Stimulation of astrocyte Na+/H+ exchange activity in response to in vitro ischemia depends in part on activation of ERK1/2.
We recently reported that Na+/H+ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na+ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by approximately 75% in NHE1+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 microM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na+ but also Ca2+ homeostasis via reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX led to a 103% increase in intracellular Ca2+ concentration ([Ca2+]i) in NHE1+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca2+]i by 73%. To further investigate changes of Ca2+ signaling, bradykinin-mediated Ca2+ release was evaluated. Bradykinin-mediated intracellular Ca2+ transient in NHE1+/+ astrocytes was increased by approximately 84% after OGD/REOX. However, in NHE1-/- astrocytes or NHE1+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca2+ release was increased by only approximately 34%. Inhibition of the reverse mode of Na+/Ca2+ exchange abolished OGD/REOX-mediated Ca2+ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na+ accumulation subsequently alters Ca2+ homeostasis via Na+/Ca2+ exchange.
Kintner
Douglas B
DB
Department of Neurosurgery, University of Wisconsin Medical School, H4/332 Clinical Sciences Center, 600 Highland Ave., Madison, Wisconsin 53792, USA.
Look
Andy
A
Shull
Gary E
GE
Sun
Dandan
D
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D012964:Sodium / Q000502:physiology; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
D027682:Cation Transport Proteins; D008565:Membrane Proteins; C490997:Slc9a1 protein, mouse; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D048049:Extracellular Signal-Regulated MAP Kinases; D048052:Mitogen-Activated Protein Kinase 3