UC San Diego

Caveolae-localized signaling microdomains are proposed sites that concentrate G protein-coupled receptors (GPCR), heterotrimeric G-proteins, GPCR/G-protein-regulated effector molecules, and gaseous enzymes involved in generating free radicals and antioxidants, such as heme oxygenase (HO) and nitric oxide synthase (NOS), in a confined region, so as to facilitate coordinated and rapid generation of second messengers and regulation of cell function. The effectiveness of these signaling cascades is dependent upon the organization and location of key signaling proteins within the plasma membrane. Caveolae, 'little caves', a subset of lipid rafts, are cholesterol- and sphingolipid-enriched 50-100 nm invaginations of the plasma membrane that contain isoforms of the structural protein caveolin (Cav-1, -2, and -3). Defining the localization and interaction of GPCR and gaseous signaling components in normal adult cardiac myocytes (CM) is thus essential for understanding signaling cascades that regulate developmental and pathological changes in the heart. Microtubules and microfilaments help regulate plasma membrane topography but their role in compartmentation of caveolar-resident signaling components involved in cAMP production has not been defined. The present study used adult rat cardiac myocytes (CM) to investigate whether (1) GPCR, G-proteins, effector enzymes, and gaseous signaling components (heme oxygenase (HO) and endothelial nitric oxide synthase (eNOS)) involved in free radical generation, localize with caveolin in both sarcolemmal and intracellular membrane regions, and (2) if cytoskeletal disruption influences localization of caveolin (Cav) and expression of caveolae, thereby altering the distribution of G protein-coupled receptor (GPCR) signaling components and cAMP production. The observed distribution of GPCRs, G-proteins, and AC with Cav-3 on both sarcolemmal and intracellular regions defines unique cellular microdomains for regulation and signaling by hormones and drugs in the adult heart. The localization of HO-1 and eNOS with caveolin, and the close apposition between caveolae and mitochondria suggests that caveolae in addition to being sites that generate gaseous signaling components (i.e. CO and NO) may also be sites for generation of antioxidants (biliverdin), and thus 'sinks' for free radicals in adult CM. Disruption microtubules and microfilaments alters the expression of caveolae, phosphorylation state of caveolins, and ability of caveolins to blunt GPCR-AC signal transduction