AJP - Cell Physiology, Vol 269, Issue 1 C257-C266, Copyright © 1995 by American Physiological Society

ARTICLES

Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase

L. D. Shrode, J. D. Klein, W. C. O'Neill and R. W. Putnam
Department of Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio 45435, USA.

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.

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