Limbic seizures induce P-glycoprotein in rodent brain: functional implications for pharmacoresistance

Abstract

The causes and mechanisms underlying multidrug resistance (MDR) in epilepsy are still elusive and may depend on inadequate drug concentration in crucial brain areas. We studied whether limbic seizures or anticonvulsant drug treatments in rodents enhance the brain expression of the MDR gene (mdr) encoding a permeability glycoprotein (P-gp) involved in MDR to various cancer chemotherapeutic agents. We also investigated whether changes in P-gp levels affect anticonvulsant drug concentrations in the brain. Mdr mRNA measured by RT-PCR increased by 85% on average in the mouse hippocampus 3-24 hr after kainic acid-induced limbic seizures, returning to control levels by 72 hr. Treatment with therapeutic doses of phenytoin or carbamazepine for 7 d did not change mdr mRNA expression in the mouse hippocampus 1-72 hr after the last drug administration. Six hours after seizures, the brain/plasma ratio of phenytoin was reduced by 30% and its extracellular concentration estimated by microdialysis was increased by twofold compared with control mice. Knock-out mice (mdr1a/b -/-) lacking P-gp protein showed a 46% increase in phenytoin concentrations in the hippocampus 1 and 4 hr after injection compared with wild-type mice. A significant 23% increase was found in the cerebellum at 1 hr and in the cortex at 4 hr. Carbamazepine concentrations were measurable in the hippocampus at 3 hr in mdr1a/b -/- mice, whereas they were undetectable at the same time interval in wild-type mice. In rats having spontaneous seizures 3 months after electrically induced status epilepticus, mdr1 mRNA levels were enhanced by 1.8-fold and fivefold on average in the hippocampus and entorhinal cortex, respectively. Thus, changes in P-gp mRNA levels occur in limbic areas after both acute and chronic epileptic activity. P-gp alterations significantly affect antiepileptic drugs concentrations in the brain, suggesting that seizure-induced mdr mRNA expression contributes to MDR in epilepsy.

Fig. 1.

Representative agarose gel showing PCR amplification products from the mouse hippocampus 24 hr after saline injection (lane 1) or 30 mg/kg kainic acid injection (lane 2). Each sample was amplified as described in Materials and Methods. Note the increased level ofmdr1 mRNA in the mouse treated with kainic acid (lane 2) compared with the control mouse (lane 1).

Fig. 2.

Time course of changes in mdr1 mRNA levels in the mouse hippocampus after kainic acid-induced seizures. Data are means ± SE (n = 5) of optical density values of gel bands representing PCR amplification products ofmdr1 mRNA normalized to the corresponding β-actin band used in each sample as an internal standard. The early time point (i.e., 3 hr) corresponds to ∼1 hr after termination of seizures. Values in treated animals are expressed as a percentage of control levels [saline-injected mice killed at the various times (n= 5 for each group) were pooled because they did not differ]. *p < 0.05; **p < 0.01 by Mann–Whitney's test.

Fig. 3.

Effect of repetitive AED treatment onmdr1 mRNA in the mouse hippocampus. Data are expressed as described in legend to Figure 2. Phenytoin (DHP; 30 mg/kg), carbamazepine (CBZ; 15 mg/kg), or their vehicle was injected intraperitoneally for 7 consecutive days as described in Materials and Methods, and mice were killed at the indicated times after the last drug administration.

Fig. 4.

Mdr1 mRNA levels in the hippocampus and entorhinal cortex of rats with spontaneous seizures. Data are means ± SE (n = 5–6) of optical density values of gel bands representing PCR amplification products ofmdr1 mRNA normalized to the corresponding β-actin band used in each sample as an internal standard. Values in spontaneously epileptic rats (SE) are expressed as a percentage of control levels [sham-stimulated rats (Sham)].HP, Stimulated hippocampus (1.8-fold increase);EC, contralateral entorhinal cortex (5.5-fold increase). **p < 0.01 versus sham by Mann–Whitney's test.