Search

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+‐releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two‐pore channels 1 (TPC1) and 2 (TPC2). NAADP‐induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony‐forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly–Phe β‐naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol‐1,4,5‐trisphosphate (InsP3)‐sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP‐induced Ca2+ release also required ER‐embedded InsP3 receptors. Finally, NAADP‐induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor‐induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine‐5′‐trisphosphate‐induced Ca2+ signaling. These findings demonstrated that NAADP‐induced TPC1‐mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs. [ABSTRACT FROM AUTHOR]

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine‐induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine‐induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+‐ and NO‐sensitive dyes, Fura‐2/AM and DAF‐FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1Rs with pyrilamine, whereas H 1R was abundantly expressed at the protein level. We further found that histamine‐induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol‐1,4,5‐trisphosphate‐ and nicotinic acid dinucleotide phosphate‐sensitive channels and maintained over time by store‐operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow. [ABSTRACT FROM AUTHOR]

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+‐dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) that was prevented by the genetic blockade of M5 muscarinic receptors (M5‐mAchRs), which was the only mAchR isoform coupled to phospholipase Cβ (PLCβ) present in hCMEC/D3 cells. A comprehensive real‐time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol‐1,4,5‐trisphosphate receptors (InsP3R3), two‐pore channels 1 and 2 (TPC1–2), Stim2, Orai1–3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3R3, TPC1–2, and store‐operated Ca2+ entry (SOCE). Ach‐induced NO release, in turn, was inhibited in cells deficient of M5‐mAchRs. Likewise, Ach failed to increase NO levels in the presence of l‐NAME, a selective NOS inhibitor, or BAPTA, a membrane‐permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5‐mAchRs. Acetylcholine induces Ca2+‐dependent nitric oxide release in human brain microvascular endothelial cells by binding to M5 muscarinic receptors. This finding sheds novel light on the mechanism by which acetylcholine triggers neurovascular coupling in the brain. [ABSTRACT FROM AUTHOR]

The main route of exposure to mercury in humans is through the diet. Consequently, the gastrointestinal mucosa is exposed to the mercurial forms, where they cause intestinal fluid accumulation, mucosal injuries and diarrhea. The relationship between inorganic mercury (HgCl2) and methylmercury (CH3HgCl) exposure and water movement in the gastrointestinal tract is still unexplored. The leading role of aquaporins (AQPs) in the rapid bidirectionalmovement of fluid in the gastrointestinal tract of mammals is well established. The present study evaluates the effect of HgCl2 and CH3HgCl exposure on AQP expression in different portions of the gastrointestinal tract of rats treated by gavage (5 mg kg-1 of mercury species, single dose, 4 days). The results show thatmercury species reducemRNA and protein levels of AQPs in different parts of the gastrointestinal tract. In the stomach, treated rats show a significant reduction of expression of AQP3 (80-90% for mRNA and 50% for protein) and AQP4 (95-99% for mRNA and 20-40%for protein). In the small and large intestine, treated rats experience a significant reduction of AQP3 and AQP7 expression. Protein contents of both AQPs are reduced in similar proportions in jejunum(AQP3: 40-50%; AQP7: 45-50%) and colon (AQP3: 35-40%; AQP7: 45-60%), regardless of the treatment. Our results indicate that some AQPs are downregulated in the rat gastrointestinal tract by mercury exposure, suggesting a possible role of AQPs in the development ofmercury gastrointestinal symptoms. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca2+ signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca2+-permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca2+ entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca2+ signals uniquely in presence of extracellular Ca2+. GSK- and PMA-induced Ca2+ elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La3+ and Gd3+ dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels. J. Cell. Physiol. 229: 95-104, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Several experiments suggest an important role for store-released Ca2+ in hair cell organs: drugs targeting IP3 and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flatmounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyRα in both ampulla and canal arm and RyRβ in the ampulla only. [ABSTRACT FROM AUTHOR]

We characterize the expression pattern of aquaporin-6 in the mouse inner ear by RT-PCR and immunohistochemistry. Our data show that in the inner ear aquaporin-6 is expressed, in both vestibular and acoustic sensory epithelia, by the supporting cells directly contacting hair cells. In particular, in the Organ of Corti, expression was strongest in Deiters' cells, which provide both a mechanical link between outer hair cells (OHCs) and the Organ of Corti, and an entry point for ion recycle pathways. Since aquaporin-6 is permeable to both water and anions, these results suggest its possible involvement in regulating OHC motility, directly through modulation of water and chloride flow or by changing mechanical compliance in Deiters' cells. In further support of this role, treating mice with salicylates, which impair OHC electromotility, dramatically reduced aquaporin-6 expression in the inner ear epithelia but not in control tissues, suggesting a role for this protein in modulating OHCs' responses. [ABSTRACT FROM AUTHOR]

