Clinicaland Experimental Pharmacology & Physiologv (1979) 6, 151-157.
EFFECT OF DITHIOTHREITOL ON HISTAMINE RECEPTORS IN RABBIT COLON AND GUINEA-PIG ILEUM
W.E . Glover School of Physiology & Pharmacology, University of New South Wales, Kensington, N.S. W., Australia (Received 2 November 1977;revision received 16 January 1978)
SUMMARY
1. Dithiothreitol, an agent which reduces disulphide linkages to sulphydryl groups, potentiated the contractile responses of the rabbit colon and guinea-pig ileum to histamine, but had no effect on the resting tone or on the responses of these preparations to acetylcholine. 2. The potentiation was greater than that produced by antagonism of histamine’s weak inhibitory action by metiamide, and still occurred after blockade of Hzreceptors with metiamide; it is concluded that DTT potentiates responses to stimulation of H1-receptorsin both preparations. 3. In this respect, these H1-receptorsresemble those in vascular smooth muscle in the rabbit but not in the guinea-pig; it is concluded that there is a tissue variation rather than a species variation in the response of H-receptors in the rabbit and guinea-pig to DTT. Key words: dithiothreitol, H1-receptor, Hrreceptor, histamine, intestinal smooth muscle, mepyramme, metiamide. INTRODUCTION Dithiothreitol (DTT), an agent which reduces disulphide linkages to sulphydryl groups, potentiates cardiovascular responses mediated by histamine H1-receptors but not by Hzreceptors in the rabbit (Carroll & Glover, 1977). However DTT, in concentrations which potentiate the contractile response of the rabbit aorta to histamine, depresses similar responses in the guinea-pig aorta (Fleisch, Krxan & Titus, 1974). These authors suggested that there are dissimilar H1-receptor subtypes in the rabbit and the guinea-pig which differ in the number or arrangement of disulphide bridges in their molecular structure. The present experiments were designed to study the effect of DTT on responses mediated by H1-receptors in the guinea-pig aorta (Fleisch, Krzan & Titus, 1974). These authors suggested that there are dissimilar Hl-receptor subtypes in the rabbit and the guinea-pig which differ in the Correspondence: Professor W. E. Glover, School of Physiology and Pharmacology, University of New South Wales, P.O. Box 1, Kensington, N.S.W.2033, Australia. 0 3 ~ ~ - ~ ~ ~ ~ / ~ ~ / 0 3 0 0 - 0 1 50 1 1979 $ 0 2 .Blackwell 00 Scientific Publications
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METHODS Short segments of rabbit colon or guinea-pig ileum were suspended in an organ bath containing Tyrode’s solution of the following composition (mmol/l): NaCl 137, NaHC03 12, NaHJ’04 0.42, KCl 2.68, CaClz 1.8, MgS04 1.05 and glucose 5.55 and aerated with 5% C02 in 0% Longitudinal contractions were recorded using a Grass FT03C transducer and a Grass polygraph. The initial tension was adjusted to 1.O g wt. All experiments were carried out at 37°C. In some experiments atropine (250 nmol/l) was added to the Tyrode’s solution to reduce spontaneous activity of the rabbit colon. Histamine or acetylcholine were added to the 20 ml organ bath in volumes of 0.1-0.5 ml and washed out after 10 s (guinea-pig ileum) or 40 s (rabbit colon). Other drugs were added to the Tyrode’s solution. Drugs The following drugs were used: actylcholine chloride (Hopkin & Williams), atropine sulphate (Abbott), dithiothreitol (Calbiochem), histamine acid phosphate (Bull), mepyramine maleate (May & Baker) and metiamide (Smith, Kline & French).
