Inhibitors caveolin-1 and protein kinase G show differential subcellular colocalization with Nitric oxide synthase

Abstract

Background: Nitric oxide synthase (NOS) is negatively regulated by protein-protein interactions with caveolin-1 before extracellular activating signals release it for nitric oxide (NO) production. Smooth muscle protein kinase G (PKG) is a down-stream effector of NO signaling for relaxation of vascular smooth muscle cells (SMC). The PKG is also found in endothelial cells and it inhibits activated NOS within endothelial cells.

Methods: We used confocal fluorescence microscopy to colocalize the inhibitors caveolin-1 and PKG with NOS in freshly isolated neonatal lamb endothelial cells in order to corroborate the speculation of their differential effects on NOS. The roles of caveolin-1 and PKG as regulators of NOS were investigated by examining their respective subcellular sites of colocalization with NOS using qualitative fluorescence immunohistochemistry and confocal microscopy.

Results: Caveolin-1 was colocalized with NOS in the plasma membrane and Golgi. The PKG1-beta isoform was colocalized with serine116 phosphorylated NOS in the cytosol and in vesicular structures seen in the endoplasmic reticulum and in the nuclear region.

Conclusion: We conclude that unlike caveolin-1, a known pre-activation inhibitor of nascent NOS, PKG may be a post-activation inhibitor of NOS, possibly important for the recycling of the spent enzyme.

Keywords: caveolin-1; nitric oxide synthase; protein kinase G.

Figure 1

Caveolin-1 and endothelial NOS (NOS-3) colocalize in the plasmalemma and golgi in untreated endothelial cells

Figure 2

Serine 116-phosphorylated nitric oxide synthase is not localized to the plasma membrane but is richly present in the endoplasmic reticulum of neonatal lamb lung microvascular endothelial cells

Figure 3

PKG 1 beta specifically colocalizes with serine 116-phosphorylated NOS in the cytosol and nucleus