Angiotensin II-induced inositol phosphate production in isolated rat zona glomerulosa and fasciculata/reticularis cells

Angiotensin II (2.5 to 250nM) induced, within 60 sec, a significant increase in [3H]inositol-labeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate in rat zona glomerulosa cells. Neither ACTH (3nM) nor K+ (8.4mM) had any effect, although aldosterone and corticosterone were significantly stimulated by all three agonists (after 30 min incubation). A similar significant dose-dependent increase in the inositol phosphates was observed with angiotensin II in zona fasciculata/reticularis cells after 30 min, but without any effect on corticosterone. In contrast ACTH significantly increased corticosterone with only a small although highly significant increase in inositol trisphosphate and inositol bisphosphate at 0.03nM ACTH. However at the higher dose (3.0nM) only inositol bisphosphate was significantly increased. These results indicate the presence on both zona glomerulosa and zona fasciculata/reticularis cells of AII receptors, which were linked to the formation of the secondary messenger, but only in the zona glomerulosa cells are associated with steroidogenesis.     

Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions

Effect of adrenergic activity on the adrenal steroidogenesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions. Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release. Both prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively. Both atenolol (beta 1 antagonist) and ICI 118-551 (beta 2 antagonist) also blocked (-)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively. Neither (-)-isoproterenol nor (+/-)-norepinephrine at concentrations of 10(-6) M potentiated aldosterone release stimulated by angiotensin II or ACTH. These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.     

Effects of prostaglandins on adrenal steroidogenesis in the rat

To elucidate the role of prostaglandins in adrenal steroidogenesis, we studied aldosterone and corticosterone responses to 3 x 10(-8) M--3 x 10(-4) M of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), prostacyclin (PGI2), and arachidonic acid (AA) in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGE2, PGF2 alpha, PGI2, and AA. Although preincubation of dispersed adrenal cells with indomethacin (3 x 10(-5) M) markedly inhibited PGE2 synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis.     

Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells

Dispersed chick adrenal cells were incubated with either ACTH, cholera toxin or forskolin. All three agents stimulated cyclic AMP accumulation and secretion of corticosterone and aldosterone by the dispersed cells. The dose-response to ACTH was similar for cyclic AMP and corticosterone but aldosterone secretion appeared to be more sensitive to ACTH stimulation. Concentrations higher than 10(-8) M of ACTH caused suppression of corticosterone output but not of cyclic AMP accumulation or aldosterone secretion. A significant cyclic AMP accumulation occurred within 30 min of exposure to ACTH whereas significant increases in steroid secretion were observed only after 30 min. An early increase (within 30 min) in cyclic AMP accumulation with both cholera toxin and forskolin was not accompanied by any significant stimulation of steroid secretion, which occurred only after 120 min. The results with the avian adrenal cells are consistent with the thesis that steroid production in the adrenocortical cells is stimulated by cyclic AMP-dependent pathways, whereas steroid release may be modulated by others.     

Rat glomerulosa cells in primary culture and E. coli lipopolysaccharide action

During endotoxic shock there is a dysfunction of the adrenal gland; both corticosterone and aldosterone secretion are altered. The aim of the present study is to use glomerulosa cells in primary culture as a target of lipopolysaccharide (LPS) action. Glomerulosa cells cultured in basal conditions are able to proliferate; bFGF and ACTH have antagonic effects, bFGF increases proliferation whereas ACTH is antimitogenic. LPS has a biphasic effect, in the short term it is antimitogenic and in the long term increases the proliferation rate. LPS inhibits ACTH-induced corticosterone secretion in a dose-dependent manner in glomerulosa cells in culture similar to that in fasciculata cells, but it does not exert an important direct effect on aldosterone secretion. These results show that LPS exerts different effects in ACTH and ANG II signal transduction pathways and in the two enzymes which catalyze the late step in the steroidogenesis, 11beta-hydroxylase and aldosterone synthase, which could be in agreement with the existence of both enzymes, regulated independently, in rat zona glomerulosa cells.     

