Protective role of vanilloid receptor type 1 in HCl-induced gastric mucosal lesions in rats.

BACKGROUND: Effects of vanilloid-receptor agonists and antagonists on HCl-induced gastric lesions in rats were investigated to elucidate the role of vanilloid receptor type 1 (VR1) in gastric mucosal defense mechanisms. METHODS: Gastric lesions in rats were evaluated after intragastric administration of 0.6 N HCl. The localization of VR1 in the stomach was investigated immunohistochemically. RESULTS: Intragastric administration of capsaicin inhibited the formation of gastric lesions in a dose-dependent manner (0.1-2.5 mg/kg). The functional VR1 antagonists ruthenium red and capsazepine markedly aggravated HCl-induced gastric lesions in rats. The gastroprotective effect of capsaicin was attenuated by ruthenium red or capsazepine. It is reported that resiniferatoxin, [6]-gingerol and lafutidine are compounds that activate VR1 and/or capsaicin-sensitive afferent neurons. These compounds significantly inhibited the formation of HCl-induced gastric lesions, and their gastroprotective effects were inhibited by treatment with ruthenium red. The immunohistochemical studies revealed that nerve fibers expressing VR1 exist along gastric glands in the mucosa, around blood vessels in the submucosa, in the myenteric plexus, and in the smooth muscle layers, especially the circular muscle layer. CONCLUSION: The results of this study suggest that VR1 plays a protective role in the gastric defensive mechanism in rats.

Pattern of distribution and co-localization of NOS and ATP in the myenteric plexus of human fetal stomach and intestine.

The pattern of distribution and co-localization of nitric oxide synthase (NOS) and quinacrine fluorescence (indicative of vesicular adenosine 5'-triphosphate, ATP), and co-localization of NADPH-diaphorase (NADPH-d) activity and NOS-immunoreactivity in the myenteric plexus of pre-term human fetal (6-17 weeks of gestation) stomach and small intestine was examined using immunohistochemical and histochemical techniques. In all stages of gestation investigated, NOS-immunoreactive and NADPH-d-reactive myenteric neurons and nerve fibres were seen in the fetal intestine and stomach. However, in fetuses of 6-10 weeks of gestation, only 15% of the NADPH-d-positive myenteric neurons were NOS-immunoreactive, whereas a 100% co-localization was found in samples of 12-17 weeks of gestation. Quinacrine fluorescent myenteric neurons and nerve fibres were found only in the fetal intestine of 12-17 weeks of gestation, of which 25% of the NADPH-d-positive myenteric neurons in these samples were quinacrine fluorescent. These findings demonstrate the presence and co-localization of markers for nitric oxide (NO)- and ATP-utilizing myenteric neurons and nerve fibres in the early stages of gestation, suggesting possible co-transmitter and/or trophic roles of ATP and NO in the process of development and maturity of human myenteric neurons. In addition, the fact that only a small percentage of NADPH-d-reactive myenteric neurons express NOS immunoreactivity at 6-10 weeks of gestation confirms that NADPH-d-reactivity does not always represent NOS activity.

Distribution and colocalization of nitric oxide synthase and calretinin in myenteric neurons of developing, aging, and Crohn's disease human small intestine.

The pattern of distribution and colocalization of nitric oxide synthase and the calcium-binding protein calretinin in myenteric neurons and nerve fibers were examined in the human small intestine from preterm fetuses (14-17 weeks of gestation), normal adults (mean age 50 years old), old age (mean age 80 years old), and Crohn's disease patients (mean age 30 years old) using NADPH-diaphorase histochemistry and immunohistochemical techniques. In all age groups investigated, NADPH-diaphorase-reactive and calretinin-immunoreactive neurons and nerve fibers were seen throughout the myenteric plexus. The highest proportion of NADPH-diaphorase-reactive neurons was found in the myenteric ganglia of old age intestines (56% of protein gene product-immunoreactive neurons) followed by fetal intestines (41%) and Crohn's intestine (30%) compared with intestines of control adults (20%). A similar trend was observed for calretinin-immunoreactive neurons where the highest proportion of immunoreactive neurons was found in the myenteric ganglia of old age intestines (28% of protein gene product-immunoreactive neurons), followed by fetal intestines (22%), and Crohn's intestines (18%) compared with intestines of control adults (9%). A colocalization of NADPH-diaphorase activity and calretinin immunoreactivity was only seen in the myenteric neurons of fetal intestines (2% of NADPH-diaphorase-reactive neurons were also calretinin-immunoreactive). The pattern of distribution of NADPH-reactive and calretinin-immunoreactive neurons in the myenteric ganglia of fetal intestine differs from that of the other age groups. In the fetal intestine, the myenteric neurons containing either calretinin or NADPH-diaphorase are distributed through out the myenteric ganglia with no specific orientation to one another. In the intestines of control adult, Crohn's, and old age patients, single large calretinin-immunoreactive neurons are surrounded by a number of small NADPH-diaphorase-positive neurons, with this feature being more prominent in intestines of old-age and Crohn's disease patients. In summary, a high number of both NADPH-diaphorase-reactive and calretinin-immunoreactive neurons were seen in the myenteric ganglia of fetal, old age, and Crohn's intestines; we discuss that there may be a role for nitric oxide and calretinin in the process of development, aging, and pathological changes in the human intestine associated with alteration in the calcium homeostasis in the myenteric neurons.

The effects of hibernation on the myenteric plexus of the golden hamster small and large intestine.

We have examined the effects of hibernation on the neurochemical composition of myenteric neurones in the small and large intestine of the golden hamster using immunohistochemical and histochemical techniques. Hibernation was induced in golden hamsters by altering the photoperiod and external ambient temperature. Age-matched hamsters kept at room temperature and those kept at 5 degrees C but which failed to hibernate were used as controls. Cell counts were carried out to examine possible changes in the numbers of cell bodies immunoreactive to all of the markers examined. The results demonstrated a significant increase during hibernation in the number of neurones immunoreactive to vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide; cell bodies positive for tyrosine hydroxylase, which were largely absent in the control animals, were prominent in the hibernating animals. There was a significant decrease in the number of neurones immunoreactive to 5-hydroxytryptamine, and no significant changes in the numbers of neurones immunoreactive to protein gene-product and nitric oxide synthase. It is suggested that selective upregulation and downregulation of myenteric neurones containing certain neurotransmitters may occur as a protective mechanism during hibernation to maintain the integrity of the muscular and mucosal layers of the intestine in the absence of luminal contents.

A neurochemical characterisation of the golden hamster myenteric plexus.

The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.

Neurochemical coding in the myenteric plexus of the upper gastrointestinal tract of hibernating hamsters.

As part of our investigation of the plasticity of autonomic nerves in physiological and pathological conditions, we have examined the effect of hibernation on the neurochemical content of myenteric nerves and nerve cell bodies of the upper gastrointestinal tract of the non-seasonal hibernator, the golden hamster. Age matched hamsters kept at room temperature and those kept at 5 degrees C but which failed to hibernate, were used as controls. Possible changes in nerve fibres and nerve cell bodies containing the general neuronal marker, protein gene product 9.5, the peptides, vasoactive intestinal polypeptide, substance P (SP) and calcitonin gene-related peptide (CGRP), the catecholamine synthesizing enzyme tyrosine hydroxylase and the enzyme responsible for synthesizing nitric oxide, nitric oxide synthase, were examined in the oesophagus, proventriculus and proximal and distal stomach of the golden hamsters using immunohistochemical techniques. The results of the present study revealed a significant increase in the number of nerve cell bodies and density of nerve fibres containing SP-immunoreactivity and increased number of CGRP-immunoreactive cell bodies but not the other markers examined in the proximal stomach and proventriculus. In contrast, there was no change in the distribution of any of the neuroactive substances examined in the myenteric plexus of the oesophagus and distal stomach. It is suggested that the change in the environment of the hibernating hamsters perturbs the normal digestive physiology in the proximal stomach and proventriculus that is reflected by the selective changes in SP- and CGRP-containing enteric nerves; these changes may be part of protective reflex mechanisms to the environmental changes resulting from hibernation, where upgrading of nerve cell bodies expressing CGRP and SP has occurred.

Neurochemical coding in the small intestine of patients with Crohn's disease.

BACKGROUND: There have been conflicting results regarding the effect of Crohn's disease on the neurochemical composition of the enteric nervous system. AIMS: To examine the effect of Crohn's disease on the neurochemical composition of enteric nerve fibres and cell bodies using whole mount preparations of human ileum. METHODS: Whole wall ileum from seven normal subjects and nine patients with Crohn's disease was used to investigate the neurochemical composition of neurones and nerve fibres in the myenteric plexus, circular muscle, and serosa layer of ileum using immunohistochemical techniques. RESULTS: Increased tyrosine hydroxylase, 5-hydroxytryptamine, and neuropeptide Y immunoreactivity was exclusively seen in the myenteric plexus. There was increased neurofilament immunoreactivity in the myenteric plexus and nerve fibres of the circular muscle layer, and thick bundles of immunoreactive nerve fibres in the serosa layer. Increased vasoactive intestinal polypeptide, nitric oxide synthase, and pituitary adenylate cyclase activating peptide immunoreactivity was seen in the myenteric plexus and nerve fibres of the circular muscle layer, and aggregates of inflammatory cells in the serosa layer of the afflicted segment of Crohn's ileum. In addition, there was a chaotic display of nerve fibres containing some of the neuroactive substances with a high frequency of enlarged varicosities in the myenteric ganglia and/or nerve fibres of the circular muscle layer of Crohn's ileum. CONCLUSION: Results show quantitative as well as qualitative changes in the neurochemical composition of enteric nerve fibres and nerve cell bodies of Crohn's ileum. These changes and the presence of nitric oxide synthase and peptides immunoreactive inflammatory cells in the serosa layer suggest that nerve-immune interactions may have a significant role in the process of the inflammatory changes seen in Crohn's ileitis.

Enteric neuropeptides in streptozotocin-diabetic rats; effects of insulin and aldose reductase inhibition.

The aim of the present study was to determine whether diabetes-induced changes in the distribution of enteric neuropeptides, could be prevented in 12-week streptozotocin-diabetic rats, by rigorous control of glycaemia, using daily adminstration of insulin, or an aldose reductase inhibitor (ponalrestat). The pattern of distribution of nerve fibres and cell bodies, containing immunoreactive vasoactive intestinal polypeptide (VIP), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P was examined in the myenteric plexus of ileum from control, untreated diabetic, insulin-treated diabetic and aldose reductase inhibitor-treated diabetic rats. The increase in VIP- and GAL-like immunoreactivity, seen in the myenteric plexus of untreated diabetic rat ileum, was not present in the myenteric plexus of ileum from insulin- and aldose reductase inhibitor-treated diabetic rats. With CGRP-like immunoreactive fibres, there was a clear decrease in the ileum of untreated diabetic rats. This was prevented by insulin treatment, but aldose reductase inhibitor treatment had no effect. No alterations in substance P-like immunoreactivity were seen in the myenteric plexus of ileum from any of the groups investigated. Generally, the similarity of effect of ponalrestat and insulin on VIP and galanin expression in this study supports a primary effect of insulin via glycaemic control. The dissimilarity of the effect of the two treatments on CGRP expression may imply a neurotrophic effect of insulin, although there are certainly consequences of hyperglycaemia other than exaggerated flux through the polyol pathway.

Increase in nitric oxide synthase and NADPH-diaphorase in the adrenal gland of streptozotocin-diabetic Wistar rats and its prevention by ganglioside.

Levels of nitric oxide synthase (NOS) and NADPH-diaphorase in adrenal glands of streptozotocin-diabetic rats of 8 and 12 weeks' duration compared with control rats were assessed with histo-chemical and biochemical techniques. Adrenal glands from streptozotocin-diabetic rats of 8 weeks' duration treated with ganglioside were examined also. In the adrenal medulla of 8-weeks- and 12-weeks-diabetic rats, NOS-immunoreactive nerve fibres were increased and decreased, respectively; additional NOS-immunoreactive and NADPH-diaphorase stained cells, which appeared to be cortical cells, were located in medulla and cortex compared with controls. Increased intensity in NADPH-diaphorase staining of the cortical cells of diabetic rats was observed also. Ganglioside treatment of the 8-weeks-diabetic rats prevented the diabetic-induced increase in NOS-immunoreactive nerve fibres. Also, it reduced most of the increase in the NOS-immunoreactive and NADPH-diaphorase stained cells and the intensity of NADPH-diaphorase staining of cortical cells. With biochemical assay, a significant increase in NOS activity was found in the adrenal glands from 8-weeks-diabetic rats, and this increase was reduced by ganglioside treatment in four out of six diabetic rats. In summary, streptozotocin-induced diabetes causes an initial increase in the levels of NOS and NADPH-diaphorase in the adrenal gland of rat, which was prevented by ganglioside treatment.

Acrylamide-induced neuropathic changes in rat enteric nerves: similarities with effects of streptozotocin-diabetes.

The effect of acrylamide intoxication (a widely used model for autonomic neuropathy) on the fluorescence intensity and density of catecholamine- and peptide-containing nerve fibres and tissue content of noradrenaline and the peptides vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P and neuropeptide Y in the enteric nerves of rat ileum was examined. Histochemical and immunohistochemical techniques were used to localize catecholamine- and peptide-containing nerve fibres. The tissue content of noradrenaline was measured using high-performance liquid chromatography, and an enzyme-linked immunosorbent assay technique was used to determine the tissue content of the peptides investigated. Acrylamide intoxication caused a significant decrease in the density of catecholamine-containing nerve fibres and tissue content of noradrenaline in the myenteric plexus of rat ileum. A decrease in tissue content and immunoreactivity of calcitonin gene-related peptide and an increase in vasoactive intestinal polypeptide was seen in the myenteric plexus of ileum from acrylamide-intoxicated rats. In the submucous plexus, the acrylamide treatment caused a decrease in calcitonin gene-related peptide immunoreactivity and an increase in vasoactive intestinal polypeptide and neuropeptide Y immunoreactivity. There was no change in either tissue content or immunoreactivity of substance P in both myenteric and submucous plexuses of the treated rat ileum. These changes have a striking similarity with those found in the enteric nerves of streptozotocin-diabetic rat ileum, suggesting the possible presence of an underlying common mechanism(s) in the development of neuropathic changes in the autonomic nerves of acrylamide-intoxicated and streptozotocin-diabetic rats.

Selective damage to sensorimotor perivascular nerves in the mesenteric vessels of diabetic rats.

The perivascular innervation of the superior mesenteric artery and vein was examined using immunohistochemical and immunoassay techniques in rats 8 weeks after induction of diabetes with streptozotocin (STZ). Increased density of innervation and fluorescence intensity was noted for substance P- and calcitonin gene-related peptide-immunoreactive nerves in the diabetic vessels. A slight increase in the density of vasoactive intestinal polypeptide-immunoreactive nerve fibers innervating the mesenteric artery was also noted. However, there was no change in the density of neuropeptide Y- and dopamine beta-hydroxylase-immunoreactive nerve fibers, although the fluorescence intensity of neuropeptide Y-immunoreactive nerve fibers was reduced in diabetic rat vessels. Immunoassays showed that the levels of substance P- and calcitonin gene-related peptide were increased > 10-fold in the diabetic mesenteric vein, while levels of neuropeptide Y and vasoactive intestinal polypeptide were unchanged. In summary, there is a marked increase in nerve fibers containing sensory neuropeptides in mesenteric vessels of STZ-induced diabetic rats, which, in view of the reported impaired sensorimotor function in these vessels, is likely to reflect a neuropathic change.

Effects of streptozotocin-diabetes on sympathetic nerve, endothelial and smooth muscle function in the rat mesenteric arterial bed.

Mesenteric arterial function was assessed in constantly perfused preparations isolated from rats 12 weeks after treatment with streptozotocin (65 mg kg-1, i.p.) to induce diabetes. Frequency-dependent vasoconstrictor responses to electrical field stimulation of sympathetic nerves (4-32 Hz, 0.1 ms, 90V, 30 s) were severely attenuated in preparations from streptozotocin-diabetic rats, although dose-dependent vasoconstrictions to the sympathetic cotransmitters noradrenaline and ATP, as well as to potassium chloride, were not significantly changed. Dose-dependent relaxations to the endothelium-dependent vasodilators acetylcholine and ATP were significantly impaired in preparations from streptozotocin-diabetic rats, although endothelium-independent vasodilatation to sodium nitroprusside was unimpaired. These results suggest 12 weeks after induction of streptozotocin-diabetes in rats there is pre-junctional impairment of sympathetic neurotransmission and impaired endothelial function of the mesenteric arteries. This is in contrast to our previous findings that at 8 weeks after induction of streptozotocin-diabetes sympathetic nerve and endothelial function is normal, although sensory-motor vasodilatation is severely attenuated. It is suggested that selective changes occur in mesenteric arterial function after streptozotocin treatment depending on the duration of diabetes; sensory-motor nerves are affected first, followed by sympathetic nerves and the endothelium, while the smooth muscle is relatively resistant to change.