RESULTS Rabbit colon In preliminary experiments it was found that, in contrast to results with rabbit jejenum, segments of proximal colon usually displayed little spontaneous activity, and responded to histamine or acetylcholine with contractions which were dose-related and reproducible provided that doses which gave maximal responseswere not used. In the presence of mepyramine (10-100 nmol/l) the responses to histamine were greatly reduced or abolished. DTT (1.0 mmol/l) had no effect on the resting tone of the preparation, but caused a marked increase in the contractile response to histamine. This potentiating effect reached a
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Fig. 1. Effect of DTT on the responses of the rabbit colon to graded doses of histamine @mol/l). (a) Control responses; (b) responses in presence of DTT (1.0 mmol/l).
DTT and histamine receptors in smooth muscle 3
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Fig. 2. Effect of DTT (1.0 mmol/l) on the responses of the rabbit colon to histamine. Each point is the mean of one or two observations on each of thirteen preparations and the vertical bars indicate the standard errors of the means. ( 0 ) Control; (0)in presence of DTT (1.O mmol/l).
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Acetylcholine (mol / I
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Fig. 3. Effect of DTT (1.0 mmol/l) on the responses of the rabbit colon to acetylcholine. Each point is the mean of one or two observations on each of six preparations and the vertical bars indicate the standard errors of the means. ( 0 ) Control; (0)in presence of DTT (1.0 mmol/l).
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maximum in 5-10 min, it was well maintained in the presence of DTT for several hours and responses decreased only very slowly after DTT was removed from the Tyrode’s solution. In thirteen experiments the effects of five graded doses of histamine were recorded before and after the addition of DTT (1 .O mmol/l) to the Tyrode’s solution. Part of a typical record is shown in Fig. 1, and the results are summarized in Fig. 2. DTT caused a parallel shift to the left of the straight part of the mean log concentration-response curve, from which it was calculated that responses were potentiated by a factor of 10.0. In the five of these experiments in which it was tested, the responses to histamine in the presence of DTT were greatly reduced by mepyramine (10-100 nmol/l). In three experiments metiamide (40 pmol/l) had no effect on the responses to histamine, but in another three experiments metiamide caused slight potentiation (X 1.25-1.5) as indicated by the parallel shift to the left of the log concentration-response curves. However, in all six of these experiments, DTT (1 .O mmol/l) added to the Tyrode’s solution in addition to the metiamide still caused marked potentiation (X 7.8-12.4) of the responses to histamine. In contrast to its effect on the responses to histamine, DTT (1.0 mmol/l) had no effect on the responses of the rabbit colon to acetylcholine. The results of the six experiments are summarised in Fig. 3. Guinea-pig ileum In each of ten experiments DTT (1 mmol/l) had no effect on the resting tone but caused an immediate, sustained and poorly reversible potentiation of the contractile response to histamine. Logconcentration relationships were studied in six experiments; one of these is illustrated in Fig. 4 and the results are summarized in Fig. 5 . DTT potentiated responses to histamine. Log-concentration relationships were studied in six experiments; one of these is tration-response curve. In five experiments the same concentration of DTT had no effect on the responses of the guinea-pig ileum to acetylcholine.
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Fig. 4. Effect
of DTT (1 mmol/l) on the responses of the guinea-pig ileum to graded doses of histamine (nmol/l). Control responses;(b) responses in presence of DTT (1 .O mmol/l).
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't 10-7
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Histamine (mol/l) Fig. 5. Effect of DTT (1 mmol/l) on the responses of the guinea-pig ileum to histamine. Each point is the mean of one or two observationson each of six preparations and the vertical bars indicate the standard errors of the means. ( 0 ) Control; (0)in presence of DTT (1.0 mmolfi).
