As a member of the multidrug-resistance associated protein (MRP) family, MRP2 affects the brain entry of different endogenous and exogenous compounds. Considering the role of this transporter at the blood-brain barrier, the regulation is of particular interest. However, there is limited knowledge regarding the factors that regulate MRP2 in neurologic disease states. Thus, we addressed the hypothesis that MRP2 might be affected by a glutamateinduced signaling pathway that we previously identified as one key mechanism in the regulation of P-glycoprotein.

Breast cancer resistance protein (BCRP) functions as a major molecular gatekeeper at the blood-brain barrier. Considering its impact on access to the brain by therapeutic drugs and harmful xenobiotics, it is of particular interest to elucidate the mechanisms of its regulation. Excessive glutamate concentrations have been reported during epileptic seizures or as a consequence of different brain insults including brain ischemia. Previously, we have demonstrated that glutamate can trigger an induction of the transporter P-glycoprotein. These findings raised the question whether other efflux transporters are affected in a comparable manner. Glutamate exposure proved to down-regulate BCRP transport function and expression in isolated porcine capillaries. The reduction was efficaciously prevented by coincubation with N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. The involvement of the NMDA receptor in the down-regulation of BCRP was further confirmed by experiments showing an effect of NMDA exposure on brain capillary BCRP transport function and expression. Pharmacological targeting of cyclooxygenase-1 and -2 (COX-1 and -2) using the nonselective inhibitor indomethacin, COX-1 inhibitor SC-560, and COX-2 inhibitor celecoxib revealed a contribution of COX-2 activity to the NMDA receptor's downstream signaling events affecting BCRP. Translational studies were performed using human capillaries isolated from surgical specimens of epilepsy patients. The findings confirmed a glutamate-induced down-regulation of BCRP transport activity in human capillaries, which argued against major species differences. In conclusion, our data reveal a novel mechanism of BCRP down-regulation in porcine and human brain capillaries. Moreover, together with previous data sets for P-glycoprotein, the findings point to a contrasting impact of the signaling pathway on the regulation of BCRP and P-glycoprotein. The effect of glutamate and arachidonic acid signaling on BCRP function might have implications for brain drug delivery and for radiotracer brain access in epilepsy patients and patients with other brain insults.

ABSTRACT:The ketogenic diet (KD) provides ketones from the degradation of free fatty acids for energy metabolism. It is a therapeutic option for pharmacoresistant epilepsies. Carnitine is the carrier molecule that transports fatty acids across the mitochondrial membrane for degradation into ketones. The integrity of this transport system is a prerequisite for an adequate ketogenic response. For monitoring of tissue metabolism with KD, we used the sampling method of s.c. microdialysis (MD), which permits minimally invasive, frequent, and extensive metabolic monitoring independent of blood tests. By using this new method, we monitored changes in carnitine metabolism induced by KD, particularly in free carnitine (C0), acetylcarnitine (C2), and hydroxybutyrylcarnitine (C4OH). Correlation of microdialysate and tissue concentrations for carnitines in vitro was about 85%. Carnitine metabolism was monitored in seven children started on a KD for pharmacoresistant epilepsy after a conventional initial fasting period. Detected metabolic changes consisted of a slight decrease in s.c. C0 and a marked increase in C2/CO and C4OH/CO levels. The levels of s.c. C4OH strongly correlate with ␤-hydroxybutyrate (␤-OHB) levels in plasma providing an additional parameter for the carnitine reserve of the body and reflect an optimal ketogenic energy supply. Subcutaneous MD allows close and extensive monitoring of metabolism with a KD. A high-fat, low-carbohydrate KD has been proven to be efficient for intractable childhood epilepsies (1). The metabolic effects of the KD are comparable to prolonged fasting. Glucose substrates are replaced by ␤-OHB, acetoacetate, and free fatty acids. Carnitine plays a major role in the degradation of fatty acids. As a trimethylated amino acid, it facilitates translocation of fatty acids into the mitochondrion and is therefore an essential cofactor in fatty acid oxidation and ketogenesis (2). In mammals, changes in the carnitine pattern in plasma and several tissues have been demonstrated with alterations in nutritional state. Studies in humans have shown a delayed decrease in plasma free carnitine and a rapid increase in long-and particularly short-chain acylcarnitines during fasting or diabetic ketosis (3)(4)(5). A study in children demonstrated that the changes in acylcarnitines during fat load (ingestion of sunflower oil) are more or less comparable to those during fasting (6). However, studies on the dynamics of carnitine metabolism, in particular C4OH, during initiation of a KD have not been reported so far.The technique of MD is a potent tool for the study of tissue metabolism. The method is based on the diffusion of substances through a semipermeable dialysis membrane implanted in the tissue of interest. It allows repeated measurement of the concentrations of tissue molecules that have crossed the membrane. Water-soluble analytes with a molecular weight below the exclusion size of the catheter cross the membrane until their concentrations in the extracellular fluid and the microdialysate are equal ...