Modulation of the Endothelial Nitric-oxide Synthase-Caveolin Interaction in Cardiac Myocytes

Abstract

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing microdomains termed caveolae. In endo-thelial cells, eNOS interacts with caveolin-1, which re-presses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 iso-form, but whether this interaction affects NO produc-tion and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and trans-fected them with wild-type (WT) eNOS or myristoyla-tion-deficient (myr ؊) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic ago-nist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr ؊ eNOS myocytes, carba-chol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible per-meabilization protocol to load intact neonatal rat myo-cytes with an oligopeptide corresponding to the caveo-lin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP eleva-tion. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate con-trol and indicate a key role for eNOS/caveolin interac-tions in the cholinergic modulation of cardiac myocyte function. The endothelial isoform of nitric-oxide synthase (eNOS) 1 , originally identified in large vessel endothelium, is now known to be expressed in numerous cell types, including cardiac myo-cytes. The eNOS enzyme is dually acylated (for review, see Refs. 1 and 2) and is thereby specifically targeted to plasma-lemmal signal-transducing microdomains termed caveolae (3, 4). In endothelial cells and cardiac myocytes, eNOS is quanti-tatively associated with caveolin (4), the structural protein of caveolae, and a stable protein-protein interaction takes place at consensus sequences present within both proteins and leads to the inhibition of the eNOS activity (5– 8). More recently, we have also documented that, in endothelial cells, when intracel-lular Ca 2ϩ concentration is increased by an agonist, a regula-tory cycle is initiated (2, 9), wherein (i) Ca 2ϩ /calmodulin acti-vates eNOS by disrupting the heteromeric complex formed between eNOS and caveolin; (ii) activated, caveolin-free eNOS is translocated from caveolae, probably associated with enzyme desensitization; (iii) when Ca 2ϩ returns to basal levels, eNOS reassociates with caveolin; and (iv) the inhibitory complex is restored to caveolae, a process facilitated by eNOS palmitoyla-tion. The role (if any) of this eNOS-caveolin regulatory cycle in cardiac myocytes, however, remains to be established. The location of eNOS in plasmalemmal caveolae and its interaction with caveolin may find several biological justifica-tions. First, the compartmentation of eNOS with other signal-ing proteins may facilitate, or improve the efficacy of, the coupling between agonist stimulation and eNOS activation. We and others have, for instance, reported that G-protein-coupled receptors known to stimulate NO production, such as the mus-carinic and bradykinin receptors, undergo rapid translocation and are targeted to caveolae upon agonist stimulation (10, 11). More recently, McDonald et al. (12) have reported the existence of a caveolar complex between the arginine transporter CAT1 and eNOS, thereby providing a mechanism for a highly effi-cient delivery of substrate to eNOS. Second, the close control of eNOS activity by caveolin is probably required to maintain a low basal production of NO and protect the cell from undesired, potentially cytotoxic bursts of NO in response to subtle in-creases in intracellular calcium. Finally, the targeting of eNOS in plasmalemmal caveolae probably facilitates paracrine sig-naling by NO, a pathway most clearly delineated in the vascu-lar wall (13) and not yet clearly established in other tissues expressing eNOS. In cardiac myocytes, eNOS is also located within plasmale-mmal caveolae, but, to date, the NO generated has only been shown to act endogenously by modulating the myocyte respon-siveness to neurohumoral or mechanical stimuli. Thus, for instance, activation of eNOS in cardiac myocytes by muscarinic * This work was supported by awards from the National Institutes of Health, the American Heart Association, and the Burroughs Wellcome Fund (to T. M.). The costs of publication of this article were defrayed in part by the payment of page charges.