HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (69) Citing Articles via Google Scholar Google Scholar Articles by SPÄT, A. Articles by HUNYADY, L. Search for Related Content PubMed PubMed Citation Articles by SPÄT, A. Articles by HUNYADY, L.

Control of Aldosterone Secretion: A Model for Convergence in Cellular Signaling Pathways

Department of Physiology and Laboratory of Cellular and Molecular Physiology, Semmelweis University and Hungarian Academy of Sciences, Budapest, Hungary

ABSTRACT

I. INTRODUCTION: MULTIPLE CONTROL OF ALDOSTERONE SECRETION—MULTIPLICITY OF SIGNAL TRANSDUCTION PATHWAYS IN THE GLOMERULOSA CELL

II. CALCIUM SIGNAL GENERATION IN GLOMERULOSA CELLS

A. Receptor-Mediated Ca2+ Release

1. ANG II receptors

2. G proteins

3. Phosphoinositide metabolism and formation of inositol phosphates

4. Ca2+ release by IP3

5. Actions of higher inositol phosphates and polyphosphoinositides

B. Ca2+ Transport Through the Plasma Membrane

1. Membrane potential and Ca2+ transport

2. Capacitative influx

3. Voltage-activated Ca2+ currents and Ca2+ channels

4. Action of ANG II on voltage-activated Ca2+ channels

5. DHP-sensitive Ca2+ channels and IP3-induced Ca2+ release

6. Ca2+ influx induced by K+

7. Na+/Ca2+ exchange

8. Ca2+-eliminating mechanisms

C. Diacylglycerol-PKC and Lipoxygenase Pathways

D. Vasopressin: A Transiently Acting Paracrine Agonist

III. EFFECT OF CYTOPLASMIC CALCIUM ON MITOCHONDRIAL FUNCTION

IV. CROSS-TALK BETWEEN CALCIUM AND cAMP-ACTIVATED PATHWAYS

A. Ca2+ Influx Evoked by ACTH

B. Ca2+-Induced Formation of cAMP

V. REGULATION AT THE LEVEL OF PLASMA MEMBRANE RECEPTORS

A. Mechanisms for Regulation of GPCRs

B. Regulation of Adrenal Angiotensin Receptors In Vivo

C. Desensitization of Glomerulosa Cells

D. Intracellular Trafficking of Angiotensin Receptors

E. Receptor Downregulation

VI. LONG-TERM EFFECTS OF ANGIOTENSIN II

A. Activation of Growth Responses by ANG II in the Glomerulosa Cell

B. Role of Receptor and Nonreceptor Tyrosine Kinases

C. ANG II-Induced Activation of MAPKs

VII. THE FINAL ACTION: ROLE OF CALCIUM IN THE CONTROL OF STEROID PRODUCTION

A. Cholesterol Transport

B. Increased Reduction of Pyridine Nucleotides in Mitochondria

C. Induction of Aldosterone Synthase

	ABSTRACT

Top Next References

	I. INTRODUCTION: MULTIPLE CONTROL OF ALDOSTERONE SECRETION—MULTIPLICITY OF SIGNAL TRANSDUCTION PATHWAYS IN THE GLOMERULOSA CELL

Top Previous Next References

 
The steroid hormone aldosterone, secreted by the glomerulosa cells of the adrenal cortex, controls sodium and potassium balance and also influences acid-base homeostasis of the vertebrate organism. Its major physiological targets are the epithelial cells, of which the most important are located in the distal nephron. It augments Na⁺ reabsorption as well as K⁺ and H⁺ excretion. Through changes in sodium balance, it influences the extracellular space and blood pressure. In addition to its epithelial actions, aldosterone influences the function of the cardiovascular system by acting on the heart, vessels, and central nervous system. Aldosterone secretion is increased during acute or chronic sodium depletion or fluid loss, erect postural position, dietary potassium loading, and tissue damage leading to hyperkalemia. In view of its essential role in maintaining extracellular fluid and thereby circulation, it is not surprising that its secretion is controlled by several factors. The list of hormonal and paracrine factors reported to exert a stimulatory effect on aldosterone production in vitro is quite long (Table 1), and the number of proposed inhibitory factors is also remarkable (Table 2). However, under physiological conditions the control of secretion is probably confined to the stimulatory factors corticotrophin (ACTH), angiotensin II (ANG II), and K⁺ and the inhibitory factor atrial natriuretic hormone (ANP). In fact, most or all increases in aldosterone secretion may be attributable to increased activity of the renin-angiotensin system and/or increased plasma level of K⁺. When sodium or fluid loss is severe, ACTH is also secreted and synergizes with ANG II or K⁺ in stimulating glomerulosa cells. ANP secretion is increased in response to sodium and/or water loading, and it in turn inhibits aldosterone secretion. The roles of other factors (shown in Tables 1 and 2) in the physiological control of aldosterone secretion may not be essential.

Signal transduction in the adrenal cortex has been studied for almost half a century. The role of cAMP in the stimulation of steroid production by pituitary trophic hormones was one of the earliest discoveries in this field, leading to the concept of second messengers (232). The formation and mode of action of cAMP have been described in several reviews. Our review focuses on the mechanism and role of Ca²⁺ signaling, paying special attention to the interaction of Ca²⁺ signaling with other signal-transducing mechanisms. The Ca²⁺ dependence of the secretory process was described by Douglas and Rubin four decades ago (142), and it is now well established that Ca²⁺ acts by inducing the exocytosis of secretory vesicles. This principle is not applicable for steroid-producing cells, which lack secretory vesicles and are devoid of an exocytotic mechanism. The steroid precursor cholesterol is stored in lipid droplets, and the rate of hormone secretion depends on the rate of hormone synthesis. However, the activation of hormone synthesis is Ca²⁺ dependent, and the regulatory mechanism involves both Ca²⁺-mediated and Ca²⁺-permitted processes.

Following the pioneering observation of Hokin and Hokin (241) of acetylcholine-induced increase in phospholipid turnover of pancreatic and brain cortical slices in 1955, systematic studies on the role of Ca²⁺ in agonist-induced biological response were initiated in 1975 by Michell's hallmark paper (355). After surveying data on the mode of action of several hormones and neurotransmitters, he hypothesized that Ca²⁺-dependent agonists activate phospholipase C and induce the hydrolysis of phosphatidylinositol and that this breakdown triggers, in turn, the influx of Ca²⁺ from the extracellular fluid. It was only later discovered that also the phosphatidylinositol derivative phosphatidylinositol 4,5-bisphosphate is cleaved (356). Subsequently, it was firmly established that the primary consequence of this breakdown is the formation of inositol 1,4,5-trisphosphate (IP₃), a water-soluble second messenger which primarily induces Ca²⁺ release from intracellular stores, rather than Ca²⁺ influx from the extracellular space (53, 437, 522).

Adrenal glomerulosa cells are a cell type in which Ca²⁺ and cAMP are equally significant in stimulation-secretion coupling. The effect of ACTH is mediated by cAMP, that of ANG II by Ca²⁺ and diacylglycerol (DAG), and that of K⁺ by Ca²⁺. ANP acts by antagonizing Ca²⁺ signaling. The basic framework of the IP₃-Ca²⁺-DAG system in adrenal glomerulosa cells was characterized in the 1980s and reviewed subsequently (32, 189, 503, 509). Therefore, classical data on the phosphoinositide-Ca²⁺-DAG system will only be reviewed concisely here. We will primarily deal with the control of Ca²⁺-releasing and influx mechanisms, the interaction of various signaling pathways, the long-term effects of Ca²⁺-mobilizing agonists, and the mechanism terminating Ca²⁺ signaling. Finally, we summarize current views on the basis of Ca²⁺-induced steroid secretion.

