MODULATION OF L-TYPE CA2-VENTRICULAR MYOCYTES( CURRENT BY EXTRACELLULAR ATP IN FERRET ISOLATED RIGHT)

1. The effects of extracellular adenosine triphosphate (ATP) on the ba sal L-type Ca2+ current (I(Ca)) were investigated in ferret isolated r ight ventricular myocytes using the gigaohm seal voltage clamp in the whole-cell and cell-attached configurations. 2. Micromolar levels of e xtracellular ATP reversibly inhibited I(Ca) in a concentration-depende nt manner, without any significant changes in the voltage dependence o f either the peak I(Ca) I-V relationship or steady-state activation cu rve. 3. In contrast, micromolar levels of extracellular ATP did signif icantly alter the inactivation characteristics of I(Ca) Ten micromolar ATP: (i) increased the degree of steady-state inactivation Of I(Ca); (ii) altered the time constants of I(Ca) inactivation at 0 mV; and (ii i) decreased the time constant of I(Ca) recovery from inactivation at -70 mV. 4. The inhibitory effect of ATP on I(Ca) was not blocked by at ropine, a muscarinic cholinergic receptor antagonist or CPDPX (8-cyclo pentyl-3,4-dipropylxanthine), an A1 adenosine receptor antagonist. In contrast, the inhibitory effect of 10 muM ATP could be nearly complete ly antagonized by 100 muM suramin, a purinergic P2 receptor antagonist . 5. The potency order of ATP analogues in inhibiting I(Ca) was 2-meth yl-thio-ATP > ATP > alpha,beta-methylene-ATP, indicating involvement o f a P2Y-type ATP receptor. 6. Pretreatment of cells with pertussis tox in (PTX) did not prevent the ATP-induced decrease in I(Ca). However, ( i) ATP produced an irreversible decrease of I(Ca) in the presence of i ntracellular GTPgammaS, and (ii) the inhibitory effect was significant ly attenuated in the presence of intracellular GDPbetaS, indicating th e involvement of a PTX-insensitive G protein in the P2y receptor-coupl ing process. 7. Neither (i) replacing extracellular Ca2+ With 1 mM Ba2 +, nor (ii) intracellular perfusion of 10 mM BAPTA for at least 30 min attenuated the inhibitory effect of ATP on the current through Ca'' c hannels, suggesting that the inhibitory effect was not obligatorily de pendent upon influx of Ca2+ or changes in [Ca2+]i. 8. Ensemble-average current behaviour constructed from cell-attached patch recordings of single L-type Ca2+ channels (110 mM BaCl2) demonstrated that when 10 m uM ATP was added to the superfusate on the outside of the patch electr ode the inhibition of I(Ca) was still observed, providing evidence for the involvement of intracellular diffusible second messenger(s). 9. B ath application of the protein kinase C (PKC) activator PMA (4beta-pho rbol-12-myristate-13-acetate) did not decrease I(Ca), and the PKC inhi bitor staurosporine did not block the ATP-induced decrease in I(Ca), i ndicating that the inhibition was not mediated by activation of PKC. 1 0. Internal perfusion of 100 muM IP3 + 7.7 muM IP4 did not alter the i nhibitory effect of ATP on I(Ca), indicating that inositol phosphates were not involved. 11. Neither (i) internal perfusion of a specific pe ptide inhibitor of protein kinase A (PKI) for 30 min, nor (ii) applica tion of an intracellular 'cAMP clamp' (1 mM cAMP + 10 muM IBMX) change d the effect of ATP on I(Ca), indicating that the cAMP-dependent prote in kinase A (PKA) pathway was not involved. 12. In ferret right ventri cular myocytes inhibition of I(Ca) by ATP occurs independently of aden osine A1 receptors. ATP inhibits I(Ca) without significantly affecting the voltage dependence of activation, but it does modify the inactiva tion gating properties. Inhibition occurs by extracellular ATP binding to a P2y receptor and subsequent activation of a PTX-insensitive G pr otein. Our results indicate that some of the conventional second messe nger systems ([Ca2+]i, PKC, inositol phosphates. cAMP-PKA) are not ess ential for the inhibitory effect of extracellular ATP on I(Ca).