Stimulatory effect of vasoactive intestinal peptide (VIP) on the secretory activity of dispersed rat adrenocortical cells. Evidence for the interaction of VIP with ACTH receptors

VIP dose-dependently increased basal, but not submaximally ACTH (10⁻¹⁰ M)-stimulated, aldosterone (ALDO) and corticosterone (B) secretion of dispersed rat capsular and inner adrenocortical cells, respectively. The maximal stimulatory effect (60–70% rise) was obtained with a VIP concentration of 10⁻⁸ M. [4-Cl-D-Phe⁶,Leu¹⁷]-VIP, a VIP-receptor antagonist (VIP-A), and corticotropin inhibiting peptide (CIP), an ACTH receptor antagonist (both 10⁻⁶ M), completely annulled VIP (10⁻⁸M)-evoked rises in basal ALDO and corticosterone secretions. The ACTH (10⁻¹⁰ M)-enhanced (about 5-fold) production of both hormones was completely reversed by CIP (10⁻⁶ M) and only partially reduced (about −30%) by VIP-A (10⁻⁶ M). The hypothesis is advanced that the weak secretagogue effect of VIP on dispersed rat capsular and inner adrenocortical cells may be due to its positive interaction with ACTH receptors.     

Cortisol and testosterone: Inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 × 10⁻⁶ and 10⁻⁴ M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 × 10⁻⁷ to 5 × 10⁻⁵ M and testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10⁻⁸ and 10⁻⁶ M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11β, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studies in our laboratory.     

Interactions between vasotocin and other corticotropic factors on the frog adrenal gland

The adrenocortical cells of the amphibian interrenal (adrenal) gland are controlled by multiple factors including neuropeptides and classical neurotransmitters. In particular, it has recently been shown that vasotocin (AVT), the amphibian counterpart of vasopressin, is a potent stimulator of frog corticosteroidogenesis. In the present study, we have investigated the possible interactions between AVT and other regulatory factors on frog interrenal tissue. When AVT (10⁻⁹ M) and serotonin (10⁻⁶ M) were infused together, a strict addition of the individual effects was observed. Similar results were obtained with concomitant infusion of AVT and vasoactive intestinal peptide or AVT and ACTH. In contrast, when AVT (10⁻⁹ M) and acetylcholine (5 × 10⁻⁵ M) were added together, the increase in corticosteroid secretion was less than additive. Dopamine induced a significant reduction of AVT-evoked stimulation of corticosterone production. These results indicate that regulatory peptides or classical neurotransmitters which participate in the control of adrenal steroidogenesis may interact on their target cell to modulate the activity of their congeners.     

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin.

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Effect of beta-endorphin on the steroid production of isolated zona glomerulosa and zona fasciculata cells

Small doses of β-endorphin (10^?11?10^?5M) decrease corticosterone production of zona fasciculata cells but fail to influence steroid production of zona glomerulosa cells. 10^?4M β-endorphin increases corticosterone production of both zones. The stimulating effect of ACTH on zona fasciculata corticosterone- and zona glomerulosa aldosterone production was decreased by β-endorphin (10^?9?10^?7M). Conclusion: β-endorphin might modulate both basal and ACTH stimulated corticosterone secretion.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Effect of carbenoxolone sodium on steroid-induced sodium transport in the toad bladder: Further studies

The licorice derivative, carbenoxolone sodium, is a potent inhibitor of the enzyme 11β-hydroxysteroid dehydrogenase. When this enzyme is suppressed or is absent, endogenous glucocorticoids induce mineralocorticoid-like sodium retention by the kidney. Carbenoxolone sodium administered in vivo to an adrenalectomized rat has also recently been shown to enhance the mineralocorticoid response to submaximal concentrations of aldosterone, deoxycorticosterone (DOC) and 11-dehydrocorticosterone (compound A). In the present studies conducted on the urinary bladder isolated from the Dominican toad, Bufo marinus, a concentration of carbenoxolone sodium shown previously to increase glucocorticoid-induced sodium transport (2.5 × 10⁻⁵ M) did not appear to alter the response to submaximal concentrations of aldosterone 10⁻⁸ M, DOC 10⁻⁷ M, or compound A 10⁻⁵ M. These findings are consistent with the view that in the whole animal carbenoxolone sodium may modify additional steroid metabolic pathways and/or physiological processes in several organs to produce the enhanced renal response to mineralocorticoids and compound A.     

