Proc. Natl. Acad. Sci. USA Vol. 75, No. 3, pp. 1605-1607, March 1978 Physiological Sciences
Calcium leakage as a cause of the high resting tension in vascular smooth muscle from the spontaneously hypertensive rat (cardiovascular physiology/a-adrenergic receptor/membrane permeability/norepinephrine)
JAMES P. NOON, PETER J. RICE, AND Ross J. BALDESSARINI Department of Psychiatry, Harvard Medical School, The Mailman Research Center, McLean Hospital, Belmont, Massachusetts 02178
Communicated by Seymour S. Kety, December 22, 1977
ABSTRACT Aortic strips from spontaneously hypertensive (SH) rats relax in calcium-free physiological medium and contract to approximately 60% of maximum when calcium is again restored to the medium. In vivid contrast, the resting tension of aortic strips from normal rats is unaffected by manipulation of the calcium concentration of the bathing medium. These findings, as well as the reduced sensitivity of aortic strips from SH rats to norepinephrine and the observation that aortic strips from SH rats relax at a faster rate in calcium-free medium in comparison with aortic strips from normal rats, are consistent with the hypothesis that vascular smooth muscle membranes from SH rats leak calcium at a rate that is only partially compensated by the calcium pump.
The contractile state of vascular smooth muscle is determined by the concentration of activator calcium ion in the cytoplasm (1). Neural or hormonal stimulation of the muscle by norepinephrine elicits the intracellular release of calcium from the sarcoplasmic reticulum and the influx of calcium from the extracellular space, both contributing to an increase in the concentration of free cytoplasmic calcium. Removal of the stimulus is followed by sequestration of calcium into the sarcoplasmic reticulum and the extrusion of calcium into the extracellular space, resulting in a reduction of the cytoplasmic concentration of free calcium, and in relaxation of the muscle
(1).
One apparent cause of hypertension in humans and animals is high peripheral resistance to the flow of blood (2, 3). At least in the early stages of hypertensive disease, high peripheral resistance results from contracted vascular smooth muscle. On the basis of the presumed relationship between cytoplasmic [Ca2+] and the contractile state and in view of the high peripheral resistance in hypertensives, one would predict that the cytoplasm of vascular smooth muscle in hypertensive humans and animals might have increased levels of calcium. In turn, increased cytoplasmic calcium levels could result from one or more of several possible pathophysiologic mechanisms: (a) sympathetic nervous system overactivity, (b) hypersensitivity of smooth muscle to adrenergic stimulation, (c) an inordinately high calcium influx, or (d) a reduced capacity to pump calcium out of the cytoplasm. Microsomes prepared from vascular smooth muscle from spontaneously hypertensive (SH) Wistar rats have recently been found to have reduced ability to sequester calcium compared to microsomes from normal Wistar rats (4-6). These reports suggest that vascular smooth muscle cells from SH rats have either an inordinately high calcium influx in the resting state or a reduced ability to pump calcium out of the cytoplasm. In either case, one might expect there to be a higher resting state cytoplasmic calcium concentration, a concomitantly high The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
resting tension in vascular smooth muscle, and high peripheral resistance in SH rats. Accordingly, we evaluated the relationship between extracellular calcium concentration and resting tension in aortic strips from SH and normal Wistar rats. MATERIALS AND METHODS Male spontaneously hypertensive (SH) rats and a closely related, but normal, strain of rats (Kyoto Wistar) (Charles River Breeding Laboratory) weighing 170-190 g were killed with a blow to the head. The thoracic aorta was quickly removed, cleared of fat and connective tissue, and cut into a helical strip (7). Helical strips from two normal and two SH rats were mounted for each experiment under 2 g of resting tension in an organ bath containing Locke's medium (in mM: NaCl, 136; KCI, 5.6; NaHCO3, 20.0; NaH2PO4, 1.2; CaC12, 1.2; glucose, 5.5) maintained at 370 and continuously bubbled with 95% 02/5% CO2. The strips were equilibrated with frequent changes of Locke's medium over a period of 3 hr before experimental changes in [Ca2+] or exposure to (-)-norepinephrine-HCI (Sigma), as described below, were begun.
