Increased blood-brain barrier (BBB)
Na-K-Cl cotransporter activity appears to contribute to
cerebral edema formation during
ischemic stroke. We have shown previously that inhibition of BBB
Na-K-Cl cotransporter activity reduces
edema and
infarct in the rat
middle cerebral artery occlusion (MCAO) model of
ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors
hypoxia, aglycemia, and
arginine vasopressin (AVP), although the mechanisms responsible are not well understood.
AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including
ischemia,
hypoxia, and aglycemia. Previous studies have shown that the
AMPK inhibitor Compound C significantly reduces
infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB
Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (
CMEC) were assessed for
Na-K-Cl cotransporter activity as
bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using
antibodies that detect total versus phosphorylated (activated) AMPK. We found that
hypoxia (7% and 2% O(2)), aglycemia, AVP, and
oxygen-
glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces
hypoxia-, aglycemia-, and AVP-induced stimulation of
CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated
kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB
Na-K-Cl cotransporter involves activation of AMPK.