Diamide decreases deformability of rabbit erythrocytes and attenuates low oxygen tension-induced ATP release

Abstract

Exposure of erythrocytes to reduced oxygen (O(2)) tension activates the heterotrimeric G-protein Gi, resulting in the accumulation of cyclic AMP (cAMP) and release of ATP. The mechanism by which exposure of erythrocytes to reduced O(2) tension activates Gi is not known. Here we investigate the hypothesis that, in rabbit erythrocytes, ATP release in response to exposure to reduced O(2) tension is linked to erythrocyte membrane deformability. If this hypothesis is correct, then decreasing the deformability of the erythrocyte membrane should decrease the release of ATP in response to reduced O(2) tension. We report that treating erythrocytes with diamide, a compound that decreases erythrocyte deformability, inhibits low O(2) tension-induced ATP release. Treating erythrocytes with diamide does not, however, interfere with cAMP accumulation or ATP release in response to a direct activator of Gi (mastoparan 7) or in response to receptor-mediated activation of Gs (the prostacyclin analog, iloprost). These results demonstrate that diamide (100 micromol/L) does not directly inhibit the signaling pathways for ATP release from rabbit erythrocytes and support the hypothesis that low O(2) tension-induced ATP release from these cells is linked to membrane deformability.

Figure 1. Effect of diamide on red cell transit time (RCTT)

Rabbit erythrocytes were incubated with 100 μM diamide for 30, 45, and 60 min and RCTT was measured (n=4). Values are the means ± SE. * p < 0.05, different from baseline ; † p<0.01, different from baseline .

Figure 2. Effect of diamide on low O₂ tension-induced ATP release from rabbit erythrocytes

Erythrocytes were incubated with 100 μM diamide or its vehicle (saline) for 30 min while exposed to gas with a composition of 15% O₂, 6 % CO₂, balance N₂ in a tonometer. The cells were then exposed sequentially to gases with compositions of 4.5 % O₂. 6% CO₂, balance N₂ and 0% O₂, 6% CO₂, balance N₂. ATP was measured 30 min after exposure to 15% O₂ and 10 min after exposure to 4.5% O₂ and 0% O₂ (n=8). The percent increase in ATP release from baseline (15% O₂) is reported. Values are the means ± SE. * p< 0.05, different from respective baseline, † p< 0.05, different from respective diamide treated group

Figure 3. Effect of diamide on mastoparan 7 (MAS 7)–induced cAMP accumulation (A) and ATP release (B) from rabbit erythrocytes

Erythrocytes were incubated with 100 μM diamide or its vehicle (saline) 30 min before the addition of MAS 7. cAMP accumulation (A) was measured 30 min after the addition of MAS 7 (30 μM, n=5) and ATP release (B) was measured 5, 10, and 15 min after the addition of MAS 7 (10 μM, n=8). For ATP release, percent increase from baseline for peak values is reported. Values are the means ± SE. † p<0.01, different from baseline; NS, not significantly different.

Figure 4. Effect of diamide on iloprost (ILO)-induced cAMP accumulation (A) and ATP release (B) from rabbit erythrocytes

Erythrocytes were incubated with 100 μM diamide or its vehicle (saline) 30 min before the addition of ILO. cAMP accumulation (A) was measured 15 min after the addition of ILO (1 μM, n=9) , and ATP release (B) was measured after 5, 10, and 15 (1 μM, n=7). For ATP release, percent increase from baseline for peak values is reported. Values are the means ± SE. † p<0.01, different from baseline; NS, not significantly different