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