DISCUSSION The guinea-pig ileum, like the rabbit ear artery (Glover, Carroll & Latt, 1973) and aorta (Carroll & Glover, 1977) contains both H1- and H2-histamine receptors. Thus, although histamine normally has a contractile action mediated by H1-receptors(Ash & Schild, 1966; Black, el al., 1972), in the presence of the H1-antagonist, mepyramine, histamine has an inhibitory effect on the response to electrical stimulation which is sensitive to the H2antagonist burimamide (Ambache, Killick& Zar, 1973). Again, as in the vascular preparations, the normal response is a balance between a predominant contractile action mediated by H1receptors and a weaker relaxing action mediated by H2-receptors,since the stimulating effect of small concentrations of histamine is potentiated by the H2-antagonist burimamide (Rocha e Silva, 1975). A similar slight potentiation of the contractile responses of the rabbit colon to histamine was produced by the H2-antagonistmetiamide in three out of six of the present experiments. The contractile response of the colon to histamine is sensitive to mepyramine, so it is concluded that the histamine receptors in the rabbit colon, like those in the guinea-pig ileum, are predominantly of the H1-type, but that there is a small and variable population of H2receptors. The variability of Hrreceptors from one piece of colon to another is not surprising, since Rocha e Silva (1975) reported a similar variability in the proportions of H1-receptors and H2-receptorsin different pieces of guinea-pig ileum. The results show that DTT caused marked potentiation of the contractile responses of both types of smooth muscle to histamine. The potentiation was much greater than that produced by antagonism of the weak inhibitory action of histamine with metiamide, and DTT still caused marked potentiation of the contractile response after blockade of HT
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receptors with metiamide. While this finding does not exclude the possibility of a concurrent action-either blockade or potentiation-on the Hz-receptor system, it can be concluded that DTT potentiates the responses to stimulation of HI-receptors in both the rabbit colon and guinea-pig ileum. In both the rabbit aorta and ear artery the potentiating action of DTT is specific for histamine contractile responses, since DTT does not potentiate the responses to other agonists (Fleisch, Krzan & Titus, 1973; Carroll & Glover, 1977). The present results show that DTT did not potentiate the contractile responses of the rabbit colon and guinea-pig ileum to acetylcholine, and it can therefore be concluded that the potentiation of the responses to histamine was not due to a non-specific action on the smooth muscle of these preparations. Fleisch et al. (1973) showed that D'IT had little effect on histamine metabolizing enzymes, and suggested that it caused potentiation by reducing a disulphide bridge at one of the steps in the sequence of events that connects the HI-receptor with the contractile mechanism. However, in a later series of experiments, Fleisch et al. (1974) found that DTT in concentrations which potentiated responses of the rabbit aorta to histamine depressed similar responses in the guinea-pig aorta, and concluded that there is a species variation in the response to DTT. In contrast, the present results show that DTT potentiates responses mediated by H1-receptors in the guinea-pig ileum. In this respect the HI-receptors in the guinea-pig ileum resemble those in the colon and aorta of the rabbit, but not those in the guinea-pig aorta. It is therefore concluded that there is a tissue variation rather than a species variation in the responsiveness of HI-receptors in the rabbit and guinea-pig to DTT. Fleisch et al. (1974) also suggested that the difference in responsiveness to DTT which they observed between the rabbit aortaand guinea-pig aorta was due to dissimilar HI-receptor subtypes, since the pA2 values for the specific HI-antagonist mepyramine were different in the two preparations, being 8.43 and 9.13 respectively. They further proposed that if such receptor subtypes do exist, they differ in the number or arrangement of disulphide bridges in their molecular structure. Previous workers have reported pA2-values for mepyramine in the guinea-pig ileum ranging from 9.3 to 9.5 (Arunlakshana & Schild, 1959), and the HI-receptors in this tissue are therefore distinctly different from those in the rabbit aorta (pA2 = 8.43), but it is not possible to tell from these data whether the receptors in the guinea-pig ileum and aorta are different. If there are different subtypes in the guinea-pigileum and aorta, it is possible that the subtypes found in the guinea-pig ileum and