The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3.

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce ß-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life.

Human adrenal glomerulosa cells express K2P and GIRK potassium channels that are inhibited by ANG II and ACTH.

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

GRK2-Mediated Crosstalk Between ß-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein ßarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. ß-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the ßAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the ßAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol's effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the ß-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a ßAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

Role of Transcription Factor Oct4 in Postnatal Development and Function of the Adrenal Cortex.

We analyzed the expression of transcriptional factor Oct4 in rat adrenal cortical cells during postnatal development. It was found that Oct4 is expressed by typical cortical cells of the zona glomerulosa, zona fasciculata, and zona reticularis in pubertal and postpubertal periods. The maximum number of Oct4+ cells was found in the zona glomerulosa. An inverse correlation between the number of Oct4+ glomerulosa cells and serum level of aldosterone both in pubertal and postpubertal periods was revealed. After puberty, the number of Oct4+ glomerulosa cells directly correlated with the number of Ki-67+ cells. A hypothesis was put forward that Oct4 is involved in postnatal morphogenesis, regeneration, and functioning of the adrenal cortex.

Endogenous Purification of NR4A2 (Nurr1) Identified Poly(ADP-Ribose) Polymerase 1 as a Prime Coregulator in Human Adrenocortical H295R Cells.

Aldosterone is synthesized in zona glomerulosa of adrenal cortex in response to angiotensin II. This stimulation transcriptionally induces expression of a series of steroidogenic genes such as HSD3B and CYP11B2 via NR4A (nuclear receptor subfamily 4 group A) nuclear receptors and ATF (activating transcription factor) family transcription factors. Nurr1 belongs to the NR4A family and is regarded as an orphan nuclear receptor. The physiological significance of Nurr1 in aldosterone production in adrenal cortex has been well studied. However, coregulators supporting the Nurr1 function still remain elusive. In this study, we performed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), a recently developed endogenous coregulator purification method, in human adrenocortical H295R cells and identified PARP1 as one of the top Nurr1-interacting proteins. Nurr1-PARP1 interaction was verified by co-immunoprecipitation. In addition, both siRNA knockdown of PARP1 and treatment of AG14361, a specific PARP1 inhibitor suppressed the angiotensin II-mediated target gene induction in H295R cells. Furthermore, PARP1 inhibitor also suppressed the aldosterone secretion in response to the angiotensin II. Together, these results suggest PARP1 is a prime coregulator for Nurr1.

Decrease in Lipid Droplets in Adrenal Cortex of Male Wistar Rats after Chronic Exposure to Energy Drinks.

Background and objectives: Energy drinks are popular non-alcoholic beverages. They are consumed in large amounts, mainly by active, young people. Although they are easily accessible and marketed as safe, numerous cases of adverse effects have been published, including cardiac arrest, arrythmias, acute hepatitis, and renal failure. The aim of the current study is the assessment of energy drink influence on the histological structure of adrenal cortex in rats. Material and Methods: 15 male young Wistar rats were equally divided into three groups: control (C), experimental (E) and reversibility control (RC). C group received water and standard rodent food ad libitum while both E and RC groups had additionally unlimited access to energy drinks. C and E groups were decapitated after 8 weeks and RC was given another 8 weeks without energy drinks. Adrenal glands were embedded in paraffin blocks and 5 µm slides were prepared and stained according to standard H&E and Masson's trichrome protocols. Additionally, immunohistochemical stainings against Ki-67, p53, CTGF and caspase-3 were prepared. Results: Decreased vacuolization and numerous pyknotic nuclei were noted in E and RC groups. Overexpression of caspase-3 was noted both subcapsular in zona glomerulosa and along sinusoids in zona fasciculata. Increased collagen deposition in zona glomerulosa and zona fasciculata of E and RC was observed. Insular and irregular overexpression of CTGF was noted. The overall picture of CTGF expression matched the Masson's trichrome. No significant difference was observed in Ki-67 expression. Conclusions: The results of the current study suggest that the stimulation is so intense that it causes significant damage to adrenal cortical cells, resulting in their apoptosis. It seems, however, that the observed effects are at least partially reversible.

