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  • Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate.

Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate.

American journal of physiology. Renal physiology (2014-07-25)
Thomas Kunit, Christian Gratzke, Andrea Schreiber, Frank Strittmatter, Raphaela Waidelich, Beata Rutz, Wolfgang Loidl, Karl-Erik Andersson, Christian G Stief, Martin Hennenberg
ABSTRACT

Smooth muscle contraction may be critical for lower urinary tract symptoms (LUTS) in patients with benign prostate hyperplasia and requires stable anchorage of the cytoskeleton to the cell membrane. These connections are regulated by focal adhesion kinase (FAK). Here, we addressed the involvement of FAK in the regulation of smooth muscle contraction in hyperplastic human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Expression of FAK and focal adhesion proteins was assessed by Western blot analysis and immunohistochemical stainings. Effects of the FAK inhibitors PF-573228 and Y-11 on contraction of prostate strips were examined in the organ bath. Expression of FAK and focal adhesion proteins (integrin-5α, paxilin, and c-Src) was detected by Western blot analysis in prostate samples. By double immunofluorescence staining with calponin and pan-cytokeratin, expression of FAK was observed in stromal and epithelial cells. Immunoreactivity for FAK colocalized with integrin-5α, paxilin, talin, and c-Src. Stimulation of prostate tissues with the α1-adrenergic agonist phenylephrine increased the phosphorylation state of FAK at Tyr³⁹⁷ and Tyr⁹²⁵ with different kinetics, which was blocked by the α1-adrenoceptor antagonist tamsulosin. Norepinephrine and phenylephrine induced concentration-dependent contractions of prostate strips. Both FAK inhibitors PF-573228 and Y-11 significantly inhibited norepinephrine- and phenylephrine-induced contractions. Finally, PF-573228 and Y-11 inhibited contractions induced by electric field stimulation, which was significant at the highest frequency. In conclusion, α1-adrenergic smooth muscle contraction or its regulation involves FAK in the human prostate. Consequently, FAK may be involved in the pathophysiology of LUTS and in current or future LUTS therapies.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3-[(1R)-1-Hydroxy-2-(methylamino)ethyl]phenol, AldrichCPR
Tamsulosin Racemate, European Pharmacopoeia (EP) Reference Standard
Phenylephrine hydrochloride, European Pharmacopoeia (EP) Reference Standard
Supelco
Phenylephrine Hydrochloride, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Tamsulosin hydrochloride, ≥98% (HPLC)
Phenylephrine, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
(R)-(−)-Phenylephrine hydrochloride, analytical standard
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
(R)-(−)-Phenylephrine hydrochloride, powder
Tamsulosin hydrochloride, European Pharmacopoeia (EP) Reference Standard
Phenylephrine hydrochloride for peak identification, European Pharmacopoeia (EP) Reference Standard
Supelco
R-(-)-Phenylephrine hydrochloride solution, 1.0 mg/mL in methanol (as free base), ampule of 1 mL, certified reference material, Cerilliant®
USP
Tamsulosin hydrochloride, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Goat Anti-Mouse IgG Antibody, Cy3 conjugate, Chemicon®, from goat