Modulation of cardiac L-type Ca2+ channels by GTP gamma S in response to isoprenaline, forskolin and photoreleased nucleotides.
1. Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosphate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2+ currents (ICa) 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 ICa by isoprenaline with an IC50 of approximately 10 microM and considerably reduced the ability of forskolin to increase ICa. In addition GTP gamma S also reduced the time-to-peak response for potentiation of ICa by forskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, Rp-adenosine-3'-5'-mono-thionophosphate (Rp-cAMPS). 3. Photoreleased GTP gamma S (PR-GTP gamma S; approximately 23 microM) generally induced a biphasic increase in ICa. This response was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximately 1 microM. 4. Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 microM GTP gamma S on isoprenaline-induced stimulation of ICa. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5. Photoreleased cyclic AMP (approximately 5 microM; PR-cyclic AMP) increased peak ICa. This effect was inhibited by dialysis of cells with Rp-cAMPS and by stimulation of ICa by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the time-to-peak for PR-cyclic AMP mediated activation of ICa but did not affect the magnitude of the response. 6. It is concluded that chronically applied GTP gamma S can (i) inhibit activation of ICa by isoprenaline by interacting with a PTX-sensitive guanosine nucleotide binding (G-) protein located upstream of adenylate cyclase (possibly Gi) and (ii) accelerate the response to cyclic AMP dependent phosphorylation possibly by interacting with a G-protein coupled directly to the channel. 7. In view of this diverse range of effects, care should be taken when using GTP gamma S to characterize G-protein-mediated events, since the resulting physiological response may be due to activation of several G-protein containing pathways.
University Laboratory of Physiology, Oxford.
R Z
RZ
Kozlowski
L J
LJ
Goodstadt
V W
VW
Twist
T
T
Powell
D000818:Animals; D015220:Calcium Channels / Q000187:drug effects*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004594:Electrophysiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology*; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects*; D007545:Isoproterenol / Q000494:pharmacology*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D010782:Photolysis; D047428:Protein Kinase Inhibitors; D013873:Thionucleotides / Q000494:pharmacology
D015220:Calcium Channels; D047428:Protein Kinase Inhibitors; D013873:Thionucleotides; D005576:Colforsin; C016957:adenosine-3',5'-cyclic phosphorothioate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D007545:Isoproterenol