Pathogenesis of Adrenal Aldosterone-Producing Adenomas Carrying Mutations of the Na+/K+-ATPase
J. Stindl, P. Tauber, C. Sterner, I. Tegtmeier, R. Warth, and S. Bandulik Medical Cell Biology, University of Regensburg, 93053 Regensburg, Germany
Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism, leading to secondary hypertension. Somatic mutations in the gene for the @1 subunit of the Na+/K+-ATPase were found in about 6% of APAs. APA-related &1 subunit of the Na+/K+-ATPase mutations lead to a loss of the pump function of the Na+/K+-ATPase, which is believed to result in membrane depolarization and Ca2+-dependent stimulation of aldosterone synthesis in adrenal cells. In ad- dition, H+ and Na+ leak currents via the mutant Na+/K+-ATPase were suggested to contribute to the phenotype. The aim of this study was to investigate the cellular pathophysiology of adenoma- associated Na+/K+-ATPase mutants (L104R, V332G, G99R) in adrenocortical NCI-H295R cells. The expression of these Na+/K+-ATPase mutants depolarized adrenal cells and stimulated aldosterone secretion. However, an increase of basal cytosolic Ca2+ levels in Na+/K+-ATPase mutant cells was not detectable, and stimulation with high extracellular K+ hardly increased Ca2+ levels in cells expressing L104R and V332G mutant Na+/K+-ATPase. Cytosolic pH measurements revealed an acidification of L104R and V332G mutant cells, despite an increased activity of the Na+/H+ ex- changer. The possible contribution of cellular acidification to the hypersecretion of aldosterone was supported by the observation that aldosterone secretion of normal adrenocortical cells was stimulated by acetate-induced acidification. Taken together, mutations of the Na+/K+-ATPase depolarize adrenocortical cells, disturb the K+ sensitivity, and lower intracellular pH but, surpris- ingly, do not induce an overt increase of intracellular Ca2+. Probably, the autonomous aldosterone secretion is caused by the concerted action of several pathological signaling pathways and incom- plete cellular compensation. (Endocrinology 156: 4582-4591, 2015)
T he mineralocorticoid aldosterone plays a central role for fluid and salt homeostasis and, thereby, for blood pressure control. An inappropriately high plasma aldo- sterone concentration causes hypertension and has further pathophysiological effects on the cardiovascular systems as well as on kidney function (1-3). Physiologically, al- dosterone synthesis in zona glomerulosa cells of the ad- renal gland is mainly stimulated by angiotensin II (ang II) and an increased plasma K+ concentration, and to a minor extent, by the adrenocorticotropic hormone. Ang II and high plasma K+ exert their action on aldosterone produc- tion principally via cell membrane depolarization and by the increase of the cytosolic Ca2+ activity, which finally stimulates the expression and the activity of steroidogenic
factors and enzymes (4). The sensitivity of glomerulosa cells to ang II and to changes of the plasma K+ concen- tration relies on the expression of background K+ chan- nels which confer a hyperpolarized resting membrane po- tential to these cells (5-12). According to that concept, mouse models with deletions of the genes coding for the 2-pore K+ channels KCNK3 and KCNK9 had a depolar- ized membrane potential of adrenocortical cells and ex- hibited partially autonomous aldosterone secretion (13- 17). In humans, so-called primary aldosteronism is believed to be causative for about 10% of the cases of secondary hypertension (18). Mutations of the K+ chan- nel KCNJ5 were identified in about 40% of aldosterone- producing adenomas (APAs) and in familial forms of PA
Abbreviations: ang II, angiotensin II; APA, aldosterone-producing adenoma; ATP1A1, Na+/ K+-ATPase @1 subunit; CYP11B2, Cytochrome P450, family 11, subfamily B, polypeptide 2; HSD3B2, hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2; NCX, Na+/Ca2+ exchanger; NHE, Na+/H+ exchanger; NMDG+, N-methyl-D-glucamine; STAR, steroidogenic acute regulatory protein.
(19). These mutations elicit a pathological Na+ and Ca2+ conductance of the KCNJ5 K+ channel leading to mem- brane depolarization (20, 21), increased cytosolic Ca2+ levels (22-26), and stimulation of aldosterone secretion (23, 25, 27).
