Phenotypic changes of morphologically identified guinea-pig myenteric neurons following intestinal inflammation.

We investigated the responses of morphologically identified myenteric neurons of the guinea-pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin-resistant component, and a prolonged after-hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca(2+)-dependent K(+) channel blocker TRAM-34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin-resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (I(h)) antagonist Cs(+). It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.

The distribution of PKC isoforms in enteric neurons, muscle and interstitial cells of the human intestine.

In many organs, different protein kinase C (PKC) isoforms are expressed in specific cell types, suggesting that the different PKCs have cell-specific roles, and also that drugs acting on a particular PKC may have effects on the whole organ that are distinguishable from drugs that target other isoforms. Previous studies of the guinea-pig and mouse intestine indicate that there are cell-specific expressions of PKC isoforms in neurons, muscle and the interstitial cells of Cajal. In the present study we have investigated the expression of different PKCs in human intestine. Immunohistochemical studies showed that the forms that are prominent in human enteric neurons are PKCs gamma and epsilon and in muscle the dominant form is PKCdelta. Neurons were weakly stained for PKCbetaI. These observations parallel findings in guinea-pig and mouse, except that in human PKCgamma-IR was not present in the same types of neurons that express it in the guinea-pig. Enteric glial cells were strongly immunoreactive for PKCalpha, which is also the major isoform in enteric glial cells of guinea-pig. In human and guinea-pig, glial cells also express PKCbetaI. Spindle-shaped cells in the mucosa were immunoreactive for PKCalpha and PKCgamma and in the muscle layers similar cells had PKCgamma-IR and PKCtheta-IR. The spindle-shaped cells were similar in morphology to interstitial cells of Cajal. Western analysis and RT-PCR confirmed the presence of the PKC isoform proteins and mRNA in the tissue. We conclude that there is cell-type specific expression of different PKCs in enteric neurons and intestinal muscle in human tissue, and that there are strong similarities in patterns of expression between laboratory animals and human, but some clear differences are also observed.

Intermediate-conductance calcium-activated potassium channels in enteric neurones of the mouse: pharmacological, molecular and immunochemical evidence for their role in mediating the slow afterhyperpolarization.

Calcium-activated potassium channels are critically important in modulating neuronal cell excitability. One member of the family, the intermediate-conductance potassium (IK) channel, is not thought to play a role in neurones because of its predominant expression in non-excitable cells such as erythrocytes and lymphocytes, in smooth muscle tissues, and its lack of apparent expression in brain. In the present study, we demonstrate that IK channels are localized on specific neurones in the mouse enteric nervous system where they mediate the slow afterhyperpolarization following an action potential. IK channels were localized by immunohistochemistry on intrinsic primary afferent neurones, identified by their characteristic Dogiel type II morphology. The slow afterhyperpolarization recorded from these cells was abolished by the IK channel blocker clotrimazole. RT-PCR and western analysis of extracts from the colon revealed an IK channel transcript and protein identical to the IK channel expressed in other cell types. These results indicate that IK channels are expressed in neurones where they play an important role in modulating firing properties.

Intermediate conductance potassium (IK) channels occur in human enteric neurons.

IK channels, which had been previously found in hemopoetically derived cells (including erythrocytes and lymphocytes) and epithelial cells, where they regulate proliferation, cell volume regulation and secretion, have only recently been discovered in neurons, where they had previously been claimed not to occur. Based on immunohistochemical detection of IK channel-like immunoreactivity, it has been reported that IK channel expression in enteric neurons is suppressed in Crohn's disease. In the present work we have investigated whether authentic IK channels are expressed by enteric neurons. Human and mouse tissue was investigated by immunohistochemistry, Western blot and RT-PCR. Immunohistochemical studies revealed IK channel-like immunoreactivity in large myenteric neurons, but not in other cell types in the external muscle layers. Many of these nerve cells had calbindin immunoreactivity. Western blots from the external muscle revealed an immunoreactive band at the molecular weight of the IK channel. Using RT-PCR, we detected a transcript corresponding to the IK channel gene in extracts from the ganglion containing layer. The sequence obtained from the RT-PCR product was identical to that previously published for the IK channel. We conclude that IK channels are expressed by human enteric neurons, including large smooth surfaced neurons that are possibly the human equivalent of the Dogiel type II neurons that express these channels in small mammals.

Projections and chemistry of Dogiel type II neurons in the mouse colon.

The physiological properties, shapes, projections and neurochemistries of Dogiel type II neurons have been thoroughly investigated in the guinea-pig intestine in which these neurons have been identified as intrinsic primary afferent neurons. Dogiel type II neurons in the myenteric ganglia of mice have similar physiological properties to those in guinea-pigs but whether other features of the neurons are similar is unknown. We have used intracellular dye-filling, retrograde tracing, immunohistochemistry and nerve lesions to determine salient features of Dogiel type II neurons of the mouse colon. Dye-filling showed that the neurons provide profuse terminal networks in the myenteric ganglia and also have axons that project towards the mucosa. Retrograde tracing and lesion studies showed that these axons provide direct innervation to the mucosa. High proportions of the neurons had immunoreactivity for calretinin, calbindin, choline acetyltransferase, the purine P2X2 receptor and calcitonin gene-related peptide (CGRP). CGRP was the most selective marker of the neurons. Following surgery to remove an area of myenteric plexus, the CGRP-immunoreactive nerve fibres in the mucosa degenerated. Thus, Dogiel type II neurons in mice have similar shapes and projections but some differences in chemistry from those in guinea-pigs. The close similarities between the two species in the shapes, projections and electrophysiology of these neurons suggest that they serve the same functions in both species.

Expression of intermediate conductance potassium channel immunoreactivity in neurons and epithelial cells of the rat gastrointestinal tract.

Recent functional evidence suggests that intermediate conductance calcium-activated potassium channels (IK channels) occur in neurons in the small intestine and in mucosal epithelial cells in the colon. This study was undertaken to investigate whether IK channel immunoreactivity occurs at these and at other sites in the gastrointestinal tract of the rat. IK channel immunoreactivity was found in nerve cell bodies throughout the gastrointestinal tract, from the esophagus to the rectum. It was revealed in the initial segments of the axons, but not in axon terminals. The majority of immunoreactive neurons had Dogiel type II morphology and in the myenteric plexus of the ileum all immunoreactive neurons were of this shape. Intrinsic primary afferent neurons in the rat small intestine are Dogiel type II neurons that are immunoreactive for calretinin, and it was found that almost all the IK channel immunoreactive neurons were also calretinin immunoreactive. IK channel immunoreactivity also occurred in calretinin-immunoreactive, Dogiel type II neurons in the caecum. Epithelial cells of the mucosal lining were immunoreactive in the esophagus, stomach, small and large intestines. In the intestines, the immunoreactivity occurred in transporting enterocytes, but not in mucous cells. Immunoreactivity was at both the apical and basolateral surfaces. A small proportion of mucosal endocrine cells was immunoreactive in the duodenum, ileum and caecum, but not in the stomach, proximal colon, distal colon or rectum. There was immunoreactivity of vascular endothelial cells. It is concluded that IK channels are located on cell bodies and proximal parts of axons of intrinsic primary afferent neurons, where, from functional studies, they would be predicted to lower neuronal excitability when opened in response to calcium entry. In the mucosa of the small and large intestine, IK channels are probably involved in control of potassium exchange, and in the esophageal and gastric mucosa they are possibly involved in control of cell volume in response to osmotic challenge.

Protein kinase C isoforms in the enteric nervous system.

C kinases (PKCs) are a family of enzymes essential for the transduction of signals in a diverse range of cell types, including neurons. The different isoforms vary in their activation requirements. Therefore, cell-specific expression of different isoforms has implications for PKC-mediated control of organ function. This study has investigated the types and distributions of PKC isoforms in the small intestine of the guinea-pig, with particular emphasis on their localisation in myenteric neurons, using immunohistochemistry and western blotting techniques. Three PKC isoforms, gamma, eta and theta, were detected in the calbindin-immunoreactive subset of intrinsic primary afferent neurons, but not in other myenteric neurons. Both gamma and theta immunoreactivities were also located in interstitial cells of Cajal. In contrast to these isoforms, immunoreactivity for PKCs lambda and epsilon was present in all myenteric neurons of the ileum. PKCalpha immunoreactivity was detected primarily in the glial network, as shown through double labelling with antibodies to the glial filament protein, S100b. Myenteric neurons were also weakly immunoreactive for this isoform. PKCdelta immunoreactivity was very highly expressed in smooth muscle, but was largely absent from neurons. Immunoreactivity for RACK1, a binding protein for PKCbeta, was detected in both calbindin-immunoreactive neurons and in smooth muscle cells. This study indicates a selective distribution of PKC isoforms to specific cell types. Isoform-specific activity of these enzymes could provide a means through which targeted modulation of intestinal function is achieved.

P2X(2) purine receptor immunoreactivity of intraganglionic laminar endings in the mouse gastrointestinal tract.

The distribution of P2X(2) purine receptor subunit immunoreactivity has been investigated in the mouse gastrointestinal tract. Immunoreactivity occurred in intraganglionic laminar endings (IGLEs) associated with myenteric ganglia throughout the gastrointestinal tract. In the esophagus, IGLEs supplied every myenteric ganglion. The proportion of ganglia supplied decreased from 85% in the stomach to 10% in the ileum, and from 50% in the caecum to 15% in the distal colon. There was substantial loss of IGLEs from myenteric ganglia of all abdominal regions after bilateral subdiaphragmatic section of the vagus nerves. IGLEs in the esophagus consisted of dense clusters of punctate immunoreactive varicosities. In the stomach and duodenum they had prominent lamellar processes and irregular, but smaller, lamellae were found in other regions. Rare immunoreactive IGLEs occurred in the submucosa of the distal colon. P2X(2) receptor immunoreactivity was on the surfaces and in the cytoplasm of a minority of nerve cells in myenteric ganglia. It is concluded that P2X(2) purine receptor immunoreactivity is a feature of IGLEs in the mouse, and that P2X receptor agonists may modulate sensitivity of the IGLEs.

Evidence that two forms of choline acetyltransferase are differentially expressed in subclasses of enteric neurons.

Cholinergic neurons have been revealed in the enteric nervous system by functional and biochemical studies but not by antibodies that provide excellent localisation of the synthesising enzyme, choline acetyltransferase (ChAT), in the central nervous system. In order to determine whether a newly described peripheral form of ChAT (pChAT) is a ChAT enzyme of enteric neurons, we have compared pChAT distribution with that of the common form of ChAT, cChAT, by quantitative analysis of the co-localisation of pChAT and cChAT with other neurochemical markers in enteric neurons of the guinea-pig ileum. We found classes of neuron with strong pChAT immunoreactivity (IR) and others with strong cChAT-IR. In myenteric ganglia, strong pChAT-IR was in calbindin-positive intrinsic primary afferent neurons (IPANs), whereas cChAT-IR of these neurons was weak. Calretinin neurons were immunoreactive for cChAT, but not pChAT. Only 4% of nitric oxide synthase (NOS) neurons (possibly interneurons) were pChAT-immunoreactive, similar to observations with cChAT. NOS-immunoreactive inhibitory motor neurons stained with neither cChAT nor pChAT antisera. In the submucosal ganglia, pChAT-IR was strongly expressed in IPANs (identified by cytoplasmic staining for the neuronal nuclear marker, NeuN) and in neuropeptide Y (NPY)-immunoreactive secretomotor neurons, but not in calretinin-immunoreactive neurons. cChAT-IR occurred weakly in submucosal IPANs and also labelled NPY- and calretinin-immunoreactive neurons. Submucosal vasoactive-intestinal-peptide-immunoreactive neurons (non-cholinergic secretomotor neurons) were not reactive for either form of ChAT.

The distribution of P2X3 purine receptor subunits in the guinea pig enteric nervous system.

Adenosine 5'-triphosphate (ATP) excites 70-90% of enteric neurons through P2X type purine receptors, and is likely to be an enteric neurotransmitter. Recent studies indicate that the P2X2 subunit is expressed by specific subgroups of enteric neurons, and that there are enteric neurons that are responsive to ATP but lack this subunit. In the present work, we have investigated whether the P2X3 subunit is similarly localised to specific subgroups of neurons, and whether these are different from the P2X2 subunit-expressing neurons. The P2X3 subunit was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines, and nerve cells of the submucosal ganglia in the small and large intestines of the guinea pig. All immunoreactivity was absorbed with the P2X3 receptor peptide against which the antiserum was raised. In myenteric ganglia of the ileum, P2X3 receptor immunoreactivity was in calretinin, enkephalin and nitric oxide synthase (NOS)-immunoreactive neurons. In submucosal ganglia, all calretinin-immunoreactive nerve cells were P2X3 receptor immunoreactive. In the submucosal ganglia of the ileum, 13 +/- 3% of neuropeptide Y (NPY)-immunoreactive neurons were also P2X3 receptor immunoreactive, whereas in the distal colon, almost all NPY-expressing nerve cells were P2X3 receptor immunoreactive. The localisation of the P2X3 subunit was largely distinct from that of the P2X2 subunit, although both subunits occur in some NOS neurons, where P2X2 and P2X3 subunits may form heteromeric receptors. Unlike the P2X2 subunit, the P2X3 subunit is not expressed in intrinsic sensory neurons in the ileum. It is concluded that the P2X3 receptor subunit is expressed in specific functional groups of neurons; the major types are excitatory and inhibitory muscle motor neurons, ascending interneurons and cholinergic secretomotor neurons.

The distribution of purine P2X(2) receptors in the guinea-pig enteric nervous system.

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.