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Nitric Oxide Contributes to Hypoxia-Reoxygenation-Induced P-Glycoprotein Expression in Rat Brain Endothelial Cells

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Abstract

Ischemia–reperfusion leads to increased levels at the blood–brain barrier of the multidrug efflux transporter, P-glycoprotein that provides protection to the brain by limiting access of unwanted substances. This is coincident with the production of nitric oxide. This present study using immortalized rat brain endothelial cells (GPNTs) examines whether following hypoxia-reoxygenation, nitric oxide contributes to the alterations in P-glycoprotein levels. After 6 h of hypoxia, both nitric oxide and reactive oxygen species, detected intracellularly using fluorescent monitoring dyes, were produced in the subsequent reoxygenation phase coincident with increased P-glycoprotein. The evidence that nitric oxide can directly affect P-glycoprotein expression was sought by applying S-nitroso-N-acetyl-dl-penicillamine that as shown increased the nitric oxide generation. Sodium nitroprusside, though more effective at increasing P-glycoprotein expression, appeared to produce different reactive species. Real time RT-PCR analysis revealed the predominant form of nitric oxide synthase in these cells to be endothelial, inhibition of which partially prevented the increase in P-glycoprotein during reoxygenation. These data indicate that the production of nitric oxide by endothelial nitric oxide synthase during reoxygenation can influence P-glycoprotein expression in cells of the blood-rat brain barrier, highlighting another route by which nitric oxide may protect the brain.

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Abbreviations

BSA:

Bovine serum albumin

DCF-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DAF-FM-DA:

4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate

EDTA:

Ethylenediaminetetraacetic acid

HR:

Hypoxia-reoxygenation

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HBSS:

Hanks balanced salt solution

HPRT:

Hypoxanthine-guanine phosphoribosyl transferase

l-NAME:

N5-[imino(nitroamino)methyl]-l-ornithine, methyl ester, monohydrochloride

l-NIO:

N (5)-(1-iminoethyl)-l-ornithine

MCAO:

Middle cerebral artery occlusion

NO• :

Nitric oxide

eNOS:

Endothelial nitric oxide synthase

PBS:

Phosphate buffered saline

PEG:

Polyethylene glycol

P-gp:

P-glycoprotein

ROS:

Reactive oxygen species

RT-PCR:

Reverse transcriptase-polymerase chain reaction

SNAP:

S-nitrosothiol S-nitroso-N-acetyl-dl-penicillamine

SNP:

Sodium nitroprusside

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Acknowledgments

During the execution of this study, SJR held a BBSRC studentship, RNM was in receipt of a Gates Scholarship, and ASG received an Erasmus exchange studentship. The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK

    Samantha J. Robertson, Ruth Mokgokong, Katarzyna D. Kania, Anne-Sophie Guedj, Stephen B. Hladky & Margery A. Barrand

  2. Institute for Medical Biology, Polish Academy of Sciences, 93232, Lodz, Poland

    Katarzyna D. Kania

  3. University of Nîmes, Nîmes, France

    Anne-Sophie Guedj

Authors
  1. Samantha J. Robertson
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  2. Ruth Mokgokong
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  3. Katarzyna D. Kania
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  4. Anne-Sophie Guedj
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  5. Stephen B. Hladky
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  6. Margery A. Barrand
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Correspondence to Margery A. Barrand.

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Robertson, S.J., Mokgokong, R., Kania, K.D. et al. Nitric Oxide Contributes to Hypoxia-Reoxygenation-Induced P-Glycoprotein Expression in Rat Brain Endothelial Cells. Cell Mol Neurobiol 31, 1103–1111 (2011). https://doi.org/10.1007/s10571-011-9711-4

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  • DOI: https://doi.org/10.1007/s10571-011-9711-4

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