The effect of calcium pump inhibitors on the response of intracellular calcium to caffeine in snail neurones.

ABSTRACT

We have measured intracellular free calcium ([Ca(2+)]i) using Fura-2 or Ca(2+)-sensitive microelectrodes in voltage-clamped neurones of the snail, Helix aspersa. Caffeine-induced transient increases in [Ca(2+)]i were normally followed by a brief fall of [Ca(2+)]i below its pre-caffeine level. We investigated the cause of this undershoot by raising [Ca(2+)]i; and by inhibiting the plasma membrane or endoplasmic reticulum Ca ATPases (PMCA or SERCA respectively). When the cell membrane potential was decreased from -60 to -25mV, steady-state [Ca(2+)]i increased. The caffeine-induced transients were smaller while the undershoots were larger than in control conditions. When the PMCA was inhibited by high pH the steady-state [Ca(2+)]i increased by 100-400nM. The caffeine-induced [Ca(2+)]i increase and the subsequent undershoot both became larger. Injection of orthovanadate, which inhibits the PMCA and increases [Ca(2+)]i, did not block either effect of caffeine. But when the SERCA was inhibited by cyclopiazonic acid the undershoot disappeared. The phosphodiesterase inhibitor IBMX did not influence the undershoot. These results suggest that the undershoot is generated by the Ca(2+)] ATPase of the stores rather than that of the plasma membrane. Since the undershoot increased as [Ca(2+)]i increased, we conclude that at higher levels of [Ca(2+)]i the stores refill more rapidly.