rabbit aorta belong to a DTT-sensitive group of subtypes, which differ from non-DTT sensitive subtypes in their molecular structure in the way suggested by Fleisch et al. (1974). If, however, the receptors in the guinea-pig ileum and aorta are identical there can be no relationship between subtypes of Hl-receptor and DTT sensitivity. In this case, DTT could act by altering an intracellular process in an excitation-contraction pathway which is specific for histamine but which is different in the two tissues. Referring to the DTT-sensitive H1-receptors in the rabbit aorta, Fleisch et al. (1973) suggested that there were two possibilities; either reduction of a disulphide bridge at some vital point increases receptor activity, or the histamine receptor system which is normally in a reduced state is oxidized in the artificial environment of the tissue bath and needs to be reduced to regain full activity. However, a further possibility is that in vivo the activity of the HI-receptor system might be modulated by the level of metabolic activity in the tissue. It is of interest that the same isolated segment of rabbit colon, like a similar segment of
DTT and histamine receptors in smooth muscle
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guinea-pig ileum or single arterial preparation, may contain both H1-receptors and H2receptors; hence, as pointed out by Rocha e Silva (1975), histamine can act as its own functional antagonist. Finding that the isolated ear artery of the rabbit contained both a-adrenoreceptors and P-adrenoreceptors (Carroll & Glover, 1973) suggested that the magnitude of the response to stimulation of the a-adrenoreceptors might be altered by simultaneous P-adrenoreceptor activity to a degree which is determined by local, humoral or nervous factors. While nothing is known about the physiological function of either H1receptors or Hrreceptors in vascular or intestinal smooth muscle, it is possible that the presence of the two types of receptor in a single tissue provides additional ways in which the response to histamine may be modulated. It is therefore proposed that in tissues where the response to histamine depends on a balance between its stimulating action mediated by HI-receptors and its inhibitory action mediated by H2-receptors, the activity of the two histamine-receptor systems may be modulated independently by alterations in the environment of the receptors. An alteration in the state of oxidation of a disulphide bridge might be one of the ways in which the H1-receptorsystem is modulated. ACKNOWLEDGMENTS
This investigation was supported by a grant from the National Health and Medical Research Council of Australia. I wish to thank Miss R. Diprose and Mrs M. de Detrich for technical assistance, and Dr W. A. M. Duncan of Smith, Kline & French for kindly supplying the me tiamide. REFERENCES Ambache, N., Killick, S.W. & Zar, M.A. (1973) Antagonism by burimamide of inhibitions induced by histamine in plexus-containing longitudinal muscle preparations from guinea-pig ileum. British Journal ofPharmacology, 48,362-363. Arunlaksh~a,0. & Schild, H.O. (1959) Some quantitative uses of drug antagonists. British Journal of Pharmacology. 14,4848. Ash, A.S.F. & Schild, H.O. (1966) Receptors mediating some actions of histamine. British Journal of Pharmacology. 27,427-439. Black, J.W., Duncan, W.A.M., Durant, CJ., Ganellm, C.R. & Parsons, E.M. (1972) Definition and antagonism of histamine H, receptors. Nafure, 236,385-390. Carroll, P.R & Glover, W.E. (1973) Beta adrenoceptors in the rabbit ear artery. Australian Journal of Experimental Biology and Medical Science, 5 1,309-324. Carroll, P.R & Glover, W.E. (1977) Modification of cardiovascular responses to histamine by dithiothreitol British Journal of Pharmacology, 59, 333-341. Fleisch, J.H., Krzan, M.C. & Titus, E. (1973) Pharmacologic receptor activity of rabbit aorta Effect of dithiothreitol and nethylmaleimide. Circulation Research, 33,284-290. Fleisch, J.H., Krzan, M.C. & Titus, E. (1974) Alterations in pharmacologic receptor activity by dithiothreitol. American Journal of Physiology, 227,1243-1247. Glover, W.E. (1977)Dithiothreitol potentiatesresponses to histamine in rabbit colon and guinea-pig ileum. Proceedings of the Australian Physiological and Pharmacological Society, 8 , 156P. Glover, W.E., Carroll, P.R & Latt, N. (1973) Histamine receptors in human temporal and rabbit ear arteries. International Symposium on Histamine H, Receptor Antagonists (eds C. I. Wood & M. A. Simkins), pp. 169-174. Smith,Kline & French, London. Rocha e Sihra, M. (1975) Concerning the presence of ‘spare receptors’ for histamine in the guinea pig ileum and in other smooth muscle structures. General Pharmacology, 6,259-266.
