Steroidogenesis in the zona glomerulosa of the adrenal cortex. II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40-50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20-30 sec of incubation with deoxycholate. The content of RNA, phospholipids, and cytochrome b5 in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.     

Autoradiographic evidence that zona glomerulosa and capsular vessels of the human adrenal cortex are provided with different subtypes of adrenomedullin receptors

Frozen sections of normal adrenal glands, obtained from patients undergoing unilateral nephrectomy for kidney cancer, were labeled in vitro with human [125I]ADM(1-52). Autoradiography and quantitative densitometry showed the presence of abundant ADM(1-52) binding sites in both zona glomerulosa (ZG) and capsular vessels, which were displaced with about the same efficiency by cold ADM(1-52) and rat ADM(1-50). The selective calcitonin gene-related peptide type 1 (CGRPI) ligand CGRP(8-37) eliminated, although less efficiently than ADMs, [125I]ADM(1-52) binding in the ZG, but not in the capsular vessels. These findings suggest the existence of different receptor subtypes for ADM in the human adrenal cortex. The CGRP(8-37)-sensitive receptors located in the ZG may mediate the well-known inhibitory effect of ADM on aldosterone secretion, while the CGRP(8-37)-insensitive receptors present in the capsular vessel may be involved in the ADM-induced rise in adrenal blood flow.     

The development of the adrenal gland zona glomerulosa in the rat. A morphological, immunohistochemical and biochemical study

We have studied the development of the adrenal gland in the rat comprising the ages ranging from 0 to 90 days after birth. The weight of the animals and that of the adrenal glands demonstrated a linear growth with time until 75 days, both in males and females. The area of the zona glomerulosa (ZG) increased in size from birth until approximately 40 days of age. After that, growth had a much smaller slope (females, r=0.84, P < 0.001; males, r=0.81, P < 0.001). Aldosterone secretion had a marked increase until 20 days of age and thereafter demonstrated a tendency for a decrease (females, r=-0.19, P < 0.02: males r=-0.26, P < 0.001). Plasma renin activity followed a trend parallel to that of aldosterone. The steroid precursor 18-OH-deoxycorticosterone (18-OH-DOC) demonstrated a different course as it increased progressively with age especially in the females (females, r=0.57, P < 0.001; males, r=0.40, P <0.001). The expression of the enzyme 3-beta-hydroxysteroid dehydrogenase (3-beta-HSD) was also studied by immunohistochemistry and it was shown to be very low at birth and starting to increase by 10 days of age. After 30/40 days of age the amount of this enzyme existing in the ZG was comparable with that of the outer zona fasciculata (ZF). We conclude that the development of the ZG in the rat has particularities that make it different from that of the rest of the cortex.     

Protein kinase C activation stimulates calcium transport in adrenal zona glomerulosa cells

Adrenal zona glomerulosa (ZG) cells produce aldosterone in response to angiotensin II and extracellular potassium through different mechanisms which involve changes in cytosolic free calcium (Cai). Protein kinase C (PKC) activation is part of the angiotensin II signalling cascade but its effects on Cai are unknown. PKC activation with 1 microM phorbol 12-myristate 13-acetate (PMA) and 8 mM Ko significantly increased the rate of calcium influx (P < 0.001). Both the PKC- and the Ko-induced calcium influx occurred via a nifedipine-sensitive pathway. When both were combined, PKC activation and 8 mM Ko were not additive over either agent alone. PKC activation and 8 mM Ko also stimulated calcium efflux (P < 0.01). When combined together PKC activation and 8 mM Ko had additive effects on calcium efflux (P < 0.05). PKC activation did not increase Cai nor the exchangeable calcium pool in contrast to 8 mM Ko which significantly increased both (P < 0.001). Thus, PKC activation in ZG cells induces a pattern of calcium transport characterized by accelerated calcium recycling across the cell membrane without increasing cell calcium content.     

Inhibitory effects of centrally administered somatostatin on the adrenal zona glomerulosa in male rats

Segmental zoster paresis (SZP) is the focal, asymmetrical neurogenic weakness which may occur in a limb affected by cutaneous zoster. We have summarized the features of this syndrome, based on a retrospective review of 8 personal and 96 published cases. Limb SZP becomes apparent in at least 3-5% of patients with cutaneous zoster, who are usually over the age of sixty and weak proximally (C5,6,7 or L2,3,4 innervated muscles). Functional motor recovery occurs in about 75% of cases, generally by 1-2 years. Limb weakness is probably due to a lesion of the ventral nerve root, in close proximity to the initiating dorsal ganglionitis. The electrodiagnostic findings, scarce in the literature, typically consist of absent compound sensory nerve action potentials in the involved limb, with less frequent reduction or loss of compound muscle action potentials. Fibrillations and positive sharp waves become detectable within 1-4 months in limb and related paraspinal muscles, decreasing or disappearing later. In addition to this radiculopathy, peripheral nerves may also occasionally become involved, manifest as mononeuropathies of the median, ulnar, long thoracic, recurrent laryngeal, and phrenic nerves. The zoster infection or consequent inflammatory response appears able to affect motor axons distally as well as proximally.     

Actions of adrenomedullin on the rat adrenal cortex

The actions of adrenomedullin on cAMP and aldosterone secretion have been investigated using an intact rat adrenal capsular preparation incubated in vitro. Adrenomedullin was found to cause a dose-dependent increase in aldosterone secretion, with a parallel increase in cAMP release. The minimum concentration of adrenomedullin required for significant stimulation of aldosterone secretion was 10nmol/l. Adrenomedullin did not affect ACTH-stimulated aldosterone secretion, but significantly inhibited endothelin-1 stimulated aldosterone secretion. We conclude that adrenomedullin is an aldosterone stimulant in the rat adrenal gland, acting through cAMP generation.     

Zonation of the adrenal cortex. I. Isolation of mitochondria from the zona glomerulosa of the bovine adrenal cortex

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described in comparison to those isolated from the zona fasciculo-reticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulo-vesicular, whereas those of mitochondria in the zona fasciculo-reticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed a condensed configuration regardless of isolation media, whereas those isolated from the later zone in a ST medium showed an orthodox configuration. When Ca2+ was added to mitochondria isolated form either zone in an STE medium in the condensed configuration, transition from the condensed to the orthodox configuration took place; the cristel membrane of mitochondria from the zona glomerulosa became tubular or tubulo-vesicular and those of mitochondria from the zona fasciculo-reticularis became vesicular. Purity of mitochondria thus obtained from the zona glomerulosa was examined by electron microscope and marker enzymes. Coupling efficiency of mitochondria was found to be remarkably affected by temperature during the isolation procedures and a choice of substrates.     

Short circuit current in rat adrenal capsule/glomerulosa

Angiotensin II (AII) and potassium are the primary regulators of aldosterone secretion by adrenal zona glomerulosa cells. Electrophysiological studies using isolated adrenal tissues and dispersed zona glomerulosa cells show that stimulation by these secretagogues results in depolarisation of the plasma membrane and the opening of voltage-sensitive ion channels. The concept that these cells act together to create a polarity within the gland has not been examined. Whole adrenal capsule/glomerulosa preparations were studied in Ussings chambers. An increase in [KCl] to either side of the capsule resulted in a concentration-related change in short-circuit current (SCC). KCl added externally caused an increase in SCC, indicating net inward movement of positive ions or net outward movement of negative ions. Internal KCl had a smaller opposite effect. Use of the non-specific potassium channel blocker tetraethylammonium chloride (TEA) resulted in an increase in SCC regardless of which side the addition was made, although on occasions the responses to TEA and internal KCl were unexpectedly reversed. The aldosterone antagonist spironolactone produced a variable change in SCC suggesting an autocrine/paracrine role for aldosterone on the adrenal cortex. No responses were observed with the addition of AII, ACTH or aldosterone, though these may be present in excess. The results suggest that ion gradients may be created by stimulation that conceivably have a role in cellular organisation.     

Role of Ca2+ in the action of adrenocorticotropin in cultured human adrenal glomerulosa cells

The present report details the role of Ca2+ in the early events of ACTH action in human adrenal glomerulosa cells. Threshold stimulations of both aldosterone and cAMP production were obtained with a concentration of 10 pM ACTH, an ED50 of 0.1 nM, and maximal aldosterone stimulation (5.5-fold increase over control) at 10 nM ACTH. ACTH also induced a sustained increase of intracellular calcium ([Ca2+]i) with maximal stimulation of 1.6 +/- 0.1-fold over control values. This increase does not involve mobilization of calcium from intracellular pools since no response was observed in Ca2+-free medium or in the presence of nifedipine, suggesting the involvement of Ca2+ influx by L-type Ca2+ channels. This was confirmed by patch clamp studies that demonstrated that ACTH stimulates L-type Ca2+ channels. Moreover, the Ca2+ ion is not required for ACTH binding to its receptor, but is essential for sustained cAMP production and aldosterone secretion after ACTH stimulation. These results indicate that, in human adrenal glomerulosa cells, a positive feedback loop between adenylyl cyclase-protein kinase A-Ca2+ channels ensures a slow but sustained [Ca2+]i increase that is responsible for sustained cAMP production and aldosterone secretion.     

