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.     

Effect of vinblastine, a potent antimicrotubular agent on steroid secretion by perifused frog adrenal glands

The role of microtubules in adrenal steroidogenesis was examined in vitro, using frog interrenal tissue. Adrenal dice from Rana ridibunda were perifused with amphibian culture medium and the effect of various antimicrotubular drugs was studied. The amounts of corticosterone and aldosterone released in the effluent perifusate were radioimmunoassayed using specific antisera. Administration of colchicine, nocodazole, and vinblastine (10(-5) M) did not affect spontaneous secretion of corticosterone and aldosterone. These results indicated that, in contrast to microfilaments which play an important role in spontaneous steroidogenesis, the microtubular system is not required for basal corticosteroid secretion. However, vinblastine (10(-5) M) was responsible for a marked decrease in ACTH-induced stimulation of corticosterone and aldosterone production. Conversely, vinblastine did not significantly alter the response of interrenal tissue to dibutyryl cAMP, forskolin and NaF, indicating that the microtubules are involved in an early step of ACTH action, namely at the level of the receptor subunit.     

Serotonin stimulates corticosteroid secretion by frog adrenocortical tissue in vitro

The mode of action of serotonin (5-HT) in the regulation of frog adrenal steroidogenesis was studied in vitro using the perifusion system technique. Graded doses of 5-HT (from 10(-8) to 10(-6) M) increased both corticosterone and aldosterone production in a dose-dependent manner. Short pulses (20 min) of 10(-6) M 5-HT, administered at 130 min intervals within the same experiment, did not cause any desensitization phenomenon. Indomethacin (IDM; 5 microM), a cyclooxygenase inhibitor which induced a dramatic decrease in the spontaneous secretion of corticosteroids, did not impair the stimulatory effect of 5-HT on corticosterone and aldosterone production. In the absence of calcium, 5-HT (10(-6) M) was still able to stimulate corticosteroid production. Dantrolene (5 x 10(-5) M), a blocker of calcium mobilization from intracellular pools which significantly inhibited the spontaneous production of corticosteroids, did not suppress 5-HT-evoked corticosteroid secretion. These results show that 5-HT, stored in adrenal chromaffin cells, may act as a paracrine factor to stimulate adrenal steroidogenesis in the frog. Our data also indicate that the mechanism of action of 5-HT does not depend on prostaglandin biosynthesis.     

Evidence for the involvement of nitric oxide in the control of steroid secretion by the frog adrenal gland

Nitric oxide (NO) has been found to modulate the response of rat, bovine and human adrenocortical cells to corticotropic factors. The aim of the present study was to investigate the possible involvement of NO in the control of corticosteroid secretion in the frog Rana ridibunda. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against NO synthase (NOS) revealed that NOS is exclusively expressed in chromaffin cells. The NO donor sodium nitroprusside (SNP) and the NO synthase inhibitor Nw-nitro- -arginine ( -NO₂Arg) did not modify the spontaneous production of corticosterone and aldosterone by perifused adrenal slices. Similarly, -NO₂Arg had no effect on the secretory responses induced by ACTH, angiotensin II (AII) and endothelin-1 (ET-1). In contrast, SNP significantly inhibited the stimulatory effects of ACTH, AII and ET-1 on corticosterone and aldosterone secretion. These data provide the first evidence for a modulatory role of NO on adrenocortical cell activity in amphibians.     

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.     

Dopamine inhibits corticosteroid secretion in frog adrenocortical cells: evidence for the involvement of prostaglandins in the mechanism of action of dopamine

We have investigated the possible involvement of arachidonic acid metabolites in dopamine-induced inhibition of adrenocortical steroidogenesis. Administration of dopamine (5 x 10(-5) M) for 20 min to perifused frog adrenal slices caused a marked reduction of the release of both prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin (PGI2). Dopamine also induced a significant inhibition of corticosterone and aldosterone secretion. A lag period of 20 min was observed between inhibition of prostanoid and corticosteroid releases. Prolonged dopamine infusion did not prevent the stimulatory effect of PGE1, PGE2 or arachidonic acid on corticosteroid secretion. These observations indicate that activation of dopaminergic receptors in adrenocortical cells is linked to an inhibition of arachidonic acid metabolism. Our data also suggest that the inhibitory effect of dopamine occurs at a step preceding arachidonic acid formation.     

