-
Brid Callaghan, Billie Hunne,
Haruko Hirayama,
Daniela M Sartor,
Trung V Nguyen,
Fe C Abogadie,
Dorota Ferens,
Peter McIntyre,
Kung Ban,
Jonathan Baell,
John B Furness,
James A Brock
[show abstract]
[hide abstract]
ABSTRACT: Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.
AJP Heart and Circulatory Physiology 08/2012; 303(8):H1011-21. · 3.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.
Cell and Tissue Research 04/2012; 348(3):397-405. · 3.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The family of two-pore domain potassium (K2P) channels is important in setting and controlling the background potassium current of excitable cells. This study examines
the localisation of the acid-sensitive channel, K2P9.1 (TASK3), in cells of the gastric mucosa. We observed K2P9.1 immunoreactivity in endocrine cells of the mucosal glands of the guinea-pig, rat and mouse but the channels were not detected
in parietal, chief, or mucous cells. K2P9.1 channel immunoreactivity was consistently co-localised with histidine decarboxylase immunopositive enterochromaffin-like
(ECL) cells, and with the majority of ghrelin immunoreactive X/A cells. Localisation in somatostatin immunoreactive D cells
was rare in the guinea-pig, and did not occur in the stomach of rat, but, in the mouse, K2P9.1 channels were observed in the majority of somatostatin-immunoreactive D cells. Conversely, sections taken from the guinea-pig
and mouse stomachs, but not rat stomach, revealed K2P9.1 in gastrin-containing G cells. These results demonstrate the presence of K2P9.1 channels in the entero-endocrine ECL, G and D cell populations of the stomach that regulate acid secretion through the
release of histamine, gastrin and somatostatin. K2P9.1 channels were located in the ghrelin X/A cells that regulate food intake.
KeywordsEntero-endocrine-TASK channels-Acid secretion-Stomach-Ghrelin
Journal of Molecular Histology 04/2012; 41(6):403-409. · 1.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ghrelin, a peptide hormone from the stomach, has been recently discovered to reduce sodium excretion from the kidney. Although the effects on the kidney suggest actions in the distal nephron, the sites of expression of ghrelin receptors have not been localised. In the present work we have used a mouse that expresses green fluorescent protein under the control of the ghrelin receptor promoter to locate sites of receptor expression in the kidney. Receptor expression was confined to the straight parts of the distal tubules and the thin limbs of the loops of Henle. No expression was detected in other structures, including the glomeruli, proximal tubules and collecting ducts. Ghrelin receptors were not found in extra-renal or intra-renal arteries, despite observations that ghrelin is a vasodilator. The distribution revealed by in situ hybridisation histochemistry was the same as that revealed by the reporter. In conclusion, ghrelin receptors have a restricted distribution in the kidney. The location in the straight parts of the distal tubules accords with observations that ghrelin promotes sodium retention.
Cell and Tissue Research 09/2011; 346(1):135-9. · 3.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the present study the relationship between tissue damage and changed electro-physiological properties of Dogiel type II myenteric neurons within the first 24 hours after induction of inflammation with trinitrobenzene sulfonate (TNBS) in the guinea-pig ileum was investigated. Treatment with TNBS causes damage to the mucosa, inflammatory responses in the mucosa and enteric ganglia and changes in myenteric neuron properties. Thus we hypothesise that the physiological changes in the myenteric neurons could be due to damage to their mucosal processes or inflammation in the vicinity of cell bodies or the processes. We found an association between hyperexcitability of myenteric Dogiel type II neurons and damage to the mucosa and its innervation at 3 and 24 h, times when there was also an inflammatory reaction. The lack of hyperexcitability in neurons from control tissues in which axons projecting to the mucosa were severed suggests that inflammation may be an important contributing factor to the neuronal hyperexcitability at the acute stage of inflammation. Despite mucosal repair and re-innervation of the mucosa before 7 days after induction of inflammation, neuronal hyperexcitability persists. Although the mechanisms underlying neuronal hyperexcitability at the acute stage of inflammation might be different from those underlying long-term changes in the absence of active inflammation in the ganglia, the persistent changes in neuronal excitability may contribute to post-inflammatory gut dysfunctions.
