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Pharmacological characterization of cardiac histamine receptors: Sensitivity to H1-receptor antagonists
European Journal of Pharmacology 27 (1974) 330-338 © North-Holland Publishing Company
PHARMACOLOGICAL
CHARACTERIZATION
OF CARDIAC HISTAMINE
R E C E P T O R S : S E N S I T I V I T Y T O H 1 - R E C E P T O R A N T A G O N I S T S 1,2 Roberto LEVI and J. Olatunde KUYE 3 Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York, N. Y. 10021, U.S.A.
Received 10 December 1973, accepted 8 April 1974 R. LEVI and J.O. KUYE, Pharmacological characterization of cardiac histamine receptors: sensitivity to H 1-receptor antagonists, European J. Pharmacol. 27 (1974) 330-338. The ability of five prototypic antihistamines (diphenhydramine, promethazine, cyclizine, chlorpheniramine and tripelennamine) to modify the cardiac actions of histamine was studied in order to determine the reactivity of cardiac histamine receptors. Histamine, as a function of dose, increases the rate, the force of contraction, the coronary flow and the time of atrioventricular conduction of the isolated guinea pig heart. Each of the five prototypic antihistamines failed to inhibit histamine-induced stimulation of cardiac rate and contractility. At the same time these antihistamines did inhibit the negative dromotropic effect of histamine. Chlorpheniramine and diphenhydramine antagonized the coronary dilating effect of histamine. These results suggest that two types of histamine receptors may be present in the guinea pig heart: Hi-receptors, at the atrioventricular node and coronary vessels; H2-receptors at the sinoatrial node and on the ventricular fibers. The activity of these prototypic antihistamines is consistent with their action exclusively on H 1-receptorsCardiac histamine receptors Cardiac effects of histamine
Antihistamines
1. Introduction In the isolated heart of the guinea pig histamine induces positive inotropic, positive chronotropic as well as negative dromotropic effects. Also, histamine increases the rate of coronary flow. All of these effects are dose dependent (Levi, 1972). The positive inotropic and chronotropic effects of histamine on isolated cardiac preparations of the guinea pig are not inhibited by tripelennamine and pyrilamine (Trende1This work was supported by U.S.P.H.S. Grant No. 1RO1 GM 20091 by a grant-in-aid from the New York Heart Association, and in part by the John Polacheck Foundation for Medical Research. 2Preliminary results were presented at the 1972 Spring Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, U.S.A. 3Post-Doctoral Trainee in Pharmacology (U.S.P.H.S. Grant No. GM 00099). Present address: c/o Delbay Pharmaceuticals Inc., 60 Orange Street, Bloomfield, New Jersey 07003, U.S.A.
Histamine
Histamine H 1-receptor antagonists
lenburg, 1960) nor inhibited by mepyramine, pyrathiazine and diphenhydramine (Bartlet, 1963). In the h e a r t - l u n g preparation of the dog, promethazine and diphenhydramine do not block the effect of histamine on heart rate and contractility, while they block completely the negative dromotropic effect of histamine (Flacke et al., 1967). Those actions of histamine which are antagonized by low concentrations of antihistamines may result from the interaction of histamine with one type of receptor (HI), whereas histamine responses not inhibited by antihistamines may result from interaction with a different histamine receptor (Ash and Schild, 1966). Thus, the cardiac actions of histamine could be mediated by more than one histamine receptor. The purpose of the present study was to define in a quantitative manner the effects of five prototypic antihistamines (diphenhydramine, tripelennamine, chlorpheniramine, cyclizine, and promethazine) on each of the actions of histamine in the isolated guinea pig heart. This information is required for the most
R. LevL J.O. Kuye, Hi-Antagonists and histamine on heart complete characterization of cardiac histamine receptors.
2. Materials and methods
2.1. Perfusion o f the isolated heart Male C a m m - H a r t l e y guinea pigs weighing between 250 and 300 g were stunned by a blow to the base of the skull. The heart was removed and m o u n t e d in a Langendorff apparatus (Levi, 1972) within a minute or less and perfused at constant pressure (40 cm of water) with oxygenated Ringer solution at 37.5°C. The composition of the Ringer solution was (mM/1): Na ÷ 160; C1- 164; K ÷ 5.6; Ca 2÷ 2.2; HCO3- 5.9; glucose 5.5. Isometric ventricular contractions, surface electrograms and coronary flow rate were recorded as previously described (Levi, 1972). Heart rate and atrioventricular conduction time ( P - R interval) were determined from the electrocardiographic tracings. Hearts were perfused for 30 to 45 min before experimentation was begun, by which time heart rate and contraction had stabilized. Histamine in increasing doses was injected intra-aortically. Histamine was dissolved in oxygenated Ringer solution kept at 37°C and the volume of all injections remained constant at
331
0.1 ml. Histamine injections were made every 20 min for a total of seven injections per experiment. In other experiments hearts were continuously perfused with a selected concentration of an antihistamine. 10 min after the beginning of this perfusion, histamine, in increasing doses, was administered intra-aortically as described above. The perfusion with the antihistamine was continued for 30 min after the last injection of histamine. The effects on the rate of coronary flow, of a constant perfusion with either histamine or histamine in combination with an antihistamine were also studied. Hearts were perfused for 10 min with a selected concentration of histamine (from 0.1 #g/ml to 10 /~g/ml). Each heart was perfused with only one concentration of histamine. When a combination histam i n e - a n t i h i s t a m i n e was studied, perfusion with the antihistamine alone was initiated 10 rain before perfusion with the combination.
2.2. Drugs Histamine dihydrochloride was purchased from Sigma Chemical Company (St. Louis, Missouri, U.S.A.); cyclizine hydrochloride, chlorpheniramine maleate, diphenhydramine hydrochloride, promethazine hydrochloride, and tripelennamine hydrochloride were gifts respectively of Burroughs Wellcome Co.
Table 1 Chronotropic, inotropic and dromotropic effects of antihistaminesa. Compound
Concentration (M)
n
Rate b
Contractionc
P-R interval c
Diphenhydramine
10- 8 10- 7 10-5 10- 7 3X 10- 6 10-7 10- s 10 - 7 10 -5 10-7 10- s
19 6 5 5 5 6 5 27 7 5 7
+15 +- 3d,e +4 + 8 -26 -+6e +10 +-4 -14_+4 e +13 _+6 -59-+ 10g +13 -+ 3e -35 -+6g +1 ± 6 -51 -+5g
-11 +- 2e - 5 -+5 -25 ± 3g -14 +-5 - 2 2 ~ 1e - 7 -+2 f -19 + 6e -6 ± 1 -30 ± 5g -5± 1 -26 +~3f
+1 + 1 0 +14 -+ 3f +3 +- 5 +3+- 2 -5 -+4 - 2 +- 4 +3 -+ 1 +5 -+ 2 +2 ± 2 +1 + 4
Promethazine Cyclizine Chlorpheniramine Tripelennamine
a Isolated hearts. b Changes in beats/rain. c Percent changes (ventricular contraction amplitude or P-R interval duration. d Means (+ S.E.) of changes observed after 10 min of peffusion with antihistamine. e0.1 > p > 0.05. f 0.025 > p > 0.01. gp < 0.01.
R. LevL J.O. Kuye, Hi-Antagonists and histamine on heart
332
(Research Triangle Park, North Carolina, U.S.A.), Schering Corporation (Bloomfield, New Jersey, U.S.A.), Parke, Davis & Co. (Detroit, Michigan, U.S.A.), Wyeth Laboratories, Inc. (Philadelphia, Pennsylvania, U.S.A.) and Ciba Pharmaceuticals Company (Summit, New Jersey, U.S.A.). All histamine doses and concentrations refer to the free base (M.W. =111).
3. Results
3.1. Effects o f antihistamines on cardiac function The effects of the five prototypic antihistamines on heart rate, ventricular contraction and P - R interval are shown in table 1. At concentrations of 10 -7 M or less antihistamines produced small but consistent increases in heart rate. At the concentration of 10-TM tripelennamine did not influence the heart rate. At the concentration of 10-s M each of the antihistamines caused a marked negative chronotropic effect. Each of the antihistamines caused a concentration dependent negative inotropic effect. At 10-s M promethazine caused contractile failure (not shown in the table). Antihistamines did not modify the duration of the P - R interval, with the exception of 10-s M diphenhydramine, which prolonged the P - R interval. The effects of antihistamines on coronary flow were studied with 10-aM diphenhydramine and 10-TM chlorpheniramine. Both drugs increased the rate of coronary flow (table 2).
3.2. Antihistamines and, the positive chronotropic effect o f histamine The dose dependent positive chronotropic effect of histamine was not blocked diphenhydramine, promethazine, chlorpheniramine or tripelennamine (figs. 1 and 2). Cyclizine at 10-s M attenuated the chronotropic response to doses of histamine from 5 to 50 gig. The positive chronotropic response to doses of 0.25 and 0.5 gig of histamine was increased by the addition of each of the antihistamines (figs. 1 and 2). Chlorpheniramine at 10-s M shifted the entire doseresponse curve for histamine to the left (fig. 2).
3.3. Antihistamines and the positive inotropic effect o f histamine As shown in fig. 3 (upper left panel) there was a progressive decrease in the force of ventricular contraction. Under these conditions histamine produced an increase in ventricular contraction (positive inotropic effect) when compared to the corresponding control (fig. 3). At a concentration of 10-s M (or at 3 )(, 10-6 M for promethazine) the antihistamines produced a significant reduction in ventricular contraction (compare control before antihistamine with response after antihistamine in fig. 3). When histamine was injected in the presence of each of the antihistamines the increase in ventricular contraction produced by histamine was greater than t h a t seen in the absence of antihistamine (fig. 3). When the concentration of the antihistamine was reduced to 10-7M (not shown in the figure) the response of the ventricular contraction
Table 2 Effect of antihistamines on the rate of coronary flowa. Compound
Diphenhydramine Chlorpheniramine
Concentration (M)
10-8 10-7
n
19 20
a Isolated hearts continuously peffused with the drug. b Means ± S.E.
c0.1>p>0.5. d 0.05 > p > 0.025.
Coronary flow rate (ml/min) Control
Treated
% increase
4.7 ± 0.17b 4.7 ± 0.18
5.1 :~ 0.18c 5.2 ± 0.17d
9.4 ± 2.1 9.8 ± 2.1
R. Levi, J.O. Kuye, Hi-Antagonists and histamine on heart
SINUS RATE
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Fig. 1. Effects of histamine and of histamine in the presence of diphenhydramine on the sinus rate and atrioventricular conduction of the isolated heart. , histamine alone; . . . . . . , histamine in the presence of diphenhydramine 10 -7 M; . . . . . . , histamine in the presence of dipfienhydramine 10-5 M. Points (means, n = 9 - 1 1 for histamine, and n = 5 - 6 for histamine in the presence of each concentration of diphenhydramine; vertical bars = S.E.) represent the maxim u m increase in sinus rate and P - R interval duration from the values immediately preceding each histamine injection. Average (+ S.E.) control values before histamine for rate and P - R interval duration were 233 + 8/min and 59.5 + 0.5 msec; 216 -+ 13/min and 58.8 + 3.3 msec in the presence of diphenhydramine 10 -7 M; 216 + 11/min and 63.7 +- 3.3 msec in the presence of diphenhydramine 10 -s M. Average (+ S.E.) control rates before the perfusion with diphenhydramine 10 -7 and 10 -s M were 211 + 9/min and 243 + 10/min.
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Fig. 2. Effects of histamine, and histamine in the presence of antihistamines, on the sinus rate of the isolated heart. histamine alone; . . . . . . , histamine in the presence of antihistamine at 10 -7 M; . . . . , histamine in the presence of antihistamine at 10 -s M (for promethazine only the concentration was 3 X 10 -6 M). Points (means, n = 9 - 1 1 for histamine, and n = 5 - 7 for histamine in the presence of each concentration of antihistamines; vertical bars = S.E.) represent the maximum increase in sinus rate from the values immediately preceding each histamine injection. Average (+ S.E.) control sinus rates before histamine were: 233 + 8/min; 241 + 7/rain and 229 + 6/rain in the presence of promethazine 10 -7 and 3 X 10 4 M; 232 + 6/min and 176 + 9/min in the presence of cyclizine 10 -7 and 10 -5 M; 238 + 11/min and 171 ± . 8/mi~ in the presence of chlorpheniramine 10 -7 and 10-5 M; 238 + 13/min and 178 + 6/min in the presence of tripelennamine 10 -7 and 10 -s M.
R. LevL J.O. Kuye, Hi-Antagonists and histamine on heart
334
to histamine was the same in the presence or in the absence of the antihistamine.
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Each of the five antihistamines antagonized in a concentration dependent fashion the histamineinduced prolongation of the P - R interval. This resuited in a downward shift of the histamine d o s e response curve (figs. 1 and 4). At doses of histamine of 25 and 50 #g second degree atrioventricular Conduction block was frequently observed. The incidence of atrioventricular block was greatly reduced by the higher concentrations of antihistamines (table 3).
3.5. Antihistamines and the histamine-induced increase in coronary flow
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3.4. Antihistamines and the negative dromotropic el. fect o f histamine
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Fig. 3. Effects of histamine, and of histamine in the presence of antihistamines, on the ventricular contraction of the isolated heart. Open bars (means, -+S.E.) represent the maximum increase in ventricular contraction amplitude from the values immediately preceding each histamine injection (closed bars, means -+S.E.). Average (± S.E.) initial control values (made equal to 100) were: 7.5 ± 1.2 g in the experiments without antihistamine, and 8.1 -+ 0.3 g before chlorpheniramine, 7.8 ± 0.5 g before diphenhydramine, 7.8 ± 0.6 g before cyclizine, 6.7 ± 0.6 g before promethazine, and 7.7 ± 0.5 g before tripelennamine.