Interrenal activity in the female lizard Lacerta vivipara J.: in vitro response to ACTH 1-39 and to [Sar1, Val5] angiotensin II (ANG II)

A perifusion system technique was developed in order to determine in vitro the respective roles of ACTH and ANG II in the regulation of adrenal steroidogenesis in the lizard Lacerta vivipara. Synthetic human ACTH 1-39, administered as 20-min pulses, stimulated corticosterone (B) and aldosterone (A) release in a dose-dependent manner. The increase in corticosterone output was higher than that in aldosterone output, leading to an enhancement of the B/A ratio. Iterative stimulations with 1 nM ACTH (20-min pulses every 120 min) led to reproducible increases in corticosterone and aldosterone release. Prolonged stimulation with 1 nM ACTH (up to 240 min) caused a sustained increase in corticosteroid release, suggesting that, in the lizard, ACTH does not induce any desensitization phenomenon. The angiotensin II analogue [Sar1, Val5] ANG II also stimulated corticosterone and aldosterone release in a dose-dependent manner; the stimulatory effects of ANG II on both steroids were very similar. These results indicate that, in lizards, ACTH plays a major role in the regulation of adrenal steroidogenesis. Since ANG II stimulates the production of gluco- and mineralocorticoids, our data raise the question of the existence of two cell types synthesizing corticosterone and aldosterone, respectively, in reptiles.     

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.     

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.     

Pharmacological profile of the tachykinin receptor involved in the stimulation of corticosteroid secretion in the frog Rana ridibunda

It has recently been shown that the adrenal gland of the frog Rana ridibunda is densely innervated by a network of fibers containing two novel tachykinins, i.e. ranakinin (the counterpart of substance P) and [Leu³, Ile⁷]neurokinin A. Both ranakinin and [Leu³, Ile⁷]neurokinin A stimulate corticosteroid secretion from frog adrenal glands in vitro. In the present study, we have investigated the pharmacological profile of the receptors involved in the stimulatory action of ranakinin on perifused frog adrenal slices. The selective NK-1 receptor antagonists [ -Pro⁴, -Trp^7,9]substance P 4–11 and CP-96,345, did not affect the stimulatory action of ranakinin. The selective NK-1 agonist substance P 6–11 had no effect on corticosteroid secretion. The non-peptidic NK-1 receptor antagonist RP 67580 significantly reduced the stimulatory effect of ranakinin on corticosterone and aldosterone secretion by 57 and 55%, respectively. In addition, the dual NK-1/NK-2 receptor antagonist FK-224 significantly inhibited the effect of ranakinin on corticosterone (−80%) and aldosterone secretion (−95%). Finally, the amphiphilic analogue of substance P, [ -Pro², -Phe⁷, -Trp⁹]substance P, had no effect on corticosteroid secretion. These data suggest that in the frog adrenal gland the stimulatory action of ranakinin on steroid secretion is mediated by a novel type of receptor which differs substantially from the mammalian NK-1 receptor subtype.     

Effect of calcium on corticosteroid secretion by isolated frog interrenal gland

The direct effect of extracellular calcium concentrations on corticosteroidogenesis has been examined in the frog, using a perifusion system technique. The release of corticosterone and aldosterone in the effluent medium was monitored by specific radioimmunoassays. Increasing concentrations of Ca2+ (from 2 to 15 mM) gave rise to a dose-related stimulation of corticosteroid release, whereas the increment of either Na+ or K+ concentrations did not modify steroid production. Iterative administration of a moderate concentration of calcium (6 mM) led to a reproducible stimulation of steroid secretion whereas the same dose infused during 6 h induced a transient rise in corticosteroid secretion followed by a plateau. The direct effect of Ca2+ on steroidogenesis was confirmed by the dose-dependent stimulation of steroid secretion induced by the calcium ionophore A 23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mM Co2+ both resulted in a significant decrease in steroid production. Conversely, the administration of verapamil (up to 10(-4) M) did not affect steroidogenesis. These results provide evidence that extracellular calcium ions are required for basal production of corticosteroids in amphibians and that Ca2+ influx does not occur through voltage-dependent channels. Since, in the frog, blood Ca2+ concentrations vary in a rather large range, these results suggest that circulating Ca2+ levels may regulate corticosteroid production in these animals.     

