Possible differences in the abilities of esophageal body and lower esophageal sphincter muscles to utilize substrates to support agonist-induced contractions ...
Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991), pp. 1057-1065
Paradoxical Relationship Between Substrates and Agonist-Induced Contractions of Opossum Esophageal Body and Sphincter In Vitro W.H. PERCY, PhD, J. SUTHERLAND, DO, and J. CHRISTENSEN, MD
Possible differences in the abilities of esophageal body and lower esophageal sphincter muscles to utilize substrates to support agonist-induced contractions were studied. Strips o f longitudinal, circular, and lower esophageal sphincter muscle from the opossum esophagus were first contracted to approximately 70% of the maximal contraction elicited by acetylcholine, histamine, or substance P. The tissues were then exhausted by exposure to 5 x 1 0 - 4 M carbachol and a 15% 02-5% C02-80% N2 gas mixture for 90 min. They were next reequilibrated with one of a number of alternative substrates and 95% 02-5% CO 2 for 3 hr. Responses to the initial agonist doses were again noted and compared to controls. The alternative substrates were : 2-deoxyglucose, glucose, fructose 1-6 diphosphate, pyruvate, lactate, acetate, butyrate, caprylate, histidine, leucine, aspartate, alanine, succinate, acetoacetone, and [3-hydroxybutyrate. Tile results obtained show qualitative differences in the ability of the three muscle types to use these substrates. More importantly, however, the ability o f any one substrate to support contractions was a function o f the agonist used to stimulate the muscle. The evidence suggests, therefore, that not all pharmacologic receptors have equal access to intracellular energy sources. KEY WORDS: acetylcholine; histamine; substance P; receptors; metabolic compartments.
The motor function of the smooth muscle part of the esophagus differs remarkably from that of other parts of the gut (1). The characteristics of the three muscle layers comprising the distal esophagus have been investigated extensively using the North American opossum (Didelphis virginiana), the only
Manuscript received November 24, 1989; revised manuscript received September 10, 1990; accepted October 1, 1990. From the Gastroenterology Research Laboratories, Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242. This work was supported by Research Grant AM 11242 from the National Institutes of Health. Address for reprint requests: W.H. Percy, Division of Gastroenterology, Winthrop-University Hospital, 259 First Street, Mineola, New York 11501.
readily available mammal having a smooth-muscled esophagus like that of primates. The differences in the operations of these three layers largely have been interpreted as representing differences in their innervation. The present study was based on the premise that differences could also exist among these layers in the properties of the smooth muscles themselves, specifically in respect to their biochemical profiles. This idea arose because certain metabolic differences already have been suggested by studies comparing the smooth muscles of the lower esophageal sphincter (LES) and the esophageal body. Included in these differences are the following: oxygen consumption by the sphincter muscle is higher than is
Digestive Diseases and Sciences. Vol. 36, No. 8 (August 1991)
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PERCY ET AL that by the esophageal body under similar degrees of stretch (2); L E S muscle contains the lactate dehydrogenase type I isozyme, which is associated with aerobic metabolism, while the muscle of the esophageal body does not (3); c y t o c h r o m e c oxidase activity in the L E S is less than it is in the esophageal body (4); and there are both quantitative and qualitative morphologic differences between the mitochondria o f L E S cells and those of the esophageal body (5). Although many intestinal smooth muscle preparations can use substrates other than glucose to maintain tone and to contract in vitro (6-14), there are considerable differences in their respective abilities to do so. In the rabbit small intestine, for example, glucose deprivation results in a reduction or loss of responses of the longitudinal muscle to acetylcholine and muscarine, while the responses of the circular muscle are resistant to this procedure. Pyruvate is a good substrate for acetylcholineinduced contractions of the longitudinal muscle but is relatively ineffective in circular muscle; lactate, in contrast, is better utilized by the circular layer than by the longitudinal (6). In guinea pig taenia coli, the energy used to produce a K+-induced rapid phasic contraction is different from that required to sustain the ensuing tonic contraction (15). The aim o f the present study, therefore, was to study the longitudinal and circular muscle layers of the esophagus and that of the lower esophageal sphincter with regard to their respective abilities to utilize substrates other than glucose to support agonist-induced contractions. The substrates chosen for this study were representatives o f a number of groups of compounds that have the potential to be used in energy-producing metabolic pathways. These groups were glycolysis intermediates, tricarboxylic acid cycle intermediates, fatty acids, amino acids and ketone bodies. The alternative substrates were made available when the different muscle types had undergone a novel metabolic exhaustion procedure. In addition, a number of these experiments were repeated in the presence of cyanide in an attempt to establish the metabolic fate of the alternative substrates. MATERIALS AND METHODS Preparation of Muscle Strips. Adult opossums of either sex, weighing 3-5 kg, were anesthetized by the intraperitoneal injection of sodium pentobarbital 30 mg/kg. The distal 5 cm of esophagus, the esophagogastric junction, and 1-2 cm of the proximal stomach were removed en
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bloc. The preparation was opened along the line of the lesser curvature of the stomach. The entire preparation then was pinned out flat, mucosal surface uppermost, in a bath containing a Krebs solution of the following composition (raM): NaC1 118.5, KCI 4.75, CaCI2 2.54, NaH2PO 4 1.19, MgSO 4 1.19, NaHCO3 25, glucose 11, gassed with 95% 02 aiad 5% CO2 at 37 • 1~ C. The lower esophageal sphincter (LES) was identified grossly as a band of tonically contracted circular muscle at the level of the mucosal junction. The mucosa was removed and muscle strips approximately 1 cm x 2 mm were cut along the longitudinal and transverse axes of the esophageal body and transversely from the LES. These three different types of muscle strips were transferred to a 1.5-ml superfusion chamber (16) in which their tension could be continuously recorded in a flowing stream of Krebs solution at 37 - 0.5~ which was, for control experiments, equilibrated with a 95% 02 and 5% CO: mixture. The flow rate through each chamber was maintained at approximately 3 ml/minute by means of a Holter peristaltic pump (Extracorporeal Medical Specialities, King of Prussia, Pennsylvania). Each strip was secured at one end and connected at the other to a Grass FTO3C force-displacement transducer. Responses were displayed on a Beckman RM Dynograph. Tension equivalent to a 2-g load (19.6 mN) was applied to each strip and an equilibration period of at least 30 min allowed. This tension has been demonstrated previously to stretch these muscle strips close to 150% of their resting length, which corresponds to approximately Lo, the optimal length for the generation of active tension (17). Individual muscle types were identified by their responses to electrical field stimulation of intrinsic nerves (10Hz, 1 msec, 120 mA, 10 sec) via platinum electrodes mounted inside the chamber (16). Each muscle type exhibits a characteristic response to electrical field stimulation that was used to confirm the identity of each strip. Sphincter strips exhibit a myogenic tone upon stretch and relaxation with field stimulation. Longitudinal strips contract during the stimulus train (the duration response) whereas transverse strips contract once at the end of each stimulus train to give a slow twitch (the off response) (17, 18). Strips showing responses corresponding to more than one muscle type were discarded; these were few. Control Responses to Agonists. Control doses of acetylcholine, histamine, and substance P that had been previously determined to produce approximately a 70% maximal contraction of each muscle type (Figure 1) were injected in a random sequence into the superfusion stream. To ensure that approximately a 70% contraction was achieved, the control contraction to each agonist was measured against a maximum contraction to that agonist each day. In order to avoid the effects of differential diffusion, doses of agonists were added to the superfusion stream in a bolus of a constant volume (100 ixl). The ability of esophageal body and LES muscle to give consistent responses to agonists administered in this fashion and over this time course has been reported previously (16). Seventy percent maximal responses were chosen for the controls so that potentiations of responses would not be obscured during the subsequent additions of agonists. For any one muscle type, there was little Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991)
SUBSTRATES AND ESOPHAGEAL MUSCLES
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Fig 1. Control log-dose-response curves for acetylcholine, histamine, and substance P on each of longitudinal (O), circular (B), and lower esophageal sphincter (0) muscles. Agonists were added to the superfusion stream in a constant volume. Doses causing approximately 70% maximal responses were based on these curves. Note that these doses vary between agonists and between muscle types. Points shown are the mean • SE of at least seven observations.
