Nitric oxide: a mediator of esophageal motor function.
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Animals↗
Biomedical subjects
J L Conklin
Publications and source records attributed to J L Conklin.
Metabolism of superoxide dismutase during the adynamic ileus of endotoxemia.
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Animals↗
Calcium release from intracellular stores and excitation-contraction coupling in intestinal smooth muscle.
Calcium release from intracellular stores plays a central role in excitation-contraction coupling of striated and smooth muscle cells. Two main intracellular calcium pools have been identified in phasic smooth muscle: (1) the inositol 1,4,5-trisphosphate-sensitive and the (2) ryanodine-sensitive calcium stores. We studied the contribution of the ryanodine-sensitive calcium stores to the excitation-contraction coupling in the intestine. The intracellular calcium concentration was measured in cultured intestinal smooth muscle cells using the fluorescent probe fura-2-AM. Isometric tension generated by the murine jejunum was recorded in vitro using force displacement transducers. The cytosolic calcium level increased significantly on cholinergic stimulation. The rise persisted in the absence of extracellular calcium. Depletion of ryanodine-sensitive calcium stores with caffeine or ryanodine blunted the response to a cholinergic agonists. Similarly, the ryanodine receptor channel blocker dantrolene significantly decreased the carbachol-induced calcium increase. We subsequently tested the effects of these pharmacological tools on the spontaneous and carbachol-induced contractions of the murine jejunum. Depletion of the ryanodine-sensitive stores and calcium release channel block both significantly decreased the contractile activity of the circular and longitudinal layer of the muscularis propria. Our data confirm the importance of intracellular calcium stores in excitation-contraction coupling of intestinal smooth muscle cells. The effects of different pharmacological tools on the intracellular calcium signal and the contractile function are consistent with other observations in phasic smooth muscle. They suggest a significant contribution of calcium release from ryanodine-sensitive stores to the calcium signal that triggers contraction.
Animals↗
The effect of ethanol on sphincter of Oddi motility in vitro.
Antioxidant enzymes are present in nerves supplying the sphincter of Oddi and regulate its motor function. Oxygen free radicals (O2.-) produce hydrogen peroxide (H2O2) by the action of superoxide dismutase (SOD). Hydroxyl radical (OH.), an important mediator of H2O2 toxicity, oxidizes ETOH. Thus, the aim of our study was determine the effects of ETOH on sphincter of Oddi motility. The sphincter of Oddi was removed from opossums and force transducers recorded tension in its transverse axis. Alcohol (ETOH) was added to the tissue bath in concentrations from 0.1 to 0.4%. OH. was generated by the addition of 0.01% H2O2. Nitric oxide production was inhibited by the addition of 0.3 mM N omega-nitro-L-arginine (L-NNA). H2O2 increased the frequency of sphincter of Oddi contractions. Concentrations of ETOH > 0.3% decreased contractile frequency; however, 0.2% ETOH alone had no affect on the basal frequency of contraction but inhibited the increase in contractile frequency caused by H2O2. L-NNA also increased the contractile frequency; however, this effect was not inhibited by ETOH. We conclude that ETOH attenuates the effect of H2O2 on sphincter of Oddi motility.
Animals↗
Pathophysiology of adynamic ileus.
We hypothesized that the inhibitory neurotransmitters nitric oxide (NO) and vasoactive intestinal peptide (VIP) may play a role in the disrupted gastrointestinal motility of endotoxemia. Strain gauge transducers on the stomach and small intestine of dogs determined interdigestive gastrointestinal motility. Tissue levels of NO synthase and VIP and serum levels of nitrite/nitrate (NO(2)-/NO(3)-) and VIP were measured. Following completion of the baseline studies, dogs were given a single dose of E. coli lipopolysaccharide, 200 microg/kg intravenously, and the studies were repeated for the next three days. Following endotoxin bolus, the migrating motor complex (MMC) was delayed for two days while serum VIP was increased on postendotoxin day 1 and serum NO(2)-/NO(3)- was increased on postendotoxin day 2. There were no changes in gut smooth muscle levels of NO synthase or VIP. We conclude that a single, sublethal dose of endotoxin results in prolongation of the MMC with distinct but independent increases in serum levels of VIP and NO(2)-/NO(3)-.
Animals↗
Effects of oxygen radicals and radical scavenging on opossum lower esophageal sphincter.
