HISTAMINE RECEPTORS IN ESOPHAGEAL SMOOTH MUSCLE OF THE OPOSSUM

Transcription

1 GASTROENTEROLOGY 70: , 1976 Copyright 1976 by The Williams & Wilkins Co. Vol. 70, No.6 Printed in U.S.A. HISTAMINE RECEPTORS IN ESOPHAGEAL SMOOTH MUSCLE OF THE OPOSSUM DAVID J. DE CARLE, M.B., B.S., M.R.A.C.P., MICHAEL J. BRODY, PH.D., AND JAMES CHRISTENSEN, M.D. Division of Gastroenterology, Department of Internal Medicine, University Hospitals, and Department of Pharmacology, University of Iowa, Iowa City, Iowa Esophageal smooth muscle was examined for histamine receptors. The effects of histamine, the histamine analogs 4-methylhistamine (4-MH) and 2-(2-pyridyl) ethylamine (PEA), the histamine receptor antagonists mepyramine and metiamide, and the histamine-releasing substance compound 48/80, on lower esophageal sphincter (LES) and esophageal body (EB) smooth muscle of the opossum were studied in a superfused tissue bath. Histamine, PEA, an HI receptor agonist, and compound 48/80 caused a dose-related increase in LES basal tension and in EB off response amplitude, the threshold for histamine being 6.7 x 10-8 M and that for PEA being 6.7 x 10-7 M. In the presence of mepyramine, an HI receptor antagonist, the effects of histamine and compound 48/80 were reversed to inhibition of both LES basal tension and EB off response amplitude, while the effect of PEA was abolished. Metiamide, an H2 receptor antagonist, did not alter responses to histamine, PEA, or compound 48/80. The H2 receptor agonist 4-methylhistamine caused a reduction in LES tension and EB off response amplitude, but caused an increase in those parameters in the presence of metiamide. A combination of mepyramine and metiamide abolished responses to all agonist drugs. The results indicate that LES and EB smooth muscle contain both excitatory HI and inhibitory H2 receptors for histamine. Endogenous histamine released from storage sites in LES and EB and exogenous histamine both preferentially activate HI receptors. Histamine, given intravenously in the monkey and in the Australian opossum, causes a fall in lower esophageal sphincter (LES) pressure. 1 The histamine analog betazole given subcutaneously in man causes a rise in LES pressure. 2 Histamine has been shown to act on two types of receptor, HI and H2 receptors, in a variety of preparations including vascular muscle, 3, 4 cardiac muscle,5 and uterine muscle. 6 H2 receptors alone are also involved in histamine-induced gastric acid secretion. 7, 8 In this study the presence and nature of histamine receptors in isolated strips of esophageal smooth muscle Received September 5, Accepted November 18, Address requests for reprints to: David J. de Carle, M.B., Division of Gastroenterology, Department of Internal Medicine, University Hospitals, Iowa City, Iowa Dr. de Carle is a recipient of the Winthrop Traveling Fellowship of the Royal Australasian College of Physicians. This research was otherwise supported by National Institutes of Health Research Grant AM Dr. C. R. Ganellin, Smith, Kline & French Laboratories, Ltd, Welwyn Garden City, England, generously provided the 4-methyl-histamine and the 2-(2-pyridyl)ethylamine dihydrochloride. Dr. R. Maxwell, Burroughs Wellcome Co., Research Triangle Park, N. C., generously provided the compound B. W. 48/80. Smith, Kline & French Laboratories, Philadelphia, Pa., generously provided the metiamide. of the North American opossum (Didelphis uirginiana) were determined by examining the effects of histamine and the HI and H2 receptor agonists, 2-(2-pyridyl) ethylamine (PEA) and 4-methylhistamine (4-MH), respectively, alone and in the presence of HI and H2 receptor antagonists. The histamine-releasing substance compound 48/80 was also used to look for the presence of stored histamine in esophageal smooth muscle, and to determine whether such endogenous histamine produces effects similar to those of administered histamine. Smooth muscle from esophageal body (EB) maintains very little active tension when stretched to 150% of initial length, and responds to electrical field stimulation by a brief contraction at the end of the stimulus train (the off response). 9 The inhibitory effects of drugs may only be seen during the development of active tension, and in this study repeated electrical field stimulation was used as a source of active tension. LES muscle maintains active tension when stretched to 150% of initial length and responds to electrical field stimulation by relaxing during the stirn ul us train. 9 In this study, inhibition during electrical field stimulation was used as measure of the capacity of LES muscle to relax. Thus, all studies were performed on EB and LES muscle simultaneously during regularly repeated electrical field stimulation. 1071

