Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y₁ receptors

Abstract

1. ADP is a potent agonist of rat and human P2Y₁ purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y₁ receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio.

2. Rat brain capillary endothelial cells of the B10 clone express P2Y₁ receptors that couple to intracellular Ca²⁺ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities.

3. ATP did not raise intracellular Ca²⁺ in B10 cells. Addition of apyrases III or VII (1 u ml⁻¹) to ATP treated cells induced large intracellular Ca²⁺ transients. Apyrases had no action in the absence of ATP.

4. A 1 u ml⁻¹ apyrase III solution generated 20 μM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y₁ receptors.

5. ATP was a full agonist of P2Y₁ receptors in the presence of 1 u ml⁻¹ apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml⁻¹ apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s.

6. Apyrase VII (1 u ml⁻¹) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve.

7. Results suggest that ATPDases prevent P2Y₁ receptor activation by degrading ADP but may contribute to P2Y₁ receptor activation by generating ADP from ATP.

Full Text

The Full Text of this article is available as a PDF (226.4 KB).