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The aim of this study was to investigate the expression of RhoA/Rho-kinase in the uterus and the effect of Rho-kinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n = 14, any procedure was not performed), vehicle group (n = 14, 0.2 ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n = 16, DHEA 6 mg/100 g in 0.2 ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCI, carbachol, and PGF2 alpha induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCI, carbachol, and PGF2 alpha induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCI, carbachol, and PGF2 alpha induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rho-kinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.

Effect of female sex hormones on the production/release of adipocyte-derived cytokines has been debatable. Furthermore, whether the cellular signaling triggered by these hormones involve Rho-kinase has not been investigated yet. Therefore, in this study, effects of 17β-estradiol and progesterone as well as the Rho-kinase inhibitor, Y-27632 on the level of adipokines such as resistin, adiponectin, leptin, TNF-α and IL-6 were investigated in 3T3-L1-derived adipocytes. Differentiation was induced in the post-confluent preadipocytes by the standard differentiation medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum together with the mixture of isobutylmethylxanthine, dexamethasone and insulin) in the presence of 17β-estradiol (10(-8)-10(-7)M), progesterone (10(-6)-10(-5)M), the Rho-kinase inhibitor, Y-27632 (10(-5)M) and their combination for 8days. Measurements of the adipokines were performed in the culturing medium by ELISA kits using specific monoclonal antibodies. 17β-estradiol elevated resistin but decreased adiponectin and IL-6 levels; however, it did not alter the concentration of leptin and TNF-α. Y-27632 pretreatment inhibited the rise of resistin and the fall of adiponectin by 17β-estradiol without any effects by its own. Progesterone did not change resistin, leptin and TNF-α level; however, it elevated adiponectin and decreased IL-6 production. Neither 17β-estradiol nor Y-27632 was able to antagonize the increase of adiponectin and the reduction of IL-6 levels by progesterone. While Y-27632 alone lowered IL-6 level, it increased leptin and TNF-α concentration without altering resistin and adiponectin. In conclusion, 17β-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation.

objective. Effect of estrogenic compounds and 17β-estradiol (E2), which induces endothelial cell motility, was investigated on ROCK-2 expression in rat coronary vascular endothelial cells (CVEC). Methods. The CVEC were isolated from the heart of Wistar rats by collagenase (0.04%) and incubated with E2 (1-100 nM), estrogen receptor α (ERα) agonist: propyl pyrazole triol (PPT, 10 nM); ERβ agonists: (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN, 10 nM) and E2-conjugate with bovine serum albumin (E2-BSA, 1 nM); and GPER1 agonist: G1 (100 nM). Furthermore, the effect of combination of E2 with estrogen receptors (ERs) antagonist and GPER1 agonist, ICI-182780 (10 µM), physiological estrogen antagonists: progesterone (P4, 10-100 nM) and testosterone (T, 10-100 nM); transcription inhibitor: actinomycin-D (1 µg/ml); GPER1 antagonist: G-15 (100 nM), superoxide dismutase, (SOD, 500 U/ml); G i/o protein inhibitor: pertussis toxin (PTX, 100 µg/ml); and epidermal growth factor receptor (EGFR) blocker: AG-1478 (10 µM) was tested. After 24h incubation, ROCK-2 and GPER1 protein expressions were detected in the CVEC by Western-blotting. results. E2, ICI-182780, and G1 but not E2-BSA significantly up-regulated ROCK-2 expression, which was suppressed by actinomycin-D, PTX, AG-1478, and G-15. However, PPT and DPN had no effects on the ROCK-2 expression. ICI-182780, P4, T or SOD did not antagonize the E2 action. GPER1 expression was demonstrated in the CVEC. Conclusions. Estrogens could up-regulate ROCK-2 in the rat CVEC through GPER1 and EGFR transactivation.

Rho/Rho-kinase signalling pathway plays a substantial role in vascular contractions. In this study, we investigated any roles of Rho/Rho-kinase pathway in the vasoconstriction of the rat conductance and capacitance vessels by hyperosmolar glucose solution. Isolated aortic, mesenteric and renal rings were suspended and exposed to hyperosmolar glucose, sucrose and NaCl in the organ chambers filled with Krebs solution gassed with 95% O2 and 5% CO2 and maintained at 37 °C. The effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-5) M), was tested on the contraction induced by hypertonic solutions. Endothelial integrity was also assessed after hyperosmolar glucose exposure. Moreover, the activity and expression of Rho-kinase (ROCK-2) as well as RhoA translocation were detected by Western blotting and enzyme-linked immunosorbent assay-based RhoA activity detection method detection kit. The vessels produced substantial contractions in response to hyperosmolar solutions. Y-27632 significantly reduced hyperosmolarity-induced vasoconstrictions (P < 0.05). Phosphorylation of myosin-phosphatase target 1 increased after hyperosmolar glucose exposure, and this phosphorylation was significantly decreased by Y-27632 (P < 0.05) in the aorta. Furthermore, RhoA translocation but not ROCK-2 expression markedly increased by hyperosmolar glucose solution. These results may indicate that hyperosmolarity could induce vasoconstriction through Rho/Rho-kinase signalling.

