"Tasting" the airway lining fluid.

Specialized epithelial cells of the respiratory tract have been termed "solitary chemosensory cells" based upon the expression of components of the canonical sweet, umami and bitter taste transduction pathway, or "brush cells" based upon their characteristic morphological feature, i.e. an apical, brush-like tuft of rigid, villin containing microvilli. Cells defined by these criteria might not match one-to-one, and a generally accepted terminology is still lacking. With respect to cellular shape, ultrastructure, expression of elements of the taste transduction cascade, innervation and synapse formation, and effects evoked upon their stimulation, it appears that chemosensory/brush in the upper respiratory tract (nasal respiratory mucosa, vomeronasal duct, auditory tube), in the olfactory mucosa, in the larynx, in the lower airways (trachea, bronchi) and in the alveolar region (rat only) each represent distinct groups. Still, they have in common to monitor the chemical composition of the mucosal lining fluid. They serve as sentinels detecting bacterial colonization or the presence of other harmful components in the mucosal lining fluid, leading to the initiation of avoidance reflexes and/or local defense mechanisms which are adapted to their anatomical localization. Free nerve endings are also responsive to inhaled irritants and further work will be needed to discriminate between the contributions of such nerve endings and chemosensory cells in chemical monitoring and defense initiation. Interestingly, there is first emerging evidence that respiratory chemosensory cells may respond to more than one canonical taste quality so that they, in analogy to polymodal nociceptors, may serve as polymodal chemosensors of potentially dangerous signals.

Cholinergic chemosensory cells in the auditory tube.

The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(ß2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCß2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.

The cholinergic system in rat testis is of non-neuronal origin.

The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three ß subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.

Real-time quantitative RT-PCR after laser-assisted cell picking.

The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.

Effect of factor XIII on endothelial barrier function.

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 +/- 7% (basal value of 5.9 +/- 0.4 x 10(-6) cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 +/- 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-D-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.

Muscarinic receptor subtypes in cilia-driven transport and airway epithelial development.

Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. Mucociliary transport and possibly airway epithelial development are governed by muscarinic acetylcholine receptors but the precise roles of the subtypes involved are unknown. This issue was addressed by determining cilia-driven particle transport, ciliary beat frequency, and the composition and ultrastructural morphology of the tracheal epithelium in M1-M5 muscarinic receptor gene-deficient mice. Knockout of M3 muscarinic receptors prevented an increase in particle transport speed and ciliary beat frequency in response to muscarine. Furthermore, the ATP response after application of muscarine was blunted. Pretreatment with atropine before application of muscarine restored the response to ATP. Additional knockout of the M2 receptor in these mice partially restored the muscarine effect, most likely through the M1 receptor, and normalised the ATP response. M1, M4 and M5 receptor-deficient mice exhibited normal responses to muscarine. None of the investigated mutant mouse strains had any impairment of epithelial cellular structure or composition. In conclusion, M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. The M1 receptor increases cilia-driven particle transport if the M3 and M2 receptors are missing. None of the receptors is necessary for epithelial development.

Impact of modulators of mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) on hypoxic pulmonary vasoconstriction.

