Permeability of porcine blood brain barrier to somatostatin analogues

Abstract

1. Transport of a fluorescent somatostatin analogue (NBD-octreotide) across freshly isolated functionally intact capillaries from porcine brain was visualized by confocal microscopy and quantitated by image analysis. 2. Luminal accumulation of NBD-octreotide showed all characteristics of specific and energy-dependent transport. Steady-state luminal fluorescence averaged 2 - 3 times cellular fluorescence and was reduced to cellular levels when metabolism was inhibited by NaCN. 3. The accumulation of NBD-octreotide in capillary lumens was inhibited in a concentration-dependent manner by unlabelled octreotide, by verapamil, PSC-833 and cyclosporin A, potent inhibitors of p-glycoprotein, and by leucotriene C(4), a strong modulator of Mrp2. Conversely, unlabelled octreotide reduced luminal accumulation of fluorescent BODIPY-verapamil on p-glycoprotein and of fluorescein-methotrexate, on Mrp2. None of the inhibitors used significantly reduced cellular accumulation of the fluorescent substrates. 4. Together, the data are consistent with octreotide being transported across the luminal membrane of porcine brain capillaries by both P-gp and Mrp2, providing further evidence that both transporters contribute substantially to the active barrier function of this endothelium.

Figure 1

Western blot detection of p-glycoprotein (A) and Mrp2 (C) in homogenates from freshly isolated porcine brain capillaries (right lanes: brain capillaries; left lanes: detection in brush border membranes from rat jejunum serving as positive controls). Immunostaining of isolated capillaries shows localization of p-glycoprotein (B) and Mrp2 (D) at the luminal membrane of the endothelial cells. Representative figures from three separate isolations.

Figure 2

(A) Accumulation of NBD-octreotide in the lumen of a freshly isolated porcine brain capillary at steady-state (30 min). (B) Luminal accumulation of fluorescent peptide is significantly reduced by 1 mM NaCN; representative for 24 measurements from three different capillary isolations.

Figure 3

Inhibition of luminal accumulation of NBD-octreotide by increasing concentrations of unlabelled octreotide. Data given as means±s.e. for 12 capillaries.

Figure 4

Excretion of NBD-octreotide into porcine brain capillary lumens in the absence (A,E) and presence of p-glycoprotein substrates/inhibitors:10 μM cyclosporin A (B), 5 μM PSC 833 (C), 50 μM verapamil (D). The Mrp2 substrate LTC₄ (0.3 μM) also reduces NBD-octreotide excretion (F). Images are representative for images from capillaries from five separate isolations.

Figure 5

Transport of the p-glycoprotein-substrate BODIPY-Verapamil (medium concentration 1 μM) and the Mrp2-substrate Fl-methotrexate (medium concentration 1 μM) into porcine brain capillary lumens in absence (control) and presence of inhibitors of p-glycoprotein and Mrp2, respectively and in presence of octreotide. Grey bars indicate luminal fluorescence intensity, white bars cellular fluorescence intensity. Data given as mean±s.e. for 10 capillaries. ^*Significantly different from control (P< 0.05).