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(Hypertension. 1995;25:785-789.)
© 1995 American Heart Association, Inc.

Articles

Lead Increases Aldosterone Production by Rat Adrenal Cells

Theodore L. Goodfriend; Dennis L. Ball; Mary E. Elliott; Cedric Shackleton

From the William S. Middleton Memorial Veterans Hospital and the Departments of Pharmacology and Medicine, University of Wisconsin, Madison (T.L.G., D.L.B., M.E.E.), and the Children's Hospital Research Institute, Oakland, Calif (C.S.).

Correspondence to Dr Theodore L. Goodfriend, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terr, Madison, WI 53705.

	Abstract

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Abstract Exposure to lead has been postulated to contribute to elevated blood pressure in humans and has been shown to raise blood pressure in animals. The mechanism of action of lead on blood pressure is unknown. We fed lead to rats in their drinking water and then examined the production of aldosterone by their adrenal cells in vitro. We also measured excretion of aldosterone and corticosterone by intact rats stimulated with corticotropin, with and without lead treatment. At a dose (273 ppm) that raised blood levels to 30 to 40 µg/dL, comparable to blood levels in exposed humans, lead induced increased aldosterone secretion in vitro and in vivo. The effect of lead was most evident when cells or animals were stimulated with aldosterone secretagogues. Experiments in vitro indicate that exposure to lead in vivo increases activity of one or more steps in the late pathway of aldosterone biosynthesis. The results suggest that the hypertensive effect of lead involves relative hyperaldosteronism and may be most evident when secretion of this hormone is stimulated.

Key Words: lead • blood pressure • aldosterone • hypertension, mineralocorticoid • corticosterone • adrenal glands

	Introduction

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Chronic exposure to low levels of environmental lead is universal in the industrialized world.¹ Based on studies of humans exposed to lead in the workplace or environment and animals fed lead in their drinking water, it has been proposed that lead is responsible for a significant proportion of human hypertension.² Several surveys of men suffering environmental exposure concluded that systolic blood pressure is a geometric function of blood lead concentration. One study found that systolic pressure was increased from 1 to 7 mm Hg for each doubling of blood lead, depending on the demographics of the population.³ Another study indicated that blood pressure was increased 1.8 mm Hg for each 10 µg/dL of blood lead.⁴ These conclusions have been disputed; some surveys of humans conclude that blood lead and blood pressure are not related.^{5 6}

The mechanism of the putative effect of lead on blood pressure is unknown. Several studies have examined the possible role of the renin-angiotensin-aldosterone axis.⁷ Campbell et al⁸ found a positive logarithmic correlation between blood lead and plasma aldosterone in humans exposed to the metal. There was no significant correlation of aldosterone with plasma renin activity, leading the authors to postulate, "The effects of lead on aldosterone may be mediated in part by an independent pathway."⁸

We postulated that lead exerts a direct effect on adrenal aldosterone production and thereby predisposes to hypertension. To test this hypothesis, we exposed rats to lead and measured aldosterone production by their adrenal cells in vitro and in response to corticotropin in vivo. We report here a potentiating effect of lead on adrenal glomerulosa responsiveness to aldosterone secretagogues.

	Methods

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Sprague-Dawley rats were given lead in their drinking water (500 ppm of lead acetate, 273 ppm metallic lead) and standard Purina chow containing 1.2% calcium. Except where noted, half of the animals in each experiment were male and half female. Animals were killed after lead exposures of 21 to 151 days. Rats were euthanatized with carbon dioxide gas followed by decapitation. Truncal blood was collected in chilled tubes containing appropriate anticoagulants. Adrenal glands were excised, placed in chilled buffered saline, trimmed to remove adherent fat, incised along one side, and the central zones expressed by gentle, downward, rolling pressure with the thumb. The outer connective tissue capsule and adhering glomerulosa cell layer were cut into three to five pieces, rinsed, then digested with collagenase and DNase to yield a suspension of glomerulosa cells.⁹ Preparation of fasciculata cells was similar to the procedure for glomerulosa cells, but the enzyme concentrations and duration of digestion were less.

Digested cells were chilled in ice and centrifuged at 270g for 10 minutes. The pellet was washed four times by suspension and centrifugation in cold incubation buffer. After the final wash, an aliquot of cells was suspended in buffer containing trypan blue to assess cell viability and number. The average yield of glomerulosa cells was significantly lower from lead-treated rats: 521 606±28 657 (SEM) in control animals and 432 329±26 971 (SEM) in lead-treated rats (P=.0004 by two-tailed t test). Viable cells constituted 80% of the total. In the capsule digest, glomerulosa cells were 92% to 97% of the total, based on the distinctive difference in cell size of the two zones. In the fasciculata digest, 98% of the adrenal cells were fasciculata.