Previously, we demonstrated that hypoxic pulmonary vasoconstriction (HPV) of intra-acinar arteries (IAA) requires mitochondrial complex II (= succinate dehydrogenase, SDH) activity (citeauthor ch41:paddenberg2006, Respir Res, 7:93, citeyear ch41:paddenberg2006). Interestingly, SDH subunits A and B have recently been described as components of a multiprotein mitochondrial ATP-sensitive potassium channel (mitoK(ATP)), together with mitochondrial ATP-binding cassette protein-1, adenine nucleotide translocator (ANT), ATP synthase, and phosphate carrier (citeauthor ch41:ardehali2004, Proc Natl Acad Sci USA, 101(32):11880-5, citeyear ch41:ardehali2004). Hence, we tested the hypothesis that such an SDH-containing mitoK(ATP) is involved in HPV. For this purpose, the impact of modulators of mitoK(ATP) on HPV of IAA was studied videomorphometrically in precision cut murine lung slices. Inhibitors of mitoK(ATP) (glibenclamide, 5-hydroxydecanoate) completely suppressed HPV, mitoK(ATP) activators (pinacidil, diazoxide) even induced vasodilatation, and ANT inhibitors (bongkrekic acid, atractyloside) attenuated HPV. This pharmacological profile differs clearly from that described for mitoK(ATP). Accordingly, co-immunoprecipitation experiments provided no evidence for association of complex II subunits SDH-A, -B and -C with ANT, ATP synthase or cytochrome c oxidase in murine heart mitochondria. Hence, it is likely that the inhibitory effects on HPV that we observed in our experiments result from modulation of several mitochondrial protein complexes independently involved in the signalling cascade such as ROS-producing complex II and ANT-regulated mitochondrial permeability transition pore.

Expression of neuropeptide Y and its receptors Y1 and Y2 in the rat heart and its supplying autonomic and spinal sensory ganglia in experimentally induced diabetes.

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.

Reduced expression of nicotinic alpha subunits 3, 7, 9 and 10 in lesional and nonlesional atopic dermatitis skin but enhanced expression of alpha subunits 3 and 5 in mast cells.

BACKGROUND: The skin cholinergic signalling system is modulated in atopic dermatitis (AD). OBJECTIVES: To investigate of the role of nicotinic acetylcholine receptors (nAChRs) in the pathogenesis of AD. METHODS: We investigated the expression and localization of nAChR alpha subunits in AD by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry of biopsies from lesional and nonlesional areas of AD skin and of skin biopsies from healthy control persons. RESULTS: Our data demonstrate the presence of mRNA and protein of the nAChR alpha subunits 3, 5, 7, 9 and 10 in keratinocytes and mast cells in healthy and AD skin. Expression of the alpha subunits 3, 7, 9 and 10 was generally reduced in the skin of patients with AD whereas mast cells in AD but not in healthy skin showed alpha3 and alpha5 subunit immunoreactivity. Differences in the subunit mRNA levels between lesional and nonlesional skin were obtained for the alpha subunits 3, 9 and 10 with higher levels of alpha3 but lower levels of alpha10 subunit mRNA in lesional areas. No differences in the expression of the alpha subunits was found between the groups of extrinsic, intrinsic or mixed AD types, between genders and between smokers and nonsmokers. CONCLUSIONS: This supports the idea that the cholinergic system is dysregulated independently from inflammation in AD and that inflammation further modulates individual nAChR subunits.

Caveolin-3 differentially orchestrates cholinergic and serotonergic constriction of murine airways.

The mechanisms of controlling airway smooth muscle (ASM) tone are of utmost clinical importance as inappropriate constriction is a hallmark in asthma and chronic obstructive pulmonary disease. Receptors for acetylcholine and serotonin, two relevant mediators in this context, appear to be incorporated in specialized, cholesterol-rich domains of the plasma membrane, termed caveolae due to their invaginated shape. The structural protein caveolin-1 partly accounts for anchoring of these receptors. We here determined the role of the other major caveolar protein, caveolin-3 (cav-3), in orchestrating cholinergic and serotonergic ASM responses, utilizing newly generated cav-3 deficient mice. Cav-3 deficiency fully abrogated serotonin-induced constriction of extrapulmonary airways in organ baths while leaving intrapulmonary airways unaffected, as assessed in precision cut lung slices. The selective expression of cav-3 in tracheal, but not intrapulmonary bronchial epithelial cells, revealed by immunohistochemistry, might explain the differential effects of cav-3 deficiency on serotonergic ASM constriction. The cholinergic response of extrapulmonary airways was not altered, whereas a considerable increase was observed in cav-3-/- intrapulmonary bronchi. Thus, cav-3 differentially organizes serotonergic and cholinergic signaling in ASM through mechanisms that are specific for airways of certain caliber and anatomical position. This may allow for selective and site-specific intervention in hyperreactive states.

Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation.

Substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) have potent proinflammatory effects in the airways. They are released from sensory nerve endings originating in jugular and dorsal root ganglia. However, the major sensory supply to the airways originates from the nodose ganglion. In this study, we evaluated changes in neuropeptide biosynthesis in the sensory airway innervation of ovalbumin-sensitized and -challenged guinea pigs at the mRNA and peptide level. In the airways, a three- to fourfold increase of SP, NKA, and CGRP, was seen 24 h following allergen challenge. Whereas no evidence of local tachykinin biosynthesis was found 12 h after challenge, increased levels of preprotachykinin (PPT)-A mRNA (encoding SP and NKA) were found in nodose ganglia. Quantitative in situ hybridization indicated that this increase could be accounted for by de novo induction of PPT-A mRNA in nodose ganglion neurons. Quantitative immunohistochemistry showed that 24 h after challenge, the number of tachykinin-immunoreactive nodose ganglion neurons had increased by 25%. Their projection to the airways was shown. Changes in other sensory ganglia innervating the airways were not evident. These findings suggest that an induction of sensory neuropeptides in nodose ganglion neurons is crucially involved in the increase of airway hyperreactivity in the late response to allergen challenge.

Tuberoinfundibular peptide of 39 residues: a new mediator of cardiac function via nitric oxide production in the rat heart.

Tuberoinfundibular peptide (TIP39) was initially identified as a neurotransmitter and agonist of the PTH2 receptor, which is expressed in the cardiovascular system. This study documents for the first time the cardiac expression and function of TIP39. Expression was analyzed via RT-PCR. Function was characterized on Langendorff-perfused rat hearts as left ventricular developed pressure (LVDP) and on isolated cells via a cell edge detection system. cGMP levels were detected with RIA. Tuberoinfundibular peptide (TIP39) mRNA was found to be constitutively expressed in coronary endothelium cells, isolated cardiomyocytes, ventricles, atria, and aorta. At first we investigated the vasodilatory properties of TIP39 (100 nM) in the presence of L-nitro-arginine (L-NA, 100 microM). Surprisingly, TIP39 had no vasodilatory effect but decreased LVDP by 35 +/- 7%. In the absence of L-NA, addition of TIP39 decreased LVDP by 8 +/- 2%. The PTH2 receptor antagonist Trp23-Tyr36-PTHrP (1-36, 100 nM) abolished this TIP39 effect in the presence of L-NA. The experiments with isolated cardiomyocytes provided similar results. TIP39 (10 nM) lowered the contraction amplitude by 6 +/- 3%. In the presence of L-NA (100 micromol/liter), TIP39 lowered the amplitude by 34 +/- 6%. cGMP concentration in cardiomyocytes was stimulated by TIP39 (10 nM) in the same range as by the nitric oxide (NO) donor SNAP (100 microM). In the presence of L-NA, this increase was abolished. These results suggest that an inhibition of endogenous NO production unmasks a profound negative inotropic effect of TIP39 that is mediated by an activation of the PTH2 receptor. The results obtained with isolated cardiomyocytes suggest that myocyte-derived NO, rather than vascular NO, is responsible for this effect. cGMP seems to be the downstream signal of produced NO.

Cardiomyopathy in streptozotocin-induced diabetes involves intra-axonal accumulation of calcitonin gene-related peptide and altered expression of its receptor in rats.

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.

Nicotinic acetylcholine receptors in rat and human placenta.

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.

Identification of cholinergic chemosensory cells in mouse tracheal and laryngeal glandular ducts.

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(ß2)), 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.

Preprotachykinin-A gene expression occurs transiently in the developing rat endocrine pancreas and can be regulated in RINm5F cells.

