Biomedical subjects

J B Furness

Publications and source records attributed to J B Furness.

At least 91 records · Page 5Linked to original sources

PubMed1996

Characterisation of substance P-induced endocytosis of NK1 receptors on enteric neurons.

Immunoreactivity for NK1 receptors is confined to specific nerve cell bodies in the guinea-pigileum, including inhibitory motor neurons and secretomotor neurons. In the present work, endocytosis of NK1 receptors in these enteric neurons was studied following addition of substance P (SP) to isolated ileum. NK1 receptors were localised with antibodies against the C-terminus of this receptor. Some preparations were incubated with SP tagged with the fluorescent label, Cy3.18, so that the fate of SP bound to receptors could be followed. Preparations were analysed by confocal microscopy. In tissue that was incubated at 4 degrees C in the absence of SP, most NK1 receptor immunoreactivity (IR) was confined to surface membranes of nerve cells. At 37 degrees C in the presence of 10(-7) M SP (plus 3 x 10(-7)M tetrodotoxin to prevent indirect activation via other neurons) the neuronal NK1 receptor was rapidly internalised. After 5 min, NK1 receptor IR was partially internalised, at 20 min NK1 receptor IR was throughout the cytoplasm and in perinuclear aggregates and at 30 min it was again at the cell surface. SP-induced NK1 receptor endocytosis was inhibited by the specific NK1 receptor antagonist, SR140333. Cy3-SP was colocalised with NK1 receptor IR and was internalised with the NK1 receptor. These results show that enteric neurons exhibit authentic NK1 receptors that are rapidly internalised when exposed to their preferred ligand.

Animals↗

PubMed1996

GABA and nitric oxide synthase immunoreactivities are colocalized in a subset of inhibitory motor neurons of the guinea-pig small intestine.

Simultaneous immunofluorescence labelling was used to determine the patterns of colocalization of immunoreactivity for gamma-aminobutyric acid (GABA-IR) with immunoreactivity for nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP) and tachykinins (TK) in nerve cells and fibres of the guinea-pig small intestine. GABA-IR nerve cell bodies were located in the myenteric plexus and varicose fibres innervated the circular and longitudinal muscle, but did not form pericellular endings in the myenteric ganglia. GABA-IR nerve cells comprised 4-5% of all nerve cells in the myenteric ganglia. Of GABA-IR myenteric nerve cells, about 85% had NOS-IR and of GABA-IR nerve fibres in both muscle layers, about 75% were NOS-IR. Conversely, 20% of NOS-IR nerve cells were GABA-IR. About 6% of GABA-IR nerve fibres innervating the circular muscle, but none innervating the longitudinal muscle, were TK-IR. Most GABA-IR fibres supplying the circular muscle, but none of those supplying the longitudinal muscle, were VIP-IR. From this study, and previous studies of projections of enteric neurons, it is concluded that most GABA-IR neurons in the guinea-pig small intestine are inhibitory motor neurons that also contain NOS-IR. A small proportion represents anally directed excitatory motor neurons that innervate the circular muscle and are also immunoreactive for TK.

Animals↗

PubMed1996

Calretinin immunoreactivity of motor neurons in the guinea-pig distal colon and taenia coli.

Calretinin is a calcium-binding protein which occurs in neurons and endocrine cells, including neurons throughout the gastrointestinal tract. Calretinin-immunoreactive (IR) neurons innervate the circular muscle in the guinea-pig distal colon and have descending as well as ascending projections. This suggests that calretinin-IR is in motor neurons, but whether it might be in excitatory or inhibitory motor neurons or both was previously undetermined. The presence of calretinin-IR in neurons innervating the taenia coli has not been previously reported. Numerous fibres in the circular muscle of the distal colon and in the taenia coli displayed immunoreactivity for calretinin. Tachykinin (TK), vasoactive intestinal peptide (VIP), calretinin, and gamma-aminobutyric acid (GABA) immunoreactivity was also in fibres innervating these targets. The abundances of these fibres was estimated to be TK > VIP > calretinin > GABA. Double label immunohistochemistry revealed the presence in both tissues of populations of calretinin-IR fibres which were also TK-IR, and fibres with calretinin and GABA-IR in the colon, but calretinin-IR fibres were never VIP-IR. TK- and VIP-IR were in separate populations of nerve fibres as were GABA- and TK-IR. It is concluded that calretinin-IR does not provide a definitive labelling of a physiologically known subgroup of motor neurons, either in the distal colon or in the taenia coli, but that calretinin is most likely to be in excitatory motor neurons.

