Human Adrenal Cells in Culture Produce Both Ouabain-like and Dihydroouabain-like Factors

Abstract Background: Ouabain-like factor (OLF) and its newly discovered reduced species, dihydroouabain-like factor (Dh-OLF), are mammalian cardenolides whose structural and functional characteristics are similar to the plant-derived compounds ouabain and dihydroouabain. These endogenous compounds are believed to be produced by the adrenals and to constitute part of an hormonal axis that may regulate the catalytic activity of the α-subunit of Na+,K+-ATPase. We developed antibodies sufficiently specific to distinguish between OLF and Dh-OLF, and in this study demonstrate the selective secretion of OLF and Dh-OLF from human H295R-1 adrenocortical cells in culture. Methods: We used reversed-phase HPLC, inhibition of Na+,K+-ATPase catalytic activity, and two enzyme immunoassays developed with antibodies specific to ouabain and dihydroouabain to purify and characterize the secretion of these two compounds by human adrenal cells in culture. Purified antisera had high titers (1 × 106 for ouabain and 8 ×105 for dihydroouabain) and were specific to their corresponding antigens. Results: Human H295R-1 cells grown in serum-free medium secreted 0.18 ± 0.03 pmol of OLF and 0.39 ± 0.04 pmol of Dh-OLF per 106 cells in 24 h. Both OLF and Dh-OLF inhibited the ouabain-sensitive catalytic activity of the sodium pump (0.03 μmol/L OLF inhibited 29% of the catalytic activity; 0.07 μmol/L Dh-OLF inhibited 17%). Stimulation of the cell culture by dibutryl cAMP increased the secretion of Dh-OLF 50% over control (unstimulated), whereas the secretion of OLF did not increase significantly. Conclusions: OLF and Dh-OLF are secreted by human adrenal cells, and antibodies specific to these two compounds can be developed, using the plant-derived counterparts as antigens. The secretion of Dh-OLF is responsive to a cAMP-dependent stimulation mechanism, whereas OLF is not. Our data suggest that either the secretory or biosynthetic pathways for production of these two compounds by human adrenal cells may have different control mechanisms or that they may be linked via a precursor–product relationship.

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Stimulation of steroidogenesis in cultured rat adrenocortical cells by unsaturated fatty acids

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1995.268.6.r1484 ◽

1995 ◽

Vol 268 (6) ◽

pp. R1484-R1490 ◽

Cited By ~ 5

Author(s):

I. Sarel ◽

E. P. Widmaier

Keyword(s):

Fatty Acids ◽

Unsaturated Fatty Acids ◽

Lipid Homeostasis ◽

Control Mechanisms ◽

Adrenocortical Cells ◽

Stimulatory Action ◽

Apparent Lack ◽

Naturally Occurring ◽

Oleic And Linoleic Acids ◽

Stimulation Of

The hypothesis that the stimulatory action of free fatty acids (FFA) in the hypothalamic-pituitary-adrenocortical (HPA) axis occurs in part at the adrenal cortex was evaluated. Pathophysiological concentrations of oleic and linoleic acids, but not stearic or caprylic acid, stimulated steroidogenesis from cultured rat adrenocortical cells (concentrations eliciting 50% of maximal responses, approximately 60 and 120 microM, respectively), with a latency of 90 min. Maximal stimulation of steroidogenesis by both acids was < 50% of that produced by adrenocorticotropic hormone (ACTH) and was blocked by cycloheximide. The maximal steroidogenic response to ACTH was inhibited approximately 50% by oleic acid. The actions of oleic and linoleic acids were not associated with an increase in adenosine 3',5'-cyclic monophosphate (cAMP) secretion but appeared to require intracellular oxidation. None of the lipids influenced cell viability or corticosterone radioimmunoassay. The latency of the steroidogenic response, the putative requirement for intracellular oxidation, and the apparent lack of involvement of cAMP suggest a mechanism of action of FFA distinct from that of ACTH, yet still requiring protein synthesis. It is concluded that the modulation of steroidogenesis by these abundant naturally occurring lipids may be an important component of the control mechanisms within the HPA pathway in disorders of lipid homeostasis (e.g., obesity, starvation, or diabetes).

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ACTH-dependent stimulation of a specific peptide in adrenocortical cells in culture

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(77)90988-3 ◽

1977 ◽

Vol 76 (4) ◽

pp. 1238-1246 ◽

Cited By ~ 16

Author(s):

A. Dazord ◽

D. Gallet ◽

J.M. Saez

Keyword(s):

Adrenocortical Cells ◽

Cells In Culture ◽

Specific Peptide ◽

Stimulation Of

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An antioxidant drug, silibinin, modulates steroid secretion in human pathological adrenocortical cells

Journal of Endocrinology ◽

10.1677/joe.0.1240341 ◽

1990 ◽

Vol 124 (2) ◽

pp. 341-345 ◽

Cited By ~ 14

Author(s):

K. Rácz ◽

J. Fehér ◽

G. Csomós ◽

I. Varga ◽

R. Kiss ◽

...

