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 acid (CPA) all inhibit the initial Ca(2+)-response to thyrotropin-releasing hormone (TRH) by depleting intracellular Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3). Treatment of GH3 pituitary cells for 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 concentrations of TG and tBHQ, but not CPA, were also found to inhibit strongly the activity of L-type calcium channels, as measured by the increase in [Ca2+]i or 45Ca2+ influx stimulated by depolarization. TG and tBHQ blocked high-K(+)-stimulated 45Ca2+ uptake, with IC50 values of 10 and 1 microM respectively. Maximal inhibition of L-channel activity was achieved 15-30 min after drug addition. Inhibition by tBHQ was reversible, whereas inhibition by TG was not. TG and CPA did not affect spontaneous [Ca2+]i oscillations when tested at concentrations adequate to deplete the IP3-sensitive Ca2+ pool. However, 20 microM TG and 10 microM tBHQ blocked [Ca2+]i oscillations completely. The effect of drugs on calcium currents was measured directly by using the patch-clamp technique. When added to the external bath, 10 microM CPA caused a sustained increase in the calcium-channel current amplitude over 8 min, 10 microM tBHQ caused a progressive inhibition, and 10 microM TG caused an enhancement followed by a sustained block of the calcium current over 8 min. In summary, CPA depletes IP3-sensitive Ca2+ stores and does not inhibit voltage-operated calcium channels. At sufficiently low concentrations, TG depletes IP3-sensitive stores without inhibiting L-channel activity, but, for tBHQ, inhibition of calcium channels occurs at concentrations close to those needed to block agonist mobilization of intracellular Ca2+.
Nelson
E J
EJ
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, NY 14642.
Li
C C
CC
Bangalore
R
R
Benson
T
T
Kass
R S
RS
Hinkle
P M
PM
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D006873:Hydroquinones / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D006873:Hydroquinones; D007211:Indoles; D013729:Terpenes; C019359:2,5-di-tert-butylhydroquinone; D019284:Thapsigargin; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D011188:Potassium; D002118:Calcium; C000543:cyclopiazonic acid