Elsevier

Nitric Oxide

Volume 16, Issue 1, February 2007, Pages 10-17
Nitric Oxide

Phosphorylation of the PKG substrate, vasodilator-stimulated phosphoprotein (VASP), in human cultured prostatic stromal cells

https://doi.org/10.1016/j.niox.2006.09.003Get rights and content

Abstract

Nitric oxide (NO) is known to regulate contractility and proliferation of cells within the prostate, however, the mechanism by which this occurs is unknown. The cGMP-dependent protein kinase (PKG) signalling pathway may be involved, and recent work has shown that activation of this pathway can be assessed by analysis of phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The aim of the current study is to characterise the expression of VASP in the human prostate and human cultured prostatic stromal cells (HCPSCs), and to investigate whether NO activates PKG in these cells. Our studies revealed that VASP is expressed, and that incubation of HCPSCs with PKG-activating cGMP-analogues or the NO-donor, SNP, caused a significant PKG-dependent increase in VASP serine-239 phosphorylation. In addition, SNP elicited a reduction in intracellular K+ in a time frame consistent with the phosphorylation of VASP and activation of PKG. These data demonstrate that VASP can be used to assess the NO/cGMP/PKG signalling pathway in HCPSCs. In addition, we demonstrate for the first time that SNP, probably via NO release, leads to phosphorylation of VASP in a manner consistent with PKG activation.

Section snippets

Ethics approval

Human tissues were obtained with the approval of the Southern Healthcare Network Ethics and Experimentation Committee (Reference number 02066A).

Human prostatic tissue

Explants of human prostate from patients (mean age 68 yrs) undergoing transurethral resection of the prostate (TURP) to treat benign prostatic hyperplasia were used to generate primary HCPSC cultures.

Primary explant cell culture

Primary cells were grown as described previously [17] on tissue culture dishes in MCDB 131 media (Sigma-Aldrich Chemical Company Castle Hill, Australia)

PKG protein expression in HCPSCs

Immunoblotting was used to confirm PKG isoform protein expression in HCPSCs. PKG Iα/β was detected in cytosolic protein fractions, while PKG II was detected in both cytosolic and particulate protein fractions of HCPSCs (n = 3; Fig. 1a and b).

VASP expression in TURP tissue and HCPSCs

Immunohistochemistry and immunocytochemistry using anti-VASP antibodies was performed on TURP tissue and HCPSCs to confirm VASP expression. Positive immunoreactivity for VASP was seen in stromal, vascular and epithelial compartments. Immunostaining of

Discussion

The current study has confirmed that PKG and VASP are expressed in HCPSCs, and that incubation with the PKG-activating cGMP-analogues, PET-cGMP and 8-pCPT-cGMP, or the NO-donor, SNP, leads to a significant increase in the phosphorylation of the PKG substrate, VASP, at serine-239. In addition, we have shown that SNP elicits a reduction in intracellular K+, which is inhibited in the presence of the soluble guanylate cyclase inhibitor, ODQ, the PKG-inhibitor, Rp-8-pCPT-cGMP and also various K+

Acknowledgments

The authors gratefully acknowledge the help of the staff at Monash Medical Centre (Moorabbin) for their help in the acquisition of human tissues, and also the support received from the Monash University Postgraduate Publication Award. The current work was supported by the National Health and Medical Research Council (Grant ID 118611).

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