology ,Biology ,Receptors, Nicotinic ,Paracrine signalling ,Adenosine Triphosphate ,In vivo ,Paracrine Communication ,medicine ,Immunology and Allergy ,Cytotoxic T cell ,Animals ,Transplantation, Homologous ,Lymphocytes ,Cation Transport Proteins ,Monocyte ,Cell Biology ,Kidney Transplantation ,Acetylcholine ,Rats ,Up-Regulation ,Transplantation ,Transplantation, Isogeneic ,medicine.anatomical_structure ,Rats, Inbred Lew ,Immunohistochemistry ,Cholinergic ,Signal Transduction - Abstract
A new role and source of the old mediator acetylcholine is described, which is produced by graft monocytes and attenuates monocytic ATP-signaling. During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti-inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half-life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT-PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double-labeling with ED1-antibody, directed to a CD68-like antigen. ACh content was measured by HPLC. [Ca2+]i was monitored by Fura-2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up-regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1-positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP-induced increase in [Ca2+]i was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up-regulate non-neuronal ACh synthesis and develop auto-/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.
- Published
- 2009
35. Administration of keratinocyte growth factor down-regulates the pulmonary capacity of acetylcholine production
- Author
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Sigrid Wilker, Wolfgang Kummer, Winfried Padberg, Veronika Grau, Ignatz Wessler, Heinz Fehrenbach, Petra Hartmann, Katrin S. Lips, and Frank Rose
- Subjects
Male ,medicine.medical_specialty ,Fibroblast Growth Factor 7 ,Cell ,Down-Regulation ,Biology ,Biochemistry ,Choline O-Acetyltransferase ,chemistry.chemical_compound ,Internal medicine ,medicine ,Animals ,RNA, Messenger ,Cation Transport Proteins ,Lung ,Surfactant homeostasis ,Epithelial Cells ,Pulmonary Surfactants ,Cell Biology ,Choline acetyltransferase ,Acetylcholine ,Recombinant Proteins ,Rats ,Choline transporter ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Rats, Inbred Lew ,Keratinocyte growth factor ,Keratinocyte ,medicine.drug - Abstract
Keratinocyte growth factor protects the lung against various injurious stimuli. The protective mechanisms, however, are not yet fully understood. The aim of this study is to determine the influence of keratinocyte growth factor on the pulmonary capacity to synthesize acetylcholine, a potent regulator of pulmonary functions which is potentially involved in lung damage. Rats were treated twice (days 1 and 2) intratracheally with keratinocyte growth factor and analyzed at day 4. The mRNA expression of choline acetyltransferase – the acetylcholine synthesizing enzyme – was analyzed by real-time RT-PCR in the lung and in isolated alveolar epithelial type II cells. Choline acetyltransferase protein was assessed by immunoblotting and immunohistochemistry. Finally, pulmonary acetylcholine content was assessed biochemically. Keratinocyte growth factor-treatment led to decreased levels of choline acetyltransferase mRNA in the lung and in isolated alveolar epithelial type II cells. Accordingly, pulmonary choline acetyltransferase protein levels were reduced and pulmonary acetylcholine content declined from 2.8 nmol (control) to 0.4 nmol acetylcholine per gram of wet weight. In conclusion, the present data show that the potent regulator of pulmonary functions, acetylcholine, is produced by the major pulmonary target cell of keratinocyte growth factor, that is alveolar epithelial type II cells. Acetylcholine synthesis is down-regulated by keratinocyte growth factor administration which might contribute to lung protection and to harmonize surfactant homeostasis under conditions of keratinocyte growth factor-induced alveolar epithelial type II cell hyperplasia.