Biphasic regulation by N, 2′-O-dibutyryl adenosine 3′, 5′-cyclic monophosphate (dbcAMP) of steroid 21-hydroxylase activity in rat hepatocytes

Steroid 21-hydroxylase activity has been identified in many tissues, including liver. But it is possible that the enzyme found in the liver is different from adrenal 21-hydroxylase. In the adrenal cortex, steroid 21-hydroxylase activity is increased by corticotropin (ACTH); the effect of ACTH is mediated by cyclic AMP (cAMP), and presumably involves a cAMP-dependent protein kinase (PKA). It is not yet clear, however, how extra-adrenal steroid 21-hydroxylase activity is regulated. In the present study, we examined the effect of N⁶, 2′-O-dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP), forskolin, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (H-8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on steroid 21-hydroxylase activity in primary cultures of rat hepatocytes to determine the nature of regulation of extra-adrenal steroid 21-hydroxylase activity. Steroid 21-hydroxylase activity in hepatocytes incubated with 10⁻¹¹M dbcAMP for 24 h was 1.6 times higher than that in control hepatocytes untreated with dbcAMP. On the other hand, steroid 21-hydroxylase activity decreased by 20 and 50% when the cells were incubated with 10⁻⁵ and 10⁻³ M dbcAMP, respectively. The stimulatory effect of 10⁻¹¹ M dbcAMP was not blocked by 10⁻⁵ M H-8 (PKA inhibitor), but the inhibitory effect of 10⁻⁵ or 10⁻³ M cAMP was. TPA did not alter the activity of steroid 21-hydroxylase. These findings indicate that the steroid 21-hydroxylase in rat liver is regulated by mechanisms different from those in the adrenal glands.     

ACTH(1–24) stimulates the migration of human monocytes in vitro

In view of the increasing evidence that a variety of stresses can influence immune responses, the direct effect of adrenocorticotropic hormone on the migration of human monocytes was studied in vitro. ACTH(1–24) significantly increased the number of migrating cells when placed in the same or the opposite compartment of the chemotaxis chamber, maximum activity being obtained at 10⁻¹⁴ and 10⁻⁸ M. The results indicate that ACTH(1–24) directly and potently stimulates the migration of human monocytes by means of a chemokinetic effect.     

Effects of prolonged treatment with adrenocorticotropin on the morphology and function of rat adrenocortical autotransplants.

Regenerated adrenocortical nodules were obtained by implanting in the musculus gracilis of rats fragments of the capsular tissue of their excised adrenal glands. Five months after operation, transplanted rats showed a slightly elevated blood concentration of adrenocorticotropin (ACTH), a moderately reduced plasma level of corticosterone (PBC) and a very low concentration of circulating aldosterone (PAC). Regenerated nodules were well encapsulated, and from the connective capsule some septa dipped into the parenchyma. Subcapsular-outer (OZ) and inner (IZ) cells were similar to those of the zona fasciculata/zona reticularis (ZF/ZR) of the normal gland; juxta-septal (JZ) cells resembled those of the zona glomerulosa (ZG). Prolonged (14 days) ACTH infusion normalized PBC and caused a conspicuous hypertrophy of transplanted tissue, which was coupled with a marked hypertrophy of ZF/ZR-like OZ and IZ cells and a notable rise in the basal in vitro production of corticosterone. Conversely, ACTH infusion strikingly lowered PAC, reduced the number of ZG-like JZ cells, and decreased both basal and stimulated secretion of 18-hydroxylated steroids by transplants in vitro.     

Effects of neuropeptides on growth of cultivated rat molar pulp fibroblasts

The effect of the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) on DNA synthesis of dental pulp cells was investigated in cells grown from molar tooth bud explants from 4–6 days old rat pups. A concentration response-assay of the proliferative response of pulpal cells was performed with SP, NPY, NKA, CGRP and VIP (0.01 to 1 nM) in the presence of EGF (10 ng/ml), hydrocortisone (0.4 μg/ml) and 3% FCS, using [³H]thymidine incorporation. The results showed that SP, NKA and CGRP, but not NPY and VIP, increased the cell number in a concentration-dependent manner, with maxima at 10⁻¹⁰ – 10⁻⁹ M (SP, NKA) and 10⁻⁷ M (CGRP). No potentiating effect was noted when cells are simultaneously stimulated with SP and CGRP. The finding that SP, NKA and CGRP have growth regulatory properties on pulpal cells in vitro suggests that sensory neuropeptides may be involved during pulpal development or in wound healing after pulpal injury.     

Progestins stimulate the differentiation of 3T3-L1 preadipocytes

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10⁻⁸ M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10⁻⁷ M. Only minimal effects were observed with testosterone and estradiol even at 10⁻⁶ M. When the cells were cultured in presence of 10⁻⁵ M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [³H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.     

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.     

Vasopressin and oxytocin modulation of melatonin secretion from rat pineal glands

The rat pineal gland is known to release melatonin in response to noradrenergic stimulation. Since vasopressin (VP)- and oxytocin (OT)-containing fibers innervate the pineal gland, the effects of VP and OT on melatonin release from perifused rat pineal glands were investigated. VP (10⁻⁷ M) and OT (10⁻⁶ M) decreased the basal melatonin secretion. No dose-dependent effect was observed. At high concentrations (10⁻⁵) these peptides potentiated the isoproterenol-induced increase of melatonin secretion. Below 10⁻⁵ M no potentiation was observed. Fragments of VP {[pGlu⁴,Cys⁶]VP(4–9)} and OT {[pGlu⁴,Cys⁶]OT(4–9)} did not display any effect on the isoproterenol-induced melatonin secretion.