Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase.
Primary rat astrocytes exposed to hyperosmotic solutions undergo Na(+)-dependent amiloride-sensitive alkalinization of 0.36 U [measured with the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein], suggesting that shrinkage-induced alkalinization is due to activation of Na+/H+ exchange (NHE). Alkalinization is maintained for at least 20 min, and is readily reversible and ATP dependent. Hyperosmotic solutions produced no increase of intracellular Ca2+ or adenosine 3',5'-cyclic monophosphate (cAMP). Loading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a Ca2+ chelator, or depleting cells of protein kinase C (PKC) had no effect on activation of NHE. Thus shrinkage-induced activation of NHE does not involve cAMP, Ca2+, or PKC. However, ML-7, an inhibitor of myosin light-chain kinase (MLCK), inhibited shrinkage-induced activation with a half-maximal inhibition of 56 microM. This activation was also inhibited by 500 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, 100 microM chlorpromazine, and 50 microM trifluoperazine, all calmodulin inhibitors. Shrinkage increased the phosphorylation of an 18-kDa protein that colocalizes with myosin light chain. Our data suggest that shrinkage-induced activation of NHE in astrocytes occurs via a novel pathway involving activation of calmodulin-dependent MLCK and phosphorylation of myosin light chain.
Shrode
L D
LD
Department of Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio 45435, USA.
Klein
J D
JD
O'Neill
W C
WC
Putnam
R W
RW
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D002147:Calmodulin; D017923:Sodium-Hydrogen Exchangers; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D002118:Calcium