Joseph H. Szurszewski

Role of nitric oxide in gastrointestinal and hepatic function and disease

A lthough the early Roman physician Galen had a predominant influence on medical thought and practice for over 14 centuries, his conception of the vascular system has been described as a physiological mistake that prevented the advancement of medical science for centuries.‘,’ Galen based his description of the vascular system on the concept of “pneuma” or spirits, a vital principle consisting of matter in a finely divided or ethereal state that flowed through the vascular and nervous systems and animated the entire organism.lv’ Many diseases were thought to owe their origin to some disturbance of these ethereal spirits. Although these ideas were fanciful, recent evidence indicates that an ethereal substance is indeed formed in many organs and that it has important roles in physiology and pathophysiology. This ethereal substance is the gas nitric oxide. In 1987, it was shown that vascular endothelial cells could synthesize NO and that this gas acted as a labile humoral-like messenger that relaxed vascular smooth muscle.3 Studies during the last 5 years have shown that NO functions as a mediator, a messenger, or a regulator of cell function in a number of physiological systems and pathophysiological states.4 This article begins with an abbreviated background on the discovery and characteristics of NO, reviews recent evidence that indicates that NO may play a role in gastrointestinal and hepatic physiology and disease, and speculates on potential clinical implications.

Decreased interstitial cell of Cajal volume in patients with slow-transit constipation ☆ ☆☆

BACKGROUND & AIMS The cause of slow-transit constipation is incompletely understood. Recent observations suggest a central role for interstitial cells of Cajal in the control of intestinal motility. The aim of this study was to determine the volume of interstitial cells of Cajal in the normal sigmoid colon and in the sigmoid colon from patients with slow transit constipation. METHODS Sigmoid colonic samples were stained with antibodies to protein gene product 9.5, c-Kit, and alpha-smooth muscle actin. Three-dimensional reconstruction of regions of interest was performed using consecutive images collected on a laser scanning confocal microscope and ANALYZE software. RESULTS Volume of interstitial cells of Cajal was significantly decreased in all layers of sigmoid colonic specimens from patients with slow-transit constipation compared with normal controls. Neuronal structures within the colonic circular smooth muscle layer were also decreased. CONCLUSIONS A decrease in the volume of interstitial cells of Cajal may play an important role in the pathophysiology of slow-transit constipation.

Pan-colonic decrease in interstitial cells of Cajal in patients with slow transit constipation

Background: Interstitial cells of Cajal (ICC) are required for normal intestinal motility. ICC are found throughout the human colon and are decreased in the sigmoid colon of patients with slow transit constipation. Aims: The aims of this study were to determine the normal distribution of ICC within the human colon and to determine if ICC are decreased throughout the colon in slow transit constipation. Patients: The caecum, ascending, transverse, and sigmoid colons from six patients with slow transit constipation and colonic tissue from patients with resected colon cancer were used for this study. Methods: ICC cells were identified with a polyclonal antibody to c-Kit, serial 0.5 μm sections were obtained by confocal microscopy, and three dimensional software was employed to reconstruct the entire thickness of the colonic muscularis propria and submucosa. Results: ICC were located within both the longitudinal and circular muscle layers. Two networks of ICC were identified, one in the myenteric plexus region and another, less defined network, in the submucosal border. Caecum, ascending colon, transverse colon, and sigmoid colon displayed similar ICC volumes. ICC volume was significantly lower in the slow transit constipation patients across all colonic regions. Conclusions: The data suggest that ICC distribution is relatively uniform throughout the human colon and that decreased ICC volume is pan-colonic in idiopathic slow transit constipation.

Electrophysiology of smooth muscle of the small intestine of some mammals.

Intracellular recordings were made from cells located in the longitudinal, inner and outer circular muscle layers of the dog, cat, rabbit, opossum and human small intestine. In whole‐thickness preparations in all five species, longitudinal muscle cells generated slow waves and spikes. However, in isolated longitudinal muscle preparations, all cells tested were electrically silent. In whole‐thickness and in isolated preparations, cells in the inner circular muscle layer generated spontaneous spikes superimposed on slow potentials. However, the occurrence of spikes and slow potentials was more regular in whole‐thickness preparations. In whole‐thickness preparations, cells in the outer circular muscle layer generated slow waves which were coupled with phasic contractions. However, in isolated outer circular muscle preparations, all cells tested were electrically silent and spontaneous phasic contractions were absent. In whole‐thickness preparations, non‐neural cells located on the serosal side of the outer circular muscle layer generated slow waves. The data suggest that spontaneous slow waves of the small intestine of the dog, cat, rabbit, opossum and human are generated in non‐neural cells located between the longitudinal and outer circular muscle layer and by non‐neural cells located between the outer and inner circular muscle layers.

