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. 2008 Jul;15(5):461-71.
doi: 10.1080/10739680701833804.

Prostacyclin analogs stimulate receptor-mediated cAMP synthesis and ATP release from rabbit and human erythrocytes

Randy S Sprague  1 , Elizabeth A Bowles, Madelyn S Hanson, Eileen A DuFaux, Meera Sridharan, Shaquria Adderley, Mary L Ellsworth, Alan H Stephenson
Affiliations

Prostacyclin analogs stimulate receptor-mediated cAMP synthesis and ATP release from rabbit and human erythrocytes

Randy S Sprague et al. Microcirculation. 2008 Jul.

Abstract

Objectives: The purpose of this study was to establish that the prostacyclin (PGI(2)) receptor (IP receptor) is present on rabbit and human erythrocytes and that its activation stimulates cyclic adenosine monophosphate (cAMP) synthesis and adenosine triphosphate (ATP) release.

Methods: The effect of incubation of erythrocytes with the active PGI(2) analogs, iloprost or UT-15C, on cAMP levels and ATP release was determined in the absence and presence of the IP receptor antagonist, CAY10441. Western analysis was used to determine the presence of the IP receptor on isolated membranes. To establish that effects of PGI(2) analogs were not due to prostaglandin E(2)(PGE(2)) receptor activation, the effect of PGE(2) on cAMP levels and ATP release was determined.

Results: Rabbit and human erythrocytes possess IP receptors. Iloprost and UT-15C stimulated increases in cAMP and ATP release that were prevented by the IP receptor antagonist, CAY10441. PGE(2) did not stimulate cAMP accumulation or ATP release and did not inhibit iloprost-induced increases in cAMP.

Conclusions: This study establishes that the IP receptor is present on rabbit and human erythrocytes and that its activation results in increases in cAMP and ATP release. These results suggest a novel mechanism by which PGI(2) and its active analogs, when administered pharmacologically, could produce vasodilation.

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Figures

Figure 1
Figure 1
A: Effect of iloprost (n = 4, open circles) and UT-15C (n = 4, closed circles) on increases in cAMP in rabbit erythrocytes (RBCs). Values are not different between agonists at any concentration. B: Effect of iloprost (n = 4) on increases in cAMP in human RBCs. Values are means ± SE.
Figure 2
Figure 2
Western immunoblots of rabbit (A) and human (B) erythrocyte membranes and platelet membranes (positive controls) probed with an antibody directed against the IP receptor. A: Lanes 1–3 = individual rabbit erythrocyte membrane preparations (representative of 7 studies) and lane 4 = rabbit platelet membranes. B: Lanes 1–3 = individual human erythrocyte membranes (representative of 14 studies) and lane 4 = human platelet membranes.
Figure 3
Figure 3
Western immunoblot of human erythrocyte and platelet membranes probed with an antibody directed against CD41. Representative of studies with 7 human erythrocyte membrane preparations.
Figure 4
Figure 4
A: Effect of iloprost (1 μM, n = 9) or its vehicle (CONTROL, saline, n = 9) on cAMP levels in rabbit erythrocytes. In some studies erythrocytes were incubated with the IP receptor antagonist CAY10441 alone (CAY, 10 μM, n = 5) or were preincubated with CAY10441 (n = 5) or the β-receptor antagonist, propranolol (PROP, 10 μM, n = 4) before the addition of iloprost. B: Effect of isoproterenol (ISO, 1 μM, n = 9) or its vehicle (CONTROL, saline, n = 9) on cAMP levels in rabbit erythrocytes. In some studies erythrocytes were incubated with the β-receptor antagonist, propranolol alone (PROP, 10 μM, n = 5) or were preincubated with PROP (n = 5) or the IP receptor antagonist CAY10441 (CAY, 10 μM, n = 4) before the addition of ISO. Values are means ± SE. *p < 0.01 compared to all other groups.
Figure 5
Figure 5
A: Effect of UT-15C (1 μM, n = 9) or its vehicle (CONTROL, saline) on cAMP levels in rabbit erythrocytes in the absence and presence of the IP receptor antagonist CAY10441 (CAY, 10 μM). B: Effect of iloprost (1 μM, n = 5) or its vehicle (CONTROL, saline) on cAMP levels in human erythrocytes in the absence and presence of the IP receptor antagonist CAY10441 (CAY, 10 μM). Values are means ± SE. *p < 0.01 compared to all other groups.
Figure 6
Figure 6
Effect of prostaglandin E2 (PGE2, 1 μM, n = 4) on cAMP levels in rabbit erythrocytes in the absence and presence of the IP receptor antagonist CAY10441 (CAY, 10 μM) or iloprost (1 μM). Values are means ± SE. ns = not significantly different. *p < 0.01 compared to all other groups.
Figure 7
Figure 7
A: Effect of UT-15C (1 μM) or its vehicle (CONTROL, saline) on ATP release from rabbit erythrocytes in the absence (open bars, n = 9) and presence (closed bars, n = 7) of the IP receptor antagonist CAY10441 (CAY, 10 μM). B: Effect of iloprost (1 μM) or its vehicle (CONTROL, saline) on ATP release from human erythrocytes in the absence (open bars, n = 7) and presence (closed bars, n = 5) of the IP receptor antagonist CAY10441 (CAY, 10 μM). Values are means ± SE. *p < 0.05 compared to CONTROL.
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