Naunyn-Schmiedeberg's Arch. Pharmacol. 309, 65 68 (1979)
Naunyn-Schmiedeberg's
Archivesof
Pharmacology 9 by Springer-Verlag 1979
Histamine Receptors in the Guinea Pig Ileum G. Bertaccini, E. M o l i n a , L. Z a p p i a , a n d J. Zseli Istituto di Farmacologia, Universitfi di Parma, Ospedale Maggiore, 1-43100 Parma, Italy
S u m m a r y . H i s t a m i n e a n d some r e l a t e d c o m p o u n d s acting selectively on H z- or H i - r e c e p t o r s were tested for their ability to c o n t r a c t the g u i n e a pig ileum, in the usual w h o l e ileum p r e p a r a t i o n a n d in the l o n g i t u d i n a l muscle p r e p a r a t i o n . The c o n c e n t r a t i o n s elicited by histamine in b o t h k i n d s o f p r e p a r a t i o n s were n o t p o t e n t i a t e d by cimetidine or m e t i a m i d e a n d were n o t inhibited by a d m i n i s t r a t i o n o f H2 r e c e p t o r selective agonists in doses w h i c h were s u b t h r e s h o l d for c o n t r a c t ing the guinea pig ileum; higher doses o f the H 2 agonists could actually p o t e n t i a t e the effect o f histamine. The results o b t a i n e d suggest t h a t H 2 receptors with relaxing effect d o n o t occur in the guinea pig ileum or at least t h a t they are n o t involved in the c o n t r a c t i o n o f the l o n g i t u d i n a l muscle layers. T h e possibility t h a t a s u b - t y p e o f H 2 r e c e p t o r s with p r o p e r t i e s different f r o m those o f the " c l a s s i c a l " H 2 r e c e p t o r s so far k n o w n , exists in the guinea pig ileum, c a n n o t be excluded.
Key words: H i s t a m i n e -- H I a n d H 2 r e c e p t o r s G u i n e a p i g ileum - C i m e t i d i n e - M e t i a m i d e .
Introduction It is generally a c c e p t e d t h a t r e c e p t o r s m e d i a t i n g the h i s t a m i n e i n d u c e d c o n t r a c t i o n on the isolated guinea pig ileum are o f the H 1 type. This c o n c e p t which was b a s e d on the c o m p e t i t i v e a n t a g o n i s m s h o w n b y m e p y r a m i n e (Ash a n d Schild, 1966), received a further c o n f i r m a t i o n b y recent e x p e r i m e n t s which s h o w e d t h a t histamine r e c e p t o r s in the guinea pig ileum are labelled b y the b i n d i n g o f 3 H - m e p y r a m i n e (Hill et al., 1977). Nevertheless p r e v i o u s p a p e r s (Bareicha a n d R o c h a e Silva, 1975, 1976) r e p o r t e d t h a t b u r i m a m i d e p o t e n tiated the h i s t a m i n e i n d u c e d c o n t r a c t i o n s on the guinea Send offprint requests to G. Bertaccini at the above address
pig ileum. This was i n t e r p r e t e d as due to the relaxing effect i n d u c e d b y H 2 r e c e p t o r s p r e s e n t in the p r e p a r a tion which are usually m a s k e d b y the o v e r w h e l m i n g presence o f H 1 receptors. T h e f i n d i n g t h a t b u r i m a m i d e besides the H 2 b l o c k i n g a c t i o n can be e n d o w e d with o t h e r effects like a true s p a s m o g e n i c activity (Bertaccini et al., 1977), a n d the availability o f new, m o r e p o t e n t a n d m o r e specific H 2 - r e c e p t o r b l o c k e r s as well as H 2r e c e p t o r selective agonists, p r o m p t e d us to investigate m o r e t h o r o u g h l y the p r o b l e m o f the occurrence o f H 2 r e c e p t o r s in the guinea pig ileum.
