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Vascular smooth muscle contraction initiated by postsynaptic α2-adrenoceptor activation is induced by an influx of extracellular calcium
205
European Journal of Pharmacology, 69 (1981) 205--208 © Elsevier/North-Holland Biomedical Press
Short communication VASCULAR SMOOTH MUSCLE CONTRACTION INITIATED BY POSTSYNAPTIC ~2-ADRENOCEPTOR ACTIVATION IS INDUCED BY AN INFLUX OF EXTRACELLULAR CALCIUM JACQUES C.A. VAN MEEL, ADRIAAN DE JONGE, HANS O. KALKMAN, BOB WILFFERT, PIETER B.M.W.M. TIMMERMANS and PIETER A. VAN ZWIETEN
Department of Pharmacy, Division of Pharmacotherapy, University of Amsterdam, Plantage Muidergracht 24, 1018 TV Amsterdam, The Netherlands Received 2 September 1980, accepted 12 November 1980
J.C.A. V A N M E E L , A. D E J O N G E , H.O. K A L K M A N , B. W I L F F E R T , P.B.M.W.M. T I M M E R M A N S and P.A. V A N Z W I E T E N , Vascular smooth muscle contraction initiated by postsynaptic a2~adrenoceptor activation is induced by an influx of extraeeUular calcium, European J. Pharmacol. 69 (1981)205--208. Vasoconstriction in pithed, normotensive rats elicited via stimulation of postsynaptic ~2-adrenoceptors by B-HT 920 was antagonized by E D T A and the calcium antagonists nifedipine, D 600 and veraparnil, whereas pressor responses to the ~l-agonist methoxamine were unaffected. This indicates that vasoconstriction in vivo initiated via postsynaptic ~2-adrenoceptors requires an influx of extracellularcalcium. Thus, the antihypertensive effect of calcium antagonists m a y be based upon a diminution of vascular tone maintained by postsynaptic c~2-adrenoceptors. Postsynaptic {~I-and ~2 -adrenoceptors Calcium antagonists
Calcium inhibition Vasoconstriction
1. Introduction Postsynaptic a ~- as well as a2-adrenoceptors are involved in drug-induced vasoconstriction in vivo (Drew and Whiting, 1979; Timmermarls et al., 1979; Docherty and McGrath, 1980; Timmermans and Van Zwieten, 1980a). When postsynaptic a2-adrenbceptors were stimulated, the hypertensive response had a slower onset than that induced by stimulation of postsynaptic a~-adrenoceptors (Timmermans and Van Zwieten, 1980b). Isotonic contractions of vascular smooth muscle consist of a fast (phasic) and a slow (tonic) component. Phasic and tonic contraction responses have been attributed to functional properties of vascular smooth muscle, reflecting the release of intracellular calcium and the utilization of extracellular calcium,
Methoxamine
B-HT 920
respectively (Golenhofen, 1976; Hester and Carrier, 1977). It seemed therefore of interest to test the hypothesis whether extracellular free calcium is a prerequisite for vasoconstriction initiated by activation of postsynaptic ~2-adrenoceptors. For this purpose we have used the so-called calcium antagonists verapamil, D 600 and nifedipine which block the transmembrane calcium flux (Fleckenstein, 1977) as well as EDTA which complexes extraceUular calcium. Selective stimulation of vascular ~:-adrenoceptors in the intact circulatory system of the pithed rat was achieved by means of B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[5,4
206 and V a n Z w i e t e n , 1 9 8 0 a , Van Meel et al., 1 9 8 0 ) . M e t h o x a m i n e was used as an agonist of a l-adrenoceptors. We have n o w investigated w h e t h e r t h e i n h i b i t i o n o f t h e inward calcium c u r r e n t can a f f e c t v a s o c o n s t r i c t o r responses initiated b y postsynaptic a2-adrenoceptor stimulation.
J.C.A. VAN MEEL ET AL.
