Abstract
General anesthetics are both neuroprotective and neurotoxic with unclear mechanisms. General anesthetics may control cell survival via their effects on autophagy by activation of type 1 inositol triphosphate receptor (InsP3R-1). DT40 or SH-SY5Y cells with only or over 99% expression of InsP3R-1 were treated with isoflurane or propofol. Cell viability was determined by MTT reduction or LDH release assays. Apoptosis was determined by measuring Caspase-3 or by TUNEL assay. Autophagy activity was determined by measuring LC3 II and P62. We evaluated mitochondrial integrity using MitoTracker Green and cytosolic ATP levels. Fura2-AM was used to measure the concentrations of cytosolic calcium ([Ca2+]c). Propofol significantly increased peak and integrated calcium response (P < 0.001) in cells with InsP3R-1 but not in cells with triple knockout of InsP3R. Both propofol and isoflurane increased autophagy induction (P < 0.05) in an mTOR- and InsP3R- activity dependent manner. Short exposure to propofol adequately activated InsP3-1 to provide sufficient autophagy for cytoprotection, while prolonged exposure to propofol induced cell apoptosis via impairment of autophagy flux through over activation of InsP3-1. Propofol damaged mitochondria and decreased cytosolic ATP. The effects of general anesthetics on apoptosis and autophagy are closely integrated; both are caused by differential activation of the type 1 InsP3R.
Highlights
General anesthetics are both neuroprotective and neurotoxic with unclear mechanisms
The average integrated Ca2+ responses calculated as the area under the curve (AUC) increase above its baseline after exposure to propofol, was significantly decreased in Triple knockout of InsP3Rs (TKO) cells compared to its wild type control (WT) control (Fig. 1c, 109999 ± 7752 vs 37540 ± 2771, P < 0.001)
In SH-SY5Y cells which predominantly express (99%) of InsP3 receptor (InsP3R)-1, propofol alone significantly elevated [Ca2+]c, an effect that was significantly attenuated by the Ca2+ chelator, BAPTA-AM, at the time when [Ca2+]c reached its peak level in the absence of BAPTA-AM (Fig. 1e, 318 ± 58 vs 140 ± 63, P < 0.001) and the average AUC increase above its baseline after exposure to propofol (Fig. 1f, 154375 ± 11 vs 26148 ± 4, P < 0.0001)
Summary
General anesthetics may control cell survival via their effects on autophagy by activation of type 1 inositol triphosphate receptor (InsP3R-1). Propofol significantly increased peak and integrated calcium response (P < 0.001) in cells with InsP3R-1 but not in cells with triple knockout of InsP3R Both propofol and isoflurane increased autophagy induction (P < 0.05) in an mTOR- and InsP3R- activity dependent manner. The effects of general anesthetics on apoptosis and autophagy are closely integrated; both are caused by differential activation of the type 1 InsP3R. TKO (a–c) or BATPA-AM at 10μM (d–f) dramatically attenuated pfl-mediated peak elevation of [Ca2+y]c (b and e), as well as the average integrated Ca2+ responses calculated as the area under the curve (AUC) (c and f), as an increase above its own baseline before addition of propofol at 200 μM. Activate InsP3R7, it is reasonable to propose that GAs may control cell survival fate by affecting autophagy via differential activation of InsP3R
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