Methods Smooth Muscle Experiments. Two sets of experiments were performed. In the first the Auerbach plexus longitudinal muscle strip preparation from guinea pig ileum (Paton and Vizi, 1969) was used. Longitudinal muscle strips weighing about 40 80 mg were set up in an organ bath of 10 ml capacity in Krebs solution at 36~ and bubbled through with 5 ~ carbon dioxide in oxygen. The Krebs solution had the following composition (mM): NaCt, 113 ; KCI, 4.7; CaC12, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO~, 25 and glucose 11.5. Responses to drugs added to the bath were recorded isometrically on a microdynamometer. In the second set of experiments the whole ileum preparation prepared in the usual way was used. Contractions were recorded with an isotonic lever and registered on a kymograph: dose-response curves were obtained in both kinds of experiments by adding to the bath single increasing doses of histamine or of the other agonists tested; drugs were allowed to act for 1 min, time interval between doses being 3 min in the absence (controls) or in the presence of cimetidine or metiamide. Atropine at a concentration of 10- 7 M was always present in the nutrient fluid. The preload of the strips was 300 mg tension; the resting tension was 250 rag. The pD 2 values reported in Table 1 were calculated according to Van Rossum (1963) using the regression line for each agonist. Statistical evaluation of the data was performed using the Student's test for paired or unpaired data when appropriate. Drugs. The following compounds were used in our investigation: histamine and atropine (Fluka); 5-methylhistamine, 5-ethylhistamine, 5-methyl-N'-methylhistamine synthetized and kindly supplied by Dr. Vitali, University of Parma. (These compounds can also be called 4-methylhistamine, 4-ethylhistamine and 4-methyl-N'-
0028-1298/79/0309/0065/$ 01.00
66
Naunyn-Schmiedeberg's Arch. Pharmacol. 309 (1979) lOO
Table 1. pD 2 values of different compounds on the guinea pig whole
#/
ileum (A) and in the longitudinal muscle preparation (B) Compound
pD 2 (A) S.E.M.
N
pD z (B) S.E.M.
N
Histamine 5-Methylhistamine 5-Ethylhistamine 2-Aminoethyl-thiazole
7.50_+0.30 4.63_+0.35 5.57_+0.35 6.53-+0.30
25 25 25 16
7.42_+0.33 4.65_+0.35 5.68_+0.30 6.37_+0.35
25 16 16 16
/
/A ii
E
/
/ //
I
E 50
?
g
N Number of experiments
/~ methylhistamine according to the numbering of the side chain of histamine which can be considered either as a 2-(4-imidazolyl)ethylamine or as a 2-(5-imidazolyl)ethylamine. Dimaprit ~ metiamide, cimetidine and 2-aminoethylthiazole (kindly supplied by Smith Kline and French, Welwyn Garden City, Herts, England).
Results
Most results were obtained on the longitudinal smooth muscle preparation; however they were very similar to those obtained on the classical whole ileum preparation. Histamine exerted the well known contracting effect starting from threshold doses of l0 -9 M and reaching the maximum effect at concentrations of 1.6 x 10 -6 M; 2-aminoethylthiazole (Durant et al., 1975) was less effective in terms of threshold doses (10-s M) but reached a maximum contraction which did not differ significantly from that of histamine; the compound 5-ethylhistamine, so far never tested in isolated guinea pig ileum, and 5-methylhistamine one of the most widely used H z selective agonists were much less active (Fig. 1). The pD2 values for these compounds in both types of preparations are reported in Table 1. No significant differences were found in the two preparations. A complete dose-response curve could not be constructed with dimaprit which did not consistently contract the ileum even in concentrations of 10- 4 M. In order to examine the possible interference of H z receptors in the contraction induced by histamine as suggested by the results of Bareicha and Rocha e Silva (1975, 1976), two sets of experiments were performed on the atropinized longitudinal muscle preparation: a) in the first, dose-response curves with histamine were constructed in the presence of metiamide or cimetidine; b) in the second, different H 2 receptor agonsists were tested for their ability to modify the contractions evoked by histamine. HN ~ H2N
\ / C-S-CH2-CH2-CH2-N / \
CH3
CHa
//
?