EFFECT OF DITHIOTHREITOL ON HISTAMINE RECEPTORS IN RABBIT COLON AND GUINEA-PIG ILEUM
W.E . Glover School of Physiology & Pharmacology, University of New South Wales, Kensington, N.S. W., Australia (Received 2 November 1977;revision received 16 January 1978)
SUMMARY
1. Dithiothreitol, an agent which reduces disulphide linkages to sulphydryl groups, potentiated the contractile responses of the rabbit colon and guinea-pig ileum to histamine, but had no effect on the resting tone or on the responses of these preparations to acetylcholine. 2. The potentiation was greater than that produced by antagonism of histamine’s weak inhibitory action by metiamide, and still occurred after blockade of Hzreceptors with metiamide; it is concluded that DTT potentiates responses to stimulation of H1-receptorsin both preparations. 3. In this respect, these H1-receptorsresemble those in vascular smooth muscle in the rabbit but not in the guinea-pig; it is concluded that there is a tissue variation rather than a species variation in the response of H-receptors in the rabbit and guinea-pig to DTT. Key words: dithiothreitol, H1-receptor, Hrreceptor, histamine, intestinal smooth muscle, mepyramme, metiamide. INTRODUCTION Dithiothreitol (DTT), an agent which reduces disulphide linkages to sulphydryl groups, potentiates cardiovascular responses mediated by histamine H1-receptors but not by Hzreceptors in the rabbit (Carroll & Glover, 1977). However DTT, in concentrations which potentiate the contractile response of the rabbit aorta to histamine, depresses similar responses in the guinea-pig aorta (Fleisch, Krxan & Titus, 1974). These authors suggested that there are dissimilar H1-receptor subtypes in the rabbit and the guinea-pig which differ in the number or arrangement of disulphide bridges in their molecular structure. The present experiments were designed to study the effect of DTT on responses mediated by H1-receptors in the guinea-pig aorta (Fleisch, Krzan & Titus, 1974). These authors suggested that there are dissimilar Hl-receptor subtypes in the rabbit and the guinea-pig which differ in the Correspondence: Professor W. E. Glover, School of Physiology and Pharmacology, University of New South Wales, P.O. Box 1, Kensington, N.S.W.2033, Australia. 0 3 ~ ~ - ~ ~ ~ ~ / ~ ~ / 0 3 0 0 - 0 1 50 1 1979 $ 0 2 .Blackwell 00 Scientific Publications
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METHODS Short segments of rabbit colon or guinea-pig ileum were suspended in an organ bath containing Tyrode’s solution of the following composition (mmol/l): NaCl 137, NaHC03 12, NaHJ’04 0.42, KCl 2.68, CaClz 1.8, MgS04 1.05 and glucose 5.55 and aerated with 5% C02 in 0% Longitudinal contractions were recorded using a Grass FT03C transducer and a Grass polygraph. The initial tension was adjusted to 1.O g wt. All experiments were carried out at 37°C. In some experiments atropine (250 nmol/l) was added to the Tyrode’s solution to reduce spontaneous activity of the rabbit colon. Histamine or acetylcholine were added to the 20 ml organ bath in volumes of 0.1-0.5 ml and washed out after 10 s (guinea-pig ileum) or 40 s (rabbit colon). Other drugs were added to the Tyrode’s solution. Drugs The following drugs were used: actylcholine chloride (Hopkin & Williams), atropine sulphate (Abbott), dithiothreitol (Calbiochem), histamine acid phosphate (Bull), mepyramine maleate (May & Baker) and metiamide (Smith, Kline & French).
RESULTS Rabbit colon In preliminary experiments it was found that, in contrast to results with rabbit jejenum, segments of proximal colon usually displayed little spontaneous activity, and responded to histamine or acetylcholine with contractions which were dose-related and reproducible provided that doses which gave maximal responseswere not used. In the presence of mepyramine (10-100 nmol/l) the responses to histamine were greatly reduced or abolished. DTT (1.0 mmol/l) had no effect on the resting tone of the preparation, but caused a marked increase in the contractile response to histamine. This potentiating effect reached a
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Fig. 1. Effect of DTT on the responses of the rabbit colon to graded doses of histamine @mol/l). (a) Control responses; (b) responses in presence of DTT (1.0 mmol/l).
