Mechanisms of aldosterone's action on epithelial Na + transport.

Aldosterone maintains total organism sodium balance in all higher vertebrates. The level of sodium reabsorption is primarily determined by the action of aldosterone on epithelial sodium channels (ENaC) in the distal nephron. Recent work shows that, in an aldosterone-sensitive renal cell line (A6), aldosterone regulates sodium reabsorption by short- and long-term processes. In the short term, aldosterone regulates sodium transport by inducing expression of the small G-protein, K-Ras2A, by stimulating the activity of methyl transferase and S-adenosyl-homocysteine hydrolase to activate Ras by methylation, and, possibly, by subsequent activation by K-Ras2A of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K), which ultimately activates ENaC. In the long term, aldosterone regulates sodium transport by altering trafficking, assembly, and degradation of ENaC.

Aldosterone induces ras methylation in A6 epithelia.

Aldosterone increases Na(+) reabsorption by renal epithelial cells: the acute actions (<4 h) appear to be promoted by protein methylation. This paper describes the relationship between protein methylation and aldosterone's action and describes aldosterone-mediated targets for methylation in cultured renal cells (A6). Aldosterone increases protein methylation from 7.90 +/- 0.60 to 20.1 +/- 0.80 methyl ester cpm/microg protein. Aldosterone stimulates protein methylation by increasing methyltransferase activity from 14.0 +/- 0.64 in aldosterone-depleted cells to 31.8 +/- 2.60 methyl ester cpm/microg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce the aldosterone-induced increase in methyltransferase activity. One of these inhibitors, the isoprenyl-cysteine methyltransferase-specific inhibitor, S-trans, trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone induces protein methylation in two molecular weight ranges: near 90 kDa and around 20 kDa. The lower molecular weight range is the weight of small G proteins, and aldosterone does increase both Ras protein 1.6-fold and Ras methylation almost 12-fold. Also, Ras antisense oligonucleotides reduce the activity of Na(+) channels by about fivefold. We conclude that 1) protein methylation is essential for aldosterone-induced increases in Na(+) transport; 2) one target for methylation is p21(ras); and 3) inhibition of Ras expression or Ras methylation inhibits Na(+) channel activity.

S-adenosyl-L-homocysteine hydrolase regulates aldosterone-induced Na+ transport.

Aldosterone-induced Na+ reabsorption, in part, is regulated by a critical methyl esterification; however, the signal transduction pathway regulating this methylation remains unclear. The A6 cell line was used as a model epithelia to investigate regulation of aldosterone-induced Na+ transport by S-adenosyl-L-homocysteine hydrolase (SAHHase), the only enzyme in vertebrates known to catabolize S-adenosyl-L-homocysteine (SAH), an end product inhibitor of methyl esterification. Sodium reabsorption was decreased within 2 h by 3-deazaadenosine, a competitive inhibitor of SAHHase, with a half inhibitory concentration between 40 and 50 microM. Aldosterone increased SAH catabolism by activating SAHHase. Increased SAH catabolism was associated with a concomitant increase in S-adenosylmethionine catabolism. Moreover, SAH decreased substrate methylation. Antisense oligonucleotide complementary to SAHHase mRNA decreased SAHHase activity and Na+ current by approximately 50%. Overexpression of SAHHase increased SAHHase activity and dependent substrate methyl esterification. Whereas basal Na+ current was not affected by overexpression of SAHHase, aldosterone-induced current in SAHHase-overexpressing cells was significantly potentiated. These results demonstrate that aldosterone induction of SAHHase activity is necessary for a concomitant relief of the methylation reaction from end product inhibition by SAH and the subsequent increase in Na+ reabsorption. Thus, regulation of SAHHase activity is a control point for aldosterone signal transduction, but SAHHase is not an aldosterone-induced protein.