Mrp1 is essential for sphingolipid signaling to p-glycoprotein in mouse blood-brain and blood-spinal cord barriers

Abstract

At the blood-brain and blood-spinal cord barriers, P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to central nervous system (CNS) pharmacotherapy. Recently, we showed that signaling through tumor necrosis factor-α (TNF-α), sphingolipids, and sphingosine-1-phosphate receptor 1 (S1PR1) rapidly and reversibly reduced basal P-glycoprotein transport activity in the rat blood-brain barrier. The present study extends those findings to the mouse blood-brain and blood-spinal cord barriers and, importantly, identifies multidrug resistance-associated protein 1 (Mrp1, Abcc1) as the transporter that mediates S1P efflux from brain and spinal cord endothelial cells. In brain and spinal cord capillaries isolated from wild-type mice, TNF-α, sphingosine, S1P, the S1PR agonist fingolimod (FTY720), and its active, phosphorylated metabolite, FTY720P, reduced P-glycoprotein transport activity; these effects were abolished by a specific S1PR1 antagonist. In brain and spinal cord capillaries isolated from Mrp1-null mice, neither TNF-α nor sphingosine nor FTY720 reduced P-glycoprotein transport activity. However, S1P and FTY720P had the same S1PR1-dependent effects on transport activity as in capillaries from wild-type mice. Thus, deletion of Mrp1 alone terminated endogenous signaling to S1PR1. These results identify Mrp1 as the transporter essential for S1P efflux from the endothelial cells and thus for inside-out S1P signaling to P-glycoprotein at the blood-brain and blood-spinal cord barriers.

Figure 1

Signaling pathway that regulates basal P-glycoprotein activity in brain capillaries. Agents that activate signaling at various points in the pathway are shown above each affected step. Identifying the molecular basis for sphingosine-1-phosphate (S1P) efflux from brain and spinal cord capillary endothelial cells (curved arrow) is the focus of the current study.

Figure 2

Tumor necrosis factor-α (TNF-α) signaling through sphingolipids and the S1P1 receptor reduce P-glycoprotein transport activity in wild-type mouse brain capillaries. (A) Representative confocal images of brain capillaries after a 60-minute incubation with 2 μmol/L NBD-CSA ((N-ɛ(4-nitrobenzofurazan-7-yl)-𝒟-Lys⁸)-cyclosporine A) and 100 ng/ml TNF-α; note the high luminal fluorescence in the control capillary and decreased luminal fluorescence in capillaries exposed to PSC833 and TNF-α. The scale bar indicates 20 μm. (B) TNF-α acts through sphingosine-1-phosphate receptor 1 (S1PR1) to reduce P-glycoprotein activity. (C) Sphingosine acts through S1PR1 to reduce P-glycoprotein activity. (D) S1P acts through S1PR1 to reduce P-glycoprotein activity. (E) FTY720 and FTY720P act through S1PR1 to reduce P-glycoprotein activity. Each bar represents the mean±s.e.m. for 8 to 15 capillaries from a single preparation (pooled tissue from 5 to 7 mice). Statistical comparisons: *** significantly lower than controls, P<0.001.

Figure 3

Sphingosine-1-phosphate (S1P) rapidly reduces P-glycoprotein transport activity. Brain capillaries from wild-type mice were incubated to steady state (60 minutes) in medium with 2 μmol/L NBD-CSA ((N-ɛ(4-nitrobenzofurazan-7-yl)-𝒟-Lys⁸)-cyclosporine A). Then, 1 μmol/L S1P was added to the medium (time 0 on graph); 30 minutes later, capillaries were washed and S1P-free medium was added. Each point represents the mean±s.e.m. for 7 to 9 capillaries from a single preparation (pooled tissue from 5 mice).

Figure 4

MK571, a nonselective multidrug resistance-associated protein 1 (Mrp) inhibitor, blocks the effects of (A) sphingosine, but not (B) sphingosine-1-phosphate (S1P), on P-glycoprotein transport activity in brain capillaries from wild-type mice. NBD-CSA, (N-ɛ(4-nitrobenzofurazan-7-yl)-𝒟-Lys⁸)-cyclosporine A. Each point represents the mean±s.e.m. for 8 to 15 capillaries from a single preparation (pooled tissue from 5 mice). Statistical comparisons: *** significantly lower than controls, P<0.001.

Figure 5

Signaling in brain capillaries from multidrug resistance-associated protein 1 (Mrp1)-null mice. Western blots showing (A) Mrp1 and (B) P-glycoprotein (P-gp) expression in mouse brain capillaries from wild-type and Mrp1-null mice. Rat brain capillaries are shown for comparison. (C) Tumor necrosis factor-α (TNF-α), (D) sphingosine, (E) sphingosine-1-phosphate (S1P), and (F) FTY720 and FTY720P signaling to P-glycoprotein in brain capillaries from Mrp1-null mice. NBD-CSA, (N-ɛ(4-nitrobenzofurazan-7-yl)-𝒟-Lys⁸)-cyclosporine A. Each point represents the mean±s.e.m. for 8 to 15 capillaries from a single preparation (pooled tissue from 5 mice). Statistical comparisons: *** significantly lower than controls, P<0.001.

Figure 6

(A) Tumor necrosis factor-α (TNF-α), (B) sphingosine, (C) sphingosine-1-phosphate (S1P), and (D) FTY720 and FTY720P signaling to P-glycoprotein in spinal cord capillaries from wild-type mice. NBD-CSA, (N-ɛ(4-nitrobenzofurazan-7-yl)-𝒟-Lys⁸)-cyclosporine A. Each point represents the mean±s.e.m. for 8 to 15 capillaries from a single preparation (pooled tissue from 5 mice). Statistical comparisons: *** significantly lower than controls, P<0.001.

Figure 7

(A) Tumor necrosis factor-α (TNF-α), (B) sphingosine, (C) sphingosine-1-phosphate (S1P), and (D) FTY720 and FTY720P signaling to P-glycoprotein in spinal cord capillaries from multidrug resistance-associated protein 1 (Mrp1)-null mice. NBD-CSA, (N-ɛ(4-nitrobenzofurazan-7-yl)-𝒟-Lys⁸)-cyclosporine A. Each point represents the mean±s.e.m. for 8 to 12 capillaries from a single preparation (pooled tissue from 5 mice). Statistical comparisons: *** significantly lower than controls, P<0.001.