RESULTS In initial experiments, aortic strips from SH rats relaxed when placed in calcium-free Locke's medium. Subsequent restoration of calcium to the bathing medium elicited an increase in the resting tension of aortic strips from SH rats (Fig. 1). Stimulation of the aortic strips from SH with (-)-norepinephrine elicited a further increase in the tension as shown in Fig. 1. Neither removal nor restoration of calcium to the bathing medium had any effect on the resting tension of aortic strips from normal Wistar rats. The increase in resting tension of aortic strips from SH rats was dependent on the extracellular [Ca2+], but this dependence was not saturable up to a high calcium concentration (11 mM) (Fig. 2). These results suggested that whatever else may contribute to hypertension in SH rats, the vascular beds of these animals are probably in a partially contracted state in the presence of physiological extracellular concentration of calcium and even in the absence of hormonal stimulation. Because of the high resting tension in aortic strips from SH rats in the presence of physiological [Ca2+] (Fig. 1), we defined maximal contraction as the maximal contractile response to norepinephrine in normal Locke's medium ([Ca2+] = 2.2 mM) measured with respect to the resting state in calcium-free medium. The relevance of this distinction lies in the fact that the results (Figs. 1-4) are expressed as the percent of maximal contraction. With respect to the resting tension in norepinephrine- and calcium-free Locke's medium, aortic strips from SH rats had a resting tension in Locke's medium containing normal [Ca2+] that was approximately 60% of maximum. In Abbreviation: SH, spontaneously hypertensive.
1605
Proc. Natl. Acad. Sci. USA 75 (1978)
Physiological Sciences: Noon et al.
1606
100 0
._ 0
F
4-i
-i
E ._ x
50 10-8 10O7 l0o9 [(-)-Norepinephrine], M
E 0
SH Normal Calcium alone
FIG. 3. Response of aortic strips from SH and normal rats to cumulatively increasing concentrations of norepinephrine applied in normal Locke's medium. Aortic strips were first equilibrated in normal Locke's medium. Thereafter, the norepinephrine concentration was increased in a stepwise manner from 10-10 to l0-5 M. Each point is the mean of six experiments ± SEM. SH rats; 0, normal
Calcium, then norepinephrine
A,
FIG. 1. Effect of physiological calcium concentrations and maximal norepinephrine concentrations on aortic strips from spontaneously hypertensive (SH) and normal rats. Results are presented as percent of maximal contraction, defined as the maximal norepinephrine-induced contractile response in medium containing physiological calcium concentrations relative to the resting tension in norepinephrine-free and calcium-free medium. Aortic strips from SH and normal rats were incubated for 30 min in calcium-free medium, then 2.2 mM calcium or 2.2 mM calcium and 1 MM norepinephrine were added as noted in the figure. Empty bars, effect of 2.2 mM calcium alone; hatched bars, effect of 1 MM norepinephrine added after 2.2 mM calcium. Each bar is the mean of 14 experiments + SEM.
agreement with several published reports (8-12), we found that aortic strips from SH rats were somewhat less sensitive to norepinephrine (the apparent mean effective concentration, EC5o was higher) than aortic strips from normal Wistar rats (Fig. 3). Moreover, aortic tissues from SH rats were somewhat less sensitive to calcium applied along with a maximally effective 10-1
100
X physiological [calcium] 1
10
' " I
rats.
concentration of norepinephrine (10 AM) than were aortic strips from normal rats (Fig. 4). Finally, aortic strips from SH rats relaxed about 30% more rapidly (t1/2 = 4.4 + 1.2 min) in calcium-free Locke's medium than did strips from normal rats (t1/2 = 6.2 + 1.9 min; n = 4, P < 0.01).
