In non – erythroid cells insulin stimulates a signal transduction pathway that results in the activation of phosphoinositide 3 – kinase (PI3K) and phosphorylation of phosphodiesterase 3 (PDE3). mechanism resulting in cAMP hydrolysis and inhibition of ATP release. We show that insulin attenuates Mas 7 – induced increases in cAMP and that selective inhibitors of PDE3 (cilostazol) or PI3K (LY294002) rescue this effect AS-605240 of insulin. In addition we exhibited that both cilostazol and LY294002 prevent insulin – induced attenuation of Mas 7 – induced ATP release. These results provide support for 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 prospects AS-605240 ultimately to decreased ATP release in response to Mas 7. Since the activation of Gi is required for reduced O2 tension – induced ATP release from erythrocytes and insulin has been shown to inhibit that release these results suggest a novel mechanism by which supraphysiological levels of plasma insulin such as those reported in humans Rabbit Polyclonal to GAK. with prediabetes could inhibit ATP release from erythrocytes. Erythrocyte – derived ATP has been shown to participate in the matching of O2 supply with demand in skeletal AS-605240 muscle mass. Thus pathological increases in circulating insulin could via activation of PDE3 inhibit ATP release from erythrocytes depriving the peripheral blood circulation of a mechanism that regulates delivery of O2 to meet tissue metabolic need. ratio indicated that a switch experienced occurred a Fisher’s LSD test was performed to identify individual differences. Results were reported as means ± the standard error of the mean (SEM). Results Effect of insulin on Mas 7-induced increases in cAMP and ATP release from human erythrocytes Consistent with previous studies incubation of erythrocytes with insulin (1 nM) experienced no effect on basal ATP release but it attenuated Mas 7 – induced ATP release from these cells (Fig.1) (37). To ensure that decreases in ATP release could not be attributed to insulin – induced decreases in erythrocyte ATP content total intraerythrocyte ATP was measured. Total ATP levels were not different in the absence and presence of 1 1 nM insulin (1.64 ± 0.17 and 1.52 ± 0.17 mM for control and insulin treated respectively). Previously we reported that increases in cAMP are required for ATP release from human erythrocytes (15). To begin to investigate the mechanism by which insulin inhibits ATP release we determined the effect of insulin on Mas 7 – induced increases in cAMP. Insulin (1 nM) experienced AS-605240 no effect on basal cAMP levels (data not shown). However as depicted in Fig. 2 insulin did attenuate Mas 7 – induced increases in cAMP. These results support the hypothesis that insulin – induced inhibition of ATP release from human erythrocytes is associated with increased cAMP hydrolysis. Physique 1 Effect of insulin on mastoparan 7 – induced ATP release from erythrocytes. Washed erythrocytes were incubated with mastoparan 7 (Mas 7 10 μM) 20 moments after the addition of either insulin (1 nM) or its vehicle (saline) (n=9). The maximal … Physique 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 … Effect of a PDE3 inhibitor on insulin-induced attenuation of cAMP accumulation within erythrocytes Insulin has been shown to antagonize the effects of cAMP by stimulating the hydrolysis of this cyclic nucleotide (25-27). Insulin stimulates cAMP hydrolysis by activating a signal transduction pathway resulting in the phosphorylation and activation of PDE3 a PDE that has been shown to be present human and rabbit erythrocytes (24). To determine if inhibition of PDE3 can rescue insulin – induced inhibition AS-605240 of increases in cAMP stimulated by Mas 7 erythrocytes were pretreated with the selective PDE3 inhibitor cilostazol (100 μM) in the absence and presence of 1 1 nM insulin (38). As shown in Table 1 100 μM cilostazol potentiated Mas 7 – induced cAMP accumulation. More importantly.