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The membrane stabilizing and β1-adrenoceptor blocking activity of (+)- and (−)-propranolol on the rat left atria
Gen. Pharmac. Vol. 21, No. 5, pp. 677-680, 1990 Printed in Great Britain. All rights reserved
0306-3623/90 $3.00+ 0.00 Copyright © 1990PergamonPress pie
THE MEMBRANE STABILIZING AND flt-ADRENOCEPTOR BLOCKING ACTIVITY OF (+)- AND (-)-PROPRANOLOL ON THE RAT LEFT ATRIA SHEILA A. DOGGRELL Department of Pharmacology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand [Tel. (649) 795780; Fax (649) 770956]
(Received 27 November 1989)
Abstract--I. The membrane stabilizing and fll-adrenoceptor blocking activities of(+)- and (-)-propranolol have been determined using the rat left atria. 2. (+)- And (-)-propranolol have membrane stabilizing activity. Thus a low concentration of (-)-propranolol, 10-aM, and a higher concentration of (+)-propranolol, 10-6M, inhibited the responses to electrical stimulation. A high concentration of (+)- and (-)-propranolol, 10-~M, also inhibited the maximal combined response to electrical stimulation and isoprenaline. 3. As competitive fit -adrenoceptor antagonists, ( - )-propranolol was over a I00 times more potent than ( + )-propranolol.
INTRODUCTION fl-Adrenoceptor antagonists (r-blockers) are commonly used in the treatment of hypertension, angina and cardiac arrhythmias. Some of the r-blockers (e.g. propranolol) have as an ancillary property the ability to cause membrane stabilization (reviewed by Fitzgerald, 1984). However the membrane stabilizing activity of propranolol is not generally considered to be clinically relevant as it occurs with concentrations of propranolol higher than those used clinically. Thus after the administration of an oral dose of propranolol to humans the plasma concentration is 6 × 10-8-6 × 10-TM for I-7 hr (Shand et al., 1970). Whereas studies with isolated animal cardiac tissues have only shown a membrane stabilizing action with ( ± ) - p r o p r a n o l o l at 10-6M (rabbit atrial preparations, Fitzgerald, 1984) and with ( + ) - and ( - ) propranolol at 3.5 and 4.5 × 10-4M, respectively (guinea-pig left atria, Barrett and Cullum, 1968). Recently I have described a method to simultaneously assess the membrane stabilizing and fl~adrenoceptor blocking activity of drugs using the rat isolated left atria (Doggrell, 1988). In this model the membrane stabilizing activity of procaine manifests itself in two ways; a depression of the response to electrical stimulation alone and, at a higher concentration, a depression of the maximal combined response to electrical stimulation and isoprenaline. Metoprolol, a r-blocker without membrane stabilizing activity, only acted as an inhibitor of the responses to isoprenaline. Analysis of the effects of ( ± ) - p r o p r a n o l o l on the responses of the left atria to electrical stimulation and to electrical stimulation and isoprenaline demonstrated that ( ± ) - p r o p r a n o l o l was a potent membrane stabilizer and flt-adrenoceptor antagonist on this preparation. There were two components to the membrane stabilizing action of ( ± ) propranolol; low concentrations, >I 3 × 10-gM, depressed the responses to electrical stimulation alone
and a high concentration (10-SM) depressed the maximal combined response to electrical stimulation and isoprenaline (Doggrell, 1988). This was the first demonstration that low, and therefore clinically relevant, concentrations of (__.)-propranolol have the manifestations of membrane stabilizing activity on cardiac tissue. The components that each of the propranolol stereoisomers contributes to the membrane stabilizing and fl~-adrenoceptor blocking actions of (_+)-propranolol on the rat left atria have not previously been described. The present communication reports the effects of ( + ) - and ( - ) - p r o p r a n o l o l on the responses of the rat left atria to electrical stimulation, and to electrical stimulation and isoprenaline. METHODS AND MATERIALS
Recording of the contractile responses of the electrically driven rat left atria in accordance with the method of Doggrell (1988), male Wistar rats (250-350 g) were stunned and exsanguinated. The heart was rapidly removed and placed in Krebs solution that was saturated with 5 0 CO 2 in oxygen, and the left atria was excised. All procedures were performed in the presence of a modified Krebs solution [composition (raM): NaCI, 116; KCI, 5.4; CaCI~, 2.5; MgCI2, 1.2; NaH2PO4, 1.2; NaHCO~, 22.0; ~glucose, 11.2; Na 2 EDTA, 0.04] which was being vigorously bubbled with 5% CO 2 in oxygen at 37°C. Each left atria was halved. Each half was mounted longitudinally between two platinum electrodes (approximately 3 crn apart, above and below the tissue) under I g tension in 5 ml organ baths containing Krebs solution (with 10- ~M guanethidine to prevent the release of noradrenaline from nerve endings and 10-~M atropine) and allowed to equilibrate for 60 rain. During the equilibration period, the tissues were washed by overflow. Tissues were electrically stimulated at 4 Hz (5 reset, 10 V). After 6 rain of stimulation, a cumulative challenge with isoprenaline was initiated. Thus atria were challenged with isoprenaline at 3 min intervals to give a final concentration
677
678
SHEILA A. DOGGRELL
of isoprenaline in the organ both successively of 3 x I0 -H, 10 -m, 3 x 10-raM etc. This cycle was continued until a maximum response was obtained with the contractile responses being recorded isometrically with force displacement transducers (Grass model F/TO3C) and displayed on a polygraph (Grass model 79B). Stimulation was then stopped. One half of the left atria remained untreated throughout while the other half of the left atria was treated with (+)-propranolol at 10 -7 M. Atria were washed rapidly in the absence or presence of drug for 60 min prior to a further period of electrical stimulation with a second cumulative challenge to isoprenaline. The treated atria were then further treated with (+)-propranolol at 10-~M. After another 60 min of rapid washing in the absence or presence of drug, a third cumulative challenge to isoprenaline of the electrically stimulated tissue was undertaken. In other studies the drugs and concentrations used were (+)-propranolol at 10 .6 and 6 x 10-6M, ( - ) - p r o p r a n o l o l at I0 g and 10-6M and ( - ) - p r o p r a n o l o l at 10 -7 and 10 5M. The contractile responses to electrical stimulation just prior to challenge with isoprenaline and the maximal contractile responses to each concentration of isoprenaline in the presence of electrical stimulation were measured.