DISCUSSION Taken together, these data suggest that calcium leaks from the extracellular medium into the cytoplasm of vascular smooth muscle cells in SH rats at a rate that is not adequately compensated by the plasma membrane calcium pump. This possibility is strongly suggested by the relaxation of aortic strips from SH rats in calcium-free Locke's medium and by their high resting tension in Locke's medium containing normal [Ca2+]. The fact that aortic strips from SH rats relaxed faster in calcium-free Locke's medium than did aortic strips from normal rats suggests that the calcium pump works efficiently in SH rats and that leakage of calcium out of the muscle from the hypertensive rats hastens the relaxation. Finally, the apparently paradoxical slight reduction in sensitivity of aortic strips from X physiological [calcium] 1 1010-2
1O-3
10
100 0
0
(U
EC so (normal rats)
SH rats 0
=
160 MM
c
50
0
-
0
'aox 50
E
I/i
E x
E
co
0
EC50 (SH rats) = 260,uM
UP go
o Normal rats
10-6
,0
e 105
.I.,.
10-4
1-' 10-3
10-2
[Calcium], M 0NO I
10-4
Hii
l
f i l
10-31-
[Calcium], M
Calcium concentration dependence of the resting tension of aortic strips from SH and normal rats. Percent of maximal contraction is as described for Fig. 1. Aortic strips were first equilibrated in calcium-free Locke's medium; the [Ca2+1 was increased stepwise from 0.022 to 11 mM. Each point is the mean of 14 experiments SEM. FIG. 2.
FIG. 4. Response of aortic strips from SH and normal rats to cumulatively increasing concentrations of calcium applied along with a maximal concentration of norepinephrine (10 AM). Aortic strips were first equilibrated in calcium-free Locke's medium and then in calcium-free Locke's medium containing 10MAM (-)-norepinephrine; the calcium-free Locke's medium containing norepinephrine elicited a small contraction that relaxed back to basal levels in 30 min or less. Thereafter, the calcium concentration was increased in a stepwise manner from 0.022 to 11 mM. Each point is the mean of six experiments + SEM. a, SH rats; 0, normal rats.
Physiological Sciences:
Noon et al.
SH rats to norepinephrine (Fig. 3) and calcium (Fig. 4) may also be related to altered calcium transport. Thus, while norepinephrine probably induces an increased rate of influx of free calcium into the cytoplasm in vascular smooth muscle from both SH and normal rats, a greater leakage of calcium out of the muscle in the SH rats would slightly reduce the concentration of free or active calcium that reaches the contractile apparatus and this might diminish the contractile response produced. The hypothesis that smooth muscle cell membranes from SH rats are leaky to calcium is supported by the observation that, compared with normal rats, smooth muscle microsomes from SH rats do not sequester calcium as well, but have much higher calcium-sensitive ATPase activities (5). The ATPase change suggests further that a membrane preparation (microsomes) from vascular smooth muscle of SH rats develops increased calcium pump activity in compensation for the leakiness of the cells to calcium. In this connection, Jones (13) has shown that vascular smooth muscle from SH rats is also leaky to Na+, K+, and Cl-, suggesting that smooth muscle membranes from hypertensive animals are nonselectively leaky to many electrolytes. Limas and Cohn (14) have shown recently that microsomes from heart muscle of SH rats also show a deficit in calcium sequestering ability as compared with normal rats, suggesting that the high basal contractility found in the vascular bed in SH rats extends to the heart muscle. In agreement with these results, we have found that heart muscle strips from SH rats (but not normal rats) relax in calcium-free medium and contract again when the physiological concentration of calcium is reestablished in the bathing medium (J. P. Noon and P. Rice, unpublished observations).
