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

Abstract

SUMMARY 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. Methods/Materials 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. Conclusion 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.

Erythrocyte: A systems model of the control of aggregation and deformability

“…There is a protein calmodulin (CaM) in erythrocytes (Nelson et al, 1983) and the processes, due to which changes of the concentration of cyclic monophosphates (cAMP, cGMP) occur. There are also ferments of their metabolism (Horga et al, 2000;Adderley et al, 2011;Bor-Kucukatay et al, 2003;Kleinbongard et al, 2006;Petrov et al, 1994Petrov et al, , 1998Tikhomirova, 2012, 2013).…”

Phosphodiesterase 5 inhibitors augment UT-15C-stimulated ATP release from erythrocytes of humans with pulmonary arterial hypertension

“…8,10,28 As depicted in Figure 5, IPR-mediated ATP release from human erythrocytes initiates a signaling pathway that requires increases in cAMP, 8,10,28 the concentration of which is determined by the balance between its synthesis by adenylyl cyclases 29 and its hydrolysis by PDE3. 12,13,30 PDE3-mediated hydrolysis of cAMP is inhibited by cGMP. 30 Inhibition of PDE5 in erythrocytes results in increases in cGMP 13 which could then inhibit PDE3 activity in these cells.…”

“…12,13,30 PDE3-mediated hydrolysis of cAMP is inhibited by cGMP. 30 Inhibition of PDE5 in erythrocytes results in increases in cGMP 13 which could then inhibit PDE3 activity in these cells. The interrelationship between the effects of prostacyclin analogs and PDE5 inhibitors on this signaling pathway is illustrated by the finding that PDE5 inhibitors augment increases in cAMP and ATP release from healthy human erythrocytes stimulated by incubation with prostacyclin analogs.…”

The effect of sildenafil on the elasticity of erythrocytes in homozygous sickle cell disease

“…[23] Although the presence of NO donor sites have been reported on erythrocyte membrane, [9][10][11] the amount of NO synthesized by sildenafil in the absence of NO or any of its donors in this study might not have been adequate to elicit the expected cGMP-mediated membrane relaxation, but possibly enough to inhibit the hyrolysis of cAMP. [13] Decreased concentrations of NO metabolites have been found in severe sickle-cell vaso-occlusive crises. Nitric oxide metabolite levels vary in sickle cell crisis (SCC) patients.…”

“…[4] The presence of PDE5 in the cytosol of erythrocytes has also been reported. [13] Cyclic GMP activates cGMP-dependent kinases that decrease intracellular calcium concentration in the smooth muscle resulting in relaxation, [14] vasodilation and increased regional blood flow. [4] The deformability of the red cell membrane is believed to be affected by both the NO produced by NO synthase as well NO-related intermediate resident within the membrane.…”