A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex.

This study aimed to elucidate the role of the AT(2) receptor (AT(2)R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGR; TGRCXmAT(2)R) that overexpresses the AT(2)R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta, and vein. As a consequence the total angiotensin II (Ang II) binding sites increased 7.8-fold in the kidney, 25-fold in the heart, and twofold in the adrenals. The AT(2)R number amounted to 82-98% of total Ang II binding sites. In the ZG of TGRCXmAT(2)R, the AT(2)R density was elevated threefold relative to wild-type (WT) littermates, whereas AT(1)R density remained unchanged. TGRCXmAT(2)R rats were viable and exhibited normal reproduction, blood pressure, and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24-h urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT(2)R at baseline. Three and 14 days of Ang II infusion (300 ng·min(-1)·kg(-1)) increased plasma aldosterone levels in WT and in TGR. These changes were completely abolished by the AT(1)R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67-positive glomerulosa cells, was stimulated by Ang II in TGR and WT rats; however, this increase was significantly attenuated in TGR overexpressing the AT(2)R. In conclusion, AT(2)R in the adrenal ZG inhibits Ang II-induced cell proliferation but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

An adrenal beta-arrestin 1-mediated signaling pathway underlies angiotensin II-induced aldosterone production in vitro and in vivo.

Aldosterone produces a multitude of effects in vivo, including promotion of postmyocardial infarction adverse cardiac remodeling and heart failure progression. It is produced and secreted by the adrenocortical zona glomerulosa (AZG) cells after angiotensin II (AngII) activation of AngII type 1 receptors (AT(1)Rs). Until now, the general consensus for AngII signaling to aldosterone production has been that it proceeds via activation of G(q/11)-proteins, to which the AT(1)R normally couples. Here, we describe a novel signaling pathway underlying this AT(1)R-dependent aldosterone production mediated by beta-arrestin-1 (betaarr1), a universal heptahelical receptor adapter/scaffolding protein. This pathway results in sustained ERK activation and subsequent up-regulation of steroidogenic acute regulatory protein, a steroid transport protein regulating aldosterone biosynthesis in AZG cells. Also, this betaarr1-mediated pathway appears capable of promoting aldosterone turnover independently of G protein activation, because treatment of AZG cells with SII, an AngII analog that induces betaarr, but not G protein coupling to the AT(1)R, recapitulates the effects of AngII on aldosterone production and secretion. In vivo, increased adrenal betaarr1 activity, by means of adrenal-targeted adenoviral-mediated gene delivery of a betaarr1 transgene, resulted in a marked elevation of circulating aldosterone levels in otherwise normal animals, suggesting that this adrenocortical betaarr1-mediated signaling pathway is operative, and promotes aldosterone production and secretion in vivo, as well. Thus, inhibition of adrenal betaarr1 activity on AT(1)Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperaldosteronism.

Biological sex modifies aldosterone's secretion at a cellular level.

Inconsistencies have been reported on the effect of sex on aldosterone (ALDO) levels leading to clinical confusion. The reasons for these inconsistencies are uncertain but include estrogen and/or its receptor modulating target gene responses to mineralocorticoid receptor activation and ALDO secretagogues' levels. This study's goal was to determine whether ALDO's biosynthesis also differed by sex. Two approaches were used. First, plasma renin activity and aldosterone were measured in rats. Both were significantly higher in males. Secondly, using rat zona glomerulosa (ZG) cells, we assessed three ex vivo areas: (1) activity/levels of early steps in ALDO's biosynthesis (StAR and CYP11A1); (2) activity/levels of a late step (CYP11B2); and (3) the status of the mineralocorticoid receptor (MR)-mediated, ultrashort feedback loop. Females had higher expression of CYP11A1 and StAR and increased CYP11A1 activity (increased pregnenolone/corticosterone levels) but did not differ in CYP11B2 expression or activity (ALDO levels). Activating the ZG's MR (thereby activating the ultrashort feedback loop) reduced CYP11B2's activity similarly in both sexes. Exvivo, these molecular effects were accompanied, in females, by lower ALDO basally but higher ALDO with angiotensin II stimulation. In conclusion, we documented that not only was there a sex-mediated difference in the activity of ALDO's biosynthesis but also these differences at the molecular level help explain the variable reports on ALDO's circulating levels. Basally, both in vivo and ex vivo, males had higher ALDO levels, likely secondary to higher ALDO secretagogue levels. However, in response to acute stimulation, ALDO levels are higher in females because of the greater levels and/or activity of their StAR/CYP11A1.

