β ₂ -Adrenergic cAMP Signaling Is Uncoupled From Phosphorylation of Cytoplasmic Proteins in Canine Heart

Abstract

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.

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

“…This suggests that in the targeted sensor, the end-point ratio in the bound state might be altered, for example, due to reduced conformational change in the sensor. An increase in local cAMP levels in the SERCA2a microdomain was not detectable after b 2 -AR stimulation, which is in line with previous studies showing no effect of b 2 -AR-cAMP on PLN phosphorylation 27 and its stringent compartmentation at the T-tubular membranes 28,29 . However, the relatively small b 2 -AR-cAMP signals are potentially difficult to resolve, especially using the less sensitive Epac1-PLN sensor, which is certainly a limitation of our study.…”

Differences in the Bioenergetic Response of the Isolated Perfused Rat Heart to Selective β 1 - and β 2 -Adrenergic Receptor Stimulation

“…To increase selectivity of the receptor stimulation, an ␣-AR antagonist, prazosin, or a ␤ 1 -AR antagonist, bisoprolol (Merck), was used for ␤ 1 and ␤ 2 -stimulated hearts, respectively, in combination with the agonists. 2,4 Figure 1 shows typical responses of RPP and MV O 2 for a ␤ 1 -and a ␤ 2 -AR-stimulated heart during HCF at baseline and during each of the 3 doses. Typical 31 P NMR spectra obtained during baseline and ␤-AR stimulation are shown in Figure 2.…”

“…Intracellular calcium ([Ca 2ϩ ] i ) levels are known to be greater during ␤ 1 -AR stimulation [1][2][3][4] and may be a more significant regulator of oxidative phosphorylation ( Figure 6, arrow 5) during ␤ 1 -AR stimulation than during ␤ 2 -AR stimulation. This regulation would be expected to be more effective when O 2 is not limiting and is consistent with the greater augmentation of energy supply and workload demand during ␤ 1 -AR stimulation under HCF compared with that under CP.…”

“…Although the importance of both the ␤ 1 -and ␤ 2 -AR subtypes has been recognized, there are substantial differences between ␤ 1 -and ␤ 2 -AR-mediated functional responses. [1][2][3][4] A recent study using canines found that cAMP signaling was coupled to PKA activation during ␤ 1 -but not during ␤ 2 -AR stimulation. 4 For ␤ 1 -AR-stimulated hearts, this led to PKA-dependent phosphorylation of the key regulatory proteins phospholamban, troponin I, and C protein and phosphorylation of the inactive b form of glycogen phosphorylase to the active a form.…”

“…In particular, greater glycogenolysis may occur during ␤ 1 -AR than ␤ 2 -AR stimulation. 4 We hypothesized that as a result of these differences in cAMP signaling, the relationship between cardiac contractile function and bioenergetics differs between ␤ 1 -and ␤ 2 -AR stimulation. Studies in the intact heart permit an evaluation of the working organ with accurate regulation of substrate and oxygen delivery without the complexity of other neurohumoral factors present in the whole organism.…”