Mutations of the Na+/K+-ATPase @1 subunit (ATP1A1) are present in about 6% of APAs (23, 25, 28- 33) but were not found in familial forms of PA. Physio- logically, the Na+/K+-ATPase exchanges 3 intracellular Na+ ions for 2 extracellular K+ ions for each ATP mole- cule hydrolyzed (34). Thereby, the Na+/K+-ATPase gen- erates the Na+ and K+ ion gradients across the plasma membrane, which are necessary to build up the cell mem- brane potential (34). Inhibition of the Na+/K+-ATPase function by ang II contributes to the stimulation of aldo- sterone secretion in rat zona glomerulosa cells (35). Phar- macological inhibition of the Na+/K+-ATPase by ouabain also stimulated aldosterone secretion (36), as well as growth of the zona glomerulosa in rats (37). By contrast, CYP11B2 (Cytochrome P450, family 11, subfamily B, polypeptide 2) mRNA expression and aldosterone secre- tion were inhibited in human adrenal glomerulosa cells (31, 38). This divergence of the ouabain effects on the aldosterone secretion is probably caused by the species- dependent ouabain sensitivity of the Na+/K+-ATPase (much lower in rat compared with human) (39). More- over, signaling functions of the Na+/K+-ATPase, which are independent of the pump function (40-45), probably modulate the action of ouabain on the aldosterone secre- tion in a species and concentration-dependent manner.
The functional Na+/K+-ATPase complex consists of a catalytic «-subunit and a regulatory ß-subunit, which acts as a chaperone permitting proper membrane integration of the a-subunit (46). In addition, different FXYD pro- teins (FXYD domain containing ion transport regulator) can associate with a-subunits and modulate the Na+/K+- ATPase activity (46). The adenoma-associated ATP1A1 mutations were shown to disrupt the pump function of the Na+/K+-ATPase (28,29). Primary cultured adenoma cells bearing ATP1A1 mutations (28) and HEK cells expressing the mutant Na+/K+-ATPase were depolarized compared with control cells (29). The expression of mutant Na+/ K+-ATPase in human adrenal cell lines increased CYP11B2 mRNA expression (29, 31) and aldosterone se- cretion (31). These functional changes of adrenal cells in- duced by ATP1A1 mutations could be caused by a loss of the pump function of the Na+/K+-ATPase and, in addi- tion, by an abnormal gain-of-function of the mutated protein (47). Electrophysiological measurements using Xenopus oocytes indicated that different mutants of the Na+/K+-ATPase cause abnormal inward leak currents carried by Na+ or H+ ions (31).
In the present study, we evaluated the putative signaling pathways linking the mutated Na+/K+-ATPase to the hy- peraldosteronism phenotype in adrenocortical NCI- H295R cells. Our data show that adrenal cells expressing mutant Na+/K+-ATPase proteins had a depolarized mem- brane potential but displayed no increase of basal cyto- solic Ca2+ activity. Instead of that, L104R and V332G mutant forms of the Na+/K+-ATPase induced the distur- bance of the pH homeostasis, which could contribute to the autonomous aldosterone secretion in APA.
Materials and Methods
Atp1a1 expression vectors
Plasmids (pMT2) containing full-length cDNA sequences en- coding the wild-type or corresponding mutant (L104R, V332G) ouabain-resistant rat Atp1a1 were originally generated by Bente Vilsen (Department of Biomedicine, Aarhus University, Aarhus, Denmark). For studies in NCI-H295R cells, cDNAs were sub- cloned into the bicistronic expression vector pIRES-CD8 (28, 48). The G99R mutant form of Atp1a1 was generated by site- directed mutagenesis of the wild-type Atp1a1 containing pIRES- CD8 plasmid (29).
Cell culture and transfection
Adrenocortical carcinoma NCI-H295R cells (CLS) were cul- tured in a commercial cell line-specific DMEM/F12 medium (MG-42; CLS) containing: 15mM HEPES, 6.25-µg/mL insulin, 6.25-µg/mL transferrin, 6.25-ng/ml selenium, 1.25-mg/mL BSA, 5.35-mg/mL linoleic acid, and 2.5% Nu-Serum I, supple- mented with antibiotics (100-U/mL penicillin and 100-µg/mL streptomycin; Life Technologies GmbH). The cells were main- tained at 37℃ under a humid atmosphere of 95% air/5% CO2.