Increase in diastolic pressure (%) 10080-
& .... i.~
----- -__~ ...... ~--_
60
*
\
40-
. ~
20-
EDTA (mg/kg)
*
2. Materials and m e t h o d s
3'0 Artificially ventilated, p i t h e d , n o r m o t e n sive, male Wistar rats weighing 2 0 0 - - 2 5 0 g were used (see T i m m e r m a n s and Van Zwieten, 1980a,b). Diastolic pressure was increased b y m e t h o x a m i n e and B-HT 920 injected i n t r a v e n o u s l y . T h e pressor effects o f b o t h agonists were studied at a dose o f 150 /~g/kg. F o r m e t h o x a m i n e as well as B-HT 920 this dose caused a pressor r e s p o n s e w h i c h a m o u n t e d t o 80% o f t h e m a x i m a l l y a t t a i n a b l e effect. M e a s u r e m e n t s w e r e m a d e 15 min a f t e r intra-arterial ( c a r o t i d a r t e r y ) t r e a t m e n t with saline ( c o n t r o l ) or d i f f e r e n t a m o u n t s o f verapamil, D 6 0 0 and n i f e d i p i n e as well as a f t e r E D T A and c a l c i u m - E D T A . T h e latter t w o agents were infused over a p e r i o d o f 15 min f o l l o w e d b y 5 min o f equilibration. Drugs used and their sources were: B-HT 9 2 0 • 2HC1 ( T h o m a e ) , D 600 • HC1 and v e r a p a m i l - H C 1 (Knoll), E D T A ( E D T A N a 2 . 2 H 2 0 ) and c a l c i u m - E D T A ( E D T A N a 2 C a . 6 H 2 0 ) (Merck), m e t h o x a m i n e . HC1 (Burroughs Wellcome) and n i f e d i p i n e (Bayer); t h e doses m e n t i o n e d r e f e r t o these dosage forms.
3. Results Mean diastolic pressure o f u n t r e a t e d p i t h e d n o r m o t e n s i v e rats a m o u n t e d t o 45.3 +0.2 m m Hg ( m e a n -+ S.E.M., n = 246). Single i n t r a v e n o u s doses o f m e t h o x a m i n e and B-HT 9 2 0 (150 /~g/kg o f each) increased diastolic pressure o f these animals b y 98.5 + 3.8 and 79.0 + 3.6 m m Hg, respectively (means + S.E.M., n = 8). Previous a d m i n i s t r a t i o n o f
6'0
lOO-
.....
9'0
t20
.....
8o-
60 4020-
*
* . . Nifedipine 1()
3'0
Jg/kg)~--~
160 360 1060
80-
.~
60-
~
40-
* ~i~..
20-
D 600 (pg/kg) 10 '--
3'0
. "~
=100 360 1000 -~
,oo 80-
"
60 40 20
* Verapami I (pg/kg) 1'0
3'0
~
160 360 10~0
Fig. 1. Increase in diastolic pressure of pithed, normotensive rats induced by intravenous B-HT 920 (m -') and methoxamine (©. . . . . . ©) (150 pg] kg of each) after intraarterial treatment with EDTA (15 rain infusion + 5 rain equilibration), nifedipine, D 600 and verapamil (injection --15 rain). The results are calculated as percent effect of saline-pretreated control responses (=100%). Symbols represent mean values -+ S.E.M. of 5--6 separate experiments. *P < 0.05 (Student's t-test) with respect to saline-pretreated control rats.
CALCIUM AND VASOCONSTRICTION
inhibited the EDTA dosedependently pressor effect of B-HT 920 (fig. 1). The hypertensive response to methoxamine was influenced to a much smaller extent (fig. 1). Equimolar quantities of calcium-EDTA did not significantly reduce the pressor responses to both CY-adrenoceptor agonists. The calcium antagonists nifedipine, D 600 and verapamil injected intraarterially 15 min before pressor effects were recorded impaired the increase in diastolic pressure induced by intravenous B-HT 920 in a dose-dependent manner to nearly complete abolishment (fig. 1). Only the higher doses of these drugs affected the vasoconstriction brought about by methoxamine.
4. Discussion The results of the present experiments point to the role of extracellular free calcium in drug-induced vasoconstriction in vivo caused by stimulation of postsynaptic 02adrenoceptors. This conclusion is based upon the observation that EDTA and specific inhibitors of the transmembrane calcium movements such as nifedipine, D 600 and verapamil (Fleckenstein, 1977) decreased the B-HT 920-induced hypertension, which is mediated by postsynaptic a,-adrenoceptors, like that of its congener B-HT 933 (Timmermans and Van Zwieten, 1980a,b; Van Meel et al., 1980). On the other hand, the hypertensive response to methoxamine, which involves postsynaptic CY ,-adrenoceptors, was hardly affected. It is submitted that the generally known tonic (slow), component of vascular smooth muscle contraction occurs with stimulation of postsynaptic a,-adrenoceptors. This leads to a transmembrane flux of extracellular calcium which activates the contractile apparatus. In contrast, vascular smooth muscle contraction initiated at CY~adrenoceptors is not directly governed by an inward current of free calcium. However, other explanations have been given to the steps linking ol- and ar,-activation to effects
207
with special reference to the regulation of cellular metabolism by catecholamines (Fain and Garcia-Sainz, 1980, and refs. quoted therein). It is most attractive to translate the present findings into a hypothesis for the mechanism by which calcium antagonists can lower arterial pressure. The blood pressure lowering effect of calcium antagonists, such as nifedipine (Olivari et al., 1979) may at least partially be due to the inhibition of the influx of extracellular calcium which is necessary to maintain that part of the vascular tone mediated by a,-adrenoceptors in the vascular wall. Accordingly, after blockade of the stimulation of inward calcium current, vascular postsynaptic a,-adrenoceptors (for instance by endogenous catecholamines) no longer contributes to the maintainance of vascular tone, so that a hypotensive effect will develop.
Acknowledgements The generous donation of B-HT 920, D 600 and verapamil, and nifedipine by Thomae, Knoll and Bayer, respectively, is gratefully acknowledged.
References Docherty, J.R. and J.C. McGrath, 1980, A comparison of pre- and postjunctional potencies of several a-adrenoceptor agonists in the cardiovascular system and anococcygeus muscle of the rat, NaunynSchmiedeb. Arch. Pharmacol. 312,107. Drew, G.M. and S.B. Whiting, 1979, Evidence for two distinct types of postsynaptic o-adrenoceptors in vascular smooth muscle in vivo, Br. J. Pharmacol. 67,207. Fain, J.N. and J.A. Garcia-Sainz, 1980, Role of phosphatidylinositol turnover in alpha1 and of adenylate cyclase inhibition in alpha? effects of catecholamines, Life Sci. 26, 1183. Fleckenstein, A., 1977, Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle, Ann. Rev. Pharmacol. Toxicol. 17, 149. Golenhofen, K., 1976, Theory of P and T systems for calcium activation in smooth muscle, in:
208 Physiology of Smooth Muscle, eds. E. Biilbring and M.F. Shuba (Raven Press, New York) p. 197. Hester, R.K. and O. Carrier, 1977, Excitation-contraction/relaxation coupling in vascular smooth muscle, in: Factors Influencing Vascular Reactivity, eds. O. Carrier and S. Shibata (Igaku-Shoin, Tokyo) p. 96. Olivari, M.T., C. Bartorelli, A. Polese, C. Fiorentini, P. Moruzzi and M.D. Guazzi, 1979, Treatment of hypertension with nifedipine, a calcium antagonistic agent, Circulation 59, 1056. Timmermans, P.B.M.W.M., H.Y. Kwa and P.A. Van Zwieten, 1979, Possible subdivision of postsynaptic a-adrenoceptors mediating pressor responses in the pithed rat, Naunyn-Schmiedeb. Arch. Pharmacol. 3 1 0 , 1 8 9 .
J.C.A. VAN MEEL ET AL. Timmermans, P.B.M.W.M. and P.A. Van Zwieten, 1980a, Postsynaptic a 1- and a2-adrenoceptors in the circulatory system of the pithed rat; selective stimulation of the a2-type by B-HT 933, European J. Pharmacol. 63,199. Timmermans, P.B.M.W.M. and P.A. Van Zwieten, 1980b, Vasoconstriction mediated by postsynaptic a2-adrenoceptor stimulation, Naunyn-Schmiedeb. Arch. Pharmacol. 313, 17. Van Meel, J.C.A., P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1980, Selectivity of some agonis~ for postsynaptic 0~1- and a2-adrenoce ptots in the vascular system of the pithed rat, Naunyn-Schmiedeb. Arch. Pharmacol. Suppl. 313, R22.
European Journal of Pharmacology, 69 (1981) 205--208 © Elsevier/North-Holland Biomedical Press
Short communication VASCULAR SMOOTH MUSCLE CONTRACTION INITIATED BY POSTSYNAPTIC ~2-ADRENOCEPTOR ACTIVATION IS INDUCED BY AN INFLUX OF EXTRACELLULAR CALCIUM JACQUES C.A. VAN MEEL, ADRIAAN DE JONGE, HANS O. KALKMAN, BOB WILFFERT, PIETER B.M.W.M. TIMMERMANS and PIETER A. VAN ZWIETEN
Department of Pharmacy, Division of Pharmacotherapy, University of Amsterdam, Plantage Muidergracht 24, 1018 TV Amsterdam, The Netherlands Received 2 September 1980, accepted 12 November 1980
J.C.A. V A N M E E L , A. D E J O N G E , H.O. K A L K M A N , B. W I L F F E R T , P.B.M.W.M. T I M M E R M A N S and P.A. V A N Z W I E T E N , Vascular smooth muscle contraction initiated by postsynaptic a2~adrenoceptor activation is induced by an influx of extraeeUular calcium, European J. Pharmacol. 69 (1981)205--208. Vasoconstriction in pithed, normotensive rats elicited via stimulation of postsynaptic ~2-adrenoceptors by B-HT 920 was antagonized by E D T A and the calcium antagonists nifedipine, D 600 and veraparnil, whereas pressor responses to the ~l-agonist methoxamine were unaffected. This indicates that vasoconstriction in vivo initiated via postsynaptic ~2-adrenoceptors requires an influx of extracellularcalcium. Thus, the antihypertensive effect of calcium antagonists m a y be based upon a diminution of vascular tone maintained by postsynaptic c~2-adrenoceptors. Postsynaptic {~I-and ~2 -adrenoceptors Calcium antagonists
Calcium inhibition Vasoconstriction
1. Introduction Postsynaptic a ~- as well as a2-adrenoceptors are involved in drug-induced vasoconstriction in vivo (Drew and Whiting, 1979; Timmermarls et al., 1979; Docherty and McGrath, 1980; Timmermans and Van Zwieten, 1980a). When postsynaptic a2-adrenbceptors were stimulated, the hypertensive response had a slower onset than that induced by stimulation of postsynaptic a~-adrenoceptors (Timmermans and Van Zwieten, 1980b). Isotonic contractions of vascular smooth muscle consist of a fast (phasic) and a slow (tonic) component. Phasic and tonic contraction responses have been attributed to functional properties of vascular smooth muscle, reflecting the release of intracellular calcium and the utilization of extracellular calcium,
Methoxamine
B-HT 920
respectively (Golenhofen, 1976; Hester and Carrier, 1977). It seemed therefore of interest to test the hypothesis whether extracellular free calcium is a prerequisite for vasoconstriction initiated by activation of postsynaptic ~2-adrenoceptors. For this purpose we have used the so-called calcium antagonists verapamil, D 600 and nifedipine which block the transmembrane calcium flux (Fleckenstein, 1977) as well as EDTA which complexes extraceUular calcium. Selective stimulation of vascular ~:-adrenoceptors in the intact circulatory system of the pithed rat was achieved by means of B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[5,4
206 and V a n Z w i e t e n , 1 9 8 0 a , Van Meel et al., 1 9 8 0 ) . M e t h o x a m i n e was used as an agonist of a l-adrenoceptors. We have n o w investigated w h e t h e r t h e i n h i b i t i o n o f t h e inward calcium c u r r e n t can a f f e c t v a s o c o n s t r i c t o r responses initiated b y postsynaptic a2-adrenoceptor stimulation.
J.C.A. VAN MEEL ET AL.