Lipopolysaccharide directly stimulates aldosterone production via toll-like receptor 2 and toll-like receptor 4 related PI(3)K/Akt pathway in rat adrenal zona glomerulosa cells.

The level of circulating endotoxin is related to the severity of cardiovascular disease. One of the indexes for the prognosis of cardiovascular disease is the plasma aldosterone level. Recently, the Toll-like receptors (TLRs), lipopolysaccharide (LPS)-regulated receptors, were found not only to mediate the inflammatory response but also to be important in the adrenal stress response. Whether LPS via TLRs induced aldosterone production in adrenal zona glomerulosa (ZG) cells was not clear. Our results suggest that LPS-induced aldosterone secretion in a time- and dose-dependent manner and via TLR2 and TLR4 signaling pathway. Administration of LPS can enhance steroidogenesis enzyme expression such as scavenger receptor-B1 (SR-B1), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage (P450scc) enzyme. LPS-induced SR-B1 and StAR protein expression are abolished by TLR2 blocker. Furthermore, we demonstrated that phosphorylation of Akt was elevated by LPS treatment and reduced by TLR2 blockers, TLR4 blockers, and LY294002 (PI(3)K inhibitor). Those inhibitors of PI(3)K/Akt pathways also abolish LPS-induced aldosterone secretion and SR-B1 protein level. In conclusion, LPS-induced aldosterone production and SR-B1 proteins expression are through the TLR2 and TLR4 related PI(3)K/Akt pathways in adrenal ZG cells.

ß-Catenin and FGFR2 regulate postnatal rosette-based adrenocortical morphogenesis.

Rosettes are widely used in epithelial morphogenesis during embryonic development and organogenesis. However, their role in postnatal development and adult tissue maintenance remains largely unknown. Here, we show zona glomerulosa cells in the adult adrenal cortex organize into rosettes through adherens junction-mediated constriction, and that rosette formation underlies the maturation of adrenal glomerular structure postnatally. Using genetic mouse models, we show loss of ß-catenin results in disrupted adherens junctions, reduced rosette number, and dysmorphic glomeruli, whereas ß-catenin stabilization leads to increased adherens junction abundance, more rosettes, and glomerular expansion. Furthermore, we uncover numerous known regulators of epithelial morphogenesis enriched in ß-catenin-stabilized adrenals. Among these genes, we show Fgfr2 is required for adrenal rosette formation by regulating adherens junction abundance and aggregation. Together, our data provide an example of rosette-mediated postnatal tissue morphogenesis and a framework for studying the role of rosettes in adult zona glomerulosa tissue maintenance and function.

Precerebellin-related genes and precerebellin 1 peptide in the adrenal gland of the rat: expression pattern, localization, developmental regulation and effects on corticosteroidogenesis.

