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Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis

2007; Springer Nature; Volume: 27; Issue: 1 Linguagem: Inglês

10.1038/sj.emboj.7601934

1460-2075

Dirk Heitzmann, Renaud Dérand, Stefan Jungbauer, Sascha Bandulik, Christina Sterner, Frank Schweda, Abeer El Wakil, Enzo Lalli, Nicolas Guy, Raymond Mengual, Markus Reichold, Ines Tegtmeier, Saı̈d Bendahhou, Celso E. Gómez-Sánchez, M. Isabel Aller, William Wisden, Achim Weber, Florian Lesage, Richard Warth, Jacques Barhanin,

Renin-Angiotensin System Studies

Article22 November 2007free access Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis Dirk Heitzmann Dirk Heitzmann Institute of Physiology, University of Regensburg, Regensburg, Germany Clinic and Policlinic for Internal Medicine II, University of Regensburg, Regensburg, Germany Search for more papers by this author Renaud Derand Renaud Derand Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Stefan Jungbauer Stefan Jungbauer Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Sascha Bandulik Sascha Bandulik Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Christina Sterner Christina Sterner Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Frank Schweda Frank Schweda Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Abeer El Wakil Abeer El Wakil Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Enzo Lalli Enzo Lalli Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Nicolas Guy Nicolas Guy Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Raymond Mengual Raymond Mengual Centre Hospitalo-Universitaire de Nice, Nice, France Search for more papers by this author Markus Reichold Markus Reichold Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Ines Tegtmeier Ines Tegtmeier Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Saïd Bendahhou Saïd Bendahhou Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Celso E Gomez-Sanchez Celso E Gomez-Sanchez Division of Endocrinology, GV Montgomery VA Medical Center, Jackson, MS, USA Search for more papers by this author M Isabel Aller M Isabel Aller Instituto de Neurociencias de Alicante, Consejo Superior de Investigaciones Cientificas-Universidad Miguel Hernández, San Juan de Alicante, Spain Search for more papers by this author William Wisden William Wisden Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Achim Weber Achim Weber Department of Pathology, University Hospital Zurich, Zurich, Switzerland Search for more papers by this author Florian Lesage Florian Lesage Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Richard Warth Corresponding Author Richard Warth Institute of Physiology, University of Regensburg, Regensburg, Germany These authors contributed equally to this work Search for more papers by this author Jacques Barhanin Jacques Barhanin Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France These authors contributed equally to this work Search for more papers by this author Dirk Heitzmann Dirk Heitzmann Institute of Physiology, University of Regensburg, Regensburg, Germany Clinic and Policlinic for Internal Medicine II, University of Regensburg, Regensburg, Germany Search for more papers by this author Renaud Derand Renaud Derand Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Stefan Jungbauer Stefan Jungbauer Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Sascha Bandulik Sascha Bandulik Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Christina Sterner Christina Sterner Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Frank Schweda Frank Schweda Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Abeer El Wakil Abeer El Wakil Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Enzo Lalli Enzo Lalli Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Nicolas Guy Nicolas Guy Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Raymond Mengual Raymond Mengual Centre Hospitalo-Universitaire de Nice, Nice, France Search for more papers by this author Markus Reichold Markus Reichold Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Ines Tegtmeier Ines Tegtmeier Institute of Physiology, University of Regensburg, Regensburg, Germany Search for more papers by this author Saïd Bendahhou Saïd Bendahhou Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Celso E Gomez-Sanchez Celso E Gomez-Sanchez Division of Endocrinology, GV Montgomery VA Medical Center, Jackson, MS, USA Search for more papers by this author M Isabel