To demonstrate the role of the rate-limiting and ATP-dependent gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) in oxidative and lactic stress and the effect of phenothiazine on PCK after stroke, a total of 168 adult male Sprague Dawley rats (3 months old, 280-300 g) underwent 2-h intraluminal middle cerebral artery occlusion (MCAO) and reperfusion for 6, 24, 48 h, or 7 days. Phenothiazine (chlorpromazine and promethazine (C+P)) (8 mg/kg) and 3-mercaptopicolinic acid (3-MPA, a PCK inhibitor, 100 μM) were administered at reperfusion onset. The effects of phosphoenolpyruvate, 3-MPA, or PCK knockdown were studied in neuronal cultures subjected to oxygen/glucose deprivation. Reactive oxygen species, lactate, phosphoenolpyruvate (PEP; a gluconeogenic product), mRNA, and protein of total PCK, PCK-1, and PCK-2 increased after MCAO and oxygen-glucose deprivation (OGD). Oxaloacetate (a gluconeogenic substrate) decreased, while PEP and glucose were increased, suggesting reactive gluconeogenesis. These changes were attenuated by phenothiazine, 3-MPA, or PCK shRNA. PCK-1 and -2 existed primarily in neurons, while the effects of ischemic stroke on the PCK expression were seen predominately in astrocytes. Thus, phenothiazine reduced infarction and oxidative/lactic stress by inhibiting PCKs, leading to functional recovery.
Keywords: Gluconeogenesis; Ischemia/reperfusion; Neuroprotection; Phenothiazine (promethazine, chlorpromazine, C+P).