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The determination of the histamine equilibrium constant for its receptor in the smooth muscle by changing the extracellular calcium concentration
EUROPEAN JOURNAL OF PHARMACOLOGY 16 (1971) 209-213 NORTH-HOLLANDPUBLISHING COMPANY
THE DETERMINATION
OF THE HISTAMINE EQUILIBRIUM
C O N S T A N T F O R I T S R E C E P T O R IN T H E S M O O T H M U S C L E BY CHANGING THE EXTRACELLULAR
CALCIUM CONCENTRATION
J M GI,~O T RICO Instttuto de Farmacologta, Faculdade de Medtcma, Ltsbon Portugal
Recewed 28 January 1971
Accepted 16 June 1971
J M GIAO T RICO, The determmatton o f the htstamme equthbrmm constant for tts receptor m the smooth muscle by changmg the extracellular calcmm concentranon, European J Pharmacol 16 (1971) 209-213 The effects of changes m the extracellular calcium concentration ([Ca ~÷] o), m the absence of magnesmm, on the h~stamme dose-effect curve, were quahtatlvely and quantItatwely studied on the xsolated longitudinal muscle stop of the guinea pig's ileum An increase of the slope of the reciprocal plot of the dose-effect curve was observed when the [Ca~÷] o was progresswely decreased, wxth low calcium concentrations (0 64 - 0 32 raM), the maximal response was also decreased It was shown that the equation described in a previous paper on the muscanmc receptor could also be apphed to the quanUtatwe study of the above mentioned effects, aUowmg for calculation of the htstamIne eqmhbrmm constant for its receptor The value determined by this method did not differ from the value obtained with the use of an ureverslble competltwe antagonist such as dlbenamlne The experimental results also suggest a common final pathway for muscle stimulation by the acUvatmn of both the histamine and muscanmc receptors Calcmm
Histamine equlhbrlum constant
Histamine receptor
1 INTRODUCTION
effect (eC[Rt] x)
In previous experiments, the role of calcium m the function of the muscarInic receptor was investigated by studying the effects of changing the calcium concentraUon in the absence of magnesium on the dose-effect curves of full and partial agonlsts (Giffo T Raco, 1971) To explain both the quahtatave and quantitative results the following hypothesis was put forward The activation of each receptor by the agonist would mobilize a certain amount of calcmm rendering it accessible, m an ionic form, to the contractfle mechamsm of the cell. This amount would be related to the intrinsic efficacy of the drug and would depend on the extracellular calcium concentrahon. The total amount of calcium thus mobihzed by a gwen concentration of the agonlst would be the limiting factor of the observed contraction From this hypothesis the following equation was derived to relate the concentration of the agomst to the reduced
y = f ( S ) = f \ x+Ke
(1)
In this equation y is the response as a percentage of its maximal value, S is the stimulus, e is the intrinsic efficacy (Furchgott, 1966) depending only on the drug, C is an efficiency factor related to the amount of calcium mobilized in the cell by the actwahon of the receptor, depending thus on the extracellular concentration of the ion, [Rt] is the total number of receptors, x represents the molar concentration of the agomst and K e its eqmhbrium constant (K e = k2/ki)
The receptors are assumed to be always functmnal, 1 e [Rt] does not depend on the extracellular calcium concentration, but the stimulus induced by the actlvaUon of the receptor ~s conditioned by this concentration The equilibrium constant does not depend
210
JM
(,tc~) T Rico, Calcium and the histamine receptor
on tile calclUln concenttatlon, as has been already demonstrated (Glao T Rico, 1971 ) When eqmactwe agomst concentrations (Xl and x2) in two different calcmm concentrations are compared, 1t may be demonstrated (Glgo T Rico, 1971) that they are related by the following equation 1 xI
=
el-e2
Cl
1
e2 Ke
c2
x2
_ + ....