prenaline were subtracted from the combined response to electrical stimulation and isoprenaline. These responses to isoprenaline were calculated as a percentage of the maximum of the first isoprenaline response curve. If the maximum to isoprenaline between treated and untreated tissues calculated in this manner were not significantly different, responses to isoprenaline were also calculated as a percentage of the individual curve to isoprenaline i.e. normalized. When responses to isoprenaline were normalized, pD 2 values, concentration-ratios and then pA 2 values were determined, pD 2 values were computed by regression line analysis on the steepest part of the response curve, which was usually over the range 20-80°/. of the maximum response. For each tissue, the concentration ratios (the antilog of the difference between the pD 2 values) were determined between each challenge with isoprenaline. The concentration ratios obtained in the presence of drugs were corrected for changes occurring in untreated tissues, pA2 values were determined by Schild plot [log (x - 1), ordinate, vs the logarithm of the molar concentration of antagonist, abscissa] using regression line analysis of individual log (x - I) values. A least squares analysis was performed to test whether the line of the Schild plot had a slope that was significantly different from 1. The pA2 values was the abscissa intercept when the slope was not significantly different from I. The individual values were compared by Student's t-test and were considered to be significantly different when P < 0.05. From these individual values, mean values + SEM were also determined.
Analysis of data The contractile responses to electrical stimulation just prior to the second and third challenges with isoprenaline were calculated as a percentage of the response to stimulation prior to the first challenge with isoprenaline. The combined responses to electrical stimulation and isoprenaline were calculated as a percentage of the maximum combined response to electrical stimulation and isoprenaline during the first challenge with isoprenaline. For each challenge with isoprenaline, the responses to electrical stimulation just prior to the challenge with iso-
Drugs The drugs used were guanethidine sulphate (donated by Ciba-Geigy), ( + ) - and ( - ) - p r o p r a n o l o l hydrochloride (donated by ICi), atropine sulphate and ( - ) - isoprenaline bitartrate (Sigma Chemical Company).
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Fig. 1. Effect o f (+)-propranolol on the contractile responses of the rat left atria to electrical stimulation and/or isoprenaline. Responses in the absence (A) and presence (A) of (+)-propranolol at 10-6M, absence ( 0 ) and presence (O) of (+)-propranolol at 6 x 10-6M and the absence (11) and presence (11) of (+)-propranolol at 10-SM. Left: responses to electrical stimulation and isoprenaline are calculated as a percentage of the maximum response o f t h e first curve to electrical stimulation. Right and top: responses to isoprenaline are calculated as a percentage of the individual curve to isoprenaline. All responses are plotted against the logarithm of the molar concentration of isoprenaline (abscissa). Each value is the mean of 6-11 atria. Vertical lines show SEM. Right and bottom: Schild regression (logarithm of the concentration-ratio minus I plotted against the logarithm of the molar concentration of antagonist) for ( + )-propranolol.
M.S.A. with ( + ) - and ( - ) - p r o p r a n o i o i
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Table I. Effects of (+)- and (-)-propranoiol on the ~ of the rat k,R atria to electrical stimulation and on the maximal respomes to electrical stimulation and itoprenaline Maximal rcsponlm' P,~'trical stimulation stimulation and itolnenaline 81 ± 5 (8) 98 + 2 (8) (ii) Control (+) Pro )ranolol, 10-6M 63 ± 8 (8)" 99 + 4(8) 62 + 7 18) 96 ± 3 (8) (iii) Control ( + )-Pro >ranolol, 6 × 10-6M 15 4- 14(8)* 90 + 4(8) Control 474. II (6) 884-4(6) (iii) ( + )-Pro )ranolol0 10- s M 5 +, 3 (I I )* 53 + 9 (I I)* Control 7s +_8 (10) 90 4- 5 (to) (ii) ( - )-Pro )ranolol, 10-SM 57 ± 7 (t0)* 97 4- 5 (10) 80 4- 8 (12) 100 4- 4 (12) (ii) Control ( - )-Pro )ranolol, 10-?M 56 4- 8 (9)* 96 4- 6 (9) Control 62 4- 12 (6) 90 4- 13 (6) (iii) ( -)-Pro )ranolol, 10-eM 26 4- 10 (10)* 100 4- 7 (10) Control 604- 10(I I) 104+5(11) (iii) ( - ).Pro )ranolol, 10-~M 0 (9) 94-6(9)* 'Calculated as a percentage of the first resImnue. (ii) and (iii) refer to the second and third responses. All values are mean ± SEM. The number in parenthesis is the number of observations. ' P < 0.05, unpaired t-test with own control.
