Adenosine release of the isolated guinea pig heart is regulated by phosphorylation of adenosine kinase

Adenosine release (Rado) is increased by inhibition of adenosine kinase (AK). We tested the hypothesis that threonine phosphorylation of AK (AL-P) influences its activity. Isolated retrograde perfused guinea pig hearts were frozen under control conditions. AK was purified using size exclusion and affinity chromatography; maximum activity (Vmax) inhibitable by iodotubercidin was studied. When purified AK was treated with alkaline phosphatase, AK activity fell to 6% of the control value while AK-P fell to 37+5%. When AK was treated with protein phosphatase lambda, AK activity fell to 40+9% and AK-P fell to 74+8%. This experiment demonstrates that phosphorylation of AK increases its activity. Hearts were also studied during norepinephrine infusion (NE) and hypoxia (HPX). Venous effluent was taken for adenosine measurement and hearts were frozen for extraction and purification of AK. AK activity (nmol/mg/min) and Rado (pg/g/min) were 19+0.7 and 56+ 7 (control), 13+0.9 and 523+ 90 (NE), and 8+0.4 and 2085+229 (HPX) respectively. Hypoxia markedly reduced AK-P. We conclude that phosphorylation of AK is a significant determinant of its activity and contributes to the regulation of adenosine release from the heart under conditions of NE infusion and hypoxia.