In situ localization of P-glycoprotein (ABCB1) in human and rat brain

Abstract

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.

Figure 1

Electron microscopy of rat brain tissue. Immunocytochemical detection of P-gp using the monoclonal anti-P-gp antibody C219 (1:10 dilution). Labeling by gold particles is found to be associated with the plasma membrane and smooth membrane invaginations (arrows) of the capillary endothelial cells (End) (A). Labeling is also present along the plasma membrane of a pericyte (Per) and in some myelinated fibers (My) (A). At higher magnification, labeling is detected along the plasma membrane and clearly associated with smooth membrane invaginations (arrows) in an endothelial cell and pericyte (B). BM, basement membrane; CL, capillary lumen; RBC, red blood cell.

Figure 2

Electron micrographs of capillary endothelial cells from rat brain tissue. Immunolabeling for P-gp by gold particles is observed in the rough endoplasmic reticulum (RER; inset and A) and Golgi complex (A) as well as along the nuclear envelope (arrowheads in B). N, nucleus.

Figure 3

Rat brain tissue. Double-labeling experiment. Glial fibrillary acidic protein (GFAP) and P-gp were simultaneously revealed on the same tissue section using specific corresponding antibodies (anti-GFAP and MRK-16) and immunogold complexes of 5 and 10 nm. GFAP labeling (10 nm) is concentrated over the bundles of neurofilaments. P-gp labeling (5-nm arrows) is associated with the plasma membrane of the astrocyte (Ast). Labeling for P-gp (arrows) is also present in endothelial cells (End).

Figure 4

Electron micrograph of human brain tissue. Immunocytochemical detection of P-gp. Labeling by gold particles is present along the plasma membrane of the brain capillary endothelial (End) cells as well as the pericyte (Per) and astrocyte (Ast). Labeling was also detected in a cytoplasmic vesicle of an endothelial cell (arrow). BM, basement membrane; L, capillary lumen.