| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
To investigate the regulatory mechanisms of Na^+-Ca^<2+> exchange under physiological conditions, we recorded Na^+-Ca^<2+> exchange current (I_<NaCa>) in the inside-out patch excised from guinea-pig ventricular myocytes (the macro patch). Applying ATP or PI(4,5)P_2 to the cytoplasmic surface of the patch membrane augmented the I_<NaCa>. Treatment of the patch membrane with PI-specific phospholipase C, which inhibits the production of PI(4,5)P_2 from PI, attenuated the ATP effect. This result suggests that the stimulatory effect of ATP is in part due to the production of PI(4,5)P_2 from PI. However, the application of inhibitors of protein tyrosine phosphatase, vanadate or bpV(phen), significantly augmented the I_<NaCa> in the presence of ATP. This result and others suggest that protein phosphorylation, probably protein tyrosine phosphorylation, is also involved in the stimulatory ATP effect on the I_<NaCa>.
To validate the report that the PKA signaling stimulates the Na^+-Ca^<2+> activity, we measured whole-cell I_<NaCa> from guinea-pig ventricular myocytes. Isoproterenol, which is an agonist of the β-adrenoceptor, activated the cAMP-dependent Cl^-current, but did not stimulate the I_<NaCa>. We concluded that the PKA signaling does not stimulate the Na^+-Ca^<2+> exchange in guinea-pig ventricular cells. The previous reports might underestimate the activation of the cAMP-dependent Cl^-current.
To study the regulatory mechanisms of NCX3, which is mainly expressed in brain and skeletal muscle, we measured the I_<NaCa> in the giant membrane patch excised from oocytes expressing NCX3. It was revealed that NCX3 possesses both the Ca^<2+>-dependent activation and inactivation, and that the increase of the I_<NaCa> by ATP is less potent in NCX3 than NCX1.
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