Bladder outlet obstruction causes up-regulation of nicotinic acetylcholine receptors in bladder-projecting pelvic ganglion neurons.

Pelvic ganglion (PG) neurons relay sympathetic and parasympathetic signals to the lower urinary tract, comprising the urinary bladder and bladder outlet, and are thus essential for both storage and voiding reflexes. Autonomic transmission is mediated by activation of the nicotinic acetylcholine receptor (nAChR) in PG neurons. Previously, bladder outlet obstruction (BOO), secondary to benign prostatic hyperplasia, was found to increase soma sizes of bladder-projecting PG neurons. To date, however, it remains unknown whether these morphological changes are accompanied by functional plasticity in PG neurons. In the present study, we investigated whether BOO alters acetylcholine receptor (nAChR) transcript expression and current density in bladder PG neurons. Partial ligation of the rat urethra for six weeks induced detrusor overactivity (DO), as observed during cystometrical measurement. In rats exhibiting DO, membrane capacitance of parasympathetic bladder PG neurons was selectively increased. Real-time PCR analysis revealed that BOO enhanced the expression of the transcripts encoding the nAChR α3 and ß4 subunits in PG neurons. Notably, BOO significantly increased ACh-evoked current density in parasympathetic bladder PG neurons, whereas no changes were observed in sympathetic bladder and parasympathetic penile PG neurons. In addition, other ligand-gated ionic currents were immune to BOO in bladder PG neurons. Taken together, these data suggest that BOO causes upregulation of nAChR in parasympathetic bladder PG neurons, which in turn may potentiate ganglionic transmission and contribute to the development of DO.

Histomorphometric and immunohistochemical study of the goat omasum during prenatal development.

This work studies the morphological changes taking place in the goat omasum during prenatal development, using scanning electron microscope, light microscopy and immunohistochemical analysis. A total of 140 goat embryos and fetuses were used, from the first stages of prenatal life until birth. Differentiation of the omasum as a separate compartment of the primitive gastric tube was observed at 35 days of prenatal life ([crown-rump length (CRL)] 3 cm, 23% gestation). By 38 days (CRL 4.3 cm, 25% gestation) the omasal wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Omasal laminae appeared in the following order: primary at 38 days (CRL 4.3 cm, 25% gestation), secondary at 50 days (CRL 7.7 cm, 33% gestation), tertiary at 59 days (CRL 12 cm, 39% gestation) and quaternary at 64 days (CRL 13.5 cm, 43% gestation). Neuroendocrine cells were detected by synaptophysin (SYP) at 52 days (CRL 8 cm, 35% gestation), while glial cell markers (glial fibrillary acidic protein - GFAP, and vimentin-VIM) were observed at 64 days (CRL 13.5 cm, 43% gestation) and 38 days (CRL 4.3 cm, 25% gestation), respectively. Sympathetic and parasympathetic nerve fibers and nerve bodies were detected via neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) at 95 days (CRL 20 cm, 63% gestation). In conclusion, prenatal development of the omasum - like that of the rumen - appears to take place somewhat earlier in goats than in sheep or cattle, but at a similar stage to that reported in deer.

Galanin expression in the mouse major pelvic ganglia during explant culture and following cavernous nerve transection.

Autonomic neurons commonly respond to injury/axotomy with an increased expression of neuropeptides including galanin and pituitary adenylyl cyclase-activating polypeptide (PACAP). The increased peptide expression may enhance neuronal survival and axonal regeneration. Using quantitative (Q) PCR and immunocytochemistry, the present study tested whether galanin expression increased in male mouse major pelvic ganglia (MPG) neurons in response to injury. Galanin transcript expression increased significantly in MPG neurons following 72 h in explant culture and 72 h after unilateral transection of the cavernous nerve. Under both conditions, the increase in galanin transcript levels was greater than the increase in PACAP transcript levels. In control MPG, galanin-IR nerve fibers formed pericellular arrangements around MPG neurons although few galanin-IR cells were evident and many of the galanin-IR cells may be small intensely fluorescent (SIF) cells. In 3-day-cultured MPGs, many more galanin-IR cells and nerve fibers were noted. The increased galanin expression was most apparent in neurons that were also immunoreactive for neuronal nitric oxide synthase, rather than tyrosine hydroxylase. Some explant-cultured MPG neurons exhibited immunoreactivity to galanin and PACAP. As reported previously for PACAP, there is an injury-induced increase in MPG galanin expression, which occurs preferentially in the parasympathetic postganglionic neurons.

