©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Variants in the P450c11AS Gene as Determinants of Aldosterone Synthase Activity in the Dahl Rat Model of Hypertension (*)

Carolyn M. Cover (1), Jia-Ming Wang (2), Elizabeth St. Lezin (2), Theodore W. Kurtz (2), Synthia H. Mellon (1)(§)

From the (1)Departments of Obstetrics, Gynecology, and Reproductive Sciences and, the (2)Laboratory Medicine, and the (3)Metabolic Research Unit, University of California, San Francisco, California 94143-0556

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Dahl salt-sensitive (S) and salt-resistant (R) rats are widely used to study genetic determinants of salt-sensitive hypertension. Differences in blood pressure under a high sodium diet in these two strains may be due to differences in the synthesis of 18-OH-11-deoxycorticosterone (18-OH DOC). This difference in 18-OH-DOC synthesis is due to mutations in the Dahl R rat's gene for P450c11 (11-hydroxylase), an adrenal enzyme involved in the synthesis of both corticosterone and 18-OH DOC from 11-deoxycorticosterone. Aldosterone/renin ratios in plasma and in the adrenals are greater in Dahl S than R rats, suggesting an altered physiologic relationship between the renin-angiotensin and aldosterone systems between these strains. We demonstrate that the mRNA for P450c11AS, (aldosterone synthase), an enzyme required for aldosterone synthesis, is identical in the Dahl S rat and in normotensive Sprague-Dawley rats, but that P450c11AS mRNA from the Dahl R rat contains 7 mutations that result in two amino acid substitutions. These two changes result in a form of P450c11AS that has a greater apparent V and lower apparent K, resulting in an enzyme that catalyzes the conversion of 11-deoxycorticosterone to aldosterone at a greater rate in Dahl R rats than the P450c11AS in Dahl S rats or Sprague-Dawley rats. Although plasma and adrenal renin are lower in Dahl S versus R rats, the regulation of P450c11AS mRNA expression in rats fed a low and high salt diet are identical in these strains. The current findings may explain both the reduced aldosterone concentrations and increased aldosterone/renin ratios previously reported in the Dahl S versus Dahl R rat.


INTRODUCTION

The etiologies of essential hypertension are unknown, but they include both environmental and genetic factors. At least one form of salt-dependent hypertension is due to a genetic lesion in the gene for 11-hydroxylase, P450c11, and aldosterone synthase, P450c11AS(1) . Both the rat and human forms of this P450c11 encode a mitochondrial protein found in the adrenal fasciculata/reticularis that synthesizes corticosterone from 11-deoxycorticosterone (DOC)()and cortisol from 11-deoxycortisol, while the rat and human forms of P450c11AS encode a mitochondrial protein found in the glomerulosa that synthesizes aldosterone from corticosterone(2, 3, 4, 5, 6, 7, 8) . In patients with glucocorticoid-remediable aldosteronism, fusion of the regulatory regions of the P450c11 gene to the coding region of the P450c11AS gene results in disordered steroid synthesis, hyperaldosteronism, and hypertension(9) . Several other rare forms of secondary hypertension also result from elevated concentrations of mineralocorticoids(10, 11) . This raises the possibility that alterations in either P450c11 or P450c11AS may contribute to the pathogenesis of other forms of salt-sensitive hypertension.

The Dahl salt-sensitive (S) rat is the most widely studied genetic model of salt-sensitive hypertension. In the Dahl S strain, supplemental dietary sodium increases blood pressure, but in the Dahl salt-resistant (R) strain, supplemental dietary sodium has little effect on blood pressure(12) . Molecular variants in the coding sequence of P450c11 have recently been identified that may contribute to the differences in blood pressure between the S and R strains(13, 14) . The P450c11 gene of the Dahl R rat differs from that of 12 commonly used rat strains, including the Dahl S rat, and contains missense mutations that alter 11-hydroxylase activity and decrease the production of 18-OH-DOC. Moreover, in genetic studies, these mutations have been found to cosegregate with reduced adrenal capacity to synthesize 18-OH-DOC and resistance to the hypertensinogenic effects of NaCl. These observations suggest that the robust salt resistance of the Dahl R rat relative to the Dahl S rat may be due in part to molecular variants in P450c11 and alterations in the activity of 11-hydroxylase. However, in rats as in other mammals, the P450c11 gene is tightly linked to P450c11AS, and therefore, blood pressure effects that cosegregate with P450c11 could also be emanating from variants in P450c11AS.