The effects o f histamine on the coronary circulation in the presence and absence of antihistamines were studied by the technique of constant perfusion (Levi, 1972). With constant perfusion of histamine the chronotropic and inotropic effects rapidly reach a plateau, thus allowing measurements of histamine changes in coronary flow rate under steady-state condition of cardiac contraction and rate. In these experiments, the effects on coronary flow of histamine, either alone or in combination with a fixed concentration of antihistamine, were determined. Histamine, in concentrations between 0.01 and 0.1 /ag/ml, caused a concentration dependent increase in coronary flow rate (fig~ 5). At histamine concentrations between 0.1 #g/ml and 10 #g/ml the increase in coronary flow rate became progressively smaller. As may be seen from fig. 5, in the presence of diphenhydramine 10-8 M the histamine-induced increase in
Table 3 Histamine-induced atrioventricular block a. Concentration of antihistamine b (M) 0 10 - 7
10- s c
Incidence 7/9 20/27 9/29
a Induced by the intra-aortic injection of a 50 #g dose of his. tamine in the isolated heart. b Diphenhydramine, promethazine, chlorpheniramine, tripel. ennamine and cyelizine. c For promethazine only the concentration was 3 X 10-6 M.
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Fig. 4. Effects of histamine, and of histamine in the presence of antihistamines, on the duration of the P - R interval in the isolated heart. , histamine alone; . . . . . . , histamine in the presence of antihistamine at 10-7 M; . . . . . . . histamine in the presence of antihistamine at 10 -s M (for promethazine only the concentration was 3 X 10 -6 M). Points (means, n = 9 - 1 1 for histamine, and n = 5 - 7 for histamine in the presence of each concentration of antihistamines; vertical bars = S.E.) represent the m a x i m u m increase in duration of the P - R interval from the values immediately preceding each histamine injection. Average (± S.E.) control values before histamine were 59.5 ± 0.5 msec; 61.0 ± 5.1 and 59.0 ± 4.0 msec in the presence of promethazine 10 -7 and 3 X 10 -6 M; 57.0 ± 3.4 and 61.0 +- 4.0 msec in the presence of cyclizine 10 -7 and 10 -s M; 62.5 ± 1.7 and 60.0 ± 2.9 msec in the presence of chlorpheniramine 10-7 and 10 -s M; 60.0 ± 0.0 and 60.0 ± 1.3 msec in the presence of tripelennamine 10 -7 and 10 -s M .
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H'STAMINt (pg/ml) Fig.-5. Effects of histamine, of histamine in combination with diphenhydramine 10 -s M, and of histamine in combination with chlorpheniramine 10 -7 M, on coronary_ flow rate of isolated hearts. Histamine was perfused alone in 28 experiments, in combination with diphenhydramine in 19 experiments and in combination with chlorpheniramine in 20 experiments. Points (means, n = 4 for histamine and n = 3 - 4 for histamine with antihistamines; vertical bars = S.E.) represent the increase in coronary flow rate after plateau level had been reached. Each heart was perf.used with only one concentration of histamine. Average (± S.E.) control coronary flow rate before histamine was 4.2 ± 0.1, and 4.7 ± 0.17 and 4.7 -+ 0.18 ml/min in the presence of diphenhydramine and chlorpheniramine.
336
R. Levi, J.O. Kuye, Hi-Antagonists and histamine on heart
coronary flow rate was reduced at all histamine concentrations. In the presence of chiorpheniramine 10 -7 M the coronary flow rate increased with increasing histamine concentrations; however, the histamine-induced effect was attenuated between histamine concentrations of 0.03 and 1/~g/ml (fig. 5). When histamine alone was perfused atrioventricular block developed at concentrations between 1 and 10 ~tg/ml. In the presence of diphenhydramine 10-8 M conduction arrhythmias occurred at histamine concentrations between 3 and 10 /ag/ml, whereas in the presence of chlorpheniramine 10 -7 M no conduction arrhythmias occurred.
4. Discussion 4.1. Effects o f antihistamines on cardiac function
The moderate increase in sinus rate at the lower concentrations of antihistamines (see table 1) may result either from direct cardiac stimulation or from potentiation of the chronotropic effects (Innes, 1958; Johnson and Kahn, 1966; Isaac and Goth, 1967) of spontaneously released (Hukovic and Muscholl, 1962) norepinephrine. A direct correlation between heart rate and the rate of coronary flow has been established in the isolated guinea pig heart (Levi, 1972). Thus, the moderate increase in coronary flow rate which we observe during continuous perfusion with diphenhydramine and chlorpheniramine (see table 2) may result from their postitive chronotropic effect. Each of the antihistamines displays a negative inotropic effect which is concentration dependent. In addition, at the higher concentrations a negative chronotropic effect is observed (see table 1). These myocardial depressant actions are most probably related to the well-known local anesthetic and quinidine-like properties of antihistamines (Dews and Graham, 1946; Dutta, 1949). 4.2. Antihistamines and the cardiac effects o f histamine
Our results clearly indicate that antihistamines do not inhibit the increase in rate and contractility induced by histamine in the isolated guinea pig heart.
This had been previously, reported for ethylenediamine, phenothiazine and ethanolamine-type antihistamines (Trendelenburg, 1960; Bartlet, 1963). We have now found that alkylamine (i.e., chlorpheniramine) and piperazine (i.e., cyclizine) antihistamines also fail to antagonize the histamine-induced stimulation of cardiac rate and contractility. We infer from these results that antihistamines from each of the five chemical classes cannot inhibit histamine-induced effects on cardiac rate and contractility. The positive chronotropic effect of low doses of histamine is potentiated by cyclizine 10-s M, whereas the effect of higher doses of histamine is slightly attenuated by cyclizine at this concentration (see fig. 2). Thus, the entire dose-response curve for the chronotropic effect of histamine is flattened. Since this occurred only with the higher concentration of cyclizine and not with the other four antihistamines, it is unlikely that the modest attenuation of the positive chronotropic effect of histamine by cyclizine is a specific antihistamine effect. Each of the antihistamines potentiates the positive chronotropic effect of the lower doses of histamine. The response to histamine is potentiated at all doses by chlorpheniramine 10-s M so that the entire doseresponse curve is shifted to the left (see fig. 2). Furthermore, each antihistamine potentiates the positive inotropic effect of histamine (see fig. 3). These findings are not surprising, since antihistamines are known to potentiate several of the other actions of histamine. These actions include the stimulation of gastric acid secretion in the cat (Howat and Schofield, 1954), the contraction of the isolated ileum of the guinea pig (Feldberg and Smith, 1954), and the vasodilatation in the perfused leg of the dog (Murray and Huston, 1952). Antihistamines may possibly act as partial agonists and/or as histamine releasers (Mota and Dias da Silva, 1960). In the isolated guinea pig heart undergoing anaphylaxis, chlorpheniramine at 10 -6 M potentiates the positive chronotropic effect of released histamine, while at 10-s M it potentiates histamine release (Levi and Capurro, 1973). Cardiac stimulation by chlorpheniramine may also result from its tyramine-like action (Davis and McNeiU, 1973). In the heart-lung preparation of the dog promethazine antagonizes the negative dromotropic effect of histamine (Flacke et al., 1967). In our preparation each of the antihistamines clearly antagonizes
