bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of thyrotropin-releasing hormone on neurons in rat dorsal motor nucleus of the vagus, in vitro
Authors: Travagli R. A. , Gillis R. A. , Vicini S.
Published: December 2017

We sought to characterize the excitatory effect of thyrotropin-releasing hormone (TRH) in dorsal motor nucleus of the vagus (DMV) motoneurons by using the patch-clamp technique in rat brain stem slices. In our initial studies we used the cell-attached recording configuration using concentrations of TRH from 1 to 30 microM. Exposure of DMV motoneurons to TRH resulted in a concentration-related increase in spontaneously occurring action potential firing rate. This was observed in 63 of 85 DMV neurons (75%) tested and was unrelated to their location rostral or caudal to the obex. Invariably, desensitization occurred to the excitatory effect of TRH. Subsequent experiments using whole cell recordings in the current-clamp mode confirmed that TRH excites DMV neurons located both rostral and caudal to the obex. In the current-clamp configuration, TRH produced depolarization; i.e., 30 microM TRH elicited a depolarization of 8.7 +/- 3.2 mV (P < 0.05, n = 7). Studies using whole cell current recordings in voltage-clamp mode indicated that TRH in a concentration-dependent manner produces a small inward current that is associated with a decrease in the input resistance of -42.5 +/- 15.6 M omega (TRH 30 microM). TRH-induced inward current was also present under conditions of inhibition of synaptic transmission (i.e., in the presence of tetrodotoxin and cobalt). We also found that TRH reduced in a concentration-dependent manner both the fast transient A-type K+ current (IA) and the Ca(2+)-dependent afterhyperpolarizing current (IAHP). Using the extracellular recording technique in the cell-attached configuration, we investigated whether any part of TRH-induced increase in firing rate was due to an increase in the synaptic release of L-glutamate or acetylcholine. Prior exposure of DMV neurons to either kynurenic acid or to atropine did not antagonize any of the excitatory effect of TRH. Finally, we observed that addition of 30 microM TRH to the perfusing solution produced an increase in spontaneously occurring excitatory postsynaptic currents (EPSCs). This occurred without any change in the amplitude of EPSCs. These results indicated that TRH-induced increase in firing of DMV neurons is due to direct postsynaptic effects to activate an inward cationic current and to counteract IA and IAHP, as well as a presynaptic effect to increase the frequency of EPSCs.
Diaphragmatic contribution to gastroesophageal competence and reflux in dogs
Authors: Martin C. J. , Dodds W. J. , Liem H. H. , Dantas R. O. , layman R. D. , Dent J.
Published: December 2017

Events associated with gastroesophageal reflux have been determined by concurrent diaphragmatic and esophageal body electromyography, video radiography, and manometry in four conscious dogs. Three characteristic phenomena occurred in parallel immediately before and during gastroesophageal reflux: 1) transient lower esophageal sphincter relaxation, 2) profound (99.5%) and selective inhibition of crural diaphragmatic activity, and 3) a previously unrecognized dorsal movement of the gastroesophageal junction (mean 1.3 cm) demonstrated by implanted radiological markers. The patterns associated with spontaneous acid and gas reflux were indistinguishable from those induced by gastric distension. Costolumbar diaphragmatic activity was stable up until the instant of sphincter opening, when there was a single costolumbar contraction of short duration and high amplitude. Esophageal shortening did not occur before reflux. Reflux that occurred after atropine-induced inhibition of lower esophageal sphincter tone to < 2 mmHg was intermittent and coincided with selective crural inhibition. These studies demonstrated that selective crural inhibition is a prerequisite for gastroesophageal reflux and suggest that the crural diaphragm is an important factor for the maintenance of gastroesophageal competence.
Vulnerability of intestinal interstitial fluid to oxidant stress
Authors: Kurtel H. , Granger D. N. , Tso P. , Grisham M. B.
Published: December 2017

The objective of this study was to quantify free radical-mediated lipid, protein, and sulfhydryl oxidation in intestinal interstitial fluid (lymph) and plasma of fasted rats. Free radicals and oxidants were generated either by thermal decomposition of 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), which yields peroxyl radicals, or by activated polymorphonuclear neutrophils (PMNs). Incubation of intestinal lymph with AAPH resulted in a time-dependent increase in the formation of thiobarbituric acid-reactive substances (TBARS; lipid peroxidation) and carbonyl content (protein oxidation). TBARS formation was completely inhibited by removal of the apo B-containing lipoproteins in lymph suggesting that very low-density lipoprotein is the major substrate for lipid peroxidation in fasted interstitial fluid. The sulfhydryl content of lymph was reduced significantly on exposure to the peroxyl radical generator. Incubation with activated PMNs revealed qualitatively similar changes in protein and sulfhydryl oxidation; however, there was no detectable TBARS formation. Exposure of plasma to AAPH produced similar increases in protein and sulfhydryl oxidation when plasma protein concentration was adjusted to that of lymph; however, TBARS formation was significantly lower compared with lymph. Incubation of dialyzed plasma with AAPH produced significantly greater amounts of TBARS. Taken together, our data suggest that plasma is more resistant to AAPH-induced lipid peroxidation than interstitial fluid and the substrate for TBARS formation in intestinal interstitial fluid is different from that of plasma.
Role of platelet-activating factor in hepatic responses after bile duct ligation in rats
Authors: Zhou W. , Chao W. , Levine B. A. , Olson M. S.
Published: December 2017