Aller M Isabel Aller Instituto de Neurociencias de Alicante, Consejo Superior de Investigaciones Cientificas-Universidad Miguel Hernández, San Juan de Alicante, Spain Search for more papers by this author William Wisden William Wisden Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK Search for more papers by this author Achim Weber Achim Weber Department of Pathology, University Hospital Zurich, Zurich, Switzerland Search for more papers by this author Florian Lesage Florian Lesage Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France Search for more papers by this author Richard Warth Corresponding Author Richard Warth Institute of Physiology, University of Regensburg, Regensburg, Germany These authors contributed equally to this work Search for more papers by this author Jacques Barhanin Jacques Barhanin Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France These authors contributed equally to this work Search for more papers by this author Author Information Dirk Heitzmann1,2, Renaud Derand3, Stefan Jungbauer1, Sascha Bandulik1, Christina Sterner1, Frank Schweda1, Abeer El Wakil3, Enzo Lalli3, Nicolas Guy3, Raymond Mengual4, Markus Reichold1, Ines Tegtmeier1, Saïd Bendahhou3, Celso E Gomez-Sanchez5, M Isabel Aller6, William Wisden7, Achim Weber8, Florian Lesage3, Richard Warth 1,9 and Jacques Barhanin3,9 1Institute of Physiology, University of Regensburg, Regensburg, Germany 2Clinic and Policlinic for Internal Medicine II, University of Regensburg, Regensburg, Germany 3Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice Sophia Antipolis, Valbonne Sophia Antipolis, France 4Centre Hospitalo-Universitaire de Nice, Nice, France 5Division of Endocrinology, GV Montgomery VA Medical Center, Jackson, MS, USA 6Instituto de Neurociencias de Alicante, Consejo Superior de Investigaciones Cientificas-Universidad Miguel Hernández, San Juan de Alicante, Spain 7Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK 8Department of Pathology, University Hospital Zurich, Zurich, Switzerland 9These authors contributed equally to this work *Corresponding author. Institute of Physiology, University of Regensburg, Universitaetsstrasse 31, NWF III—VKL, Regensburg 93053, Germany. Tel.: +49 941 943 2894; Fax: +49 941 943 2896; E-mail: [email protected] The EMBO Journal (2008)27:179-187https://doi.org/10.1038/sj.emboj.7601934 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1−/− mice to analyze the role of this channel in adrenal gland function. Task1−/− exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial ‘low-renin’ hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1−/− mice are the first demonstration of the causative role of a potassium channel in development/differentiation. Introduction The mineralocorticoid aldosterone is the major regulator of extracellular fluid and salt balance thereby controlling arterial blood pressure. The major target organ of aldosterone is the kidney, where it promotes Na+ retention and K+ secretion in the aldosterone-sensitive distal nephron. Dysregulation of aldosterone secretion leading to hyperaldosteronism is causal for about 3% of the cases of arterial hypertension (Nussberger, 2003). Moreover, aldosterone contributes to several aspects of cardiac fibrosis, cardiovascular dysfunction as well as progressive renal disease (Ibrahim and Hostetter, 2003; Remuzzi et al, 2005). The importance of aldosterone as clinical risk factor has been highlighted by recent clinical trials (Aldosterone Evaluation Study, (RALES); EPlerenone Heart failure and SUrvival Study (EPHESUS)) demonstrating the beneficial effects of mineralocorticoid receptor antagonists in patients with heart failure (Chai and Danser, 2006). The mechanisms regulating aldosterone secretion in glomerulosa cells of the adrenal cortex have been studied for already half a century. The most important physiological stimuli for aldosterone secretion are angiotensin II, high plasma K+, and, for a minor part, ACTH. Binding of angiotensin II to AT1 receptors stimulates phospholipase C, which in turn triggers InsP3-dependent Ca2+ release from intracellular stores. The immediate phase of aldosterone secretion is followed by sustained secretion, which is dependent on membrane depolarization and activation of T- and L-type Ca2+ channels. High plasmatic K+ concentrations stimulate exclusively the sustained phase of aldosterone secretion via influx of Ca2+ through voltage-dependent Ca2+ channels (Lotshaw, 2001). In this respect, the glomerulosa cell appears to be a unique sensor of extracellular K+ (Spat and Hunyady, 2004): increases of the extracellular K+ concentration by approximately 1 mmol/l are sufficient to double aldosterone secretion; maximal secretion occurs at 8 mmol/l of extracellular K+ (Lotshaw, 2001). The basis for this unique sensitivity for plasma potassium concentration is a very high background K+ conductance, which makes the membrane voltage strictly follow the K+ equilibrium potential: at low extracellular K+, the membrane voltage is hyperpolarized; upon relatively small increases in extracellular K+, membrane depolarization between −80 and −70 mV suffices to activate T-type Ca2+ channels in rat glomerulosa cells (Szabadkai et al, 1999; Lotshaw, 2001). The relative contributions of molecularly identified K+ channels to membrane depolarization and stimulation of aldosterone secretion have not been fully elucidated. Studies from genetically modified mice have pointed to a role of voltage-dependent KCNE1/KCNQ1 channels for the regulation of aldosterone secretion (Arrighi et al, 2001). Moreover, Ca2+-activated MaxiK channels have been proposed as K+ channels limiting aldosterone secretion (Sausbier et al, 2005). These K+ channels are not constitutively open at rest and therefore cannot underlie the background conductance necessary to confer the exquisite extracellular K+ sensitivity to glomerulosa cells. Electrophysiological recordings of glomerulosa cells have emphasized the importance for leak-type K+ channels of the 2P domain (K2P) family (Czirjak et al, 2000). The human K2P channel family comprises 15 different members, which are characterized by their typical structure of four transmembrane domains and a tandem of two pore-forming loops (Lesage and Lazdunski, 2000). These channels exhibit little voltage dependence, remain open at negative membrane potentials, and can be modulated by a variety of physical and chemical stimuli such as external pH, membrane stretch, protein kinase A- or protein kinase C-dependent phoshorylation, and PiP2 membrane depletion. Recently, task1 (KCNK3) and task3 (KCNK9), two K2P channels, were reported to be the dominant background channels in rat glomerulosa cells (Czirjak et al, 2000; Czirjak and Enyedi, 2002). In bovine glomerulosa cells, trek1, another K2P channel, is thought to be a major channel setting the membrane potential (Enyeart et al, 2004). The situation appears confusing mainly because the channels are poorly characterized beyond electrophysiological methods. However, a pivotal role of TASK1 is strongly suggested by the unusual high abundance of its messenger RNA in adrenal glands of several species including human and mouse (http://symatlas.gnf.org/SymAtlas/). In this study, we have investigated the specific contribution of task1 K+ channels to the control of aldosterone secretion using the task1 knockout (task1−/−) mouse as a model. We provide evidence that task1−/− mice have a strongly impaired mineralocorticoid homeostasis resulting in salt retention, arterial hypertension associated with low plasma renin activity. Surprisingly, this phenotype was restricted to the female gender. This pathology was caused by abnormal zonation of the adrenal cortex in female mice. In young male mice, the same zonation defect was observed, which however regressed after puberty. Age- and gender-dependent task3 expression might rescue the adrenal gland abnormalities. Results Plasma aldosterone, renin, and K+ levels On normal salt diet, adult task1−/− mice had a higher plasma aldosterone concentration than wild-type and heterozygous mice (task1+/+ 394±73 ng/l, n=52; task1+/− 235±44 ng/l, n=22; task1−/− 2361±452 ng/l, n=55). This hyperaldosteronism was restricted to the female gender as males displayed normal aldosterone values (Figure 1). To assess the response upon physiological regulators of aldosterone secretion, the salt intake was modified by varying the salt content of the diet. The response to high and low K+ intake as well as low Na+ was similar in heterozygous and wild-type mice of either sex. By contrast, female task1−/− mice exhibited a total loss of the physiological control of aldosterone secretion; plasma aldosterone concentration was neither stimulated by high K+ and low Na+ diet nor was it decreased by low K+ diet (Figure 1A). In female task1−/− mice, invariable high plasma concentrations of