R. Crowe

Purinergic innervation of the guinea-pig urinary bladder.

1 A number of criteria for considering adenosine 5′‐triphosphate (ATP) as a neurotransmitter in the guinea‐pig urinary bladder have been examined. In addition, the effect of tachyphylaxis to ATP on the response to non‐adrenergic, non‐cholinergic nerve stimulation has been re‐examined. 2 Quinacrine fluorescence histochemistry revealed a population of nerve fibres, ganglion cells, and nerve bundles in the bladder which were not seen in either the iris or vas deferens, where adrenergic and cholinergic nerves predominate. The distribution and morphology of the quinacrine‐positive nerves in the bladder were different from those observed with catecholamine fluorescence and cholinesterase histochemistry, and were unaffected by chemical sympathectomy. 3 Release of ATP from the bladder during stimulation of intramural excitatory nerves, in the presence of atropine and guanethidine increased to 3–12 times prestimulation levels. Tetrodotoxin abolished both the contractile response and the increase in ATP release resulting from intramural nerve stimulation. There was no increase in ATP release during contraction resulting from direct muscle stimulation following nerve paralysis with tetrodotoxin. 4 Sympathectomy with 6‐hydroxydopamine did not affect release of ATP in response to intramural nerve stimulation. 5 Release of ATP was dependent on the concentration of calcium ion in the medium. 6 Contractions in response to non‐adrenergic, non‐cholinergic intramural nerve stimulation were closely mimicked by ATP, but not by acetylcholine or histamine. 7 Adenosine and dipyridamole reduced the contractions to both ATP and non‐cholinergic nerve stimulation. 8 2–2′‐Pyridylisatogen was not a specific blocker of either ATP or intramural nerve stimulation in the guinea‐pig bladder. 2‐Substituted imidazolines initiated spontaneous activity making it impossible to assess any blocking action that they may have had. 9 Prostaglandins (E1 E2 and F2a) gave weak, slow contractions and an increase in spontaneous activity. Both the response to ATP and non‐adrenergic, non‐cholinergic nerve stimulation were greatly potentiated in the presence of prostaglandins. 10 In the presence of indomethacin the response to non‐adrenergic, non‐cholinergic nerve stimulation was virtually abolished following desensitization to ATP.

MYENTERIC PLEXUS IN STREPTOZOTOCIN TREATED RATS. NEUROCHEMICAL AND HISTOCHEMICAL EVIDENCE FOR DIABETIC NEUROPATHY IN THE GUT

Adrenergic, cholinergic, and serotoninergic nerves were studied in the myenteric plexus of ileum and colon from streptozotocin-treated rats, an animal model of juvenile-onset diabetes. In view of clinical reports implicating diabetic autonomic neuropathy as the cause of gastrointestinal dysfunction in diabetes mellitus, neurochemical and histochemical techniques were used to study changes in the innervation of the gut. In the myenteric plexus of the ileum from diabetic animals, adrenergic nerves displayed signs of degeneration and the brightness of fluorescence in serotoninlike immunoreactive nerves was lower. Cholinergic nerves, however, did not display any signs of reduction in the ileum, and both choline acetyltransferase and acetylcholinesterase activities per centimeter were increased. In contrast, in the proximal colon 8 wk after induction of diabetes, neurochemical assays revealed significant increases in noradrenaline and serotonin levels as well as choline acetyltransferase activity, although no obvious changes in the pattern of innervation could be detected histochemically. The results indicate that changes do occur in the innervation of the gut of the streptozotocin-diabetic model shortly after the induction of diabetes, although they differ significantly in the ileum and colon; these may be of relevance to the types of gastrointestinal dysfunction displayed in human diabetes.

