Assessment of the Mechanism of Astrocyte Swelling Induced by the Macrolide Immunosuppressant Sirolimus Using Multinuclear Nuclear Magnetic Resonance Spectroscopy

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Abstract
The toxic effect of the macrolide immunosuppressant sirolimus on cell metabolism of primary astrocytes was studied by multinuclear NMR spectroscopy of viable cells and perchloric acid (PCA) extracts and compared to the effects of the immunosuppressant cyclosporine. The addition of 5 mg/L sirolimus (5.5 μmol/L) induced swelling of primary astrocytes to 110% of the original volume. Alteration in astrocyte volume in the presence of sirolimus was accompanied by reduction of the following important cell osmolytes and amino acid metabolites: myo-inositol, −58 ± 12% (mean ± standard deviation, n = 5); taurine, −44 ± 5%; glutamine, −13 ± 2%; compared with control. Sirolimus altered glucose metabolism and partially inhibited the tricarboxylic acid (TCA) cycle: ΣTCA/Σglycolyse = 1.36 ± 0.09 (control, n = 3), 0.96 ± 0.08 (with sirolimus). The increased concentration of phosphodiesters by sirolimus addition (glycerophosphoethanolamine, 52 ± 18%; glycerophosphocholine, 61 ± 14%; compared with control, n = 5) indicated disorders in phospholipid metabolism of cellular membranes. Addition of sirolimus led to a decline of the energy state in astrocytes: the concentration of phosphocreatine (PCr) decreased to 75% of control value within 60 min of perfusion with sirolimus and the nucleotide triphosphate (NTP) concentration to 85% within 90 min (n = 3). The effect of sirolimus on the cell metabolism of astrocytes equals that of the immunosuppressants cyclosporine and tacrolimus, the neurotoxicity of which is well-established in clinical studies. The results of this in vitro study indicate that sirolimus possesses neurotoxic potential as well.