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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 α-subunits of nicotinic acetylcholine receptors (nAChR). Eight different neuronal nAChR α-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 α-subunits are expressed in the human and rat placenta. Immunohistochemically, subunits α2–5, α7, α9 and α10 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. [Copyright &y& Elsevier]

Abstract: The integrity of gamete transport, fertilization, and early embryonic development in the oviduct are essential prerequisites for successful reproduction. Although the basic mechanisms of gamete transport, gamete interaction, and early embryogenesis are known in most mammals, the interactions between gametes and oviductal epithelium as well as the communication between the early embryo and the female reproductive tract remain to be elucidated. Recent techniques of live cell imaging such as digital videomicroscopy and confocal fluorescence microscopy are valuable tools that provide actual new insights into these interactions. By applying these techniques, the mechanisms of sperm transport, sperm storage, oocyte transport, gamete interaction, and early embryo-maternal crosstalk can be analyzed under in vivo or in situ conditions. Detailed knowledge of these very early and important processes creates the basis to develop new therapeutic concepts for subfertility and infertility and to improve the techniques of assisted reproduction. The current review will focus on a short description of recent techniques of live cell imaging in the reproductive tract followed by an overview of actual observations during the early events of reproduction. [Copyright &y& Elsevier]

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, α10, and its potential interaction partner, α9 (Elgoyhen et al., 2001), are expressed in these ganglia. All neurons of rat dorsal root ganglia, but no glial cells, expressed both α9 and α10 mRNA in in situ hybridization, and exhibited α10 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 α9 and α10 nicotinic receptor subunits, thereby providing the first example of neuronal expression of this receptor subunit pair. [Copyright &y& Elsevier]

Specialized epithelial cells in the respiratory tract such as solitary chemosensory cells and brush cells sense the luminal content and initiate protective reflexes in response to the detection of potentially harmful substances. The majority of these cells are cholinergic and utilize the canonical taste signal transduction cascade to detect “bitter” substances such as bacterial quorum sensing molecules. Utilizing two different mouse strains reporting expression of choline acetyltransferase (ChAT), the synthesizing enzyme of acetylcholine (ACh), we detected cholinergic cells in the submucosal glands of the murine larynx and trachea. These cells were localized in the ciliated glandular ducts and were neither found in the collecting ducts nor in alveolar or tubular segments of the glands. ChAT expression in tracheal gland ducts was confirmed by in situ hybridization. The cholinergic duct cells expressed the brush cell marker proteins, villin and cytokeratin-18, and were immunoreactive for components of the taste signal transduction cascade (Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel = TRPM5, phospholipase C β2 ), but not for carbonic anhydrase IV. Furthermore, these cells expressed the bitter taste receptor Tas2r131, as demonstrated utilizing an appropriate reporter mouse strain. Our study identified a previously unrecognized presumptive chemosensory cell type in the duct of the airway submucosal glands that likely utilizes ACh for paracrine signaling. We propose that these cells participate in infection-sensing mechanisms and initiate responses assisting bacterial clearance from the lower airways. [ABSTRACT FROM AUTHOR]

To date, there is only a fragmentary understanding of the fundamental mechanisms of airway mucociliary transport. Application of the latest measurement techniques can aid in deciphering the complex interplay between ciliary beat and airway surface liquid (ASL) transport. In the present study, direct, quasi-simultaneous measurements of the cilia-induced fluid and bead transport were performed to gain a better insight into both transport mechanisms. In this study cilia-induced periciliary liquid (PCL) transport is measured by means of micro Particle Image Velocimetry (µPIV) with neutrally buoyant tracers. Particle Tracking Velocimetry (PTV) with heavier polystyrene-ferrite beads is performed to simulate particle transport. Contrary to recent literature, in which the presence of mucus was deemed necessary to maintain periciliary liquid (PCL) transport, effective particle and fluid transport was measured in our experiments in the absence of mucus. In response to muscarine or ATP stimulation, maximum fluid transport rates of 250 µm/s at 15 µm distance to the tracheal epithelia were measured while bead transport rates over the epithelia surfaces reached 200 µm/s. We estimated that the mean bead transport is dominated by viscous drag compared to inertial fluid forces. Further more, mean bead transport velocities appear to be two orders of magnitude larger compared to bead sedimentation velocities. There fore, beads are expected to closely follow the mean PCL flow in non-ciliated epithelium regions. Based on our results, we have shown that PCL transport can be directly driven by the cilia beat and that the PCL motion may be capable of driving bead transport by fluid drag. [ABSTRACT FROM AUTHOR]

