INHIBITION OF L-TYPE CALCIUM-CHANNEL ACTIVITY BY THAPSIGARGIN AND 2,5-T-BUTYLHYDROQUINONE, BUT NOT BY CYCLOPIAZONIC ACID

Thapsigargin (TG), 2,5-t-butylhydroquinone (tBHQ) and cyclopiazonic ac id (CPA) all inhibit the initial Ca2+-response to thyrotropin-releasin g hormone (TRH) by depleting intracellular Ca2+ pools sensitive to ino sitol 1,3,5-trisphosphate (IP3). Treatment of GH(3) pituitary cells fo r 30 min with 5 nM TG, 500 nM tBHQ or 50 nM CPA completely eliminated the TRH-induced spike in intracellular free Ca2+ ([Ca2+](i)). Higher c oncentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the i ncrease in [Ca2+](i) or Ca-45(2+) influx stimulated by depolarization. TG and tBHQ blocked high-K+-stimulated Ca-45(2+) uptake, with IC50 va lues of 10 and 1 mu M respectively. Maximal inhibition of L-channel ac tivity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not a ffect spontaneous [Ca2+](i) oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 mu M TG and 10 mu M tBHQ blocked [Ca2+], oscillations completely. The effect o f drugs on calcium currents was measured directly by using the patch-c lamp technique. When added to the external bath, 10 mu M CPA caused a sustained increase in the calcium-channel current amplitude over 8 min , 10 mu M tBHQ caused a progressive inhibition, and 10 mu M TG caused an enhancement followed by a sustained block of the calcium current ov er 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low con centrations, TG depletes IP3-sensitive stores without inhibiting L-cha nnel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.