- Email: [email protected]
Expression of the epithelial Na+ channel and other components of an aldosterone response pathway in human adrenocortical cells
European Journal of Pharmacology 613 (2009) 176–181
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Expression of the epithelial Na+ channel and other components of an aldosterone response pathway in human adrenocortical cells☆ Timothy J. Burton ⁎, Georgina Cope, Jing Wang, Joalice C. Sim, Elena A.B. Azizan, Kevin M. O'Shaughnessy, Morris J. Brown Clinical Pharmacology, University of Cambridge, Addenbrooke's Hospital, Box 110, Hills Road, Cambridge, CB2 2QQ, UK
a r t i c l e
i n f o
Article history: Received 27 November 2008 Received in revised form 19 March 2009 Accepted 1 April 2009 Available online 14 April 2009 Keywords: Epithelial Na+ channel Aldosterone Adrenal cortex
a b s t r a c t We have unexpectedly found expression of the epithelial Na+ channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na+ on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for α-, β- and γ-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11β hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting α-, β- and γ-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K+ and angiotensin II, suppression by both Na+ and high-concentration 5-(N-ethyl-N-isopropyl) amiloride (EIPA, blocker of Na+–H+ exchange) and no change with benzamil (ENaC blocker). 22Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Aldosterone stimulates Na+ reabsorption across epithelial cells by favourably changing the electrochemical gradients across epithelial cell membranes through the up-regulation of ion channels and transporters (Bonvalet, 1998). One key target is the amiloridesensitive epithelial Na+ channel (ENaC) that is located in the apical membrane of epithelial cells and forms the rate-limiting step in transepithelial Na+ transport (Garty and Palmer, 1997). ENaC is expressed primarily in Na+ absorbing epithelia such as the distal nephron, colon, sweat glands and airways (Garty and Palmer, 1997). However it is also expressed in non-epithelial cells such as osteoblasts, lymphocytes and hair follicles (Brouard et al., 1999; Bubien et al., 2001; Kizer et al., 1997). The regulation of ENaC activity by aldosterone is complex and tissue dependent involving changes in channel subunit synthesis, the number of channels expressed at the plasma membrane and channel open probability (Gormley et al., 2003). In many ENaCexpressing tissues Nedd4-2 (neuronal precursor cell-expressed and developmentally down-regulated protein 4-2) and Sgk-1 (serum and ☆ This work has not been presented previously. Sources of support: British Heart Foundation Programme Grant RG/02/007, MRC Studentship. ⁎ Corresponding author. Tel.: +44 1223 762577; fax: +44 1223 762576. E-mail address: [email protected] (T.J. Burton). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.04.005
glucocorticoid-inducible kinase type 1) have a pivotal role in determining the number of ENaC channels at the membrane and responsiveness to aldosterone (Chen et al., 1999; Henry et al., 2003). In distal tubular epithelial cells, for example, mineralocorticoid receptor activation induces Sgk-1 expression which in turn leads to phosphorylation and inhibition of Nedd4-2 thereby increasing surface expression of ENaC (Loffing et al., 2001). In this system the presence of 11β-hydroxysteroid dehydrogenase type II confers mineralocorticoid specificity by preventing activation of the mineralocorticoid receptor by cortisol (Albiston et al., 1995). We have unexpectedly found expression of the three subunits of ENaC in human adrenocortical cells. We therefore tested the hypothesis that these cells contain all the same components as the classical renal aldosterone response pathway. Secondarily, we have also tested the hypothesis that ENaC is an important route of Na+ uptake into adrenocortical cells and may modulate aldosterone secretion. 2. Material and methods 2.1. Human tissue collection Adrenal tissue was obtained from patients undergoing adrenalectomy for adrenal causes of hypertension (Conn's syndrome and
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
phaeochromocytoma). We obtained informed consent from each patient and local ethical approval. Tissue was dissected from histologically normal regions of adrenal cortex and from adjacent cortical tumour. 2.2. Culture of H295R cells NCI-H295R adrenocortical cells obtained from ATCC were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U penicillin, 0.1 mg/ml streptomycin 0.4 mM L-glutamine and ITS (insulin–transferrin– sodium selenite media) at 37 °C in 5% CO2. Cells were maintained at 37 °C in 5% CO2 and studied between passage numbers 5 and 10.
177
spironolactone, fludrocortisone and potassium canrenoate) were made when cell confluence equalled 50–70%. Drugs were dissolved in either DMEM or Kreb's solution. The volume of medium added per well was 0.5 ml. Kreb's solution (mM: NaHCO3 26, NaCl 83–153, KCl 4–14, CaCl2 1.1, MgSO4 1.3, NaH2PO4 1.4, glucose 10, N-methyl-Dglucamine chloride 0–70, pH = 7.4) was used in experiments designed to vary [Na+] and osmolarity. Media bathing the cells was harvested after 24 h. Aldosterone concentrations were determined by 125I radioimmunoassay using a commercially available Coat-A-Count kit (Diagnostic Products Corp, LA, CA, USA). Aldosterone concentrations were normalised to total cell protein, determined by extraction of protein with RIPA buffer and BCA™ protein assay (Pierce Biotechnology, Illinois, USA). 2.6. Measurements of Na+ uptake by H295R cells
2.3. RT-PCR
22
Total RNA was extracted from normal adrenal cortex, Conn's adenoma tissue and H295R cells using Fast Prep homogenisation and phenol:chloroform extraction with TRIzol reagent (Invitrogen™). Yield was determined spectrophotometrically and quality assessed by formamide agarose gel electrophoresis. RNA was converted into cDNA by first strand synthesis primed by random hexamers using Superscript III protocol (Invitrogen™). 1 ng of cDNA was used in each PCR reaction using specific primers (designed to span introns) against α-, β- and γ-ENaC, the mineralocorticoid receptor, Nedd4L, Sgk-1 and 11β-HSD type II. Annealing temperatures were optimized using an appropriate gradient based on melting temperatures. Resultant fragments were resolved on 3% agarose gels containing ethidium bromide and visualised by UV transillumination. Fragments produced were compared to a 100 bp ladder. 2.4. Western blotting Cells were lysed by addition of RIPA buffer (NaCl 150 mM, Tris base 50 mM, Triton X-100 1% (v/v), protease inhibitor cocktail, pH 7.4). Protein derived from the lysate was quantified and an equal volume of loading buffer (62.5 mM Tris–HCl, 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue, pH = 6.8) added. 20 μg protein aliquots were denatured for 10 min at 95 °C prior to loading on gels. Polyacrylamide gels (8% (w/v) acrylamide, 375 mM Tris–HCl (pH = 8.8), 0.1% (w/v) sodium dodecyl sulphate (SDS), 0.05% (w/v) ammonium persulphate and 0.05% (v/v) N,N,N,N'-tetra-methylethylenediamine) were used to resolve proteins loaded adjacent to a Precision plus protein marker to assess electrophoretic conditions. Samples were resolved at 150 V for 90 min and transferred onto PVDF membrane at 20 V for 60 min. The membrane was blocked overnight in 1 × PBS containing 5% Marvel and 0.5% Tween. Membranes were washed for 3 × 15 min in 1 × PBS with 0.5% Tween. Blots were probed for ENaC subunits using the following primary antibodies: Raised in
Target
Concentration
Rabbit Goat Rat Rabbit
ENaC α (H-95 Santa Cruz) ENaC β (C-20 Santa Cruz) ENaC γ (y31A Alpha Diagnostics) Rho-GDI (Santa Cruz) — as internal control
1:500 1:1000 1:1000 1:1000
Na uptake assays were performed according to published protocols (Girardi et al., 2004). H295R cells grown in 24-well plates were pre-incubated for 20 min at room temperature in NH+ 4 loading buffer containing 30 mM NH4Cl, 90 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 20 mM HEPES–Tris, pH 7.4. The NH+ 4 loading buffer was then removed, and cells were incubated for 30 min at room temperature with an NH+ 4 -free solution containing 1 μCi/ml 22Na and 1 mM NaCl, 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 20 mM HEPES–Tris, pH 7.4. All test drugs were present during the 20-min preincubation period and the 30-min Na+ uptake period. Uptake was terminated by washing cells three times with ice-cold radionuclidefree NH+ 4 -free solution (pH 7.4). Cells were solubilized in 0.2 ml of RIPA buffer and aspirated into scintillation vials. 22Na uptake was measured with a Cobra™II Auto-Gamma counter (Perkin Elmer). Values were normalised to total cell protein. 2.7. Statistical analysis All data are presented as mean + SEM. Comparison between 2 groups is by 2-tailed t tests and between multiple groups by 2-way ANOVA with repeated measures. Statistical relationships not otherwise stated are to be assumed nonsignificant (P N 0.05). The authors had full access to and take responsibility for the integrity of the data. 2.8. Drugs All drugs and chemical components of solutions not otherwise stated were purchased from Sigma (St Louis, Missouri,USA). 3. Results 3.1. RT-PCR Expression of mRNA transcripts for α-, β- and γ-ENaC is shown in Fig. 1. All three transcripts are expressed in human kidney (positive
Blots were washed as previously and incubated for 45 min with a species-appropriate HRP-coupled secondary antibody at a concentration of 1:1500. After repeated washing blots were visualised using ECL detection (Pierce) and autoradiography film. 2.5. Measurements of aldosterone release by H295R cells Cells were incubated in 24-well plates. Drug additions (KCl, angiotensin II, benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA),
Fig. 1. RT-PCR showing expression of α-, β- and γ-ENaC in human kidney, adrenal and H295R cells. Bands for adrenal are shown from 2 subjects including normal cortex (2 left lanes) and Conn's adenoma (2 right lanes). A 100 bp ladder is shown for product size determination.
178
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
Fig. 2. RT-PCR showing expression of Nedd-4L, Sgk-1 the mineralocorticoid receptor (MR) and 11β-HSD type II in human kidney (Kid) normal adrenal cortex (Adr) and Conn's adenoma (Conn).
control), human adrenal and the H295R cell line. ENaC mRNA is present in both normal adrenal and Conn's adenoma tissue. We have demonstrated expression of ENaC mRNAs in adrenocortical tissue from 10 subjects, 8 with Conn's syndrome (expression shown in adenoma and normal cortex) and 2 with phaeochromocytomas. Data from two subjects is shown in Fig. 1. Expression of α- and β-ENaC is more consistent than for γ-ENaC. The mRNAs for other components of an aldosterone response pathway including the mineralocorticoid receptor, Nedd4L, Sgk-1 and 11β-HSD type II are also expressed in human adrenal tissue. Expression in normal adrenal, adenoma and kidney (positive control) is shown in Fig. 2. Data is shown for two subjects; we have demonstrated consistent expression of the mineralocorticoid receptor, Nedd4L, Sgk-1 and 11β-HSD type II in adenoma and normal adrenocortical tissue from the 10 subjects previously outlined.
Fig. 4. H295R cells in 24-well plates were treated with fludrocortisone (fludro), spironolactone (spiro) or potassium canrenoate at doses of 10− 8 M (− 8) or 10− 6 M (− 6) in the presence of different concentrations of angiotensin II (ang II). Control wells were exposed only to ang II and the appropriate solvent control. Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+ SEM) are given for 6 wells. Significant dose-dependent effects were seen with ang II (P b 0.001, t-test) but not with other drug treatments (P = NS, ANOVA).
3.2. Western blotting Western blotting demonstrates expression of α-, β- and γ-ENaC subunit proteins in human adrenal and human kidney (Fig. 3). α-ENaC appears as a doublet at 70 and 97 kDa whereas β- and γ-ENaC appear
Fig. 3. Western blot showing expression of α-, β- and γ-ENaC subunits in kidney (Kid) normal adrenal cortex (Adr) and Conn's adenoma (Conn). Rho-GDI was used as an internal control. Subunit sizes (in kDa), as determined by a Precision plus protein marker, are indicated by arrows.
Fig. 5. H295R cells were treated with different concentrations of EIPA or benzamil in the presence of varying concentrations of extracellular K+ (K = 4, 9 or 14 mM). Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+SEM) are given for 5 experiments. Benzamil had no effect (P = NS, ANOVA) on basal aldosterone release or aldosterone release evoked by a rise in extracellular K+ of 5 or 10 mM. EIPA at doses ≥10 μM inhibited K-stimulated aldosterone release (P b 0.01, ANOVA with Bonferroni correction).
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
179
as single bands at 80 kDa. This is consistent with the known molecular weights and variable glycosylation status of ENaC subunits (Canessa et al., 1994). Data is representative of Western blots from 5 subjects.
3.3. Aldosterone release by H295R cells We have investigated if the components of an aldosterone response pathway in H295R cells modulate aldosterone release. Fig. 4 shows the effects of the mineralocorticoid agonist fludrocortisone and mineralocorticoid antagonists, spironolactone and potassium canrenoate, on 24 h aldosterone release at different angiotensin II concentrations. Angiotensin II elicits a dose-dependent increase in aldosterone release (significant at 10− 10 M and maximal at 10− 6 M, P b 0.001, t-test). Addition of fludrocortisone, spironolactone or potassium canrenoate (at 10− 8 and 10− 6 M) had no effect on angiotensin II stimulated aldosterone release (P = NS, n = 6, ANOVA). K+-stimulated aldosterone release was similarly unaffected by the three mineralocorticoid receptor agonists/antagonists (data not shown). The effect of amiloride analogues on 24 h aldosterone release in response to K+ stimulation is shown in Fig. 5. Benzamil, a potent inhibitor of ENaC, had no effect (P = NS, n = 5, ANOVA) on basal aldosterone release or aldosterone release evoked by a rise in extracellular K+ of 5 or 10 mM. To corroborate previous work the effects of EIPA, an inhibitor of ENaC and Na+–H+ exchange (NHE), on K+ stimulated aldosterone release were assessed. At concentrations around its Ki for either ENaC or NHE, EIPA had no effect. EIPA at doses ≥10 μM inhibited K-stimulated aldosterone release (P b 0.01, n = 5, ANOVA with Bonferroni correction). Angiotensin-stimulated aldoster-
Fig. 7. H295R cells were treated with different concentrations of extracellular Na+ in the presence of 14 mM extracellular K+. Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+ SEM) are given for 5 experiments. As shown in Fig. 6, a reduction in extracellular Na+ concentration from 160 to 120 mM significantly enhanced aldosterone release (P b 0.01, ANOVA). This effect was abolished if osmotic changes were corrected for by replacement of Na+ with N-methyl-D-glucamine. EIPA (10− 5 M) significantly inhibited aldosterone release (P b 0.01, ANOVA), but did not block the response to osmolarity.
