help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Makara, J. K.
Right arrow Articles by Spät, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Makara, J. K.
Right arrow Articles by Spät, A.
Endocrinology Vol. 141, No. 5 1705-1710
Copyright © 2000 by The Endocrine Society

ARTICLES

Effect of Osmolarity on Aldosterone Production by Rat Adrenal Glomerulosa Cells1

Judit K. Makara, Gábor L. Petheö, Attila Tóth and András Spät

Department of Physiology and Laboratory of Cellular and Molecular Physiology, Semmelweis University Medical School, H-1444, Budapest, Hungary

Address all correspondence and requests for reprints to: Dr. András Spät, Department of Physiology, Semmelweis University Medical School, P.O. Box 259, H-1444, Hungary. E-mail: spat{at}puskin.sote.hu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effect of osmotic changes on aldosterone production, [Ca2+]i and voltage-gated Ca2+ currents, was studied in cultured rat glomerulosa cells. Alteration of osmolarity by sucrose addition in the 250–330 mosM range did not influence aldosterone production per se, but it substantially affected K+-stimulated aldosterone production. Hyposmosis markedly increased the hormone response evoked by raising [K+] from 3.6 to 5 mM, whereas hyperosmosis had a mild decreasing effect. Cytoplasmic [Ca2+]i, measured in single glomerulosa cells, did not show detectable change in response to either hyposmotic or hyperosmotic exposure, but the [Ca2+]i signal evoked by elevation of [K+] to 5 mM was augmented in hyposmotic solution. The osmosensitivity of the transient (T)-type and long-lasting (L)-type voltage-gated Ca2+ currents was studied using the nystatin-perforated voltage-clamp technique. Lowering osmolarity to 250 mosM significantly increased the amplitude of the T-type current, and it had a transient augmenting effect on L-type current amplitude. Hyperosmotic solution (330 mosM) reduced L-type current amplitude but did not evoke significant change in T-type current. These results indicate that the responsiveness of rat glomerulosa cells to physiological elevation of [K+] is remarkably influenced by changes in osmolarity by means of modulating the function of voltage-gated Ca2+ channels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MOST IMPORTANT physiological stimuli of aldosterone secretion by adrenocortical glomerulosa cells are extracellular K+, angiotensin II (AII), and ACTH. Elevation of [K+] evokes plasma membrane depolarization and Ca2+ influx through voltage-gated Ca2+ channels (VGCCs), AII acts by activating Ca2+ release from intracellular stores followed by Ca2+ influx from the extracellular space, and ACTH exerts its effect by stimulating cAMP formation (1). Besides these well-known regulators, clinical observations and studies conducted in vivo suggest that aldosterone secretion may be directly modulated by alterations of plasma [Na+] or osmolarity, as well. Hyponatremia, induced by hemodialysis in anephric man (2) in intact or nephrectomized dogs (3), or induced by peritoneal dialysis in rats after pharmacological inhibition of the renin-angiotensin system (RAS) and ACTH secretion (4), increased plasma concentration of aldosterone (PAC). In peritoneally dialysed, dexamethasone-treated rats with functioning RAS, the plasma concentration of Na+ (122–142 mM) correlated negatively with PAC, and the ratio of PAC-to-PRA rose steeply below 132 mM Na+, suggesting that either hyponatremia increases the sensitivity of glomerulosa cells to AII or factors other than RAS may also contribute to the induction of hyperaldosteronism (5). In water-deprived dogs, dehydration was followed by increased PRA without increased PAC (6), and the authors concluded that the induced hypernatremia or hyperosmosis reduced the sensitivity of the adrenal cortex to AII. Though certainly this is a possibility, the significance of K+ loss, observed in this study, should also be considered. In a clinical study on adipsic diabetes insipidus, the ratio of PAC to plasma renin concentration showed negative correlation with plasma [Na+] and [osm] (7), also suggesting a role of [Na+] or [osm] in the control of aldosterone secretion.

Although several in vitro studies investigated the direct regulatory effect of Na+ concentration on the adrenal gland or isolated glomerulosa cells (8, 9, 10), much less interest was focused on the possible role of osmotic concentration changes. However, studies performed on canine adrenal glands (11, 12) and cultured bovine glomerulosa cells (13) demonstrated the regulatory effect of osmolarity on aldosterone production. In these studies, aldosterone production increased in hyposmotic and decreased in hyperosmotic environment, and hyposmosis enhanced, whereas hyperosmosis suppressed the stimulatory effect of K+ and AII. In spite of the potential significance of osmotic effects, these observations have not been appropriately considered in the literature. Therefore, in the present study, we examined the effect of osmotic concentration on the function of rat glomerulosa cells, with special attention to the behavior of plasma membrane ion channels under osmotic stress. Our results show that hyposmosis significantly augments the stimulatory effect of physiological elevation of [K+] on aldosterone production. An increase in the amplitude of voltage-gated Ca2+ currents is an essential component of this response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation and culture
Wistar rats were used with the approval (No. 17/1998) of the Animal Care and Ethics Committee of the Semmelweis University. All procedures followed legal and institutional guidelines for animal care. Rat glomerulosa cells were prepared from the adrenal capsular tissue of rats (200–300 g) kept on a standard semisynthetic diet (100 mmol kg-1 Na+, 120 mmol kg-1 K+) with collagenase digestion, as described (14). Freshly digested cells were plated onto polylysine-coated 24-well tissue culture dishes (for aldosterone measurements) or polylysine-coated glass coverslips (for [Ca2+]i and patch-clamp measurements) and were kept in an incubator in 5% CO2 at 37 C in a mixture (38:62, vol/vol) of modified Krebs-Ringer bicarbonate solution and M199 [final concentrations: 146 mM Na+, 3.6 mM K+, 0.5 mM Mg2+ (pH 7.4), 290 mosM] for 3 h (aldosterone measurements), 1 day ([Ca2+]i measurements), or 2 days (patch-clamp measurements).

Aldosterone production
After a preincubation of 3 h, glomerulosa cells (~2 x 105 cells/well, equivalent to one adrenal) were exposed to different osmolarities, ranging from 250–330 mosM, with control (3.6 mM) or elevated (5 mM) [K+] for 1 h. After 1 h incubation, the media (500 µl/well) were removed, and aldosterone was measured directly by RIA, as described (15).

Measurement of cytoplasmic [Ca2+] in single cells
Cells plated on glass coverslips were loaded with Indo-1 acetoxymethyl esther (2 µM, TefLabs, Austin, TX) in cell culture medium for 45–60 min in a CO2-incubator at 37 C. Test solutions (see below) were applied by a gravity-driven perfusion system located at about 50 µm from the cells. Fluorescence was monitored at 30 C on the stage of a Diaphot 300 inverted microscope (Nikon, Tokyo, Japan), applying a monochromator and two photomultipliers (PTI, South Brunswick, NJ), using the ratio mode. Excitation was performed at 355 nm; emission wavelengths were 400 and 500 nm. The intracellular [Ca2+] was calculated from the formula [Ca2+]i = KD x ß x [(R-Rmin)/(Rmax-R)] (16), where R is the ratio F400/F500, ß is the ratio of the 500 nm fluorescence signal at zero and saturating [Ca2+], and KD is the dissociation constant of Indo-1 for Ca2+ (230 nM). Rmin, Rmax, and ß were determined by calibration in Indo-loaded cells (n = 5). Rmin was measured in the presence of 10 µM ionomycin in a nominal Ca2+-free extracellular solution, and Rmax was obtained by adding 10 mM Ca2+. Resting [Ca2+]i, measured with Indo-1 (204 ± 28 nM, n = 54) was higher than that measured in the same cell preparations with Fura-2 (132 ± 25 nM, n = 9).

Electrophysiological recordings
For patch-clamp measurements, the nystatin-perforated voltage-clamp method was applied. Nystatin was freshly dissolved in dimethylsulfoxide and added to the pipette solution at a final concentration of 240 µg/ml. Pipettes were pulled from hard borosilicate glass (B120–90-10, Sutter, Novato, CA) using a P-87 puller (Sutter) and fire-polished. Pipette resistance ranged between 3 and 7 Mohms when filled with the pipette solution, containing (in mM) 140 Cs-gluconate, 5 MgCl2, 1 EGTA, and 10 HEPES (pH 7.3). Composition of the extracellular test solutions is given below. The perfusion system was the same as for Ca2+ measurements. All experiments were carried out at 30 C. The pipette was mounted on the headstage of an Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA) or an RK-400 patch-clamp amplifier (Biologic Science Instruments, Claix, France). Seals with Gohm resistance only were used. Recordings were started when series resistance reached a value less than 40 Mohms. Currents were low-pass filtered at 1 kHz (-3 decibel) with an eight-pole Bessel filter, and digitally sampled at 4 kHz by a Digidata 1200 interface board (Axon). Experiments, data storage, and analysis were performed with P-Clamp software, version 6.0 (Axon). No leak correction was made in the current analysis.

We studied transient (T)- and long-lasting (L)-type Ba2+ currents in the same cells, using 1000-msec-step depolarizations from the -100-mV holding potential to -20 mV in every 15 sec. Depolarization steps elicited two inward currents. The current, which activated and inactivated rapidly and reached a maximal value within the first 30 msec of the step, was considered as T-type current. Amplitude of this current was measured at the peak. The second inward current, activating more slowly to reach its maximum only after 200 msec, represented L-type current. We determined L-type current amplitude by averaging maximal sustained current values in a 100–150-msec-long time period.

The amplitude of the T-type current showed decay during repeated activations at isosmotic conditions. The decrease in current amplitude, as related to that in the first activating episode (IX/I1), was described by the equation: y = (1.008) - 0.361 x log10(X) fitted by a statistical software (Statistica 4.5, Statsoft, Tulsa, OK), where X is the episode number. The equation is based on measurements in eight cells from four independent cell preparations (r = 0.84). Control values for T-type current were calculated with extrapolation from this equation. The amplitude of the L-type current showed no consequent changes in control solution; therefore, control current amplitude was considered as the mean current of the three preceding episodes before changing osmolarity. The effect of osmotic changes was statistically analyzed 30 sec and 60 sec after starting osmotic stimulation.

Test solutions
Osmotically different test solutions were prepared from hyposmotic basal solutions containing (in mM), for [Ca2+]i measurements: 121 NaCl, 3.6 KCl, 2 CaCl2, 0.5 MgCl2, 10 HEPES, and 11 glucose (pH 7.4, 245 mosM); and for patch-clamp studies of Ca2+ currents: 90 N-methyl-D-glucamine (NMDG)-Cl, 10 BaCl2, 10 tetraethylammonium (TEA)-Cl, 3.6 KCl, 2 NaCl, 0.5 MgCl2, 10 HEPES, 11 glucose (pH 7.4, 240 mosM). For aldosterone measurements, the hyposmotic basal medium was prepared as the cell culture medium but with lower Na+ concentration (121 instead of 146 mM, 250 mosM). Osmolarity of the solutions was raised with sucrose as needed to adjust a final osmolarity of 250, 270, 290, 310, or 330 mosM, checked by a freezing-point osmometer (MicroOsmometer 3MO, Advanced Instruments, Norwood, MA).

Statistical analysis
Data are expressed as means ± SE. Statistical significance was estimated in aldosterone measurements by two-way ANOVA, in current measurements by ANOVA for repeated measures, and in Ca2+ measurements by Student’s paired t test or ANOVA for repeated measures, using a statistical software (Statistica 4.5 or 5.1, Statsoft). A P < 0.05 was considered significant for all tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of osmolarity on aldosterone production
Basal aldosterone production by rat glomerulosa cells, incubated in 290 mosM medium for 1 h, was 0.22 ± 0.02 pmol/105 cells/h, which increased to 0.94 ± 0.12 pmol/105 cells/h in response to elevation of [K+] from the control 3.6 mM to 5 mM. Variations of osmotic concentration, in the range of 250–330 mosM, evoked no significant change in aldosterone production at 3.6 mM K+. However, the effect of 5 mM K+ was influenced by osmotic concentration: hyposmosis markedly enhanced the K+-stimulated aldosterone production, whereas hyperosmosis decreased the response to K+ (Fig. 1Go). The interaction between the effect of osmolarity and K+ was significant (n = 4, P < 0.01).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Interaction between the effect of [osm] and [K+] on aldosterone production. Different osmotic concentrations, in the range of 250–330 mosM, were applied with 3.6 (square) or 5 mM (circle) [K+]. Aldosterone (aldo) production was measured after 1 h incubation. Means ± SE of four independent experiments are shown.

 
Effect of osmolarity on [Ca2+]i
Resting [Ca2+]i in single glomerulosa cells, estimated with Indo-1, was 204 ± 28 nM (n = 54). Lowering osmolarity from the control 290 mosM to 250 mosM induced no changes in [Ca2+]i in 22 of the total 25 cells where hyposmosis was applied. Even when the exposure to the osmotic change lasted more than 5 min, neither hyposmosis (Fig. 2AGo, n = 4) nor hyperosmosis (Fig. 2BGo, n = 3) evoked detectable change in [Ca2+]i. The responsiveness of the cells was maintained, as indicated by the Ca2+ signal produced by 8.6 mM K+ at the end of the experiments.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Effect of osmolarity on [Ca2+]i in single glomerulosa cells. Changes in [Ca2+]i were monitored fluorimetrically with Indo-1. Isosmotic (290 mosM) solution was replaced with either hyposmotic (250 mosM; A) or hyperosmotic (330 mosM; B)solution. Osmotic concentrations were adjusted by changing sucrose concentration. After restoring 290 mosM, 8.6 mM K+ was applied to test the responsiveness of the cells. Representative traces for four (A) and three (B) cells, obtained from two cell preparations, are shown.

 
Effect of osmolarity on the K+-induced [Ca2+]i response
After checking the [Ca2+]i responsiveness of the cells to 5 mM K+, we examined this response parameter in hyposmotic or hyperosmotic environment. At control osmotic concentration (290 mosM), 5 mM K+ increased [Ca2+]i from the control value of 229 ± 38 by 22 ± 6 nM (n = 15, P < 0.01). The stimulation was then repeated at 250 or 330 mosM, and again at 290 mosM. In the hyposmotic solution (Fig. 3AGo, n = 8), the response attained 67 ± 22 nM; whereas under the restored isosmotic conditions, it returned to 31 ± 9 nM. The response at 250 mosM was significantly greater (P < 0.01) than that under isosmotic conditions examined either before or after the hyposmotic phase. When osmolarity was reduced only after K+-induced sustained elevation of [Ca2+]i developed, hyposmosis further augmented [Ca2+]i within a few seconds (Fig. 3BGo, n = 4). Hyperosmosis failed to influence K+-induced Ca2+ signal (Fig. 3CGo, n = 7).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Effect of osmolarity on the [Ca2+]i response evoked by 5 mM K+. Osmotic concentration is shown on the top of the panels. Elevations of [K+] from the control (3.6 mM) to 5 mM are indicated by horizontal bars. A, Effect of lowering osmolarity to 250 mosM (representative trace for eight similar experiments performed on two cell preparations); B, osmolarity was reduced to 250 mosM after stimulation with K+ (representative trace for four similar experiments on three cell preparations); C, effect of increasing osmolarity to 330 mosM (representative trace for seven experiments performed on 4 cell preparations).

 
Effect of osmolarity on VGCCs
The interaction between the effect of osmolarity and K+ on hormone production, as well as on Ca2+ concentration, suggests that osmotic changes may influence the function of VGCCs that are activated by depolarization at elevated [K+]. To study the effect of osmolarity on VGCCs, the nystatin-perforated voltage-clamp method was applied. After obtaining the perforated whole-cell condition, cells were superfused with isosmotic (290 mosM) control solution containing 10 mM TEA and 10 mM Ba2+, to decrease K+ conductance and increase the current through VGCCs. The pipette solution contained Cs+ instead of K+ to further eliminate outward potassium currents. Consecutive depolarizing steps from -100 to -20 mV were applied, in every 15 sec, to activate the T-type and long-lasting (L) Ba2+ currents. All studied cells possessed T-type current, whereas L-type current could be detected in 81% of the cells. After obtaining at least three control curves, the isosmotic external solution was replaced by an osmotically different solution (250 or 330 mosM). The currents at 30 sec and 60 sec of perfusion with hypo or hyperosmotic solutions were compared with control current values that were calculated as described in Materials and Methods.