Animals↗

PubMed1996

Distribution of neurokinin-2 receptors in the guinea-pig gastrointestinal tract.

The distribution of neurokinin-2 (NK2) tachykinin receptors was investigated by immunohistochemistry in the guinea-pig oesophagus, stomach, small and large intestine. Receptor immunoreactivity occurred at the surfaces of smooth muscle cells throughout the digestive tract. Nerve fibre varicosities in enteric ganglia were also immunoreactive. In myenteric ganglia, these varicosities were most numerous in the ileum, frequent, but less dense, in the proximal colon and caecum, rare in the distal colon, extremely infrequent in the rectum and duodenum, and absent from the stomach and oesophagus. Reactive varicosities were rare in the submucous ganglia. Reactive nerve fibres in the mucosa were only found in the caecum and proximal colon. Strong NK2 receptor immunoreactivity was also found on the surfaces of enterocytes at the bases of mucosal glands in the proximal colon. Receptors were not detectable on the surfaces of nerve cells or on non-terminal axons. Reactivity did not occur on nerve fibres innervating the muscle. Denervation studies showed that the immunoreactive varicosities in the myenteric plexus of the ileum were the terminals of descending interneurons. Immunoreactivity for nitric oxide synthase was colocalised with NK2 receptor (NK-R) immunoreactivity in about 70% of the myenteric varicosities in the small intestine. Bombesin immunoreactivity occurred in about 30% of NK2-R immunoreactive varicosities in the small intestine.

Animals↗

PubMed1996

Experimental basis for realistic large-scale computer simulation of the enteric nervous system.

1. The enteric nervous system is perhaps the most accessible part of the mammalian nervous system in which it is feasible to attempt large scale computer simulation that is based closely on experimentally determined data. Here we summarize the data obtained for simulation of motility reflexes in the guinea-pig small intestine. 2. The chemistry, morphology and connectivity of each type of neuron involved in intrinsic reflexes have been investigated and most classes of neurons are physiologically well characterized. This includes primary sensory neurons, ascending and descending interneurons and motor neurons to circular and longitudinal muscle. 3. The responses of primary sensory neurons and the physiology of synaptic transmission from sensory neurons to interneurons and motor neurons, from interneurons to interneurons and from interneurons to motor neurons have been recorded during reflexes and in some cases the pharmacology of transmission has also been investigated. 4. Computer simulation, in which the activities of up to 30,000 neurons are modelled, produces patterns of activity that closely mimic those recorded in physiological experiments.

Animals↗

PubMed1996

Direct measurement of the release of ATP and its major metabolites from the nerve fibres of the guinea-pig taenia coli.

1. The release of adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate and adenosine from guinea-pig taenia coli in response to electrical stimulation of intramural nerves was measured directly using high performance liquid chromatography separation and fluorometric detection. 2. Purines were released in a frequency-dependent manner by trains of transmural electrical pulses at 1-30 Hz. 3. Electrically evoked release of ATP was abolished by tetrodotoxin (10(-6)mol/L) but was not affected by nicardipine (10(-6)mol/L). 4. The release of purines was reduced in the presence of atropine. 5. Pituitary adenylyl cyclase-activating peptide did not evoke the release of any purines and did not modify the electrically evoked release of purines. 6. The results suggest that ATP and its major metabolites are released from a neuronal source, possibly the enteric inhibitory nerves, in the guinea-pig taenia coli.

Adenosine↗

PubMed1996

Analysis of contributions of acetylcholine and tachykinins to neuro-neuronal transmission in motility reflexes in the guinea-pig ileum.