Background Information: Celiac disease is a chronic inflammatory disorder of the small bowel induced in genetically susceptible subjects by gluten ingestion. Diarrhoea, weight loss and malabsorption represent the major clinical presentation of the disease. Here we examined the possible alteration in the expression and localization of water channels [AQPs (aquaporins)] and some solute transporters in duodenal mucosa of celiac disease patients. Duodenal biopsies from untreated celiacs, treated celiacs, healthy controls and disease controls were considered in the present study. The expressions of some AQPs and transporter mRNAs in human duodenal biopsies were determined by semi-quantitative RT-PCR (reverse transcription PCR) and real-time RT-PCR. The localization of AQPs 3, 7 and 10 and of SGLT1 (Na+/glucose co-transporter 1), PEPT1 (H+/oligopeptide transporter 1) and NHE3 (Na+/H+ exchanger 3) was evaluated by immunohistochemistry., Results: AQPs 3, 7, 10 and 11, SGLT, PEPT and NHE, CFTR (cystic fibrosis transmembrane conductance regulator) and NKCC (Na-K-2Cl co-transporter) mRNAs were expressed in duodenal biopsies of healthy controls, treated celiac patients and disease controls. The expression of transcripts was virtually absent in duodenal biopsies of untreated celiac disease patients except for CFTR and NKCC. In healthy controls, immunohistochemistry revealed a labelling in the apical membrane of surface epithelial cells of the duodenum. The immunolabelling was heavily reduced or absent in untreated celiac patients, while it was normal in patients consuming a gluten-free diet for at least 12 months., Conclusions: Our results indicate that the main routes for water and solute absorption are deficient in celiac disease and may play a role in the onset of malabsorption symptoms.

Background Information: In the gastrointestinal tract of mammals, water can either be secreted with digestive juices or absorbed by the small and large intestine. Transcellular water movement can be mediated by the transmembrane protein family of AQPs (aquaporins), as has also been recently identified in the gastrointestinal tract. However, the localization, expression and functioning of AQPs in the gastrointestinal tract have not been completely characterized. For the present study, we investigated: (1) the expression of AQP7 in some portions of rat gastrointestinal tract by semiquantitative reverse transcriptase-PCR and by immunoblotting and (2) the cellular and subcellular localization of AQP7 by immunohistochemistry., Results: AQP7 mRNA and proteins were highly expressed in the small intestine, weakly in the caecum, colon and rectum and were absent in the stomach. Immunoblotting analysis using rat gastrointestinal tract membrane fractions showed two major bands corresponding to a molecular mass of approx. 34 and 40 kDa for the AQP7 protein. No bands were observed when the anti-AQP7 antibody was preadsorbed with the immunizing peptide. Immunohistochemistry revealed strong AQP7 labelling in the surface epithelial cells of duodenum, jejunum, ileum, caecum, colon and rectum, whereas weak or no labelling was observed in the crypt cells. The labelling was manifest particularly in the apical membrane but intracellular staining was also observed., Conclusions: The results indicate that AQP7 is present in the small and large intestine. The higher expression of AQP7 protein at the apical pole of the superficial epithelial cells suggests its involvement in rapid fluid movement through the villus epithelium.

We examined whether protein kinase C (PKC) modulates the transport systems involved in bicarbonate movements across the plasma membranes of rat jejunum. Results of enzymatic assays provide evidence that under basal conditions conventional PKC (cPKC) is present in both basolateral membranes (BLMs) and apical (brush border) membranes (BBMs) of the enterocyte. In BLMs the basal expression of the kinase is low compared to expression in BBMs; however, treatment with Ca(2+) and phorbol 12-myristate 13-acetate (PMA) causes a significant increase, thus suggesting an asymmetrical kinase translocation. To explore the effect of PKC activation on membrane-bound transport mechanisms, 'in vitro' phosphorylated membrane vesicles were used to perform uptake studies. Results suggest that PKC activation exerts an inhibitory effect on the basolateral Cl(-)-HCO(3)(-) antiporter, whereas the basolateral HCO(3)(-) conductive pathway seems to be stimulated and Cl(-) conductance unaffected. The apical, but not basolateral, Na(+)-H(+) exchanger is inhibited by PKC activation. The specificity of the response to PKC was confirmed by using the kinase inhibitor staurosporine or the inactive phorbol ester 4-alpha-PMA. The inhibition of both apical Na(+)-H(+) and basolateral Cl(-)-HCO(3)(-) exchange activities suggests that the overall action of PKC causes a reduction of transepithelial bicarbonate transport.