J
Log molar concentration
Fig. 1. Dose response curves for histamine (A A), 2-aminoethylthiazole (A A); 5-ethylhistamine (n v1) and 5-methylhistamine (-I). Values refer to the mean of the values obtained from 4 to 5 experiments on the longitudinal muscle preparation (continuous lines). The interrupted lines refer to the whole ileum preparation for which the single values were not reported
1~176 1 o~
so
5.4 x l O -10
5.4
1.6
xlO - 9 -" x l O - 8
5.4
1.6
5,4
1.6
x l O -B
xlO -7
xlO -6
xlO -6
Fig. 2. Contractions of the longitudinal muscle from guinea pig ileum after administration of different doses of histamine in the absence (white columns) or in the presence (black columns) of cimetidine 4 x 10 6 M. Mean responses of 3 to 4 preparations; vertical bars are standard errors
a) In our experimental conditions neither cimetidine ( 4 • 10 - 6 M ) (Fig. 2) nor metiamide (10 -5 M) (two
experiments) were able to affect the doese-response curves to histamine which did not differ significantly from those obtained in absence of the H 2 blockers, b) In the case of Hz receptor agonists results were as follows: 5-methyl-histamine and dimaprit were unable to reduce the contractions provoked by histamine when administered both immediately before the amine and at the time of maximum contraction. This was observed up to concentration of 1 . 3 x 1 0 - 6 M (four experiments); when higher concentrations were used, a potentiation of the effect of histamine but never an inhibition could be observed. 5-ethylhistamine which in
G. Bertaccini et al. : Histamine Receptors in the Guinea Pig Ileum
67
a previous investigation (Bertaccini et al., 1978) was found to possess a good, rather selective, stimulant action o n H 2 receptors, behaved as the methyl derivative. The same was true for 5-methyl-N'methylhistamine which, owing to the paucity of the material at our disposal, was not tested as a stimulant compound in this investigation but was previously reported (Bertaccini et al., 1976) to be an excellent H z selective agonist and to retain on the guinea pig ileum only 0.2 % of the activity of histamine. Similar results were obtained when the contractions of the longitudinal muscle preparation were induced by the Ha receptor selective agonist, 2-aminoethylthiazole (two experiments).
the classic H1 receptor blockers. However even in the presence of this artificial transformation of H a into H 2 receptors, stimulation of H 2 receptors gave rise to the usual dose-dependent contraction and not relaxation of the ileum. A relaxant effect of histamine in the presence of mepyramine was observed by Ambache et al. (1973) on the neurogenic atropine-resistant tetanic spasms elicited on the guinea pig ileum by field stimulation. This effect, however, was blocked burimamide but was not mimicked by selective H 2 agonists. Thus it cannot be related to the classic H 2 receptors. Provided that all the known H 2 inhibitors, and not only burimamide, are able to block this relaxant activity it may be suggested that a sub-type of H 2 receptors is involved in this phenomenon. Evidence for the existence of a sub-type of H 2 receptor in the guinea pig ileum was recently suggested by Fjalland (1979); however it was based on the clonidine (considered a H 2 agonist)-induced suppression of electrically-induced twitches, and of different antagonistic activity ratio of cimetidine and burimamide on the guinea pig ileum versus the isolated atria. According to very recent data (Chand and De Roth, 1978) the chicken ileum (circular muscle layer) contains Hz-inhibitory histamine receptors and this statement is based on the potentiation by metiamide on the contractions induced by histamine and not on those evoked by carbachol. Apart from the species differences which can largely explain different results, the lack of experiments performed with H z receptor selective agonists must again be pointed out.
Discussion
Our experiments tend to exclude the occurrence of "classical" H 2 receptors mediating a relaxing effect on the longitudinal muscle from guinea pig ileum, which was suggested by other investigations (Bareicha and Rocha e Silva, 1975, 1976). In fact metiamide and cimetidine the two most effective H e receptor blockers so far available, failed to modify the dose-response curve to histamine in the longitudinal muscle preparation. This is in accordance with data by Black et al. (1973); Kenakin et al. (1974) and Parsons (1977): these authors found that the H z receptor blockers do not affect the response to histamine up to concentrations of 10 -4 M and they can actually inhibit in a non competitive way, but never potentiate, the contracting effect of histamine. Another evidence against the presence of H 2 rec e p t o r s with relaxing activity was that based on the experiments performed with the H E receptors selective agonists: dimaprit, 5-methyl-histamine, 5-methyl-N'methylhistamine and 5-ethylhistamine added to the bath before the stimulants as well as during the maximum muscle contraction, failed to inhibit the spasmogenic effect of histamine or of 2-aminoethylthiazole but could actually potentiate it. 5-methylhistamine and 5-ethylhistamine were, in our experiments, sufficiently active as Ha agonists to exert a contracting effect on the guinea pig ileum though doses 100 to 1000 times as high as those of histamine had to be used. Conversely, dimaprit was apparently devoid of H 1 stimulant effects as already observed by Parsons et al. (1977). Another interesting and quite peculiar finding is again not in favour of the hypothesis of a relaxing effect induced through stimulation of H 2 receptors : Kenakin et al. (1974) showed a temperature-dependent interconversion of histamine H a into H z receptors and by lowering the temperature of the nutrient fluid down to 12~ they obtained a dose-response curve to histamine which was shifted to the right by metiamide and not by
Acknowledgements. This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Roma.
