S-Adenosyl-l-homocysteine Hydrolase Regulates Aldosterone-induced Na+ Transport

Abstract

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 byS-adenosyl-l-homocysteine hydrolase (SAHHase), the only enzyme in vertebrates known to catabolizeS-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 μm. 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. 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 byS-adenosyl-l-homocysteine hydrolase (SAHHase), the only enzyme in vertebrates known to catabolizeS-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 μm. 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. Endocrine regulation of NaCl and water reabsorption at the collecting tubule of the kidney is the major site in vertebrates for maintaining normal blood pressure. Aldosterone is the primary hormone modulating salt reabsorption across renal epithelial tissue. Aldosterone increases cell entry of Na+ from the urine across the apical plasma membrane by regulating the activity of the 4 pS, amiloride-sensitive, Na+-selective channel (ENaC 1The abbreviations used are: ENaC, epithelial Na+ channel; βENaC, β subunit (one of three) of the heteromultimeric ENaC; SAHHase, S-adenosyl-l-homocysteine hydrolase; SAH, S-adenosyl-l-homocysteine; DZA, 3-deazaadenosine; AdoMet, S-adenosylmethionine; AFC, N-acetyl-S-farnesyl-l-cysteine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.1The abbreviations used are: ENaC, epithelial Na+ channel; βENaC, β subunit (one of three) of the heteromultimeric ENaC; SAHHase, S-adenosyl-l-homocysteine hydrolase; SAH, S-adenosyl-l-homocysteine; DZA, 3-deazaadenosine; AdoMet, S-adenosylmethionine; AFC, N-acetyl-S-farnesyl-l-cysteine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.; see Refs. 1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1036) Google Scholar, 2Verrey F. J. Membr. Biol. 1995; 144: 93-110Crossref PubMed Scopus (161) Google Scholar, 3Eaton D.C. Becchetti A. Ma H. Ling B.N. Kidney Int. 1995; 48: 941-949Abstract Full Text PDF PubMed Scopus (66) Google Scholar). Similar to other steroids, aldosterone regulates cell activity by modifying gene expression. Whereas the end result of aldosterone action, increased Na+ reabsorption, is well known, the aldosterone-induced proteins and resulting signal transduction pathways remain poorly described and controversial. Provocative studies by Sariban-Sohraby and colleagues (4Sariban-Sohraby S. Burg M. Wiesmann W.P. Chiang P.K. Johnson J.P. Science. 1984; 225: 745-746Crossref PubMed Scopus (87) Google Scholar) and Wiesmannet al. (5Wiesmann W.P. Johnson J.P. Miura G.A. Chaing P.K. Am. J. Physiol. 1985; 248: F43-F47Crossref PubMed Google Scholar) established a correlation between methyl esterification and acute, aldosterone-induced Na+transport. Several subsequent studies support further the hypothesis that a critical protein methyl esterification, in part, signals the acute actions of aldosterone (3Eaton D.C. Becchetti A. Ma H. Ling B.N. Kidney Int. 1995; 48: 941-949Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 6Blazer-Yost B.L. Hughes C.L. Nolan P.L. Am. J. Physiol. 1997; 272: C1928-C1935Crossref PubMed Google Scholar, 7Ismailov I.I. McDuffie J.H. Sariban-Sohraby S. Johnson J.P. Benos D.J. J. Biol. Chem. 1994; 269: 22193-22197Abstract Full Text PDF PubMed Google Scholar, 8Sariban-Sohraby S. Fisher R.S. Abramow M. J. Biol. Chem. 1993; 268: 26613-26617Abstract Full Text PDF PubMed Google Scholar). The substrate of this critical methylation remains controversial and is an active area of research. Results from our laboratory suggest that aldosterone increases methyl esterification of p21ras in renal epithelial cells (9Al-Baldawi N.F. Stockand J.D. Eaton D.C. FASEB J. 1998; 21 (abstr.): 2447Google Scholar). A study by Rokaw et al. (10Rokaw M.D. Wang J.