A fluorescence-activated cell sorter analysis of the relationship between protein kinase G and endothelial nitric oxide synthase.

The Anatomical Record : Advances in Integrative Anatomy and Evolutionary Biology
Theresa A John, J Usha Raj

Abstract

The process of regulation of NOS after production of nitric oxide is not yet delineated. Protein kinase G may exert a feedback regulation of this enzyme. We used diaminofluorescein assays to detect changes in basal nitric oxide production caused by modulators of protein kinase G activity in freshly isolated ovine lung microvascular endothelial cells. We also used fluorescence activated cell sorter analysis (FACS) to determine molecular and phosphorylation changes caused by PKG activation with 8-Br-cGMP. The PKG activator, 8-Br-cGMP (100 μM) produced a shift in the basal NO production curve downward. The inhibition began within 5 min and was sustained over 4.5 hr. The two protein kinase G inhibitors 100 μM Rp-8-Br-PET-cGMPS and 50 nM guanosine 3'-5'-cyclic monophosphoro thionate-8-Br-Rp isomer Na salt and the cGMP inhibitor 4 μM Rp-8-pCPT-cGMPS all enhanced NO production as seen by the upward shift in the basal NO curve. Conversely, the PKG activator drug, 100 μM guanosine-3'-5'-cyclic monophosphate-β-phenyl-1NF-ethano-8-bromo sodium salt decreased NO production causing a downward shift in the basal curve. FACS analysis revealed that 5 μM 8-Br-cGMP in <5 min caused an increase in N-terminal labeling of NOS and a decrease in both...Continue Reading

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Citations

May 21, 2013·Journal of Molecular and Cellular Cardiology·George VaniotisBruce G Allen

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