Keyword(s):

Antioxidant Property ◽

High Concentration ◽

Dual Effect ◽

Adrenocortical Cells ◽

Adrenal Cells ◽

Aldosterone Producing Adenoma ◽

Dose Dependent ◽

Cytochrome P 450 ◽

Corticosteroid Secretion ◽

Stimulation Of

ABSTRACT Because human adrenocortical cells from different adrenal disorders exhibit pathologically altered corticosteroid synthesis, and free radical mechanisms may induce pathological changes in the activities of corticosteroid biosynthetic enzymes (cytochrome P-450), we examined the effect of an antioxidant, silibinin, on basal and ACTH-stimulated secretion of several corticosteroids in isolated adrenal cells from an aldosterone-producing adenoma, atrophied adrenal tissues surrounding the adenoma, and hyperplastic adrenals from Cushing's syndrome. In the presence of a high concentration (100 μmol/l) of silibinin, variably diminished secretion of basal aldosterone, corticosterone, cortisol, 18-OH-corticosterone and 11-deoxycorticosterone was found. In contrast, the addition of 0·01 μmol silibinin/l, which failed to produce a clear effect on basal corticosteroid secretion, resulted in a potentiation of ACTH-stimulated secretion of several corticosteroids in the adenomatous and hyperplastic adrenocortical cells. These results suggest that the dose-dependent dual effect of silibinin on corticosteroid secretion may be attributed to corresponding changes in the activities of cytochrome P-450 enzymes, and that stimulation of ACTH-induced corticosteroidogenesis by silibinin is presumably due to the antioxidant property of the drug. Journal of Endocrinology (1990) 124, 341–345

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Faculty Opinions recommendation of MRGPRX2 is negatively targeted by SCF and IL-4 to diminish pseudo-allergic stimulation of skin mast cells in culture.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733793504.793571843 ◽

2020 ◽

Author(s):

Toshiaki Kawakami

Keyword(s):

Mast Cells ◽

Cells In Culture ◽

Stimulation Of

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Endothelial Stimulation of Sodium Pump in Cultured Vascular Smooth Muscle

Hypertension ◽

10.1161/01.hyp.26.1.177 ◽

1995 ◽

Vol 26 (1) ◽

pp. 177-185 ◽

Cited By ~ 8

Author(s):

Juliana Redondo ◽

Concepción Peiró ◽

Leocadio Rodríguez-Mañas ◽

Mercedes Salaices ◽

Jesús Marín ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Sodium Pump ◽

Stimulation Of

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Role of pro-opiomelanocortin-derived peptides in the regulation of steroid production by human fetal adrenal cells in culture

Journal of Endocrinology ◽

10.1677/joe.0.0970357 ◽

1983 ◽

Vol 97 (3) ◽

pp. 357-367 ◽

Cited By ~ 15

Author(s):

Andrew Baird ◽

K. W. Kan ◽

Samuel Solomon

Keyword(s):

Peptide Hormones ◽

The Other ◽

Dehydroepiandrosterone Sulphate ◽

Steroid Production ◽

Adrenal Cells ◽

The Third ◽

Cells In Culture

Synthetic (1–39)ACTH, (1–24)ACTH, (18–39)ACTH, α-MSH, met-enkephalin and α-, βand γ-endorphin were tested for their ability to stimulate steroidogenesis by human fetal adrenal cells in culture. Adrenal cells were incubated with peptide hormones for two periods of 24 h. On the third day of the experiment the cells were incubated with progesterone (4 μg/2 ml) for 8 h. At the doses tested only (1–39)ACTH, (1–24)ACTH and α-MSH stimulated steroidogenesis. None of the other peptides had any corticotrophic effect on the formation of cortisol, corticosterone or dehydroepiandrosterone sulphate (DHAS). At the highest doses tested, α-MSH (100 μg/2 ml) had a corticotrophic effect that was not different from that obtained with 20 ng (1–39)ACTH or (1–24)ACTH. At the lower doses (0·2–2 μg/2 ml), α-MSH stimulated the formation of DHAS (P<0·01) without stimulating the formation of cortisol.

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Magnesium stimulation of catalytic activity of horse liver aldehyde dehydrogenase. Changes in molecular weight and catalytic sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)70631-0 ◽

1980 ◽

Vol 255 (17) ◽

pp. 8206-8209

Author(s):

K. Takahashi ◽

H. Weiner

Keyword(s):

Molecular Weight ◽

Catalytic Activity ◽

Aldehyde Dehydrogenase ◽

Horse Liver ◽

Catalytic Sites ◽

Liver Aldehyde Dehydrogenase ◽

Stimulation Of

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Interleukin-8 Synthesis, Regulation, and Steroidogenic Role in H295R Human Adrenocortical Cells

Endocrinology ◽

10.1210/en.2005-0951 ◽

2006 ◽

Vol 147 (2) ◽

pp. 891-898 ◽

Cited By ~ 22

Author(s):

Damian G. Romero ◽

Gaston R. Vergara ◽

Zheng Zhu ◽

Gina S. Covington ◽

Maria W. Plonczynski ◽

...

Keyword(s):

Immune System ◽

Angiotensin Ii ◽

Adrenal Gland ◽

Interferon Γ ◽

Cell Culture Supernatant ◽

Adrenocortical Cells ◽

Adrenal Cells ◽

Endothelin 1 ◽

H295r Cells ◽

First Time

The adrenal gland secretes several cytokines, and cytokines modulate steroid secretion by this gland. In this study, a survey of cytokine production by H295R human adrenocortical cells demonstrated that these cells secreted IL-2, IL-4, IL-8, IL-10, IL-13, and TNFα but not IL-5, IL-12, or interferon-γ. IL-8 was the IL secreted at higher concentration. IL-8 secretion, its regulation, and role in steroidogenesis were further studied. Secreted ILs and steroids were measured by ELISA in cell culture supernatant. IL-8 mRNA was quantified by real-time RT-PCR. H295R cells and human adrenal gland expressed IL-8 mRNA. Angiotensin II, potassium, endothelin-1, IL-1α, IL-1β, TNFα, and Escherichia coli lipopolysaccharide dose-dependently increase IL-8 secretion by H295R cells after 24 h incubation. IL-6 had no effect on IL-8 secretion. Angiotensin II time-dependently increased IL-8 secretion by H295R cells up to 48 h. Angiotensin II caused a biphasic increase in IL-8 mRNA expression with a peak 6 h after stimulation. TNFα synergized angiotensin II, potassium, and IL-1α-mediated IL-8 secretion. IL-8 did not modify aldosterone or cortisol secretion by H295R cells under basal or stimulated (angiotensin II or potassium) conditions. In conclusion, it is demonstrated for the first time that human adrenal cells expressed and secreted IL-8 under the regulation of angiotensin II, potassium, endothelin-1, and immune peptides. Adrenal-secreted IL-8 is one point of convergence between the adrenal gland and the immune system and may have relevance in physiological and pathophysiological conditions associated with increased levels of aldosterone secretagogues and the immune system.

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Swelling-activated transport of taurine in cultured gill cells of sea bass: physiological adaptation and pavement cell plasticity

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.90615.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. R1149-R1160 ◽

Cited By ~ 17

Author(s):

Martine Avella ◽

Olivier Ducoudret ◽

Didier F. Pisani ◽

Philippe Poujeol

Keyword(s):

Sea Bass ◽

Physiological Adaptation ◽

Dimensional Structure ◽

Taurine Transport ◽

Hypotonic Shock ◽

Cells In Culture ◽

Membrane Stretch ◽

Gill Cells ◽

Fold Stimulation ◽

Stimulation Of

We have investigated volume-activated taurine transport and ultrastructural swelling response of sea bass gill cells in culture, assuming that euryhaline fish may have developed particularly efficient mechanisms of salinity adaptation. In vivo, when sea basses were progressively transferred from seawater to freshwater, we noticed a decrease in blood osmotic pressure. When gill cells in culture were subjected to 30% hypotonic shock, we observed a five-fold stimulation of [3H]taurine efflux. This transport was reduced by various anion channel inhibitors with the following efficiency: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid > DIDS = diphenylamine-2-carboxylic acid. With polarized gill cells in culture, the hypotonic shock produced a five-fold stimulation of apical taurine transport, whereas basolateral exit was 25 times higher. Experiments using ionomycin, thapsigargin, BAPTA-AM, or removal of extracellular calcium suggested that taurine transport was regulated by external calcium. The inhibitory effects of lanthanum and streptomycin support Ca2+ entry through mechanosensitive Ca2+ channels. Branchial cells also showed hypotonically activated anionic currents sensitive to DIDS and NPPB. Similar pharmacology and time course suggested the potential existence of a common pathway for osmosensitive taurine and Cl− efflux through volume-sensitive organic osmolyte and anion channels. A three-dimensional structure study revealed that respiratory gill cells began to swell only 15 s after hypoosmotic shock. Apical microridges showed membrane outfoldings: the cell surface became smoother with a progressive disappearance of ridges. Therefore, osmotic swelling may not actually induce membrane stretch per se, inasmuch as the microridges may provide a reserve of surface area. This work demonstrates mechanisms of functional and morphological plasticity of branchial cells during osmotic stress.

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