Modulation of the endothelial nitric-oxide synthase-caveolin interaction in cardiac myocytes. Implications for the autonomic regulation of heart rate.
The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing microdomains termed caveolae. In endothelial cells, eNOS interacts with caveolin-1, which represses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and transfected them with wild-type (WT) eNOS or myristoylation-deficient (myr-) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr- eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to the caveolin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP elevation. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the cholinergic modulation of cardiac myocyte function.
Feron
O
O
Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. feron@mint.ucl.ac.be
Dessy
C
C
Opel
D J
DJ
Arstall
M A
MA
Kelly
R A
RA
Michel
T
T
D000215:Acylation; D000818:Animals; D000831:Animals, Newborn; D001341:Autonomic Nervous System / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D051244:Caveolin 3; D022461:Caveolins; D002451:Cell Compartmentation / Q000502:physiology; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D006152:Cyclic GMP / Q000096:biosynthesis; D003864:Depression, Chemical; D006339:Heart Rate / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011485:Protein Binding; D051381:Rats
C495548:Cav3 protein, mouse; C495549:Cav3 protein, rat; D051244:Caveolin 3; D022461:Caveolins; D018679:Cholinergic Agonists; D008565:Membrane Proteins; D002217:Carbachol; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C496316:Nos3 protein, rat; D006152:Cyclic GMP