Investigation of the anticancer effect of newly synthesized palladium conjugate Schiff base metal complexes on non-small cell lung cancer cell line and mouse embryonic fibroblast cell line.

Lung cancer remains one of the leading causes of death worldwide. Due to the side effects of chemotherapeutic agents on normal cells and the development of resistance by cancer cells, there is an urgent need for alternative new pharmacological agents. Palladium (Pd)-conjugated Schiff base (SB) compounds represent an alternative approach with promising potential applications in cancer treatment. This study aims to identify novel therapeutic agents on A549 cells through the synthesis and characterization of Schiff base conjugated-Palladium complexes (Pd-L1 and Pd-L2). Additionally, it seeks to elucidate the mechanism of action of these compounds on both the A549 and NIH/3T3 cell lines. In the present study, two new Pd-L1 and Pd-L2 were synthesized for the first time and characterized mainly by single crystal X-ray diffraction and 1H, 13C, 31P NMR techniques. The cytotoxic effect of the compounds was evaluated by MTT assay on A549 and NIH/3T3 cell lines for 24 and 48 h. Cisplatin was used as a positive control group. Based on the cytotoxicity results, the complexes were evaluated for their anticancer activities against A549 cell lines for 48 h through reactive oxygen species (ROS), cell cycle, apoptotic, and necrotic cell analyses. The most potent cytotoxic effects were determined for Pd-L1 (IC50: 23.33 µM), Pd-L2 (IC50: 3.19 µM), and cisplatin (IC50: 33.27 µM) on A549 cells (p < 0.05). The compounds exhibited a significant cytotoxic effect at lower concentrations on A549 cells compared to NIH/3T3 cells (p < 0.05). All compounds showed a significant increase in ROS levels in A549 cells compared to the control group (p < 0.05). While necrosis and apoptosis was observed in A549 cells treated with cisplatin, induction of apoptosis was effective in cell death for A549 cells treated with Pd-L1 and Pd-L2 (p < 0.05). Additionally, it was observed that the compounds inhibited cell proliferation in the G0/G1 and G2/M cell cycle phases (p < 0.05). All compounds induced cell cycle arrest and cell death in A549 cells by increasing ROS levels. The results obtained in the present study could advance the utilization of the compounds as anticancer agents.

Effects of the Rho/Rho-Kinase Pathway on Perfusion Pressure in the Isolated-Perfused Rat Hind Limb Vascular Bed.

BACKGROUND: Rho/ROCK signaling has been demonstrated to be involved in the vascular reactivity of many arterial networks. However, RhoA expression and the contribution of Rho/ROCK pathway to the control of perfusion pressure have not been investigated in the rat hind limb vascular bed as a skeletal muscle vascular network. AIMS: To investigate the contribution of the Rho/ROCK pathway in the control of perfusion pressure in the isolated-perfused rat hind limb vascular bed. STUDY DESIGN: Animal experimentation. METHODS: Two Rho inhibitors (atorvastatin and C3 exoenzyme) and ROCK inhibitors (Y-27632 and fasudil) were tested on the phenylephrine-elevated perfusion pressure in the isolated-perfused rat hind limb vascular bed. Furthermore, we sought the expression of RhoA protein in the femoral, popliteal and saphenous arteries as well as quadriceps and gastrocnemius muscles by Western blotting. RESULTS: The ROCK inhibitors Y-27632 and fasudil (both 10-8 to 10-5 M) induced substantial vasodilatations. The maximum vasodilatations induced by Y-27632 and fasudil (both at 10-5 M) were 84.0 ± 6.9% and 76.9 ± 6.9%, respectively (P = .091). Y-27632 was not more potent than fasudil, as the EC50 values for Y-27632 and fasudil were 0.7 ± 2.1 µM and 2.5 ± 2.4 µM, respectively (P = .177). Atorvastatin (10-7 to 10-4 M) and C3 exoenzyme (3 × 10-8 M) also produced vasodilatation (maximum vasodilatation; 20.3 ± 1.7% and 13.7 ± 3.6%, respectively). The EC50 value for atorvastatin was 94.9 ± 1.2 µM. The western blot analysis showed that the femoral, saphenous, and popliteal arteries, as well as the gastrocnemius and quadriceps muscles, express RhoA protein. CONCLUSION: The Rho/ROCK pathway contributes significantly to the control of perfusion pressure in the rat hind limb vascular bed.

The effect of testosterone replacement therapy on bladder functions, histology, apoptosis, and Rho-kinase expression in bladder outlet obstruction and hypogonadism rat model

Background/aim: The effect of testosterone replacement therapy was investigated on bladder functions, histology, apoptosis as well as Rho-kinase expression in the rat bladder outlet obstruction (BOO) and hypogonadism models. Materials and methods: 30 mature male rats divided into 4 groups: sham group (n = 8), BOO group (n = 8), BOO + orchiectomy group (n = 7), BOO + orchiectomy + testosterone (T) treatment group (n = 7). Cystometric findings, apoptosis index, Rho-kinase (ROCK-2) expression, and smooth muscle/collagen ratio were compared. Results: BOO did not change ROCK-2 expression level, compared to sham group (P > 0.05). However, when compared to BOO group (P < 0.01), BOO + orchiectomy led ROCK-2 increase. The testosterone treatment failed to reverse the up-regulation of ROCK-2 induced by orchiectomy although it tended to lower ROCK-2 level. Compared to sham group (P = 0.002), changes in maximal bladder capacity and leak point pressure were higher (P = 0.026, P = 0.001), and bladder compliance was lower in BOO group. Also, the apoptosis index was different between the two groups (P = 0.380). Smooth muscle/collagen ratio was higher in BOO + orchiectomy + T group than in BOO + orchiectomy group (P = 0.010). Conclusions: The research draws attention to alternating treatment approaches in case of the presence of hypogonadism and BOO.

RhoA, ROCK-1, and ROCK-2 Gene Expression and Polymorphisms in Cholesteatoma Patients.

BACKGROUND: The aim of this study was to evaluate the gene polymorphism and expressions of Rho-A, ROCK-1, and ROCK-2 in cholesteatoma. METHODS: In this study, 120 healthy control group patients and 120 cholesteatoma patients were enrolled. Venous blood was taken from all of the cholesteatoma and control group patients. The genotyping of ROCK-1(G/T)rs35996865, ROCK-2(A/C)rs10178332, and Rho-A(A/T)rs2177268 polymorphisms was performed using predesigned TaqMan SNP Genotyping Assays. Assays-on-Demand SNP genotyping kit was used for the realtime polymerase chain reaction. The expression levels of Rho-A(Hs00357608_m1), ROCK-1(Hs01127699_m1), and ROCK-2(Hs00178154_m1) genes were determined. RESULTS: The expression of Rho-A, ROCK-1, and ROCK-2 was lower in cholesteatoma patients than in the control group. There was no difference in Rho-AAT/TT and ROCK-1GT/TT variation in cholesteatoma patients compared to the control. However, ROCK-2 AC/CC variance was lower in cholesteatoma patients. CONCLUSION: The expression of Rho-A, ROCK-1, and ROCK-2 genes may be decreased in cholesteatoma. Furthermore, since ROCK-2 AC/CC genotype is also lower in cholesteatoma, having C allele seems to decrease the risk of developing this disease.

Suppressive effect of Rho-kinase inhibitors Y-27632 and fasudil on spike-and-wave discharges in genetic absence epilepsy rats from Strasbourg (GAERS).

Rho/Rho-kinase (ROCK) signaling contributes to neuroinflammation, epileptogenesis, and seizures in convulsive-type epilepsies. However, this pathway has not been investigated in absence epilepsy. We investigated RhoA activity in genetic absence epilepsy rats from Strasburg (GAERS) and the effects of ROCK inhibitors Y-27632 and fasudil on spike-and-wave discharges (SWDs) of GAERS. ROCK level and activity were measured by Western blot analysis in the brain areas involved in absence seizures (i.e., cortex and thalamus) and hippocampus. Male GAERS were stereotaxically implanted with bilateral cortical electrodes for electroencephalogram (EEG) recordings and/or guide cannula into the right ventricle. ROCK inhibitors were administered by intraperitoneal injection (1-10 mg/kg for Y-27632 or fasudil) or intracerebroventricular injection (7-20 nmol/5 µl for Y-27632 or 10-100 nmol/5 µl for fasudil). EEG was recorded under freely moving conditions. Compared with Wistar rats, GAERS exhibited increased RhoA activity in the somatosensory cortex but not in the thalamus or hippocampus. The single systemic administration of Y-27632 and fasudil partially suppressed the duration and frequency of absence seizure, respectively. However, local brain administration caused a widespread suppressive effect on the total seizure duration, number of seizures, and the average individual seizure length. In summary, Rho/ROCK signaling may be involved in the pathophysiology of absence epilepsy. Furthermore, ROCK inhibitors can control the expression of absence seizure in GAERS, thus indicating that Y-27632 and fasudil have the potential to be used as novel anti-absence drugs.

