Joseph A. Murray

The effects of recombinant human hemoglobin on esophageal motor function in humans

Abstract 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.

Celiac disease and recurrent pancreatitis

BACKGROUNDnCeliac disease is associated with pancreatico-biliary disease. Postulated mechanisms include reduced gallbladder emptying due to impaired cholecystokinin release and pancreatitis due to malnutrition. We hypothesize that celiac disease may also be associated with pancreatico-biliary abnormalities due to duodenal inflammation and papillary stenosis.nnnMETHODSnOver a 48-month period, 169 patients referred for possible sphincter of Oddi dysfunction who underwent pancreatico-biliary manometry were tested for gliadin and endomysial antibodies. Duodenal and papillary biopsies were preformed in those patients who were positive.nnnRESULTSnCeliac disease was diagnosed in 12 (7.1%; 3 men, 9 women). The mean age was 61 years as compared with 37 years for those patients without celiac disease. All of the celiac patients had been referred for recurrent abdominal pain and/or idiopathic pancreatitis. Ten had idiopathic recurrent pancreatitis with elevated amylase and lipase levels. Two of these patients also had mildly elevated liver function tests associated with the abdominal pain. Only 3 of 12 patients had a prior diagnosis of celiac disease. These 12 patients had manometric evidence of stenosis and histologic evidence of periampullary inflammation as well as histologic changes consistent with celiac disease. In 10 of 12 patients sphincterotomy or extension of a prior papillotomy was performed. Two patients were treated with a gluten-free diet alone.nnnCONCLUSIONSnWe describe 12 patients with papillary stenosis and celiac disease. In 9 cases the celiac disease was a new diagnosis. Celiac disease should be considered in the etiology of papillary stenosis or idiopathic recurrent pancreatitis.

Characterization and mediation of inhibitory junction potentials from opossum lower esophageal sphincter

BACKGROUNDnActivating nonadrenergic, noncholinergic (NANC) nerves of the lower esophageal sphincter (LES) hyperpolarizes and relaxes its circular smooth muscle. This relaxation is mediated by nitric oxide (NO) or an NO-containing compound. These studies were undertaken to compare the electrophysiological responses of circular smooth muscle from the LES and esophagus in response to NANC nerve stimulation and to test the hypothesis that NO mediates LES hyperpolarization.nnnMETHODSnThe transmembrane potential difference was recorded with glass microelectrodes. Nerve-mediated membrane responses were evoked by electrical pulses of 0.5 msec duration and 50 V amplitude.nnnRESULTSnResponses of LES muscle differed from those of the esophageal muscle. The duration of hyperpolarization was much longer in sphincteric muscle. The depolarization that followed hyperpolarization of esophageal muscle was not observed in sphincteric muscle. NG-nitro-L-arginine, an inhibitor of NO synthase, attenuated the nerve-induced hyperpolarization. L-arginine, the substrate for NO synthase, antagonized the effect of NG-nitro-L-arginine. Exogenous NO hyperpolarized of the smooth muscle membrane.nnnCONCLUSIONSnThese data support the hypothesis that NO or an NO-like compound may mediate nerve-induced hyperpolarization of the opossum LES.

Characterization of nitric oxide synthase in the opossum esophagus

BACKGROUND/AIMSnNitric oxide mediates the nonadrenergic, noncholinergic neural control of esophageal motor function. The purpose of this study was to characterize the NO synthase found in the muscularis propria of the opossum esophagus and determine its distribution along the esophagus.nnnMETHODSnEsophageal muscle was homogenized in HEPES buffer and ultracentrifuged. The supernatant was exposed to [3H]L-arginine. The [3H]L-citrulline produced by NO synthase was separated from [3H]L-arginine with a Dowex AG 50 W-X8 column (Biorad, Hercules, CA). Assays were performed in the presence and absence of Ca2+ or reduced nicotinamide adenine dinucleotide phosphate (NADPH). The distribution of NO synthase activity along the esophagus was determined.nnnRESULTSnThe apparent Michaelis constant and maximum velocity of NO synthase were 7.5 +/- 1.4 mumol/L L-arginine and 76.0 +/- 17.3 pmol.mg protein-1.min-1, respectively. The enzyme required both Ca2+ and NADPH for activity. Smooth muscle tissue from the lower esophageal sphincter and the esophageal body 1-2 cm or 5-6 cm above the lower esophageal sphincter differed little in enzymatic activity, ranging from 0.97 to 1.27 pmol.mg wet wt-1.min-1. Striated muscle had less activity with 0.40 pmol.mg wet wt-1.min-1.nnnCONCLUSIONSnThese data indicate the presence of a constitutive NO synthase in the esophagus of the opossum.

