The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 3 831-834
Copyright © 1997 by The Endocrine Society
Impaired Rapid Mineralocorticoid Action on Free Intracellular Calcium in Pseudohypoaldosteronism1
Fernando Gamarra,
Gunter Simic-Schleicher,
Rudolf M. Huber,
Axel Ulsenheimer,
Peter C. Scriba,
Ursula Kuhnle and
Martin Wehling
Institute of Clinical Pharmacology (M.W.), Faculty of Clinical
Medicine at Mannheim, University of Heidelberg, Mannheim; Medizinische
Klinik (F.G., R.M.H., A.U., P.C.S., M.W.), Klinikum Innenstadt,
University of Munich, Munich; Universitätskinderklinik (G.S-S.),
Abteilung für pädiatrische Endokrinologie, University of
Essen, Essen; v-Hauner‘sche Kinderklinik (U.K.), University of Munich,
Munich, Federal Republic of Germany
Address correspondence and requests for reprints to: Professor Martin Wehling, MD, Institute of Clinical Pharmacology, Faculty of Clinical Medicine at Mannheim, University of Heidelberg, Klinikum Mannheim, 68135 Mannheim, Federal Republic of Germany.
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Abstract
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Earlier observations on impaired in vitro effects of
aldosterone on lymphocytic sodium and potassium pointed to the
involvement of a defective nongenomic rather than genomic effector in
pseudohypoaldosteronism. In this study, we investigated nongenomic
aldosterone action in five patients with pseudohypoaldosteronism with
regard to a rapid increase of free intracellular calcium
[Ca2+]i in cultured nasal epithelial cells,
assumably reflecting calcium influx through calcium channels. Patients
were defined by episodes of salt loss despite high plasma aldosterone
and renin levels. Four unaffected members of the families and four
independent subjects served as controls. Considering an
aldosterone-induced increase of [Ca2+]i by at
least 10 nm as positive response, only 12% of cells from patients were
responsive compared with 25% in normal subjects (P
< 0.05). In terms of absolute changes, mean increase of
[Ca2+]i was 1.6 ± 1.1 nm in the
patients (range - 1–4) and 9.5 ± 2.7 nm (P
< 0.025) in the controls (range 1–25). Basal
[Ca2+]i was not different between both groups
(167 ± 5 vs. 169 ± 8 nm, mean ±
SE).
These findings show an impaired nongenomic mineralocorticoid effector
in patients with pseudohypoaldosteronism, which is in line with a
defective sodium channel as shown recently by molecular cloning, and
also with the fact that the classical, genomic intracellular receptor
is structurally normal in these patients.
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Introduction
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PSEUDOHYPOALDOSTERONISM (PHA) is a rare
condition in which salt and water losses occur despite high plasma
levels of aldosterone, with an end organ resistance postulated as early
as 1956 (1). To identify the pathogenesis of this disease, known
mechanisms of mineralocorticoid action have been checked for
abnormalities in PHA. In the classic model of genomic steroid action,
steroids bind to intracellular receptors that act as ligand-dependent
transcription factors. The pathway of genomic steroid action involving
transcription, translation, and protein synthesis is characterized by
late onset effects preceded by a latency of 1–4 h. Recently, rapid
in vitro effects of aldosterone on sodium, potassium, and
calcium concentrations and cell volume of human mononuclear leukocytes
(HML) (2, 3, 4) and on the activity of the sodium-proton-exchanger of the
cell membrane in HML and vascular smooth muscle cells (VSMC) (5, 6, 7)
have been demonstrated in our laboratory. These nongenomic effects
suggested the existence of distinct receptors that were subsequently
described in plasma membranes from HML and pig kidney (8, 9). The
phosphoinositide pathway and free intracellular calcium
[Ca2+]i appear to be involved in
intracellular signalling in HML, VSMC, and endothelial cells (10, 11).
The involvement of classic Type I receptors was originally proposed in
PHA, in that reduced numbers of receptors had been found in peripheral
lymphocytes (12), although subsequent molecular analysis of these
receptors (13, 14, 15) did not identify abnormalities in their primary
structure. Furthermore, the impaired in vitro effects of
aldosterone on lymphocyte sodium and potassium reported earlier (16, 17) were pharmacologically incompatible with and fairly rapid for an
exclusive involvement of genomic mechanisms, and, thus, suggested an
involvement of the nongenomic effector. This assumption has been
supported by a recent demonstration of mutations in amiloride-sensitive
sodium channels in the autosomal recessive form of PHA (18),
potentially representing a nongenomic effector. Defective sodium
channels and, therefore, intracellular sodium may be linked to
intracellular calcium by the sodium-calcium-exchanger of the cell
membrane (11). In the present paper, rapid, nongenomic aldosterone
effects on intracellular calcium were quantified in cultured nasal
epithelial cells by single cell imaging of Fura2-fluorescence.
