The characteristics, capacity and receptor regulation of inositol uptake in 1321N1 astrocytoma cells

Abstract

The uptake of inositol into 1321N1 astrocytoma cells was studied by measurement of the accumulation of free [3H]inositol within the intracellular pool. Uptake occurs via a saturable transporter with apparent Km for inositol approximately 40 microM and Vmax approximately 180 pmol/min per mg of protein, which permits intracellular inositol concentrations to exceed those of the medium by a factor of approximately 500. At extracellular concentrations up to 500 microM, inositol uptake is highly dependent (> or = 85%) on the presence of Na+ in the medium, and at physiological extracellular inositol concentrations, allows inositol to achieve an intracellular concentration of approximately 20 mM, indicating an active process driven by the Na+ gradient. Despite this, uptake was only minimally impaired or was unaffected by ouabain (1 mM) or dinitrophenol (1 mM). Consistent with a carrier-mediated mechanism, uptake was competitively blocked by phlorhizin (K1 approximately 125 microM). Uptake was also inhibited by carbachol and histamine, which act respectively via muscarinic and H1 receptors in these cells to stimulate phospholipase C. Inhibition by carbachol was dose-dependent (EC50 approximately 3-30 microM) and blocked by atropine. Inhibition by carbachol (1 mM) was non-competitive, resulting from approximately 50% decrease in the Vmax for uptake without affecting the Km and was persistent over 30-90 min. Inhibition by carbachol and histamine was independent of extracellular Ca2+ and was reproduced by phorbol ester, but not by Ca2+ ionophore or stimulation of adenylate cyclase. These results imply that receptors which couple to phospholipase C may mediate inhibition of inositol uptake via protein kinase C. The data are discussed in relation to inositol homoeostasis in resting and stimulated cells.