Vagus nerve stimulation protects against ventricular fibrillation independent of muscarinic receptor activation.

AIMS: The role of the vagus in the ventricle is controversial, although the vagus can protect against ventricular fibrillation (VF) via nitric oxide (NO). This study aims to determine whether the mechanisms involved are dependent on post-ganglionic release and muscarinic receptor activation. For this purpose, NO release and electrophysiological effects of vagus nerve stimulation (VNS) were evaluated in relation to acetylcholine and vasoactive intestinal peptide (VIP). In addition, the role of the coronary endothelium and afferent nerves was tested. METHODS AND RESULTS: Using the isolated innervated rabbit heart, we measured ventricular NO release using 4,5-diaminofluorescein (DAF-2) fluorescence and ventricular fibrillation threshold (VFT) during VNS after muscarinic, ganglionic, and VIP inhibition [atropine, hexamethonium, and VIP (6-28), respectively] and after Triton-X endothelial functional dysfunction. The vagal-mediated increases in NO and VFT were not significantly affected (P> 0.05) during (i) atropine perfusion [increase in NO: 196.8 ± 35.2 mV (control) vs. 156.1 ± 20.3 mV (atropine) and VFT 3.1 ± 0.5 mA (control) vs. 2.7 ± 0.4 mA (atropine)], (ii) VIP inhibition-increase in NO: 243.0 ± 42.4 mV (control) vs. 203.9 ± 28.5 mV [VIP(6-28)] and VFT 3.3 ± 0.3 mA (control) vs. 3.9 ± 0.6 mA [VIP(6-28)], or (iii) after endothelial functional dysfunction [increase in NO: 127.7 ± 31.7 mV (control) vs. 172.1 ± 31.5 mV (Triton-X) and VFT 2.6 ± 0.4 mA (control) vs. 2.5 ± 0.5 mA (Triton-X)]. However, the vagal effects were inhibited during ganglionic blockade [increase in NO: 175.1 ± 38.1 mV (control) vs. 0.6 ± 25.3 mV (hexamethonium) and VFT 3.3 ± 0.5 mA (control) vs. -0.3 ± 0.3 mA (hexamethonium)]. CONCLUSIONS: We show that the vagal anti-fibrillatory action in the rabbit ventricle occurs via post-ganglionic efferent nerve fibres, independent of muscarinic receptor activation, VIP, and the endothelium. Together with our previous publications, our data support the possibility of a novel ventricular nitrergic parasympathetic innervation and highlight potential for new therapeutic targets to treat ventricular dysrhythmias.

Complex innervation patterns of the conus arteriosus in the heart of the longnose gar, Lepisosteus osseus.

Anatomical and functional studies of the autonomic innervation in the conus arteriosus of the garfishes are lacking. This study reveals that the conus arteriosus of the longnose gar is primarily myocardial in nature, but additionally, large numbers of smooth muscle cells are present in the subendocardium. A well-developed system of adrenergic, cholinergic, substance P (SP) and neuronal nitric oxide synthase (nNOS) positive nerve terminals are found in the wall of the conus arteriosus. Coronary blood vessels running in the adventitia receive a rich supply of nNOS positive nerve fibers, thus suggesting their importance in the nitrergic control of blood flow in the conus arteriosus. The present data show that the patterns of autonomic innervation of the garfish conus arteriosus are more complex than previously appreciated.

Identification of mu- and kappa-opioid receptors as potential targets to regulate parasympathetic, sympathetic, and sensory neurons within rat intracardiac ganglia.

Recent interest has been focused on the opioid regulation of heart performance; however, specific allocation of opioid receptors to the parasympathetic, sympathetic, and sensory innervations of the heart is scarce. Therefore, the present study aimed to characterize such specific target sites for opioids in intracardiac ganglia, which act as a complex network for the integration of the heart's neuronal in- and output. Tissue samples from rat heart atria were subjected to RT-PCR, Western blot, radioligand-binding, and double immunofluorescence confocal analysis of mu (M)- and kappa (K)-opioid receptors (ORs) with the neuronal markers vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and substance P (SP). Our results demonstrated MOR- and KOR-specific mRNA, receptor protein, and selective membrane ligand binding. By using immunofluorescence confocal microscopy, MOR and KOR immunoreactivity were colocalized with VAChT in large-diameter parasympathetic principal neurons, with TH-immunoreactive small intensely fluorescent (SIF) cells, and on nearby TH-IR varicose terminals. In addition, MOR and KOR immunoreactivity were identified on CGRP- and SP-IR sensory neurons throughout intracardiac ganglia and atrial myocardium. Our findings show that MOR and KOR are expressed as mRNA and translated into specific receptor proteins on cardiac parasympathetic, sympathetic, and sensory neurons as potential binding sites for opioids. Thus, they may well play a role within the complex network for the integration of the heart's neuronal in- and output.

Remodeling of the guinea pig intrinsic cardiac plexus with chronic pressure overload.

Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted approximately 20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a twofold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators.

Arachnoid cyst causing third cranial nerve palsy manifesting as isolated internal ophthalmoplegia and iris cholinergic supersensitivity.

An 8-month-old boy presented with anisocoria, a sluggishly reactive right pupil, and cholinergic supersensitivity as the only signs of what proved months later to be compressive third cranial nerve palsy due to an arachnoid cyst. Tonic constriction and dilation, segmental iris sphincter palsy, aberrant regeneration phenomena, ductional deficits, and ptosis were absent. The initial diagnosis was postganglionic internal ophthalmoplegia attributed to a viral ciliary ganglionopathy. Nineteen months later, he had developed an incomitant exodeviation and a supraduction deficit. Brain MRI revealed a mass consistent with an arachnoid cyst compressing the third cranial nerve in the right interpeduncular cistern. Resection of the cyst led to a persistent complete third cranial nerve palsy. This is the second reported case of prolonged internal ophthalmoplegia in a young child as a manifestation of a compressive third cranial nerve palsy. Our patient serves as a reminder that isolated internal ophthalmoplegia with cholinergic supersensitivity is compatible with a preganglionic compressive third nerve lesion, particularly in a young child.

Atrial sympathetic and parasympathetic nerve sprouting and hyperinnervation induced by subthreshold electrical stimulation of the left stellate ganglion in normal dogs.

BACKGROUND: Subthreshold electrical stimulation of the left stellate ganglion (LSG) can induce nerve sprouting and sympathetic hyperinnervation in canine ventricles. It is unclear whether a similar neural plasticity involving both sympathetic and parasympathetic innervation also exists in the atria. METHODS AND RESULTS: We applied subthreshold electrical stimulation at 20 Hz (0.45 ms pulse width) or 5 Hz (1.9 ms pulse width) to the LSG in 6 normal mongrel dogs. After 41+/-9 days, the hearts were harvested and the right and left atrium stained for synaptophysin (SYN), growth-associated protein 43 (GAP43), sympathetic nerve markers tyrosine hydroxylase (TH), and parasympathetic marker choline acetyltransferase (ChAT). Tissues from 6 additional healthy dogs were used as controls. The hearts from dogs with LSG electrical stimulation had a higher density of nerve structures immunopositive to the SYN, GAP43, TH, and ChAT (P<.01) in both right and left atria. Nerve density was equal in right and left atria. There were more TH-positive nerve structures than ChAT-positive nerve structures (P<.01) for both right and left atria. No atrial arrhythmia was observed at the second surgery. CONCLUSIONS: Continuous subthreshold electrical stimulation to the LSG induces both sympathetic and parasympathetic hyperinnervation in both right and left atria in normal dogs.