1. The roles of acetylcholine (ACh) and tachykinins in neuro-neuronal transmission during ascending excitatory and descending inhibitory reflexes were studied by recording intracellular reflex responses of the circular muscle to physiological stimuli. Experiments were carried out in opened segments of guinea pig ileum in an organ bath that was partitioned so that three regions could be independently exposed to drugs. 2. Ascending excitatory reflexes evoked by either distension from the serosal side or compression of the mucosa were depressed by 55% and 85%, respectively, in the presence of hexamethonium (200 microM) and by 30% and 45%, respectively, by a desensitizing concentration of the selective NK3 receptor agonist, senktide (1 microM), in the chamber in which reflexes were initiated. Together, hexamethonium and senktide abolished responses to compression. A residual response to distension persisted. This was abolished by hyoscine (1 microM). 3. Hexamethonium (200 microM) abolished ascending reflexes when applied to the region between the stimulus and the recording sites, or to the recording chamber. 4. Descending reflex responses were reduced by 35% by synaptic blockade in the stimulus chamber with physiological saline containing 0.1 mM Ca2+ plus 10 mM Mg2+. Senktide (1 microM) in the stimulus chamber reduced distension reflexes to the same extent as synaptic blockade, whereas hexamethonium (200 microM) and hyoscine (1 microM) depressed responses by less than 20%. Responses to compression were reduced by 40% by senktide alone, while senktide and hexamethonium together reduced responses by 60%, an effect similar to synaptic blockade. Under these conditions, hyoscine in the stimulus chamber restored reflexes evoked by distension, but did not alter those evoked by mucosal compression. 5. Total synaptic blockade in the intermediate chamber, between stimulus and recording sites, reduced descending reflex responses by more than 90%. In contrast, hexamethonium (200 microM) had no effect and hyoscine (1 microM) reduced only the responses to distension (by 30%). Senktide (1 microM) depressed responses to both stimuli by approximately 80%. 6. Application of hexamethonium (200 microM) to the recording chamber depressed descending reflex responses to distension applied in the near stimulation chamber by 15%, but had no effect on responses to compression in the near chamber or to either stimulus applied in the far chamber. 7. Descending reflexes evoked by near chamber stimuli were unaffected by hyoscine (1 microM) or senktide (1 microM) applied to the recording chamber; hyoscine enhanced reflexes evoked by compression in the far chamber by 50%. 8. For the ascending excitatory reflex pathway, it is concluded that transmission from sensory neurones is mediated by ACh acting via both nicotinic and muscarinic receptors, and by tachykinins acting at NK3 receptors. Transmission from ascending interneurones appears to be predominantly via nicotinic receptors. The descending inhibitory pathways are more complex, and while transmission from sensory neurones involves nicotinic, muscarinic and NK3 receptor-dependent components, transmission from descending interneurones to inhibitory motor neurones is neither cholinergic nor due to tachykinins acting via NK3 receptors.

Acetylcholine↗

PubMed1995

Chemical coding of neurons that project from different regions of intestine to the coeliac ganglion of the guinea pig.

The chemical codings of neurons that project from the small intestine, caecum, proximal colon, distal colon and rectum to the coeliac ganglion of the guinea pig were investigated. The coeliac ganglion was injected with the retrogradely transported dye Fast Blue, and each of the regions was examined 6 days later in wholemounts that had been prepared for immunohistochemical localisation of pairs of antigens. In both the small and large intestines, all intestinofugal neurons were immunoreactive (IR) for choline acetyltransferase (ChAT). In each region of the large intestine, the largest population, representing 50-60% of retrogradely labelled neurons in each region, was immunoreactive for ChAT, bombesin (BN), calbindin (Calb) and nitric oxide synthase (NOS). Most intestinofugal neurons in the small intestine contain bombesin and VIP-IR along with ChAT-IR but none contain either Calb or NOS. Thus, nerve endings of enteric origin in the coeliac ganglion that contain NOS-IR or Calb-IR come from the large intestine and those with bombesin-IR but not NOS-IR are from the small intestine. The gastric wall was injected with Fast Blue in order to label noradrenergic (NA) neurons in the coeliac ganglion and to determine, by localisation of NOS and bombesin-IR, whether they receive inputs from the small and large intestine. Some NA neurons received inputs from the large intestine (and perhaps also from the small intestine) and some received inputs exclusively from the small intestine. Most NA neurons that received intestinofugal inputs had the chemical code NA/-; some were immunoreactive for somatostatin (NA/SOM neurons), but those with IR for neuropeptide Y (NA/NPY) rarely received intestinofugal inputs.