R. Levi, ZO. Kuye, Hi-Antagonists and histamine on heart
the impairment of atrioventricular conduction caused by histamine. The slope of the dose-response curve for the prolongation of the P - R interval is decreased in a concentration dependent fashion (see figs. 1 and 4). This shift does not meet the criterion of the competitive antagonism (Goldstein et al., 1968)since neither the slope of the dose-response curve nor the maximum response coincide with those obtained with histamine alone. However, since the response is the resultant of multiple components, and antihistamines selectively affect one or more of the components, the possible competitive nature of this antagonism could be obscured. In the isolated guinea pig heart histamine prolongs the P - R interval by acting directly on the atrioventricular node and also indirectly by way of its concomitant positive chronotropic effect (Levi, 1972). Antihistamines not only do not antagonize the positive chronotropic effect of histamine, but actually potentiate this effect at the lower doses of histamine. Consequently, the inhibition of the negative dromotropic effect of histamine does not result in a parallel shift of the dose-response curve. The antagonism of the negative dromotropic effect of histamine also results in a lower incidence of atrioventricular block in the presence of antihistamines (see table 3). Furthermore, it is evident that diphenhydramine and chlorpheniramine increase the threshold for histamine-induced atrioventricular block as a function of their concentration (see fig. 5). This antiarrhythmic action is not a quinidine-like effect but rather a specific effect of antihistamines, because it results from the inhibition of histamine at the atrioventricular node. The well-known quinidine-like actions of antihistamines would be expected to result in a further slowing of atrioventricular conduction by prolongation of the refractory period of the node (Dews and Graham, 1946; Winbury and Alworth, 1959). As previously reported (Levi, 1972), the increase in rate of coronary flow caused by histamine is concentration dependent, but the increments become progressively smaller with the impairment of atrioventricular conduction (see fig. 5). Both diphenhydramine 10-aM and chlorpheniramine 10-TM antagonize the coronary-dilating effect of histamine. Chlorpheniramine, but not diphenhydramine, prevents the occurrence of atrioventricular block. This is probably due to a difference in concentration rather than to a difference in activity. The concentration-response
337
curves for histamine-induced increases in the rate of coronary flow in the presence of diphenhydramine and chlorpheniramine are superimposable up to the point where conduction arrhythrnia develops in the presence of diphenhydramine (see fig. 5). Prevention of arrhythmia by chlorpheniramine allows greater coronary flow increments at higher concentrations of histamine. The failure of diphenhydramine to prevent atrioventricular block at these higher concentrations of histamine reduces the increments in rate of coronary flow. Thus, the concentration-response curve for histamine in the presence of diphenhydramine parallels histamine alone, while the curve for chlorpheniramine continues in a linear fashion. It is probable that diphenhydramine and chlorpheniramine competitively antagonize the coronary-dilating effect of lower concentrations of histamine. This is illustrated by the parallel shift of a portion of the concentration-response curve for histamine in the presence of chlorpheniramine and diphenhydramine (see fig. 5). At higher concentrations of histamine the shift of the curve produced by diphenhydramine is complicated by the presence of arrhythmias. In conclusion, our results clearly indicate that: (1) each of the five prototypic antihistamines fail to inhibit the histamine-induced increase in cardiac rate and contractility; (2) each of the antihistamines inhibits the negative dromotropic effect of histamine; (3) chlorpheniramine and diphenhydramine antagonize the coronary-dilating effect of histamine. Thus, our results suggest that two types of histamine receptors may be present in the guinea pig heart: HI -receptors (Ash and Schild, 1966), at the atrioventricular node and coronary vessels; H2-receptors (Black et al., 1972), at the sinoatrial node and on the ventricular fibers. This hypothesis is supported by the observations that: (a) promethazine, chlorpheniramine and diphenhydramine antagonize the effect of histamine at the atrioventricular node in the same decreasing order of potency (see dose-response curves for the lower concentrations of these antihistamines in figs. 1 and 4) as in the guinea pig ileum (pA2 and pAl o values calculated by Marshall, 1955) where histamine effect is mediated by H~-receptors (Ash and Schild, 1966; Black et al., 1972); (b) 2-methyl histamine (H~-receptor agonist; Black et al., 1972; GaneUin, 1973) slows atrioventricular conduction without increasing rate and contractility in the isolated guinea
338
R. Levi, J.O. Kuye, Hi-Antagonists and histamine on heart
pig heart (Levi and Lee, 1974); (c) burimamide, the newly synthesized H2-histamine receptor blocker (Black et al., 1972), competitively inhibits the positive chronotropic (Black et al., 1972; Capurro and Levi, 1973; Levi and Capurro, 1973) and positive inotropic (Capurro and Levi, 1973; Levi and Lee, 1974; McNeill and Verma, 1974) but not the negative dromotropic effect of histamine (Levi and Lee, 1974) in the guinea pig heart; (d) 4-methyl histamine (H2-receptor agonist; Black et al., 1972; Ganellin, 1973) selectively increases sinus rate and ventricular contractility without impairing atrioventricular conduction in the guinea pig heart (Levi and Lee, 1974). The identification and quantitative characterization of each type of cardiac histamine receptor should provide important new information on the role of histamine in cardiac function in normal and pathological states.
Acknowledgments We are grateful to Drs. Charles E. Inturrisi and Norine Capurro for helpful criticism in the preparation of the manuscript.
References Ash, A.S.F. and H.O. Schild, 1966, Receptors mediating some actions of histamine, Brit. J. Pharmacol. 27,427. Bartlet, L.A., 1963, The action of histamine on the isolated heart, Brit. J. Pharmacol. 21,450. Black, J.W., W.A.M. Duncan, C.J. Durant, C.R. Ganellin and E.M. Parsons, 1972, Definition and antagonism of histamine H2-receptors, Nature (London) 236,385. Capurro, N. and R. Levi, 1973, Anaphylaxis in the isolated guinea pig heart: selective inhibition by burimamide of the positive inotropic and chronotropic effects of released histamine, Brit. J. Pharmacol. 48,620. Davis, R.S. and J.H. McNeill, 1973, The cardiac effects of cocaine and certain antihistamines and antidepressants, Arch. Intern. Pharmacodyn. 201,262. Dews, P.B. and J.D.P. Graham, 1946, The antihistamine substance 2786 R.P., Brit. J. Pharmacol. 1,278. Dutta, N.K., 1949, Some pharmacological properties common to antihistamine compounds, Brit. J. Pharmacol. 4, 281. Feldberg, W. and A.N. Smith, 1954, The role of histamine release for the motor effects of histamine liberators on the guinea-pig's ileum preparation, J. Physiol. (London) 124,219.
