Differential Targeting of β-Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae

Abstract

Differential modes for ␤ 1 -and ␤ 2 -adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in ␤ 2 -ARs, G␣ i , protein kinase A RII␣ subunits, caveolin-3, and flotillins (caveolin functional homologues); ␤ 1 -ARs, m 2 -muscarinic cholinergic receptors, G␣ s , and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RI␣ subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface ␤ 2 -ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different ␤ 2 -AR expression levels), indicating that the fidelity of ␤ 2 -AR targeting to caveolae is maintained over a physiologic range of ␤ 2 -AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of ␤ 2 -ARs (but not ␤ 1 -ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domainspecific differences in the stoichiometry of individual elements in the ␤-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.Catecholamines act through cardiac ␤-adrenergic receptors (␤-ARs) 1 to influence the contractile state of the heart. The direct inotropic and chronotropic support provided by cardiac ␤-ARs represents a critical compensatory mechanism to preserve cardiac function during stress and/or states associated with circulatory compromise. In the hearts of most mammalian species, the physiologic effects of catecholamines are mediated by the predominant ␤ 1 -AR subtype (75-80% of the total ␤-ARs), which activates a signaling pathway involving the G sdependent stimulation of adenylyl cyclase leading to the accumulation of cAMP and protein kinase A-dependent phosphorylation of key target proteins. Cardiomyocytes also express ␤ 2 -ARs that support contractile function. Until quite recently, most studies of ␤ 2 -AR signaling in cardiomyocytes were wedded to the concept that ␤ 2 -ARs signal to the G s /cAMP pathway in a manner that is essentially equivalent to the pathway activated by ␤ 1 -ARs. However, there is evidence that ␤ 2 -ARs are not functionally redundant, including the findings that ␤ 2 -ARs ...

Lateral Diffusion of Gαs in the Plasma Membrane Is Decreased after Chronic but not Acute Antidepressant Treatment: Role of Lipid Raft and Non-Raft Membrane Microdomains

“…This was observed with chronic antidepressant treatment (Chen and Rasenick, 1995a;Zhang and Rasenick, 2010) as well as with cholesterol chelation by methyl-b-cyclodextrin Head et al, 2006;Rybin et al, 2000) or with caveolin depletion . It is noteworthy, however, that although raft disruption has similar effects on GFP-Ga s and GFPGa i1 , chronic antidepressant treatment affects only Ga s (Figure 4a).…”

“…Previous studies suggest that increased Ga s association with adenylyl cyclase may underlie antidepressant regulation of cAMP Rasenick, 1995a, 1995b;Menkes et al, 1983;Ozawa and Rasenick, 1991). Furthermore, translocation of Ga s to non-raft membrane fractions following raft disruption results in increased coupling to adenylyl cyclase , and this is unique to Ga s Head et al, 2006;Rybin et al, 2000). Those earlier experiments relied on lipid raft preparations from lysed tissue and cells rather than intact, living cells.…”

Cytoplasmic cAMP concentrations in intact cardiac myocytes

“…In ventricular myocytes, the stimulatory effect can be explained by the observation that these cells express adenylyl cyclase types 4 and/or 7 (AC4/7) in addition to AC5/6, and the fact that M 2 R activation of G i can actually stimulate AC4/7 whereas it inhibits AC5/6 (3). However, AC5/6 is predominantly found in caveolar membrane fractions, whereas AC4/7 is extracaveolar (7,21,27). This suggests that M 2 R activation inhibits and stimulates cAMP production in distinctly different microdomains.…”

Characterization of Secretory Type IIA Phospholipase A2 (sPLA2-IIA) as a Glycyrrhizin (GL)-Binding Protein and the GL-Induced Inhibition of the CK-II-Mediated Stimulation of sPLA2-IIA Activity in Vitro.

“…This correlation will be elucidated by the in vivo experiments for direct detection of the specific phosphorylation of cellular sPLA 2 -IIA with membrane-associated CK-II 17) in interleukin-1 (IL-1)/tumor necrosis factor (TNF)-stimulated cells. This possibility is strongly supported by the following recent reports: (i) sPLA 2 -IIA is the most widely distributed isozyme in humans and rats, 20) and is extremely induced by proinflammatory stimuli, such as bacterial endotoxin, IL-1 and TNF 21) ; (ii) this isozyme is localized in the punctate and perinuclear domains in IL-1/TNF-stimulated cells and is accumulated in the caveolae of cytokine-stimulated cells; 22,23) and (iii) caveolae are highly enriched in caveolins (structural proteins), b 2 -adrenergic receptor, cAMP-dependent protein kinase (A-kinase) 24) and receptor tyrosine kinases, such as platelet-derived growth factor receptor 25) and ErbB receptor, 26) corresponding signal transduction for metabolic and mitogenic effects, and functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways. 27) For understanding clearly the biochemical mechanism involved in the GL-induced anti-inflammatory effect in vivo, further analytical studies will be required (i) to confirm a GL-sensitive sPLA 2 among at least five isozymes (sPLA 2 -IIA, sPLA 2 -IIC, sPLA 2 -IID, sPLA 2 -IIE and sPLA 2 -IIF) 28,29) of the sPLA 2 -II family in various clinical materials, such as synovial fluid of patients with rheumatoid arthritis and sera of patients with autoimmune diseases; (ii) to characterize the regulatory mechanism of the GL-sensitive sPLA 2 by membrane-associated CK-II or other protein kinases [A-kinase, Ca 2ϩ /phospholipid-dependent protein kinase (C-kinase), mitogen-activated protein kinase (MAP-kinase) and receptor tyrosine kinases] in vivo and in vitro; and (iii) to determine the suppressive effects of GL, GA and oGA on the induction of a GL-sensitive sPLA 2 in the cytokine-mediated response at the cellular level.…”