Before the experiment, 1 × 106 cells were transfected with 5 µg of pIRES-CD8 plasmids containing wild-type or mutant rat Atp1a1 using an electroporation system (NEON; Life Technol- ogies GmbH). Electroporation was done according to the man- ufacturer using 1 pulse 40 milliseconds at 1100 V. After elec- troporation, cells were cultured in the serum containing medium (1.25-mg/mL BSA and 2.5% Nu-Serum I) but without antibiot- ics on fibronectin/collagen-coated glass cover slips or without cover slips on 35-mm Falcon dishes (Corning Life Sciences). Next, cells were analyzed 24 or 48 hours after electroporation, as indicated in the figure legends. The expression of the rat Atp1a1 protein in transfected NCI-H295R cells was verified by Western blotting using a custom made rat-specific antibody and protein lysate from rat kidney as a positive control (Supplemen- tal Figure 1). For patch-clamp, pH, and Ca2+ measurements, transfected cells were identified using anti-CD8-coated dyna- beads (Life Technologies GmbH).
Patch clamp measurements
Perforated patch recordings were performed at room temperature on NCI-H295R cells using an EPC 10 amplifier (Heka). Patch pipettes with 6-8 MQ were used for the record- ings. The patch pipette solution contained: 95mM K-gluconate, 30mM KCl, 4.8mM Na2HPO4, 1.2mM NaH2PO4, 5mM glu-
cose, 2.38mM MgCl2, 0.726mM CaCl2, 1mM EGTA, 3mM ATP (pH 7.2), and 100-µg/mL Nystatin (Sigma-Aldrich Chemie GmbH) were added to the pipette solution before use. The ex- tracellular Ringer-type control solution contained: 145mM NaCl, 0.4mM KH2PO4, 1.6mM K2HPO4, 5mM glucose, 1mM MgCl2, 1.3mM CaCl2, and 5mM HEPES (pH 7.4). For some measurements the K+ concentration was increased, whereas Na+ was reduced to the same extent.
Ca2+ measurements
Cytosolic-free Ca2+ activity was measured using the ratio- metric fluorescent Ca2+ sensitive dye fura 2 (Life Technologies GmbH). NCI-H295R cells were loaded at room temperature for 45 minutes with 1.5 uM fura 2-AM in the presence of 1x Power Load permeabilizing reagent (Life Technologies GmbH). The control extracellular Ringer-type solution contained: 145mM NaCl, 0.4mM KH2PO4, 1.6mM K2HPO4, 5mM glucose, 1mM MgCl2, 1.3mM CaCl2, and 5mM HEPES (pH 7.4). For some experiments, the K+ concentration was increased, whereas Na+ was reduced to the same extent. Mean fluorescence ratios of emission at 490-530 nm after excitation at 340 and 380 nm were calculated for single transfected cells using Axiovision software (Zeiss). All the experiments were performed at 37℃ temperature.
pH measurements
Relative cytosolic pH levels were measured using the ratio- metric fluorescent pH sensitive dye BCECF-AM (Life Technol- ogies GmbH). NCI-H295R cells (grown on glass cover slips) were loaded at 37℃ for 20 minutes with 1p.M BCECF-AM. Mean fluorescence ratios of emission at 535 nm after excitation at 495 and 440 nm were calculated for single transfected cells using MetaFluor software (Visitron Systems GmbH). Measure- ments were performed at 37℃. The extracellular Ringer-type control solution contained: 145mM NaCl, 0.4mM KH2PO4, 1.6mM K2HPO4, 5mM glucose, 1mM MgCl2, 1.3mM CaCl2, and 5mM HEPES (pH 7.4). For some measurements the K+ concentration was increased, whereas Na+ was reduced to the same extent or bath Na+ was replaced by N-methyl-D-gluca- mine (NMDG+).
Aldosterone measurements
Cell culture medium from 1 × 106 transfected NCI-H295R cells was collected 24 or 48 hours (as indicated in the figures) after electroporation. Aldosterone concentration was measured in the medium using a specific enzyme-linked immunosorbent assays (IBL). The remaining cells were washed twice with PBS, and then lysed in PBS containing 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% IGEPAL CA-630, and protease inhibitors. Protein concentration of the cell lysate was measured using a standard Bradford assay (Bio-Rad). The total amount of aldosterone in the medium was normalized against the total amount of cellular protein.
RNA isolation
Total RNA from NCI-H295R cells was isolated using a col- umn based kit according to the manual (NucleoSpin RNA; Ma- cherey). The RNA concentration was quantified using a pho- tometer (Nanodrop; PEQLAB Biotechnologie GmbH). Quality
of the RNA used for reverse transcription was tested by agarose electrophoresis.
Real-time PCR
Reverse transcription with Moloney Murine Leukemia Virus Reverse Transcriptase (Promega GmbH) and random primers (Fermentas GmbH) was perfomed using 1-µg total RNA. Real- time PCR of cDNA samples was performed on a LightCycler 480 device (Roche) using specific and intron-spanning or intron- flanking primers (Life Technologies GmbH), and a SYBR Green Master Mix (Roche). Target gene expression levels in cDNA samples were quantified relative to ß-actin expression under con- sideration of PCR efficiencies. The specificity of PCR amplifi- cations was verified by agarose electrophoresis and melting curve analysis. Primer sequences are listed in Supplemental Table 1.
Substances
The next substances were used: cariporide (inhibitor of the Na+/H+ exchangers [NHEs]; Santa Cruz Biotechnology, Inc), stock solution was prepared in Dimethyl sulfoxide; ang II (Sig- ma-Aldrich), stock solution was prepared in deionized water.
Statistics
Statistical significance was tested using Student’s t test (for paired or unpaired samples as appropriate). All data were pre- sented as mean ± SEM, P < . 05 was accepted as significant; n refers to the number of cells (calcium, pH, and patch clamp measurements) or dishes (real-time PCR, ELISA) analyzed.
Results
Increased expression of steroid hormone- producing enzymes and increased aldosterone production in ATP1A1 mutant adrenal cells
The functional consequences of adenoma-associated mu- tations of the @1-Na+/K+-ATPase isoform (ATPase, Na+/K+ transporting, «1 polypeptide, ATP1A1) were an- alyzed in human adrenocortical carcinoma NCI-H295R cells expressing wild-type or mutated rat Atp1a1. Expres- sion of CYP11B2 mRNA (aldosterone synthase) was in- creased by 1.5- to 2-fold in mutant Na+/K+-ATPase-ex- pressing cells compared with wild-type cells (Figure 1A). In addition, mRNA expression of HSD3B2, which is the enzyme for the production of the precursor progesterone, was increased (Figure 1A). The effects of the mutant Na+/ K+-ATPase on the mRNA expression of other key pro- teins involved in the regulation and catalysis of the steroid hormone synthesis (NR4A2, STAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1) were rather small and changes were not significant in all tested mutant variants of the Na+/K+-ATPase (Supplemental Figure 2A).
In parallel to the higher expression of CYP11B2 mRNA, aldosterone production was increased by 20%- 40% in mutant Na+/K+-ATPase-expressing cells (Figure
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1B). This small effect size is most likely due to the low per- centage of strongly transfected cells (10%-20%) leading to the underestimation of the mutation-induced increase.
Membrane depolarization, normal basal Ca2+ activity, and altered K+ sensitivity of ATP1A1 mutant adrenal cells
The effect of mutant Na+/K+-ATPase on the cell membrane potential of adrenal NCI-H295R cells was measured at dif- ferent extracellular K+ concentrations. Wild-type-express-
ing cells had a hyperpolarized mem- brane potential of -69 mV under physiological K+ concentration (Fig- ure 1C). By contrast, mutant Na+/K+- ATPase-expressing cells were depolar- ized under control condition. The depolarization was strongest in L104R and V332G mutant cells, which displayed a shift of the mem- brane potential by 20 mV (Figure 1C). An increase of extracellular K+ to 15mM depolarized wild-type and G99R mutant cells by 22 and 14 mV, respectively. L104R and V332G mu- tant cells hardly showed a further de- polarization at 15mM extracellular K+.
Next, the effect of mutants of the Na+/K+-ATPase on cytosolic Ca2+ activity was examined. Under con- trol conditions, Ca2+ levels were not different in NCI-H295R cells ex- pressing the L104R, V332G, or G99R mutants of Na+/K+-ATPase compared with cells expressing the wild-type protein (Figure 1D). Stim- ulation with 15mM K+ bath solu- tion induced a strong Ca2+ increase in wild-type and G99R mutant-ex- pressing cells. By contrast, the re- sponse to K+ was almost abolished in L104R- and V332G-expressing cells (Figure 1D).
Acidification and disturbed pH homeostasis in ATP1A1 mutant adrenal cells
Next, we examined whether the proton leak through the mutant Na+/K+-ATPase protein, proposed in the literature (31, 47), has an effect on the pH homeostasis of adrenal NCI-H295R cells. Under control ex- tracellular solution with pH 7.4, NCI-H295R cells expressing the L104R or V332G mutants of Na+/K+- ATPase had an acidified cytosolic pH compared with wild-type cells (Figure 2A). Cytosolic pH of cells ex- pressing the G99R mutant was not altered. Challenging wild-type or mutant Na+/K+-ATPase-expressing cells by changing extracellular pH to 7 and 8 provoked a decrease and an increase of cytosolic pH, respectively (Figure 2B). However, the response of L104R and V332G mutant cells
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was stronger compared with G99R mutant and wild-type cells leading to a marked acidification at pH 7 and an alkalization above wild-type level at pH 8 (Figure 2B).
Physiologically, stimulation of adrenal cells with high K+ induces cytosolic acidification (49). This effect is be- lieved to be a consequence of the activation of Ca2+- ATPases, which import H+ during Ca2+ export (50, 51). Also in our experiments, cytosolic pH in NCI-H295R cells expressing wild-type Na+/K+-ATPase decreased upon stimulation with 15mM K+ (Figure 2C). By contrast, mu- tant L104R and V332G cells showed, instead of the phys- iological decrease, an increase of cytosolic pH under high K+ solution (Figure 2C). G99R mutant-expressing cells, however, were not different from wild-type cells.
Interestingly, NCI-H295R cells expressing the adenoma- associated mutant KCNJ5L168R K+ channel also displayed an acidified cytosolic pH under control condition (Supple- mental Figure 3A). However, the rate of intracellular pH
changes in presence of extracellular pH 7 and pH 8 was not different be- tween wild-type and mutant KCNJ5- expressing cells (Supplemental Figure 3B).
Next, the possible relevance of in- tracellular acidosis as an independent stimulus of aldosterone synthesis was tested. Untransfected NCI-H295R cells were exposed for 24 hours to 20mM extracellular Na+ acetate re- sulting in an intracellular acidification (Figure 3A) similar to the one observed in L104R and V332G mutant Na+/ K+-ATPase-expressing cells (Figure 2A). Na+ acetate increased the mRNA expression of CYP11B2 and the aldo- sterone secretion (within 48 h) (Figure 3, B and C). However, the effects of Na+ acetate were not as strong as the effect of 15mM extracellular K+, which strongly stimulated CYP11B2 expression and aldosterone secretion.
Role of the Na+/H+ exchanger (NHE) activity for the phenotype of mutant ATP1A1 cells
In addition to the described pro- ton leak, the activity of proton ex- trusion pathways, for example via the plasma membrane NHEs, could modulate the phenotype of Na+/K+- ATPase mutant cells. To test the con- tribution of NHE, we measured pH under conditions which inhibited the NHE-dependent H+ export. Removal of Na+ from the extracellular solution (Na+ replaced by NMDG+) led to a further decrease of cy- tosolic pH in L104R mutant cells, whereas pH in wild-type cells remained stable (Figure 4A; V332G and G99R were not analyzed in this subset of experiments). Direct inhibition of NHE activity by 100uM cariporide (a specific blocker of NHE (52)) further acidified L104R mutant cells but had no effect in wild-type cells (Figure 4B). The pathological increase of cytosolic pH under 15mM K+ observed in mutant Na+/ K+-ATPase cells (Figure 2C) was absent when NHE was blocked (Figure 4B).
Discussion
APAs are a major cause of primary aldosteronism leading to secondary hypertension (53). Somatic mutations of dif-
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ferent genes coding for ion channels (KCNJ5 [20, 23, 25, 29, 54, 55], CACNA1D [31, 56], and ATPases [ATP1A1 and ATP2B3] [25, 28, 29, 31]) have been identified in APAs.
In the present study, we analyzed the cellular patho- physiology of adenoma-associated mutations of the ATP1A1. Adrenal NCI-H295R cells expressing L104R and V332G mutant forms of the Na+/K+-ATPase dis- played a depolarized membrane voltage, impaired K+ sen- sitivity, but no overt increase of basal Ca2+. Moreover, the mutant cells displayed a cytosolic acidification that ap- pears to contribute to the increased aldosterone secretion.
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Mutant ATP1A1 lead to depolarization of adrenal cells and increase aldosterone secretion without altering the basal cytosolic Ca2+ level
Up to now, 6 different somatic mutations of ATP1A1 have been * identified in APA (G99R, L104R, V332G, del100_104, EETA963S, 48h stim. and del102_103) (25, 28, 29, 31). The ATP1A1 mutations analyzed in the present study (G99R, L104R, and V332G) are structurally close to a residue (E334) in the Na+/K+- ATPase protein, which is important for ion binding and for conforma- tional changes of the pump during the transport process (57). Hence, the mutated residues disrupt the catalytic activity of the Na+/K+- ATPase and prevent the exchange of Na+ and K+ ions (28,29). Normally, the Na+/K+-ATPase activity is a prerequisite for the hyperpolarized resting cell membrane potential of adrenal cells. Therefore, a loss of the pump function of the mutant Na+/K+-ATPase is sup- posed to cause membrane depolarization. According to this, primary cultured adrenal adenoma cells bearing ATP1A1 mutations as well as HEK cells and Xenopus oocytes expressing mutant Na+/K+-ATPase displayed a depolarized membrane potential compared with control cells (28, 29, 31).
Here, we tested the effect of the L104R, V332G, and G99R mutant Na+/K+-ATPase on the membrane poten- tial and on the aldosterone production in the adrenocor- tical NCI-H295R cell model (Figure 1) (27, 55, 58-60). The degree of depolarization correlated with the level of CYP11B2 expression and aldosterone secretion: L104R mutant cells exhibited the strongest depolarization and CYP11B2 expression and produced highest amounts of aldosterone (Figure 1). Furthermore, the mRNA expres- sion of HSD3B2 (an enzyme that produces a precursor of aldosterone and is stimulated by ang II) (61, 62) was increased in mutant cells. By contrast, the mRNA ex- pression of STAR, which is also stimulated by ang II (62, 63) was only very weakly increased in L104R and V332G mutant cells (Supplemental Figure 2A). Because the function of STAR is also modulated by nontran- scriptional effects (64, 65), we measured STAR protein levels. However, in agreement with the mRNA expres- sion, there was only a mild increase of STAR protein amounts in cells expressing mutant Na+/K+-ATPase (Supplemental Figure 2B).
As in the above mentioned studies (28, 29, 31), we used the rat Atp1a1 cDNA, because the lower ouabain sensi- tivity of the rat Na+/K+-ATPase (39) allowed us to test the pump function of the transfected rat Na+/K+-ATPase while blocking the endogenous human Na+/K+-ATPase. Without ouabain treatment (control condition with ac- tive endogenous human Na+/K+-ATPase), the expres- sion of the L104R, V332G, and G99R mutant rat Na+/K+-ATPase caused an increase of intracellular Na+ and a decrease of K+ (Supplemental Figure 4A). This was probably caused by a gain-of-function in terms of the sug- gested leak currents (31, 47). On the other hand, also a loss of the pump function could change the Na+ and K+ con- centrations, in case the overexpressed mutant Na+/K+- ATPase interfered with the endogenous Na+/K+-ATPase activity via concurrent binding of ß- and y-subunits. Treatment with ouabain (10µM for 1 h) led to a strong increase of the intracellular Na+ concentration and a de- crease of the intracellular K+ concentration in mutant rat Na+/K+-ATPase-expressing cells thereby inverting the Na+/K+ ratio (Supplemental Figure 4B). Obviously, the expression of the mutant Na+/K+-ATPase did not com- pensate for the inhibition of the endogenous human Na+/ K+-ATPase, which supports the reported loss of the pump function of the mutant Na+/K+-ATPase (28, 29). The Na+ and K+ concentrations also changed in cells express- ing the wild-type rat Na+/K+-ATPase, but the Na+/K+ ratio was not inverted. The compensation by the expres- sion of the wild-type rat Na+/K+-ATPase appeared in- complete, most likely because of the low percentage of strongly transfected cells.
The Na+/K+-ATPase @-subunits from rat and humans share only 97% identity, which causes differences in the sensitivity to ouabain and probably also influences protein-protein interactions. Therefore, the cellular pa- thology caused by the expression of the mutant Na+/ K+-ATPase from rat is possibly different compared with the mutant human protein. On the other hand, the regions bearing the adenoma-associated mutations are strongly conserved among species, because they play an important role for the catalytic transport cycle of the Na+/K+- ATPase. Thus, the deleterious effects of the mutations on the pump function leading to membrane depolarization and increased aldosterone secretion are presumably equal in the human and rat protein.
Surprisingly, despite the strong membrane depolariza- tion and the increased aldosterone secretion, the basal cy- tosolic Ca2+ levels in cells with mutants of the Na+/K+- ATPase appeared to be normal (Figure 1D). Different compensatory mechanisms could account for that phenomenon:
1) Reduced function of Ca2+ channels during chronic depolarization.
2) Intracellular acidification was observed to depress T-type Ca2+ channel currents (66).
3) Activation of Ca2+ export pathways (via Na+/Ca2+ exchanger [NCX] or Ca2+-ATPase) (67, 68), which could compensate for the Ca2+ influx leading to ap- parently normal cytosolic Ca2+ levels.
In contrast to mutant Na+/K+-ATPase-expressing cells, adrenal cells expressing mutant KCNJ5 exhibited increased Ca2+ levels under control condition (22-24, 26). In these cells, the compensatory mechanisms limiting cytosolic Ca2+ activity might be overridden by the path- ological Ca2+ permeability of the mutant KCNJ5 (21) and by a stronger membrane depolarization (22).
Disturbed pH homeostasis of mutant ATP1A1 cells: Implications for the phenotype?
Activation of the Ca2+-dependent signaling is probably not the only factor driving aldosterone secretion and proliferation in adrenal cells expressing different APA-as- sociated mutated genes. Molecular simulations and electro- physiological measurements using Xenopus oocytes sug- gested abnormal inward leak currents caused by mutant Na+/K+-ATPase (31, 47). This gain of function probably contributes to the pathophysiology of mutants of the Na+/ K+-ATPase. The L104R mutant Na+/K+-ATPase was proposed to exhibit a strong H+ leak, whereas the V332G mutant was proposed to have no such leak (47). Until now, the functional consequences of these leak currents of mu- tant Na+/K+-ATPases were not tested in adrenal cells.
Using adrenal NCI-H295R cells, we observed that L104R mutant cells had an acidified cytosolic pH despite of strongly enhanced NHE activity (Figures 2A and 4). Variation of the extracellular pH induced stronger changes of the cytosolic pH in L104R cells, pointing to a pathological H+ leak (Figure 2A). In contrast to the pre- diction based on molecular simulations, also the V332G mutant exhibited evidence for an H+ leak and disturbed pH regulation (Figure 2, A and B). Interestingly, high ex- tracellular K+ concentrations caused an abnormal in- crease of the intracellular pH in L104R and V332G mu- tant cells (Figure 2C). These data are in agreement with electrophysiological measurements of Azizan et al (31), who have shown partial inhibition of the leak currents through the mutated Na+/K+-ATPase by K+ ions.
Are the alterations in pH regulation causative for the increased aldosterone secretion of mutant Na+/K+- ATPase-expressing cells? Cytosolic acidification of un- transfected NCI-H295R cells by acetate led to an increase of CYP11B2 mRNA expression and aldosterone produc-
tion (Figure 3). Therefore, the disturbance of the pH homeostasis probably contributes to the phenotype of mu- tant Na+/K+-ATPase-expressing cells. In vivo, tumori- genesis-associated anaerobic glycolysis (69) and the H+ permeability of the mutant Na+/K+-ATPase could cause a more pronounced acidification aggravating autonomous aldosterone production.
Pathophysiology of G99R mutant of the Na+/K+-ATPase
The overall phenotype of NCI-H295R cells expressing the G99R mutant Na+/K+-ATPase was rather mild com- pared with the L104R and V332G mutant cells. The G99R mutant-expressing NCI-H295R cells did not show the ab- normal acidification, found in mutant L104R and V332G cells, and, they increased the cytosolic Ca2+ level upon stimulation with high K+ as the wild-type-expressing cells did. In addition, electrophysiological measurements on HEK cells (29) indicate that, in contrast to the L104R and V332G mutant Na+/K+-ATPase (Supplemental Figure 5) (28), the G99R mutant protein does not exhibit an ab- normal Na+ conductance. Nevertheless, NCI-H295R cells expressing the G99R mutant Na+/K+-ATPase showed an increased CYP11B2 mRNA expression and increased aldosterone production, compared with control cells. Probably, other mechanisms contribute to the pa- thology of the G99R mutant. The Na+/K+-ATPase mod- ulates the activity of the src kinase (70), which stimulates aldosterone secretion in NCI-H295R cells (71). In addi- tion, the Na+/K+-ATPase binds to the lipid raft-associated cholesterol-binding protein caveolin-1, and thereby af- fects distribution of cholesterol between the plasma mem- brane and intracellular compartments (44). Interestingly, the mutated G99R residue lies adjacent to the caveolin-1 binding motif of the Na+/K+-ATPase (72). Moreover, the Na+/K+-ATPase was shown to modulate the IP3R-depen- dent Ca2+ release from the endoplasmatic reticulum (73), as well as the function of the Ca2+ activated maxi K+ channel (45). Thereby, adenoma-associated mutations of the Na+/K+-ATPase, including the mutant G99R, could induce further cellular alterations by these pump indepen- dent functions.
Conclusions
The cellular pathophysiology of adrenal cells bearing mu- tations of the Na+/K+-ATPase appears to be complex and multifaceted (Figure 5). The L104R and V332G mutants of the Na+/K+-ATPase depolarized the membrane of ad- renocortical NCI-H295R cells and impaired K+ sensitiv- ity, but they did not cause a rise of the resting Ca2+ activity
A
K+t
Ca
2+
2K+
ang II
wildtype ATP1A1
depol.
ATP
3Na
3Na*
K+
Ca
2+
NCX
Ca
2+
NHE
H+
+ [Ca2+]t
PMCA
Na
Ca ER
2H+
aldosterone synthase
B
2K+
H+ Na+
Ca
2+
wildtype ATP1A1
mutant ATP1A1
depol.
ATP
4
P
NCX
3Na*
3Na
K+
Ca 2+
Na++
?
?
Ca
2+
NHE
H+
PMCA
Na
pH+
[Ca2+]?
2H+
proliferation+
?
aldosterone synthase
as observed in cells carrying mutant forms of the K+ chan- nel KCNJ5. Interestingly, L104R and V332G mutant cells displayed a pathological H+ leak leading to intracellular acidification. This acidification probably contributes to the stimulation of aldosterone secretion. Further studies are required to elucidate the interplay between patholog- ical ion homeostasis and complex signaling pathways that underlie autonomous aldosterone secretion and adenoma formation in adrenocortical cells.
Note added by author in proof
After acceptance of the manuscript, further experi- ments indicated that acetate treatment of NCI-H295R cells for 24 hours not always acidified cytosolic pH but resulted in variable pH effects, probably due to secondary cellular compensation. Nevertheless, acetate consistently increased CYP11B2 expression, suggesting that acetate might stimulate aldosterone secretion also independently of its direct effect on cytosolic pH, eg, via metabolic signals or activation of the NHEs.
Acknowledgments
Address all correspondence and requests for reprints to: Dr Sas- cha Bandulik, Medizinische Zellbiologie, Universitaetsstrasse 31, 93053 Regensburg, E-mail: sascha.bandulik@ur.de.
The work was supported by the Deutsche Forschungsgemein- schaft Grant FOR1086 (to R.W. and S.B.).
Disclosure Summary: The authors have nothing to disclose.
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