Flacke, W., D. Atanackovic, R.A. Gillis and M.H. Alper, 1967, The actions of histamine on the mammalian heart, J. Pharmacol. Exptl. Therap. 155,271. Ganellin, C.R., 1973, Conformation of histamine derivatives. 3. A relationship between conformation and pharmacological activity, J. Med. Chem. 16,620. Goldstein, A., L. Aronow and S.M. Kalman, 1968, Principles of Drug Action, The Basis of Pharmacology (Harper and Row, New York). Howat, H.T. and B. Schofield, 1954, The effect of urogastrone, enterogastrone and mepyramine maleate on gastric and pancreatic secretion, J. Physiol. (London) 123, 1. Hukovic, S. and E. Muscholl, 1962, Die Noradrenalin-Freisetzung aus dem isolierten Herzen dutch Sympatische Nervenreizung, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 243,348. Innes, I.R., 1958, Sensitization of the heart and nictitating membrane of the cat to sympathomimetic amines by antihistamine drugs, Brit. J. Pharmacol. 13, 6. Isaac, L. and A.Goth, 1967, The mechanism of the potentiation of norepinephrine by antihistaminics, J. Pharmacol. Exptl. Therap. 156,463. Johnson, G.L. and J.B. Kahn, 1966, Cocaine and antihistamine compounds: comparison of effects of some cardiovascular actions of norepinephrine, tyramine and bretylium, J. Pharmacol. Exptl. Therap. 152,458. Levi, R., 1972, Effects of exogenous and immunologicaliy released histamine on the isolated heart: a quantitative comparison, J. Pharmacol. Exptl. Therap. 182,227. Levi, R. and N. Capurro, 1973, Histamine H2-receptor antagonism and cardiac anaphylaxis, International Symposium on Histamine H2-Receptor Antagonists, eds. C.J. Wood and M.A. Simkins (Smith Kline & French, Ltd., London) p. 175. Levi, R. and C.-H. Lee, 1974, Characterization of cardiac histamine receptors by means of selective Hl- and H2agonists and antagonists, Federation Proc. 33, 585. Marshall, P.B., 1955, Some chemical and physical properties associated with histamine antagonism, Brit. J. Pharmacol. 10,270. McNeill, J.H. and S.C. Verma, 1974, Blockade by burimamide of the effects of histamine and histamine analogs on cardiac contractility, phosphorylase activation and cyclic adenosine monophosphate, J. Pharmacol. Exptl. Therap. 188,180. Mota, I. and W. Dias da Silva, 1960, The anti-anaphylactic and histamine-releasing properties of the antihistamines. Their effect on the mast cells, Brit. J. Pharmaeol. 15,396. Murray, J.R. and M.J. Huston, 1952, The effect of diphenhydramine HC1 (Benadryl HC1) on skeletal muscle, Arch. Intern. Pharmacodyn. 89,204. Trendelenburg, U., 1960, The action of histamine and 5-hydroxytryptamine on isolated mammalian atria, J. Pharmacol. Exptl. Therap. 130,450. Winbury, M.M. and B.L. Alworth, 1959, Suppression of experimental atrial arrhythmias by several antihistamines, Arch. Intern. Pharmacodyn. 122,318.
PHARMACOLOGICAL
CHARACTERIZATION
OF CARDIAC HISTAMINE
R E C E P T O R S : S E N S I T I V I T Y T O H 1 - R E C E P T O R A N T A G O N I S T S 1,2 Roberto LEVI and J. Olatunde KUYE 3 Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York, N. Y. 10021, U.S.A.
Received 10 December 1973, accepted 8 April 1974 R. LEVI and J.O. KUYE, Pharmacological characterization of cardiac histamine receptors: sensitivity to H 1-receptor antagonists, European J. Pharmacol. 27 (1974) 330-338. The ability of five prototypic antihistamines (diphenhydramine, promethazine, cyclizine, chlorpheniramine and tripelennamine) to modify the cardiac actions of histamine was studied in order to determine the reactivity of cardiac histamine receptors. Histamine, as a function of dose, increases the rate, the force of contraction, the coronary flow and the time of atrioventricular conduction of the isolated guinea pig heart. Each of the five prototypic antihistamines failed to inhibit histamine-induced stimulation of cardiac rate and contractility. At the same time these antihistamines did inhibit the negative dromotropic effect of histamine. Chlorpheniramine and diphenhydramine antagonized the coronary dilating effect of histamine. These results suggest that two types of histamine receptors may be present in the guinea pig heart: Hi-receptors, at the atrioventricular node and coronary vessels; H2-receptors at the sinoatrial node and on the ventricular fibers. The activity of these prototypic antihistamines is consistent with their action exclusively on H 1-receptorsCardiac histamine receptors Cardiac effects of histamine
Antihistamines
1. Introduction In the isolated heart of the guinea pig histamine induces positive inotropic, positive chronotropic as well as negative dromotropic effects. Also, histamine increases the rate of coronary flow. All of these effects are dose dependent (Levi, 1972). The positive inotropic and chronotropic effects of histamine on isolated cardiac preparations of the guinea pig are not inhibited by tripelennamine and pyrilamine (Trende1This work was supported by U.S.P.H.S. Grant No. 1RO1 GM 20091 by a grant-in-aid from the New York Heart Association, and in part by the John Polacheck Foundation for Medical Research. 2Preliminary results were presented at the 1972 Spring Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, U.S.A. 3Post-Doctoral Trainee in Pharmacology (U.S.P.H.S. Grant No. GM 00099). Present address: c/o Delbay Pharmaceuticals Inc., 60 Orange Street, Bloomfield, New Jersey 07003, U.S.A.
Histamine
Histamine H 1-receptor antagonists
lenburg, 1960) nor inhibited by mepyramine, pyrathiazine and diphenhydramine (Bartlet, 1963). In the h e a r t - l u n g preparation of the dog, promethazine and diphenhydramine do not block the effect of histamine on heart rate and contractility, while they block completely the negative dromotropic effect of histamine (Flacke et al., 1967). Those actions of histamine which are antagonized by low concentrations of antihistamines may result from the interaction of histamine with one type of receptor (HI), whereas histamine responses not inhibited by antihistamines may result from interaction with a different histamine receptor (Ash and Schild, 1966). Thus, the cardiac actions of histamine could be mediated by more than one histamine receptor. The purpose of the present study was to define in a quantitative manner the effects of five prototypic antihistamines (diphenhydramine, tripelennamine, chlorpheniramine, cyclizine, and promethazine) on each of the actions of histamine in the isolated guinea pig heart. This information is required for the most
R. LevL J.O. Kuye, Hi-Antagonists and histamine on heart complete characterization of cardiac histamine receptors.
2. Materials and methods
2.1. Perfusion o f the isolated heart Male C a m m - H a r t l e y guinea pigs weighing between 250 and 300 g were stunned by a blow to the base of the skull. The heart was removed and m o u n t e d in a Langendorff apparatus (Levi, 1972) within a minute or less and perfused at constant pressure (40 cm of water) with oxygenated Ringer solution at 37.5°C. The composition of the Ringer solution was (mM/1): Na ÷ 160; C1- 164; K ÷ 5.6; Ca 2÷ 2.2; HCO3- 5.9; glucose 5.5. Isometric ventricular contractions, surface electrograms and coronary flow rate were recorded as previously described (Levi, 1972). Heart rate and atrioventricular conduction time ( P - R interval) were determined from the electrocardiographic tracings. Hearts were perfused for 30 to 45 min before experimentation was begun, by which time heart rate and contraction had stabilized. Histamine in increasing doses was injected intra-aortically. Histamine was dissolved in oxygenated Ringer solution kept at 37°C and the volume of all injections remained constant at
331
0.1 ml. Histamine injections were made every 20 min for a total of seven injections per experiment. In other experiments hearts were continuously perfused with a selected concentration of an antihistamine. 10 min after the beginning of this perfusion, histamine, in increasing doses, was administered intra-aortically as described above. The perfusion with the antihistamine was continued for 30 min after the last injection of histamine. The effects on the rate of coronary flow, of a constant perfusion with either histamine or histamine in combination with an antihistamine were also studied. Hearts were perfused for 10 min with a selected concentration of histamine (from 0.1 #g/ml to 10 /~g/ml). Each heart was perfused with only one concentration of histamine. When a combination histam i n e - a n t i h i s t a m i n e was studied, perfusion with the antihistamine alone was initiated 10 rain before perfusion with the combination.
2.2. Drugs Histamine dihydrochloride was purchased from Sigma Chemical Company (St. Louis, Missouri, U.S.A.); cyclizine hydrochloride, chlorpheniramine maleate, diphenhydramine hydrochloride, promethazine hydrochloride, and tripelennamine hydrochloride were gifts respectively of Burroughs Wellcome Co.
Table 1 Chronotropic, inotropic and dromotropic effects of antihistaminesa. Compound
Concentration (M)
n
Rate b
Contractionc
P-R interval c
Diphenhydramine
10- 8 10- 7 10-5 10- 7 3X 10- 6 10-7 10- s 10 - 7 10 -5 10-7 10- s
19 6 5 5 5 6 5 27 7 5 7
+15 +- 3d,e +4 + 8 -26 -+6e +10 +-4 -14_+4 e +13 _+6 -59-+ 10g +13 -+ 3e -35 -+6g +1 ± 6 -51 -+5g
-11 +- 2e - 5 -+5 -25 ± 3g -14 +-5 - 2 2 ~ 1e - 7 -+2 f -19 + 6e -6 ± 1 -30 ± 5g -5± 1 -26 +~3f
+1 + 1 0 +14 -+ 3f +3 +- 5 +3+- 2 -5 -+4 - 2 +- 4 +3 -+ 1 +5 -+ 2 +2 ± 2 +1 + 4
Promethazine Cyclizine Chlorpheniramine Tripelennamine
a Isolated hearts. b Changes in beats/rain. c Percent changes (ventricular contraction amplitude or P-R interval duration. d Means (+ S.E.) of changes observed after 10 min of peffusion with antihistamine. e0.1 > p > 0.05. f 0.025 > p > 0.01. gp < 0.01.
R. LevL J.O. Kuye, Hi-Antagonists and histamine on heart
332
(Research Triangle Park, North Carolina, U.S.A.), Schering Corporation (Bloomfield, New Jersey, U.S.A.), Parke, Davis & Co. (Detroit, Michigan, U.S.A.), Wyeth Laboratories, Inc. (Philadelphia, Pennsylvania, U.S.A.) and Ciba Pharmaceuticals Company (Summit, New Jersey, U.S.A.). All histamine doses and concentrations refer to the free base (M.W. =111).
3. Results
3.1. Effects o f antihistamines on cardiac function The effects of the five prototypic antihistamines on heart rate, ventricular contraction and P - R interval are shown in table 1. At concentrations of 10 -7 M or less antihistamines produced small but consistent increases in heart rate. At the concentration of 10-TM tripelennamine did not influence the heart rate. At the concentration of 10-s M each of the antihistamines caused a marked negative chronotropic effect. Each of the antihistamines caused a concentration dependent negative inotropic effect. At 10-s M promethazine caused contractile failure (not shown in the table). Antihistamines did not modify the duration of the P - R interval, with the exception of 10-s M diphenhydramine, which prolonged the P - R interval. The effects of antihistamines on coronary flow were studied with 10-aM diphenhydramine and 10-TM chlorpheniramine. Both drugs increased the rate of coronary flow (table 2).
3.2. Antihistamines and, the positive chronotropic effect o f histamine The dose dependent positive chronotropic effect of histamine was not blocked diphenhydramine, promethazine, chlorpheniramine or tripelennamine (figs. 1 and 2). Cyclizine at 10-s M attenuated the chronotropic response to doses of histamine from 5 to 50 gig. The positive chronotropic response to doses of 0.25 and 0.5 gig of histamine was increased by the addition of each of the antihistamines (figs. 1 and 2). Chlorpheniramine at 10-s M shifted the entire doseresponse curve for histamine to the left (fig. 2).
3.3. Antihistamines and the positive inotropic effect o f histamine As shown in fig. 3 (upper left panel) there was a progressive decrease in the force of ventricular contraction. Under these conditions histamine produced an increase in ventricular contraction (positive inotropic effect) when compared to the corresponding control (fig. 3). At a concentration of 10-s M (or at 3 )(, 10-6 M for promethazine) the antihistamines produced a significant reduction in ventricular contraction (compare control before antihistamine with response after antihistamine in fig. 3). When histamine was injected in the presence of each of the antihistamines the increase in ventricular contraction produced by histamine was greater than t h a t seen in the absence of antihistamine (fig. 3). When the concentration of the antihistamine was reduced to 10-7M (not shown in the figure) the response of the ventricular contraction
Table 2 Effect of antihistamines on the rate of coronary flowa. Compound
Diphenhydramine Chlorpheniramine
Concentration (M)
10-8 10-7
n
19 20
a Isolated hearts continuously peffused with the drug. b Means ± S.E.
c0.1>p>0.5. d 0.05 > p > 0.025.
Coronary flow rate (ml/min) Control
Treated
% increase
4.7 ± 0.17b 4.7 ± 0.18
5.1 :~ 0.18c 5.2 ± 0.17d
9.4 ± 2.1 9.8 ± 2.1
R. Levi, J.O. Kuye, Hi-Antagonists and histamine on heart
SINUS RATE
140
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Fig. 1. Effects of histamine and of histamine in the presence of diphenhydramine on the sinus rate and atrioventricular conduction of the isolated heart. , histamine alone; . . . . . . , histamine in the presence of diphenhydramine 10 -7 M; . . . . . . , histamine in the presence of dipfienhydramine 10-5 M. Points (means, n = 9 - 1 1 for histamine, and n = 5 - 6 for histamine in the presence of each concentration of diphenhydramine; vertical bars = S.E.) represent the maxim u m increase in sinus rate and P - R interval duration from the values immediately preceding each histamine injection. Average (+ S.E.) control values before histamine for rate and P - R interval duration were 233 + 8/min and 59.5 + 0.5 msec; 216 -+ 13/min and 58.8 + 3.3 msec in the presence of diphenhydramine 10 -7 M; 216 + 11/min and 63.7 +- 3.3 msec in the presence of diphenhydramine 10 -s M. Average (+ S.E.) control rates before the perfusion with diphenhydramine 10 -7 and 10 -s M were 211 + 9/min and 243 + 10/min.