- Published
- 2007
36. Caveolin-3 and eNOS colocalize and interact in ciliated airway epithelial cells in the rat
- Author
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Peter König, Katrin S. Lips, Wolfgang Kummer, Ana-Maria Filip, Uwe Pfeil, and Gabriela Krasteva
- Subjects
Nitric Oxide Synthase Type III ,Caveolin 3 ,Blotting, Western ,Apical cell ,Biology ,Biochemistry ,Cell membrane ,Caveolae ,medicine ,Basal body ,Animals ,Cilia ,RNA, Messenger ,Rats, Wistar ,DNA Primers ,Base Sequence ,Reverse Transcriptase Polymerase Chain Reaction ,Endoplasmic reticulum ,Antibodies, Monoclonal ,Epithelial Cells ,Cell Biology ,Immunohistochemistry ,Cell biology ,Rats ,Choline transporter ,Trachea ,Microscopy, Electron ,medicine.anatomical_structure ,Respiratory epithelium - Abstract
In ciliated airway epithelial cells endothelial nitric oxide synthase as well as several other membrane bound proteins are located in the apical cell pole. To date, mechanisms that serve to target and to keep these proteins in this region are unknown. Endothelial nitric oxide synthase is known to target to caveolae by interaction with caveolin-1 or caveolin-3. Since caveolin-1 is found only in a subpopulation of ciliated cells at the basolateral cell membrane, we examined if caveolin-3 could be responsible for the apical localization of endothelial nitric oxide synthase in ciliated cells. We used real-time RT-PCR, laser-assisted microdissection, Western blotting and double-labeling immunohistochemistry to examine the presence of caveolin-3 in the airway epithelium of the rat. Indeed, we found caveolin-3-mRNA as well as protein in ciliated cells throughout the trachea and the bronchial tree. Caveolin-3-immunoreactivity was confined to the apical region and was colocalized with endothelial nitric oxide synthase and the high affinity choline transporter in a compartment distinct from the plasma membrane at the light microscopic level. No caveolae were found in the apical plasma membrane of ciliated cells but a tubulovesicular network was present in the apical region that reached up to the basal bodies of the cilia and was in close contact with mitochondria. Co-immunoprecipitation of caveolin-3 with endothelial nitric oxide synthase verified that both proteins interact in airway ciliated cells. These findings indicate that caveolin-3 is responsible to keep endothelial nitric oxide synthase in a membrane compartment in the apical region of ciliated cells.
- Published
- 2006
37. Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse
- Author
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Rainer Viktor Haberberger, Sibel Akinci, Ignatz Wessler, Wolfgang Kummer, Jürgen Wess, Alfred H. Schinkel, Silke Wiegand, Katrin S. Lips, and Hermann Koepsell
- Subjects
Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Serotonin ,Organic Cation Transport Proteins ,Bronchoconstriction ,Bronchi ,Respiratory Mucosa ,Biology ,In Vitro Techniques ,chemistry.chemical_compound ,Mice ,Internal medicine ,Muscarine ,Muscarinic acetylcholine receptor ,medicine ,Animals ,Tissue Distribution ,Acetylcholine receptor ,lcsh:RC705-779 ,Mice, Knockout ,Receptor, Muscarinic M3 ,Receptor, Muscarinic M2 ,Research ,Organic Cation Transporter 1 ,Organic Cation Transporter 2 ,lcsh:Diseases of the respiratory system ,Epithelium ,Acetylcholine ,Trachea ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Knockout mouse ,Respiratory epithelium ,medicine.symptom ,medicine.drug - Abstract
Background It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs. Methods We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry. Results Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 ± 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 ± 3.6 %, n = 11). In wild-type mice, 5-HT (1 μM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 μM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 μM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle. Conclusion The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.
- Published
- 2005
38. Expression of the cholinergic gene locus in the rat placenta
- Author
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Katrin S. Lips, Wolfgang Kummer, Reinhard Vollerthun, and Uwe Pfeil
- Subjects
Histology ,Placenta ,Vesicular Acetylcholine Transport Proteins ,Blotting, Western ,Fluorescent Antibody Technique ,Biology ,Synaptic vesicle ,Choline O-Acetyltransferase ,Parasympathetic Nervous System ,Pregnancy ,Vesicular acetylcholine transporter ,mental disorders ,medicine ,Animals ,Rats, Wistar ,Cholinergic neuron ,Molecular Biology ,In Situ Hybridization ,Neurons ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Regulation, Developmental ,Membrane Transport Proteins ,Trophoblast ,Cell Biology ,Immunohistochemistry ,Choline acetyltransferase ,Molecular biology ,Rats ,Medical Laboratory Technology ,medicine.anatomical_structure ,nervous system ,embryonic structures ,Cholinergic ,Female ,Acetylcholine ,medicine.drug - Abstract
High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the "cholinergic gene locus" in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.