2 1072 DE CARLE ET AL. Vol. 70, No.6 Methods Twenty opossums of either sex, weighing between 2 kg and 5. 1 kg, were anesthetized with intraperitoneal sodium pentobarbital. The stomach and esophagus were removed en bloc and opened along the greater curve of the stomach. The mucosa was removed by sharp dissection. Transverse strips of muscle 1.5 by 0.2 cm were cut from the esophagogastric junction and from the esophagus 1.5 cm proximal to that strip. Silk threads (000) were tied to both ends of the strips and the strips were suspended in an organ bath. One end of each strip was fixed and the other end was attached to an isometric force-displacement transducer (Grass model IT.03) connected to a strip-chart recorder (Beckman type RM Dynograph). The order of strips in the bath was randomly varied and only one pair of strips from each animal was tested. The bath, which has been described in detail previously,' consists of a small plexiglass block with a central channel. At 3-cm intervals along the channel there are platinum rings connected to the exterior to allow regional field stimulation. Krebs solution, containing sodium mm, potassium 4.6 mm, calcium 2.5 mm, chloride mm, phosphate 1.2 mm, and bicarbonate 21.9 mm, and aerated with 95% O 2, 5% CO 2, was pumped through the bath from a constant temperature reservoir at 6.0 ml per min. Temperature was maintained at 33.5 to 34.5 C. The strips were stretched to 150% of their initial length and simultaneously field-stimulated with 2-sec trains of rectangular pulses of 50 v and 0.5 msec, at 10 Hz. As previously reported, these parameters are in the optimal range for excitation of the two responses sought. 9 The trains were repeated every 30 sec. Strips showing predominantly relaxation during the stimulus train were considered to consist of LES muscle. Strips from EB showed off responses only. Under control conditions, the responses remained constant over several hours. Serial dilutions of agonist drugs in Krebs solutions were made fresh daily. Ten-microliter amounts of these solutions were injected into the superfusion stream immediately proxi- mal to the organ bath. The same concentration was injected on several occasions intermittently to look for tachyphylaxis, and the sequence of concentrations was repeated after several hours to discover time-dependent changes in sensitivity. The concentrations given in the dose-response curves are the injected concentrations (as the base). The dilution occurring in the bath was determined by spectrophotometric analysis at 555 nm of phenol red injected in the same volume as the test drugs. Antagonist drugs were dissolved in Krebs solution and superfused from the reservoir. The drugs used were histamine dihydrochloride 10-6 M to 10-2 M (lcn-k & K Laboratories, Inc., Cleveland, Ohio), 4-methylhistamine 10-5 M to 10-2 M, 2-(2-pyridyl)ethylamine dihydrochloride 10-5 M to 10-2 M, metiamide 10-5 M, mepyramine 10-5 M (Pfaltz and Bauer, Inc., Stamford, Conn.), compound 48/ g per ml, and tetrodotoxin 10-7 M (Sigma Chemical Co., St. Louis, Mo.). Alteration in amplitude of the off response is calculated as the change in amplitude expressed as a percentage of the control amplitude. Inhibition of LES basal tension is expressed as a percentage of the maximal inhibition achieved by electrical field stimulation, assumed to represent complete relaxation. The basal tension maintained by individual LES strips varied between approximately 6 and 9 g. Thus, increase in tension is expressed as a percentage of basal tension rather than an absolute value. Results Histamine caused a rise in LES basal tension (fig. 1). This effect was dose-related, with a threshold of 10-5 M, whereas a maximum rise of 187% occurred at 5 x 10-3 M (fig. 2). This represents an increase in t ~, ",.e ~ approximately 15 g. Dilution curves using phenol red showed U 1 J that the maximum concentration achieved in the bath was 6.7 x 10-3 of the