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 concentrat ion 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 d eveloped slowly, the effect reaching a maximum in about 1.6 min after appli cation of ATP. The rank order of agonist potency in enhancing I-K was 2-met hylthio-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. T hese features are consistent with operation of P-2Y-type purinoceptors. ATP produced a further increase in I-K stimulated maximally either by isoprena line (1 mu M) through protein kinase A (PKA) or by 12-O-tetradecanoylphorbo l 13-acetate (TPA, 100 nM) through protein kinase C (PKC), while 1-(5-isoqu inolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 10 mu M) did not affect the ATP response, suggesting that PKA and PKC do not mediate the res ponse. 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 phospha tase 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 ana logue of genistein, had little effect on it, although both genistein or dai dzein alone decreased I-K. It is hypothesized that tyrosine phosphorylation plays a role in the signalling pathway involved in the enhancement of card iac I-K by P-2Y-purinergic stimulation.