The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 ؎ 0.3 pmol/oocyte) generated a slow inward current (2.3 ؎ 0.1 A). During hyperpolarizing pulses, the permeability for ATP 4 -was more than 4000 times higher than that for Cl -. The sensitivity to GdCl 3 (0.2 mM) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP.
A field-stimulated electric organ generated an electric field and release of a large quantity of ATP. Oocytes injected with mRNA from a Torpedo electric organ released ATP when stimulated with carbamylcholine. We set up a configuration recording that let us simultaneously monitor the oocyte ionic currents and ATP release from a single oocyte. Hyperpolarization pulses applied to Xenopus oocytes induced an inward current and release of ATP independently of the expression of the ClC-0 chloride channel. The current was insensitive to substitution of ions in the extracellular saline solution, either anions or cations. The reversal potential of the current was established between +40 and +100 mV, which is in the same range as the equilibrium potential of ATP -4 . There was a correlation between the magnitude of the inward current and the amount of ATP released. The inward current closed slowly. During this closing period there was still release of ATP, which is sensitive to the holding potential. Positive potentials cut the amount of ATP released. The evoked current and ATP release were both sensitive to inhibitors of E-NTPDase activity, such as suramin, 4-acetamido-4′isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′diisothiocyanatostilbene-2′-disulfonic acid (DIDS), and a high concentration of calcium and gadolinium ions. The expression of CD39 increased the size of the hyperpolarizing induced current. By reverse transcription polymerase chain reaction, we found a protein homologous to CD39. The relationship between the results obtained in the electric organ and oocytes is that, in both cases, stimulation or hyperpolarization causes a change in the intracellular ion concentration, producing an osmotic stress, which, in turn, triggers ATP release. Stimulation or hyperpolarization may alter the intercellular ion concentration, leading to osmotic stress and the release of ATP in both electric organ and oocytes. Drug Dev.
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