CA-2+ DEPENDENCE OF ALPHA-ADRENERGIC EFFECTS ON THE CONTRACTILE PROPERTIES AND CA-2+ HOMEOSTASIS OF CARDIAC MYOCYTES

alpha-Adrenergic stimulation is known to enhance myocardial contractility. Adult rat left ventricular myocytes bathed in 1 mM [Ca2+] (Ca0) and electrically stimulated at 0.2 Hz responded to alpha-adrenergic stimulation with 50-mu-M phenylephrine and 1-mu-M propranolol with an increase in twitch amplitude to 177.1 +/- 25.6% of control (mean +/- SEM). In contrast, when cell Ca2+ loading was increased by bathing cells in 5 mM Ca0, alpha-adrenergic stimulation decreased twitch amplitude to 68.6 +/- 8.2% of control. Time-averaged cytosolic [Ca2+] of cells in 1.0 mM Ca0 is enhanced via an increase in the frequency of electrical stimulation. When myocytes were stimulated at 2 Hz in 1 mM Ca0, alpha-adrenergic stimulation did not increase twitch amplitude (103.8 +/- 12.4% of control). In myocytes loaded with the Ca2+ probe indo-1, alpha-adrenergic effects during stimulation at 0.2 Hz (an increase in twitch amplitude in 1 mM Ca0 and a decrease in twitch amplitude in 5 mM Ca0) were associated with similar changes in the indo-1 transient. In 5 mM Ca0, spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) occurred in the diastolic interval between twitches (2.9 +/- 1.4 spontaneous SR Ca2+ oscillations/min; n = 7); alpha-adrenergic stimulation abolished these oscillations in six of seven cells. Thus, an increase in the frequency of spontaneous diastolic SR Ca2+ release (i.e., Ca2+ overload) is not the mechanism for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. In experiments with unstimulated myocytes, we determined whether the effect of alpha-adrenergic stimulation on cell Ca2+ homeostasis and oscillatory SR Ca2+ release observed in 5 mM Ca0 occurs only during electrical stimulation, when voltage-dependent currents are operative, or also at rest. Unstimulated rat ventricular myocytes in 5 mM Ca0 exhibit oscillatory SR Ca2+ release; alpha-adrenergic stimulation decreased the frequency of these oscillations to 53.9 +/- 8.9% of control, and this effect was blocked by 1-mu-M prazosin. In unstimulated indo-1-loaded myocytes alpha-adrenergic stimulation decreased the resting indo-1 fluorescence ratio in 5 mM Ca0, whereas it had no effect in 1 mM Ca0. Additional experiments were aimed at defining a role for Ca2+-activated, phospholipid-dependent protein kinase C (PKC) for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. Short-term preexposure to 0.1-mu-M 4-beta-phorbol 12-myristate 13-acetate (PMA) has been shown to maximally activate PKC. PMA, in 5 mM Ca0, decreased contraction and indo-1 transient amplitudes and prevented any additional negative inotropic action by alpha-adrenergic stimulation. Additionally, staurosporine (5 nM), a PKC inhibitor, abolished the alpha-adrenergic-stimulated decrease in twitch amplitude in 5 mM Ca0. Thus, an increase in cytosolic [Ca2+] achieved via an increase in Ca0 or by rapid pacing leads to effects of alpha-adrenergic stimulation on the contractile properties and Ca2+ homeostasis of rat ventricular myocytes that are different from those observed in lower Ca0. These results may be explained with a [Ca2+]-dependent enhancement of the activity of PKC, an enzyme that is activated during alpha-adrenergic stimulation of the myocardium.