Role of platelet-activating factor (PAF) as a potential mediator of hepatic pathophysiology was investigated using a rat model of obstructive jaundice. Over a 1-wk course of bile duct ligation, a sixfold increase in tissue levels of PAF (1.57 +/- 0.43 ng/g vs. control 0.24 +/- 0.08 ng/g) occurred in the liver, whereas no change was observed in PAF levels in plasma. Concomitantly, endotoxin was detected in portal blood drawn from jaundiced rats, and antagonism of the putative effect of endotoxin by neomycin plus polymyxin B reduced local PAF concentrations in livers from jaundiced animals. Induction of neutropenia failed to alter the elevated hepatic PAF concentrations. Moreover, a large quantity of PAF was released spontaneously from Kupffer cells isolated from livers derived from jaundiced rats but not from endothelial cells or hepatocytes from the same animals. An in vitro study using cultured Kupffer cells from normal rats indicated that Kupffer cells secreted a significant amount of PAF in response to lipopolysaccharide challenge; pretreatment of cells with polymyxin B prevented this stimulated PAF release. Treatment of animals with either of two PAF receptor antagonists (BN 52021 and WEB 2170) partially prevented the increase in tissue levels of eicosanoids and O2-derived free radicals and partially alleviated liver injury as judged by the appearance of glutamate-pyruvate transaminase in the plasma of jaundiced rats. The present study indicates 1) that endogenous PAF may be an important signaling mediator for the hepatic inflammatory alterations associated with short-term bile duct ligation and 2) that the interaction of Kupffer cells with portal endotoxin is the mechanism by which PAF is produced locally.
Mechanisms of neurotensin-induced inhibition in rat ileal smooth muscle
Authors: Allescher H. D. , Fick H. , Schusdziarra V. , Classen M.
Published: December 2017

The aim of the present study was to determine the mechanisms of neurotensin-induced inhibition in ileal smooth muscle. Isolated rat ileal smooth muscle strips were stimulated in an organ bath using carbachol (CCH) or by KCl depolarization. Neurotensin produced a concentration-dependent inhibition of muscle contraction [mean inhibitory concentration (IC50): 2.8 x 10(-9) M], which was not blocked by phentolamine (10(-6) M), hexamethonium (10(-4) M), indomethacin (10(-6) M), nordihydroguaretic acid (10(-6) M), or tetrodotoxin (10(-6) M). The inhibitory effect of neurotensin during CCH stimulation was blocked concentration dependently in the presence of the K(+)-channel blocker apamin. By contrast, other K(+)-channel blockers such as 9-aminoacridine (10(-6) M to 3 x 10(-5) M), 4-aminopyridine (10(-4) M to 5 x 10(-3) M), tetraethylammonium (10(-4) M to 10(-1) M), or glibenclamide (10(-5) M) were ineffective. The presence of the Ca(2+)-channel antagonist nitrendipine (IC50: 2.4 x 10(-9) M) or verapamil (IC50: 1.1 x 10(-7) M) also blocked the neurotensin inhibitory effect. Ileal contraction, induced by the Ca(2+)-channel activator BAY K 8644 (10(-7) M), was completely inhibited by neurotensin. After depletion of internal Ca2+ stores by repetitive stimulation with CCH and caffeine in Ca(2+)-free buffer, reintroduction of external Ca2+ restored neurotensin inhibition of the contraction induced by CCH. These results demonstrate that the inhibitory effect of neurotensin in rat ileum longitudinal muscle is apamin sensitive and cannot be observed in the presence of the Ca(2+)-channel blockers nitrendipine or verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential expression of early response genes, c-jun, c-fos, and jun B, in A5 cells
Authors: Yeh C. K. , Ambudkar I. S. , Kousvelari E.
Published: December 2017

We examined the expression of c-fos, c-jun, and jun B after activation of different signal transduction pathways in the A5 rat salivary epithelial cell line. Stimulation of beta-adrenergic receptors by isoproterenol, or addition of 8-bromoadenosine 3',5'-cyclic monophosphate, induces the expression of c-fos and jun B by a protein kinase A-mediated pathway. Phorbol 12-myristate 13-acetate (PMA) induces the expression of all three genes, but with different kinetics. While c-fos and jun B mRNA levels increase early (1 h) after stimulation and transiently, those of c-jun remain higher than control even after stimulation for 8 h and return to basal levels by 24 h. Inhibitors of protein kinase C block the effect of PMA on c-fos, c-jun, and jun B expression, indicating that these genes are also regulated by a protein kinase C-mediated mechanism in A5 cells. Increases in cytosolic Ca2+ by A23187 or ionomycin induce only the expression of c-fos gene. This induction is abolished when A5 cells are loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid before treatment with the ionophores, or when serum is excluded from the incubation medium. Exclusion of serum from the medium does not change the effects of isoproterenol or PMA on c-fos, c-jun, or jun B. These results strongly suggest that serum factors act synergistically with Ca2+ to induce c-fos expression in A5 cells. The studies presented here indicate that different signal transduction pathways operate in A5 cells for the induction of c-fos, c-jun, and jun B genes.
Stimulation of Cl- and HCO3- secretion by intramural cholinergic neurons in guinea pig antrum in vitro
Authors: Suzuki A. G. , Kameyama J. , Tsukamoto M. , Kaneko K. , Suzuki Y.
Published: December 2017

Regulation of Cl- and HCO3- secretion by intramural cholinergic neurons was investigated in guinea pig antrum in vitro. Sheet preparations composed of the mucosa and the submucosa were mounted between Ussing chambers and bathed with buffer-free solution on the luminal surface and with HCO3(-)-CO2 solution on the serosal side. Short-circuit current (Isc), unidirectional fluxes of 36Cl and 22Na, and the luminal alkalinization rate (JOHSL) were determined. Electrical stimulation of the preparations elicited increases in both JOHSL and Isc, which were inhibited by tetrodotoxin (TTX) and atropine. Physostigmine also evoked TTX- and atropine-sensitive increases in JOHSL and Isc. Similar increases in JOHSL and Isc were observed when the muscarinic agonist bethanechol chloride (BCh) was added to the serosal side. The responses to BCh were not affected by TTX. The BCh-induced increase in JOHSL was largely abolished by removal of HCO3- or Na+ and addition of ouabain (serosal side) but was neither sensitive to Cl- removal nor associated with 22Na secretion. The increase in Isc induced by BCh was associated with the increase in 36Cl secretion and was inhibited by removal of Cl- or Na+ and by addition of bumetanide or ouabain (both, serosal side). These results suggest that the submucosal cholinergic neurons are involved via muscarinic receptors in the stimulation of epithelial HCO3- and Cl- secretion. For both HCO3- and Cl-, the cellular and membrane mechanisms of secretion induced by muscarinic stimulation, although not entirely clear, appear to be different from those occurring under baseline conditions.
Control of gastric corpus chromogranin A messenger RNA abundance in the rat
Authors: Dimaline R. , Evans D. , Forster E. R. , Sandvik A. K. , Dockray G. J.
Published: December 2017

Enterochromaffin-like cells in the corpus mucosa of the stomach produce histamine in response to gastrin; chromogranin A (CGA) is often used as a morphological marker for these cells, but its functional significance in the gastric mucosa is largely unknown. We have examined whether CGA mRNA abundance in the rat corpus is controlled by endogenous gastrin. In rats fasted for up to 48 h, there was a progressive decline in plasma gastrin and CGA mRNA; refeeding of fasted rats produced a prompt increase in plasma gastrin and an increase in CGA mRNA that was significant after 4 h. Treatment of fasted rats with omeprazole to inhibit acid secretion increased plasma gastrin and CGA mRNA levels. The increased CGA mRNA associated with omeprazole or refeeding was reversed by treatment of rats with the gastrin/cholecystokinin B antagonist CI-988 and gastrin antibody, respectively. The results suggest that CGA production in enterochromaffin-like cells of the rat stomach is part of the functional response of these cells to circulating gastrin.
pH-dependent nonlysosomal proteolysis contributes to lethal anoxic injury of rat hepatocytes
Authors: Bronk S. F. , Gores G. J.
Published: December 2017

Our aim was to test the hypothesis that pH-dependent nonlysosomal proteolysis is a key mechanism culminating in lethal anoxic injury of rat hepatocytes. Although lysosomal proteolysis was suppressed during anoxia, total nonlysosomal proteolysis was increased twofold compared with aerobic controls. Extracellular acidosis inhibited total nonlysosomal proteolysis and improved cell survival during anoxia. Indeed, we found a direct highly significant linear relationship between cell death and total nonlysosomal proteolysis as modulated by changes in the extracellular pH (r = 0.99, P < 0.01). Glycolytic generation of ATP from fructose during anoxia suppressed total nonlysosomal proteolysis and improved cell survival. An increase in a pH-dependent calpain-like protease activity was also identified during anoxia, but calpain-like protease activity only accounted for 16% of total nonlysosomal protease activity. In addition, the calpain protease inhibitor Cbz-Leu-Leu-Tyr-CHN2 only partially protected against cell killing despite complete inhibition of calpain-like protease activity. These data suggest that pH-dependent total nonlysosomal proteolysis contributes to lethal cell injury during anoxia. However, calpain protease activity only partially contributes to total nonlysosomal protease activity and cell death.
Pilocarpine and carbachol exhibit markedly different patterns of Ca2+ signaling in rat pancreatic acinar cells
Authors: Yule D. I. , Essington T. E. , Williams J. A.
Published: December 2017

