Protein kinase G inhibits basal and stimulated nitric oxide synthase activity in neonatal ovine lung microvascular endothelial cells.
Protein kinase G (PKG) is abundant in neonatal ovine lung microvascular endothelial cells (LMVECs) but its various functions are not known. To test the hypothesis that PKG plays a role in feedback regulation of nitric oxide (NO) synthesis, we studied the effects of modulators of PKG signaling on real time NO release from LMVECs microcultures in 96-well clusters. We used 0.5 - 2.43 microM DAF FM and DAF-FM diacetate to measure amount of NO present in the cells and in the cell bathing medium. We found a dose-response relationship between 8-Br-cGMP (0.02 - 2 microM), a stimulator of PKG activity, and inhibition of basal NO production. The time-course of the effect of 2 microM 8-Br-cGMP on NO production exhibited a parallel shift downwards in the presence of PKG receptor inhibitor, 100 ng/ml DT-2, indicating that 8-Br-cGMP acts through PKG to inhibit NOS. PKG also decreased stimulated NO production: acetylcholine produced raw fluorescence of 59999 +/- 702 and in the presence of 1 mM 8-Br-cGMP the value was 20645 +/- 292 (p < 0.0001) while carbachol produced raw fluorescence of 60600 +/- 890 and in the presence of 1 mM 8-Br-cGMP was 30442 +/- 2000 (p < 0.01). The PKG inhibitor 125 nM guanosine 3'-5'-cyclic-monophosphorothionate-8-Br-Rp isomer increased basal NO production (p < 0.01). NO synthase inhibitor, L-NNA reversed this effect (p < 0.05) as well as that of another PKG inhibitor 25 microM Rp-8-Br-PET-cGMPS but enhanced the effect of 25 microM 8-Br-cGMP. Both basal and stimulated NO production is regulated by the downstream activation of PKG by NO-induced 8-Br-cGMP production in endothelial cells.
Department of Pharmacology, Lagos State University College of Medicine, Ikeja, Lagos, Nigeria. theresaadebola@yahoo.com
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D000818:Animals; D006152:Cyclic GMP / Q000031:analogs & derivatives; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism; D008168:Lung / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012756:Sheep
C104224:8-bromo-beta-phenylethenoguanosine 3',5'-cyclic monophosphate; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP