Adrenal Glomerulosa Cells Research Articles

TASK-3 dominates the background potassium conductance in rat adrenal glomerulosa cells.

In a preceding study we showed that the highly negative resting membrane potential of rat adrenal glomerulosa cells is related to background potassium channel(s), which belong to the two-pore domain channel family. TWIK-related acid-sensitive K+ channel (TASK-1) expression was found in glomerulosa tissue, and the currents elicited by injection of glomerulosa mRNA (I(glom)) or TASK-1 cRNA (I(TASK-1)) showed remarkable similarity in Xenopus laevis oocytes. However, based on the different sensitivity of these currents to acidification, we concluded that TASK-1 may be responsible for a maximum of 25% of the weakly pH-dependent glomerulosa background K+ current. Here we demonstrate that TASK-3, a close relative of TASK-1, is expressed abundantly in glomerulosa cells. Northern blot detected TASK-3 message in adrenal glomerulosa, but not in other tissues. Quantitative RT-PCR experiments indicated even higher mRNA expression of TASK-3 than TASK-1 in glomerulosa tissue. Similarly to the glomerulosa background current, the current expressed by injection of TASK-3 cRNA (I(TASK-3)) was less acid-sensitive than I(TASK-1). Ruthenium red in the micromolar range inhibited I(glom) and I(TASK-3), but not I(TASK-1). Like I(TASK-1), I(TASK-3) was inhibited by stimulation of AT1a angiotensin II receptor coexpressed with the potassium channel. The high level of expression and its pharmacological properties suggest that TASK-3 dominates the resting potassium conductance of glomerulosa cells.

Mitochondria respond to Ca2+ already in the submicromolar range: correlation with redox state

Rapid formation of high-Ca2+ perimitochondrial cytoplasmic microdomains has been shown to evoke mitochondrial Ca2+ signal and activate mitochondrial dehydrogenases, however, the significance of submicromolar cytoplasmic Ca2+ concentrations in the control of mitochondrial metabolism has not been sufficiently elucidated. Here we studied the mitochondrial response to application of Ca2+ at buffered concentrations in permeabilized rat adrenal glomerulosa cells, in an insulin-producing cell line (INS-1/EK-3) and in an osteosarcoma cell line (143BmA-13). Mitochondrial Ca2+ concentration was measured with the fluorescent dye rhod-2 and, using an in situ calibration method, with the mitochondrially targeted luminescent protein mt-aequorin. In both endocrine cell types, mitochondrial Ca2+ concentration increased in response to elevated cytoplasmic Ca2+ concentration (between 60 and 740 nM) and an increase in mitochondrial Ca2+ concentration could be revealed already at a cytoplasmic Ca2+ concentration step from 60–140 nM. Similar responses were observed in the osteosarcoma cell line, although a clearcut response was first observed at 280 nM extramitochondrial Ca2+ only. As examined in glomerulosa cells, graded increases in cytoplasmic Ca2+ concentration were associated with graded increases in the reduction of mitochondrial pyridine nucleotides, consistent with Ca2+-dependent activation of mitochondrial dehydrogenases. Our data indicate that in addition to therecognized role of high-Ca2+ cytoplasmic microdomains, also small Ca2+ signals may influence mitochondrialmetabolism.

Key role of P38 mitogen-activated protein kinase and the lipoxygenase pathway in angiotensin II actions in H295R adrenocortical cells.

Angiotensin II (ANG II) can activate the mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases in several cell types. We have previously shown that the 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism is a mediator of ANG II-induced aldosterone synthesis in adrenal glomerulosa cells. To evaluate the role of MAPK activation in ANG II and the effects of LO on aldosterone synthesis, experiments were performed using the human adrenocortical cell line H295R, which secretes aldosterone in response to ANG II. MAPK activities were determined by Western immunoblotting using specific antibodies to their activated phosphorylated forms. ANG II led to a dose-dependent increase in extracellular signal-regulated kinase (ERK1/2) activity in these cells, with a peak at 5 min and lasting up to 3 h. The effects of ANG II were blocked by the ANG-II Type 1 receptor antagonist losartan. A specific 12-LO product, 12(S)-hydroxyeicosatetraenoic acid (12-HETE), had no direct effect on ERK activity. However, both ANG II and 12-HETE led to significant dose-dependent increases in p38 MAPK activity with peak effects at 5 min. By contrast, the 15-LO product, 15-HETE, had no effect on p38 MAPK activity. Furthermore, two dissimilar 12-LO inhibitors, CDC and baicalein, blocked ANG II-induced p38 MAPK activation. ANG II significantly increased aldosterone release, and this effect was inhibited by the LO inhibitor baicalein, as well as a specific p38 MAPK inhibitor, SB202190, but not by PD098059, a specific inhibitor of the ERK activator MEK. In summary, in H295R cells, ANG II activated ERK and p38 MAPKs, ANG II-induced p38 MAPK was mediated by 12-LO activation, and ANG II-induced aldosterone synthesis was prevented by 12-LO- and p38 MAPK-specific inhibitors. These results suggest, for the first time, that activation of p38 MAPK, either directly or via LO activation, participates in aldosterone's stimulatory effects of ANG II in adrenal cells.