The effects of the partial muscarinic agonist pilocarpine on physiological responses were investigated in rat pancreatic acinar cells and compared with carbachol, a full muscarinic agonist, together with previous results using JMV-180, a partial agonist of CCK-A receptors. Pilocarpine was found to stimulate amylase release from isolated pancreatic acini in a concentration-dependent manner. At a maximal concentration (10 microM), pilocarpine was only capable of stimulating 63% of the secretion stimulated by a maximal concentration of carbachol. Moreover pilocarpine did not induce a decrease in secretion at supramaximal concentrations as does carbachol. In acini loaded with fura-2, superfusion of pilocarpine resulted exclusively in generation of intracellular Ca2+ concentration ([Ca2+]i) oscillations at all concentrations tested (0.3 microM-1 mM), in marked contrast to high concentrations of full agonists, which result in a biphasic sustained increase in [Ca2+]i. In common with low concentrations of other secretagogues that stimulate [Ca2+]i oscillations, pilocarpine at all concentrations was only able to stimulate a very small increase in phosphoinositide (PI) hydrolysis. In acini previously incubated with [3H]inositol, pilocarpine was shown to stimulate PI hydrolysis 27% above basal, compared with 872% for carbachol. To ascertain if this small degree of PI hydrolysis seen with pilocarpine is responsible for the generation of [Ca2+]i oscillations, an inhibitor of phospholipase C-linked processes, U-73122, which has been shown to inhibit Ca2+ oscillations induced by carbachol and CCK but not JMV-180 was tested. This agent rapidly inhibited pilocarpine-stimulated oscillations, indicating that in contrast to JMV-180, oscillations induced by pilocarpine are the result of PI hydrolysis.
Inhibition of gastric acid secretion by oxyntomodulin and its 19-37 fragment in the conscious rat
Authors: Jarrousse C. , Carles-Bonnet C. , Niel H. , Sabatier R. , Audousset-Puech M. P. , Blache P. , Kervran A. , Martinez J. , Bataille D.
Published: December 2017

Oxyntomodulin (Oxm) is a hormone, released from the intestine during digestion. Its target tissue is the gastric mucosa, where it inhibits acid secretion. It contains the 29-amino acid glucagon moiety, extended at its COOH-terminal end by an octapeptide. The glucagon moiety contains a basic doublet (Arg17-Arg18). Our working hypothesis was that the mode of action of Oxm may imply a processing of the molecule at the Arg-Arg doublet, releasing Oxm-(19-37). We compared the effect of Oxm with that of Oxm-(19-37) on gastric acid secretion in the conscious rat provided with a chronic gastric fistula. The acid secretion was plateau stimulated by a perfusion of either pentagastrin or histamine. Whereas Oxm or Oxm-(19-37) had no effect on basal acid secretion, both peptides inhibited pentagastrin (0.5 micrograms.kg-1.h-1)- and histamine (0.4 mg.kg-1.h-1)-stimulated acid secretion in a dose-dependent manner. When the metabolic clearance rate for each peptide was taken into account, the 19-37 fragment was as potent as the whole Oxm, regardless of the type of stimulant. When the dose of pentagastrin was increased from 0.175 to 1.1 micrograms.kg-1.h-1, the extent of inhibition induced by Oxm (40 pmol/kg) also increased. In contrast, when the dose of histamine was increased from 0.25 to 1.2 mg.kg-1.h-1, the extent of inhibition induced by Oxm (40 pmol/kg) decreased. Oxm-(19-37) (70-140 pmol/kg) displayed the same behavior as the whole molecule under both types of stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of nitric oxide as an inhibitory neurotransmitter in the canine pyloric sphincter
Authors: Bayguinov O. , Sanders K. M.
Published: December 2017

Experiments were performed to test the hypothesis that enteric inhibitory neurotransmission in pyloric muscles is mediated by NO. Junction potentials were recorded with intracellular microelectrodes from cells near the myenteric and submucosal surfaces of the circular muscle layer. Inhibitory junction potentials (IJPs) were apamin sensitive and were reduced by arginine analogues [NG-nitro-L-arginine-methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA)]. The effects of arginine analogues were reversed by L-arginine. Inhibition of IJPs unmasked excitatory JPs (EJPs) in the myenteric region and increased excitability of cells in the submucosal region. IJPs were also reduced by oxyhemoglobin. As with arginine analogues, reduction in IJPs increased EJP amplitude. Combination of L-NAME and oxyhemoglobin completely blocked IJPs, suggesting that NO, or an NO-containing compound, mediated the enteric inhibitory nerve responses. Exogenous NO hyperpolarized membrane potential, and these responses were also reduced by apamin. The magnitude of the responses to a given dose of NO was similar in cells of the myenteric and submucosal regions, suggesting that relatively smaller IJPs in submucosal cells may be due to a lower density of enteric inhibitory innervation in the submucosal region. The effects of NO were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and M & B 22948, a specific cGMP phosphodiesterase inhibitor, suggesting that the hyperpolarization response to NO may be mediated by enhanced production of cGMP. IJPs were also prolonged by M & B 22948. IJPs and NO disrupted normal electrical rhythmicity in cells in the myenteric region. This may provide a basis for inhibitory effects of enteric inhibitory nerve stimulation on sphincter pressure in pyloric canal in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Lipid peroxidation of the brush-border membrane: membrane physical properties and glucose transport
Authors: Jourd'Heuil D. , Vaananen P. , Meddings J. B.
Published: December 2017

During inflammatory conditions, peroxidation of biological membranes often takes place. Deleterious physiological consequences, in terms of membrane function, could theoretically be mediated by either direct oxidative attack upon integral membrane proteins or by indirectly altering the lipid environment surrounding these proteins. To address this issue, in vitro peroxidation of guinea pig brush-border membrane vesicles was induced by incubation of the vesicles with ferrous sulfate and ascorbic acid. We found that ongoing peroxidative attack initiates lipid peroxidation and radically alters the physical properties of the membrane lipid bilayer in a well-defined and regional manner. Peroxidation of microvillous membrane produced an increasingly rigid membrane. Coupled with these alterations was a fivefold reduction in maximal rates of sodium-dependent glucose transport that appeared to have a multifactorial origin. Approximately one-third of this reduction was secondary to altered membrane physical properties and was reversible by fluidizing the vesicles and returning membrane physical properties to normal. The remaining reduction in glucose transport activity was not responsive to membrane fluidization and was presumably related to direct damage of the transport protein.
Hepatic vein, hepatic parenchymal, and inferior vena caval mechanoreceptors with phrenic afferents
Authors: Kostreva D. R. , Pontus S. P.
Published: December 2017