In the Dahl S rat, plasma levels of aldosterone are lower than in the Dahl R rat(15, 16, 17, 18) , and, therefore, it has been assumed that genetically determined alterations in aldosterone biosynthesis do not contribute to the strain differences in blood pressure. The lower aldosterone concentrations in Dahl S versus Dahl R rats have been proposed to be a physiologic consequence of the lower renin concentrations in Dahl S rats(19) . However, in Dahl S rats, the ratio of aldosterone/renin in the adrenals and in the plasma is greater than in Dahl R rats(18, 20) . This raises the possibility that in Dahl S and Dahl R rats, differences in aldosterone biosynthesis may involve something more than just differences in plasma renin activity. Although the P450c11 gene has been well characterized in Dahl S and R rats, little is known about the structure or function of P450c11AS in this model. In the current studies in Dahl S and R rats, we cloned, sequenced, and expressed P450c11AS cDNAs from these strains and studied the regulation of P450c11 and P450c11AS mRNAs. Our results suggest that strain differences in the structure and regulation of P450c11AS may be related to the strain differences in aldosterone concentrations and aldosterone/renin ratios.


MATERIALS AND METHODS

Cloning and Sequencing P450c11AS cDNAs from Dahl-resistant and Dahl-sensitive Rat Adrenals

Reverse transcription and polymerase chain reaction were used to prepare and clone P450c11AS cDNAs from the adrenals of three Dahl R and three Dahl S rats. Rats were treated with furosemide (2 mg for 2 days) to induce a sodium diuresis and stimulate P450c11AS synthesis, and with dexamethasone (20 µg/animal) to suppress P450c11 mRNA synthesis(3) . RNA isolated by guanidinium isothiocyanate solubilization and CsCl gradient ultracentrifugation was reverse transcribed using with the Superscript cDNA Synthesis Kit (Life Technologies Inc.). Our polymerase chain reaction amplification of cDNA used two sets of P450c11AS-specific primers so that the amplified products contained overlapping sequences. The first polymerase chain reaction amplified exons 1-4 and used a 5` primer that included the first 6 amino acids of exon 1 (5`-GGATGGCAATGGCTCTCAGGGTGACA-3`) and a 3` primer that spanned the first 6 amino acids of exon 5 (5`-CTTCCAGATACATCTGTTGG-3`). An EcoRI cloning site was added at the 5` end of the 5` primer. The 3` primer has 3`-terminal mismatches for P450c11 and is specific for P450c11AS. The second polymerase chain reaction amplified exon 4-9, and used a 5` primer specific for P450c11AS (nucleotides 651-672(21) ) (5`-CCCTGGTAGCCTGAAGTTCATC-3`) and a 3` primer (nucleotides 1526-1547 (21)) that distinguished P450c11 from P450c11AS (5`-AGAAGTAGGCCGGTGGACTGAT-3`). An XbaI cloning site was added to the 5` end of the 3` primer. Twenty nanograms of cDNA were amplified for 30 cycles using the following conditions: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min. Amplified fragments were digested with XbaI and HindIII, purified by agarose gel electrophoresis, ligated, and cloned into M13 and sequenced by dideoxy sequencing (Sequenase, U. S. Biochemical Corp.). All but the first 18 nucleotides of the coding sequence could be compared.

Expression of P450c11AS cDNAs after Transfection into MA-10 Cells

Full-length P450c11AS cDNAs from both Dahl S and Dahl R rats were cloned into pECE (22) and were transfected into mouse Leydig MA-10 cells by calcium phosphate precipitation. After 48 h, the cells were incubated with varying concentrations of cold corticosterone for 24 h. Enzymatic activity was assayed by determining the 0-48 h time courses for aldosterone production using 10M 11-deoxycorticosterone in the incubation medium or by determining aldosterone production using 10-10M 11-deoxycorticosterone in the medium for 24 h. Medium was analyzed for aldosterone by RIA (ICN Biochemicals, Costa Mesa, CA).

