cGMP-phosphodiesterase antagonists inhibit Ca2+-influx in Dictyostelium discoideum and bovine cyclic-nucleotide-gated-channel.
We used antagonists of cGMP-phosphodiesterases to examine the role of cGMP for light-scattering oscillations and cAMP-induced Ca(2+)-influx in Dictyostelium discoideum, however, SCH 51866 (cis-5,6a,7,8,9,9a-hexahydro-2-[4-(trifluoromethyl)phenylmethyl]-5-methyl-cyclopent[4,5]imidazo[2,1-b]purin-4(3H)-one) and sildenafil citrate (1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1-H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazine citrate) were poor inhibitors of cGMP-hydrolysis. Instead, SCH 51866 (IC(50) = 16 microM) and sildenafil, blocked chemoattractant (cAMP)-induced Ca(2+)-influx as determined with a Ca(2+)-specific electrode. SCH 51866 (150 microM) affected neither spontaneous cGMP transients during light-scattering-oscillations nor cAMP-mediated K(+)-efflux. SCH 51866 and sildenafil are competitive inhibitors of cGMP phosphodiesterases. However, the activity of cGMP-dependent protein kinase Ialpha (PKGIalpha) was not altered by SCH 51866 (150 microM). By contrast, patch-clamp measurements of bovine cone cGMP-gated-channels (cyclic-nucleotide-gated-channel, CNGA3), stably expressed in human embryonic kidney cells, HEK 293 cells, revealed reversible, competitive and dose-dependent inhibition of sodium currents by SCH 51866 (IC(50) = 25 microM) and sildenafil, but not by another inhibitor of cGMP-phosphodiesterases, UK 114,542. The possibility that D. discoideum cells also express a cGMP-regulated channel is supported by our finding that LY 83583 (6-(phenylamino)-5,8-quinolinedione) (35 microM), known to inhibit cyclic-nucleotide-gated-channels as well as guanylyl-cyclases, reduced cAMP-induced Ca(2+)-influx in D. discoideum, but did not affect cAMP-induced cGMP accumulation. Utilizing a PDED null strain that exhibits a prolonged and elevated cGMP transient following receptor activation, we found that the inhibition of Ca(2+)-influx by SCH 51866 in the wildtype was absent in the mutant. Our results show that SCH 51866 and sildenafil are antagonists of a Ca(2+)-permeable channel (CNGA3) and that both compete with cGMP for a regulatory site of Ca(2+)-influx in D. discoideum.
Lusche
Daniel F
DF
Department of Biology, University of Konstanz, Germany. Daniel.Lusche@web.de
Kaneko
Hiroshi
H
Malchow
Dieter
D
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D018408:Patch-Clamp Techniques; D010879:Piperazines / Q000494:pharmacology; D011687:Purines; D000068677:Sildenafil Citrate; D013450:Sulfones
D007093:Imidazoles; D007473:Ion Channels; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D000068677:Sildenafil Citrate; D000242:Cyclic AMP; D017869:Cyclic GMP-Dependent Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C096501:SCH 51866; D006152:Cyclic GMP; D002118:Calcium