Different inhibitors of the Ca2+/calmodulin‐stimulated phosphodiesterase 1 family have been described and used for the examination of phosphodiesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA. In addition, the measured Km values (cAMP, 1.7 µm; cGMP, 1.3 µm) are characteristic of phosphodiesterase 1C. We demonstrate that treatment with the Ca2+ ionophore ionomycin increases intracellular Ca2+ in a concentration‐dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca2+ concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca2+‐activated phosphodiesterase 1C. The reduction of forskolin‐stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration‐dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8‐methoxymethyl‐3‐isobutyl‐1‐methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering‐Plough (named compounds 31 and 30). We demonstrate that up to 10 µm 8‐methoxymethyl‐3‐isobutyl‐1‐methylxanthine and vinpocetine had no effect on the reduction of forskolin‐stimulated cAMP levels by ionomycin, whereas the more selective and up to 10 000 times more potent phosphodiesterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited the ionomycin‐induced decline of forskolin‐induced cAMP at nanomolar concentrations. Thus, our data indicate that SCH51866 and compounds 31 and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in contrast to the weakly selective and low‐potency phosphodiesterase inhibitors 8‐methoxymethyl‐3‐isobutyl‐1‐methylxanthine and vinpocetine. A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase 1 inhibitors. 8‐Methoxymethyl‐3‐isobutyl‐1‐methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesterase 1‐mediated functions.

01 Mar 2007·British Journal of OphthalmologyQ2 · MEDICINE

Selective blockade of phosphodiesterase types 2, 5 and 9 results in cyclic 3'5' guanosine monophosphate accumulation in retinal pigment epithelium cells

Q2 · MEDICINE

Article

Author: Markerink-van Ittersum, M ; Kijlstra, A ; La Heij, E C ; Hendrikse, F ; de Vente, J ; Diederen, R M H

AIMTo investigate which phosphodiesterase (PDE) is involved in regulating cyclic 3'5' guanosine monophosphate breakdown in retinal pigment epithelium (RPE) cells.METHODScGMP content in the cultured RPE cells (D407 cell line) was evaluated by immunocytochemistry in the presence of non-selective or isoform-selective PDE inhibitors in combination with the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). mRNA expression of PDE2, PDE5 and PDE9 was studied in cultured human RPE cells and rat RPE cell layers using non-radioactive in situ hybridisation.RESULTSIn the absence of PDE inhibitors, cGMP levels in cultured RPE cells are very low. cGMP accumulation was readily detected in cultured human RPE cells after incubation with Bay60-7550 as a selective PDE2 inhibitor, sildenafil as a selective PDE5 inhibitor or Sch51866 as a selective PDE9 inhibitor. In the presence of PDE inhibition, cGMP content increased markedly after stimulation of the particulate guanylyl cyclase. mRNA of PDE2,PDE5 and PDE9 was detected in all cultured human RPE cells and also in rat RPE cell layers.CONCLUSIONSPDE2, PDE5 and PDE9 have a role in cGMP metabolism in RPE cells.

01 Apr 2005·European Journal of PharmacologyQ3 · MEDICINE

cGMP-phosphodiesterase antagonists inhibit Ca2+-influx in Dictyostelium discoideum and bovine cyclic-nucleotide-gated-channel

Q3 · MEDICINE

Article

Author: Malchow, Dieter ; Kaneko, Hiroshi ; Lusche, Daniel F.

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.

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