In all mammalian species examined to date, there is a 10 mV or more gradient in resting membrane potential across the wall of the gastric antrum, small intestine and colon, and an even larger ...gradient along the long axis of the stomach. These voltage gradients, which may be considered biological rheostats, are central to the ability of circular smooth muscle to vary the strength of contraction from weak to propulsive and occluding. In this short review, we consider recent data that support the hypothesis that carbon monoxide generated in interstitial cells of Cajal is a hyperpolarizing factor for circular smooth muscle and the root of the essential voltage gradients.
The neurotransmitter(s) that generates the fast component of the inhibitory junction potential (IJP-F) in human jejunal circular smooth muscle is not known. The aim of this study was to determine the ...role of ATP and purinergic receptors in the generation of the IJP-F in human jejunal circular smooth muscle strips. The P2-receptor antagonist suramin (100 microM) reduced the IJP-F by 28%. Apamin (1 microM) reduced the IJP-F by 25%. Desensitization of muscle strips with the putative P2x-receptor agonist alpha, beta-methylene ATP (alpha,beta-MeATP, 100 microM) decreased the IJP-F by 44%, and desensitization with the putative P2y-receptor agonist adenosine 5'-O-2-thiodiphosphate (ADPbetaS) completely abolished the IJP-F. Desensitization with the putative P2y-receptor agonist 2-methylthioATP had no effect on the IJP-F. Exogenous ATP evoked a hyperpolarization with a time course that matched the IJP-F. The ATP-evoked hyperpolarization was reduced by apamin and suramin, reduced by desensitization with alpha,beta-MeATP (69% decrease), and abolished by desensitization with ADPbetaS. These data suggest that the IJP-F in human jejunal circular smooth muscle is mediated in part by ATP through an ADPbetaS-sensitive P2 receptor.
Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin ...in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca2+ increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology.
Carbon monoxide (CO) is proposed as a physiological messenger. CO activates cGMP and has a direct effect on potassium channels. Both actions of CO lead to hyperpolarization of a cell's resting ...membrane potential, suggesting that CO may function as a hyper-polarizing factor, although direct evidence is still lacking. Here we take advantage of the known membrane potential gradient that exists in the muscle layers of the gastrointestinal tract to determine whether CO is an endogenous hyperpolarizing factor. We find that heme oxygenase-2-null mice have depolarized smooth muscle cells and that the membrane potential gradient in the gut is abolished. Exogenous CO hyperpolarizes the membrane potential. Regions of the canine gastrointestinal tract that are more hyperpolarized generate more CO and have higher heme oxygenase activity than more depolarized regions. Our results suggest that CO is a critical hyperpolarizing factor required for the maintenance of intestinal smooth muscle membrane potential and gradient.
The binding of Steel factor (SF) to c-kit initiates a signaling pathway essential for development and maintenance of interstitial cells of Cajal (ICC). Soluble and membrane-bound isoforms of SF are ...expressed in the gastrointestinal tract, but the role for either isoform in supporting ICC development is unknown. The aim of this study was to determine the role of SF in supporting ICC in culture. ICC were cultured from dissociated mouse jejunum and grown with fibroblast cell lines that produced either soluble, membrane-bound or membrane-restricted SF. ICC were identified and counted by c-kit immunoreactivity. The number of c-kit immunoreactive cells was greater in the coculture system compared with cultures grown without SF-producing fibroblasts. All forms of SF-producing fibroblasts increased ICC number in culture but physical separation of the fibroblasts from the c-kit immunoreactive cells, the addition of exogenous SF to the culture medium, or fibroblast-conditioned media did not. These results are consistent with the hypothesis that the membrane-bound form of SF preferentially contributes to expression of c-kit-positive ICC under cell culture conditions.
Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of Cajal (ICC) and myenteric neurons. In animal models of diabetic ...gastroparesis, development of delayed gastric emptying has been associated with loss of macrophages that express cytoprotective markers and reduced networks of ICC. Mice with long-term diabetes and normal gastric emptying have macrophages that express anti-inflammatory markers and have normal gastric ICC. Mice homozygous for the osteopetrosis spontaneous mutation in the colony-stimulating factor 1 gene (Csf1op/op) do not have macrophages; when they are given streptozotocin to induce diabetes, they do not develop delayed gastric emptying. We investigated whether population of the gastric muscularis propria of diabetic Csf1op/op mice with macrophages is necessary to change gastric emptying, ICC, and myenteric neurons and investigated the macrophage-derived factors that determine whether diabetic mice do or do not develop delayed gastric emptying.
Wild-type and Csf1op/op mice were given streptozotocin to induce diabetes. Some Csf1op/op mice were given daily intraperitoneal injections of CSF1 for 7 weeks; gastric tissues were collected and cellular distributions were analyzed by immunohistochemistry. CD45+, CD11b+, F4/80+ macrophages were dissociated from gastric muscularis propria, isolated by flow cytometry and analyzed by quantitative real-time polymerase chain reaction. Cultured gastric muscularis propria from Csf1op/op mice was exposed to medium that was conditioned by culture with bone marrow–derived macrophages from wild-type mice.
Gastric muscularis propria from Csf1op/op mice given CSF1 contained macrophages; 11 of 15 diabetic mice given CSF1 developed delayed gastric emptying and had damaged ICC. In non-diabetic Csf1op/op mice, administration of CSF1 reduced numbers of gastric myenteric neurons but did not affect the proportion of nitrergic neurons or ICC. In diabetic Csf1op/op mice given CSF1 that developed delayed gastric emptying, the proportion of nitrergic neurons was the same as in non-diabetic wild-type controls. Medium conditioned by macrophages previously exposed to oxidative injury caused damage to ICC in cultured gastric muscularis propria from Csf1op/op mice; neutralizing antibodies against IL6R or TNF prevented this damage to ICC. CD45+, CD11b+, and F4/80+ macrophages isolated from diabetic wild-type mice with delayed gastric emptying expressed higher levels of messenger RNAs encoding inflammatory markers (IL6 and inducible nitric oxide synthase) and lower levels of messenger RNAs encoding markers of anti-inflammatory cells (heme oxygenase 1, arginase 1, and FIZZ1) than macrophages isolated from diabetic mice with normal gastric emptying.