References Ambache, N., Killick, S. W., Aboo Zar, M.: Antagonism by burimamide of inhibitions induced by histamine in plexuscontaining longitudinal muscle preparations from guinea-pig ileum. Br. J. Pharmacol. 48, 3 6 2 - 3 6 3 P (1973) Ash, A. S. F., Schild, H. O. : Receptors mediating some actions of histamine. Br. J. Pharmacol. 2"7, 427-439 (1966) Bareicha, I., Rocha e Silva, M.: Occurrence of H2-receptors for histamine in the guinea-pig intestine. Biochem. Pharmacol. 24, 1215-1219 (1975) Bareicha, I., Rocha e Silva, M. : H 1- and Ha-receptors for histamine in the ileum of the guinea pig. Gen. Pharmacol. 7, 103-106 (1976) Bertaceini, G., Impicciatore, M., Vitali, T. : Biological activities of a new specific stimulant of histamine H z receptors. I1 Farmaco Ed. Sci. 31, 935-938 (1976) Bertaccini, G., Coruzzi, G., Molina, E., Chiavarini, M.: Action of histamine and related compounds on the pyloric sphincter of the rat. Rendic. Gastroenterol. 9, 163--168 (1977) Bertaccini, G., Coruzzi, G., Vitali, T. : Effect of histamine and related compounds on the papillary muscle of the guinea pig. Pharmacol. Res. Commun. 10, 747-757 (1978)
68 Black, J. W., Duncan, W. A. M., Emmett, J. C., Ganellin, C. R., Hesselbo, T., Parsons, M. E., Wyllie, J. H.: Metiamide - An orally active histamine H2-receptor antagonist. Agents and Actions 3/3, 133-137 (1973) Chand, N., De Roth, L.: Occurrence of H2-inhibitory histamine receptors in chicken ileum. Eur. J. Pharmacol. 52, 143-145 (1978) Durant, G. J., Ganellin, C. R., Parsons, M. E. : Chemical differentiation of histamine H1- and Hz-receptor agonists. J. Med. Chem. 18, 905-909 (1975) Fjalland, B. : Evidence for the existence of another type of histamine Hz-receptor in guinea-pig ileum. J. Pharm. Pharmacol. 31, 5 0 51 (1979) Hill, S. J., Young, J. M., Marrian, D. H. : Specific binding of 3Hmepyramine to histamine Ha receptors in intestinal smooth muscle. Nature 270, 361- 362 (1977) Kenakin, T. P., Krueger, C. A., Cook, D. A.: Temperaturedependent interconversion of histamine Ha and H2 receptors in guinea pig ileum. Nature 252, 54-55 (1974)
Naunyn-Schmiedeberg's Arch. Pharmacol. 309 (1979) Parsons, M. E. : The antagonism of histamine H2-receptors in vitro and in vivo with particular reference to the actions of cimetidine. Cimetidine: second international symposium on histamine Hzreceptor antagonists, pp. 13-20. Amsterdam-Oxford: Royal College of Physicians, Exeerpta Medica 1977 Parsons, M. E., Owen, D. A. A., Durant, G. J., Ganellin, C. R.: Dimaprit-S- 3-(N-N-dimethylamino)propyl isothiourea a highly specific histamine Hz-receptor agonist. Part. I. Pharmacology, Agents and Actions 7, 3 1 - 37 (1977) Paton, W. D. M., Vizi, E. S. : The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip. Br. J. Pharmacol. 35, 10-29 (1969) Van Rossum, J. M. : Cumulative dose-response curves. Arch. Int. Pharmacodyn. 143, 299-330 (1963)
Received March 5/Accepted July 2, 1979
Naunyn-Schmiedeberg's
Archivesof
Pharmacology 9 by Springer-Verlag 1979
Histamine Receptors in the Guinea Pig Ileum G. Bertaccini, E. M o l i n a , L. Z a p p i a , a n d J. Zseli Istituto di Farmacologia, Universitfi di Parma, Ospedale Maggiore, 1-43100 Parma, Italy