Increase in diastolic pressure (%) 10080-
& .... i.~
----- -__~ ...... ~--_
60
*
\
40-
. ~
20-
EDTA (mg/kg)
*
2. Materials and m e t h o d s
3'0 Artificially ventilated, p i t h e d , n o r m o t e n sive, male Wistar rats weighing 2 0 0 - - 2 5 0 g were used (see T i m m e r m a n s and Van Zwieten, 1980a,b). Diastolic pressure was increased b y m e t h o x a m i n e and B-HT 920 injected i n t r a v e n o u s l y . T h e pressor effects o f b o t h agonists were studied at a dose o f 150 /~g/kg. F o r m e t h o x a m i n e as well as B-HT 920 this dose caused a pressor r e s p o n s e w h i c h a m o u n t e d t o 80% o f t h e m a x i m a l l y a t t a i n a b l e effect. M e a s u r e m e n t s w e r e m a d e 15 min a f t e r intra-arterial ( c a r o t i d a r t e r y ) t r e a t m e n t with saline ( c o n t r o l ) or d i f f e r e n t a m o u n t s o f verapamil, D 6 0 0 and n i f e d i p i n e as well as a f t e r E D T A and c a l c i u m - E D T A . T h e latter t w o agents were infused over a p e r i o d o f 15 min f o l l o w e d b y 5 min o f equilibration. Drugs used and their sources were: B-HT 9 2 0 • 2HC1 ( T h o m a e ) , D 600 • HC1 and v e r a p a m i l - H C 1 (Knoll), E D T A ( E D T A N a 2 . 2 H 2 0 ) and c a l c i u m - E D T A ( E D T A N a 2 C a . 6 H 2 0 ) (Merck), m e t h o x a m i n e . HC1 (Burroughs Wellcome) and n i f e d i p i n e (Bayer); t h e doses m e n t i o n e d r e f e r t o these dosage forms.
3. Results Mean diastolic pressure o f u n t r e a t e d p i t h e d n o r m o t e n s i v e rats a m o u n t e d t o 45.3 +0.2 m m Hg ( m e a n -+ S.E.M., n = 246). Single i n t r a v e n o u s doses o f m e t h o x a m i n e and B-HT 9 2 0 (150 /~g/kg o f each) increased diastolic pressure o f these animals b y 98.5 + 3.8 and 79.0 + 3.6 m m Hg, respectively (means + S.E.M., n = 8). Previous a d m i n i s t r a t i o n o f
6'0
lOO-
.....
9'0
t20
.....
8o-
60 4020-
*
* . . Nifedipine 1()
3'0
Jg/kg)~--~
160 360 1060
80-
.~
60-
~
40-
* ~i~..
20-
D 600 (pg/kg) 10 '--
3'0
. "~
=100 360 1000 -~
,oo 80-
"
60 40 20
* Verapami I (pg/kg) 1'0
3'0
~
160 360 10~0
Fig. 1. Increase in diastolic pressure of pithed, normotensive rats induced by intravenous B-HT 920 (m -') and methoxamine (©. . . . . . ©) (150 pg] kg of each) after intraarterial treatment with EDTA (15 rain infusion + 5 rain equilibration), nifedipine, D 600 and verapamil (injection --15 rain). The results are calculated as percent effect of saline-pretreated control responses (=100%). Symbols represent mean values -+ S.E.M. of 5--6 separate experiments. *P < 0.05 (Student's t-test) with respect to saline-pretreated control rats.
CALCIUM AND VASOCONSTRICTION
inhibited the EDTA dosedependently pressor effect of B-HT 920 (fig. 1). The hypertensive response to methoxamine was influenced to a much smaller extent (fig. 1). Equimolar quantities of calcium-EDTA did not significantly reduce the pressor responses to both CY-adrenoceptor agonists. The calcium antagonists nifedipine, D 600 and verapamil injected intraarterially 15 min before pressor effects were recorded impaired the increase in diastolic pressure induced by intravenous B-HT 920 in a dose-dependent manner to nearly complete abolishment (fig. 1). Only the higher doses of these drugs affected the vasoconstriction brought about by methoxamine.
