title: 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.
  
  
  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.
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, MO, USA. adderleys@ecu.edu
authors.forename: Shaquria P
authors.initials: SP
authors.lastname: Adderley
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Thuet
authors.forename: Meera
authors.initials: M
authors.lastname: Sridharan
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Bowles
authors.forename: Alan H
authors.initials: AH
authors.lastname: Stephenson
authors.forename: Mary L
authors.initials: ML
authors.lastname: Ellsworth
authors.forename: Randy S
authors.initials: RS
authors.lastname: Sprague
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000494:pharmacology; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D004912:Erythrocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011688:Purinones / Q000494:pharmacology; D011817:Rabbits; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology; D014748:Vinca Alkaloids / Q000494:pharmacology
chemical_list: D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D011688:Purinones; D014748:Vinca Alkaloids; C091861:spermine nitric oxide complex; D013096:Spermine; C013983:vinpocetine; D000242:Cyclic AMP; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C011145:zaprinast; D006152:Cyclic GMP; D007545:Isoproterenol