THE CHARACTERISTICS, CAPACITY AND RECEPTOR REGULATION OF INOSITOL UPTAKE IN 1321N1 ASTROCYTOMA-CELLS

The uptake of inositol into 1321N1 astrocytoma cells was studied by me asurement of the accumulation of free [H-3]inositol within the intrace llular pool. Uptake occurs via a saturable transporter with apparent K (m) for inositol approximately 40 muM and V(max.) approximately 180 pm ol/min per mg of protein, which permits intracellular inositol concent rations to exceed those of the medium by a factor of approximately 500 . At extracellular concentrations up to 500 muM, inositol uptake is hi ghly dependent (greater-than-or-equal-to 85%) on the presence of Na+ i n the medium, and, at physiological extracellular inositol concentrati ons, allows inositol to achieve an intracellular concentration of appr oximately 20 mM, indicating an active process driven by the Na+ gradie nt. 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 phlorrhizin (K 1 approximately 125 muM). Uptake was also inhibited by carbachol and h istamine, which act respectively via muscarinic and H-1 receptors in t hese cells to stimulate phospholipase C. Inhibition by carbachol was d ose-dependent (EC50 approximately 3-30 muM) and blocked by atropine. I nhibition by carbachol (1 mM) was non-competitive, resulting from appr oximately 50% decrease in the V(max.) for uptake without affecting the K(m) and was persistent over 30-90 min. Inhibition by carbachol and h istamine was independent of extracellular Ca2+ and was reproduced by p horbol ester, but not by Ca2+ ionophore or stimulation of adenylate cy clase. These results imply that receptors which couple to phospholipas e C may mediate inhibition of inositol uptake via protein kinase C. Th e data are discussed in relation to inositol homoeostasis in resting a nd stimulated cells.