	II. CALCIUM SIGNAL GENERATION IN GLOMERULOSA CELLS

Top Previous Next References

 
The two most important physiological stimuli of aldosterone secretion, ANG II and extracellular K⁺ (reviewed in Ref. 570), exert their effects through generating cytoplasmic Ca²⁺ signal. The octapeptide ANG II is formed from the plasma protein angiotensinogen by the sequential action of two proteases, renin and angiotensin-converting enzyme. Under resting conditions its plasma concentration in humans and rats is between 10 and 60 pM (309, 492). ANG II level rises above 100 pM in response to administration of furosemide or dietary sodium depletion, whereas it may attain several hundred picomoles per liter following hemorrhage or water deprivation (337, 492). Plasma levels between 1 and 2 nM were measured under severe pathological conditions (e.g., malignant hypertension) (337).

Renin expression and ANG II production also occur in adrenal glomerulosa cells (371, 372). Within the adrenal cortex, immunolabeling for renin and prorenin is not limited to the steroid-producing cells of the zona glomerulosa but is also present in chromaffin cells, which occur singly or in groups throughout the whole cortex (45, 594). In this latter cell type the expression of angiotensinogen mRNA and the presence of ANG II in chromaffin granules were also shown (594), suggesting a paracrine action of ANG II released from vicinal chromaffin cells. The adrenal expression of renin mRNA is activated by the recognized physiological stimuli of aldosterone secretion, namely, ANG II (593), ACTH (576), and K⁺ (102, 281, 576). Transcription of the (pro)renin gene is also enhanced in dietary sodium depletion (566). In transgenic rats termed TGR(mREN2)27, the murine ren-2 renin gene, originally detected in the submaxillary gland, is expressed predominantly in the adrenal cortex. Although plasma renin and ANG II concentrations are low in these animals, aldosterone secretion is high, indicating that intra-adrenal production of renin can effectively stimulate the glomerulosa cells by stimulating local ANG II production (419). Sodium restriction also increases adrenal renin activity and mRNA in such transgenic rats (480). The observation that the AT₁-type ANG II receptor antagonist DuP 753 attenuates K⁺-stimulated and ACTH-stimulated aldosterone production (209) suggests that the intra-adrenal renin-angiotensin system functions as a local amplifier of systemic stimuli. Renin, in addition to acting as a soluble enzyme, has been found to bind to a 350-amino acid membrane protein. This binding results not only in increased catalytic activity, but also in the activation of mitogen-activated protein kinases (MAPKs) within the respective cell (385). No data are yet available as to whether this binding protein is expressed in the adrenal cortex. Additional studies are required to assess the tissue concentration of ANG II within the glomerulosa zone. Such data would be essential for the evaluation of the physiological relevance of experimental results obtained with exogenous ANG II.

ANG II-stimulated aldosterone production by incubated rat glomerulosa cells exhibits a biphasic dose-response curve. Steroidogenesis is stimulated in the physiological concentration range of 10^–11 M, maximal effect is attained at 10^–9 M, above which level hormone output is reduced (97, 230, 323). In superfusion system ANG II increases aldosterone production of isolated cells by two orders of magnitude (24, 447).

Aldosterone secretion in vivo (76) and production in vitro (74, 180, 238, 563) are stimulated by increases in K⁺ concentration as small as a few tenths of millimolar. Maximal hormone production is attained by [K⁺] around 8–10 mM both in capsular (glomerulosa) tissue (225, 368) and cell suspension experiments (323, 432), whereas decreased hormone production can be observed at [K⁺] between 10 and 20 mM (33, 442). The falling phase of the K⁺-aldosterone dose-response curve is due to the rapid decay of aldosterone output, which follows in time the steep onset of the response (28).

ANG II is a classical Ca²⁺-mobilizing ligand: it induces Ca²⁺ release from IP₃-sensitive intracellular stores, and this release is followed by Ca²⁺ influx from the extracellular space. The mode of action of K⁺ is quite different, because its primary site of action is a voltage-operated Ca²⁺ channel. Nevertheless, how the exceptional sensitivity of glomerulosa cell to K⁺ is achieved is a question still only partially elucidated. In the forthcoming sections we first describe the primary actions of the two agonists and later deal with additional actions that ensure the fine-tuning of regulation.

A. Receptor-Mediated Ca²⁺ Release

Glomerulosa cells do not constitute a homogeneous population. Expression of aldosterone synthase is confined to the two or three outer cell layers of the rat zona glomerulosa (221, 396, 418). The function of glomerulosa cells not expressing aldosterone synthase is not clear; moreover, presently no method is available for correlating aldosterone production and Ca²⁺ response at the single-cell level. Individual cells also differ in their response to different stimuli. Although the majority of the cells generate a Ca²⁺ signal in response to both K⁺ and ANG II, only a smaller percentage of the cells respond to vasopressin (AVP) (447) and still less to ACTH (551). Immunocell blot assay data suggest that 20–25% of the glomerulosa cells release ANG II (103).

This cellular heterogeneity is also reflected by the variable Ca²⁺ response of single cells to ANG II and K⁺ (119) (Fig. 1). However, some general characteristics of the response may be observed. Similar to other Ca²⁺-mobilizing agonists (47, 545), ANG II evokes an oscillating signal at physiological concentrations (usually below 300 pM). The hormone concentration affects the frequency rather than the amplitude of the single spikes. Gradual confluence of the spikes occurs at higher concentrations of the peptide, and the Ca²⁺ response becomes sustained in the nanomolar angiotensin II range (rat, Refs. 447–449, 466; bovine cells, Refs. 107, 269, 479). Also, the onset of the response exhibits a concentration-dependent delay (447, 466). Oscillatory Ca²⁺ signals show a gradual decrease in spiking frequency and/or amplitude after a few minutes (269, 433), which results in a gradually decreasing Ca²⁺ signal in cell suspension, where the response of 100,000 or more cells is averaged. (The underlying mechanism may also account for the declining [Ca²⁺] during the sustained phase of the Ca²⁺ signal in single cells exposed to higher concentration of ANG II.) Ca²⁺ spiking is maintained in the absence of extracellular Ca²⁺ for at least 20 min, indicating that oscillating Ca²⁺ release occurs from the IP₃-sensitive intracellular store (479). The frequency of Ca²⁺ oscillations is reduced by nifedipine, suggesting that voltage-activated Ca²⁺ channels have a supporting role during the process (479). Several models have been proposed for the mechanism of Ca²⁺ oscillation in other cell types (for review, see Refs. 178, 490), but none of these has been thoroughly tested for steroid-secreting cells.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Effect of angiotensin II (ANG II) applied at a physiological concentration of 150 or 300 pM (37°C) on cytoplasmic and mitochondrial [Ca2+]. Cytoplasmic [Ca2+] was monitored with Fura Red (red), and changes in mitochondrial [Ca2+] were followed with rhod 2 (blue) by confocal microscopy in MitoTracker Green-preloaded single glomerulosa cells. The y-axis shows the fluorescence ratio of (Fo-F)/Fo for Fura Red and (F-Fo)/Fo for rhod 2, where F is the actual fluorescence intensity and Fo is the averaged fluorescence intesity of the control period. Note the heterogeneity of the Ca2+ response of individual cells. (Recordings courtesy of A. Spät.)

When cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) is examined in cell suspension, i.e., a statistical average of several hundred thousand cells is monitored, the Ca²⁺ signal induced by low concentration of ANG II is characterized by a slow increase followed by a gradual decrease, whereas that induced by supraphysiological concentration consists of an initial peak followed by a smaller plateau (24, 255, 301). If extracellular Ca²⁺ is chelated in a superfusion system after the onset of ANG II-induced aldosterone production, hormone output falls below resting levels (503). This phenomenon indicates that the maintenance of [Ca²⁺]_c at a suprabasal level is essential for maintaining hypersecretion of aldosterone (269, 300, 435, 449).

1. ANG II receptors

ANG II is the major physiological regulator of aldosterone secretion, cell growth, and proliferation of glomerulosa cells. The effects of ANG II in glomerulosa cells and other target tissues are mediated by binding to heptahelical, G protein-coupled receptors. Two different binding sites of ANG II, termed AT₁ and AT₂, were identified in the rat adrenal cortex, and the AT₁ receptor was found to be the predominant isoform (17, 140, 579), whereas the bovine cortex contains almost exclusively AT₁ receptors (17). Immunocytochemical localization of the receptors in the adult rat confirmed that glomerulosa cells contain AT₁ receptors, whereas AT₂ is expressed in the medulla (177, 501). Nonpeptide receptor antagonists have demonstrated that AT₁ receptors mediate the ANG II-induced enhanced formation of IP₃ and depolarization (217), inhibition of adenylyl cyclase (17), stimulation of aldosterone production (17, 217), and the growth-promoting effect of ANG II in glomerulosa cells (see sect. VIA). However, AT₂ receptors appear to activate protein phosphatases, the nitric oxide-cGMP system, and phospholipase A₂ (390)_. AT₂ receptors inhibit cell growth and stimulate apoptosis, and the expression of this receptor is increased during fetal growth and tissue regeneration (132, 360). In contrast to other cloned mammalian AT₁ receptors, rat and mouse AT₁ receptors exist as two distinct subtypes, termed AT_1A and AT_1B. They are 95% identical in their amino acid sequences and have very similar ligand binding and activation properties, but differ in tissue distribution and transcriptional regulation. Although most ANG II target tissues express predominantly AT_1A receptor mRNA, in the adrenal and the pituitary glands the AT_1B message is the major subtype (132, 192). In contrast to pituitary cells, in rat and murine adrenal cells the mRNA of AT_1A receptors can also be detected (144, 192).

Competition curves of ANG II in radioligand-binding assays are characterized by a slope factor significantly lower than 1. These curves were originally explained by the presence of high- and low-affinity AT₁ receptor-binding sites. Depending on the experimental conditions in rat and bovine glomerulosa cells, the high-affinity sites have dissociation constant (K_d) values around or below 1 nM, whereas the affinity of the low-affinity site is 1 order of magnitude lower (73, 133). Both uncoupling of the receptor from G proteins, by nonhydrolyzable GTP analogs (see references in Ref. 132) and desensitization of the receptor, markedly reduces the number of high-affinity sites (see sect. VC). Although the two-state model is now generally considered to be an oversimplification of the receptor activation process (257), the effects of G protein coupling and/or receptor phosphorylation on binding affinity are considered to be general features of many G protein-coupled receptors (GPCRs).

2. G proteins

Catt and co-workers (194) described the inhibition of the binding of ANG II by guanine nucleotides as early as 1974. The underlying mechanism of this phenomenon was elucidated by the discovery of heterotrimeric G proteins 6 years later (464). Of the different heterotrimeric G proteins in glomerulosa cells, G_i, the inhibitory G protein of adenylyl cyclase, was the first to be identified on the basis of pertussis toxin-induced ADP-ribosylation of a 41-kDa protein (162). Immunoblotting revealed G_i (and the related G_o) in bovine glomerulosa cells (344). These observations were in harmony with the previous finding that ANG II reduces, rather than enhances, ACTH-induced and serotonin-induced cAMP production in rat glomerulosa cells (43, 230, 463) and adenylyl cyclase activity of membrane preparations (587). G_i seems to couple the activated ANG II receptor with T-type voltage-activated Ca²⁺ channels in bovine and L-type Ca²⁺ channels in both rat and bovine glomerulosa cells (see sect. IIB).

The primary effect of calcium-mobilizing agonists, such as ANG II, is the hydrolysis of phosphoinositides by a phosphoinositide-specific phospholipase C (PLC). Although -subunits derived from G_i/o may activate the ₂-isoform of PLC (454), inhibition of G_i by pertussis toxin failed to influence the ANG II-induced and AVP-induced activation of phosphoinositide metabolism (36, 162, 204, 296) or aldosterone production (230). Therefore, it may be assumed that the ₂-isoform of PLC is not expressed in the glomerulosa cell; however, nonhydrolyzable GTP analogs increased the formation or potentiated the ANG II-induced formation of IP₃ by permeabilized glomerulosa cells and glomerulosa membrane preparations (36, 162, 473). These observations indicated the involvement of another G protein in the action of ANG II. It was demonstrated in 1992 that the two pertussis toxin-insensitive G proteins, G_q and G₁₁ (the G_q family of heterotrimeric G proteins), mediate the effects of ANG II and AVP on PLC (210). Few data are available on G_q in adrenocortical cells. It was found that the G_q/G₁₁ protein is associated with the cytoskeleton and that this association is essential for the translocation of cytosolic G protein to the plasma membrane (121). As shown with the application of a specific antibody, _q/11 mediates the inhibitory effect of ANG II on Ca²⁺-dependent K⁺ (BK) channels (408).

The third heterotrimeric G protein participating in the control of steroid-producing cell types is G_s, the stimulator of adenylyl cyclase. The presence of _s, the guanyl nucleotide binding subunit of G_s, in glomerulosa cells was demonstrated by Western blot analysis. This also showed that a 1-min exposure to ACTH induced a large but transient translocation of _s from the cytoskeleton to the plasma membrane (122).

The site of action of heterotrimeric G proteins in the glomerulosa cell is summarized in Figure 2.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Site of action of heterotrimeric G proteins in the glomerulosa cell. Mcr2, melanocortin type 2 receptor. The red arrows indicate stimulatory actions, and the green arrows indicate inhibitory actions. (Inhibition of adenylyl cyclase by Gi may not be effective in bovine cells; see sect. IVB.)

3. Phosphoinositide metabolism and formation of inositol phosphates

Enhancement of phosphoinositide turnover by ANG II, but not by the two other physiological stimuli of the glomerulosa cell, K⁺ and ACTH, was described two decades ago (153, 245, 580). The effect of ANG II did not require extracellular Ca²⁺ (246), indicating that reduction of the radioactively labeled phosphatidylinositol pool was not secondary to ANG II-induced Ca²⁺ influx.

It was R. H. Michel who first presented data showing that agonist stimulation resulted in a rapid, phosphodiesterase-catalyzed breakdown of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate (PIP₂) that did not appear to be a response to changes in [Ca²⁺]_c (356). Subsequent work by Berridge, Irvine, Schulz, and co-workers (52, 522) definitely confirmed in 1983 that Ca²⁺-mobilizing agonists induce the breakdown of PIP₂, resulting in the formation of the water-soluble, Ca²⁺-releasing second messenger IP₃ and DAG. Shortly after this landmark discovery, the ANG II-induced rapid breakdown of PIP₂ (161, 172, 289) and the ensuing formation of IP₃ (18, 161, 188, 473) were observed in bovine and rat glomerulosa cells.

Whereas the ACTH-induced cAMP response does not evoke the formation of IP₃ (161, 172), vasopressin (22, 204, 589), endothelin (588), and acethylcholine (293), all acting in a paracrine mode within the adrenal gland, enhance the formation of IP₃. The hydrolyzing enzyme, the phosphoinositide-specific PLC, is activated not only by hormonal or neurotransmitter stimuli (through G_q), but also by Ca²⁺ (158, 366). The K⁺-induced moderate breakdown of PIP₂ (218) is compatible with such a Ca²⁺ action.

Although ANG II- (18, 161) and AVP-induced increases in IP₃ (204) peak within 10–15 s and decrease thereafter, a second phase of enhanced PIP₂ breakdown may last as long as stimulation is persisting. In the presence of lithium, an inhibitor of inositol polyphosphate 1-phosphatase and of inositol monophosphatase, labeled inositol phosphates, exhibit continuous accumulation, indicating the sustained activation of PLC (161, 289). In contrast to the initial formation of IP₃ (246, 473), the sustained phase of ANG II-stimulated IP₃ formation is dependent on Ca²⁺ influx (248, 590). This observation may be accounted for by the Ca²⁺ requirement of PLC activity (158, 375) and is consistent with the Ca²⁺ requirement of the sustained (but not the initial) phase of ANG II-stimulated aldosterone production (503).

The sustained phase of stimulation with ANG II is characterized not only by increased formation of inositol phosphates but also by a change in the ratio of different inositol phosphate fractions. Dephosphorylation by 5-phosphomonoesterase is the prevailing metabolic route of IP₃ in resting cells and is also a significant pathway in ANG II-stimulated cells (18). Another metabolic route is the sequential phosphorylation of IP₃ to inositol 1,3,4,5-tetrakisphosphate (IP₄) by IP₃ 3-kinase (262) and dephosphorylation to its biologically inactive product, inositol 1,3,4-trisphosphate. Ca²⁺ signaling may activate IP₃ -kinase (59), leading to increased formation of IP₄ and inositol 1,3,4-trisphosphate, as also observed in bovine glomerulosa cells (18, 23, 475). In the course of sustained stimulation, inositol 1,3,4-trisphosphate is consecutively converted into inositol 1,3,4,6-tetrakisphosphate (23, 250), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,4,5,6-tetrakisphosphate (25). An alternative pathway of the pentakisphosphate metabolism, which seems to be significant only after several hours of stimulation, is the formation of two further inositol tetrakisphosphate stereoisomers (1,4,5,6- and 3,4,5,6-P₄) (20).

Of these compounds, increased labeling of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate has been found in rat glomerulosa cells exposed to ANG II (19, 505) or AVP (204). The formation of an unspecified pentakisphosphate was also detected, and its labeling was not influenced by AVP within 30 min (204). The small number of glomerulosa cells that can be isolated from rat adrenal (a few hundred thousands per rat) has discouraged further physiologal studies on higher inositol polyphosphate metabolism in these cells.

4. Ca²⁺ release by IP₃

Before the availability of Ca²⁺-sensitive fluorescent dyes, Ca²⁺ release from an intracellular particle into the cytoplasm in response to hormones and neurotransmitters was revealed by the application of isotope flux techniques. Similar to the effect of acetylcholine on the salivary gland (387), ANG II was found to enhance Ca²⁺ efflux from rat glomerulosa cells, irrespective of the presence of extracellular Ca²⁺ (27). These observations indicated that these agonists induce intracellular Ca²⁺ release and that their primary action does not require Ca²⁺ influx. Moreover, in ANG II-stimulated glomerulosa cells the applied isotope flux technique revealed a nonmitochondrial origin of the released Ca²⁺ (27). IP₃-induced Ca²⁺ release from a nonmitochondrial intracellular store was first demonstrated in permeabilized pancreatic acinar cells, in 1983 (522). This observation was soon confirmed in permeabilized bovine glomerulosa cells (289) and in insulinoma microsomes (436). High-affinity IP₃-binding sites were described in 1986 by Spät and coworkers in permeabilized peritoneal polymorphonuclears and hepatocytes (507) and liver microsomes (510). The concentration dependence of binding and Ca²⁺ release were comparable, showing that the binding sites represented biologically active receptors. Whereas Scatchard analysis revealed both high-affinity and low-affinity binding compartments in liver microsomes (426, 510), only a single (high-affinity) compartment of binding sites was observed in adrenocortical microsomes (37, 94, 201, 403). It is possible that the lack of low-affinity binding sites in the adrenal cortex was caused by Ca²⁺-induced conversion of low-affinity to high-affinity binding sites (367, 427).

In glomerulosa cells, in harmony with other cell types, the K_d of IP₃ binding to the (high-affinity) receptor (10^–9 M) is about two orders of magnitude lower than the EC₅₀ of IP₃-induced Ca²⁺ release (201). This discrepancy may be accounted for by the different experimental conditions (temperature and absence or presence of Mg-ATP) of binding and transport studies. The redox state of thiol groups also influences the binding of the ligand (203). It has also been proposed that Ca²⁺ release by IP₃ in other cell types depends on the occupancy of low-affinity binding sites (426). It is possible that the IP₃ concentration in confined regions of stimulated cells might attain 10^–6 to 10^–5 M, which may induce maximal Ca²⁺ release also from low-affinity receptors (reviewed in Ref. 546).

Fractionation of liver homogenates (510) as well as electron microscopic studies on cerebellar Purkinje cells (486) confirmed the localization of the IP₃ receptor in the endoplasmic reticulum (ER). That IP₃ acted on the ER was supported later by several studies, among others on adrenocortical membrane fractions (94, 201, 403). Comparison of the effect of IP₃ and thapsigargin on glomerulosa cells indicated that only a subpopulation of ER vesicles can be regarded as IP₃ sensitive (159, 465). These data were in harmony with the results of subfractionation studies on liver that suggested that the receptor may not be evenly distributed in the whole reticulum but concentrated in specialized vesicles, marked with the calcium-binding protein calreticulin (166). In intact exocrine cells (421) and neurons (51), the ER appeared to be a continuous membrane system that has a number of regional specializations. Provided that also in intact glomerulosa cells ER is luminally continuous, IP₃ receptors may cluster in confined regions that could appear as a separate population of vesicles after homogenization.

IP₃ receptors have been identified in the plasma membrane of T lymphocytes (304). However, these receptors probably differ from the ER receptor, since activation of such nonselective cation channels in the plasma membrane would cause depolarization; that is not the case. IP₃ receptors in the plasma membrane were also reported in adrenocortical cells (474). Observations on adrenocortical cells (202) and liver homogenates (166, 471) strongly suggest that IP₃ receptor detected in the plasma membrane fraction is localized in ER vesicles, loosely attached to the plasma membrane by cytoskeletal elements. Additional IP₃ binding sites, described in the nuclear envelope and in the Golgi apparatus, as well as in secretory vesicles, (presently) have no known relevance in steroid-producing cells, and therefore we refer to a recent review (405).

