DIFFERENT EFFECTS OF ALPHA-ADRENERGIC AND BETA-ADRENERGIC STIMULATION ON CYTOSOLIC PH AND MYOFILAMENT RESPONSIVENESS TO CA2+ IN CARDIAC MYOCYTES

Alpha-adrenergic stimulation (alpha-AS) and beta-adrenergic stimulation (beta-AS) of the myocardium are associated respectively with an increase and a decrease in myofilament responsiveness to Ca2+. We hypothesized that changes in cytosolic pH (pH(i)) may modulate these opposite actions of alpha-AS and beta-AS. The effects of alpha-AS (50-mu-M phenylephrine and 1-mu-M nadolol) and beta-AS (0.05-mu-M isoproterenol) on contraction and either cytosolic Ca2+ (Ca(i)) or pH(i) were assessed in adult rat ventricular myocytes bathed in bicarbonate buffer (pH 7.36+/-0.05). In cells loaded with the ester derivative (AM form) of indo-1, the 410/490-nm ratio of emitted fluorescence indexed Ca(i). Myofilament responsiveness to Ca2+ was assessed by the relaxation phase of the length-indo-1 fluorescence relation during a twitch. Alpha-AS and beta-AS shifted this relation in opposite directions, indicating that alpha-AS increased and beta-AS decreased myofilament responsiveness to Ca2+. In addition, the positive inotropic action of alpha-AS was associated with an increased Ca+ transient amplitude in 50% of the myocytes (n=12), whereas beta-AS always increased Ca(i) (n=5). In cells loaded with the fluorescent pH(i) probe SNARF-1 AM, the emitted 590/640-nm fluorescence is a measure of pH(i). The effect of alpha-AS on the extent of cell shortening during the twitch (ES) was expressed as the percentage of resting cell length. Both ES and pH(i) were assessed in myocytes bathed in 1.5 mM [Ca2+] and stimulated at 0.5 Hz (control ES, 7.4+/-1.5%; control pH(i), 7.11+/-0.05; n=10). Alpha-AS enhanced both ES (DELTA-ES, 1.8+/-0.6%; p<0.05) and pH(i) (DELTA-pH(i), 0.06+/-0.01; p<0.005), and there was a significant correlation between DELTA-ES and DELTA-pH(i) (r=0.76, p < 0.05). A similar effect of alpha-AS on pH(i) was observed in the absence of electrical stimulation (n=8). The alpha-AS-induced enhancement of ES and pH(i) was abolished by 10-mu-M ethylisopropylamiloride, a Na+-H+ exchange inhibitor (n=7). In additional experiments, myocytes were preincubated either with 0.2-mu-M 4-beta-phorbol 12-myristate 13-acetate (n=8) or with 5 nM staurosporine (n=8), which have been shown to downregulate and inhibit Ca2-activated phospholipid-dependent protein kinase C, respectively. In either group, alpha-AS had no effect on pH(i) and decreased ES to almost-equal-to 60% of control. In myocytes bathed in 0.5 mM [Ca2+] and stimulated at 0.2 Hz, beta-AS enhanced ES (control ES as percentage of resting cell length, 2.8+/-1.0%; DELTA-ES, 8.1+/-1.5%; n=7; p<0.005) but had no effect on pH(i) either during electrical stimulation (n=7) or at rest (n=11). In summary, under the conditions of this study, alpha-AS enhances myofilament response to Ca2+ and increases pH(i) via protein kinase C-mediated activation of Na+-H+ exchange. Cytosolic alkalinization contributes to the effect of alpha-AS to augment contraction amplitude. An enhanced Ca(i) transient occurs in 50% of the myocytes and is not an absolute requirement for the positive inotropic action of alpha-AS. In contrast, beta-AS does not affect pH(i), and cytosolic acidification is not the mechanism for the beta-AS-induced decrease in myofilament responsiveness to Ca2+.