(2)
c ~ and c2 are the values of C at those calcmm levels If one of the calcmm concentrations is considered as normal or standard, the value of C at this level is taken a s u m t y (Cl = 1) If we make C = c 2 / c l eq (2) takes the general form 1
I-C
xl = CK~
+
1 1 -. --. c
(3)
x2
C represents tire value of tile efficiency factor as related to the one m the normal calcium concentration (taken as umty) It was demonstrated for several full and partlat muscarlnlC agomsts that C is directly related to the extlacellular calcium concentration and that the value of K e as calculated from eq (3) does not differ from the value obtained by the classical method using 1> reversible competitive antagonists, such as dlbenamine and benzyhlchohne nmstard In this papel we present the results obtained when eq (~) was ,lpphed to tire determulatlon of the hlstamme-leceptol equlhblnJm constant m tile isolated strlt~ of the longitudinal muscle ot the gult!ea pig's ileum This study was made to obtain a nrore detailed knowledge ol lilt_ functional behav|om of tire tnstamine receptor and to see ~f lh,: ~elat)onslnps totmd between the calcluin and the nmscarnnc leceptor could also be verllted with a dffl,,ent receptor Some plehinmaly results x~,ele presented to the Socledadc Portuguesa de Far,n,lc,)log,a
2 MATERI&LS AND METtIODS The experunental method has already been lully described (Oiao T Rico. 1971 ) so that only its prmc> oal aspects ~111 be Desented m,xs qhe expelunents were made on the Isolated strip
of the longitudinal nmscle of the guinea Dg's ileum (Rang, 1964) The muscle was eqmhbrated at 37°C m a magnesium-free Krebs-Henselmt solution of the following composition (raM) NaCI, 113, KC1, 4 7, CaCI2, 2 5 5 , NaHCO3, 2 4 7 6 , KH2PO4, 1 19, glucose, 11 56 All reagents were analytical grade and were dissolved m boroslhcate-glass-dlstllled water Both the organ bath and the Krebs reservoir were bubbled with 95% 0 2 - 5 % CO2 The muscle was mounted m a 10 ml organ bath under a resting tension of 20 mg and its contractions were ~sometncally recorded with a FTA-I-1 Sanborn transducer coupled to a career pre-amphfler and a photographm recorder The preparation was eqmhbrated for 1 hr m the normal calcium (2 55 raM) Krebs solution and then stimulated w~th a standard dose of histamine until the responses had a constant amphtude The contlol dose effect curve was then obtained The Krebs solution was afterwards successwely substituted by others with decreasing calcmm concentrations 128 raM, 0 64 raM, 0 32 mM Whenever the calcmm concentratmn was changed the muscle was reeqmhbrated for 2 0 - 3 0 mm until the responses to histamine were stablhzed A dose effect curve was determined m each of these low calcmm concentrations Tile dose-effect curves were obtained by applying the histamine with an automatic syringe m isolated doses every 2 mm The agonlst was washed out by overflow The use of alr automatic syringe has many advantages, specially fol a good reproduclblhtv of the results These advantages compensate for the lnmted range of concentrations that 1t is possible to use Very tngh tustamme concentrations were also avoided to prevent the desensmz,mon of the receptor The reciprocal ot the ef)ects versus the leclprocal of the concentratam, ol the agomst graptucal plot was used throughout this study, because 11 was found tllat )t stralghtenco the dose effect turves making tot easy determination of the maximal effect and of the reciprocals of tile equlactlve concentrations ~ specml stress was put on the points lying m the tmddle zone of the dose- effect craves because the expelunental error of the small responses is emphasized by this type of graphical plot and because the responses to the hlgJlest agonlst concentrations are too closely packed together To blmphfy compallson between the lesults ol the several experiments the responses of the muscle v~ere plotted as percentage of the maxmml effect
JM
Gt~o T Rtco, Calcium and the htstamme receptor
As the dose-effect curves were stratghtened by the reciprocal plot used, the reciprocals of the eqmactlve concentrations In the several calcium concentrations, or after the several dlbenamlne apphcatlons, were read directly from this graph The histamine equlhbrlum constant was calculated with eq (3) using the remprocals of the equlactwe concentrations in the several calcmm concentrations For comparison the equthbnum constant of the histamine was also calculated w~th the classical method employing Dlbenamlne (N,N-&benzyl-3-chloroethylamine) already used as an irreversible competmve antagonist of histamine by Furchgott (1966) This drug was applied in a concentratmn of 2 × 10 -6 M for successive periods of 10 mm each After each period the muscle was washed every 2 mm during 15 mm and then stimulated w~th histamine tdl the responses became constant Only then a dose-effect curve was determined Drugs used histamine phosphate, B D H , dibenamlne, S K & F