The effect of hyposmosis (250 mosM) on T-type Ba2+ current is shown in Fig. 4Go, A and B (n = 5). The amplitude of the T-current significantly increased in hyposmosis, by 37 ± 17% after 60 sec. The L-type current amplitude measured in the same cells (Fig. 4Go, C and D; n = 5) was augmented 30 sec after starting hyposmotic exposure. However, in some cells, this effect proved to be transient; and at 60 sec, no more remarkable change in current amplitude could be demonstrated.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. Effect of hyposmosis on voltage-gated Ba2+ currents. Currents were elicited with 1000-msec voltage steps, from -100 mV holding potential to -20 mV, every 15 sec. Bath solution contained 10 mM Ba2+ and 10 mM TEA; pipette solution contained Cs+ instead of K+. T- and L-type currents were distinguished by their different kinetics, as described in Materials and Methods. A, Traces of the T-type Ba2+ current in a representative cell (a) before and (b) during lowering osmolarity from 290 to 250 mosM and (c) after return to 290 mosM. The first 100-msec period of the step is presented. The corresponding voltage protocol is shown on the top. B, Peak transient currents of the experiment shown in A are plotted as a function of time. Protocol of changing osmolarity is shown at the top. The corresponding traces of A are indicated. Similar results were obtained in five experiments performed on three cell preparations. C, Traces of the L-type Ba2+ current in another representative cell (a) before and (b) during lowering osmolarity from 290 to 250 mosM and (c) after return to 290 mosM. Voltage protocol is shown above. D, 100-msec periods at the maximum sustained current values (dotted box) in the experiment shown in Panel C were averaged and plotted as a function of time. Representative for five experiments. ms, Milliseconds; s, seconds.

 
Elevated osmotic concentration (330 mosM) evoked no significant change in T-current amplitude (Fig. 5Go, A and B; n = 9); although in the first episode after starting the hyperosmotic exposure, we observed a mild increase of current amplitude. Because this increase appeared immediately after changing the isosmotic to hyperosmotic solution, and was not persistent, it may be attributed to a mechanical artifact. Significant decrease in L-current amplitude was observed on the same cell population. The current decreased by 29 ± 8%, 60 sec after applying hyperosmosis (Fig. 5Go, C and D; n = 7; in two further cells, L-current was negligible). The changes in L-type current amplitude were usually only partially reversible after restoring 290 mosM.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Effect of hyperosmosis on voltage-gated Ba2+ currents. Voltage protocol and solutions are described in the legend of Fig. 4Go. A, Traces of the T-type Ba2+ current (a) before, and (b) during increasing osmolarity from 290 to 330 mosM and (c) after return to 290 mosM. B, Peak transient currents of the experiment shown in Panel A were plotted in function of time. Hyperosmosis was applied as shown on the top protocol. Similar results were obtained in nine experiments performed on three cell preparations. C, Traces of the L-type Ba2+ current in another representative cell (a) before and (b) during increasing osmolarity from 290 to 330 mosM and (c) after return to 290 mosM. D, Maximum sustained current values of the experiment shown in Panel C were plotted in function of time as described in the legend for Fig. 5Go. Representative for seven cells, on two cell preparations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The secretion of hormones participating in the control of salt-water balance was shown to be influenced by changes in extracellular osmolarity. In addition to vasopressin, this is true for renin (17) and ANP (18), but the idea that osmotic concentration plays a role in the control of aldosterone secretion has not been appropriately considered. Early clinical observations (2, 7) and in vivo experiments (2, 4, 5) suggested that besides the well-known stimuli like K+, AII, and ACTH, aldosterone secretion may be influenced also by changes in plasma osmolarity. In these studies, [Na+] and the osmolarity of the blood was markedly altered, and an unexpected dissociation of aldosterone secretion and the activity of the RAS was observed. The results could not be explained by changes in [K+] and ACTH levels, suggesting a direct regulatory effect of either [Na+] or osmolarity on aldosterone secretion. The differentiation between these two possibilities required in vitro examination. The direct modulatory effect of [Na+] was investigated in several studies (8, 9, 10), but the participation of this mechanism in the physiological control of aldosterone secretion remained debated. Less attempt was made to explore the potential direct influence of osmotic changes on the production of aldosterone. In studies performed by Schneider et al. on perfused canine adrenal glands (11) and on isolated bovine glomerulosa cells (13), hyposmosis increased both basal and K+- or AII-stimulated aldosterone production, whereas hyperosmosis had an opposite effect. The stimulation of hormone production by hyposmosis was shown to be mediated by Ca2+ influx. Hyposmosis failed to evoke its effect in canine adrenal glands at lowered extracellular [Ca2+] or in the presence of nifedipine (12); whereas in bovine glomerulosa cells, hyposmosis increased 45Ca2+ uptake and evoked biphasic Ca2+ signal, which was abolished in the absence of extracellular Ca2+ (13). However, the exact cellular mechanism whereby hyposmosis provokes Ca2+ influx remained unknown.

