Pharmacology of a Ca(2+)-influx pathway activated by emptying the intracellular Ca2+ stores in HL-60 cells: evidence that a cytochrome P-450 is not involved.
In HL-60 cells, inhibition of the endoplasmic-reticular Ca2+ pump by thapsigargin leads to the emptying of this intracellular Ca2+ store and a subsequent activation of plasma-membrane Ca2+ influx through a non-voltage-dependent pathway. The elevated intracellular free Ca2+ concentration ([Ca2+]i) produced and maintained by this Ca2+ inflow was used to examine the potency of various compounds to inhibit this influx mechanism. As expected, specific blockers of known Ca2+ channels, such as nifedipine, omega-conotoxin GVIA and ryanodine were without effect. The less selective inhibitors La3+, SKF-96365 and L-651,582, which are thought to inhibit both voltage-dependent and voltage-independent Ca2+ channels, decreased [Ca2+]i back to resting levels, with pIC50 values of 5.2, 5.9 and 6.2 respectively. It has been proposed that a cytochrome P-450 is involved in activating Ca(2+)-influx pathways in thymocytes, neutrophils and platelets. Consistent with this idea, the imidazole cytochrome P-450 inhibitors miconazole, econazole, clotrimazole and ketoconazole inhibited the thapsigargin-elevated [Ca2+]i with pIC50 values of 7.1, 7.1, 7.1 and 5.8 respectively. The high affinity of imidazoles for cytochromes P-450 is due to co-ordinate binding to the haem. This interaction is greatly decreased in 2-substituted imidazoles. We examined whether the inhibition of Ca2+ influx was due to an interaction of the inhibitor imidazole nitrogen with the haem iron of the putative cytochrome P-450 by comparing the activity of two compounds, identical except that one was methylated at the imidazole 2-position. They were found to block thapsigargin-activated Ca2+ influx with equal potency. These results strongly suggest that a cytochrome P-450 is not involved in the activation of the Ca2+ influx produced by emptying the intracellular Ca2+ stores.
Koch
B D
BD
Institute of Pharmacology, Syntex Discovery Research, Palo Alto, CA 94304.
Faurot
G F
GF
Kopanitsa
M V
MV
Swinney
D C
DC
D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D065607:Cytochrome P-450 Enzyme Inhibitors; D007093:Imidazoles; D013729:Terpenes; D019284:Thapsigargin; D003577:Cytochrome P-450 Enzyme System; D000252:Calcium-Transporting ATPases; D002118:Calcium