Nitric oxide mediates esophageal peristalsis and lower esophageal sphincter (LES) relaxation. Superoxide produced with inflammation inactivates nitric oxide. Superoxide is cleared in biological systems by superoxide dismutase. We tested the hypothesis that superoxide and the superoxide scavenging system modulate LES function. Transverse strips of muscle from the opossum LES relaxed when stimulated by an electrical field. Diethyldithiocarbamite was used to inhibit copper/zinc superoxide dismutase. Xanthine and xanthine oxidase were used to generate superoxide. Xanthine with xanthine oxidase or diethyldithiocarbamite alone had no effect on the LES. However, xanthine/xanthine oxidase and diethyldithiocarbamite reduced LES relaxation 34.1% and increased its resting tone 71.2%. Superoxide dismutase did not affect LES function, but protected the tissue from the effects of diethyldithiocarbamite and xanthine/xanthine oxidase. These studies are consistent with the hypothesis that superoxide acts by inactivating nitric oxide and suggest that these antioxidant enzyme systems may play a role in the maintenance of LES function.
Animals↗
Intestinal motility during hypoxia and reoxygenation in vitro.
Ischemia-reperfusion injury leads to profound functional and structural alterations of the gastrointestinal tract. We developed an in vitro model of reperfusion injury to study the changes in intestinal motility during hypoxia followed by reoxygenation. We recorded the spontaneous motor activity of intestinal rings from the proximal mouse jejunum, using force displacement transducers. In addition to the rhythmic contractions, we studied the contractile response to transmural stimulation of intrinsic nerves. During hypoxia, the frequency of the spontaneous contractions and the resting tension decreased. While 29% of the tissues still responded to neural stimulation after 15 min of hypoxia, electrical field stimulation did not evoke any response after 60 min of hypoxia. Reoxygenation resulted in a transient increase in the baseline tension and an initial normalization of the spontaneous rhythmic contractions, which subsequently became irregular. The percentage of tissues that recovered their ability to respond to electrical field stimulation 10 min after reoxygenation decreased from 100% after 15 min of hypoxia to 47% after 60 min of hypoxia. The administration of the antioxidant glutathione prevented the functional abnormalities seen 10 min after reoxygenation. The pharmacological inhibition of Cu,Zn superoxide dismutase exacerbated the functional reoxygenation damage. Conversely, the overexpression of this radical-scavenging enzyme in transgenic mice increased the likelihood of functional recovery. Reoxygenation in a calcium-free solution also prevented prolonged functional damage of the muscle rings. We conclude that hypoxia-reoxygenation significantly alters intestinal motility. The generation of reactive oxygen species and disruptions in the calcium homeostasis play an important role in the pathogenesis of reoxygenation damage. Interventions that alter the intracellular redox state or affect the secondary changes in the intracellular calcium concentration can prevent or blunt the effects of reoxygenation injury on intestinal motility.
Animals↗
Effects of calcitonin gene-related peptide on opossum esophageal smooth muscle.
BACKGROUND & AIMS: Peristaltic contraction of the esophagus and relaxation of the lower esophageal sphincter (LES) are mediated by nitric oxide (NO.)-producing myenteric nerves. NO synthase immunoreactivity and calcitonin gene-related peptide (CGRP) immunoreactivity colocalize in these nerves. CGRP relaxes the LES and decreases the amplitude of esophageal peristalsis. The aim of this study was to test the hypothesis that CGRP plays a role in nerve-induced responses of the esophagus. METHODS: Smooth muscle strips from the LES and the body of the esophagus were stimulated by an electrical field to evoke nerve-mediated responses. RESULTS: CGRP decreased the amplitude and lengthened the latency of the off-response, and it relaxed the LES. Tetrodotoxin did not block the CGRP-induced LES relaxation. The CGRP antagonist CGRP 8-37 inhibited the effects of exogenous CGRP. It increased the amplitude and shortened the latency of the off-response, and it increased the resting tone of the LES. CGRP 8-37 did not inhibit LES relaxation or esophageal contraction. N omega-nitro-L-arginine abolished nerve-induced relaxation of the LES muscle and the esophageal off-response but not the relaxation produced by CGRP. CONCLUSIONS: These data support the hypothesis that CGRP may decrease the LES basal tone and modulate the timing and amplitude of the esophageal off-responses.
Animals↗
The role of antioxidant enzymes in the control of opossum sphincter of Oddi motility.