by the 1 28 mM calcmm concentratmn but decreased with lower calcium levels (fig 1) Fig 2 illustrates the double remprocal plot when eq (3) is applied to the experimental data given in fig 1 It may be seen that the graphical plot of the remprocals of the histamine equlachve concentrahons in the several low calcmm levels, as compared with the normal one, are straight hnes and thus eq (3) allows the calculation of the values of C and K e All the values of C and K e were individually determined at each calcium level m every experiment and the results were later analysed stahstlcally Fig 3 shows the relationship between the value of C, as given by eq (3), and the extracellular calcium concentration The value of C in the normal calcmm level was taken as umty and marked * on the graph to differentiate it from the experimental points The value of C in the 128 mM calcium levels was 0 5 1 4 + - 0 0 6 8 , In the 0 6 4 m M calcmm concentration, 0 259-+ 0 029 and In the lowest calcium level (0 32 mM), 0 135 + 0 028 The calcmm concentrations were also represented as fractions of the normal concentration The continuous line shows the slope of the straight line calculated between the experimental values of C and the fractional concentrations of the calcmm by the least squares method Its equahon is C=- 0 0 0 6 + 1 012 [Ca2+]o The interrupted
3 RESULTS When the calcium concentration of the Krebs solutmn was progresswely decreased, the dose-effect curves of the histamine showed a gradual decrease m the responses to the agonlst as revealed by an mcrease m the slope of the rectprocal plot of the dose-effect curve The maximal effect was not usually affected
211
I
,$
1 "/. Em
sxl~. 4O
x)OS 3 xl~) 2. xl2~
Ix1~J'Hlsta mine "]
Fig 1 Histamine d o s e - r e s p o n s e curves Reciprocal plot Effect of decreasing the calcium concentration o f the magnesmm-free Krebs solutmn Curve A [Ca2÷]0 = 2 55 mM, Curve B [Ca2÷]o = 1 2 8 m M , CurveC [Ca 2+]0 = 0 6 4 r a M , CurveD [Ca ~÷]o = 0 3 2 m M
1
W
1~xlO 5
28~1o S
Fig 2 Graphical plot of eq (3) as apphed to the experiments shown In fig 1 The hnes B,C,D relate the reciprocals of the agomst eqmactwe concentrations m the normal (2 55 mM) and respectwely the 1 28 mM, 0 64 mM and 0 32 mM calCltlm concentrations
212
J M (;t&) T Rico Cahmm and the histamine receptor Table 1 Equlhbrlum constant (K e = k2/kl) values of h]stamlne as determined by changing the calcium concentration m the bathmg solution and by the use of an irreversible competitive antagomst dlbenamme The results are given as mean ± S E M
1,000¸ C
0,750,
(N 10 -6 M)
Histamine - eqmbbrmm constant [Ca2*] 0 variation
Dlbenamlne
0 5O0.
Ke = 4 . 1 3 ± 0 3 7
(19)
Ke = 4 . 7 9 ± 0 . 2 7
(10)
* In brackets the number of observations. 0,250-
__~O'~o
,)8
,)4
(0,32mM) (O,64mM)
,)2
i
(1,28 mM )
(2,55mM)
Fig 3 Graph of C = 3' ([Ca 2*]o) as determmed with histamine The continuous line is the best fit straight hne calculated by the least squares method The interrupted line is the straight hne that passes through the origin, calculated by a constrained least squares method (for detads, see text)
hne shows the hne that passes through the origin, calculated by a constrained least squares m e t h o d Its equat]on is C =- 1 032 [Ca 2÷] o The value o f the intercept o f the first hne does not differ significantly f r o m zero ( p > 0 2), also the slopes o f b o t h the lines do not differ f r o m each other ( p > 0 2) These results can be superimposed on those o b t a i n e d with the study of the nruscarmm receptor, and show that the value o f C is p r o p o r t i o n a l to the c a l c m m c o n c e n t r a t i o n in the bathing solution
In table 1, the values o f the e q u l h b r l u m constant ( K e = k 2 / k l ) o f the h i s t a m i n e - r e c e p t o r Interaction o b t a i n e d by variation o f the extracellular calcium c o n c e n t r a t i o n , are c o m p a r e d with those obtained by the classical dlbenamlne m e t h o d The difference is not statlstmally significant ( p > 0 2) An analysis o f variance o f the results obtained by the c a l c m m variation m e t h o d (table 2) did not show any difference b e t w e e n the results o b t a i n e d at the various c a l c m m levels nor b e t w e e n the animals used
4 DISCUSSION In this paper, the results obtained by studying qualitatively and quantxtatwely the effects of changes in the extracellular calcium c o n c e n t r a t i o n , m the absence of magnesium, on the histamine d o s e - e f f e c t curves are presented The Isolated longitudinal muscle strip o f the guinea pig's ileum (Rang, 1964) was used as the test organ.
