Identification of cytosolic phosphodiesterases in the erythrocyte: a possible role for PDE5

Abstract

Background: Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP.

Material/methods: Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP).

Results: Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP.

Conclusions: PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes.

Figure 1

Identification of PDE4D isoforms in human erythrocytes. Erythrocyte cytosol preparations were incubated with an affinity purified primary antibody generated against the C-terminus of all PDE4D variants (representative of 5 individual samples, 40 μg protein per lane).

Figure 2

(A) Identification of the PDE3A isoform in human erythrocytes. Erythrocyte cytosol preparations were incubated with a goat polyclonal primary antibody directed against the N-terminus of PDE3A (representative of 8 individual samples, 40 μg protein per lane). (B) Identification of the PDE3A isoform in human erythrocytes. Erythrocyte cytosol preparations were incubated with a goat polyclonal primary antibody against the C-terminus of PDE3A (representative of 8 individual samples, 40 μg protein per lane).

Figure 3

Identification of the PDE5A isoform in human erythrocytes. Erythrocyte cytosol preparations were incubated with an affinity purified primary antibody against the C-terminus of PDE5A (representative of 5 individual samples, 40 μg protein per lane).

Figure 4

Effect of the dbcGMP (10 μM) on cAMP levels in rabbit erythrocytes (n=4). Erythrocytes were incubated with dbcGMP for 30 min. Values are means ±SE. * different from control (N′,N-dimethylformamide, DMF), (p<0.01); ** different from all other values (P<0.01).

Figure 5

Effect of YC1 (100 μM) & spermine NONOate (NO, 100 nM) or the combination of YC1 and NO in the presence of either zaprinast (ZAP, 10 μM) or vinpocetine (VIN, 30 μM) on cGMP levels in rabbit erythrocytes (n=4). Erythrocytes were incubated with either ZAP or VIN for 30 min prior to stimulation with YC1 and spermine NONOate for 20 min. Values are means ±SE. * different from control (N′,N-dimethylformamide, DMF), (p<0.01); ** different from all other values (P<0.05).

Figure 6

Effect of the dbcGMP (10 μM) on isoproterenol (ISO, 1 μM)-induced increases in cAMP levels in rabbit erythrocytes (n=9). Erythrocytes were incubated with dbcGMP for 30 min prior to stimulation with ISO for 30 min. Values are means ±SE. * different from control (saline), (p<0.01).

Figure 7

Panel A: Effect of YC1 (100μM) and spermine NONOate (NO, 100 nM) on un-stimulated (control) and isoproterenol (ISO, 1 μM)-induced increases in cAMP in the absence and presence of vinpocetine (VIN, 30 μM) or zaprinast (10 μM) in rabbit erythrocytes (n=5). Erythrocytes were incubated with YC1 and spermine NONOate alone or with VIN or ZAP for 30 min prior to stimulation with ISO for 30 min. Values are means ±SE. * different from control (N′,N-dimethylformamide, DMF), (p<0.01); ** different from all other values (P<0.01). Panel B: Effect of YC1 (100 μM) and spermine NONOate (NO, 100 nM) as well as zaprinast (10 μM) on isoproterenol (ISO, 1 μM)-induced increases in human erythrocytes (n=7). Erythrocytes were incubated with YC1 and spermine NONOate alone, ZAP alone or the combination for 30 min prior to stimulation with ISO for 30 min. Values are means ±SE. * different from control (N′,N-dimethylformamide, DMF), (p<0.01); ** different from all other values (P<0.01).