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 Ca2+-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 (IC50 = 16 μM) and sildenafil, blocked chemoattractant (cAMP)-induced Ca2+-influx as determined with a Ca2+-specific electrode. SCH 51866 (150 μM) 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 Iα (PKGIα) was not altered by SCH 51866 (150 μM). 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 (IC50 = 25 μM) 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 μM), known to inhibit cyclic-nucleotide-gated-channels as well as guanylyl-cyclases, reduced cAMP-induced Ca2+-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 Ca2+-influx by SCH 51866 in the wildtype was absent in the mutant. Our results show that SCH 51866 and sildenafil are antagonists of a Ca2+-permeable channel (CNGA3) and that both compete with cGMP for a regulatory site of Ca2+-influx in D. discoideum.