Inhibitory action of mibefradil on calcium signaling and aldosterone synthesis in bovine adrenal glomerulosa cells

Mibefradil is a new cardiovascular drug with peculiar Ca++ antagonistic properties. The most remarkable feature of mibefradil is its unique relative selectivity for T type calcium channels, a property that has been proposed to explain in part the beneficial pharmacological and clinical profiles of this drug. In adrenal glomerulosa cells, aldosterone biosynthesis and secretion in response to angiotensin II or extracellular potassium is dependent on a sustained influx of Ca++ through T type Ca++ channels. The effect of mibefradil on the steroidogenic function of glomerulosa cells was therefore investigated. Using the patch clamp technique, we found that mibefradil inhibits selectively and in a concentration-dependent manner (IC50 = 3 microM)++ T type currents in bovine glomerulosa cells. In addition to this tonic (voltage independent) inhibition, the drug also induced a shift of the steady-state inactivation curve of these channels toward hyperpolarized voltages, contributing to its efficacy to prevent Ca++ influx into the cell through T type channels. Concomitantly, mibefradil reduced the cytosolic calcium responses to potassium and angiotensin II (as assessed with fluorescent probes), without affecting the capacitative Ca++ influx, and inhibited pregnenolone and aldosterone formation. This inhibition of steroidogenesis was not exclusively due to mibefradil action on voltage-operated Ca++ channels, because this agent also partially reduced steroid synthesis induced by adrenocorticotropic hormone or forskolin, two activators of the cyclic AMP pathway. In conclusion, mibefradil is highly effective in adrenal glomerulosa cells in reducing T type channel activity and aldosterone biosynthesis, two actions that should contribute to the beneficial effect of the drug in the treatment of hypertension.     

Duality of the voltage-dependent calcium influx in adrenal glomerulosa cells

Both T- and L-type calcium channels are expressed in bovine adrenal glomerulosa cells and both channels are sensitive to moderate depolarizations of the cell membrane induced by angiotensin II (AngII) or physiological concentrations of extracellular K+. These channels present distinct pharmacology, L-type channels being more sensitive to dihydropyridines, whereas T channels are inhibited by lower concentrations of mibefradil, a new type of calcium antagonist currently used for treating hypertension. The activity of these channels is also differently modulated by AngII, which inhibits T channels through activation of protein kinase C and L channels through a Pertussis toxin-sensitive G protein. Finally, whereas the activity of L-type channels is directly reflected on the levels of the cytosolic calcium concentration ([Ca2+]c), T-type channels are more closely related to the control of steroidogenesis, possibly through a kind of "calcium pipeline" linking the plasma membrane to the mitochondria. In conclusion, two types of calcium channels, with distinct functions and differential modulation by AngII, are activated by agonists of aldosterone biosynthesis in adrenal glomerulosa cells. Most importantly, these channels have distinct sensitivities to currently used antihypertensive therapeutic drugs.     

Adenosine increases interleukin 6 release and decreases tumour necrosis factor release from rat adrenal zona glomerulosa cells, ovarian cells, anterior pituitary cells, and peritoneal macrophages

The health benefits associated with dietary fiber have resulted in it now being used in virtually all food product categories, including many products which are manufactured using extrusion processing. The objective of the present study was to determine if extrusion processing affected phytic acid, and soluble and insoluble fiber contents. The effect of screw speeds of 50, 70, and 100% of maximum rotations per minute (% MRPM) on these components was investigated. A BI-EX Model DNDG-62/20D co-rotating intermeshing self-cleaning twin-screw extruder, manufactured by Bühlerag, CH-9240, Uzwil, Switzerland, was used to process wheat, oat and rice brans. It was found that extrusion did not affect the insoluble fiber content of wheat bran; however, a decrease in this component was observed in rice and oat brans. The effect on rice bran insoluble fiber was greatest at screw speeds of 50 and 70% MRPM. This occurred in oat bran at 50% MRPM. Soluble fiber content increased in all brans after extrusion, except ER100. For oat and rice bran soluble fibers, the greatest increase occurred at 50 and 70% MRPM, while for wheat bran this occurred at 70 and 100% MRPM. Extrusion did not affect the phytate content of the cereal brans.     

