Pflfigers Arch (1992) 421:397-399

Joumal of Physiology 9 Springer-Verlag 1992

Short communication

1- and fl-adrenergic interactions on L-type calcium current in cardiac myocytes M. Boutjdir, M . Restivo, Y. Wei, and N. EI-Sherif Cardiology Division, Department of Medicine, State University of New York, Health Science Center and Veterans Administration Medical Center, 800 poly Place, Brooklyn, New York, 11209, USA

:Received March 31, 1992/Received after revision June 15, 1992/Accepted June 23, 1992

Abstract We investigated the mechanism by which al-adrenergie activation regulates basal and stimulated whole cell L-type Ca current (ICa) in rat ventricular myocytes using the physiological neurotransmitter, norepinephrine (hiE, 10 ~M). Stimulation Of aradrenoeeptors, achieved by NE + 10/zM esmolol (a B-receptor antagonist), had no significant effect on basal ICa. aradrenergic activation had a marked inhibitory effect on ICa elevated by/3 activation (N-E+ I#M) prazosin, an arreceptor antagonist) or activation of adenylyl eyclase by forskolin (25/zM); the inhibitory effect was reversible upon washout. However, ax-adrenergic stimulation had no significant effect on ICa previously increased by intracellular application of cAMP (25/xM). The inhibitory effect seen on Ica elevated by NE showed no significant shift of either I-V or inactivation curves. It is unlikely that the inhibitory effect of aladrenergic stimulation on NE or forskolin-elevated Ica is mediated through activation of Ca-dependent protein kinase C or changes in intraeellular free Ca (pCa=8.5, EGTA 5 mM) or cAMP-dependent phosphodiesterase. We conclude that al-adrenergie inhibition of/3adrenergic stimulated-Ica is probably mediated through an as yet unknown G-protein. This inhibitory effect could serve as a regialatory feedback mechanism in physiological and pathophysiological settings. Key Words: Cardiac myocytes, Norepinephrine, Calcium channels

Introduction Calcium channels are vital to several functions in the cardiovascular system. The sympathetic nervous system exerts an important modulatory effect on Ca channels through both cq and/3adrenoceptors [13,15]. While the/3-adrenergic component of sympathetic regulation has received a great deal of attention [7], little attention has been directed to the interaction of al and 13adrenoceptors on Ca channels. The present study reveals that, in rat ventricular myocytes, norepinephrine (NE) exerts an inhibitory effect through a~-adrenoceptors on/3-adrenergic stimulated Ca current (Ica). The observation that cq-adrenergic activation exerts an inhibitory effect on B-adrenergic or adenylyl-cyclase stimulated Ica is of major physiological and pathophysiologicai significance in the heart. Preliminary results have been reported [1].

Methods Cardiac ventricular myoeytes were obtained from Wistar rat hearts (200-250g). Cells were isolated according to the method of Wittenberg et al [16]. Control external solution contained (in mM): NaC1 132, CsC1 20, CaC12 1.8, MgC12 1.8, NaI-I2PO40.6, Dextrose 5, NaPyruvate 5, Tetrodotoxin 50 I~M, 4-Aminopyridine 5, HEPES 10, adjusted to pH=7.4 with NaOH. Solutions were applied to the exterior of the cell by placing the cell at the opening of a 250 #m Offprint requests to: M. Boutjdir

