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. 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

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

WOS: 000253874000005PubMed ID: 18287881Rho 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 (similar to 16 h) with an NO generator, A-23187 (10(-7) to 10(-6) M) NO donors, such as sodium nitroprusside (10(-7) to 10-6 M) glyceryl trinitrate (10(-7) to 10-6 M), 2,2'-(hydroxynitro- sohydrazono)bis-ethanimine (10(-7) to 10(-6) M), and NaNO2 (10(-4) to 10(-3) M) or a nitric oxide synthase (NOS) inhibitor, N-G-nitro-L-arginme methylester (2x10(-4) M), or two ROCK inhibitors, (-+)(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-5) M) and fasudil (10(-5) 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 mu M in control and 4.0 +/- 0.93 mu M 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

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

WOS: 000249953400037PubMed ID: 17711509To 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). 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 Rhokinase, 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. 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. 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