Animals↗

PubMed1995

Transcription and translation of two glutamate decarboxylase genes in the ileum of rat, mouse and guinea pig.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter, synthesised from glutamate by glutamate decarboxylase (GAD), in the central nervous system. Two forms of GAD, designated GAD 65 and GAD 67, are encoded by distinct genes and have been demonstrated in the mammalian brain. GABA has been postulated to be synthesised in neurons of the enteric nervous system (ENS), but evidence for its role as an enteric neurotransmitter is equivocal. We therefore aimed to determine whether GAD 65 and GAD 67 messenger RNAs (mRNAs) and proteins were expressed in the ileum of mice, rats and guinea pigs. Using an RNase protection assay, both GAD 65 and GAD 67 mRNAs were detected in the rodent small intestine. Antisera specific for GAD 65 or GAD 67, used in immunoblot analyses, revealed GAD 65-like and GAD 67-like immunoreactivity in rat and guinea pig ileum. Anti-GAD 65 antisera detected a major band of 65 kDa. Anti-GAD 67 antisera detected a major band of 55 kDa, which probably represented a breakdown product, and a minor band of 67 kDa. Analysis of immunoblot extracts of rat and guinea pig ileum revealed more GAD 67-like than GAD 65-like immunoreactivity. GAD enzymatic activity was high in the rat and guinea-pig brain, and low in the whole and dissected ileum. These results demonstrate that both GAD 65 and GAD 67 genes are transcribed and translated in the ileum of three rodent species and lend indirect support to the postulate that GABA is synthesised by neurons of the ENS and intestinal endocrine cells.

Amino Acid Sequence↗

PubMed1995

Ultrastructural examination of the targets of serotonin-immunoreactive descending interneurons in the guinea pig small intestine.

Serotonin neurons are descending interneurons in the myenteric plexus of the guinea pig small intestine. Preembedding single- and double-label immunocytochemistries at the ultrastructural level were used to identify the targets of these serotonin interneurons. Serial ultrathin sections were taken through a myenteric ganglion that had been processed for serotonin immunocytochemistry. The ganglion contained the cell bodies of 69 neurons, including 2 serotonin neurons and 6 neurons with the ultrastructural features of Dogiel type II cells. For each cell body in the ganglion, the number of serotonin inputs (synapses and close contacts) was determined. About 59% of the cell bodies did not receive any serotonin inputs. The most abundant serotonin terminals were related to two targets: other serotonin descending interneurons and a population of neurons with Dogiel type I morphology, but whose neurochemistry and function is unknown. The serotonin inputs to the serotonin cell bodies were located predominantly on the lamellar dendrites. Each of the Dogiel type II neurons received 3 or fewer serotonin inputs, and none of the serotonin inputs to Dogiel type II neurons formed a synapse. Overall, about 40% of the serotonin inputs formed synapses. The serotonin inputs to neurons that received many serotonin inputs were more likely to show synaptic specializations than serotonin inputs to neurons that received few serotonin inputs. Inhibitory motor neurons contain nitric oxide synthase (NOS). At the light microscope level, serotonin nerve fibers do not form dense pericellular baskets around NOS cell bodies. To determine whether there are serotonin inputs to NOS neurons, serial ultrathin sections were taken through a myenteric ganglion that had been processed for preembedding double-label immunocytochemistry, in which the NOS neurons were labeled with peroxidase-diaminobenzidine and the serotonin neurons with silver-intensified 1 nm gold. Only 1 out of 9 NOS cells examined in serial section received more than 5 serotonin inputs. The results suggest that, in the guinea pig small intestine, the serotonin descending interneurons are not an essential element of the descending inhibitory reflex.

Animals↗

PubMed1995

Characterisation of neurons with nitric oxide synthase immunoreactivity that project to prevertebral ganglia.

Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40-70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatin-immunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic nerve cell bodies in the intermediolateral column and dorsal commissural nucleus from T12 to L2 were labelled from the inferior mesenteric ganglion. Nearly 70% of neurons at each level were NOS immunoreactive. Thus, two sources of NOS terminals in prevertebral ganglia have been identified, intestinofugal neurons of the large, but not the small intestine, and sympathetic preganglionic neurons.

Amidines↗

PubMed1995

Histochemical, pharmacological, biochemical and chromatographic evidence that pituitary adenylyl cyclase activating peptide is involved in inhibitory neurotransmission in the taenia of the guinea-pig caecum.