Intrinsic neuronal control of the pyloric sphincter of the lamb.

To better understand the local neuronal network of the gastro-duodenal junction in ruminants, we identified the components of the enteric nervous system (ENS) innervating the pyloric sphincter (PS) of the lamb abomasum. The neurons were labelled after injecting the tracer Fast Blue (FB) into the wall of the PS, and the phenotype of the FB-labelled neurons was immunohistochemically investigated using antibodies against nitric oxide synthase (NOS), choline acetyltransferase (ChAT), tachykinin (substance P) and tyrosine hydroxylase (TH). The FB-labelled abomasal myenteric plexus (MP) neurons, observed up to 14cm from the PS, were NOS-immunoreactive (IR) (82+/-12%), ChAT-IR (51+/-29%), SP-IR (61+/-33%), and also TH-IR (2%). The descending nitrergic neurons were also SP-IR (64%) and ChAT-IR (21%); the cholinergic descending neurons were SP-IR (3%). The FB-labelled duodenal neurons were located only in the MP, up to 8cm from the sphincter and were ChAT-IR (79+/-16%), SP-IR (32+/-18%), NOS-IR (from 0 to 2%), and also TH-IR (4+/-3%). The cholinergic ascending neurons were also SP-IR (60%) whereas no ChAT-IR cells were NOS-IR. The findings of this research indicate that the sheep PS is innervated by long-projecting neurons of the abomasal and duodenal ENS.

Control by cholinergic mechanisms.

In the respiratory tract acetylcholine is neurotransmitter in ganglia and postganglionic parasympathetic nerves, but in addition is paracrine mediator released from various non-neuronal cells. Almost every cell type present in the respiratory tract expresses nicotinic and muscarinic receptors and therefore appears to be a target for acetylcholine. The present review describes the mechanisms of synthesis and release of acetylcholine from neuronal and non-neuronal cells and the differential control mechanisms. The different cholinoceptors, multiple nicotinic and muscarinic receptors and their signalling are outlined and their involvement in the modulation of the function of various target cells, smooth muscles, nerves, surface epithelial, secretory cells, fibroblasts and inflammatory cells is discussed in detail.

Peptidergic sensory and parasympathetic fiber sprouting in the mucosa of the rat urinary bladder in a chronic model of cyclophosphamide-induced cystitis.

In this study, we used a well-established animal model to investigate changes in the peptidergic and parasympathetic innervation of the bladder following chronic bladder inflammation. Adult female Sprague-Dawley rats were injected with either 70 mg/kg cyclophosphamide diluted in saline, i.p., once every 3 days or saline. After 10 days, all animals were tested for urinary frequency and number of low volume voids, as well as symptoms of spontaneous pain. At the end of 12 days, all animals were perfused with histological fixatives and the urinary bladders processed for immunofluorescence using antibodies against calcitonin gene-related peptide and the vesicular acetylcholine transporter as markers, respectively, of peptidergic primary afferent fibers and parasympathetic efferent fibers. We show that animals treated with cyclophosphamide had inflamed bladders and displayed high urinary frequency as well as some indicators of spontaneous pain, such as piloerection and a rounded-back posture. Furthermore, they had a significant increase in the density of both parasympathetic and peptidergic sensory fibers in the bladder mucosa and an increase in peptidergic sensory fibers in the detrusor muscle. Based on these results, we suggest that peripheral sprouting of parasympathetic and peptidergic fibers could be a mechanism responsible for sensitization of the bladder, leading to urinary symptoms. Since we observed that the parasympathetic and peptidergic fibers often wrapped around one another and that their varicosities were very close, these two fiber populations may be interacting with each other to lead to and maintain sensitization. Future studies are required to establish the role of this fiber sprouting in bladder symptoms.

Loss of purinergic P2X receptor innervation in human detrusor and subepithelium from adults with sensory urgency.

