Dual actions of phorbol esters on cytosolic free Ca2+ concentrations and reconstitution with ionomycin of acute thyrotropin-releasing hormone responses.
We have used phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), to study the actions of protein kinase C (a TPA receptor) on cytosolic free Ca2+ concentrations [( Ca2+]i) and hormone secretion in rat pituitary cells (GH cells), and to elucidate the role of diacylglycerol (a protein kinase C activator) in thyrotropin-releasing hormone (TRH) action. TPA had a dual action on [Ca2+]i, inducing a stimulatory phase from 300 (basal) to 420 nM, which was interrupted in 30-60 s by an inhibitory phase which transiently lowered [Ca2+]i to 240 nM and rose in 3-10 min to yield the stimulatory phase. TPA-mediated changes in [Ca2+]i were induced by other phorbol esters and mezerein but not by phorbol or activators of kinases different from protein kinase C. Both phases of TPA action on [Ca2+]i were abolished by 5-min pretreatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (1.33 mM) or Ca2+ channel antagonists (verapamil or nifedipine). TPA also enhanced the rate of sustained hormone secretion without inducing a burst of hormone release (unlike TRH). Also, stimulation of secretion by TPA was not inhibited by Ca2+ channel antagonists and was resistant (10%) to EGTA. Simultaneous addition of TPA with the ionophore ionomycin (100 nM) reconstituted a TRH-like spike, nadir and plateau of [Ca2+]i. Ionomycin generated the spike in [Ca2+]i by releasing TRH-sensitive Ca2+ stores, while TPA induced the nadir (inhibitory phase), and a nifedipine/verapamil-sensitive plateau of [Ca2+]i (stimulatory phase). Concurrent (but not separate) addition of ionomycin and TPA also reconstituted a TRH-like burst of hormone secretion. These and previous results indicate that activation of protein kinase C by TPA or diacylglycerol (which is elevated by TRH) and a simultaneous spike in [Ca2+]i are required for burst secretion. Diacylglycerol may also mediate the TRH-induced nadir and plateau of [Ca2+]i; the latter process contributes to Ca2+-dependent stimulation of steady secretion by TRH.
Albert
P R
PR
Tashjian
A H
AH
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000032:analysis*; D002352:Carrier Proteins; D002478:Cells, Cultured; D003600:Cytosol / Q000032:analysis*; D004224:Diterpenes; D004533:Egtazic Acid / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D015759:Ionomycin; D010704:Phorbols / Q000494:pharmacology*; D010902:Pituitary Gland / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C; D011494:Protein Kinases / Q000502:physiology; D051381:Rats; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000187:drug effects; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004224:Diterpenes; D004987:Ethers; D007328:Insulin; D010704:Phorbols; D011955:Receptors, Drug; D011971:Receptors, Immunologic; D013729:Terpenes; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; C011309:mezerein; D004533:Egtazic Acid; D015759:Ionomycin; D013973:Thyrotropin-Releasing Hormone; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D002118:Calcium