Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system.

Hyperpolarisation-activated non-specific cation currents (Ih currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the Ih current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having Ih currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with Ih currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the Ih and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent Ih, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.

The opioid system in the gastrointestinal tract.

Mu-, delta- and kappa-opioid receptors (ORs) mediate the effects of endogenous opioids and opiate drugs. Here we report (1) the distribution of muOR in the guinea-pig and human gastrointestinal tract in relation to endogenous ligands, to functionally distinct structures in the gut and to deltaOR and kappaOR; and (2) the ligand-induced muOR endocytosis in enteric neurones using in vitro and in vivo models. In the guinea pig, muOR immunoreactivity is confined mainly to the myenteric plexus. MuOR myenteric neurones are most numerous in the small intestine, followed by the stomach and the proximal colon. MuOR immunoreactive fibres are dense in the muscle layer and the deep muscular plexus, where they are in close association with interstitial cells of Cajal. This distribution closely matches the pattern of enkephalin. MuOR enteric neurones comprise functionally distinct populations of neurones of the ascending and descending pathways of the peristaltic reflex. In human gut, muOR immunoreactivity is localized to myenteric and submucosal neurones and to immune cells of the lamina propria. DeltaOR immunoreactivity is located in both plexuses where it is predominantly in varicose fibres in the plexuses, muscle and mucosa, whereas kappaOR immunoreactivity appears to be confined to the myenteric plexus and to bundles of fibres in the muscle. MuOR undergoes endocytosis in a concentration-dependent manner, in vitro and in vivo. Pronounced muOR endocytosis is observed in neurones from animals that underwent abdominal surgery that has been shown to induce delay in gastrointestinal transit. We can conclude that all three ORs are localized to the enteric nervous system with differences among species, and that muOR endocytosis can be utilized as a means to visualize enteric neurones activated by opioids and sites of opioid release.

First localisation of somatostatin sst(4) receptor protein in selected human brain areas: an immunohistochemical study.

Somatostatin is known to have diverse neurophysiological effects in the mammalian CNS. To date, genes for five different receptors, termed sst(1-5), have been isolated. Recently several reports have been published on the localisation of the individual receptor protein in the rat CNS, but their localisation in the human CNS remains largely unknown. Until now little information about the function of the sst(4) receptor is available, and there is a lack of receptor specific agonists and antagonists. Here, we report for the first time the immunohistochemical localisation of the sst(4) receptor in selected human brain areas using an anti-peptide antibody raised against a carboxy-terminal portion of the receptor protein. Strong receptor immunoreactivity was found in several brain regions, including the hippocampal formation, the cerebellar cortex and the medulla. Further immunohistochemical labelling was observed in the cerebral cortex, the red nucleus and the globus pallidus. Somatodendritic as well as axonal staining was observed. Specific signals were entirely absent following antibody pre-adsorption with the synthetic peptide. The results are in good agreement with the previously published immunohistochemical localisation of the sst(4) receptor in the rat brain. This is the first immunohistochemical study of the localisation of the sst(4) receptor in the human brain, and implicates this receptor in the function of higher centres of the human nervous system.

Immunohistochemical localization of the somatostatin sst(4) receptor in rat brain.

The biological actions of the neuromodulator somatostatin are mediated through a family of G-protein-coupled receptors, of which five members, sst(1-5), have been identified. Although the messenger RNA distribution of the sst(4) receptor has been reported, no information about the distribution of the receptor protein in the central nervous system is available. We have therefore raised a polyclonal peptide antibody against a rat carboxy-terminal sst(4) peptide. The selectivity of the affinity-purified antibody was demonstrated by western blotting of membrane proteins isolated from Chinese hamster ovary-K1 cells expressing the recombinant sst(4) receptor and from the rat hippocampus. This resulted in both cases in the identification of a single band of approximately 42,000 mol. wt. Furthermore, the sst(4) receptor antibody selectively labelled Chinese hamster ovary-K1 cells expressing the recombinant sst(4) receptor in immunocytochemistry. No cross-reactivity was observed with other recombinant somatostatin receptors. Immunohistochemistry on adult rat brain sections showed the sst(4) receptor to have a widespread distribution. This included labelling of cell bodies as well as processes in the cerebral cortex, hippocampus and several nuclei in the brainstem. All signals were absent following antibody preabsorption with the synthetic sst(4) peptide. This study provides the first detailed analysis of the distribution of sst(4) receptor protein in the rat brain.

Upregulation of connexin 26 is a feature of keratinocyte differentiation in hyperproliferative epidermis, vaginal epithelium, and buccal epithelium.

In epidermis, it has been suggested, intercellular communication through gap junctions is important in coordinating cell behavior. The connexins, may facilitate selective assembly or permeability of gap junctions, influencing the distribution of metabolites between cells. Using immunohistochemistry, we have compared the distribution of connexins 26 and 43 with that of proliferating cells (Ki67 labeling) in normal epidermis, hyperplastic epidermis (tape-stripped epidermis, psoriatic lesions, and viral warts), and vaginal and buccal epithelia. Connexin 43 was abundant in spinous layers of all epidermal specimens and in vaginal and buccal epithelia. Connexin 26 was absent from the interfollicular and interductal epidermis of normal hair-bearing skin, and nonlesional psoriatic epidermis but present at very low levels in plantar epidermis. Connexin 26 was prominent in lesional psoriatic epidermis and viral warts and in vaginal and buccal epithelia. In three independent experiments connexin 26 appeared in a patchy intercellular distribution in the basal epidermis within 24 h of tape stripping, proceeding to more extensive distribution in basal and suprabasal layers by 48 h. The increase in connexin 26 preceded that in cell proliferation. In vaginal epithelium, buccal epithelium, and viral warts connexin 26 was restricted mainly to suprabasal, nonproliferating cells. In psoriatic lesional epidermis connexin 26 was also located mainly in suprabasal, nonproliferating cells. Connexin 26 was present in a patchy distribution in the basal layer of psoriatic lesional epidermis, but double labeling for connexin 26 and Ki67 showed that many connexin 26 positive basal cells were nonproliferative, suggesting that connexin 26 may be related to differentiation rather than to proliferation. These observations would be consistent with a role for connexin 26 containing gap junctions during both early and later stages of keratinocyte differentiation in hyperplastic epidermis and in vaginal and buccal epithelia.