Mansoor I. Tiwana

Pharmacologic Specificity of Nicotinic Receptor-Mediated Relaxation of Muscarinic Receptor Precontracted Human Gastric Clasp and Sling Muscle Fibers within the Gastroesophageal Junction

Relaxation of gastric clasp and sling muscle fibers is involved the transient lower esophageal sphincter relaxations underlying the pathophysiology of gastroesophageal reflux disease (GERD). These fibers do not contribute tone to the high-pressure zone in GERD patients, indicating their role in pathophysiology. This study identifies some mediators of the nicotine-induced relaxation of muscarinic receptor precontracted gastric clasp and sling fibers. Muscle strips from organ donors precontracted with bethanechol were relaxed with nicotine and then rechallenged after washing and adding inhibitors tetrodotoxin (TTX), the nitric-oxide synthase inhibitor l-nitro-arginine methyl ester (l-NAME), the β-adrenoceptor antagonist propranolol, the glycine receptor antagonist strychnine or ginkgolide B, and the GABAA receptor antagonist bicuculline or 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide [(gabazine) SR95531]. TTX only inhibited clasp fiber relaxations. l-NAME and propranolol inhibited, and ginkgolide B was ineffective in both. SR95531 was ineffective in clasp fibers and partially effective in sling fibers. Strychnine and bicuculline prevented relaxations with low potency, indicating actions not on glycine or GABAA receptors but more consistent with nicotinic receptor blockade. Bethanechol-precontracted fibers were relaxed by the nitric oxide donor S-nitroso-N-acetyl-dl-penicillamine and by the β-adrenergic agonist isoproterenol (clasp fibers only) but not by the glycine receptor agonist taurine or glycine or the GABAA agonist muscimol. These data indicate that nicotinic receptor activation mediates relaxation via release of nitric oxide in clasp and sling fibers, norepinephrine acting on β-adrenoceptors in clasp fibers, and GABA acting on GABAA receptors in sling fibers. Agents that selectively prevent these relaxations may be useful in the treatment of GERD.

Quantitation of the contractile response mediated by two receptors: M2 and M3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle.

Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M2 and M3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M2 and M3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M2 and M3 receptors to contraction is presented. Using this new method of analysis, it was found that the M2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction.

Comparison of human and porcine gastric clasp and sling fiber contraction by M2 and M3 muscarinic receptors

To compare the gastroesophageal junction of the human with the pig, M(2) and M(3) receptor densities and the potencies of M(2) and M(3) muscarinic receptor subtype selective antagonists were determined in gastric clasp and sling smooth muscle fibers. Total muscarinic and M(2) receptors are higher in pig than human clasp and sling fibers. M(3) receptors are higher in human compared with pig sling fibers but lower in human compared with pig clasp fibers. Clasp fibers have fewer M(3) receptors than sling fibers in both humans and pigs. Similar to human clasp fibers, pig clasp fibers contract significantly less than pig sling fibers. Analysis of the methoctramine Schild plot suggests that M(2) receptors are involved in mediating contraction in pig clasp and sling fibers. Darifenacin potency suggests that M(3) receptors mediate contraction in pig sling fibers and that M(2) and M(3) receptors mediate contraction in pig clasp fibers. Taken together, the data suggest that both M(2) and M(3) muscarinic receptors mediate the contraction in both pig clasp and sling fibers similar to human clasp and sling fibers.

Minimally invasive measurement of esophageal variceal pressure and wall tension (with video)