(+)-Propranoiol
RESULTS P r e v i o u s l y I h a v e d e m o n s t r a t e d t h a t direct muscle s t i m u l a t i o n c o n t r a c t s t h e rat left a t r i a a n d t h a t t h e force o f c o n t r a c t i o n is i n c r e a s e d by isoprenaline. A l s o t h e r e s p o n s e s to electrical s t i m u l a t i o n are successively s m a l l e r in t h e s e c o n d a n d t h i r d p e r i o d s o f s t i m u l a t i o n w h e r e a s t h e m a x i m a l r e s p o n s e to electrical s t i m u l a t i o n a n d i s o p r e n a l i n e is n o t significantly different b e t w e e n the p e r i o d s o f s t i m u l a t i o n (Doggrell, 1988). In the p r e s e n t s t u d y ( + ) - a n d ( - ) - p r o p r a n o l o l (~< 1 0 - 5 M ) h a d n o effect o n t h e resting t o n e o f t h e rat left atria.
D a t a for ( + ) - p r o p a n o l o l a t >t 1 0 - 6 M is s h o w n in T a b l e 1 a n d Fig. 1. ( + ) - P r o p r a n o l o i a t 1 0 - 7 M h a d n o effect o n t h e r e s p o n s e s o f the r a t left a t r i a to electrical s t i m u l a t i o n o r o n t h e r e s p o n s e s to electrical s t i m u l a t i o n a n d i s o p r e n a l i n e [n = 1 i, n u m b e r o f tissues t r e a t e d w i t h ( + ) - p r o p r a n o l o l at 1 0 - ~ M = 11; d a t a n o t shown]. H i g h e r c o n c e n t r a t i o n s o f ( + ) p r o p r a n o l o l , 10 -6, 6 x 10 -6 a n d 1 0 - S M , i n h i b i t e d the r e s p o n s e s to electrical s t i m u l a t i o n a l o n e by 22, 76 a n d 8 9 % , respectively. T h e m a x i m a l c o m b i n e d r e s p o n s e to electrical s t i m u l a t i o n a n d i s o p r e n a l i n e was r e d u c e d
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Fig. 2. Effect of ( - )-propranolol on the contractile responses of the rat left atria to electrical stimulation and/or isoprenaline. Responses in the absence (<3) and presence (~l) of ( - ) - p r o p r a n o l o l at 1 0 - ' M , absence (~') and presence ( V ) of (-)-propranolol at 10-TM, absence ( A ) and presence ( A ) of ( - ) - p r o p r a n o l o l at 10-6M and the absence (1"7) and presence ( I ) of ( - ) - p r o p r a n o l o l at 10-SM. Left: responses to electrical stimulation and isoprenaline are calculated as a percentage of the maximum response of the first curve to electrical stimulation and isoprenaline. Right and top: responses to isoprenaline are calculated as a percentage of the maximum response of the individual curve to isoprenaline. All responses are plotted against the logarithm of the molar concentration of isoprenaline (abscissa). Each value is the mean of 9-10 atria. Vertical lines show SEM. Right and bottom: Schild regression (logarithm of the concentration-ratio minus 1 plotted against the logarithm of the molar concentration of antagonist) for (-)-propranolol
680
SHEILAA. DOGGRELL
by ( + ) - p r o p r a n o l o l at 1 0 5 M but not at 10 -6 or 6 × 10-6M. The isoprenaline response curves in the absence and presence of ( + ) - p r o p r a n o l o l at 10- 6 and 6 x 10-6M were normalized. F r o m the normalized data, the p D 2 values were significantly reduced from 8.92 4- 0.24 (8) [Mean 4- SEM] in the absence to 7.76 4- 0.18 in the presence of ( + ) - p r o p r a n o l o l at 10 6M and from 8.484-0.14 (8) in the absence to 6.67 + 0.09 (8) in the presence of ( + ) - p r o p r a n o l o l at 6 × 10-6M. On submitting the data to &child regression analysis, the slope of the line was 0.98 which was not significantly different from I and the pA 2 for ( + ) - p r o p r a n o l o l was 6.95. ( - )-Propranolol Data shown in Table i and Fig. 2. ( - ) - P r o p r a o nolol, 10 -s, 10 -7, 10 -6 and 10-SM, inhibited the responses to electrical stimulation alone by 27, 30, 58 and 100%, respectively. The maximal combined response to electrical stimulation and isoprenaline was reduced by ( - ) - p r o p r a n o l o l at 10-SM but not by lower concentrations, ~< 10-6M. The isoprenaline response curves in the absence and presence of ( - ) propranolol at 10 -8, 10 -7 and 10-6M were normalized; the pD2 values were significantly reduced from 8.93 4- 0.24 (10) in the absence to 7.50 4- 0.10 (10) in the presence of ( - ) - p r o p r a n o l o l at 10-SM, from 8.91 4-0.30 (12) in the absence to 6.564-0.11 (9) in the presence of ( - ) - p r o p r a n o l o l at 10-7M, and from 9.20 4- 0.24 (6) in the absence to 5.52 + 0.10 (10) in the presence of ( - ) - p r o p r a n o l