FIGURE 2.

cAMP, PKA, and PDE3 inhibition increase SERCA2 activity and Ca²⁺ uptake in human SR fractions. A, as described under “Methods,” after incubation of SR fractions (20 μg) in the absence or presence of the indicated concentrations of ATP and cAMP without or with cilostamide (Cil) or rolipram (Rol), endogenous PLB, phosphorylated PLB, and β-actin were detected after SDS-PAGE and immunoblotting. Data are representative of three experiments. In these and other Western blots PLB is predominantly monomeric, most likely due to the heating of samples before electrophoresis under reducing conditions with Laemmli SDS sample buffer. B, bar graph summarizing pSer16PLB/PLB total ratios. Cilostamide significantly enhanced the effect of 0.1 μm cAMP on phosphorylation of PLB. *, p < 0.01 versus control (n = 3 independent experiments). C, PDE activity in human cardiac SR fractions was assayed as described under “Methods” and expressed as specific activity (pmol of cAMP hydrolyzed/min/mg). Results are presented as the mean ± S.E. (n = 3 preparations). PDE3 activity was determined as the cilostamide-sensitive fraction, and PDE4 activity was determined as the rolipram-sensitive fraction. PDE3 activity was significantly higher than PDE4 activity (*, p < 0.001). D, after incubation of SR fractions without or with the indicated concentrations of cAMP (0–10 μm), ⁴⁵Ca²⁺ uptake was measured in the presence of 0.5 μm free Ca²⁺ as described under “Methods.” Results are presented as % increase due to cAMP, with basal Ca²⁺ uptake (9.4 ± 0.8 nmol/mg/min, n = 3) taken as 100%. cAMP significantly enhanced ⁴⁵Ca²⁺ uptake (*, p < .01; **, p < 0.04). E, after incubation of SR fractions with or without cAMP (3 μm) in the presence or absence of cilostamide (1 μm), ⁴⁵Ca²⁺ uptake (0.5 μm free Ca²⁺) was assayed as described under “Methods.” Results are presented as the mean ± S.E. (n = 3). Cilostamide significantly enhanced the effect of cAMP on ⁴⁵Ca²⁺ uptake (*, p < .02). F, Ca²⁺ uptake (upper panel) and SERCA activity (lower panel) were assayed in reaction mixtures containing Mg²⁺ and ATP in the presence or absence of rPKAc as described under “Methods.” Results are presented as nmol (Ca²⁺ or P_i)/min/mg (mean ± S.E.) (n = 3). rPKAc significantly enhanced ⁴⁵Ca²⁺ uptake and SERCA2 activity (*, p < .001).