-M. Edinger R.S. Weisz O.A. Hui D. Middleton P. Shyonsky V. Berdiev B.K. Ismailov I. Eaton D.C. Benos D.J. Johnson J.P. J. Biol. Chem. 1998; 273: 28746-28751Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) provides evidence that βENaC also may be an important substrate of aldosterone-induced methylation. Although methylation is a component of aldosterone regulation of Na+transport, the signal transduction controlling this covalent modification remains to be described. The enzymes directly regulating substrate methylation are methyltransferase and methylesterase catalyzing methyl esterification and methyl ester hydrolysis, respectively. Protein methylation is analogous to protein phosphorylation both being molecular switches controlling protein activity/locale in a reversible manner. The predominant, intracellular methyl-donating molecule is S-adenosyl-l-methionine (AdoMet). Methyltransferase catalyzes the transfer of a methyl moiety from AdoMet onto a substrate with the end product S-adenosyl-l-homocysteine (SAH) being formed. This end product is a potent feedback inhibitor of most transmethylation reactions involving AdoMet as the methyl donor (11Ueland P.M. Pharmacol. Rev. 1982; 34: 223-253PubMed Google Scholar). The observation by Sariban-Sohraby et al. (4Sariban-Sohraby S. Burg M. Wiesmann W.P. Chiang P.K. Johnson J.P. Science. 1984; 225: 745-746Crossref PubMed Scopus (87) Google Scholar) that aldosterone increases methylation of both protein and lipid is consistent with the notion that a general regulator of transmethylation reactions is the control point for mineralocorticoid-induced methylation. Thus, regulation of cellular SAH concentration may be an important site for controlling methylation relevant to aldosterone signal transduction. The only enzyme known to hydrolyze SAH in vertebrates isS-adenosyl-l-homocysteine hydrolase (SAHHase; EC3.3.1.1; see Ref. 11Ueland P.M. Pharmacol. Rev. 1982; 34: 223-253PubMed Google Scholar). This enzyme reversibly metabolizes SAH with hydrolysis resulting in production of adenosine andl-homocysteine. The cDNA encoding SAHHase has been identified in various species including Xenopus laevis(12Seery L.T. McCabe B.D. Schoenber D.R. Whitehead A.S. Biochem. Biophys. Res. Commun. 1994; 205: 1539-1546Crossref PubMed Scopus (3) Google Scholar). Pharmacological and molecular inhibitors of SAHHase and overexpression of SAHHase were used to test directly the notion that SAHHase activity regulates Na+ reabsorption. Since cellular SAH levels as set by hydrolase activity may regulate Na+ transport through modulation of substrate methyl esterification, the hypothesis that aldosterone increases SAHHase activity with concomitant increases in methylation also was tested. The results of the current study demonstrate that the natriferic actions of aldosterone are dependent on SAHHase activity with aldosterone-induced SAH hydrolysis promoting substrate methyl esterification and subsequent induction of Na+ reabsorption. Regulation of SAHHase activity by aldosterone was shown to be one control site for mineralocorticoid-induced methylation and Na+reabsorption. Amphibian distal tubule principal cells (A6 cells; American Type Culture Collection, Rockville, MD) were maintained in 4% CO2 at 26 °C in a mixture of Coon's F-12 (3 parts) and Leibovitz's L-15 (7 parts) media (Irving Scientific, Santa Ana, CA) supplemented with 104 mm NaCl, 0.6% penicillin, 1.0% streptomycin, 10% (v/v) fetal bovine serum, 25 mmNaHCO3, and 1.5 μm aldosterone (complete media). Basic media were without aldosterone and fetal bovine serum. Single-stranded cDNA from A6 cells was created using the Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA) and poly(A)+ mRNA harvested with the FastTrack 2.0 Kit (Invitrogen, Carlsbad, CA). An X. laevis SAHHase full-length clone was amplified from this cDNA using a polymerase chain reaction in conjunction with specific primers developed from the reported X. laevis SAHHase sequence (see Ref. 12Seery L.T. McCabe B.D. Schoenber D.R. Whitehead A.S. Biochem. Biophys. Res. Commun. 1994; 205: 1539-1546Crossref PubMed Scopus (3) Google Scholar; GenBankTM accession number L35559) as follows: forward primer, 5′-TTCACCATGTCTGACAAACTGTCC-3′; reverse primer, 5′-TGGAGGAAGATTGGTAAAAGAAAGC-3′. to to polymerase chain reaction a consistent with was and The was sequence from this was to that reported by al. L.T. McCabe B.D. Schoenber D.R. Whitehead A.S. Biochem. Biophys. Res. Commun. 