Medicine, Faculty of
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Roskelley, Calvin
University of British Columbia
The zones of the adrenal cortex contain distinct subpopulations of cells which share a common mesodermal origin and steroidogenic template. How zonal specializations are overlaid upon this template is unknown. During adrenocortical cytogenesis zonal phenotypes are expressed transiently as a cell moves inward from a subcapsular position toward the medulla. In this study, homogeneous populations of rat adrenocortical parenchymal cells were maintained and manipulated in vitro. This was done in an effort to determine the mechanism responsible for zone-specific differential responsiveness to the trophic hormone angiotensin II (Ang II). In addition, the effects of ras oncogene expression and parenchymal/stromal interactions on overall steroidogenic differentiation were assessed. Initially, a method was devised to separate rat adrenocortical cells by density into populations which retain zone specific properties in primary culture. Two different parenchymal populations were obtained. They were designated FASC (1 .034g/mI, 18.0µ cell diameter) and GLOM (1.069g1m1, 11.7 µ. cell diameter). In freshly isolated cell suspensions the physical characteristics and differential steroidogenic responses to adrenocorticotropin and angiotensin II suggested that FASC cells originated predominantly from the zona fasciculata and GLOM cells from the zona glomerulosa. In primary culture, the two populations exhibited different morphologies. FASC cells retained lipid and formed cohesive epithelial monolayers that remained stationary for three weeks. GLOM cells were initially epithelial but rapidly lost lipid, spread, assumed fibroblastic shapes and expressed vimentin. Both cell types were positive for the steroidogenic cytochrome P-450 side chain cleavage enzyme (P-45Oscc). Therefore, the morphological changes observed in GLOM cultures were due to modulation, not fibroblastic overgrowth. Ang II increased steroidogenesis in GLOM cells but not in FASC cells. To determine the mechanism responsible for this zonal specialization, Ang Il-mediated signal transduction was closely examined. Ang II significantly increased the production of inositol 1,4,5 triphosphate in both cell types. In contrast, the two cell types exhibited very different intracellular release dependent increases in free intracellular calcium ([Ca²⁺]i). Ang II induced [Ca²⁺]i increases of >50nM in 90% of individual GLOM cells, but in only 28% of FASC cells. Also, in populations, Ang II induced dose dependent [Ca²⁺]i increases in GLOM but not FASC cells. Importantly, calcium ionophore treatment increased [Ca2+]i and steroidogenesis in both cell types. These results suggest that FASC cells lack a steroidogenic response to Ang II, at least in part, because of an interruption of the signalling pathway at the level of intracellular calcium release. Therefore, alterations within a specific signal transduction pathway are responsible for one of the cellular phenotypes associated with zonal specialization in the rat adrenal cortex. GLOM and FASC cells were infected with Kirsten murine sarcoma virus, which contains an activated form of the ras oncogene. FASC cells exhibited no discernable morphologic change after infection. In GLOM cultures, discrete foci did not appear. Instead, transient cellular multilayers formed from which rounded cells emerged. After selective passaging these cells (designated KGLOM) acquired a transformed morphology, proliferated rapidly in both 10% and 1% serum, grew to high saturation densities, and they were tumorigenic. They also expressed P-45Oscc and the viral ras protein, p21. In addition, KGLOM cells produced endpoint steroids under conditions in which GLOM cells did not. KGLOM steroidogenesis could be inhibited by lovastatin, a pharmacological inhibitor of p21 ras function. Therefore, expression of the ras oncogene induces transformation and enhances the steroidogenic differentiation of rat glomerulosa cells. This dual function of ras p21 likely reflects the state of the signal transduction pathways present within steroidogenic cells. In longterm culture, FASC and GLOM cells de-differentiated and lost their steroidogenic characteristics. This did not occur in a third density isolated population that consisted of a mixture of cell types (designated MIX). In primary culture, MIX populations formed highly proliferative cellular multilayers that were composed of stromal fibroblasts, endothelial cells and parenchymal cells. From these multilayers rounded cells emerged that contained lipid and the steroidogenic enzymes Δ5,3ßhydroxysteroid dehydrogenase (Δ5,3ß-HSD) and P-45Oscc. This phenomenom was associated with a 10 fold increase in the number of Δ5,3ß-HSD containing cells during the first 21 days in culture. Rounded cells were selectively passaged into secondary culture where they continued to proliferate and remained steroidogenic. Thus, primary MIX cultures provide a microenvironment that produces proliferating, steroidogenic cells independently of trophic hormone treatment. This unique model may prove useful in the quest to further identify specific inducers that are responsible for adrenocortical cytogenesis in general and steroidogenic differentiation in particular.
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RAT ADRENOCORTICAL CELL DIFFERENTIATION: EFFECTS OF SIGNALTRANSDUCTION ALTERATIONS, rasONCOGENE EXPRESSION,AND PARENCHYMAL/STROMAL INTERACTIONSbyCALVIN ROSKELLEYB.Sc., The University of Victoria, 1983A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTSFOR ThE DEGREE OF DOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Faculty of Medicine, Department of Anatomy)We accept this thesis as conforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMay 1992© Calvin D. RoskelleySignature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives, It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)_____________________Department of ANATOMYThe University of British ColumbiaVancouver, CanadaDate June 1, 1992DE-6 (2/88)Signature(s) removed to protect privacyIIABSTRACTThe zones of the adrenal cortex contain distinct subpopulations of cells whichshare a common mesodermal origin and steroidogenic template. How zonal specializationsare overlaid upon this template is unknown. During adrenocortical cytogenesis zonalphenotypes are expressed transiently as a cell moves inward from a subcapsularposition toward the medulla. In this study, homogeneous populations of rat adrenocorticalparenchymal cells were maintained and manipulated in vitro. This was done in an effortto determine the mechanism responsible for zone-specific differential responsiveness tothe trophic hormone angiotensin II (Ang II). In addition, the effects of ras oncogeneexpression and parenchymal/stromal interactions on overall steroidogenicdifferentiation were assessed.Initially, a method was devised to separate rat adrenocortical cells by densityinto populations which retain zone specific properties in primary culture. Two differentparenchymal populations were obtained. They were designated FASC (1 .034g/mI, 1 8.Oj.i.cell diameter) and GLOM (1.069g1m1, 11.711. cell diameter). In freshly isolated cellsuspensions the physical characteristics and differential steroidogenic responses toadrenocorticotropin and angiotensin II suggested that FASC cells originatedpredominantly from the zona fasciculata and GLOM cells from the zona glomerulosa. Inprimary culture, the two populations exhibited different morphologies. FASC cellsretained lipid and formed cohesive epithelial monolayers that remained stationary forthree weeks. GLOM cells were initially epithelial but rapidly lost lipid, spread, assumedfibroblastic shapes and expressed vimentin. Both cell types were positive for thesteroidogenic cytochrome P-450 side chain cleavage enzyme (P-45Oscc). Therefore,the morphological changes observed in GLOM cultures were due to modulation, notfibroblastic overgrowth.IIIAng II increased steroidogenesis in GLOM cells but not in FASC cells. To determinethe mechanism responsible for this zonal specialization, Ang Il-mediated signaltransduction was closely examined. Ang II significantly increased the production ofinositol 1,4,5 triphosphate in both cell types. In contrast, the two cell types exhibitedvery different intracellular release dependent increases in free intracellular calcium([Ca2]i). Ang II induced [Ca2]i increases of >5OnM in 90% of individual GLOM cells,but in only 28% of FASC cells. Also, in populations, Ang II induced dose dependent[Ca2]i increases in GLOM but not FASC cells. Importantly, calcium ionophoretreatment increased [Ca2+]i and steroidogenesis in both cell types. These resultssuggest that FASC cells lack a steroidogenic response to Ang II, at least in part, because ofan interruption of the signalling pathway at the level of intracellular calcium release.Therefore, alterations within a specific signal transduction pathway are responsible forone of the cellular phenotypes associated with zonal specialization in the rat adrenalcortex.GLOM and FASC cells were infected with Kirsten murine sarcoma virus, whichcontains an activated form of the ras oncogene. FASC cells exhibited no discernablemorphologic change after infection. In GLOM cultures, discrete foci did not appear.Instead, transient cellular multilayers formed from which rounded cells emerged. Afterselective passaging these cells (designated KGLOM) acquired a transformed morphology,proliferated rapidly in both 10% and 1% serum, grew to high saturation densities, andthey were tumorigenic. They also expressed P-45Oscc and the viral ras protein, p21.In addition, KGLOM cells produced endpoint steroids under conditions in which GLOMcells did not. KGLOM steroidogenesis could be inhibited by lovastatin, a pharmacologicalinhibitor of p21 ras function. Therefore, expression of the ras oncogene inducestransformation and enhances the steroidogenic differentiation of rat glomerulosa cells.This dual function of ras p21 likely reflects the state of the signal transduction pathwayspresent within steroidogenic cells.ivIn longterm culture, FASC and GLOM cells de-differentiated and lost theirsteroidogenic characteristics. This did not occur in a third density isolated populationthat consisted of a mixture of cell types (designated MIX). In primary culture, MIXpopulations formed highly proliferative cellular multilayers that were composed ofstromal fibroblasts, endothelial cells and parenchymal cells. From these multilayersrounded cells emerged that contained lipid and the steroidogenic enzymes A5,3B-hydroxysteroid dehydrogenase (A5,313-HSD) and P-45Oscc. This phenomenom wasassociated with a 10 fold increase in the number of z5,3I3-HSD containing cells duringthe first 21 days in culture. Rounded cells were selectively passaged into secondaryculture where they continued to proliferate and remained steroidogenic. Thus, primaryMIX cultures provide a microenvironment that produces proliferating, steroidogeniccells independently of trophic hormone treatment. This unique model may prove usefulin the quest to further identify specific inducers that are responsible for adrenocorticalcytogenesis in general and steroidogenic differentiation in particular.VTABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTS vLIST OF TABLES xLiST OF FIGURES xiLIST OF APPENDICES xvLIST OF ABBREVIATIONS xviACKNOWLEDGEMENTS x i xI. INTRODUCTION 11.Rationale 12. The Rat Adrenal Cortex 2i) Morphology 2ii) Development 4iii) Steroidogenesis 6iv) Steroidogenic Regulation 7a)ACTH 7b)Angll 8C) The ras Oricogene 93. Rat Adrenocortical Cells in vitro 13II. MATERIALS AND METHODS 1 91. Production of Rat Adrenocortical Cells 19i) Animals 19ii) Adrenocortical Fibroblast Migration from Explants 19viiii)Collagenase Dissociation of Rat Adrenocortical Cells 202. Separation of Rat Adrenocortical Cell Types 21i) Differerential Substratum Adhesion Separation 21ii) Differential Morphological Response to cAMP 22iii) Dissection of Adrenocortical Capsules from the Inner Portion of the Gland 22iv) Discontinuous Percoll Density Gradient Separation 23a) Gradient Preparation 23b) Cell Separation and Density Estimation 243. Tissue Culture 244. Differentiation Markers: Histochemistry 26i) Cytoplasmic Lipid 26ii) A5,3r3-hydroxysteroid dehydrogenase (z\5,313-HSD) 26iii) Catecholamines 275. Differentiation Markers: Immunofluorescence 27i) Cytochrome P-450 side chain cleavage enzyme (P-45Oscc) 28ii) Keratin 28iii) Double Staining for Vimentin and P-45Oscc 29iv) Factor VIII 306. Time Lapse Microscopy 307. Electron Microscopy 318. Steroidogenesis 31i) Acid-ethanol fluorescence 31ii) Radloimmunoassay (RIA) 329. Inositol Phosphate Determination 33i) Analysis of inostol phosphates 33ii) Analysis of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 34vii10. Free Intracellular Calcium ([Ca2]i) Determination 35i) Culture of cells 35ii) Fura-2 loading 36iii) Fluorescence measurement 36iv) Experimental Protocol 37v) Calculation of [Ca2]i 3811. Kirsten Murine Sarcoma Virus (KiMSV) Isolation, Infection and OncogenicTransformation 39i) KiMSV isolation and titration 39ii) KiMSV infection 40iii) Selective passaging of morphologically altered cells 40iv) Serum independence 41v) Tumorigenicity 4112. p21 ras Expression 42i) Immunofluorescence 42ii) Immunoprecipitation 4213. Lovastatin Treatment of KGLOM cells 43Ill. Results 441. Preliminary Attempts at Rat Adrenocortical Cell Separation 44i) Whole Gland Dissociation of the Rat CorLex 44ii) Differential Substratum Adhesion of Collagenase DissociatedAdrenocorLical Cells in Primary Culture 46iii) cAMP-Induced Rounding and Detachment of Parenchymal Cells fromCollagenase Dissociated Adrenocortical Cells in Primary Culture 47iv) Dissection of the Adrenal Cortex 50a) Capsular Glands (containing zona glomerulosa) 50b) De-capsulated Glands (containing zonae fasciculata and reticularis) 52viii2. Percoll Density Gradient Separation of Rat Adrenocortical Cells 52i) Cell Separation into 7 Visible Bands 53ii) Preliminary characterization of separated cells 563. Characterization of FASC (fasciculata) and GLOM (glomerulosa) Populations 61i) Steroidogenesis 61a) In Suspension 61b) In Primary Culture 63c) In Secondary Cultureii) Primary Culture Morphology 66iii) Phenotypic Markers in Primary Culture 70a) cAMP Induced Retraction 70b) Cytoplasmic Lipid 70C) Ultrastructure 70d) P-4SOscc Expression 71e) Cytokeratin Expression 71f) Vimentin Expression 72g) Catecholamine Activity 804. Angiotensin Il-mediated Signal Transduction in GLOM and FASC Cells 82i) Ang Il-Mediated Steroidogenesis 82ii) Inositol Phosphate Accumulation 83a) InsPi, lnsP2 lnsP3 83b) lns(1,4,5)P3 83iii) [Ca2]i in Cell Groups 88iv) [Ca2]i in Single Cells 92v) Calcium lonophore-Mediated Steroidogenesis 93ix5. ras Oncoprotein Induced Transformation and Differentiation of FASC and GLOM cells 99i) KiMSV Infection and Selection of Morphologically Altered Cells 99ii) Transformation Associated Parameters of KGLOM(KiMSV infected glomerulosa) Cells 101a) Transformation in vitro 101b)Tumourigenicity in vivo 102iii) Steroidogenic Differentiation of KGLOM Cells 110iv) Inhibition of p21 ras function in KGLOM Cells 1136. Steroidogenic Differentiation of a Mixed Parenchymal-Stromal (MIX) CellPopulation in Culture 119i) Characterization 119ii) Primary Culture Morphology 120iii) Steroidogenic Differentiation in Primary Culture 124iv) Secondary Culture Morphology 130v) Steroidogenic Differentiation in Secondary Culture 130IV. DISCUSSION 1361. Preliminary Attempts at Parenchymal Cell Separation 1362. Percoll Density Gradient Parenchymal Cell Separation 1383. in vitro Characterization of FASC and GLOM Cells 1414. Ang Il-Mediated Signal Transduction in FASC and GLOM Cells 1435. Expression of the ras Oncogene in GLOM Cells 1506. Parenchymal Cell Proliferation and Differentiation in MIX Cultures 1567. Conclusions 159V. REFERENCES 162xLIST OF TABLESTable 1: Characteristics of Rat Zona Glomerulosa and Zona Fasciculata Cells 1 8Table 2: Density Gradient Separation 55Table 3: Intermediate Filament Expression in FASC and GLOM Cells 79Table 4: KGLOM Tumour Histopathology 1 08xiLIST OF FIGURESFig 1: Collagenase dissociated cells from whole adrenal cortices 45Fig 2: Differential adhesion and cAMP treatment in primary culture 4 8Fig 3: Factor VIII immunofluorescence of adrenocortical endothelial cellcolonies obtained by differential adhesion 4 9Fig 4: Adrenocortical cells derived from decapsulated (a,c) or capsular (b,d)glands 51Fig 5: Representative experiment of separated cell bands obtained from thepercoll density gradient 54Fig 6: t5,313-hydroxysteroid dehydrogenase (A5,313-HSD) staining of cellsderived from bands separated on percoll gradient 5 9Fig 7: In vivo origin of FASC and GLOM cells 6 0Fig 8: Steroidogenesis in freshly isolated cell FASC and GLOM suspensions 62Fig 9: FASC and GLOM corticosterone production in primary culture 64Fig 10: FASC and GLOM cell steroidogenesis before and after passaging inculture 65Fig 11: FASC and GLOM morphology in primary culture 6 7Fig 12: Seeding efficiencies and growth in primary culture of FASC and GLOMcells 68Fig 13: cAMP mediated morphological response of FASC and GLOM cells inprimary culture 69Fig 14: Lipid content of FASC and GLOM cells in primary culture 73Fig 15: Transmission electron microscopy of FASC (a,c) and GLOM (b,d)cellsin primary culture 74Fig 16: P-45Oscc expression of FASC and GLOM cells in primary culture 75xiiFig 17: Cytokeratin expression of FASC and GLOM cells in primary culture 76Fig 18: P-45Oscc and vimentin expression in FASC and GLOM cells 77Fig 19: P-45Oscc and vimentin expression in GLOM cells and rat lungfibroblasts (RLF) 78Fig 20: Comparison of morphology and catecholamine expression in adrenalmedullary cells (a,c,e) and FASC cells (b,d,f) in primary culture 8 1Fig 21: Ang Il-mediated steroidogenesis in freshly isolated suspensions ofGLOM and FASC cells 84Fig 22: Ang Il-mediated steroidogenesis of GLOM and FASC cells in primaryculture 85Fig 23: Ang fl-mediated inositol phosphate production 8 6Fig 24: Ang Il-mediated lns(1,4,5)P3 production 87Fig 25: Ang Il-mediated changes in [Ca2]i in groups of FASC and GLOM cellsin primary culture 89Fig 26: Examples of [Ca2]i changes in GLOM and FASC cell groups 9 0Fig 27: Comparison of Ang II and Thapsigargin induced [Ca 2]i changes in cellgroups 91Fig 28: Examples of [Ca2]i changes in individual GLOM and FASC cells 94Fig 29: Ang Il-mediated [Ca2]i changes in individual GLOM cells in theabsence of extracellular calcium (A) and in the presence ofverapamil (B) 95Fig 30: Frequency distribution of increases in [Ca2]i in single GLOM andFASC cells after 1 OnM Ang II treatment 9 6Fig 31: Ang Il-mediated [Ca2]i responses in an individual GLOM cell (A) anda Yl mouse adrenal cortical tumour cell (B) 9 7Fig 32: Calcium ionophore-mediated steroidogenesis of GLOM and FASC cells inprimary culture 98xiiiFig 33: Morphology of KiMSV infected GLOM cells 1 00Fig 34: p21 ras expression in KGLOM cells (p2) 1 03Fig 35: p21 ras expression in uninfected GLOM cells (p2) 1 04Fig 36: p21 ras extraction in KGLOM cells (p5) 1 05Fig 37: p21 ras immunoprecipitation in GLOM and KGLOM cells (p5) 1 06Fig 38: Proliferation of GLOM and KGLOM cells (p5) 1 07Fig 39: KGLOM tumour histopathology 1 09Fig 40: P-45Oscc expression in KGLOM cells 111Fig 41: Corticosterone production by GLOM and KGLOM cells (p2) 11 2Fig 42: Corticosterone production in lovastatin treated early primary GLOMcultures 11 5Fig 43: Corticosterone production in lovastatin treated late primary GLOMcultures 11 6Fig 44: Corticosterone production in lovastatin treated KGLOM cultures 11 7Fig 45: p21 localization in lovastatin treated KGLOM cells 11 8Fig 46: Morphology of MIX adrencocortical cell population in primaryculture 121Fig 47: Growth of FASC, GLOM and MIX cells in primary culture 1 22Fig 48: Time lapse microscopy of proliferating MIX cells in primaryculture 123Fig 49: Corticosterone production by FASC, GLOM and MIX cells in primaryculture 126Fig 50: Fluorogenic steroid production by FASC, GLOM and MIX cells inprimary culture 1 27Fig 51: A5,313-Hydroxysteroid dehydrogenase (A5,3I3-HSD) activity of FASC,GLOM and MIX cells in primary culture 1 28xivFig 52: Differentiation marker expression of multilayered MIX primarycultures in situ 1 29Fig 53: Morphology of MIX adrencocortical cell population in secondaryculture 132Fig 54: P-45Oscc expression in MIX (p1) cells 133Fig 55: Response to cAMP and P-45Oscc expression in heterogeneous MIX(p1) cultures 134Fig 56: Steroidogenesis of passage 2 GLOM and MIX cultures 1 35xvLIST OF APPENDICESAppendix A: Percoll Gradient Preparation 1 77Appendix B: Ins(1,4,5)P3 Determination 179Appendix C: p21 ras Immunoprecipitation 1 82xviLIST OF ABBREVIATIONSCTh adrenocorticotropic hormoneAldo aldosteroneAMCA 7-amino-4 methylcoumarin-3-acetic acidAng II angiotensin IICAMP 3’-5’ cyclic adenosine monophosphateFE cAMP response elementcs corticosteroneDG diacylglcerolDHEA dehydroepiandrostenedioneDMEM/F12 Dulbecco’s modified essential medium/Ham’s F12 medium (1:1)DMSO dimethylsulfoxideEOM extracellular matrixepidermal growth factorRTA ethylenebis(oxyethelenenitrilo)-tetraacetic acidFASC percoll separated cells derived from the rat zona fasciculataFBS fetal bovine serumGTPase activating proteinGLY.4 percoll separated cells derived from the rat zona glomerulosaGscL G protein a-subunit that stimulates adenylate cyclaseGTP guanosine trisphosphateHBSS Hanks’ balanced salt solutionHEPES N-2-hydroxyethylpiperazine-N’-2-ehanesulfonic acidHMG CoA reductase 3-hydroxy-3-methytglutaryl-co-enzyme AHS horse serumIGF-1 insulin-like growth factor-IxviilgG immunoglobulinI ns( 1 ,4,5) P3 inositol (1 ,4,5)-trisphosphatelnsP1 inositol-monophosphatelnsP2 inositol-bisphosphatelnsP3 inositol-trisphosphateIP ionophoreKGLOM KiMSV infected/transformed GLOM cellsKiMSV Kirsten murine sarcoma virusKNRK K1MSV infected/transformed NRK cellsMIX percoll separated cells containing a mixture of parenchymal,stromal and endothelial cellsNAD nicotinamide adenine dinucleotideNE-i neurofibromatosis gene #1I%12F nerve growth factorNRK normal rat kidney cellsP-450C2 1 cytochrome p-450 C21 -hydroxylaseP-45O cytochrome P-450 side chain cleavage enzymeP-4501 1 B cytochrome P-450 11f3-hydroxylase45017a cytochrome P-450 l7cx-hydroxylasePAS protein A sepharosePBS phosphate buffered salinePC-i 2 adrenomedullary tumour cell linePDGF platelet derived growth factorPKC protein kinase CPLC phosphoilpase Cp(n) passage number in pre-senescent cultureRIA radioimmunoassayRIPA Buffer radiolabeled immunoprecipitation assay bufferSDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresisTCA trichioroacetic acidTPA 1 2-O-tetradecanoylphorbol-13-acetate (phorbol ester)TRE TPA (PKC) response elementYl adrenocortical tumour cell lineA5,3 13-H SD A5,313-hydroxysteroid dehydrogenaseFITC fluoroscein isothiocyanatexviiixixACKNOWLEDGEMENTSI would like to thank the members of my research committee for their expert guidance andassistance throughout the lifespan of this project. These include Drs. Nelly Auersperg,Bruce Crawford, Keith Humphries and Peter Leung. Also, the help of Dr. KenBaimbridge was instrumental in carrying out the calcium studies. Special thanks go toNelly for her wisdom, enthusiasm and friendship from start to finish. Nelly, I’ve never onceregretted sending you that pleuripotent letter, not for a minute.The faculty, staff and graduate students of the UBC department of Anatomy were a greatreservoir of help and support. It was an exciting and stimulating place to be, and I will lookback with great fondness on my days and nights spent in the Freidman building.Specifically, I would like to express gratitude to department head Dr. Charles Sloneckerand student advisors Drs. Bill Ovalle and Joanne Emmerman. In addition, Drs. Wayne yogIand Carl Friz have made sure I will always remain a gross anatomist in structure, if notfunction. The technical assistance of Judith Black, Carolyn Burr and Sarah Maines wasgreatly appreciated. Also, both Dr. Jie Pan and Mr. lan MacLaren were experts with ideasas well as experiments.Heartfelt thanks go to my family, especially my Mom and Dad. They made things possiblein a million different ways, from Jimmy Cricket right through graduate school. And finally,to Christine Williams, whose patience and understanding were never ending. Chris, I’llnever be able to repay you completely, but I’ve got a lifetime to try. Maybe if I could findyou Zuzu’s petals, or Allie’s mit, that just might do it.—1—I. INTRODUCTION1. RationaleVertebrate development is continuously driven by three processes: growth,differentiation and morphogenesis (Steinberg, 1963>. Utilizing arbitrary, oftenterminal, endpoints these processes have been successfully studied in a number of celllineages hi However, there are few well defined models available for the detailedexamination of the ways in which these processes act to alter celluar phenotypes atspecific stages within a particular developmental lineage.In the rat, the ‘adrenocyte’ develops along a spatial and temporal continuum as itleaves the region of the capsule and migrates centripitally through the adrenal cortex(Gottschau, 1883; lannacone and Weinberg, 1987). Therefore, cells at different stageswithin this developmental lineage are found within separate anatomical compartments ofthe endocrine gland. In the present study, 2 homogeneous adrenocortical cell populationsfrom different anatomical compartments were isolated and maintained in culture. Theproliferative potential, steroidogen ic differentiation, and morphologic characteristics ofthe two parenchymal cell types were then compared to each other in an effort todetermine how, and by what means, subtle differences in developmental staging affect thecellular phenotype. For example, as an adrenocyte moves inward from the zonaglomerulosa to the zona fasciculata it no longer responds to the trophic hormoneangiotensin II (Ang II; Haning et al., 1970; Tait et al., 1974; Douglas et al., 1978;Braley et al., 1986; Whitley et al., 1987). Utilizing the 2 isolated populations, whichare derived from these zones, an attempt was made to define the molecular mechanismresponsible for this developmentally driven alteration in the differentiation state.With increasing time in primary culture, particularly in cells derived from therat adrenal cortex, overt steroidogenic function is lost (O’Hare and Neville, 1 973a;-2-Ryback and Ramachandran, 1981a; Payet et al.,1984). Efforts were made here toprolong this tissue-specific differentiative state in both primary and secondary culture.In one situation, expression of the ras oncogene, which also transforms the cells totumorigenicity, was used. In another, steroidogenic differentiation in mixedparenchymal/stromal cultures was assayed. Interestingly, in each case, the phenotypesobserved appeared to be associated with specific stages of adrenocortical development.2. The Rat Adrenal Cortexi MorphologyThe adrenal gland is located retroperitoneally in a fat pad just superior to thekidney. This small endocrine gland consists of two parts, the mesodermally derivedcortex and the medulla, which is derived from neural crest (Nussdorfer, 1986). Thecortex is a major producer of steroid hormones, while the medulla secretescatecholamines. The adrenal is highly vascularized, receiving branches from theinferior phrenic, celiac and renal arteries. After passing through the connective tissuecapsule of the gland these branches give rise to a subcapsular plexus of sinusoidalcapillaries that sends longtitudinal branches plunging through the cortex. The latterempty into venous sinusoids within the medulla. Thus the blood supply flows from theoutside inward.As is the case in most mammalian species, the parenchymal cells of the ratadrenal cortex are arranged into 3 morphologically distinct zonae, the glomerulosa,fasciculata and reticularis (ldleman, 1970).The outer zona glomerulosa occupies about 10-15% of the gland volume(Nickerson, 1976; Mazzocchi et al., 1977). The parenchymal cells are small (600--3-700 jim3), have a high nuclear to cytoplasmic ratio and they are grouped into sphericalclusters that are in intimate contact with the subcapsular capillary plexus. Glomerulosacells contain an abundant smooth endoplasmic reticulum (SER), well developed Golgiapparatus, elongated mitochondria with tubular or tubulovescicular cristae, and smalllipid droplets that occupy about 5% of the cytoplasmic volume. The rat zona glomerulosais the principal source of the mineralocorticoid aldosterone.The broad, centrally located zona fasciculata contains large cells (1300-2000jim3) that are arranged in radial columns around the longtitudinal capillary network(Nussdorfer et aL, 1974). These columns are approximately 30-40 cells long and oftenonly 2-3 cells wide. Fasciculata cells contain an abundant SER, well developed Golgi,many round or oval mitochondria of varying size with tubulovescicular or vescicularchristae, and numerous lipid vesicles that occupy up to 25% of the cytoplasmic volume.The rat zona fasciculata is the principal source of the glucocorticoid corticosterone.The zona reticularis arises gradually from the fasciculata and ends abruptly atthe adrenal medulla. The cells are intermediate in size (800-1350 jim3) and arearranged in a haphazard array (Malendowicz, 1974; Conran and Nickerson, 1979).Reticularis cells contain a very abundant SER that occupies up to 50% of the cytoplasmicvolume, a small and sparse Golgi apparatus, round or ovoid mitochondria with vescicularcristae, and lipid vesicles that occupy about 10% of the cytoplasmic volume. Throughoutthis zone a small percentage of cells are constantly deleted by a process that involves aninitial separation from neighbours, followed by severe nuclear/cytoplasmiccondensation, and finally ingestion by perisinusoidal histiocytes (Wyllie et al., 1973).Functionally, it is difficult to separate the rat reticularis from the fasciculata as bothzones produce corticosterone. In some mammalian species the reticularis producesadrenal androgens such as androstenedione, although this activity is limited in the rat(Bell et al., 1978).-4-ii) DevelopmentAt about mid-gestation in the developing rat embryo the cells of the ceolomicepithelium located between the gastric mesentery and the urogenital ridge undergo awave of proliferation and begin to migrate into the underlying stroma just superior tothe developing mesonephros (Idleman, 1970). These mesodermal cells condense to formthe adrenocortical primordia. At day 15 post-conception (PC), there is the evidence ofsteroidogenic differentiation as inner and outer cortical zones begin to develop (Daikokuet al., 1976). At day 17 PC, the outer adrenocortical zone begins to morphologicallyresemble the zona glomerulosa, consisting of small cells arranged in clusters thatcontain numerous mitoses. The inner zone consists of irregularly grouped cords of largepolygonal cells whose mitochondria have tubulovescicular christae. This is the earlyzonae fasciculata/reticularis (Nussdorfer 1986). Also at day 17 PC, a connective tissuecapsule is discernable, the cells of which have steroidogenic characteristics as theyexpress the steroidogenic enzyme z5,3l3-hydroxysteroid dehydrogenase (A5,313-HSD)(Farcnik and Auersperg, 1984). It is only at this point, day 17 or 18 PC, that thepituitary begins to secrete ACTH and true zonation begins in earnest (Jost, 1975). Atbirth (Day 22-23 PC), all three adrenocortical zones are present although the zonareticularis is scanty and intermingled with the neual crest-derived adrenal medulla.There is no fetal adrenal cortex in the rat as there is in a number of other mammalianspecies, including human. Three days after birth the steroidogenic properties of theconnective tissue capsule begin to diminish. Six weeks after birth, when the three zonaeare fully formed and functional, the capsule is no longer steroidogenic.Scanning electron microscopy suggests that the parenchymal cells of the adrenalcortex are arranged as a tunneled continuum within and across each of the zones (Mottaet al., 1979). In chimeric rats, individual clones form radial patterns that span theentire cortex, from the connective tissue capsule to the medulla (lannacone and-5-Weinberg, 1987). Thus, parenchymal cells from all three zones must have a commoncellular origin. Most mitoses occur in the outer regions of the cortex and 1 hour after3H thymidine injection 95% of the labelled cells are found in the subcacapsular stromaor the zona glomerulosa (Wright and Voncina, 1977; Zajicek et al., 1986). Theselabelled cells move slowly inward at the rate of approximately 0.25 cell diameters perday until they reach the inner region of the zona reticularis where they degenerate. Thisdirected movement may be driven by the cytogenesis taking place in the outer portion ofthe gland, and the cytolysis taking place in the inner portion. Taken together these dataindicate that the Gottschau’s archetypical cell migration hypothesis (1883) accuratelydepicts adrenocortical cytogenesis. This theory was deduced purely from an examinationof adrenocortical histology. It states that the adrenocortical zonae represent 3 separatebut related states of the “adrenocyte”, and that each of these states is expressedtransiently as a cell moves inward from a subcapsular position towards the medulla. Inaddition to the observational evidence cited above, this hypothesis has also beensupported by experiment. For example, after an enucleation procedure that removes thewhole cortex except for a thin layer of sub-capsular cells, a functional three-zonedgland regenerates after an initial period of proliferation followed by differentiation(Ingle and Higgins, 1938; Taki and Nickerson, 1985).Therefore, it is very likely that cells of the zona glomerulosa give rise to zonafasciculata cells. There may even be a spatial and developmental midpoint between thesetwo cell types within the zona intermedia. The zona intermedia is a very thin layer of 3-5 layers of small cells located between the glomerulosa and the fasciculata. The cellshave mitochondria similar to glomerulosa cells and SER similar to fasciculata cells(Nickerson, 1976).-6-iii) Steroidogenesis (Waterman and Simpson, 1985; Simpson and Waterman, 1988)All steroids originate with a single precursor, cholesterol. This is eithersynthesized de novo within adrenal cells from acetate or it is derived from the plasmavia lipoproteins (Hall, 1984). Cholesterol, in either a free or esterified form, is oftenstored in adrenocortical lipid inclusions. All steroidogenic pathways are initiated by theside chain cleavage reaction that takes place in the inner mitochondrial membrane andyields pregnenelone by cleaving a 6 carbon aliphatic tail from the cholesterol molecule.This reaction is catalyzed by the mixed function oxidase, cholesterol side chain cleavagecytochrome P-450 (P-4SOscc), which obtains its reduction equivalents from NADPHvia a flavoprotein (adrenodoxin reductase) and an iron-sulfur protein (adrenodoxin).The major steroidogenic pathways in the rat adrenal cortex are as follows.Pregenelone leaves the mitochondria and travels to the SER where it is converted toprogesterone by the combination dehydrogenase/isomerase iX5,3 13-hydroxysteroiddehydrogenase (A5,3f3-HSD). Progesterone remains in the SER and is hydroxylated atposition 21 by a C21 hydroxylase (P-450C21) to produce deoxycorticosterone. Thisweak glucocorticoid then moves to the mitochondria where it is converted into thepowerful glucocorticoid, corticosterone, by a second hydroxylase that acts at the 1113postition (P-450i 1 ). Corticosterone production occurs in all 3 zones of the cortex butproduction is highest in the fasciculata. Aldosterone, the major endpointmineralocorticoid is then produced in the mitochondria of glomerulosa cells by an 18-hydroxylation of corticosterone. This reaction may also be catalyzed by P-450i 1 13.Corticosterone is the major endpoint glucocorticoid in this species. Cortisol isnot produced due to the low activity of17 hydroxylase (P-45O17)in the rat adrenalcortex. This decreased activity also limits the production of adrenal androgens.When all of the above steroidogenic enzymes are introduced into a non-descriptfibroblast line by multiple transfection, and are expressed constituitively under the-7influence of strong heterologous promoters, the cells produce endpoint steroids (Mathewet al. 1990). When cholesterol is the substrate, steroid production is limited as thecholesterol is not efficiently transported to the mitochondria and P-45Oscc. In contrast,when pregnenelone is the substrate steroid, production is greatly increased. Thesefindings indicate that tissue specific adrenocortical function is dependent uponsteroiciogenic gene expression and cholesterol transport into the mitochondria. Otheraspects of steroid synthesis, such as the movement of steroid intermediates between themitochondria and the SER, are not tissue specific as they occurred independently in thefibroblast line.iv) Steroidogenic RegulationTrophic hormones which regulate steroidogenic differentiation act at the cellsurface, thereby activating receptor specific signal transduction pathways. The rasoncogene, which also appears to regulate steroidogenic differentiation, likely bypassesthe cell surface receptor and impinges directly upon signal pathways.a ACTHOne of the major regulators of overall adrenal steroidogenesis is the pituitaryhormone ACTH (Simpson et al., 1990). This peptide binds to a cell surface receptor thatactivates adenylate cyclase via the cholera toxin sensitive stimulatory G protein (Gs).This results in the production of the second messenger cAMP and the subequent activationof the cAMP dependent protein kinase A. There are two temporally separablesteroidogenic responses to this signal pathway activation. The acute response occursrapidly within minutes and involves the transport of cholesterol from cytoplasmic lipidinclusions to the mitochondria (Jefcoate et al., 1987). This transport is dependent on-8-adrenal-specific cholesterol carrier protein(s) present in the cytoplasm. The movementof cholesterol into the mitochondria brings it into the vicinity of P-45Oscc and therebyleads to the production of pregenelone and the subsequent production of endpoint steroids.Chronically, over a period of hours or days, ACTH-mediated cAMP productionleads to increased expression of steroidogenic enzymes. This was first observed inhyphosectomized rats where the levels of steroid hydroxylases decreased unless theywere restored by the administration of exogenous ACTH (Levy et al., 1959; Purvis etal., 1973). ACTH stimulation of steroid gene expression occurs principally at thetranscriptional level. The genes for steroid hydroxylases contain a variety of cAMPresponse elements (CRE) in their 5’ regulatory regions. These CREs bind adrenalspecific transcription factors in response to elevated cAMP. This process is crucial forincreased transcription (Simpson et al., 1990). cAMP-mediated upregulation of geneexpression is also enhanced, and over the longterm may be dependent upon, activation ofthe insulin-like growth factor-I (IGF-1) receptor, which is a tyrosine kinase(Hornsby et al., 1985; Veldhuis et aI., 1986; Naseerudin and Hornsby, 1990).b) AnQ IIAnother major adrenocortical secretagogue is angiotensin II (Ang II). In the rat,this octapeptide acts principally to increase aldosterone production in the zonaglomerulosa (Haning et al., 1970; Tait et al., 1974; Douglas et al., 1978; Braley et aL,1986; Whitley et al.; 1987). Ang II binds to a cell surface receptor and activates a Gprotein complex (Barrett et al., 1989). This stimulates phosphatidyl inositol specificphospholipase C (PLC), thereby producing the second messengers diacylglycerol (DAG)and inositol 1,4,5-trisphosphate (lns(1,4,5)P3). A rapid lns(1,4,5)P3-mediatedintracellular calcium release is critical in initiating the steroidogenic response (Kojimaet al., 1984), while a subsequent influx of calcium across the plasma membrane along-9-with the DAG-mediated stimulation of protein kinase C (PKC) are important inmaintaining this response (Kojima et al., 1985; Alkon and Rasmussen, 1988).Therefore, when this signal transduction pathway is activated, an increase inintracellular calcium ([Ca2+]i) is required for both the acute and chronic steroidogenicresponses. In addition, the activation of PKC is needed for the maintenance of the chronicresponse. It is not yet clear if this kinase always acts directly to upregulate steroidenzyme gene expression, or if it impinges upon the cAMP pathway via signal pathwaycrosstalk (Brami et al., 1987; Woodcock, 1989). For example, while only the P45Oscc gene has been shown to contain a classical PKC responsive element (TRE),transcription of the P-450c21 and P-450i 1 B genes are altered after PKC stimulationby 12-O-tetradecanolyphorbol-13-acetate (TPA; Deutsch et at., 1988; Chang et al.,1991).C) The ras OncoçieneExpression of the ras oncogene appears to effect steroidogenic differentiationpositively (Auersperg et al., 1981,1990; Amsterdam et at., 1988). There are 3mammalian ras genes, H, K and N-ras. They are highly homologous and encode forproteins of approximately 180 amino acids that have a molecular weight of 21 kD (p21ras; Barbacid, 1987). Like G protein a subunits, p21 ras proteins bind GTP, and haveintrinsic GTPase activity (Shih et al., 1980, Willingham et al., 1983; McGrath et at.,1984). They also cycle between the cytoplasm and inner face of the plasma membrane.The biologically active form of p21 ras molecules are bound to GTP and associated withthe plasma membrane (Trahey and McCormick, 1987). Retroviral ras oncogenes havepoint mutations at amino acid #12 which is located in one of the domains involved in GTPbinding. The result of this mutation is decreased GTPase activity. It is this characteristicthat most often correlates with the gene’s transforming ability. (Gibbs et a)., 1984).- 10 -Similar dominant, activating mutations have been found in a wide variety of humantumours (Barbacid, 1987). A second point mutation is found in the v-Kras gene ofKirsten Murine Sarcoma Virus (KiMSV). This mutation is at amino acid #59 and allowsfor phosphorylation of the molecule (Shih et al., 1982). All of this information suggeststhat p21 ras molecules are involved in signal transduction and that the viral forms mayact constitutively because of their reduced ability to hydrolyze GTP (Macara, 1991).Unlike the situation in yeast, ras proteins do not act directly as G proteins foradenylate cyclase in mammalian cells (Toda et al., 1984; Beckner et al., 1985).However, adenylate cyclase activity is altered in ras-transformed mammalian cell lines.The nature of these alterations is highly variable depending on the cell type. In a numberof fibroblast lines, ras-transformation reduces basal and agonist stimulated adenylatecyclase activities (Beckner, 1984; Saltorelli et al., 1985; Levitski et al., 1986).However, the physiologic state of the cell can alter the nature of the ras effect. Forexample, in serum deprived fibroblasts, activated ras expression increases cAMPproduction via inactivation of the inhibitory G protein (Franks et al., 1987). Activatedp21 ras expression can also have variable effects on c-AMP production in differentiatedcells. For example, in one thyroid epithelial cell line ras transformation decreasesadenylate cyclase activity (Colletta et al., 1988), while in another line cAMP productionis increased (Spina et al., 1987).ras p21 also impinges upon other signalling pathways, including those initiatedby PLC. In a number of fibroblast lines, basal and agonist stimulated levels of lnsP3 andDAG are increased after ras transformation (Chiarugi et al., 1985; Fleischman et al.,1986; Hancock et al., 1988). Y13-259, a neutralizing ras antibody, is able to blockthis increase, suggesting that it is p21 ras specific rather than associated withtranformation in general (Chiarugi et al., 1986). As with adenylate cyclase, the effectsof ras p21 on PLC activity are not straightforward. Wakelam and co-workers found thatras-transformation increases agonist stimulated PLC activity in sub-confluent, but not— 11confluent, 3T3 cultures (Wakelam et al., 1986; Wakelam, 1988). They suggest thecoupling of p21 ras to PLC may be reduced by the increased accumulation of autocrinegrowth factors that occurs when the cultures are crowded. In certain situations,activated ras expression increases DAG production with no change in lnsP3 levels,suggesting an effect upon PLC subtypes with specificities for substrates other thanphosphatidyl inositol, perhaps phosphatidyl ethanolamine or phosphatidyl choline (Lacalet al., 1987a; Wolfman and Macara, 1987; Morris et al., 1989). There is also evidencethat p21 functions both proximally and distally to PLC activation (Yu et al., 1988;Smith et al., 1990). Regardless, stimulation of this pathway leads to the activation ofPKC which has been implicated in a number of cellular activities including mitogenesis,determination, differentiation, secretion and cytoskeletal re-arrangements (N ishizuka,1988). Functional PKC is required for ras-mediated transformation (Lacal et al.,1 987b), and fibroblast lines that overexpress PKC have an increased susceptibility tothat transformation (Hsio et al., 1989). PKC also influences G protein activity, therebystimulating or inhibiting receptor mediated adenylate cyclase, depending on the cell type(Katada et al., 1985; Zick et al., 1986; Rozengurt et al., 1987). Thus, ras-mediatedactivation of PKC could lead to altered cAMP levels via signal pathway crosstalk. Thismight help explain many of the ras associated effects on adenylate cyclase describedabove.In certain situations, tyrosine kinases and PLC are linked in normal cells. Forexample, the platelet derived growth factor (PDGF) and epidermal growth factor (EGF)receptors are both transmembrane tyrosine kinases that phosphorylate and stimulatePLC after binding their respective ligands (Meisenhelder et al., 1989; Nishibe, 1990).Interestingly, microinjection of neutralizing antibodies to either ras p21 or PLCprevents tyrosine kinase induced mitogenesis (Smith et al., 1986,1990). This suggeststhat ras p21 may act to link tyrosine kinases to PLC. The interaction between p21 andreceptor tyrosine kinases may occur indirectly via the GTPase activating protein (GAP).- 12 -GAP associates with the GTP bound form of p21, and in so doing regulates ras function byincreasing GTPase activity more than 100 fold (Trahey and McCormick, 1987). GAPalso physically associates with, and may regulate the function of, tyrosine kinase growthfactor receptors (Molloy et al., 1989; Anderson et al., 1990; Ellis et al., 1990;Kazlauskas et al., 1990). It appears to do so via a regulatory domain known as SH2,which is found in a number of other proteins that act within signal transductionpathways (Heldin, 1991; Koch et al., 1991). Thus, GAP could act as a lateral effectorwithin the signal transduction network, regulating both the signal and its transduction.There are actually a number of proteins with GAP like activity. These include alternatelyspliced forms and genic homologues such as NF-1, which is mutated in the diseaseneurofibromatosis (Bollag and McCormick et al., 1991). Also, there is a group ofguanine nucleotide exchange factors that associate with ras p21 (Downward et al., 1990;Wolfman and Macara, 1990), as well as an inhibitor of GTPase activity (GIP, Tsai et al.,1990). Consequently, the ability of ras to act upon numerous transduction pathwaysmay be enhanced by the multiplicity of proteins that regulate its biochemical state. Howthese regulatory proteins are affected by the oncogenic activation of p21 ras, whicheliminates its GTP/GDP switching, may be important. For example, the associationbetween GAP and activated p21/GTP is long lived, presumably because GTPase activitycannot be increased (Macara 1991). Thus, activated ras could act as a sink for GAP,which would diminish the regulatory interactions between GAP and receptor tyrosinekinases.Most investigators have used proliferation or oncogenic transformation asendpoints when studying ras function. However, ras also has profound effects ondifferentiation. These effects are not always negative. For example, v-ras enhancestissue specific differentiation in adrenomedullary tumour cells (PC12), thyroidmedullary carcinoma cells, and small cell lung carcinoma cells (Noda et al., 1985;Nakagawa et al., 1987; Mabry et al., 1988). In the case of PC12 cells, this- 13 -differentiative effect occurs via a PLC stimulation that is normally initiated by nervegrowth factor (Sassone-Corsi et al., 1989; Kremer et al., 1991). Activated ras can alsoenhance differentiation in some non-tumour cell types. For example activated rasexpression induces adipocyte differentiation in pre-adipocytes by activating the signalpathway that is normally initiated by insulin, whose receptor is a tyrosine kinase(Benito et al., 1991). These tissue specific effects suggest that ras p21 can act upondifferentiation associated signal transduction pathway templatesH that are constructedduring the process of normal development. If this is indeed the case, ras expressionshould induce a common set of changes in cells with a similar differentiated phenotypeand developmental history . This appears to be the case with steroidogenic cells of therat.in 3jy, cellular ras is often expressed in differentiated, non-proliferativetissues. For example, ovarian and adrenocortical cells contain c-ras p21 (Chesa et al.,1987; Furth et al., 1987). nyj.trn, expression of oncogenic ras is associated withsteroid production in immortalized ovarian granulosa cells (Amsterdam et al., 1988;Suh and Amsterdam, 1990) and in normally non-steroidogenic ovarian surfaceepithelial cells (Pan et al., 1991). Oncogenic ras also enhances the steroidogeniccharacteristics of explant-derived adrenocortical fibroblasts (Auersperg et al., 1981,1990; Wiebe et al., 1987). Presumably, the ras protein elicits these effects by actingwithin those signal transduction pathways that are associated with normal steroidogenicdifferentiation.3. Rat Adrenocortical Cells in vitroAdrenocortical explants produce large numbers of outwardly migratingfibroblasts. In FBS supplemented medium these cells are myofibroblastic in growthpattern, ultrastructure and extracellular matrix production (Slavinski et al., 1 974,- 14 -1976; Slavinski-Turley and Auersperg, 1978; Turley, 1980). They do however, have avery limited steroidogenic potential that is increased after ACTH treatment. When theyare maintained in HS-supplemented medium these cells modulate to a more epithelialform and acquire parenchymal characteristics which include: the accumulation ofcytoplasmic lipid; expression of the steroidogenic enzyme A5,3f3-HSD; and increasedsteroid production both in the absence and presence of ACTH. Thus, these adrenalfibroblasts have a dual stromal/parenchymal potential. These cells likely originate fromthe connective tissue capsule of the gland (Bressler, 1973; Ryback and Ramachandran,1981a). capsular fibroblasts express 5,36-HSD during fetal development butnot in the adult animal, suggesting that capsular cells have the potential to expressparenchymal characteristics early in development but that this potential is suppressedafter birth (Farcnik and Auersperg, 1984). Therefore, the expression of parenchymalcharacteristics by adrenal fibroblasts in culture may actually be representative of anearly developmental state of the cells.The ras oncogene is able to transform explant derived adrenocortical fibroblasts(Auersperg et al., 1977; Auersperg 1978). These transformed cells grow to highsaturation densities, are serum and anchorage independent, and form locally invasivetumours in immunosuppressed rats. Depending on the particular transformed line, thesetumours are either sarcomas or carcinomas, or sometimes show elements of both(Auersperg et al., 1981). This ability to express two tumour phenotypes may reflectthe bipotential nature of the target cells prior to transformation. In