Intrinsic neurones of the gall bladder modulate its function. Nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) are present in gall bladder neurones and nitric oxide and VIP modulate its epithelial functions. As an extensive extrinsic innervation of the gall bladder is also present, the source of the epithelial innervation is unclear. In this study the source of the gall bladder epithelial innervation is defined. Immunoreactivity for VIP, NOS, substance P (SP), calcitonin gene related peptide (CGRP) and tyrosine hydroxylase (TH) in organotypic cultured and freshly fixed gall bladder were compared. Retrograde tracing in vitro from the epithelium was used to identify putative intrinsic secretomotor neurones, which were then characterized by immunohistochemistry. Abundant spinal afferent and sympathetic innervation of the gall bladder epithelium was demonstrated by CGRP/SP and TH immunohistochemistry, respectively. The intrinsic secretomotor innervation of the epithelium is derived exclusively from neurones of the subepithelial plexus. A majority of these neurones were immunoreactive for NOS. Some of the NOS-immunoreactive neurones of the subepithelial plexus also contained VIP and/or SP. Gall bladder subepithelial plexus neurones, containing NOS and/or VIP/SP, innervate the epithelium, as do extrinsic neurones.

Electrical field stimulation (EFS) of dog gallbladder strips induced a frequency-dependent contractile response followed by an off-relaxation that was turned into a pure inhibitory response after atropine pretreatment. Guanethidine reduced the atropine-induced relaxing responses, so an adrenergic mechanism can partially account for the nerve-mediated gallbladder relaxation. However, guanethidine pretreatment also revealed a nonadrenergic noncholinergic (NANC) relaxation induced by EFS, which was frequency independent. NANC relaxations were reduced by L-arginine methyl ester (L-NAME, 100 micromol L-1), a nitric oxide synthase inhibitor (D-p-Cl-Phe6, Leul7; 10 micromol L-1), a vasoactive intestinal peptide (VIP) receptor antagonist, and an inhibitor of haem oxygenase, (copper protoporphyrin IX; CuPP-IX; 10 micromol L-1), suggesting that nitric oxide (NO), VIP and carbon monoxide (CO), respectively, are released in response to EFS. Immunoreactivities for haem oxygenase-2 (HO-2) and VIP, and histochemical staining for NADPH diaphorase were observed in nerve cell bodies and fibres, demonstrating the presence of CO, VIP and NO as putative NANC neurotransmitters in dog gallbladder. These data support the hypothesis that NO, VIP and CO contribute to NANC relaxation of the canine gallbladder.

Atrial natriuretic peptide (ANP) plays a part in the regulation of volume homeostasis and possibly, in the pathophysiology of water and electrolyte disorder. Patients with serious burn injuries risk huge body fluids losses, which are compensated for by perfusion. Blood volume and the renin and aldosterone system are also disturbed. This study measured plasma ANP and vasoactive intestinal polypeptide (VIP) in patients with >20% total burned surface area (TBSA), at admission and 24 h post-admission.Eleven patients (mean age 46.5 years, 8 males) with a mean TBSA of 34.5% were sampled. Standard treatment was given. Eleven closely age-matched volunteers were used as controls. A specific ELISA method suitable for the measurement of ANP and VIP was used.ANP was higher (p<0.0001), while VIP was lower (p=NS) in patients' samples compared to controls. While the level of VIP was higher at 24 h post-admission, mean ANP level remained about the same. The increased levels of plasma ANP may result from volaemic disturbances during resuscitation, low VIP levels, the increase in pulmonary resistance or post-burn stress.

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.

In guinea-pig gallbladder epithelium, cAMP converts electroneutral HCO3- secretion into an electrogenic process. The effects of blood side Ba2+ (5 mmol/l) on HCO3- transport were investigated in vitro, using pH-stat and voltage clamp techniques to determine unidirectional fluxes of HCO3- and transepithelial electrical characteristics. Serosal, not mucosal addition of Ba2+ elevated short-circuit current (Isc), transepithelial potential difference, and tissue conductance; it inhibited the absorptive HCO3- flux while leaving the secretory flux unchanged. The Isc effect of Ba2+ was inhibited or prevented by tetrodotoxin; D- and L-propranolol; the Cl- channel blocker 4-N-methyl-N-phenylaminothiophene-3-carboxylic acid; the intracellular Ca2+ antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)ocytl ester; noradrenaline, by a yohimbine-sensitive action; somatostatin; HCO3(-)-free solutions. Thus Ba2+ appeared to release a neurotransmitter that gives rise to cAMP synthesis sufficient to turn part of electroneutral HCO3- secretion electrogenic. In a search for the involved signalling pathways, the H1-receptor antagonist, cetirizine, largely and hexamethonium, atropine, atenolol, indomethacin, and trifluoperazine entirely failed to antagonize the Isc effect of Ba2+. Similarly, carbachol, dobutamine, salbutamol, and serotonin were unable to mimic the action of Ba2+ and Isc effects of histamine were small and short-lived. By contrast, vasoactive intestinal peptide (VIP; 3 x 10(-7) mol/l) completely transformed HCO3- secretion into an electrogenic process. The VIP receptor antagonist (4Cl-DPhe6, Leu17) VIP, delayed and reduced the Isc responses to Ba2+ and VIP. As guinea-pig gallbladder epithelial cells possess cAMP-coupled VIP receptors close to VIPergic neurons, Ba2+ is likely to act by releasing VIP from neural terminals.