injected concentration, and that it was achieved 15 sec after injection. This gives a calculated threshold dose for histamine of 6.7 x 10-8 M and a maximal effect at a concentration of 3.3 x 10-5 M. Histamine also caused a dose-related rise in the amplitude of the offresponse (fig. 1). The threshold dose of histamine for this effect was 5 x 10-5 M, giving a calculated threshold concentration of 3.3 x 10-7 M. The maximal effect was 41.8% and occurred at 5 x 10-3 (fig. 2), giving a calculated maximal concentration of 3.3 x 10-5 M. Neither mepyramine, 10-5 M, nor metiamide, 10-5 M, caused a sustained or reproducible change in basal LES tension or in the amplitude of the off response. During infusion of mepyramine, 10-5 M, histamine caused a fall both in LES basal tension and in off response amplitude (fig. 1). These effects were dose-related, with threshold doses for both effects of 5 x 10-5 M and maximal effect at 12 gm [ Ogm J'---''--_''_-' g 16 m[ ; -\. -~, '--- ~' \ -" ' :-~ \ ~ i Ogm ' \. ~ t ". '" '" \i \I Histamine 10-3 M ~ MINUTE--', Sitmukis StQnol "r... {- ----,-- -r----,,---,---,--,----, 6 9 m : \[. ~.. OQffi, 1 r. 'L ~ i 1,1\. (' ~ I. ; ~,'--, ' \ L :'-- : '-'! ',i \ ", '... I I 1'1 \J \, I., \. J " ~. I Histamine 5.,10-3 M Stimulus Signal - - r , ' r r/ -- r~ \,_ r. _, FIG. 1. The responses of LESand EB muscle to histamine are shown before (A) and during infusion of mepyramine 10-5 M (B). Histamine caused an increase in both LES basal tension (lower tracing) and off response amplitude (upper tracing). During mepyramine infusion histamine caused a fall in both LES basal tension and off response amplitude. All parameters returned to preinjection levels within 5 min of injection. The LES tension transducer sensitivity was doubled in (B) to make the inhibition more obvious.

3 June 1976 ESOPHAGEAL HISTAMINE RECEPTORS X 10-3 M. The maximum inhibition of LES basal tension was 89.3%, while that of off response amplitude was 57.0% (fig. 3). Metiamide, 10-5 M, alone had no effect on the response to histamine. When combined with mepyramine, 10-5 M, however, it abolished all responses to histamine. The combination of mepyramine, metiamide, and histamine was tested on six pairs of strips. Tetrodotoxin 10-7 M abolished the responses to electrical field stimulation but did not alter either the excitatory or inhibitory effects of histamine on LES basal tension. The effect of histamine on EB in the presence of tetrodotoxin could not be assessed because the off responses were abolished. These effects were assessed in four pairs of strips. PEA caused a dose-related rise in LES basal tension with a threshold dose of 10-4 M and an increase of 123.4% at 10-2 M, the highest concentration tested. PEA also caused a rise in off response amplitude with a threshold of 10-4 M and a 46% increase at 10-2 M (fig. 4). Both these effects of PEA were abolished by mepyramine 10-5 M. No inhibitory effects were seen with PEA. 4-MH caused a fall in both LES basal tension and off response amplitude, with a threshold of 5 x 10-4 M and a maximal effect at 10-2 M. The maximum effect on LES basal tension was 25% and on the off response amplitude, 29%. Metiamide 10-5 M altered the response to 4-MH to a small dose-related rise in LES basal tension and off response amplitude. The effect of 4-MH before and after metiamide was tested in five pairs of strips. Compound 48/80, 10-4 g, when injected into the perfusion stream, caused a rise in both LES basal tension and off response amplitude. There was marked tachyphylaxis, so that a reproducible dose-response curve could not be obtained. The response to 10-4 gin 10 Ill, giving a final concentration in the bath of6.7 x 10-5 g per ml, was similar to the effect of 10-3 M histamine. Mepyramine 10-5 M altered response to compound 48/ '" 120 ~ 100 ~ ~ LESBosolTension --e Off-Response Amplitude 40 L _-r "' ~ 1 ~ ~ 5xl0 ~ ~ xl Molar Concentration of Injected Histamine FIG. 2. The effects of histamine on.les basal tension and off response amplitude are shown as dose-response curves. Each point represents the mean of six strips and SEM are shown as bars. Mean maximum increase in LES basal tension is 187%, whereas mean maximum increase in off response amplitude is 41.8%. g, o ~ -50 ~ LESBosoiTensiOfI.---. Off-Response Amplitude x-'-10"C;.-1..L ~ ~ ' 0.