Animals

Dahl salt-resistant (SR/Jr strain, hereafter referred to as R rats) and Dahl salt-sensitive rats (SS/Jr, hereafter referred to as S rats) were obtained from Harlan-Sprague-Dawley Inc., prior to the occurrence of genetic contamination in the Dahl strain (23). They were divided into four groups and received one of four regimens for 7 days(3) . Control animals were fed rat chow and water ad libitum. Dexamethasone-treated animals received daily subcutaneous injections of 20 µg of dexamethasone in 100 µl of normal saline and were fed rat chow and water ad libitum. Sodium-restricted animals (low salt) received 2 mg of furosemide subcutaneously for 2 days and were fed sodium-deficient rat chow (background sodium, 0.0033%) (Teklad, Madison, WI) and water ad libitum. Sodium-replete animals (high salt) received 2% NaCl in their drinking water and rat chow ad libitum. After 7-days of each treatment, the rats were anesthetized with metaphane and killed by decapitation at 1300 h. Pairs of adrenals were frozen separately in liquid nitrogen.

Preparation of RNA

-RNA was extracted from adrenals by guanidinium isothiocyanate homogenization, purified by cesium chloride ultracentrifugation as described previously(3) , and quantitated by determining the optical density at 260 and 280 nm.

Preparation of RNA Probes

Three different cRNA probes were prepared by transcription of various cDNA fragments cloned in pKS (Stratagene, La Jolla, CA) to analyze the abundance of P450c11 and P450c11AS mRNAs in the adrenal. The P450c11 probe was a 280-base RNA probe containing 219-bases of rat P450c11 cDNA (corresponding to nucleotides 213-432(24) ) that hybridizes to both P450c11 and to P450c11AS mRNAs. When hybridized to normal rat P450c11 mRNA, a fragment of 219 bases is protected, and when hybridized to normal rat P450c11AS mRNA, a fragment of 192 bases is protected(3) . A 290-base RNA probe containing 185-bases of rat P450c11AS cDNA (corresponding to bases 809-994(5, 24) ). A 260-base RNA probe containing a 155-base pair rat P450c11 cDNA fragment (corresponding to bases 839-994(5, 24) ). To analyze P450scc mRNA, a 241-base pair EcoRI-PvuII fragment of rat P450scc cDNA(25, 26) was cloned in pKS, generating a 296-base RNA probe that protects a 233-base RNA fragment. As a control, a 195-base rat actin RNA probe containing a 149-base pair rat actin cDNA fragment (corresponding to nucleotides 2066-2216 of rat cytoplasmic -actin(27) ) was synthesized by reverse transcription polymerase chain reaction of rat liver RNA(5) . RNA probes were synthesized as described previously (28).

RNA Analysis

RNA was analyzed by RNase protection assays (3, 5). One microgram of RNA from the adrenal was precipitated with 500,000 cpm of P-labeled cRNA probe and was resuspended in 20 µl of hybridization buffer containing 80% formamide, 400 mM NaCl, 40 mM Pipes, pH 6.4, 1 mM EDTA. Samples were heated to 95 °C for 5 min and incubated at 42 °C overnight. Samples were then diluted 10-fold with buffer containing 300 mM NaCl, 10 mM Tris-Cl, pH 7.9, 5 mM EDTA, and 20 µg/ml RNase A. Samples were incubated for 30 min at 42 °C, extracted once with phenol/chloroform, and precipitated with ethanol. RNase-resistant double-stranded fragments were separated on 6% polyacrylamide, 7.5 M urea/sequencing gels using P-labeled MspI-digested pBR322 DNA as molecular weight markers. The gels were dried onto filter paper and exposed to x-ray film under an intensifying screen at -70 °C.

Steroid Analysis

Medium was saved from transfection assays and was analyzed for aldosterone by RIA using a commercially available kit (ICN Biochemicals, Costa Mesa, CA).


