Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+‐K+‐Cl‐ cotransporter

Abstract

We investigated the role of Na⁺‐K⁺‐Cl^‐ cotransporter (NKCC1) in conjunction with Na⁺/Ca²⁺ exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca²⁺ homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen‐glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na⁺]_cyt which was accompanied by an elevation in [Ca²⁺]_cyt. Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX‐induced rise in [Na⁺]_cyt and [Ca²⁺]_cyt. Moreover, OGD significantly increased Ca²⁺_ER accumulation. Following REOX, a biphasic change in Ca²⁺_ER occurred with an initial release of Ca²⁺_ER which was sensitive to inositol 1,4,5‐trisphosphate receptor (IP₃R) inhibition and a subsequent refilling of Ca²⁺_ER stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca²⁺_ER. A similar result was obtained with inhibition of reversed mode operation of NCX (NCX_rev). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho‐form of the alpha subunit of eukaryotic initiation factor 2 (p‐eIF2α), and cleaved caspase 12 proteins. Pre‐treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p‐eIF2α. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX_rev may be involved in ischemic cell damage in part via disrupting ER Ca²⁺ homeostasis and ER function.