aldosterone were paralleled by reduced plasma renin activity (Figure 2A), which is indicative for primary hyperaldosteronism. Aldosterone stimulates K+ secretion and Na+ reabsorption in its target tissues. In female mice, plasma K+ concentration was reduced (Figure 2B); plasma Na+ concentration was not different between the genotypes. However, aldosterone-regulated Na+ transport through the epithelial Na+ channels (ENaC) was increased in the distal colon mucosa of female task1−/− mice (Figure 2C). Figure 1.Aldosterone levels in task1−/− mice. Plasma aldosterone levels of adult female (A) and male (B) mice on various salt diets are shown. In female task1+/+ (+/+) and task1+/− (+/−) mice, aldosterone concentrations are not different from each other under the three diet conditions (P=0.679), whereas they are considerably higher (P 0.1). The numbers of female mice per genotype were 11–27 (+/+), 13–15 (+/−), and 17–32 (−/−). Corresponding numbers for males were 10–27 (+/+), 3–9 (+/−), and 9–23 (−/−). NK: normal K+ (0.75%) diet; HK: high-K+ (3%) diet; LK: low-K+ (0.05%) diet; LNa: low-Na+ (<0.005%) diet. *Indicates statistically different from normal K+ diet (NK) or between the groups as indicated. NS: not significantly different. Download figure Download PowerPoint Figure 2.Primary hyperaldosteronism-associated symptoms under normal diet condition. (A) Female task1−/− mice showed a strongly decreased plasma renin activity, which was paralleled by reduced renin-specific immunostaining in female task1−/− kidney (right picture) compared with wild type (left picture) (n=20). In males, renin activities were similar for both genotypes (n=15). (B) Hypokalemia in female task1−/− mice (n=14); normokalemia in male mice (n=15). (C) Ussing chamber experiments of distal colonic mucosa. As a measure of ENaC-dependent Na+ reabsorption, amiloride-sensitive equivalent short circuit current (Isc) was increased in female task1−/− mice (left hand side; n=14). An original experiment showing the effect of luminal amiloride (10 μM) on transepithelial voltage (Vte) is depicted on the right hand side. *Indicates statistically different from task1+/+ mice. Download figure Download PowerPoint Blood pressure and heart rate Since enhanced salt retention in hyperaldosteronism can cause arterial hypertension, we assessed blood pressure by tail cuff measurements. Systolic blood pressure values of male task1−/− were not significantly different from those of male wild-type mice. In female task1−/− mice, however, systolic arterial blood pressure was 15 mmHg higher than in wild-type mice (Figure 3A). This increase in blood pressure was paralleled by a significant bradycardia, suggesting that the elevated blood pressure was not caused by a higher sympathetic nerve tone. In male mice, no difference in the heart rate was observed (Figure 3B). Figure 3.Hyperaldosteronism and hypertension in task1−/− mice. (A) In tail cuff measurements, female task1−/− mice displayed an increased systolic blood pressure compared with task1+/+ mice (n=20). Male task1−/− only tended to have a higher blood pressure (P=0.067; n=20). (B) Heart rate was lower in female task1−/− compared with task1+/+ mice (n=20). In male mice, the heart rate was not different between the genotypes (P=0.20; n=20). (C) Inhibition of the mineralocorticoid receptor by canrenoate normalized systolic blood pressure of female task1−/− mice (n=12, paired experiments). *Indicates statistically different. Download figure Download PowerPoint To further evaluate the contribution of increased aldosterone concentration in female task1−/− mice as cause for the arterial hypertension, the effect of the aldosterone receptor blocker canrenoate was tested in another set of experiments. Under control conditions, female task1−/− mice displayed higher systolic blood pressure values than wild-type mice. Next, canrenoate was added to the drinking water (canrenoate 1.25 g/l) for 10 days. From days 6–10, arterial blood pressure was determined. Canrenoate led to a decrease of systolic blood pressure in task1−/− but not in wild-type mice, thereby diminishing the difference in blood pressure between the genotypes (Figure 3C). These data confirm that the arterial hypertension of female task1−/− mice is caused by aldosterone-induced expansion of the extracellular volume. Localization of aldosterone-producing cells in the adrenal cortex The high aldosterone plasma concentrations of female