Receptor crosstalk: effects of prolonged carbachol exposure on beta 1-adrenoceptors and adenylyl cyclase activity in neonatal rat ventricular myocytes.
Supersensitivity of adenylyl cyclase after exposure to inhibitory agonists is a general means of cellular adaptation. We hypothesized that such "crosstalk" between muscarinic cholinergic agonists, beta 1-adrenoceptors, and adenylyl cyclase may be an important mechanism of cardiac adaptation to interventions that enhance vagal activity. We used primary cultures of neonatal rat ventricular myocytes and measured beta-adrenoceptors by radioligand binding and adenylyl cyclase activity by a single column method. Carbachol induced a time- and dose-dependent reversible decrease in cell surface beta 1-adrenoceptors. The peak effect occurred after 20 h of exposure to 100 microM carbachol which caused a decrease in the maximum number of binding sites for the beta-adrenoceptor antagonist 3H-CGP-12177 from 42.3 +/- 3.4 to 33.0 +/- 2.6 fmol/mg protein (n = 12, P < 0.03) without a change in antagonist affinity. Loss of cell surface receptors was prevented by atropine and by the protein kinase C inhibitor H7. The decrease in cell surface receptors was not accompanied by receptor internalization as assessed by equilibrium binding experiments in a cytosolic fraction using 125I-iodocyanopindolol. In contrast to the well-known acute inhibitory effects of carbachol on adenylyl cyclase activation, prolonged carbachol exposure preserved (-)-isoprenaline-stimulated adenylyl cyclase activity and enhanced postreceptor stimulated adenylyl cyclase activity. Carbachol did not further enhance adenylyl cyclase activity after pretreatment with pertussis toxin. The protein kinase C inhibitor chelerythrine prevented the carbachol induced enhancement of forskolin-stimulated adenylyl cyclase activity. We conclude that prolonged incubation with carbachol in rat neonatal ventricular myocytes causes a reduction in cell surface beta 1-Adrenoceptor density. beta 1-Adrenoceptor-mediated adenylyl cyclase activity is preserved and postreceptor-mediated adenylyl cyclase activity is augmented. Our data suggest that carbachol-stimulated protein kinase C activity may play a key role in the prolonged muscarinic regulation of adenylyl cyclase activity.
Paraschos
A
A
Cardiology Section, Veterans Affairs Medical Center, San Francisco, CA 94121.
Karliner
J S
JS
D000262:Adenylyl Cyclases / Q000187:drug effects* / Q000378:metabolism*; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology; D006321:Heart / Q000187:drug effects* / Q000502:physiology*; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects; D018721:Muscarinic Agonists; D009206:Myocardium / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure*; D011412:Propanolamines / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D018342:Receptors, Adrenergic, beta-1 / Q000187:drug effects* / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D014316:Tritium
D000319:Adrenergic beta-Antagonists; D018721:Muscarinic Agonists; D011412:Propanolamines; D018342:Receptors, Adrenergic, beta-1; D011976:Receptors, Muscarinic; D014316:Tritium; D001285:Atropine; D002217:Carbachol; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; C033394:CGP 12177