The Rho kinase inhibitor Y-27632 increases erythrocyte deformability and low oxygen tension-induced ATP release

Abstract

Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 μM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 μM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.

Fig. 1.

Western immunoblot showing the distribution of RhoA in membrane (M) and cytosol (C) of healthy human erythrocytes (A). Membrane and cytosolic fractions of healthy human erythrocytes were incubated with a RhoA polyclonal antibody generated against an internal region of RhoA. Twenty-five micrograms of total protein were loaded per lane, and values are reported as means ± SE, n = 3 experiments (B).

Fig. 2.

Effect of Y-27632 on erythrocyte deformability. Deformability of healthy human erythrocytes was determined in the absence and presence of Y-27632. Healthy human erythrocyte deformability was measured before and 30 min after incubation with Y-27632 or its vehicle, saline. Deformability values taken before incubation with either vehicle or Y-27632, 8.35 ± 0.16 and 8.39 ± 0.06 red cell transit time (RCTT), respectively, were not different between the two groups. A decrease in RCTT reflects an increase in erythrocyte deformability. Values are reported as means ± SE. *Different from baseline (P < 0.05), n = 9.

Fig. 3.

Effect of Y-27632 on low O₂ tension-induced ATP release from erythrocytes [red blood cells (RBC)]. Healthy human erythrocytes were equilibrated with a gas mixture containing 15% O₂-6% CO₂-balance nitrogen (Po₂ = 107 ± 1 mmHg) in the absence (filled bars) or presence (open bars) of Y-27632. After a 30-min incubation period, baseline erythrocyte ATP release was established (normoxia). The gas mixture was changed to 0% O₂-6% CO₂-balance nitrogen (Po₂ = 11 ± 1 mmHg), and ATP release from erythrocytes was measured (low O₂). During the course of the experiment, temperature (37°C), pH (7.37 ± 0.02), and Pco₂ (36 ± 0.4) were held constant. Values are reported as means ± SE. *Different from baseline (P < 0.05). †Different from all other values (P < 0.01), n = 13.

Fig. 4.

Effect of Y-27632 on mastoparan 7-induced ATP release from erythrocytes. Healthy human erythrocytes were incubated with vehicle (saline) or 1 μM Y-27632 for 30 min. Mastoparan 7 (10 μM) was added to the erythrocyte suspension. Baseline ATP values for vehicle and Y-27632 were 24.8 ± 4.0 and 27.6 ± 6.4 nmol/4 × 10⁸ erythrocytes, respectively, and ATP values after mastoparan 7 stimulation for vehicle and Y-27632 were 53.2 ± 7.2 and 60.0 ± 16.0 nmol/4 × 10⁸ erythrocytes, respectively. While each stimulated value was significantly different (P < 0.01) from its baseline value, there was no significant difference between the two groups. NS, not significant. The peak ATP value is reported as percent change from baseline ± SE, n = 5.

Fig. 5.

Effect of diamide in the absence and presence of Y-27632 on erythrocyte deformability. Healthy human erythrocyte deformability was measured before and after 30 min incubation with 100 μM diamide. Diamide-stiffened erythrocytes were then treated for an additional 30 min with 1 μM Y-27632. Values are reported as means ± SE. †Different from all other values (P < 0.01), n = 5.

Fig. 6.

Effect of diamide in the absence and presence of Y-27632 on low O₂ tension-induced ATP release from erythrocytes. Healthy human erythrocytes were equilibrated under normoxic conditions in the presence of vehicle (saline), diamide, and diamide plus Y-27632. Baseline ATP release was not different among the vehicle-treated, diamide-treated, and diamide- plus Y-27632-treated erythrocytes (30.2 ± 3.6, 36.6 ± 4.40, and 33.0 ± 4.5 nmol/4 × 10⁸ erythrocytes, respectively). Exposure to low O₂ tension increased ATP release from vehicle-treated and diamide- plus Y-27632-treated erythrocytes (47.5 ± 3.7 and 50.7 ± 11.2 nmol/4 × 10⁸ erythrocytes, respectively), but not from diamide-treated erythrocytes (46.4 ± 2.50 nmol/4 × 10⁸ erythrocytes). During the course of the experiment, temperature (37°C), pH (7.32 ± 0.01), and Pco₂ (41 ± 0.6 mmHg) were held constant. Values are reported as changes from baseline ± SE. *Different from baseline values (P < 0.05), n = 7.