Heme Oxygenase-1 Protects Interstitial Cells of Cajal from Oxidative Stress and Reverses Diabetic Gastroparesis

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.

Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells

Gut microbiota alterations have been described in several diseases with altered gastrointestinal (GI) motility, and awareness is increasing regarding the role of the gut microbiome in modulating GI function. Serotonin [5‐hydroxytryptamine (5‐HT)] is a key regulator of GI motility and secretion. To determine the relationship among gut microbes, colonic contractility, and host serotonergic gene expression, we evaluated mice that were germ‐free (GF) or humanized (HM; ex‐GF colonized with human gut microbiota). 5‐HT reduced contractile duration in both GF and HM colons. Microbiota from HM and conventionally raised (CR) mice significantly increased colonic mRNAs Tph1 [(tryptophan hydroxylase) 1, rate limiting for mucosal 5‐HT synthesis; P < 0.01] and chromogranin A (neuroendocrine secretion; P < 0.01), with no effect on monoamine oxidase A (serotonin catabolism), serotonin receptor 5‐HT4, or mouse serotonin transporter. HM and CR mice also had increased colonic Tph1 protein (P < 0.05) and 5‐HT concentrations (GF, 17 ± 3 ng/mg; HM, 25 ± 2 ng/mg; and CR, 35 ± 3 ng/mg; P < 0.05). Enterochromaffin (EC) cell numbers (cells producing 5‐HT) were unchanged. Short‐chain fatty acids (SCFAs) promoted TPH1 transcription in BON cells (human EC cell model). Thus, gut microbiota acting through SCFAs are important determinants of enteric 5‐HT production and homeostasis.—Reigstad, C. S., Salmonson, C. E., Rainey, III, J. F., Szurszewski, J. H., Linden, D. R., Sonnenburg, J. L., Farrugia, G., Kashyap, P. C. Gut microbes promote colonic serotonin production through an effect of short‐chain fatty acids on enterochromaffin cells. FASEB J. 29, 1395‐1403 (2015). www.fasebj.org

Nitric oxide mediates inhibitory nerve input in human and canine jejunum

BACKGROUND Nitric oxide (NO) may be an inhibitory neurotransmitter in the intestinal muscle. The present study examined its role in human and canine jejunum. METHODS Mechanical and intracellular electrical activity were recorded simultaneously from the circular muscle layer. RESULTS 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. CONCLUSIONS NO mediates neural inhibition in circular muscle of both human and canine jejunum through different mechanisms.

Effect of nitric oxide on circular muscle of the canine small intestine.

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.

Immunization with neuronal nicotinic acetylcholine receptor induces neurological autoimmune disease.

Neuronal nicotinic AChRs (nAChRs) are implicated in the pathogenesis of diverse neurological disorders and in the regulation of small-cell lung carcinoma growth. Twelve subunits have been identified in vertebrates, and mutations of one are recognized in a rare form of human epilepsy. Mice with genetically manipulated neuronal nAChR subunits exhibit behavioral or autonomic phenotypes. Here, we report the first model of an acquired neuronal nAChR disorder and evidence for its pertinence to paraneoplastic neurological autoimmunity. Rabbits immunized once with recombinant alpha3 subunit (residues 1-205) develop profound gastrointestinal hypomotility, dilated pupils with impaired light response, and grossly distended bladders. As in patients with idiopathic and paraneoplastic autoimmune autonomic neuropathy, the severity parallels serum levels of ganglionic nAChR autoantibody. Failure of neurotransmission through abdominal sympathetic ganglia, with retention of neuronal viability, confirms that the disorder is a postsynaptic channelopathy. In addition, we found ganglionic nAChR protein in small-cell carcinoma lines, identifying this cancer as a potential initiator of ganglionic nAChR autoimmunity. The data support our hypothesis that immune responses driven by distinct neuronal nAChR subtypes expressed in small-cell carcinomas account for several lung cancer-related paraneoplastic disorders affecting cholinergic systems, including autoimmune autonomic neuropathy, seizures, dementia, and movement disorders.

CD206-Positive M2 Macrophages That Express Heme Oxygenase-1 Protect Against Diabetic Gastroparesis in Mice

BACKGROUND & AIMS Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic [NOD]) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay. METHODS Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction. RESULTS Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206(+) M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206(+)/HO1(+) M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206(+)/HO1(+) M2 macrophages. CONCLUSIONS CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.