Abstract
In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.
Highlights
In the renal medulla during antidiuresis, the extratcoenli-c medium from 300 to 915 mosmkg H,O and accumulate lular fluid becomes hyperosmotic
MAPKK appears to bae convergence concentrated, theosmolality in the renal medulla increases to point for two pathways, one originating at receptor and nonover 1000 mosmkg H,O [1].The Madin-Darby canine kidney receptor tyrosine kinases where information travels through (MDCK)' epithelial cell line is considered to have characteris- ~21"" tRoaf-1 and tMo APKK and the other originatiantg tics of distal nephron segments and can tolerate extremes of G-protein-coupled signaling systems throughMEKK WAl"AP
Tyrosine phosphorylation of p42 myristate 13-acetate pretreatmentin the gen-activated protein kinase (MAPK) was in- lalitystimulates phospholipase C, MAPKK, MAPK, and S6 creased i n a nosmolality-dependent manner inanti-phosphoty- kinase activity and increasesphosphorylation of Raf-1 kinase, rosine immunoprecipitants probed with anti-MAPK antibody p42 MAPK, and pp9WSk,in MDCK cells
Summary
Cell Culture and Preparation of Extracts-MDCK cells originally purchased from the AmericaTn ype Culture Collection (Rockville, MD). 5.2 plof 4.8 pg/ml MAPK protein (UBI), and phosphorylation reactions 10 p~ l-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7) for 30 min were initiated by adding 8.3 pl of the kinase assay buffer described before hyperosmotic incubation. After incubatioant 30 "C for 10 min, myelin incubated MDCK cells in the presence of 20 1.1~genistein or 1 mM basic protein phosphorylation was initiatbeyd adding 4 polf 0.33 mg/ml herbimycin for 30 minbefore hyperosmotic incubation. Inositol 1,4,btriphosphate Measurements-MDCK cells were incuassay, levels of phosphate incorporation measured inthe absence of bated with hypertonic medium (600 mosmkg H,O by adding NaCl or exogenous MAPK protein were subtracted from values in the presenceraffinose) for 5 min at 37"C.
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