Co-localization of vasoactive intestinal peptide (VIP) and enkephalins in chromaffin cells of the adrenal gland of amphibia. Stimulation of corticosteroid production by VIP

Recent studies have shown that biologically active peptides and monoaminergic neurotransmitters coexist in certain neuronal cell populations. Using the immunofluorescence technique, we have examined the localization of enkephalins, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase in the adrenal gland of the frog Rana ridibunda. Most chromaffin cells which stained for tyrosine hydroxylase contained VIP-like immunoreactivity, whereas methionine- (Met-) and leucine- (Leu-) enkephalin-like immunoreactivity was detected in about 40% of the cells revealed by the anti-tyrosine hydroxylase serum. No VIP- or enkephalin-like immunoreactive nerve fibres were observed. Since in the frog, the chromaffin cells are in close contact with the adrenocortical (interrenal) tissue, a possible action of VIP and opiates on corticosteroidogenesis has been investigated. At doses 10(-6) and 10(-5) M, 20-min infusions of synthetic porcine or chicken VIP elicited a significant increase in corticosterone and aldosterone production by perifused frog adrenals, in a dose-dependent manner. As compared to ACTH, VIP was several orders of magnitude less effective in stimulating corticosteroid production. Morphine, Met- and Leu-enkephalins (10(-5) M) had no effect on spontaneous secretion of corticosteroids. In addition, Met- and Leu-enkephalins (10(-5) M) did not alter the production of corticosterone induced by ACTH. THese results suggest that VIP contained in the chromaffin cells of the frog adrenal gland may exert a local action in stimulating corticosteroid production by the interrenal tissue.     

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.     

Development of a simplified perifusion system of rat zona glomerulosa. Effect of cytochalasin B on spontaneous and ACTH-stimulated corticosteroidogenesis

The aim of the present study was to develop a perifusion technique for rat adrenal glomerulosa slices as a model to study the kinetics of corticosteroid production in vitro. Perifusion of adrenal tissue with increasing concentration of ACTH (3.16 X 10(-12) to 10(-9) M) led to a dose-related stimulation of corticosterone and aldosterone secretion. Administration of cytochalasin B did not alter the basal secretion of corticosterone and aldosterone. In addition, cytochalasin B did not modify the response of glomerulosa tissue to ACTH. The results indicate that the perifusion model for glomerulosa fragments may provide valuable information, concerning the kinetics of steroid production and its regulation at the cellular level.     

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.     

Effects of TMB-8 and dantrolene on ACTH- and angiotensin-induced steroidogenesis by frog interrenal gland: evidence for a role of intracellular calcium in angiotensin action

The influence of intracellular calcium on the steroidogenic response of adrenocortical tissue to ACTH and angiotensin has been studied in the frog, using a perifusion system technique. The release of corticosterone, aldosterone and prostaglandins in the effluent medium was monitored by specific radioimmunoassays. TMB-8 and dantrolene, two potential blockers of calcium mobilization from intracellular pool(s), were tested. Dantrolene (5 X 10(-5) M) significantly reduced basal and angiotensin-induced corticosterone and aldosterone production but had little effect on ACTH-evoked steroid release. Conversely TMB-8 (10(-4) M) profoundly depressed spontaneous as well as ACTH- and angiotensin II-induced corticosteroid secretion, suggesting that this compound may affect not only calcium mobilization from the endoplasmic reticulum pool but also calcium influx. Adrenal glands perifused with both dantrolene and calcium-free medium showed no response to angiotensin II. Conversely, in calcium-free conditions and in the presence of dantrolene, angiotensin II still caused an increase in prostaglandin synthesis. Taken together, these results indicate that 1) dantrolene is a more specific agent than TMB-8 in inhibiting calcium mobilization from intracellular pool(s); 2) ACTH increases corticosteroidogenesis without inducing mobilization of intracellular calcium; 3) angiotensin II stimulates both the efflux of calcium from the endoplasmic reticulum and the influx of calcium through the plasma membrane; 4) calcium is required after prostaglandin production in the steroidogenic response of frog interrenal gland to angiotensin II.     