RESULTS

Cloning P450c11AS cDNA from Dahl S and Dahl R Rats

The sequence of the P450c11AS cDNA (from nucleotides 1 to 1547) from the Dahl S rat was identical to that of the outbred Sprague-Dawley rat and thus predicted identical amino acid sequences. However, the sequence of the P450c11AS cDNA from the Dahl R rat contained seven nucleotide differences. Five of these seven differences were silent mutations, but two changed the encoded amino acid sequence of P450c11AS (): Glu 136 Asp and Gln 251 Arg. Both of these substitutions in the Dahl R P450c11AS sequence generate the same amino acids found at the corresponding residue of the P450c11 gene.

Expression of P450c11AS cDNAs and Analysis of Their Activities

We cloned P450c11AS cDNAs from the Dahl S and Dahl R rats in the SV40 eukaryotic expression vector, pECE(22) . These plasmids were then transfected into mouse Leydig MA-10 cells, which do not normally express P450c11AS, but which contain the mitochondrial electron transfer proteins adrenodoxin and adrenodoxin reductase that are needed for P450c11AS activity. P450c11AS from the Dahl R and Dahl S rats synthesized aldosterone from corticosterone (Fig. 1). However, the amounts of aldosterone synthesized were radically different. The Dahl R P450c11AS synthesized >1000 times more aldosterone than did the Dahl S P450c11AS. This was not due to differences in the transfection efficiencies of the plasmids containing each cDNA, since all were co-transfected with a probe for RSV-luciferase and corrected for transfection efficiency. This difference was also not due to differential expression and stability of the different forms of P450c11AS mRNA in the transfected MA-10 cells, as RNase protection assays showed that cells transfected with both cDNA plasmids expressed P450c11AS mRNA to the same degree (not shown). These data suggest that the proteins encoded by these mRNAs are inherently different enzymatically.


Figure 1: RIA analysis of aldosterone secreted from MA-10 cells transfected with pECE-P450c11AS expression vectors. One microgram of pECE-P450c11AS or pECE-P450c11AS was co-transfected into MA-10 cells with 0.5 µg of RSV-luciferase as an internal control for transfection efficiency. Forty eight hours after transfection, cells were incubated for 24 hr with 10M 11-deoxycorticosterone. Aldosterone from the medium was analyzed in duplicate, and was corrected for transfection efficiency. Data from the P450c11AS cDNA expression vector are shown in the black bar. Control cells transfected with the pECE vector alone had aldosterone concentrations of less than 0.02 pg/ml. Results are ± S.E. from three separate transfections, each done in triplicate.



To characterize the enzymatic activity of each of these forms of P450c11AS, we incubated transfected MA-10 cells with varying concentrations of 11-deoxycorticosterone for various periods of time (Fig. 2). All samples were co-transfected with a vector for RSV-luciferase to correct for transfection efficiency. At all DOC concentrations and at all times, P450c11AS from Dahl R rats converted more DOC to aldosterone than did P450c11AS from Dahl S rats. Lineweaver-Burk analysis of the enzymatic conversion of DOC to aldosterone indicated an apparent V of 0.0044 pg of aldosterone/ml/min for the Dahl S P450c11AS and an apparent V of 4.57 pg of aldosterone/ml/min for the Dahl R P450c11AS. Similarly, Lineweaver-Burk analysis of the enzymatic conversion of DOC to aldosterone indicated an apparent K of 19.26 µM for the Dahl S enzyme and an apparent K of 2.15 µM for the Dahl R P450c11AS. Thus, the P450c11AS from the Dahl R rat works faster and at lower substrate concentrations than does the Dahl S P450c11AS enzyme.


Figure 2: RIA analysis of aldosterone. MA-10 cells transfected with 1 µg of pECE-P450c11AS expression vectors and 0.5 µg of RSV-luciferase were incubated with varying concentrations of 11-deoxycorticosterone (A and B) or with 10M 11-deoxycorticosterone for varying times (C). Experiments were performed as described in the legend to Fig. 1. Data from the P450c11AS cDNA expression vector are in panelsA and D and are shown as closedcircles in panelC; data from the P450c11AS cDNA expression vector are in panelsB and E and are shown as opencircles in panelC. Lineweaver-Burk replots of the data from A and B are shown in D and E. Aldosterone from the medium was analyzed in duplicate by RIA and was corrected for transfection efficiency. Results are ± S.E. from three separate transfections, each done in triplicate.