o l at 10-6M. On submitting the data to Schild regression analysis, the slope of the line was 1.07 which was not significantly different from 1 and the pA 2 for ( - ) - p r o p r a n o l o l was 9.37. DISCUSSION In the only previous study of the manifestations of membrane stabilizing activity with the propanolol stereoisomers on a cardiac tissue (Barrett and Cullum, 1968), it was demonstrated that the stereoisomers were equipotent membrane stabilizers of the guinea-pig left atria and that high concentrations (4 x 10-4M) were required to exert this action. We have previously shown that low concentrations of (___)-propranolol ( i > 3 × 10-9M) have membrane stabilizing activity on the rat left atria which manifests itself as a depression of the response to electrical stimulation (Doggrell, 1988). This was an indication that one or both of the propranolol stereoisomers must be a more potent membrane stabilizer on the rat that guinea-pig atria. The present study demonstrates that both of the propranolol stereoisomers have membrane stabilizing activity on the rat left atria and that the stereoisomers were not equipotent on this tissue. Thus the threshold for membrane stabilizing activity with ( - ) - p r o p r a n o l o l was 10-~M and with ( + ) - p r o p r a n o l o l was 10-6M. A second component to the membrane stabilizing activity of (4-)-propranolol on the rat left atria was
observed with a high concentration (10-5 M), a depression of the maximal combined response to electrical stimulation and isoprenaline (Doggrell, 1988). The present study illustrates that both ( + ) - and ( - ) - p r o p r a n o l o l exert this second component of membrane stabilizing activity on the rat left atria at 10-SM with the ( - ) - s t e r e o i s o m e r having a greater effect than the (+)-stereoisomer. It has previously been reported that ( - ) - p r o p r a nolol is over a 100 times more potent than ( + ) - p r o pranolol as a flj-adrenoceptor antagonist (guinea-pig atria, Barrett and Cullum, 1968). The present study confirms this; thus, on the rat left atria, ( - ) - p r o p r a nolol (pA 2 = 9.37) was over a 100 times more potent than ( + ) - p r o p r a n o l o l (pA2 = 6.95) as a /~t-adrenoceptor antagonist. In conclusion, the present study has demonstrated that at low, and therefore clinical relevant concentrations, ( - ) - p r o p r a n o l o l is a membrane stabilizer and a fl~-adrenoceptor antagonist. At higher concentrations ( + ) - p r o p r a n o l o l is also a membrane stabilizer and a/3~ -adrenoceptor antagonist on the rat left atria. SUMMARY
I. The effects of (+)- and (-)-propranolol on the responses of the rat left atria to electrical stimulation, and to electrical stimulation and isoprenaline have been studied. 2. (+)-Propranolol at 10-6M inhibited the responses to electrical stimulation, and at 10-SM inhibited the maximal combined response to electrical stimulation and isoprenaline. (+)-Propranolol was a competitive antagonist of the responses to isoprenaline with a pA 2 of 6.95. 3. (-)-Propranolol at 10-~M inhibited the responses to electrical stimulation, and at 10 -5 M inhibited the maximal combined response to electrical stimulation and isoprenaline. The responses to isoprenaline were inhibited in a competitive manner by (-)-propranolol with a pA 2 of 9.37. 4. These results illustrate that on the rat left atria low concentrations of (-)-propranolol have membrane stabilizing and /~t-adrenoceptor blocking activity. High concentrations of ( + )-propranolol also have membrane stabilizing and fl~-adrenoceptor blocking activity on this preparation. Acknowledgement--This study was supported by a Project Grant from the National Heart Foundation of New Zealand. REFERENCES
Barrett A. M. and Cullum V. A. (1968) The biological properties of the optical isomers of propranolol and their effects on cardiac arrhythmias. Br. J. Pharmac. 34, 43 -55. Doggrell S. A. (1988) Simultaneous assessment of membrane-stabilizing and/1-adrenoceptor blocking activity of drugs with the rat isolated left atria. J. Pharmac. Meth. 19, 93-107. Fitzgerald J. D. (1984) /~-Adrenoceptor antagonists. In Handbook of Hypertension Vol. 3. Pharmacology of Antihypertensive Drugs (Edited by van Zwieten P. A.), pp. 249-306. Elsevier, Amsterdam. Shand D. G., Nuckolls E. M. and Oates J. A. (1970) Plasma propranolol levels in adults, with observation in four children. Clin. Pharmac. Ther. 11, 112-120.
0306-3623/90 $3.00+ 0.00 Copyright © 1990PergamonPress pie
THE MEMBRANE STABILIZING AND flt-ADRENOCEPTOR BLOCKING ACTIVITY OF (+)- AND (-)-PROPRANOLOL ON THE RAT LEFT ATRIA SHEILA A. DOGGRELL Department of Pharmacology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand [Tel. (649) 795780; Fax (649) 770956]
(Received 27 November 1989)
Abstract--I. The membrane stabilizing and fll-adrenoceptor blocking activities of(+)- and (-)-propranolol have been determined using the rat left atria. 2. (+)- And (-)-propranolol have membrane stabilizing activity. Thus a low concentration of (-)-propranolol, 10-aM, and a higher concentration of (+)-propranolol, 10-6M, inhibited the responses to electrical stimulation. A high concentration of (+)- and (-)-propranolol, 10-~M, also inhibited the maximal combined response to electrical stimulation and isoprenaline. 3. As competitive fit -adrenoceptor antagonists, ( - )-propranolol was over a I00 times more potent than ( + )-propranolol.
INTRODUCTION fl-Adrenoceptor antagonists (r-blockers) are commonly used in the treatment of hypertension, angina and cardiac arrhythmias. Some of the r-blockers (e.g. propranolol) have as an ancillary property the ability to cause membrane stabilization (reviewed by Fitzgerald, 1984). However the membrane stabilizing activity of propranolol is not generally considered to be clinically relevant as it occurs with concentrations of propranolol higher than those used clinically. Thus after the administration of an oral dose of propranolol to humans the plasma concentration is 6 × 10-8-6 × 10-TM for I-7 hr (Shand et al., 1970). Whereas studies with isolated animal cardiac tissues have only shown a membrane stabilizing action with ( ± ) - p r o p r a n o l o l at 10-6M (rabbit atrial preparations, Fitzgerald, 1984) and with ( + ) - and ( - ) propranolol at 3.5 and 4.5 × 10-4M, respectively (guinea-pig left atria, Barrett and Cullum, 1968). Recently I have described a method to simultaneously assess the membrane stabilizing and fl~adrenoceptor blocking activity of drugs using the rat isolated left atria (Doggrell, 1988). In this model the membrane stabilizing activity of procaine manifests itself in two ways; a depression of the response to electrical stimulation alone and, at a higher concentration, a depression of the maximal combined response to electrical stimulation and isoprenaline. Metoprolol, a r-blocker without membrane stabilizing activity, only acted as an inhibitor of the responses to isoprenaline. Analysis of the effects of ( ± ) - p r o p r a n o l o l on the responses of the left atria to electrical stimulation and to electrical stimulation and isoprenaline demonstrated that ( ± ) - p r o p r a n o l o l was a potent membrane stabilizer and flt-adrenoceptor antagonist on this preparation. There were two components to the membrane stabilizing action of ( ± ) propranolol; low concentrations, >I 3 × 10-gM, depressed the responses to electrical stimulation alone
and a high concentration (10-SM) depressed the maximal combined response to electrical stimulation and isoprenaline (Doggrell, 1988). This was the first demonstration that low, and therefore clinically relevant, concentrations of (__.)-propranolol have the manifestations of membrane stabilizing activity on cardiac tissue. The components that each of the propranolol stereoisomers contributes to the membrane stabilizing and fl~-adrenoceptor blocking actions of (_+)-propranolol on the rat left atria have not previously been described. The present communication reports the effects of ( + ) - and ( - ) - p r o p r a n o l o l on the responses of the rat left atria to electrical stimulation, and to electrical stimulation and isoprenaline. METHODS AND MATERIALS
Recording of the contractile responses of the electrically driven rat left atria in accordance with the method of Doggrell (1988), male Wistar rats (250-350 g) were stunned and exsanguinated. The heart was rapidly removed and placed in Krebs solution that was saturated with 5 0 CO 2 in oxygen, and the left atria was excised. All procedures were performed in the presence of a modified Krebs solution [composition (raM): NaCI, 116; KCI, 5.4; CaCI~, 2.5; MgCI2, 1.2; NaH2PO4, 1.2; NaHCO~, 22.0; ~glucose, 11.2; Na 2 EDTA, 0.04] which was being vigorously bubbled with 5% CO 2 in oxygen at 37°C. Each left atria was halved. Each half was mounted longitudinally between two platinum electrodes (approximately 3 crn apart, above and below the tissue) under I g tension in 5 ml organ baths containing Krebs solution (with 10- ~M guanethidine to prevent the release of noradrenaline from nerve endings and 10-~M atropine) and allowed to equilibrate for 60 rain. During the equilibration period, the tissues were washed by overflow. Tissues were electrically stimulated at 4 Hz (5 reset, 10 V). After 6 rain of stimulation, a cumulative challenge with isoprenaline was initiated. Thus atria were challenged with isoprenaline at 3 min intervals to give a final concentration
677
678
SHEILA A. DOGGRELL
of isoprenaline in the organ both successively of 3 x I0 -H, 10 -m, 3 x 10-raM etc. This cycle was continued until a maximum response was obtained with the contractile responses being recorded isometrically with force displacement transducers (Grass model F/TO3C) and displayed on a polygraph (Grass model 79B). Stimulation was then stopped. One half of the left atria remained untreated throughout while the other half of the left atria was treated with (+)-propranolol at 10 -7 M. Atria were washed rapidly in the absence or presence of drug for 60 min prior to a further period of electrical stimulation with a second cumulative challenge to isoprenaline. The treated atria were then further treated with (+)-propranolol at 10-~M. After another 60 min of rapid washing in the absence or presence of drug, a third cumulative challenge to isoprenaline of the electrically stimulated tissue was undertaken. In other studies the drugs and concentrations used were (+)-propranolol at 10 .6 and 6 x 10-6M, ( - ) - p r o p r a n o l o l at I0 g and 10-6M and ( - ) - p r o p r a n o l o l at 10 -7 and 10 5M. The contractile responses to electrical stimulation just prior to challenge with isoprenaline and the maximal contractile responses to each concentration of isoprenaline in the presence of electrical stimulation were measured.