Dogs were anesthetized with pentobarbital sodium and placed on positive-pressure ventilation. The right phrenic nerve and/or its C5 branch were prepared for afferent recording. The hepatic veins, hepatic parenchyma, diaphragm, and inferior vena cava were studied for mechanoreceptors using light pressure and stroking as the stimuli. Mechanosensitive areas were found in the hepatic veins, hepatic parenchyma of the right medial lobe, and inferior vena cava. The hepatic vein and inferior vena caval receptors are located in the same 1- to 2-cm region as the sphincters that are found in these vessels. This study presents the first experimental evidence for the existence of hepatic vein receptors, hepatic parenchymal receptors, and inferior vena caval mechanoreceptors with phrenic afferents in the dog. These sensory areas of the circulation may be involved in the neural control of venous return as well as mediating changes in intrahepatic and portal venous blood pressure during normal respiration.
Autoradiographic characterization of angiotensin II receptor subtypes in rat intestine
Authors: Sechi L. A. , Valentin J. P. , Griffin C. A. , Schambelan M.
Published: December 2017

Angiotensin II is known to regulate motility and ion and water absorption in the intestine. These effects are presumed to be mediated by angiotensin II (ANG II) receptors that are present in both mucosal and muscular layers throughout the intestine. To evaluate tissue density and distribution of ANG II receptor subtypes (AT1 and AT2), we performed an in situ autoradiographic study on jejunum, ileum, and colon of Sprague-Dawley rats. Tissue sections (10 microns) were incubated with 500 pM 125I-[Sar1,Ile8]ANG II, fixed with paraformaldehyde vapors, and coated with photographic emulsion. Binding specificity was verified by competition with unlabeled [Sar1]ANG II (10 microM). AT1 and AT2 receptor distribution was characterized by competition with the nonpeptide antagonists losartan (10 microM) and PD123177 (10 microM), respectively, and the density of receptors was quantified by counting the silver grains overlying the different layers of intestinal wall. Specific binding was moderately abundant in the mucosa and the muscularis of both jejunum and ileum, whereas no binding was present in the submucosa and the serosa. Losartan inhibited 86% of radioligand binding to the mucosa in both jejunum and ileum, whereas PD123177 inhibited only 10%. The combination of the two compounds inhibited 96% of specific binding. In the colon, binding was significantly more abundant in the muscularis than in the mucosa. In this segment, losartan inhibited 90% and PD123177 16% of specific binding to muscularis. The combination of these compounds reduced binding by 97%. Thus the predominant ANG II receptor in all intestinal segments is AT1, but a small population of AT2 receptors also seems to be present.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of ammonium ion and ammonia on function and morphology of in vitro frog gastric mucosa
Authors: Yanaka A. , Muto H. , Ito S. , Silen W.
Published: December 2017

The effects of ammonium ion (NH+4) and ammonia (NH3) on function and morphology of gastric epithelial cells were studied in intact sheets of in vitro frog (Rana catesbeiana) gastric mucosa. Luminal 115 mM NH4Cl at luminal pH 8.0 (calculated [NH3] 2.7 mM), but not at 5.0 (calculated [NH3] 3 microM) induced 1) an increase in intracellular pH (pHi) in oxynticopeptic cells (OPC) and decreases in transmucosal potential difference (PD) and electrical resistance (R) in resting tissues, 2) a decrease in histamine-stimulated H+ secretion and an increase in H+ backdiffusion after removal of luminal NH4Cl, and 3) augmented acidification of OPC during luminal acidification. Serosal 30 mM NH4Cl at serosal pH 7.2 (calculated [NH3] 0.47 mM) induced 1) an increase in pHi in OPC and inhibition of the alkalinization of OPC after removal of ambient Cl-, 2) a decrease in PD associated with the increase in R and decrease in short-circuit current, effects attenuated by serosal 15 mM K+, accentuated by 0.2 mM Ba2+, and abolished by removal of ambient Cl-, 3) a sudden drop of PD in resting, but not in stimulated tissues, effects prevented by high serosal pH (7.8), serosal HCO3-, or removal of luminal Cl-, 4) a decrease in histamine-stimulated H+ secretion and an increase in H+ backdiffusion after removal of NH4Cl, and 5) augmented acidification of OPC during luminal acidification. These results suggest that 1) luminal NH3, but not NH+4, increases backdiffusion of H+ from the lumen to the mucosa, 2) serosal NH3 and/or NH+4 induces depolarization of OPC and decreases electrogenic Cl- transport, thereby attenuating the activity of the basolateral Cl(-)-HCO3- exchanger in OPC, and 3) both of these effects contribute to the augmented acidification of OPC during exposure to high luminal [H+].
Mechanoreceptors sensitive to flow at the gastroduodenal junction of the cat
Authors: Malbert C. H. , Leitner L. M.
Published: December 2017