Abstract: Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT–PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3β4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, β2–4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR – α3∗, α6∗ and α7∗ – and of additional combinations with β-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n =1367) were eGFP–nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP–nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP–nAChRα3 were observed, with eGFP–nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations. [Copyright &y& Elsevier]

Abstract: Aim: The airway epithelial surface is constantly exposed to inhaled environmental factors and pathogens. Bitter “tasting” bacterial products such as quorum sensing molecules (QSM) can be detected by solitary chemosensory cells of the upper respiratory tract. Recently, we have shown that tracheal brush cells are cholinergic chemosensory cells affecting the respiration upon stimulation with bitter substances. Here, we explore the hypothesis that tracheal brush cells are capable of detection of bacterial products such as QSM resulting in changes in respiration and in induction of local effects, e.g. regulation of mucociliary clearance. Main methods: Functional analyses of respiration were performed in the trachea using a newly established model for investigation of respiration in spontaneously breathing anesthetized mice upon isolated tracheal stimulation. Influence of N-3-oxododecanoyl-homoserine lactone (3-OxoC12-HSL) on cilia-driven particle transport speed (PTS) in the airways was investigated in acutely excised and submerged mouse tracheae. Key findings: 3-OxoC12-HSL, a Pseudomonas aeruginosa quorum sensing autoinducer, caused a drop in the respiratory rate 2min after the application at the mucosal surface. The 3-OxoC12-HSL-induced effect on respiration was abolished by inhibition of nicotinic receptors with mecamylamine and by removal of the respiratory epithelium. At the same concentration, 3-OxoC12-HSL enhanced significantly PTS on the mucosal surface. Significance: We conclude that cholinergic airway epithelial cells sense bacterial QSM in the airway lining fluid and communicate this to the CNS via ACh release and nicotinic stimulation of sensory neurons. In addition, QSM enhance PTS. [Copyright &y& Elsevier]

Abstract: Cell bodies of afferent neurons located in lumbosacral dorsal root ganglia (DRG) provide Aδ- and C-fibres to the urinary bladder, reporting bladder wall tension, volume and noxious stimuli. Recent studies suggested an involvement of muscarinic acetylcholine receptors (mAChRs) not only in detrusor contractility but also in modulating afferent function, and this has been linked to the beneficial effects of muscarinic antagonists in the treatment of overactive bladder. Here, we aimed to determine the inventory of mAChR subtypes expressed by bladder afferent neurons in the mouse. Bladder afferent neurons were identified by retrograde neuronal tracing using Fast Blue (FB) or 1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorhydrate (DiI) injection into the detrusor muscle. DRG L6-S1 were recognized as the major location of bladder afferent perikarya with an additional smaller peak at L1/L2. Retrogradely labelled bladder afferents located in DRG L4-S2 were subjected to immunohistochemistry or to laser-assisted microdissection with subsequent RT-PCR to study expression of mAChRs subtypes M1R–M5R. Immunolabelling for mAChR subtype M2R, validated on DRG from M2R gene-deficient mice, demonstrated this subtype on 35% of FB-labelled bladder afferents. RT-PCR demonstrated expression of subtypes M2R, M3R and M4R, but not of M1R and M5R, in pooled samples (30 section profiles each) of laser microdissected DiI-labelled bladder afferent cell bodies. In conclusion, bladder afferent neurons express different subtypes of mAChRs (M2R, M3R and M4R). Thus, processing of sensory information from the bladder appears to be under direct cholinergic control. [Copyright &y& Elsevier]

