Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment.
Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.
Pruss
R M
RM
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Stauderman
K A
KA
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004744:Enkephalin, Methionine / Q000378:metabolism; D004745:Enkephalins / Q000096:biosynthesis*; D007473:Ion Channels / Q000378:metabolism*; D007811:Lanthanum / Q000494:pharmacology; D009539:Nicotinic Acids / Q000494:pharmacology; D010069:Oxadiazoles; D011189:Potassium Chloride / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004745:Enkephalins; D007473:Ion Channels; D009539:Nicotinic Acids; D010069:Oxadiazoles; D012333:RNA, Messenger; C028521:lanthanum chloride; C049834:PN 202-791; D004744:Enkephalin, Methionine; D011189:Potassium Chloride; D007811:Lanthanum; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium