1 a1-Adrenoceptor stimulation of rat left ventricular papillary muscle produced a triphasic inotropic response: an initial transient positive inotropic effect (PIE) followed by a transient negative inotropic effect (NIE) and a sustained PIE. 2 The protein kinase C inhibitor, staurosporine, at concentrations ranging from 30nm to 100nm inhibited the sustained PIE, but had no significant effect on the transient PIE and NIE. 3 H-7, 1-(5-isoquinoline sulphonyl)-2-methylpiperazine, a less specific inhibitor of protein kinase C than staurosporine, at a concentration of 100UM inhibited both the transient NIE and the sustained PIE without affecting the transient PIE. 4 Amiloride, an inhibitor of Na+/H + exchange, at concentrations ranging from 0.1 mm to 1 mm inhibited the sustained PIE and, at higher concentrations, also inhibited the transient NIE. 5 An amiloride analogue, 5-(N-methyl-N-isobutyl)amiloride (MIBA), inhibited only the sustained PIE with an IC50 of 0.3 Mm which is approximately two orders of magnitude lower than amiloride.6 The receptor-linked stimulation of Na+/H+ exchange through protein kinase C activation may be a mechanism for ac-adrenoceptor-mediated sustained PIE.
Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.
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