Immunohistochemical localization of type 2 inositol 1,4,5‐trisphosphate receptor to the nucleus of different mammalian cells

The inositol 1,4,5-trisphosphate receptor (InsP3R) is a ligand-gated Ca2+ channel responsible for the release of Ca2+ from intracellular stores in the response of a wide variety of cells to external stimuli. Molecular cloning studies have revealed the existence of three types of InsP3R encoded by distinct genes. In the study presented here, we used selective anti-InsP3R antibodies to determine the intracellular location of each InsP3R subtype in bovine aortic endothelial cells, bovine adrenal glomerulosa cells, and COS-7 cells. InsP3R1 was found to be widely distributed throughout the cytosol and most abundantly in the perinuclear region identified as the endoplasmic reticulum (co-localization with protein disulfide isomerase). The intracellular location of InsP3R3 was similar to that of InsP3R1. Surprisingly, InsP3R2 was found mostly associated to the cell nucleus. This observation was made with two antibodies recognizing different epitopes on InsP3R2. Binding studies revealed the presence of a high affinity-binding site for [3H] InsP3 on purified nuclei from bovine adrenal cortex. Confocal images showed that InsP3R2 was not confined to the nuclear envelope but was distributed relatively uniformly within the nucleus. Our results demonstrate that the three types of InsP3R are not similarly distributed within a specific cell type. Our results also suggest the existence of an intranuclear membrane network on which InsP3R2 is abundantly expressed.

Elevated K + induces myristoylated alanine-rich C-kinase substrate phosphorylation and phospholipase D activation in glomerulosa cells

Elevated extracellular potassium concentrations ([K +] e) are known to stimulate aldosterone secretion from adrenal glomerulosa cells in vivo and in vitro. The mechanism is thought to involve depolarization-elicited activation of voltage-dependent calcium channels and an increase in calcium influx. Until now protein kinase C (PKC) was thought not to play a role in the steroidogenic response to elevated [K +] e. In this report, we provide evidence in bovine adrenal glomerulosa cells to suggest that elevated [K +] e increases PKC activity, as shown by an enhancement in the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS). Elevated [K +] e-induced MARCKS phosphorylation was delayed and transient and was not the result of a local production of angiotensin II (AngII). MARCKS phosphorylation in response to elevated [K +] e was not accompanied by phosphoinositide hydrolysis but was inhibited by a selective PKC inhibitor. Elevated [K +] e also activated phospholipase D (PLD) in a delayed but sustained manner. We propose that the observed PLD activation mediates the elevated [K +] e-induced MARCKS phosphorylation via PKC, although other factors may modulate this phosphorylation event.

Angiotensin II promotes selective uptake of high density lipoprotein cholesterol esters in bovine adrenal glomerulosa and human adrenocortical carcinoma cells through induction of scavenger receptor class B type I.

Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 +/- 15% of controls after 2 h and to 260 +/- 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 +/- 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells.

Angiotensin II Promotes Selective Uptake of High Density Lipoprotein Cholesterol Esters in Bovine Adrenal Glomerulosa and Human Adrenocortical Carcinoma Cells Through Induction of Scavenger Receptor Class B Type I

Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175± 15% of controls after 2 h and to 260 ± 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 ± 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells.

Aldosterone increases T-type calcium currents in human adrenocarcinoma (H295R) cells by inducing channel expression.