Role of rho-kinase (ROCK) in tonic but not phasic contraction in the frog stomach smooth muscle.

AIMS: Rho/Rho-kinase (ROCK) signaling has extensively been shown to take part in mammalian smooth muscle contractions in response to diverse agents yet its role in the contraction of amphibian smooth muscle has not been investigated. Therefore, we aimed to explore any role of this pathway in the contractions of frog stomach smooth. MAIN METHODS: The strips were prepared and suspended in organ baths filled with Ringer solution. Changes in the circular strips of the frog stomach muscle length were recorded isotonically with a force transducer in organ baths. KEY FINDINGS: Carbachol (CCh) exerted both phasic and tonic contractions. In contrast, atropin abolished all types of contractions by CCh. The phasic contractions were suppressed by a Ca2+ channel blocker, nifedipine but not by the ROCK inhibitor, Y-27632. However, the tonic contractions were markedly attenuated by Y-27632. Selective M1 receptor blocker, pirenzepin, selective M3 receptor blocker and DAMP had no effects on CCh-elicited contractions. On the other hand, selective M2 receptor blocker, AF-DX suppressed all types of contractile activity by CCh. SIGNIFICANCE: These data suggest that M2 receptor activation could mainly mediate CCh-induced phasic and tonic contractions, and ROCK seems to be involved in the CCh-induced tonic but not phasic contractions of the frog stomach smooth muscle.

Rho/rho-kinase signalling in chronically alcohol-fed mice.

BACKGROUND/AIM: The effects of chronic ethanol consumption on male sexual function are debateable, and its effects on the Rho/Rho-kinase pathway are unknown. Therefore, we investigated smooth muscle reactivity and Rho/Rho-kinase signalling in the corpus cavernosum of ethanol-fed mice. MATERIALS AND METHODS: Ethanol was added to drinking water at 5% concentration in the first 2 days with 5% increment every subsequent 2 days, up to a final concentration of 20% for 45 days. Corpora cavernosa were isolated and a cumulative dose-response curve to phenylephrine was obtained. Acetylcholine (10-6 M), electrical field stimulation (EFS, 40 V, 8-16 Hz) and Y-27632 (10-6-10-5 M)-induced relaxations were compared in the control and ethanol groups. RESULTS: Blood ethanol levels in the control and ethanol-treated mice were 1.5 ± 0.3 mg/dL and 37.4 ± 4.1 mg/dL (P < 0.001), respectively. Phenylephrine-induced contractile responses were potentiated in the ethanol group, with pD2 values of 4.92 ± 0.18 and 5.71 ± 0.21 (P < 0.01) in the corpus cavernosum obtained from control and alcohol-fed mice, respectively. The relaxant responses to acetylcholine and EFS, but not Y-27632, were significantly augmented in the corpus cavernosum obtained from ethanol-treated mice. However, expression and activation of Rho-kinase remained unchanged in the alcohol group. CONCLUSION: Ethanol drinking may increase sensitivity to both vasoconstrictors and vasodilators in the mouse corpus cavernosum without any alteration in Rho/Rho-kinase signalling.

The Role of Rho/Rho-Kinase Pathway in the Pathogenesis of Cholesteatoma.

