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. 2016 Jan;66(1):85-92.
doi: 10.1007/s12576-015-0400-9. Epub 2015 Sep 21.

The role of the RhoA/ROCK pathway in gender-dependent differences in gastric smooth muscle contraction

Othman Al-Shboul  1
Affiliations

The role of the RhoA/ROCK pathway in gender-dependent differences in gastric smooth muscle contraction

Othman Al-Shboul. J Physiol Sci. 2016 Jan.

Abstract

Gender-related differences in various gastric functions and diseases have been reported, with women having a higher prevalence of gastrointestinal disturbances than men. The aim of this study was to investigate sex-dependent differences in activation of the Rho-associated protein kinase (ROCK; RhoA/Rho kinase) pathway and muscle contraction in the stomach using single gastric smooth muscle cells (GSMC) from male and female Sprague-Dawley rats. Expression of ROCK1 and ROCK2 protein and acetylcholine (ACh)-induced activation of RhoA and ROCK were measured using a specifically designed enzyme-linked immunosorbent assay and activity assay kits, respectively. Contraction of a single GSMC was measured by scanning micrometry in the presence or absence of the ROCK inhibitor Y27632 dihydrochloride. ACh-induced activation of RhoA and ROCK and subsequent contraction were greater in male rats than in female rats but neither was related to differences in the expression of ROCK1 or ROCK2 or total RhoA amount. Most important, Y27632 inhibited and abolished differences in ACh-induced contraction in both sexes. In conclusion, increased ACh-induced contraction in the GSMC of male rats is attributable to greater RhoA/ROCK activation independent of differences in the expression of ROCK isoforms or total RhoA.

Keywords: Contraction; Gastrointestinal system; Rho kinase; Smooth muscle.

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Conflict of interest statement

The author declares that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
Identification of rat gastric smooth muscle cells (GSMC). a Freshly isolated GSMC were of variable length and had a spindle-shaped form by phase contrast microscopy. Cells were viewed under a 20× objective of an inverted Nikon TMS-f microscope, and the image was captured with a Canon digital camera. Average length of resting SMC isolated from male and female rats was 113.53 ± 4 and 114.39 ± 3  µm, respectively. b Immunohistochemical staining of paraffin-embedded rat smooth muscle using anti-h1-calponin antibody at 1/100 dilution
Fig. 2
Fig. 2
Acetylcholine (ACh)-induced activity of Rho-associated protein kinase (ROCK) in GSMC isolated from male and female rats. ROCK activity is expressed as absorbance at 450 nm. Treatment of GSMC with ACh (0.1 µM) significantly increased ROCK activity in the cells of both sexes. Note that the ACh-induced activation of ROCK was significantly higher in cells isolated from male rats than in those isolated from female rats (*P < 0.01 vs. female; n = 10 males, n = 7 females)
Fig. 3
Fig. 3
Expression of ROCK1 and ROCK2 proteins in GSMC of male and female rats. ROCK1 (a) and ROCK2 (b) protein expression level is expressed as absorbance at 450 nm. The values shown are representative of at least four independent experiments performed in triplicate. Note that there was no significant difference in both ROCK1 and ROCK2 protein levels in the GSMC of male and female rats (P > 0.05; n = 11 males, n = 7 females)
Fig. 4
Fig. 4
Acetylcholine-induced RhoA activation in GSMC of male and female rats. a Activated RhoA is expressed as absorbance at 450 nm. Analysis using the RhoA G-LISA™ Activation Assay kit revealed increased levels of activated RhoA in response to ACh (0.1 µM) in both male and female GSMC. Note that ACh-induced activation of RhoA was significantly greater in male cells versus female. b Total RhoA protein was similar in both male and female GSMC (*P < 0.01 vs. female, n = 10 males, n = 7 females)
Fig. 5
Fig. 5
Acetylcholine-induced contraction of GSMC isolated from male and female rats. GSMC of male and female rats were stimulated with ACh (0.1 µM) and viewed under a Nikon microscope. Images of treated and non-treated single cells were acquired and cell contraction measured. ACh caused muscle cell contraction in GSMC from both male and female rats. Note that contraction in response to ACh was significantly greater in male cells than in female ones (*P < 0.01 vs. female; n = at least 50 cells from 10 different rats of each sex)
Fig. 6
Fig. 6
Effect of the ROCK inhibitor Y27632 on ACh-induced contraction in GSMC of male and female rats. GSMC of male and female rats were stimulated with ACh (0.1 µM) in the presence or absence of 1 µM Y27632 and then viewed under a Nikon microscope. Images of treated and non-treated single cells were acquired and cell contraction measured. The ROCK inhibitor significantly inhibited ACh-induced contraction in GSMC of both male and female rats. Importantly, Y27632-induced inhibition of gastric muscle contraction abolished the sex differences in the strength of ACh-induced contractions (*P < 0.01 vs. male ACh, **P < 0.01 vs. female ACh; n = at least 50 cells from 10 different rats of each sex)
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