Insulin inhibits human erythrocyte cAMP accumulation and ATP release: role of phosphodiesterase 3 and phosphoinositide 3-kinase

Abstract

In non-erythroid cells, insulin stimulates a signal transduction pathway that results in the activation of phosphoinositide 3-kinase (PI3K) and subsequent phosphorylation of phosphodiesterase 3 (PDE3). Erythrocytes possess insulin receptors, PI3K and PDE3B. These cells release adenosine triphosphate (ATP) when exposed to reduced O(2) tension via a signaling pathway that requires activation of the G protein, Gi, as well as increases in cAMP. Although insulin inhibits ATP release from human erythrocytes in response to Gi activation by mastoparan 7 (Mas 7), no effect on cAMP was described. Here, we investigated the hypothesis that insulin activates PDE3 in human erythrocytes via a PI3K-mediated mechanism resulting in cAMP hydrolysis and inhibition of ATP release. Incubation of human erythrocytes with Mas 7 resulted in a 62 +/- 7% increase in cAMP (n = 9, P < 0.05) and a 306 +/- 69% increase in ATP release (n = 9, P < 0.05), both of which were attenuated by pre-treatment with insulin. Selective inhibitors of PDE3 (cilostazol) or PI3K (LY294002) rescued these effects of insulin. These results support the hypothesis that insulin activates PDE3 in erythrocytes via a PI3K-dependent mechanism. Once activated, PDE3 limits Mas 7-induced increases in intracellular cAMP. This effect of insulin leads, ultimately, to decreased ATP release in response to Mas 7. Activation of Gi is required for reduced O(2) tension-induced ATP release from erythrocytes and this ATP release has been shown to participate in the matching of O(2) supply with demand in skeletal muscle. Thus, pathological increases in circulating insulin could, via activation of PDE3 in erythrocytes, inhibit ATP release from these cells, depriving the peripheral circulation of one mechanism that could aid in the regulation of the delivery of O(2) to meet tissue metabolic need.

Figure 1

Effect of insulin on mastoparan 7 – induced ATP release from erythrocytes. Washed erythrocytes were incubated with mastoparan 7 (Mas 7, 10 µM) 20 minutes after the addition of either insulin (1 nM) or its vehicle (saline) (n=9). The maximal ATP release in response to Mas 7 treatment was reported. Values are the means ± SE. * = greater than all other values (P < 0.05).

Figure 2

Effect of cilostazol or LY294002 on mastoparan 7 – induced cAMP increases in erythrocytes in the absence and presence of insulin. Washed erythrocytes were incubated with cilostazol (Cilo, 100 µM) or LY294002 (LY, 10 µM) for 30 min in the absence or presence of insulin (1 nM) or its vehicle (saline) and then cAMP increases were stimulated by mastoparan 7 (Mas 7, 30 µM). (n=9). cAMP levels were determined 30 minutes after the addition of Mas 7. Values are the means ± SE. * = greater than all other values (P < 0.05).

Figure 3

Effect of cilostazol on mastoparan 7 – induced ATP release from human erythrocytes in the absence and presence of insulin. Washed erythrocytes were incubated with cilostazol (Cilo, 100 µM) for 30 min in the absence or presence of insulin (1 nM) or its vehicle (saline) and then ATP release was induced by mastoparan 7 (Mas 7, 10 µM) (n=8). The maximal ATP release in response to Mas 7 treatment was reported. Values are the means ± SE. * = greater than all other values (P < 0.05), NS = no significant difference between values.

Figure 4

Effect of LY294002 on mastoparan 7 – induced ATP release from human erythrocytes in the absence and presence of insulin. Washed erythrocytes were incubated with LY294002 (LY, 10 µM) for 30 min in the absence or presence of insulin (1 nM) or its vehicle (saline) and then ATP release was induced by mastoparan 7 (Mas 7, 10 µM) (n=8). The maximal ATP release in response to Mas 7 treatment was reported. Values are the means ± SE. * = greater than all other values (P < 0.05), NS = no significant difference between values.

Figure 5

Proposed mechanism by which insulin activates PDE3 in erythrocytes, inhibiting ATP release from these cells. G_αi, heterotrimeric G protein Gi; Mas 7, mastoparan 7; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; CFTR, cystic fibrosis transmembrane conductance regulator; PI3K, phosphoinositide – 3 kinase; PDE3B, phosphdiesterase 3B; AMP, adenosine monophosphate