Purinergic P2X receptors associated with the parasympathetic nerves supplying human bladder smooth muscle (detrusor) are implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalised with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder and in adults with the urodynamics findings of sensory urgency (SU) to determine how receptor distribution varied in patients with a small bladder capacity. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody (SV2) against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for haematuria (n=22, age 33-88 years) and adults with sensory urgency at cystoscopy/cystodistension (n=11, age 37-70 years). Normal adult specimens contained detrusor muscle innervated by parasympathetic nerves possessing large varicosities (1.2 microm) distributed along their length. These mostly all showed colocalised patches of presynaptic P2X(1,2,3,5) subtypes while presynaptic subtypes P2X(4,6,7) were present in only 6-18% of varicosities. Detrusor nerve varicosities from SU patients revealed general loss of all presynaptic P2X subtypes with the proportion containing receptors reducing to only 0.5-5% depending on P2X subtype. The same loss was recorded from the sensory nerves in the surrounding lamina propria. This specific loss of P2X receptors may impair control of detrusor distension and contribute to the pathophysiology of sensory urgency.

Responses of hepatic glucose output to noxious mechanical stimulation of the skin in anaesthetised rats.

Responses of hepatic glucose output (HGO) to noxious mechanical stimulation of different skin areas were investigated in anaesthetised rats with central nervous system intact or acutely spinalized at the thoracic 1-2 (T1-T2) level by focusing on the involvement of the sympathetic and parasympathetic nerves in the responses of HGO. We measured HGO with a microdialysis probe implanted into the left lateral lobe of the liver. Pinching was applied to bilateral skin areas of the abdomen and hindlimb for 10 min. Atropine was injected in order to block the action of the parasympathetic nerves, whereas phentolamine and propranolol were injected in order to block the action of the sympathetic nerves. The HGO started to increase immediately after the cessation of pinching of the abdomen and the hindlimb, and lasted for 30 min. The increase of HGO was observed during stimulus period in animals pretreated with atropine, and totally abolished in animals pretreated with phentolamine and propranolol. The responses of HGO to abdominal pinching, but not to hindlimb pinching, remained after spinal cord transection at the T1-T2 level. The present results suggest that HGO is regulated as a reflex response via both sympathetic and parasympathetic nerves by noxious mechanical stimulation of the skin. Furthermore, it was shown that relative contribution of the spinal and supraspinal organization to the somato-HGO responses was dependent on the skin areas stimulated.

Effect of cryoinjury on the contractile parameters of bladder strips: a model of impaired detrusor contractility.

In anesthetized Sprague-Dawley rats, the bladder was exposed and cryoinjury was induced by abruptly freezing the serosal side of the bladder wall with a chilled aluminum rod previously placed on dry ice (-40 degrees C). Five days later, the rats were euthanized, and strips were prepared from the area adjacent to the injury. Neurally and alpha,beta methylene-ATP (alpha,beta m-ATP; 50 microM)-evoked contractions were measured in bladder strips from cryoinjured or intact bladders prepared from sham-operated rats. Cryoinjured bladder strips produced significantly lower contractile forces than intact strips to electrical stimulation at higher (10-40 Hz) frequencies. The maximal rate of the neurally evoked contractions was slower in the cryoinjured bladders. The contractile response to alpha,beta m-ATP was smaller in the cryoinjured preparations indicating that the changes may have also occurred at the postjunctional site. In addition, atropine was more effective at inhibiting the neurally evoked contractions in the cryoinjured bladder strips suggesting that a cholinergic dominance occurs after cryoinjury. It is concluded that cryoinjury is a viable method of causing a defined, reproducible injury to the urinary bladder resulting in impaired function of both the cholinergic transmission and the smooth muscle. The bladder cryoinjury can be used as a model for studying impaired bladder compliance and detrusor contractility as well as treatments that may improve bladder function such as tissue engineering.

Nitric oxide synthase I and VIP-activated signaling are affected in salivary glands of NOD mice.

The autoimmune sialadenitis developed by non-obese diabetic (NOD) mice is considered a suitable model to study the ethiopathogenic mechanisms leading to sicca symptoms in Sjögren's syndrome (SS). Evidence supporting a neural rather than immune origin of the secretory dysfunction has been provided. As both nitric oxide and vasoactive intestinal peptide (VIP) are common messengers to nervous and immune systems mediating secretory and inflammatory responses, we examined nitric oxide synthase (NOS) activity with special focus on VIP-mediated effects in salivary glands of NOD mice. We found a decreased NOS activity and expression in major salivary glands of NOD mice with respect to control mice. In addition, there was a deficient VIP-activated signaling associated with a reduced saliva and amylase secretion in response to VIP. Our results support the hypothesis of an impaired balance of neuroimmune interactions in salivary glands as early events to take place in the progressive loss of secretory function of NOD mice.

Early and late pathogenesis of natural scrapie infection in sheep.

The pathogenesis of scrapie infection was studied in sheep carrying the PrP(VRQ)/PrP(VRQ) genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie-associated prion protein PrP(Sc), a marker for scrapie infectivity The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyer's patches of the caudal jejunum and ileum, the so-called gut-associated lymphoid tissues (GALT). At the same time, PrP(Sc) was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyer's patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non-GALT-related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8-T10, respectively.

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia.

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurones of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), or the desensitising agonist, alpha,beta-methylene ATP. In contrast, EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 microM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 microM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gamma S, reversibly depressed EPSPs amplitude. The inhibitory effects of ATP and ATP gamma S on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurones from rat submandibular ganglia, focal application of ATP (100 microM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 microM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurones to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability.

Effects of nociceptin on cardiac norepinephrine and acetylcholine release evoked by ouabain.

We investigated whether the novel peptide, nociceptin, modulates neuronal transmission at autonomic nerve endings. Using a cardiac dialysis technique, the effects of locally applied nociceptin on cardiac acetylcholine (ACh) and norepinephrine (NE) release were examined in anesthetized cats. Dialysis probes were implanted in the left ventricular wall, with the concentration of dialysate NE or ACh serving as an indicator of NE or ACh output at cardiac sympathetic or parasympathetic nerve endings. Locally applied ouabain evoked increases in NE and ACh output. Nociceptin suppressed the ouabain induced ACh increment. The ouabain induced NE release was not altered by nociceptin. However, in the presence of desipramine (a NE uptake inhibitor), nociceptin suppressed the ouabain-induced NE release. Inhibition by nociceptin of ouabain-induced release of NE or ACh was blocked by pretreatment with nocistatin (a nociceptin action blocking peptide). Nociceptin-induced inhibition of ACh or NE release is attributable to pre-synaptic modulation rather than a reversal of the ouabain effect. These findings demonstrate that nociceptin inhibits cardiac autonomic neurotransmission via a presynaptic opioid receptor-like1(ORL1) receptor.

Autonomic regulation of cystatin S gene expression in rat submandibular glands.

Innervation of rat submandibular and parotid glands by the autonomic nervous system regulates saliva volume, its rate of secretion and its composition. The autonomic nervous system also plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of specific sets of genes. Rat cystatin S, a member of family 2 of the cysteine proteinase inhibitor superfamily, is expressed in submandibular and parotid glands of human and rat. In the rat, cystatin S gene expression is tissue- and cell type-specific, is temporally regulated during postnatal development, and not observed in adult animals. The beta-adrenergic agonist isoproterenol (IPR) induces hypertrophic and hyperplastic enlargements of rat salivary glands and the expression of a number of genes including cystatin S. Sympathectomy reduces, but does not completely block, IPR-induced expression of the cystatin S gene in submandibular glands of adult female rats, indicating the participation of sympathetic factor(s) in its regulation. Bilateral parasympathectomy also reduces IPR-induced cystatin S gene expression, suggesting a role of the parasympathetic nervous system in its regulation. Experiments described in this paper suggest that similar factor(s) arising from both the sympathetic and parasympathetic branches of the autonomic nervous system simultaneously participate in IPR-induced cystatin S gene expression in submandibular glands.