On the mechanism of the enhancement of delayed rectifier K+ current by extracellular ATP in guinea-pig ventricular myocytes

The effects of extracellular adenosine 5'-triphosphate (ATP) on the delayed rectifier K+ current (I-K) were studied in guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique. ATP increased I-K concentration dependently with a concentration eliciting a half-maximal response of 1.86 mu M and a maximal increase of about 1.8-fold. The enhancement of I-K developed slowly, the effect reaching a maximum in about 1.6 min after application of ATP. The rank order of agonist potency in enhancing I-K was 2-methylthio-ATP greater than or equal to ATP>>alpha,beta-methylene-ATP. The ATP response was attenuated in guanosine 5'-O-(2-thiodiphosphate) (GDP beta S)-loaded cells, but was not affected by pertussis toxin (PTX)-pre-treatment, indicating that a PTX-insensitive G protein is involved in the response. These features are consistent with operation of P-2Y-type purinoceptors. ATP produced a further increase in I-K stimulated maximally either by isoprenaline (1 mu M) through protein kinase A (PKA) or by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM) through protein kinase C (PKC), while 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 10 mu M) did not affect the ATP response, suggesting that PKA and PKC do not mediate the response. ATP irreversibly enhanced I-K in cells loaded with adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S, 5 mM) or okadaic acid (10 mu M), a phosphatase inhibitor, suggesting that a phosphorylation step is present after the receptor stimulation. Genistein, an inhibitor of tyrosine phosphorylation, suppressed the ATP response significantly, while daidzein, an inactive analogue of genistein, had little effect on it, although both genistein or daidzein alone decreased I-K. It is hypothesized that tyrosine phosphorylation plays a role in the signalling pathway involved in the enhancement of cardiac I-K by P-2Y-purinergic stimulation.