Platelet activating factor (PAF) and other lysophospolipids are released during conditions of ischemia/reperfusion. We have previously shown that PAF enhances the activity of ecto-5'-nucleotidase (CD73) in endothelial and other cells. Furthermore we provided evidence in isolated mouse heart experiments (Langendorff) that PAF dose dependently affects heart function and activates cardiac CD73. We now asked the question whether nitric oxide and adenosine might be involved in the regulation of the hemodynamic responses during PAF application potentially via cGMP or cAMP signaling. In isolated perfused mouse hearts infusion of low doses of PAF (< 500 nM) induced a significant rise in coronary flow (from 11.41.2 to 15.22.1 ml/(min*g)) and systolic ventricular pressure (from 726 mmHg to 1187 mmHg) after 20 min PAF infusion. Changes were completely reversible upon discontinuation of PAF application. To test the involvement of nitric oxide pharmacological block (N^G-monomethyl-L-arginine, L-NMMA 100 mM) of nitric oxide synthases (NOS) and genetically eNOS deficient mice were employed. In both groups the positive inotropic effect of PAF was strongly inhibited (by 85% in L-NMMA group and 72% in eNOS-/- group). Similarly the rise in coronary flow was blunted (by 94% in L-NMMA group and 56% in eNOS-/- group). The potential involvement of adenosine was addressed by selectively blocking A_2A receptors with SCH58612 (100 nM). Presence of this antagonist also significantly attenuated the PAF induced positive inotropic effect (23% reduction of systolic ventricular pressure rise), while the effect on coronary flow remained unchanged. To further explore whether PAF changes cardiac adenosine production, we measured CD73 activity by the conversion of etheno-AMP (e-AMP) to e-adenosine and adenine nucleotide release in isolated perfused hearts from wild type mice. PAF infusion (500 nM) continuously elevated e-AMP conversion to e-adenosine (within 25 min by 20%, within 80 min by 40%). This increase persisted after discontinuation of PAF infusion. The enhanced conversion of e-AMP was absent when PAF was infused in the presence of the selective CD73 blocker (adenosine 5'-(a,ß-methylene) diphosphate, AOPCP 50 mM) or in CD73 deficient mice. Furthermore, in wild type hearts PAF induced a transient release of native adenosine (366130%), ADP (1600900%) and AMP (905450%).

From this data we conclude that PAF exerts complex regulatory functions in mouse heart. The inotropic response is partly mediated via NO and adenosine signaling. Extracellular adenosine production by CD73 is specifically augmented and AMP, the substrate of CD73, is enhanced due to a steep rise of adenine nucleotide release.