Table 2 Analysis of variance of the K e values determined for histamine by changing the calcium concentratmn m the bathing fired Source of variation
df
s.s.
m s.
F
p
Between animals
8
18 4901
2 3112
1.140
> 0.2
Between calcium levels
2
3 6475
1.8237
1 445
> 0.2
Error
8
21.0794
2.6349
Total
18
43.2170
JM
GdIo T Rtco, Calctum and the htstamme receptor
The method has already been apphed to a study o1 the muscarmic receptor (Gi~o T Raco, 1971)so that only relevant aspects of the method are described The theory underlying the suggested hypothesis on the relationship between the calcium and the receptor was then also fully described and discussed The main purpose of this study was to determine whether the method developed for the study of the muscarmlc receptor of the smooth muscle could be applied to a different kind of receptor The experimental conditions and the experimental design were the same as previously used (Gi~o T. Rico, 1971) It was observed that the decrease m the extracellular calcium concentration induced an increase in the slope of the reciprocal of the effects versus reciprocal of the histamine concentrations graphical plot (fig 1) A decrease in the maximal effect was also observed in the two lower calcium levels (0 64 mM and 0.32 raM) The quantitative study of these modifications of the dose-effect curve showed that the equation derived from the results obtained in the muscarimc receptor could be applied to the present experiments (fig 2), allowing the calculation of the equilibrium constant of histamine This value was confirmed with the use of an irreversible competitive antagonist, dlbenamme, following the classical method (table 1) The maximal effect of histamine decreased with the two lower calcium concentrations (0.64 mM and 0 32 raM), while the acetylchohne maximal effect was only infrequently affected by the lowest calcium concentration (Gl$o T. Rico, 1971) The suggested hypothesis, that the activation of the receptor mobilizes a certain amount of calcium (dependen,t on the drug and on the extracellular calcium concentration) rendering it accessible, in an lomc form, to the muscle cell contractile mechanism, and that this amount of calcium is the limiting factor of the reduced response, explains the difference between these two full agonlsts if it is assumed that acetylcholme mobilizes more calcium than histamine following each receptor activation The change in the slope of the dose-effect curve reciprocal plot, observed when the calcium concentration was progressively decreased, was similar to the change observed with the muscarlnic agonists in the same experimental conditions This similarity is emphasized by the quantitative study The experiments just described show a linear relationship be-
213
tween the efficiency of the stimulus and the extracellular calcium concentration within the range of concentrations used This relationship is similar to that already observed in the muscarlnic receptor under the same experimental con&tlons The graph given in fig. 3 can indeed be superimposed on that obtained with acetylchohne (Gi~o T Rico, 1971), and its equation is analogous to the ones obtained for the muscarlnlC agomsts This similarity between the results determined under the same special experimental conditions by the activation of two different kinds of receptors may suggest that, although the receptor sites for the two types of agonlsts are different, there is a common final pathway for muscle cell stimulation. A similar hypothesis has already been suggested by Schlld (1964) for the acetylchohne and epinephrine receptors The agreement between the equilibrium constant values as determined by the calcium variation method, and the values determined by the classical method using dlbenamme, also favours the validity of the hypothesis underlying eq (3) Table 2 shows that the equdlbrlum constant value is independent of the actual extracellular calcium concentration, as had also been observed with the muscarlnlC agomsts The hypothesis previously suggested, to explain qualitatively and quantitatively the relationship between calcium and muscarmic receptor, may also be applied to the histamine receptor in the smooth muscle of the guinea pig's ileum