In studies of Csf1op/op and wild-type mice with diabetes, we found delayed gastric emptying to be associated with increased production of inflammatory factors, and reduced production of anti-inflammatory factors, by macrophages, leading to loss of ICC.
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Neuron activity and insulin release were measured simultaneously from 33 preparations of intrapancreatic canine ganglia and pancreatic parenchyma adjacent to the ganglia. The electrical activity of ...single neurons of the ganglia was recorded with intracellular microelectrodes, and insulin release from the attached islets was determined with an enzyme-linked immunosorbent assay. Insulin release was 62 +/- 18 fmol preparation/min in the presence of 10 mmol/l glucose and pulsatile (3.7 +/- 0.4 min/pulse). Corresponding measurements of neuronal electrical activity showed a stable membrane potential of -53.5 +/- 0.6 mV. Short, high-frequency (20 Hz) preganglionic nerve stimulation evoked action potentials and, in 46% of the preparations, a threefold rise in the insulin secretory rate associated with increased amplitude of the insulin pulses. The effects were blocked by 10 micromol/l tetrodotoxin (TTX). In other preparations, continuous low-frequency (0.05-0.5 Hz) preganglionic nerve stimulation evoked action potentials and, in 50% of the preparations, a gradual increase of insulin release associated with augmentation of insulin pulse amplitude without alteration of the duration. The effects were blocked by 50 micromol/l hexamethonium (HEX). In the remaining preparations, no change in insulin release was observed during nerve stimulation. In the absence of stimulation, neither TTX nor HEX affected the membrane potential or insulin secretion. These first simultaneous measurements of intrapancreatic ganglion activity and insulin secretion are consistent with amplitude modulation of pulsatile insulin secretion induced by changes in electrical activity in a population of intrapancreatic ganglion neurons.
Background & Aims: The interstitial cell (IC) network may be of fundamental importance in regulating gastrointestinal motility. Intestinal smooth muscle cells are depolarized in the absence of ICs, ...and there are no spontaneous slow waves. The messenger molecules between IC network and smooth muscle are unknown. Exogenous administration of CO relaxes the opossum internal anal sphincter and the guinea pig ileum, and it modulates potassium current and membrane potential of circular smooth muscle cells of the human jejunum. The aim of this study was to determine whether heme oxygenase (HO)-1 and HO-2, enzymes that catalyze the production of CO, are present in the IC network of the mouse small intestine.
Methods: Antibodies specific for c-Kit, HO-1, and HO-2 were used for immunohistochemistry. Confocal images were obtained and were volume rendered, and the images were converted into three-dimensional images.
Results: HO-2–like but not HO-1–like immunoreactivity was found in IC networks associated with the myenteric plexus and the deep muscular plexus.
Conclusions: HO-2 but not HO-1 is present in the IC cell network of the mouse small intestine. The enzymatic activity of HO-2 will result in the endogenous production of CO in IC networks of the mouse small intestine.
GASTROENTEROLOGY 1998;114:239-244
Nitric oxide (NO) may be an inhibitory neurotransmitter in the intestinal muscle. The present study examined its role in human and canine jejunum.
Mechanical and intracellular electrical activity ...were recorded simultaneously from the circular muscle layer.
In the human jejunum, nerve stimulation inhibited mechanical activity and evoked an inhibitory junction potential that consisted of an initial fast hyperpolarization followed by a late sustained hyperpolarization. NO inhibited mechanical activity and evoked a dose-dependent hyperpolarization that mimicked the late hyperpolarization. In the canine jejunum, nerve stimulation inhibited mechanical activity and evoked an inhibitory junction potential that consisted of only a fast hyperpolarization. NG-Monomethyl-L-arginine and NG-nitro-L-arginine attenuated nerve-mediated inhibition of mechanical activity in both species. However, the effect of the synthase inhibitors on inhibitory junction potentials differed in the two species. In canine jejunum, both inhibitors reduced the amplitude of the initial fast hyperpolarization. In human jejunum, both inhibitors reduced only the late sustained hyperpolarization.
NO mediates neural inhibition in circular muscle of both human and canine jejunum through different mechanisms.
Abdominal prevertebral ganglion neurones receive excitatory synaptic input from intestinofugal neurones. To better understand the physiological significance of this input, we examined the ...relationship between synaptic input to mouse superior mesenteric ganglion (SMG) neurones and intracolonic pressure and volume changes that accompany spontaneous colonic contractions in vitro. Electrical activity was recorded intracellularly from SMG neurones in ganglia attached to a segment of distal colon. The majority of neurones examined received ongoing fast excitatory potentials (F‐EPSPs). F‐EPSP frequency increased when the colon was distended with fluid and during spontaneous increases in colonic volume that accompanied colonic relaxation. In contrast, F‐EPSP frequency in SMG neurones decreased when the colon emptied, and remained at a reduced frequency until the colon refilled and volume increased. Nicotinic blockade of the colon abolished spontaneous colonic contractions and reduced or abolished synaptic input to SMG neurones, suggesting that most of the synaptic input arose from second or higher order neurones. Retrograde labelling identified cell bodies of intestinofugal neurones in myenteric ganglia. Most had short, club‐like dendritic processes and appeared uni‐axonal. These results show that mechanosensory intestinofugal afferent nerves monitor intracolonic volume changes.
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