Action of vasoactive intestinal peptide (VIP) on amphibian adrenocortical function,in vitro

Vasoactive intestinal peptide (VIP) is located in chromaffin cells of the frog adrenal gland and is able to stimulate corticosteroid secretion in amphibians. In the present study we have investigated the possible involvement of prostaglandins, microfilaments and calcium in the mechanism of action of VIP on frog adrenocortical tissue. Rana ridibunda interrenal dice were perifused with amphibian culture medium for more than 10 hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. In the presence of indomethacin (5 μM), a specific blocker of prostaglandin biosynthesis, the spontaneous secretion of corticosteroids was markedly reduced (80%) but the stimulatory effect of VIP was not altered. The administration of the microfilament disrupting agent cytochalasin B (50 μM) inhibited both spontaneous and VIP-induced corticosteroid secretion. In the absence of calcium, the spontaneous level of corticosteroid was reduced to about 60% but VIP was still able to stimulate corticosteroid secretion. From these data we conclude that the integrity of the cytoskeleton is required for the secretory response of adrenocortical cells to VIP, whereas neither prostaglandins nor calcium are involved in VIP-induced adrenocortical stimulation.     

Formation of 11 beta-hydroxysteroids requires the integrity of the microfilament network in adrenocortical cells

In order to determine the role of microfilaments in adrenal steroidogenesis, we have studied the effect of cytochalasin B, a microfilament-disrupting agent, on the kinetics of [3H] pregnenolone conversion to labelled metabolites by frog interrenal tissue in vitro. Cytochalasin B (5 x 10(-5)M) induced a 50 to 70% decrease in corticosterone, 18-hydroxycorticosterone and aldosterone biosynthesis while the formation of progesterone and 11-desoxycorticosterone was not affected. These results suggest that microfilaments interfere in the conversion of 11-desoxycorticosterone to corticosterone probably by controlling the movement of 11-desoxycorticosterone from the reticulum to the mitochondria.     

Role of calcium in stimulus-secretion coupling on isolated frog interrenal gland

The influence of extracellular calcium concentration on the steroidogenic response to ACTH and to the angiotensin II analogue [Sar1-Val5]AII has been studied in the frog, using a perfusion system technique. The release of corticosterone and aldosterone in the effluent medium was measured by specific radioimmunoassays. In calcium-free medium the stimulatory effect of ACTH (10(-9) M) was completely abolished whereas the response to dbcAMP (5 mM) was unchanged indicating that the role of calcium takes place before the formation of cAMP. Conversely, in the absence of calcium, angiotensin II (10(-7) M) was still able to stimulate corticosterone and aldosterone production. Addition of Co2+ (4 mM), a calcium antagonist, to the perfusion medium, inhibited partially the response of adrenal tissue to ACTH, dbcAMP and angiotensin. The voltage-dependent calcium channel blocker verapamil (10(-6) induced a dose-related inhibition of the corticotropic effect of ACTH. At the higher dose (10(-4) M), verapamil totally inhibited the stimulation of corticosterone and aldosterone production induced by ACTH. By contrast, at the same dose it did not alter the stimulatory effect of forskolin (2.4 X 10(-7)M) on corticosterone output, but significantly diminished forskolin-induced aldosterone response. Similarly, angiotensin-stimulated corticosterone production was slightly inhibited by 10(-4) M verapamil, whereas aldosterone response to angiotensin was totally abolished, indicating that verapamil may act intracellularly to block the conversion of corticosterone to aldosterone. Taken together, these results indicate that, in amphibians extracellular calcium is essential for the action of ACTH, either for the binding of the hormone to its receptor and/or for the transduction of the information from hormone-receptor complex to the adenylate cyclase moiety and that the mechanism of action of angiotensin does not involve calcium uptake by adrenocortical cells.     

Adrenocortical response to corticotropin is inhibited by gamma 3-MSH antisera in normotensive and spontaneously hypertensive rats

In order to further investigate the coordinate action of pro-corticotropin/endorphin-derived peptides on adrenal steroidogenesis, we have evaluated the effects of highly specific antisera to synthetic rat gamma 3-MSH (1-27) peptide (gamma 3-MSH Ab) on corticosterone, 18-hydroxycorticosterone and aldosterone responses to ACTH (1-24) in chronically cannulated spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). Antisera eliminated the ACTH induced rise of all three corticosteroids. It had no effect on basal corticosteroid levels. Our results offer further evidence that the potentiating action of gamma 3-MSH may play an important role in modulating ACTH induced steroidogenesis.