difference in the respective amplitudes of the maximum contractions to the three agonists under study. Procedure for Exhaustion of Tissues. The technique employed to exhaust the tissues and then to provide an alternative substrate was a modification of the techniques of Coe et al (19) and Percy et al (12), based on observations by Feldberg and Solandt (6) regarding the optimum conditions for tissue exhaustion. After control responses to each agonist in a normal (11 mM) glucose environment were obtained, the superfusion solution was replaced with a glucose-free Krebs solution containing 5 • 10 -5 M carbachol and gassed with a 15% 0 2 - 8 0 % N 2 - 5 % C O 2 mixture. Carbachol was chosen for this procedure because of its stability in solution when compared to such other agonists as acetylcholine or histamine. Decreasing the available oxygen by 80% does not by itself damage the tissue (20) and was employed to ensure that both aerobic and anaerobic pathways had the potential to be utilized during the carbachol-induced contraction. Throughout the following 90 min, the sustained maximal contraction produced by carbachol declined to zero. At this time the tissue was refractory to any of acetylcholine, histamine, or substance P at their control doses. No
responses could be elicited subsequently to any agonist by reintroduction of 95% 02 and 5% CO2 alone. The Krebs--carbachol superfusion then was replaced with a carbachol- and glucose-free Krebs containing one of the alternative substrates at a concentration of 11 mM and gassed with 95% 0 2 and 5% CO2. The pH of all solutions ranged from 7.2 to 7.5. In the case of acetate, butyrate, and succinate, this was achieved by the addition of 1 N NaOH (12). The concentration chosen for the alternative substrates (11 mM) was based on the concentration of glucose in the control Krebs solution. This allowed all substrates to be compared on an equimolar basis. Tissues were equilibrated under these conditions for a further 180 min when the responses to the initial doses of acetylcholine, histamine, and substance P were noted again and expressed as a percentage of the control 70% response. A schematic representation of this protocol is shown in Figure 2. Only the peak contraction amplitudes were compared. There was no obvious difference in the rates at which these peaks were achieved relative to controls following incubation with any substrate. In preliminary experiments with glucose, it was determined that optimum recovery from substrate depletion was achieved within 180 min. Similar observations have been made using the distal colonic muscularis mucosae of
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Schematic representation of tissue exhaustion and substrate substitution protocol. The peak amplitude of the final agonist-induced contraction was compared to and expressed as a percentage of the control 70% response. Digestive Diseases and Sciences, Vol. 36, No. 8 (August 1991)
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PERCY ET AL the opossum (12). This time, therefore, was used as the end point for experiments with other substrates. This long equilibration time also was employed in an attempt to minimize differences between substrates due to the rates at which they are transported across cell or mitochondrial membranes or become incorporated into different biochemical pathways. The importance of these factors has been recognized previously (21). In a second series of experiments, one representative from each substrate group was chosen, but the final superfusion medium contained not only 11 mM substrate but also NaCN (10 -3 M), a concentration previously shown to depress the active tension or evoked responses of LES and esophageal body strips, respectively (20). Where appropriate, statistical analysis of results was carried out using a non-paired Student's t test. The number of observations in each group ranged from 5 to 16. The 15 substrates used in this study were: the glucose analog 2-deoxyglucose; the glycolysis intermediates glucose, fructose 1-6 diphosphate (trisodium salt), pyruvic acid (sodium salt), and lactic acid; the fatty acids acetic acid (CH3CH2COOH), butyric acid (CH3(CH2)3COOH), and caprylic acid (CH3(CH2)7COOH)-sodium salt; the amino acids: /-histidine (free base), l-leucine, /-aspartic acid, and /-alanine; the tricarboxylic acid intermediate succinic acid-sodium salt and the ketone bodies acetoacetone (ethyl ester) and 13-hydroxybutyrate-sodium salt. Drugs. The drugs used in these experiments were: acetylcholine chloride (Sigma, St. Louis, Missouri) carbamylcholine chloride (carbachoI) (Sigma), histamine acid phosphate (British Drug Houses, Poole, UK), sodium cyanide (Aldrich Chemical Co. Milwaukee, Wisconsin), and substance P (Sigma). RESULTS
Dose-Response Curves. Figure 1 shows control log-dose-response curves for each o f acetylcholine, histamine, and substance P on longitudinal, circular, and L E S muscle. The doses producing 70% maximal contractions were based on these curves. Effects of Alternative Substrates. No alternative substrate was found to cause either a contractile or a relaxation response in any muscle layer upon being introduced into the superfusion medium. Only in the case of 2-deoxyglucose were no responses achieved for any muscle layer with any agonist. The recovery of each type o f tissue following incubation with the remaining alternative substrates in shown in Figures 3-5. Recoveries of contractile response are expressed as a percentage o f the control 70% response to the individual agonists. E a c h figure shows the response to one agonist on each of longitudinal muscle (upper), circular muscle (center), and L E S muscle (lower). In no case was there a consistent change in the nature o f the response to each agonist with the
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