OBJECTIVE: To assess the role of Rho/Rho-kinase pathway in the pathogenesis of cholesteatoma. MATERIALS AND METHODS: Thirty-eight patients with cholesteatoma, who had gone mastoidectomies were enrolled in this prospective study. Cholesteatomas matrix (CM) and a piece of the external ear canal skin (EECS as control) were taken and transferred to the liquid nitrogen and kept at -86 °C for Rho A and Rho-kinase (ROCK) analysis with Western blotting and commercial ELISA kits (Cell Biolabs Inc., San Diego, CA). The tissues were homogenized by an appropriate ice-cold lysis buffer. Following centrifugation, the supernatant was taken and total protein amount was detected by the Bradford method. Thereafter, tissue homogenates were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis electrophoresis then transferred to nitrocellulose membrane where it was treated with specific monoclonal primary antibody against to ROCK-2 and HRP-conjugated seconder antibody, respectively. The protein blots were visualized with commercial x-ray film and dansitometrically analyzed by the Scion Image Program (Cell Biolabs Inc., San Diego, CA). In another series of experiments, Rho-kinase activities were assessed by ROCK-2 ELISA kits. RESULTS: There were no statistical differences in Rho A translocation between CM and EECS. However, ROCK activity was found to be lower in CM than EECS as detected by ELISA kits. Furthermore, ROCK protein expression was also significantly lower in CM than EECS as demonstrated by Western blotting. CONCLUSION: Given Rho-kinase could take essential roles in cell differentiation, the results of this study implicate that down-regulated Rho-kinase could be responsible for the keratinocyte undifferentiation seen in cholesteatoma pathogenesis.

Effects of 17ß-estradiol and progesterone on the production of adipokines in differentiating 3T3-L1 adipocytes: Role of Rho-kinase.

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 17ß-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 17ß-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. 17ß-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 17ß-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 17ß-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, 17ß-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation.

The functional significance of the rho/rho-kinase pathway in human erythrocytes.

OBJECTIVE: Erythrocyte deformability, which can be influenced by various intracellular signaling mechanisms, such as nitric oxide, cAMP, cGMP, and protein kinases, is the most important physiological factor providing the blood flow in microcirculation. However, the functional significance of the Rho/Rho-kinase pathway, which contributes cell shape changes and the reorganization of the actin cytoskeleton, has yet to be explored in erythrocytes. Therefore, we examined the influence of several activators and inhibitors of Rho/Rho-kinase signaling on human erythrocyte deformability. MATERIALS AND METHODS: RhoA and ROCK-2 proteins were studied by western blotting. Influences of 2 Rho-kinase inhibitors, fasudil and Y-27632 (both 10-7 to 10-4 M), on erythrocyte deformability was determined by ektacytometer at various shear stresses (0-30 Pa) in the presence or absence of a known Rho activator, lysophosphatidic acid (LPA, 10-5 to 5x10-5 M, 1-15 min). RESULTS: LPA incubation reduced deformability with concomitant RhoA-GTP inhibition. Y-27632 and fasudil also decreased deformability, but had no effect on LPA-induced reduction of deformability. Rho inhibitor C3 had no effect on RhoA activation. Reduction in RhoA activation was induced by sub-hemolytic mechanical stress. CONCLUSION: Our findings may indicate that the Rho/Rho-kinase pathway could contribute to the regulation of deformability of human erythrocytes.

Rho-kinase levels in testicular ischemia-reperfusion injury and effects of its inhibitor, Y-27632, on oxidative stress, spermatogenesis, and apoptosis.

OBJECTIVE: To investigate testicular Rho-kinase levels and the effects of its inhibitor, Y-27632, on oxidative stress, spermatogenesis, and apoptosis in testicular ischemia-reperfusion rat model. METHODS: The study included 29 adult Wistar-Albino male rats weighing 150-200 g. The rats were divided into 3 groups. Group 1 underwent sham operation (n = 10). In group 2, left testicular torsion-detorsion was performed (n = 9). In group 3, Rho-kinase inhibitor Y-27632 (5 mg/kg) was injected intraperitoneally 30 minutes before detorsion (n = 10). Two months later, bilateral orchiectomy was performed in all the groups. Rho-kinase levels by Western blotting, apoptosis with terminal deoxynucleotidyl transferase dUTP nick end labeling method, testicular damage and spermatogenesis with modified Johnsen score, testicular total antioxidative status, and total oxidative status were measured. RESULTS: In the torsion-detorsion (T/D) group, Rho-kinase level increased significantly, compared with the sham group (P = .025). In the Y-27632 treatment group, Johnsen scores were significantly higher, and apoptosis indexes were significantly lower, compared with the T/D group (P = .001). Significantly higher total antioxidative status levels and lower total oxidative status levels were observed in the Y-27632 treatment group, compared with the T/D group (P = .001 and P = .002, respectively). CONCLUSION: Testicular ischemia-reperfusion significantly increased Rho-kinase levels in rats, and administration of Rho-kinase inhibitor, Y-27632, before detorsion might prevent ischemia-reperfusion injury.

