Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes

doi:10.1016/0960-0760(95)00132-J

The Journal of Steroid Biochemistry and Molecular Biology

Volume 54, Issues 5–6, September 1995, Pages 227-235

https://doi.org/10.1016/0960-0760(95)00132-J Get rights and content

Abstract

The purpose of the present study was to characterize the transport of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) into hepatocytes at physiological and pharmacological concentrations. Hepatocytes were isolated from female Sprague-Dawley rats by collagenase perfusion. Uptake of [³H]DHEA and [³H]DHEAS at increasing concentrations (3.5 nM-100 μM) was measured by the rapid filtration technique at 30 s intervals up to 120 s. The uptake of DHEAS by hepatocytes was saturable (K_m = 17.0 μM; V_max = 3.7 nmol/min/mg cell protein). In contrast, a specific saturable transport system for DHEA could not be detected in rat hepatocytes. It is suggested that DHEA enters the cell by diffusion. The uptake of DHEAS could be inhibited by antimycin A, carbonylcyanide-m-chlorophenylhydrazone, and dinitrophenol (inhibitors of the mitochondrial respiratory chain), by dinitrofluorobenzene and p-hydroxymercuribenzoate (NH₂- and SH-blockers, respectively), and by monensin (Na⁺-specific ionophore). No inhibition was seen in the presence of ouabain (inhibitor of Na⁺-K⁺-ATPase) and phalloidin (inhibitor of cholate transport and actin-blocker). Interestingly, DHEAS uptake was inhibited by bile acids (cholate, taurocholate and glycocholate). Conversely, [³H]cholate uptake was strongly inhibited by DHEAS, which indicates a competition for the same carrier. Replacement of sodium ion with choline markedly decreased uptake velocity at pharmacological DHEAS concentrations. The results suggest that DHEAS uptake is a saturable, energy-dependent, carrier-mediated, partially Na⁺-dependent process, and that DHEAS may be taken up via the multispecific bile acid transport system.

