Eugene Clark

Effect of First Part of Duodenum on Gastric Emptying in Dogs: Response to Acid, Fat, Glucose, and Neural Blockade

Five dogs were prepared, each with a gastric and duodenal fistula (5 cm distal to the pylorus), to study the inhibitory role of the first 5 cm of the duodenum on gastric emptying. The basic design of the experiments was to instill the test meal (300 ml at 37 degrees C, containing phenol red 40 mg 1(-1)) into the stomach and collect it at 1- or 2-min intervals for 10 or 20 min from the duodenal fistula. As the test meal emptied from the stomach it bathed the first 5 cm of duodenum and thus stimulated the appropriate receptor. A Foley catheter with an inflated balloon prevented passage into the second part of the duodenum. Test meals of hypertonic glucose (15%, 865 milliosmoles kg-1) or 20 and 80 mM of sodium oleate emptied at the same rate as water when allowed to bathe the first 5 cm of duodenum, whereas test meals of 100 mM of HCl were slowed. In further studies using neural blocking agents, the emptying of water meals was slowed with subcutaneous atropine sulfate (0.03 and 0.15 mg kg-1), intravenous hexamethonium chloride (10 mg kg-1), and norepinephrine bitartrate (0.04 mg kg-1). The emptying of 100 to 120 mM HCl meals was slowed by subcutaneous atropine sulfate (0.03 and 0.15 mg kg-1), intravenous norepinephrine bitartrate (0.04 mg kg-1), and the intravenous alpha-receptor blocking agents phenoxybenzamine HCl (2 mg kg-1) and phentolamine (2 mg kg-1), was unaffected by intravenous hexamethonium chloide (10 mg kg-1), and was unchanged (1.0 mg kg-1) or slightly slowed (2.0 mg kg-1) by the beta-receptor blocker propranolol. In contrast, acid test meals were emptied at the same rate as water when treated with intravenous guanethidine monosulfate (2 mg kg-1) or intramuscular reserpine (1 mg kg-1), indicating that the acid inhibition was mediated by an adrenergic mechanism. The emptying of water meals was unchanged by these two drugs. The authors suggest that the first 5 cm of duodenum contain receptors for inhibition of emptying of acid but not for fat or hypertonic glucose. Furthermore, the neural blocking studies indicate that the inhibitory effect of acid in the first part of the duodenum is an adrenergic mechanism which appears to be neither alpha nor beta-receptor-mediated.

Characterization of nitric oxide synthase in the opossum esophagus

BACKGROUND/AIMS Nitric 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. METHODS Esophageal 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. RESULTS The 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. CONCLUSIONS These data indicate the presence of a constitutive NO synthase in the esophagus of the opossum.

Biphasic relaxation of the opossum lower esophageal sphincter: roles of NO., VIP, and CGRP.

Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO ⋅) are thought to mediate lower esophageal sphincter (LES) relaxation. Transverse muscle strips from the opossum LES were used to test this hypothesis. Electrical field stimulation (EFS) produced a biphasic LES relaxation: a rapid component during the stimulus was more prominent at lower stimulus frequencies, and a sustained component was more prominent at higher frequencies. N ω-nitro-l-arginine and hemoglobin inhibited the rapid component but affected the sustained component less. Exogenous VIP decreased LES tone. A number of purported VIP antagonists blocked neither VIP-induced nor EFS-induced relaxation of the LES. The calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) did not alter EFS-induced LES relaxation. EFS-induced relaxation of opossum LES muscle is biphasic, and the initial, rapid component of the relaxation is mediated primarily by NO ⋅. The mediator of the sustained component was not identified.Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO.) are thought to mediate lower esophageal sphincter (LES) relaxation. Transverse muscle strips from the opossum LES were used to test this hypothesis. Electrical field stimulation (EFS) produced a biphasic LES relaxation: a rapid component during the stimulus was more prominent at lower stimulus frequencies, and a sustained component was more prominent at higher frequencies. N(omega)-nitro-L-arginine and hemoglobin inhibited the rapid component but affected the sustained component less. Exogenous VIP decreased LES tone. A number of purported VIP antagonists blocked neither VIP-induced nor EFS-induced relaxation of the LES. The calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) did not alter EFS-induced LES relaxation. EFS-induced relaxation of opossum LES muscle is biphasic, and the initial, rapid component of the relaxation is mediated primarily by NO. The mediator of the sustained component was not identified.

Expression of inducible nitric oxide synthase in the lower esophageal sphincter of the endotoxemic opossum.

Background: Endotoxin modulates esophageal motor function by increasing nitric oxide (NO) production. The aims of this study were to examine inducible nitric oxide synthase (iNOS) induction in the lower esophageal sphincter (LES) of endotoxemic opossums and to investigate the effects of aminoguanidine (AG), a selective inhibitor of iNOS, on plasma nitrite/nitrate levels and on iNOS protein and mRNA expression after exposure to lipopolysaccharide (LPS). Methods: Before and 12 h after the intravenous administration of LPS and/or AG, plasma nitrite/nitrate levels were determined. The iNOS protein and mRNA expression were investigated in the tissues taken from the LES by Western blot and reverse-transcriptase polymerase chain reaction (RT-PCR). Results: Plasma nitrite/nitrate levels were significantly increased by LPS. The increase in plasma nitrite/nitrate produced by LPS was significantly decreased by AG. Western blot and RT-PCR demonstrated that iNOS expression was markedly increased by LPS, and attenuated slightly by AG. Conclusions: These studies support the hypothesis that endotoxin increases NO production by the induction of iNOS protein and mRNA.

Surface charge, fluidity, and calcium uptake by rat intestinal brush-border vesicles

Biological membrane outer surfaces are negatively charged and interact with positively charged calcium ion during calcium uptake. Positively charged polycations such as polyarginine bind to membranes with high affinity, displacing bound calcium from the membrane. We tested the effect of polyarginine on uptake of calcium by brush-border membrane vesicles and examined the responses in terms of membrane fluidity by electron paramagnetic resonance (EPR). Polyarginine inhibited the saturable component of calcium uptake by a mechanism combining inhibition characteristics of strontium (competitive) and magnesium (non-competitive). Unlike the inhibition of non-saturable calcium uptake by strontium and magnesium, polyarginine increased kD, the rate constant for non-saturable calcium uptake, by a concentration dependent mechanism. These effects of polyarginine on calcium uptake were associated with decreased membrane fluidity at the uptake temperature. These findings are consistent with a role for surface negative charge in determining both saturable and non-saturable calcium uptake. Increased membrane fluidity is associated with decreased saturable and increased non-saturable calcium uptake. Although increased fluidity might be involved in the increased kD for non-saturable uptake, the concentration-specific stimulating effect of polyarginine suggests a gating mechanism.

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.