one release was also inhibited by high dose EIPA but not by benzamil (data not shown). The effects of extracellular Na+ concentration on basal and stimulated aldosterone release are shown in Fig. 6. A reduction in extracellular Na+ concentration from 160 to 120 mM significantly enhanced K+ and angiotensin II stimulated aldosterone release (P b 0.01, n = 5, ANOVA). Basal aldosterone levels were unaffected by changes in extracellular Na+ concentration over this range. By substituting Na+ with N-methyl-D-glucamine we have shown that the modulation of aldosterone release by extracellular Na+ is a consequence of changes in osmolarity (Fig. 7). Although EIPA at a dose of 10− 5 M significantly inhibits aldosterone release at all Na+ concentrations, it does not block the response to extracellular Na+/ osmolarity. The response to osmolarity is similarly unaffected by benzamil (data not shown).
3.4.
22
Na+ uptake by H295R cells
22
Na+ uptake into H295R cells in the presence of different concentrations of inhibitors is shown in Fig. 8. 30-min 22Na uptake was significantly inhibited by EIPA (IC50 of 0.1 μM) and amiloride (P b 0.01, n = 5, ANOVA). Benzamil was without effect at concentrations ≤10 μM (P = NS, n = 5, ANOVA).
Fig. 6. H295R cells were treated with different concentrations of extracellular Na+ in the presence of varying concentrations of extracellular K+ (K = 4, 9 or 14 mM) or angiotensin II (ang 10− 10, 10− 8 or 10− 6 M). Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+ SEM) are given for 5 experiments. A reduction in extracellular Na+ concentration from 160 to 120 mM significantly enhanced K+ and angiotensin II stimulated aldosterone release (P b 0.01, ANOVA). Basal aldosterone levels were unaffected by changes in extracellular Na+ concentration over this range.
Fig. 8. 30-min 22Na uptake (normalised for cell protein) was measured in H295R cells following incubation with different concentrations of amiloride, benzamil and EIPA. Mean values (+S.E.M.) are given for 5 experiments. Over the concentration range used, benzamil had no effect on 22Na uptake. EIPA significantly inhibited 22Na uptake with an IC50 of 0.1 μM (P b 0.01, ANOVA). Amiloride also produced significant inhibition (P b 0.01, ANOVA), although was less potent than EIPA.
180
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
4. Discussion This paper documents that the components of an aldosterone response pathway are present in human adrenal cortex at the mRNA level. Although a novel claim, it has previously been reported that mineralocorticoid receptor mRNA is present in H295R cells (Lesouhaitier et al., 2001). Here a role for the mineralocorticoid receptor in negative feedback regulation of aldosterone secretion was suggested by the finding that spironolactone increases pregnenolone secretion, an indirect measure of aldosterone synthesis. We show that ENaC subunit proteins are expressed in normal adrenal cortex and Conn's adenoma. Although we use the term ‘normal adrenal cortex’, the majority of adrenal tissue used in these experiments was obtained from Conn's patients in whom adenomas can develop on a background of micronodular hyperplasia (ChungPark et al., 2003). However the expression of ENaC in adrenocortical tissue from two patients with phaeochromocytoma supports the notion that ENaC is expressed in normal adrenal cortex. Currently we lack immunohistochemical data on the localisation of ENaC subunit proteins within adrenocortical cells. In other ENaCexpressing tissues, heterogeneous expression patterns have been noted for different subunits. For example, in rat kidney cortical collecting duct cells, α-ENaC is localised mainly in a zone in the apical domain, whereas β- and γ-ENaC are found throughout the cytoplasm (Hager et al., 2001). Antibodies used in immunohistochemistry to date have been raised against non-human ENaC. We will study intracellular localisation when specific human antisera become available. In H295R cells, pharmacological manipulation of an aldosterone response pathway does not influence aldosterone release. This immortalised cell line, established from an invasive primary adrenocortical carcinoma, is well characterised and displays many phenotypic characteristics of zona glomerulosa cells (Rainey et al., 1994). As previously demonstrated H295R cells increase aldosterone secretion in response to extracellular K+ and angiotensin II with maximal responses seen at supra-physiological concentrations (Rainey et al., 1994). We have shown that mineralocorticoid receptor agonists and antagonists do not influence basal, K+-stimulated or angiotensin IIstimulated aldosterone release in H295R cells over a 24 h period. In contrast, previous work has shown that 20 μM spironolactone increases basal and K+-stimulated 24 h pregnenolone release in H295R cells by ~4-fold (Lesouhaitier et al., 2001). Possible explanations are the very high spironolactone concentrations or measurement of different markers of corticosteroid biosynthesis. High doses of spironolactone (3–30 μM) have actions unrelated to mineralocorticoid receptor antagonism through, for example, binding to the Na+/K+ATPase (Sorrentino et al., 1996). We have used benzamil to investigate the effects of ENaC inhibition in view of its high affinity (Ki = 3 × 10− 8 M) and selectivity for ENaC over other Na+ transporters such as the Na+–H+ exchanger (Yu et al., 1993). This is important in that one isoform of the Na+–H+ exchanger (NHE-2) is abundant in adrenal tissue (Ghishan et al., 1995). It has previously been shown that the amiloride analogue EIPA, at doses from 10–100 μM, inhibits angiotensin II-stimulated aldosterone production in bovine glomerulosa cells (van der Bent et al., 1993). This effect was attributed to inhibition of Na+–H+ exchange, however such high concentrations are more likely to inhibit Na+–Ca2+ exchange pathways. We have corroborated this finding in H295R cells showing that over a similar dose range EIPA inhibits both K+stimulated and angiotensin II-stimulated aldosterone release. However, we have shown no effect of benzamil on aldosterone synthesis arguing against an important role for ENaC in the regulation of aldosterone production in H295R cells under the conditions studied. We considered the possibility that an adrenal aldosterone response pathway may be influenced by extracellular Na+ concentration. It has long been recognised that a rise in extracellular Na+ within the physiological range of plasma Na+ concentrations (130–150 mM)
