Compartmentalized cAMP regulates receptor-mediated ATP release from erythrocytes.

摘要

ATP released from RBCs in response to activation of the Gs coupled prostacyclin (IPR) or beta-adrenergic (beta2AR) receptor can contribute to the local control of vascular resistance via activation of purinergic receptors on the vascular endothelium stimulating release of vasodilators, such as nitric oxide and prostacyclin. A signal transduction pathway for ATP release has been proposed and includes Gs, adenylyl cyclase (AC), protein kinase A (PKA), and the cystic fibrosis transmembrane conductance regulator. Importantly, activation of either receptor results in increases in cAMP and ATP release.;Cyclic nucleotide regulation is critical due to the involvement of cAMP and cGMP in many signaling pathways. The level of cyclic nucleotides is determined by their synthesis via ACs or guanylyl cyclases (GCs) and degradation via phosphodiesterases (PDEs). Since increases in cAMP are required for ATP release, identification of individual PDEs that regulate cAMP increases and the proteins that regulate PDE activity would provide insight into regulation of ATP release.;RBCs possess PDE activity that is regulated via phosphorylation by specific protein kinases as well as cGMP. However, no isoform-specific PDE activity has been associated with the IPR or beta2AR in RBCs. Moreover, the mechanisms for regulation of PDE activity in RBCs have not been elucidated. We hypothesize that in RBCs increases in cAMP are localized and regulated by distinct PDEs. Subsequently, these PDEs are regulated by distinct kinases as well as cGMP.;In support of this hypothesis, we demonstrate that in RBCs, cAMP increases associated with IPR activation are regulated by PDE3, while PDEs 2 and 4 regulate cAMP increases associated with beta2AR activation. Additionally, we establish that, PKC as well as PKA regulates PDE3 while PKA only regulates PDE4 activity. We also provide evidence that activation of soluble GC and inhibition of PDE5 increases cAMP levels associated with activation of the beta2AR. Finally, we demonstrate that an increase in cAMP not localized to a signal pathway inhibits ATP release.;All together these data provide evidence for the localization of cyclic nucleotide signaling in RBCs and suggest that the RBC is a possible therapeutic target for the treatment of vascular disease.