Endothelial cell nitric oxide inhibits aldosterone synthesis in zona glomerulosa cells: modulation by oxygen

The regulation of aldosterone synthesis by endogenous nitric oxide (NO) was examined in cultured cells of the adrenal cortex. Endothelial NO synthase (eNOS) was detected by Western blot in cultured adrenal endothelial cells (ECs) but not in zona glomerulosa (ZG) cells or adrenal fibroblasts. Neither inducible (iNOS) nor neuronal NOS (nNOS) isoforms were detected in the cells. Only ECs had NOS activity and converted [(3)H]L-arginine to [(3)H]L-citrulline. Angiotensin II (ANG II, 100 nM) increased EC production of nitrate/nitrite by 2.4-fold. Coincubation with ECs or treatment with DETA nonoate increased the fluorescence of ZG cells loaded with an NO-sensitive dye, diaminofluorescein 2 diacetate (DAF-2 DA). DETA nonoate inhibited ANG II (1 nM) and potassium (10 mM) -stimulated aldosterone release in a concentration-related manner. This inhibitory effect of NO was enhanced >10-fold by decreasing the oxygen concentration from 21 to 8%. Coincubation of EC and ZG cells in 8% oxygen inhibited ANG II-induced aldosterone release, and inhibition was reversed by blockade of NOS. These findings indicate that adrenal EC-derived NO inhibits aldosterone release by cultured ZG cells and that the sensitivity to NO inhibition is increased at low oxygen concentrations.     

Effects of prostaglandins on adrenal steroidogenesis in the rat

To elucidate the role of prostaglandins in adrenal steroidogenesis, we studied aldosterone and corticosterone responses to

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of prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), prostacyclin (PGI₂), and arachidonic acid (AA) in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGE₂, PGF_2α, PGI₂, and AA. Although preincubation of dispersed adrenal cells with indomethacin ( ) markedly inhibited PGE₂ synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis.     

Adrenocortical responses to adrenocorticotrophin in the rat

The time course of plasma adrenocorticotrophin (ACTH), adrenal cyclic AMP, adrenal corticosterone, and plasma corticosterone was measured in male Sprague-Dawley rats whose endogenous release of ACTH had been blocked (1) following rapid injections of 100 and 300 ng ACTH/100 g body weight, i.v., (2) during prolonged infusions at rates of 1, 2, and 4 ng ACTH/min per 100 g body weight, and (3) after termination of 30-min infusions at rates extending from 0.06 to 8 ng ACTH/min per 100 g body weight. Following injections, the time course of the variables is similar to the one simulated from our models of adrenal cortical secretion, including the simulation of an intermediate variable of our models of the adrenal cortex cell which was presumed to correspond to cyclic AMP. However, during prolonged infusions there is an unexpected overshoot of adrenal cyclic AMP content whereas adrenal and plasma corticosterone concentrations rise to a steady-state value without overshoot. The total amount of cyclic AMP gradually increases following the three increasing infusion rates of ACTH whereas similar levels of plasma corticosterone concentrations are reached at steady state; therefore the saturation of the adrenal cortical secretion is due to a step ulterior to cyclic AMP formation in the steroidogenesis. After 30-min infusions, plasma corticosterone concentration reaches its maximal value following a rate of ACTH input which evokes only a 4-fold increase in adrenal cyclic AMP content; however, there is a 250-fold increase of adrenal cyclic AMP with respect to control value following the higher rates of infusion of ACTH.     

Magnesium ion: a possible physiological regulator of aldosterone production

We examined the direct effect of magnesium ion on aldosterone production by adrenal cells using collagenase-dispersed zona-glomerulosa cells in rats. The effects of magnesium on aldosterone production stimulated by angiotensin II or ACTH were also investigated. Both magnesium sulphate (MgSO4) and magnesium chloride (MgCl2) (0 to 2 mM) decreased aldosterone production in a dose-dependent manner. In comparison with magnesium-free medium, 2 mM MgSO4 inhibited aldosterone production by 73% and MgCl2 by 65%. In addition, MgSO4 showed an inhibitory effect on aldosterone production stimulated by angiotensin II (10pM to 10nM), whereas it had no significant effect on aldosterone production due to ACTH stimulation (10pM to 10nM). These data suggest that magnesium has an inhibitory action on aldosterone production in vitro and may be a physiological regulator of aldosterone production.     

Des-Asp-1-angiotensin II. possible role in mediating the renin--angiotensin response in the rat.

Angiotensin II and its heptapeptide fragment, Des-Asp-1-angiotensin II, produced a striking increase in aldosterone secretion in rats pretreated with dexamethasone and morphine to reduce ACTH release. 1-Sar-8-Ala-angiotensin II (10 mug/kg min-1) given simultaneously with angiotensin II (1 mug/min) blocked the aldosterone response to angiotensin II in rats pretreated to reduce ACTH release. In contrast, 1-Sar-8-Ala-angiotensin II at the same dose failed to block the steroid response to Des-Asp-1-angiotensin II (1 mug/min) but a larger dose of 50 mug/kg min-1 of the angiotensin II antagonist blocked completely both the aldosterone and the corticosterone responses to 1 mug/min of Des-Asp-1-angiotensin II. From these data it is suggested that the heptapeptide has a higher affinity for zona glomerulosa receptors than the octapeptide and that Des-Asp-1-angiotensin II mediates, at least in part, the steroidogenic response to the renin-angiotensin system in the rat. The pressor response to Des-Asp-1-angiotensin II was approximately 50% of that produced by the octapeptide in the rat, and 1-Sar-8-Ala-angiotensin II was as effective in partially blocking the pressor response to the octapeptide as in inhibiting the heptapeptide. The present observations indicate a dissociation of adrenal cortex and peripheral arteriolar receptors in their affinity for angiotensin.     

In vitro study of frog adrenal function--IX. Evidence against the involvement of lipoxygenase metabolites in the control of steroid production

The possible role of arachidonic acid metabolites of the lipoxygenase pathway in the regulation of steroidogenesis was studied in vitro using perifused frog interrenal (adrenal) glands. Graded doses of arachidonic acid (10(-6)-10(-4)M) increased the production of corticosterone and aldosterone in a dose-dependent manner. In the presence of indomethacin (5 X 10(-6)M), the effect of arachidonic acid on steroid secretion was totally abolished. Nordihydroguaiaretic acid (NDGA: 10(-6)M), a lipoxygenase inhibitor, did not alter the spontaneous secretion of corticosteroids and did not impair the stimulatory effect of arachidonic acid. In the presence of NDGA, both ACTH and angiotensin II were still able to stimulate corticosteroid production. Our data support the view that arachidonic acid metabolites play an important role in the regulation of amphibian steroidogenesis. Moreover, the results show that the lipoxygenase pathway is not involved in the spontaneous secretion of corticosteroids and in angiotensin II- or ACTH-induced steroidogenesis.     

The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K(+) ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K(+) concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K(+) to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K(+) concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K(+) and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.     

Comparative in vitro responses of fetal, pregnant and non-pregnant rabbits' adrenal glands to steroidogenic agents