Proc. Natl. Acad. Sci. USA 75 (1978)
1607
We therefore suggest, on the basis of our present observations, that the membranes of vascular smooth muscle (and probably heart muscle also) leak calcium at a rate that is only partially compensated by the plasma membrane calcium pump and that, as a consequence, the [Ca2+] in the cytoplasm of these muscle cells is high enough to maintain their high resting tension. This work was partially supported by U.S. Public Health Service Grants MH-16674 and MH-25515 and National Institute of Mental Health Career Investigator Award MH-74370 to R.J.B. 1. Bohr, D. F. (1973) Circ. Res. 32,665-672. 2. Tobia, A. J., Walsh, G. M., Todepalli, A. S. & Lee, J. Y. (1974) Blood Vessels 11, 287-294. 3. Lund-Johansen, P. (1967) Acta Med. Scand. Suppl. 482, 1-34. 4. Moore, L., Hurwitz, L., Davenport, G. R. & Landon, E. J. (1975) Biochim. Biophys. Acta 413, 432-443. 5. Webb, R. C. & Bhalla, R. C. (1976) J. Mol. Cell. Cardiol. 8, 651-661. 6. Wei, J. W., Janis, R. A. & Daniel, E. E. (1976) Blood Vessels 13, 293-308. 7. Furchgott, R. F. & Bhadrakom, S. (1953) J. Pharmacol. Exp. Ther. 108, 129-140. 8. Clineschmidt, B. V., Geller, R. G., Govier, W. C. & Sjoerdsma, A. (1970) Eur. J. Pharmacol. 10, 45-50. 9. Field, F. P., Janis, R. A. & Triggle, D. F. (1972) Can. J. Physiol. Pharmacol. 50, 1072-1079. 10. Grollman, A. & Krishnamurty, V. S. R. (1973) Arch. Int. Pharmacodyn. Ther. 203,376-387. 11. Shibata, S., Kurahashi, K. & Kuchii, M. (1973) J. Pharmacol. Exp. Ther. 185,406-417. 12. Hansen, T. R. & Bohr, D. F. (1975) Circ. Res. 36,590-598. 13. Jones, A. W. (1973) Circ. Res. 33,563-572. 14. Limas, C. J. & Cohn, J. N. (1977) Circ. Res. Suppl. I 40, 6269.
Calcium leakage as a cause of the high resting tension in vascular smooth muscle from the spontaneously hypertensive rat (cardiovascular physiology/a-adrenergic receptor/membrane permeability/norepinephrine)
JAMES P. NOON, PETER J. RICE, AND Ross J. BALDESSARINI Department of Psychiatry, Harvard Medical School, The Mailman Research Center, McLean Hospital, Belmont, Massachusetts 02178
Communicated by Seymour S. Kety, December 22, 1977
ABSTRACT Aortic strips from spontaneously hypertensive (SH) rats relax in calcium-free physiological medium and contract to approximately 60% of maximum when calcium is again restored to the medium. In vivid contrast, the resting tension of aortic strips from normal rats is unaffected by manipulation of the calcium concentration of the bathing medium. These findings, as well as the reduced sensitivity of aortic strips from SH rats to norepinephrine and the observation that aortic strips from SH rats relax at a faster rate in calcium-free medium in comparison with aortic strips from normal rats, are consistent with the hypothesis that vascular smooth muscle membranes from SH rats leak calcium at a rate that is only partially compensated by the calcium pump.
The contractile state of vascular smooth muscle is determined by the concentration of activator calcium ion in the cytoplasm (1). Neural or hormonal stimulation of the muscle by norepinephrine elicits the intracellular release of calcium from the sarcoplasmic reticulum and the influx of calcium from the extracellular space, both contributing to an increase in the concentration of free cytoplasmic calcium. Removal of the stimulus is followed by sequestration of calcium into the sarcoplasmic reticulum and the extrusion of calcium into the extracellular space, resulting in a reduction of the cytoplasmic concentration of free calcium, and in relaxation of the muscle
(1).
One apparent cause of hypertension in humans and animals is high peripheral resistance to the flow of blood (2, 3). At least in the early stages of hypertensive disease, high peripheral resistance results from contracted vascular smooth muscle. On the basis of the presumed relationship between cytoplasmic [Ca2+] and the contractile state and in view of the high peripheral resistance in hypertensives, one would predict that the cytoplasm of vascular smooth muscle in hypertensive humans and animals might have increased levels of calcium. In turn, increased cytoplasmic calcium levels could result from one or more of several possible pathophysiologic mechanisms: (a) sympathetic nervous system overactivity, (b) hypersensitivity of smooth muscle to adrenergic stimulation, (c) an inordinately high calcium influx, or (d) a reduced capacity to pump calcium out of the cytoplasm. Microsomes prepared from vascular smooth muscle from spontaneously hypertensive (SH) Wistar rats have recently been found to have reduced ability to sequester calcium compared to microsomes from normal Wistar rats (4-6). These reports suggest that vascular smooth muscle cells from SH rats have either an inordinately high calcium influx in the resting state or a reduced ability to pump calcium out of the cytoplasm. In either case, one might expect there to be a higher resting state cytoplasmic calcium concentration, a concomitantly high The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
resting tension in vascular smooth muscle, and high peripheral resistance in SH rats. Accordingly, we evaluated the relationship between extracellular calcium concentration and resting tension in aortic strips from SH and normal Wistar rats. MATERIALS AND METHODS Male spontaneously hypertensive (SH) rats and a closely related, but normal, strain of rats (Kyoto Wistar) (Charles River Breeding Laboratory) weighing 170-190 g were killed with a blow to the head. The thoracic aorta was quickly removed, cleared of fat and connective tissue, and cut into a helical strip (7). Helical strips from two normal and two SH rats were mounted for each experiment under 2 g of resting tension in an organ bath containing Locke's medium (in mM: NaCl, 136; KCI, 5.6; NaHCO3, 20.0; NaH2PO4, 1.2; CaC12, 1.2; glucose, 5.5) maintained at 370 and continuously bubbled with 95% 02/5% CO2. The strips were equilibrated with frequent changes of Locke's medium over a period of 3 hr before experimental changes in [Ca2+] or exposure to (-)-norepinephrine-HCI (Sigma), as described below, were begun.
RESULTS In initial experiments, aortic strips from SH rats relaxed when placed in calcium-free Locke's medium. Subsequent restoration of calcium to the bathing medium elicited an increase in the resting tension of aortic strips from SH rats (Fig. 1). Stimulation of the aortic strips from SH with (-)-norepinephrine elicited a further increase in the tension as shown in Fig. 1. Neither removal nor restoration of calcium to the bathing medium had any effect on the resting tension of aortic strips from normal Wistar rats. The increase in resting tension of aortic strips from SH rats was dependent on the extracellular [Ca2+], but this dependence was not saturable up to a high calcium concentration (11 mM) (Fig. 2). These results suggested that whatever else may contribute to hypertension in SH rats, the vascular beds of these animals are probably in a partially contracted state in the presence of physiological extracellular concentration of calcium and even in the absence of hormonal stimulation. Because of the high resting tension in aortic strips from SH rats in the presence of physiological [Ca2+] (Fig. 1), we defined maximal contraction as the maximal contractile response to norepinephrine in normal Locke's medium ([Ca2+] = 2.2 mM) measured with respect to the resting state in calcium-free medium. The relevance of this distinction lies in the fact that the results (Figs. 1-4) are expressed as the percent of maximal contraction. With respect to the resting tension in norepinephrine- and calcium-free Locke's medium, aortic strips from SH rats had a resting tension in Locke's medium containing normal [Ca2+] that was approximately 60% of maximum. In Abbreviation: SH, spontaneously hypertensive.
1605
Proc. Natl. Acad. Sci. USA 75 (1978)
Physiological Sciences: Noon et al.