inner diameter capillary [2] with a flow rate of ~- 10/zl/min. Patch electrodes were fiUed (0.8-1.6 Mfl) with control internal solution containing (in raM): CsC1 139.8, I~EGTA 5, MgC12 4, CaC12 0.062 (pCa 8.5)[2], Na~-creatine phosphate 5, HEPES 10, Na2ATP 3.1, adjusted to pH 7.1 with KOH, To record Ica, all K currents were blocked with intracellular and extracellular Cs and 4aminopyridine. The fast Na current was blocked with 50 #M tetrodotoxin. Cells were depolarized every 4 or 8 seconds from -80 mV to -50 mV for 100 msec and then to 0 mV for 200 msec (figure 1A) or from -80 mV to 0 mV for 200 msec. ICa was measured as the difference between peak current and leak current measured at the end of 200 msec pulse (figure 1A), using a custom data acquisition and analysis system [2] or pCLAMP system (Axon Instruments, Foster City, CA). Current-voltage relationships and inactivation curves were determined by voltage clamp protocols as previously described [2]. Inhibition of Ica was considered steady when peak Ica remain unchanged over 20-40 consecutive pulses. All experiments were performed at room temperature (21-24 oC). Forskolin was prepared as a stock solution of 10 mM in anhydrous ethanol. An appropriate amount of ethanol was added to the external solution so that the same ethanol concentration was present in all solutions tested. All drugs were from Sigma Chemicals Co. (St. Louis, MO) unless otherwise indicated. Norepinephrine bitartrate was from Winthrop Pharmaceuticals (NY, NY). Esmolol hydrochloride was from Du-Pont Pharmaceuticals (Wilmington, DE). Prazosin was kindly supplied by Pfizer Laboratory Division (N-Y, NY). Collagenase was from Boehringer Mannhein Biochemicals (Indianapolis, IN). Results Effect of oq-adrenergie activation on basal and sthnulated ICa When ventricular myocytes (n=8) were superfused with NE 10/~M + 10 tzM esmolol to achieve al-adrenergie activation, no significant effect was seen on basal ICa. To investigate the al-and B-adrenergic interaction on ICa, cells (n = 11) were first superfused by NE 10/~M+ 1/~M prazosin to achieve a B-adrenergic effect, then by NE without prazosin for an al-adrenergic effect. Figure 1B illustrates a typical experiment in which B-adrenergic stimulated ICa was markedly inhibited by ~radrenergie activation; this effect could be repeated in the same cell. The average percentage inhibition of Ica was 45+_13 % ( n = l l ) . To bypass the B-receptor and its coupled Gs-protein, forskolin (25/zM) was used to activate adenylyl cyclase and increase ICa. Figure 1C shows a representative experiment in which ICa previously stimulated by forskolin was also inhibited upon a~adrenergic activation (N-E 10 tzM+ esmolol 10/~M), similar to the 6ffects observed with NE-elevated Ica; this effect was also reversible upon return to forskolin. The average percentage inhibition of ICa was 43+ 15 % (n=7). To exclude a possible interaction of aradrenergic activation and adenylyl cyclase, we also investigated the effect of cq-adrenergie activation on ICa stimulated by direct intmcellular application of

398

B

A

-eo_J C.A)

0mV

k

I

Calcium Current (pA) lOOO 800

/.,/,,e~,.

600

I

250

/ ' ~ l l ,

I

0

lCa

: Control

Control

:

,%,._

~.~-,~.-~ 200-

NE(10pM)*Pz(IlJM)

NE(101JM)*Pz(lpM)

0

200

II115~

NE(IOUM)

/

400"

I

100

NE(IOIJM)

0

300

2

4

6

8

10

12

14

16

Time (rain)

D

C Calcium Current (pA) 1800

Calcium Current (pA) 2800-

Formkolln*NE*Eamolol

1600

:'~"-"~-~

2400-

1400 1200

2000-

l"

1000'

1800

t

.

=a00

9

For,,kolln

800.

200

4OO

Forllkolln

:

1200

6 0 0 Control 4 0 0 ,,,-,.,.,~

NE*EIImolol

25 uM cAMP

0 O

2

4

6

8

10

12

14

16

Time (mln)

0

0

2

4

6 Time (min)

8

10

12

Figure 1: Effects of cq-adrenergic activation on stimulated ICa. Panel A illustrates peak ICa measurement. The effects of al-adrenergic activation on ICa previously elevated by g-adrenergic stimulation (norepinephrine, NE i0 #M + prazosin, Pz 1/~M) are shown in panel B; by forskolin 25/~M in panel C and by adenosine 3',5'monophosphate (cAMP, 25 t~M) in panel D during the periods indicated. Panels B, C, and D were taken from 3 ~different representative experiments. cyclic adenosine 3',5'-monophosphate (cAMP 25/zM) thereby bypassing adenylyl cyclase activation. Figure 1D shows that cAMPelevated ICa was not inhibited by ax-adrenergic activation suggesting Nat ~he inhibitory effect was upstream in the cAMP cascade. The overall change in ICa was -7___21% (n=13). Voltage dependence of the inhibitory effect of ~wadrenergic activation on norepinephrine elevated-ICa The voltage dependence of the cq-adrenergic inhibition on NE stimulated-Iea was examined by I-V relationship and inactivation curves. Figure 2A and 2B shows that cq-adrenergic activation inhibited ICa stimulated by NE (10#M)+prazosin (1/LM) without causing any shift of either the I-V or inactivation curves. This indicates that the inhibitory effect was independent of membrane potential. Discussion The present study reveals that the physiological neurotransmitter nor-epinephrine exerts an inhibitory effect (al-adrenergic effect) on NE or forskolin-stimulated ICa in rat ventricular myocytes. This inhibitory effect appears to be mediated through a G-protein that inhibits adenylyl cyclase. Effects of oq-adrenergic activation on basal ICa In this study, ~l-adrenergic activation did not have any significant effect on basal ICa. These results are similar to those reported by Hescheler et al [8] and Terzic et al [14] who using patch clamping techniques, showed that phenylephrine had no effect on ICa.

Hartmann et al [6], also using the patch clamp technique, showed that ICa increased by phenylephrine was completely blocked by propranolol indicating a/3 and not an cq-adrenergic effect. However, Bruckner and Scholz [3] reported that phenylephrine in the presence of propranolol increased maximal inward ICa in voltage clamped bovine ventricular trabeculae. This discrepancy could be related to the technical errors inherent to voltage-clamp techniques and/or to the species and tissue-dependence of aladrenergic stimulation. al- and g-adrenergie interaction on ICa The absence of an inhibitory effect on cAMP-elevated'Ica by cqadrenergic activation suggests that the site of inhibition is upstream in the cAMP cascade and it is unlikely that the effect was mediated through activation of cAMP-dependent phosphodiesterase[4]. However, lower concentrations of cAMP than the one used in this study should be tested to completely rule out a phosphodiesterase effect. Recently, Danziger et al[5] studied the interactive al and Badrenergic actions of NE on the extent and velocity of shortening, and contraction in rat cardiac myocytes; they found that Badrenergic effects were significantly inhibited by ~l-adrenergie stimulation. They suggested a negative feedback intrinsic to the action of norepinephrine stimulation in the heart. The precise mechanism(s) whereby cq-adrenergic stimulation opposes the gadrenergic effects is yet unknown. Keung et al[10] reported that al-inhibition of Ica in guinea pig ventricular cells did not require a Gi protein, it is therefore likely that the inhibitory effect could be mediated by an interaction between the ~l-adrenoceptor and another type of G -protein (s) leading to inhibition of adenylyl cyelase[9].

399 --e.--Control --..e--NE( 10pM)+Pz(1 9--a.--NE(10!JM)

A "_.

8"

'2

_"

:.

-"

References

pM)

1. Boutjdir M, Restivo M, Wei Y, E1-Sherif N (1992) a 1- and i3adrenergic interactions on L-type Ca current in cardiac myocytes. Biophys J 61:A303.

"-.

2. Boutjdir M, Mery PF, Hanf R, Shrier A, Fischmeister R (1990) High affinity forskolin inhibition of L-Type Ca current in cardiac cells. Mol Pharmacol 38:758-765.

-200.

3. Bruckner R, Scholz H (1984) Effects of a-adrenoceptor stimulation with phenylephrine in the presence of propranolol on force of contraction, slow inward current and cyclic AMP content in bovine heart. Br J Pharmacol 82:223-232.

-400' U -800'

u

4. Buxton ILO, Brunton LL (1986) a-adrenergic receptors in rat ventricular myocytes: Characteristics and linkage to cAMP metabolism. Am J Physiol 251:H307-H313.

-800

-IO00

.,;o

-~;o

-~o

-;o -~o

;

2'0 " ,'0 6'o

Voltage (mV)

B

5. Danziger RS, Sakai M, Lakatta EG, Hansford RG (1990) Interactive a-and 13-adrenergicactions of norepinephrine in rat cardiac myocytes. J Mol Cell Cardiol 22:111-123. 6. Hartmann HA, Mazzoca NI, Kleiman liB, Houser ST (1988) Effects of phenylephrine on calcium current and contractility of feline ventricular myocytes. Am J Physiol 255:H1173-H1180.

i

7. Hartzell HC (1988) Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systems. Prog Biophys Mol Biol 52:165-247. 0.8 .

M)+Pz(I llM)

.=