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• W1870049952 abstract "This study examined the in vivo relationship between expression of the HDL receptor scavenger receptor class B (SR-BI) and corresponding structural changes in the rat adrenocortical cell microvillar compartment. Using hormonal stimulation and withdrawal protocols, we were able to manipulate adrenal SR-BI levels and carry out qualitative and quantitative measurements correlating SR-BI expression with microvillar mass and microvillar channel formation. Young male rats were used as controls or treated with adrenocorticotropin hormone (ACTH) (24 h), 17α-ethinyl estradiol (17α-E2) (5 days), or dexamethasone (DEX) (24 h). Quantitative Western blot analysis and immunocytochemistry indicated that ACTH and 17α-E2 treatment greatly increased SR-BI expression in the adrenal (especially in the microvillar compartment of adrenocortical cells), whereas DEX treatment led to a decrease of SR-BI by all measurements. At the same time, striking ultrastructural changes occurred in the adrenocortical cell microvillar compartment: e.g., microvillar area and microvillar channel formation and complexity dramatically increased (compared with control values) after ACTH or 17α-E2 treatment, whereas the same values declined after DEX treatment.These measurements illustrate the exceptional flexibility and responsiveness of the microvillar compartment to hormonal stimuli, and suggest that regulation of SR-BI expression and structural configuration of the surface of steroidogenic cells goes hand in hand. This study examined the in vivo relationship between expression of the HDL receptor scavenger receptor class B (SR-BI) and corresponding structural changes in the rat adrenocortical cell microvillar compartment. Using hormonal stimulation and withdrawal protocols, we were able to manipulate adrenal SR-BI levels and carry out qualitative and quantitative measurements correlating SR-BI expression with microvillar mass and microvillar channel formation. Young male rats were used as controls or treated with adrenocorticotropin hormone (ACTH) (24 h), 17α-ethinyl estradiol (17α-E2) (5 days), or dexamethasone (DEX) (24 h). Quantitative Western blot analysis and immunocytochemistry indicated that ACTH and 17α-E2 treatment greatly increased SR-BI expression in the adrenal (especially in the microvillar compartment of adrenocortical cells), whereas DEX treatment led to a decrease of SR-BI by all measurements. At the same time, striking ultrastructural changes occurred in the adrenocortical cell microvillar compartment: e.g., microvillar area and microvillar channel formation and complexity dramatically increased (compared with control values) after ACTH or 17α-E2 treatment, whereas the same values declined after DEX treatment. These measurements illustrate the exceptional flexibility and responsiveness of the microvillar compartment to hormonal stimuli, and suggest that regulation of SR-BI expression and structural configuration of the surface of steroidogenic cells goes hand in hand. Tissues of the rat that require large amounts of exogeneous cholesterol for steroid hormone production (e.g., adrenal, ovary, and trophic hormone sensitized testes) obtain much of this cholesterol from circulating HDL. The major cholesterol uptake process used by these tissues is known as the selective cholesteryl ester (CE) uptake pathway (1Glass C. Pittman R.C. Weinstein D.B. Steinberg D. Dissociation of tissue uptake of cholesteryl ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesteryl ester to liver, adrenal, and gonad..Proc. Natl. Acad. Sci. USA. 1983; 80: 5435-5439Crossref PubMed Scopus (422) Google Scholar), a term that indicates that HDL-CEs are released directly into cells without cell internalization of the whole lipoprotein particle (2Reaven E. Chen Y-D.I. Spicher M. Azhar S. Morphological evidence that high density lipoproteins are not internalized by steroid-producing cells during in situ organ perfusion..J. Clin. Invest. 1984; 74: 1384-1397Crossref PubMed Scopus (94) Google Scholar, 3Reaven E. Chen Y-D.I. Spicher M. Hwang S-F. Mondon C.E. Azhar S. Uptake of low density lipoprotein by rat tissues: special emphasis on the luteinized ovary..J. Clin. Invest. 1986; 77: 1971-1984Crossref PubMed Scopus (35) Google Scholar, 4Pittman R.C. Knecht T.P. Rosenbaum M.S. Taylor Jr., C.M. A nonendocytic mechanism for the selective uptake of high density lipoprotein-associated cholesterol esters..J. Biol. Chem. 1987; 262: 2443-2450Abstract Full Text PDF PubMed Google Scholar, 5Azhar S. Stewart D. Reaven E. Utilization of cholesterol-rich lipoproteins by perfused rat adrenals..J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar, 6Gwynne J.T. Mahaffee D.D. Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester..J. Biol. Chem. 1989; 264: 8141-8150Abstract Full Text PDF PubMed Google Scholar, 7Reaven E. Tsai L. Azhar S. Cholesterol uptake by the 'selective’ pathway of ovarian granulosa cells: early intracellular events..J. Lipid Res. 1995; 36: 1602-1617Abstract Full Text PDF PubMed Google Scholar, 8Reaven E. Tsai L. Azhar S. Intracellular events in the “selective” transport of lipoprotein-derived cholesteryl esters..J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Although details of the molecular mechanisms by which cells can selectively internalize large quantities of neutral lipids are not yet clear, some aspects of the pathway have been established. Most importantly, it is known that a regulatable HDL receptor protein scavenger receptor class B type I (SR-BI) is found on the cell surface of a variety of rodent high cholesterol-requiring tissues (steroidogenic tissues, liver) (9Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat..J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (469) Google Scholar, 10Rigotti A. Edelman E.R. Seifert P. Iqbal S.N. DeMatto R.B. Temel R.E. Krieger M. Williams D.L. Regulation by adrenocorticotropic hormone of the in vivo expression of scavenger receptor class B type I (SR-BI), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland..J. Biol. Chem. 1996; 271: 33545-33549Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 11Wang N. Weng W. Breslow J.L. Tall A.R. Scavenger receptor BI (SR-BI) is up-regulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock-out mice as a response to depletion of cholesterol stores: in vivo evidence that SR-BI is a functional high density lipoprotein receptor under feedback control..J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 12Azhar S. Nomoto A. Leers-Sucheta S. Reaven E. Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model..J. Lipid Res. 1998; 39: 1616-1628Abstract Full Text Full Text PDF PubMed Google Scholar, 13Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian model..Endocrinology. 1998; 139: 2847-2856Crossref PubMed Scopus (0) Google Scholar, 14Reaven E. Lua Y. Nomoto A. Temel R. Williams D.L. van der Westhuyzen D.R. Azhar S. The selective pathway and a high-density lipoprotein receptor (SR-BI) in ovarian granulosa cells of the mouse..Biochim. Biophys. Acta. 1999; 1436: 565-576Crossref PubMed Scopus (41) Google Scholar, 15Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis..J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 16Fluiter K. van der Westhuijzen D.R. and T.J. van Berkel C. In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells..J. Biol. Chem. 1998; 273: 8434-8438Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and that cells that express high levels of SR-BI can efficiently obtain lipoprotein derived CEs for use in hormone or product synthesis (7Reaven E. Tsai L. Azhar S. Cholesterol uptake by the 'selective’ pathway of ovarian granulosa cells: early intracellular events..J. Lipid Res. 1995; 36: 1602-1617Abstract Full Text PDF PubMed Google Scholar, 8Reaven E. Tsai L. Azhar S. Intracellular events in the “selective” transport of lipoprotein-derived cholesteryl esters..J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 12Azhar S. Nomoto A. Leers-Sucheta S. Reaven E. Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model..J. Lipid Res. 1998; 39: 1616-1628Abstract Full Text Full Text PDF PubMed Google Scholar, 14Reaven E. Lua Y. Nomoto A. Temel R. Williams D.L. van der Westhuyzen D.R. Azhar S. The selective pathway and a high-density lipoprotein receptor (SR-BI) in ovarian granulosa cells of the mouse..Biochim. Biophys. Acta. 1999; 1436: 565-576Crossref PubMed Scopus (41) Google Scholar, 15Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis..J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 17Temel R.E. Trigatti B. DeMatto R.B. Azhar S. Krieger M. Williams D.L. Scavenger receptor class, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells..Proc. Natl. Acad. Sci. USA. 1997; 94: 13600-13605Crossref PubMed Scopus (209) Google Scholar, 18Sehayek E. Ono J.G. Shefer S. Nguyen L.B. Wang N. Batta A.K. Salen G. Smith J.D. Tall A.R. Breslow J.L. Biliary cholesterol excretion: a novel mechanism that regulates dietary cholesterol absorption..Proc. Natl. Acad. Sci. USA. 1998; 95: 10194-10199Crossref PubMed Scopus (153) Google Scholar, 19Mardones P. Quinones V. Amigo L. Moreno M. Miquel J.F. Schwarz M. Miettinen H.E. Trigatti B. Krieger M. VanPatten S. Cohen D.E. Rigotti A. Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice..J. Lipid Res. 2001; 42: 170-180Abstract Full Text Full Text PDF PubMed Google Scholar). Indeed, work from several laboratories using different adrenal, ovarian, testicular, or liver tissue models show an exceptionally tight association between expression of SR-BI protein and the direct measure of selective CE uptake under changing physiological conditions, including hormone stimulation, hormone inhibition, lipid levels, SR-BI over-expression, or genetic ablation (11Wang N. Weng W. Breslow J.L. Tall A.R. Scavenger receptor BI (SR-BI) is up-regulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock-out mice as a response to depletion of cholesterol stores: in vivo evidence that SR-BI is a functional high density lipoprotein receptor under feedback control..J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 12Azhar S. Nomoto A. Leers-Sucheta S. Reaven E. Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model..J. Lipid Res. 1998; 39: 1616-1628Abstract Full Text Full Text PDF PubMed Google Scholar, 15Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis..J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 20Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism..Proc. Natl. Acad. Sci. USA. 1997; 94: 12610-12615Crossref PubMed Scopus (756) Google Scholar, 21Vieira-van Bruggen D. Kalkman I. van Gent T. van Tol A. Jansen H. Induction of adrenal scavenger receptor BI and increased high density lipoprotein-cholesteryl ether uptake in vivo inhibition of hepatic lipase..J. Biol. Chem. 1998; 273: 32038-32041Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 22Wang N. Arai T. Ji Y. Rinninger F. Tall A.R. Liver-specific overexpression of scavenger receptor BI decreases levels of very low density lipoprotein apoB, low density lipoprotein apoB, and high density lipoprotein in transgenic mice..J. Biol. Chem. 1998; 273: 32920-32926Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 23Azhar S. Luo Y. Medicherla S. Reaven E. Up-regulation of selective cholesteryl ester uptake pathway in mice with deletion of low-density lipoprotein function..J. Cell. Physiol. 1999; 180: 190-202Crossref PubMed Scopus (28) Google Scholar, 24Spady D.K. Kearney D.M. Hobbs H.H. Polyunsaturated fatty acids up-regulate hepatic scavenger receptor BI (SR-BI) expression and HDL cholesteryl ester uptake in the hamster..J. Lipid Res. 1999; 40: 1384-1394Abstract Full Text Full Text PDF PubMed Google Scholar, 25Sun Y. Wang N. Tall A.R. Regulation of adrenal scavenger receptor-BI expression by ACTH and cellular cholesterol pools..J. Lipid Res. 1999; 40: 1799-1805Abstract Full Text Full Text PDF PubMed Google Scholar, 26Ueda Y. Gong E. Royer L. Cooper P.N. Francone O.L. Rubin E.M. Relationship between expression levels and atherogenesis in scavenger receptor class, type I transgenics..J. Biol. Chem. 2000; 275: 20368-20373Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 27Greene D.J. Skeggs J.W. Morton R.E. Elevated triglyceride content diminishes the capacity of high density lipoprotein to deliver cholesteryl esters via the scavenger receptor class B type I (SR-BI)..J. Biol. Chem. 2001; 276: 4804-4871Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). It is of interest that rodent steroidogenic cells that express high levels of SR-BI in vivo are endowed with an intricate surface microvillar system specialized for the trapping of lipoproteins (2Reaven E. Chen Y-D.I. Spicher M. Azhar S. Morphological evidence that high density lipoproteins are not internalized by steroid-producing cells during in situ organ perfusion..J. Clin. Invest. 1984; 74: 1384-1397Crossref PubMed Scopus (94) Google Scholar, 3Reaven E. Chen Y-D.I. Spicher M. Hwang S-F. Mondon C.E. Azhar S. Uptake of low density lipoprotein by rat tissues: special emphasis on the luteinized ovary..J. Clin. Invest. 