S u m m a r y . H i s t a m i n e a n d some r e l a t e d c o m p o u n d s acting selectively on H z- or H i - r e c e p t o r s were tested for their ability to c o n t r a c t the g u i n e a pig ileum, in the usual w h o l e ileum p r e p a r a t i o n a n d in the l o n g i t u d i n a l muscle p r e p a r a t i o n . The c o n c e n t r a t i o n s elicited by histamine in b o t h k i n d s o f p r e p a r a t i o n s were n o t p o t e n t i a t e d by cimetidine or m e t i a m i d e a n d were n o t inhibited by a d m i n i s t r a t i o n o f H2 r e c e p t o r selective agonists in doses w h i c h were s u b t h r e s h o l d for c o n t r a c t ing the guinea pig ileum; higher doses o f the H 2 agonists could actually p o t e n t i a t e the effect o f histamine. The results o b t a i n e d suggest t h a t H 2 receptors with relaxing effect d o n o t occur in the guinea pig ileum or at least t h a t they are n o t involved in the c o n t r a c t i o n o f the l o n g i t u d i n a l muscle layers. T h e possibility t h a t a s u b - t y p e o f H 2 r e c e p t o r s with p r o p e r t i e s different f r o m those o f the " c l a s s i c a l " H 2 r e c e p t o r s so far k n o w n , exists in the guinea pig ileum, c a n n o t be excluded.
Key words: H i s t a m i n e -- H I a n d H 2 r e c e p t o r s G u i n e a p i g ileum - C i m e t i d i n e - M e t i a m i d e .
Introduction It is generally a c c e p t e d t h a t r e c e p t o r s m e d i a t i n g the h i s t a m i n e i n d u c e d c o n t r a c t i o n on the isolated guinea pig ileum are o f the H 1 type. This c o n c e p t which was b a s e d on the c o m p e t i t i v e a n t a g o n i s m s h o w n b y m e p y r a m i n e (Ash a n d Schild, 1966), received a further c o n f i r m a t i o n b y recent e x p e r i m e n t s which s h o w e d t h a t histamine r e c e p t o r s in the guinea pig ileum are labelled b y the b i n d i n g o f 3 H - m e p y r a m i n e (Hill et al., 1977). Nevertheless p r e v i o u s p a p e r s (Bareicha a n d R o c h a e Silva, 1975, 1976) r e p o r t e d t h a t b u r i m a m i d e p o t e n tiated the h i s t a m i n e i n d u c e d c o n t r a c t i o n s on the guinea Send offprint requests to G. Bertaccini at the above address
pig ileum. This was i n t e r p r e t e d as due to the relaxing effect i n d u c e d b y H 2 r e c e p t o r s p r e s e n t in the p r e p a r a tion which are usually m a s k e d b y the o v e r w h e l m i n g presence o f H 1 receptors. T h e f i n d i n g t h a t b u r i m a m i d e besides the H 2 b l o c k i n g a c t i o n can be e n d o w e d with o t h e r effects like a true s p a s m o g e n i c activity (Bertaccini et al., 1977), a n d the availability o f new, m o r e p o t e n t a n d m o r e specific H 2 - r e c e p t o r b l o c k e r s as well as H 2r e c e p t o r selective agonists, p r o m p t e d us to investigate m o r e t h o r o u g h l y the p r o b l e m o f the occurrence o f H 2 r e c e p t o r s in the guinea pig ileum.
Methods Smooth Muscle Experiments. Two sets of experiments were performed. In the first the Auerbach plexus longitudinal muscle strip preparation from guinea pig ileum (Paton and Vizi, 1969) was used. Longitudinal muscle strips weighing about 40 80 mg were set up in an organ bath of 10 ml capacity in Krebs solution at 36~ and bubbled through with 5 ~ carbon dioxide in oxygen. The Krebs solution had the following composition (mM): NaCt, 113 ; KCI, 4.7; CaC12, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO~, 25 and glucose 11.5. Responses to drugs added to the bath were recorded isometrically on a microdynamometer. In the second set of experiments the whole ileum preparation prepared in the usual way was used. Contractions were recorded with an isotonic lever and registered on a kymograph: dose-response curves were obtained in both kinds of experiments by adding to the bath single increasing doses of histamine or of the other agonists tested; drugs were allowed to act for 1 min, time interval between doses being 3 min in the absence (controls) or in the presence of cimetidine or metiamide. Atropine at a concentration of 10- 7 M was always present in the nutrient fluid. The preload of the strips was 300 mg tension; the resting tension was 250 rag. The pD 2 values reported in Table 1 were calculated according to Van Rossum (1963) using the regression line for each agonist. Statistical evaluation of the data was performed using the Student's test for paired or unpaired data when appropriate. Drugs. The following compounds were used in our investigation: histamine and atropine (Fluka); 5-methylhistamine, 5-ethylhistamine, 5-methyl-N'-methylhistamine synthetized and kindly supplied by Dr. Vitali, University of Parma. (These compounds can also be called 4-methylhistamine, 4-ethylhistamine and 4-methyl-N'-
0028-1298/79/0309/0065/$ 01.00
66
Naunyn-Schmiedeberg's Arch. Pharmacol. 309 (1979) lOO
Table 1. pD 2 values of different compounds on the guinea pig whole
#/
ileum (A) and in the longitudinal muscle preparation (B) Compound
pD 2 (A) S.E.M.
N
pD z (B) S.E.M.
N
Histamine 5-Methylhistamine 5-Ethylhistamine 2-Aminoethyl-thiazole
7.50_+0.30 4.63_+0.35 5.57_+0.35 6.53-+0.30
25 25 25 16
7.42_+0.33 4.65_+0.35 5.68_+0.30 6.37_+0.35
25 16 16 16
/
/A ii
E
/
/ //
I
E 50
?
g
N Number of experiments
/~ methylhistamine according to the numbering of the side chain of histamine which can be considered either as a 2-(4-imidazolyl)ethylamine or as a 2-(5-imidazolyl)ethylamine. Dimaprit ~ metiamide, cimetidine and 2-aminoethylthiazole (kindly supplied by Smith Kline and French, Welwyn Garden City, Herts, England).
Results
Most results were obtained on the longitudinal smooth muscle preparation; however they were very similar to those obtained on the classical whole ileum preparation. Histamine exerted the well known contracting effect starting from threshold doses of l0 -9 M and reaching the maximum effect at concentrations of 1.6 x 10 -6 M; 2-aminoethylthiazole (Durant et al., 1975) was less effective in terms of threshold doses (10-s M) but reached a maximum contraction which did not differ significantly from that of histamine; the compound 5-ethylhistamine, so far never tested in isolated guinea pig ileum, and 5-methylhistamine one of the most widely used H z selective agonists were much less active (Fig. 1). The pD2 values for these compounds in both types of preparations are reported in Table 1. No significant differences were found in the two preparations. A complete dose-response curve could not be constructed with dimaprit which did not consistently contract the ileum even in concentrations of 10- 4 M. In order to examine the possible interference of H z receptors in the contraction induced by histamine as suggested by the results of Bareicha and Rocha e Silva (1975, 1976), two sets of experiments were performed on the atropinized longitudinal muscle preparation: a) in the first, dose-response curves with histamine were constructed in the presence of metiamide or cimetidine; b) in the second, different H 2 receptor agonsists were tested for their ability to modify the contractions evoked by histamine. HN ~ H2N
\ / C-S-CH2-CH2-CH2-N / \
CH3
CHa
//
?