Mechanisms of action of endothelin-1 in rat adrenal

Displacement curves of 125I-Endothelim-1 (ET-1) binding to rat adrenal cells with unlabeled ET-1, and the ET-1 receptor-related peptides sarafotoxin and BQ-123, show that rat adrenal cortex possess, as its bovine counterpart, two different receptors to ET-1 named ET-A and ET-B. Binding of ET-1 to its rat adrenal receptors stimulates i) aldosterone production, in vivo and in vitro ii) calcium influx, which is mediated through voltage dependent- and receptor operated- calcium channels, iii) cholesterol uptake, iv) stimulation of Na+/K+-ATPase and iv) diacylglycerol production. While the last effect is mediated through ET-A receptors the others involve binding of ET-1 to ET-B receptors. Finally, ouabain potentiates the ET-1-mediated stimulation of aldosterone production, suggesting that the effect of the peptidic hormone on Na+/K+-ATPase could act as a negative feedback mechanism.     

Enhancement of potassium-, and angiotensin II-stimulated aldosterone production by the calcium chelator EGTA in bovine adrenal glomerulosa cells in vitro

OBJECTIVE: The aim of this study was to determine the influence of renal impairment on the single-dose pharmacokinetics of temocapril and its pharmacologically active metabolite, temocapril diacid. METHODS: A single oral dose of 20 mg temocapril hydrochloride was given after an overnight fast to eight healthy (control) subjects (group A, n = 8) with a mean baseline creatinine clearance (CLCR) of 115.2 ml.min-1 and to three groups of patients with decreased renal function (mean CLCR 56.9 ml in group B, n = 8, 30.0 ml.min-1 in group C, n = 8 and 15.4 ml.min-1 in group D, n = 5). RESULTS: The mean peak concentration and median time to peak concentration for both temocapril and its diacid metabolite as well as the man area under the curve (AUC0-infinity) for temocapril did not differ significantly between groups. The mean AUC0-infinity for temocapril diacid increased only two- to threefold from group A to D. The mean terminal elimination half-life (t1/2) for temocapril diacid was prolonged in subjects with impaired renal function. However, prolongation of mean t1/2 and increase in AUC0-infinity did not parallel the decrease of mean renal clearance for temocapril diacid. CONCLUSION: The results suggest the existence of an alternative pathway in addition to the renal excretion of temocapril, e.g. via the bile. This pathway substantially contributes to the elimination of the active metabolite, temocapril diacid, in patients with decreased renal function. Nonetheless, to avoid any risks, the dose of temocapril hydrochloride in patients with moderate to severe renal impairment should be reduced.     

Internalization of the type I angiotensin II receptor (AT1) is required for protein kinase C activation but not for inositol trisphosphate release in the angiotensin II stimulated rat adrenal zona glomerulosa cell

A specific antibody, 6313/G2, to the N-terminus of the angiotensin II type I (AT1) receptor causes retention of the AT1 receptor in the plasma membrane of rat adrenal zona glomerulosa cells and stimulates steroidogenesis and inositol trisphosphate (IP3) release. Its effects are not significantly additive with those of angiotensin. In contrast, 6313/G2 completely inhibits angiotensin induced translocation of protein kinase C to the membrane fraction, although alone it has no effect. The data suggest that IP3 linked events, such as steroidogenesis, do not require receptor internalization, but protein kinase C activation does. They also confirm that protein kinase C activation is not required for stimulation of steroidogenesis in rat dispersed glomerulosa cells.     

Calcium stimulates intramitochondrial cholesterol transfer in bovine adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II (Ang II) stimulates aldosterone synthesis through rises of cytosolic calcium ([Ca2+]c). The rate-limiting step in this process is the transfer of cholesterol to the inner mitochondrial membrane, where it is converted to pregnenolone by the P450 side chain cleavage enzyme. The aim of the present study was to examine the effect of changes in [Ca2+]c and of Ang II on intramitochondrial cholesterol distribution. Freshly prepared bovine zona glomerulosa cells were submitted to a cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM). Mitochondria were isolated and subfractionated into outer membranes (OM), inner membranes (IM), and contact sites (CS). Cholesterol content was determined by the cholesterol oxidase assay. Stimulation of intact cells with Ca2+ led to a marked decrease in cholesterol content of OM (to 54 +/- 24% of controls, n = 5) and to a concomitant increase of cholesterol in CS and IM (to 145 +/- 14%, n = 5). When glomerulosa cells were exposed to Ang II, a marked increase of cholesterol in CS occurred (to 172 +/- 16% of controls, n = 5). No significant changes were detected in OM cholesterol, suggesting a stimulation of cholesterol supply to the mitochondria in response to Ang II. Cycloheximide specifically and significantly reduced Ca2+-activated cholesterol transfer to CS and IM. In conclusion, our data indicate that one of the main functions of the Ca2+ messenger is to increase cholesterol supply to the P450 side chain cleavage enzyme by enhancing endogenous intermembrane cholesterol transfer to a mitochondrial site containing the enzymes responsible for the initial steps of the steroidogenic cascade.