Precerebellin (Cbln)-related peptides are known to modulate the secretory activity and growth of the adrenal gland. However, precise expression of the Cbln-related genes and Cbln1 peptide in the adrenal remains unclear. Therefore, we investigated, using RT-PCR, QPCR, Western blotting, immunohistochemistry and hormonal assays, their expression in the adrenals of adult rats and in the course of postnatal ontogenesis. Of the 4 known Cblns, Cbln(1-3) mRNAs were found in the adrenal gland of the adult male rats. Expression patterns of Cbln1 and 3 were similar to each other and different from that of Cbln2. Highest expression of the Cbln1 and 3 genes was observed in the zona glomerulosa (ZG), lower expression was noted in the fasciculata/reticularis and lowest expression was observed in the adrenal medulla. Expression of these genes was also present in freshly isolated rat adrenocortical cells. On the contrary, by means of classic RT-PCR, we demonstrated the presence of mRNAs of CBLN(1-4) in the human adrenal gland. In the rat, highest expression of the Cbln1 and 3 genes was found at postnatal day 2 and was somewhat lower at day 90. On the contrary, expression of the Cbln2 gene was low in adrenals of 2-day-old rats and notably higher at the remaining time points studied (up to day 360). Cerebellin (CER)-like immunoreactivity was observed in the membranes of the adrenal ZG cells, while in the medulla, immunoreactive substances were localized primarily in the cytoplasm of chromaffin cells. Cbln1-like immunoreactivity was present mainly in the cortex of the gland, and reaction products were noted both in the membranes and cytoplasm of adrenocortical cells. Semiquantitative evaluation of Cbln1 protein expression in compartments of the adrenal gland of the adult rat revealed a higher concentration of Cbln1 protein in the cortex than in the medulla of studied rats. We also found that both CER and desCER stimulated basal aldosterone secretion by freshly isolated ZG cells. Thus, the present study provided precise data on expression of the Cbln-related genes and Cbln1 peptide in the adrenal gland of the rat and on their developmental regulation. We also found that, contrary to the human adrenal, in the rat adrenal gland, the Cbln4 gene was not expressed at the mRNA level.

Neuromedin U enhances proliferation of ACTH-stimulated adrenocortical cells in the rat.

Neuromedin U (NMU) is a brain-gut peptide involved in the regulation of the hypothalamic-pituitary-adrenal axis and adrenocortical cell proliferation. In this study, we investigated the effects of NMU8 (three subcutaneous injections of 6.0 nmol/100 g, 24, 16 and 8 h before autopsy) on the adrenal glands of rats treated for 2 or 4 days with a low (2 microg/100 g body weight/24 h) or a high (8 microg) dose of adrenocorticotropic hormone (ACTH). As revealed by RT-PCR, ACTH treatment did not prevent expression of NMUR1 in rat adrenal cortex. At day 4 of ACTH administration, the weight of adrenals was lower than at day 2. NMU8 administration prevented ACTH-induced increases of adrenal weight at day 2 of the experiment. ACTH plasma concentrations were increased in all ACTH-administered rats. NMU8 administration increased ACTH plasma concentration at day 2 of the lower ACTH dose-treated group while it reduced the ACTH plasma level at day 4 in the higher ACTH dose-administered rats. In all groups of ACTH-treated rats, NMU8 changed neither aldosterone nor corticosterone plasma concentrations. In the zona glomerulosa (ZG), NMU8 increased metaphase index at days 2 and 4 in the lower ACTH dose-treated group and had no statistically significant effect in rats treated with the higher ACTH dose. In the zona fasciculata (ZF), NMU8 administration increased metaphase index at day 2 in the lower ACTH dose-treated group but reduced metaphase index at day 4 in the higher dose ACTH-administered rats. NMU8 reduced the number of cells per unit area both in ZG and ZF at day 2 in the higher ACTH dose-treated rats. In the remaining groups NMU8 did not produce statistically significant changes in the number of cells per unit area. Thus, our findings demonstrate that exogenous NMU may stimulate proliferation primarily of the cortical ZG cells in rats administered with ACTH, although at high doses of exogenous corticotropin an opposite effect occurred.

Histone demethylase LSD1 deficiency and biological sex: impact on blood pressure and aldosterone production.