Plasticity in the myenteric plexus of the rat ileum after long-term sympathectomy.

To investigate the effect of chronic sympathectomy on the innervation of a tissue with an extensive intrinsic component, 1-week-old rat pups were treated with 50 mg/kg guanethidine for 3 weeks, a treatment shown to produce complete and long-lasting sympathectomy, and the ileum examined. Changes in the levels of noradrenaline, neuropeptide Y, calcitonin gene-related peptide, substance P and vasoactive intestinal polypeptide in the external muscle layers containing the myenteric plexus of the ileum were determined between 6 and 20 weeks of age. After sympathectomy, noradrenaline levels were initially depleted (3% of age-matched controls at 6 weeks, P < 0.001, and 18% of age-matched controls at 12 weeks, P < 0.001), but were not significantly reduced at 20 weeks (67% of age-matched controls). Such increases in noradrenaline content with time after sympathectomy did not occur in the mesenteric vein (levels in 20-week-old sympathectomized rats were 2% of the control values (P < 0.001). In the myenteric plexus, catecholamine fluorescent nerve fibers were seen in the 12-week-old sympathectomized rats, although tyrosine hydroxylase-immunoreactivity was absent. Guanethidine sympathectomy had no effect on the neuropeptide levels in 6-week-old rat ileum but there was a selective increase at 20 weeks; the levels of calcitonin gene-related peptide and substance P were increased (X3, P < 0.001 and X1.6, P < 0.05, respectively) while vasoactive intestinal polypeptide and neuropeptide Y levels were unchanged. Short-term sympathectomy (destruction of sympathetic nerve terminals by acute 6-hydroxydopamine treatment) had no affect on noradrenaline or peptide levels in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Innervation of the rat gastrointestinal sphincters: changes during development and aging.

The effect of age on the adrenergic and peptidergic innervation of the lower oesophageal, pyloric and ileocaecal sphincters of the rat was investigated using immunohistochemical techniques. The distribution of nerve fibres containing the neuronal protein, growth associated protein-43, was also studied to determine the integrity of the enteric nervous system during development and aging. The four age groups examined were 2-3 days, 6 weeks, 3 months and 25 months old rats. Using protein gene product 9.5 antibody (a non-specific general neuronal marker), it was revealed that the myenteric ganglia in all sphincter regions were compactly arranged and were smaller in size at neonatal stage getting more spaced out and larger in size with age. There was no obvious change in the structure of the neutral elements with age. In the lower oesophageal sphincter, calcitonin gene-related peptide- and substance P-like immunoreactive nerve fibres showed notable changes in density and fluorescence intensity with age, decreasing and increasing, respectively, with no obvious change in vasoactive intestinal polypeptide- and growth-associated protein-like immunoreactivity. A slight increase in dopamine-beta-hydroxylase-like immunoreactivity was seen in old age. In the pyloric sphincter, there was an increase in calcitonin gene-related peptide- and substance P-like immunoreactivity with a less notable increase in dopamine-beta-hydroxylase-like immunoreactivity. A decrease in vasoactive intestinal polypeptide- and growth-associated protein-43-like immunoreactivity in the circular muscle of the sphincter was seen in old age. In the ileocaecal sphincter there was a marked increase in growth associated protein-43-, vasoactive intestinal polypeptide-, dopamine-beta-hydroxylase and substance P-like immunoreactivity. There was a decrease in the density of calcitonin gene-related peptide-like immuno-reactive nerve fibres in old age. In summary, two main conclusions can be drawn from the results of the present study. First, there was an age-related differential change in the density of immunoreactive nerve fibres containing various neuroactive substances. This indicates a level of plasticity of the various enteric nerve types and may reflect the degree of importance of the different neurotrasmitters in the physiological activities of the specific sphincter.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of age on NADPH-diaphorase-containing myenteric neurones of rat ileum and proximal colon.

The effect of age on the distribution of NADPH-diaphorase-containing neurones was investigated in the myenteric plexus of ileum and proximal colon of embryonic day-19 rats, as well as in rats at postnatal day 4, 6 months and 26 months. The mean percentage of NADPH-diaphorase-stained neurones per ganglion was established using protein gene product 9.5(protein found in most if not all neurones)-immunostained neurones as 100%. The results revealed that there was a significant relative increase in NADPH-diaphorase-positive neurones with increasing age in the myenteric plexus of proximal colon with nearly all protein gene product 9.5-immunoreactive neurones staining for NADPH-diaphorase in 26-month-old rats. This was in marked contrast with the ileum, where no significant relative increase in NADPH-diaphorase-positive neurones was seen in aged rats. The implications of these findings in relation to programmed cell survival and cell death are discussed.

Effects of acrylamide on cotransmission in perivascular sympathetic and sensory nerves.

The effects of chronic administration of acrylamide on sympathetic and sensory nerves were examined in the mesenteric artery of rabbits. The noradrenaline (NA) content of the artery was significantly decreased and the total contractile response to electrical field stimulation (4-64 Hz) markedly reduced in the acrylamide group. This was not due to an impairment of the contractility of the smooth muscle or to alterations in the postjunctional receptors. At 16 Hz, only the purinergic component of sympathetic cotransmission was significantly reduced by acrylamide. At 64 Hz, both the purinergic and the adrenergic components were significantly decreased. Field stimulation of the artery pretreated with guanethidine and precontracted with NA produced a frequency-dependent relaxation which was prevented by capsaicin and thus mediated by perivascular sensory nerves. In contrast to its effects on sympathetic cotransmission, acrylamide resulted in a trend, although not significant, towards increased responses at each frequency studied (2-16 Hz). 2-Methylthio-ATP (2Me-S-ATP) caused significantly greater relaxation following acrylamide treatment while vasodilator responses to calcitonin gene-related peptide and substance P were unchanged. It is concluded that, in addition to its known action in producing neuropathy in myelinated somatic motor and sensory nerves, acrylamide causes damage to unmyelinated perivascular sympathetic fibres. Purinergic mechanisms may be particularly susceptible to acrylamide since both the purinergic component of sympathetic vasoconstriction and the relaxation in response to 2Me-S-ATP were affected by acrylamide treatment.

Evidence for coexistence of ATP and nitric oxide in non-adrenergic, non-cholinergic (NANC) inhibitory neurones in the rat ileum, colon and anococcygeus muscle.

The possible coexistence of the two non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitters, adenosine 5'-triphosphate and nitric oxide in the myenteric plexus was investigated using whole-mount preparations of rat ileum, proximal colon and anococcygeus muscle. The presence of adenosine 5'-triphosphate in neurones was examined using the quinacrine fluorescence technique. After localizing and taking photographs of quinacrine-fluorescent neurones and nerve fibres, the same tissues were then fixed and processed for NADPH-diaphorase activity, a marker for nitric oxide-containing neurones. We have demonstrated for the first time that almost all quinacrine-fluorescent myenteric neurones in the proximal colon are also NADPH-diaphorase reactive, while only a subpopulation of quinacrine-fluorescent neurones in ileum and anococcygeus muscle were also NADPH-diaphorase reactive.

Noradrenergic-nitrergic interactions in the rat anococcygeus muscle: evidence for postjunctional modulation by nitric oxide.

