Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP - PubMed (original) (raw)

Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP

Madelyn S Hanson et al. Am J Physiol Heart Circ Physiol. 2008 Aug.

Abstract

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.

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Figures

Fig. 1.

Fig. 1.

Identification of phosphodiesterase 3B (PDE 3B) in rabbit and human erythrocyte membranes. A: human erythrocyte membranes and purified human PDE3B were probed with a polyclonal antibody generated against the NH2 terminus of human PDE3B. B: rabbit and human erythrocyte membranes were probed with either a polyclonal antibody directed against the COOH terminus of human PDE3B (rabbit erythrocyte membranes, representative of 6 studies) or a polyclonal antibody generated against the NH2 terminus of human PDE3B (human erythrocyte membranes, representative of 14 studies).

Fig. 2.

Fig. 2.

Effect of 3-isobutyl-1-methylxanthine (IBMX; 10 μM) on iloprost (Ilo)-induced levels of cAMP in rabbit and human erythrocytes. A: washed erythrocytes from rabbits (n = 5) were incubated with Ilo (1 μM) in the presence of 10 μM IBMX or its vehicle [_N,N_-dimethylformamide (DMF)]. B: washed erythrocytes from humans (n = 8) were incubated with Ilo (1 μM) in the presence of 10 μM IBMX or its vehicle (DMF). In both A and B, cells were incubated with IBMX or its vehicle (DMF) for 30 min, and then cAMP increases were stimulated by the addition of Ilo for 15 min. Values are means ± SE. P < 0.05; *different from control and inhibitor alone; **greater than all other values. RBCs, red blood cells.

Fig. 3.

Fig. 3.

Effect of inhibitors of PDE3 on Ilo-induced increases in cAMP in rabbit erythrocytes. A: washed erythrocytes from rabbits (n = 6) were incubated with Ilo (1 μM) in the presence of 20 μM milrinone (Mil) or its vehicle (DMF). B: washed erythrocytes from rabbits (n = 6) were incubated with Ilo (1 μM) in the presence of 10 μM cilostazol (Cilo) or its vehicle (DMF). Cells were incubated with Mil, Cilo, or DMF for 30 min, and then cAMP increases were stimulated by the addition of Ilo for 15 min. Values are means ± SE. P < 0.05; *different from control and inhibitor alone; **greater than all other values.

Fig. 4.

Fig. 4.

Effect of PDE3 inhibitors on Ilo-induced levels of cAMP in human erythrocytes. A: washed erythrocytes from humans (n = 6) were incubated with Ilo (1 μM) in the presence of 20 μM Mil or its vehicle (DMF). B: washed erythrocytes from humans were incubated with Ilo (1 μM) in the presence of 10 μM (n = 12), 30 μM (n = 9), or 100 μM (n = 6) Cilo or its vehicle (DMF). C: washed erythrocytes from humans (n = 4) were incubated with Ilo (1 μM) in the presence of 30 μM cilostamide (C'mide) or its vehicle (DMF). Cells were incubated with Mil, Cilo, C'mide, or DMF for 30 min, and then cAMP increases were stimulated by the addition of Ilo for 15 min. Values are means ± SE. P < 0.05; *different from control and inhibitor alone (A and C) or corresponding Ilo (B); **greater than all other values. D: human erythrocyte membranes and human platelet membranes (representative of 7 studies) were probed with a monoclonal antibody generated against integrin αIIb (CD41).

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