This study aimed to determine the effect of pluripotent astrocytic stem cells (PASCs) and fibroblast growth factor-2 (FGF-2) on cognitive function in neonatal rats with hypoxic-ischemic brain injury (HIBI).The study was performed on 7-d-old rats that were randomly divided into four groups. All rats, except those in the sham group, were kept in a hypoxic chamber containing 8% oxygen for 2 h after the ligation of the right carotid artery. Next, 5 d after HIBI was induced, PASCs were administered to the motor cortex, and FGF-2 was administered intraperitoneally to group AF; PASCs were administered to the motor cortex, and salt solution buffered with phosphate was administered intraperitoneally to group A; and fresh cell culture solution (medium) was administered to group M. Immunofluorescence was used to localize the administered PASCs in the brains of rats from groups A and AF. The Morris water maze tank (MWM) test was performed to assess the rats' cognitive functions at week 12. The rats that were administered PASCs were observed for the development of neoplasms and autopsies were performed after 30 months.PASCs migrated to damaged brain regions surrounding the hippocampus in groups A and AF. The mean platform finding time (PFT) significantly decreased over time in each group on day 1-4 of MWM testing (p 0.001). On day 2-4, the mean PFT was shortest in group S followed by group AF. In group A, the PFT was significantly longer than in group S on day 3-4 (p = 0.01 and 0.007, respectively). On day 5 of the MWM test, the time spent in the eastern quadrant (which previously contained the platform) was longest in group S followed by groups AF, A, and M; however, the differences between groups were not significant (p = 0.51). After 30 months, none of the rats in groups A or AF had benign or malignant neoplasms.Following the administration of PASCs in rats with experimentally induced HIBI, PASCs migrated to the injured brain regions; however, treatment with PASCs did not have a positive effect on cognitive function. The administration of FGF-2 together with PASCs resulted in positive cognitive results, although not at the level of significance.

To investigate the antimuscarinic effect of oxybutynin in the rat detrusor muscle after estrogen pretreatment because, to our knowledge, no study has been done on the interaction of estrogen with antimuscarinic drugs. Estrogen has been shown to affect muscarinic receptors in the detrusor muscle of animals. In addition, oxybutynin has been shown to block muscarinic receptors in the bladder.Estradiol benzoate (150 microg/kg) or saline was given subcutaneously to virgin female Wistar albino rats (n = 6, each group) for 10 consecutive days. On the 11th day, isolated detrusor muscle strips were taken, and acetylcholine (ACh)-induced contractions were evaluated in the absence or presence of oxybutynin (10 and 100 nM).ACh induced concentration-dependent contractions in the detrusor muscle. In the estradiol-pretreated group, the maximum of the ACh-induced contractions was diminished compared with that in the control group (P0.05). Oxybutynin (10 and 100 nM) inhibited ACh-induced contractions competitively (pK(B) 8.85). In the estradiol-pretreated group, the concentration-response curve to ACh was shifted further to the right in the presence of oxybutynin (100 nM).We have demonstrated for the first time that oxybutynin further inhibits ACh-induced and muscarinic receptor-mediated contractions in rat detrusor muscle after pretreatment with estrogen.

In human internal mammary artery (IMA), testosterone induces vasodilation that shows marked variability among patients. We aimed to investigate the relationship of this variability with cardiovascular risk factors. Cumulative relaxations to testosterone after precontraction with KCl were examined in IMA segments from patients with identified cardiovascular risk factors such as hypercholesterolemia, diabetes, hypertension, smoking, age, gender, body mass index (BMI), and number of occluded vessels. Testosterone responses were significantly diminished in subjects with 3 compared with 1 risk factor. Hypercholesterolemia independently influenced testosterone responses by significantly decreasing its maximum, and smoking significantly decreased the sensitivity to testosterone. Thus, the variability observed in testosterone-induced vascular relaxations may in part be related to differences in risk factors present among the individuals studied.

Relaxant responses to two Rho-kinase inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil, were compared in the corpus cavernosum obtained from diabetic and non-diabetic mice. Streptozotocin (100 mg kg(-1) day(-1), for 2 days) induced diabetes with a blood glucose level of 318+/-55.4 mg dl(-1); whereas it was 85.4+/-4.1 mg dl(-1) in control mice (P0.05). Electrical field stimulation (40 V, 0.5 ms, 1, 2, 4, 8, 16 Hz for 15 s) and acetylcholine-induced relaxations were markedly attenuated in the corpus cavernosum from streptozotocin-diabetic mice whereas responses to Y-27632 (10(-9)-3 x 10(-5) M) and fasudil (10(-9)-3 x 10(-5) M) were not altered. EC(50) values for Y-27632 were 2.98+/-0.89 and 4.19+/-2.71 microM in the corpus cavernosum from control and diabetic mice, respectively (P0.05). The values for fasudil were 7.42+/-4.91 and 3.53+/-1.41 microM in the corpus cavernosum from control and diabetic mice, respectively (P0.05). These results may suggest that, in diabetes, the relaxant effects of the Rho-kinase inhibitors may not be changed and thus, they may have a beneficial therapeutic effect in diabetic erectile dysfunction.

PubMedID: 14757149 Rho-kinase expression was investigated in the rat mesenteric artery and the effects of its inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil (HA-1077), were examined on the increase in perfusion pressure induced by two different receptor agonists, namely the ?-adrenoceptor agonist, phenylephrine and, the endothelin ETA and ETB receptor agonist, endothelin-1. Y-27632 and fasudil produced a concentration-dependent decrease in perfusion pressure. There was no difference between the concentration-response lines of these two inhibitors. The maximum decrease in the perfusion pressure induced by 10-5 M Y-27632 was 85.8±3. 7% when the tone was increased by phenylephrine. However, it was 48.1±5.4% (P