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

J Neurochem. 1996 Jan;66(1):105-13. doi: 10.1046/j.1471-4159.1996.66010105.x.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
  • Adrenal Medulla / cytology
  • Adrenal Medulla / drug effects*
  • Adrenal Medulla / metabolism
  • Alkaloids / pharmacology
  • Animals
  • Benzophenanthridines
  • Biological Transport / drug effects
  • Calcimycin / pharmacology
  • Calcium / metabolism*
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
  • Carbazoles / pharmacology
  • Catecholamines / metabolism*
  • Cattle
  • Cells, Cultured / drug effects
  • Enzyme Inhibitors / pharmacology*
  • Indole Alkaloids
  • Isoquinolines / pharmacology*
  • Phenanthridines / pharmacology
  • Piperazines / pharmacology*
  • Protein Kinase C / antagonists & inhibitors
  • Staurosporine
  • Substrate Specificity

Substances

  • Alkaloids
  • Benzophenanthridines
  • Carbazoles
  • Catecholamines
  • Enzyme Inhibitors
  • Indole Alkaloids
  • Isoquinolines
  • Phenanthridines
  • Piperazines
  • Calcimycin
  • KN 62
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
  • N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide
  • staurosporine aglycone
  • chelerythrine
  • Protein Kinase C
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Staurosporine
  • Calcium