We have reported previously that the rat insulinoma cell lines, RINm5F and RINr1046-38, express the preprotachykinin(PPT)-A gene, which encodes the tachykinin peptides, substance P and neurokinin A. Because endocrine cells of the adult rat pancreas do not appear to express PPT-A, we investigated whether the gene is expressed by rat pancreatic endocrine cells during development. We used immunohistochemistry, employing different substance P and neurokinin A antibodies, to show that many endocrine cells of the fetal and neonatal rat pancreas synthesise these products of PPT-A gene expression. Colabeling experiments revealed that a significant number of both insulin-containing and non-insulin-containing cells express tachykinins. After postnatal day 20, the number of tachykinin-immunoreactive pancreatic islet cells declines and, as already reported, none were detected in the adult rat pancreas. The transient expression of PPT-A by the developing endocrine pancreas is a novel finding. Substance P and neurokinin A are known to have trophic actions and may serve as growth factors during pancreatic islet development. PPT-A gene expression by RINm5F and RINr1046-38 cells is further evidence that these cells resemble developing pancreatic endocrine cells. They are potentially valuable as unique models for studying the regulation of tachykinin biosynthesis. We provide evidence in this study, using quantitative PPT-A messenger RNA analysis, that PPT-A expression in RINm5F cells may be up-regulated by activation of protein kinase C, down-regulated by activation of glucocorticoid receptors, and is not significantly affected by changes in intracellular cAMP levels.

Hypoxic upregulation of tyrosine hydroxylase gene expression is paralleled, but not induced, by increased generation of reactive oxygen species in PC12 cells.

Oxygen sensing was investigated in rat pheochromocytoma PC12 cells. They respond to hypoxia with an increased intracellular generation of reactive oxygen species (ROS), measured by oxidation of dihydrorhodamine 123. This increase is abolished by intracellular superoxide scavenging by Mn(III)-tetrakis(1-methyl-4-pyridyl)-porphyrin, and reduced or absent in the presence of the flavoprotein/complex I inhibitors, diphenyl-eneiodonium and rotenone. The same inhibitors, but neither intra- nor extracellular (superoxide dismutase) superoxide scavenging, abolish the hypoxia-induced increase in tyrosine hydroxylase (TH) gene expression. Thus, ROS production increases in PC12 cells during hypoxia, but this is not the cause of hypoxic TH mRNA upregulation that involves a flavoprotein.

Evidence for an esophageal origin of VIP-IR and NO synthase-IR nerves innervating the guinea pig trachealis: a retrograde neuronal tracing and immunohistochemical analysis.

Nonadrenergic noncholinergic (NANC) relaxations of the guinea pig trachea are thought to be mediated by vasoactive intestinal peptide (VIP) and nitric oxide (NO). Physiological studies have indicated that the parasympathetic ganglion neurons mediating NANC relaxations of the guinea pig trachea but not the ganglion neurons mediating cholinergic contractions are in some way associated with the adjacent esophagus. In the present study, we attempted to locate precisely the noncholinergic parasympathetic ganglia innervating the trachealis. Two days after injection of the retrograde neuronal tracer DiI into the trachealis of organotypic cultures of the guinea pig trachea and esophagus, neurons within the myenteric plexus of the esophagus or closely associated with the outer striated longitudinal muscle layers of the esophagus were labeled. Subsequent immunohistochemical analyses revealed that a majority of the retrogradely labeled neurons possessed VIP immunoreactivity (IR) or NO synthase (NOS)-IR or had VIP-IR nerve fibers associated with their cell bodies. By contrast, no labeling of esophageal neurons was seen when the tissue between the trachea and esophagus had been disrupted by blunt dissection prior to tracer injection or when the cultures were treated with the axonal transport inhibitor colchicine. The results of these experiments provide the first direct evidence that VIP-IR and NOS-IR neurons intrinsic to the guinea pig esophagus project axons to the adjacent trachealis. Based on their location and phenotype and the results of our previous studies, it is likely that these neurons are the postganglionic parasympathetic neurons mediating NANC relaxations of the trachealis.