The Journal of Physiology 01/2011; 589(Pt 2):325-39. · 4.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The family of two-pore domain potassium (K(₂P)) channels is important in setting and controlling the background potassium current of excitable cells. This study examines the localisation of the acid-sensitive channel, K(₂P)9.1 (TASK3), in cells of the gastric mucosa. We observed K(₂P)9.1 immunoreactivity in endocrine cells of the mucosal glands of the guinea-pig, rat and mouse but the channels were not detected in parietal, chief, or mucous cells. K(₂P)9.1 channel immunoreactivity was consistently co-localised with histidine decarboxylase immunopositive enterochromaffin-like (ECL) cells, and with the majority of ghrelin immunoreactive X/A cells. Localisation in somatostatin immunoreactive D cells was rare in the guinea-pig, and did not occur in the stomach of rat, but, in the mouse, K(₂P)9.1 channels were observed in the majority of somatostatin-immunoreactive D cells. Conversely, sections taken from the guinea-pig and mouse stomachs, but not rat stomach, revealed K(₂P)9.1 in gastrin-containing G cells. These results demonstrate the presence of K(₂P)9.1 channels in the entero-endocrine ECL, G and D cell populations of the stomach that regulate acid secretion through the release of histamine, gastrin and somatostatin. K(₂P)9.1 channels were located in the ghrelin X/A cells that regulate food intake.
Journal of molecular histology 12/2010; 41(6):403-9. · 1.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The late afterhyperpolarizing potential (AHP) that follows the action potential in intrinsic primary afferent neurons of the gastrointestinal tract has a profound influence on their firing patterns. There has been uncertainty about the identity of the channels that carry the late AHP current, especially in guinea pigs, where the majority of the physiological studies have been made. In the present work, the late AHP was recorded with intracellular microelectrodes from myenteric neurons in the guinea pig small intestine. mRNA was extracted from the ganglia to determine the identity of the guinea pig intermediate conductance potassium (I(K)) channel gene transcript. The late AHP was inhibited by two blockers of I(K) channels, TRAM34 (0.1-1 microM) and clotrimazole (10 microM), and was enhanced by the potentiator of the opening of these channels, DC-EBIO (100 nM). Action potential characteristics were unchanged by TRAM34 or DC-EBIO. The full sequence of the gene transcript and the deduced amino acid sequence were determined from extracts including myenteric ganglia and from bladder urothelium, which is a rich source of I(K) channel mRNA. This showed that the guinea pig sequence has a high degree of homology with other mammalian sequences but that the guinea pig channel lacks a phosphorylation site that was thought to be critical for channel regulation. It is concluded that the channels that carry the current of the late afterhyperpolarizing potential in guinea pig enteric neurons are I(K) channels.
Journal of Neurophysiology 04/2007; 97(3):2024-31. · 3.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 5-Hydroxytryptamine (5-HT) is an endogenous stimulant of intestinal propulsive reflexes. It exerts its effects partly through 5-HT4 receptors; 5-HT4 receptor agonists that are stimulants of intestinal transit are in clinical use. Both pharmacological and recent immunohistochemical studies indicate that 5-HT4 receptors are present on enteric neurons but the specific neurons that express the receptors have not been determined. In the present work, we describe the characterization of an anti-5-HT4 receptor antiserum that reveals immunoreactivity for enteric neurons and other cell types in the gastrointestinal tract. With this antiserum, 5-HT4 receptor immunoreactivity has been found in the muscularis mucosae of the rat oesophagus, a standard assay tissue for 5-HT4 receptors. It is also present in the muscularis mucosae of the guinea-pig and mouse oesophagus. In guinea-pig small intestine and rat and mouse colon, 5-HT4 receptor immunoreactivity occurs in subpopulations of enteric neurons, including prominent large neurons. Double-staining has shown that these large neurons in the guinea-pig small intestine are also immunoreactive for two markers of intrinsic primary afferent neurons, cytoplasmic NeuN and calbindin. Some muscle motor neurons in the myenteric ganglia are immunoreactive for this receptor, whereas it is rarely expressed by secretomotor neurons. Immunoreactivity also occurs in the interstitial cells of Cajal but is faint in the external muscle. Expression of the protein and mRNA has been confirmed in extracts containing enteric neurons. The observations suggest that one site of action of 5-HT4 receptor agonists is the intrinsic primary afferent neurons.
