The protein phosphatase inhibitor calyculin-A affects catecholamine secretion and granular distribution in cultured adrenomedullary chromaffin cells.
Calyculin-A, a potent inhibitor of types 1 and 2A protein phosphatases, increases basal catecholamine secretion in cultured chromaffin cells with a maximum effect observed at 100 nM. This effect was increased by forskolin and the calmodulin antagonist W7, but was modified neither by phorbol esters nor the protein kinase inhibitor, H7. The effect of the toxin, calyculin-A, on basal secretion was completely prevented by the protein kinase inhibitor K252a. In digitonin-permeabilized cells calyculin-A induced an increase in basal release, but, in contrast, it partially reduced calcium-induced secretion. Analysis of total proteins revealed that calyculin-A treatment of the cells increased the level of phosphorylation of different protein bands. Examination of the Triton X-100-insoluble fraction revealed a clear increase in the phosphorylation level of various proteins, including vimentin. Calyculin-A provoked a rapid morphological change in chromaffin cells in the same range of concentration (50-300 nM). Cells became rounder and were partially detached from the substratum forming clusters, this effect was also blocked by K252a. Transmission electron microscopy of calyculin-A-treated cells showed an increase in the proportion of chromaffin granules located closer to the membrane. These results suggest that calyculin-A induces changes both in the catecholamine secretory response and in the cytoskeletal elements of chromaffin cells by protein phosphorylation.
Departamento de Neuroquímica, Facultad de Medicina, Universidad de Alicante, Spain.
L M
LM
Gutierrez
J L
JL
Quintanar
J
J
Rueda
S
S
Viniegra
J A
JA
Reig
D000313:Adrenal Medulla / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D001481:Basal Metabolism; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000187:drug effects*; D004072:Digitonin; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008387:Marine Toxins; D010080:Oxazoles / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000187:drug effects / Q000378:metabolism; D012636:Secretory Rate / Q000187:drug effects
D002395:Catecholamines; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D011506:Proteins; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D004072:Digitonin