title: Muscarinic inhibitory and stimulatory regulation of the L-type Ca2+ current is not altered in cardiac ventricular myocytes from mice lacking endothelial nitric oxide synthase.
abstract: Using conventional and perforated patch-clamp techniques, the inhibitory and stimulatory effects of acetylcholine (ACh) on beta-adrenergic regulation of the L-type Ca2+ current (ICa) were studied in ventricular myocytes from wild-type mice (WT) and from mice lacking endothelial nitric oxide synthase (eNOS or NOS3; NOS3-KO mice). To validate the direct comparison of ACh effects on beta-adrenergic responses, the sensitivity of ICa to the beta-adrenergic agonist isoprenaline (Iso) was studied in both WT and NOS3-KO mouse myocytes. ICa sensitivity to Iso was not found to be significantly different in WT and NOS3-KO myocytes: Iso increased ICa with an EC50 of 4.9 and 3.7 nM in WT and NOS3-KO myocytes, respectively. ACh-induced inhibition of ICa did not significantly differ in ventricular myocytes from WT and NOS3-KO mice. ACh (10 microM) inhibited the stimulatory effect of 3 nM Iso by 39 and 35% in WT and NOS3-KO myocytes, respectively. Exposure to and subsequent washout of ACh in the continuous presence of submaximally stimulating concentrations of Iso (1-3 nM) resulted in a transient rebound stimulation of ICa in both WT and NOS3-KO mouse myocytes. The magnitude of the stimulatory effect of ACh did not significantly differ in WT and NOS3-KO mice. These results indicate that nitric oxide (NO) generated by NOS3 does not significantly affect the beta-adrenergic responsiveness of ICa. The results also confirm previous work indicating that NO generated by NOS3 is not obligatory for muscarinic inhibition of the beta-adrenergically regulated ICa in ventricular myocytes. Finally these results demonstrate for the first time that NO generated by NOS3 is not involved in muscarinic rebound stimulation of ICa in ventricular myocytes.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4970, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Belevych
authors.forename: R D
authors.initials: RD
authors.lastname: Harvey
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D018408:Patch-Clamp Techniques; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism
chemical_list: D000318:Adrenergic beta-Agonists; D020746:Calcium Channels, L-Type; D011976:Receptors, Muscarinic; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D007545:Isoproterenol; D000109:Acetylcholine