Cell and Tissue Research 10/2006; 325(3):413-22. · 3.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Intermediate-conductance, calcium-activated, potassium (IK) channels were first identified by their roles in cell volume regulation, and were later shown to be involved in control of proliferation of lymphocytes and to provide a K+ current for epithelial secretory activity. Until now, there has been no systematic investigation of IK channel localization within different epithelia. IK channel immunoreactivity was present in most epithelia, where it occurred in surface membranes of epithelial cells. It was found in all stratified epithelia, including skin, cornea, oral mucosa, vaginal mucosa, urothelium and the oesophageal lining. It occurred in the ducts of fluid-secreting glands, the salivary glands, lacrimal glands and pancreas, and in the respiratory epithelium. A low level of expression was seen in serous acinar cells. It was also found in other epithelia with fluid-exchange properties, the choroid plexus epithelium, the ependyma, visceral pleura and peritoneum, bile ducts and intestinal lining epithelium. However, there was little or no expression in vascular endothelial cells, kidney tubules or collecting ducts, lung alveoli, or in sebaceous glands. It is concluded that the channel is present in surface epithelia (e.g. skin) where it has a cell-protective role against osmotic challenge, and in epithelia where there is anion secretion that is facilitated by a K+ current-dependent hyperpolarization. It was also in some epithelial cells where its roles are as yet unknown.
Journal of Anatomy 03/2006; 208(2):219-29. · 2.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Neurochemistry 10/2004; 90(6):1414-22. · 4.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Autonomic Neuroscience 06/2004; 112(1-2):93-7. · 1.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cell and Tissue Research 12/2003; 314(2):179-89. · 3.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Oligophrenin-1 is a RhoGTPase-activating protein (RhoGAP) that is involved in the regulation of shape changes in dendritic spines, and outgrowth of axons and dendrites in the brain. These changes in neuronal morphology are central to the mechanisms of plasticity, learning, and memory. Although the enteric nervous system also exhibits long-term changes in neuronal function, the expression and involvement of oligophrenin-1 has not previously been investigated. We show by RT-PCR analysis that oligophrenin-1 mRNA is expressed in the myenteric plexus (MP) of the guinea pig ileum. Sequencing of RT-PCR products showed that guinea pig oligophrenin-1 mRNA is 98% and 87% homologous to human and mouse oligophrenin-1, respectively, except that a 42 bp sequence is absent from the guinea pig mRNA. This 42 bp sequence codes for a sequence of 14 amino acids located near the carboxy-terminal end of the RhoGAP domain in the human sequence. An antibody that recognizes human oligophrenin-1 identified a 91 kDa protein band in rat and mouse brain lysates and in guinea pig sciatic nerve, and a 36 kDa protein band in both purified enteric ganglion cell and brain lysate from guinea pig. Oligophrenin-1 is localized specifically to neurons and varicose axons in the MPs and submucosal plexuses (SMPs) of the guinea pig and rat, but is not detectable in glial cells, smooth muscle, or other cell types. These findings indicate that oligophrenin-1 is expressed in the enteric nervous system, where it may regulate morphological changes in axons and dendrites, and thus modulate neuronal connectivity.
The Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology 09/2003; 273(2):671-6.
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Histochemie 08/2003; 120(1):51-61. · 2.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cell and Tissue Research 01/2003; 314(2):179-189. · 3.11 Impact Factor
