Effects of genetic variation in the aldosterone synthase (CYP11B2) gene on enzyme function.

OBJECTIVE: Evidence suggests that high levels of aldosterone lead to hypertension and increased risk of cardiovascular disease. Around 15% of patients with essential hypertension have a raised aldosterone to renin ratio (ARR) suggesting that aldosterone production is inappropriately high in relation to its principal agonist angiotensin II. This may be due to increased activity of aldosterone synthase caused by genetic variation in the CYP11B2 gene. We screened the coding region of human CYP11B2 for genetic variants and tested their effects on function in vitro. PROTOCOL: Normotensive subjects (n = 69) were screened for sequence variants in the coding region of CYP11B2 by single-stranded conformation polymorphism (SSCP) analysis and sequencing. The effects of nonsynonymous variants on enzyme activity were assessed in JEG-3 cells transiently transfected with wild-type or variant expression plasmids. The conversion of the substrate 11-deoxycorticosterone (DOC) to corticosterone (B) and aldosterone was measured. RESULTS: Twenty variants were detected in CYP11B2 and eight analysed functionally (Arg87Gly, Asn281Thr, Gly288Ser, Lys296Asn, Asp335Asn, Gln404Arg, Ala414Pro and His439Tyr). Corticosterone synthesis was unaltered and aldosterone synthesis reduced in variant Arg87Gly; Asn281Thr increased corticosterone and decreased aldosterone production; Gly288Ser increased corticosterone production and abolished aldosterone production; Lys296Asn reduced both corticosterone and aldosterone production; Asp335Asn increased corticosterone synthesis but did not affect aldosterone production. Variants Gln404Arg, Ala414Pro and His439Tyr showed increases in both corticosterone and aldosterone synthesis compared to the wild-type. CONCLUSION: The study confirms the genetic variability of the CYP11B2 gene and provides us with additional valuable structure-function information.

Phenotypic consequences of variation across the aldosterone synthase and 11-beta hydroxylase locus in a hypertensive cohort: data from the MRC BRIGHT Study.

BACKGROUND: Aldosterone is an important cardiovascular hormone; 15% of hypertensive subjects have alteration in aldosterone regulation, defined by a raised ratio of aldosterone to renin (ARR). Studies of the aldosterone synthase gene (CYP11B2) have focused on a single nucleotide polymorphism in the 5'promoter region (-344 C/T). In normotensive subjects, the T allele associates with raised levels of the 11-deoxysteroids, deoxycorticosterone and 11-deoxycortisol which are substrates for 11beta-hydroxylase, encoded by the adjacent and homologous gene, CYP11B1. We have speculated that this altered 11beta-hydroxylase efficiency leads to increased ACTH drive to the adrenal gland to maintain cortisol production and reported herein the association between the -344 C/T single nucleotide polymorphism (SNP) and adrenal steroid production in subjects with essential hypertension. METHODS: The CYP11B2-344 C/T polymorphism was genotyped and urinary excretion of adrenal steroid metabolites was measured (by GCMS) in 511 unrelated hypertensives from the Medical Research Council (MRC) British Genetics of Hypertension (BRIGHT) study. RESULTS: Thirty-five per cent of subjects were homozygous for the -344T allele whilst 16% were CC homozygotes. There was no difference in cortisol excretion rate between the two genotype groups but the index of adrenal 11beta-hydroxylation (ratio of tetrahydrodeoxycortisol/total cortisol) was significantly higher in the TT group (P < 0.005) than in the CC group. Excretion rates of the major urinary metabolite of aldosterone (tetrahydroaldosterone) correlated strongly with the ACTH-regulated steroids, cortisol (r = 0.437, P < 0.0001) and total androgen metabolites (r = 0.4, P < 0.0001) in TT but not CC subjects. CONCLUSIONS: Hypertensives homozygous for the -344 T allele of CYP11B2 demonstrate altered 11beta-hydroxylase efficiency (CYP11B1); this is consistent with the hypothesis of a genetically determined increase in adrenal ACTH drive in these subjects. The correlation between excretion of aldosterone and cortisol metabolites and suggests that, in TT subjects, ACTH exerts an important common regulatory influence on adrenal corticosteroid production in subjects with hypertension.

Effects of 18-hydroxylated steroids on corticosteroid production by human aldosterone synthase and 11beta-hydroxylase.

