Functional role of PKC in contraction of cultured human prostatic stromal cells

Abstract

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI-17 was over-expressed, PDBu induced a large contraction, but the agonist-induced contraction did not become larger than expected. In alpha-toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI-17 over-expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI-17 and also that the PKC-CPI-17 pathway does not appear to play a major role in the agonist-induced contraction even when CPI-17 was over-expressed.

RhoA/Rho kinase‐mediated Ca2+ sensitization in the contraction of human prostate

“…In CHPSCs ring experiments, we used ET-1 as the representative contractile agonist. As we previously reported, 8 CHPSCs did not respond to a-stimulant, a major contractile agonist in the human prostate, 10,11 probably due to some change in the receptor phenotype during culture. On the other hand, ET-1 has been reported to be one of the agents that cause the contraction of the human prostate.…”

“…The preparation of CHPSCs and the production of a reconstituted ring preparation made of CHPSCs have been previously described. 8 The ring preparation so obtained was mounted between two tungsten wires, one of which was fixed while the other one was attached to a force transducer (UL2; Minebea Co., Nagano, Japan). These rings were placed in a normal physiological saline solution (PSS) consisting of the following composition (mM): NaCl, 123; KCl, 4.7; CaCl 2 , 1.25; MgCl 2 , 1.2; KH 2 PO 4 , 1.2; NaHCO 3 , 15.5; D-glucose 11.5; pH 7.4.…”

“…Isolation of RhoA cDNA by RT-PCR and gene transfer using a baculovirus vector was performed as previously described. 8 All tension experiments were performed at room temperature.…”

“…A Western blot analysis was performed as previously described. 8 Protein extract was separated by SDS-PAGE and subjected to immunoblotting with anti-RhoA (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ROCK I (1:250, BD Biosciences, San Jose, CA), or anti-ROCK II (1:500, BD Biosciences) antibodies and the signals were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Buckinghamshire, England). The transferred membrane was finally stained with Coomassie brilliant blue and the bands corresponding to actin were evaluated.…”

“…6,7 We also recently reported that the isometric tension development of cultured human prostatic stromal cells (CHPSCs) could be measured using the collagen gel technique and a high efficiency gene transfer into CHPSCs could be achieved. 8 Using these methods, the functional role of RhoA/ROCK in the contraction of human prostate was examined.…”