Molecular and Functional Characterization of P-Glycoprotein In Vitro
The blood-brain barrier (BBB) physically and metabolically functions as a neurovascular interface between the brain parenchyma and the systemic circulation, and regulates the permeability of several endogenous substrates and xenobiotics in and out of the central nervous system. Several membrane-associated transport proteins, such as P-glycoprotein (P-gp), multidrug resistance-associated proteins, breast cancer resistance protein, and organic anion transporting polypeptides, have been characterized at the BBB and identified to play a major role in regulating the brain bioavailability of several pharmacological agents. This chapter reviews several well-established techniques for the study of the molecular expression, cellular localization, and functional activity of transport proteins in primary and immortalized cell culture systems of the BBB. In particular, we describe the molecular characterization of P-gp/MDR1 at the transcript level using semiquantitative polymerase chain reaction (PCR), at the protein level using immunoblotting, and at the cellular level using immunofluorescence. In addition, the uptake/efflux and transepithelial flux studies, which characterize P-gp transport activity, are described.
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