Detection of P450c11, P450c11AS, and P450c11ASmRNAs with a Single Probe

We used a P450c11 RNA probe that detects both P450c11 and P450c11AS mRNAs, but distinguishes them by size on polyacrylamide gels(3, 5, 29) . The nucleotide sequences of the cloned genes and cDNAs for P450c11 (24, 30) and P450c11AS (21, 30) are known. We chose a region corresponding to bases 213-432 (24) of P450c11 for the probe (Fig. 3). The probe contains 219 bases of P450c11 sequences and 61 bases of vector sequences. Hybridization of this probe to P450c11 mRNA from either Dahl salt-sensitive or -resistant rats yields a protected fragment of 219 bases. (Mutation at base 379 in the Dahl R P450c11 mRNA from a CU does not result in an RNase A-sensitive site.) In the region of DNA used as probe, there are eight differences between the sequence of P450c11AS and P450c11 used as probe, and six differences between the sequence of P450c11AS and P450c11 used as probe (Fig. 3). However, only the differences at bases 405 and 429 (underlined in Fig. 3) result in a mismatched RNA hybrid that is sensitive to RNase A digestion(3, 29) . Thus P450c11AS mRNA from both normal Sprague-Dawley and from Dahl S rats will protect a probe fragment of 192 bases (from base 213 to base 405). In the Dahl R rat, there are mutations at bases 405 and 408 that change the base to the corresponding base in P450c11 and thus do not create mismatched base pairs. However, the P450c11AS sequence in both the Dahl R and S rats differs from the sequence of P450c11 at base 429. This RNase A-sensitive mismatch results in the protection of a fragment of 216 bases. This is not seen in the Dahl S rat, since the smaller, 192 base fragment, lacking this region, is generated. Thus, our RNase protection assay can distinguish the mRNAs for P450c11AS from Dahl S and R rats.


Figure 3: Alignments of P450c11, P450c11AS (P450c11AS) and P450c11AS (P450c11AS) mRNA sequences with the P450c11 cRNA used as probe in RNase protection assays. The sequences are from nucleotide 213-432, as numbered by Nonaka et al. (24). Differences in eight nucleotides in the P450c11AS sequence and differences in six nucleotides in the P450c11AS sequence are noted below the c11 sequence. Nucleotides 405 and 408 in boldface show the mutation in the P450c11AS sequence. The lines under nucleotides 405 and 429 show the P450c11AS:P450c11 mismatched bases that are cleaved by RNase A digestion. The singleunderlined base 405 in P450c11AS is sensitive to RNase A digestion, resulting in a 192-nucleotide protected fragment. The doubleunderlined base 429 in P450c11AS is sensitive to RNase A digestion, resulting in a 216-nucleotide protected fragment.



Regulation of P450c11 and P450c11AS mRNAs in Dahl sensitive and Dahl resistant Rats

To determine if there was differential regulation of the genes encoding the P450c11 and P450c11AS mRNAs, we analyzed RNA isolated from Dahl S and Dahl R rats given different dietary and hormonal treatments. Rats were given either a high or low salt diet, which is known to alter P450c11AS mRNA abundance(3, 31, 32) , or they were given injections of dexamethasone, which is known to alter the abundance of the mRNA for P450c11 but not for P450c11AS (3).

Both Dahl S and Dahl R rats given a normal diet produce much more P450c11 mRNA than P450c11AS mRNA (Fig. 4). This is similar to what is seen in normotensive Sprague-Dawley rats(3) . When given a low salt diet, both strains of Dahl rats increase the abundance of P450c11AS mRNA without affecting expression of P450c11 mRNA. The degree to which P450c11AS mRNA is induced by a low salt diet is the same in the Dahl S and Dahl R rats and is similar to that in normotensive Sprague-Dawley rats(3) .


Figure 4: RNase protection assay of RNA isolated from adrenals of Dahl S and R rats given a low salt (LS) or high salt (HS) diet, or normal rat chow (C). 2 µg of adrenal RNA was combined with 1 10 cpm of P-labeled P450c11 and actin cRNA probes, hybridized overnight, digested with RNase A, and separated on 5% acrylamide. 7.5 M urea sequencing gels. Markers (M) are P-labeled MspI pBR322 DNA fragments. The lane t contained 50 µg of tRNA and P-labeled probe.