- Published
- 2004
39. Nicotinic acetylcholine receptors in rat and human placenta
- Author
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Reinhard Vollerthun, Wolfgang Kummer, Katrin S. Lips, Uwe Pfeil, Sergei A. Grando, and Dörthe Brüggmann
- Subjects
medicine.medical_specialty ,Placenta ,Biology ,Receptors, Nicotinic ,Nicotine ,Acetylcholine binding ,Syncytiotrophoblast ,Pregnancy ,Internal medicine ,medicine ,Animals ,Humans ,RNA, Messenger ,Rats, Wistar ,Receptor ,Acetylcholine receptor ,Reverse Transcriptase Polymerase Chain Reaction ,Obstetrics and Gynecology ,Rats ,Endocrinology ,medicine.anatomical_structure ,Nicotinic agonist ,Reproductive Medicine ,Fluorescent Antibody Technique, Direct ,embryonic structures ,Female ,Acetylcholine ,Developmental Biology ,medicine.drug - Abstract
Smoking during pregnancy causes low birth weight, premature delivery, neonatal morbidity, and mortality. Nicotine is a main pathogenic compound of cigarette smoke, and depresses active amino-acid uptake by human placental villi. It binds to the acetylcholine binding site of the alpha-subunits of nicotinic acetylcholine receptors (nAChR). Eight different neuronal nAChR alpha-subunits have been identified in mammals. Here, we investigated their localisation and distribution in the human and rat placenta by RT-PCR and immunofluorescence. The mRNAs of all alpha-subunits are expressed in the human and rat placenta. Immunohistochemically, subunits alpha2-5, alpha7, alpha9 and alpha10 are localised in different combinations in rat cytotrophoblast, human and rat syncytiotrophoblast, vascular smooth muscle cells, endothelial cells, Hofbauer cells, human amnion epithelium and rat visceral yolk sac epithelium. Thus, all human and rat placental cell types exhibit receptor subunits with binding sites for the endogenous ligand ACh and nicotine. ACh is suggested to be an important placental signalling molecule that, through stimulation of nAChR, controls the uptake of nutrients, blood flow and fluid volume in placental vessels, and the vascularisation during placental development. Chronic stimulation of nAChR by nicotine might result in unbalanced receptor activation or functional desensitisation followed by the known pathological effects of smoking.
- Published
- 2004
40. Nicotinic receptor mediated stimulation of NO-generation in neurons of rat thoracic dorsal root ganglia
- Author
-
S. Papadopolou, Katrin S. Lips, Petra Hartmann, Wolfgang Kummer, and Rainer Viktor Haberberger
- Subjects
Nicotine ,Nitric Oxide Synthase Type III ,alpha7 Nicotinic Acetylcholine Receptor ,Nitric Oxide Synthase Type II ,Stimulation ,Nicotinic Antagonists ,Nitric Oxide Synthase Type I ,Biology ,Receptors, Nicotinic ,Nitric Oxide ,chemistry.chemical_compound ,Dorsal root ganglion ,Ganglia, Spinal ,Mecamylamine ,medicine ,Animals ,Channel blocker ,Neurons, Afferent ,RNA, Messenger ,Rats, Wistar ,Cells, Cultured ,Acetylcholine receptor ,Methyllycaconitine ,General Neuroscience ,Cell biology ,Rats ,medicine.anatomical_structure ,Nicotinic agonist ,nervous system ,chemistry ,Female ,Fluorescein ,Neuron ,Nitric Oxide Synthase ,Neuroscience ,medicine.drug - Abstract
The contribution of nicotinic acetylcholine receptors (nAChRs) to stimulation of NO-production was investigated in isolated rat dorsal root ganglion (DRG) neurons utilizing an NO-sensitive fluorescent indicator 4,5-diaminofluorescein-diacetate (DAF-2DA) and appropriate channel blockers. RT-PCR and immunohistochemical analysis of NOS isoforms in cultured neurons revealed the expression of eNOS in the vast majority of neurons, nNOS in about 5–10%, and iNOS only exceptionally. Application of nicotine resulted in an abrupt increase in DAF-2T fluorescence in 65% of neuronal cell bodies that was fully sensitive to the general nAChR antagonist mecamylamine. Methyllycaconitine reduced the number of nicotine-sensitive neurons and the extent of NO-generation. Thus, α7- and/or α9/10-nAChRs are required for nicotine-induced NO-production in a subpopulation of DRG neurons, and appear to be partially involved in the remaining, larger subpopulation.
- Published
- 2003
41. Nicotinic receptor alpha 7-subunits are coupled to the stimulation of nitric oxide synthase in rat dorsal root ganglion neurons
- Author
-
Rainer Viktor Haberberger, Katrin S. Lips, Michael Henrich, and Wolfgang Kummer
- Subjects
Histology ,alpha7 Nicotinic Acetylcholine Receptor ,Receptors, Drug ,TRPV Cation Channels ,Stimulation ,Cell Communication ,Nicotinic Antagonists ,Mecamylamine ,Receptors, Nicotinic ,Nitric Oxide ,Dorsal root ganglion ,Ganglia, Spinal ,medicine ,Animals ,Molecular Biology ,Cyclic GMP ,Acetylcholine receptor ,Neurons ,omega-N-Methylarginine ,biology ,Cell Biology ,Anatomy ,Bungarotoxins ,Sensory neuron ,Cell biology ,Rats ,Nitric oxide synthase ,Medical Laboratory Technology ,medicine.anatomical_structure ,Nicotinic agonist ,NG-Nitroarginine Methyl Ester ,nervous system ,Verapamil ,biology.protein ,Calcium Channels ,Nitric Oxide Synthase ,Acetylcholine ,medicine.drug - Abstract
In dorsal root ganglia (DRG) intraganglionic communication takes place both among neurons and between neurons and satellite cells. One diffusible substance involved in this signalling is nitric oxide (NO), and acetylcholine (ACh) is a candidate for the stimulation of intraganglionic NO synthesis. DRG neurons react to ACh-receptor stimulation with NO-dependent cGMP production. Here, we investigated the role of the alpha 7-subunit containing Ca(2+)-permeable nicotinic ACh receptors (nAChR) in this process. The alpha 7-nAChR mRNA and the protein were expressed in virtually all lumbar DRG neurons as evidenced by laser-assisted cell picking and oligo cell RT-PCR, in situ hybridisation and immunohistochemistry. Strong alpha 7-nAChR immunoreactivity was present in vanilloid receptor 1-immunoreactive, i.e. nociceptive, neurons. A neuronal production of NO in response to nicotine could be demonstrated in DRG slice preparations utilising the NO-sensitive fluorescent indicator diaminofluorescein diacetate (DAF-2DA). This stimulation of NO production was sensitive to inhibition of alpha 7-nAChR by mecamylamine and alpha-bungarotoxin, to inhibition of nitric oxide synthase (NOS) with L-NAME and L-NMMA, and to the blockade of voltage-operated Ca(2+) channels by verapamil. The results show the presence of the alpha 7-nAChR subunit in nociceptive rat DRG neurons and provide evidence for its coupling to NOS activation, indicating a role of this pathway in the intraganglionic communication in sensory ganglia.
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- 2003
42. Expression of the high-affinity choline transporter, CHT1, in the rat trachea
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Katrin S. Lips, Wolfgang Kummer, Rainer Viktor Haberberger, Uwe Pfeil, Veronika Grau, and Lars Eberling
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Pulmonary and Respiratory Medicine ,Clinical Biochemistry ,Respiratory Mucosa ,Biology ,Vesicular acetylcholine transporter ,medicine ,Animals ,Rats, Wistar ,Molecular Biology ,In Situ Hybridization ,DNA Primers ,Tracheal Epithelium ,Base Sequence ,Reverse Transcriptase Polymerase Chain Reaction ,Membrane Transport Proteins ,Cell Biology ,respiratory system ,Apical membrane ,Molecular biology ,Choline acetyltransferase ,Immunohistochemistry ,Epithelium ,Rats ,Choline transporter ,Trachea ,medicine.anatomical_structure ,Cholinergic ,Acetylcholine ,medicine.drug - Abstract
The rate limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline by the high-affinity choline transporter (CHT1). Here, we investigated the distribution of CHT1 in the rat trachea. CHT1-mRNA was detected by reverse transcriptase-polymerase chain reaction in trachea without epithelium, abraded tracheal mucosa, and in epithelial cells obtained by laser-assisted cell-picking. Accordingly, CHT1-mRNA could also be detected in tracheal epithelial cells by in situ hybridization. Recently obtained polyclonal rabbit and guinea-pig antisera against a synthetic peptide corresponding to amino acid residues 29-40 of the rat CHT1 sequence localized CHT1 protein in combination with antisera against the vesicular acetylcholine transporter in cholinergic fibers innervating tracheal glands and the tracheal muscle. In case of the tracheal epithelium, CHT1 was restricted to the apical membrane of the ciliated cells, as demonstrated by confocal laser scanning and electron microscopy using an affinity-purified CHT1 antiserum. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favor of ACh synthesis being fueled by choline uptake via CHT1 after release and breakdown of ACh at the luminal surface. Accordingly, cholinergic regulation of tracheal epithelial function is governed by local release and recycling of ACh by ciliated cells.