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Subjects and Methods
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Patients and controls
Five patients and eight controls were examined. Informed consent
was obtained from each patient or the patient‘s parent. The patients
were from four families. Two families were related by the affected
mothers (1a and 1b - Table 1
). The pattern of
inheritance was autosomal dominant in families 1a and 1b and autosomal
recessive or a new mutation in families 2 and 3. Laboratory evaluation
and clinical features (hypotension) were compatible with PHA type
I.
Three patients presented to local hospitals by a typical salt losing
crisis during the first year of life (numbers 1, 7, and 8). The
underlying disease was finally diagnosed by determination of
mineralocorticoid receptor binding (12) in mononuclear leukocytes
(number 1) and/or determination of aldosterone and plasma renin
activity under clinically stable basal conditions. These patients were
subsequently treated with NaCl. In the family of patient 1,
investigation identified several other patients by determination of
mineralocorticoid binding. Of these patients, two cousins of number 1
were examined in the present study. To avoid false categorization based
only on determination of mineralocorticoid receptor binding in
mononuclear leukocytes, we determined in these patients (numbers 2 and
3) aldosterone and plasma renin activity under basal conditions and
4 h after 1 mg/kg body weight furosemide iv (maximum dose 10 mg).
Aldosterone was determined by RIA (Sorin, Dusseldorf, Germany) and
plasma renin activity was determined by RIA (Abbott, Wiesbaden,
Germany) in one assay each. Values above 350 pg/mL for aldosterone and
5.7 ng/mL·h for plasma renin activity under basal and 680 pg/mL and
6.5 ng/mL·h after furosemide were considered pathological (19, 20).
As controls, the nasal epithelium from eight controls (of whom four
were related to the patients) was used. Three subjects (numbers 4, 5,
and 6) were unaffected sisters of patient 1, as determined by
mineralocorticoid receptor binding (20). To exclude latent PHA despite
normal mineralocorticoid receptor levels, aldosterone and plasma renin
activity was determined under basal conditions and 4 h after 1
mg/kg body weight furosemide iv (maximum dose 10 mg). As none showed
pathological levels in any parameter, they were considered unaffected.
The fourth related person was the mother (number 9) of patient number
7. She was completely healthy and had no history of PHA and was
therefore assigned to the control group. No other member of family,
including the father of patient number 7, was affected by the disease.
Comparisons were calculated with presence of the mother of patient
number 7 in either group. Three other controls were attending the
outpatient clinic for nonrelated disease, and one was the healthy son
of one of the investigators.
Materials
Aldosterone was from Fluka (Buchs, Switzerland),
Fura2-acetoxymethylester (Fura2-AM) from Molecular Probes (Eugene,
MO), human placental collagen type VI and dithiothreitol from Sigma
(Deisenhofen, Germany), anti-cytokeratin antibody AE1/AE3 from
Boehringer (Mannheim, Germany), trypsin-EDTA (x10) from Gibco BRL
(Eggenstein, Germany).
Epithelial cells were prepared from nasal swipes by trypsinization,
dissipated in bronchial epithelial growth medium (PromoCell,
Heidelberg, Germany) supplemented with 5% newborn fetal calf serum
(Life Technologies GmbH, Eggenstein, Germany) and grown on 22 mm cover
slips coated with human placental collagen type VI. Medium was
exchanged every other day. Cells were used at subconfluence, which was
achieved between days 5 and 61 mainly depending on the number of
adherent cells at the beginning. The patient/control status of the
donor did not affect culture success. Specimens were stained by the
anticytokeratin antibody AE1/AE3 immunohistochemically to confirm the
epithelial origin of the cultured cells and used only if positive. From
13 donors studied, 58 (37%) of the total number of attempted cultures
were successfully measured by antibody characterization and
subconfluence. By phase-contrast microscopy, cultured cells showed
features typical for human nasal epithelial cells as previously
described (21). Imaging of free intracellular calcium
[Ca2+]i was performed in single cells that
were washed 2 times with 2 mL PSS-buffer (135 mmol/L NaCl, 5 mmol/L
KCl, 1.8 mmol/L CaCl2, 0.8 mmol/L MgCl2, 10
mmol/L HEPES, 5.5 mmol/L glucose, pH 7.4) to remove serum and loaded
with 4 µmol/L Fura2-AM from a 0.2% stock solution in dimethyl
sulfoxide for 40 min at 37 C. At the end of the loading period and
again immediately before the experiment, cells were washed with
PSS-buffer (2 x 2 mL). They were placed in a thermostatically
controlled ring chamber (37 C) holding 0.4 mL of incubation fluid. Cell
imaging of [Ca2+]i was performed using a Till
Photonics dual wavelength imaging system (Till Photonics GmbH,