Enteric nerves in diabetic rats: Increase in vasoactive intestinal polypeptide but not substance P

The distribution of vasoactive intestinal polypeptide (VIP) and substance P-like immunoreactivities was studied by immunohistochemistry in the myenteric plexus and circular muscle layer of the ileum and proximal colon of rats 8 wk after induction of diabetes with streptozotocin. A consistent increase was observed in fluorescence intensity of VIP-like immunoreactivity in the nerve fibers, and intensely stained cell bodies were significantly more frequent in the myenteric plexus of the ileum (p less than 0.001) from diabetic animals. Some varicosities of VIP-like immunoreactive fibers in the myenteric plexus appeared to be enlarged. Vasoactive intestinal polypeptide-like immunoreactivity was increased and VIP-like immunoreactive nerves appeared thicker in the circular muscle layer of both diabetic ileum and proximal colon. The VIP levels were measured biochemically in tissue consisting of the smooth muscle layers and myenteric plexus. A significant increase in the VIP content per centimeter of intestine was found in both the ileum (p less than and proximal colon (p less than 0.01) from diabetic rats. In contrast, no apparent change in substance P innervation was observed immunohistochemically in the myenteric plexus and circular muscle layer of either diabetic ileum or proximal colon when compared with controls. The results are discussed in relation to the symptoms of autonomic neuropathy of the gut in diabetes.

Vasoactive intestinal polypeptide levels in sigmoid colon in idiopathic constipation and diverticular disease

The distribution in the bowel wall of vasoactive intestinal polypeptide-, neuropeptide Y-, and substance P-containing nerve cell bodies and nerve fibers has been described in human sigmoid colon by immunohistochemical examination. In patients with chronic idiopathic constipation, diverticular disease, and in controls (of tissue taken from patients with carcinoma, from a site distant from the tumor that appeared macroscopically normal), the concentrations of vasoactive intestinal polypeptide, neuropeptide Y, and substance P have been measured by immunoassay in the following preparations of sigmoid colon: mucosa, whole colonic wall with mucosa dissected away, circular muscle, and taenia coli. In idiopathic constipation, the vasoactive intestinal polypeptide content of the whole wall minus mucosa was reduced when compared with controls (P less than 0.05) but was unaltered in the mucosa, circular muscle, and taenia coli. In diverticular disease, the vasoactive intestinal polypeptide content of the mucosa and whole wall minus the mucosal layer was increased when compared with control tissue (P less than 0.05 and P less than 0.02, respectively) but was unaltered in the circular muscle and taenia coli. Substance P and neuropeptide Y levels in all layers of colonic wall were unaltered in these two diseases. The disturbances in the normal neural content of vasoactive intestinal polypeptide in the bowel wall in idiopathic constipation and diverticular disease may initiate or contribute to the functional changes seen in these disorders.

Serotonin and 5-Hydroxyindoleacetic Acid Are Increased in the Sigmoid Colon in Severe Idiopathic Constipation

The distribution of serotonin and dopamine beta-hydroxylase was examined in sigmoid colon specimens from patients with severe idiopathic constipation and control patients with carcinoma of the rectum or colon. Specimens were divided into three regions: (a) the mucosa; (b) the myenteric and submucosal plexuses with the longitudinal and circular smooth muscles; and (c) the circular smooth muscle, for biochemical analysis of serotonin and 5-hydroxyindoleacetic acid (total indoles) and noradrenaline. In both groups of patients, serotonin- and dopamine beta-hydroxylase-like immunoreactivity was localized in nerves in the myenteric and submucosal plexuses, and a sparse innervation was observed in the circular muscle. In addition, intense serotonin-like fluorescence was present in a large number of enterochromaffin cells in the mucosa. Total indole levels were significantly increased in the mucosa (p less than 0.02) and circular muscle (p less than 0.05) of the constipated patients. In contrast, no changes in noradrenaline levels were observed in any of the regions studied. Altered levels of total indoles may thus contribute to severe idiopathic constipation. Analysis of biopsy specimens could be a useful tool in clinical diagnosis and future investigations of diseases of the gut.

Vasoactive Intestinal Polypeptide-like Immunoreactive Nerves in Diabetic Penis: A Comparison Between Streptozotocin-treated Rats and Man

Vasoactive intestinal polypeptide (VIP) has been demonstrated by immunofluorescence histochermistry in nerves in human and rat penile tissue. A reduction in VIP-like immunoreactivity in nerves was revealed in tissue from streptozotocin-diabetic rats and a human diabetic with impotence. These results suggest that an impairment in the VIP-ergic innervation in penile tissue may be an important factor in the development of impotence in diabetes. They also support the view that the streptozotocin-treated rat is a useful experimental model for diabetic autonomie neuropathy.

EVIDENCE FOR PURINERGIC INNERVATION OF THE ANOCOCCYGEUS MUSCLE

1 Fluorescence histochemical localization of quinacrine (which binds to adenosine 5′‐triphosphate (ATP)) revealed nerve fibres running singly and in bundles in both rat and rabbit anococcygeus muscle. Single neurone cell bodies and ganglia containing between 2 and 50 cells were also observed. 2 Catecholamine fluorescence studies revealed a dense adrenergic ground plexus, but no adrenergic ganglion cells were detected. No acetylcholinesterase‐positive nerve fibres or ganglion cells were seen in the rat. 3 When the tone was raised with guanethidine, a relaxation in response to field stimulation was revealed, which was unaffected by atropine but blocked by tetrodotoxin. 4 Release of ATP increased 3 to 6 times above background during stimulation of these non‐adrenergic, non‐cholinergic, inhibitory nerves. 5 Neither quinacrine staining nor the release of ATP during inhibitory nerve stimulation was affected by 6‐hydroxydopamine treatment, which abolished catecholamine fluorescence. 6 Exogenous ATP produced relaxation in high tone preparations of the rabbit anococcygeus muscle. ATP produced either contraction or a small relaxation followed by a contraction of the rat anococcygeus muscle, but treatment with low concentrations of the prostaglandin synthesis inhibitor indomethacin, converted the contraction to a relaxation. 7 These data are consistent with the view that the anococcygeus muscle is innervated by purinergic inhibitory nerves.

Intramural neurones of the guinea-pig urinary bladder: histochemical localization of putative neurotransmitters in cultures and newborn animals

Histochemical methods have been used to study the distribution of putative neurotransmitters in the urinary bladder of newborn guinea-pigs and in cultures of intramural ganglia. Following the nicotinamide adenine dinucleotide (NADH)-diaphorase reaction which specifically labels nerve cell bodies, up to 66 ganglia were observed in stretch preparations of the newborn urinary bladder. Each ganglion contained 2-50 nerve cell bodies. Vasoactive intestinal polypeptide was localized in a few nerve cell bodies of intramural ganglia both in in situ and culture preparations. In the in situ preparations it was widely distributed in nerve fibres to the muscle, being most dense at the base of the bladder, and in some mucosal epithelial cells. Somatostatin was contained in numerous neuronal cell bodies in the detrusor muscle both in situ and in culture. Extensively distributed varicose fibres were found in culture and in the muscle, submucous and mucosal layers in situ. Substance P immunofluorescence was demonstrated in a few neuronal cell bodies in ganglia both in situ and in vitro, particularly in those of the mucosa at the base of the bladder. In the in situ preparations varicose nerve fibres containing substance P were seen in the muscle coats with greatest density in the bladder base. Met-enkephalin-immunoreactive nerve cell bodies were not seen either in situ or in culture. Nerve fibres in in situ preparations were found largely enveloping neuronal cell bodies within the ganglia. Neither serotonin-immunoreactive nor catecholamine-containing neuronal cell bodies were seen in the in situ bladder preparation. However, some nerve cell bodies in culture showed positive staining, possibly as a result of selective uptake of serotonin and catecholamine known to be contained in foetal calf serum in the culture medium or possibly as the result of increased synthetic activity in certain neurones in the culture situation. In whole-mount stretch preparations, no serotonin-immunoreactive nerve fibres were seen, but catecholamine-containing small intensely fluorescent cells and nerve fibres were observed. Acetylcholinesterase-positive nerve cell bodies and nerve fibres were observed both in in situ and culture preparations of the bladder. Quinacrine-positive nerve cell bodies (as an indicator of purinergic neurones) were found in numerous intramural neurones examined. in situ; however, under the culture conditions used, non-selective staining of all cell types occurred.

COMPARATIVE PHARMACOLOGICAL AND HISTOCHEMICAL EVIDENCE FOR PURINERGIC INHIBITORY INNERVATION OF THE PORTAL VEIN OF THE RABBIT, BUT NOT GUINEA‐PIG

1 Intramural nerve stimulation elicited a powerful relaxation of the longitudinal muscle of the rabbit portal vein in the presence of atropine and guanethidine, but not of the guinea‐pig portal vein. 2 Intramural nerve stimulation of the rabbit portal vein produced a 13 fold increase in release of 3H‐adenyl compounds after preloading with [3H]‐adenosine. About 50% of this release was abolished by guanethidine. All release was abolished by tetrodotoxin. No significant release of radioactive compounds was observed during intramural nerve stimulation of the guinea‐pig portal vein in the presence of guanethidine, although there was a 6 fold increase in release of radioactivity in the absence of drugs. 3 Histochemical studies using quinacrine, which binds ATP showed a fine fluorescent nerve plexus, nerve bundles, and ganglion cells in the rabbit portal vein, but not in the guinea‐pig portal vein. This plexus was still present after chemical sympathectomy with 6‐hydroxydopamine. 4 Adenosine 5′‐triphosphate (ATP) relaxed the rabbit portal vein, but usually produced a biphasic response, consisting of a contraction followed by a relaxation, of the guinea‐pig portal vein. 5 Prostaglandins E1 and E2 caused contraction of the rabbit portal vein. Indomethacin, a prostaglandin synthesis inhibitor, potentiated the relaxations of the rabbit portal vein produced by both non‐adrenergic, non‐cholinergic nerve stimulation and ATP. 6 High concentrations of antazoline and phentolamine, which antagonize purinergic responses in the guinea‐pig taenia coli, caused a loss of basal tone so that it was not possible to assess their effects on the responses of the portal vein to either non‐adrenergic, non‐cholinergic nerve stimulation, or ATP. 7 Comparison of the results on the portal vein of the rabbit and guinea‐pig provides support for the view that: (i) quinacrine fluorescence can be used to localize purinergic nerves and that the rabbit portal vein is supplied by these nerves; (ii) ATP is released from adrenergic nerve fibres, although, based on histochemical analysis, about 3 to 7 times less than is released from purinergic nerve fibres.

The innervation of the human prostate gland : the changes associated with benign enlargement

Different regions of the prostate gland, namely prostatic capsule, peripheral prostate and central prostate (subdivided into proximal (near the bladder neck), distal (near the verumontanum) and midway between these areas) were obtained from 32 obstructed (stable obstructed, n = 8; unstable obstructed, n = 13; acute retention, n = 11) and five control patients. The innervation of these tissues was studied both histochemically to localise acetylcholinesterase activity and immunohistochemically for dopamine-beta-hydroxylase, 5-hydroxytryptamine, vasoactive intestinal polypeptide, neuropeptide Y, leu- and met-enkephalin, calcitonin gene-related peptide, substance P and somatostatin. In control patients the greatest density of nerves was found in the proximal central prostate, followed by the anterior capsule and distal central prostate, with the least density in the peripheral prostate. The greatest density of nerves were acetylcholinesterase positive and immunoreactive to neuropeptide Y followed (in decreasing order) by nerves immunoreactive to: vasoactive intestinal polypeptide and dopamine beta-hydroxylase; leu-enkephalin and 5-hydroxytryptamine; calcitonin gene-related peptide; met-enkephalin; substance P; somatostatin. In addition a group of periacinar 5-hydroxytryptamine-immunoreactive cells and ganglia containing acetylcholinesterase, dopamine beta-hydroxylase and all of the peptides studied except somatostatin were identified. In the prostate gland from obstructed patients there was a significant reduction in the density of acetylcholinesterase-positive nerves (p less than 0.001) when compared with the controls. A similar trend was found for dopamine beta-hydroxylase, 5-hydroxytryptamine and all of the putative neuropeptides in most areas of the prostate, the most notable exceptions being in the peripheral prostate, with an increase in dopamine beta-hydroxylase- and leu-enkephalin-immunoreactive nerves in all three groups of obstructed patients an an increase in vasoactive intestinal polypeptide- and calcitonin gene-related peptide-immunoreactive nerves in those presenting in urinary retention. The functional significance of these findings is discussed.