The IP₃ receptor type cloned first (IP₃R1) contains 2,749 amino acid residues, corresponding to a molecular mass of 313 kDa. It consists of three functional domains, the cytosolic NH₂-terminal ligand-binding domain, a COOH-terminal channel-forming domain, and an interconnecting coupling domain. The COOH-terminal 550 amino acid residues contain six membrane-spanning regions, which form the ion-conducting pore. The receptor expressed in the brain may differ from that expressed in peripheral tissues by splice variance: the former containing, and the latter missing, a 40-amino acid segment (SII) in the coupling domain. IP₃ receptors and ryanodine receptors (RyR) share 40% amino acid sequence identity in their COOH-terminal, pore-forming domains and show some sequence identity in the NH₂-terminal, cytosolic ligand binding domains (for review, see Refs. 55, 542). Following the cloning and characterization of IP₃R1, two further types (IP₃R2 and IP₃R3) have been identified. The central nervous system contains almost exclusively IP₃R1 [SII(+) splice variant], while several peripheral cell types express more than one type of IP₃R (reviewed in Refs. 55, 405, 542). Simultaneous with the observation of the coexpression of more than one type of IP₃R in peripheral tissue, including the whole adrenal (containing both cortex and medulla) (384), we reported that rat glomerulosa cells express the mRNAs of all three types of IP₃R (165). About four times more IP₃R1 mRNA was expressed than IP₃R2 mRNA, and the SI segment was present in nearly 50% of the IP₃R1 receptors. Somewhat less than 5% of the total IP₃R was expressed as type 3. Interestingly, sodium restriction for 1 wk, a strong stimulus of aldosterone secretion by glomerulosa cells, failed to influence the ratio of expression of the different receptor types or spliced variants (165).

The receptor affinity for IP₃ has a rank order of IP₃R2 > IP₃R1 > IP₃R3 (364, 384). However, the membrane environment may modify the properties of the receptor (425). Different receptor types may exhibit different downregulation or rates of receptor degradation (498, 585). The receptor has a tetrameric structure of 1.2 kDa (for review, see Refs. 55, 174), and different receptor isoforms may constitute heterotetramers (272). Since the three receptor types differ in their affinity for IP₃ as well as in their sensitivity to controlling factors (see below), the diverse composition of these heterotetrameric receptors may provide more versatile control of IP₃ receptor function.

Physiological concentrations of Ca²⁺-mobilizing agonists usually evoke oscillatory Ca²⁺ signals (see sect. IIA). Although no uniformly valid model for different cell types has yet been presented, oscillations are obviously evoked by positive and negative feedback control of [Ca²⁺]_c by Ca²⁺ itself. Ca²⁺ variously activates different isoforms of PLC, protein kinase C (PKC), plasmalemmal Ca²⁺-ATPase, adenylyl cyclase, and cAMP phosphodiesterase, and cell type-specific expression of these isoforms contributes to the complexity of Ca²⁺ signal generation. Nevertheless, the major factor responsible for Ca²⁺ oscillations is probably the dual effect of Ca²⁺ on IP₃R itself. Elevation of [Ca²⁺]_c within the lower physiological range (up to 300 nM) increases, and further elevation, still in the physiological range, decreases the sensitivity of IP₃R1 to IP₃ (260). Gradual elevation of [IP₃] from 20 nM to 180 µM shifts the peak of the Ca²⁺-dependence curve of channel open probability from 100 nM to 1 µM (275). The biphasic effect of Ca²⁺ on channel activity is extremely important in determining the pattern of Ca²⁺ signaling. The Ca²⁺-dependent enhancement of IP₃R activity implies that Ca²⁺-induced Ca²⁺ release (CICR), similar to that occurring through RyRs, may also occur through the IP₃R provided that [Ca²⁺]_c is <300 nM (55). This process may have an essential role in the generation of cytoplasmic Ca²⁺ oscillations and waves (48). The dependence of the nadir of the bell-shaped Ca²⁺-dependence curve on [IP₃] ensures the coregulation of channel function by Ca²⁺ and IP₃, allowing the generation of short-lasting Ca²⁺ transients at low levels of IP₃, while maintaining high [Ca²⁺]_c during prolonged and intense stimulation of the cell. Ca²⁺ can directly facilitate IP₃-induced Ca²⁺ release (at Glu²¹⁰⁰ in IP₃R1) (56, 224, 365, 405). Calmodulin-dependent kinase II (CaMKII), on the other hand, phosphorylates the IP₃R and potentiates the Ca²⁺-releasing effect of IP₃, thus exerting positive feedback on IP₃R function (83, 598). High [Ca²⁺]_c may reduce IP₃ binding (510). This inhibition, which may be attributed to an increase in the K_d of IP₃ binding to the receptor (367), is independent of calmodulin. However, calmodulin probably exerts tonic inhibition on the IP₃R under physiological conditions (357, 389, 405, 406). In contrast to the bell-shaped Ca²⁺ sensitivity IP₃R1, peaking at 300 nM Ca²⁺, IP₃R2 displays a sigmoidal increase in open probability of the release channel up to 1 µM, and [Ca²⁺]_c exerts a moderate inhibitory effect only above 1 µM (452, but see Ref. 364). Observations on the Ca²⁺ dependence of IP₃R3 are conflicting, since increasing (212), unaltered (364), and decreasing open probability at high [Ca²⁺] were reported (333, 363). The predominant expression of IP₃R1 in the glomerulosa cell is compatible with the observed high affinity of IP₃ binding; moreover, the bell-shaped Ca²⁺ dependence of IP₃-induced Ca²⁺ release is to be expected.

Simultaneous knock-out of the three types of IP₃R in B lymphocytes abolishes agonist-induced Ca²⁺ signals. However, signal generation is maintained if any of the three isoforms can function (523). Nevertheless, when only a single isoform was expressed, different patterns of Ca²⁺ signals were observed. IP₃R2 was required for sustained Ca²⁺ oscillations, and IP₃R1 mediated less regular Ca²⁺ oscillations, while IP₃R3 generated monophasic Ca²⁺ transients (364).

The threshold and pattern of IP₃-induced Ca²⁺ signals are significantly modified by other signal transduction mechanisms. The receptor is phosphorylated in its coupling domain by protein kinase A (PKA), PKC, and CaMKII. The number, and even the site, of the phosphorylation consensus sites varies in the different receptor types (for review, see Refs. 405, 546). In the nonneural [SII(–)] splice variant of IP₃R1, that is predominantly expressed in glomerulosa cells (165), low cAMP concentrations preferentially phosphorylate Ser¹⁵⁸⁹ and therefore potentiate IP₃-induced Ca²⁺ release (80, 215). Substantial phosphorylation of the IP₃R with PKC, as well as augmentation of IP₃ potency in stimulating ⁴⁵ Ca release, were detected after pharmacological inhibition of the Ca²⁺/calmodulin-dependent phosphatase calcineurin, known to be anchored to IP₃R (83). Phosphorylation of IP₃R by PKC and dephosphorylation by calcineurin were reported also in bovine glomerulosa cells (430). The potentiation by PKC of IP₃ action at low [Ca²⁺]_c and the calcineurin-mediated inhibition of IP₃ action at higher [Ca²⁺]_c may contribute to the bell-shaped Ca²⁺-sensitive response to IP₃. However, in bovine glomerulosa cells a persistent high-conductance state was observed in the phosphorylated state of the release channel, suggesting that PKC-induced phosphorylation causes cessation of ANG II-induced Ca²⁺ oscillations (430). Tyrosine phosphorylation and cGMP-dependent phosphorylation of IP₃R were detected in stimulated T lymphocytes and vascular smooth muscle cells, respectively (reviewed in Ref. 405), but no data are available for glomerulosa cells.