The possibility that pituitary adenylyl cyclase-activating peptide (PACAP) is an inhibitory neurotransmitter has been investigated in the taenia of the guinea-pig caecum. The action of PACAP on muscle contractility and its ability to alter levels of adenosine-3':5'-cyclic monophosphate (cyclic AMP) and guanosine-3':5'-cyclic monophosphate (cyclic GMP) were investigated. PACAP-1-27 was an effective agonist, giving relaxations comparable in magnitude to isoproterenol; its EC50 was 3.4 x 10(-7) M. PACAP (10(-6) M) caused an almost two-fold increase in cyclic AMP levels; but the level of cyclic GMP was not affected. The relaxation caused by PACAP was slow in onset, with a latency of 5.8 +/- 0.8 s and reached a maximum at 9.1 +/- 1.1 s after onset. The relaxation was significantly reduced by apamin (10(-6) M) and suramin (10(-4) M) but was not reduced by tetrodotoxin (10(-7) M). Relaxation of the taenia coli caused by electrical stimulation of the inhibitory nerves was greatly reduced by apamin but only slightly reduced by suramin. PACAP-like immunoreactivity (-IR) was localised immunohistochemically in varicose nerve fibres within the taenia coli and in the underlying myenteric plexus and circular muscle. Approx. 50% of vasoactive intestinal peptide (VIP)-IR nerve fibres in the taenia also had immunoreactivity for PACAP; conversely, almost all PACAP-IR fibres were immunoreactive for VIP. PACAP-IR and substance P (SP)-IR were generally in separate fibres; only about 5% of SP-IR fibres were PACAP-IR. Radioimmunoassay revealed tissue concentrations of PACAP-1-27 and PACAP-1-38 of 1.0 +/- 0.1 and 2.1 +/- 0.3 (SEM) pmol/g wet weight of tissue, respectively. Material with PACAP-1-27 immunoreactivity co-eluted with authentic PACAP-1-27 on gel filtration chromatography, and PACAP-1-38 immunoreactivity also co-eluted with the authentic peptide. This study provides structural, chemical and pharmacological evidence that PACAP could be involved in inhibitory neurotransmission to the taenia coli of the guinea-pig caecum.

Animals↗

PubMed1995

Electrophysiological analysis of the actions of pituitary adenylyl cyclase activating peptide in the taenia of the guinea-pig caecum.

The actions of pituitary adenylyl cyclase activating peptide (PACAP) on membrane potential and conductance were investigated in the taenia of the guinea-pig caecum. The possible role of PACAP in inhibitory transmission was also investigated. Membrane potentials of smooth muscle cells were measured by intracellular microelectrodes, in the presence of hyoscine and nifidepine (both 10(-6)M. To determine conductance changes, current was passed from external plate electrodes using the technique of Abe and Tomita (1968). PACAP-27 caused a concentration dependent hyperpolarization of the muscle with a maximum of 12-15 mV at 10(-6)M. The hyperpolarization caused by PACAP was associated with a substantial increase in membrane conductance. The hyperpolarization was abolished by apamin (10(-6)M), a blocker of small conductance, calcium-dependent, potassium channels, and was reduced to about 50% by suramin (10(-4)M), which is an antagonist of P2 receptors for purines. The hyperpolarization was not reduced by tetrodotoxin (2 x 10(-6)M), suggesting PACAP acts directly on the muscle. With continued exposure to PACAP, the hyperpolarization decayed back to resting membrane potential after several minutes, possibly due to receptor desensitization. Inhibitory junction potentials (IJPs) were markedly reduced in amplitude in the period of presumed receptor desensitization to PACAP, were abolished by tetrodotoxin, but were not affected by suramin. Apamin abolished the IJP and revealed a small excitatory junction potential. This study implies that PACAP released from nerve fibres in the taenia caeci hyperpolarizes the muscle via an opening of apamin-sensitive potassium channels. The action is probably through type I PACAP receptors.

Animals↗

PubMed1995

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine.

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guinea-pig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin- and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.

Animals↗

PubMed1995

Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon.

Pituitary adenylyl cyclase activating peptide (PACAP) is a novel hypothalamic peptide that is widely distributed in neurons, including those of the gastrointestinal tract. In this study, a polyclonal antiserum directed against PACAP-27 was used to investigate the localisation of PACAP throughout the gut and to determine the projections of PACAP-immunoreactive (IR) neurons in the guinea-pig small and large intestines. PACAP-IR fibres were seen in the myenteric and submucous plexuses, in the longitudinal and circular muscle layers and around blood vessels of the submucosa throughout the gut. In both the small and large intestine, PACAP-IR cell bodies, most with Dogiel type-I morphology, were seen in the myenteric ganglia following colchicine treatment. Lesion studies (myotomy and myectomy operations) revealed that PACAP-IR interneurons projected anally in the ileum and colon. Myectomy operations resulted in a loss of PACAP-IR fibres in the circular muscle under the operation, whereas PACAP-IR fibres remained in the submucosa and around blood vessels. Following extrinsic denervation of the ileum, the number of PACAP-IR fibres in the submucosal ganglia and around blood vessels decreased. This suggests that a portion of PACAP-IR fibres supplying the submucosal ganglia and blood vessels have an extrinsic source. To investigate this, immunohistochemical studies were performed on sympathetic and dorsal root ganglia. Numerous reactive cells were seen in the dorsal root ganglia, but none was seen in sympathetic pre- or paravertebral ganglia.