In adrenal glomerulosa cells, low-threshold voltage-activated (T-type) calcium channels are known to play a crucial role in coupling physiological variations of extracellular potassium to aldosterone biosynthesis. On the other hand, aldosterone itself has been recently shown to regulate Ca(2+) currents in its target cells. In the present study, we have investigated the effect of aldosterone on Ca(2+) channels of the steroidogenic human adrenocarcinoma cell line, using both electrophysiological and molecular techniques. Cell incubation with aldosterone (1 microM) for 24 h increased by 39% the density of T-type calcium currents, as assessed with the patch clamp technique. This effect of aldosterone was not related to a modification of T channel activation and inactivation properties. In contrast, L-type calcium currents remained unaffected by aldosterone treatment. The mineralocorticoid receptor antagonist, spironolactone, blunted the aldosterone-induced increase in T-type calcium current. By RT-PCR, we detected in human adrenocarcinoma cells the presence of mRNA coding for the alpha(1) subunits of three different calcium channels: the alpha(1)H isoform of T channels and the alpha(1)C and alpha(1)D isoforms of the L channels. The presence of mRNA coding for the mineralocorticoid receptor was also found in these cells. Aldosterone treatment induced a 36% increase of mRNA coding for alpha(1)H, as assessed by real-time PCR. This aldosterone-evoked stimulation of mRNA expression was maximal at 24-48 h and reversed by spironolactone, suggesting a receptor-mediated genomic effect of aldosterone. Pregnenolone production in response to KCl stimulation was increased after aldosterone treatment, in parallel to T channel expression, confirming the essential role of these channels in the steroidogenic response to potassium. Taken together, these data indicate that, in human adrenocarcinoma cells, aldosterone increases, through an autocrine pathway, the expression of T-type calcium channels and therefore modifies the ability of these cells to respond to steroidogenic agonists.

Inhibition of TASK-1 potassium channel by phospholipase C.

The two-pore-domain K(+) channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca(2+)-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M(1) muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5'-O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca(2+) concentration, and diacylglycerol) do not mediate the inhibition. Unlike the G(q)-coupled receptors, stimulation of the G(i)-activating M(2) muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-beta(2) (which is responsive also to G(i) beta gamma-subunits) renders M(2) receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.

Aldosterone as a determinant of cardiovascular and renal dysfunction.

Aldosterone is a major regulator of extracellular fluid volume and the principal determinant of potassium metabolism 1,2,3,4,5. These effects are mediated by the binding of aldosterone to the mineralocorticoid receptor in target tissues, primarily the kidney. Volume is regulated through a direct effect on the collecting duct, where aldosterone promotes sodium retention and potassium excretion. The reabsorption of sodium ions produces a fall in the transmembrane potential, thus enhancing the flow of positive ions (such as potassium) out of the cell into the lumen. The reabsorbed sodium ions are transported out of the tubular epithellium into the renal interstitial fluid and from there into the renal capillary circulation. Three primary mechanisms control aldosterone release—the renin-angiotensin system, potassium, and adrenocorticotropic hormone. The renin-angiotensin system controls extracellular fluid volume via regulation of aldosterone secretion. In effect, the renin-angiotensin system keeps the circulating blood volume constant by causing aldosterone-induced sodium retention during volume deficiency and by decreasing aldosterone-dependent sodium retention when volume is ample. In recent years there has been a radical shift in our view of aldosterone's effects on the heart, the vasculature and the kidney6,7,8,9. Aldosterone's endocrine properties have taken on a broader perspective, involving non-classic actions in non-epithelial cells found in non-classic target tissues6, 10,11,12,13,14,15. The traditional concept, that aldosterone is synthesized only in the adrenal glomerulosa cell and acts almost exclusively on the kidney to modify sodium and potassium homoeostasis, needs to be expanded. There is increasing evidence that aldosterone can have an effect on vascular remodelling and collagen formation, and a non-genomic action to modify endothelial function. Among the most intriguing effects of aldosterone are its impact on fibrosis and activity associated with a cell surface receptor in certain target tissues, including endothelial cells 6, 7, 16,17,18,19. These actions contribute substantially to the pathophysiology of congestive heart failure (CHF), as well as progressive renal dysfunction. This new information has increased interest in the development of an antagonist to block aldosterone's effect, not just because of its diuretic action but primarily because of its potential cardiovascular and renal protective effects. In this review I consider the broad spectrum of non-genomic effects of aldosterone. It is becoming increasingly evident that these effects, occurring independently of haemodynamic factors, contribute to enhanced cardiovascular risk manifested by congestive heart failure and progressive renal disease. I also discuss the clinical trials with selective aldosterone receptor antagonists that are currently underway in patients with left ventricular hypertrophy, essential hypertension and systolic hypertension, and in those who have experienced myocardial infarction. Such trials will enhance our understanding of the role of aldosterone in the pathophysiology of cardiovascular disease. Also, selective aldosterone receptor antagonism holds promise for a reduction in cardiovascular and renal disease morbidity and mortality, and for enhancement of patient wellbeing.

UNILATERAL ADRENAL HEMORRHAGE IN A PATIENT WITH ADDISON’S DISEASE

A 44-year-old white man was admitted to the hospital with acute right upper quadrant abdominal pain radiating to the right scapula area. He had been diagnosed with primary Addison’s disease 21⁄2 years previously based on an inability to stimulate adrenal cortisone production. He was unable to suppress adrenocorticotropic hormones adequately and was placed on three times daily maintenance doses (20, 10 and 20 mg.), as well 0.05 mg. fludrocortisone acetate orally daily and 50 mg. dihydroepiandosterone orally daily. The patient had been diagnosed in infancy with hypothyroidism and was taking 0.05 mg. levothyroxine sodium orally daily. Etiology of the adrenal and thyroid insufficiency was believed to be chronic autoimmune disease. His son had undergone parathyroid adenoma excision at 8 years old, and thyroid insufficiency was diagnosed in his mother and maternal aunt. History included chronic intermittent right upper quadrant abdominal pain radiating to the back 1 year in duration. Abdominal magnetic resonance imaging (MRI) 21⁄2 years earlier had revealed no adrenal masses. Another MRI of the brain and pituitary gland 8 months later because of increased adrenocorticotropic hormones was normal. On hospital admission for acute abdominal pain, unenhanced and enhanced abdominal computerized tomography demonstrated a 3 3 10 cm. solid mass with fluid accumulation in the perirenal space thought to represent adrenal hemorrhage. A left lower pole renal cyst was also identified. MRI was consistent with right adrenal hemorrhage (fig. 1). Laboratory evaluations included normal serum epinephrine, dopamine and catecholamine, as well as normal 24-hour urine vanillylmandelic acid. Serum chemistry evaluations, prothrombin time, partial thromboplastin time and platelet counts were normal. Right adrenalectomy was performed through an 11th rib incision. The adrenal mass was cystic and on sectioning contained old blood (fig. 2). The histological sections revealed only scant foci of residual adrenal glomerulosa cell tissue with no evidence of malignancy. Convalescence was uneventful and the patient has not had further episodes of abdominal pain. However, 3 months after hospital discharge he was diagnosed with sigmoid colon carcinoma and underwent successful resection of the tumor. DISCUSSION

Angiotensin II priming of aldosterone secretion with agents that enhance Ca 2+ influx

We have previously shown that angiotensin II (AngII) is able to prime, or sensitize, the secretory response of cultured bovine adrenal glomerulosa cells to the Ca 2+ channel agonist, BAY K8644. We examined the ability of AngII to prime glomerulosa cells to an elevated extracellular K + level, a physiological agonist that also triggers Ca 2+ influx. K + (9 mM) elicited enhanced secretion in AngII-primed cells compared to those with no prior exposure to the hormone, suggesting that AngII can sensitize glomerulosa cells to respond to increases in extracellular K +. The potential involvement of protein kinase C (PKC) in priming was investigated by determining whether enhanced Ca 2+ influx could maintain the AngII-induced phosphorylation of the endogenous PKC substrate, myristoylated, alanine-rich C kinase substrate (MARCKS). Incubation with the AngII antagonist, saralasin, for 30 min following an AngII exposure reduced the AngII-induced increase in MARCKS phosphorylation. 100 nM BAY K8644 together with saralasin was unable to maintain AngII-stimulated MARCKS phosphorylation. On the other hand, phosphorylation of the steroidogenic acute regulatory (StAR) protein was sustained with saralasin exposure, both in the presence and absence of BAY K8644. This observation suggests that persistent StAR phosphorylation/activation may play a role in priming.

FK506 blocks intracellular Ca2+ oscillations in bovine adrenal glomerulosa cells.

The inositol 1,4,5-trisphosphate (InsP(3)) receptor is a ligand-gated Ca(2+) channel playing an important role in the control of intracellular Ca(2+). In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP(3) receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP(3) receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP(3) receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca(2+) induced by a submaximal dose of InsP(3). We also showed that FK506 blocks intracellular Ca(2+) oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca(2+) concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP(3) receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP(3) receptor in the regulation of intracellular Ca(2+) oscillations and show that FK506, by maintaining the phosphorylated state of the InsP(3) receptor, causes important changes in the Ca(2+) oscillatory process.

