Androgens induce nongenomic stimulation of colonic contractile activity through induction of calcium sensitization and phosphorylation of LC20 and CPI-17.
We show that androgens, testosterone and 5alpha-dihydrotestosterone (DHT), acutely (approximately 40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC(20)) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC(20) phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC(20) by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways.
González-Montelongo
María C
MC
Laboratory of Membrane Physiology and Biophysics, Department of Animal Biology, University of La Laguna, Tenerife 38206, Spain.
Marín
Raquel
R
Gómez
Tomás
T
Marrero-Alonso
Jorge
J
Díaz
Mario
M
D000577:Amides / Q000494:pharmacology; D000728:Androgens / Q000494:pharmacology*; D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D003106:Colon / Q000187:drug effects* / Q000502:physiology; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D013196:Dihydrotestosterone / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018120:Finasteride / Q000494:pharmacology; D005485:Flutamide / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008954:Models, Biological; D008962:Models, Theoretical; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011944:Receptors, Androgen / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013739:Testosterone / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000577:Amides; D000728:Androgens; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D011725:Pyridines; D011944:Receptors, Androgen; D013196:Dihydrotestosterone; C108830:Y 27632; D003609:Dactinomycin; D013739:Testosterone; D018120:Finasteride; D005485:Flutamide; D003513:Cycloheximide; C016299:chelerythrine; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D002118:Calcium