1994; 205: 1539-1546Crossref PubMed Scopus (3) Google Scholar) and was consistent with the the full-length clone for shown in in of using the a protein of for a were used for of A6 and the for only in the that for A6 cells was increased to cells were maintained in using and used for A6 cell was by to of the A6 cells were used for of affected cell Antisense oligonucleotide was complementary to and the to of oligonucleotide was to the sequence of the X. the only sequence with to as described by a for was SAHHase. of to A6 cells a to that described by this laboratory B.N. Eaton D.C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). A6 cell with were for h to with in Aldosterone was for 4 h in the of of poly(A)+ from A6 cells was by on to and with of were by and of were and and on a h was using A to that reported by Palmer and J. Biol. Chem. Full Text PDF PubMed Google Scholar) and et al. Biochem. J. 1985; PubMed Scopus Google Scholar) was used to SAHHase cell was by cells 2 with in cells of mm mm mm were by at for at 4 The resulting maintained at 4 °C was used as the of SAHHase with enzyme activity being within 2 h the of adenosine SAHHase activity in cell for the of SAH to activity was in the in the reaction 25 2 The reaction was with of reaction of mm SAH, of A6 cell and 2 of adenosine The reaction was of and at resulting from SAH to were using an The of was used to was by hydrolysis of methyl in a to that described by et al. S. J.P. J. S. A. PubMed Scopus Google Scholar). was used as a methyl AdoMet as a methyl and A6 cell as the of to with cells were at 4 °C by in 50 mm mm were by at for at 4 of was in 50 of supplemented with and μm for the reaction was with 50 of substrate was by in of The was and to hydrolysis only methyl are was with a The methyl donor and end product (SAH) were using of A6 cells were maintained in media for to cell media were supplemented with of Inc., CA). Aldosterone was for 4 h and cells in 50 mm as were by on AdoMet and SAH, of a mixture of mm AdoMet, and SAH was in with was identified using were used in to as follows: and to of were from the and was using the of and A6 cells were on μm and to in the of was by to were with media for to Na+ transport to a This the of the acute from actions of were with aldosterone with without for 4 and and were using a with current was using Aldosterone-induced current in this was and by were from and were by the This also the specific X. laevis SAHHase primers used for polymerase chain protein concentration was established using the Protein Kit are as the was using test for and as a of in conjunction with the test was A was in the and of the actions of on Na+ transport in A6 cells maintained in current inhibition within 2 h 2 were and and were significantly at h for of and and current at 2 h across with was not with Moreover, at 2 h were with at 2 The half inhibitory concentration for was between 40 and 50 μm. current inhibition by at μm only 4 current levels to current at h for the μm was significantly cells with The of SAHHase inhibition on aldosterone-induced current and cellular of AdoMet and SAH are shown in The in current by aldosterone in the and of are shown in A and in I. of aldosterone for 4 h to and for h significantly increased by the of and μm DZA, aldosterone significantly increased current and with however, current in the of was significantly with that in the of of by aldosterone in and A6 cells with with aldosterone in to of cells with control respectively. is in in this are not 4 other control control as control as A6 cells with with aldosterone in to of cells with control respectively. is in in this are not other control as in a the in AdoMet and SAH levels in A6 cells in to aldosterone in the and of the SAHHase AdoMet levels significantly decreased by in to aldosterone significantly decreased SAH levels by the of aldosterone-induced AdoMet catabolism was significantly with levels being only the concentration to of with SAH significantly increased is significantly with that in the of These demonstrate that inhibition of SAHHase activity SAH resulting in a concomitant in AdoMet Regulation of methyltransferase activity in A6 cells by end product inhibition was of in to enzymes in cell was significantly by SAH from a control of to not shown in a 4 that aldosterone increases SAHHase activity in cell but not increase significantly mRNA SAHHase was by shown in 4 A in of aldosterone for 4 h to and cells significantly increased SAHHase activity from to aldosterone-induced SAHHase activity was significantly to of to cell enzyme in and that mRNA