Histochemical staining was used to demonstrate that intramural neurons of the gallbladder contain NADPH-diaphorase, and therefore are likely to produce nitric oxide. A subset of the neurons in the gallbladders of the guinea pig, gerbil, opossum, dog, and human stained positively for the enzyme. In the guinea pig, all neurons that were immunoreactive for vasoactive intestinal peptide (VIP), also contained NADPH-diaphorase. Furthermore, neurons that were immunoreactive for neuropeptide Y, which have been shown to be immunoreactive for substance P and somatostatin as well, rarely contained NADPH-diaphorase. It is suggested that the VIP/NADPH-diaphorase-containing neurons represent intrinsic inhibitory motor neurons of the gallbladder, and that these neurons may have a role in the relaxation of the muscularis during gallbladder filling.

Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.

The neuropeptide vasoactive intestinal peptide was localized to taste buds of the posterior tongue regions of hamsters and rats by immunocytochemical techniques. Tissue sections, taken from foliate and circumvallate papillae, generally revealed taste buds in which all cells were immunoreactive; however, occasionally some taste buds were found to contain highly reactive individual cells adjacent to non-reactive cells. Additionally, some non-reactive taste buds were observed. Taste buds that displayed vasoactive intestinal peptide-like immunoreactivity usually had a tendency for much darker staining at the apical ends of the cells than the basal ends, suggesting a polar cytoplasmic distribution of the peptide. The multi-functional roles of vasoactive intestinal peptide in other physiological systems combined with both its cytoplasmic localization in taste cells and the known histochemistry/ultrastructure of taste cells raises interesting speculations of this peptide's function in gustation that include secretion, stimulation of a second messenger system, and neuromodulation.

The effect of vagal stimulation in chloralose-anesthetized cats on release of vasoactive intestinal polypeptide into the jejunal lumen and portal venous blood was tested simultaneously, and the effect of atropine and hexamethonium was investigated to elucidate the regulatory mechanisms involved in the release. Vagal stimulation caused a significant increase in vasoactive intestinal polypeptide concentrations in the luminal perfusates. A significant concomitant increase was seen in portal plasma. Gel filtration chromatography of luminal and portal samples demonstrated that the vasoactive intestinal polypeptide coeluted with synthetic porcine vasoactive intestinal polypeptide. Vasoactive intestinal polypeptide infusion at 80 and 160 pmol/kg.min produced portal plasma levels of at least 3000 pM but did not increase vasoactive intestinal polypeptide concentrations in the luminal perfusates. Thus, luminal vasoactive intestinal polypeptide originates from gastrointestinal tissue rather than by transduction from the circulation. Vagally induced release of vasoactive intestinal polypeptide into the lumen and portal plasma was not abolished by atropine but was totally suppressed by hexamethonium. The regulatory mechanisms controlling the parallel release of vasoactive intestinal polypeptide into both the jejunal lumen and portal circulation are identical and involve a non-muscarinic process which is under cholinoceptive, nicotinic control.

The evidence for, and possible roles of, inhibitory and excitatory non-adrenergic, non-cholinergic (NANC) nerves supplying smooth muscle, and the effects of putative transmitter candidates are considered for each of three main regions of the upper gastrointestinal tract: (A) the smooth muscle portion of the oesophagus and the oesophagogastric junction, (B) the stomach (fundus, body and antrum) and gastroduodenal junction and (C) the biliary tract and choledochoduodenal junction. The major points from human tissues are as follows: 1. Inhibitory (NANCI) nerves appear to be present in the muscularis externa of oesophagus, stomach and duodenum, with greater density in the circular than in the longitudinal muscle. 2. NANCI nerves are present in high density at the oesophagogastric and choledochoduodenal junctions. They may also be present at the gastroduodenal junction. The gall-bladder may have a very sparse NANCI innervation. 3. Excitatory (NANCE) nerves appear to be present throughout the upper gastrointestinal tract. 4. Many candidates need at present to be considered for the role of NANCE transmitter(s) in the human upper gastrointestinal tract but substance P still seems a likely contender for this role. 5. Fewer candidates are at present generally available for the role of NANCI transmitter(s), with VIP and ATP being leading contenders. However, in the human upper gastrointestinal tract the evidence for ATP is not good, and VIP still remains the favourite candidate except in the gall-bladder, where its role remains to be elucidated.

The normal fluid absorption across the gallbladder mucosa is, in experimental cholecystitis, changed to an active net fluid secretion. This fluid secretion, studied in anesthetized cats, is not abolished by extrinsic gallbladder denervation and is unaffected by atropine but is strongly reduced by intraarterial tetrodotoxin or intraluminal administration of lidocaine hydrochloride. Intravenous somatostatin or hexamethonium administration also reduce this secretion. Indomethacin, known to abolish this fluid secretion, did not further reduce it when administered after nerve blocking agents in the present study. These data demonstrate that the prostaglandin-induced gallbladder fluid secretion in experimental cholecystitis is influenced by intramural nerves. It is suggested that gallbladder inflammation is associated with prostaglandin-induced activation of intrinsic nerves which may stimulate the epithelial cells to fluid secretion. In the obstructed gallbladder, this secretion causes gallbladder distension by increasing the intraluminal pressure. This mechanism may have a key role in the pathophysiology of acute cholecystitis.