Acidosis alters the phosphorylation of Ser(16) and Thr(17) of phospholamban in rat cardiac muscle

The effect of acidosis on the phosphorylation of Ser(16) and Thr(17) of phospholamban in rat cardiac muscle has been investigated using phosphorylation-site-specific antibodies to this protein. Ventricular myocytes were stimulated at 0.5 Hz for 5 min, in either control (pH 7.4) or acid (pH 6.5) physiological salt solution, in the absence or presence of isoprenaline. Site-specific phosphorylation of phospholamban was determined by Western blotting. Acidosis reduced phosphorylation of Ser(16) in the absence of isoprenaline, but did not alter the isoprenaline-induced phosphorylation of Ser(16). In contrast, acidosis increased Thr(17) phosphorylation in the absence and presence of isoprenaline. Buffering intracellular Ca2+ ([Ca2+](i)) with BAPTA inhibited the increase in Thr(17) phosphorylation during acidosis but had no effect on Ser(16) phosphorylation. We conclude that acidosis can alter the phosphorylation of Ser(16) and Thr(17) by inhibition of protein kinase A, and by an acidosis-induced increase in [Ca2+](i) and the subsequent activation of a Ca2+/calmodulin-dependent protein kinase, respectively. The possible effect of these changes in phosphorylation on the activity of the Ca2+-ATPase of the cardiac sarcoplasmic reticulum during acidosis is discussed.