identified by a not increase in to shown in 4 and in the of to mRNA not significantly in to aldosterone for 4 h with the and being and of SAHHase activity by aldosterone in and 4 control and control and are and cells with μm oligonucleotide for h to cells with control control and in a are and cells with μm oligonucleotide for h to cells with control of complementary to the of the sequence the site SAHHase activity and aldosterone-induced in that the SAHHase activity in cells with aldosterone for 4 h and oligonucleotide was not significantly from the SAHHase activity in cells with 4 the SAHHase activity in cells that were with aldosterone for 4 h and was significantly cells with aldosterone and cells with Aldosterone-induced current also was by but not and aldosterone significantly by an increase to the of cells with to with significantly decreased aldosterone-induced current by approximately to that and cells with media to significantly current Overexpression of SAHHase in cells increased hydrolase activity and methylation A that the hydrolase activity in cells of is significantly the in Moreover, the methylation of AFC, in of in from cells is significantly with the in control with of A6 shown in increased both mRNA and SAHHase shown in the A6 cells with mRNA with control are in The of for to is significantly increased in control of in that to SAHHase activity in the of aldosterone and is increased in A6 cells with Aldosterone significantly increased activity in to The activity in to aldosterone for SAHHase-overexpressing cells was significantly with that for control that the activity in control was not that in Overexpression of SAHHase in to enzyme activity aldosterone-induced and SAHHase not a significantly basal current with control and cells but a significantly increased current to aldosterone 4 Aldosterone significantly increased current by and in not shown in this and A6 cell respectively. The aldosterone-induced current in SAHHase-overexpressing cells was significantly with that in control and Aldosterone-induced current in was with inhibitor a in Na+ the of aldosterone and current in decreased from basal levels by in the of aldosterone for control and were and respectively. These are not significantly to in the of A of the current by aldosterone to A6 cells SAHHase and in the of molecular and SAHHase inhibitors is shown in I. Aldosterone for 4 h significantly increased current in control A6 the without of Aldosterone also increased current in cells and cells with and for and cells and cells were not from and aldosterone-induced current was decreased with control cells and respectively. Aldosterone increased current in with control cells and control The regulation of SAHHase activity by aldosterone in the of and molecular inhibitors of SAHHase and in A6 cells is in Aldosterone increased activity in control and cells; however, the activity in cells was with and in cells with aldosterone and was with that in cells with aldosterone in to decreased SAHHase activity with The current study directly the SAHHase in the signal transduction by aldosterone in increased Na+ reabsorption. Aldosterone increased the activity of SAHHase through Moreover, as shown by both and molecular induction of SAHHase activity was critical for aldosterone to increase and Na+ reabsorption across renal epithelial The current results also demonstrate that overexpression of SAHHase aldosterone-induced current and enzyme overexpression is to the natriferic actions of the Thus, results support the hypothesis that the critical methylation for aldosterone signal transduction is regulated by SAH catabolism with SAHHase activity being the control in is a of the critical enzymes and for the aldosterone-induced transmethylation reaction that Na+ channel Sariban-Sohraby and colleagues (4Sariban-Sohraby S. Burg M. Wiesmann W.P. Chiang P.K. Johnson J.P. Science. 1984; 225: 745-746Crossref PubMed Scopus (87) Google Scholar) and Wiesmann et al. (5Wiesmann W.P. Johnson J.P. Miura G.A. Chaing P.K. Am. J. Physiol. 