1606
100 0
._ 0
F
4-i
-i
E ._ x
50 10-8 10O7 l0o9 [(-)-Norepinephrine], M
E 0
SH Normal Calcium alone
FIG. 3. Response of aortic strips from SH and normal rats to cumulatively increasing concentrations of norepinephrine applied in normal Locke's medium. Aortic strips were first equilibrated in normal Locke's medium. Thereafter, the norepinephrine concentration was increased in a stepwise manner from 10-10 to l0-5 M. Each point is the mean of six experiments ± SEM. SH rats; 0, normal
Calcium, then norepinephrine
A,
FIG. 1. Effect of physiological calcium concentrations and maximal norepinephrine concentrations on aortic strips from spontaneously hypertensive (SH) and normal rats. Results are presented as percent of maximal contraction, defined as the maximal norepinephrine-induced contractile response in medium containing physiological calcium concentrations relative to the resting tension in norepinephrine-free and calcium-free medium. Aortic strips from SH and normal rats were incubated for 30 min in calcium-free medium, then 2.2 mM calcium or 2.2 mM calcium and 1 MM norepinephrine were added as noted in the figure. Empty bars, effect of 2.2 mM calcium alone; hatched bars, effect of 1 MM norepinephrine added after 2.2 mM calcium. Each bar is the mean of 14 experiments + SEM.
agreement with several published reports (8-12), we found that aortic strips from SH rats were somewhat less sensitive to norepinephrine (the apparent mean effective concentration, EC5o was higher) than aortic strips from normal Wistar rats (Fig. 3). Moreover, aortic tissues from SH rats were somewhat less sensitive to calcium applied along with a maximally effective 10-1
100
X physiological [calcium] 1
10
' " I
rats.
concentration of norepinephrine (10 AM) than were aortic strips from normal rats (Fig. 4). Finally, aortic strips from SH rats relaxed about 30% more rapidly (t1/2 = 4.4 + 1.2 min) in calcium-free Locke's medium than did strips from normal rats (t1/2 = 6.2 + 1.9 min; n = 4, P < 0.01).
DISCUSSION Taken together, these data suggest that calcium leaks from the extracellular medium into the cytoplasm of vascular smooth muscle cells in SH rats at a rate that is not adequately compensated by the plasma membrane calcium pump. This possibility is strongly suggested by the relaxation of aortic strips from SH rats in calcium-free Locke's medium and by their high resting tension in Locke's medium containing normal [Ca2+]. The fact that aortic strips from SH rats relaxed faster in calcium-free Locke's medium than did aortic strips from normal rats suggests that the calcium pump works efficiently in SH rats and that leakage of calcium out of the muscle from the hypertensive rats hastens the relaxation. Finally, the apparently paradoxical slight reduction in sensitivity of aortic strips from X physiological [calcium] 1 1010-2
1O-3
10
100 0
0
(U
EC so (normal rats)
SH rats 0
=
160 MM
c
50
0
-
0
'aox 50
E
I/i
E x
E
co
0
EC50 (SH rats) = 260,uM
UP go
o Normal rats
10-6
,0
e 105
.I.,.
10-4
1-' 10-3
10-2
[Calcium], M 0NO I
10-4
Hii
l
f i l
10-31-
[Calcium], M
Calcium concentration dependence of the resting tension of aortic strips from SH and normal rats. Percent of maximal contraction is as described for Fig. 1. Aortic strips were first equilibrated in calcium-free Locke's medium; the [Ca2+1 was increased stepwise from 0.022 to 11 mM. Each point is the mean of 14 experiments SEM. FIG. 2.
FIG. 4. Response of aortic strips from SH and normal rats to cumulatively increasing concentrations of calcium applied along with a maximal concentration of norepinephrine (10 AM). Aortic strips were first equilibrated in calcium-free Locke's medium and then in calcium-free Locke's medium containing 10MAM (-)-norepinephrine; the calcium-free Locke's medium containing norepinephrine elicited a small contraction that relaxed back to basal levels in 30 min or less. Thereafter, the calcium concentration was increased in a stepwise manner from 0.022 to 11 mM. Each point is the mean of six experiments + SEM. a, SH rats; 0, normal rats.