As expected, there is no effect on the expression of P450c11 mRNA in either the S or R rat in rats given a high salt diet (Fig. 5). Using a greater amount of RNA (20 µg rather than 2 µg) and using a probe specific for P450c11AS mRNA (bases 809-994) (5) that does not contain any differences between P450c11AS from the S and R rats, we could see that in both Dahl S and R rats given a high salt diet, there is an equivalent reduction in the abundance of P450c11AS mRNA (Fig. 5).


Figure 5: RNase protection assay of RNA isolated from adrenals of Dahl S and R rats given a low or high salt diet, or injections of dexamethasone (Dex). 20 µg of adrenal RNA was combined with 1 10 cpm of P-labeled P450c11AS and actin cRNA probes, hybridized overnight, digested with RNase A, and separated on 5% acrylamide. 7.5 M urea sequencing gels. Lanes Dex (1) and Dex (3) are samples from both Dahl-sensitive and -resistant rats treated with dexamethasone, but whose P450c11 mRNA responses were different (see Fig. 6) Markers (M) are P-labeled MspI pBR322 DNA fragments. The lane tRNA contained 50 µg of tRNA and P-labeled probe. Lane probes contains the P450c11AS and actin cRNA probes.



Regulation of expression of both P450c11 and P450c11AS mRNAs by dexamethasone was also studied ( Fig. 5and Fig. 6). In both the S and R rat, like in Sprague-Dawley rats, dexamethasone selectively decreases the expression of P450c11 mRNA, without affecting expression of P450c11AS mRNA. In some cases, both Dahl S and Dahl R rats are equally resistant to dexamethasone treatment, and P450c11 mRNA is unaffected (Fig. 6, Dex3, sensitive; Dex1, resistant). This apparent lack of regulation by dexamethasone is similar in frequency (about 1 in 3 rats) to that seen in Sprague-Dawley rats(33) .()This lack of P450c11 mRNA regulation was not indicative of abnormal regulation of P450c11AS mRNA in either Dahl S or Dahl R rats (Fig. 5), since P450c11AS mRNA from neither rat was affected by dexamethasone treatment. Thus, there is no difference in the regulation of P450c11 or P450c11AS mRNAs by dexamethasone between strains of Dahl rats, or between normotensive Sprague-Dawley rats and Dahl rats.


Figure 6: RNase protection assay of RNA isolated from adrenals of Dahl S and R rats given dexamethasone injections. 2 µg of adrenal RNA was analyzed from each sample, as described in the legend to Fig. 4. Three animals from each experimental group are shown. Samples from Dahl sensitive and resistant rats given dexamethasone injections, dexamethasone 1 and 3 from each group, show different responses in P450c11 mRNA regulation, and were also analyzed using a P450c11AS-specific probe (see Fig. 5).




DISCUSSION

It is well established that in Dahl S rats, plasma and adrenal concentrations of both renin and aldosterone are lower than in Dahl R rats(18, 20) . This would suggest that disordered aldosterone production is not an important determinant of the greater blood pressure in S rats versus R rats. It has been proposed that the decreased concentrations of aldosterone in Dahl S rats versus R rats are a physiologic consequence of the decreased renin concentrations(19) . However, in Dahl S rats, the aldosterone/renin ratios in the plasma and in adrenals are greater than in Dahl R rats. This raises the possibility that reduced aldosterone concentrations in Dahl S rats are not just a consequence of reduced renin concentrations.

In the current study, we have found that the amount of adrenal P450c11AS mRNA is similar in S rats versus R rats and that in both strains, NaCl depletion increases P450c11AS mRNA and NaCl loading decreases P450c11AS mRNA to the same extent. Thus, the reduced plasma concentrations of aldosterone in S rats versus R rats cannot be readily attributed to physiologic reductions in P450c11AS gene expression caused by reduced plasma concentrations of renin. The finding that P450c11AS mRNA levels are similar in Dahl S and R rats despite the fact that plasma and adrenal renin levels are known to be reduced in the S strain suggests that Dahl S and R rats may differ with respect to the transcriptional regulation and or message stability of P450c11AS. Enhanced transcription or message stability of P450c11AS in Dahl S rats versus R rats could account for similar levels of P450c11AS message despite reduced renin levels in the S strain. This would serve to explain the greater aldosterone/renin ratios that have been reported in Dahl S rats versus R rats.