1985; 248: F43-F47Crossref PubMed Google Scholar) were the to the of aldosterone-induced Na+ transport with the methylation These that methyl as AdoMet, the actions of inhibition of transmethylation induction of transport by aldosterone that a critical methylation was necessary for Na+ reabsorption. aldosterone increased transmethylation of of including both proteins and that the regulated methylation in general but not regulate a specific transmethylation The current results that one through aldosterone regulates methylation is by the activity of SAHHase. results demonstrate that aldosterone increases Na+ transport by the activity of SAHHase through an as An aldosterone-induced increase in hydrolysis of SAH increases the of most transmethylation reactions by end product feedback Several other also that and activity are regulated by transmethylation (3Eaton D.C. Becchetti A. Ma H. Ling B.N. Kidney Int. 1995; 48: 941-949Abstract Full Text PDF PubMed Scopus (66) Google B.L. Hughes C.L. Nolan P.L. Am. J. Physiol. 1997; 272: C1928-C1935Crossref PubMed Google Scholar, 7Ismailov I.I. McDuffie J.H. Sariban-Sohraby S. Johnson J.P. Benos D.J. J. Biol. Chem. 1994; 269: 22193-22197Abstract Full Text PDF PubMed Google Scholar, 8Sariban-Sohraby S. Fisher R.S. Abramow M. J. Biol. Chem. 1993; 268: 26613-26617Abstract Full Text PDF PubMed Google however, the substrate for this aldosterone-induced methylation remains unclear. results from our laboratory suggest that methylation of p21ras is increased by aldosterone (9Al-Baldawi N.F. Stockand J.D. Eaton D.C. FASEB J. 1998; 21 (abstr.): 2447Google and results from the laboratory of Johnson (10Rokaw M.D. Wang J.-M. Edinger R.S. Weisz O.A. Hui D. Middleton P. Shyonsky V. Berdiev B.K. Ismailov I. Eaton D.C. Benos D.J. Johnson J.P. J. Biol. Chem. 1998; 273: 28746-28751Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) support the notion that βENaC also is an methylation decreased aldosterone-induced SAH hydrolysis and SAHHase activity in and respectively. This is consistent with that reported by (11Ueland P.M. Pharmacol. Rev. 1982; 34: 223-253PubMed Google Scholar, P.K. Pharmacol. Google Scholar). Similar to DZA, oligonucleotide complementary to the the site of mRNA decreased enzyme activity The activity of SAHHase as by SAH hydrolysis in A6 cell 4 was to activity reported in of J. H. Biochem. J. PubMed Scopus Google Scholar) and bovine kidney D. J. S. H. Kidney Res. PubMed Scopus Google however, activity was with of and J. H. Biochem. J. PubMed Scopus Google Scholar). Aldosterone for 4 h to A6 cell SAHHase activity to a to that in J. Biochem. Biophys. PubMed Scopus (3) Google Scholar). This induction of activity that SAHHase is regulated by aldosterone signal transduction, an observation consistent with that reported by and J.D. Biochem. Biophys. PubMed Scopus Google Scholar) that regulate SAHHase activity in epithelial tissue. The for modulation of SAHHase by however, remains to be for increased activity in to aldosterone as follows: increased enzyme increased activity through modification of the and relief of the enzyme from regulation by increased of an as Aldosterone to significantly increase SAHHase mRNA to the that SAHHase enzyme activity increased that an increase in enzyme concentration was The observation that activity was in from cells with aldosterone with cells of SAHHase has been of end product inhibition by the of adenosine of a protein of consistent with that reported for SAHHase (11Ueland P.M. Pharmacol. Rev. 1982; 34: 223-253PubMed Google Scholar, D. J. S. H. Kidney Res. PubMed Scopus Google Scholar). Overexpression of SAHHase increased enzyme activity in both and A6 cells The observation that A6 cells of aldosterone increase in SAHHase mRNA levels with control but only the activity the notion that regulated by aldosterone control enzyme Moreover, aldosterone to cells an on SAHHase activity that from regulating protein increased activity through other These results that aldosterone increases SAHHase activity through a signal transduction in regulation of the The primary sequence of SAHHase phosphorylation for both and a number of for Although the of SAHHase regulation by aldosterone remains to be is that this enzyme is regulated by as phosphorylation be to test SAHHase is regulated by the aldosterone-induced protein has been shown to be regulated in to aldosterone S. A. D. F. D. J. Am. 1998; (abstr.): Scholar). is well established that SAH is an end product inhibitor of most transmethylation reactions involving AdoMet as the methyl donor (11Ueland P.M. Pharmacol. Rev. 1982; 34: 223-253PubMed Google Scholar). Sariban-Sohraby and colleagues (4Sariban-Sohraby S. Burg M. Wiesmann W.P. Chiang P.K. Johnson J.P. Science. 