Gräfelfing, Germany) attached to a Zeiss Aviovert 35 (Zeiss,
Hanau, Germany) inverted fluorescence microscope with a fluor 40/1.30
oil immersion objective. The imaging camera was a AE2000 system from
General Scanning GmbH, (Planegg, Germany). Excitation wavelengths were
separated by a dichroic mirror at 340 and 380 nm and emitted light was
collected at 510 nm. Integration times were 0.1 sec at an excitation
wavelength of 340 nm and 0.06 sec at 380 nm, with a time increment of 6
sec. Autofluorescence was determined in each experiment by the addition
of 4 mm MnCl2 and 5 µm ionomycin to quench
intracellularly located dye. The autofluorescence level was subtracted
from each reading before calculation of
[Ca2+]i. Ionomycin-evoked responses of
[Ca2+]i served as positive controls of cell
viability. The system was calibrated by the method of Grynkiewicz et
al. (22), and the following equation was used for the calculation of
[Ca2+]i:
 |
where R is the fluorescence ratio for the excitation wavelengths
of 340 and 380 nm, Rmin the ratio for Fura 2 acid in
solution at zero calcium (10 mM EGTA) and Rmax
the ratio at 1.8 mM calcium, Sf2 and
Sb2 the fluorescences at 0 and 1.8 mmol/L calcium for an
excitation wavelength of 380 nm. After addition of 50 µL PSS-buffer,
baseline stability was checked for 1–2 min and the experiment canceled
if spontaneous fluctuations of the fluorescence ratio were seen. At
times indicated, aldosterone (50 µL) was added from a stock solution
(10 mmol/L) in ethanol. At a final steroid concentration of 10 nmol/L,
the ethanol concentration was 0.001% without effect on
[Ca2+]i. [Ca2+]i
was analyzed on serial images in a region of interest (ROI) in the
perinuclear region of the cell with the operator blind to patient
status.
Values are given as mean ± SE. The two-tailed
t- test for unpaired data was used (significance at
P < 0.05)
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Results
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Basal [Ca2+]i in nasal epithelial cells
was not different for the patients and the controls (167 ± 5
vs. 169 ± 8 nm) ranging from 107–253 nm in the
control group and from 133–193 in the patient group. Switching person
number 9—the mother of patient number 7—to the affected group did not
essentially change the results.
A typical response of [Ca2+]i to 10 nm
aldosterone in cells from normal subjects is shown in Fig. 1a, and in cells from affected patients in Fig. 1b. To
exclude the influence of spontaneous fluctuations in
[Ca2+]i, cells with changes in
[Ca2+]i more than 10 nm in the run-in phase
(addition of buffer alone) were excluded from the determinations
(approximately 10%). In turn, only increases of
[Ca2+]i by more than 10 nm were considered
significantly different from spontaneous fluctuations and termed
"responding". This cut off limit is defined by changes in
[Ca2+]i in a preparation of adherent human
monocytes that are considered nonresponding (Wehling M., unpublished
data). In these cells [Ca2+]i changes with or
without aldosterone are statistically not different from zero (change
of calcium 1 ± 5 nm, n = 44, mean ± SD).
Thus, 10 nm is close to the upper 2 x
SD[Ca2+]i fluctuations in
nonresponding cells. A similar range was observed for epithelial cells
in the patients studied here (see below).
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Figure 1. Perinuclear region of interest (ROI) of free
intracellular calcium in cultured nasal epithelial cells from a normal
subject (A) and from a patient with pseudohypoaldosteronism (B) after
addition of buffer (PSS) and 10 nm aldosterone.
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When categorized by this criteria, mean percentage of responding cells
was 12% of cells (range 4.5–25%, n = 106) from patients
compared with 25% (range from 3.7–37.5%, n = 146) in normal
subjects (P < 0.05). Thus, a considerable overlap was
observed. Switching of person number 9 (the mother of patient number 7)
to the affected group did not affect the results. In terms of absolute
changes, mean increase of [Ca2+]i was
1.6 ± 1.1 nm in the patients (individual range - 1–4) and
9.5 ± 2.7 nm (P < 0.025) in the controls
(individual range 1–25).
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Discussion
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The main finding of the present study is the demonstration of an
impaired nongenomic effect of aldosterone on
[Ca2+]i in cultured nasal epithelial cells
from patients with PHA. Setting a cutoff limit at values rarely
exceeded by spontaneous fluctuations should eliminate false results
caused by still uncontroled variables such as state of the culture or
even minute changes in cuvette temperature. There were no abnormalities
of nasal cells from the patients by morphological or immunological
means, although heterogeneity is a well-known feature of cell culture
reflecting differences in the proliferative state or subpopulations.