task1−/− mice pointed to an increased secretion by aldosterone-producing cells in the zona glomerulosa of the adrenal cortex. Interestingly, immunofluorescence experiments using an antibody directed against the aldosterone synthase (CYP11B2) disclosed a sex-dependent effect of the task1 knockout on adrenocortical zonation. In wild-type mice of either sex, the aldosterone synthase-specific staining was localized exclusively in the glomerulosa cells of the outer layer of the adrenal cortex. In adrenal glands of female task1−/− mice, however, the zona glomerulosa was virtually absent and aldosterone synthase-positive cells were observed in deeper zones of the adrenal cortex (Figure 4, right upper panels). In contrast, adult male task1−/− mice did not show abnormal zonation patterns (Figure 4, right lower panels). High K+ diet is known to drastically increase aldosterone synthase expression in glomerulosa cells. Thus, we checked whether high K+ diet could unmask responsive glomerulosa-like cells in adrenal glands from female task1−/− mice. Aldosterone synthase-specific staining was strongly increased after K+-rich diet in female task1+/+ mice. In female task1−/− mice, no such increase could be observed (Figure 5A). Clearly, as observed for aldosterone itself, the aldosterone synthase expression is independent of the salt diet in female task1−/− mice. Western blot analysis of aldosterone synthase (Figure 5B) and real-time PCR experiments (data not shown) further supported this conclusion. Figure 4.Mislocalization of aldosterone synthase in adrenal glands of task1−/− mice is sex-dependent. In adult task1+/+ mice of either sex and in male task1−/− mice, aldosterone synthase staining is restricted to the zona glomerulosa cells of the outer adrenal cortex. In female task1−/− mice (right upper picture), however, the regular aldosterone synthase staining is disrupted and broadened to the inner parts of adrenal cortex. The right part of each panel shows higher magnification pictures of the adrenal cortex (aldosterone synthase in green, differential interference contrast in gray scale, nuclear staining with HOE33342 in blue). Download figure Download PowerPoint Figure 5.Effect of K+-rich diet on aldosterone synthase expression. (A) In female task1+/+ mice (left side), high-K+ diet strongly increased aldosterone synthase-specific staining in glomerulosa cells. In female task1−/− mice (right side), high-K+ diet did not change localization and abundance of aldosterone synthase. (B) Western blot analysis of aldosterone synthase (cyp11b2) protein abundance in adrenal glands at normal K+ diet (NK) and high-K+ diet (HK). The upper trace shows a typical blot (the bands of higher molecular weight are considered non-specific), and the lower trace shows the quantification of aldosterone synthase protein expression (normalized to beta-actin; task1+/+, n=8; task1−/−, n=8). *Indicates statistically different from normal K+ diet (NK). Download figure Download PowerPoint The mislocalization of aldosterone synthase in the zona fasciculata raised the question whether aldosterone secretion could be under the control of ACTH instead of angiotensin II. Dexamethasone (6 mg/kg, s.c.) was administered to female mice for 3 days to decrease plasma ACTH concentrations. This treatment virtually suppressed aldosterone in female task1+/+ and task1−/− mice (Figure 6). Therefore, hyperaldosteronism of female task1−/− mice was fully glucocorticoid-remediable. Figure 6.Glucocorticoid-remediable hyperaldosteronism in female task1−/− mice. Plasma aldosterone concentration has been measured in task1+/+ and task1−/− mice before (con) and after (dexa) administration of dexamethason (n=6). *Indicates statistically different from task1+/+ mice. Download figure Download PowerPoint Corticosterone synthesis in adrenal glands Next we investigated whether the replacement of the typical zona fasciculata by aldosterone-producing cells in female task1−/− mice was paralleled by an impairment of corticosterone synthesis. To this end, expression of the corticosterone-producing enzyme 11beta-hydroxylase (CYP11B1) in adrenal glands was determined by semiquantitative real-time PCR. No difference in 11beta-hydroxylase expression was observed between task1−/− and task1+/+, but female mice showed a threefold higher expression level than male mice (data not shown), which is in agreement with previously published data (Bielohuby et al, 