Localization of P-glycoprotein at the nuclear envelope of rat brain cells

https://doi.org/10.1016/j.bbrc.2007.06.176Get rights and content

Abstract

P-Glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.

Section snippets

Antibodies

The murine monoclonal P-gp antibodies C219 and MRK16 were purchased from ID Laboratories (London, ON) and Kamiya Biomedical (Seattle, WA), respectively. The C219 antibody recognizes a conserved intracellular epitope (VQEALD) on all human and rodent isoforms of P-gp [20] while the MRK-16 antibody recognizes a conserved extracellular epitope on all drug-transporting isoforms of human and rat P-gp protein [20]. Protein A–gold was prepared with 10 mm gold particle according to our previously

Nuclear localization of P-gp by immunofluorescence

Immunofluorescence using P-gp monoclonal C219 antibody revealed a signal at the plasma membranes of RBE4 and MLS-9 cells respectively (Fig. 1). Signal was also detected subcellularly in the cytoplasm, as well as at the nuclear envelope of both cell types. No signal was detected in the control samples in which incubation with the primary antibody was substituted with PBS (data not shown).

Morphology of isolated nuclei

To determine the nuclear localization of P-gp in RBE4 and MLS-9 cells, nuclei were first isolated and the

Discussion

Drug efflux transport proteins are primarily expressed at the plasma membrane of endothelial cells and exert their action by pumping substrate drugs out of the cell [4]. The role of P-gp mediated cellular drug resistance in brain pathologies is compelling because this transport protein is present at the BBB and restricts access of drugs to the brain parenchyma. However, brain parenchymal cells including neurons and glial (microglia, astrocytes) cells can also express P-gp [27]. Thus, once a

Acknowledgments

This work is supported by a grant from the Canadian Institutes of Health Research (MOP 56976) and the Ontario HIV Treatment Network, Ontario Ministry of Health. The authors would like to thank Ms. Manisha Ramaswamy and Ms. Diane Gingras for their excellent technical assistance and Dr. Jeff Henderson for the help and advice with the undertaking of the fluorescence microscopy studies.

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