REFERENCES Furchgott, R.F., 1966, The use of #-haloalkylammes m the differentiation of receptors and in the determination of dissociation constants of receptor-agomst complexes, Advan Drug Res 3, 21-56 Furchgott, R F. and F Bursztyn, 1967, Comparison of dissociation constants and of relative efficaoes of selected agonists acting on parasympathetic receptors, Ann N.Y Acad Sci 144,882-898. Gl~o T Rico, J M., 1971, The mfluence of calcmm on the actlvaty of the full and partml muscarmic agonists European J Pharmacoi 13, 218-229 Rang, H.P, 1964, Stimulant actions of volatile anaesthetics on smooth muscle, Bnt J Pharmacol Chemotherap 22, 356 -365 Schlld, H O., 1964, Calcmm and the effects of drugs on depolarized smooth muscle, Proc Second Intern Pharmacol Meeting 6, 95-104
THE DETERMINATION
OF THE HISTAMINE EQUILIBRIUM
C O N S T A N T F O R I T S R E C E P T O R IN T H E S M O O T H M U S C L E BY CHANGING THE EXTRACELLULAR
CALCIUM CONCENTRATION
J M GI,~O T RICO Instttuto de Farmacologta, Faculdade de Medtcma, Ltsbon Portugal
Recewed 28 January 1971
Accepted 16 June 1971
J M GIAO T RICO, The determmatton o f the htstamme equthbrmm constant for tts receptor m the smooth muscle by changmg the extracellular calcmm concentranon, European J Pharmacol 16 (1971) 209-213 The effects of changes m the extracellular calcium concentration ([Ca ~÷] o), m the absence of magnesmm, on the h~stamme dose-effect curve, were quahtatlvely and quantItatwely studied on the xsolated longitudinal muscle stop of the guinea pig's ileum An increase of the slope of the reciprocal plot of the dose-effect curve was observed when the [Ca~÷] o was progresswely decreased, wxth low calcium concentrations (0 64 - 0 32 raM), the maximal response was also decreased It was shown that the equation described in a previous paper on the muscanmc receptor could also be apphed to the quanUtatwe study of the above mentioned effects, aUowmg for calculation of the htstamIne eqmhbrmm constant for its receptor The value determined by this method did not differ from the value obtained with the use of an ureverslble competltwe antagonist such as dlbenamlne The experimental results also suggest a common final pathway for muscle stimulation by the acUvatmn of both the histamine and muscanmc receptors Calcmm
Histamine equlhbrlum constant
Histamine receptor
1 INTRODUCTION
effect (eC[Rt] x)
In previous experiments, the role of calcium m the function of the muscarInic receptor was investigated by studying the effects of changing the calcium concentraUon in the absence of magnesium on the dose-effect curves of full and partial agonlsts (Giffo T Raco, 1971) To explain both the quahtatave and quantitative results the following hypothesis was put forward The activation of each receptor by the agonist would mobilize a certain amount of calcmm rendering it accessible, m an ionic form, to the contractfle mechamsm of the cell. This amount would be related to the intrinsic efficacy of the drug and would depend on the extracellular calcium concentrahon. The total amount of calcium thus mobihzed by a gwen concentration of the agonlst would be the limiting factor of the observed contraction From this hypothesis the following equation was derived to relate the concentration of the agomst to the reduced
y = f ( S ) = f \ x+Ke
(1)
In this equation y is the response as a percentage of its maximal value, S is the stimulus, e is the intrinsic efficacy (Furchgott, 1966) depending only on the drug, C is an efficiency factor related to the amount of calcium mobilized in the cell by the actwahon of the receptor, depending thus on the extracellular concentration of the ion, [Rt] is the total number of receptors, x represents the molar concentration of the agomst and K e its eqmhbrium constant (K e = k2/ki)
The receptors are assumed to be always functmnal, 1 e [Rt] does not depend on the extracellular calcium concentration, but the stimulus induced by the actlvaUon of the receptor ~s conditioned by this concentration The equilibrium constant does not depend
210
JM
(,tc~) T Rico, Calcium and the histamine receptor
on tile calclUln concenttatlon, as has been already demonstrated (Glao T Rico, 1971 ) When eqmactwe agomst concentrations (Xl and x2) in two different calcmm concentrations are compared, 1t may be demonstrated (Glgo T Rico, 1971) that they are related by the following equation 1 xI
=
el-e2
Cl
1
e2 Ke
c2
x2
_ + ....
(2)
c ~ and c2 are the values of C at those calcmm levels If one of the calcmm concentrations is considered as normal or standard, the value of C at this level is taken a s u m t y (Cl = 1) If we make C = c 2 / c l eq (2) takes the general form 1
I-C
xl = CK~
+
1 1 -. --. c
(3)
x2
C represents tire value of tile efficiency factor as related to the one m the normal calcium concentration (taken as umty) It was demonstrated for several full and partlat muscarlnlC agomsts that C is directly related to the extlacellular calcium concentration and that the value of K e as calculated from eq (3) does not differ from the value obtained by the classical method using 1> reversible competitive antagonists, such as dlbenamine and benzyhlchohne nmstard In this papel we present the results obtained when eq (~) was ,lpphed to tire determulatlon of the hlstamme-leceptol equlhblnJm constant m tile isolated strlt~ of the longitudinal muscle ot the gult!ea pig's ileum This study was made to obtain a nrore detailed knowledge ol lilt_ functional behav|om of tire tnstamine receptor and to see ~f lh,: ~elat)onslnps totmd between the calcluin and the nmscarnnc leceptor could also be verllted with a dffl,,ent receptor Some plehinmaly results x~,ele presented to the Socledadc Portuguesa de Far,n,lc,)log,a
2 MATERI&LS AND METtIODS The experunental method has already been lully described (Oiao T Rico. 1971 ) so that only its prmc> oal aspects ~111 be Desented m,xs qhe expelunents were made on the Isolated strip
of the longitudinal nmscle of the guinea Dg's ileum (Rang, 1964) The muscle was eqmhbrated at 37°C m a magnesium-free Krebs-Henselmt solution of the following composition (raM) NaCI, 113, KC1, 4 7, CaCI2, 2 5 5 , NaHCO3, 2 4 7 6 , KH2PO4, 1 19, glucose, 11 56 All reagents were analytical grade and were dissolved m boroslhcate-glass-dlstllled water Both the organ bath and the Krebs reservoir were bubbled with 95% 0 2 - 5 % CO2 The muscle was mounted m a 10 ml organ bath under a resting tension of 20 mg and its contractions were ~sometncally recorded with a FTA-I-1 Sanborn transducer coupled to a career pre-amphfler and a photographm recorder The preparation was eqmhbrated for 1 hr m the normal calcium (2 55 raM) Krebs solution and then stimulated w~th a standard dose of histamine until the responses had a constant amphtude The contlol dose effect curve was then obtained The Krebs solution was afterwards successwely substituted by others with decreasing calcmm concentrations 128 raM, 0 64 raM, 0 32 mM Whenever the calcmm concentratmn was changed the muscle was reeqmhbrated for 2 0 - 3 0 mm until the responses to histamine were stablhzed A dose effect curve was determined m each of these low calcmm concentrations Tile dose-effect curves were obtained by applying the histamine with an automatic syringe m isolated doses every 2 mm The agonlst was washed out by overflow The use of alr automatic syringe has many advantages, specially fol a good reproduclblhtv of the results These advantages compensate for the lnmted range of concentrations that 1t is possible to use Very tngh tustamme concentrations were also avoided to prevent the desensmz,mon of the receptor The reciprocal ot the ef)ects versus the leclprocal of the concentratam, ol the agomst graptucal plot was used throughout this study, because 11 was found tllat )t stralghtenco the dose effect turves making tot easy determination of the maximal effect and of the reciprocals of tile equlactlve concentrations ~ specml stress was put on the points lying m the tmddle zone of the dose- effect craves because the expelunental error of the small responses is emphasized by this type of graphical plot and because the responses to the hlgJlest agonlst concentrations are too closely packed together To blmphfy compallson between the lesults ol the several experiments the responses of the muscle v~ere plotted as percentage of the maxmml effect
JM
Gt~o T Rtco, Calcium and the htstamme receptor
As the dose-effect curves were stratghtened by the reciprocal plot used, the reciprocals of the eqmactlve concentrations In the several calcium concentrations, or after the several dlbenamlne apphcatlons, were read directly from this graph The histamine equlhbrlum constant was calculated with eq (3) using the remprocals of the equlactwe concentrations in the several calcmm concentrations For comparison the equthbnum constant of the histamine was also calculated w~th the classical method employing Dlbenamlne (N,N-&benzyl-3-chloroethylamine) already used as an irreversible competmve antagonist of histamine by Furchgott (1966) This drug was applied in a concentratmn of 2 × 10 -6 M for successive periods of 10 mm each After each period the muscle was washed every 2 mm during 15 mm and then stimulated w~th histamine tdl the responses became constant Only then a dose-effect curve was determined Drugs used histamine phosphate, B D H , dibenamlne, S K & F
by the 1 28 mM calcmm concentratmn but decreased with lower calcium levels (fig 1) Fig 2 illustrates the double remprocal plot when eq (3) is applied to the experimental data given in fig 1 It may be seen that the graphical plot of the remprocals of the histamine equlachve concentrahons in the several low calcmm levels, as compared with the normal one, are straight hnes and thus eq (3) allows the calculation of the values of C and K e All the values of C and K e were individually determined at each calcium level m every experiment and the results were later analysed stahstlcally Fig 3 shows the relationship between the value of C, as given by eq (3), and the extracellular calcium concentration The value of C in the normal calcmm level was taken as umty and marked * on the graph to differentiate it from the experimental points The value of C in the 128 mM calcium levels was 0 5 1 4 + - 0 0 6 8 , In the 0 6 4 m M calcmm concentration, 0 259-+ 0 029 and In the lowest calcium level (0 32 mM), 0 135 + 0 028 The calcmm concentrations were also represented as fractions of the normal concentration The continuous line shows the slope of the straight line calculated between the experimental values of C and the fractional concentrations of the calcmm by the least squares method Its equahon is C=- 0 0 0 6 + 1 012 [Ca2+]o The interrupted
3 RESULTS When the calcium concentration of the Krebs solutmn was progresswely decreased, the dose-effect curves of the histamine showed a gradual decrease m the responses to the agonlst as revealed by an mcrease m the slope of the rectprocal plot of the dose-effect curve The maximal effect was not usually affected
211
I
,$
1 "/. Em
sxl~. 4O
x)OS 3 xl~) 2. xl2~
Ix1~J'Hlsta mine "]
Fig 1 Histamine d o s e - r e s p o n s e curves Reciprocal plot Effect of decreasing the calcium concentration o f the magnesmm-free Krebs solutmn Curve A [Ca2÷]0 = 2 55 mM, Curve B [Ca2÷]o = 1 2 8 m M , CurveC [Ca 2+]0 = 0 6 4 r a M , CurveD [Ca ~÷]o = 0 3 2 m M
1
W
1~xlO 5
28~1o S
Fig 2 Graphical plot of eq (3) as apphed to the experiments shown In fig 1 The hnes B,C,D relate the reciprocals of the agomst eqmactwe concentrations m the normal (2 55 mM) and respectwely the 1 28 mM, 0 64 mM and 0 32 mM calCltlm concentrations
212
J M (;t&) T Rico Cahmm and the histamine receptor Table 1 Equlhbrlum constant (K e = k2/kl) values of h]stamlne as determined by changing the calcium concentration m the bathmg solution and by the use of an irreversible competitive antagomst dlbenamme The results are given as mean ± S E M
1,000¸ C
0,750,
(N 10 -6 M)
Histamine - eqmbbrmm constant [Ca2*] 0 variation
Dlbenamlne
0 5O0.
Ke = 4 . 1 3 ± 0 3 7
(19)
Ke = 4 . 7 9 ± 0 . 2 7
(10)
* In brackets the number of observations. 0,250-
__~O'~o
,)8
,)4
(0,32mM) (O,64mM)
,)2
i
(1,28 mM )
(2,55mM)
Fig 3 Graph of C = 3' ([Ca 2*]o) as determmed with histamine The continuous line is the best fit straight hne calculated by the least squares method The interrupted line is the straight hne that passes through the origin, calculated by a constrained least squares method (for detads, see text)
hne shows the hne that passes through the origin, calculated by a constrained least squares m e t h o d Its equat]on is C =- 1 032 [Ca 2÷] o The value o f the intercept o f the first hne does not differ significantly f r o m zero ( p > 0 2), also the slopes o f b o t h the lines do not differ f r o m each other ( p > 0 2) These results can be superimposed on those o b t a i n e d with the study of the nruscarmm receptor, and show that the value o f C is p r o p o r t i o n a l to the c a l c m m c o n c e n t r a t i o n in the bathing solution
In table 1, the values o f the e q u l h b r l u m constant ( K e = k 2 / k l ) o f the h i s t a m i n e - r e c e p t o r Interaction o b t a i n e d by variation o f the extracellular calcium c o n c e n t r a t i o n , are c o m p a r e d with those obtained by the classical dlbenamlne m e t h o d The difference is not statlstmally significant ( p > 0 2) An analysis o f variance o f the results obtained by the c a l c m m variation m e t h o d (table 2) did not show any difference b e t w e e n the results o b t a i n e d at the various c a l c m m levels nor b e t w e e n the animals used
4 DISCUSSION In this paper, the results obtained by studying qualitatively and quantxtatwely the effects of changes in the extracellular calcium c o n c e n t r a t i o n , m the absence of magnesium, on the histamine d o s e - e f f e c t curves are presented The Isolated longitudinal muscle strip o f the guinea pig's ileum (Rang, 1964) was used as the test organ.
