Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral microvessels: Implications for P-glycoprotein trafficking

Abstract

P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.

Modulation of Opioid Transport at the Blood-Brain Barrier by Altered ATP-Binding Cassette (ABC) Transporter Expression and Activity

“…This is consistent with previous immunogold immunocytochemistry studies that demonstrated P-gp localization at the nuclear envelope in microvascular endothelial cells in human and rat brain [122]. The trafficking mechanism that has been identified and characterized by our group involves phosphorylation of caveolin-1, which is the primary signal that causes enhanced recruitment of P-gp to the plasma membrane in response to acute inflammatory pain [121]. This highly significant finding is supported by our previous work, which demonstrates that P-gp colocalizes with caveolar proteins (i.e., caveolin-1, cavin-1, and cavin-2) in membrane fractions derived from intact brain microvessels [123].…”

“…The complexity of P-gp-mediated transport regulation at the BBB in pain is emphasized by knowledge that pain triggers increased P-gp trafficking from the endothelial cell nucleus to the plasma membrane [21,120,121]. Using our detergent-free subcellular fractionation method [109], we demonstrated that P-gp traffics from the nucleus and increases P-gp protein expression and activity at the plasma membrane in response to the CIP stimulus (Figure 3) [120].…”

Structure, Function, and Regulation of the Blood-Brain Barrier Tight Junction in Central Nervous System Disorders

“…(C) Cross-section of a microvessel labeled with lectin (red), an antibody to the tight junction (TJ) protein claudin-5 (green), and DAPI (blue). Modified with permission from Tome et al (2018).…”

Functional NHE1 expression is critical to blood brain barrier integrity and sumatriptan blood to brain uptake

“…Rat cortical microvessels were isolated as previously described [42,43]. Briefly, rats were anesthetized with intraperitoneal injection of 80:10 mg/kg ketamine:xylazine, decapitated and the brains placed in ice-cold buffer A (136.9 mM NaCl; 2.7 mM KCl; 1 mM CaCl 2 ; 1.5 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 ; 0.5 mM MgCl 2 ; 5 mM glucose; 1 mM sodium pyruvate, pH7.4) supplemented with Roche EDTA-free Complete Protease Inhibitor cocktail, Sigma protease inhibitor cocktail and 2 mM phenylmethylsulfonyl fluoride.…”