Background & Aims Diabetic gastroparesis (delayed gastric emptying) is a well-recognized complication of diabetes that causes considerable morbidity and makes glucose control difficult. Interstitial ...cells of Cajal, which express the receptor tyrosine kinase Kit, are required for normal gastric emptying. We proposed that Kit expression is lost during diabetic gastroparesis due to increased levels of oxidative stress caused by low levels of heme oxygenase-1 (HO-1), an important cytoprotective molecule against oxidative injury. Methods Gastric emptying was measured in nonobese diabetic mice and correlated with levels of HO-1 expression and activity. Endogenous HO-1 activity was increased by administration of hemin and inhibited by chromium mesoporphyrin. Results In early stages of diabetes, HO-1 was up-regulated in gastric macrophages and remained up-regulated in all mice that were resistant to development of delayed gastric emptying. In contrast, HO-1 did not remain up-regulated in all the mice that developed delayed gastric emptying; expression of Kit and neuronal nitric oxide synthase decreased markedly in these mice. Loss of HO-1 up-regulation increased levels of reactive oxygen species. Induction of HO-1 by hemin decreased reactive oxygen species, rapidly restored Kit and neuronal nitric oxide synthase expression, and completely normalized gastric emptying in all mice. Inhibition of HO-1 activity in mice with normal gastric emptying caused a loss of Kit expression and development of diabetic gastroparesis. Conclusions Induction of the HO-1 pathway prevents and reverses cellular changes that lead to development of gastrointestinal complications of diabetes. Reagents that induce this pathway might therefore be developed as therapeutics.
The purpose of this study was to determine the non‐adrenergic non‐cholinergic inhibitory neurotransmitter in pig jejunum. Intracellular electrical activity was recorded from circular smooth muscle ...cells. Inhibitory junction potentials (IJPs) evoked by electrical field stimulation were inhibited by tetrodotoxin (1 μmol L−1), ω‐conotoxin GVIA (0.1 μmol L−1) tetrodotoxin, apamin (1 μ mol L−1), 1‐6‐((17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl)amino)hexyl‐1H‐pyrrole‐2,5‐dione (U‐73122; 10 μmol L−1) but not by N ω‐nitro‐l‐arginine (l‐NNA; 100 μ mol L−1), haemoglobin (10 μ mol L−1), 1H‐1,2,4oxadiazolo4,3‐aquinoxalin‐1‐one (ODQ; 10 μ mol L−1) or 9‐(tetrahydro‐2‐furyl)adenine (SQ‐22536; 10 μmol L−1). S‐nitroso‐N‐acetylpenicillamine (SNAP) hyperpolarized the membrane potential. This was inhibited by ODQ (3 μmol L−1) and charybdotoxin (0.1 μ mol L−1). Adenosine‐5‐triphosphate (ATP; 100 μ mol L−1) and 2‐methylthio ATP (2‐MeS‐ATP; 100 μmol L−1) did not hyperpolarize the membrane potential and 6‐N‐N‐diethyl‐β‐ γ ‐dibromomethylene‐d‐adenosine‐5′‐triphosphate (ARL67156; 100 μ mol L−1) did not modify IJPs. Carbon monoxide (CO; 10%) and tricarbonyl dichlororuthenium dimer (Ru(CO3Cl2)2; 100 μ mol L−1) hyperpolarized the membrane potential however zinc, copper and tin protoporphyrin IX (100 μmol L−1) did not alter IJPs. Vasoactive intestinal peptide (VIP) hyperpolarized the membrane potential but 4‐Cl‐d‐Phe6‐Leu17‐VIP (1 μmol L−1) did not modify IJPs. Pituitary adenylate cyclase activating peptide (PACAP)38 (0.5 μ mol L−1) hyperpolarized the membrane potential. This was inhibited by apamin (1 μmol L−1) but not by tetrodotoxin (1 μ mol L−1). Pituitary adenylate cyclase activating peptide6‐38 (1 μ mol L−1) inhibited IJPs. These data suggest that inhibitory neurotransmission in pig jejunum is mediated partly by PACAP.
This contribution to the centennial commemorative issue of the American Journal of Physiology: Gastrointestinal and Liver Physiology identifies some of the important studies of spontaneous electrical ...and motor activity in the gastrointestinal tract published in the Journal between 1898 and 1996. Emphasis is given to the contributions made by Walter B. Cannon, Walter C. Alvarez, Emil Bozler, C. Ladd Prosser, and James Christensen.
Recent evidence suggests that carbon monoxide (CO) may be a neurotransmitter, similar to nitric oxide (NO) in the enteric nervous system. The distribution of haem oxygenase (HO), the biosynthetic ...enzyme for CO, has been determined in the enteric nervous system of animals, but little is known about the distribution of HO in human gastrointestinal tract. The present study investigated the expression of HO and its colocalization with NO synthase (NOS), the biosynthetic enzyme for NO, in human antrum and jejunum. HO isoforms were identified using immunohistochemistry and NOS was identified by immunohistochemistry or NADPH‐d histochemistry. HO‐2 immunoreactive (IR) cell bodies in enteric ganglia and nerve fibres in longitudinal and circular muscle were found in both antrum and jejunum. Co‐localization of HO‐2 and NOS was about 40% in HO‐2 containing cell bodies of myenteric ganglia and only 10% or less in cell bodies of submucous ganglia. HO‐1 immunoreactivity was not detected in antrum or jejunum. The results suggest that CO is produced in human enteric ganglion neurones and indicate a possible role of CO as a neurotransmitter and possible interaction between HO and NOS pathways in inhibitory neurotransmission in the human gastrointestinal tract.
1. Experiments were designed to determine in circular muscle of the canine jejunum whether exogenous nitric oxide (NO) mimics
the non-adrenergic, non-cholinergic inhibitory junction potential (NANC ...IJP), and whether changes in the availability of endogenous
NO affects IJP amplitude. 2. Mechanical and intracellular electrical activity were recorded simultaneously from circular muscle
of the canine jejunum. Electrical field stimulation evoked NANC IJPs and inhibited spontaneous contractions. 3. Infusions
of NO solutions evoked immediate dose-dependent and transient hyperpolarizations and transiently inhibited spontaneous contractions.
NO-evoked hyperpolarizations were unaffected by atropine, propranolol, phentolamine and tetrodotoxin. 4. The maximum IJP amplitude
and the maximum amplitude of NO-evoked hyperpolarization were similar. 5. NG-Mono-methyl-L-arginine (L-NMMA), which inhibits
synthesis of NO from L-arginine, reduced IJP amplitudes but did not reduce the response to exogenous NO. L-Arginine, but not
D-arginine, reversed the effect of L-NMMA on IJP amplitude. 6. Oxyhaemoglobin, which binds and inactivates NO, reduced IJP
amplitude and abolished the response to exogenous NO. 7. Exogenous NO mimicked the effects of NANC inhibitory input. Reducing
the availability of endogenous NO reduced NANC inhibitory input. 8. It was concluded that NO mediates NANC neural inhibition
and may act as a NANC inhibitory neurotransmitter in the canine jejunum.
Key points
Ano1, a Ca2+‐activated Cl− channel, is expressed in interstitial cells of Cajal (ICC) throughout the gut. We report here that it is required to maintain coordinated Ca2+ transients within ...myenteric ICC of mouse small intestine. Ca2+ transients in Ano1 WT mice were rhythmic and coordinated whereas uncoordinated Ca2+ transients were seen in knockout mice.
Ca2+ transients were un‐coordinated following pharmacological block of Ano1 in WT mice using niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid and 4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid disodium salt. Transient knockdown of Ano1 in organotypic cultures with short hairpin RNA to Ano1 in WT tissues also caused loss of coordinated Ca2+ transients.
Contractility of Ano1 knockout mouse intestinal segments in organ bath experiments was significantly decreased, less coordinated and non‐rhythmic. Spatiotemporal maps from knockout mouse small intestine also showed loss of phasic contractile activity.
This study provides important information on the basic mechanisms driving coordinated contractile activity in the gastrointestinal tract.
Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca2+‐activated Cl− channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca2+ transients between myenteric ICC (ICC‐MY) of small intestine. First, we found spontaneous Ca2+ transients in ICC‐MY in both Ano1 WT and knockout (KO) mice. However, Ca2+ transients within the ICC‐MY network in Ano1 KO mice were uncoordinated, while ICC‐MY Ca2+ transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca2+ transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA‐mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca2+ transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca2+ transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non‐rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca2+ transients in ICC‐MY.
Diabetes affects many organs including the stomach. Altered number and function of interstitial cells of Cajal (ICC), the gastrointestinal pacemaker cells, underlie a number of gastrointestinal ...motility disorders, including diabetic gastroparesis. In the muscle layers, ICC selectively express Ano1, thought to underlie classical Ca2+-activated Cl− currents. Mice homozygous for Ano1 knock-out exhibit abnormal ICC function and motility. Several transcripts for Ano1 are generated by alternative splicing of four exons. Here, we report expression levels of transcripts encoded by alternative splicing of Ano1 gene in gastric muscles of patients with diabetic gastroparesis and nondiabetic control tissues. Expression of mRNA from two alternatively transcribed exons are significantly different between patients and controls. Furthermore, patients with diabetic gastroparesis express mRNA for a previously unknown variant of Ano1. The 5′ end of this novel variant lacks exons 1 and 2 and part of exon 3. Expression of this variant in HEK cells produces a decreased density of Ca2+-activated Cl− currents that exhibit slower kinetics compared with the full-length Ano1. These results identify important changes in expression and splicing of Ano1 in patients with diabetic gastroparesis that alter the electrophysiological properties of the channel. Changes in Ano1 expression in ICC may directly contribute to diabetic gastroparesis.
Interstitial cells in the deep muscular plexus (ICC‐DMP) are thought to be essential for neurotransmission in the circular muscle. There is evidence for gap junctions within the ICC‐DMP network and ...between ICC‐DMP and muscle cells; however, there is no evidence for functional coupling via these gap junctions. In addition, the innervation of individual ICC‐DMP has not been studied. We investigated these questions by injecting the dye Lucifer yellow into ICC‐DMP of guinea‐pig ileum. Nerves were labelled immunohistochemically for protein gene product 9.5. Cells were imaged by confocal microscopy. Most (79%) of the dye‐injected ICC‐DMP were coupled to one to five other ICC‐DMP, and 86% of them were coupled to one to five circular muscle cells. Octanol effectively blocked all coupling. Incubation in pH 6.8–7.0 reduced ICC–ICC coupling to 49% and ICC–muscle coupling to 32%. In contrast, pH 7.8–7.9 increased ICC–ICC and ICC–muscle coupling to 100%. Most ICC somata (95%) and processes (60%) were in close proximity with both nerve fibres and smooth muscle cells. These results provide direct evidence for functional coupling within the ICC‐DMP network, and between this network and cells of the outer circular muscle layer and showed that coupling can be affected by pH.
Carbon monoxide (CO) has been postulated to be a messenger in the gastrointestinal tract. The aims of this study were to determine the distribution of heme oxygenase (HO), the source for endogenous ...CO in the canine jejunum, and to determine the effects of CO on jejunal circular smooth muscle cells. HO-2 isoform was present in a population of myenteric and submucosal neuronal cell bodies, in nerve fibers innervating the muscle layers, and in smooth muscle cells. HO-1 isozyme was not detected in the canine jejunum. Exogenous CO increased whole cell current by 285 +/- 86%, hyperpolarized the membrane potential by 8.5 +/- 2.9 mV, and increased guanosine 3',5'-cyclic monophosphate (cGMP) levels in smooth muscle cells. 8-Bromo-cGMP also increased the whole cell current. The data suggest that endogenous activity of HO-2 may be a source of CO in the canine jejunum and that exogenously applied CO can modulate intestinal smooth muscle electrical activity. It is therefore reasonable to suggest a role for endogenously produced CO as a messenger in the canine jejunum.
PDF