Role of membrane depolarization and T-type Ca 2+ channels in angiotensin II and K + stimulated aldosterone secretion

The hypothesis that Ca 2+ influx necessary for angiotensin II (AngII) and K + stimulation of aldosterone secretion is primarily mediated by membrane depolarization and activation of T-type Ca 2+ channels was examined in isolated rat adrenal glomerulosa cells. Perforated-patch clamp recordings of membrane potential ( V m) demonstrated that AngII and K + induce concentration-dependent depolarizations capable of activating T channels and, at high K + and AngII concentrations, activating L channels and inactivating T channels. K +-induced depolarizations were stable and readily reversible. V m was proportional to K + concentration, exhibiting a linear slope of 53.7 mV per 10-fold increase in K +. AngII-induced depolarizations were complex, consisting of a slow maintained component superimposed with small amplitude depolarizing fluctuations. Slow oscillations in V m were occasionally observed in response to 10 −9 M AngII or greater. The slow, maintained component of depolarization coincided with inhibition of K + conductance. Neither rapid fluctuations nor slow oscillations in V m were blocked by mibefradil or other treatments that inhibit voltage-gated Ca 2+ channels. Perforated-patch clamp experiments also demonstrated that AngII (10 −8 M) inhibited L channels by 45.6% without affecting T channels. Thus AngII activates T channels by depolarization rather than T channel modulation in rat cells. The concentration dependencies of mibefradil inhibition of T channels and AngII- and K +-induced aldosterone secretion were compared. Under whole-cell patch clamp mibefradil induced a concentration-dependent inhibition of T channels, exhibiting a K app of 0.62 μM. Mibefradil inhibition was use-dependent but mibefradil neither acted as an open channel blocker nor significantly affected T channel inactivation or activation. Mibefradil inhibited K +- and AngII-induced secretion at concentrations similar to that for T channel inhibition; at high concentrations (10 μM) mibefradil inhibited AngII-induced secretion by 88% and completely inhibited K +-induced secretion. The IC 50 for K +-induced secretion was dependent on K + concentration, increasing from 0.2 μM for 6 mM K + to 2.5 μM for 10 mM K + or greater. Mibefradil exhibited an IC 50 of 1.1 μM for inhibition of secretion at all AngII concentrations examined (0.1, 1.0, and 10 nM). Mibefradil also exhibited multiple nonspecific effects, which complicated the assessment of T channel function, including; inhibition of leak and voltage-dependent K + conductances, inhibition of Ca 2+-independent aldosterone secretion, and inhibition of secretion under conditions expected to completely inactivate T channels (10 nM AngII or 20 mM K +). In summary, these results indicate that voltage-gated T channels represent the primary Ca 2+ influx pathway activated by physiological concentrations of AngII and K + but other Ca 2+ influx pathways must mediate aldosterone secretion induced by high K + or AngII concentrations.

Differential effects of agonists of aldosterone secretion on steroidogenic acute regulatory phosphorylation

The steroidogenic acute regulatory (StAR) protein mediates cholesterol transport within the mitochondria, and its phosphorylation is believed to be required for steroidogenesis. Increased extracellular potassium concentrations (K +), angiotensin II (AngII), and adrenocorticotropic hormone (ACTH) induce aldosterone secretion from bovine adrenal glomerulosa cells. We hypothesized that, although these agonists act via different signaling pathways, StAR phosphorylation should be common to their action. We studied the effects of K +, AngII, and ACTH, at concentrations that yield comparable secretory responses, on StAR phosphorylation. All three agents induced significant increases in StAR phosphorylation although the response to ACTH was less than that of AngII and K +. In cells stimulated with the protein kinase C (PKC) agonist 12-tetradecanoylphorbol 13-acetate (TPA), the Ca 2+ channel agonist BAY K8644, and the adenylate cyclase agonist forskolin, TPA caused a small but statistically significant increase in StAR phosphorylation while BAY K8644 and forskolin had no significant effect. Interestingly, the combination of TPA and BAY K8644 produced a larger increase in StAR phosphorylation than the agents alone. We conclude that in cultured bovine adrenal glomerulosa cells the PKC signaling pathway is most effective at inducing StAR phosphorylation but that there is no simple correlation between this event and aldosterone production.

Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells.

Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (K(o)) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive (86)Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing K(o) from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca(o)) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing K(o) from 4 to 10 mM in the absence of Ca(o) stimulated the sodium pump approximately 30% and did not increase intracellular free calcium concentration ([Ca(2+)](i)). In some experiments, addition of 1.8 mM Ca(o) in the presence of 4 mM K(o) increased [Ca(2+)](i) above the levels observed in the absence of Ca(o) and stimulated the sodium pump up to 100%. Ca-dependent stimulation of the sodium pump by K(o) and Ca(o) was inhibited by isradipine (10 microM), a blocker of L- and T-type calcium channels, by compound 48/80 (40 microg/ml) and calmidizolium (10 microM), which inhibits calmodulin (CaM), and by KN-62 (10 microM), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 microM), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Ca(o) increased [Ca(2+)](i) to the level observed in the presence of 10 mM K(o) and 1.8 mM Ca(o) and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Ca(o) was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that K(o) stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Ca(o). The resulting increase in [Ca(2+)](i) may stimulate the sodium pump by activating CaM and/or CaMKII.

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II.

The effect of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca(2+) channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of approximately 9 pS in 110 mM BaCl(2) or CaCl(2). In the cell-attached patch, elevation of bath Ca(2+) from 150 nM to 1 microM raised intracellular Ca(2+) in K(+)-depolarized (140 mM) cells and evoked an increase in the LVA Ca(2+) channel probability of opening (NP(o)) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP(o) elicited by elevated Ca(2+) concentration. Furthermore, administration of a mutant recombinant CaMKIIalpha exhibiting cofactor-independent activity in the absence of elevated Ca(2+) produced a threefold elevation in LVA channel NP(o). These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca(2+).

Effect of high extracellular phosphate concentration on arachidonic acid production by parathyroid tissue in vitro.

Recent in vivo and in vitro studies show that high phosphate directly stimulates parathyroid hormone (PTH) secretion. However, little is known about the intracellular signaling system involved in the regulation of PTH secretion by extracellular phosphate. High extracellular calcium is coupled to the activation of phospholipase A(2) (PLA(2)) and the formation of arachidonic acid (AA), a potent inhibitor of PTH release. The present study was designed to evaluate whether a high phosphate concentration has an effect on the PLA(2)-AA pathway in parathyroid cells. In vitro experiments were performed in parathyroid tissue obtained from normal rats and dogs. AA production was measured in parathyroid tissue in response to 1- and 4-mM phosphate concentration and after addition of PLA(2) to the medium. To determine whether the effect of phosphate on AA production in parathyroid cells was tissue specific, separate experiments were performed to test the effect of phosphate in rat adrenal glomerulosa cells, which are known to increase AA production in response to angiotensin II. The effect of sulfate, an ion with chemical characteristics similar to phosphate, on PTH secretion was also evaluated. In parathyroid tissue, a high phosphate concentration decreased the high calcium-induced AA production. This effect of phosphate was associated with an increase in PTH secretion. The addition of AA reversed the stimulatory effect of phosphate on PTH secretion. In another type of AA-responsive tissue, the adrenal glomerulosa, a high phosphate concentration did not affect the production of AA when stimulated by angiotensin II. In a normal phosphate concentration, the addition of PLA(2) stimulated AA production and decreased the PTH secretion. However, in a 4-mM phosphate concentration, the addition of PLA(2) did not reduce PTH secretion and did not stimulate AA production. Finally, sulfate did not affect PTH secretion. In conclusion, a high phosphate concentration affects the production of AA by parathyroid tissue. This effect of phosphate may be the mechanism by which a high phosphate concentration stimulates PTH secretion.

Essential Role of Dynamin in Internalization of M2Muscarinic Acetylcholine and Angiotensin AT1AReceptors

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.

Involvement of tyrosine kinase in citrate-stimulated aldosterone production in bovine glomerulosa cells.

The present study was designed to assess whether citrate stimulates aldosterone production by isolated bovine adrenal glomerulosa cells in vitro. When the cells were incubated with graded concentrations of citrate up to 4.0 mM, basal aldosterone production was significantly elevated, with a gradual reduction of extracellular ionized calcium concentration. Without citrate, however, adding increasing amounts of calcium chloride to a calcium-free medium did not reproduce the citrate's effect on basal aldosterone production. Genistein, an inhibitor of tyrosine kinases, inhibited the citrate (4 mM)-induced aldosterone production in a dose-dependent manner, with 89.8% of inhibition at a concentration of 10 microM. When the cells were exposed to citrate (4 mM) for 5, 10, and 30 min, tyrosine in Mr 105,000 endogenous protein was dominantly phosphorylated. This study demonstrates for the first time that citrate stimulates aldosterone production in bovine adrenal glomerulosa cells in vitro and also suggests a crucial involvement of protein tyrosine kinase in the steroidogenic action of citrate in the cells.