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.     

Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells

Immunoreactive-adrenomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non-functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochromocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 ± 1.2 pmol/g ww (mean ± SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/10⁵ cells/24h, mean ± SEM, n = 4). On the other hand, immunoreactive-adrenomedullin was not detectable in the extract of SW-13 cells (<0.09 fmol/10⁵ cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

Effects of glucagon and glucagon-like peptide-1 on glucocorticoid secretion of dispersed rat adrenocortical cells.

The effects of glucagon and glucagon-like peptide-1 (GLP-1) on the secretory activity of rat adrenocortical cells have been investigated in vitro. Neither hormones affected basal or agonist-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells or basal corticosterone production of zona fasciculata-reticularis (inner) cells. In contrast, glucagon and GLP-1 partially (40%) inhibited ACTH (10(-9) M)-enhanced corticosterone secretion of inner cells, maximal effective concentration being 10(-7) M. The effect of 10(-7) M glucagon or GPL-1 was suppressed by 10(-6) M Des-His1-[Glu9]-glucagon amide (glucagon-A) and exendin-4(3-39) (GPL-1-A), which are selective antagonists of glucagon and GLP-1 receptors, respectively. Glucagon and GLP-1 (10(-7) M) decreased by about 45-50% cyclic-AMP production by dispersed inner adrenocortical cells in response to ACTH (10(-9) M), but not to the adenylate cyclase activator forskolin (10(-5) M). Again this effect was blocked by 10(-6) M glucagon-A or GLP-1-A. The exposure of dispersed inner cells to 10(-7) M glucagon plus GLP-1 completely suppressed corticosterone response to ACTH (10(-9) M). However, they only partially inhibited (by about 65-70%) both corticosterone response to forskolin (10(-5) M) or dibutyryl-cyclic-AMP (10(-5) M) and ACTH (10(-9) M)-enhanced cyclic-AMP production. Quantitative HPLC showed that 10(-7) M glucagon or GLP-1 did not affect ACTH-stimulated pregnenolone production, evoked a slight rise in progesterone and 11-deoxycorticosterone release, and markedly reduced (by about 55%) corticosterone secretion of dispersed inner adrenocortical cells. In light of these findings the following conclusion are drawn: (i) glucagon and GLP-1, via the activation of specific receptors, inhibit glucocorticoid response of rat adrenal cortex to ACTH; and (ii) the mechanism underlying the effect of glucagon and GLP-1 is probably two-fold, and involves both the inhibition of the ACTH-induced activation of adenylate cyclase and the impairment of the late steps of glucocorticoid synthesis.     

The inhibitor of phospholipase-A2, AACOCF3, stimulates steroid secretion by dispersed human and rat adrenocortical cells

AACOF3 is a trifluomethylketone analog of arachidonic acid, which inhibits phospholipase-A2 (PLA2). AACOCF3 was found to concentration-dependently increase basal aldosterone and corticosterone secretion by dispersed rat zona glomerulosa and zona fasciculata/reticularis cells, respectively, as well as aldosterone and cortisol production by dispersed human adrenocortical cells. Maximal effective concentration was 10(-5) M, and elicited about 2.5-3.0-fold rises in steroid output. 10(-5) M AACOCF3 also enhanced submaximally (10(-15)/10(-12) M), but not maximally (10(-9) M) ACTH-stimulated hormonal secretion. Quantitative HPLC showed that 10(-5) M AACOCF3 evokes similar increases (from 2.0- to 3.0-fold) in the basal release of the entire spectrum of adrenocortical steroids (i.e. both intermediate and definitive products of steroid synthesis), thereby suggesting that AACOCF3 acts on the early steps of steroid synthesis. Accordingly, when pregnenolone metabolism is prevented by cyanoketone, 10(-5) M AACOCF3 increased by about 8-10-fold the production of this steroid. In conclusion, we have demonstrated a side-effect of AACOCF3, which may become relevant in studies where this chemical is used to inhibit PLA2 in tissues able to convert cholesterol to pregnenolone.     