DTT and histamine receptors in smooth muscle 3
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Fig. 2. Effect of DTT (1.0 mmol/l) on the responses of the rabbit colon to histamine. Each point is the mean of one or two observations on each of thirteen preparations and the vertical bars indicate the standard errors of the means. ( 0 ) Control; (0)in presence of DTT (1.O mmol/l).
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Acetylcholine (mol / I
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Fig. 3. Effect of DTT (1.0 mmol/l) on the responses of the rabbit colon to acetylcholine. Each point is the mean of one or two observations on each of six preparations and the vertical bars indicate the standard errors of the means. ( 0 ) Control; (0)in presence of DTT (1.0 mmol/l).
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maximum in 5-10 min, it was well maintained in the presence of DTT for several hours and responses decreased only very slowly after DTT was removed from the Tyrode’s solution. In thirteen experiments the effects of five graded doses of histamine were recorded before and after the addition of DTT (1 .O mmol/l) to the Tyrode’s solution. Part of a typical record is shown in Fig. 1, and the results are summarized in Fig. 2. DTT caused a parallel shift to the left of the straight part of the mean log concentration-response curve, from which it was calculated that responses were potentiated by a factor of 10.0. In the five of these experiments in which it was tested, the responses to histamine in the presence of DTT were greatly reduced by mepyramine (10-100 nmol/l). In three experiments metiamide (40 pmol/l) had no effect on the responses to histamine, but in another three experiments metiamide caused slight potentiation (X 1.25-1.5) as indicated by the parallel shift to the left of the log concentration-response curves. However, in all six of these experiments, DTT (1 .O mmol/l) added to the Tyrode’s solution in addition to the metiamide still caused marked potentiation (X 7.8-12.4) of the responses to histamine. In contrast to its effect on the responses to histamine, DTT (1.0 mmol/l) had no effect on the responses of the rabbit colon to acetylcholine. The results of the six experiments are summarised in Fig. 3. Guinea-pig ileum In each of ten experiments DTT (1 mmol/l) had no effect on the resting tone but caused an immediate, sustained and poorly reversible potentiation of the contractile response to histamine. Logconcentration relationships were studied in six experiments; one of these is illustrated in Fig. 4 and the results are summarized in Fig. 5 . DTT potentiated responses to histamine. Log-concentration relationships were studied in six experiments; one of these is tration-response curve. In five experiments the same concentration of DTT had no effect on the responses of the guinea-pig ileum to acetylcholine.
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Fig. 4. Effect
of DTT (1 mmol/l) on the responses of the guinea-pig ileum to graded doses of histamine (nmol/l). Control responses;(b) responses in presence of DTT (1 .O mmol/l).
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't 10-7
10-8
Histamine (mol/l) Fig. 5. Effect of DTT (1 mmol/l) on the responses of the guinea-pig ileum to histamine. Each point is the mean of one or two observationson each of six preparations and the vertical bars indicate the standard errors of the means. ( 0 ) Control; (0)in presence of DTT (1.0 mmolfi).