1986; 77: 1971-1984Crossref PubMed Scopus (35) Google Scholar, 5Azhar S. Stewart D. Reaven E. Utilization of cholesterol-rich lipoproteins by perfused rat adrenals..J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar, 28Reaven E. Boyles J. Spicher M. Azhar S. Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary..Arteriosclerosis. 1988; 8: 298-309Crossref PubMed Google Scholar, 29Reaven E. Spicher M. Azhar S. Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells..J. Lipid Res. 1989; 30: 1551-1560Abstract Full Text PDF PubMed Google Scholar, 30Reaven E. Shi X-Y. Azhar S. Interaction of lipoproteins with isolated ovary plasma membranes..J. Biol. Chem. 1990; 265: 19100-19111Abstract Full Text PDF PubMed Google Scholar). We refer to this general region of the steroidogenic cell as the microvillar compartment and the specialized space created between adjacent microvilli as microvillar channels (28Reaven E. Boyles J. Spicher M. Azhar S. Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary..Arteriosclerosis. 1988; 8: 298-309Crossref PubMed Google Scholar, 29Reaven E. Spicher M. Azhar S. Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells..J. Lipid Res. 1989; 30: 1551-1560Abstract Full Text PDF PubMed Google Scholar, 30Reaven E. Shi X-Y. Azhar S. Interaction of lipoproteins with isolated ovary plasma membranes..J. Biol. Chem. 1990; 265: 19100-19111Abstract Full Text PDF PubMed Google Scholar). Often, inverted microvilli form double-membrane channels within the peripheral cytoplasm of the cells as they make close contact with the invaginated portion of the adjacent cytoplasm (15Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis..J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 28Reaven E. Boyles J. Spicher M. Azhar S. Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary..Arteriosclerosis. 1988; 8: 298-309Crossref PubMed Google Scholar, 29Reaven E. Spicher M. Azhar S. Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells..J. Lipid Res. 1989; 30: 1551-1560Abstract Full Text PDF PubMed Google Scholar). Electron microscopic immunocytochemistry techniques show heavy labeling for SR-BI specifically in regions corresponding to such microvilli and microvillar channels (13Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian model..Endocrinology. 1998; 139: 2847-2856Crossref PubMed Scopus (0) Google Scholar, 15Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis..J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar), and there is no longer any doubt that the cell surface localization of SR-BI is specifically localized to the microvillar compartment including the microvillar channels, and that a tissue exhibiting a microvillar compartment expressing high levels of SR-BI is active in selective CE uptake (5Azhar S. Stewart D. Reaven E. Utilization of cholesterol-rich lipoproteins by perfused rat adrenals..J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar, 13Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian model..Endocrinology. 1998; 139: 2847-2856Crossref PubMed Scopus (0) Google Scholar, 15Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis..J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar). In rat steroidogenic tissues, one finds that the microvillar channels are often filled with spherical particles the size of rat HDL (15Reaven E. Zhan L. Nomoto A. Leers-Sucheta S. Azhar S. Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis..J. Lipid Res. 2000; 41: 343-356Abstract Full Text Full Text PDF PubMed Google Scholar, 28Reaven E. Boyles J. Spicher M. Azhar S. Evidence for surface entrapment of cholesterol-rich lipoproteins in luteinized ovary..Arteriosclerosis. 1988; 8: 298-309Crossref PubMed Google Scholar, 29Reaven E. Spicher M. Azhar S. Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells..J. Lipid Res. 1989; 30: 1551-1560Abstract Full Text PDF PubMed Google Scholar). Results from a recent study using a heterologous insect cell system (31Reaven E. Leers-Sucheta S. Nomoto A. Azhar S. Expression of scavenger receptor class B type I (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system..Proc. Natl. Acad. Sci. USA. 2001; 98: 1613-1618Crossref PubMed Scopus (61) Google Scholar) has added new meaning to the relationship between SR-BI, microvillar channels, and “selective” lipoprotein-CE uptake. It had previously been assumed that preformed microvillar channel structures in cells secondarily acquire SR-BI protein after a stimulus (in most cases, a hormonal stimulus) activating the selective uptake of lipoprotein CEs and steroidogenesis. The over-expression of recombinant SR-BI in baculovirus infected insect ovary cells (Sf 9) altered this story. It became clear (even in such a primitive cell system) that the expression of SR-BI by itself was the stimulus for double membrane formation. That is, non-infected or infected control Sf 9 cells do not express SR-BI, show microvillar channels, or internalize CEs. However, in baculovirus infected Sf 9 cells expressing high levels of SR-BI, new double membraned channels are induced, and these membranes, in turn, facilitate the binding of exogenously provided HDL and selective HDL-CE uptake. It was of interest that the newly formed double membraned structures were most often observed as a complex network of channels within the cell peripheral cytoplasm. In the current effort, we examined the functional relationship between SR-BI expression and microvillar channel formation in a physiologically responsive steroidogenic tissue, the rat adrenal. Using hormonal stimulation and withdrawal protocols, we have been able to manipulate adrenal SR-BI expression, permitting qualitative and quantitative correlations to be made between adrenal SR-BI levels and adrenocortical cell microvillar channel formation and function. [1,2-3H(N)]corticosterone, 40–60 Ci (1.48–2.22 TBq/mmol) was purchased from NEN Life Science Products (Boston, MA). Bovine plasma fibronectin, corticosterone, 17α-ethinyl estradiol (17α-E2), and N6,2′-O-dibutylyladenosine 3′,5′-cyclic monophosphate (Bt2cAMP) were purchased from Sigma Chemical Co. (St. Louis, MO). Collaborative Biomedical Products (Bedford, MA) supplied insulin and transferrin. Electrochemiluminescence kit Western blotting detection reagents were purchased from KPL (Gaithersburg, MD). Long acting adrenocorticotropin hormone (ACTH) gel preparation (HP Acthar gel) was the product of Armour Pharmaceuticals, (Kankakee, IL). The anti-rat SR-BI and anti-rat apolipoprotein (apo)A-I antibodies used have been described and characterized in this laboratory (12Azhar S. Nomoto A. Leers-Sucheta S. Reaven E. Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model..J. Lipid Res. 1998; 39: 1616-1628Abstract Full Text Full Text PDF PubMed Google Scholar, 31Reaven E. Leers-Sucheta S. Nomoto A. Azhar S. Expression of scavenger receptor class B type I (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system..Proc. Natl. Acad. Sci. USA. 2001; 98: 1613-1618Crossref PubMed Scopus (61) Google Scholar). All other reagents used were of analytical grade. Four groups of 3-month-old male Sprague-Dawley rats (Charles Rivers Laboratories, Hollister, CA) were studied: 1) non-stressed controls (C), 2) rats treated with long-acting ACTH gel (10 IU sc every 8 h for 24 h period), 3) rats treated with 17α-E2, 10 mg/kg BW sc every 24 h for 5 days, and 4) rats treated with dexamethasone (DEX) (a single injection 100 μg, sc) for a 24 h period. The expression of SR-BI protein in whole adrenal tissues and cultured adrenocortical cells was assessed by Western blot analysis using previously described methodology (12Azhar S. Nomoto A. Leers-Sucheta S. Reaven E. Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model..J. Lipid Res. 1998; 39: 1616-1628Abstract Full Text Full Text PDF PubMed Google Scholar, 13Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian model..Endocrinology. 1998; 139: 2847-2856Crossref PubMed Scopus (0) Google Scholar). Briefly, adrenal tissues were homogenized in 10 vol of buffer (20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 0.25 M sucrose, 1 mM phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 20 μg/ml aprotinin, and 5 μg/ml pepstatin), centrifuged (800 × g) for 10 min, and supernatant centrifuged for 60 min at 100,000 × g. The resulting pellet was washed with buffer to remove floating lipids and membranes were used for immunoblotting of SR-BI. Similarly, cultured adrenocortical cells were washed twice in ice-cold phosphate-buffered saline, and lyzed in lysis buffer [50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% Triton X-100 (v/v), 0.5% deoxycholate (v/v), 1% SDS (w/v), 5 mM EDTA, and 1 mM dithiothreitol]. Following incubation at 37°C for 15 min, each lysate was sonicated briefly to disrupt chromatin (DNA) and then used for SR-BI immunoblotting. Samples were mixed with equal volumes of 2× Laemmli sample buffer [120 mM Tris-HCl, pH 6.8, 2% SDS (w/v), 10% sucrose (w/v), and 1% 2-mercaptoethanol] and subjected to 7% SDS-PAGE. For each sample, a constant amount of protein (5–20 μg) was loaded on the gel. Protein standards (Myosin, 200 kDa; β-galactosidase, 116.3 kDa; Phosphorylase b, 97.4 kDa; BSA, 66.2 kDa; and ovalbumin, 45 kDa) were also loaded on the gel. After electrophoretic separation, the proteins were transferred to Immobilon PVDF membranes (Millipore Corp., Bedford, MA) using standard techniques. The protein blots were incubated with anti-rat SR-BI for 2 h at room temperature, then probed with peroxidase-labeled mouse anti-rabbit IgG and visualized using the ECL system. The resulting radiographic chemiluminescence was visualized for different time points (1–10 min), and appropriate films were subjected to densitometric scanning. Adrenocortical cells from hypocholesterolemic (17α-E2 treated) animals were used to determine if the addition of exogenous lipoproteins could downregulate SR-BI expression. Adrenocortical cells were isolated by digestion with collagenase and DNase as previously described from this laboratory (32Azhar S. Wang H. Tsai L. Reaven E. Okadaic acid interferes with lipoprotein-supported corticosterone production in adrenal cells..Biochem. Biophys. Res. Commun. 1991; 179: 726-733Crossref PubMed Scopus (18) Google Scholar), maintained in serum free Dulbecco's modified Eagles medium: nutrient mixture F-12 (1:1) (32Azhar S. Wang H. Tsai L. Reaven E. Okadaic acid interferes with lipoprotein-supported corticosterone production in adrenal cells..Biochem. Biophys. Res. Commun. 1991; 179: 726-733Crossref PubMed Scopus (18) Google Scholar) for 24 h prior to the experimental protocol comparing untreated cells (basal) with those given apoE-free human high density lipoproteins (hHDL3) (500 μg protein/ml), Bt2cAMP (2.5 mM), or hHDL3 + Bt2cAMP for a 24 h interval. Aliquots of culture media were assayed for secreted corticosterone by the radioimmunoassay (RIA) technique as previously described (5Azhar S. Stewart D. Reaven E. Utilization of cholesterol-rich lipoproteins by perfused rat adrenals..J. Lipid Res. 1989; 30: 1799-1810Abstract Full Text PDF PubMed Google Scholar, 32Azhar S. Wang H. Tsai L. Reaven E. Okadaic acid interferes with lipoprotein-supported corticosterone production in adrenal cells..Biochem. Biophys. Res. Commun. 1991; 179: 726-733Crossref PubMed Scopus (18) Google Scholar). hHDL3 used in the cell culture studies were isolated as described previously (30Reaven E. Shi X-Y. Azhar S. Interaction of lipoproteins with isolated ovary plasma membranes..J. Biol. Chem. 1990; 265: 19100-19111Abstract Full Text PDF PubMed Google Scholar), and the procedure of Markwell et al. was used to quantify protein content of hHDL3 (33Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples..Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5328) Google Scholar). The DNA content of the cells was quantified fluorimetrically (34West D.C. Sattar A. Kumar S. A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells..Anal. Biochem. 1985; 147: 289-295Crossref PubMed Scopus (261) Google Scholar) while protein in membrane fractions was determined by a modification of the procedure as described by Peterson (35Peterson G.L. A simplification of the protein assay method of Lowry et al which is more generally applicable..Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7132) Google Scholar). Serum cholesterol concentrations were determined according to the procedure of Tercyak (36Tercyak A.M. Determination of cholesterol and cholesterol esters..J. Nutr. Biochem. 1991; 2: 281-292Crossref Scopus (36) Google Scholar). Serum corticosterone was extracted with methylene chloride and assayed by RIA (5Azhar S. St" @default.
• W1870049952 created "2016-06-24" @default.
• W1870049952 creator A5039155716 @default.
• W1870049952 creator A5049078893 @default.
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• W1870049952 date "2002-06-01" @default.
• W1870049952 modified "2023-09-27" @default.
• W1870049952 title "Hormonal regulation of adrenal microvillar channel formation" @default.
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