suppresses aldosterone release, an effect most notable if steroidogenesis is maximised by high extracellular K+ (Balla et al., 1981; Enyedi and Spat, 1981). Our data confirm that in H295R cells, a reduction in Na+ concentration over this range increases K+-stimulated and angiotensin II-stimulated aldosterone release, but not basal release. We have shown, through Na+ substitution with N-methyl-D-glucamine that this is a direct consequence of osmotic change. Our findings are consistent with those in rat adrenal glomerulosa cells where alteration of osmolarity by sucrose addition in the 250–330 mosM range does not influence aldosterone production per se, but substantially affects K+-stimulated aldosterone production (Makara et al., 2000). At the time, the effect of hyposmosis was attributed to activation of voltage-gated Ca2+ channels. It now seems more likely to be due to the mechanosensitive properties of two-pore domain K+ channels which regulate adrenal aldosterone secretion (Davies et al., 2008). Experiments measuring 22Na+ uptake confirm that ENaC is not the major route of Na+ entry into H295R cells. Under low Na+ conditions, ENaC is inhibited by both benzamil and amiloride with an IC50 in the 0.01 μM range. Our finding that Na+ entry into H295R cells is inhibited by EIPA N amiloride N benzamil is more in keeping with a Na+–H+ exchange portal. In conclusion, our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. However, studies in H295R cells suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are now required to determine the importance of an adrenal aldosterone response pathway in primary cultures of adrenal cells. We speculate that an adrenal aldosterone-response pathway may mediate negative feedback of aldosterone on its own release, and that this pathway may be altered in some patients with hypertension. For example, patients with low renin hypertension have inappropriately high levels of aldosterone and the explanation for this is currently unclear (Wambach et al., 1984). Future studies in primary adrenal cells may clarify this enigma. References Albiston, A.L., Smith, R.E., Obeyesekere, V.R., Krozowski, Z.S., 1995. Cloning of the 11 beta HSD type II enzyme from human kidney. Endocr. Res. 21, 399–409. Balla, T., Nagy, K., Tarjan, E., Renczes, G., Spat, A., 1981. Effect of reduced extracellular sodium concentration on the function of adrenal zona glomerulosa: studies in conscious rats. J. Endocrinol. 89, 411–416. Bonvalet, J.P., 1998. Regulation of sodium transport by steroid hormones. Kidney Int. Suppl. 65, S49–56. Brouard, M., Casado, M., Djelidi, S., Barrandon, Y., Farman, N., 1999. Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation. J. Cell Sci. 112, 3343–3352. Bubien, J.K., Watson, B., Khan, M.A., Langloh, A.L., Fuller, C.M., Berdiev, B., et al., 2001. Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes. J. Biol. Chem. 276, 8557–8566. Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.D., Rossier, B.C., 1994. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367, 463–467. Chen, S.Y., Bhargava, A., Mastroberardino, L., Meijer, O.C., Wang, J., Buse, P., et al., 1999. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc. Natl. Acad. Sci. U. S. A. 96, 2514–2519. Chung-Park, M., Yang, J.T., McHenry, C.R., Khiyami, A., 2003. Adenomatoid tumor of the adrenal gland with micronodular adrenal cortical hyperplasia. Hum. Pathol. 34, 818–821. Davies, L.A., Hu, C., Guagliardo, N.A., Sen, N., Chen, X., Talley, E.M., Carey, R.M., Bayliss, D.A., Barrett, P.Q., 2008. TASK channel deletion in mice causes primary hyperaldosteronism. Proc. Natl. Acad. Sci. U. S. A. 105, 2203–2208. Enyedi, P., Spat, 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. Garty, H., Palmer, L.G., 1997. Epithelial sodium channels: function, structure, and regulation. Physiol. Rev. 77, 359–396. Ghishan, F.K., Knobel, S.M., Summar, M., 1995. Molecular cloning, sequencing, chromosomal localization, and tissue distribution of the human Na+/H+ exchanger (SLC9A2). Genomics 30, 25–30. Girardi, A.C., Knauf, F., Demuth, H.U., Aronson, P.S., 2004. Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells. Am. J. Physiol. Cell Physiol. 287, C1238–1245. Gormley, K., Dong, Y., Sagnella, G.A., 2003. Regulation of the epithelial sodium channel by accessory proteins. Biochem. J. 371, 1–14.
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181 Hager, H., Kwon, T.H., Vinnikova, A.K., Masilamani, S., Brooks, H.L., Frokiaer, J., et al., 2001. Immunocytochemical and immunoelectron microscopic localization of alpha-, beta-, and gamma-ENaC in rat kidney. Am. J. Physiol. Renal. Physiol. 280, F1093–1106. Henry, P.C., Kanelis, V., O'Brien, M.C., Kim, B., Gautschi, I., Forman-Kay, J., et al., 2003. Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel. J. Biol. Chem. 278, 20019–20028. Kizer, N., Guo, X.L., Hruska, K., 1997. Reconstitution of stretch-activated cation channels by expression of the alpha-subunit of the epithelial sodium channel cloned from osteoblasts. Proc. Natl. Acad. Sci. U. S. A. 94, 1013–1018. Lesouhaitier, O., Chiappe, A., Rossier, M.F., 2001. Aldosterone increases T-type calcium currents in human adrenocarcinoma (H295R) cells by inducing channel expression. Endocrinology 142, 4320–4330. Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B.C., et al., 2001. Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK. Am. J. Physiol. Renal. Physiol. 280, F675–682. Makara, J.K., Petheo, G.L., Toth, A., Spat, A., 2000. Effect of osmolarity on aldosterone production by rat adrenal glomerulosa cells. Endocrinology 141, 1705–1710.
181
Rainey, W.E., Bird, I.M., Mason, J.I., 1994. The NCI-H295 cell line: a pluripotent model for human adrenocortical studies. Mol. Cell Endocrinol. 100, 45–50. Sorrentino, R., Cirino, G., Calignano, A., Mancuso, F., Sorrentino, L., Andriuoli, G., Pinto, A., 1996. Increase in the basal tone of guinea pig thoracic aorta induced by ouabain is inhibited by spironolactone canrenone and potassium canrenoate. J. Cardiovasc. Pharmacol. 28, 519–525. van der Bent, V., Demole, C., Johnson, E.I., Rossier, M.F., Python, C.P., Vallotton, M.B., Capponi, A.M., 1993. Angiotensin-II induces changes in the cytosolic sodium concentration in bovine adrenal glomerulosa cells: involvement in the activation of aldosterone biosynthesis. Endocrinology 133, 1213–1220. Wambach, G., Meiners, U., Bonner, G., Konrads, A., Helber, A., 1984. Cardiovascular and adrenal sensitivity to angiotensin II in essential hypertension. Klin. Wochenschr. 62, 1097–1101. Yu, F.H., Shull, G.E., Orlowski, J., 1993. Functional properties of the rat Na/H exchanger NHE-2 isoform expressed in Na/H exchanger-deficient Chinese hamster ovary cells. J Biol Chem 268, 25536–25541.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Expression of the epithelial Na+ channel and other components of an aldosterone response pathway in human adrenocortical cells☆ Timothy J. Burton ⁎, Georgina Cope, Jing Wang, Joalice C. Sim, Elena A.B. Azizan, Kevin M. O'Shaughnessy, Morris J. Brown Clinical Pharmacology, University of Cambridge, Addenbrooke's Hospital, Box 110, Hills Road, Cambridge, CB2 2QQ, UK
a r t i c l e
i n f o
Article history: Received 27 November 2008 Received in revised form 19 March 2009 Accepted 1 April 2009 Available online 14 April 2009 Keywords: Epithelial Na+ channel Aldosterone Adrenal cortex
a b s t r a c t We have unexpectedly found expression of the epithelial Na+ channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na+ on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for α-, β- and γ-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11β hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting α-, β- and γ-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K+ and angiotensin II, suppression by both Na+ and high-concentration 5-(N-ethyl-N-isopropyl) amiloride (EIPA, blocker of Na+–H+ exchange) and no change with benzamil (ENaC blocker). 22Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Aldosterone stimulates Na+ reabsorption across epithelial cells by favourably changing the electrochemical gradients across epithelial cell membranes through the up-regulation of ion channels and transporters (Bonvalet, 1998). One key target is the amiloridesensitive epithelial Na+ channel (ENaC) that is located in the apical membrane of epithelial cells and forms the rate-limiting step in transepithelial Na+ transport (Garty and Palmer, 1997). ENaC is expressed primarily in Na+ absorbing epithelia such as the distal nephron, colon, sweat glands and airways (Garty and Palmer, 1997). However it is also expressed in non-epithelial cells such as osteoblasts, lymphocytes and hair follicles (Brouard et al., 1999; Bubien et al., 2001; Kizer et al., 1997). The regulation of ENaC activity by aldosterone is complex and tissue dependent involving changes in channel subunit synthesis, the number of channels expressed at the plasma membrane and channel open probability (Gormley et al., 2003). In many ENaCexpressing tissues Nedd4-2 (neuronal precursor cell-expressed and developmentally down-regulated protein 4-2) and Sgk-1 (serum and ☆ This work has not been presented previously. Sources of support: British Heart Foundation Programme Grant RG/02/007, MRC Studentship. ⁎ Corresponding author. Tel.: +44 1223 762577; fax: +44 1223 762576. E-mail address: [email protected] (T.J. Burton). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.04.005
glucocorticoid-inducible kinase type 1) have a pivotal role in determining the number of ENaC channels at the membrane and responsiveness to aldosterone (Chen et al., 1999; Henry et al., 2003). In distal tubular epithelial cells, for example, mineralocorticoid receptor activation induces Sgk-1 expression which in turn leads to phosphorylation and inhibition of Nedd4-2 thereby increasing surface expression of ENaC (Loffing et al., 2001). In this system the presence of 11β-hydroxysteroid dehydrogenase type II confers mineralocorticoid specificity by preventing activation of the mineralocorticoid receptor by cortisol (Albiston et al., 1995). We have unexpectedly found expression of the three subunits of ENaC in human adrenocortical cells. We therefore tested the hypothesis that these cells contain all the same components as the classical renal aldosterone response pathway. Secondarily, we have also tested the hypothesis that ENaC is an important route of Na+ uptake into adrenocortical cells and may modulate aldosterone secretion. 2. Material and methods 2.1. Human tissue collection Adrenal tissue was obtained from patients undergoing adrenalectomy for adrenal causes of hypertension (Conn's syndrome and
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
phaeochromocytoma). We obtained informed consent from each patient and local ethical approval. Tissue was dissected from histologically normal regions of adrenal cortex and from adjacent cortical tumour. 2.2. Culture of H295R cells NCI-H295R adrenocortical cells obtained from ATCC were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U penicillin, 0.1 mg/ml streptomycin 0.4 mM L-glutamine and ITS (insulin–transferrin– sodium selenite media) at 37 °C in 5% CO2. Cells were maintained at 37 °C in 5% CO2 and studied between passage numbers 5 and 10.
177
spironolactone, fludrocortisone and potassium canrenoate) were made when cell confluence equalled 50–70%. Drugs were dissolved in either DMEM or Kreb's solution. The volume of medium added per well was 0.5 ml. Kreb's solution (mM: NaHCO3 26, NaCl 83–153, KCl 4–14, CaCl2 1.1, MgSO4 1.3, NaH2PO4 1.4, glucose 10, N-methyl-Dglucamine chloride 0–70, pH = 7.4) was used in experiments designed to vary [Na+] and osmolarity. Media bathing the cells was harvested after 24 h. Aldosterone concentrations were determined by 125I radioimmunoassay using a commercially available Coat-A-Count kit (Diagnostic Products Corp, LA, CA, USA). Aldosterone concentrations were normalised to total cell protein, determined by extraction of protein with RIPA buffer and BCA™ protein assay (Pierce Biotechnology, Illinois, USA). 2.6. Measurements of Na+ uptake by H295R cells
2.3. RT-PCR
22
Total RNA was extracted from normal adrenal cortex, Conn's adenoma tissue and H295R cells using Fast Prep homogenisation and phenol:chloroform extraction with TRIzol reagent (Invitrogen™). Yield was determined spectrophotometrically and quality assessed by formamide agarose gel electrophoresis. RNA was converted into cDNA by first strand synthesis primed by random hexamers using Superscript III protocol (Invitrogen™). 1 ng of cDNA was used in each PCR reaction using specific primers (designed to span introns) against α-, β- and γ-ENaC, the mineralocorticoid receptor, Nedd4L, Sgk-1 and 11β-HSD type II. Annealing temperatures were optimized using an appropriate gradient based on melting temperatures. Resultant fragments were resolved on 3% agarose gels containing ethidium bromide and visualised by UV transillumination. Fragments produced were compared to a 100 bp ladder. 2.4. Western blotting Cells were lysed by addition of RIPA buffer (NaCl 150 mM, Tris base 50 mM, Triton X-100 1% (v/v), protease inhibitor cocktail, pH 7.4). Protein derived from the lysate was quantified and an equal volume of loading buffer (62.5 mM Tris–HCl, 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue, pH = 6.8) added. 20 μg protein aliquots were denatured for 10 min at 95 °C prior to loading on gels. Polyacrylamide gels (8% (w/v) acrylamide, 375 mM Tris–HCl (pH = 8.8), 0.1% (w/v) sodium dodecyl sulphate (SDS), 0.05% (w/v) ammonium persulphate and 0.05% (v/v) N,N,N,N'-tetra-methylethylenediamine) were used to resolve proteins loaded adjacent to a Precision plus protein marker to assess electrophoretic conditions. Samples were resolved at 150 V for 90 min and transferred onto PVDF membrane at 20 V for 60 min. The membrane was blocked overnight in 1 × PBS containing 5% Marvel and 0.5% Tween. Membranes were washed for 3 × 15 min in 1 × PBS with 0.5% Tween. Blots were probed for ENaC subunits using the following primary antibodies: Raised in
Target
Concentration
Rabbit Goat Rat Rabbit
ENaC α (H-95 Santa Cruz) ENaC β (C-20 Santa Cruz) ENaC γ (y31A Alpha Diagnostics) Rho-GDI (Santa Cruz) — as internal control
1:500 1:1000 1:1000 1:1000
Na uptake assays were performed according to published protocols (Girardi et al., 2004). H295R cells grown in 24-well plates were pre-incubated for 20 min at room temperature in NH+ 4 loading buffer containing 30 mM NH4Cl, 90 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 20 mM HEPES–Tris, pH 7.4. The NH+ 4 loading buffer was then removed, and cells were incubated for 30 min at room temperature with an NH+ 4 -free solution containing 1 μCi/ml 22Na and 1 mM NaCl, 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 20 mM HEPES–Tris, pH 7.4. All test drugs were present during the 20-min preincubation period and the 30-min Na+ uptake period. Uptake was terminated by washing cells three times with ice-cold radionuclidefree NH+ 4 -free solution (pH 7.4). Cells were solubilized in 0.2 ml of RIPA buffer and aspirated into scintillation vials. 22Na uptake was measured with a Cobra™II Auto-Gamma counter (Perkin Elmer). Values were normalised to total cell protein. 2.7. Statistical analysis All data are presented as mean + SEM. Comparison between 2 groups is by 2-tailed t tests and between multiple groups by 2-way ANOVA with repeated measures. Statistical relationships not otherwise stated are to be assumed nonsignificant (P N 0.05). The authors had full access to and take responsibility for the integrity of the data. 2.8. Drugs All drugs and chemical components of solutions not otherwise stated were purchased from Sigma (St Louis, Missouri,USA). 3. Results 3.1. RT-PCR Expression of mRNA transcripts for α-, β- and γ-ENaC is shown in Fig. 1. All three transcripts are expressed in human kidney (positive
Blots were washed as previously and incubated for 45 min with a species-appropriate HRP-coupled secondary antibody at a concentration of 1:1500. After repeated washing blots were visualised using ECL detection (Pierce) and autoradiography film. 2.5. Measurements of aldosterone release by H295R cells Cells were incubated in 24-well plates. Drug additions (KCl, angiotensin II, benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA),
Fig. 1. RT-PCR showing expression of α-, β- and γ-ENaC in human kidney, adrenal and H295R cells. Bands for adrenal are shown from 2 subjects including normal cortex (2 left lanes) and Conn's adenoma (2 right lanes). A 100 bp ladder is shown for product size determination.
178
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
Fig. 2. RT-PCR showing expression of Nedd-4L, Sgk-1 the mineralocorticoid receptor (MR) and 11β-HSD type II in human kidney (Kid) normal adrenal cortex (Adr) and Conn's adenoma (Conn).
control), human adrenal and the H295R cell line. ENaC mRNA is present in both normal adrenal and Conn's adenoma tissue. We have demonstrated expression of ENaC mRNAs in adrenocortical tissue from 10 subjects, 8 with Conn's syndrome (expression shown in adenoma and normal cortex) and 2 with phaeochromocytomas. Data from two subjects is shown in Fig. 1. Expression of α- and β-ENaC is more consistent than for γ-ENaC. The mRNAs for other components of an aldosterone response pathway including the mineralocorticoid receptor, Nedd4L, Sgk-1 and 11β-HSD type II are also expressed in human adrenal tissue. Expression in normal adrenal, adenoma and kidney (positive control) is shown in Fig. 2. Data is shown for two subjects; we have demonstrated consistent expression of the mineralocorticoid receptor, Nedd4L, Sgk-1 and 11β-HSD type II in adenoma and normal adrenocortical tissue from the 10 subjects previously outlined.
Fig. 4. H295R cells in 24-well plates were treated with fludrocortisone (fludro), spironolactone (spiro) or potassium canrenoate at doses of 10− 8 M (− 8) or 10− 6 M (− 6) in the presence of different concentrations of angiotensin II (ang II). Control wells were exposed only to ang II and the appropriate solvent control. Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+ SEM) are given for 6 wells. Significant dose-dependent effects were seen with ang II (P b 0.001, t-test) but not with other drug treatments (P = NS, ANOVA).
3.2. Western blotting Western blotting demonstrates expression of α-, β- and γ-ENaC subunit proteins in human adrenal and human kidney (Fig. 3). α-ENaC appears as a doublet at 70 and 97 kDa whereas β- and γ-ENaC appear
Fig. 3. Western blot showing expression of α-, β- and γ-ENaC subunits in kidney (Kid) normal adrenal cortex (Adr) and Conn's adenoma (Conn). Rho-GDI was used as an internal control. Subunit sizes (in kDa), as determined by a Precision plus protein marker, are indicated by arrows.
Fig. 5. H295R cells were treated with different concentrations of EIPA or benzamil in the presence of varying concentrations of extracellular K+ (K = 4, 9 or 14 mM). Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+SEM) are given for 5 experiments. Benzamil had no effect (P = NS, ANOVA) on basal aldosterone release or aldosterone release evoked by a rise in extracellular K+ of 5 or 10 mM. EIPA at doses ≥10 μM inhibited K-stimulated aldosterone release (P b 0.01, ANOVA with Bonferroni correction).
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
179
as single bands at 80 kDa. This is consistent with the known molecular weights and variable glycosylation status of ENaC subunits (Canessa et al., 1994). Data is representative of Western blots from 5 subjects.
3.3. Aldosterone release by H295R cells We have investigated if the components of an aldosterone response pathway in H295R cells modulate aldosterone release. Fig. 4 shows the effects of the mineralocorticoid agonist fludrocortisone and mineralocorticoid antagonists, spironolactone and potassium canrenoate, on 24 h aldosterone release at different angiotensin II concentrations. Angiotensin II elicits a dose-dependent increase in aldosterone release (significant at 10− 10 M and maximal at 10− 6 M, P b 0.001, t-test). Addition of fludrocortisone, spironolactone or potassium canrenoate (at 10− 8 and 10− 6 M) had no effect on angiotensin II stimulated aldosterone release (P = NS, n = 6, ANOVA). K+-stimulated aldosterone release was similarly unaffected by the three mineralocorticoid receptor agonists/antagonists (data not shown). The effect of amiloride analogues on 24 h aldosterone release in response to K+ stimulation is shown in Fig. 5. Benzamil, a potent inhibitor of ENaC, had no effect (P = NS, n = 5, ANOVA) on basal aldosterone release or aldosterone release evoked by a rise in extracellular K+ of 5 or 10 mM. To corroborate previous work the effects of EIPA, an inhibitor of ENaC and Na+–H+ exchange (NHE), on K+ stimulated aldosterone release were assessed. At concentrations around its Ki for either ENaC or NHE, EIPA had no effect. EIPA at doses ≥10 μM inhibited K-stimulated aldosterone release (P b 0.01, n = 5, ANOVA with Bonferroni correction). Angiotensin-stimulated aldoster-
Fig. 7. H295R cells were treated with different concentrations of extracellular Na+ in the presence of 14 mM extracellular K+. Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+ SEM) are given for 5 experiments. As shown in Fig. 6, a reduction in extracellular Na+ concentration from 160 to 120 mM significantly enhanced aldosterone release (P b 0.01, ANOVA). This effect was abolished if osmotic changes were corrected for by replacement of Na+ with N-methyl-D-glucamine. EIPA (10− 5 M) significantly inhibited aldosterone release (P b 0.01, ANOVA), but did not block the response to osmolarity.
one release was also inhibited by high dose EIPA but not by benzamil (data not shown). The effects of extracellular Na+ concentration on basal and stimulated aldosterone release are shown in Fig. 6. A reduction in extracellular Na+ concentration from 160 to 120 mM significantly enhanced K+ and angiotensin II stimulated aldosterone release (P b 0.01, n = 5, ANOVA). Basal aldosterone levels were unaffected by changes in extracellular Na+ concentration over this range. By substituting Na+ with N-methyl-D-glucamine we have shown that the modulation of aldosterone release by extracellular Na+ is a consequence of changes in osmolarity (Fig. 7). Although EIPA at a dose of 10− 5 M significantly inhibits aldosterone release at all Na+ concentrations, it does not block the response to extracellular Na+/ osmolarity. The response to osmolarity is similarly unaffected by benzamil (data not shown).
3.4.
22
Na+ uptake by H295R cells
22
Na+ uptake into H295R cells in the presence of different concentrations of inhibitors is shown in Fig. 8. 30-min 22Na uptake was significantly inhibited by EIPA (IC50 of 0.1 μM) and amiloride (P b 0.01, n = 5, ANOVA). Benzamil was without effect at concentrations ≤10 μM (P = NS, n = 5, ANOVA).
Fig. 6. H295R cells were treated with different concentrations of extracellular Na+ in the presence of varying concentrations of extracellular K+ (K = 4, 9 or 14 mM) or angiotensin II (ang 10− 10, 10− 8 or 10− 6 M). Aldosterone release was determined at 24 h by radio-immunoassay and values normalised for cell protein. Mean values (+ SEM) are given for 5 experiments. A reduction in extracellular Na+ concentration from 160 to 120 mM significantly enhanced K+ and angiotensin II stimulated aldosterone release (P b 0.01, ANOVA). Basal aldosterone levels were unaffected by changes in extracellular Na+ concentration over this range.
Fig. 8. 30-min 22Na uptake (normalised for cell protein) was measured in H295R cells following incubation with different concentrations of amiloride, benzamil and EIPA. Mean values (+S.E.M.) are given for 5 experiments. Over the concentration range used, benzamil had no effect on 22Na uptake. EIPA significantly inhibited 22Na uptake with an IC50 of 0.1 μM (P b 0.01, ANOVA). Amiloride also produced significant inhibition (P b 0.01, ANOVA), although was less potent than EIPA.
180
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181
4. Discussion This paper documents that the components of an aldosterone response pathway are present in human adrenal cortex at the mRNA level. Although a novel claim, it has previously been reported that mineralocorticoid receptor mRNA is present in H295R cells (Lesouhaitier et al., 2001). Here a role for the mineralocorticoid receptor in negative feedback regulation of aldosterone secretion was suggested by the finding that spironolactone increases pregnenolone secretion, an indirect measure of aldosterone synthesis. We show that ENaC subunit proteins are expressed in normal adrenal cortex and Conn's adenoma. Although we use the term ‘normal adrenal cortex’, the majority of adrenal tissue used in these experiments was obtained from Conn's patients in whom adenomas can develop on a background of micronodular hyperplasia (ChungPark et al., 2003). However the expression of ENaC in adrenocortical tissue from two patients with phaeochromocytoma supports the notion that ENaC is expressed in normal adrenal cortex. Currently we lack immunohistochemical data on the localisation of ENaC subunit proteins within adrenocortical cells. In other ENaCexpressing tissues, heterogeneous expression patterns have been noted for different subunits. For example, in rat kidney cortical collecting duct cells, α-ENaC is localised mainly in a zone in the apical domain, whereas β- and γ-ENaC are found throughout the cytoplasm (Hager et al., 2001). Antibodies used in immunohistochemistry to date have been raised against non-human ENaC. We will study intracellular localisation when specific human antisera become available. In H295R cells, pharmacological manipulation of an aldosterone response pathway does not influence aldosterone release. This immortalised cell line, established from an invasive primary adrenocortical carcinoma, is well characterised and displays many phenotypic characteristics of zona glomerulosa cells (Rainey et al., 1994). As previously demonstrated H295R cells increase aldosterone secretion in response to extracellular K+ and angiotensin II with maximal responses seen at supra-physiological concentrations (Rainey et al., 1994). We have shown that mineralocorticoid receptor agonists and antagonists do not influence basal, K+-stimulated or angiotensin IIstimulated aldosterone release in H295R cells over a 24 h period. In contrast, previous work has shown that 20 μM spironolactone increases basal and K+-stimulated 24 h pregnenolone release in H295R cells by ~4-fold (Lesouhaitier et al., 2001). Possible explanations are the very high spironolactone concentrations or measurement of different markers of corticosteroid biosynthesis. High doses of spironolactone (3–30 μM) have actions unrelated to mineralocorticoid receptor antagonism through, for example, binding to the Na+/K+ATPase (Sorrentino et al., 1996). We have used benzamil to investigate the effects of ENaC inhibition in view of its high affinity (Ki = 3 × 10− 8 M) and selectivity for ENaC over other Na+ transporters such as the Na+–H+ exchanger (Yu et al., 1993). This is important in that one isoform of the Na+–H+ exchanger (NHE-2) is abundant in adrenal tissue (Ghishan et al., 1995). It has previously been shown that the amiloride analogue EIPA, at doses from 10–100 μM, inhibits angiotensin II-stimulated aldosterone production in bovine glomerulosa cells (van der Bent et al., 1993). This effect was attributed to inhibition of Na+–H+ exchange, however such high concentrations are more likely to inhibit Na+–Ca2+ exchange pathways. We have corroborated this finding in H295R cells showing that over a similar dose range EIPA inhibits both K+stimulated and angiotensin II-stimulated aldosterone release. However, we have shown no effect of benzamil on aldosterone synthesis arguing against an important role for ENaC in the regulation of aldosterone production in H295R cells under the conditions studied. We considered the possibility that an adrenal aldosterone response pathway may be influenced by extracellular Na+ concentration. It has long been recognised that a rise in extracellular Na+ within the physiological range of plasma Na+ concentrations (130–150 mM)
suppresses aldosterone release, an effect most notable if steroidogenesis is maximised by high extracellular K+ (Balla et al., 1981; Enyedi and Spat, 1981). Our data confirm that in H295R cells, a reduction in Na+ concentration over this range increases K+-stimulated and angiotensin II-stimulated aldosterone release, but not basal release. We have shown, through Na+ substitution with N-methyl-D-glucamine that this is a direct consequence of osmotic change. Our findings are consistent with those in rat adrenal glomerulosa cells where alteration of osmolarity by sucrose addition in the 250–330 mosM range does not influence aldosterone production per se, but substantially affects K+-stimulated aldosterone production (Makara et al., 2000). At the time, the effect of hyposmosis was attributed to activation of voltage-gated Ca2+ channels. It now seems more likely to be due to the mechanosensitive properties of two-pore domain K+ channels which regulate adrenal aldosterone secretion (Davies et al., 2008). Experiments measuring 22Na+ uptake confirm that ENaC is not the major route of Na+ entry into H295R cells. Under low Na+ conditions, ENaC is inhibited by both benzamil and amiloride with an IC50 in the 0.01 μM range. Our finding that Na+ entry into H295R cells is inhibited by EIPA N amiloride N benzamil is more in keeping with a Na+–H+ exchange portal. In conclusion, our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. However, studies in H295R cells suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are now required to determine the importance of an adrenal aldosterone response pathway in primary cultures of adrenal cells. We speculate that an adrenal aldosterone-response pathway may mediate negative feedback of aldosterone on its own release, and that this pathway may be altered in some patients with hypertension. For example, patients with low renin hypertension have inappropriately high levels of aldosterone and the explanation for this is currently unclear (Wambach et al., 1984). Future studies in primary adrenal cells may clarify this enigma. References Albiston, A.L., Smith, R.E., Obeyesekere, V.R., Krozowski, Z.S., 1995. Cloning of the 11 beta HSD type II enzyme from human kidney. Endocr. Res. 21, 399–409. Balla, T., Nagy, K., Tarjan, E., Renczes, G., Spat, A., 1981. Effect of reduced extracellular sodium concentration on the function of adrenal zona glomerulosa: studies in conscious rats. J. Endocrinol. 89, 411–416. Bonvalet, J.P., 1998. Regulation of sodium transport by steroid hormones. Kidney Int. Suppl. 65, S49–56. Brouard, M., Casado, M., Djelidi, S., Barrandon, Y., Farman, N., 1999. Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation. J. Cell Sci. 112, 3343–3352. Bubien, J.K., Watson, B., Khan, M.A., Langloh, A.L., Fuller, C.M., Berdiev, B., et al., 2001. Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes. J. Biol. Chem. 276, 8557–8566. Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.D., Rossier, B.C., 1994. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367, 463–467. Chen, S.Y., Bhargava, A., Mastroberardino, L., Meijer, O.C., Wang, J., Buse, P., et al., 1999. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc. Natl. Acad. Sci. U. S. A. 96, 2514–2519. Chung-Park, M., Yang, J.T., McHenry, C.R., Khiyami, A., 2003. Adenomatoid tumor of the adrenal gland with micronodular adrenal cortical hyperplasia. Hum. Pathol. 34, 818–821. Davies, L.A., Hu, C., Guagliardo, N.A., Sen, N., Chen, X., Talley, E.M., Carey, R.M., Bayliss, D.A., Barrett, P.Q., 2008. TASK channel deletion in mice causes primary hyperaldosteronism. Proc. Natl. Acad. Sci. U. S. A. 105, 2203–2208. Enyedi, P., Spat, 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. Garty, H., Palmer, L.G., 1997. Epithelial sodium channels: function, structure, and regulation. Physiol. Rev. 77, 359–396. Ghishan, F.K., Knobel, S.M., Summar, M., 1995. Molecular cloning, sequencing, chromosomal localization, and tissue distribution of the human Na+/H+ exchanger (SLC9A2). Genomics 30, 25–30. Girardi, A.C., Knauf, F., Demuth, H.U., Aronson, P.S., 2004. Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells. Am. J. Physiol. Cell Physiol. 287, C1238–1245. Gormley, K., Dong, Y., Sagnella, G.A., 2003. Regulation of the epithelial sodium channel by accessory proteins. Biochem. J. 371, 1–14.
T.J. Burton et al. / European Journal of Pharmacology 613 (2009) 176–181 Hager, H., Kwon, T.H., Vinnikova, A.K., Masilamani, S., Brooks, H.L., Frokiaer, J., et al., 2001. Immunocytochemical and immunoelectron microscopic localization of alpha-, beta-, and gamma-ENaC in rat kidney. Am. J. Physiol. Renal. Physiol. 280, F1093–1106. Henry, P.C., Kanelis, V., O'Brien, M.C., Kim, B., Gautschi, I., Forman-Kay, J., et al., 2003. Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel. J. Biol. Chem. 278, 20019–20028. Kizer, N., Guo, X.L., Hruska, K., 1997. Reconstitution of stretch-activated cation channels by expression of the alpha-subunit of the epithelial sodium channel cloned from osteoblasts. Proc. Natl. Acad. Sci. U. S. A. 94, 1013–1018. Lesouhaitier, O., Chiappe, A., Rossier, M.F., 2001. Aldosterone increases T-type calcium currents in human adrenocarcinoma (H295R) cells by inducing channel expression. Endocrinology 142, 4320–4330. Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B.C., et al., 2001. Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK. Am. J. Physiol. Renal. Physiol. 280, F675–682. Makara, J.K., Petheo, G.L., Toth, A., Spat, A., 2000. Effect of osmolarity on aldosterone production by rat adrenal glomerulosa cells. Endocrinology 141, 1705–1710.
181
Rainey, W.E., Bird, I.M., Mason, J.I., 1994. The NCI-H295 cell line: a pluripotent model for human adrenocortical studies. Mol. Cell Endocrinol. 100, 45–50. Sorrentino, R., Cirino, G., Calignano, A., Mancuso, F., Sorrentino, L., Andriuoli, G., Pinto, A., 1996. Increase in the basal tone of guinea pig thoracic aorta induced by ouabain is inhibited by spironolactone canrenone and potassium canrenoate. J. Cardiovasc. Pharmacol. 28, 519–525. van der Bent, V., Demole, C., Johnson, E.I., Rossier, M.F., Python, C.P., Vallotton, M.B., Capponi, A.M., 1993. Angiotensin-II induces changes in the cytosolic sodium concentration in bovine adrenal glomerulosa cells: involvement in the activation of aldosterone biosynthesis. Endocrinology 133, 1213–1220. Wambach, G., Meiners, U., Bonner, G., Konrads, A., Helber, A., 1984. Cardiovascular and adrenal sensitivity to angiotensin II in essential hypertension. Klin. Wochenschr. 62, 1097–1101. Yu, F.H., Shull, G.E., Orlowski, J., 1993. Functional properties of the rat Na/H exchanger NHE-2 isoform expressed in Na/H exchanger-deficient Chinese hamster ovary cells. J Biol Chem 268, 25536–25541.