Different effects of α- and β-adrenergic stimulation on cytosolic pH and myofilament responsiveness to Ca²⁺ in cardiac myocytes

α-Adrenergic stimulation (α-AS) and β-adrenergic stimulation (β-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 α-AS and β-AS. The effects of α-AS (50 μM phenylephrine and 1 μM nadolol) and β-AS (0.05 μM 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. α-AS and β-AS shifted this relation in opposite directions, indicating that α-AS increased and β-AS decreased myofilament responsiveness to Ca²⁺. In addition, the positive inotropic action of α-AS was associated with an increased Ca(i) transient amplitude in 50% of the myocytes (n=12), whereas β-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 α-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). α-AS enhanced both ES (ΔES, 1.8±0.6%; p<0.05) and pH(i) (ΔpH(i), 0.06±0.01; p<0.005), and there was a significant correlation between ΔES and ΔpH(i) (r=0.76, p<0.05). A similar effect of α-AS on pH(i) was observed in the absence of electrical stimulation (n=8). The α-AS-induced enhancement of ES and pH(i) was abolished by 10 μM ethylisopropylamiloride, a Na⁺-H⁺ exchange inhibitor (n=7). In additional experiments, myocytes were preincubated either with 0.2 μM 4β-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, α-AS had no effect on pH(i) and decreased ES to ≃60% of control. In myocytes bathed in 0.5 mM [Ca²⁺] and stimulated at 0.2 Hz, β- AS enhanced ES (control ES as percentage of resting cell length, 2.8±1.0%; ΔES, 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, α-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 α-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 α-AS. In contrast, β-AS does not affect pH(i), and cytosolic acidification is not the mechanism for the β-AS-induced decrease in myofilament responsiveness to Ca²⁺.