ATP exerts a concentration-dependent effect on the function of IP₃R. Applied at micromolar concentrations, it enhances the binding of IP₃ (508, but see Ref. 583) and potentiates the open probability of the IP₃-gated Ca²⁺ channel (for review, see Refs. 55, 405). IP₃R1, the dominant receptor isoform in glomerulosa cells, exhibits greater affinity for ATP and greater ATP sensitivity of IP₃-induced Ca²⁺ release than IP₃R3 (331, 332). Competitive inhibition of the binding of IP₃ was observed with supramicromolar concentrations of ATP in several cell types (391, 512, 582), including adrenocortical cells (94, 474). This competition may be an additional and significant factor in the 10^–7 M EC₅₀ of IP₃-induced Ca²⁺ release (in ATP-containing media), as opposed to the 10^–9 M K_d of IP₃ binding. The question may be raised: how can the potentiating effect of low, and the inhibitory effect of high concentration of ATP on IP₃-induced Ca²⁺ release be reconciled? We assume that following Ca²⁺ release the activated Ca²⁺-ATPase would reduce ATP concentration around the ER. The ensuing disinhibition of IP₃R would facilitate the generation of Ca²⁺ signal.

When describing the control of the different types of IP₃R, emphasis was placed on that of IP₃R1, which is predominantly expressed in glomerulosa cells. IP₃R3 constitutes a minor fraction within the IP₃R pool (165), and its significance has yet to be studied. In this respect the Trp binding site (72) should be kept in mind, since it may be essential for capacitative Ca²⁺ influx (see sect. IIB).

Significant expression of IP₃R in rat adrenal fasciculata-reticularis cells (secreting the glucocorticoid corticosterone) could not be detected. The IP₃R mRNA content was <0.5 molecules/fasciculata cell, while it was 300 copies/glomerulosa cell (524). Since the rat fasciculata cell would be unique among nucleated cells in lacking IP₃R, this unexpected result certainly requires confirmation by other laboratories. Even so, it seems to account for data showing that ANG II is capable of increasing the formation of IP₃ in these cells (468, 581) but fails to evoke Ca²⁺ signals (77, 584) or increased production of corticosterone (581).

5. Actions of higher inositol phosphates and polyphosphoinositides

There is a notable contrast between the number of identified highly phosphorylated inositol phosphates and our knowledge of their role. There are sporadic observations on the effect of a given compound in a special cell type, but confirmation of these observations in other cell types is usually missing. The most studied compound is the first metabolite of IP₃, inositol 1,3,4,5-tetrakisphosphate. It potentiated the Ca²⁺-releasing effect of IP₃ in neuroblastoma (193) and L1210 lymphoma cells (126) as well as in pituitary microsomes (512). It was also found to synergize with IP₃ in inducing Ca²⁺ influx in sea urchin eggs (263) and in exocrine acinar cells (420). It was also proposed that capacitative Ca²⁺ influx is triggered by the interaction of IP₃Rs in the ER vesicle and the vicinal, plasmalemmal IP₄ receptors (261). Adrenocortical microsomes also contain high-affinity binding sites for IP₄ (168), but their function has not been examined. Anti-secretagogue (555) and hemodynamic effects of other inositol tetra- and pentakisphosphate analogs (556) have no relevance for our subject. Data on the modulation of chromatin-remodeling complexes by inositol polyphosphates (494) are not yet available for adrenocortical cells.

It was recognized only in recent years that PIP₂, in addition to being the precursor of the second messengers IP₃ and DAG, controls the function of several membrane-associated proteins. However, phosphoinositides also regulate protein targets through direct binding to specific phosphoinositide-binding domains (266). These domains include pleckstrin homology (PH) domains, Fab1P, YOTB, Vps27p, EEA1 (FYVE) domains, Phox homology (PX) domains and epsin NH₂-terminal homology (ENTH) domains, and other less well-defined lipid-binding structures.

The PH domain is a conserved region of 100–120 amino acids that was first identified in pleckstrin but also present in many other proteins. PH domains have a central role in the formation of molecular complexes involved in signaling and trafficking of receptors. These domains can be categorized into four groups, which preferentially bind phosphatidylinositol 3,4,5-P₃ [PI(3,4,5)P₃] (e.g., Bruton tyrosine kinase), PI(4,5)P₂ (e.g., PLC1, pleckstrin-N, spectrin), or PI(3,4)P₂ (e.g., Akt/PKB) or show no clear ligand specificity (e.g., dynamin) (see references in Ref. 266). The affinity of such domains for specific phosphoinositide ligands was sufficient to monitor ANG II-induced changes in membrane lipid composition using GFP-tagged PH domains (558, 561), and the lipid binding of the PH domain of dynamin is required for -arrestin-dependent AT₁ receptor endocytosis (see references in sect. VD). However, the latter effect cannot be explained by the role of the PH domain in the targeting of dynamin, since the lipid binding is not required for the proper targeting of dynamin to clathrin-coated structures (531). Therefore, the interaction with phosphoinositides can regulate the function or the submicroscopical orientation of PH domain-containing proteins.

FYVE domains bind PI(3)P and are present in proteins involved in the processing of endosomal vesicles (e.g., EEA1) and regulation of actin cytoskeleton (e.g., Fgd1). Inhibition of phosphatidylinositol (PI) 3-kinase has a marked effect on the trafficking of AT₁ receptors expressed in HEK 293 cells by interfering with the proper localization of FYVE domain-containing endosomal proteins (249). PX domains, which also bind 3-phosphoinositides, were first identified in two cytosolic components of NADPH oxidase (p47^phox and p40^phox), but they also occur in a variety of other proteins associated with signaling (e.g., class II PI 3-kinase, phospholipase D) and membrane trafficking (e.g., sorting nexins). Finally, the ENTH domain, which was first identified in epsin, is present in proteins that participate in endocytosis via clathrin-coated pits, and the intact phosphoinositide binding of this domain was shown to be required for clathrin-mediated endocytosis (see references in Ref. 266).

In addition to these well-defined protein folds, the lipid binding domains of plasmalemmal Ca²⁺ pumps, the Na⁺/Ca²⁺ exchanger, Na⁺/H⁺ exchangers, the RyR (for review, see Ref. 237) and the background K⁺ channel TASK (see sect. IIB) may also be relevant to the adrenocortical actions of ANG II. The role of lipid rafts in the plasma membrane in the colocalization of the receptor of the Ca²⁺-mobilizing hormone, PIP₂,G_q/11 and PLC, as well as the transport proteins, is currently being elucidated.

B. Ca²⁺ Transport Through the Plasma Membrane

ANG II-induced Ca²⁺ influx was one of the most contradictory subjects of aldosterone research. Species differences, inappropriately applied experimental techniques, and application of ANG II at concentrations one or two orders of magnitude higher than required for maximal aldosterone production have all delayed the recognition of the physiological role and mechanism of ANG II action.

Several early studies, applying the ⁴⁵Ca flux technique, failed to detect increased Ca²⁺ influx in response to ANG II. This failure may be due to neglecting the significance of measuring the initial rate of isotope uptake. Yet, this technique could reveal the ANG II-induced reduction of the exchangeable Ca²⁺ pool in bovine (107, 154, 289) and rat glomerulosa cells (27). The loss of cell Ca²⁺ is due to increased efflux of Ca²⁺, also detectable in a Ca²⁺-free medium (27, 157, 176, 301, 584). This Ca²⁺ efflux is due to the extrusion of Ca²⁺, released from the IP₃-sensitive store, by the Ca²⁺-activated plasmalemmal Ca²⁺-ATPase. The subsequently verified increase of Ca²⁺ influx in ANG II-stimulated cells is not in contradiction with the sustained efflux; together they perform continuous Ca²⁺ cycling across the plasma membrane. Although a high concentration of ANG II was applied in all the aforementioned studies, in a single report on the effect of ANG II at low concentration no significant change in the exchangeable Ca²⁺ pool could be revealed (107).

The frequently observed dependence of the ANG II-induced sustained Ca²⁺ signal on extracellular Ca²⁺ (89, 301, 467) has been regarded as evidence in favor of ANG II-induced Ca²⁺ influx. The successful demonstration of ANG II-induced increase in the initial influx rate of ⁴⁵Ca confirmed this interpretation in rat (95, 503) as well as bovine glomerulosa cells (159, 290, 301).

Calcium influx immediately following Ca²⁺ release is brought about by the depletion of IP₃-sensitive calcium stores and, subsequently, the slowly developing depolarization activates voltage-operated Ca²⁺ influx. Ca²⁺ is extruded from the cytoplasm by means of the plasmalemmal and ER Ca²⁺ pumps. In addition, Na⁺/Ca²⁺ exchange may modify [Ca²⁺]_c.

1. Membrane potential and Ca²⁺ transport

A major factor controlling Ca²⁺ transport through the plasma membrane is the membrane potential (E_m). Together with the concentration gradient it determines the driving force of Ca²⁺ transport and also regulates the activity of a set of Ca²⁺ channels. Considering that the two major physiological stimuli of aldosterone secretion, K⁺ and ANG II, depolarize the glomerulosa cell, we first discuss the control of E_m in these cells. E_m depends on the distribution of diffusible ions and their selective conductance accross the membrane. The Na⁺-K⁺ and Ca²⁺ pumps are responsible for the uneven distribution of the major cations. The greater the contribution of opened K⁺ channels to the total conductance of the cell membrane, the more the membrane potential approaches the equilibrium potential of K⁺ (E_K), which is in the range of 70–90 mV (inside negative) in excitable cells. Accordingly, a reduction of (the absolute value of) E_K following elevation of extracellular [K⁺] ([K⁺]_o), decrease of intracellular [K⁺], inhibition of Na⁺-K⁺-ATPase, or closure of K⁺ channels will depolarize the cell. This depolarization may, in turn, activate voltage-operated channels.

[K⁺]_o under resting conditions in the rat is 3.5–4 mM (75, 513). At such K⁺ levels E_m may exceed –80 mV, as measured with microelectrodes (445) or by patch-clamp technique (321, 324, 563). This highly negative E_m is due to a dominant K⁺ conductance (g_K), as confirmed by the perforated patch technique, which avoids the rundown of susceptible ionic currents. The linear relationship between log [K⁺]_o and E_m had a slope of 53.7 mV/10-fold change in [K⁺] (323) that almost attains the value predicted by the Nernst equation for a membrane with K⁺ conductance only (58 mV/decade). These findings are consistent with the lack of Na⁺ channel expression in the glomerulosa cell.

The primary step in the energy-dependent generation of membrane potential is the build-up of a transmembrane K⁺ concentration gradient by the Na⁺-K⁺-ATPase ("sodium pump"). Furthermore, the exchange of three cytoplasmic Na⁺ for two extracellular K⁺ also contributes to the generation of intracellular electronegativity ("pump potential"). Reducing the activity of the sodium pump dissipates the K⁺ gradient and results in depolarization. It was revealed a quarter of a century ago that ANG II decreased rather than increased the potassium content of rat glomerulosa cells (329, 526), suggesting that ANG II inhibits the pump. Although ANG II had no significant effect on ouabain-sensitive or potassium-stimulated ATPase activity in adrenal capsular membranes (139, 175), damage of the signal-transducing pathway in such membrane preparations cannot be ruled out. In fact, as shown in our laboratory (213), the uptake of ⁸⁶Rb, a surrogate for K⁺, is abolished by ouabain, the conventional inhibitor of the Na⁺-K⁺ pump. Half-maximal inhibition was attained at 80 µM, indicating that of the two forms of Na⁺-K⁺-ATPase present in rat tissues, the -isoenzyme is the predominant form in the rat glomerulosa cell. The isotope uptake is dependent on cytoplasmic [Na⁺], and it attains half-maximal rate at 10 mM, corresponding again to the -isoenzyme form. Maximal uptake rate is reached at 25 mM Na⁺, which corresponds to the measured physiological level of cytoplasmic Na⁺ (557) and decreases again above 50 mM. The transport activity is 4 and 20 times higher than that in fasciculata cells and hepatocytes, respectively. ANG II reduces the ouabain-sensitive ⁸⁶Rb uptake, i.e., the activity of Na⁺-K⁺-ATPase, and the observed IC₅₀ of ANG II (300 pM) suggests that the inhibitory effect of the peptide has physiological relevance.

ANG II exerts its inhibitory effect on Na⁺-K⁺-ATPase through AT₁ receptors (213). A recent observation suggests that ANG II inhibits the pump through activating a tyrosine phosphatase (596), whereas activation of the pump by Ca²⁺ through CaMKII (597) may serve as negative feedback during intense stimulation with ANG II. When ouabain is applied at submillimolar concentrations, the drug induces a continuous increase in [Ca²⁺]_c without any detectable lag time, and this increase can be abolished with nifedipine (213). At similar concentrations the drug also stimulates the production of aldosterone, again requiring extracellular Ca²⁺ and activable voltage-operated Ca²⁺ channels (213, 488, 527, 595). These data suggest that during ANG II-induced stimulation of aldosterone secretion, inhibition of Na⁺-K⁺-ATPase contributes to the depolarization and Ca²⁺ signal generation in glomerulosa cells.

In addition to Na⁺-K⁺-ATPase, K⁺ conductance is of great significance in setting E_m. Patch-clamp studies revealed inwardly rectifying, outwardly (delayed) rectifying, Ca²⁺-activated as well as background K⁺ current in glomerulosa cells. The ion channel through which the current is conducted has been identified in only a few cases. Of major concern is the estimation of the physiological significance of the observed currents/channels.

Only ion channels that are active in the range of the resting membrane potential may play a role in its fine setting. Members of the superfamily of inwardly rectifying K⁺ channels (386, 481) display a large inward current at potentials negative to E_K (occurring only under laboratory conditions) and a small and gradually diminishing outward current as E_m is shifted from E_K to more positive values. It is this latter K⁺ efflux by which E_m approaches the more negative E_K. Inwardly rectifying K⁺ current, with characteristics of Kir 2, has been observed in the membrane patch of rat and bovine glomerulosa cells (564). Kir 2 passes through the ubiquitous IRK channels and, in contrast to other families of inwardly rectifying channels, does not require G protein for activation and is insensitive to the ADP/ATP ratio. Nonetheless, the physiological significance of Kir 2 in glomerulosa cells has been questioned by the lack of inward rectification in macroscopic current under whole cell configuration (79, 321).

The previously mysterious background (or leak) K⁺ current, which, by definition, is also capable of permeating K⁺ in the range of resting E_m, would also be capable of generating highly negative E_m. Such a current was described in glomerulosa cells by Lotshaw in 1997 (322). Since that time the respective channel has been identified and termed TASK, an acronym for TWIK (tandem of P domains in a weak inward rectifying K⁺ channel)-like, acid-sensitive K⁺ channel (148). The expression of this two-pore domain K⁺ channel in rat glomerulosa cell has been demonstrated by Enyedi and co-workers in our laboratory (129). The zona glomerulosa exhibits the highest density of TASK mRNA among all tissues hitherto examined. In Xenopus oocytes injected with glomerulosa mRNA, the expressed K⁺ current had a pharmacology comparable with that of the authentic TASK current (129). More recently, the mRNA of the subsequently described TASK-3 (284, 451) was also found in rat glomerulosa cells (128). Interestingly, the glomerulosa cell is exclusive among several examined cell types in expressing TASK-3. TASK-1 and TASK-3 are coexpressed (the latter in larger amount) and form heterodimers. A fine-tuning of channel control may be achieved by the formation of heterodimers by the two subtypes (127).