Vagal afferent activity was recorded from the cervical vagus simultaneously with intraluminal pressure and flow rate at the proximal duodenum, in cats anesthetized with pentobarbital sodium. Thirty-seven mechanoreceptors that adapted slowly to maintained pressure changes were found to be sensitive to the flow of saline through the pylorus. The passage of liquid was associated with an increase of pressure equal to one-tenth of that necessary to trigger a response of the receptors during the distension of a small balloon. The frequency of discharge was increased by the passage of liquid for positive back pressures < or = 1.5 kPa but not when back pressure was negative. The relationship between the discharge of the receptor and the flow rate was complex and showed an hysteresis. We conclude that there exist slowly adapting mechanoreceptors sensitive to distension of the duodenum that can also sense the passage of liquid.
Substance P levels in experimental ileitis in guinea pigs: effects of misoprostol
Authors: Miller M. J. , Sadowska-Krowicka H. , Jeng A. Y. , Chotinaruemol S. , Wong M. , Clark D. A. , Ho W. , Sharkey K. A.
Published: December 2017

Substance P, a neuropeptide mediator of inflammation, was quantified during the evolution of trinitrobenzene sulfonic acid (TNBS)-induced ileitis in guinea pigs. Ileitis was induced by a single intraluminal injection of TNBS (30 mg/kg in 50% ethanol). Misoprostol, a prostaglandin E1 analogue, was administered parenterally (30 mg/kg sc twice daily) in a group of TNBS-treated animals. Control guinea pigs received intraluminal saline (sham) or 50% ethanol (TNBS vehicle). Guinea pigs were evaluated at day 1, 3, 7, 14, or 30 after ileitis induction for substance P content (radioimmunoassay) and distribution (immunohistochemistry), morphology, myeloperoxidase (MPO) activity, and protein leak into the gut lumen. TNBS administration caused an increase in ileal MPO activity that peaked at day 7 and increased mucosal leak of protein. Misoprostol attenuated the granulocyte infiltration (MPO) response to TNBS but exacerbated the mucosal leak of protein. Substance P levels in whole ileal segments were unaltered from baseline on day 1 in all groups. On day 3 a marked decrease in ileal substance P content was evident in the TNBS and TNBS + misoprostol groups. As early as day 1, immunohistochemistry suggested that the decreased substance P content was confined to the mucosa and submucosa, because myenteric plexus staining was not reduced. Loss of staining in the perivascular nerves was particularly marked. Substance P content and distribution returned to baseline by day 30 post-TNBS, although MPO activity remained slightly elevated. We concluded that TNBS ileitis is associated with a marked reduction in mucosal and submucosal substance P content in parallel with the inflammatory response. Although misoprostol attenuated granulocyte infiltration in this model, it did not prevent the disturbances in enteric substance P or mucosal protein leak.
Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells
Authors: Wang J. Y. , McCormack S. A. , Viar M. J. , Wang H. , Tzen C. Y. , Scott R. E. , Johnson L. R.
Published: December 2017

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)
The human sucrase-isomaltase gene directs complex patterns of gene expression in transgenic mice
Authors: Markowitz A. J. , Wu G. D. , Birkenmeier E. H. , Traber P. G.
Published: December 2017

Sucrase-isomaltase (SI) is an enterocyte-specific gene that is expressed in complex developmental and spatial patterns. In this study, we examine the ability of regulatory elements within the human SI (hSI) gene to direct appropriate cell lineage and spatial patterns of expression in transgenic mice. Transgenic mouse lines were established using a construct containing bases -3424 to +54 of the hSI gene linked to the human growth hormone (hGH) structural gene. In each transgenic line, hGH mRNA and protein were expressed only in the small intestine and colon. In contrast to the endogenous mouse SI (mSI) gene, which was expressed along the entire length of the small intestine, hGH mRNA expression was predominantly found in the distal jejunum and ileum, with very low levels in more proximal portions of the small intestine. However, the pattern of transgene expression along the small intestinal crypt-villus axis was identical to the pattern of the endogenous mSI gene. These results suggest that regulatory elements necessary for intestine-specific transcription and differential expression along the intestinal crypt-villus axis are included in the 5'-flanking region of the hSI gene. Furthermore, these data suggest that different DNA regulatory regions regulate transcription along the horizontal intestinal axis. In the colon, there was aberrant expression of hGH in a subpopulation of enteroendocrine cells that contained peptide tyrosine tyrosine (PYY). This suggests that there are DNA regulatory elements, missing in the transgene construct, which normally suppress expression of the endogenous mSI gene in these cells. Taken together, these findings define the SI gene as a useful model for studies of differentiation, cell lineage determination, and mechanisms of complex spatial gene expression in the intestine.
VIP-mediated G protein-coupled Ca2+ influx activates a constitutive NOS in dispersed gastric muscle cells
Authors: Murthy K. S. , Zhang K. M. , Jin J. G. , Grider J. R. , Makhlouf G. M.
Published: December 2017