4. Discussion The results of the present experiments point to the role of extracellular free calcium in drug-induced vasoconstriction in vivo caused by stimulation of postsynaptic 02adrenoceptors. This conclusion is based upon the observation that EDTA and specific inhibitors of the transmembrane calcium movements such as nifedipine, D 600 and verapamil (Fleckenstein, 1977) decreased the B-HT 920-induced hypertension, which is mediated by postsynaptic a,-adrenoceptors, like that of its congener B-HT 933 (Timmermans and Van Zwieten, 1980a,b; Van Meel et al., 1980). On the other hand, the hypertensive response to methoxamine, which involves postsynaptic CY ,-adrenoceptors, was hardly affected. It is submitted that the generally known tonic (slow), component of vascular smooth muscle contraction occurs with stimulation of postsynaptic a,-adrenoceptors. This leads to a transmembrane flux of extracellular calcium which activates the contractile apparatus. In contrast, vascular smooth muscle contraction initiated at CY~adrenoceptors is not directly governed by an inward current of free calcium. However, other explanations have been given to the steps linking ol- and ar,-activation to effects
207
with special reference to the regulation of cellular metabolism by catecholamines (Fain and Garcia-Sainz, 1980, and refs. quoted therein). It is most attractive to translate the present findings into a hypothesis for the mechanism by which calcium antagonists can lower arterial pressure. The blood pressure lowering effect of calcium antagonists, such as nifedipine (Olivari et al., 1979) may at least partially be due to the inhibition of the influx of extracellular calcium which is necessary to maintain that part of the vascular tone mediated by a,-adrenoceptors in the vascular wall. Accordingly, after blockade of the stimulation of inward calcium current, vascular postsynaptic a,-adrenoceptors (for instance by endogenous catecholamines) no longer contributes to the maintainance of vascular tone, so that a hypotensive effect will develop.
Acknowledgements The generous donation of B-HT 920, D 600 and verapamil, and nifedipine by Thomae, Knoll and Bayer, respectively, is gratefully acknowledged.
References Docherty, J.R. and J.C. McGrath, 1980, A comparison of pre- and postjunctional potencies of several a-adrenoceptor agonists in the cardiovascular system and anococcygeus muscle of the rat, NaunynSchmiedeb. Arch. Pharmacol. 312,107. Drew, G.M. and S.B. Whiting, 1979, Evidence for two distinct types of postsynaptic o-adrenoceptors in vascular smooth muscle in vivo, Br. J. Pharmacol. 67,207. Fain, J.N. and J.A. Garcia-Sainz, 1980, Role of phosphatidylinositol turnover in alpha1 and of adenylate cyclase inhibition in alpha? effects of catecholamines, Life Sci. 26, 1183. Fleckenstein, A., 1977, Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle, Ann. Rev. Pharmacol. Toxicol. 17, 149. Golenhofen, K., 1976, Theory of P and T systems for calcium activation in smooth muscle, in:
208 Physiology of Smooth Muscle, eds. E. Biilbring and M.F. Shuba (Raven Press, New York) p. 197. Hester, R.K. and O. Carrier, 1977, Excitation-contraction/relaxation coupling in vascular smooth muscle, in: Factors Influencing Vascular Reactivity, eds. O. Carrier and S. Shibata (Igaku-Shoin, Tokyo) p. 96. Olivari, M.T., C. Bartorelli, A. Polese, C. Fiorentini, P. Moruzzi and M.D. Guazzi, 1979, Treatment of hypertension with nifedipine, a calcium antagonistic agent, Circulation 59, 1056. Timmermans, P.B.M.W.M., H.Y. Kwa and P.A. Van Zwieten, 1979, Possible subdivision of postsynaptic a-adrenoceptors mediating pressor responses in the pithed rat, Naunyn-Schmiedeb. Arch. Pharmacol. 3 1 0 , 1 8 9 .
J.C.A. VAN MEEL ET AL. Timmermans, P.B.M.W.M. and P.A. Van Zwieten, 1980a, Postsynaptic a 1- and a2-adrenoceptors in the circulatory system of the pithed rat; selective stimulation of the a2-type by B-HT 933, European J. Pharmacol. 63,199. Timmermans, P.B.M.W.M. and P.A. Van Zwieten, 1980b, Vasoconstriction mediated by postsynaptic a2-adrenoceptor stimulation, Naunyn-Schmiedeb. Arch. Pharmacol. 313, 17. Van Meel, J.C.A., P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1980, Selectivity of some agonis~ for postsynaptic 0~1- and a2-adrenoce ptots in the vascular system of the pithed rat, Naunyn-Schmiedeb. Arch. Pharmacol. Suppl. 313, R22.