The in vitro secretion of aldosterone and corticosterone by the adrenal glands of fetal (day 30), pregnant and non-pregnant rabbits was examined under basal and stimulated conditions. In general, non-pregnant animals basally secreted less aldosterone than either pregnant or fetal rabbits, whereas basal corticosterone secretion by pregnant animals exceeded that of either fetal or non-pregnant animals. At similar doses of adrenocorticotropin (ACTH), fetal and pregnant adrenal glands produced comparatively more aldosterone than non-pregnant animals, while corticosterone secretion was accelerated to a greater degree in fetal rabbits than in the other groups. Angiotensin II had its greatest effect on the aldosterone secretory rates of fetal and non-pregnant animals without affecting corticosterone secretion in any group. Elevated potassium (K+) enhanced the secretory rates of aldosterone and corticosterone in fetal animals, while increasing only aldosterone secretion in non-pregnant rabbits. Serotonin accelerated aldosterone secretion in all animals, whereas it increased corticosterone secretion only in non-pregnant animals. These results suggest that (1) in fetal rabbits, the secretory rates of both aldosterone and corticosterone are regulated primarily by ACTH and to a much lesser extent by angiotensin II and K+, (2) the corticosterone secretory rates of pregnant and non-pregnant rabbits are controlled mainly by ACTH, and (3) aldosterone secretion by non-pregnant animals is regulated primarily by angiotensin II and secondarily by ACTH and K+, while in pregnant animals ACTH may be the primary regulator of aldosterone secretion as it is in the fetus.     

Involvement of cycloheximide-sensitive mediators in the steroidogenic action of adrenocorticotropin and angiotensin II

The involvement of short-lived proteins in the steroidogenic action of corticotropic peptides has been investigated in vitro by means of a perifusion technique using frog adrenal glands. Graded concentrations of cycloheximide (10(-7) M to 10(-5) M) led to a dose-related inhibition of corticosterone and aldosterone production. The perifusion model gives detailed information on the kinetics of the inhibitory effect of cycloheximide. This effect was rapidly observed (the lag period was about 15 min), maximum inhibition being obtained 25 min after the end of administration of the protein synthesis inhibitor. Whatever the concentration of cycloheximide, corticosteroid output returned to basal values 2 h after the onset of cycloheximide infusion. Stimulation of steroidogenesis by ACTH and angiotensin II was totally inhibited by cycloheximide (10(-6) M) indicating that the synthesis of a labile protein was required for the adrenal response to corticotropic peptides. In addition, the stimulatory effect of cAMP and PGE1, which are considered to be the second messengers of ACTH and angiotensin II in amphibian interrenal gland, was blocked by cycloheximide. Taken together, these data suggest that a labile protein is involved in an early step of corticosteroid biosynthesis in the frog.     

Aldosterone and corticosterone stimulation by ACTH in isolated rat adrenal glomerulosa cells: interaction with vasopressin

An interaction between ACTH and vasopressin on steroidogenesis was observed in isolated rat adrenal zona glomerulosa cell preparations. 1. The presence of 10(-11) M vasopressin further increased by 52% the output of aldosterone produced by 10(-12) M ACTH on those cells. 2. At a pharmacological concentration of ACTH (10(-7) M), the aldosterone output was increased 5 fold while the addition of 10(-12) M or 10(-8) M vasopressin decreased it by 17% and 48% respectively. 3. Vasopressin also produced a dose-dependent inhibition of the stimulatory effect of ACTH on the output of corticosterone. 4. We have thus shown for the first time, that vasopressin acts directly on adrenal zona glomerulosa cell preparations to modify the aldosterone output by modulating the action of ACTH. It is postulated that, in addition to other known aldosterone regulating factors, ACTH and vasopressin might synergistically act to regulate the secretion of aldosterone in vivo.     

Stress-hyperresponsive period in postnatal development of hypothalamic-hypophyseal-adrenal system in mice

OBJECTIVE OF THE STUDY: to compare response to stress, activity of adenylatcyclase and enzymes of steroidogenesis in adrenal cells in 3-week and adult (15-week) mice. In 3-week mice, basal and stressor level of blood corticosterone was increased. Their response to stress was faster developing as compared with adult mice. In vitro, 3-week mice had enhanced adrenal sensitivity to ACTH, corticosterone release in response to ACTH, exogenous cAMP and progesterone; and the cAMP level in the cells stimulated with ACTH and forscoline. Thus, in 3-week mice, a stress-hyperresponsive period occurs due to increased release of corticosterone and adrenal sensitivity to ACTH. The reason of this involves enhanced activity of adenylatcyclase and intracellular enzymes of steroidogenesis.     

TREK-1 K+ channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells

Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K(+) channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K(+) channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K(+) current and a noninactivating background K(+) current with properties that collectively identify it as bTREK-1. The outwardly rectifying K(+) current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K(+) current by 82.1 +/- 6.1% and depolarized AZG cells by 31.6 +/- 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K(+) current by 73.8 +/- 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca(2+) current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca(2+)-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K(+) channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca(2+) entry and aldosterone secretion.     

Role of prostacyclin on steroidogenesis by frog interrenal gland

The role of prostacyclin (PGI₂) on amphibian adrenal steroidogenesis was studied in perifused interrenal fragments from adult male frogs. Exogenous PGI₂ (3×10⁻⁸ M to 3×10⁻⁵ M) and, in a lesser extent, 6-keto-PGF_1α increased both corticosterone and aldosterone production in a dose-related manner. Short pulses (20 min) of 0.88 μM PGI₂ administered at 90 min intervals within the same experiment did not induce any desensitization phenomenon. A prolonged administration (6 h) of PGI₂ gave rise to an important increase in steroid production followed by a decline of corticosteroidogenesis. Indomethacin (IDM, 5 μM) induced a marked reduction of the spontaneous secretion of corticosteroid which confirmed the involvement of endogenous PGs in the process of corticosteroid biosynthesis. The IDM-induced blockade of corticosterone and aldosterone secretion was totally reversed by administration of exogenous PGI₂ in our model. Angiotensin II (AII) induced a massive release of 6-keto-PGF_1α, the stable metabolite of PGI₂. The increase of 6-keto-PGF_1α preceded the stimulation of corticosterone and aldosterone secretions. In contrast, the administration of ACTH did not modify the release of 6-keto-PGF_1α. These results indicate that PGI₂ might be an important mediator of adrenal steroidogenesis in frog. They confirm that the corticosteroidogenic actions of ACTH and AII are mediated by different mechanisms.     

Effects of angiotensin, prostaglandin E2 and indomethacin on the early and late steps of aldosterone biosynthesis in isolated adrenal cells

The involvement of prostaglandins in the regulation of aldosterone biosynthesis was investigated in isolated adrenal glomerulosa cells. Cells were treated with cyanoketone to inhibit the 3 beta-hydroxy-steroid dehydrogenase and isolate the early step of aldosterone synthesis and the late step. Angiotensin II and PGE2 stimulated the synthesis of aldosterone in a concentration-related manner. The stimulation by both compounds was inhibited by indomethacin, a prostaglandin synthesis inhibitor. Indomethacin also inhibited arachidonic acid-stimulation of 6-keto PGF1 alpha synthesis, whereas cyanoketone was without effect. Both angiotensin II and PGE2 stimulated the synthesis of pregnenolone (the early step) in a concentration-related manner. At higher concentrations, angiotensin II also stimulated the conversion of [3H]corticosterone to [3H]aldosterone (the late step). PGE2 did not alter the late step significantly. Indomethacin had no effect on either biosynthetic step when added alone. However, it inhibited the angiotensin- and PGE2-stimulated pregnenolone synthesis by 41 and 59%, respectively (P less than 0.05). Indomethacin did not alter angiotensin stimulation of the conversion of corticosterone to aldosterone. These findings indicate that PGE2 increases the synthesis of aldosterone by stimulating the conversion of cholesterol to pregnenolone. Indomethacin inhibits angiotensin II- and PGE2-induced steroidogenesis at the same early biosynthetic step. These findings suggest that indomethacin may act by a mechanism other than a reduction in the concentration of prostaglandins.