Vasoactive intestinal peptide (VIP) and peptide histidine-isoleucine (PHI) receptors and the signaling pathways to which they are coupled were characterized in dispersed gastric smooth muscle cells. Radioligand binding using 125I-labeled VIP and PHI identified 4 classes of receptors: VIP-preferring and PHI-preferring receptors recognized by both ligands and readily desensitized by the preferred ligand, and VIP-specific and PHI-specific receptors recognized by only 1 ligand and resistant to desensitization. All except VIP-specific receptors were coupled to adenylate cyclase. VIP-specific receptors mediated a G protein-coupled Ca2+ influx that led to activation of NO synthase (NOS), NO-dependent activation of soluble guanylate cyclase, and activation of guanosine 3',5'-cyclic monophosphate (cGMP) kinase resulting in muscle relaxation. The entire cascade was blocked by Ca2+ channel and/or calmodulin antagonists. The NOS inhibitor NG-nitro-L-arginine abolished L-[3H]citrulline (coproduct of NO synthesis) and cGMP generation and partly inhibited (52 +/- 4%) relaxation. The components of response mediated by VIP-specific receptors (increase in [Ca2+]i, L-[3H]citrulline, and cGMP) were preserved after desensitization. Insertion of guanosine 5'-O-(beta-thio)diphosphate into reversibly permeabilized muscle cells abolished responses mediated by VIP-preferring and VIP-specific receptors. VIP stimulated both adenosine 3',5'-cyclic monophosphate (cAMP)-kinase and cGMP-kinase activities consistent with stimulation of cAMP and cGMP. Both kinases contributed to relaxation that was partly inhibited by cAMP-kinase [H-89 and (R)-p-adenosine 3',5'-cyclic monophosphorothioate] and cGMP-kinase (KT-5823) inhibitors and abolished by a combination of the 2 types of inhibitors. We conclude that VIP-specific receptors mediate a G protein-coupled Ca2+ influx leading to activation of a constitutive Ca2+/calmodulin-dependent NOS and generation of NO, which is partly responsible for relaxation in smooth muscle.
Effects of the somatostatin analogue octreotide on rectal afferent nerves in humans
Authors: Plourde V. , Lembo T. , Shui Z. , Parker J. , Mertz H. , Tache Y. , Sytnik B. , Mayer E.
Published: December 2017

Somatostatin (Som) administered intrathecally to humans has been shown to exert potent analgesic effects on somatic pain, and anecdotal evidence suggests that Som may also relieve visceral pain. In the current study, we used rectal balloon distension in seven healthy volunteers to evaluate the effect of the Som analogue octreotide (Oct; 1.25 microgram/kg sc) on four pathways mediated by different visceral afferents that originate in the rectum: conscious perception, receptive relaxation, reflex inhibition of internal anal sphincter, and a viscerosomatic reflex. Rectal mechanoreceptors were stimulated either by distending the rectum tonically (volume ramp at 20-40 and 400 ml/min) or phasically (intermittent pressure steps of 60 s duration). Pressure thresholds for nonnoxious and noxious sensations in response to slow tonic distension were increased in the presence of rectal lidocaine (20 ml of 2% solution), whereas those to phasic distension were unaffected. Oct significantly increased pressure and volume thresholds for nonnoxious and noxious sensations in response to slow tonic distension but did not further increase thresholds in the presence of intrarectal lidocaine. In contrast, no effect of Oct on rectal sensations was observed during rapid tonic or phasic distension. Oct had no effect on any of the monitored reflex responses. The effect of Oct on rectal sensation in the concentration used in this study was not associated with changes in the rectal wall pressure-volume relationship during any distension protocol. These findings indicate that the inhibitory effect of Oct on rectal sensation is likely to represent a direct effect on a subset of extrinsic primary afferent neurons, with receptive fields in the mucosa
Actions of nitric oxide-generating sodium nitroprusside in myenteric plexus of guinea pig small intestine
Authors: Tamura K. , Schemann M. , Wood J. D.
Published: December 2017

Sodium nitroprusside (NaNP) was used as a donor of nitric oxide (NO) to investigate actions of NO on electrical and synaptic behavior of single myenteric neurons in guinea pig small intestine. NaNP (10 microM-1 mM) did not affect resting membrane properties of the neurons, except for an occasional decrease in input resistance and hyperpolarization attributable to suppression of excitatory transmitter release. NaNP did not alter fast nicotinic neurotransmission but suppressed noncholinergic slow excitatory postsynaptic potentials (slow EPSPs) in a concentration-dependent manner. Pretreatment with either methylene blue or oxyhemoglobin reduced the inhibitory action of NaNP on the slow EPSPs. Slow EPSP-like responses to microejected substance P or 5-hydroxytryptamine were unaffected by NaNP. The nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester, did not affect resting membrane excitability or excitatory synaptic events in any of the myenteric neurons. The results suggest that NO may not be released extensively as a neurotransmitter at synapses within the myenteric plexus. If myenteric neurons are exposed to NO released from nonneural sources, then the principal action is expected to be presynaptic inhibition of slow synaptic excitation.
Pharyngeal, esophageal, and proximal gastric responses associated with vomiting
Authors: Lang I. M. , Sarna S. K. , Dodds W. J.
Published: December 2017