The aim of our study was to investigate the effect of osmolarity on aldosterone production by rat glomerulosa cells and to explore the cellular events participating in its action. Osmotic changes significantly modified the aldosterone response to physiological elevation of [K+] from the control 3.6 mM to 5 mM. Hyposmosis enhanced markedly (whereas hyperosmosis suppressed slightly) the response. However, cells incubated at control [K+] did not respond to changes in osmotic concentration between 250 and 330 mosM. Potassium ion exerts its effect on aldosterone production by elevating cytoplasmic [Ca2+]. Measurements of [Ca2+]i were concordant with these observations, because hyposmosis alone did not change [Ca2+]i but potentiated the Ca2+ signal evoked by 5 mM [K+]. It is important to emphasize that the applied 5 mM K+ falls within the physiological range of [K+] and that the osmotic concentrations examined in the present study may occur also in humans under strenuous physical exercise, under environmental stress, or in pathological conditions.

Exposure to hyposmosis evokes an increase of [Ca2+]i in several cell types (as reviewed in Refs. 19, 20). Ca2+ may originate from intracellular stores; but in most cases, it is generated by Ca2+ influx from the extracellular space (19). The activation of mechanosensitive ion channels, described in many cell types (21), may contribute to the development of the Ca2+ signal either directly by being permeable to Ca2+ (22), or indirectly by causing depolarization and Ca2+ influx through VGCCs. The increase of [Ca2+]i can be involved in the activation of volume-restoring mechanisms, which bring about regulatory volume decrease after cell swelling in a persistent hyposmotic environment (19, 23). It is not known whether rat glomerulosa cells possess volume regulatory mechanisms. Our present observations, however, cannot be explained by any of the above described mechanisms, because hyposmosis alone did not have any effect either on [Ca2+]i or on aldosterone production. This is in contrast with studies performed on canine adrenal glands and bovine glomerulosa cells, where low osmolarity alone increased Ca2+ influx and aldosterone secretion, raising the possibility that a direct or indirect stretch-activated Ca2+ influx mechanism exists in those cells.

Although aldosterone production and [Ca2+]i were unaffected by hyposmosis, the Ca2+- and hormone response to 5 mM K+ were augmented significantly in our study. This strong interaction indicates that osmolarity influences the development of the cytoplasmic Ca2+ signal evoked by K+. The accepted mechanism whereby K+ increases [Ca2+]i is depolarization of the cell membrane and a subsequent Ca2+ influx through VGCCs (1), although a Ca2+ influx pathway activated by K+ as a ligand was also proposed to contribute to the unique sensitivity of glomerulosa cells to K+ (24). In the present study, we tested the possibility that hyposmosis alters the function of VGCCs. The patch-clamp experiments presented here clearly show that the amplitude of both the T- and L-type currents are influenced by osmotic changes: hyposmosis increases (and hyperosmosis decreases) the current flowing through VGCCs. Similarly to glomerulosa cells, hyposmosis or cell inflation increased, whereas hyperosmosis decreased the amplitude of L-type Ca2+ current in myocytes (25, 26, 27). We are not aware of studies on the effect of osmotic changes on other VGCC types within the osmotic range applied in our study. The cytoskeleton was a possible candidate to transfer the swelling-induced mechanical stress to the plasma membrane in myocytes (28), although changes in intracellular ion concentration or ionic strength may also be of significance. The mechanism underlying this phenomenon in rat glomerulosa cells is presently not known.

