Iloprost- and isoproterenol-induced increases in cAMP are regulated by different phosphodiesterases in erythrocytes of both rabbits and humans

Abstract

Activation of the G protein G(s) results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (Ilo), a PGI(2) analog, and isoproterenol (Iso), a beta-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways.

Fig. 1.

A: effect of 3-isobutyl-1-methylxanthine (IBMX; 10 μM) on iloprost (Ilo; 1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 7). Erythrocytes were incubated with IBMX for 30 min before addition of Ilo for 15 min. B: effect of IBMX (10 μM) on isoproterenol (Iso; 1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 7). Erythrocytes were incubated with IBMX for 30 min before addition of Iso for 30 min. Values are means ± SE. *Different from respective control (P < 0.05); †different from all other values (P < 0.01). RBC, red blood cell.

Fig. 2.

A: effect of cilostazol (Cilo; 10 μM) on Ilo (1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 7). Erythrocytes were incubated with Cilo for 30 min before addition of Ilo for 15 min. B: effect of Cilo (10 μM) on Iso (1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 4). Erythrocytes were incubated with Cilo for 30 min before addition of Iso for 30 min. Values are means ± SE. *Different from respective control (P < 0.05); †different from all other values (P < 0.01). NS, not statistically different.

Fig. 3.

A: effect of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 30 μM) on Ilo (1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 7). Erythrocytes were incubated with EHNA for 30 min before addition of Ilo for 15 min. B: effect of EHNA (30 μM) on Iso (1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 8). Erythrocytes were incubated with EHNA for 30 min before addition of Iso for 30 min. Values are means ± SE. *Different from respective control (P < 0.05); †different from all other values (P < 0.01).

Fig. 4.

A: identification of PDE2A in rabbit erythrocyte membranes. Rabbit erythrocyte membranes were probed with a monoclonal antibody generated against amino acids 869-912 in the N terminus of human PDE2A (representative of 7 individual membrane preparations). B: identification of PDE2A in human erythrocyte membranes. Human erythrocyte membranes were probed with a polyclonal antibody directed to an internal region of human PDE2A (representative of 8 individual membrane preparations).

Fig. 5.

A: effect of rolipram (Rol; 20 μM) on Ilo (1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 7). Erythrocytes were incubated with Rol for 30 min before stimulation with Ilo for 15 min. B: effect of Rol (20 μM) on Iso (1 μM)-induced increases in cAMP in rabbit erythrocytes (n = 9). Erythrocytes were incubated with Rol for 30 min before addition of Iso for 30 min. Values are means ± SE. *Different from respective control (P < 0.05); †different from all other values (P < 0.05).

Fig. 6.

Effect of Rol (10 μM; n = 10) and EHNA (10 μM; n = 8) alone and in combination (n = 9) on Iso (1 μM)-induced increases in cAMP in rabbit erythrocytes. Erythrocytes were incubated with inhibitors for 30 min before addition of Iso for 30 min. Values are means ± SE. *Different from respective control (P < 0.05); †different from all other values (P < 0.01).

Fig. 7.

Effect of EHNA (30 μM) or Rol (20 μM) on Iso (1 μM)-induced increases in cAMP in human erythrocytes (n = 7). Erythrocytes were incubated with either Rol or EHNA for 30 min before addition of Iso for 30 min. Values are means ± SE. *Different from respective control (P < 0.05); †different from all other values (P < 0.01).

Fig. 8.

Effect of Cilo (30 μM) on Ilo (10 nM)-induced increases in ATP release from human erythrocytes (n = 9). Erythrocytes were incubated with Cilo for 30 min before addition of Ilo. Values are means ± SE. *Different from Ilo alone (P < 0.01).

Fig. 9.

Effect of EHNA (30 μM) and Rol (20 μM) on Iso (1 μM)-induced increases in ATP release from human erythrocytes (n = 18). Erythrocytes were incubated with EHNA and Rol for 30 min before addition of Iso. Values are means ± SE. *Different from Iso alone (P < 0.01).

Fig. 10.

Model of PDE activity localized with the β₂-adrenergic receptor (β₂AR) and PGI₂ receptor (IPR) in rabbit and human erythrocytes. Activation of the IPR leads to generation of cAMP in a restricted cAMP pool that is hydrolyzed by PDE3. In contrast, activation of the β₂AR leads to generation of a distinct cAMP pool that is regulated by the activities of both PDE2 and PDE4. AC, adenylyl cyclase; G_s, heterotrimeric G protein.