P-glycoprotein activity regulation at the blood–brain barrier through activation of endothelin-1 signaling. Twenty-hours after endothelin-1 (ET-1) intracerebroventricular (icv) treatment, digoxin, a P-glycoprotein (P-gp) substrate, was administered intravenously in adult brain (A) and juvenile brain (B) during four hours (steady state (ss) achievement) at a concentration of 1 mg/kg/h. At the end of the infusion period, blood was sampled, brain was isolated and the administered substrate was quantified using mass spectrometry. P-gp transport activity was estimated by the ratio between the substrates’ unbound brain concentrations and unbound plasma concentrations. Unbound plasma digoxin concentration in adult (C) and juvenile rat (D) was also calculated. ET-1 induces an enhanced transporter activity of P-gp at the adult blood–brain barrier (BBB), whereas no significant change occurred in P-gp activity at the juvenile BBB. Data represent the mean concentration for five rat brains/plasma. Variability is given by bars ± S.E.M. Data were analyzed using Student’s t-test. Statistical comparisons: *P <0.05.