We conclude that T- and L-type voltage-gated Ca2+ currents in rat glomerulosa cells are influenced by extracellular osmolarity. The change in the function of VGCCs in hyposmosis and hyperosmosis presumably plays a role in the altered sensitivity of glomerulosa cells to the physiological elevation of [K+]. This phenomenon may have special significance in man during physical exercise, under environmental stress, and in several pathological states.


   Acknowledgments
 
The skillful technical assistance of Ms. Anikó Rajki and Ms. Erika Kovács is highly appreciated. Aldosterone antibody was a generous gift from Prof. G. P. Vinson (London, UK). We express our thanks to Dr. Péter Enyedi for his valuable comments.


   Footnotes
 
1 This work was supported by grants from the Hungarian Council for Medical Sciences (ETT Grant No. 528/96) and National Science Foundation (OTKA Grant No. T 026173). Back

Received December 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Spät A, Enyedi P, Hajnóczky G, Hunyady L 1991 Generation and role of calcium signal in adrenal glomerulosa cells. Exp Physiol 76:859–885[Medline]
  2. McCaa RE, Bower JD, McCaa CS 1973 Relative influence of acute sodium and volume depletion on aldosterone secretion in nephrectomized man. Circ Res 33:555–562[Abstract/Free Full Text]
  3. McCaa RE, Young DB, Guyton AC, McCaa CS 1974 Evidence for a role of an unidentified pituitary factor in regulating aldosterone secretion during altered sodium balance. Circ Res 34–35:I-15–I-25
  4. Spät A, Nagy K, Tarján É 1979 Hyperaldosteronism in the sodium-depleted rat: mechanism of aldosterone stimulation by peritoneal dialysis with glucose solution. J Endocrinol 82:17–25[Abstract]
  5. Balla T, Nagy K, Tarján É, Renczes G, Spät A 1981 Effect of reduced extracellular sodium concentration on the function of adrenal zona glomerulosa: studies in conscious rats. J Endocrinol 89:411–416[Abstract]
  6. Zucker A, Gleason SD, Schneider EG 1982 Renal and endocrine response to water deprivation in dog. Am J Physiol 242:R296–R302
  7. Schalekamp MA, Donker SC, Jansen-Goemans A, Fawzi TD, Muller A 1976 Dissociation of renin and aldosterone during dehydration: studies in a case of diabetes insipidus and adipsia. J Clin Endocrinol Metab 43:287–294[Abstract]
  8. Lobo MV, Marusic ET, Aguilera G 1978 Further studies on the relationship between potassium and sodium levels and adrenocortical activity. Endocrinology 102:1061–1068[Abstract]
  9. Enyedi P, Spät A 1981 Effect of reduced extracellular sodium concentration on the function of adrenal zona glomerulosa: studies on isolated glomerulosa cells from the rat. J Endocrinol 89:417–421[Abstract]
  10. Taylor Jr RE, Radke KJ, Kramer RE, Schneider EG 1988 Dissociation of osmotic and ionic modulation of aldosterone secretion. Am J Physiol 254:E243–E247
  11. Schneider EG, Radke KJ, Ulderich DA, Taylor RE 1985 Effect of osmolality on aldosterone secretion. Endocrinology 116:1621–1626[Abstract]
  12. Radke KJ, Clendenin RE, Taylor Jr RE, Schneider EG 1989 Calcium dependence of osmolality-, potassium-, and angiotensin II-induced aldosterone secretion. Am J Physiol 256:E760–E764
  13. Wang W, Hayama N, Robinson TV, Kramer RE, Schneider EG 1992 Effect of osmolality on cytosolic free calcium and aldosterone secretion. Am J Physiol 262:E68–E75
  14. Spät A, Balla I, Balla T, Cragoe Jr EJ, Hajnóczky G, Hunyady L 1989 Angiotensin II and potassium activate different calcium entry mechanisms in rat adrenal glomerulosa cells. J Endocrinol 122:361–370[Abstract]
  15. Szabadkai G, Várnai P, Enyedi P 1999 Selective inhibition of potassium-stimulated rat adrenal glomerulosa cells by ruthenium red. Biochem Pharmacol 57:209–218[CrossRef][Medline]
  16. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
