Contributions in astrocytes of SMIT1/2 and HMIT to myo-inositol uptake at different concentrations and pH

myo-Inositol is important for cell signaling both in cytoplasm and in intracellular organelles. It is required in the plasma membrane and cytoplasm for maintained synthesis of the second messengers, inositoltrisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol bisphosphate (PIP2), and in organelles as precursor for synthesis of complex signaling phospholipids and inositolphosphates from IP3 and PIP2. myo-Inositol must be taken up into the cell where its is used, because neither neurons nor astrocytes synthesize it. It is also an osmolyte, taken up in response to surrounding hyperosmolarity and released during hypo-osmolarity. There are three myo-inositol transporters, the Na+-dependent SMIT1 and SMIT2, and HMIT, which co-transports myo-inositol with H+. Their relative expressions in astrocytes and neurons are unknown. Uptake kinetics for myo-inositol in astrocytes has repeatedly been determined, but always on the assumption of only one component, leaving kinetics for the individual transporters unknown. This paper demonstrates that astrocytes obtained directly from the brain express SMIT1 and HMIT, but little SMIT2, and that all three transporters are expressed in neurons. Cultured mouse astrocytes show a high-affinity/low-capacity myo-inositol uptake (Vmax: 60.0 ± 3.0 pmol/min per mg protein; Km: 16.7 ± 2.6 μM), mediated by SMIT1 and perhaps partly by SMIT2. It was determined in cells pre-treated with HMIT-siRNA and confirmed by specific inhibition of SMIT. However at physiologically relevant myo-inositol concentrations most uptake is by a lower-affinity/higher-capacity uptake, mediated by HMIT (Vmax: 358 ± 60 pmol/min per mg protein; Km: 143 ± 36 μM) and determined by subtraction of SMIT-mediated from total uptake. At high myo-inositol concentrations, its uptake is inhibited by incubation in medium with increased pH, and increased during intracellular acidification with NH4Cl. This is in agreement with literature data for HMIT alone. At low concentration, where SMIT1/2 activity gains importance, myo-inositol uptake is reduced by ammonia-induced intracellular acidification, consistent with the transporter’s pH sensitivity reported in the literature.

► Normal mouse astrocytes mainly express myo-inositol transporters HMIT and SMIT1 mRNA.
► HMIT and SMIT Vmax and Km were determined separately in cultured mouse astrocytes.
► At physiologically relevant myo-inositol concentrations most uptake is by HMIT.
► Intracellular alkalinization inhibits uptake at 100 μM but stimulates it at 10 μM.