Vasoconstriction induced by G1, a G-protein-coupled oestrogen receptor1 (GPER-1) agonist, in the isolated perfused rat kidney.

Vascular effects of the G protein-coupled oestrogen receptor1 (GPER-1) agonist, G1 (10(-7)-5×10(-6) M), the main oestrogenic hormone, 17ß-estradiol (10(-9)-10(-4) M), the NR3A1 agonist, PPT (10(-8)-10(-5) M), the NR3A2 agonist DPN (10(-8)-10(-5) M), and the classical oestrogen receptor blocker but also a GPER agonist, ICI-182780 (10(-8)-3×10(-6) M), were investigated on the perfusion pressure in the isolated rat kidney. To seek cellular mechanisms involved in GPER-1-induced signalling we tested several compounds including the inhibitors of Rho-kinase (ROCK) (Y-27632), tyrosine kinase (genistein), p38MAPK (SB203580), p44/42MAPK (PD98059), protein kinase C (PKC) (GF109203X), Jun-kinase (JNK) (SP600125), phosphatidylinositol-3-kinase (PI3K) (LY294002), Ca(2+) channels (nifedipine), GPER-1 (G15) and epidermal growth factor (EGF) receptor kinase (AG-1478). Moreover, the effect of saponin (50mg/ml) that was used for endothelium removal was explored on G1-elicited vascular action. G1, 17ß-estradiol and ICI-182780 but not PPT and DPN induced vasoconstrictions in basal renal perfusion pressure. In contrast, G1 promoted vasodilatation when the perfusion pressure was elevated in advance by phenylephrine. G1-elicited vasoconstriction was not modified by endothelial removal; however, it was markedly inhibited by GPER-1 antagonist, G15. The vasoconstrictor response to G1 was also significantly attenuated by Y-27632, PD98059, SB203580, GF109203X, genistein, AG-1478, and nifedipine, but not LY294002 and SP600125. Western blotting indicated the expression of GPER-1 in renal artery, medulla and cortex of rat kidney. In conclusion, GPER-1 could substantially modulate vascular responses through a variety of signalling pathways including ROCK, PKC, p38 MAPK, p42/44 MAPK, tyrosine kinase, EGF receptor kinase and VOCC but not JNK or PI3K in isolated perfused rat kidney.

Hyperosmolar glucose induces vasoconstriction through Rho/Rho-kinase pathway in the rat aorta.

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.

Proton pump inhibitors omeprazole, lansoprazole and pantoprazole induce relaxation in the rat lower oesophageal sphincter.

OBJECTIVES: We aimed to investigate effects of the proton pump inhibitors (PPIs) omeprazole, lansoprazole and pantoprazole, which are currently used for the treatment of hyperacidity and gastro-oesophageal reflux, on the reactivity of the isolated rat lower oesophageal sphincter. METHODS: Omeprazole, lansoprazole and pantoprazole (all 10(-9) -10(-3) m, cumulatively) were tested on carbachol-induced (10(-6) m) contraction. In addition, the effects of PPI preincubation (all 10(-3) m) on the contractions induced by cumulative carbachol (10(-9) -10(-5) m), angiotensin-2 (10(-9) -10(-5) m) or electrical field stimulation (EFS; 40 V, 32 Hz, 1 ms, 10 s) were assessed. Finally, the effects of PPI on the spontaneous contractile activity of the tissue were also evaluated. KEY FINDINGS: PPI relaxed precontracted lower oesophageal sphincter in a concentration-dependent manner and suppressed carbachol-, angiotensin- and EFS-induced contractions. Furthermore, PPI attenuated spontaneous contractile activity of the tissue. CONCLUSIONS: Omeprazole, lansoprazole and pantoprazole had a suppressor effect on lower oesophageal sphincter contractions.

CDP-choline-induced contractions in the mouse gastric fundus through purinoceptors and Rho/Rho-kinase signalling.

AIMS: This study aimed to investigate the effects of cytidine-5'-diphosphocholine (CDP-choline), an endogenous lipid precursor, on the reactivity of the mouse gastric fundus and to determine the mechanism(s) mediating its effects. MAIN METHODS: Possible contractile effect of CDP-choline (10(-5)-10(-2)M) was investigated in the absence and presence of a muscarinic receptor antagonist, atropine (3 × 10(-6)M), an acetylcholine esterase inhibitor, physostigmine (10(-6)M), a Na(+) channel blocker, tetrodotoxin (TTX, 3 × 10(-6)M), a Rho-kinase inhibitor, Y-27632 (10(-5) M), a purinoceptor antagonist, suramin (2 × 10(-4)M), a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine (L-NA, 3 × 10(-4)M), a Ca(2+) channel blocker, nifedipine (10(-6)M), an α(7) nicotinic receptor antagonist, methyllycaconitine citrate (MLA, 10(-6)M) and a G protein (G(i/o)) inhibitor, pertussis toxin (PTX, 2 µg/ml). The metabolites of CDP-choline, namely choline (10(-4)-10(-2)M), cytidine 5'-triphosphate (CTP, 10(-5)-10(-2)M), cytidine (10(-5)-10(-2)M) and cytidine monophosphate (CMP, 10(-3)-10(-2)M) were also tested. Besides, phosphorylation of MYPT1, which indicates Rho-kinase activity, was also detected. KEY FINDINGS: CDP-choline produced contractions in a concentration-dependent manner. The contractions were not affected by atropine, physostigmine, TTX, PTX, MLA or L-NA. However, Y-27632, suramin or nifedipine partly reduced these contractions. CDP-choline increased phosphorylation of MYPT1. Among CDP-choline metabolites, cytidine had no contractile effects. However, choline induced considerable contractions, which were sensitive to atropine. CMP and CTP had also contractile activity, comparable to that of CDP-choline. SIGNIFICANCE: These results suggest that CDP-choline produced contraction through, at least in part, purinoceptors and Rho/Rho-kinase signalling in the mouse gastric fundus.

Nebivolol, but not metoprolol, improves endothelial function of the corpus cavernosum in apolipoprotein e-knockout mice.

To determine the effects and underlying mechanisms of treatment with the beta-receptor blockers nebivolol and metoprolol on penile endothelial function in apolipoprotein E (ApoE)-/- mice, wild-type (WT) and ApoE-/- mice were fed with a cholesterol-rich diet for 7 weeks. ApoE-/- mice were treated with nebivolol (10 mg/kg/day) or metoprolol (90 mg/kg/day). Endothelial function of aortic and corpora cavernosal tissue was assessed by pharmacological stimulation with carbachol (endothelium dependent) or glycerol trinitrate (endothelium independent) in organ bath experiments. Atherosclerotic lesion formation was evaluated with oil-red staining, and modulation of reactive oxygen species (ROS) production was determined with lipid peroxidation. Heart rate, but not blood pressure, was decreased in nebivolol- and metoprolol-treated ApoE-/- mice (p < 0.01) compared with controls and WT mice without significant intergroup differences. Atherosclerotic lesion formation in the aortic root was increased in ApoE-/- mice (p < 0.01) with a more significant improvement in nebivolol- (p < 0.01) compared with metoprolol-treated mice (p < 0.05). Endothelium-dependent relaxation of the corpora cavernosa was significantly impaired in ApoE-/- mice (p < 0.05), which improved in nebivolol- versus metoprolol-treated mice. Efficacy of endothelium-dependent relaxation was comparable in aortic and penile tissue. Quantification of ROS production via lipid peroxidation revealed a significant reduction of superoxide anion production in nebivolol-treated (p < 0.05) but not metoprolol-treated mice compared with ApoE-/- controls. Nebivolol improves penile endothelial function as a surrogate of erectile function in ApoE-/- mice. These effects may be related to a reduction of ROS production, which is independent of heart rate reduction, because metoprolol did not increase endothelial function.

Nitric oxide does not downregulate Rho-kinase (ROCK-2) expression in rat coronary endothelial cells.

Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (approximately 16 h) with an NO generator, A-23187 (10 to 10 M), NO donors, such as sodium nitroprusside (10 to 10 M), glyceryl trinitrate (10 to 10 M), 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (10 to 10 M), and NaNO2 (10 to 10 M) or a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methylester (2 x 10 M), or two ROCK inhibitors, (+)-(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10 M) and fasudil (10 M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 +/- 0.32 microM in control and 4.0 +/- 0.93 microM and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells.

Urocortin induces endothelium-dependent vasodilatation and hyperpolarization of rat mesenteric arteries by activating Ca2+-activated K+ channels.