In glucocorticoid-suppressible hyperaldosteronism, 11beta- hydroxylase activity is impaired. A chimeric enzyme formed from the control elements of 11beta-hydroxylase and the structural elements of aldosterone synthase is expressed ectopically in the zona fasciculata, thus exposing cortisol to aldosterone synthase. Increased quantities of 18-hydroxycortisol and 18-oxocortisol are synthesized, which, it has been suggested, might have a local inhibitory effect on the normal 11beta-hydroxylase. The effects of these compounds and also of 18-hydroxydeoxycorticosterone were tested in cells stably transfected with CYP11B1 and CYP11B2, the genes encoding 11beta-hydroxylase and aldosterone synthase, respectively. Neither 18-hydroxycortisol nor 18-oxocortisol affected the efficiency of use of 11-deoxycorticosterone or 11-deoxycortisol as substrates by the enzymes. 18-Hydroxydeoxycorticosterone significantly reduced the conversion rate of 11-deoxycorticosterone to corticosterone and that of 11-deoxycortisol to cortisol by both enzymes, but the production rate of 18- hydroxycorticosterone and aldosterone by aldosterone synthase increased. Aldosterone synthase was able to convert 18-hydroxydeoxycorticosterone to 18-hydroxycorticosterone and aldosterone, although its affinity for this substrate was lower (4.76 micromol/liter) than that for 11-deoxycorticosterone (0.11 micromol/liter). 11beta-Hydroxylase was unable to convert 18- hydroxydeoxycorticosterone to 18-hydroxycorticosterone. 18-Hydroxycortisol and 18-oxocortisol are not, therefore, the cause of lower 11beta-hydroxylase activity in glucocorticoid- suppressible hyperaldosteronism. 18-Hydroxydeoxycorticosterone can be converted to aldosterone, but its local concentration in man and its K(m) suggest that it is unlikely to be important.

Effect of salivary components on volatile partitioning from solutions.

Partition of fourteen volatile compounds, representing the diverse physicochemical properties of aroma compounds, was measured by static equilibrium headspace in solutions containing the components of artificial saliva, either singly or in mixtures. Comparison of a bovine salivary mucin and pig gastric mucin showed no significant difference in partition behavior of the volatiles, so gastric mucin was used. Mucin viscosity changed with pH, but binding of volatile compounds did not show a marked dependence on pH. All combinations of the salivary components were tested for their effect on partition. Three types of behavior were noted. Partition of some compounds was unaffected by mucin, and with other compounds mucin decreased partition, whereas another group showed a decrease with mucin that was affected by the presence of salivary salts and sugar. When volatiles or sugar were added to a mucin solution, the final headspace concentration depended on the order of addition, indicating some competition for binding. These solute-mucin effects are discussed in relation to mucin structure and behavior in solution.

Polymorphic differences from normal in the aldosterone synthase gene (CYP11B2) in patients with primary hyperaldosteronism and adrenal tumour (Conn's syndrome).

OBJECTIVE: The hypertension of Conn's syndrome is due to autonomous aldosterone production by a unilateral adrenocortical adenoma. The source of tumour initiation and the reasons for excess aldosterone production as opposed to cortisol are not known, although variations in the promoter region of the gene coding for aldosterone synthase (CYP11B2) might account for the altered rate of aldosterone secretion. DESIGN: In a series (n = 27) of well-characterized Conn's syndrome cases, the aldosterone synthase gene (CYP11B2) was screened by single-strand conformational polymorphism (SSCP) for differences from the consensus sequence. RESULTS: No new mutations were found. The frequencies of two previously described linked polymorphisms, one a change of -344C to T in a putative steroidogenic factor-1 (SF-1) binding site and the other an exchange of intron 2 for that of CYP11B1 (conversion) were measured in tumour and genomic DNA. The frequency of the SF-1 T allele (P < 0.0001) and the conversion allele (P < 0.001) were markedly different between the Conn's syndrome group and the normal controls. However, the frequency did not differ between tumour and genomic DNA in the patient group. CONCLUSION: While it is unlikely that this difference from normal is related to tumour growth, these genotypes may predispose the tumour to aldosterone production.

An influence of variation in the aldosterone synthase gene (CYP11B2) on corticosteroid responses to ACTH in normal human subjects.

OBJECTIVE: Previous evidence suggests that the efficiency of 11beta-hydroxylase is at least partly heritable and also that it may be mildly impaired in essential hypertension. In both cases, assessment of activity was based on the response of 11-deoxycorticosterone (DOC) and 11-deoxycortisol to ACTH. The gene (CYP11B1) coding for this enzyme is highly homologous with and lies a relatively short distance downstream from the gene coding for aldosterone synthase (CYP11B2) on chromosome 8. Two polymorphisms of CYP11B2 have been described. The first involves a change of -344C to T in a putative steroidogenic factor-1 (SF-1) binding site and the other, the intron conversion, an exchange of intron 2 for that of CYP11B1. These polymorphisms are in linkage dysequilibrium. Their effects on 11beta-hydroxylation were studied. METHODS AND RESULTS: Normal subjects (n = 135) were genotyped and those homozygous for either or both the polymorphisms were given ACTH (250 microg, i.v.). Plasma was sampled before and 30 minutes after administration. Basal concentrations of DOC, corticosterone, 11-deoxycortisol and cortisol and responses of corticosterone and cortisol to ACTH were not affected by genotype. However, the responses of DOC (P = 0.002 and P = 0.001, respectively) and 11-deoxycortisol (P = 0.025 and P = 0.002, respectively) were significantly greater in subjects homozygous for SF-1 T and/or intron conversion than in those homozygous for SF-1 C and/or normal intron. CONCLUSIONS: These results indicate different 11beta-hydroxylase efficiencies. Thus, variation in CYP11B2 appears to affect the product of CYP11B1. The mechanism is unclear. The close proximity of the two genes may lead to competition for transcription factors or specific differences in intron 2 may affect transcription. Alternatively, the polymorphisms may be acting as markers for adjacent functional genetic variations.

Effects of glucose and amino acids on free ADP in betaHC9 insulin-secreting cells.

Stimulation of insulin release by glucose is widely thought to be coupled to a decrease in the activity of ATP-sensitive K+ channels (KATP channels) that is caused by a decreased concentration of free ADP. To date, most other investigators have reported only on total cellular ADP concentrations, even though only a small fraction of all ADP is free and only the free ADP affects KATP channels. We tested the hypothesis that amino acids elicit insulin release via a decrease in the activity of KATP channels owing to a decrease in the level of free ADP. We estimated the concentration of free ADP in betaHC9 hyperplastic insulin-secreting cells based on the cell diameter and on luminometric measurements of ATP, phosphocreatine, and total creatine. The concentration of free ADP fell exponentially as the concentration of glucose increased. A physiological mixture of amino acids greatly stimulated insulin release at 0-30 mmol/l glucose but affected the concentration of free ADP only to a minor degree and significantly so only at < or = 2 mmol/l glucose. In the presence of 2-deoxyglucose and NaN3, amino acids were unable to stimulate insulin release. When KATP channels were held open with diazoxide (and the plasma membrane partially depolarized with high extracellular KCl), amino acids still stimulated insulin release. We conclude that amino acid-induced insulin release depends on two components: a yet-unknown amino acid sensor and KATP channels, which serve to attenuate hormone release when cellular energy stores are low. We propose that glucose-induced insulin release may be regulated similarly by two components: glucokinase and KATP channels.

alpha-adducin and angiotensin I-converting enzyme polymorphisms in essential hypertension.

This study focused on two genes that have previously been implicated in hypertension and may influence renal sodium handling, adducin, and angiotensin I-converting enzyme (ACE). We compared their polymorphic frequencies and interaction in patients with essential hypertension (n=128) and individually age- and gender-matched normotensive control subjects. The alpha-adducin G460W polymorphism was genotyped by DNA amplification and restriction digestion. The ACE I/D polymorphism was assayed by a triple-primer method, with a "nested" polymerase chain reaction primer situated completely within the insertion sequence of the I: allele. The distributions of genotypes and alleles for the two polymorphisms were not significantly different between the case and control populations, and the cross-classification of cases by alpha-adducin and ACE genotype gave a distribution similar to that of control subjects. We have previously reported that the distributions of genotypes for two linked polymorphisms in the aldosterone synthase gene (one in the steroidogenic factor-1 [SF-1] binding site and the other an intronic conversion [IC]) were significantly different between this cohort of essential hypertensives and matched control subjects. The cross-classification of cases by alpha-adducin and SF-1, alpha-adducin and IC, ACE and SF-1, and ACE and IC genotype gave a distribution similar to that of control subjects. Hence, no evidence was found to suggest an association between either the alpha-adducin G460W or the ACE I/D polymorphism and hypertension in a careful case-control study. Furthermore, the alpha-adducin G460W, ACE I/D, and aldosterone synthase SF-1 and IC polymorphisms do not appear to interact in our hypertensive population.

Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP.

