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Hyperglycemia‐induced Elevation of NKCC1 Expression in Blood Brain Barrier Endothelial Cells: Greater Effect of Intermittent Versus Sustained Exposures
Author(s) -
Klug Nicholas R,
Raposa Jesse T,
Riley Michelle A,
O'Donnell Martha E
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1224.20
Subject(s) - medicine , mannitol , ischemia , endocrinology , blood–brain barrier , cerebral edema , cotransporter , osmotic concentration , chemistry , central nervous system , sodium , biochemistry , organic chemistry
Diabetics with accompanied hyperglycemia (HG) suffer from exacerbated cerebral edema and worsened neurological outcomes following ischemic stroke. We have demonstrated previously that hypoxia and other factors present during cerebral ischemia stimulate cerebral microvascular endothelial cell (CMEC) Na‐K‐Cl (NKCC) cotransport and Na/H exchange (NHE) activities. We also found that inhibition of these blood‐brain barrier (BBB) Na transporters reduces brain Na uptake and edema in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. In more recent studies focusing on diabetic stroke, we demonstrated that sustained 6‐hour to 7‐day HG exposures increase expression and activity of CMEC NKCC and NHE, resulting in more pronounced responses to ischemic factor stimulation. Further, we found that inhibition of BBB NKCC and NHE effectively reduces edema and infarct in hyperglycemic rats subjected to MCAO. Diabetics often experience postprandial excursions of blood glucose. Studies have provided evidence that while sustained HG can induce stress/injury responses in endothelial cells, intermittent HG can exaggerate those responses. The present study was conducted to investigate the effects of intermittent HG exposures on BBB Na transporter expression and activity. For this, cultured bovine CMEC were exposed daily over 2 to 5 days to HG medium (DMEM with 30 mM glucose, 320 mOsm) or to osmotic control medium (DMEM with 30 mM mannitol, 320 mOsm) for 6 hours, followed by exposure to normoglycemic medium (DMEM with 5 mM glucose, 295 mOsm) for 18 hours. Quantitative Western blot analysis was then used to assess NKCC1 protein abundance in CMEC lysates. We found that CMEC NKCC1 abundance increased in response to intermittent HG exposures in a graded manner with larger increases observed following greater number of successive exposures. No change in NKCC1 abundance occurred with repeated intermittent exposures to normoglycemic medium. In addition, our results show that 5 days of intermittent HG exposures caused a 124% increase in NKCC1 abundance compared to 40% and 65% increases following sustained 2‐day and 7‐day exposures, respectively. In the present study we also investigated the effects of varying HG levels on CMEC Na transporter abundance. Here, we exposed CMEC for 48 hours to HG media containing 10, 20 and 30 mM glucose, to osmotic control media (with mannitol) or to normoglycemic medium (with 5 mM glucose). We found that NKCC1 and NHE1 protein abundances exhibited a graded response to increasing HG levels. Our findings support the hypothesis that BBB endothelial NKCC1 and NHE1 are highly sensitive to increasing levels of HG and, in particular, that clinically relevant repeated intermittent exposures result in more pronounced BBB Na transporter responses than sustained exposures. Support or Funding Information Supported by NIH, ADA and AHA