Given that P450c11AS mRNA levels are similar in Dahl S and R rats, how does one account for the lower plasma levels of aldosterone reported in the S strain? In transfection studies, we found that the inherent enzymatic activity of aldosterone synthase is lower in the S rat versus the R rat, owing to a lower apparent V and a greater apparent K of the Dahl S enzyme. This could contribute to the lower plasma concentrations of aldosterone in S rats versus R rats in the face of equivalent amounts of P450c11AS mRNA. Reduced translational efficiency of P450c11AS mRNA from the Dahl S versus R rat could also contribute to lower aldosterone synthase activity and lower plasma levels of aldosterone in the S strain versus R strain despite equivalent amounts of P450c11AS mRNA. The transfection studies suggest that the S form of aldosterone synthase is nearly 1000-fold less active than the R form of aldosterone synthase. This 1000-fold difference in aldosterone synthase activity is far greater than the modest 2-fold differences in plasma and adrenal aldosterone levels reported between Dahl S and R rats(18, 20) . This presumably reflects the fact that in addition to the inherent enzymatic properties of aldosterone synthase, other factors (e.g. substrate availability, translational efficiency of P450c11AS mRNA, aldosterone clearance rate, etc.) also influence circulating and tissue levels of aldosterone.

The P450c11AS cDNA sequence of the Dahl S rat is identical to the P450c11AS cDNA and genomic sequence published for a Sprague-Dawley rat (21, 30), whereas the P450c11AS sequence of the Dahl R rat differs by seven nucleotides, two of which alter the predicted protein sequence of aldosterone synthase. This is similar to what was found for the P450c11 cDNA sequence of the Dahl S and R rats, as the P450c11 cDNA sequence of the Dahl S rat was identical to that from a Sprague-Dawley rat, and the sequence from the Dahl R rat was different from both. However, in the case of P450c11AS, the nucleotide changes result in amino acid changes that substantially increase enzymatic activity, while those for P450c11 result in changes that slightly decrease enzymatic activity.

The observation of specific amino acid residues associated with substantial differences in aldosterone synthase activity in Dahl S and R rats recently prompted us to investigate whether similar variations could alter the enzymatic activity of human P450c11AS. When the mutations found in the Dahl R P450c11AS were made at the corresponding residues in the human P450c11AS, they resulted in 50-80% more aldosterone production than the wild-type P450c11AS cDNA(34) . Thus, identification of variants affecting aldosterone synthase activity in rats could be relevant to the molecular control of aldosterone synthase in humans. This raises the possibility that in humans, naturally occurring variation in the corresponding regions of P450c11AS might contribute to inherited variation in aldosterone production.

  
Table: Amino acid substitutions in P450c11AS from the Dahl resistant rat



FOOTNOTES

*
This work was supported by National Insitutes of Health Grants HD 27970 (to S. H. M.), Core Grant HD-11979 (to the Department of Obstetrics and Gynecology and the Reproductive Endocrinology Center, University of California, San Francisco), and grants from the American Heart Association, National Center #91019540 (to S. H. M.) and California affiliate 91-115 and 93-222 (to S. H. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Ob/Gyn & Repro. Sci., University of California, Box 0556, San Francisco, California 94143-0556. Tel: 415-476-5329; Fax: 415-753-3271.

The abbreviations used are: DOC, 11-deoxycorticosterone; RIA, radioimmunoassay; S, salt-sensitive; R, salt-resistant; Pipes, 1,4-piperazinediethanesulfonic acid; RSV, Rous sarcoma virus.

C. Cover and S. Mellon, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Walter L. Miller and members of his laboratory for providing the data on the activity of the human P450c11AS synthetic mutations prior to publication. We also thank Susanna Bair for performing the kinetic assays and for overall excellent technical assistance.


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