Sensory ganglia as a target of autonomic and sensory nerve fibres in the guinea-pig.

The distribution of neuropeptide- (neuropeptide Y, substance P, vasoactive intestinal peptide) and catecholamine-synthesizing enzyme-immunoreactive axons in guinea-pig trigeminal, nodose, and cervical dorsal root ganglia was studied by double-labelling immunofluorescence in controls and after extirpation of either the cervical sympathetic trunk or the stellate ganglion; tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactive terminals in dorsal root ganglia were ultrastructurally investigated. Six neurochemically identifiable axons innervated the trigeminal ganglion, five kinds were found in the nodose and dorsal root ganglia. Two of them (catecholaminergic with and without neuropeptide Y) were of sympathetic origin and, besides their termination at arteries, provided a direct innervation of capsule cells of the trigeminal and cervical dorsal root ganglia facing the subarachnoid space. Varicosities which were interpreted as being of sensory origin were equally numerous in all ganglia, whereas those being likely of parasympathetic origin decreased in numbers from the trigeminal to the dorsal root and nodose ganglia. It is concluded that the sensory ganglia are the target of postganglionic sympathetic, parasympathetic and primary afferent neurons, each of which are specifically organized with respect to the neurochemical phenotype and inter- and intraganglionic distribution. Among other targets, these "nervi gangliorum" appear to be intimately linked to the ganglionic capsular cells and meningeal sheaths facing the liquor spaces.

Chemoreceptor A-fibres in the human carotid body contain tyrosine hydroxylase and neurofilament immunoreactivity.

Previous retrograde tracing studies on rat and guinea-pig showed a projection of sensory tyrosine hydroxylase-immunoreactive neurons to the region of the carotid bifurcation via the carotid sinus nerve. In the present study, focussing on the sensory innervation of the human carotid body, antisera to tyrosine hydroxylase and other catecholamine synthesizing enzymes were applied for an immunohistochemical investigation of carotid bodies obtained at autopsy. In addition, an array of antisera directed to non-enzyme antigens known to be present in viscero-afferent neurons were incorporated in the study. The glomic lobules consisting of glomus cells and sustentacular cells contained a variable number of enzyme-immunoreactive glomus cells. Arteries were supplied by nerve fibres displaying the full phenotype of sympathetic noradrenergic axons, i.e. immunoreactivity to tyrosine hydroxylase, aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. The glomic lobules, however, were densely innervated by tyrosine hydroxylase-immunoreactive axons lacking immunoreactivity to aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. These fibres reacted with neurofilament 160kD-antibody but were devoid of immunoreactivity to all neuropeptides tested (calcitonin gene-related peptide, somatostatin, substance P). Ultrastructurally, tyrosine hydroxylase/neurofilament 160kD-immunoreactive axons gave rise to large axonal swellings filled with mitochondria and vesicles, and established extensive contacts to glomus cells. Nerve bundles surrounded by a perineural sheath contained both myelinated (2.0-2.8 microns in diameter) and unmyelinated (0.14-3.0 microns) tyrosine hydroxylase-immunoreactive axons. Most of the unmyelinated immunoreactive axons were running singularly within a Schwann cell-sheath. Judged from the pattern of immunoreactivities as well as their preterminal and terminal ultrastructure, tyrosine hydroxylase-immunoreactive axons innervating glomus cells are of sensory origin. Although final proof by retrograde tracing cannot be presented in man, this conclusion is supported by experimental evidence in laboratory animals. The myelinated immunoreactive axons correspond to chemoreceptor A-fibres whereas the classification of the large unmyelinated immunoreactive axons has yet to be established. The lack of immunoreactivity to the dopamine-synthesizing enzyme, aromatic-L-amino-acid-decarboxylase, in this fibre type does not support the view of dopamine being the primary transmitter of chemoreceptor afferents.