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Fig. 2. Effects of histamine, and histamine in the presence of antihistamines, on the sinus rate of the isolated heart. histamine alone; . . . . . . , histamine in the presence of antihistamine at 10 -7 M; . . . . , histamine in the presence of antihistamine at 10 -s M (for promethazine only the concentration was 3 X 10 -6 M). Points (means, n = 9 - 1 1 for histamine, and n = 5 - 7 for histamine in the presence of each concentration of antihistamines; vertical bars = S.E.) represent the maximum increase in sinus rate from the values immediately preceding each histamine injection. Average (+ S.E.) control sinus rates before histamine were: 233 + 8/min; 241 + 7/rain and 229 + 6/rain in the presence of promethazine 10 -7 and 3 X 10 4 M; 232 + 6/min and 176 + 9/min in the presence of cyclizine 10 -7 and 10 -5 M; 238 + 11/min and 171 ± . 8/mi~ in the presence of chlorpheniramine 10 -7 and 10-5 M; 238 + 13/min and 178 + 6/min in the presence of tripelennamine 10 -7 and 10 -s M.
R. LevL J.O. Kuye, Hi-Antagonists and histamine on heart
334
to histamine was the same in the presence or in the absence of the antihistamine.
No Antihistamine
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Each of the five antihistamines antagonized in a concentration dependent fashion the histamineinduced prolongation of the P - R interval. This resuited in a downward shift of the histamine d o s e response curve (figs. 1 and 4). At doses of histamine of 25 and 50 #g second degree atrioventricular Conduction block was frequently observed. The incidence of atrioventricular block was greatly reduced by the higher concentrations of antihistamines (table 3).
3.5. Antihistamines and the histamine-induced increase in coronary flow
40
g
3.4. Antihistamines and the negative dromotropic el. fect o f histamine
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The effects o f histamine on the coronary circulation in the presence and absence of antihistamines were studied by the technique of constant perfusion (Levi, 1972). With constant perfusion of histamine the chronotropic and inotropic effects rapidly reach a plateau, thus allowing measurements of histamine changes in coronary flow rate under steady-state condition of cardiac contraction and rate. In these experiments, the effects on coronary flow of histamine, either alone or in combination with a fixed concentration of antihistamine, were determined. Histamine, in concentrations between 0.01 and 0.1 /ag/ml, caused a concentration dependent increase in coronary flow rate (fig~ 5). At histamine concentrations between 0.1 #g/ml and 10 #g/ml the increase in coronary flow rate became progressively smaller. As may be seen from fig. 5, in the presence of diphenhydramine 10-8 M the histamine-induced increase in
Table 3 Histamine-induced atrioventricular block a. Concentration of antihistamine b (M) 0 10 - 7
10- s c
Incidence 7/9 20/27 9/29
a Induced by the intra-aortic injection of a 50 #g dose of his. tamine in the isolated heart. b Diphenhydramine, promethazine, chlorpheniramine, tripel. ennamine and cyelizine. c For promethazine only the concentration was 3 X 10-6 M.
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Fig. 4. Effects of histamine, and of histamine in the presence of antihistamines, on the duration of the P - R interval in the isolated heart. , histamine alone; . . . . . . , histamine in the presence of antihistamine at 10-7 M; . . . . . . . histamine in the presence of antihistamine at 10 -s M (for promethazine only the concentration was 3 X 10 -6 M). Points (means, n = 9 - 1 1 for histamine, and n = 5 - 7 for histamine in the presence of each concentration of antihistamines; vertical bars = S.E.) represent the m a x i m u m increase in duration of the P - R interval from the values immediately preceding each histamine injection. Average (± S.E.) control values before histamine were 59.5 ± 0.5 msec; 61.0 ± 5.1 and 59.0 ± 4.0 msec in the presence of promethazine 10 -7 and 3 X 10 -6 M; 57.0 ± 3.4 and 61.0 +- 4.0 msec in the presence of cyclizine 10 -7 and 10 -s M; 62.5 ± 1.7 and 60.0 ± 2.9 msec in the presence of chlorpheniramine 10-7 and 10 -s M; 60.0 ± 0.0 and 60.0 ± 1.3 msec in the presence of tripelennamine 10 -7 and 10 -s M .
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H'STAMINt (pg/ml) Fig.-5. Effects of histamine, of histamine in combination with diphenhydramine 10 -s M, and of histamine in combination with chlorpheniramine 10 -7 M, on coronary_ flow rate of isolated hearts. Histamine was perfused alone in 28 experiments, in combination with diphenhydramine in 19 experiments and in combination with chlorpheniramine in 20 experiments. Points (means, n = 4 for histamine and n = 3 - 4 for histamine with antihistamines; vertical bars = S.E.) represent the increase in coronary flow rate after plateau level had been reached. Each heart was perf.used with only one concentration of histamine. Average (± S.E.) control coronary flow rate before histamine was 4.2 ± 0.1, and 4.7 ± 0.17 and 4.7 -+ 0.18 ml/min in the presence of diphenhydramine and chlorpheniramine.
336
R. Levi, J.O. Kuye, Hi-Antagonists and histamine on heart
coronary flow rate was reduced at all histamine concentrations. In the presence of chiorpheniramine 10 -7 M the coronary flow rate increased with increasing histamine concentrations; however, the histamine-induced effect was attenuated between histamine concentrations of 0.03 and 1/~g/ml (fig. 5). When histamine alone was perfused atrioventricular block developed at concentrations between 1 and 10 ~tg/ml. In the presence of diphenhydramine 10-8 M conduction arrhythmias occurred at histamine concentrations between 3 and 10 /ag/ml, whereas in the presence of chlorpheniramine 10 -7 M no conduction arrhythmias occurred.
4. Discussion 4.1. Effects o f antihistamines on cardiac function
The moderate increase in sinus rate at the lower concentrations of antihistamines (see table 1) may result either from direct cardiac stimulation or from potentiation of the chronotropic effects (Innes, 1958; Johnson and Kahn, 1966; Isaac and Goth, 1967) of spontaneously released (Hukovic and Muscholl, 1962) norepinephrine. A direct correlation between heart rate and the rate of coronary flow has been established in the isolated guinea pig heart (Levi, 1972). Thus, the moderate increase in coronary flow rate which we observe during continuous perfusion with diphenhydramine and chlorpheniramine (see table 2) may result from their postitive chronotropic effect. Each of the antihistamines displays a negative inotropic effect which is concentration dependent. In addition, at the higher concentrations a negative chronotropic effect is observed (see table 1). These myocardial depressant actions are most probably related to the well-known local anesthetic and quinidine-like properties of antihistamines (Dews and Graham, 1946; Dutta, 1949). 4.2. Antihistamines and the cardiac effects o f histamine
Our results clearly indicate that antihistamines do not inhibit the increase in rate and contractility induced by histamine in the isolated guinea pig heart.