prenaline were subtracted from the combined response to electrical stimulation and isoprenaline. These responses to isoprenaline were calculated as a percentage of the maximum of the first isoprenaline response curve. If the maximum to isoprenaline between treated and untreated tissues calculated in this manner were not significantly different, responses to isoprenaline were also calculated as a percentage of the individual curve to isoprenaline i.e. normalized. When responses to isoprenaline were normalized, pD 2 values, concentration-ratios and then pA 2 values were determined, pD 2 values were computed by regression line analysis on the steepest part of the response curve, which was usually over the range 20-80°/. of the maximum response. For each tissue, the concentration ratios (the antilog of the difference between the pD 2 values) were determined between each challenge with isoprenaline. The concentration ratios obtained in the presence of drugs were corrected for changes occurring in untreated tissues, pA2 values were determined by Schild plot [log (x - 1), ordinate, vs the logarithm of the molar concentration of antagonist, abscissa] using regression line analysis of individual log (x - I) values. A least squares analysis was performed to test whether the line of the Schild plot had a slope that was significantly different from 1. The pA2 values was the abscissa intercept when the slope was not significantly different from I. The individual values were compared by Student's t-test and were considered to be significantly different when P < 0.05. From these individual values, mean values + SEM were also determined.
Analysis of data The contractile responses to electrical stimulation just prior to the second and third challenges with isoprenaline were calculated as a percentage of the response to stimulation prior to the first challenge with isoprenaline. The combined responses to electrical stimulation and isoprenaline were calculated as a percentage of the maximum combined response to electrical stimulation and isoprenaline during the first challenge with isoprenaline. For each challenge with isoprenaline, the responses to electrical stimulation just prior to the challenge with iso-
Drugs The drugs used were guanethidine sulphate (donated by Ciba-Geigy), ( + ) - and ( - ) - p r o p r a n o l o l hydrochloride (donated by ICi), atropine sulphate and ( - ) - isoprenaline bitartrate (Sigma Chemical Company).
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Fig. 1. Effect o f (+)-propranolol on the contractile responses of the rat left atria to electrical stimulation and/or isoprenaline. Responses in the absence (A) and presence (A) of (+)-propranolol at 10-6M, absence ( 0 ) and presence (O) of (+)-propranolol at 6 x 10-6M and the absence (11) and presence (11) of (+)-propranolol at 10-SM. Left: responses to electrical stimulation and isoprenaline are calculated as a percentage of the maximum response o f t h e first curve to electrical stimulation. Right and top: responses to isoprenaline are calculated as a percentage of the individual curve to isoprenaline. All responses are plotted against the logarithm of the molar concentration of isoprenaline (abscissa). Each value is the mean of 6-11 atria. Vertical lines show SEM. Right and bottom: Schild regression (logarithm of the concentration-ratio minus I plotted against the logarithm of the molar concentration of antagonist) for ( + )-propranolol.
M.S.A. with ( + ) - and ( - ) - p r o p r a n o i o i
679
Table I. Effects of (+)- and (-)-propranoiol on the ~ of the rat k,R atria to electrical stimulation and on the maximal respomes to electrical stimulation and itoprenaline Maximal rcsponlm' P,~'trical stimulation stimulation and itolnenaline 81 ± 5 (8) 98 + 2 (8) (ii) Control (+) Pro )ranolol, 10-6M 63 ± 8 (8)" 99 + 4(8) 62 + 7 18) 96 ± 3 (8) (iii) Control ( + )-Pro >ranolol, 6 × 10-6M 15 4- 14(8)* 90 + 4(8) Control 474. II (6) 884-4(6) (iii) ( + )-Pro )ranolol0 10- s M 5 +, 3 (I I )* 53 + 9 (I I)* Control 7s +_8 (10) 90 4- 5 (to) (ii) ( - )-Pro )ranolol, 10-SM 57 ± 7 (t0)* 97 4- 5 (10) 80 4- 8 (12) 100 4- 4 (12) (ii) Control ( - )-Pro )ranolol, 10-?M 56 4- 8 (9)* 96 4- 6 (9) Control 62 4- 12 (6) 90 4- 13 (6) (iii) ( -)-Pro )ranolol, 10-eM 26 4- 10 (10)* 100 4- 7 (10) Control 604- 10(I I) 104+5(11) (iii) ( - ).Pro )ranolol, 10-~M 0 (9) 94-6(9)* 'Calculated as a percentage of the first resImnue. (ii) and (iii) refer to the second and third responses. All values are mean ± SEM. The number in parenthesis is the number of observations. ' P < 0.05, unpaired t-test with own control.