BACKGROUND There is no simple method to measure intravariceal pressure in patients with esophageal varices. OBJECTIVE Our purpose was to develop a new noninvasive technique to measure resting intravariceal pressure and wall tension. DESIGN A model was developed. A long balloon (varix) was fitted inside an airtight cylinder (esophagus). Fluid ran through the model varices to maintain 5 different constant pressures. An endoscope was placed in the model esophagus, and pressure was increased by air insufflation. The endoscopy and pressure readings from the esophagus and varix were recorded continuously until variceal collapse. SETTING Patient studies were done in an endoscopy suite with the patient under fentanyl and midazolam sedation. PATIENTS Esophageal pressure was measured during air insufflation in patients with varices until the varices collapsed. EUS was used to measure radius and wall thickness to calculate wall tension. RESULTS In the varix model, the mean (SD) intraluminal esophageal pressures at variceal flattening for the model varices at 5, 10, 15, 20, and 25 mm Hg were 5.69 (0.34), 11 (0.32), 15.72 (0.51), 21.55 (0.63), and 25.8 (0.14) mm Hg. The correlation between actual and measured variceal pressure in the model at variceal flattening was r = 0.98. In the patients, a total of 10 varices in 3 patients were evaluated. The mean (SD) for the varices in each subject was 12.16 (2.4), 23.2 (1.3), and 6.5 (2.2) mm Hg for subjects 1, 2, and 3, respectively. CONCLUSION Standard endoscopy with air insufflation and manometry can be used as an accurate, simple, and reproducible method to measure intravariceal pressure.

T1686 Gastric Clasp and Lower Esophageal Circular Muscle Fibers From GERD Patients With Barrett's Esophagitis Have a Decreased Contractile Response to Cholinergic Stimulation

Background: We recently showed that the gastric sling/clasp muscle complex does not contribute to the high pressure zone in GERD patients indicating a role in pathophysiology. Aim: We determined the cholinergic contractile response in clasp, sling and lower esophageal circular (LEC) fibers. Methods: Stomach and esophagi were obtained from 17 human transplant donors: 11 with no GERD history, 4 with probable GERD (proton pump inhibitor use) and 2 with definite GERD (Barretts esophagitis). The contractile response to increasing carbachol concentrations was determined. Muscarinic receptor density was measured by subtype selective immunoprecipitation of [3]H-QNB binding. Results: Clasp and LEC fibers from definite GERD have decreased maximal contractile response. Contractility is increased in sling fibers of both definite and probable GERD. Concentrations of carbachol higher than 100 μM induce relaxations that are decreased in clasp fibers of probable and definite GERD. Relaxations are greater in sling fibers from definite GERD and higher in LEC from probable and definite GERD. Total and M2 receptor density is statistically lower in the GERD than non-GERD specimens in both clasp and sling fibers and M3 density is statistically lower in GERD than non-GERD clasp fibers. Conclusion: This suggest that a myogenic defect in clasp fibers may be involved in GERD pathophysiology in the organ donors with Barretts esophagitis. The greater contractile response in the sling fibers from GERD donors suggest a possible compensatory mechanism for the lack of contractility of the clasp muscle fibers.

T1238 Comparison of Muscarinic Receptor Subtypes Mediating Contraction of Human and Pig Gastric Clasp and Sling Fibers

This study determined how closely the contractile physiology of the pig gastroesophageal junction follows the human. We obtained human tissue from organ transplant donors and pig tissue from a slaughterhouse. Total, M-2 and M-3 receptor density was determined by subtype specific immunoprecipitation. Total and M-2 are higher in pig than human. M-3 receptors are 2 fold higher in human than pig sling and over 2 fold lower in human than pig clasp fibers. The methoctramine and darifenacin potency to inhibit bethanechol contractions, calculated by classic Schild analysis, indicates that both M-2 and M-3 receptors cause contraction which violates the assumption of one receptor causing the effect. An analysis method relating dual occupation of M-2 andM-3 receptors to the contractile response was developed based on the published Ka values of M-2 and M-3 receptors for bethanechol of 170 μM and 110 μM respectively, and mass-action binding which, at equilibrium, gives receptor occupation = [A][R] / ([A] + Ka), where [A] denotes the agonist concentration, [R] is the receptor concentration and Ka is the agonist dissociation constant (reciprocal of affinity). Three dimensional plots for M-2 and M-3 occupation and contractile response are shown in the figure. Although the M-3 receptor subtype density is different between human and pig, the physiology of the contractile response is similar. This indicates that the pig may be a good model for human gastroesophageal junction physiology. Muscarinic receptor density (fMol/mg solubile protein)