Effects of cooling and ARL 67156 on synaptic ecto-ATPase activity in guinea pig and mouse vas deferens

We have studied the influence of temperature and ARL 67156 on ATP hydrolysis in mouse and guinea pig vas deferens in order to explore the properties of the enzymatic inactivation mechanism proposed to regulate purinergic neurotransmission at the sympathetic neuromuscular junction of smooth muscle. The ectonucleotidase activity was determined by using the malachite green method to measure the inorganic phosphate (Pi) liberated with ATP used as a substrate. ATP hydrolysis in both species was found to be insensitive to ouabain (100 μM), sodium azide (1 mM), sodium vanadate (100 μM) and β-glycerophosphate (10 mM) and was also found to depend on Ca2+ and Mg2+. VMAX of the ectonucleotidase activity for guinea pig and mouse vas deferens was 958.4±66.3 and 79.7±8.5 pmol/min/mg, while KM was 625.1±45.2 and 406.0±29.0 μM, respectively. Cooling the tissues from 35 to 25 °C reduced the enzyme activity significantly (P<0.01) by 52.7±9.2% in guinea pig vas deferens and 34.9±5.3% in mouse vas deferens. ARL 67156 (100 μM), the specific ecto-ATPase inhibitor, caused a reduction in enzyme activity in guinea pig and mouse vas of 54.1±16.4% and 53.0±7.6%, respectively (P<0.01). The degree of inhibition of ATP hydrolysis by lowered temperature and 100 μM ARL 67156 correlates well with the reported potentiation and prolongation of junction potentials under these conditions. It is concluded that ecto-ATPase or a closely related ectonucleotidase plays an important role in the physiological regulation of purinergic neurotransmission.