(+)-Propranoiol
RESULTS P r e v i o u s l y I h a v e d e m o n s t r a t e d t h a t direct muscle s t i m u l a t i o n c o n t r a c t s t h e rat left a t r i a a n d t h a t t h e force o f c o n t r a c t i o n is i n c r e a s e d by isoprenaline. A l s o t h e r e s p o n s e s to electrical s t i m u l a t i o n are successively s m a l l e r in t h e s e c o n d a n d t h i r d p e r i o d s o f s t i m u l a t i o n w h e r e a s t h e m a x i m a l r e s p o n s e to electrical s t i m u l a t i o n a n d i s o p r e n a l i n e is n o t significantly different b e t w e e n the p e r i o d s o f s t i m u l a t i o n (Doggrell, 1988). In the p r e s e n t s t u d y ( + ) - a n d ( - ) - p r o p r a n o l o l (~< 1 0 - 5 M ) h a d n o effect o n t h e resting t o n e o f t h e rat left atria.
D a t a for ( + ) - p r o p a n o l o l a t >t 1 0 - 6 M is s h o w n in T a b l e 1 a n d Fig. 1. ( + ) - P r o p r a n o l o i a t 1 0 - 7 M h a d n o effect o n t h e r e s p o n s e s o f the r a t left a t r i a to electrical s t i m u l a t i o n o r o n t h e r e s p o n s e s to electrical s t i m u l a t i o n a n d i s o p r e n a l i n e [n = 1 i, n u m b e r o f tissues t r e a t e d w i t h ( + ) - p r o p r a n o l o l at 1 0 - ~ M = 11; d a t a n o t shown]. H i g h e r c o n c e n t r a t i o n s o f ( + ) p r o p r a n o l o l , 10 -6, 6 x 10 -6 a n d 1 0 - S M , i n h i b i t e d the r e s p o n s e s to electrical s t i m u l a t i o n a l o n e by 22, 76 a n d 8 9 % , respectively. T h e m a x i m a l c o m b i n e d r e s p o n s e to electrical s t i m u l a t i o n a n d i s o p r e n a l i n e was r e d u c e d
(-)-PIIOPRANQLOL
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Fig. 2. Effect of ( - )-propranolol on the contractile responses of the rat left atria to electrical stimulation and/or isoprenaline. Responses in the absence (<3) and presence (~l) of ( - ) - p r o p r a n o l o l at 1 0 - ' M , absence (~') and presence ( V ) of (-)-propranolol at 10-TM, absence ( A ) and presence ( A ) of ( - ) - p r o p r a n o l o l at 10-6M and the absence (1"7) and presence ( I ) of ( - ) - p r o p r a n o l o l at 10-SM. Left: responses to electrical stimulation and isoprenaline are calculated as a percentage of the maximum response of the first curve to electrical stimulation and isoprenaline. Right and top: responses to isoprenaline are calculated as a percentage of the maximum response of the individual curve to isoprenaline. All responses are plotted against the logarithm of the molar concentration of isoprenaline (abscissa). Each value is the mean of 9-10 atria. Vertical lines show SEM. Right and bottom: Schild regression (logarithm of the concentration-ratio minus 1 plotted against the logarithm of the molar concentration of antagonist) for (-)-propranolol
680
SHEILAA. DOGGRELL
by ( + ) - p r o p r a n o l o l at 1 0 5 M but not at 10 -6 or 6 × 10-6M. The isoprenaline response curves in the absence and presence of ( + ) - p r o p r a n o l o l at 10- 6 and 6 x 10-6M were normalized. F r o m the normalized data, the p D 2 values were significantly reduced from 8.92 4- 0.24 (8) [Mean 4- SEM] in the absence to 7.76 4- 0.18 in the presence of ( + ) - p r o p r a n o l o l at 10 6M and from 8.484-0.14 (8) in the absence to 6.67 + 0.09 (8) in the presence of ( + ) - p r o p r a n o l o l at 6 × 10-6M. On submitting the data to &child regression analysis, the slope of the line was 0.98 which was not significantly different from I and the pA 2 for ( + ) - p r o p r a n o l o l was 6.95. ( - )-Propranolol Data shown in Table i and Fig. 2. ( - ) - P r o p r a o nolol, 10 -s, 10 -7, 10 -6 and 10-SM, inhibited the responses to electrical stimulation alone by 27, 30, 58 and 100%, respectively. The maximal combined response to electrical stimulation and isoprenaline was reduced by ( - ) - p r o p r a n o l o l at 10-SM but not by lower concentrations, ~< 10-6M. The isoprenaline response curves in the absence and presence of ( - ) propranolol at 10 -8, 10 -7 and 10-6M were normalized; the pD2 values were significantly reduced from 8.93 4- 0.24 (10) in the absence to 7.50 4- 0.10 (10) in the presence of ( - ) - p r o p r a n o l o l at 10-SM, from 8.91 4-0.30 (12) in the absence to 6.564-0.11 (9) in the presence of ( - ) - p r o p r a n o l o l at 10-7M, and from 9.20 4- 0.24 (6) in the absence to 5.52 + 0.10 (10) in the presence of ( - ) - p r o p r a n o l o l at 10-6M. On submitting the data to Schild regression analysis, the slope of the line was 1.07 which was not significantly different from 1 and the pA 2 for ( - ) - p r o p r a n o l o l was 9.37. DISCUSSION In the only previous study of the manifestations of membrane stabilizing activity with the propanolol stereoisomers on a cardiac tissue (Barrett and Cullum, 1968), it was demonstrated that the stereoisomers were equipotent membrane stabilizers of the guinea-pig left atria and that high concentrations (4 x 10-4M) were required to exert this action. We have previously shown that low concentrations of (___)-propranolol ( i > 3 × 10-9M) have membrane stabilizing activity on the rat left atria which manifests itself as a depression of the response to electrical stimulation (Doggrell, 1988). This was an indication that one or both of the propranolol stereoisomers must be a more potent membrane stabilizer on the rat that guinea-pig atria. The present study demonstrates that both of the propranolol stereoisomers have membrane stabilizing activity on the rat left atria and that the stereoisomers were not equipotent on this tissue. Thus the threshold for membrane stabilizing activity with ( - ) - p r o p r a n o l o l was 10-~M and with ( + ) - p r o p r a n o l o l was 10-6M. A second component to the membrane stabilizing activity of (4-)-propranolol on the rat left atria was
observed with a high concentration (10-5 M), a depression of the maximal combined response to electrical stimulation and isoprenaline (Doggrell, 1988). The present study illustrates that both ( + ) - and ( - ) - p r o p r a n o l o l exert this second component of membrane stabilizing activity on the rat left atria at 10-SM with the ( - ) - s t e r e o i s o m e r having a greater effect than the (+)-stereoisomer. It has previously been reported that ( - ) - p r o p r a nolol is over a 100 times more potent than ( + ) - p r o pranolol as a flj-adrenoceptor antagonist (guinea-pig atria, Barrett and Cullum, 1968). The present study confirms this; thus, on the rat left atria, ( - ) - p r o p r a nolol (pA 2 = 9.37) was over a 100 times more potent than ( + ) - p r o p r a n o l o l (pA2 = 6.95) as a /~t-adrenoceptor antagonist. In conclusion, the present study has demonstrated that at low, and therefore clinical relevant concentrations, ( - ) - p r o p r a n o l o l is a membrane stabilizer and a fl~-adrenoceptor antagonist. At higher concentrations ( + ) - p r o p r a n o l o l is also a membrane stabilizer and a/3~ -adrenoceptor antagonist on the rat left atria. SUMMARY
I. The effects of (+)- and (-)-propranolol on the responses of the rat left atria to electrical stimulation, and to electrical stimulation and isoprenaline have been studied. 2. (+)-Propranolol at 10-6M inhibited the responses to electrical stimulation, and at 10-SM inhibited the maximal combined response to electrical stimulation and isoprenaline. (+)-Propranolol was a competitive antagonist of the responses to isoprenaline with a pA 2 of 6.95. 3. (-)-Propranolol at 10-~M inhibited the responses to electrical stimulation, and at 10 -5 M inhibited the maximal combined response to electrical stimulation and isoprenaline. The responses to isoprenaline were inhibited in a competitive manner by (-)-propranolol with a pA 2 of 9.37. 4. These results illustrate that on the rat left atria low concentrations of (-)-propranolol have membrane stabilizing and /~t-adrenoceptor blocking activity. High concentrations of ( + )-propranolol also have membrane stabilizing and fl~-adrenoceptor blocking activity on this preparation. Acknowledgement--This study was supported by a Project Grant from the National Heart Foundation of New Zealand. REFERENCES
Barrett A. M. and Cullum V. A. (1968) The biological properties of the optical isomers of propranolol and their effects on cardiac arrhythmias. Br. J. Pharmac. 34, 43 -55. Doggrell S. A. (1988) Simultaneous assessment of membrane-stabilizing and/1-adrenoceptor blocking activity of drugs with the rat isolated left atria. J. Pharmac. Meth. 19, 93-107. Fitzgerald J. D. (1984) /~-Adrenoceptor antagonists. In Handbook of Hypertension Vol. 3. Pharmacology of Antihypertensive Drugs (Edited by van Zwieten P. A.), pp. 249-306. Elsevier, Amsterdam. Shand D. G., Nuckolls E. M. and Oates J. A. (1970) Plasma propranolol levels in adults, with observation in four children. Clin. Pharmac. Ther. 11, 112-120.