Androgens Induce Nongenomic Stimulation of Colonic Contractile Activity through Induction of Calcium Sensitization and Phosphorylation of LC20 and CPI-17

Abstract

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.

Effect of Gonadal Hormones on Colonic Contractile Activity in Albino Rats

“…Our results are in controversy with Gonzáles-Montelongo et al who found that physiological concentrations (10nmol) of testosterone had a stimulating effect on spontaneous contractility in longitudinal colonic smooth muscle of male Swiss CD1 mice [44]. The reason for the contradictory results could be a dose dependent action of TEST: In our study, we used supraphysiological concentrations.…”

Histologic Evaluation of Human Benign Prostatic Hyperplasia Treated by Dutasteride: A Study by Xenograft Model With Improved Severe Combined Immunodeficient Mice

“…28 Regarding the relation between DHT and RhoA, a study using mouse colonic longitudinal smooth muscle showed that DHT elicits RhoA activation and translocation to the plasma membrane. 29 Thus, our findings that decreased DHT in the prostate tissue induced low expression of RhoA stand to reason. We suggest that low activity of RhoA may inhibit the contraction of human prostatic smooth muscle and decrease intraurethral pressure.…”

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

“…99 Androgens have recently been shown to potentiate ileal and colonic smooth muscle contractions by a non-genomic mechanism involving stimulation of polyamine synthesis which induces calcium sensitization of contractile machinery by ROCK and PKC activation along with increased CPI-17 phosphorylation. 100 Angiotensin IIinduced contractions of rat LES and IAS smooth muscle cells are inhibited by Clostridium botulinum C3 exozyme, HA-1077, and Y-27632, suggesting a role for ROCK. 101 It has been reported that thromboxane A2 and prostaglandin F2α, the end products of the renin-angiotensin system and arachidonic acid pathways, increase IAS tone via activation of RhoA/ROCK and increased MYPT1 T696 and MLC S19 phosphorylation.…”