Effects of hyperosmolality on Na, K-ATPase gene expression in vascular smooth muscle cells.
Cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas were exposed to hyperosmotic media to determine the effects on Na, K-ATPase alpha1- and beta1-mRNA expression. Hyperosmotic media (500 mOsm/kgH2O) supplemented with glucose or mannitol increased alpha1-mRNA levels threefold at 24 hr and beta1-mRNA levels sevenfold at 12 hr. In sharp contrast, hyperosmotic urea medium had no effect at any time. Both the protein synthesis inhibitor cycloheximide and the RNA transcription inhibitor actinomycin D reduced alpha1- and beta1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) had no effect on the alpha1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Hyperosmotic glucose or mannitol media (500 mOsm/kgH2O) significantly increased alpha1- and beta1-subunit protein levels and Na, K-ATPase activity, whereas hyperosmotic urea medium had no effect. Transfection experiments with the 5'-flanking sequences of the alpha1- or beta1-subunit genes linked to the luciferase reporter gene revealed that hyperosmolar glucose medium increased luciferase activity 2.9- and 3.7-fold, respectively. Similarly, hyperosmotic mannitol medium increased such activity 2.7- and 3.4-fold, respectively. These results demonstrate that: (i) hyperosmolality induced by the poorly permeating solutes (glucose and mannitol) stimulates alpha1- and beta1-mRNA accumulation, alpha1- and beta1-subunit protein accumulation, and Na, K-ATPase activity, whereas the rapidly permeating solute (urea) has no effect; (ii) the upregulation of alpha1- and beta1-mRNA in response to hyperosmotic glucose or mannitol media requires, at least in part, de novo synthesis of intermediate regulatory proteins; (iii) the hyperosmolality-induced alpha1-mRNA upregulation occurs through PKC- and TK-independent mechanisms, whereas the hyperosmolality-induced beta1-mRNA upregulation occurs through activation of PKC and TK; and (iv) hyperosmolality induced by glucose or mannitol increases promoter activities of the alpha1- and beta1-subunit genes.
Muto
S
S
Departments of Nephrology and Biology, Jichi Medical School, Minamikawachi, Japan.
Ohtaka
A
A
Nemoto
J
J
Kawakami
K
K
Asano
Y
Y
D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D000254:Sodium-Potassium-Exchanging ATPase