Abstract
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.
Highlights
Dual Actionsof Phorbol Esterson Cytosolic Free Ca2’ Concentrations and Reconstitution withIonomycin of Acute Thyrotropin-releasing Hormone Responses*
N,N,N’,N’-tetraacetic acid (EGTA) (1.33mM) or Ca” secretion, we have studied the actions of phorbol esters on channelantagonists(verapamil or nifedipine)
The plateau phaseof [Ca2+Iriesults from another action of TRH which closes voltage-dependent K+ channels, depolarizing the cell, activating the voltage-dependent Ca2+channel (18, 40). This pathway accounts for about half of the TRH-mediated plateau of elevated [Ca2+Ii,the other half appears to be due to a TRH-induced, nifedipine/ verapamil-resistant Caz+influx (18,46). It remained unclear what mediator triggered the TRHinduced nadirin [Ca2++I(iwhich attains sub-basal levels in some preparations) and theclosure of the voltage-dependent K+channels, for a spike in [Ca2+];pseer(e.g. after ionomycin) induces neither (18).We report here that when GH cells are treated simultaneously with phorbol ester and ionomycin, the spike, nadir and a nifedipine-sensitive plateau in [Ca2+Ii are reconstituted with time course and magnitude indistinguishable from TRH-induced changes in [Ca2+Ii.activation of protein kinase C by endogenous 1,2-diacylglycerolappears to mediate both the TRH-induced nadir in [Ca"]; and the nifedipine/verapamil-sensitiveinflux of [Ca2+Ii(possibly via closure of voltage-dependent K+ channels)
Summary
Dual Actionsof Phorbol Esterson Cytosolic Free Ca2’ Concentrations and Reconstitution withIonomycin of Acute Thyrotropin-releasing Hormone Responses*. We have used phorbol esters, such as 12-O-tetra- phospholipid-dependent kinase, protein kinase C (3-6).Acdecanoyl phorbol 13-acetate (TPA), to study the ac- tivation of protein kinase C mediates the phorbol estertions of protein kinaseC (a TPA receptor)on cytosolic induced phosphorylation of numerous proteins (3, 7-16). Free Caa+concentrations ([Ca2+Ii)and hormone secre- These phosphorylation events may mediate some or allof the tion in rat pituitary cells (GH cells), and to elucidate biological actions of phorbol esters including their ability to htitpsmironhnuihaeeepdyant[ztsrhiCateeeoelyrdalydledre2fuoilr+inotoaotnh]frlwmbieo3awdeu0pcire3sat-tie6itnrc0oinde0y-m0onrl[ietguCn(slllbbedayaboayaauctyns+osecapir]reanlh[yiotd)nCogolptrbai(hh2onbazyao4ho+p4sr0ilemoIr2bio.nto0,iohitMTtrnenoenPdiaerranAyucn~p(ctd-TpihkimwvnhRr,oianogaehrHtassbdiocaeeso)irahealiswstncetewohCtid3sifmiaot-cc1esknuhhar0i.licasnanttTnratitamoagvePsnnrereaiAdyn-ss-tor)sceeaa1tefcsc8iftltuIm)eelintsvcrweusi(toltel1yyuao9(,trhn2e,a(a218nscvs0,t,2deeeu)22lc.edl3)urnie)gTse.htersihIoaadoonenwfnnsGcdtetG(hcehH3ee,Hhp,l’balrm7scocvu-ep9eedetl,lcoullsshssct1t,,shati3eimnpeaos)ihn.suscaomsllsoraposbfstenoeoPocaclfiPRlifaehiRLstcsoettLrrred(eamr2caisp4enon)rphnd.ootaeIotfvneGsreirsernHacefltiorcteksrteptleipniienctohtuarnooesisrtret(taibu1norodC7yonl,-. Different from protein kinase C. different from protein kinase C Both phases of TPA action on [Ca2+Iwr ere abolished by 6-min pretreatment ies (17, 18) we related changes in hormone secretion to changes in [Ca”]i induced by TRH. To investigate further weithylene glycol bis(8-aminoethyl ether)- the mechanism of protein kinase C activation of hormone
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