MODULATION OF CARDIAC L-TYPE CA2-GAMMA-S IN RESPONSE TO ISOPRENALINE,FORSKOLIN AND PHOTORELEASED NUCLEOTIDES( CHANNELS BY GTP)

1 Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosp hate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2 + currents (I-Ca) by isoprenaline and forskolin and also by GTP gamma S and cyclic AMP released intracellularly by flash-photolysis of their caged derivatives. 2 GTP gamma S prevented enhancement of I-Ca by iso prenaline with an IC50 of approximate to 10 mu M and considerably redu ced the ability of forskolin to increase I-Ca. In addition GTP gamma S also reduced the time-to-peak response for potentiation of I-Ca by fo rskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, R(p)-adenosine-3'-5'-mono-thionophosp hate (R(p)-cAMPS). 3 Photoreleased GTP gamma S (PR-GTP gamma S; approx imate to 23 mu M) generally induced a biphasic increase in I-Ca This r esponse was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximate to 1 mu M. 4 Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 mu M GTP gamma S on isoprenaline-induced stimulation of I-Ca. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5 Photoreleased cycli c AMP (approximate to 5 mu M; PR-cyclic AMP) increased peak I-Ca. This effect was inhibited by dialysis of cells with R(p)-cAMPS and by stim ulation of I-Ca by the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the ti me-to-peak for PR-cyclic AMP mediated activation of I-Ca but did not a ffect the magnitude of the response. 6 It is concluded that chronicall y applied GTP gamma S can (i) inhibit activation of I-Ca by isoprenali ne by interacting with a PTX-sensitive guanosine nucleotide binding (G -) protein located upstream of adenylate cyclase (possibly G(i)) and ( ii) accelerate the response to cyclic AMP dependent phosphorylation po ssibly by interacting with a G-protein coupled directly to the channel . 7 In view of this diverse range of effects, care should be taken whe n using GTP gamma S to characterize G-protein-mediated events, since t he resulting physiological response may be due to activation of severa l G-protein containing pathways.