Propofol induces neural progenitor cell (NPC) damage through excessive autophagy by inositol 1,4,5-trisphosphate (InsP₃) and/or ryanodine receptors. (A) Average calcium response to 200 μM propofol (Pfl, arrow) in NPCs cultured in normal medium or medium pretreated with 200 nm of the inositol 1,4,5-trisphosphate receptor antagonist, xestospongin C (Xc), 20 μM of the ryanodine receptor antagonist, dantrolene (Dan), or 5 μM of the intracellular calcium chelator, bis-N,N,N',N'-tetraacetic acid-acetoxy methylester (BAPTA-AM). Changes in Fura-2 AM intensities from at least 15 single cells in three separate experiments were measured to assess the F₃₄₀/F₃₈₀ ratio, a representative indicator for the cytosolic calcium concentration ([Ca²⁺]_c). The F₃₄₀/F₃₈₀ ratios were normalized to baseline. (B) Pretreatment with Xc, Dan, or BAPTA-AM decreased the peak amplitudes and the areas under the curve (AUC) of the propofol-evoked [Ca²⁺]_c signals. ****P < 0.0001 compared with propofol administration alone. (C) Average calcium response to 200 μM propofol (arrow) in NPCs cultured in normal medium or medium pretreated with inositol 1,4,5-trisphosphate production inhibitor lithium (Li; 2 or 10 mM). (D) Pretreatment of 2 or 10 mM Li significantly decreased the peak amplitudes and AUC of propofol-evoked [Ca²⁺]_c signals. ****P < 0.0001 compared with propofol administration alone. (E–G) Cell viability, determined with the 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay, detected inhibition of cell damage induced by 200 μM propofol for 24 h after cotreatment with Xc and Dan (E), pretreatment with Li for 3 days (F), and cotreatment with BAPTA-AM (G). The data in (E, F) are expressed as the means ± SD from at least three separate experiments and analyzed by one-way ANOVA followed by Tukey multiple comparison tests. **P < 0.01, ***P < 0.001, ****P < 0.0001. (H) A representative immunoblot of LC3II of three separate experiments from cells cultured with 200 μM propofol for 24 h with or without Xc. (I) Dan and the quantitative analysis of LC3II/β-actin ratio (means ± SD). Cotreatment with Xc or Dan alleviated the propofol-induced elevation in the autophagy biomarker, LC3II. (J) Similarly, the NPC cytotoxicity induced by 200 μM propofol for 24 h was mitigated by cotreatment with 5 nM of the autophagy inhibitor 3-methyladenine (3-MA). (K, L) Cotreatment with (K) autophagy inducer rapamycin (Rap; 100 nM) or (L) autophagy flux inhibitor bafilomycin (Baf; 1 nM) promoted NPC damage induced by 200 μM propofol for 24 h. The data in (I, J) are expressed as the means ± SD from at least three separate experiments and analyzed by one-way ANOVA followed by Tukey multiple comparison tests. **P < 0.01, ****P < 0.0001.