-; 'xl L 67". --'10:;-3--5-x.1..10:;- 3---'1O.2 Molar Concentration of Injected Histamine FIG. 3. The effects of histamine on LES basal tension and off response amplitude, during infusion of mepyramine 10-5 M, are shown as dose-response curves. Each point represents the mean of five strips and SEM are shown as bars. The mean maximum inhibition of LES basal tension is 89.3%, whereas mean maximum inhibition of off response amplitude is 57.0%. '" tj '" " ~ 60 ~ LES Basal Tension.. Off - Respor:s'] Amplitude O ~ ~ ~ ~ ~ ~ ~ ~ ~ Molar Concentration of Injected PEA FIG. 4. The effects of PEA on LES basal tension and off response amplitude are shown as dose-response curves. Each point represents the mean of five strips and SEM are shown as bars. The mean maximum increase in LES basal tension is 123%, whereas the mean maximum increase in off response amplitude is 46.%. to inhibition of both LES basal tension and off response amplitude (fig. 5). The effect of mepyramine was tested at least 30 min after the previous injection of compound 48/80, at which time tachyphylaxis was not apparent. The effect of mepyramine on the response to compound 48/80 was tested in four pairs of strips. With all agents except compound 48/80, tachyphylaxis was not seen and time-dependent changes in sensitivity of the strips were trivial. None of the agents except tetrodotoxin altered the inhibitory response of LES muscle to electrical field stimulation. Discussion The histamine analog betazole given subcutaneously to man causes a rise in LES pressure. 10 In the Australian opossum and the monkey, however, histamine given

4 _ ~ > O.. I 1074 DE CARLE ET AL. 12 g g m[,"--,,"--,,"--, 1"--,,r---,,r-/l h ('-, I'--t I'--t!'---, I 16 m[-, (\-, I ~,I\..., \., r.. _ :\_, ~ LILlI... Og J V V V V V V V V V V V V V 0 ~ V ~ \ J V ~ v, m ~ Vol. 70, No.6 I I ) i fi j i.,, " : ii,---j I ~ I ~ U I ~ I\---.J L J,1\, \ 'I" I I',! 12 g m[ I I.1 I I 1ft l I ~ j 12 g m[ A L ~ ~ Ogm Ogm ---' ~, - -, , - -, ~ ~ t t Compound 48/80 Compound 48/80 10"2gm/ml 10-2 gm/ml MINUTE: I N U T E ~ 1, FIG. 5. The responses of LES and EB smooth muscle to compound 48/80 before (A) and during infusion of mepyramine 10-5 M (B) are shown. Before mepyramine, compound 48/80 caused an increase in both LES basal tension (upper tracing) and EB off response amplitude (lower tracing). During mepyramine infusion c o m p 48/80 o u n caused ~ a fall in both LES basal tension and off response amplitude. intravenously causes a fall in LES pressure. 1 This discrepancy suggests that LES muscle may contain both excitatory and inhibitory histamine receptors. The differing results seen in man and other animals may be due to species-dependent differences, difference in the route of administration, or pharmacological differences between betazole and histamine. Recently H2 receptors which mediate inhibitory responses have been described in a variety of smooth muscle preparations. In some instances it has been necessary to block HI receptor effects of histamine before demonstrating H2 receptormediated inhibition. Histamine-stimulated gastric acid secretion is mediated through H2 receptors. 7, B The fact that gastrointestinal mucosa contains H2 receptors suggests that these receptors may also be present III gastrointestinal smooth muscle. The stimulation of LES and EB smooth muscle by histamine was abolished by mepyramine, an HI receptor antagonist,ll suggesting that this effect is mediated by HI receptors. PEA, reported to be a specific HI receptor agonist, 12 caused an increase in LES basal tension and off response amplitude, and this response was also abolished by mepyramine. This also suggests that esophageal smooth muscle layer contains HI receptors which are excitatory. In smooth muscle strips from LES, the response to histamine was not altered by tetrodotoxin in a concentration which abolished nerve-mediated responses. This suggests that the receptors are in the muscle itself rather than in the intramural nerves. In EB muscle the effect of tetrodotoxin on responses to histamine could not be assessed, so the exact site of the receptors could not be determined. By analogy with LES muscle it seems likely that they are in the muscle rather than the intramural nerves. The inhibition of LES basal tension and off response amplitude by histamine during mepyramine superfusion was blocked by