Nerve-mediated nitric oxide production by opossum lower esophageal sphincter

Antagonists of nitric oxide synthesis inhibit nerve-induced hyperpolarization and relaxation of muscle from the opossum lower esophageal sphincter. These studies test the hypothesis that nitric oxide is released during stimulation of intrinsic esophageal nerves. The intrinsic nerves were stimulated with an electrical field (10-sec trains of 1-msec, 30-V pulses delivered at 10 Hz). Nitric oxide production was measured with a DASIBI model 2108 Chemiluminescence NO Analyzer.NG-Nitro-l-arginine, an inhibitor of NO synthase, antagonized nerve-induced relaxation the lower esophageal sphincter. Nerve stimulation increased NO production from 0.50±0.04 nmol/min/100 mg tissue to 0.87±0.07 nmol/min/100 mg tissue (P<0.01).NG-nitro-l-arginine inhibited both basal (0.030±0.09 nmol/min/100 mg tissue,P<0.05 vs baseline) and stimulated (0.38±0.10 nmol/min/100 mg tissue,P<0.01 vs stimulated). These studies support the hypothesis that nerve stimulation releases nitric oxide from the lower esophageal sphincter.

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 anin 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 cyanomethemologbin (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 contractionin vivo. It increased the baseline amplitude, the frequency, and the peak amplitudes of contractionsin 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.

Roles of Triton X-100 in NADPH-diaphorase histochemistry

Triton X-100 is widely but not universally used in NADPH-diaphorase histochemical staining. We investigated its effect on the staining and examined nitroblue diformazan (NBF) production under the influence of Triton X-100. Exposure of opossum esophagus, intestine, and colon tissues to Triton X-100 before staining enhanced staining of nerve cells and fibers and suppressed staining of non-neural structures. Long exposures and high concentrations nearly abolished the staining of non-neural structures and decreased the staining of nerves. The use of an incubation medium containing Triton X-100 achieved the best staining of nerve cells and fibers. Addition of Triton X-100 to the incubation medium changed its color from yellow to purple; in the presence of tissues, this color change occurred much more quickly. Spectral analysis showed that Triton X-100 increases the rate of NBF formation in the presence of tissue supernatant. Triton X-100 increases it less in the absence of tissue supernatant. Therefore, Triton X-100 improves the histochemical staining, probably by catalyzing the activity of NADPH-diaphorase, by keeping the extracellular NBF in solution and thus suppressing the staining of non-neural structures, and by increasing the permeability of cell membranes.

Vasoactive intestinal contractor: Localization in the opossum esophagus and effects on motor functions

BACKGROUND/AIMSnVasoactive intestinal contractor (VIC), an endothelinlike peptide and a putative gastrointestinal hormone, contracts gastrointestinal smooth muscle. The aim was to study VIC in relation to esophageal function.nnnMETHODSnIntramural nerves in opossum esophageal smooth muscle strips were stimulated in the presence of various concentrations of VIC and were stained for VIC immunoreactivity.nnnRESULTSnVIC caused an atropine-resistant increase in the amplitude of nerve-induced contractions of the circular muscle. VIC alone contracted longitudinal muscle, and this effect was nearly eliminated by 1 mmol/L atropine. VIC caused an atropine-resistant increase in the resting tone of the lower esophageal sphincter muscle, but it did not affect nerve-induced relaxation of that muscle. VIC-immunoreactive nerve fibers occurred in the longitudinal muscle layer, in the muscularis mucosae, and around the ducts of esophageal glands. A few such fibers were found in the circular muscle layer. Nerve fibers and cell bodies of the myenteric plexus showed VIC immunoreactivity. In the stomach, immunoreactive nerve fibers occurred in muscularis mucosae and circular muscle but not in longitudinal muscle.nnnCONCLUSIONSnVIC is localized in neuronal elements of the opossum esophagus and excites contractions in esophageal smooth muscle.

Effects of Oxygen Radicals and Radical Scavenging on Opossum Lower Esophageal Sphincter

Nitric oxide mediates esophageal peristalsis andlower esophageal sphincter (LES) relaxation. Superoxideproduced with inflammation inactivates nitric oxide.Superoxide is cleared in biological systems by superoxide dismutase. We tested thehypothesis that superoxide and the superoxide scavengingsystem modulate LES function. Transverse strips ofmuscle from the opossum LES relaxed when stimulated by an electrical field. Diethyldithiocarbamite wasused to inhibit copper/zinc superoxide dismutase.Xanthine and xanthine oxidase were used to generatesuperoxide. Xanthine with xanthine oxidase ordiethyldithiocarbamite alone had no effect on the LES. However,xanthine/xanthine oxidase and diethyldithiocarbamitereduced LES relaxation 34.1% and increased its restingtone 71.2%. Superoxide dismutase did not affect LESfunction, but protected the tissue from the effects ofdiethyldithiocarbamite and xanthine/xanthine oxidase.These studies are consistent with the hypothesis thatsuperoxide acts by inactivating nitric oxide and suggest that these antioxidant enzyme systemsmay play a role in the maintenance of LESfunction.

Acute and chronic diarrhea. How to keep laboratory testing to a minimum.

Preview Whether the cause of diarrhea is relatively common (eg, contaminated water) or more rare (eg, carcinoid syndrome), the result is disconcerting. Since the list of tests available to aid in diagnosis is almost endless, physicians need to follow a systematic plan to avoid enormous laboratory charges. Drs Talal and Murray offer a pragmatic approach to differential diagnosis that is suited to the primary care setting.