The motor activities of the pharynx, esophagus, and proximal stomach associated with vomiting were characterized and quantified in 25 awake chronically instrumented dogs. These motor events were correlated temporally with motor responses of the gastrointestinal and respiratory tracts. Vomiting was stimulated by apomorphine or UK-14304, and motor activities of striated and smooth muscles were recorded by electromyography and strain-gauge transducers, respectively. We found responses that began 1) before retching: increased swallow frequency, relaxation of the lower esophageal sphincter (LES) and proximal stomach, and tonic contraction of the cricopharyngeus and cervical esophagus; 2) during retching: rhythmic relaxation and contraction of the pharynx and cervical esophagus 180 degrees out of phase with retching; and 3) during vomitus expulsion: relaxation of the pharynx and cervical esophagus but strong contraction of the geniohyoideus and a retrograde contraction of the cervical esophagus and pharynx. The increased pharyngoesophageal tone occurred in an all-or-none fashion independent of vomiting or the gastrointestinal correlates of vomiting. Cervical vagal blockade increased swallow frequency but did not alter the other pharyngoesophageal responses associated with vomiting. We concluded that the motor events of the pharynx and esophagus play a significant role in gastrooral evacuation during vomiting and that these motor events (except LES relaxation) are not controlled by subnodose vagal pathways.
Topography of normal and high-amplitude esophageal peristalsis
Authors: Clouse R. E. , Staiano A.
Published: December 2017

Topographic plots were created from esophageal manometric tracings in 12 asymptomatic volunteers and 10 symptomatic patients with high-amplitude peristaltic contraction waves (nutcracker esophagus) to identify segmental contraction differences between the two groups. Median waves at each centimeter of esophageal length were spatially interconnected using a computerized gridding and plotting system, and a characteristic peristaltic contraction from the proximal esophageal body through the lower sphincter was constructed for each subject. Contour plots in the volunteers revealed three amplitude troughs dividing the peristaltic contraction into four sequential segments: the skeletal muscle body, the proximal and distal segments of the smooth muscle body, and the lower sphincter region. In nutcracker-esophagus patients, only the first and third troughs could be identified, since augmented contraction in the distal smooth muscle segment blurred separation of the smooth muscle body segments. Volume measurements under the topographic plots showed no significant intergroup differences in contraction of the skeletal muscle region or lower sphincter but modest increase in contraction of the proximal smooth muscle segment [1.7 times normal, 95% confidence interval (CI) 1.2-2.0; P = 0.002 comparing 2 groups] and marked increase in the distal smooth muscle segment (2.7 times normal, 95% CI 2.0-3.3; P < 0.0001). These data indicate that normal peristalsis through the esophageal body and lower sphincter includes four separate contraction segments that can be distinguished with intraluminal manometry and the topographic analysis method. The nutcracker esophagus primarily affects the distal segment in the smooth muscle body.
Nitric oxide-mediated excitatory effect on neurons of dorsal motor nucleus of vagus
Authors: Travagli R. A. , Gillis R. A.
Published: December 2017

The purpose of our study was to explore whether nitric oxide was involved as an intercellular messenger in the dorsal motor nucleus of the vagus (DMV). To achieve this purpose we examined DMV motoneurons of the rat in vitro with the use of the extracellular cell-attached recording technique. The motoneurons, in general, exhibit a spontaneous discharge and when exposed to NO-producing drugs (i.e., 3-300 microM L-arginine and 10-100 microM S-nitroso-N-acetylpenicillamine) exhibit a concentration-related increase in their spontaneous firing rate. Because NO activates soluble guanylate cyclase and increases guanosine 3',5'-cyclic monophosphate (cGMP), we tested dibutyryl-cGMP (30-300 microM) and found that it also excites DMV neurons. Perfusion of the DMV neurons with N omega-nitro-L-arginine (300 microM), an inhibitor of NO synthase (NOS), and with NO scavenger, reduced hemoglobin (1 microM), counteracted the excitatory effect of L-arginine and N-methyl-D-aspartate (NMDA). Perfusion of the preparation with LY-83583 (10 microM), an inhibitor of guanylate cyclase, also counteracted the effects of L-arginine and NMDA. These data indicate that NOS is present in DMV neurons, and that the excitatory effect of NMDA on these neurons is due in part to formation of NO and the resulting accumulation of cGMP in DMV neurons.
Role of nitric oxide-related inhibition in intestinal function: relation to vasoactive intestinal polypeptide
Authors: Daniel E. E. , Haugh C. , Woskowska Z. , Cipris S. , Jury J. , Fox-Threlkeld J. E.
Published: December 2017

This study examined the role of nitric oxide (NO) in tonic inhibition of motor activity in isolated, perfused canine ileal segments. Brief addition of N omega-nitro-L-arginine methyl ester (L-NAME) to the perfusate caused, after a delay, a concentration-dependent persistent increase in tonic and phasic activity of circular muscle. This increased motor activity was prevented or reversed by addition of L- but not D-arginine to the perfusate. Removal of Ca2+ or addition of 10(-7) M omega-conotoxin (GVIA) to the perfusate markedly reduced this response. The motor activity induced by L-NAME was accompanied by loss of distal inhibition and enhanced excitation to low-frequency field stimulation. L-NAME infusion significantly reduced tonic vasoactive intestinal polypeptide (VIP) output, sodium nitroprusside increased VIP output, but L-arginine infusion did not restore VIP output. Atropine (10(-7) M) and/or hexamethonium (10(-4) M) reduced the motor response to L-NAME by 75%. Atropine reduced and hexamethonium nearly abolished VIP output. We conclude that there is tonic Ca(2+)-dependent NO output from perfused intestinal segments dependent on nerves with N-Ca channels, that NO acts to inhibit muscle directly and by inhibiting release of excitatory mediators, and that this output is the primary inhibitory determinant of contractile activity.