Human risk allele carriers of lysine-specific demethylase 1 (LSD1) and LSD1-deficient mice have salt-sensitive hypertension for unclear reasons. We hypothesized that LSD1 deficiency causes dysregulation of aldosterone's response to salt intake resulting in increased cardiovascular risk factors (blood pressure and microalbumin). Furthermore, we determined the effect of biological sex on these potential abnormalities. To test our hypotheses, LSD1 male and female heterozygote-knockout (LSD1+/-) and WT mice were assigned to two age groups: 18 weeks and 36 weeks. Plasma aldosterone levels and aldosterone production from zona glomerulosa cells studied ex vivo were greater in both male and female LSD1+/- mice consuming a liberal salt diet as compared to WT mice consuming the same diet. However, salt-sensitive blood pressure elevation and increased microalbuminuria were only observed in male LSD1+/- mice. These data suggest that LSD1 interacts with aldosterone's secretory response to salt intake. Lack of LSD1 causes inappropriate aldosterone production on a liberal salt diet; males appear to be more sensitive to this aldosterone increase as males, but not females, develop salt sensitivity of blood pressure and increased microalbuminuria. The mechanism responsible for the cardiovascular protective effect in females is uncertain but may be related to estrogen modulating the effect of mineralocorticoid receptor activation.

Role of SCTR/AT1aR heteromer in mediating ANGII-induced aldosterone secretion.

The involvement of secretin (SCT) and its receptor (SCTR) in angiotensin II (ANGII)-mediated osmoregulation by forming SCTR/ angiotensin II type 1 receptor (AT1R) heteromer is well established. In this study, we demonstrated that SCTR/AT1R complex can mediate ANGII-induced aldosterone secretion/release through potentiating calcium mobilization. Through IHC and cAMP studies, we showed the presence of functional SCTR and AT1R in the primary zona glomerulosa (ZG) cells of C57BL/6N (C57), and functional AT1R and non-functional SCTR in SCTR knockout (SCTR-/-) mice. Calcium mobilization studies revealed the important role of SCTR on ANGII-mediated calcium mobilization in adrenal gland. The fluo4-AM loaded primary adrenal ZG cells from the C57 mice displayed a dose-dependent increase in intracellular calcium influx ([Ca2+]i) when exposed to ANGII but not from the SCTR-/- ZG cells. Synthetic SCTR transmembrane (TM) peptides STM-II/-IV were able to alter [Ca2+]i in C57 mice, but not the mice with mutated STM-II/-IV (STM-IIm/IVm) peptides. Through enzyme immunoassay (EIA), we measured the aldosterone release from primary ZG cells of both C57 and SCTR-/- mice by exposing them to ANGII (10nM). SCTR-/- ZG cells showed impaired ANGII-induced aldosterone secretion compared to the C57 mice. TM peptide, STM-II hindered the aldosterone secretion in ZG cells of C57 mice. These findings support the involvement of SCTR/AT1R heterodimer complex in aldosterone secretion/release through [Ca2+]i.

In adrenal glomerulosa cells, angiotensin II inhibits proliferation by interfering with fibronectin-integrin signaling.

Angiotensin II (Ang II), through the Ang II type 1 receptor subtype, inhibits basal proliferation of adrenal glomerulosa cells by inducing the disruption of actin stress fiber organization. This effect is observed in cells cultured on plastic or on fibronectin. The aim of the present study was to investigate how Ang II may interfere with extracellular matrix/integrin signaling. In cells treated for 3 d with echistatin (EC) (a snake-venom RGD-containing protein that abolishes fibronectin binding to alpha(5)beta(1) or alpha(v)beta(3) integrins), basal proliferation decreased by 38%, whereas Ang II was unable to abolish basal proliferation. In cells grown on fibronectin, Ang II decreased binding of paxillin to focal adhesions and, similarly to EC, induced a rapid dephosphorylation of paxillin (1 min), followed by an increase after 15 min. Fibronectin enhanced RhoA/B and Rac activation induced by Ang II, an effect abolished by EC. Under basal conditions, paxillin was more readily associated with RhoA/B than with Rac. Stimulation with Ang II induced a transient decrease in RhoA/B-associated paxillin (after 5 min), with a return to basal levels after 10 min, while increasing Rac-associated paxillin. Finally, results reveal that glomerulosa cells are able to synthesize and secrete fibronectin, a process by which cells can stimulate their own proliferative activity when cultured on plastic. Together, these results suggest that Ang II acts at the level of integrin-paxillin complexes to disrupt the well- developed microfilament network, a condition necessary for the inhibition of cell proliferation and initiation of steroidogenesis.

Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells.

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37 degrees C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca2+ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes.

Obligatory Metabolism of Angiotensin II to Angiotensin III for Zona Glomerulosa Cell-Mediated Relaxations of Bovine Adrenal Cortical Arteries.

Hyperaldosteronism is associated with hypertension, cardiac hypertrophy, and congestive heart failure. Steroidogenic factors facilitate aldosterone secretion by increasing adrenal blood flow. Angiotensin (Ang) II decreases adrenal vascular tone through release of zona glomerulosa (ZG) cell-derived vasodilatory eicosanoids. However, ZG cell-mediated relaxation of bovine adrenal cortical arteries to Ang II is not altered by angiotensin type 1 or 2 receptor antagonists. Because traditional Ang II receptors do not mediate these vasorelaxations to Ang II, we investigated the role of Ang II metabolites. Ang III was identified by liquid chromatography-mass spectrometry as the primary ZG cell metabolite of Ang II. Ang III stimulated ZG cell-mediated relaxation of adrenal arteries with greater potency than did Ang II. Furthermore, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by aminopeptidase inhibition, and Ang III-stimulated relaxations persisted. Ang IV had little effect compared with Ang II. Moreover, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by an Ang III antagonist but not by an Ang (1-7) antagonist. In contrast, Ang II and Ang III were equipotent in stimulating aldosterone secretion from ZG cells and were unaffected by aminopeptidase inhibition. Additionally, aspartyl and leucyl aminopeptidases, which convert Ang II to Ang III, are the primary peptidase expressed in ZG cells. This was confirmed by enzyme activity. These data indicate that intra-adrenal metabolism of Ang II to Ang III is required for ZG cell-mediated relaxations of adrenal arteries but not aldosterone secretion. These studies have defined an important role of Ang III in the adrenal gland.

Gene expression profile in rat adrenal zona glomerulosa cells stimulated with aldosterone secretagogues.

The mineralocorticoid aldosterone, mainly produced by the adrenal gland, is essential for life, but an abnormally excessive secretion causes severe pathological effects including hypertension and target organ injury in the heart and kidney. The aim of this study was to determine the gene regulatory network triggered by aldosterone secretagogues in a nontransformed cell system. Freshly isolated rat adrenal zona glomerulosa cells were stimulated with the two main aldosterone secretagogues, angiotensin II and potassium, for 2 h and subjected to whole genome expression studies using multiple biological and bioinformatics tools. Several genes were differentially expressed by ANG II (n = 133) or potassium (n = 216). Genes belonging to the nucleic acid binding and transcription factor activity categories were significantly enriched. A subset of the most regulated genes was confirmed by real-time RT-PCR, and then their expression was analyzed in time curve studies. Differentially expressed genes were grouped according to their time response expression pattern, and their promoter regions were analyzed for common regulatory transcription factor binding sites. Finally, data mining with gene promoters, transcription factors, and literature databases was performed to generate gene interaction networks for either ANG II or potassium. This paper provides for the first time a complete study of the genes that are regulated, and the interaction between them, by aldosterone secretagogues in rat adrenal cells. Increasing our knowledge of adrenal physiology and gene regulation in nontransformed cell systems could lead us to a better approach for the discovery of candidate genes involved in pathological conditions of the adrenal cortex.