Table 2 Analysis of variance of the K e values determined for histamine by changing the calcium concentratmn m the bathing fired Source of variation
df
s.s.
m s.
F
p
Between animals
8
18 4901
2 3112
1.140
> 0.2
Between calcium levels
2
3 6475
1.8237
1 445
> 0.2
Error
8
21.0794
2.6349
Total
18
43.2170
JM
GdIo T Rtco, Calctum and the htstamme receptor
The method has already been apphed to a study o1 the muscarmic receptor (Gi~o T Raco, 1971)so that only relevant aspects of the method are described The theory underlying the suggested hypothesis on the relationship between the calcium and the receptor was then also fully described and discussed The main purpose of this study was to determine whether the method developed for the study of the muscarmlc receptor of the smooth muscle could be applied to a different kind of receptor The experimental conditions and the experimental design were the same as previously used (Gi~o T. Rico, 1971) It was observed that the decrease m the extracellular calcium concentration induced an increase in the slope of the reciprocal of the effects versus reciprocal of the histamine concentrations graphical plot (fig 1) A decrease in the maximal effect was also observed in the two lower calcium levels (0 64 mM and 0.32 raM) The quantitative study of these modifications of the dose-effect curve showed that the equation derived from the results obtained in the muscarimc receptor could be applied to the present experiments (fig 2), allowing the calculation of the equilibrium constant of histamine This value was confirmed with the use of an irreversible competitive antagonist, dlbenamme, following the classical method (table 1) The maximal effect of histamine decreased with the two lower calcium concentrations (0.64 mM and 0 32 raM), while the acetylchohne maximal effect was only infrequently affected by the lowest calcium concentration (Gl$o T. Rico, 1971) The suggested hypothesis, that the activation of the receptor mobilizes a certain amount of calcium (dependen,t on the drug and on the extracellular calcium concentration) rendering it accessible, in an lomc form, to the muscle cell contractile mechanism, and that this amount of calcium is the limiting factor of the reduced response, explains the difference between these two full agonlsts if it is assumed that acetylcholme mobilizes more calcium than histamine following each receptor activation The change in the slope of the dose-effect curve reciprocal plot, observed when the calcium concentration was progressively decreased, was similar to the change observed with the muscarlnic agonists in the same experimental conditions This similarity is emphasized by the quantitative study The experiments just described show a linear relationship be-
213
tween the efficiency of the stimulus and the extracellular calcium concentration within the range of concentrations used This relationship is similar to that already observed in the muscarlnic receptor under the same experimental con&tlons The graph given in fig. 3 can indeed be superimposed on that obtained with acetylchohne (Gi~o T Rico, 1971), and its equation is analogous to the ones obtained for the muscarlnlC agomsts This similarity between the results determined under the same special experimental conditions by the activation of two different kinds of receptors may suggest that, although the receptor sites for the two types of agonlsts are different, there is a common final pathway for muscle cell stimulation. A similar hypothesis has already been suggested by Schlld (1964) for the acetylchohne and epinephrine receptors The agreement between the equilibrium constant values as determined by the calcium variation method, and the values determined by the classical method using dlbenamme, also favours the validity of the hypothesis underlying eq (3) Table 2 shows that the equdlbrlum constant value is independent of the actual extracellular calcium concentration, as had also been observed with the muscarlnlC agomsts The hypothesis previously suggested, to explain qualitatively and quantitatively the relationship between calcium and muscarmic receptor, may also be applied to the histamine receptor in the smooth muscle of the guinea pig's ileum
REFERENCES Furchgott, R.F., 1966, The use of #-haloalkylammes m the differentiation of receptors and in the determination of dissociation constants of receptor-agomst complexes, Advan Drug Res 3, 21-56 Furchgott, R F. and F Bursztyn, 1967, Comparison of dissociation constants and of relative efficaoes of selected agonists acting on parasympathetic receptors, Ann N.Y Acad Sci 144,882-898. Gl~o T Rico, J M., 1971, The mfluence of calcmm on the actlvaty of the full and partml muscarmic agonists European J Pharmacoi 13, 218-229 Rang, H.P, 1964, Stimulant actions of volatile anaesthetics on smooth muscle, Bnt J Pharmacol Chemotherap 22, 356 -365 Schlld, H O., 1964, Calcmm and the effects of drugs on depolarized smooth muscle, Proc Second Intern Pharmacol Meeting 6, 95-104