2007). Additionally, plasma corticosterone concentration was similar in both genotypes (female task1+/+ 391±35 μg/l, n=20; female task1−/− 341±26 μg/l, n=41). Dynamics of adrenal cortex zonation The following series of experiments was aimed at elucidating possible mechanisms underlying the gender differences of adrenal cortex zonation in task1−/− mice. Obviously, mechanisms compensating for the absence of TASK1 were present in adult male task1−/− but absent in female task1−/− mice. Adrenal glands from mice of both sexes were compared before the age of puberty. Immunofluorescence of adrenal glands of mice at postnatal day 18 disclosed a normal zonation pattern of wild-type mice of either sex but abnormal zonation in male and female task1−/− mice (Figure 7A). Figure 7.Age and sex dependence of adrenocortical zonation. (A) In 18-day-old task1+/+ mice of either sex, aldosterone synthase-specific staining is restricted to glomerulosa cells of outer adrenal cortex (left pictures). In task1−/− mice of the same age, aberrant aldosterone synthase-specific staining is found in the mid-region of adrenal cortex (right pictures). The staining in the adrenal medulla is considered non-specific. (B) Effects of castration, estradiol (estr.), and testosterone (test.) treatment on aldosterone synthase localization. Male mice were castrated at the age of 5 weeks, followed by treatment with or without estradiol benzoate (4 μg/g/day, s.c., for 5 weeks). In male task1+/+, castration (with or without estradiol treatment) did not affect adrenocortical zonation. Sham-operated male task1−/− showed normal zonation, too (upper panel). In male task1−/−, castration prevented normal zonation. Estradiol appeared to reduce aldosterone synthase expression without having clear effects on zonation patterns. In female task1−/− mice, treatment with testosterone for 3 weeks induced normal zonation, highlighting the importance of androgens for adrenocortical rezonation in task1−/− mice (lower panel). Download figure Download PowerPoint To elucidate the possible role of sex hormones on adrenocortical zonation of task1−/− mice in more detail, aldosterone synthase localization was determined in male mice after castration. Castration was performed at the age of 5 weeks (when male task1−/− mice are not yet zonated) followed by an estrogen treatment. At the age of 10 weeks, castrated task1+/+ mice showed normal zonation irrespective of the treatment with or without estrogen (Figure 7B). Sham-operated task1−/− mice also showed normal zonation. However, castration prevented normal zonation in task1−/− mice. Treatment with estrogens did not enforce abnormal aldosterone synthase expression; if at all, high-dose estrogen treatment reduced aldosterone synthase expression without clearly restoring normal zonation pattern. These data suggested that the compensatory mechanisms allowing restoration of adrenocortical zonation in male task1−/− mice were androgen-dependent. To further evaluate this hypothesis, 4-week-old female task1−/− mice were treated with testosterone, and adrenocortical zonation was analyzed by aldosterone synthase-specific immunofluorescence. In fact, aldosterone synthase localization in female task1−/− mice was redirected to the zona glomerulosa after testosterone treatment (Figure 7B). Electrical properties of adrenocortical cells To evaluate the contribution of task1 channels for the whole-cell conductance, primary cultured adrenocortical cells of adult male task1−/− and task1+/+ mice were examined by the patch-clamp technique. Cells from task1−/− mice exhibited a more depolarized membrane voltage at resting conditions. In contrast to task1+/+ cells, task1−/− cells displayed a non-significant change of whole-cell conductance upon acid extracellular pH. However, the membrane voltage, which is more sensitive to small changes of K+ conductance, depolarized significantly at pH 6, suggesting the presence of other acid-sensitive K+ channels (e.g., homomeric task3 channels) in task1−/− cells (Figure 8A and B). Angiotensin II inhibited the whole-cell conductance and depolarized the membrane of task1+/+ and task1−/− cells. Interestingly, the inhibition of the whole-cell conductance by angiotensin II was more pronounced than by acidic extracellular pH, indicating that angiotensin II—besides TASK-like channels—inhibits acid-insensitive channels, too. The task1-related K+ channel task3 is also expressed in adrenal glands.