J
Log molar concentration
Fig. 1. Dose response curves for histamine (A A), 2-aminoethylthiazole (A A); 5-ethylhistamine (n v1) and 5-methylhistamine (-I). Values refer to the mean of the values obtained from 4 to 5 experiments on the longitudinal muscle preparation (continuous lines). The interrupted lines refer to the whole ileum preparation for which the single values were not reported
1~176 1 o~
so
5.4 x l O -10
5.4
1.6
xlO - 9 -" x l O - 8
5.4
1.6
5,4
1.6
x l O -B
xlO -7
xlO -6
xlO -6
Fig. 2. Contractions of the longitudinal muscle from guinea pig ileum after administration of different doses of histamine in the absence (white columns) or in the presence (black columns) of cimetidine 4 x 10 6 M. Mean responses of 3 to 4 preparations; vertical bars are standard errors
a) In our experimental conditions neither cimetidine ( 4 • 10 - 6 M ) (Fig. 2) nor metiamide (10 -5 M) (two
experiments) were able to affect the doese-response curves to histamine which did not differ significantly from those obtained in absence of the H 2 blockers, b) In the case of Hz receptor agonists results were as follows: 5-methyl-histamine and dimaprit were unable to reduce the contractions provoked by histamine when administered both immediately before the amine and at the time of maximum contraction. This was observed up to concentration of 1 . 3 x 1 0 - 6 M (four experiments); when higher concentrations were used, a potentiation of the effect of histamine but never an inhibition could be observed. 5-ethylhistamine which in
G. Bertaccini et al. : Histamine Receptors in the Guinea Pig Ileum
67
a previous investigation (Bertaccini et al., 1978) was found to possess a good, rather selective, stimulant action o n H 2 receptors, behaved as the methyl derivative. The same was true for 5-methyl-N'methylhistamine which, owing to the paucity of the material at our disposal, was not tested as a stimulant compound in this investigation but was previously reported (Bertaccini et al., 1976) to be an excellent H z selective agonist and to retain on the guinea pig ileum only 0.2 % of the activity of histamine. Similar results were obtained when the contractions of the longitudinal muscle preparation were induced by the Ha receptor selective agonist, 2-aminoethylthiazole (two experiments).
the classic H1 receptor blockers. However even in the presence of this artificial transformation of H a into H 2 receptors, stimulation of H 2 receptors gave rise to the usual dose-dependent contraction and not relaxation of the ileum. A relaxant effect of histamine in the presence of mepyramine was observed by Ambache et al. (1973) on the neurogenic atropine-resistant tetanic spasms elicited on the guinea pig ileum by field stimulation. This effect, however, was blocked burimamide but was not mimicked by selective H 2 agonists. Thus it cannot be related to the classic H 2 receptors. Provided that all the known H 2 inhibitors, and not only burimamide, are able to block this relaxant activity it may be suggested that a sub-type of H 2 receptors is involved in this phenomenon. Evidence for the existence of a sub-type of H 2 receptor in the guinea pig ileum was recently suggested by Fjalland (1979); however it was based on the clonidine (considered a H 2 agonist)-induced suppression of electrically-induced twitches, and of different antagonistic activity ratio of cimetidine and burimamide on the guinea pig ileum versus the isolated atria. According to very recent data (Chand and De Roth, 1978) the chicken ileum (circular muscle layer) contains Hz-inhibitory histamine receptors and this statement is based on the potentiation by metiamide on the contractions induced by histamine and not on those evoked by carbachol. Apart from the species differences which can largely explain different results, the lack of experiments performed with H z receptor selective agonists must again be pointed out.
Discussion
Our experiments tend to exclude the occurrence of "classical" H 2 receptors mediating a relaxing effect on the longitudinal muscle from guinea pig ileum, which was suggested by other investigations (Bareicha and Rocha e Silva, 1975, 1976). In fact metiamide and cimetidine the two most effective H e receptor blockers so far available, failed to modify the dose-response curve to histamine in the longitudinal muscle preparation. This is in accordance with data by Black et al. (1973); Kenakin et al. (1974) and Parsons (1977): these authors found that the H z receptor blockers do not affect the response to histamine up to concentrations of 10 -4 M and they can actually inhibit in a non competitive way, but never potentiate, the contracting effect of histamine. Another evidence against the presence of H 2 rec e p t o r s with relaxing activity was that based on the experiments performed with the H E receptors selective agonists: dimaprit, 5-methyl-histamine, 5-methyl-N'methylhistamine and 5-ethylhistamine added to the bath before the stimulants as well as during the maximum muscle contraction, failed to inhibit the spasmogenic effect of histamine or of 2-aminoethylthiazole but could actually potentiate it. 5-methylhistamine and 5-ethylhistamine were, in our experiments, sufficiently active as Ha agonists to exert a contracting effect on the guinea pig ileum though doses 100 to 1000 times as high as those of histamine had to be used. Conversely, dimaprit was apparently devoid of H 1 stimulant effects as already observed by Parsons et al. (1977). Another interesting and quite peculiar finding is again not in favour of the hypothesis of a relaxing effect induced through stimulation of H 2 receptors : Kenakin et al. (1974) showed a temperature-dependent interconversion of histamine H a into H z receptors and by lowering the temperature of the nutrient fluid down to 12~ they obtained a dose-response curve to histamine which was shifted to the right by metiamide and not by
Acknowledgements. This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Roma.
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Received March 5/Accepted July 2, 1979