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- 2003
43. Expression of the high-affinity choline transporter CHT1 in epithelia
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Rainer Viktor Haberberger, Lars Eberling, Katrin S. Lips, Wolfgang Kummer, Uwe Pfeil, and Veronika Grau
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Male ,Biology ,General Biochemistry, Genetics and Molecular Biology ,medicine ,Animals ,Humans ,Cilia ,RNA, Messenger ,General Pharmacology, Toxicology and Pharmaceutics ,Rats, Wistar ,Tracheal Epithelium ,Reverse Transcriptase Polymerase Chain Reaction ,Membrane Transport Proteins ,Epithelial Cells ,General Medicine ,Apical membrane ,Molecular biology ,Choline acetyltransferase ,Rats ,Choline transporter ,Trachea ,HaCaT ,Cholinergic Fibers ,Respiratory epithelium ,Cholinergic ,Female ,Acetylcholine ,medicine.drug - Abstract
Uptake of choline by the high-affinity choline transporter CHT1 is the rate-limiting step in neuronal acetylcholine (ACh) synthesis. Here, we investigated by RT-PCR, in-situ hybridisation, immunohistochemistry, and Western blotting whether CHT1 is also expressed in cholinergic epithelia. CHT1-mRNA and -protein were detected in keratinocytes of human skin, rat skin and tongue, the human keratinocyte cell line HaCaT, and the ciliated cells of the rat tracheal epithelium. Immunohistochemically, CHT1 was predominantly localized to the epithelial cell membranes, in case of ciliated tracheal cells it was restricted to the apical membrane. This is the first study to demonstrate the expression of CHT1 in non-neuronal cells. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favour of ACh synthesis being fuelled by choline uptake via CHT1 in these epithelia.
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- 2003
44. Coexpression of alpha 9 and alpha 10 nicotinic acetylcholine receptors in rat dorsal root ganglion neurons
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Katrin S. Lips, Wolfgang Kummer, and Uwe Pfeil
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Male ,Guinea Pigs ,Molecular Sequence Data ,Biology ,Receptors, Nicotinic ,Ganglion type nicotinic receptor ,Dorsal root ganglion ,Ganglia, Spinal ,medicine ,Animals ,Amino Acid Sequence ,RNA, Messenger ,Rats, Wistar ,Acetylcholine receptor ,Neurons ,General Neuroscience ,Sensory neuron ,Cell biology ,Rats ,Nicotinic acetylcholine receptor ,Protein Subunits ,medicine.anatomical_structure ,Nicotinic agonist ,Biochemistry ,Cholinergic ,Female ,Rabbits ,Alpha-4 beta-2 nicotinic receptor - Abstract
Previous binding studies have suggested the presence of a so far unknown nicotinic acetylcholine receptor subunit in dorsal root ganglia (Pugh et al., 1995). Here, we investigated whether the most recently identified subunit, alpha10, and its potential interaction partner, alpha9 (Elgoyhen et al., 2001), are expressed in these ganglia. All neurons of rat dorsal root ganglia, but no glial cells, expressed both alpha9 and alpha10 mRNA in in situ hybridization, and exhibited alpha10 immunoreactivity using a newly raised antibody. These findings were confirmed by RT-PCR and western blotting. The data show that dorsal root ganglion neurons coexpress alpha9 and alpha10 nicotinic receptor subunits, thereby providing the first example of neuronal expression of this receptor subunit pair.
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- 2002
45. Multiple nicotinic acetylcholine receptor alpha-subunits are expressed in the arterial system of the rat
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Uwe Pfeil, Katrin S. Lips, Wolfgang Kummer, Dörthe Brüggmann, and Rainer Viktor Haberberger
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Pathology ,medicine.medical_specialty ,Histology ,Vascular smooth muscle ,Protein subunit ,Gene Expression ,Aorta, Thoracic ,Biology ,In Vitro Techniques ,Receptors, Nicotinic ,medicine ,Animals ,Protein Isoforms ,Aorta, Abdominal ,Rats, Wistar ,Receptor ,Molecular Biology ,Aorta ,Acetylcholine receptor ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Biology ,Arteries ,Ligand (biochemistry) ,Immunohistochemistry ,Cell biology ,Rats ,Endothelial stem cell ,Medical Laboratory Technology ,Nicotinic acetylcholine receptor ,Protein Subunits ,Nicotinic agonist ,RNA - Abstract
Neuronal nicotinic acetylcholine receptors (nAChRs) are hetero- and homopentamers built up by nine different alpha-subunits and three different beta-subunits. The subtype composition within the receptor determines ligand specificity, affinity and cation permeability. In this study we focused on the distribution of the ligand binding alpha-subunits in the rat arterial system by means of RT-PCR and immunohistochemistry. Subtypes alpha3, alpha5, alpha7 and alpha10 were found to be expressed by endothelial cells, suggesting that they are equipped both with calcium-preferring (alpha7 homopentamers) and monovalent cation-preferring (heteropentamers containing alpha3- and alpha5-subunits) nAChR channels. All alpha-subtypes except alpha9 were expressed by vascular smooth muscle cells with a highly specific distribution pattern along the vascular tree. While every alpha-subunit except alpha9 was detected in the thoracic aorta, intrapulmonary arterial branches contained only alpha7 immunoreactivity, and other vascular beds held intermediate positions with respect to the extent of alpha-subunit expression. Current knowledge does not allow to correlate these distribution patterns to specific functions, but it can be anticipated that at least some components of nAChR-mediated signalling in the arterial wall are highly specific for individual arteries.
