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

Abstract

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.

Keywords: C3; Erythrocyte deformability; Fasudil; Lysophosphatidic acid; Rho-kinase; RhoA; Y-27632.

Figure 1. Western blotting for RhoA (A) and Rho-kinase (ROCK-2) (B) in human erythrocytes. Both cytosolic and membranous fractions for RhoA (MW: 24 kDa) and cytosolic fraction for ROCK-2 (MW: 160 kDa) were studied. A, B, and C are erythrocytes isolated from different volunteers.

Figure 2. Effect of lysophosphatidic acid (10-5, 2x10-5, 5x10-5 M) on shear stress for half-maximal deformation (SS1/2) at different time periods. Data are mean ± SE, n=8. Statistical analyses were done by Wilcoxon signed ranks test (*: p<0.05, difference from control).

Figure 3. The potential effect of Y-27632 (A) and fasudil (B) on LPA-induced deformability. Data are mean ± SE, n=9. Statistical analysis was done by Wilcoxon signed ranks test (*: p<0.05, difference from control; +: p<0.05, difference from LPA).

Figure 4. Effect of lysophosphatidic acid (10-5 M) on RhoA activation of erythrocytes. Rho-GTP levels were measured with G-LISA activation assay kit and expressed as % RhoA activation. Data are mean ± SE, n=8. Statistical analysis was done by one-sample t-test (*: p<0.05, difference from control).

Figure 5. Rho-GTP level in erythrocytes treated with exoenzyme C3 transferase (3 µg/mL). Rho-GTP levels were measured with G-LISA activation assay kit and expressed as % RhoA activation. Data are mean ± SE, n=5. Statistical analysis was done by one-sample t-test (p=0.0526).

Figure 6. A) Rho-GTP level in erythrocytes exposed to mechanical stress (120 Pa, 30 s). Rho-GTP levels were measured with G-LISA activation assay kit and expressed as % RhoA activation. Data are mean ± SE, n=4. Statistical analyses were done by one-sample t-test (**: p<0.01, difference from control). B) Mechanical stress induced deterioration of deformability. Data are mean ± SE, n=8. Statistical analysis was done by paired t-test (*: p<0.05, difference from control).