Inhibitory effects of digoxin and ouabain on aldosterone synthesis in human adrenocortical NCI‐H295 cells

Abstract

The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI-H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)-stimulated release of aldosterone by NCI-H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11beta-hydroxylase, P450c11beta) to cortisol were reduced by digoxin or ouabain. The basal and 22-hydroxy-cholesterol (a membrane-permeable cholesterol, substrate of cytochrome P450 side-chain cleavage enzyme, P450scc)-stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II-stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI-H295 cells. Digoxin or ouabain (10(-6) M, 24 h)-treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10(-6) M digoxin or ouabain, the digoxin- or ouabain-induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11beta and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI-H295 cells.

Adrenal toxicology: a strategy for assessment of functional toxicity to the adrenal cortex and steroidogenesis

“…Li and Wang, 2005), functional corticotrophin-releasing hormone (CRH) receptors (Willenberg et al, 2005) and respond to forskolin and isobutyl methylxanthine cyclic AMP induction and dibutyryl cyclic AMP by stimulated corticosteroid synthesis (Xu et al, 2003). They also respond to angiotensin II by production of aldosterone (Kau et al, 2005), vasoactive intestinal peptide (VIP) by production of cortisol (Nicol et al, 2004), have functional atrial natriuretic peptide (ANP) receptors (Bodart et al, 1996), have functional luteinizing hormone (LH)/chorionic gonadotrophin (hCG) receptors (Rao et al, 2004) and respond to activin A (activins and inhibins are related glycoproteins that modulate pituitary follicle stimulating hormone (FSH) and consequently sex steroid production) by decreased sex steroid secretion (Vanttinen et al, 2003). H295R cells are also reported to respond to tumour necrosis factor which increases steroidogenesis (Mikhaylova et al, 2007) and epidermal growth factor and prostaglandins (Watanabe et al, 2006).…”

“…Although these mechanisms are largely relevant to in vivo adrenal toxicology and consequent pathology, effective and relevant in vitro models, such as the H295R cell line, have only very recently been developed and these are now expanding the range of known adrenocortical toxicants and molecular mechanisms of action (e.g. Ohno et al, 2002;Sanderson et al, 2002Sanderson et al, , 2004Li et al, 2004;Hilscherova et al, 2004;Voets et al, 2004;Zhang et al, 2005;Kau et al, 2005;MullerVieira et al, 2005;Li and Wang, 2005;Blaha et al, 2006;Lin et al, 2006;Xu et al, 2006;Hecker et al, 2006;Gracia et al, 2006;Canton et al, 2006;Imagawa et al, 2006;Oskarsson et al, 2006;Sanderson, 2006; see discussion in Harvey and Everett, 2003).…”

Digoxin and ouabain increase the synthesis of cholesterol in human liver cells

“…Their radioactivity was measured by liquid scintillation, using a Tri-Carb Liquid Scintillation Analyzer (PerkinElmer, Waltham, MA). Cholesterol and ubiquinone synthesis was expressed as fmol [ 3 …”

“…For instance, digoxin has been shown to regulate the transcription of several genes, such as cytochrome P450 CYP3A4, multidrug resistance-1 and steroid xenobiotic receptor, by unknown mechanisms [2]. Moreover, both digoxin and ouabain modulate the synthesis of corticosteroid hormones in human and rat adrenal glands [3 -5], by regulating the activity of several enzymes, such as the steroidogenic acute regulatory protein and the cytochrome P450 side chain cleavage enzyme [3,5]. These results are in keeping with other experimental observations, showing that the synthesis of steroid hormones is often controlled by the end products of the pathway or by endogenous/exogenous steroid compounds [6].…”

Ins and Outs of Aldosterone-Producing Adenomas of the Adrenal

“…The classic ATPase inhibitors, ouabain and digoxin, do not cause sustained increases in aldosterone production from human cells-if anything, the opposite. 12 The question whether particular mutations activate an inward current, or block an outward current, may seem more exciting to a few scientists than to clinicians managing hypertension. But this would miss the point that novel gain-of-function mutations can confirm long-standing hypotheses and disclose unexpected mechanisms regulating normal physiology.…”