Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes

The effects of the cholinergic muscarinic agonist carbachol (CCh) on the basal L-type calcium current(>) I-Ca.L> in ferret right ventricular (RV) myocytes were studied using whole cell patch clamp. CCh produced two major effects: (i) in all myocytes, extracellular application of CCh inhibited I-Ca.L in a reversible concentration-dependent manner; and (ii) in many (but not all) myocytes, upon washout CCh produced a significant transient stimulation of I-Ca,I-L ('rebound stimulation'). Inhibitory effects could be observed at 1 x 10(-10) M CCh. The mean steady-state inhibitory concentration-response relationship was shallow and could be described with a single Hill equation (maximum inhibition = 34.5 %, IC50 = 4 x 10(-8) m, Hill coefficient n = 0.60). Steady-state inhibition (I or 10,am CCh) had no significant effect on I-Ca,I-L selectivity or macroscopic (i) activation characteristics, (ii) inactivation kinetics, (iii) steady-state inactivation or (iv) kinetics of recovery from inactivation. Maximal inhibition of nitric oxide synthase (NOS) activity (preincubation of myocytes in I MM L-NMMA (N-G-monomethyl-L-arginine) + 1 MM L-NNA (N-G-nitro-L-arginine) for 2-3 h plus inclusion of I MM L-NMMA + I MM L-NNA in the patch pipette solution) produced no significant attenuation of the CCh-mediated inhibition of I-Ca,I-L Protocols involving (i) the nitric oxide (NO) scavenger PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide; 200 muM), (ii) imposition of a 'cGMP clamp' (100 muM 8-Bromo-cGMP), and (iii) inhibition of soluble guanylyl cyclase (ODQ (1H-[1,2,4,] oxadiazolo(4,3,-a)quinoxalin-1-one), 50 muM) all failed to attenuate CCh-mediated inhibition of I-Ca,I-L. While CCh consistently inhibited basal I-Ca,I-L in all RV myocytes studied, not all myocytes displayed rebound stimulation upon CCh washout. However, there was no difference between CCh-mediated inhibition of I-ca,I-L between these two RV myocyte types, and in myocytes displaying rebound stimulation neither ODQ nor 8-Bromo-cGMP (8-Br-cGMP) altered the effect. We conclude that NO production, activation of soluble guanylyl cyclase, or changes in intracellular cGMP levels are not obligatorily involved in muscarinic-mediated modulation of basal I-Ca,I-L in ferret RV myocytes.