This had been previously, reported for ethylenediamine, phenothiazine and ethanolamine-type antihistamines (Trendelenburg, 1960; Bartlet, 1963). We have now found that alkylamine (i.e., chlorpheniramine) and piperazine (i.e., cyclizine) antihistamines also fail to antagonize the histamine-induced stimulation of cardiac rate and contractility. We infer from these results that antihistamines from each of the five chemical classes cannot inhibit histamine-induced effects on cardiac rate and contractility. The positive chronotropic effect of low doses of histamine is potentiated by cyclizine 10-s M, whereas the effect of higher doses of histamine is slightly attenuated by cyclizine at this concentration (see fig. 2). Thus, the entire dose-response curve for the chronotropic effect of histamine is flattened. Since this occurred only with the higher concentration of cyclizine and not with the other four antihistamines, it is unlikely that the modest attenuation of the positive chronotropic effect of histamine by cyclizine is a specific antihistamine effect. Each of the antihistamines potentiates the positive chronotropic effect of the lower doses of histamine. The response to histamine is potentiated at all doses by chlorpheniramine 10-s M so that the entire doseresponse curve is shifted to the left (see fig. 2). Furthermore, each antihistamine potentiates the positive inotropic effect of histamine (see fig. 3). These findings are not surprising, since antihistamines are known to potentiate several of the other actions of histamine. These actions include the stimulation of gastric acid secretion in the cat (Howat and Schofield, 1954), the contraction of the isolated ileum of the guinea pig (Feldberg and Smith, 1954), and the vasodilatation in the perfused leg of the dog (Murray and Huston, 1952). Antihistamines may possibly act as partial agonists and/or as histamine releasers (Mota and Dias da Silva, 1960). In the isolated guinea pig heart undergoing anaphylaxis, chlorpheniramine at 10 -6 M potentiates the positive chronotropic effect of released histamine, while at 10-s M it potentiates histamine release (Levi and Capurro, 1973). Cardiac stimulation by chlorpheniramine may also result from its tyramine-like action (Davis and McNeiU, 1973). In the heart-lung preparation of the dog promethazine antagonizes the negative dromotropic effect of histamine (Flacke et al., 1967). In our preparation each of the antihistamines clearly antagonizes
R. Levi, ZO. Kuye, Hi-Antagonists and histamine on heart
the impairment of atrioventricular conduction caused by histamine. The slope of the dose-response curve for the prolongation of the P - R interval is decreased in a concentration dependent fashion (see figs. 1 and 4). This shift does not meet the criterion of the competitive antagonism (Goldstein et al., 1968)since neither the slope of the dose-response curve nor the maximum response coincide with those obtained with histamine alone. However, since the response is the resultant of multiple components, and antihistamines selectively affect one or more of the components, the possible competitive nature of this antagonism could be obscured. In the isolated guinea pig heart histamine prolongs the P - R interval by acting directly on the atrioventricular node and also indirectly by way of its concomitant positive chronotropic effect (Levi, 1972). Antihistamines not only do not antagonize the positive chronotropic effect of histamine, but actually potentiate this effect at the lower doses of histamine. Consequently, the inhibition of the negative dromotropic effect of histamine does not result in a parallel shift of the dose-response curve. The antagonism of the negative dromotropic effect of histamine also results in a lower incidence of atrioventricular block in the presence of antihistamines (see table 3). Furthermore, it is evident that diphenhydramine and chlorpheniramine increase the threshold for histamine-induced atrioventricular block as a function of their concentration (see fig. 5). This antiarrhythmic action is not a quinidine-like effect but rather a specific effect of antihistamines, because it results from the inhibition of histamine at the atrioventricular node. The well-known quinidine-like actions of antihistamines would be expected to result in a further slowing of atrioventricular conduction by prolongation of the refractory period of the node (Dews and Graham, 1946; Winbury and Alworth, 1959). As previously reported (Levi, 1972), the increase in rate of coronary flow caused by histamine is concentration dependent, but the increments become progressively smaller with the impairment of atrioventricular conduction (see fig. 5). Both diphenhydramine 10-aM and chlorpheniramine 10-TM antagonize the coronary-dilating effect of histamine. Chlorpheniramine, but not diphenhydramine, prevents the occurrence of atrioventricular block. This is probably due to a difference in concentration rather than to a difference in activity. The concentration-response
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curves for histamine-induced increases in the rate of coronary flow in the presence of diphenhydramine and chlorpheniramine are superimposable up to the point where conduction arrhythrnia develops in the presence of diphenhydramine (see fig. 5). Prevention of arrhythmia by chlorpheniramine allows greater coronary flow increments at higher concentrations of histamine. The failure of diphenhydramine to prevent atrioventricular block at these higher concentrations of histamine reduces the increments in rate of coronary flow. Thus, the concentration-response curve for histamine in the presence of diphenhydramine parallels histamine alone, while the curve for chlorpheniramine continues in a linear fashion. It is probable that diphenhydramine and chlorpheniramine competitively antagonize the coronary-dilating effect of lower concentrations of histamine. This is illustrated by the parallel shift of a portion of the concentration-response curve for histamine in the presence of chlorpheniramine and diphenhydramine (see fig. 5). At higher concentrations of histamine the shift of the curve produced by diphenhydramine is complicated by the presence of arrhythmias. In conclusion, our results clearly indicate that: (1) each of the five prototypic antihistamines fail to inhibit the histamine-induced increase in cardiac rate and contractility; (2) each of the antihistamines inhibits the negative dromotropic effect of histamine; (3) chlorpheniramine and diphenhydramine antagonize the coronary-dilating effect of histamine. Thus, our results suggest that two types of histamine receptors may be present in the guinea pig heart: HI -receptors (Ash and Schild, 1966), at the atrioventricular node and coronary vessels; H2-receptors (Black et al., 1972), at the sinoatrial node and on the ventricular fibers. This hypothesis is supported by the observations that: (a) promethazine, chlorpheniramine and diphenhydramine antagonize the effect of histamine at the atrioventricular node in the same decreasing order of potency (see dose-response curves for the lower concentrations of these antihistamines in figs. 1 and 4) as in the guinea pig ileum (pA2 and pAl o values calculated by Marshall, 1955) where histamine effect is mediated by H~-receptors (Ash and Schild, 1966; Black et al., 1972); (b) 2-methyl histamine (H~-receptor agonist; Black et al., 1972; GaneUin, 1973) slows atrioventricular conduction without increasing rate and contractility in the isolated guinea
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R. Levi, J.O. Kuye, Hi-Antagonists and histamine on heart
pig heart (Levi and Lee, 1974); (c) burimamide, the newly synthesized H2-histamine receptor blocker (Black et al., 1972), competitively inhibits the positive chronotropic (Black et al., 1972; Capurro and Levi, 1973; Levi and Capurro, 1973) and positive inotropic (Capurro and Levi, 1973; Levi and Lee, 1974; McNeill and Verma, 1974) but not the negative dromotropic effect of histamine (Levi and Lee, 1974) in the guinea pig heart; (d) 4-methyl histamine (H2-receptor agonist; Black et al., 1972; Ganellin, 1973) selectively increases sinus rate and ventricular contractility without impairing atrioventricular conduction in the guinea pig heart (Levi and Lee, 1974). The identification and quantitative characterization of each type of cardiac histamine receptor should provide important new information on the role of histamine in cardiac function in normal and pathological states.
Acknowledgments We are grateful to Drs. Charles E. Inturrisi and Norine Capurro for helpful criticism in the preparation of the manuscript.
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