Simvastatin and GGTI-2133, a geranylgeranyl transferase inhibitor, increase erythrocyte deformability but reduce low O(2) tension-induced ATP release

Abstract

Statin drugs inhibit 3-hydroxy-3-methylglutaryl CoA reductase, which reduces the synthesis of both cholesterol and isoprenoids (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), with the latter being lipid molecules responsible for the posttranslational modification of small GTP-binding proteins such as Rho. Effects of statins, independent of lowering blood cholesterol levels, are thought to occur by inhibition of Rho/Rho kinase. The Rho kinase inhibitor Y-27632 has been reported to increase both erythrocyte deformability and low O2 tension-induced ATP release. Here, we tested the hypothesis that by inhibiting Rho/Rho kinase, simvastatin would increase both erythrocyte deformability and low O2 tension-induced ATP release. Male Sprague-Dawley rats were divided into two groups, control or simvastatin treated [simvastatin-supplemented chow (0.02%)], for 4 wk. Simvastatin treatment increased rat erythrocyte deformability compared with controls (n = 6, P < 0.05). However, erythrocytes of simvastatin-treated rats (n = 9, P < 0.05) exhibited impaired low O2 tension-induced ATP release. Similarly, the geranylgeranyl transferase inhibitor GGTI-2133 (10 μM) also increased deformability and impaired low O2 tension-induced ATP release in healthy human erythrocytes (P < 0.05). Interestingly, ATP release in response to mastoparan 7 (n = 7, P < 0.05), which directly activates Gi, and isoproterenol (n = 5, P < 0.05), which signals through Gs, was not altered by incubation with GGTI-2133. These results suggest that although statins increase erythrocyte deformability, likely by inhibiting geranylgeranylation, the finding that both statins and a geranylgeranyl transferase inhibitor attenuated low O2 tension-induced ATP release demonstrates that factors in addition to erythrocyte deformability are critical for ATP release in response to this physiological stimulus.

Fig. 1.

Simvastatin increases rat erythrocyte deformability. Erythrocytes isolated from rats treated with simvastatin displayed a lower red (blood) cell (RBC) transit time (RCTT) than erythrocytes from control rats, demonstrating that simvastatin enhanced erythrocyte deformability. Values are reported as means ± SE; n = 6. *P < 0.05.

Fig. 2.

The geranylgeranyl transferase inhibitor GGTI-2133 increased healthy human erythrocyte deformability. RCTT was determined before and after a 30-min incubation with GGTI-2133 or its vehicle [dimethyl formamide (DMF)]. Baseline RCTT measurements taken before incubation with either vehicle or GGTI-2133 were 8.48 ± 0.23 and 8.68 ± 0.19, respectively. After 30 min, RCTT measurements in the presence of vehicle or GGTI-2133 were 8.62 ± 0.15 and 8.48 ± 0.19, respectively. Values are reported as percent changes of RCTT from baseline ± SE; n = 9. *P < 0.05.

Fig. 3.

Inhibition of low O₂ tension-induced ATP release from erythrocytes after treatment with simvastatin and GGTI-2133. A: control (n = 10) and simvastatin-treated (n = 11) rat erythrocytes were equilibrated with 15% O₂-6% CO₂-balance nitrogen (normoxia; Po₂: 107 ± 2 mmHg, Pco₂: 38 ± 0.4mmHg, pH 7.37 ± 0.02). Baseline ATP values obtained for control rats and simvastatin-treated rats were 23.5 ± 4.7 and 21.7 ± 3.0 nmol/4 × 10⁸ erythrocytes, respectively. Erythrocytes were then exposed to 0% O₂-6% CO₂-balance nitrogen (reduced O₂; Po₂: 17 ± 1 mmHg, Pco₂: 38 ± 0.5 mmHg, pH 7.4 ± 0.02). ATP values for control and simvastatin-treated rats were 35.8 ± 7.0 and 23.8 ± 3.7 nmol/4 × 10⁸ erythrocytes, respectively. The percent change in ATP release in response to low O₂ was significantly increased for erythrocytes from control rats compared with those obtained from simvastatin-treated rats. Values are reported as percent changes in ATP release from normoxia ± SE; n = 9 for the control group and 8 for the simvastatin-treated group. *P < 0.05, significantly different from the simvastatin-treated group. B: healthy human erythrocytes were equilibrated in the presence of vehicle or GGTI-2133 with 15% O₂-6% CO₂-balance nitrogen (normoxia; Po₂: 112 ± 1 mmHg, Pco₂: 39 ± 0.6 mmHg, pH 7.33 ± 0.02). Values for ATP release during normoxia were 32.5 ± 4.6 and 35.7 ± 4.3 nmoles/10⁸ erythrocytes for vehicle and GGTI-2133, respectively. ATP values obtained after exposure to reduced O₂ (Po₂: 8 ± 1 mmHg, Pco₂: 40 ± 0.5 mmHg, pH 7.36 ± 0.02) were 53.5 ± 6.1 and 37.0 ± 3.8 nmol ATP/10⁸ RBCs for vehicle and GGTI-2133, respectively. Low O₂ tension-induced ATP release in the absence of GGTI-2133 was significantly different from all other values. Values are reported as percent changes in ATP release from normoxia ± SE; n = 5. *P < 0.05, significantly different from normoxia.

Fig. 4.

Simvastatin does not inhibit mastoparan 7 (Mas 7)-induced ATP release from erythrocytes. Baseline erythrocyte ATP release was measured from control and simvastatin-treated rats and was not different between the two groups. Mas 7 stimulated ATP release similarly from both control and simvastatin-treated rat erythrocytes. Values are reported as means ± SE; n = 6 for the simvastatin-treated group and 5 for the control group. *P < 0.05, significantly different from baseline.

Fig. 5.

GGTI-2133 does not inhibit Mas 7- or isoproterenol-induced ATP release from healthy human erythrocytes. A: healthy human erythrocytes were incubated with vehicle (DMF) or GGTI-2133 for 30 min. Baseline ATP values were not different between the two groups. Mas 7 stimulated ATP release in both the absence and presence of GGTI-2133. After Mas 7 incubation, ATP release was not different between erythrocytes treated with GGTI-2133 and vehicle. Values are reported as means ± SE; n = 7. *P < 0.05, significantly different from baseline. B: baseline values for ATP release were obtained from healthy human erythrocytes incubated with vehicle (DMF) or GGTI-2133 for 30 min. After incubation with isoproterenol (1 μM), ATP release was significantly increased in the presence and absence of GGTI-2133. Values are reported as means ± SE; n = 5. *P < 0.05, significantly different from baseline.