Differentiated Human NT2-N Neurons Possess a High Intracellular Content of myo-Inositol *†James E. Novak, ‡R. Scott Turner, *§Bernard W. Agranoff, and *†Stephen K. Fisher *Neuroscience Laboratory, Mental Health Research Institute, and Departments of †Pharmacology, ‡Neurology, and §Biological Chemistry, University of Michigan, Ann Arbor, Michigan, U.S.A. Abstract: myo-Inositol plays a key role in signal trans- duction and osmotic regulation events in the CNS. De- spite the known high concentrations of inositol in the human CNS, relatively little is known about its distribution within the different cell types. In this report, inositol ho- meostasis was studied in NT2-N cells, a unique cell cul- ture model of human CNS neurons. Differentiation of precursor NT2 teratocarcinoma cells into NT2-N neurons by means of retinoic acid treatment resulted in an in- crease in inositol concentration from 24 to 195 nmol/mg of protein. After measurement of intracellular water spaces, inositol concentrations of 1.6 and 17.4 mM were calculated for NT2 and NT2-N cells, respectively. The high concentrations of inositol in NT2-N neurons could be explained by (1) an increased uptake of inositol (3.7 vs. 1.6 nmol/mg of protein/h, for NT2-N and NT2 cells, re- spectively) and (2) a decreased efflux of inositol (1.7%/h for NT2-N neurons vs. 9.0%/h for NT2 cells). Activity of inositol synthase, which mediates de novo synthesis of inositol, was not detected in either cell type. The obser- vation that CNS neurons maintain a high intracellular concentration of inositol may be relevant to the regulation of both phosphoinositide signaling and osmotic stress events in the CNS. Key Words: Neuronal differentia- tion—Inositol efflux—Osmotic stress—Phlorizin—Inosi- tol uptake—Inositol synthase. J. Neurochem. 72, 1431–1440 (1999). myo-Inositol serves at least two major physiological roles in the CNS. First, it is a precursor of phospholipids such as the 3-phosphoinositides, which have been impli- cated in adhesion, growth, vesicular trafficking, and cell survival (Toker and Cantley, 1997), as well as the quan- titatively major polyphosphoinositides, including phos- phatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, which mediate signal transduction (Berridge and Irvine, 1989) and cytoskeletal rearrange- ment (Carpenter, 1996). Second, free inositol is involved in volume regulation during persistent osmotic stress (Nakanishi et al., 1989; Isaacks et al., 1994; Strange et al., 1994; Reeves and Cammarata, 1996). Whereas acute osmotic regulation is mediated by changes in electrolyte balance, chronic osmotic stress is countered by changes in the concentration of organic osmolytes such as inositol (Gullans and Verbalis, 1993). Disorders of neuronal inositol metabolism are be- lieved to lead to neurological and psychiatric disease. For example, the pathogenesis of Down syndrome could be related to the expression of multiple gene copies of the Na + /myo-inositol transporter and a high concentration of inositol in the fetal CSF (Berry et al., 1995; Acevedo et al., 1997). Increased cerebral inositol concentrations also result from persistent hypernatremia (Lee et al., 1994), the rapid correction of which can lead to fatal cerebral edema (Gullans and Verbalis, 1993). Conversely, condi- tions such as diabetic peripheral neuropathy (Yorek et al., 1994; Karihaloo et al., 1997; Sima et al., 1997) and some cases of neural tube defect (Cockroft, 1988; Greene and Copp, 1997) have been linked to a deficiency of inositol and an attenuation of phosphoinositide signaling. It has been speculated that the therapeutic effectiveness of Li + in the treatment of bipolar disorder derives from its ability to deplete inositol stores and consequently to inhibit inositol lipid synthesis in those neurons that ex- hibit abnormally high rates of phosphoinositide turnover (Berridge et al., 1989; Einat et al., 1998). Despite the clinical significance of neuronal inositol homeostasis, the concentration of inositol in CNS neu- rons remains uncertain. Relatively high concentrations of inositol have been reported for giant neurons of Deiters’ nucleus (Sherman et al., 1977) and the molecular layer of cerebellar flocculus (Godfrey et al., 1982). In contrast, based on experiments with dissected layers of cerebellar cortex (Stewart et al., 1969), whole sections of cerebral cortex (Godfrey, 1989), and neuroblastoma and glioma cells (Glanville et al., 1989), it has been concluded that concentrations of inositol in glia may exceed those in Received August 13, 1998; revised manuscript received November 5, 1998; accepted November 10, 1998. Address correspondence and reprint requests to Dr. S. K. Fisher at Neuroscience Laboratory, University of Michigan, 1103 E. Huron St., Ann Arbor, MI 48104-1687, U.S.A. Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; phloretin, 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone; phlorizin, phloretin 2'--D-glucoside dihydrate. 1431 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry