m-Calpain activation/depletion is associated with androgen-induced reduction of protein kinase C and proliferation of male accessory sex organ smooth muscle cells.
OBJECTIVE
In the guinea pig seminal vesicle smooth muscle (SVM), androgen-dependent proliferation and terminal differentiation appear to be coupled to protein kinase C (PKC). This is based on the observations that both the soluble (cytosolic) enzyme and the Triton X-100 solubilizable form of the particulate enzyme were reduced during proliferation but were androgen-resistant in the amitotic state of adults. The purpose of the present investigation was to determine if the reduction in PKC activity was linked to the translocation of the activated enzyme to acceptor sites in the Triton X-100 insoluble fraction of the cell or reflected enzyme depletion due to proteolysis by androgen-dependent activation of the u- and/or m-calpains.


METHODS
SVM was harvested from treated animals, homogenized and separated into soluble and particulate components. The particulate material was further fractionated into Triton X-100 soluble and insoluble fractions. PKC activity was determined in all fractions using radioactive ATP. Cultures of pure smooth muscle cells from both human prostate and SVM were also employed to assess the role of calpain in smooth muscle growth.


RESULTS
During androgen-induced proliferation, instead of a translocation of PKC activity to the Triton X-100 insoluble particulate fraction of the cell, PKC activity in this fraction was significantly reduced. The m-calpain was the only isoform detected in SVM. At the peak of androgen-induced DNA synthesis in pre-pubertal castrate animals, m-calpain decreased 45% whereas in proliferative resistant SVM of adult castrates the protease was not significantly affected by androgen treatment. In pure smooth muscle cultures from the SVM as well as human prostate glands calpeptin the cell permeable inhibitor of calpains produced a concentration-dependent inhibition (IC50 approximately equal to 35 microM) of cellular proliferation.


CONCLUSIONS
Given that biochemical assays of calpain quantify the residual proenzyme and that upon activation calpain is rapidly degraded, our findings indicate that m-calpain activation occurs in association with androgen-induced degradation of PKC and SMC proliferation. Thus in vitro inhibition of m-calpain activation is antiproliferative. The relative resistance of m-calpain in adult SVM may be an important component of the terminal differentiation process in normal smooth muscle.
Gebrosky
N
N
Department of Urology, Robert C. Byrd Health Sciences Center, Morgantown, West Virginia 26506, USA.
Cole
D
D
Stetter-Neel
C
C
Durham
J
J
Mawhinney
M
M
D000728:Androgens / Q000502:physiology; D000818:Animals; D002154:Calpain / Q000502:physiology*; D002455:Cell Division; D004533:Egtazic Acid; D005837:Genitalia, Male / Q000166:cytology* / Q000378:metabolism*; D006168:Guinea Pigs; D008297:Male; D017830:Octoxynol; D011493:Protein Kinase C / Q000096:biosynthesis*; D012669:Seminal Vesicles / Q000166:cytology* / Q000378:metabolism*
D000728:Androgens; D004533:Egtazic Acid; D017830:Octoxynol; D011493:Protein Kinase C; D002154:Calpain