Physiological Sciences:
Noon et al.
SH rats to norepinephrine (Fig. 3) and calcium (Fig. 4) may also be related to altered calcium transport. Thus, while norepinephrine probably induces an increased rate of influx of free calcium into the cytoplasm in vascular smooth muscle from both SH and normal rats, a greater leakage of calcium out of the muscle in the SH rats would slightly reduce the concentration of free or active calcium that reaches the contractile apparatus and this might diminish the contractile response produced. The hypothesis that smooth muscle cell membranes from SH rats are leaky to calcium is supported by the observation that, compared with normal rats, smooth muscle microsomes from SH rats do not sequester calcium as well, but have much higher calcium-sensitive ATPase activities (5). The ATPase change suggests further that a membrane preparation (microsomes) from vascular smooth muscle of SH rats develops increased calcium pump activity in compensation for the leakiness of the cells to calcium. In this connection, Jones (13) has shown that vascular smooth muscle from SH rats is also leaky to Na+, K+, and Cl-, suggesting that smooth muscle membranes from hypertensive animals are nonselectively leaky to many electrolytes. Limas and Cohn (14) have shown recently that microsomes from heart muscle of SH rats also show a deficit in calcium sequestering ability as compared with normal rats, suggesting that the high basal contractility found in the vascular bed in SH rats extends to the heart muscle. In agreement with these results, we have found that heart muscle strips from SH rats (but not normal rats) relax in calcium-free medium and contract again when the physiological concentration of calcium is reestablished in the bathing medium (J. P. Noon and P. Rice, unpublished observations).
Proc. Natl. Acad. Sci. USA 75 (1978)
1607
We therefore suggest, on the basis of our present observations, that the membranes of vascular smooth muscle (and probably heart muscle also) leak calcium at a rate that is only partially compensated by the plasma membrane calcium pump and that, as a consequence, the [Ca2+] in the cytoplasm of these muscle cells is high enough to maintain their high resting tension. This work was partially supported by U.S. Public Health Service Grants MH-16674 and MH-25515 and National Institute of Mental Health Career Investigator Award MH-74370 to R.J.B. 1. Bohr, D. F. (1973) Circ. Res. 32,665-672. 2. Tobia, A. J., Walsh, G. M., Todepalli, A. S. & Lee, J. Y. (1974) Blood Vessels 11, 287-294. 3. Lund-Johansen, P. (1967) Acta Med. Scand. Suppl. 482, 1-34. 4. Moore, L., Hurwitz, L., Davenport, G. R. & Landon, E. J. (1975) Biochim. Biophys. Acta 413, 432-443. 5. Webb, R. C. & Bhalla, R. C. (1976) J. Mol. Cell. Cardiol. 8, 651-661. 6. Wei, J. W., Janis, R. A. & Daniel, E. E. (1976) Blood Vessels 13, 293-308. 7. Furchgott, R. F. & Bhadrakom, S. (1953) J. Pharmacol. Exp. Ther. 108, 129-140. 8. Clineschmidt, B. V., Geller, R. G., Govier, W. C. & Sjoerdsma, A. (1970) Eur. J. Pharmacol. 10, 45-50. 9. Field, F. P., Janis, R. A. & Triggle, D. F. (1972) Can. J. Physiol. Pharmacol. 50, 1072-1079. 10. Grollman, A. & Krishnamurty, V. S. R. (1973) Arch. Int. Pharmacodyn. Ther. 203,376-387. 11. Shibata, S., Kurahashi, K. & Kuchii, M. (1973) J. Pharmacol. Exp. Ther. 185,406-417. 12. Hansen, T. R. & Bohr, D. F. (1975) Circ. Res. 36,590-598. 13. Jones, A. W. (1973) Circ. Res. 33,563-572. 14. Limas, C. J. & Cohn, J. N. (1977) Circ. Res. Suppl. I 40, 6269.