Animals↗

PubMed1995

Do vasoactive intestinal peptide (VIP)- and nitric oxide synthase-immunoreactive terminals synapse exclusively with VIP cell bodies in the submucous plexus of the guinea-pig ileum?

In the submucous plexus of the guinea-pig ileum, previous light-microscopic studies have revealed that vasoactive intestinal peptide (VIP)-immunoreactive and nitric oxide synthase (NOS)-immunoreactive terminals are found predominantly in association with VIP-immunoreactive nerve cell bodies. In this study, double-label immunohistochemistry at the light-microscopic level demonstrated co-localization of NOS-immunoreactivity and VIP-immunoreactivity in axon terminals in submucous ganglia. About 90% of nerve fibres with NOS-immunoreactivity or VIP-immunoreactivity were immunoreactive for both antigens; only about 10% of labelled varicosities contained only NOS-immunoreactivity or VIP-immunoreactivity. The VIP/NOS varicosities were more often seen in the central parts of the ganglia, close to the VIP-immunoreactive cell bodies. Ultrastructural immunocytochemistry with antibodies to VIP was used to determine if NOS/VIP terminals synapse exclusively with VIP-immunoreactive nerve cell bodies. We examined the targets of VIP-immunoreactive boutons in two submucous ganglia from different animals. Serial ultrathin sections were taken through the ganglia after they had been processed for VIP immunocytochemistry. For each cell body, the number of VIP inputs (synapses and close contacts) was determined. The number of VIP-immunoreactive synapses received by the cell bodies of submucous neurons varied from 0-4 and the number of VIP-immunoreactive close contacts varied from 3-10. There was no significant difference between VIP-immunoreactive nerve cell bodies and non-VIP nerve cell bodies in the number of VIP-immunoreactive synapses and close contacts they received. Thus, the implication from light microscopy that NOS/VIP terminals end predominantly on VIP nerve cells was not vindicated by electron microscopy.

Animals↗

PubMed1995

Analysis of connections between nitric oxide synthase neurons in the myenteric plexus of the guinea-pig small intestine.

In the myenteric plexus of the guinea-pig ileum, a sub-population of descending interneurons contains nitric oxide synthase. Final neurons in descending motility pathways, inhibitory circular muscle motor neurons, also contain nitric oxide synthase. In this study we used ultrastructural immunocytochemistry to determine whether nitric oxide synthase descending interneurons provide inputs to all nitric oxide synthase neurons. The presence of nitric oxide synthase inputs to 35 nitric oxide synthase nerve cells from three animals was examined. Nine nerve cells from one ganglion were studied in serial section. Every nerve cell received inputs (close contacts and synapses) from nitric oxide synthase terminals. The number of inputs to the nine serially sectioned neurons ranged from 13 to 45. The inputs were found in about equal numbers on the cell bodies and the dendrites. There was no significant correlation between the size of nitric oxide synthase neurons and the number of nitric oxide synthase inputs they received. There was also no correlation between the number of nitric oxide synthase inputs and the number of 5-hydroxytryptamine inputs (determined in a previous study) received by nitric oxide synthase neurons. Random sections through an additional 26 nitric oxide synthase neurons (seven in the same ganglion and 19 from another two myenteric ganglia from different animals) were examined and nitric oxide synthase synapses and close contacts were observed on each neuron. Nitric oxide synthase interneurons and motor neurons are morphologically indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMed1995

Nitric oxide synthase is localized predominantly in the Golgi apparatus and cytoplasmic vesicles of vascular endothelial cells.

The localization of nitric oxide synthase (NOS) in vascular endothelial cells of submucosal blood vessels from the guinea-pig ileum was examined using NADPH diaphorase histochemistry at the light microscopic level, and endothelial NOS immunohistochemistry at the light and electron microscopic level. The pattern of staining observed following NADPH diaphorase histochemistry and endothelial NOS immunohistochemistry was identical. Endothelial cells of the arterioles, capillaries and venules showed small patches of intense, perinuclear staining. Under the electron microscope, endothelial NOS immunoreactivity was found predominantly in association with the Golgi apparatus and with the membranes of some vesicles. Small regions of the plasma membrane and the rough endoplasmic reticulum also showed some immunoreactivity. The presence of NOS in the Golgi apparatus and in vesicles raises the possibility that NOS may be exteriorized by endothelial cells, and hence that nitric oxide is synthesized extracellularly.

Animals↗