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α1-Adrenoceptors induce Ca influx and intracellular Ca release in isolated rabbit aorta
European Journal of Pharmacology, 84 (1982) 233-235
233
Elsevier Biomedical Press
Rapid communication c%-ADRENOCEPTORS INDUCE Ca INFLUX AND INTRACELLULAR Ca RELEASE IN I S O L A T E D RABBIT AORTA C. CAUVIN *, R. LOUTZENHISER, O. HWANG and C. VAN BREEMEN
Department of Pharmacology, University of Miami, School of Medicine, Miami, Florida, U.S.A. Received 2 September 1982, accepted 6 September 1982
Activation of vascular smooth muscle of the rat with a2-adrenoceptor selective agonists has been reported to be more susceptible to inhibition by Ca antagonists than is that with oq-adrenoceptor selective agonists (Van Meel et al., 1981 ; Godfraind and Miller, 1982). These observations have led to the hypothesis that oL2-adrenoceptor stimulation releases intracellularly stored Ca, while a~-adrenoceptor stimulation induces Ca influx into vascular smooth muscle. De Mey and Vanhoutte (1981), however, have reached the opposite conclusion from studies of isolated canine arteries and veins, wherein verapamil was more effective in inhibiting contractile responses to methoxamine, a relatively selective a~-adrenoceptor agonist, than to norepinephrine (NE), which is less cq-selective. Contrary to both these conclusions, we have reported that NE-induced contraction of rabbit aorta, which involves both Ca influx and intracellular Ca release, appears to be mediated primarily if not exclusively by stimulation of a~-adrenoceptors (Van Breemen et al., 1982). In this tissue prazosin (an al-antagonist) was 1000 fold more potent than yohimbine (an a2-antagonist ) in blocking NEinduced contraction. In the present communication, we have further investigated this problem by examining the effects of yohimbine and prazosin upon NE-stimulated Ca influx and release of intracellular Ca. The thoracic aorta was removed and aortic rings were prepared as previously described (Van Breemen et al., 1982). NE-stimulated 45Ca influx * To whom all correspondence should be addressed: Dept. of Pharmacology, University of Miami (R-189). Miami. FL 33101, U.S.A. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
was determined by pulse labeling the Ca which enters smooth muscle cells during a 90 s time period in the presence and absence of 10 -6 M N E in physiological salt solution (PSS; Na Hepes; Van Breemen et al., 1982). The tissues were then washed for 45 min in ice-cold Ca-free PSS (containing 2 mM EGTA) to remove extracellular 45Ca before counting the tissues. Intracellular Ca release was determined by measuring the maximal contractile response to N E (10 - 6 M ) in the absence of extracellular Ca (after preincubation in Ca-free PSS containing 2 mM EGTA for 10 min). We have shown that the Ca free N E contraction and stimulation of 45Ca efflux have the same dependency on [NE] (Van Breemen et al., 1982). The rings were returned to PSS for at least 15 min before this sequence was repeated. The effects of prazosin and yohimbine on NE-stimulated contraction, Ca influx, and intracellular Ca release were determined in each case by preincubating tissues for I0 min in appropriate solutions containing the specific 6antagonist concentrations before administration of NE. Fig. 1 shows that prazosin is three orders of magnitude more potent than yohimbine in the inhibition of contraction, Ca influx and Ca release. It is, moreover, clear that neither the al-antagonist prazosin nor the a2-antagonist yohimbine is able to differentiate between the o~-receptor-coupled processes of Ca influx and intracellular Ca release. These results, therefore, do not support the concept that oq- and a2-adrenoceptor subtypes are linked specifically to Ca influx or Ca release, but rather indicate that one subtype can mediate both processes. Although the differences between our conclusion and those of Van Meel et al. (1981),
234
PRAZOSIN+
I ( ) e M NE
YOHIMBINE * 1()6M NE
lOO
.ol :::::o" ~-'z0
,olea. .....
60
Ca influx ~
"
_d
//,
/
//
//
s •
-,-
z #
i/
40
e"S:" /
/
/ f
o
0
10 "e
1 (~e
10 -7
[ ANTAGONIST
l(~e
I
I
10 "s
10 -4
] M
Fig. I. Prazosin and yohimbine inhibition of norepinephrine (NE) induced contraction (in normal PSS), intracellular Ca release (as indicated by contractions in Ca-free PSS containing 2 mM EGTA), and 45Ca influx (measured during a 90 s exposure to 45Ca containing media). Each point on the contraction curves represents the mean of at least two determinations from each of four animals. The 45Ca influx determinations were made on 5 tissues from each of 3 rabbits for each antagonist.
Godfraind and Miller (1982) and De Mey and Vanhoutte (1981) may have resulted from species variation in ~-adrenoceptor mechanisms of activation of vascular smooth muscle, we feel that alternative explanations may reconcile the differences among our results. We have recently shown that the sensitivity of NE-induced Ca influx in rabbit aorta to inhibition by diltiazem and nisoldipine decreases dramatically as the degree of smooth muscle activation by N E is increased (Van Breemen et al., 1982). Thus, in order to draw conclusions regarding possible differences between the Ca delivery mechanisms coupled to oq- and e%-receptors, it is necessary to compare equieffective doses of the corresponding agonists. This was not done in the studies of Godfraind and Miller (1982) and De Mey and Vanhoutte (1981), wherein the less efficacious agonists were always more susceptible to Ca antagonist inhibition. Van Meel et al. (1981) did compare equieffective doses of eq- and o~2-agonists in the pithed rat and found that the in vivo vasoconstrictor effects of the %-agonist were more susceptible to Ca antagonists. However, the
e%-induced vasoconstriction in this model is largely inhibited by captopril, leading these investigators to conclude that it is primarily mediated through the renin-angiotensin system (De Jonge et al., 1981). Therefore, no conclusions regarding the susceptibility of vascular e%-adrenoceptor effects to inhibition by Ca antagonists can be drawn from these studies. Hence, we would like to suggest that differences in sensitivities to Ca antagonists seen when various ~x1- and e%-adrenoceptor agonists are used for activation may be a reflection of differences in the degree of activation of the receptor activated Ca channel rather than an indication that the two adrenoceptor subtypes utilize different Ca pools.
Acknowledgements Our thanks to Barbara Bradie for typing the manuscript. This work was supported by National Institutes of Health Grants HE 25359 and HL 07188.
235
References De Jonge, A., B. Wilffert, H.O. Kalkman, J.C.A. Van Meel, J.M.C. Thoolen, P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1981, Captopril impairs the vascular smooth muscle contraction mediated by postsynaptic a2-adrenoceptors in the pithed rat, European J. Pharmacol. 74, 385. De Mey, J. and P. Vanhoutte, 1981, Uneven distribution of postjunctional a l p h a : and alpha2-1ike adrenoceptors in canine arterial and venous smooth muscle, Circ. Res. 48, 875.
Godfraind, T. and R.C. Miller, 1982, ~xI- and a2-adrenoceptor stimulation and Ca fluxes in isolated rat aorta, Arch. Int. Pharmacodyn. Ther. 256, 171. Van Meel, J.C.A., A. De Jonge, H.O. Kalkman, B. Wilffert, P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1981, Organic and inorganic calcium antagonists reduce vasoconstriction in vivo mediated by postsynaptic a2-adrenoceptors, Naunyn-Schmiedeb. Arch. Pharmacol. 316, 288. Van Breemen, C., O. Hwang and C. Cauvin, 1982, Ca antagonist inhibition of norepinephrine stimulated Ca influx in vascular smooth muscle, in: International Symposium on Calcium Modulators (Elsevier/North-Holland, Amsterdam).
233
Elsevier Biomedical Press
Rapid communication c%-ADRENOCEPTORS INDUCE Ca INFLUX AND INTRACELLULAR Ca RELEASE IN I S O L A T E D RABBIT AORTA C. CAUVIN *, R. LOUTZENHISER, O. HWANG and C. VAN BREEMEN
Department of Pharmacology, University of Miami, School of Medicine, Miami, Florida, U.S.A. Received 2 September 1982, accepted 6 September 1982
Activation of vascular smooth muscle of the rat with a2-adrenoceptor selective agonists has been reported to be more susceptible to inhibition by Ca antagonists than is that with oq-adrenoceptor selective agonists (Van Meel et al., 1981 ; Godfraind and Miller, 1982). These observations have led to the hypothesis that oL2-adrenoceptor stimulation releases intracellularly stored Ca, while a~-adrenoceptor stimulation induces Ca influx into vascular smooth muscle. De Mey and Vanhoutte (1981), however, have reached the opposite conclusion from studies of isolated canine arteries and veins, wherein verapamil was more effective in inhibiting contractile responses to methoxamine, a relatively selective a~-adrenoceptor agonist, than to norepinephrine (NE), which is less cq-selective. Contrary to both these conclusions, we have reported that NE-induced contraction of rabbit aorta, which involves both Ca influx and intracellular Ca release, appears to be mediated primarily if not exclusively by stimulation of a~-adrenoceptors (Van Breemen et al., 1982). In this tissue prazosin (an al-antagonist) was 1000 fold more potent than yohimbine (an a2-antagonist ) in blocking NEinduced contraction. In the present communication, we have further investigated this problem by examining the effects of yohimbine and prazosin upon NE-stimulated Ca influx and release of intracellular Ca. The thoracic aorta was removed and aortic rings were prepared as previously described (Van Breemen et al., 1982). NE-stimulated 45Ca influx * To whom all correspondence should be addressed: Dept. of Pharmacology, University of Miami (R-189). Miami. FL 33101, U.S.A. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
was determined by pulse labeling the Ca which enters smooth muscle cells during a 90 s time period in the presence and absence of 10 -6 M N E in physiological salt solution (PSS; Na Hepes; Van Breemen et al., 1982). The tissues were then washed for 45 min in ice-cold Ca-free PSS (containing 2 mM EGTA) to remove extracellular 45Ca before counting the tissues. Intracellular Ca release was determined by measuring the maximal contractile response to N E (10 - 6 M ) in the absence of extracellular Ca (after preincubation in Ca-free PSS containing 2 mM EGTA for 10 min). We have shown that the Ca free N E contraction and stimulation of 45Ca efflux have the same dependency on [NE] (Van Breemen et al., 1982). The rings were returned to PSS for at least 15 min before this sequence was repeated. The effects of prazosin and yohimbine on NE-stimulated contraction, Ca influx, and intracellular Ca release were determined in each case by preincubating tissues for I0 min in appropriate solutions containing the specific 6antagonist concentrations before administration of NE. Fig. 1 shows that prazosin is three orders of magnitude more potent than yohimbine in the inhibition of contraction, Ca influx and Ca release. It is, moreover, clear that neither the al-antagonist prazosin nor the a2-antagonist yohimbine is able to differentiate between the o~-receptor-coupled processes of Ca influx and intracellular Ca release. These results, therefore, do not support the concept that oq- and a2-adrenoceptor subtypes are linked specifically to Ca influx or Ca release, but rather indicate that one subtype can mediate both processes. Although the differences between our conclusion and those of Van Meel et al. (1981),
234
PRAZOSIN+
I ( ) e M NE
YOHIMBINE * 1()6M NE
lOO
.ol :::::o" ~-'z0
,olea. .....
60
Ca influx ~
"
_d
//,
/
//
//
s •
-,-
z #
i/
40
e"S:" /
/
/ f
o
0
10 "e
1 (~e
10 -7
[ ANTAGONIST
l(~e
I
I
10 "s
10 -4
] M
Fig. I. Prazosin and yohimbine inhibition of norepinephrine (NE) induced contraction (in normal PSS), intracellular Ca release (as indicated by contractions in Ca-free PSS containing 2 mM EGTA), and 45Ca influx (measured during a 90 s exposure to 45Ca containing media). Each point on the contraction curves represents the mean of at least two determinations from each of four animals. The 45Ca influx determinations were made on 5 tissues from each of 3 rabbits for each antagonist.
Godfraind and Miller (1982) and De Mey and Vanhoutte (1981) may have resulted from species variation in ~-adrenoceptor mechanisms of activation of vascular smooth muscle, we feel that alternative explanations may reconcile the differences among our results. We have recently shown that the sensitivity of NE-induced Ca influx in rabbit aorta to inhibition by diltiazem and nisoldipine decreases dramatically as the degree of smooth muscle activation by N E is increased (Van Breemen et al., 1982). Thus, in order to draw conclusions regarding possible differences between the Ca delivery mechanisms coupled to oq- and e%-receptors, it is necessary to compare equieffective doses of the corresponding agonists. This was not done in the studies of Godfraind and Miller (1982) and De Mey and Vanhoutte (1981), wherein the less efficacious agonists were always more susceptible to Ca antagonist inhibition. Van Meel et al. (1981) did compare equieffective doses of eq- and o~2-agonists in the pithed rat and found that the in vivo vasoconstrictor effects of the %-agonist were more susceptible to Ca antagonists. However, the
e%-induced vasoconstriction in this model is largely inhibited by captopril, leading these investigators to conclude that it is primarily mediated through the renin-angiotensin system (De Jonge et al., 1981). Therefore, no conclusions regarding the susceptibility of vascular e%-adrenoceptor effects to inhibition by Ca antagonists can be drawn from these studies. Hence, we would like to suggest that differences in sensitivities to Ca antagonists seen when various ~x1- and e%-adrenoceptor agonists are used for activation may be a reflection of differences in the degree of activation of the receptor activated Ca channel rather than an indication that the two adrenoceptor subtypes utilize different Ca pools.
Acknowledgements Our thanks to Barbara Bradie for typing the manuscript. This work was supported by National Institutes of Health Grants HE 25359 and HL 07188.
235
References De Jonge, A., B. Wilffert, H.O. Kalkman, J.C.A. Van Meel, J.M.C. Thoolen, P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1981, Captopril impairs the vascular smooth muscle contraction mediated by postsynaptic a2-adrenoceptors in the pithed rat, European J. Pharmacol. 74, 385. De Mey, J. and P. Vanhoutte, 1981, Uneven distribution of postjunctional a l p h a : and alpha2-1ike adrenoceptors in canine arterial and venous smooth muscle, Circ. Res. 48, 875.
Godfraind, T. and R.C. Miller, 1982, ~xI- and a2-adrenoceptor stimulation and Ca fluxes in isolated rat aorta, Arch. Int. Pharmacodyn. Ther. 256, 171. Van Meel, J.C.A., A. De Jonge, H.O. Kalkman, B. Wilffert, P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1981, Organic and inorganic calcium antagonists reduce vasoconstriction in vivo mediated by postsynaptic a2-adrenoceptors, Naunyn-Schmiedeb. Arch. Pharmacol. 316, 288. Van Breemen, C., O. Hwang and C. Cauvin, 1982, Ca antagonist inhibition of norepinephrine stimulated Ca influx in vascular smooth muscle, in: International Symposium on Calcium Modulators (Elsevier/North-Holland, Amsterdam).