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- 2002
46. Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit
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Katrin S. Lips, Rainer Viktor Haberberger, Wolfgang Kummer, and Uwe Pfeil
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Male ,Histology ,Vesicular Acetylcholine Transport Proteins ,Vesicular Transport Proteins ,Nerve Tissue Proteins ,Biology ,Plasma Membrane Neurotransmitter Transport Proteins ,Thoracic Vertebrae ,Pathology and Forensic Medicine ,Motor Endplate ,Tongue ,Vesicular acetylcholine transporter ,medicine ,Animals ,Axon ,Rats, Wistar ,Muscle, Skeletal ,Motor Neurons ,Symporters ,Reverse Transcriptase Polymerase Chain Reaction ,Skeletal muscle ,Membrane Transport Proteins ,Ganglia, Parasympathetic ,Cell Biology ,Immunohistochemistry ,Acetylcholine ,Cell biology ,Rats ,Choline transporter ,Motor unit ,medicine.anatomical_structure ,nervous system ,Spinal Cord ,Cholinergic ,Female ,Carrier Proteins ,Neuroscience ,medicine.drug - Abstract
The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.
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- 2001
47. Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration
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Katrin S. Lips, Martin Fronius, Jürgen Lohmeyer, Zbigniew Zaslona, Uwe Pfeil, Hjalmar Kurzen, Gitte Jositsch, Wolfgang Kummer, Zbigniew Mikulski, Petra Hartmann, Wolfgang Clauss, and Veronika Grau
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Male ,Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Stimulation ,Biology ,Receptors, Nicotinic ,Calcium in biology ,Nicotine ,Mice ,610 Medical sciences Medicine ,Adenosine Triphosphate ,Cytosol ,Internal medicine ,Macrophages, Alveolar ,medicine ,Animals ,Patch clamp ,Rats, Wistar ,Receptor ,Acetylcholine receptor ,lcsh:RC705-779 ,Research ,lcsh:Diseases of the respiratory system ,Cell biology ,Rats ,Mice, Inbred C57BL ,Nicotinic agonist ,Endocrinology ,Calcium ,Female ,Ionotropic effect ,medicine.drug - Abstract
Background Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. Methods Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. Results Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. Conclusions Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.
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48. Expression of the High-affinity Choline Transporter CHT1 in Rat and Human Arteries
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Klaus Kuchelmeister, Katrin S. Lips, Christoph Rimasch, Ruediger C. Braun-Dullaeus, Uwe Pfeil, Rainer Viktor Haberberger, Wolfgang Kummer, Rupert Schmidt, and K Reiners
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0301 basic medicine ,Male ,medicine.medical_specialty ,Histology ,Vascular smooth muscle ,Endothelium ,Blotting, Western ,Fluorescent Antibody Technique ,Biology ,Choline ,03 medical and health sciences ,medicine.artery ,Internal medicine ,medicine ,Animals ,Humans ,Rats, Wistar ,Cells, Cultured ,Aged ,Aorta ,030102 biochemistry & molecular biology ,Reverse Transcriptase Polymerase Chain Reaction ,Membrane Transport Proteins ,Muscle, Smooth ,Arteries ,Middle Aged ,Molecular biology ,Choline acetyltransferase ,Rats ,Endothelial stem cell ,Choline transporter ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Cell culture ,Autoradiography ,Female ,Endothelium, Vascular ,Anatomy ,Acetylcholine ,medicine.drug - Abstract
The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.
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