Abstract: Diabetic cardiomyopathy, involving both cardiomyocytes and the sensory and autonomic cardiac innervation, is a major life-threatening complication in diabetes mellitus. Here, we induced long-term (26–53 weeks) diabetes in rats by streptozotocin injection and analyzed the major cardiac neuropeptide signaling system, neuropeptide Y (NPY) and its receptors Y1R and Y2R. Heart compartments and ganglia supplying sympathetic (stellate ganglion) and spinal sensory fibers (upper thoracic dorsal root ganglia=DRG) were analyzed separately by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Ventricular, but not atrial innervation density by NPY-immunoreactive fibers was diminished, and preproNPY expression was transiently (26 weeks) reduced in left atria, but remained unchanged in sympathetic neurons and was not induced in DRG neurons. In all ganglia and heart compartments, Y1R expression dominated over Y2R, and Y1R-immunoreactivity was observed on cardiomyocytes and neuronal perikarya. Atrial, but not ventricular Y1R expression was up-regulated after 1 year of diabetes. Collectively, these data show that a disturbance of the cardiac NPY–Y1R/Y2R signaling system develops slowly in the course of experimentally induced diabetes and differentially affects atria and ventricles. This is in parallel with the clinically observed imbalances of the cardiac autonomic innervation in diabetic cardiac autonomic neuropathy. [Copyright &y& Elsevier]

Abstract: Calcitonin gene-related peptide (CGRP) is a vasorelaxant and positive inotropic and chronotropic peptide that binds to the calcitonin receptor-like receptor. In the heart, upon stimulation CGRP is released from sensory nerve terminals and improves cardiac perfusion and function. In the present study, we investigated alterations in the components of the CGRP signaling system during development of diabetic cardiomyopathy. Rats received a single injection of streptozotocin. Four, 8, and 16 weeks thereafter cardiac CGRP content (radioimmunoassay), calcitonin receptor-like receptor expression (by real-time RT-PCR), and CGRP and calcitonin receptor-like receptor tissue distribution (immunohistochemistry) were assessed. CGRP content of atria and ventricles progressively increased during the 4 months following streptozotocin-treatment, while the distribution of CGRP-immunoreactive fibers was not visibly altered. Conversely, cardiac expression of calcitonin receptor-like receptor initially (4 weeks after treatment) increased but then gradually declined to 47% of control levels in both atria after 16 weeks. These quantitative changes were not associated with altered cellular distribution patterns (primarily in venous and capillary endothelium). Since sensory neurons have been reported to decrease expression of the CGRP precursor in the course of diabetes, the intra-axonal accumulation of CGRP observed here reflects impaired release, which, coupled with the down-regulation of its cognate receptor, calcitonin receptor-like receptor, may contribute to the well-documented impairment of cardioprotective functions in diabetes. [Copyright &y& Elsevier]

Recently, we have demonstrated that sensory neurons of rat lumbar dorsal root ganglia (DRG) respond to hypoxia with an activation of endothelial nitric oxide (NO) synthase (eNOS) resulting in enhanced NO production associated with mitochondria which contributes to resistance against hypoxia. Extracellular calcium is essential to this effect. In the present study on rat DRG slices, we set out to determine what types of calcium channels operate under hypoxia, and which upstream events contribute to their activation, thereby focusing upon mitochondrial complex II. Both the metallic ions Cd2+ and Ni2+, known to inhibit voltage-gated calcium channels and T-type channels, respectively, and verapamil and nifedipine, typical blocker of L-type calcium channels completely prevented the hypoxic neuronal NO generation. Inhibition of complex II by thenoyltrifluoroacetone at the ubiquinon binding site or by 3-nitropropionic acid at the substrate binding site largely diminished hypoxic-induced NO production while having an opposite effect under normoxia. An additional blockade of voltage-gated calcium channels entirely abolished the hypoxic response. The complex II inhibitor malonate inhibited both normoxic and hypoxic NO generation. These data show that complex II activity is required for increased hypoxic NO production. Since succinate dehydrogenase activity of complex II decreased at hypoxia, as measured by histochemistry and densitometry, we propose a hypoxia-induced functional switch of complex II from succinate dehydrogenase to fumarate reductase, which subsequently leads to activation of voltage-gated calcium channels resulting in increased NO production by eNOS. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]