Urocortin, a member of corticotropin releasing factor (CRF) peptide family, has positive chronotropic and inotropic effects on heart and also shows a vasodilatory effect. However, the mechanism underlying its vasodilatory effect has yet to be elucidated. Endothelium-dependent relaxation of resistance arteries is mainly achieved by activation of K+ channels. Therefore, we investigated possible role of K+ channels and hyperpolarization for the vasodilatory effect of urocortin using the isolated perfused rat mesenteric arteries. Urocortin (0.2 nM) produced a slow-onset decrease in the perfusion pressure of the mesenteric vascular bed, which was elevated by an alpha1-adrenoceptor agonist, phenylephrine (2-4 microM). Urocortin also hyperpolarized the main mesenteric artery. Removal of endothelium with saponin treatment considerably inhibited the relaxation and hyperpolarization induced by urocortin. In contrast, the hyperpolarization was not significantly changed by cyclooxygenase inhibitor, indomethacin (1 microM) and/or nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (100 microM). Urocortin-induced relaxation was not affected by the combination of a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), indomethacin and N(omega)-nitro-L-arginine. However, the relaxation and hyperpolarization were abolished by high extracellular potassium concentration (40 mM) or by a large conductance Ca(2+)-activated K+ channel blocker, charybdotoxin (1 nM). Glibenclamide (1 microM), an ATP-dependent K+ channel inhibitor, did not affect the relaxation and hyperpolarization. These results suggest that urocortin causes endothelium-dependent relaxation and hyperpolarization of rat mesenteric arteries, probably through the activation of charybdotoxin sensitive Ca2+-activated K+ channels. These findings also indicate an essential role of the endothelium for the urocortin-elicited vascular relaxation and hyperpolarization.

Role of Rho-kinase in contractions of ureters from rabbits with unilateral ureteric obstruction.

OBJECTIVE: To investigate the expression of two isoforms of Rho-kinase (ROCK) and its functional role in the pathophysiological control of smooth muscle contraction in rabbits with unilateral ureteric obstruction (UUO). MATERIAL AND METHODS: Left UUO was created in 14 rabbits and eight other rabbits (controls) had sham operations. After 2 weeks all the rabbits were killed. Ureteric strips suspended in an organ bath were used for functional studies and the effects of Y-27632, a specific inhibitor of Rho-kinase, on spontaneous contractions and electrical field stimulation (EFS; 50 V, 1 ms, 16 Hz, for 20 s), carbachol- (10(-7)-10(-4)m), phenylephrine- (10(-7)-10(-4)m) and KCl- (50 mm) induced contractions were analysed. Western blotting was used to determine expression levels of Rho-kinase protein in the ureters of UUO and control rabbits. RESULTS: In the functional analysis, the contractions induced by EFS, KCl, phenylephrine and carbachol in the ureteric strips from rabbits with UUO were significantly greater than those from the control rabbits. Y-27632 considerably suppressed the ureter contractile responses in both UUO and control rabbits. Western blot analysis showed that both ROCK-1 and ROCK-2 proteins were expressed in the rabbit ureter. In accordance with the functional studies, the expression levels of both ROCK-1 and ROCK-2 were significantly greater in the ureters of UUO rabbits than in the controls. CONCLUSIONS: Y-27632 suppressed ureteric contractions in the rabbits with UUO. Western blot analysis also confirmed greater expression levels of ROCK-1 and ROCK-2 in the ureters of UUO rabbits. It is important to elucidate by which mechanisms the Rho-kinase pathway affects ureteric function after obstruction.

Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with allergic contact dermatitis.

Angiotensin-converting enzyme (ACE) plays an important role in the physiological control of blood pressure and inflammation. Insertion/deletion (I/D) polymorphism of the gene for ACE was investigated in relation to cardiovascular, cerebrovascular, neurodegenerative and inflammatory diseases. The purpose of the present study was to investigate the possible association between allergic contact dermatitis and insertion/deletion polymorphism of the ACE gene. A total of 90 patients with allergic contact dermatitis and 160 control persons were enrolled in the present study. ACE I/D genotypes were determined by the polymerase chain reaction. Allelic frequencies and genotype distribution of the ACE I/D polymorphism in the patient group were significantly different from control group (ACE II genotype 30.0% versus 17.5%, P = 0.022; ACE I allele 51.7% versus 39.4%, P = 0.008). Our data suggest that the ACE polymorphism could be a risk factor for patients with allergic contact dermatitis.