1. The distribution of NADPH-diaphorase positive and catecholamine-containing nerve structures, and functional noradrenergic-nitrergic interactions, were studied in the rat anococcygeus muscle. 2. The morphological findings demonstrated NADPH-diaphorase positive neurons mostly as aggregates in intramural ganglia, nerve tracts and few single nerve fibres forming plexus-like structures. 3. The nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NOARG) inhibited concentration-dependently the nitrergic relaxation, an effect reversed by L-arginine. The drug had dual effects on noradrenergic contractile responses: at lower concentrations (0.1-10 microM) it decreased the amplitude of contractions and this was not affected by L-arginine; higher concentrations (50-500 microM) potentiated the contractions, an effect that was prevented by L-arginine. 4. The electron acceptor, nitro blue tetrazolium (NBT) produced a rapid inhibition of the noradrenergic contractile responses (EC50 0.178 +/- 0.041 microM). The drug decreased the tone of the preparations. However, it potentiated concentration-dependently the nitrergic relaxations. 5. NBT (1 microM) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)-donor sodium nitroprusside (SNP, 0.01-50 microM). However, the effect of NBT (0.1-10 microM) on the electrically induced relaxation was significantly decreased by L-NOARG (10 and 50 microM). The inhibition was of a non-competitive type. 6. Neither L-NOARG (100 microM) nor NBT (1 microM) had any effect on the spontaneous or electrically-induced release of 3H-radioactivity from the tissues preincubated in [3H]-noradrenaline. 7. It is concluded that L-arginine-NO pathway can modulate noradrenergic transmission in the rat anococcygeus muscle at postjunctional, but not prejunctional site(s).

A developmental study of the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder.

A histochemical investigation of age-related changes that occur with respect to the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder was carried out. In all age groups examined (embryonic stages day 34 and 52, 2 to 4-day old, 6-month old and 2-year old), the mean percentage of NADPH-diaphorase-positive neurons per ganglion was obtained by taking the number of neurons that were immunoreactive to protein gene product 9.5 (a general neuronal marker) as 100%. In addition, the possible co-existence of NADPH-diaphorase and nitric oxide synthase in the ganglionated plexus of 2 to 4-day old and 6-month old guinea-pig gallbladder was investigated. NADPH-diaphorase was not present in the ganglionated plexus of the gallbladder at embryonic day 34. At embryonic day 52, all the protein gene product 9.5-immunoreactive neurons showed positive staining to NADPH-diaphorase; this dropped to a minimum at 2-4 days (26.7%), rose slightly at 6 months (33.6%), and finally returned close to the 100% value at 2 years. In the gallbladders of 2-year old guinea-pigs, some (3 out of 10) ganglia were devoid of protein gene product 9.5-immunoreactive neurons, but NADPH-diaphorase-stained granules were found within the ganglia. However, all those neurons that were immunopositive to protein gene product 9.5 also expressed NADPH-diaphorase. Moreover, NADPH-diaphorase-positive neurons in the gallbladder of 2 to 4-day-old and 6-month-old guinea-pigs were found to express nitric oxide synthase.

An ultrastructural study of NADPH-diaphorase and nitric oxide synthase in the perivascular nerves and vascular endothelium of the rat basilar artery.

This is the first report on the ultrastructural distribution of nicotinamide adenine dinucleotide phosphate-diaphorase activity and neuronal isoform (Type I) of nitric oxide synthase immunoreactivity in perivascular nerves (axons) and vascular endothelial cells. In the Sprague-Dawley rat cerebral basilar artery, positive labelling for nicotinamide adenine dinucleotide phosphate-diaphorase and nitric oxide synthase was localized in axons and the endothelium. Over half (approximately 53%) of the axon profiles examined were positive for nicotinamide adenine dinucleotide phosphate-diaphorase. Labelling of nicotinamide adenine dinucleotide phosphate-diaphorase activity in the axons and endothelial cells was mostly distributed in patches within the cytoplasm. In endothelial cells, a relation between the nicotinamide adenine dinucleotide phosphate-diaphorase-labelling and cytoplasmic vesicle-like structures was seen. In both axons and the endothelium, nitric oxide synthase immunoreactivity was seen throughout the cell cytoplasm and in association with the membranes of mitochondria, endoplasmic reticulum and cytoplasmic/synaptic vesicles (the lumen/content of the vesicles was negative for nitric oxide synthase). Also, microtubules were labelled in nitric oxide synthase positive axon profiles. The nitric oxide synthase-positive axon varicosities were characterized by the presence of spherical agranular vesicles with a diameter of 40-50 nm. Approximately 30% of the axon profiles examined were positive for nitric oxide synthase. The nicotinamide adenine dinucleotide phosphate-diaphorase-positive endothelial cells (approximately 20% of all observed endothelial cell profiles) were more frequently seen than those positive for nitric oxide synthase (approximately 7%). It is suggested that nitric oxide released from both perivascular nerves and endothelial cells may be involved in vasomotor control of cerebral circulation.