Superoxide rapidly oxidizes nitric oxide (NO) to form peroxynitrite, thus terminating the biological activity of NO. The aims of our study were to determine if superoxide alters the motor function of the sphincter of Oddi and to localize the antioxidant enzymes in the sphincter of Oddi. Immunostaining was performed and enzyme activities were measured in the sphincter of Oddi. In physiological experiments, force-displacement transducers recorded tension in the spontaneously contracting sphincter of Oddi and after electrical field stimulation (EFS) of precontracted sphincter of Oddi. Superoxide was generated by the addition of xanthine with xanthine oxidase, superoxide radicals were scavenged by the addition of superoxide dismutase (SOD), and catalase or SOD was inhibited by diethyldithiocarbamic acid. Immunostaining demonstrated SOD and catalase immunoreactivity in ganglia situated at the serosal surface of the circular muscle. Total SOD activity was 202 +/- 12 U/mg. Generation of superoxide or inhibition of SOD increased the contractile frequency and decreased relaxation after EFS. We conclude that superoxide alters sphincter of Oddi motor function, and the presence of superoxide scavenging enzymes in enteric plexuses suggests that they may regulate sphincter of Oddi neuromuscular function by clearing endogenous superoxide.
Animals↗
Reactive oxygen species and calcium homeostasis in cultured human intestinal smooth muscle cells.
Reactive oxygen species (ROS) significantly alter cell function. We examined the effects of hydrogen peroxide (H2O2) and xanthine/xanthine oxidase (X/XO) on isolated intestinal muscle cells. We assessed cell viability with the exclusion dye trypan blue and assayed the effects of H2O2 and X/XO on the intracellular redox state with the fluorescent probe 2',7'-dichlorofluorescein. Intracellular calcium concentration was measured in cells loaded with fura 2-acetoxymethyl ester, and we recorded whole membrane currents with conventional patch-clamp methods. Cells remained viable after a 5-min exposure to H2O2 and X/XO. H2O2 and X/XO led to a significant rise of the intracellular concentration of ROS. H2O2 (270 microM to 2.7 mM) as well as X/XO (0.25-16 mU; 0.5 mM xanthine) significantly increased intracellular calcium concentrations. Depletion of intracellular calcium with ryanodine or thapsigargin did not abolish the effect of ROS on the intracellular calcium concentration. In the absence of external calcium or in the presence of the calcium channel blocker nifedipine, H2O2 and X/XO still increased the intracellular calcium level. Thus calcium influx and calcium release from internal stores contributed to this rise in cytosolic calcium. Catalase and superoxide dismutase blunted or completely abolished the changes in calcium concentration elicited by H2O2 and X/XO. Exposure to ROS resulted in a rapid decline of the membrane resistance without significant changes in voltage-sensitive ion currents. We conclude that ROS disrupt the calcium homeostasis of cells at concentrations that do not lead to immediate cell death. The resulting elevation in cytosolic free calcium will activate a variety of biochemical reactions and may thus contribute to the cytotoxicity of reactive oxygen molecules.
Acetylcholine↗
Effect of hydroxyl radical (OH.) on sphincter of Oddi motility.
Antioxidant enzymes are present in sphincter of Oddi nerves and regulate sphincter of Oddi motor function mediated by NO-releasing nerves. Oxygen free radicals (O2-.) produce hydrogen peroxide (H2O2) by the action of superoxide dismutase (SOD). Hydroxyl radical (OH.) has been shown to play an important role as a mediator of H2O2 toxicity. The aims of our study were to determine the effects of H2O2 on sphincter of Oddi motility and if these effects are mediated by OH.. Adult opossums were sacrificed and the sphincter of Oddi removed and placed in a tissue bath containing oxygenated Krebs solution. Force transducers recorded tension in a transverse orientation at two sites along the sphincter of Oddi specimen. H2O2 was added into the tissue bath at concentrations from 0.01 to 0.5%. O2-. radicals were inhibited by the addition of SOD, while OH. was scavenged by the addition of alcohol (ETOH) or dimethyl sulfoxide (DMSO). H2O2 produced a dose-dependent increase in baseline amplitude, frequency, and peak amplitude of contractions. The effect of 0.01% H2O2 on sphincter of Oddi contractile frequency was inhibited by 0.2% ETOH and DMSO, but not by SOD. We conclude that H2O2 has profound effects on sphincter of Oddi motility and that the actions of H2O2 are probably mediated through OH..
Animals↗
Gastrointestinal transit during endotoxemia: the role of nitric oxide.