DISCUSSION The guinea-pig ileum, like the rabbit ear artery (Glover, Carroll & Latt, 1973) and aorta (Carroll & Glover, 1977) contains both H1- and H2-histamine receptors. Thus, although histamine normally has a contractile action mediated by H1-receptors(Ash & Schild, 1966; Black, el al., 1972), in the presence of the H1-antagonist, mepyramine, histamine has an inhibitory effect on the response to electrical stimulation which is sensitive to the H2antagonist burimamide (Ambache, Killick& Zar, 1973). Again, as in the vascular preparations, the normal response is a balance between a predominant contractile action mediated by H1receptors and a weaker relaxing action mediated by H2-receptors,since the stimulating effect of small concentrations of histamine is potentiated by the H2-antagonist burimamide (Rocha e Silva, 1975). A similar slight potentiation of the contractile responses of the rabbit colon to histamine was produced by the H2-antagonistmetiamide in three out of six of the present experiments. The contractile response of the colon to histamine is sensitive to mepyramine, so it is concluded that the histamine receptors in the rabbit colon, like those in the guinea-pig ileum, are predominantly of the H1-type, but that there is a small and variable population of H2receptors. The variability of Hrreceptors from one piece of colon to another is not surprising, since Rocha e Silva (1975) reported a similar variability in the proportions of H1-receptors and H2-receptorsin different pieces of guinea-pig ileum. The results show that DTT caused marked potentiation of the contractile responses of both types of smooth muscle to histamine. The potentiation was much greater than that produced by antagonism of the weak inhibitory action of histamine with metiamide, and DTT still caused marked potentiation of the contractile response after blockade of HT
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receptors with metiamide. While this finding does not exclude the possibility of a concurrent action-either blockade or potentiation-on the Hz-receptor system, it can be concluded that DTT potentiates the responses to stimulation of HI-receptors in both the rabbit colon and guinea-pig ileum. In both the rabbit aorta and ear artery the potentiating action of DTT is specific for histamine contractile responses, since DTT does not potentiate the responses to other agonists (Fleisch, Krzan & Titus, 1973; Carroll & Glover, 1977). The present results show that DTT did not potentiate the contractile responses of the rabbit colon and guinea-pig ileum to acetylcholine, and it can therefore be concluded that the potentiation of the responses to histamine was not due to a non-specific action on the smooth muscle of these preparations. Fleisch et al. (1973) showed that D'IT had little effect on histamine metabolizing enzymes, and suggested that it caused potentiation by reducing a disulphide bridge at one of the steps in the sequence of events that connects the HI-receptor with the contractile mechanism. However, in a later series of experiments, Fleisch et al. (1974) found that DTT in concentrations which potentiated responses of the rabbit aorta to histamine depressed similar responses in the guinea-pig aorta, and concluded that there is a species variation in the response to DTT. In contrast, the present results show that DTT potentiates responses mediated by H1-receptors in the guinea-pig ileum. In this respect the HI-receptors in the guinea-pig ileum resemble those in the colon and aorta of the rabbit, but not those in the guinea-pig aorta. It is therefore concluded that there is a tissue variation rather than a species variation in the responsiveness of HI-receptors in the rabbit and guinea-pig to DTT. Fleisch et al. (1974) also suggested that the difference in responsiveness to DTT which they observed between the rabbit aortaand guinea-pig aorta was due to dissimilar HI-receptor subtypes, since the pA2 values for the specific HI-antagonist mepyramine were different in the two preparations, being 8.43 and 9.13 respectively. They further proposed that if such receptor subtypes do exist, they differ in the number or arrangement of disulphide bridges in their molecular structure. Previous workers have reported pA2-values for mepyramine in the guinea-pig ileum ranging from 9.3 to 9.5 (Arunlakshana & Schild, 1959), and the HI-receptors in this tissue are therefore distinctly different from those in the rabbit aorta (pA2 = 8.43), but it is not possible to tell from these data whether the receptors in the guinea-pig ileum and aorta are different. If there are different subtypes in the guinea-pigileum and aorta, it is possible that the subtypes found in the guinea-pig ileum and rabbit aorta belong to a DTT-sensitive group of subtypes, which differ from non-DTT sensitive subtypes in their molecular structure in the way suggested by Fleisch et al. (1974). If, however, the receptors in the guinea-pig ileum and aorta are identical there can be no relationship between subtypes of Hl-receptor and DTT sensitivity. In this case, DTT could act by altering an intracellular process in an excitation-contraction pathway which is specific for histamine but which is different in the two tissues. Referring to the DTT-sensitive H1-receptors in the rabbit aorta, Fleisch et al. (1973) suggested that there were two possibilities; either reduction of a disulphide bridge at some vital point increases receptor activity, or the histamine receptor system which is normally in a reduced state is oxidized in the artificial environment of the tissue bath and needs to be reduced to regain full activity. However, a further possibility is that in vivo the activity of the HI-receptor system might be modulated by the level of metabolic activity in the tissue. It is of interest that the same isolated segment of rabbit colon, like a similar segment of
DTT and histamine receptors in smooth muscle
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guinea-pig ileum or single arterial preparation, may contain both H1-receptors and H2receptors; hence, as pointed out by Rocha e Silva (1975), histamine can act as its own functional antagonist. Finding that the isolated ear artery of the rabbit contained both a-adrenoreceptors and P-adrenoreceptors (Carroll & Glover, 1973) suggested that the magnitude of the response to stimulation of the a-adrenoreceptors might be altered by simultaneous P-adrenoreceptor activity to a degree which is determined by local, humoral or nervous factors. While nothing is known about the physiological function of either H1receptors or Hrreceptors in vascular or intestinal smooth muscle, it is possible that the presence of the two types of receptor in a single tissue provides additional ways in which the response to histamine may be modulated. It is therefore proposed that in tissues where the response to histamine depends on a balance between its stimulating action mediated by HI-receptors and its inhibitory action mediated by H2-receptors, the activity of the two histamine-receptor systems may be modulated independently by alterations in the environment of the receptors. An alteration in the state of oxidation of a disulphide bridge might be one of the ways in which the H1-receptorsystem is modulated. ACKNOWLEDGMENTS
This investigation was supported by a grant from the National Health and Medical Research Council of Australia. I wish to thank Miss R. Diprose and Mrs M. de Detrich for technical assistance, and Dr W. A. M. Duncan of Smith, Kline & French for kindly supplying the me tiamide. REFERENCES Ambache, N., Killick, S.W. & Zar, M.A. (1973) Antagonism by burimamide of inhibitions induced by histamine in plexus-containing longitudinal muscle preparations from guinea-pig ileum. British Journal ofPharmacology, 48,362-363. Arunlaksh~a,0. & Schild, H.O. (1959) Some quantitative uses of drug antagonists. British Journal of Pharmacology. 14,4848. Ash, A.S.F. & Schild, H.O. (1966) Receptors mediating some actions of histamine. British Journal of Pharmacology. 27,427-439. Black, J.W., Duncan, W.A.M., Durant, CJ., Ganellm, C.R. & Parsons, E.M. (1972) Definition and antagonism of histamine H, receptors. Nafure, 236,385-390. Carroll, P.R & Glover, W.E. (1973) Beta adrenoceptors in the rabbit ear artery. Australian Journal of Experimental Biology and Medical Science, 5 1,309-324. Carroll, P.R & Glover, W.E. (1977) Modification of cardiovascular responses to histamine by dithiothreitol British Journal of Pharmacology, 59, 333-341. Fleisch, J.H., Krzan, M.C. & Titus, E. (1973) Pharmacologic receptor activity of rabbit aorta Effect of dithiothreitol and nethylmaleimide. Circulation Research, 33,284-290. Fleisch, J.H., Krzan, M.C. & Titus, E. (1974) Alterations in pharmacologic receptor activity by dithiothreitol. American Journal of Physiology, 227,1243-1247. Glover, W.E. (1977)Dithiothreitol potentiatesresponses to histamine in rabbit colon and guinea-pig ileum. Proceedings of the Australian Physiological and Pharmacological Society, 8 , 156P. Glover, W.E., Carroll, P.R & Latt, N. (1973) Histamine receptors in human temporal and rabbit ear arteries. International Symposium on Histamine H, Receptor Antagonists (eds C. I. Wood & M. A. Simkins), pp. 169-174. Smith,Kline & French, London. Rocha e Sihra, M. (1975) Concerning the presence of ‘spare receptors’ for histamine in the guinea pig ileum and in other smooth muscle structures. General Pharmacology, 6,259-266.
