Background —Recent studies of β-adrenergic receptor (β-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to β 2 -AR versus β 1 -AR stimulation. Methods and Results —The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to β-AR subtype stimulation. In addition, many of these parameters and L-type Ca 2+ current ( I Ca ) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by β 1 -AR stimulation could explain the resultant modulation of cardiac function, substantial β 2 -AR–mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that β 2 -AR–stimulated increases in both I Ca and contraction were abolished by PKA inhibition. Thus, the β 2 -AR–directed cAMP/PKA signaling modulates sarcolemmal L-type Ca 2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. Conclusions —These results indicate that the dissociation of β 2 -AR signaling from cAMP regulatory systems is only apparent and that β 2 -AR–stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.

Cyclic AMP-rnediat~ phospho~la~on of calcium channel subunits was studied in vitro and in vivo in preparations from dog heart. Calcium channels in native cardiac membranes were phosphorylated by CAMP-dependent protein kinase (PKA) solubilized with digitonin and subsequently immunoprecipitated using a polyclonal antibody generated against the deduced carboxy-terminal sequence of the cardiac /I subunit. A 62 kDa protein was identified as the major PKA-substrate in the immunoprecipitates. In the intact myocardium, this putative p subunit was found to be phosphorylated in response to CAMP elevating agents. In contrast, no phosphorylation of a protein with an electrophoretic mobility similar to the a, subunit was detected, although 1,4diiydropyridine receptor sites were recovered in the immunoprecipitates. Thus, we suggest that IX&mediated phospho~lation of the b subunit is the major mechanism for @-adrenergic regulation of cardiac L-type calcium channel activity.

Phosphorylation of phospholamban (PLB), a membrane-bound 15 kDa protein and troponin I (TNI) was studied in isolated perfused rat hearts by using the back-phosphorylation technique with [32P]ATP catalysed by an excess of exogenous catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, followed by protein separation. This standardized method allows the quantitative detection of protein phosphorylation specifically stimulated by cAMP. In control hearts the extent of specific phosphorylation was equivalent to 3.3 nmol of PLB and 11.0 mumol of TNI per g of cardiac tissue. In hearts freeze-clamped 30 s after exposure to isoprenaline (10 pM-10 microM), there was a dose-dependent decrease in phosphate incorporation in vitro, indicating a phosphorylation of the respective proteins in vivo. A differential sensitivity of TNI and PLB phosphorylation towards the beta-adrenergic agonist and the subsequent increase in tissue cAMP was found, favouring TNI phosphorylation. K0.5 values for isoprenaline were 2.94 +/- 0.04 nM and 4.46 +/- 0.24 nM for PLB and the 15 kDa protein, but 0.13 +/- 0.01 nM for TNI phosphorylation in the intact tissue. At an isoprenaline-induced increase in cAMP less than 3 pmol/mg of protein there was no or only a small increase in PLB phosphorylation, whereas TNI phosphorylation was nearly maximal. By plotting phosphorylation data against changes in contractile parameters a strong correlation was obtained for TNI (r = 0.95), assuming a linear relationship. For PLB a complex relationship is likely to exist. Our data (i) indicate a functional compartmentalization of the cAMP signal cascade and (ii) confirm that phosphorylation of TNI rather than of PLB is related to changes in mechanical myocardial responses.

The aim of this study was to examine the mechanism(s) underlying the reduced isoproterenol-induced positive inotropic and lusitropic effects in hypertrophied hearts. Chronic beta-adrenergic stimulation (2.4 mg isoproterenol.kg-1. day-1 for 4 days) induced cardiac hypertrophy by 33 +/- 2% in rats. A parallel downregulation of phospholamban (PLB) and sarcoplasmic reticulum Ca2(+)-ATPase (SERCA2) protein expression by 49 and 40%, respectively, was observed, whereas troponin I (TNI) and C protein remained unchanged. In papillary muscles from chronically beta-adrenergically stimulated rats, the isoproterenol-induced positive inotropic and lusitropic effects, as well as adenosine 3',5'-cyclic monophosphate (cAMP) accumulation, were attenuated compared with those in control animals. Acute exposure to isoproterenol induced phosphate incorporation into PLB, TNI, and C protein of 48 +/- 4.6, 55 +/- 5.0, and 27 +/- 4.9 pmol/mg homogenate protein, respectively, in control animals. In the hypertrophied hearts, phosphate incorporation into PLB was reduced by 76%, whereas phosphate incorporation into TNI or C protein remained unchanged. In conclusion, chronic beta-adrenergic stimulation reduced the isoproterenol-stimulated positive inotropic and lusitropic effects in papillary muscles, which were accompanied by 1) diminished cAMP formation, 2) attenuation of cAMP-mediated PLB phosphorylation, and 3) downregulation of PLB and SERCA2 protein.

In canine myocardium, the beta-subunit of the L-type Ca2+ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217-222, 1993). We have assessed the identity of the beta-subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca2+ channel beta-subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the beta-subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dtmax. The results imply the involvement of a PKA-mediated phosphorylation of the Ca2+ channel beta-subunit in the adrenergic stimulation of intact canine myocardium.