We hypothesized that the disrupted gastrointestinal transit that occurs during endotoxemia is mediated by nitric oxide (NO) and that the inhibition of NO synthesis will normalize intestinal transit and gastric emptying. To determine the effects of endotoxin and steroids on the activity of gastrointestinal smooth muscle NO synthase, rats underwent placement of an intravenous (iv) line and then were given Escherichia coli lipopolysaccharide (LPS) 10 mg/kg/iv; LPS, 10 mg/kg/iv + dexamethasone, 3 mg/kg/iv; or saline. The activity of nitric oxide synthase in the stomach, small intestine, and colon were determined by measuring the conversion of L-[3H]arginine to L-[3H]citrulline. To determine intestinal transit and gastric emptying, gavage feedings of nonabsorbable liquid markers were given and rats divided into eight groups: 0.9% NaCl, 1 ml/hr x 5 hr (control); LPS, 10 mg/kg/iv; LPS + N-omega-nitro-L-arginine methyl ester (L-NAME), 10 mg/kg/hr x 5 hr; LPS + N-omega-nitro-D-arginine methyl ester (D-NAME), 10 mg/kg/hr x 5 hr; LPS + L-arginine, 100 mg/kg/hr x 5 hr; LPS + L+NAME + L-arginine; LPS + N-omega-nitro-L-arginine (L-NNA) 10 mg/kg/hr; or LPS + L-NNA + L-arginine. LPS increased the enzymatic activity of both the constitutive and the inducible forms of NO synthase in the small intestine and fundus of the stomach. The acceleration of intestinal transit produced by endotoxemia was reversed with both L-NAME and L-NNA but not with D-NAME. Endotoxemia slowed gastric emptying but this effect was not reversed with either L-NAME or L-NNA. We conclude that NO plays a major role in mediating the rapid intestinal transit during endotoxemia.
Animals↗
Effects of recombinant human hemoglobin on opossum sphincter of Oddi motor function in vivo and in vitro.
Nitric oxide (NO) acts as a nonadrenergic, noncholinergic inhibitor neurotransmitter that regulates sphincter of Oddi (SO) motor function. Hemoglobin blocks NO activity by binding it after it is synthesized. We hypothesized that recombinant human hemoglobin (rHb1.1) affects SO motor function by scavenging NO. Under anesthesia, 12 opossums underwent biliary tract manometry. Following a stabilization period, six animals were given rHb1.1 (0.28 g/kg over 30 min), while six received bovine albumin (0.28 g/kg over 30 min). Recordings were made during the infusion and for 3 hr after the infusion. In an in vitro preparation, force transducers were used to record spontaneous contractions at two sites along the sphincter segment. After a control period, rHb1.1 (0.1 mM) or cyanomethemoglobin (0.1 mM) was added to the tissue bath and recordings continued for another 2 hr. Recombinant human hemoglobin decreased the frequency of contractions, increased resting tone, and blocked the relaxation phase of contraction in vivo. It increased the baseline amplitude, the frequency, and the peak amplitudes of contractions in vitro. Albumin or cyanomethoglobin, which are unable to bind NO, had little effect on SO motor activity. We conclude that rHb1.1 may alter SO motor function by binding endogenous NO.
Analysis of Variance↗
Effects of prostaglandins E2 and F2 alpha on electromyogram of cat colon in vitro.
Prostaglandins cause diarrhea, and their production by the gut increases in diarrheal states. We studied the effects of PGF2 alpha and PGE2 on the electromyogram recorded from the cat colon in vitro to determine if these prostaglandins might produce electromyographic changes similar to those seen in diarrheal states. PGF2 alpha decreased slow wave frequency and uncoupled slow wave propagation in the proximal colon. It increased the frequency of migrating spike bursts. PGE2 had no effect on slow waves, but increased the frequency of the migrating spike burst. PGF2 alpha produced electromyographic changes similar to those recorded from the colon of cats with spontaneous diarrhea or after exposure to diarrhea-producing agents such as ricinoleate or quinidine. Some diarrhea-producing agents are likely to act by increasing prostaglandin production.
Animals↗
Antioxidant enzymes in intramural nerves of the opossum esophagus.