Cell suspensions were diluted to 200 000 cells per milliliter in incubation buffer for each experiment. Incubation buffer contained two parts of medium 199 with bicarbonate (Sigma Chemical Co), diluted with a saline solution and supplemented with bovine serum albumin (Sigma). The final concentrations of electrolytes in the incubation buffer (mmol/L) were NaCl 119, KCl 3.6, MgSO₄ 1.2, CaCl₂ 2.54, sodium acetate 0.4, NaH₂PO₄ 1.2, and NaHCO₃ 17.5 (pH 7.4). Other concentrations were HEPES 7.44 mmol/L, glucose 11 mmol/L, and bovine serum albumin 1 mg/L. One hour before starting the experimental incubations, the dilute suspension of cells was placed in a 37°C water bath under oxygen/carbon dioxide. Incubations to assess steroidogenesis were for 2 hours at 37°C under 95% O₂/5% CO₂. Aldosterone and corticosterone were measured by radioimmunoassay with antibodies and radiolabeled steroids supplied by Diagnostic Products Corp.

Arterial blood pressure was measured in nine animals receiving lead and nine controls. With rats under pentobarbital anesthesia (45 mg/kg IP), a polyethylene catheter was inserted into the right femoral artery and tunneled under the skin to the posterior thorax. Pressures were measured on the third and fourth days after surgery.

We measured binding of radioactive monoiodo-angiotensin II to glomerulosa cells using published methods.¹⁰ Plasma renin activity was measured by radioimmunoassay of generated angiotensin I in blood obtained at the time of decapitation. Whole blood was submitted to lead assay by flameless atomic absorption spectroscopy in the Wisconsin State Laboratory of Hygiene, Madison, Wis.

Aldosterone production was measured in vivo by collecting 24-hour urine and assaying for aldosterone. After 4 days of acclimatization to individual metabolic cages, rats received daily subcutaneous injections of 1.2 U of corticotropin (H.P. Acthar Gel, Rhone-Poulenc Rorer). Injections and urine collections were continued for 14 days. Urine was acidified and extracted before aldosterone assays.¹¹

Statistical analyses are described in the legends to the Table and Figs 1 through 3.

View this table: [in this window] [in a new window]   Table 1. Effects of Lead on Aldosterone Production by Rat Adrenal Glomerulosa Cells In Vitro

View larger version (21K): [in this window] [in a new window]   Figure 1. Line graph shows effects of lead in the drinking water on responses of adrenal glomerulosa cells to agonists added in vitro. Data from three experiments are pooled. In each experiment, one group of five animals served as controls, and another group of five animals were fed 273 ppm lead as lead acetate in their drinking water for 47, 54, and 77 days after weaning. Adrenals were removed and the glomerulosa zones digested and incubated with agonists as described in the text. Aldosterone in the supernatants was measured by radioimmunoassay. To pool the results, each value was converted to a percentage of the highest aldosterone production achieved with any stimulus on that day. Brackets indicate SEM. Statistical analysis by curve-fitting was performed. There was no difference in the maximum slopes of the lead-treated and control curves with either agonist. The results for lead-exposed compared with control at the two highest doses of each agonist were different (P<.001) by the extra sum of squares test. ACTH indicates adrenocorticotropic hormone (corticotropin); AII, angiotensin II.

View larger version (28K): [in this window] [in a new window]   Figure 2. Bar graphs show the effects of lead on aldosterone and corticosterone production by adrenal cells in vitro. Data from three experiments are pooled. The lead-treated animals were exposed to 273 ppm lead as lead acetate in their drinking water for 47, 54, and 77 days after weaning. Adrenals were removed, glomerulosa and fasciculata zones were digested separately, and a mixture of the two zones was incubated as described in the text. Supernatants were assayed by radioimmunoassays of untreated aliquots. Brackets indicate SEM. Statistical significance was tested with a two-tailed t test. The aldosterone responses to stimulation were different when lead-treated and control cells (C) were compared (**P=.007 with angiotensin [A II] and *P=.03 with corticotropin), but the corticosterone values from lead-treated and control cells were not different. ACTH indicates adrenocorticotropic hormone (corticotropin).