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Endogenous steroids, including sex hormones and bile acids, are a group of essential compounds with various biological functions. In this study, we developed an LC-MS method that simultaneously measures 14 sex hormones and metabolites (SH) and 32 bile acids (BA) in rat plasma. Multiple innovative approaches were applied to increase the sensitivity and specificity, including optimization of the mobile phases, gradients, and dynamic multiple reaction monitoring (DMRM) transitions. The method was validated and applied on plasma samples from pregnant rats before and 0.5 h after oral glucose tolerance test (OGTT) at gestational days 0.5 and 18.5. Results showed that the method was applicable, and 9 SH and 30 BA were measurable in the samples. In summary, this method is applicable in studies on SH and BA in rat plasma, and may also be used on other matrix and species.
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The liver is the primary site of drug metabolism in the body. Typically, metabolic conversion of a drug results in inactivation, detoxification, and enhanced likelihood for excretion in urine or feces. Sulfation, glucuronidation, and glutathione conjugation represent the three most prevalent classes of phase II metabolism, which may occur directly on the parent compounds that contain appropriate structural motifs, or, as is usually the case, on functional groups added or exposed by phase I oxidation. These three conjugation reactions increase the molecular weight and water solubility of the compound, in addition to adding a negative charge to the molecule. As a result of these changes in the physicochemical properties, phase II conjugates tend to have very poor membrane permeability, and necessitate carrier-mediated transport for biliary or hepatic basolateral excretion into sinusoidal blood for eventual excretion into urine. This review summarizes sulfation, glucuronidation, and glutathione conjugation reactions, as well as recent progress in elucidating the hepatic transport mechanisms responsible for the excretion of these conjugates from the liver. The discussion focuses on alterations of metabolism and transport by chemical modulators, and disease states, as well as pharmacodynamic and toxicological implications of hepatic metabolism and/or transport modulation for certain active phase II conjugates. A brief discussion of issues that must be considered in the design and interpretation of phase II metabolite transport studies follows.
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Dehydroepiandrosterone (DHEA), produced from cholesterol in the adrenals, is the most abundant steroid in our circulation. It is present almost entirely as the sulfate ester, but the free steroid is the form that serves as a precursor of estrogens and androgens, as well as 7‐ and 16‐oxygenated dervatives. Mammalian tissues reduce the 17‐keto Group of DHEA to produce androstenediol—a weak estrogen and full‐fledged androgen. Its androgen activity is not inhibited by the anti‐androgens commonly used to treat prostate cancer. It is probably responsible for the growth of therapy‐resistant prostate cancer. DHEA is hydroxylated at the 7α position, and this derivative is oxidized by 11β‐hydroxysteroid dehydrogenase to form 7‐keto DHEA. The latter is reduced by the same dehydrogenase to form 7β‐hydroxy DHEA. When fed to rats, each of the latter three steroids induce the formation of two thermogenic enzymes in the liver. The late‐term human fetus produces relatively large amounts of 16αhydroxy DHEA, which serves the mother as a precursor of estriol.
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Dehydroepiandrosterone (DHEA) has been reported to have diverse effects on overall physiology, although its mechanism of action and specific receptor are not yet known. We have used the immortalized, clonal GT1–7 hypothalamic neurons to study DHEA effects on gonadotropin-releasing hormone (GnRH) gene expression. DHEA (10⁻⁴ M) downregulates GnRH transcription by 39, 70 and 83% at 24, 36, and 48 h, respectively, while DHEA-sulphate had no effect. Hydroxyflutamide a specific androgen receptor (AR) antagonist, and cyproterone acetate or trilostane, both inhibitors of 3β-hydroxysteroid dehydrogenase/▵4,5 isomerase, the rate-limiting enzyme for the conversion of DHEA to sex steroids, did not affect the ability of DHEA to downregulate GnRH gene expression. We found that GT1–7 cells did not express aromatase, thereby precluding conversion to estrogen. Analysis of [¹⁴C] DHEA metabolism by thin layer chromatography indicates that the main metabolites produced are 7α- and 7β-hydroxy DHEA, and 7-oxo DHEA, although these steroids were not able to repress GnRH gene expression alone. Cell viability studies indicated that the transcriptional repression observed is not due to GT1–7 cell death. Interestingly, SV40 T-antigen mRNA levels, under the control of 2.3 kb of the rat GnRH gene 5′ regulatory region, are also repressed by DHEA. Our studies indicate that DHEA has direct effects on GnRH transcription that appear to be unique from those observed after conversion to other steroidogenic compounds.
Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Gα<inf>i2,3</inf>
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Many of these actions are reported at pharmacological concentrations, or with more prolonged incubations than we have used in our studies (60). In these situations the effects of lipophilic steroids on the plasma membrane (61-63) or the direct effects of intracellular steroid (64) may be of greater importance than in our study. Furthermore, many of the effects of high circulating levels of DHEA in vivo cannot be differentiated from the effects of DHEA metabolites, including estradiol or testosterone.
The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [³H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (K_d = 48.7 pm) and saturable (B_max = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5′-O-(3-thio)triphosphate (GTPγS) inhibited [³H]DHEA binding to plasma membranes by 69%, and DHEA increased [³⁵S]GTPγS binding by 157%. DHEA stimulated [³⁵S]GTPγS binding to Gα_i2 and Gα_i3, but not to Gα_i1 or Gα_o. Pretreatment of plasma membranes with antibody to Gα_i2 or Gα_i3, but not to Gα_i1, inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Gα_i2 and Gα_i3. These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.
A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liver-specific human organic anion transporting polypeptide and identification of rat and human hydroxymethylglutaryl-CoA reductase inhibitor transporters
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A novel human organic transporter, OATP2, has been identified that transports taurocholic acid, the adrenal androgen dehydroepiandrosterone sulfate, and thyroid hormone, as well as the hydroxymethylglutaryl-CoA reductase inhibitor, pravastatin. OATP2 is expressed exclusively in liver in contrast to all other known transporter subtypes that are found in both hepatic and nonhepatic tissues. OATP2 is considerably diverged from other family members, sharing only 42% sequence identity with the four other subtypes. Furthermore, unlike other subtypes, OATP2 did not transport digoxin or aldosterone. The rat isoform oatp1 was also shown to transport pravastatin, whereas other members of the OATP family, i.e.rat oatp2, human OATP, and the prostaglandin transporter, did not. Cis-inhibition studies indicate that both OATP2 and roatp1 also transport other statins including lovastatin, simvastatin, and atorvastatin. In summary, OATP2 is a novel organic anion transport protein that has overlapping but not identical substrate specificities with each of the other subtypes and, with its liver-specific expression, represents a functionally distinct OATP isoform. Furthermore, the identification of oatp1 and OATP2 as pravastatin transporters suggests that they are responsible for the hepatic uptake of this liver-specific hydroxymethylglutaryl-CoA reductase inhibitor in rat and man.

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Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes

Abstract

Adv. Cancer Res.

Adv. Enzyme Regul.

Arch. Biochem. Biophys.

Cancer Res.

Biochem. Biophys. Res. Commun.

Eur. J. Biochem.

Cytobiologie

Biometrics

J. Pharmac. Exp. Ther.

J. Pharmac. Exp. Ther.

Dehydroepiandrosterone (DHEA): important or very important? The last 30 years saga

Dehydroepiandrosterone and dehydroepiandrosterone sulfate: their relation to cardiovascular disease

Epidemiol. Rev.

Sex differences in dehydroepiandrosterone metabolism in the rat: different plasma levels following ingestion of DHEA-supplemented diet and different metabolite patterns in plasma, bile and urine

Horm. Metab. Res.

Physiological importance of dehydroepiandrosterone

Lancet

Biologic activity of dehydroepiandrosterone sulfate in man

J. Clin. Endocr. Metab.

Novel dehydroepiandrosterone analogues with enhanced biological activity and reduced side effects in mice and rats

Cancer Res.

Inhibition by dehydroepiandrosterone of liver preneoplastic foci formation in rats after initiation-selection in experimental carcinogenesis

Toxic. Path.