1984; 225: 745-746Crossref PubMed Scopus (87) Google Scholar) that and SAH the acute actions of aldosterone as by Na+ across apical membrane from A6 Moreover, of aldosterone increased both lipid and protein transmethylation in a in epithelia that aldosterone affected a general control point regulating of methylation. Eaton et al. (3Eaton D.C. Becchetti A. Ma H. Ling B.N. Kidney Int. 1995; 48: 941-949Abstract Full Text PDF PubMed Scopus (66) Google Scholar) shown through channel that regulates activity through regulating the transmethylation Although of studies support the hypothesis that SAHHase activity regulates substrate methyl esterification, this was not the of of studies and only a inhibitor was Moreover, was used only at a concentration in of of not be and was control of SAHHase was a for regulating transmethylation reactions relevant to aldosterone-induced Na+ transport in epithelial The current study is the to investigate directly the SAHHase in regulation of aldosterone-induced methylation and Na+ The results of the current study that aldosterone to A6 cells increases both SAH and AdoMet of SAHHase by decreased SAH to an that cellular in to aldosterone AdoMet by methyltransferase increased SAH, not be by an SAHHase. This notion also for the in AdoMet in the of with increased SAH methyltransferase activity AdoMet catabolism. Methyltransferase activity in A6 cell also was decreased by end product inhibition that SAHHase activity through regulation of cellular SAH levels control substrate methylation. Overexpression of SAHHase in an increase in both SAHHase activity and substrate methyl esterification These results are consistent with aldosterone of the end product inhibitor of methyl esterification SAH, resulting in a concomitant increase in AdoMet in A6 Thus, in epithelial one site controlling substrate methylation is modulation of SAHHase of with aldosterone for 4 h current in a of to Na+ transport within 2 h in a 4 Na+ current in the with to with current levels line by The for inhibition of and aldosterone-induced transport are consistent with also that Na+ transport in and A6 epithelia (4Sariban-Sohraby S. Burg M. Wiesmann W.P. Chiang P.K. Johnson J.P. Science. 1984; 225: 745-746Crossref PubMed Scopus (87) Google Scholar, W.P. Johnson J.P. Miura G.A. Chaing P.K. Am. J. Physiol. 1985; 248: F43-F47Crossref PubMed Google Scholar). consistent with our that aldosterone-induced Na+ reabsorption are the of Eaton et al. (3Eaton D.C. Becchetti A. Ma H. Ling B.N. Kidney Int. 1995; 48: 941-949Abstract Full Text PDF PubMed Scopus (66) Google Scholar) that of for h aldosterone-induced of in The results of the current study that of Na+ reabsorption to 4 h be by of to a potent inhibitor of SAHHase. is that DZA, also is a substrate for SAHHase in the (11Ueland P.M. Pharmacol. Rev. 1982; 34: 223-253PubMed Google was to 4 Since for is that of I. Chiang P.K. Biochem. Biophys. PubMed Scopus Google this current Since is in results from current are consistent with this support for the hypothesis that aldosterone Na+ in part, by regulating SAH hydrolysis is by and overexpression Antisense oligonucleotide but not decreased both aldosterone-induced enzyme activity and Na+ current that active SAHHase is necessary for salt reabsorption. Overexpression of SAHHase increased enzyme activity in an Moreover, overexpression of SAHHase aldosterone-induced current but not basal that increased SAHHase activity was to signal increased The current results support the hypothesis that a critical methyl esterification in to aldosterone signals the increase in activity at the apical membrane of epithelial Since methyltransferase and SAHHase are by SAH the enzyme is a site for regulating methyl esterification. The results of the current study demonstrate that aldosterone increase SAHHase activity to the subsequent increase in both methyl esterification and Na+transport. Thus, regulation of SAHHase activity by aldosterone-induced signal transduction is a control site for induction of methylation and subsequent Na+ reabsorption. J. for support and of A6 cells in