Recent studies of b-adrenoceptor (b-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling pathway is activated in the same manner in response to b2- AR versus b1-AR stimulation. The present study compared in the intact dog the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects, the extent of cAMP accumulation, PKA activation and PKA dependent phosphorylation of key effector proteins in response to b-AR subtype stimulation. Additionally, many of these parameters and L-type Ca2+ current (ICa) were also measured in single canine ventricular myocytes. The results indicate that while the cAMP/PKA dependent phosphorylation cascade activated by b1-AR stimulation could explain the resultant modulation of cardiac function, substantial chronotropic, inotropic and lusitropic responses following b2-AR stimulation occurred in the absence of global PKA activation and phosphorylation of non-sarcolemmal proteins, including phospholamban (PLB), troponin I (Tn I), C protein and glycogen phosphorylase kinase. However, in single canine myocytes we found that the b2-AR effect to augment contraction was primarily due to augmentation of ICa, and that b2-AR-stimulated increases in both ICa and contraction were abolished by the PKA inhibitors, H-89 and Rp-cAMPS. Thus, the b2-AR-directed cAMP/PKA signaling does modulate sarcolemmal L-type Ca2+ channels, but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. These results indicate that the dissociation of b2-AR signaling from cAMP regulatory systems is only apparent, and that b2-AR-stimulated cAMP/PKA signaling is localized to subsarcolemmal space and uncoupled from phosphorylation of major non-sarcolemmal regulatory proteins involved in excitation-contraction coupling.