We present methods to measure ATP, phosphocreatine, and total creatine (the sum of creatine and phosphocreatine) in alkaline cell extracts. Knowledge of these parameters, together with the known equilibrium constants for the creatine kinase and adenylate kinase-catalyzed reactions, allows one to estimate the levels of free ADP and free AMP inside cells. The enzymatic assays for the above-mentioned metabolites all lead up to the production of ATP, which is measured luminometrically with the ATP-dependent oxidation of luciferin catalyzed by firefly luciferase. To determine phosphocreatine, endogenous ATP is first destroyed, and phosphocreatine is then quantitatively reacted with exogenous ADP to form ATP. Total creatine is measured after quantitative conversion of creatine to phosphocreatine with a large excess of exogenous ATP, conversion of all ATP to ADP, and final reaction of phosphocreatine with ADP to form ATP. We used 5-microl samples in 0.5-ml microcentrifuge tubes and subsequent 5-microl additions of analytical reagents. We expect that the volumes can be changed easily. We tested the methods with glucagon- and insulin-secreting cells. Estimates of free ADP and AMP are expected to be useful in many different areas of research, such as cellular energy metabolism, purine nucleotide metabolism, adenine nucleotide gating of ion channels, and release of vasoactive or angiogenic factors.

Aldosterone excretion rate and blood pressure in essential hypertension are related to polymorphic differences in the aldosterone synthase gene CYP11B2.

Significant correlation of body sodium and potassium with blood pressure (BP) may suggest a role for aldosterone in essential hypertension. In patients with this disease, the ratio of plasma renin to plasma aldosterone may be lower than in control subjects and plasma aldosterone levels may be more sensitive to angiotensin II (Ang II) infusion. Because essential hypertension is partly genetic, it is possible that altered control of aldosterone synthase gene expression or translation may be responsible. We compared the frequency of 2 linked polymorphisms, one in the steroidogenic factor-1 (SF-1) binding site and the other an intronic conversion (IC), in groups of hypertensive and normotensive subjects. In a larger population, the relationship of aldosterone excretion rate to these polymorphisms was also evaluated. In 138 hypertensive subjects, there was a highly significant excess of TT homozygosity (SF-1) over CC homozygosity compared with a group of individually matched normotensive control subjects. The T allele was significantly more frequent than the C allele in the hypertensive group compared with the control group. Similarly, there was a highly significant relative excess of the conversion allele over the "wild-type" allele and of conversion homozygosity over wild-type homozygosity in the hypertensive group compared with the control group. In 486 subjects sampled from the North Glasgow Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) population, SF-1 and IC genotypes were compared with tetrahydroaldosterone excretion rate. Subjects with the SF-1 genotypes TT or TC had significantly higher excretion rates than those with the CC genotype. The T allele was associated with higher excretion rates than the C allele. However, no significant differences were found in excretion rate between subjects of different IC genotype. Urinary aldosterone excretion rate may be a useful intermediate phenotype linking these genotypes to raised BP. However, no causal relationship has yet been established, and it is possible that the polymorphisms may be in linkage with other causative mutations.

Adjuvant vigabatrin in refractory epilepsy: a ceiling to effective dosage in individual patients?

A double-blind, randomized, cross-over study of additional vigabatrin (gamma-vinyl-GABA, VGB, 1.0 g twice daily for 6 weeks, followed by 1.5 g twice daily for 6 weeks) and matched placebo was undertaken in 24 patients with refractory epilepsy. Nineteen completed the trial satisfactorily. Fewer seizure days were reported during VGB treatment [placebo 41, VGB 23, p < 0.05, 95% confidence interval (CI) -1.5 to -14]. An overall reduction in median seizure numbers failed to reach statistical significance (n = 19; placebo 52, VGB 32, NS, 95% CI -18 to +24). Subgroup analysis, however, showed a significant reduction in partial seizures (n = 17) with 2 g VGB daily (placebo 22, VGB 13, p < 0.05, 95% CI -0.5 to -16.5), but not with higher dosage (placebo 28, VGB 22, NS, 95% CI -18 to +11). A deterioration in control of partial seizures as compared with the equivalent placebo phase was observed when patients were changed from 2 to 3 g/day VGB (2 g VGB 13, 3 g VGB 22, p = 0.05, 95% CI 0 to +20). Loss of efficacy was noted in 3 patients, and seizure control worsened slightly in 5 others. One previously resistant patient developed a therapeutic response, and 2 other patients reported an additional useful reduction in seizures. In the remaining 8 patients, seizure frequency did not change. VGB did not appear to benefit tonic-clonic seizures. Serum VGB concentrations were higher during treatment with 3 g (15.5 +/- 8.9 mg/L) daily than with 2 g (13.5 +/- 11.2 mg/L). No important alterations were noted in the concentrations of concomitantly administered antiepileptic drugs (AEDs) throughout the trial. VGB is useful adjuvant therapy for treatment of partial seizures. There may be a ceiling to effective dosage. This demands individual dose titration for each patient.