View larger version (22K): [in this window] [in a new window]   Figure 3. Line graphs show the effects of lead on urinary aldosterone (A) and corticosterone (B) excretion by rats. Each group comprised 10 female rats housed in individual metabolic cages. The experimental group drank water containing 500 ppm lead acetate, which supplies 273 ppm of lead. Urine was collected each day and assayed for aldosterone and corticosterone by radioimmunoassay after acid hydrolysis. Statistical analysis by two-factor ANOVA, repeated on time, showed that aldosterone excretion by the two groups was different during the 4 days before corticotropin (P=.02) and during 11 days of corticotropin (P=.005) but not on the last day of acclimatization to new cages. Time-by-treatment interactions were tested and found to be nonsignificant, and therefore individual means were not compared. Corticosterone excretion values by lead-treated and by control rats were not different from each other. ACTH indicates adrenocorticotropic hormone (corticotropin).

	Results

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Aldosterone synthesis was greater in adrenal glomerulosa cells from lead-treated rats compared with controls. Results of 14 experiments from five cohorts are presented in the Table. The effect was seen with as little as 21 days of exposure (starting at weaning) and remained evident during 151 days of exposure (starting at weaning). We did not observe a difference between males and females in two experiments in which the sexes were tested separately. All experiments were pooled in the tabulation. As shown in the Table, the difference in aldosterone production between cells from lead-treated animals and controls was observed when cells were incubated in the absence of stimulus or in the presence of angiotensin II, dibutyryl cyclic AMP, corticotropin, or increased potassium.

The potentiating effects of lead treatment on adrenal responses in vitro to corticotropin and angiotensin II were examined in greater detail. Fig 1 shows dose-response curves for corticotropin and angiotensin II. Data for a maximal dose of angiotensin II are presented in the Table but were not obtained in the experiments depicted in Fig 1. At the higher doses of these two agonists, cells from animals exposed to lead responded with more aldosterone production than cells from control animals.

Because the antibody used for radioimmunoassay was not absolutely specific to aldosterone, we subjected the incubation supernatants to further analysis. An extract of pooled incubation medium from cells derived from lead-treated rats was concentrated, fractionated by high-performance liquid chromatography, and tested by immunoassay. The fractions with the highest immunoassayable aldosterone were characterized by gas chromatography/mass spectrometry. The results showed a predominance of aldosterone in the fractions with the immunoreactivity (data not shown).

The effect of lead on steroidogenesis in vitro was specific to aldosterone biosynthesis. There was no potentiating effect of lead on the production of corticosterone by isolated adrenal cells (Fig 2).

The effect of lead on steroid excretion by intact rats was examined. Compared with controls, rats fed lead for 6 weeks after weaning excreted more aldosterone in their urine when they were stressed by transfer to metabolic cages or given corticotropin (Fig 3). Again, the effect was specific to aldosterone. Corticosterone excretion by the two groups of rats did not differ before or during corticotropin administration. Under basal conditions, on the fifth day after transfer to new cages, control and lead-treated rats showed no significant difference in aldosterone excretion.

Mean whole-blood lead levels in 15 rats fed 273 ppm metallic lead as lead acetate and Purina chow for 100 days were 37±2 µg/dL. These values are similar to those observed in humans exposed to lead in the workplace, where hypertension has been associated with the metal.² The levels in control animals were all less than 5 µg/dL.

The only visible manifestation of lead toxicity was a slower rate of weight gain in some experiments. In one, after 100 days of exposure, rats ingesting lead weighed 10% (male) or 12% (female) less than controls. In a second experiment, after 76 days of exposure, female rats ingesting lead weighed 6% less than controls. In a third experiment, there was no difference in weight after 90 days.

We measured direct arterial blood pressure in 18 unanesthetized animals using indwelling femoral artery catheters. After 12 weeks of lead exposure, blood pressure was increased. In controls, mean arterial pressure (±SEM) was 105±5 mm Hg; in animals ingesting 273 ppm, it was 123±4 mm Hg (P<.05). Plasma renin activity in blood drawn at time of euthanasia was 11.3±2.6 ng angiotensin I per milliliter per hour in lead-exposed rats and 10.3±0.8 in controls (n=7).

To examine possible effects of lead on receptors, we measured binding of labeled angiotensins to adrenal cells from lead-treated and control rats. Lead treatment had no effect on saturable binding of iodinated angiotensin II or on the binding of a type 2 angiotensin receptor ligand, CGP 42,112.¹²

To study the site at which aldosterone biosynthesis is potentiated by lead, adrenal cells were incubated with added corticosterone. Cells from lead-treated rats converted 37% more corticosterone to aldosterone than cells from controls (Table). This indicates that the effect of lead in the adrenal cells is on the later steps of aldosteronogenesis.

	Discussion

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Our results show that lead in the drinking water induced selective hyperresponsiveness of rat adrenal glomerulosa cells. Hypersecretion of aldosterone was seen in vitro when glomerulosa cells from lead-treated rats were stimulated with angiotensin II, corticotropin, K⁺, or cyclic AMP. Hypersecretion was also seen in intact rats subjected to the stress of cage change or parenteral corticotropin.

Cells from lead-treated rats produced more aldosterone than controls when corticosterone was added as an exogenous precursor. This result and the hyperresponsiveness to a variety of stimuli, including K⁺, suggest that lead increases one or more steps in the "late pathway" of aldosterone synthesis. In contrast, corticosterone biosynthesis in vitro and corticosterone excretion in vivo were not increased by lead treatment. These results indicate that the metal affects one or more steps unique to aldosterone synthesis, such as 18-oxidation by aldosterone synthase, or has a predilection for cells of the adrenal zona glomerulosa.

Although the effects of lead could be observed when adrenal cells were removed from the lead-exposed animals, that does not prove that the change was caused by a direct action of lead on the adrenal gland. Lead may have altered one or more factors that regulate glomerulosa cell growth and development. For example, lead could have caused renal damage and increased plasma renin activity.^{7 13} Chronic excess circulating renin can cause hypertrophy of the zona glomerulosa and increased responsiveness to aldosterone secretagogues. Four observations argue against that explanation of our results. First, plasma renin activity in blood collected at euthanasia was the same in rats exposed to lead and in controls. Second, microscopic examination of adrenal glands from lead-exposed rats showed no hypertrophy of the zona glomerulosa. Third, there was no evidence of upregulation of angiotensin receptors, as might be expected from high-renin activities. Finally, the yield of glomerulosa cells in digests of adrenal capsules of lead-treated rats was, on average, 17% less than the yield from control capsules. Although plasma renin activities measured at time of euthanasia are not necessarily representative of the integrated stimulus to the adrenal gland and our sample size is small, the four independent observations fail to support the proposition that lead affected the adrenals indirectly via renin release. It is still possible that the effect of lead on the adrenal was mediated by an alteration of levels of corticotropin, serum electrolytes, atrial peptides, or other blood-borne factors that regulate glomerulosa cells.

There are several possible explanations for disagreements about an effect of lead on blood pressure in humans: (1) blood lead is not a perfect reflection of exposure, (2) epidemiological studies may omit important factors that can affect blood pressure, and (3) large surveys may miss effects on subgroups within a very heterogeneous population. Experiments with animals support the notion that individuals respond to lead very differently. Perry et al¹⁴ observed a wide range of blood pressures in individual rats exposed to the same amount of the metal. It may well be that individuals respond differently to adrenal effects of lead. We observed a wide range of lead-induced increases in aldosterone excretion in response to corticotropin, reflected in the size of the standard error bars in Fig 3. There are no published experiments in which the variable responses to lead of blood pressure and aldosterone secretion were measured and correlated, and we did not perform those measurements in our animals.

If the pressor effect of lead is mediated by aldosterone secretion, the pathogenesis of hypertension would include the level of adrenal stimulation, responsiveness of the kidney to aldosterone, salt intake, and the ability of the cardiovascular system to compensate for increased extracellular volume. Future epidemiological tests of the relationship between lead and blood pressure might address these variables. Our results suggest that the level of hypothalamic-pituitary activation and the intake of salt may be especially critical to exposing a pressor effect of lead.

	Acknowledgments

 
This study was supported by the Department of Veterans Affairs and by National Institutes of Health grant DK34400 (Dr Shackleton). The authors acknowledge with thanks Bruce Frohlich, Paul Brake, and Michele Larsen for measuring blood pressure, Richard Roman for measuring rat plasma renin activities, and Colin Jefcoate and Frank Siegel for collaboration and advice from the beginning of these experiments. Statistical analysis was performed by Thomas Havighurst, Department of Medical Statistics, University of Wisconsin. Editorial assistance was by Susi Nehls.

	References

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Goodfriend, T. L. Articles by Shackleton, C. Search for Related Content PubMed PubMed Citation Articles by Goodfriend, T. L. Articles by Shackleton, C. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB LEAD, ELEMENTAL