metiamide, an H2 receptor antagonist. This suggests that the inhibitory response is mediated by H2 receptors. The direct inhibitory effect of 4-MH, a selective H2 receptor agonist,12 also suggests that the esophageal smooth muscle layer contains inhibitory H2 receptors. As with the excitatory receptors, it seems likely that these receptors are in the muscle. Compound 48/80 is known to release histamine from tissue mast cell stores, but its potency varies widely depending on the organ system studied, 13, 14 presumably because of variation in mast cell density. In this study, compound 48/80 had effects like those of exogenous histamine and its effects were antagonized by histamine receptor antagonists. It thus seems likely that compound 48/80 was releasing histamine from sites of storage and that the marked tachyphylaxis seen was due to depletion of those stores. It has been observed histologically, using toluidine blue staining, that LES and EB of the opossum exhibit numerous metachromatic mast cells. These probably serve as the source of histamine released by compound 48/80 (unpublished observations). If histamine acts on both HI and H2 receptors, it might be expected that antagonism of the inhibitory H2 receptor would augment the excitatory response seen in the absence of antagonists, but this was not the case in this study. It has been reported that in vascular smooth muscle, H2 receptor blockade alone does not alter the response to histamine. 4 This suggests that histamine has a high affinity for HI receptors, and may be explained on the basis of the molecular configuration of histamine. It has been suggested that tautomeric compounds such as 4-MH have a high affinity for H2 receptors,12 whereas nontautomeric compounds such as PEA have a high affinity for HI receptors. If histamine exists primarily in a nontautomeric hydrogen-bonded form, it would tend to interact strongly w.ith HI receptors. 4 This study has shown that the smooth muscle layer of the opossum EB and LES contain both excitatory HI and inhibitory H2 receptors. Both exogenously applied and endogenously released histamine appear to interact preferentially with HI receptors. Concentrations of mepyramine and metiamide which completely blocked responses to exogenous histamine did not alter the response of either LES or EB to electrical field stimulation. Thus it seems unlikely that endogenous histamine release plays a role in these responses. The physiological role of the receptors described remains unclear. REFERENCES l. de Carle, DJ, Glover, WE: Independence of gastrin and histamine receptors in the lower esophageal sphincter of the monkey and possum (abstr). J Physiol (Lond) 245:78P-79P, Castell, DO, Harris, LD: Hormonal control of gastroesophageal sphincter strength. N Engl J Med 282: , 1970

5 June 1976 ESOPHAGEAL HISTAMINE RECEPTORS Black, JW, Duncan, WAM, Durant, CV: Definition and antagonism of histamine H 2-receptors. Nature 236: , Powell, JR, Brody, MJ: Identification and specific blockade of two receptors for histamine in the cardiovascular system. J Pharmacol Exp Ther 196:1-14, Ercan, ZS, Bokesoy, TA, Turker, RK: A study of the histamine H2 receptors in heart muscle and coronary vessels. Eur J Pharmacol 27: , Blyth, DI: Some effects of histamine on the depolarized rat uterus. Br J Pharmacol 49: , Grossman, MI, Konturek, SJ: Inhibition of acid secretion in dog by metiamide: a histamine antagonist acting on H2 receptors. Gastroenterology 66: , Konturek, SJ: Antagonism of histamine H2 receptors and gastric secretion. Scand J Gastroenterol 8: , Christensen, J, Conklin, JL, Freeman, BW: Physiologic specialization at the esophagogastric junction in three species. Am J Physio\ 225: , Farrell, RL, Nebel, OT, McGuire, AT, et al: The abnormal lower oesophageal sphincter in pernicious anaemia. Gut 14: , Ash, ASF, Schild, HD: Receptors mediating some actions of histamine. Br J Pharmacol 27: , Durant, GJ, Ganellin, DR, Parsons, ME: Chemical differentiation of histamine H,- and H,-receptor agonists. Abstract of papers presented at 168th Am Chern Soc Natl Mtg, Atlantic City, N. J. Abstract no. MEDI 29, Paton, WDM: Compound 48/80: a potent histamine liberator. BrJ Pharmacol 6: , Paton, WDM, Schachter, M: The influence of an antihistamine drug on the release of histamine in the unanaesthetized dog. Br J Pharmacol 6: , 1951