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24 April 2011

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

Shaquria P. AdderleyABCDEF, Kelly M. ThuetBE, Meera SridharanADE, Elizabeth A. BowlesAE, Alan H. StephensonADE, Mary L. EllsworthEG, Randy S. SpragueACDEG

DOI: 10.12659/MSM.881763

Med Sci Monit 2011; 17(5): CR241-247


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.

Keywords: Isoproterenol - pharmacology, Isoenzymes - metabolism, Erythrocytes - enzymology, Cytosol - enzymology, Cyclic Nucleotide Phosphodiesterases, Type 5 - metabolism, Cyclic Nucleotide Phosphodiesterases, Type 4 - metabolism, Cyclic Nucleotide Phosphodiesterases, Type 3 - metabolism, Cyclic GMP - pharmacology, Cyclic AMP - metabolism, Animals, Phosphodiesterase Inhibitors - pharmacology, Purinones - pharmacology, Rabbits, Spermine - pharmacology, Vinca Alkaloids - pharmacology


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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750