The Met-enkephalin analog D-Ala2-Met-enkephalinamide decreases the adrenocortical response to ACTH in dispersed rat adrenal cells

The effects of the Met-enkephalin analog D-Ala²-Met-enkephalinamide (DALA) on basal and ACTH-stimulated corticosterone secretion from dispersed adrenal cells were investigated. Low doses (10^?10 and 10^?12 M) of DALA resulted in no apparent alteration in the response to ACTH (8×10^?9, 3.2×10^?8 or 1.6×10^?7 M). High doses of DALA (10^?8 and 10^?6 M) produced a decline in the steroidogenic response to ACTH. The opiate receptor antagonist naloxone (10^?4–10^?10 M) did not influence the basal production of corticosterone or the stimulating action exerted by ACTH. However, the presence of naloxone reversed the blocking action on corticosterone production that was exerted by DALA. These findings indicate that enkephalins may decrease adrenocortical responsiveness to ACTH.     

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R, leptin expression and cytokine secretion by human hematopoietic malignant cells

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.     

Seasonal alterations in adrenocortical cell function associated with stress-responsiveness and sex in the eastern fence lizard (Sceloporus undulatus)

We characterized steroidogenic properties of dispersed adrenocortical cells from field-active male and female eastern fence lizards (Sceloporus undulatus) to investigate whether alterations in cell function could, in part, explain seasonal variation in baseline and stress-induced plasma corticosterone (B). Lizards were collected during the breeding and postbreeding seasons and shortly prior to hibernation. Dispersed cells in vitro produced B, aldosterone (ALDO), and progesterone in response to 8-Br-cAMP, 25-(OH)cholesterol, adrenocorticotropin (ACTH; as little as 100 fM), and angiotensin II. Maximal progesterone, B, and ALDO responses to ACTH were roughly 1000%, 500%, and 100% greater than corresponding basal values. Angiotensin II was an effective steroidogenic stimulant but much less so than ACTH. Corticosteroid production exhibited considerable steroid-specific variation among seasons. Maximal ACTH-induced B production was lower in the postbreeding season than at either of the other two measurement points, essentially opposite to the pattern for ALDO. Males and females generally produced B at similar rates, but ALDO and progesterone showed numerous sex differences that usually covaried between the two steroids. Cellular sensitivity to 25-(OH)cholesterol and angiotensin II showed few sex differences or seasonal changes. In contrast, sensitivity to ACTH decreased markedly from the breeding to the postbreeding season in males, corresponding to the decrease in stress-responsiveness, and in both sexes was considerably lower prior to hibernation than during the breeding season. Under some conditions, plasma B may be limited by the production capacity of adrenocortical cells. In summary, seasonal variations in body condition, reproductive activity, and baseline and stress-induced plasma B may be attributed at least in part to alterations in adrenocortical cell steroidogenic function.     

Functional and structural differences between cultured outer and inner layer cells of bovine adrenal cortex

Summary Outer and inner layer cells of bovine adrenal cortex were cultured separately to compare cellular structural characteristics and functional differences. Outer layer cells were polygonal in shape with radially distributed lipid droplets in the cytoplasm, and produced mainly aldosterone and cortisol. The aldosterone production increased upon stimulation with angiotensin II or dibutyryl-cAMP. In contrast, inner layer cells were spindle-shaped and had fine diffused lipid droplets. They produced four times as much cortisol as outer layer cells but no aldosterone. Cortisol production increased with ACTH or dibutyryl-cAMP stimulation.When stimulated by ACTH or by dibutyryl-cAMP, both types of adrenocortical cells showed cellular retraction whereby the number of cytoplasmic lipid droplets decreased and microvilli on the cellular surface increased. At the same time, the transverse distribution of actin fibers disappeared and the microtubules changed their distribution pattern from circular to radial. Stimulation by angiotensin II, on the other hand, brought no marked structural changes.These results indicate that, in functional terms, the outer layer cells and the inner layer cells in this culture system reflect zona glomerulosa and zona fasciculata-reticularis, respectively.     

