| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Extracellular ATP has been demonstrated to increase the amplitude of the delayed rectifier K^+ current (l_k) in a concentration-dependent manner with a half-maximal concentration (EC_<50>) of 0.95 muM and maximal increase of about a factor of 2 in guinea-pig atrial myocytes (Matsuura et al, J Physiol, 1996). In the present research project we characterized this stimulatory effects of ATP on l_K in isolated cardiac myocytes using the whole-cell patch-clamp technique. We first addressed the question whether extracellular ATP potentiates the rapid (l_<Kr>), the slow (l_<Ks>), or both components of l_K. An envelope of tails test and pharmacological experiments using E-4031 revealed that ATP selectively potentiates l_<Ks>, with no measurable effects on l_<Ks>, in guinea-pig atrial myocytes. We then investigated the signal transduction mechanism mediating the stimulatory effects of ATP on l_<1Ks> in guinea-pig atrial and ventricular myocytes. An agonist potency order of ATP<greater than or e
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qual> adenosine 5'-O-(3-thiotriphosphate) (ATP-gammaS)>ADP>>adenosine indicates an involvement of a P2-purinoceptor. This l_<Ks> response to ATP was attenuated by intracellular loading of guanosine 5'-O-(2-thiodiphosphate (GDP-betaS, 1 mM), but was not affected by pertussis toxin (PTX)-pretreatment, indicating that a PTX-insensitive G protein was involved in the response. ATP irreversibly enhanced l_<Ks> in cells loaded with 5 mM ATP-gammaS, suggesting that the response involved a protein phosphorylation possibly by a protein kinase which can utilize ATP-gammaS as a phosphate donor. ATP produced a further increase in lKs stimulated maximally either by isoprenaline (1 muM) via protein kinase A (PKA) or by 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) through protein kinase C (PKC), indicating that PKA and PKC did not mediate the response. This stimulatory effect of ATP on l_<Ks> was partially suppressed by genistein (50 muM) but not influenced by the same concentration of daidzein, suggesting that a tyrosine protein phosphorylation was, at least in part, involved in the response. Thus, the present research project revealed that extracellular ATP potentiates lKs by activating some kind of tyrosine protein kinases through a stimulation of G-protein coupled P2-purinoceptor (P2gamma-purinoceptor). Less
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