Only 60% of cultured VSMC produce a calcium signal in response to
aldosterone (11). Cell viability was always tested, but subtle changes
of culture growth caused by secondary effects of the underlying disease
cannot be excluded with certainty, possibly affecting the percentages
of responding cells.
With regard to sodium and potassium content, the HML model has been
applied earlier to various clinical situations in patients with
disturbances of the sodium and water balances. At the level of net
changes of these cellular electrolytes after one hour, abnormalities in
the response to aldosterone have been found in patients with PHA,
primary and secondary aldosteronism, and essential hypertension (16, 23, 24).
In seven patients with PHA, unrelated to those included here, the
effects of aldosterone on intracellular sodium and potassium have been
studied and compared with normal controls in whom aldosterone prevents
the loss of sodium and potassium in vitro (16). In those
patients, intracellular sodium and potassium decreased normally in the
absence of aldosterone. With aldosterone added to the incubation medium
intracellular sodium and potassium were not different from values
obtained without aldosterone. Baseline values of sodium and potassium
before the incubation were within the normal range. From these findings
we concluded that, after the critical period of the disease during the
first months of life, intracellular sodium and potassium may be
maintained at normal levels by mechanisms unrelated to the action of
mineralocorticoids. Though not known in detail, these mechanisms are
rapidly reversible as significant wash-out of the sodium increasing
effect is observed within 1 h of incubation.
In addition, the families of these seven patients (index cases) were
studied. In the first family studied, two siblings were affected by the
disease and had a reduced number of mineralocorticoid type I receptors
on HML. The parents, who were first cousins, had no history of disease
and normal receptor data, but in the mother, the response of HML
electrolytes to aldosterone was abnormal. In the second family, the
mother of a child with PHA, the mother’s sister, and her son had low
numbers of type I receptors. Only the aunt of the index case had an
uncertain history of the disease. The mineralocorticoid effector
mechanism was abnormal in both children and in both mothers studied. In
a third family, the effector defect was present only in HML of the
father. In three further families the abnormality of the effector
mechanism was detected in HML of the patient‘s mother (17).
The discrepancy between findings about HML type I receptors and
aldosterone effects in patients with PHA may indicate that the
molecular base of the disease might include abnormalities of
aldosterone membrane receptors and thus of the rapid effects of
aldosterone. These rapid effects could be responsible for the decreased
influx of sodium into cells. So far, only effects at 1 h have been
studied in these patients, which may therefore include genomic as well
as nongenomic mechanisms.
Subsequently, these nongenomic aldosterone effects have been analyzed
in detail, and it was shown that free intracellular calcium is an
important second messenger involved. The aim of the present study was
therefore to investigate rapid aldosterone effects on calcium as an
indicator of nongenomic actions in patients with PHA. On-line
registrations of free intracellular calcium in single cells for 5–10
min appear to be more sensitive and reliable than 1-h determinations of
calcium requiring repeated washes of the cells. In single cells,
regions of interest may be studied that might be chosen to give maximum
effects. Unfortunately, HML as used in previous studies are not
adherent to cover slips and thus may not be studied as movement
artifacts otherwise occur. Nasal epithelium was used as it may be
easily obtained from the donors and can be cultured on cover slips. To
our knowledge, mineralocorticoid action has not been investigated in
nasal epithelia before, but was expected to exist as it does in other
epithelial tissues such as glands, colon, and of course, renal
epithelia. Thus, it should be noted that the system utilized here for
the first time provides an easy access to single epithelial cells in
culture for the study of mineralocorticoid action ex
vivo.
The approach chosen here was further supported by results of the
molecular analysis of classic type I receptors, which by all means were
normal with regard to their primary structure (13, 14, 15), and by recent
results in which a mutation of the sodium channel was found to be
essentially involved in the pathogenesis of the autosomal recessive
form of this disease (18). Sodium influx through these channels is
likely to be involved in rapid aldosterone action (2) and possibly
linked to [Ca2+]i by the
sodium-calcium-exchanger of the cell membrane (3, 11). Under these
assumptions, low mineralocorticoid receptor binding measured in
symptomatic patients with PHA (as shown in family 1) may reflect an
epiphenomenon of the disease indicating downregulation in response to
elevated aldosterone levels (19, 20).
The results presented here underline the importance of the nongenomic
mineralocorticoid effector in the pathogenesis of PHA and may represent
the functional correlate of mutations in the sodium channel at the
level of rapid aldosterone action.
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Acknowledgments
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We thank Ms. K. Sippel for expert technical assistance.
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Footnotes
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1 The study was supported by the Deutsche Forschungsgemeinschaft (We
1184/4–2, Sc 4/9–4). 
Received August 12, 1996.
Revised November 8, 1996.
Accepted November 12, 1996.
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