Several K⁺ currents or channels, active in depolarized cells only, have also been described in the glomerulosa cell. Noninactivating delayed rectifier K⁺ current (inducing K⁺ efflux from depolarized cells) was described in rat, bovine, and human glomerulosa cells (79, 407, 409, 564). This current probably corresponds to KvLQT1, the slow component of the delayed outward K⁺ current (I_Ks) in the heart. It is conducted by the channel KCNQ1, the regulatory subunit of which (KCNE1) has been detected in glomerulosa cells (13). In view of the voltage dependence of their activation, the delayed rectifier channels may not modify the resting E_m, but their activation could limit the extent of depolarization by allowing the efflux of K⁺. In harmony with this prediction, knock-out of the KCNE1 gene resulted in enhanced responsiveness of the plasma aldosterone concentration to K⁺ loading (13).

Transient outward (A-type) K⁺ current was observed in rat, bovine, and human glomerulosa cells (407, 409, 564). Ca²⁺-dependent maxi-K⁺ (BK) channels were also described in the rat (321, 408, 564). In view of the voltage dependence of their activation, the A-type and the BK channels may not modify the resting E_m. On the other hand, ANG II, probably acting through G_q/11, inhibits the Ca²⁺-dependent K⁺ channel (408), an action that could augment the depolarization triggered by some other mechanism. ANP, probably acting via cGMP, increases K⁺ conductance by facilitating the function of BK channels (191). The ensuing hyperpolarization may represent one of the means by which the peptide antagonizes the secretagogue action of Ca²⁺-mobilizing hormones.

Any agent that decreases K⁺ conductance evokes the shift of E_m from the strongly negative E_K towards the positive equilibrium potential of Na⁺ and Ca²⁺. The first evidence that ANG II induces membrane depolarization was obtained with intracellular microelectrode recording of cat adrenocortical slices as early as the 1970s (383). Later studies provided evidence that a significant component of this depolarization is caused by the reduction of g_K. In microelectrode studies a biphasic E_m response to ANG II was observed in rat glomerulosa cells (446). A brief hyperpolarization was followed by a long-lasting depolarization, characterized by a large decrease in cell conductance. The reversal potential of the response suggested that E_m followed changes in g_K. Subsequent ⁸⁶Rb flux measurements confirmed that ANG II inhibits g_K in rat glomerulosa cells (217). The primary site of action of ANG II is the AT₁ receptor, and its effect is mimicked neither by ionomycin-induced elevation of [Ca²⁺]_c nor by pharmacological activation of PKC, suggesting that inhibition of K⁺ conductance by ANG II is a membrane-delimited process. An initial increase, followed by a prolonged decrease in g_K, was observed by means of ⁸⁶Rb flux studies also in bovine glomerulosa cells. [Surprisingly, the decrease in g_K was found to be Ca²⁺ dependent (320).] The significance of the ANG II-induced decrease in K⁺ conductance is shown by the observation that the K⁺ ionophore valinomycin significantly reduced ANG II-stimulated aldosterone production (495).

Subsequently, in the patch-clamp era, ANG II was found to inhibit each type of the hitherto described K⁺ channels. However, only inhibition of channels active at resting E_m may be responsible for triggering depolarization. Of the two channel types meeting this requirement in glomerulosa cell, Kir 2 displayed reduced single-channel activity in membrane patches exposed to ANG II (276). However, as previously mentioned, this current has not yet been found in whole cells. The background K⁺ current, corresponding to the later described TASK, was inhibited by physiological concentrations of ANG II in rat glomerulosa cells. At the moderately elevated physiological concentration of 10^–10 M, ANG II reduced the leak membrane conductance (measured between –127 and –87 mV) by 50% and shifted E_m by 6 mV to positive direction. The extent of depolarization was 18 and 32 mV on average at 10^–9 and 10^–8 M ANG II, respectively (323). A stable level of current inhibition required 10 min at concentrations below 100 pM ANG II and at least 5 min at higher concentrations (321).

ANG II also inhibits the TASK channels responsible for background K⁺ current. ANG II added to isolated glomerulosa cells or to oocytes injected with glomerulosa mRNA inhibited the K⁺ current. ANG II also inhibited the TASK current in oocytes, coexpressing TASK and the AT_1a angiotensin II receptor. These observations provided firm evidence that TASK exerts a significant role in the generation of the strongly negative resting membrane potential in glomerulosa cells and that ANG II may depolarize the cell by inhibiting these channels (129). Although the inhibition of TASK-3 current by ANG II is smaller than that of TASK-1 current (128), it still may have physiological significance, especially in view of the modified characteristics of the TASK-1/TASK-3 heterodimers (127). Maneuvers applied to activate phospholipase C, G_q, and protein kinase C, or to increase the concentration of cytoplasmic Ca²⁺ and IP₃, led to the conclusion that the action of ANG II (or other Ca²⁺-mobilizing hormones) is mediated by the breakdown of PIP₂ in the plasma membrane (130).

ANG II inhibits the delayed rectifier K⁺ current (79, 276). The Ca²⁺-dependent maxi K⁺ channels are also inhibited by ANG II, and in this case the effect is mediated by a pertussis toxin-insensitive G protein (408). Considering that depolarization induced by ANG II levels occurring in vivo is probably not sufficient for the activation of these channel types, their role in the physiological action of ANG II awaits confirmation.

Activation of nonselective cation channels also results in depolarization. Such a channel, with a nearly linear slope conductance between –80 and 0 mV under quasi-physiological ionic conditions, has been described by Lotshaw and Li (324). The channel was also permeable to Ca²⁺, exhibiting a P_Ca/P_Na of 4 (with 110 mM Ca²⁺ on the extracellular side). It would be worthwhile to examine the relation of this current with the dihydropyridine (DHP)-insensitive background Ca²⁺ currents observed in glomerulosa cells by applying the cell-attached mode of the patch-clamp technique, but with pipette solutions lacking monovalent cations (149). In cell-attached patches ANG II (1 nM) increased the opening probability of the nonselective cation channel, rendering cation influx and depolarization possible in the intact cell. In fact, using the nystatin-perforated patch technique, depolarization began in 2 min and was characterized by a large increase in input resistance, indicating the decrease in g_K. However, transient depolarizations of variable amplitude were superimposed on the slow depolarization, and membrane conductance during these voltage transients exhibited large increases. Considering that Ca²⁺ was omitted from the pipette solution, the increased conductance indicated the activation of nonselective cation channels. Summarizing, these channels may contribute to signal generation by ANG II by two means: cation influx contributes to the voltage-dependent activation of Ca²⁺ channels; moreover, Ca²⁺ influx per se may participate in Ca²⁺ signaling. (For further studies of channel structure, see Ref. 325.)

The participation of the Na⁺-K⁺-ATPase and ion channels in hormonally induced resetting of E_m is summarized in Figure 3.

View larger version (21K): [in this window] [in a new window]   FIG. 3. The participation of the Na+-K+-ATPase and ion channels in hormonally induced resetting of membrane potential (Em). Nonspecif., nonspecific cation channel; delayed rect. K+, delayed rectifier K+ channel; big K, Ca2+-activated maxi (BK) K+ channel. The red arrows indicate stimulatory actions, and the green arrows indicate inhibitory hormonal actions.

Because changes in Cl^– conductance are involved in the depolarization or repolarization of various cell types, their function in glomerulosa cells is also summarized. Apart from a Ras-dependent chloride current activated by ACTH (105), we are not aware of any report on Cl^– current in