Hyperglycemia‐Induced Alteration of Brain Microvascular Endothelial Intracellular Ca Response to Ischemic Factors: Role of TRPV4 Channels

Abstract

Cerebral edema is exacerbated in hyperglycemic ischemic stroke through poorly understood mechanisms. We showed previously that hypoxia and other factors present during the early hours of ischemic stroke stimulate blood‐brain barrier (BBB) Na‐K‐Cl cotransport (NKCC1) and Na/H exchange (NHE1) activities, leading to increased secretion of Na, Cl and water from blood into brain across a still intact BBB and consequent edema formation. More recently we found that BBB endothelial cells exposed to high glucose (6 hr to 7 d) exhibit increased abundance of NKCC and NHE and greater stimulation of the transporter activities by ischemic factors compared to cells maintained in normoglycemic conditions. Further, we found that the more robust edema formation observed in hyperglycemic rats is significantly attenuated by bumetanide and HOE‐642, inhibitors of NKCC1 and NHE1 activities. The present study was conducted to investigate signaling events underlying these effects of hyperglycemia on BBB Na transporters and ischemia‐induced cerebral edema with the overall goal of identifying additional therapeutic targets for diabetic ischemic stroke. Compelling evidence suggests that intracellular Ca (Cai) signaling mechanisms are likely to be involved. Exposure to high glucose is known to alter Cai dynamics in several cell types, including non‐brain endothelial cells. In addition, stimulation of NKCC and/or NHE activity by increases in Cai has been demonstrated in a variety of cell types. In recent studies we found that exposing BBB endothelial cells to high glucose for 48 hours resulted in elevated resting levels of Cai and significantly increased the abundance of the Transient Receptor Potential V4 (TRPV4) channel. The present study was conducted to further evaluate the effects of hyperglycemia on BBB endothelial cell Cai and to investigate whether TRPV4 plays a role in hyperglycemia‐induced alteration of Cai dynamics in the cells. Bovine cerebral microvascular endothelial cells (CMEC) were exposed for 24 hr to hyperglycemic (HG, 30mM glucose), normoglycemic (NG, 5mM glucose) or osmotic control media (5mM glucose + 12.5 mM NaCl) then loaded with 5μM Cal‐520‐AM and imaged after exposure (1 hr) to normoxia or hypoxia (7% O2). Calcium levels were normalized to maximal non‐saturated calcium signal following the addition of 8μM ionomycin. We found that HG CMEC exhibited a 4‐fold increase in [Cai] when exposed to hypoxia compared to a 2‐fold in NG CMEC. This effect was abolished in the presence Ca‐free media or the TRPV4 inhibitor HC067047 (1μM). Western blot evaluation of CMEC lysates following exposure to HG media (6 to 48 hr) revealed significant increases in TRPV4 abundance after 24 and 48 hr but no effect of osmotic control medium (NG media with added 12.5 mM NaCl). Using conventional perforated patch‐clamp methods and a 1‐second voltage ramp (−100 mV to +100 mV) protocol, we also found that the TRPV4 agonist GSK1016790A activated an outwardly rectifying current in the cells that was blocked by the antagonist (HC067047; 1μM), indicating the presence of TRPV4 activity in the cells. Our findings indicate that hyperglycemia exposures cause more robust hypoxia‐induced increases in BBB endothelial cell [Cai] in a TRPV4‐dependent manner.Support or Funding InformationSupported by NINDS and the American Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.