ATP ACTIVATES CATIONIC CURRENTS AND MODULATES THE CALCIUM CURRENT THROUGH GTP-BINDING PROTEIN IN RABBIT PORTAL-VEIN

1. Effects of adenosine 5'-triphosphate (ATP) on ionic currents of dispersed smooth muscle cells of the rabbit portal vein were investigated using the voltage-clamp procedure. 2. ATP (greater-than-or-equal-to 300-mu-M) produced transient and maintained inward currents. The former was inactivated within a few seconds, but the latter lasted more than several minutes. The transient but not the maintained current was blocked by pre-treatment with alpha,beta-methylene adenosine 5'-triphosphate (AMP-CPP). The amplitude of the latter was increased by ATP in a concentration-dependent manner. The following investigations were made on the ionic mechanism of the ATP-induced maintained inward current. 3. In 2.5 mM-Ca2+-containing tetraethylammonium chloride (TEA-Cl) solution (2.5 mM-Ca2+-TEA+ solution), the reversal potential for the ATP-induced inward current was close to the Cl- equilibrium potential, and in 140 mM-Na+ (nominally Ca2+-free or 0.3 mM-EGTA-containing) solution, the reversal potential was coincident with the Na+ equilibrium potential. 4. In 2.5 mM-Ca2+-TEA+ solution but not in 140 mM-Na+ solution and in physiological salt solution (PSS), niflumic acid (10-mu-M), a Cl- channel blocker, and Cl- -deficient perfusate in the pipette markedly inhibited the ATP-induced inward current. These results imply that in 2.5 mM-Ca2+-TEA+ solution the ATP-activated ion channel may admit Ca2+ which then accelerates the Ca2+-dependent Cl- current, but in 140 mM-Na+ solution and in PSS this channel may admit only Na+. 5. Intracellular perfusion of guanosine 5'-O-(3-thio triphosphate (GTP-gamma-S) did not provoke the current, but significantly increased the amplitude of the ATP-induced inward current in 2.5 mM-Ca2+-TEA+, 140 mM-Na+ and 2.5 mM-Ba2+-containing TEA+ (2.5 mM-Ba2+-TEA+) solutions. On the other hand, intracellular perfusion of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) reduced the amplitude of the ATP-induced inward current in the above solutions. 6. A low concentration of ATP (30-mu-M) transiently augmented the amplitude of the voltage-dependent Ca2+ current recorded in both 2.5 mM-Ca2+-TEA+ solution and PSS, but a high concentration of ATP (3 mM) consistently inhibited the voltage-dependent Ca2+ current in both solutions (4 mM-EGTA in the pipette). Such inhibition was partly prevented by application of 20 mM-EGTA in the pipette. In 140 mM-Na+ solution (with 0.3 mM-EGTA), ATP (3 mM) slightly inhibited by permeation of the Na+ (Na+ current) through the voltage-dependent Ca2+ channel. 7. In 140 mM-Na+ solution (with 0.3 mM-EGTA), 2.5 mM-Ba2+-TEA+ solution and PSS, neither GTP-gamma-S nor GDP-beta-S in the pipette modified the amplitude of the voltage-dependent inward current. However, GTP-gamma-S pronounced but GDP-beta-S prevented the inhibition of the voltage-dependent inward current induced by ATP. 8. Pertussis toxin (300 ng/ml) inhibited the ATP-induced inward current, and attenuated the ATP-induced inhibition of the voltage-dependent inward current in 2.5 mM-Ba2+-TEA+ solution and PSS. 9. These results indicate that ATP activates two different receptor-operated currents: AMP-CPP-sensitive (transient component) and -resistant (maintained component) currents. The latter appears to permeate only Na+ in PSS. The results also suggest that ATP transiently activates and then inhibits the voltage-dependent Ca2+ channel through mechanisms which involve functions of the GTP-binding protein and Ca2+-mediated inactivation process.