  17. Skott O 1986 Calcium and osmotic stimulation in renin release from isolated rat glomeruli. Pflugers Arch 406:485–491[CrossRef][Medline]
  18. Gibbs DM 1987 Noncalcium-dependent modulation of in vitro atrial natriuretic factor release by extracellular osmolality. Endocrinology 120:194–197[Abstract]
  19. McCarty NA, O’Neil RG 1992 Calcium signaling in cell volume regulation. Physiol Rev 72:1037–1061[Abstract/Free Full Text]
  20. Pierce SK, Politis AD 1990 Ca2+-activated cell volume recovery mechanisms. Annu Rev Physiol 52:27–42[CrossRef][Medline]
  21. Morris CE 1990 Mechanosensitive ion channels. J Membr Biol 113:93–107[CrossRef][Medline]
  22. Christensen O 1987 Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels. Nature 330:66–68[CrossRef][Medline]
  23. Hoffmann EK, Dunham PB 1995 Membrane mechanisms and intracellular signalling in cell volume regulation. Int Rev Cytol 161:173–262[Medline]
  24. Várnai P, Petheö GL, Makara JK, Spät A 1998 Electrophysiological study on the high K+ sensitivity of rat glomerulosa cells. Pflugers Arch 435:429–431[CrossRef][Medline]
  25. Xu WX, Kim SJ, So I, Kang TM, Rhee JC, Kim KW 1996 Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach. Pflugers Arch 432:159–164[CrossRef][Medline]
  26. Matsuda N, Hagiwara N, Shoda M, Kasanuki H, Hosoda S 1996 Enhancement of the L-type Ca2+ current by mechanical stimulation in single rabbit cardiac myocytes. Circ Res 78:650–659[Abstract/Free Full Text]
  27. Langton PD 1993 Calcium channel currents recorded from isolated myocytes of rat basilar artery are stretch sensitive. J Physiol (Lond) 471:1–11[Abstract/Free Full Text]
  28. Xu WX, Kim SJ, So I, Kim KW 1997 Role of actin microfilament in osmotic stretch-induced increase of voltage-operated calcium channel current in guinea-pig gastric myocytes. Pflugers Arch 434:502–504[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
D. G. Romero, M. W. Plonczynski, B. L. Welsh, C. E. Gomez-Sanchez, M. Y. Zhou, and E. P. Gomez-Sanchez
Gene expression profile in rat adrenal zona glomerulosa cells stimulated with aldosterone secretagogues
Physiol Genomics, December 19, 2007; 32(1): 117 - 127.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. Tiwari, R. K. Packer, X. Hu, Y. Sugimura, J. G. Verbalis, and C. A. Ecelbarger
Increased renal {alpha}-ENaC and NCC abundance and elevated blood pressure are independent of hyperaldosteronism in vasopressin escape
Am J Physiol Renal Physiol, July 1, 2006; 291(1): F49 - F57.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
J. Song, X. Hu, O. Khan, Y. Tian, J. G. Verbalis, and C. A. Ecelbarger
Increased blood pressure, aldosterone activity, and regional differences in renal ENaC protein during vasopressin escape
Am J Physiol Renal Physiol, November 1, 2004; 287(5): F1076 - F1083.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
A. SPAT and L. HUNYADY
Control of Aldosterone Secretion: A Model for Convergence in Cellular Signaling Pathways
Physiol Rev, April 1, 2004; 84(2): 489 - 539.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. K. Makara, P. Koncz, G. L. Petheo, and A. Spat
Role of Cell Volume in K+-Induced Ca2+ Signaling by Rat Adrenal Glomerulosa Cells
Endocrinology, November 1, 2003; 144(11): 4916 - 4922.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. Startchik, D. Morabito, U. Lang, and M. F. Rossier
Control of Calcium Homeostasis by Angiotensin II in Adrenal Glomerulosa Cells through Activation of p38 MAPK
J. Biol. Chem., June 28, 2002; 277(27): 24265 - 24273.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Makara, J. K.
Right arrow Articles by Spät, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Makara, J. K.
Right arrow Articles by Spät, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals