Activation of PKC isoform beta(I) at the blood-brain barrier rapidly decreases P-glycoprotein activity and enhances drug delivery to the brain

Abstract

P-glycoprotein is an ATP (adenosine triphosphate)-driven drug efflux transporter that is highly expressed at the blood-brain barrier (BBB) and is a major obstacle to the pharmacotherapy of central nervous system diseases, including brain tumors, neuro-AIDS, and epilepsy. Previous studies have shown that P-glycoprotein transport activity in rat brain capillaries is rapidly reduced by the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) acting through protein kinase C (PKC)-dependent signaling. In this study, we used isolated rat brain capillaries to show that the TNF-alpha-induced reduction of P-glycoprotein activity was prevented by a PKCbeta(I/II) inhibitor, LY333531, and mimicked by a PKCbeta(I/II) activator, 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). Western blotting of brain capillary extracts with phospho-specific antibodies showed that dPPA activated PKCbeta(I), but not PKCbeta(II). Moreover, in intact rats, intracarotid infusion of dPPA potently increased brain accumulation of the P-glycoprotein substrate, [(3)H]-verapamil without compromising tight junction integrity. Thus, PKCbeta(I) activation selectively reduced P-glycoprotein activity both in vitro and in vivo. Targeting PKCbeta(I) at the BBB may prove to be an effective strategy for enhancing the delivery of small molecule therapeutics to the brain.

Figure 1

LY333531 prevents rapid loss of P-glycoprotein transport activity (steady-state luminal NBD-CSA fluorescence) induced by tumor necrosis factor (TNF)-α and endothelin-1 (ET-1) in rat brain capillaries. (A) Representative confocal images of capillaries showing reduced luminal accumulation with exposure to 5 μmol/L PSC833 or 1 mmol/L NaCN. (B) Quantitative image analysis of luminal NBD-CSA accumulation in capillary lumens showing significant decreases with exposure to 5 μmol/L PSC833 or 1 mmol/L NaCN. (C) Representative confocal images of brain capillaries exposed for 1 h to 100 ng/mL TNF-α or 100 ng/mL TNF-α plus 10 nmol/L LY333531. (D) A volume of 10 nmol/L LY333531 blocks the effects of 100 ng/mL TNF-α or 100 nmol/L ET-1 on P-glycoprotein activity. (E) LY333531 blocks the effects of PMA on P-glycoprotein activity. Pooled data from 3 to 5 experiments, each involving measurements of 10 to 15 capillaries within each treatment are shown. Statistical comparisons: ^*Significantly different from control, P<0.05; ^***Significantly different from control, P<0.001.

Figure 2

12-Deoxyphorbol-13-phenylacetate-20-acetate (dPPA) reduces P-glycoprotein transport activity. (A) Dose response. (B) Reversal of dPPA effect by LY333531. The half-maximal effective concentration (EC₅₀) for LY333531 is 6 nmol/L. (C) Lack of effect of dPPA on Mrp2-mediated Texas red accumulation in capillary lumens. Texas red accumulation was reduced when metabolism was inhibited by NaCN and when tight junctions were disrupted by hypertonic mannitol. Pooled data from 3 to 5 experiments, each involving measurements of 10 to 15 capillaries within each treatment are shown. Statistical comparisons: ^***Significantly different from control, P<0.001.

Figure 3

Phosphorylation and membrane/aqueous distribution of protein kinase C (PKC)β_I (A, C) and PKCβ_II (B, D) in fractions derived from control and tumor necrosis factor (TNF)-α-treated (10 ng/mL for 30 mins) brain capillaries. Panels A and B show western blots for total PKC isoform levels. Panels C and D show blots for phosphorylated isoforms. Representative blots, and quantified band intensities normalized to actin, involving measurements from 5 to 10 separate experiments are shown. Statistical comparisons: ^***Significantly different from control, P<0.001.

Figure 4

Phosphorylation and membrane/aqueous distribution of protein kinase C (PKC)β_I (A, C) and PKCβ_II (B, D) in fractions derived from control and 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA)-treated (10 nmol/L for 30 mins) brain capillaries. Panels A and B show western blots for total PKC isoform levels. Panels C and D show blots for phosphorylated isoforms. Representative blots, and quantified band intensities normalized to actin, involving measurements from 5 to 10 separate experiments are shown. Statistical comparisons: ^***Significantly different from control, P<0.001.

Figure 5

P-glycoprotein transport activity (A) and protein expression (B) in brain capillaries after 6 h of exposure to 10 ng/mL tumor necrosis factor (TNF)-α or 10 nmol/L 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). (A) Pooled data from 5 to 7 experiments, each involving measurements of 10 to 15 capillaries within each treatment are shown. (B) Representative confocal images of capillaries immunostained for P-glycoprotein (green), counterstained with propidium iodide (red). Numbers under the images show the corresponding relative fluorescence of P-glycoprotein from 10 to 15 capillaries in each treatment, representing 3 independent experiments. Statistical comparisons: ^**Significantly different from control, P<0.01; ^***Significantly different from control, P<0.001.

Figure 6

In situ brain perfusion with [³H]-verapamil or [¹⁴C]-sucrose. (A) Brain distribution of [¹⁴C]-sucrose was not increased in response to 8 μmol/L cyclosporine A (CSA) or 1 μmol/L 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). A bolus of 1.3 mol/L mannitol increased [¹⁴C]-sucrose accumulation. (B) Perfusion with 8 μmol/L CSA increased [³H]-verapamil accumulation. (C) Perfusion with dPPA increased [³H]-verapamil accumulation. Statistical comparisons: ^*Significantly different from control, P<0.05; ^***Significantly different from control, P<0.001 (data shown are n>5 rats in each treatment group).