Effects of protein kinase inhibitors on morphology and function of cultured bovine adrenal chromaffin cells: KN-62 inhibits secretory function by blocking stimulated Ca2+ entry.
In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and 45Ca2+ uptake. Treatment of chromaffin cells with 10 microM K-252a, 50 microM H-7, or 50 microM H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca2+/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC50 value of 0.32 microM), but had no effect on cell morphology. The reduction of secretory function induced by 1 microM KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated 45Ca2+ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated 45Ca2+ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca2+ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.
Division of Pharmacology, Ohio State University, College of Pharmacy, Columbus 43210, USA.
J A
JA
Maurer
B W
BW
Wenger
D B
DB
McKay
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives; D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D053119:Benzophenanthridines; D001692:Biological Transport / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002227:Carbazoles / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D026121:Indole Alkaloids; D007546:Isoquinolines / Q000494:pharmacology*; D010617:Phenanthridines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D019311:Staurosporine; D013379:Substrate Specificity
D000470:Alkaloids; D053119:Benzophenanthridines; D002227:Carbazoles; D002395:Catecholamines; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D007546:Isoquinolines; D010617:Phenanthridines; D010879:Piperazines; D000001:Calcimycin; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; C049985:staurosporine aglycone; C016299:chelerythrine; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019311:Staurosporine; D002118:Calcium