| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Infusion of the G protein activator, AIF complex, into the chromaffin cell results in transient generation of a nonselective cation current (Ins). The maximum amplitude of Ins and the half decay time (T_<1/2>) both incerased as the concentration of intracellular Mg^<2+> ([Mg^<2+>]) decreased from 1 mM to 12muM.The addition of alkaline phosphatase to AIF-ocntaining pipette solution mimicked the effect of an increase in [Mg^<2+>], whereas the addition of vanadate, a nonselective phosphatase inhibitor, reproduced the effect of a decrease in [Mg^<2+>]. Application of muscarine induced a transient generation of INs in the presence of AIF complex but in the absence, it resulted in a sustained production of INs. These results suggest that intracellular application of AIF complex induced not only activation, but also deactivation of NS channels and that the deactivation is mediated by Mg^<2+> -dependent phosphatase. To obtain further evidence for involvement of phosphorylation in channel activ
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ation, ATP in the AIF solution was removed or was replaced with AMP-PNP.Unexpectedly, maximum amplitudes of AIF-induced INs or T_<1/2> for deactivation was not significantly altered in either case. When endogenous ATP was depleted with cyanide, however, the generation of INs entirely depended on the concentration of ATP in the AIF solution. The non-hydrolysable ATP analogs AMP-PNP and ATPgammaS,GTP,ITP,and UTP do not have this supporting action of ATP.The results indicate that phosphorylation of the channel or regulator participates in channel activation. Thus, we investigated Ca^<2+>-dependence of current generation to identify the protein kinase involved. When Ca^<2+> in perfusate was replaced with Mg^<2+>, muscarine-induced INS decreased to half in 36% of cells tested. This incidence of INS diminution was increased to 73% with the addition of cyclopiazonic acid to the Ca^<2+>-free solution. On the other hand, addition of A-23187 in perfusate enhanced muscarine-induced INS by 30%. These results suggest that intracellular Ca^<2+> has a faciltating action, but is not prerequisite for INS generation. Less
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