Superoxide radical (O2-.) combines with nitric oxide (NO) to form peroxynitrite, thereby nullifying the biological activity of NO. Superoxide dismutase (SOD) prevents this reaction by converting O2-. to H2O2. We tested the hypotheses that the antioxidant enzymes catalase (CAT), Mn SOD, and Cu/Zn SOD are present in enteric neurons of the opossum esophagus, and that O2-. alters esophageal motor function. Immunostaining demonstrated CAT, Mn SOD, and Cu/Zn SOD immunoreactivity in interganglionic nerve bundles and ganglia of the myenteric and submucosal plexuses. Western blot analysis confirmed the presence of these enzymes in homogenates of esophageal muscularis propria, and enzyme assays demonstrated Cu/Zn SOD and Mn SOD activities of 262 and 73 U/mg protein, respectively. Both diethyldithiocarbamic acid, an inhibitor of Cu/Zn SOD, and xanthine (X) with xanthine oxidase (XO), which generate O2-., shortened the latency of the nerve-mediated contraction of circular esophageal muscle, the off response, by 20.2 and 23.4%, respectively. SOD alone did not affect the latency, but it inhibited the effect of X with XO on the latency. Antioxidant enzymes found in intramural esophageal nerves may play a role in regulating NO-mediated neuromuscular communication in the esophagus.
Animals↗
Nongenomic effects of progesterone on human intestinal smooth muscle cells.
Previous experiments demonstrated that progesterone affects intestinal smooth muscle cells through genomic and nongenomic pathways. We hypothesized that the nongenomic effect was mediated by changes in membrane excitability. We studied the effects of progesterone and other steroid hormones on a human intestinal smooth muscle cell line, using the whole cell patch-clamp technique. Ionic currents were elicited through steps from -70 mV to various test potentials. Progesterone dose-dependently reduced calcium currents. The decrease in inward current was partly due to a shift in the steady-state inactivation to more hyperpolarized potentials. This effect did not involve gene transcription, since it was not blocked by the progesterone antagonist ZK-98-299. The progesterone analogue 5-beta-dihydroprogesterone also decreased calcium currents, whereas its stereoisomer, 5-alpha- dihydroprogesterone, did not affect the properties of voltage-sensitive ion channels. Similarly, estradiol and dexamethasone did not alter inward currents. We conclude that progestins exert their nongenomic effects on intestinal smooth muscle cells by decreasing calcium currents. The change in the calcium signal may contribute to the reduction in muscle contraction observed after progesterone.
Cells, Cultured↗
Effect of nitroblue tetrazolium on NO synthase and motor function of opossum esophagus.
Nitric oxide mediates neuromuscular events in the opossum esophagus. The NADPH diaphorase stain is used to localize nitric oxide synthase-containing enteric neurons. Cells stain by the NADPH diaphorase technique because they reduce nitroblue tetrazolium to the visible formazan. The effects of nitroblue tetrazolium on neuromuscular function and nitric oxide synthase of esophageal muscle were studied. The NADPH diaphorase stain was performed. Nitroblue tetrazolium inhibited lower esophageal sphincter relaxation, abolished the latency gradient of the off response, and inhibited nitric oxide synthase. The NADPH diaphorase technique stained myenteric plexus nerve cell bodies and nerve processes. Nitroblue tetrazolium is not a nonspecific muscle or nerve toxin, as nerve-mediated cholinergic responses, responses to exogenous nitric oxide, and responses to myogenic stimulation were maintained after nitroblue tetrazolium abolished the off response and lower esophageal sphincter relaxation. Nitroblue tetrazolium inhibits nitric oxide-mediated events and nitric oxide synthase. It stains neurons in the esophageal myenteric plexus.
Animals↗
The effects of recombinant human hemoglobin on esophageal motor functions in humans.
BACKGROUND & AIMS: Nitric oxide controls lower esophageal sphincter (LES) relaxation and esophageal peristalsis in opossums, but its role in the control of esophageal motility in humans is not defined. Hemoglobin inactivates NO by binding it. Recombinant human hemoglobin (rHb1.1) was used to test the hypothesis that NO mediates esophageal motor functions in humans. METHODS: rHb1.1 or human serum albumin was administered intravenously to fasting male volunteers. Esophageal manometric studies were performed before, during, and up to 6 hours after the infusion. RESULTS: rHb1.1 increased the velocities of peristaltic contractions to produce simultaneous contractions in 6 of 9 subjects. It increased the amplitude and duration of contractile waves in the esophagus. There was no consistent effect on the resting tone of the LES, but LES relaxation was inhibited. Spontaneous, simultaneous high-pressure contractions occurred in 8 of 9 subjects. Lower retrosternal chest pain during swallowing was observed in 4 subjects. CONCLUSIONS: rHb1.1 interfered with esophageal peristalsis and LES relaxation. It precipitated esophageal spasm in some subjects. These data support the hypothesis that the timing of smooth muscle esophageal peristalsis and LES relaxation are mediated by NO. They suggest that some disorders of esophageal motor function may result from defects in NO neuromuscular communication.
Adult↗