Effects of cell-type specific leptin receptor mutation on leptin transport across the BBB

The functions of leptin receptors (LRs) are cell-type specific. At the blood-brain barrier, LRs mediate leptin transport that is essential for its CNS actions, and both endothelial and astrocytic LRs may be involved. To test this, we generated endothelia specific LR knockout (ELKO) and astrocyte specific LR knockout (ALKO) mice. ELKO mice were derived from a cross of Tie2-cre recombinase mice with LR-floxed mice, whereas ALKO mice were generated by a cross of GFAP-cre with LR-floxed mice, yielding mutant transmembrane LRs without signaling functions in endothelial cells and astrocytes, respectively. The ELKO mutation did not affect leptin half-life in blood or apparent influx rate to the brain and spinal cord, though there was an increase of brain parenchymal uptake of leptin after in situ brain perfusion. Similarly, the ALKO mutation did not affect blood-brain barrier permeation of leptin or its degradation in blood and brain. The results support our observation from cellular studies that membrane-bound truncated LRs are fully efficient in transporting leptin, and that basal levels of astrocytic LRs do not affect leptin transport across the endothelial monolayer. Nonetheless, the absence of leptin signaling at the BBB appears to enhance the availability of leptin to CNS parenchyma. The ELKO and ALKO mice provide new models to determine the dynamic regulation of leptin transport in metabolic and inflammatory disorders where cellular distribution of LRs is shifted.     

Preferential effects of leptin on CD4 T cells in central and peripheral immune system are critically linked to the expression of leptin receptor

Leptin can enhance thymopoiesis and modulate the T-cell immune response. However, it remains controversial whether these effects correlate with the expression of leptin receptor, ObR. We herein addressed this issue by using in vivo animal models and in vitro culture systems. Leptin treatment in both ob/ob mice and normal young mice induced increases of CD4 SP thymocytes in thymus and CD4 T cells in the periphery. Interestingly, expression of the long form ObR was significantly restricted to DN, DP and CD4 SP, but not CD8 SP thymocytes. Moreover, in the reaggregated DP thymocyte cultures with leptin plus TSCs, leptin profoundly induced differentiation of CD4 SP but not CD8 SP thymocytes, suggesting that the effects of leptin on thymocyte differentiation might be closely related to the expression of leptin receptor in developing thymocytes. Surprisingly, ObR expression was markedly higher in peripheral CD4 T cells than that in CD8 T cells. Furthermore, leptin treatment with or without IL-2 and PHA had preferential effects on cell proliferation of CD4 T cells compared to that of CD8 T cells. Collectively, these data provide evidence that the effects of leptin on differentiation and proliferation of CD4 T cells might be closely related to the expression of leptin receptor.     

Investigations into the mechanism of hormone release by rat adrenocortical cells

Summary Vinblastine treatment blocks corticosterone release from rat adrenal zona fasciculata without impairing hormone synthesis, and induces the formation of acid phosphatase-positive granular clumps at the juxta-sinusoidal pole of the cells. Autoradiography shows that ACTH administration to vinblastine-treated animals mobilizes the 3H-cholesterol stored in the lipid droplets and leads to a noticeable labelling of the granular clumps. The possible significance of these granules is discussed.     

Cholecystokinin synthesizes and secretes leptin in isolated canine gastric chief cells

It is well recognized that a product of obese (ob) locus and body weight control hormone, leptin, acts on both short-term satiety for meal-induced termination of food intake (gastric phase) and long-term satiety for energy expenditure via the hypothalamus. The considerable sources of leptin are chief cells for gastric phase and adipocytes for the long-term satiety. The objective of this study was to demonstrate if CCK enhances leptin synthesis and secretion in isolated canine gastric chief cells. Confocal immunofluorescence studies showed that the CCK-A receptor and leptin were colocalized in the endoplasm. Western blotting demonstrated that canine chief cells expressed the leptin peptide and its protein level was enhanced by CCK treatment. An ELISA further showed that CCK dose-dependently secreted leptin from isolated canine chief cells. This was reproduced by the high-affinity CCK-A receptor agonist, CCK-OPE. These results indicate that canine chief cells synthesize and secrete leptin in response to CCK via the high-affinity state of the CCK-A receptor.     

Effects of chronic hypogonadism on corticosterone secretion and cyclic AMP production in male rat adrenocortical cells

AIM: To determine the secretion of corticosterone (CCS) both in vivo and in vitro during different intervals after orchidectomy in male rats. METHODS: Three- and 12-month-old rats had been orchidectomized 0, 3, 6, or 9 months before decapitation. RESULTS: Orchidectomy increased the concentrations of plasma CCS, the basal release of CCS, and the adenosine 3', 5'-cyclic monophosphate (cAMP) production in rat zona fasciculata reticularis (ZFR) cells. The forskolin/3-isobutyl-l-methylxanthine-stimulated releases of CCS and cAMP production by ZFR cells were higher in rats with chronic hypogonadism. The CCS release from ZFR cells of orchidectomized rat was not altered by 8-bromo-cAMP treatment. Orchidectomy enhanced the stimulatory effect of deoxycorticosterone on CCS release in ZFR cells. CONCLUSION: These results suggest that orchidectomy-related increases of CCS secretion in rats are associated with an increase of adenylate cyclase activity, cAMP generation, and 11-beta-hydroxylase activity in ZFR cells.     

The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary The choroid plexus consists of the choroidal epithelium, a derivative of the neural tube, and the choroidal stroma, which originates from the embryonic head mesenchyme. This study deals with epithelio-mesenchymal interactions of these two components leading to the formation of the organ. Grafting experiments of the prospective components have been performed using the quail-chicken marker technique. Prospective choroidal epithelium of quail embryos, forced to interact with mesenchyme of the body wall of chicken embryos, gives rise to a choroid plexus showing normal morphogenesis and differentiation. The choroidal epithelium induces the differentiation of organtypical fenestrated capillaries, which are highly permeable to intravenously injected horseradish peroxidase. The choroidal epithelium of the grafts constitutes a blood-cerebrospinal fluid barrier. On top of the choroidal epithelium, there are epiplexus cells displaying a typical ultrastructure. The experimental results show that these cells do not originate from the transplanted neural epithelium. Prospective choroidal stroma of chicken embryos does not exert a choroid plexus-inducing influence upon a quail embryo's neural epithelium isolated from parts of the brain that normally do not develop a choroid plexus. The experiments show that the choroidal epithelial cells are determined at least three days before the first organ anlage is detectable.This work was supported by the Deutsche Forschungsgemeinschaft (grant Ch 44/7-1)     

Dual effect of antimitotic drugs on steroid secretion in mouse adrenocortical Y-1 tumor cells

Mouse adrenocortical Y-1 tumor cells were examined in a monolayer culture and their steroid secretion was measured. The Y-1 cells constantly released a small amount of steroids in the absence of adrenocorticotropin (ACTH). When synthetic ACTH (tetracosactide acetate) was added to the medium, an increase in the steroid secretion of approximately 5-fold was observed. The Y-1 cells also showed a typical cytoplasmic retraction in response to ACTH. Incubation of the cells with an antimitotic drug, colchicine, prior to ACTH-stimulation resulted in a 30-50% reduction in ACTH-induced steroid secretion. Under the conditions used in these experiments, viable numbers of cell and of total amount of protein per dish were not measurably changed, indicating that the condition was not lethal. Another antimitotic drug, colcemid, caused similar reactions, while lumicolchicine showed no effect. This suggests that the disruption of the microtubular system is the main cause of the inhibition. On the other hand, the ACTH-independent secretion was slightly enhanced by colchicine. The enhancement was also observed in prolonged incubation with colchicine, a condition which caused death in some of the cells.