Laburpena
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 μM 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 μM), a stimulator of PKG activity, and inhibition of basal NO production. The time-course of the effect of 2 μM 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 59999702 and in the presence of 1 mM 8-BrcGMP the value was 20645292 (p<0.0001) while carbachol produced raw fluorescence of 60600890 and in the presence of 1 mM 8-Br-cGMP was 304422000 (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 μM Rp-8-Br-PET-cGMPS but enhanced the effect of 25 μM 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.
Keywords: Protein kinase G, nitric oxide, nitric oxide synthase, cGMP, microvascular endothelial cells
Résumé
La protéine kinase G (PKG) est abondante dans les cellules endothéliales des poumons microvasculaires chez les ovins nouveaux nés. Mais ses multiples fonctions ne sont pas connues. Pour tester les hypothèses selon lesquels le PKG joue un rôle dans la régulation feedback de la synthèse de l’oxyde nitrique (NO), nous avons étudié les effets des modulateurs de PKG signalant au temps exact ou NO est libéré des cellules micro cultures LMVECs dans 96-micro plates. Nous avons utilise 0.5 – 2.43 μM DAF FM et DAF-FM di- acétate pour mesurer le nombre de NO présent dans les cellules et le milieu de culture. Nous avons trouve une dose de réponse en relation entre 8-Br-cGMP (0.02 - 2 μM), l’activité du stimulateur de PKG et l’inhibition de la production de NO de base. La durée de l’effet de 2μM 8-BrcGMP sur la production de NO a démontré un recul parallèle en présence de l’inhibiteur récepteur PKG. 100ng/ml DT-2 indiquant que 8-Br-CGMP agit a travers le PKG pour inhiber le NOS. Le PKG réduit également la production de NO stimule : l’acétylcholine a produit une fluorescence brute de 59999_+702 et en présence de 1mM 8-Br-cGMP la valeur était 20645 292 (P<0.0001) alors que le carbachol a produit une fluorescence brute de 60600 890 et en présence de 1mM 8-Br-cGMP était 304422000 (p<0.0001). L’’inhibiteur du PKG 125 nM guanosine 3’-5’-monophosphorothionate cyclique-8-Br-Rp isomère augmentait la production de base de NO (P<0.01) l’inhibiteur de la synthase NO, L-NNA ont renverse cet effet (P<0.05) aussi bien que d’autre inhibiteur PKG 25 μM Rp-8-Br-PET-cGMPS mais ont amélioré l’effet du 25 μM 8-Br-cGMP. La production de NO de base et celle stimule est régulé par l’activation de la libération de PKG par le NO induit, la production de 8-Br-cGMP dans les cellules endothéliales.
Correspondence: Dr. T.A. John, Department of Pharmacology, Lagos State University College of Medicine, PMB 21266, Ikeja, Lagos, Nigeria. E-mail: theresaadebola@yahoo.com
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