culture, the rastransformed fibroblasts contain lipid, constitutively convert pregnenelone toprogesterone (E5,3f3-HSD activity), and express a number of other steroidogenicenzymes (Auersperg et al., 1981, 1990; Wiebe et al.1 987). These cells also respondmorphologically to treatment with cAMP in a manner similar to that observed in anumber of steroidogenic cell types. In addition, the production and elaboration of anextracellular matrix is reduced (Auersperg et al., 1990). Thus, in adrenocortical- 15 -fibroblasts, ras oncogene expression is associated with the enhancement of parenchymalcharacteristics, and the reduction of stromal ones. As has been noted, this phenotype maybe representative of an early developmental state of these cells.During development, individual clones move from the adrenal capsule inwardacross the entire cortex until they degenerate in the zona reticularis near the medulla(I. 2. ii). This suggests that as an ‘adrenocyte’ moves inward, its stromal potentialbecomes gradually restricted as its parenchymal potential becomes fully expressed.Therefore, unlike explant-derived fibroblasts, there may be a population ofadrenocortical cells with the potential to exhibit the full complement of stromal andparenchymal characteristics at the same time. Presumably these cells would be locatedcloser to the capsule than the medulla. A good candidate would be a population of cellsderived from the sub-capsular zona glomerulosa.The zona glomerulosa can be separated from the zonae fasciculata/reticularis bydissection (for summary of cellular characteristics, see Table 1). This involvesremoving the capsule with the adherent glomerulosa from the rest of the cortex (Haninget al., 1970). This is the most common method for separating adrenocortical zones in therat. Single cell suspensions can then be obtained by subjecting the separated tissues tocollagenase digestion. Ninety-five percent of zona fasciculata/reticularis cells obtainedby this method contained numerous lipid droplets. In primary culture they producecorticosterone and responded strongly to ACTH stimulation, both by increasing theirsteroid production and retracting from the substratum (O’Hare and Neville, 1973a,b;Ramachandran and Suyama, 1975; Ryback and Ramachandran,1981a). Nevertheless, thesteroidogenic differentiation of these cells decreases rapidly with increasing time inculture (Ryback and Ramachandran, 1981b). Initially, they form confluent epithelialmonolayers that exhibit very little growth (Neville and O’Hare, 1973b). However, after1-2 weeks fibroblasts begin to proliferate which eventually overgrow the entireculture. Addition of the fibroblastic inhibitor d-valine (Ryback and Ramachandran,- 16 -198Th), or addition of a mixture of HS and FBS on a fibronectin substratum reduces thefibroblast growth in these cultures (McAllister and Hornsby, 1987), but they do noteliminate the decrease in steroidogenic differentiation. Therefore, decapsulationprovides a population of fasciculata/reticularis cells that are well suited to short termexaminations of parenchymal growth and differentiation. For long term study, at least inthe rat, this method may not be so useful. In bovine adrenocortical cells steroidogenicdifferentiation can be maintained for much longer periods (Hornsby et al., 1980, 1987,Hornsby, 1991).Collagenase dissociation of adrenocortical capsules produces a population of cellsthe majority of which are small and contain scanty amounts of cytoplasmic lipid (Haninget al., 1970). These cells are also steroidogenic and respond to ACTH with increasedcorticosterone and aldosterone production. Unlike in rat fasciculatata/reticularispopulations these cells also respond to Ang II (Barrett et al., 1989). In culture, thecells spread, flatten and proliferate rapidly (Hornsby et al., 1974; Ryback andRamachanciran, 1981a; McAllister and Hornsby, 1987). These authors refer to theproliferating cells in capsular preparations as adrenal “fibroblasts”. Prior to thepresent study it had not been determined whether these cells are true stromal cells, suchas those that migrate from adrenocortical explants (Slavinski et al., 1974, 1976), or ifthey are actually modulating parenchymal cells derived from the definitive zonaglomerulosa.In this study, morphologically homogeneous parenchymal cell populations devoidof stromal or endothelial cell contaminants have been isolated by density gradientcentrifugation. In particular, cellular sub-populations derived from the zonaglomerulosa (GLOM) and zona fasciculata (FASC) were used to determine whetherstromal and parenchymal characteristics could be expressed simultaneously in culture.These cells were also used to carry out an unambiguous investigation of the mechanismresponsible for differential zonal responses to Ang II. In addition, these density isolated- 17sub-populations were used in mass transformation experiments with the ras oncogene,as they ensured an unambiguous pool of parenchymally derived target cells.- 18 -TABLE 1Characteristics of Rat Zona Glomerulosa and Zona Fasciculata CellsCharacteristic Glomerulosa Fasciculataposition in gland subcapsular centralcell size 7-12 urn 14-24 urnlipid content scanty abundantcell density ‘1.O7 g/ml -1.O4 g/rnlmajor steroid aldosterone corticosteroneACTH response + + + + + +Ang II response ++++-- 19 -II. MATERIALS AND METHODS1. Production of Rat Adrenocortical Cells.i) AnimalsThroughout these experiments 8-16 wk. old male Fischer rats (housebred,strain 344), were used to obtain adrenocortical cells for experimentation. Males werechosen to eliminate steroidogenic fluctuations that occur during the female eostrouscycle. Animals were sacrificed at approximately the same time each day (10 am. to 12noon) to minimize daily fluctuations in steroid output. At no time were the animals kepton a high or low salt diet as such changes can also effect steroid production, particularlyof mineralocorticoids (Taki and Nickerson, 1985). The animals were kept on a l2hrlight dark cycle and fed a diet of purina rat chow and water without restrictions.ii) Adrenocortical Fibroblast Migration From Explants (Slavinskl et al., 1974, 1976;Slavinski-Turley and Auersperg, 1978)Animals were lightly anaesthetized with either chloroform or halothane (MTCPharmaceuticals, Caimbridge Ont.) and killed by cervical dislocation. Adrenal glandswere then removed, trimmed of adherent fat, minced into pieces of approximately 1mm3 and placed in 35mm tissue culture dishes (1 gland per dish) whose bottoms hadbeen scratched to provide an adherent surface. In certain experiments, adrenocorticalcapsules and de-capsulated glands were minced separately . The minced tissue was left- 20 -for 5-10 mm and then tissue culture medium was added, consisting of Waymouth’s 752(GIBCO) supplemented with 25% Fetal Bovine Serum (FBS, Hyclone).The cultures were incubated in a humidified atmosphere containing 5%C02 at37°C. They were left undisturbed for 3-5 days at which time approximately 40-60% ofthe explants had adhered to the the dish. The culture medium was then changed toWaymouth’s supplemented with 10% FBS and changed as needed thereafter. During thefirst week in culture fibroblastic cells began to migrate from the explants.iii) Collagenase dissociation of Rat Adrenocortical Cells (Haning et al.,1970)Whole adrenal glands were hemisected and the medulla was squeezed out usingblunt ended forceps. The tissue was then minced to 1 mm3. In certain experiments eitheradrenal capsules or inner glands were used. The minced tissue was then placed in acollagenase digestion solution. Typically, tissue from 8 glands was placed in 30 ml ofHBSS (pH 7.2) containing 5 mg/mI BSA (Fraction V, Sigma), 3 mg/mI collagenase typeII (Sigma), 0.05 g/ml DNAse type I (Sigma) and 0.5 mg/mI soybean trypsin inhibitor.Collagenase type II was first developed for the isolation of adipocytes (Rodbell, 1964).This enzyme has very little tryptic activity which is harmful to lipid-containing cellssuch as those derived from the adrenocortical parenchyma. As an additional precautiontrypsin inhibitor was also added to the digestion solution. DNAse was added to preventcell clumping due to DNA that was released from damaged and dead cells during theprocedure.The tissue supension in digestion medium was gassed for 1 mm with 95% 02/5%C02 and incubated in a lOOmI spinner flask (Bellco, Vineland NJ) at 50 rpm for 1 hourat 37°C. This produced friable tissue pieces that were then dissociated by pipetting 50-75X in a 5 ml glass serological pipette. Dissociated cells were removed and theremaining tissue was further incubated in digestion solution for 15 mm followed by- 21pipetting. This procedure was continued until all of the tissue was dissociated. Theresulting cell suspensions were pooled, filtered through multilayered nitex cloth(lOOjim mesh size) to remove cell clumps, centrifuged twice (200X g, 5 mm), and resuspended in tissue culture medium which consisted of Dulbecco’s modified essentialmedium mixed 1:1 v:v with Ham’s F12 medium (DMEM/F12, Sigma or GIBCO).Typically the majority of the cells were single (>80%) or in small aggregates of 2-4cells.2. Separation of Rat Adrenocortical Cell TypesA number of methods were used in an effort to separate the steroidogenic cells ofthe adrenocortical parenchyma from contaminating stromal and endothelial cells.i) Differential Substratum Adhesion SeparationDifferent cell types have different capabilities for attaching to tissue cultureplastic. These differences can be exploited to separate specific cell types from mixedpopulations (Folkman et al., 1979).Cell suspensions obtained by collagenase dissociation of whole adrenocorticeswere seeded into 35 mm tissue culture dishes (approximately 1 Xl cells per dish)for 15-180 minutes. At the end of the prescribed period, the medium was removed andthe unattached cells were re-seeded into new tissue culture dishes. The two populationswere then cultured and observed morphologically for one week.- 22 -ii) Differential Morphological Response to cAMP.Many steroidogenic cell types in culture respond to cAMP treatment byretracting from the substratum and rounding up (Gill et al., 1980; Amsterdam et al.,1989; Hornsby et al., 1989). Therefore, we treated primary cultures derived fromcollagenase dissociation of whole adrenocortices with 8Br-cAMP (0.5 mM, 72 hrs,Sigma) to induce rounding of the parenchymal cell population.cAMP caused many of the cells in 1 week old primary cultures to round severely.These rounded cells were detached from the substratum by gentle flushing with mediumusing a pasteur pipette. These cells were then centrifuged (200 Xg, 5 mm) and replated into secondary culture utilizing medium that did not contain cAMP.iii) Dissection of Adrenocortical Capsules (zona glomerulosa from the Inner Portion ofthe Gland (zonae fasciculata and reticularis.This procedure has been described previously and is the most commonly usedmethod for separating rat adrenocortical cell types (Haning et al., 1970; Tait et al.,1974; Hornsby et al., 1974, Barrett et al. 1989).Whole adrenal glands were trimmed of fat and placed on HBSS soaked filter paper.Under a dissecting microscope an incision through the capsule was then made along oneface of the gland from pole to pole. The capsule was then peeled off of the rest of the gland.This provided two fractions, the adrenal capsule with the adherent zona glomerulosa andthe inner portion of the gland which predominantly contains the zona fasciculata and thezona reticularis. These glandular fractions were utilized in explant, collagenasedissociation, and percoll gradient experiments.- 23 -iv) Discontinuous Percoll Density Gradient SeparationUtilizing serum albumin gradients, the densities of rat fasciculata cells and zonaglomerulosa cells were estimated previously to be 1 .040 g/ml and 1 .072 g/mlrespectively (Tait et al., 1974). However, since fasciculata cells are large andglomerulosa cells are small it is very difficult to separate these cell types in albumingradients because the rate of cell movement depends on both cell density and cell size.Thus, cells with a low density and large size (ie. fasciculata) move through the gradientat a similar rate as cells with a high density and small size (ie. glomerulosa).In the following experiments we have therefore utilized percoll gradients whichseparate fasciculata and glomerulosa cells solely on the basis of density. In addition,discontinuous gradients were utilized so that density interfaces could be constructed withvalues similar to those determined previously in the serum albumin gradients.a) Gradient PreparationStock isosmotic percoll (SIP) was prepared by mixing 9 parts percoll(Pharmacia, Uppsala Sweden, density- 1.128 g/ml) with 1 part lox DME/F12(Appendix A). This was diluted with 1X DME/F12 containing 0.5% BSA to producesolutions of 15,25,30,35,40,45,50, and 65% SIP. These solutions were layered uponone another from heaviest to lightest in a round bottom plastic centrifuge tube to producean eight step discontinuous density gradient with seven density interfaces that was stablefor several hours at room temperature.- 24 -b) Cell Separation and Density EstimationCollagenase dissociated cell suspensions, derived from either wholeadrenocortices, glandular capsules, de-capsulated glands or supra-renal fatpads wereplaced atop the gradient and the tube centrifuged at 1000 Xg for 20 minutes. Typically, acell suspension derived from 8-20 glands (approximately 4-10 X 106 cells) was addedto the gradient in a maximum volume of 1.5 ml. Separated cells, visible as 7 bands,were collected by pipetting, diluted in DME/F1 2 medium (1:5 v:v) to reduce the densityof the medium, and centrifuged (200 Xg 5 mm) to remove the percoll. Allmanipulations involving percoll were carried out at room temperature.The density of each cell band was estimated by measuring its distance from themeniscus and then extrapolating back to a standard curve that was constructed usingcolour coded density marker beads (Pharmacia) which were separated in parallelgradients. Cells isolated from the 25-30% Percoll interface (Band 2, 1 .034 g/ml)were designated FASC (derived from the zona fasciculata). Cells isolated from the 50-65% interface (Band 7, 1 .069 g/ml) were designated GLOM (derived from the zonaglomerulosa). Cells isolated from the 40-45% interface (Band 5, 1.057 g/ml) weredesignated MIX (contained a mixture of stromal, endothelial and parenchymal cells).In addition, rat adrenal medullary cells isolated using the method of Livett(1984) were subjected to percoll gradient separation. These cells were used as acatecholamine positive control for comparison with FASC and GLOM cells.3. Tissue Culture.Collagenase dissociated cells were routinely cultured in DME/F12 mediumsupplemented with 15% HS and 2.5% FBS (Hyclone, Logan UT, USA) at 37°C in ahumidified atmosphere of 5% C02 in air. Medium was changed 48 hrs after plating and- 25 -subsequently on every third or fourth day. When these culture conditions were variedfor specific experiments it is so noted in the text. Keeping in mind the mesodermalorigin of the adrenal cortex, we attempted to provide an environment conducive to themaintenance of parenchymal function while minimizing the possibility of stromalovergrowth. Therefore, the high HS/low FBS supplement combination used in theseexperiments was chosen for three reasons: (1) it allows for the maintainence ofsteroidogenesis by Yl adrenocortical tumour cells (Yasumura et al., 1966) and primarycultures of collagenase dissociated cells from whole rat adrenal cortices (Leonard et al.1983); (2) pre-senescent stromal adrenocortical cells derived from explants modulatefrom a myofibroblastic state in FBS to a steroidogenic, epithelial state in HS (Slavinskiet al., 1974, 1976); (3) HS and FBS together have been reported to inhibitadrenocortical fibroblast overgrowth in culture (McAllister and Hornsby, 1987).Cells were passaged either with trypsin (1:250 in Ca2/Mg free HBSS), orwith collagenase (1 mg/mI in Ca2/Mg containing HBSS), or by gentle agitationusing a pasteur pipette when rounded, loosely adherent cells were removed selectivelyfrom the substratum.Cells were counted using a hemocytometer or an Artek semi-automated cellcounter (Artek Corp. East Farmingdale NY, USA). In certain experiments the cellnumber was estimated by determining the amount of DNA per culture using thefluorescent DNA-binding dye bisbenzimide (Mates et al., 1986; Sigma). Fluorescencewas measured using an Aminco-Bowmann Spectrophotometer. After assaying cultures ofknown cell number, it was determined that rat adrenocortical cells containedapproximately 9 pg DNA per cell.- 26 -4. Differentiation Markers: HistochemistryAdrenocortical cells separated on percoll gradients were assayed histochemicallyfor a number of differentiation markers, both in suspension and in primary culture.i) Cytoplasmic LipidCytoplasmic lipid could be seen as areas of refractility within percoll separatedadrenocortical cells, especially when viewed with phase microscopy. The presence ofcytoplasmic lipid was confirmed by staining primary cultures with the lipophilic dyeOil Red 0 (Culling, 1974). Cultures were rinsed with HBSS, rapidly frozen and thawed3 times on dry ice and then stained with 0.5% oil red 0 in 60% isopropyl alcohol for 15minutes. The cultures were then rinsed in dH2O and the nuclei counterstained withhemotoxylin. Lipid then appears as red cytoplasmic vesicles.ii) A5.3B-hydroxysteroid dehydrogenase (5.3B-HSD)A5,313-HSD activity was assayed in percoll separated adrenocortical cells usingdehydroepiandrostenedione (DHEA) as substrate (Levy et al., 1959; Farcnik andAuersperg, 1984).Cells on glass coverslips were rapidly freeze-thawed on dry ice and incubated inPBS (pH 7.2) containing 0.05 mg/mI DHEA, 0.13 mg/mI nitroblue tetrazolium dye, and0.38 mg/mI nicotinamide adenine dinucleotide (NAD, all Sigma) for 1 hour at 37°C. TheDHEA is converted to androstenedione by z5,3l3-HSD activity. This produces a reductionequivalent that is carried to nitroblue tetrazolium by the NAD. The result is thereduction of the dye which produces insoluble, blue fomazan deposits within the cells.- 27Cells were scored as z5,3I3-HSD positive if the formazan deposits obscured the nucleus.Yl mouse adrenocortical tumour cells (Yasamura et al., 1966), pre-senescent rat lungfibroblasts, and crude rat testicular cell suspensions were used as tissue specificpositive (Yl and Leydig cells) and negative (fibroblasts and developing germ cells)controls.iii) CatecholaminesTo determine if adrenal medullary cells were present as contaminants, primarycultures were stained for the presence of catecholamines using the sucrose-phosphateglyoxylic (SPG) method (de Ia Torre and Surgeon, 1976).Cells were grown on coverslips for 4-7 days, rinsed in PBS, and dipped rapidly3 times in the SPG solution which contained 1% w:v glyoxylic acid (Sigma) and 0.200 Msucrose in PBS (pH of 7.4). The coverslips were then air dried, heated to 80°C for 5mm, and mounted in mineral oil. Catecholamine staining was viewed by epifluorescence.5. Differentiation Markers: ImmunofluorescencePercoll separated adrenocortical cells in culture were fixed and stained for anumber of differentiation markers that were identified by immunofluorescence. Afterstaining the coverslips were mounted in gelvatol, (pH 7.2, Rodriques and Kienhardt,1960), viewed by epifluorescence and photographed with Kodak Tn X Pan film (forblack and white, ASA 800), or with Kodak Ektachrome 400 (for colour, ASA 800).- 28 -i) Cytochrome P-450 side chain cleavage enzyme (P-45Oscc).P-45Oscc is a mitochondrial enzyme that converts cholesterol to pregnenelone.It is found only in steroidogenic cells (Goldring et al., 1986).Cells on glass coverslips were fixed in formalin or 3.6% paraformaldehyde,blocked with 0.1M glycine and permeabilized in 1% triton X-100 (Sigma) for 4minutes; next the cells were incubated with a rabbit polyclonal antibody raised againstrat P-45Oscc (1:20 dilution, courtesy of J. Orly, Jerusalem, Israel) for 15 mm. atroom temperature, washed thoroughly in PBS and incubated with the second antibody, arhodamine labelled goat anti-rabbit IgG (1:20, Heavy and Light chain specific, Cappel,West Chester PA, USA). Normal rabbit serum was used as an immunological control andfull antibody staining of rat lung fibroblasts was used as a tissue specific negativecontrol. The mitochondrial location of P-4SOscc aided in determining specificity as thisresulted in small definitive fluorescent bodies in positive cells rather than a diffusestaining in negative cells. The exception was in rounded cells where cell thickness madeit difficult to pick out individual positive mitochondria.ii) KeratinKeratins are a class of intermediate filament proteins that are expressed in anumber of epithelial cell types (Steinert and Liem, 1990). The presence of keratins hasbeen noted in the adrenal cortex of some species, including human (Henzen-Logmans etal., 1988), but not in the rat.FASC and GLOM cells were cultured on glass coverslips for either 4 or 10 days.Three different antibodies were used in an effort to determine if any keratin was presentin these cells. Two antibodies (CK-49, CK-55) were mouse monoclonals raised againstlow molecular weight rat keratins found in many glandular epithelia (Germain et al.,- 29 -1988; courtesy N. Marceau, Laval Que.). A third antibody (RAK), was a rabbitpolyclonal that reacts with a number of different rat keratins (O’Guin et al., 1985;courtesy T.T. Sun, New York, USA, REF).The cells were fixed in methanol (-20°C), permeabilized with MeOH:acetone(1:1, -20°C) for 5 mm., air dried and then rehydrated in PBS; next the cells wereincubated with one of either CK-49 (1:100), CK-55 (1:200) or RAK (1:50) for 30mm at 37°C, washed in PBS and incubated with the appropriate second antibody,rhodamine labelled goat anti-mouse lgG (1:20, Jackson Labs, for CK-49 and CK-55),or goat anti-rabbit lgG (1:20, Cappel, for RAK) for 30 mm at 37°C.Mouse lgG (for CK-49, CK-55) and normal rabbit serum (for RAK) were usedas immunological controls. The rat ovarian surface epithelial cell line ROSE199(Hornby and Auersperg 1992) was used as a species and tissue specific positive controlcell line.IH) Double Staining for Vimentin and P-45OsccVimentin is an intermediate filament protein that is expressed in manymesodermally derived cells including stromal fibroblasts (Steinert and Liem, 1990).FASC and GLOM cells were cultured on glass coverslips for 3 or 9 days, fixed inmethanol (-20°C), permeabilized with MeOH:acetone (1:1, -20°C), air dried andrehydrated in PBS; next they were incubated with the rabbit anti-rat P-45Osccpolyclonal antibody (1:20 dilution, courtesy J. Orly, Jerusalem, Israel) and a mouseanti-rat vimentin monoclonal antibody (1:50, courtesy N. Marceau, Laval Que.) for 30mm at 37°C; finally, they were washed with PBS and incubated with the secondantibodies, rhodamine labelled goat anti-rabbit lgG (1:20, Cappel) and AMCA labelledswine anti-mouse lgG (1:20, heavy and light chain specific, Jackson lmmunoresearch,West Grove PA, USA).- 30 -Normal rabbit serum (for anti P-45Oscc) and mouse IgG (for anti-vimentin)were used as cytochemical controls. Full antibody staining of rat lung fibroblasts wasused as a tissue specific positive control for vimentin, and a simultaneous negativecontrol for P-45Oscc.iv) Factor VIIIFactor VIII, one of the components of the blood clotting cascade is found in manyendothelial cell types (Folkman et al. 1979).Rapidly adherent cells from whole adrenocortices were cultured on glasscoverslips for 14 days, at which time endothelial cell colonies were readily visible. Thecells were fixed in methanol (-20°C), air dried and rehydrated in PBS; next they wereincubated with a rabbit polyclonal antibody raised against human factor VIII (1:25,Calbiochem) for 60 mins at room temperature, washed in PBS and incubated withrhodamine labelled goat anti-rabbit lgG (1:20, Cappel) for 45 mins at roomtemperature.Normal rabbit serum was used as an immunological control. Human umbilicalvein endothelial cells were used as a tissue specific positive control.6. Time Lapse MicroscopyMixed parenchymal-stromal populations (MIX) in primary and secondaryculture were kept in sealed flasks, mounted on the stage of an inverted phase contrastmicroscope (Leitz, Fluorovert), and observed through the camera port using a videocamera equipped with mechanical focus (Panasonic, model #VW-1 850). The image wasthen collected and recorded on a time lapse video recorder at 1/120 real time(Panasonic, model #TL-6050). The temperature was maintained at 37°C by using a- 31thermo-regulatable air curtain (Sage Instruments, White Plains NY). Routinely,individual fields were recorded for periods of 48-72 h. For detailed morphologicalexamination, single fields were photographed periodically for periods of up to 72 hours.7. Electron MicroscopyThe ultrastructure of FASC and GLOM cells was examined by transmissionelectron microscopy.After 14 days in primary culture, the cells were scraped from the substratum,fixed with 2.5% gluteraldehyde in Millonig’s Buffer (pH 7.2) for 60 mm at 4°C,washed, and post fixed with 1% 0s04 for 30 mm; next they were dehydrated in a gradedethanol series and embedded in epon. Sections were stained with uranyl acetate/leadcitrate and examined on a Zeiss EM-i 0 electron microscope.8. SteroidogenesisSteroidogenesis was assayed by two methods: (1) fluorescence after treatmentwith acid-ethanol was used to give an overall indication of steroid production; (2)radioimmunoassay was used to specifically measure corticosterone (CS) and aldosterone(Aldo).i) Acid-ethanol fluorescenceA number of 3-keto, 4-ene steroids, including 20a-dihydroprogesterone andcorticosterone, fluoresce after treatment with acid-ethanol (Kowal and Fiedler 1968;Neville and O’Hare 1973a). The steroid present in conditioned tissue culture mediumwas extracted into 10 volumes of dichloromethane and then mixed 1:5 (v:v) with cold- 32 -ethanol/sulphuric acid (35:65 v:v). The mixture was shaken vigorously and left to standfor 60 mm. The fluorescence in the acqueous layer was then measured using an Amino-Bowman spectrofluorometer (activating wavelength-468 jim, emitting wavelength-525nm). Corticosterone (Calbiochem, La Jolla CA, USA) in tissue culture medium was usedas the standard. Results were expressed as amount of fluorogenic steroid/time/culturedish or cell number, depending on the experiment.ii) Radioimmunoassay (RIA)Competitive RIA was used to specifically measure the production of CS and Aldo.To assay steroidogenesis in suspension prior to culture, percoll-separated cellswere counted and immediately suspended in 1.0 ml aliquots of 0.5% BSA in DME/F1 2(4.2mM Kj. Parallel tubes were supplemented with or without trophic hormones atthe indicated concentrations. The cell suspensions were then placed in 1.5 ml plasticmicrofuge tubes and rotated at 30 rpm at 37°C for the indicated time. Cells were thenremoved by centrifugation (800 Xg, 10 minutes, 4°C) and the medium frozen at -30°Cuntil assayed. In culture, cells were treated with or without trophic hormones for thetime indicated. The conditioned medium was then collected and frozen at -30°C untilassay. The cells were then counted or their number was estimated by DNA assay.CS was ethanol-extracted to remove serum associated CS binding proteins andassayed by RIA using an antiserum raised against corticosterone-3-carboxymethyl-oxime:BSA (ICN, Costa Mesa CA, USA). Aldo was assayed by RIA directly from the mediumusing antiserum raised against aldosterone-18,21-dihemisuccinate (ICN).Steroid standard or experimental samples were diluted to 500 il in PBS (pH7.0) containing 1mg/mi gelatin (Sigma) . 3H steroid tracer (NEN, Boston MA, USA)and steroid antibody were then mixed 1:1, and 200 jil was added to the steroid/PBSsolution. 12-15,000 c.p.m. of 3H tracer was used per sample and a concentration of- 33 -antibody was used that was capable of binding 30-50% of the tracer in the absence ofcold steroid. The steroid/tracer/antibody mixture was incubated for 4 hours at 4°C.Unbound steroid was then removed from the solution by adding 200 jil of charcoal-coateddextran (charcoal:dextran, 10:1), mixing for 20 mm., and then clearing bycentrifugation (2000 Xg, 10 mm). Antibody bound steroid left in the aqueous phase wasthen mixed with ACS scintillation fluid (Amersham, Oakville Orit.) and counted for 5 miiiin a 6-counter.The amount of steroid per sample was determined by extrapolating the percent oftracer bound back to a standard curve that was constructed using known quantities of recrystallized corticosterone and aldosterone (Calbiochem). Fresh tissue culture mediumwas always assayed twice per assay and subtracted as a blank.9. Inositol Phosphate DeterminationAngiotensin II (Ang II) binds to a cell surface receptor and stimulates the activityof phosphatidyl inositol specific phospholipase C resulting in the increased production ofinositol phosphates (Barrett et al., 1989).I) Analysis of Inositol PhosphatesTwo day old GLOM and FASC cultures (2.5 X 1 cells/i 6mm well) were labelledwith myo-2-3H-in sitol (5j.tCi/well, Dupont-NEN, Mississauga Canada) for 24 hrs,washed and pre-incubated for 30 minutes in serum-free medium with 10mM LiCI2.HBSS (200 III, with 10 mM HEPES, pH 7.2), with or without 100 nM angiotensin II(Ang II), was then added to cells and incubated at 37°C for either 3 or 10 mm.hnciibations were terminated with 1 .Oml cold (-20°C) methanol. Radiolabelled inositol- 34 -phosphates were then extracted into the aqueous phase of acidifiedchloroform/methanol/water (10:5:3, v:v:v). The extracts were neutralized and theinositol phosphates were separated by ion exchange chromatography on 1 .Oml Dowexcolumns (AG1-X8 resin, 200-400 mesh, formate form, Bio-Rad, Richmond CA) usingsequential washes of 0.1M formic acid containing 0.2 M (InsPi), 0.4 M (lnsP2), and1.0 M (lnsP3) ammonium formate (Downes and Mitchell, 1981; Leung et al., 1986).The relative amount of inositol phosphate per fraction was then determined by by liquidscintillation counting.ii) Analysis of Inositol 1 .4.5-trisphosphate (lns(1 .4.5)Pp).Freshly isolated suspensions or 2 days old cultures of GLOM and FASC cells (2-4X 105 cells/i 6mm well) were rinsed and maintained in serum free medium for 4 hrsprior to experiment to reduce baseline variation. The cells were then incubated in HBSS(125 iii, with 10 mM HEPES, pH 7.2> with or without lOOnM Ang II or 25 pM calciumionophore (Br-A23187) for the indicated times at 37°C.lncubations were terminated and the intracellular inositol phosphates werereleased into the medium by treatment with percloric acid (Appendix B). The sampleswere cooled, centrifuged and neutralized with KOH. lns(i,4,5)P3 was then assayedutilizing a commercially available, competitive, isomer specific, radioligand bindingassay (Palmer et al., 1989; Amersham, Arlington Heights IL).lns(1 ,4,5)P3 standards (0.19-25 pm), or aliquots from experimentalsamples, were incubated in Tris buffer (pH 9.0) containing 3H lns(1 ,4,5)P3 tracerand a slurry of lns(1,4,5)P3 binding protein. The binding protein was then centrifugedout of this mixture, resuspended in dH2O and the amount of tracer bound determined byliquid scintillation counting. The binding protein bound 25-40% of the tracer in the- 35 -absence of cold standard or sample. The amount of lns(1 ,4,5)P3 in the sample wasestimated by extrapolating back to a standard curve constructed utilizing the % bindingobtained in the presence of known quantities of lns(1 ,4,5)P3 standards. 20 pMIns(1,4,5)P3 in HBSS was extracted, assayed and then used to estimate the percentrecovery. In 4 of 5 experiments this value was >80%.10. Free Intracellular ([Ca2]i) DeterminationAng Il-induced lns(1,4,5)P3 production triggers a release of calcium fromintracellular stores in rat zona glomerulosa cells, causing an increase in [Ca2+]i that iscritical for the stimulation of steroidogenesis (Barrett et al. 1989).GLOM and FASC cells were assayed for levels of [Ca2]i in primary culture usingan optical method that involved loading the cells with the fluorescent calcium binding dyeFura-2 (Grynkewicz et aL, 1985, Wang et al., 1989). Specifically, at excitationwavelengths of 350 and 380 nm, the fluorescence emissions vary inversely dependingon the amount of calcium bound to the dye. Therefore, by recording emission levels atthese wavelengths in Fura-2 loaded cells, [Ca2]i can be estimated.i) Culture of CellsGLOM and FASC cells (approximately 1 X cells) were maintained in primaryculture on round 18 mm glass coverslips for 4-12 days. It was necessary to culture thecells for at least 4 days as this ensured that they were sufficiently attached to thecoverslip to prevent washing away during the assay. We found no difference in [Ca2]iresponses at either extreme of this time period. However, after two weeks or more inculture the [Ca2]i responses, particularly to Ang II, began to decline.- 36 -ii) Fura-2 LoadingThe cultures were loaded with the aceto-methyl ester derivative of Fura-2(Fura-2AM). In this form the dye is lipophilic, and is thus able to diffuse freely into thecells. Fura-2AM in DMSO was added to HBSS (with 10mM HEPES, pH 7.2) to a finalconcentration of 5jiM. The non-ionic detergent plurionic F-127 (0.25% v:v, MolecularProbes) and fetal bovine serum (5%) were added as dispersing agents. Cells oncoverslips were then loaded in this mixture for 90 mm at room temperature.Cytoplasmic esterase conversion of the Fura-2AM to the free, Ca2+ responsive, form ofthe dye (Fura-2) occurred during this period. Examination of the cells with 360nMexcitation showed even, diffuse cytoplasmic fluorescence. The use of this loading regimenwas critical in preventing non-cytoplasmic compartmentalization of the dye, whichcould be seen in lipid vesicles and mitochondria if the cells were loaded at 37°C orwithout dispersing agents. Cells exhibiting this compartmentalization were not used forthe assay as they did not respond to calcium ionophore treatment.iii’ Fluorescence MeasurementThe coverslips were mounted cells down atop a laminar flow-through chamber.The chamber was inserted into a stainless steel holder and mounted onto the stage of aZeiss Jena Jenalumar microscope equipped for epifluorescence. Light from a 200 wattmercury arc lamp was passed first through one of three diffential interference filters(350, 363 or 380 nm ÷/-5nm) inserted in a rotating computer operated turret, thenthrough a 400 nm dichroic mirror, and finally through a bOX apochromat oilimmersion lens before reaching the cells. A circular field diaphragm in the light pathwas used to restrict the area of illumination to colonies of 10-30 cells or to a singlecell. The fluorescent light emitted by the cell(s) was passed back through the dichroic- 37mirror, an interference filter (490 nm cutoff), and then deflected to a camera portmounted with a photomultiplier. At the photomultiplier the emitted light was convertedto DC voltage, and digitized. The ratio of 350/380 nm fluorescence was obtained on a 1 .8or 5.0 sec. time base, stored on a computer, and later converted to [Ca2+]i (Il.10.v.).iv) Experimental ProtocolA 400 iI chamber and holder were designed to provide a warmed (36 +/-0.5°C)and continuous laminar flow at a rate of 2m1/min. HBSS (1 .2 mM calcium) was used asthe perfusate. Ang II was delivered directly into the chamber in 75 il aliquots through ashort port via a cannulated syringe. Use of a non-toxic coloured dye indicated that thebolus reached the cells on the coverslip within a few seconds and then was washed out ofthe chamber within 1 minute. Therefore, Ang II treatment was rapid and transient. Allsingle cells which did not respond to Ang II with [Ca2]i increases of >5OnM weretreated with the non-fluorescent calcium ionophore Br-A231 87 (25j.tM) to show thatthe cells had been properly loaded with Fura-2 and that the dye had not becomecompartmentalized into non-cytoplasmic pools.For calcium free experiments, HBSS without calcium and 1mM EGTA wasperfused through the chamber. Ang II was added in aliquots made up in calcium freemedium. In other experiments, the calcium channel blocker verapamil (50 liM, Sigma)was added to calcium containing HBSS and perfused through the chamber. Also,thapsigargin (Thastrup et al., 1990; Calbiochem), which induces intracellular calciumrelease independently of lns(1 ,4,5)P3 was added to calcium containing and calcium freeHBSS. It was flooded into the chamber and then the flow was stopped, so that a staticconcentration of 200nM was reached.- 38 -v) Calculation of [Ca2]j[Ca2]i was calculated using the following formula (Grynkiewicz et al., 1985).[Ca2ji= kd(Beta)(R-Rmjn)/(R-Rmax)Where:kd = the rate constant for the association of fura-2 with cytosolic free calcium: 224 nM.B = ratio of the values: 380 nm fluorescence in calcium free HBSS /380 nmfluorescence in calcium containing HBSS.R = experimentally determined ratio of the values 350 nm/380 nm fluorescence.Rmin = ratio of the values 350 nm1380 nm in calcium free HBSS.Rmax = ratio of the values 350 nm/380 nm in calcium containing HBSS.These values were determined by first perfusing the chamber with calcium freemedium, stopping the flow and adding calcium ionophore (Br-A23187, 25ii.M). 350nM/380 nM ratios of cell groups or individual cells recorded at this time representedRmin. HBSS with calcium (1 .3mM) was then perfused into the chamber and 350nM/380 nM ratios recorded taken again which now represented Rmax. The 380 nmreadings in each medium were then used to obtain B.Typical calibration values obtained in these experiments were:GLOM: 13=7.65, Rmin=0.63, Rmax=2.5.FASC: 3=8.20, Rmin=0.45, Rmax=2.7.- 39 -11. Kirsten Murine Sarcoma Virus (KIMSV) Isolation. Infection and OncogenicTransformation.FASC and GLOM cells in primary culture were infected with KiMSV, whichcontains a virally activated Ki-ras gene (Shih et al., 1982). The cells were thenmonitored for morphologic changes. Morphologically altered cells were selected,passaged into secondary culture and assayed for changes in growth, serum independence,tumourigenicity and differentiation.i) KiMSV Isolation and TitrationK1MSV transformed normal rat kidney cells (KNRK) served as the virusproducer line (Roy-Burman and Klement, 1975). This virus is a replication defective Ctype oncogenic retrovirus (Teich et al., 1982). The non-acutely transforming Maloneymurine leukemia virus is present as the helper virus.KNRK cells were grown to subconfluence in T-75 flasks in 10% FBS containingWaymouth’s medium. Medium was conditioned for 24 hr, chilled to 4°C and centrifugedat 3000 Xg for 30 mm to remove cells and debris. The supernatant was furthercentrifuged at 50,000 Xg for 60 mm to pellet the virus. The pellet was then resuspendedin tissue culture medium at one tenth the original volume to give a 10 X virusconcentration. The concentrated viral isolates were pooled and frozen in liquid N2.Focus forming efficiency was evaluated in cultures of normal rat kidney cells(NRK). Subconfluent NRK cultures were infected with serial dilutions of K1MSV in 5%heat inactivated calf serum (56°C, 30 mm) in Waymouth’s medium for 24 hours. Themedium was then changed and after 6 days the cultures were fixed and the number of focicounted. Foci appeared as discrete areas of piled up, disorganized, spindle shaped cells- 40 -against a monolayered background of flattened, morphologically normal NRK cells. Usingthis assay it was estimated that the virus pool contained 4.4 X105 FFU/ml.ii K1MSV InfectionFreshly isolated FASC and GLOM cells were seeded into 35 mm dishes at 3different cell densities (3 X 1.2 X105, 5 X i0 cells per dish). To these dishes1.0 ml KiMSV containing medium (10 X virus concentration) was added for 2 hours.This procedure was repeated daily for the first 3 days in culture. Polybrene, a fusogenwhich has been used to facilitate viral infection of explant adrenocortical fibroblasts(MacAuley et al., 1986), was not used here because in preliminary experiments itappeared to be toxic to primary FASC and GLOM cultures.iii) Selective Passaging of Morphologically Altered CellsDuplicate FASC cultures from 3 experiments and GLOM cultures from 2experiments were infected with KiMSV. In no instance did KiMSV infection result in anymorphologic changes in FASC cultures.KiMSV infected GLOM cultures did not give rise to discrete foci. Instead, after 1-2 weeks, diffuse areas of transient cellular multilayering appeared. In these multilayedareas refractile cells often emerged which were very loosely adherent to the underlyingsubstratum. These cells were detached from the multilayer by gentle flushing withmedium using a pasteur pipette. The medium was then collected, centrifuged (200 X g, 5mm) and the cells were resuspended in fresh medium and replated into secondaryculture. These cells were designated KGLOM cells.41 -lv) Snnsn IndnpendwmeKGLOM and unlfected GLOM cells were maintained until passage 5 bysubculturlng at a ratio of 1t The cells were then plated In 16mm wells (1X104cellslwell) and cultured In DMEMIFI2 medium supplemented with eIther 10% or 1% FBS.Cell numbers were then estimated by determining the amount of DNA per culture at days2, 4, 6, 8 and 10.v) TumorlçienldtyTumorigeniclty testing was carried out essentIally as described previously(Auersperg at al., 1977, 1981). Male Fischer rats (aged 6 weeks) wereImmunosuppressed by exposure to a Cobalt-60 radiation source (400 rads) 36 hoursprior to acinilnlsntlon of KGLOM cells. KGLOM cells In peseagelO were trypsindissocIated and suspended In serum free DMEWdF12 medIum. 2 X106 KGLOM cells werethen Injected subcutaneously or Intraperltoneally. In addition, 1X106cells wereinjected Into the adrenal fat —, the adrenal gland, or under the kidney capsule. After 7-12 days the animals were killed and the tumour masses were excised. At this time thesizes of the tumours were recorded. The tumoure were then fixed In phosphate bufferedformalin (pH 72), embedded In paraffin, sectioned and stained with hematoxylln andeosln for histopathologlcal examination.- 42 -12. p21 ras Expressioni) ImmunofluorescenceGLOM and KGLOM cells were grown to subconfluence on glass coverslips, fixed in3.6 % paraformaldehyde and washed in PBS. The cells were then incubated with a ratmonoclonal antibody raised against p21 ras (30 gig/mI, Y13-259, Oncogene Science,Manhasset NY, USA) for 30 mins at room temperature, washed in PBS, and incubatedwith FITC labelled goat anti-rat lgG (1:80, heavy and light chain specific, Cappel) for45 mm at room temperature.Rat lgG was used as an immunological control. KNRK cells were used as a v-Kiras expressing positive control (Myrdal et al., 1985). KGLOM cells were also treatedwith 1% triton X-100 (Sigma) for 30 sec to 4 mm, prior to immunfluorescence assayto determine if this detergent treatment extracted the membrane associated p21 ras.ii) ImmunoprecipitationThis procedure was carried out essentially as described previously (Shih et al.,1982; Auersperg et al., 1990).GLOM and KGLOM cells (p5) were grown to subconfluence in 60 mm dishes,labelled for 24 hours with 35 methionine, and lysed (Appendix C). Lysates wereincubated with protein A coated sepharose (Pharmacia) linked to rabbit anti-rat lgG(Cappel) in the presence and absence of the anti-ras antibody Y13-259. Theimmunoprecipitated p21 ras was then separated by SDS-PAGE, and visualized byautoradiography.KNRK and NRK cells were utilized as v-Ki-ras positive and negative controlsrespectively.- 43 -13. Lovastatin Treatment of KGLOM cellsIn an attempt to determine if the maintenance of steroidogenic differentiation inKGLOM cells was v-ras specific, we treated the cells with lovastatin, a pharmacologicinhibitor of p21 function.Lovastatin is a fungal metabolite derived from Aspergillus terreus (Alberts etal., 1980). It inhibits the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA reductase). Therefore, it blocks cholesterol biosynthesis at the level of mevalonateproduction and is used clinically for treating hypercholesterolemia. This inhibition ofmevalonate production also blocks the synthesis of isoprenoids such as farnesyl. Asfarnesylation of the ras protein is essential for its localization to the cytoplasmic face ofthe plasma membrane, treatment of cells with lovastatin blocks the biologic function ofp21 ras (Schafer et al., 1989).GLOM and KiGLOM cells were treated with lovastatin (.5-50 jiM, Merck Frosst,Missasauga Orit.) for 8 or 26hr in the presence or absence of ACTH (10 nM) or 8Br-cAMP (0.5 mM). Pregnenolone (100 ng/ml) was also added to the medium to overcomethe lovastatin associated inhibition of cholesterol synthesis. At the end of the incubationperiod the medium was assayed for corticosterone content by RIA. The cells were thentrypsin dissociated and the cell number estimated by DNA assay.- 44 -Ill. RESULTS1. Preliminary Attempts at Rat Adrenocortical Cell SeparationOur initial goal was to produce homogeneous populations of steroidogenic ratadrenocortical cells that were free of capsule derived fibroblasts, other stromal cellsand endothelial cell contamination. In so doing a pool of steroidogenic cells would beacquired that could be utilized for assays of in ijfl differentiation and oncogenictransformation without resorting to cloning. Cloning was not possible here due to theshort lifespan of the differentiated phenotype of rodent derived steroidogenic cells inii.trn (O’Hare and Neville, 1 973a,b; Hornsby et al., 1974; Ryback and Ramachandran,1981a,b; Payet et al., 1984).i) Whole Gland Dissociation of the Rat Adrenal CortexCollagenase dissociation of the whole adrenal cortex followed by filtrationthrough 100 jtm filters produced approximately 5 X cells per gland. These cellswere examined microscopically in suspension just prior to culture. The majority of thecells were single and contained lipid, which is a characteristic of steroidogenicadrenocortical parenchymal cells (Haning et al., 1970; Tait et al., 1974). However,the amount of lipid per cell and the size of the cell varied widely and there were always afew cells present which did not contain lipid (Fig la).- 45 -Fig 1: Collagenase dissociated cells from whole adrenal cortices.a) Cells at the time of dissociation. The cells are spherical, of varying, diameter and mostcontain refractile cytoplasmic lipid (phase microscopy; bar=1 00 jim).b) Primary culture at day 7. A central epithelial island of adrenocortical cells is shown.It is not readily apparent at this magnification, but at higher power cells with thisepithelial morphology often can be seen to contain retractile lipid inclusions. At theperiphery of the field the cells are spread and fibroblastic. (phase microscopy;bar=100 jim).c) Fluorogenic steroid production in primary and passage 1 culture with or without 24hr ACTH stimulation. The cultures were assayed at confluence in 35 mm dishes.Ill Basal, EIl 108M, 107M, • 106M ACTH. The data represent the amount ofsteroid per dish per 24 hr (each in triplicate +1- SEM).F.:r&i’4.s*.k9!‘ars‘E’ean.4(ng)-L 0 0 010FluorogenicSteroidI\).0)0)000000000-v-‘ w -‘ -U (I) C’) 03 a CD0- 46 -Primary cultures derived from these suspensions contained a mixture ofmorphologically distinct cell types. These included: epithelial regions made up ofretractile lipid containing cells; large areas of flattened fibroblastic cells; andoccasional endothelial cell colonies. Often the borders between the epithelial islands andfibroblastic areas were not sharp (Fig 1 b). Instead, at the periphery of the epithelialisland the cells gradually spread and lost some of their lipid. This suggested that somephenotypic modulation between an epithelial and fibroblastic morphology had taken placein primary culture.Primary cultures of whole gland-derived adrenocortical cells weresteroidogenic. They produced fluorogenic steroid basally and responded to ACTHtreatment by greatly increasing their steroid production (Fig lc). However, afterenzymatic dissociation and passaging the cells produced little steroid and no longerresponded to ACTH.ii) Differential Substratum Adhesion of Collagenase Dissociated AdrenocorLical Cells inPrimary Culture.We attempted to separate stromal and endothelial cells from parenchymaladrenocortical cells by differential substratum adhesion (Folkman et al., 1979). Cellsuspensions derived from the whole adrenal cortex were plated into tissue culture dishesfor varying time periods up to 180 mm. Unattached cells were then transferred to newdishes. In this way we attempted to leave behind rapidly attaching fibroblasts andendothelial cells in the first dish and transfer only the lipid containing steroidogeniccells to the second dish.Significant numbers of endothelial and fibroblastic cells were left behind in thefirst dish (Fig 2 a,b). The endothelial cells had a cobblestone, epithelial morphology andthe cells did not contain lipid. These cells expressed the clotting factor VIII (Fig 3 a,b),- 47 -while the fibroblastic cells did not (Fig 3 e,f). There were also cells left behind in thefirst dish which contained lipid. However, the lipid was present in scanty amounts suchthat the nucleus could be seen.The great majority of the cells transferred to the second dish contained largeamounts of lipid that obscured the nucleus (Fig 2c). In primary culture these cellsformed epithelial areas in which the cells retained there lipid. However, a smallpercentage of fibroblastic cells were always carried over to these second dishes, therebycontaminating the lipid containing parenchymal cells (Fig 2d).iii) cAMP-Induced Rounding and Detachment of Parenchymal Cells from CollagenaseDissociated Adrenocortical Cells in Primary CulturePrimary cultures derived from collagenase dissociated whole adrenocorticeswere treated with 8Br-cAMP (0.5 mM, 72 hrs). The majority of the cells in thesecultures responded by retracting from the substratum and rounding up (Fig 2e). Theserounded cells could be easily detached from the culture dish by gentle agitation. This leftbehind an underlying bed of fibroblastic cells still firmly attached to the substratum(Fig 2f).cAMP-induced retraction and rounding is a characteristic of most steroidogeniccell types in culture (Gill et al., 1980; Amsterdam et al., 1989; Hornsby et al., 1989).Thus, we reasoned that the rounded, detached cells in our cAMP treated whole glandcultures represented an enriched population of adrenocortical parenchymal cells.However, upon re-plating into secondary culture these cells. did not re-attach to aplastic substratum, either in the presence or absence of cAMP, which suggested that afibroblastic feeder layer may be required for attachment.- 48 -Fig 2: Differential adhesion and cAMP treatment in primary culture.(a-d) Differential Adhesion: (a.cl day 2: (b.d) day 10.Cells from whole glands were collagenase dissociated and then seeded into tissueculture dishes for 3 hrs (first dish). The non-adherent cells were then passaged to asecond dish. The cells were then cultured for 10 days.Cells which adhered rapidly in the first dish are seen in a) & b). At day 2,endothelial cell colonies are visible as well as cells with varying amounts of refractilecytoplasmic lipid (a). At day 10, an endothelial colony formed of lipid-free epithelialcells is present, along with numeruous elongated fibroblasts and some lipid containingcells (b).Cells which were transferred to a second dish are seen in c) & d). At day 2, thegreat majority of the cells contained large amounts of lipid that obscured the nucleus.The dark unucleated cells are red blood cells (C). At Day 10, most cells are epithelial andcontain lipid although there are some fibroblastic cells present (d).e. Whole gland derived cells in primary culture treated with cAMP.In (e), cells were cultured for one week and then treated for 4 days with 8Br-cAMP (0,5 mM). Many of the cells are rounded and refractile (compare with Fig lb). In(f), cAMP treated cultures have been flushed with a pasteur pipette to remove theloosely attached rounded cells, leaving behind an underlying fibroblastic cells which didnot round in response to cAMP treatment.Phase microscopy; bar=50 jim.Yb- 49 -Fig 3: Factor VIII immunofluorescence of adrenocortical endothelial cell coloniesobtained by differential adhesion.Cells in primary culture (Day 10) were stained with a polyclonal antibody for thesurface antigen factor VIII which is found on adrencortical endothelial cells (Folkman etal., 1979).An endothelial colony stained with antibody against factor VIII. The cells arepositive.An endothelial colony stained with normal rabbit serum. The cells are negative.fl Fibroblastic adrenocortical cells were stained with the antibody against factor VIII.The cells are negative (a,c,e immunofluorescence; b,d,f phase; bar=20 jim).Lfl9 rw:I- 50 -iv) Dissection of the Adrenal CortexThe capsule of the rat adrenal cortex can be separated from the inner portion ofthe gland by dissection. The capsular portions of the gland have been shown to containmost of the zona glomerulosa and the inner de-capsulated portion of the gland containsthe zonae fasciculata and reticularis (Hanirig et al., 1970).a) Capsular Glands (containing zona glomerulosa)When adrenocortical capsules were minced and cultured as explants theyproduced outgrowths of outwardly migrating fibroblastic cells that lined up in parallelarrays (Fig 4b). These explant derived cells have been characterized previously andthey are not parenchymally derived (Slavinski et al., 1974, 1976) Instead, theyappear to be stromal cells with some parenchymal potential depending on the cultureconditions (Turley, 1980, Auersperg et al., 1990).When adrenocortical capsules were collagenase dissociated they yieldedapproximately 1.8 X cells per gland. Presumably, the bulk of these cells werederived from the parenchyma of the zona glomerulosa. In primary culture most of thecells were fibroblastic (Fig 4d). Whether these fibroblastic cells represented stromalcell overgrowth or parenchymal cell modulation was not clear.- 51 -Fig 4: Adrenocortical cells derived from decapsulated (a.c) or capsular (b.d glands.Adrenocortices were dissected to separate the connective tissue capsules (with adherentzona glomerulosa) from the inner decapsulated portion of the gland that contains thezonae fasciculata and reticularis.a.b) Dissected tissue cultured as explants for 14 days.In (a), decapsulated glands produced little fibroblastic outgrowth from the lipid ladenexplant in top left corner of figure. In (b) there was vigorous fibroblastic outgrowthfrom the capsular explant.c.d Dissected tissue collagenase dissociated and resulting cells cultured for 14 days.In (c), the cells from decapsulated glands are predominantly epithelial and containcytoplsamic lipid, although a fibroblastic area is visible. In (d), most of the cells arefibroblastic and they do not contain cytoplasmic lipid. (phase microscopy; bar=50 jim).p- 52 -b. De-capsulated Glands (containing zonae fasciculata and reticularis)When the inner, de-capsulated portions of the adrenal cortex were cultured asexplants they formed slightly adherent clumps of lipid filled tissue. Very fewfibroblastic cells migrated out from these explants (Fig 4a).When de-capsulated adrenocortices were collagenase dissociated, they yieldedapproximately 2.5 X i05 cells per gland. Presumably, the bulk of these cells werederived from the parenchyma of the zonae fasciculata and reticularis. In primaryculture, many of these cells did not attach to the tissue culture plastic. Most of the cellsthat did attach contained large amounts of lipid and formed epithelial monolayers duringthe first few days in primary culture. However, after 2 weeks occasional endothelial celland fibroblastic colonies appeared (Fig 4c). After 3-4 weeks the cultures were oftenovergrown with fibroblasts.It was readily apparent that our preliminary attempts at adrenocorticalparenchymal cell separation had not been wholly successful. Thus, we attempted toseparate these cells on the basis of density.2. Percoll Density Gradient Separation of Rat Adrenocortical CellsDue to the wide variation in lipid content and cell size, adrenocorticalparenchymal cells derived from the different adrenocortical zona have differentdensities. Therefore, we attempted to separate these cell types by density usingdiscontinuous percoll gradients.- 53 -i) Cell Separation into 7 Visible BandsWhole adrenocortices were collagenase dissociated and filtered throughmultilayered nitex cloth (100 jim mesh) to remove undigested tissue and cell clumps.The resulting suspension, which consisted of single cells with occasional doublets andtriplets, was placed atop an eight step discontinuous percoll density gradient (4-10Xl 06 cells per gradient). The parameters of the percoll gradient were chosen to givemaximum separation of fasciculata and glomerulosa cells based on the estimated celldensities obtained using serum albumin gradients (Tait et al., 1974).After centrifugation, 7 cell bands were visible at the density interfaces withinthe gradient. The bands were numbered 1 through 7 relative to their position from topto bottom of the tube. These numbered bands did not include an opaque region thatremained atop the gradient and a reddish sediment that formed below the gradient at thebottom of the tube.Cell band density was determined by extrapolating back to a standard curve thatwas obtained from a parallel tube containing colour coded marker beads of knowndensity. An example density determination from a representative experiment is provided(Fig 5). The density of each cell band was highly reproducible between experiments(Table 2).- 54 -Fig 5: Representative experiment of separated cell bands obtained from the percolldensity gradient.A: Standard curveThe distance travelled by density marker beads in the percoll gradient was plottedagainst the known density of the beads (n). Cell band distance was then plotted on thegraph to determine the density by extrapolation (a). Cell bands are numbered 1through 7 in order of increasing density.B. Summary of marker bead migration and density.Six different colour coded marker beads of known density were added atop the percollgradient and centrifuged into it. Each bead type settled at its isopycnic point, and thedistance from the top of the gradient (meniscus) was determined. These values wereutilized to construct the standard curve in (A).C. Summary of cell band migration and density.The distance travelled by each cell band from the meniscus was determined. This distancewas then used to determine cell band density by extrapolation from the standard curve in(A).-54a-1.101.081.061.041.021.00ABDensity Marker Beads80Bead Distance Density# (mm) (g/mI)1 4 1.0162 18 1.0333 28 1.0484 39 1.0625 50 1.0776 65 1.087CEstimated Cell Band DensityBand Distance Density# (mm) (g/mI)1 10 1.0252 16 1.0323 22 1.0404 28 1.0475 35 1.0556 42 1.0647 47 1.070EC)>Cl)ci)U0 20 40 60Distance from meniscus (mm)- 55 -TABLE 2Density Gradient SeparationBanda Density Cells#/Band Cell Diameterb t\5,3,f3HSDc# (gIml) % of Total (urn) % positive(step) n=4 exp n=4exp n=3 exp >20 Fields1 1.021 +/-0.003 7.2 +/-2.0 19.9 +1-0.6 51 +/-292 1.034 +/-0.002 10.6 +1-3.9 18.0 +1-0.6 86 +1- 73 1.044 +/-0.002 15.1 +1-5.1 16.6 +1-0.9 65 +/-154 1.051 +/-0.001 20.9 +1-9.0 N.D.d N.D.5 1.057 +/-0.001 23.3 +/-4.3 14.0 +1-1.1 60 +/-156 1.061 +/-0.001 15.1 +1-7.4 N.D. N.D.7 1 .069 +/-0.002 7.8 +/-2.5 1 1 .7 +1-0.7 80 +1- 5Characterization of collagenase-dissociated adrenocortical cellsfrom whole glands after separation on an 8-step discontinuousdensity gradient. Cells were present in visible bands at each of the 7density interfaces between steps (means +1- SD).a A few cells and debris were found atop the gradient, erythrocytes were found belowthe gradient.b Each experiment 20-50 cells/band were evaluated.C Each field examined contained 50 or more cells.d Not Done- 56 -ii) Preliminary Characterization of Separated Cells in SuspensionThe visible cell bands and the material atop and below the gradient were washedfree of percoll, counted for nucleated cells, and examined microscopically. In theopaque, un-numbered fraction that sat atop the gradient there was cellular debris, freelipid and a few lipid containing cells. The sediment from the bottom of the tube consistedexclusively of red blood cells. Each of the 7 numbered bands located within the gradientconsisted almost entirely of nucleated cells. The upper bands (#1-2) consisted mostlyof single cells. The middle bands (#3-6) contained the greatest number of cells as apercentage of the total, and contained a wide variety of single cells, small aggregates, andclumps of up to 20-30 cells. The lowest band (#7) contained single cells and smallaggregates of less than 6-8 cells. The cell aggregates found in bands 3-7 were notpresent when the cell suspension was originally loaded onto the percoll gradient.Moving from top to bottom in the gradient, the density of each numbered bandincreased. In general, as the band density increased the average cell size decreased(Table 2).Cytoplasmic lipid is often used to store cholesterol esters and cholesterolprecursors which are crucial for the production of pregnenelone (Hall 1984). Themicrosomal enzyme z5,3t3-hydroxysteroid dehydrogenase (A5,313-HSD) then convertspregnenelone to progesterone in steroidogenic cells. Thus, the presence of cytoplasmiclipid and A5,313-HSD activity are two general markers of adrenocortical parenchymalcells. All the populations separated on the percoll gradient contained individual cells thatwere both lipid and 5,3I3-HSD positive. However, there were vast differences in therelative number of doubly positive cells in each population.The population with the lowest density, Band 1 (1.021g/ml), containedpredominantly large, lipid filled cells. Suprisingly, only 51% of these cells were- 57A5,313-HSD positive (Table 2, Fig 6a). We reasoned that the non-steroidogenic lipidcontaining cells in this band might be contaminating adipocytes, which often adhere tothe capsule when the adrenal is dissected out from the supra-renal fat pad. This wasconfirmed by collagenase dissociation of adipose tissue from the fat pad followed bypercoll gradient separation. Most of the adipose cells remained atop the gradient (ie.density less than 1 .021 g/ml). However, of those fat pad derived cells that did enter thegradient, greater than 90% separated in Band #1 (1.021 g/ml).Mid-density adrenocortical populations, such as Band 5 (1 .057g/ml), containedslightly higher proportions of A5,313-HSD positive cells than Band 1 (60%). However,morphologically the cells of Band 5 were extremely heterogeneous and the15,36-HSDnegative cells did not contain lipid (Fig 6c). Based on cell suspension observations andprimary culture results (III. 6.), we believe that most of these negative cells werecontaminating stromal and endothelial cells. Therefore, band #5 was designated “MIX”because it contains a mixture of cell types. All of the mid-range bands (bands #3-6)were heterogeneous and appeared to be contaminated to varying degrees with stromalcells. Therefore, they were not characterized fully as to size and z\5,313-HSD staining.The density gradient parameters used in this study provided two populationswhich contained predominantly z\5,313-HSD positive cells. These populations, band #2and band #7, were found at opposite ends of the density gradient and thus differeddrastically in size and lipid content.Band 2 contained large, low density cells (18.Ojim mean diameter, 1.034g/ml)that were packed with lipid inclusions. 86% of these cells were t5,313-HSD positive(Fig 6b). Band 7 contained small cells with a high nuclear to cytoplasmic ratio anddensity (11 .7i.tm mean diameter, 1 .069g/ml). Band 7 cells also contained smallcytoplasmic lipid vesicles. 80% of these cells were 5,3B-HSD positive (Fig 6d).Adrenocortical capsules were dissected from inner portions of the gland,collagenase dissociated and separated on the percoll gradients. Band 2 originated- 58 -predominantly from the inner glands (zonae fasciculata/reticularis), and band 7originated predominantly from the capsules (zona glornerulosa) (Fig 7). Therefore,Band #2 was designated as “FASC” (fasciculata) and band #7 was designated as “GLOM”(glomerulosa).- 59 -Fig 6: z\5.313-hydroxysteroid dehydrogenase (&5.313-HSD) staining of cells derivedfrom bands separated on the percoll gradientCells obtained from various bands on the percoll gradient were plated onto coverslips andstained histochemically for A5,38-HSD. Enzymatic activity is represented by darkformazan deposits.a) Band 1: The cells are large and lipid filled; The upper cells are A5,313-HSD positivewhile the lower cells are negative.b) Band 2 (FASC): The cells are large and lipid filled. All are positive for A5,313-HSD.c) Band 5 (MIX): The cells are variable in size and15,3i3-HSD staining.d) Band 7 (GLOM): The cells are small and they are A5,313-HSD positive.e) Rat lung fibroblasts: All of the cells are z5,3l3-HSD negative (negative control).f) Collagenase dispersed whole testis: Most of the cells are negative including the longtailed spermatic cells. Occasional steroidogenic leydig cells are positive (positivecontrol).Light microscopy; bar=15 pPm.-o-o0- 60 -Fig 7: In vivo origin of FASC and GLOM cellsWhole adrenal cortices were dissected into two parts: inner fractions with zonaefasciculata/reticularis; and capsular fractions with zona glomerulosa and connectivetissue capsule. Each fraction was dissociated and cells were loaded onto separate densitygradients. After centrifugation cell bands were collected and counted. Inner fractions() contained approximately 9X as many FASC cells as the capsular fractions (n).Capsular fractions contained approximately 8X as many GLOM cells as the innerfractions. Data represent means +1- SEM from 3 experiments.%Cells/Band0- 000__I___•I•(-I) 0C- 61 -3. Characterization of FASC (fasciculata) and GLOM (glomerulosa) Populations.Due to their homogeneous nature and abundance of z5,313-HSD positive cellsFASC and GLOM cells were chosen for in depth characterization both in suspension and inculture.i) Steroidogenesisa) In SuspensionIn suspension, immediately after percoll density separation, both FASC and GLOMcells were steroidogenic. They produced the rat endpoint glucocorticoid corticosterone(CS) and mineralocorticoid aldosterone (Aldo).FASC cell suspensions produced 12.4 +1-4.7 ng CS and 142 +1- 42 pg AldoiiO5 cellsl2hrs. GLOM cell suspensions produced 9.7 +1- 1.3 ng CS and 291 +1- 87pg Aldo/1 cellsl2hrs (means +1- SEM from 6 experiments each done in duplicate ortriplicate).A 2hr incubation with a high dose of ACTH (1 OnM) resulted in increased CS andAldo production by both FASC and GLOM cells (Fig 8). ACTH-mediated CS stimulation wasmuch higher in FASC than in GLOM. In contrast, a 2hr incubation with AnglI (lOOnM)resulted in a much higher stimulation of steroid production by GLOM than FASC (Fig 8).Therefore, while both cell types were responsive to ACTH, FASC cells were considerablymore so. In contrast, only GLOM cells responded strongly to Ang II.- 62 -Fig 8: Steroidogenesis in freshly isolated cell FASC and GLOM suspensions.Cells were treated in suspension with (a), or without (la) trophic hormones (10 nMACTH, or 100 nM Ang II) for 2 hrs. Corticosterone (CS) and aldosterone (Aldo) wereassayed by RIA. As the basal values varied considerably between experiments, data areexpressed as a ratio of treated vs. basal levels. Therefore, basal values are expressed as1. Absolute values are noted in the text. Data represent means +1.. SEM from 3experiments, each in duplicate or triplicate).Ang II and high basal production of Aldo exhibited by GLOM cells is characteristic of cellsderived from the zona glomerulosa.RatioIncreasevsBasalRatioIncreasevsBasalF)C’).Ciio.01C)000C)C)I>-n•I•II•I______1=II’3 p)__________I1C 0C’,C-)C,)C-)- 63 -b In Primary CultureBoth FASC and GLOM cells remained basally and inducibly steroidogenic inprimary culture. Cells were treated acutely (2hr), and chronically (24 hr), with ACTH10 nM to determine if their response was altered after long term trophic hormoneexposure. This dose of ACTH was previosly found to be maximal for cells in primaryculture (Fig ic). For Ang II responses in primary culture see Ill. 4.At day 4 in primary culture, ACTH treatment increased CS productiondramatically in both FASC and GLOM cultures (Fig 9). In FASC cultures, acute ACTHstimulation was maximal, and chronic stimulation did not result in a further increase.This was not the case in GLOM cultures where chronic ACTH treatment resulted in agreater production of CS than did acute treatment. In addition, after chronic ACTHtreatment FASC and GLOM cells now produced similar amounts of corticosterone.At day 14 in culture, FASC and GLOM cultures remained steroidogenic. However,acute ACTH treatment produced much smaller increases in CS production than at day 4,and chronic ACTH treatment further increased CS production in both cell types. Thisdecreased acute steroidogenic potential suggested that some de-differentiation wasoccuring in both cell types with increasing time in culture.c) In Secondary CultureAfter passaging, both FASC and GLOM cells no longer responded to ACTH treatmentwith increased fluorogenic steroid production (Fig 10).- 64 -Fig 9: FASC and GLOM corticosterone production in primary culture.FASC and GLOM cells were seeded at approximately 1 X 1 cells in 16mm wells.Cultures at day 4 and day 14 were not treated with trophic hormone ( basal, ED) for2hrs, or with lOnM ACTH for 2hrs () or 24hrs (•). Media were collected andassayed by RIA for corticosterone (CS). Cultures were then trypsinized and the cellswere counted. All data were normalized for 2hr time periods. Data represent CS/i Ocells /2hr; means +1- SEM; each in triplicate. All ACTH treated cultures producedsignificantly greater amounts of CS (p<.05) compared to untreated cultures.-64a-0)CCl)04003002001000!FASC GLOM FASC GLOM- 65 -Fig 10: FASC and GLOM cell steroidogenesis before and after passaging in culture.FASC and GLOM cells were seeded in 35 mm dishes and assayed for fluorogenic steroidproduction in primary culture (passage 0) or after trypsinization (passages 1-3). Ineach passage, medium was collected and assayed after 24 hrs in the absence (IE orpresence of lOnM ACTH (s). Data represent fluorogenic steroid/dish/24hrs; means+/-SEM; each in triplicate.FluorogenicSteroid(ng)FluorogenicSteroid(ng)-o (0 (0 CD CD z p-1’)o•)o000000000000-n Cr) C)F’)0)000000000000001Iv,c.)C)- 66 -ii) Primary Culture MorphologyIn the first few days after seeding into culture, both FASC and GLOM cells hadmorphologies which have been previously described for adrenocortical parenchymalcells (O’Hare and Neville, 1973 a,b; Hornsby et al., 1974; Ryback and Ramachandran1981 a,b). The cells were round and contained a central nucleus surrounded bycytoplasmic lipid (Fig 11). FASC cells were larger and contained much more lipid thanGLOM cells. In addition, FASC cells adhered less efficiently to the substratum than GLOMcells (Fig 12a). Through the first 3 days in culture, both FASC and GLOM culturesremained as homogeneous, parenchymal cell clusters, and there was no evidence ofstromal or endothelial cell contamination (Fig 11).With increasing time in primary culture, FASC and GLOM cells exhibiteddifferent morphologies. During the first week in culture, FASC cells spread slightly butmaintained much of their lipid and formed cohesive, epithelial monolayers. GLOM cellswere initially epithelial but after one week in culture they had lost much of their lipid,flattened, and took on bipolar fibroblastic shapes. The difference in morphology of thetwo cell types at day 9 in culture was striking (Fig 11). Through 21 days in primaryculture, FASC cells remained epithelial and were quite stationary, while GLOM cells lostcontact and began to divide (Fig 12b).- 67 -Fig 11: FASC and GLOM morphology in primary culture.FASC (b,d) and GLOM (a,c) cells were photographed live at day 2 (a,b) and day 10 (c,d)in primary culture. At day 2, FASC cells were large and filled with lipid that oftenobscured the nucleus (b), while GLOM cells were much smaller and contained less lipid(a). At this time there was no evidence of fibroblast contamination in either population.At day 10, FASC cells formed epithelial monolayers and the cells retained much of theirlipid (d), while GLOM cells flattened, spread, lost their lipid and became fibroblastic inmorphology (b)Phase microscopy, bar=50 urn.‘44- 68 -Fig 12: Seeding efficiencies and growth in primary culture of FASC and GLOM cells.A. Seeding efficiency1 X FASC (EJ ) and GLOM cells () were seeded in 35mm dishes. After 48hr,unattached cells were collected and counted. By subtraction, the cells remaining wereconverted to % attached. Means +1- SEM of three experiments, each in triplicate.B. Growth in primary culture. 1 X FASC (C) or GLOM () cells were seeded in16mm wells. At the times indicated, triplicate cultures were trypsinized and counted.Data represent means, all SEM < 10%, each time point is in triplicate. The results arerepresentative of 3 experiments.copeqos,ieo%coCoc\J00>Uc’J0000000)coCDLf)oi.x)0Niieo- 69 -Fig 13: cAMP mediated morpholoQical responses of FASC and GLOM cells in primaryculture.Sub-confluent primary cultures (day 7) of density separated cells were treated with8Br-cAMP (0.5mM) for 48 hours. FASC cells (a) showed prominent retraction fromthe substratum (compare with Fig lid). GLOM cells (b) showed very little, if any,retraction (compare with Fig lic).Phase contrast microscopy (x200).- 70 -iii) Phenotypic Markers in Primary Culturea) cAMP RetractionIn culture, many steroidogenic cells respond to treatment with ACTH, or itssecond messenger cAMP, by retracting from the substratum (Gill et al., 1980). 8Br-cAMP (0.5mM) treatment resulted in a marked retraction of FASC cells (Fig 13a).However, 8Br-cAMP treatment resulted in very little retraction of GLOM cells (Fig13b).b) Cytoplasmic LipidCytoplasmic lipid, which is found in many steroidogenic cell types, wasvisualized by Oil Red 0 staining of FASC and GLOM cells at day 14 in culture.Epithelial FASC cells contained numerous, large cytoplasmic lipid vescicles (Fig14). Fibroblastic GLOM cells contained only scanty numbers of small lipid vescicles.c) UltrastructureThe ultrastructure of FASC and GLOM cells at day 14 in primary culture wasexamined by electron microscopy. FASC cells contained numerous large lipid droplets inthe cytoplasm, a well developed smooth endoplasmic reticulum and numerous globlularmitochondria with tubulo-vescicular christae (Fig 15). All of these features have beendescribed previously in steroidogenic cells (Nussdorfer, 1986).GLOM cells contained large elongated mitochondria with tubulo-vescicularchristae along with promiment smooth endoplasmic reticulum, and golgi complexes (Fig15). These features are not found in adrenocortical stromal fibroblasts derived from- 71explant cultures (Slavinski-Turley and Auersperg, 1978). In addition, GLOM cellslacked features associated with stromal fibroblasts including: prominent distended roughendoplasmic reticulum; filament bundles with focal thickenings; and large amounts ofextracellular matrix.d) P-45Oscc ExpressionTo determine definitively whether or not the morphological changes seen in GLOMcultures were due to adrenocorticaf parenchymal cell plasticity or to stromalovergrowth, we probed the cultures at day 9 for expression of P-45Oscc byimmunofluorescence. This mitochondrial enzyme converts cholesterol to pregneneloneand is only expressed in steroidogenic cells (Goldring et al., 1986).Both the epithelial FASC cells and the fibroblastic GLOM cells expressed P45Oscc, indicating a parenchymal origin for both cell types (Fig 16). In epithelial FASCcells, P-45Oscc positive mitochondria were compact and globular (Fig 1 6g). Incontrast, the P-45Oscc positive mitochondria of fibroblastic GLOM cells were elongatedand attenuated (Fig 16h).e) Cytokeratin ExpressionCytokeratins (CK) make up a family of intermediate filaments that are expressedin a number of epithelial cell types (Steinert and Liem, 1990). We used 3 differentantibodies to probe FASC and GLOM cells in primary culture for CK expression byimmunofluorescence. These experiments were carried out both before (day 4), and after(daylO), phenotypic modulation of GLOM cells. Two antibodies were monoclonal inorigin and were directed against CK 49 kD, and CK 55 kD, both of which are expressed in- 72 -a number of glandular epithelia (Germain et al., 1988). The other antibody, RAK waspolyclonal in origin and reacts with several different rat CKs (O’Guin et at., 1985).There was no evidence of keratin positivity in either FASC or GLOM cells ateither time in culture using all three of these antibodies (Table 3, Fig 17). In order toensure that these results were truly negative we utilized rat ovarian surface epithelialcells as a tissue specific positive control in these experiments.f) Vimentin ExpressionVimentin is an intermediate filament protein that is expressed in manymesodermal and stromal cell types (Steinert and Liem, 1990). Utilizing antibodiesdirected against rat vimentin and rat P-45Oscc, we probed FASC and GLOM cellssimultaneously for the expression of these antigens by immunfluorescence. Theseexperiments were carried out before (day 3), and after (day 9), the phenotypicmodulation of GLOM cells.Epithelial FASC cells expressed P-45Oscc but not vimentin at both day 3 and day9 in primary culture (Table 3, Fig 18). When GLOM cells were still epithelial at day 3in primary culture they expressed P-45Oscc but not vimentin. However, afterphenotypic modulation to a fibroblastic form at day 9, GLOM cells expressed both P45Oscc and vimentin (Fig 18,19). Vimentin was found in filaments that traversed thelength of the fibroblastic GLOM cells. The expression of P-45Oscc and vimentin in thesame fibroblastic cells of GLOM cultures indicated that these cells were of parenchymalorigin but that they had the potential to express at least one feature ofmesodermal/stromal cells. Stromal lung fibroblasts expressed vimentin but not P45Oscc (Fig 19).- 73 -Fig 14: Lipid content of FASC and GLOM cells in primary culture.Subconfluent primary cultures (day 10) were stained with oil red 0 to showcytoplasmic lipid. FASC cells (a) contained many large lipid vesicles while the vesiclesin GLOM cells (b) were small and scanty.Light microscopy; bar=50 lim.p- 74 -Fig 15: Transmission electron microscopy of FASC (a.c) and GLOM (b.d)cells inprimary culture.Subconfluent primary cultures (day 14) were scraped from the tissue culture dishfixed and processed for electron microscopy. In FASC cells, large cytoplasmic lipidinclusions are visible (a) as well as globular mitochondria with tubulovescicularcristae (a,b). In GLOM cells, a prominent Golgi apparatus and smooth endoplasmicreticulum can be seen (b) as well as an elongated mitochondrion with tubular christae(d). Bar=O.5 Itm.I’1•’.7e__’cJ- 75 -Fig 16: P-45Oscc expression of FASC and GLOM cells in primary culture.Subconfluent cells in primary culture (day 9) were treated with ACTH (lOnM) for 24hrs. Cells were then fixed and stained for P-45Oscc by immunofluorescence.a.b FASC: The cells are epithelial in morphology and positive for P-45Oscc whichappears as punctate regions in the cytoplasm as the antigen is present in themitochondria. The large negative areas are nuclei, while the smaller negative areas arecytoplasmic lipid.c.d GLOM: The cells are fibroblastic and all of the cells are positive for P-45Oscc.e.f FASC: Normal rabbit serum (no first antibody) control. There is no mitochondrialstaining.y.h FASC and GLOM: High power view of FASC (g) and GLOM (h) cells showing P-45Osccpositive mitochondria. Notice that the mitochondria in the epithelial GLOM cells tend tobe globular while those in the fibroblastic GLOM cells are elongated (compare withfigure 15 c,d).a,c,e,g,h fluorescence; b,d,f phase microscopy; bar=20 jim.4-4-p- 76 -Fig 17: Cytokeratin in FASC and GLOM cells in primary culture.Subconfluent cultures (day 10) were fixed and stained for keratin using a polyclonalantibody (RAK) which reacts with several rat keratins.a.b GLOM: The cells are fibroblastic and there is no evidence of intermediate filamentstaining in the cytoplasm. However, staining of the nucleoli was first antibody specificas normal rabbit serum staining was negative (not shown).c.d FASC: The cells are epithelial and there is no evidence of intermediate filamentstaining.e.f ROSE 199: This is a keratin positive cell line which was derived from the rat ovariansurface epithelium (Hornby et al. 1992). The cells are epithelial and the keratinpositive intermediate filaments form a cytoplasmic cage around the nucleus.a,c,e fluorescence microscopy; b,d,f phase microscopy; bar=25 jim.ta.cir- 77 -Fig 18: P-45Oscc and vimentin expression in FASC and GLOM cells.Subconfluent cultures (Day 9) were treated for 24 hours with lOnM ACTH and thenfixed and double stained for P-45Oscc (a,b) and the intermediate filament vimentin(c,d). Photography consisted of individual exposures for each fluorochrome.a.c.e GLOM: The cells are fibroblastic and positive for both P-45Oscc (a) andfilamentous vimentin (b).b.d.f FASC: The cells are epithelial and are positive for P-45Oscc (b) but not forvimentin (d). The slight punctate staining in (d) is fluorescent bleed through from theP-45Oscc wavelength (rhodamine) to the vimentin wavelength (AMCA).a-d fluorescence microscopy; e,f phase microscopy; bar=25 jim.,.;t.,.I t •4tra‘0;.,I-‘4.’ tt- 78 -Fig 19: P-45Oscc and vimentin expression in GLOM cells and rat lung fibroblasts(R LF).Cells at various times in culture (as noted below) were treated for 24 hr with lOnMACTH and then were fixed and double stained for vimentin and P-45Oscc. Colourphotographs were taken using a double exposure of both the vimentin (AMCA-blue) andP-45Oscc (rhodamine-red) wavelengths.GLOM cells at Day 3 are still epithelial and they are just beginning to spread atedges of the colony. The cells are P-45Oscc positive (Red mitochondria) but vimentinnegative (blue).Middle: GLOM cells at Day 9 have modulated to a fibroblastic form. They are now positivefor both P-45Oscc (red mitochondria) and vimentin (blue intermediate filaments).Bottom: RLF in primary culture do not produce steroids (not shown). The cells arevimentin positive (blue intermediate filaments) and P-45Oscc negative.- 79 -TABLE 3Intermediate Filament Expression in FASC and GLOM CellsCELLS KERATINa VIMENTINb. CK49 CK55 RAKFASC Day 3-4 - - - -FASC Day 9-10 - - - -GLOM Day 3...4c - - - -GLOM Day 910d - - -ROSE 199k ++ - ÷Fibroblastsg ND- NDFASC and GLOM cells in primary culture were assayed forintermediate filament expression by immunofluorescence.a Keratin expression was assayed using 3 different antibodies that react with ratkeratins. CK49 and CK 55 are monoclonals raised against specific keratin species thatare found in glandular epithelia; RAK reacts with a number of different keratins.b Vimentin expression was assayed using a monoclonal antibody raised against ratvimentin.C At days 3-4 in culture both FASC and GLOM cells were epithelial in morphology.d At days 9-10 in culture FASC cells remained epithelial while GLOM cells hadmodulated to a fibroblastic form.e Vimentin positive GLOM cells also expressed P-45Oscc.ROSE 199 is a rat surface epithelial cell line that was used as a positive control forkeratin expression.g Fibroblasts were explanted from the rat lung. They were used as a positive control forvimentin expression.h Vimentin positive fibroblasts were P-45Oscc negative.- 80 -y) Catecholamine ActivityThere was a concern that adrenal medullary cells might be a contaminant of theadrenocortical parenchymal populations, particularly FASC. Therefore, primarycultures at days 4-7 were probed histochemically for catecholamine expressionAdrenal medullary cells were isolated and cultured separately as they do notsurvive the dissociation method used here for dissociating the adrenocortical cells (Bellet al., 1978, Livett et al. 1984). Adrenomedullary cells would not attach to uncoatedplastic, but only to collagen-coated plates. Also, after separation on the percoll gradientthe overwhelming majority of the cells had a different density (Band 6, 1.061 g/ml)than either FASC or GLOM cells. These cells were uniformly phase bright and they werepositive for catecholamine expression (Fig 20). In contrast, FASC cells, which are alsophase bright but mottled because of the presence of large amounts of cytoplasmic lipid,were not catecholamine positive.- 81Fig 20: Comparison of morphology and catecholamine staining in adrenal medullarycells (a.c.e) and FASC cells (b.d.f) in primary culture.a.b phase microscopy: Adrenal medullary cells (a) are uniformly phase bright and aresmaller than FASC cells (b) which are heterogeneously phase bright because of anabundance of cytoplasmic lipid vesicles. a,b bar=20 jim.c-f catecholamine staining: Adrenal medullary cells (c,e) and FASC cells in primaryculture (day 2) were fixed and stained for catecholamine expression by histochemistry.Medullary cells are positive, exhibiting intense fluorescence (the small negative cellsare red blood cells). FASC cells are negative. c,d fluorescence microscopy; e,f phasemicroscopy; bar=20 jim.81r.% -‘I..1’IzL.r .‘-II- 82 -4. Angiotensin Il-mediated Siynal Transduction in GLOM and FASC Cells.In the rat, cells of different adrenocortical zones have differing responses to thetrophic hormone angiotensin II (Ang II; Barrett et al., 1989). For example, Ang IIincreases steroidogenesis in capsule derived zona glomerulosa cells but not infasciculata/reticularis cells derived from decapsulated glands (Haning et al., 1970;Douglas et al., 1978; Braley et al., 1986). Utilizing the homogeneous GLOM (zonaglomerulosa) and FASC (zona fasciculata) cell populations, an attempt was made toelucidate the basis for this developmentally associated phenotypic difference by closelyexamining Ang Il-mediated signal transduction in the two cell types.i) Any Il-mediated steroidogenesisIn suspension, immediately after density isolation, GLOM cells produced muchhigher basal levels of aldosterone (Aldo) while FASC cells produced higher basal levelsof corticosterorie (CS) (Fig 21). Aldo and CS are the major endpoint mineralo- andglucocorticoids respectively in the rat (Nussdorfer, 1986). GLOM cells in suspensionresponded strongly to a 4 hour Ang II treatment with increased CS and ALDO production(Fig 21). Ang II had little effect on steroidogenesis in FASC cell suspensions, except for asmall CS increase at high Ang II doses.In primary culture (day 4), Ang II continued to increase steroidogenesis in GLOMcells while FASC cells remained refractory to the effects of the trophic hormone (Fig22). Therefore, the differential steroidogenic response of GLOM and FASC cells to Ang IIwas maintained in culture. This allowed us to examine signal transduction in culturewhere individual, morphologically identifiable, cells could be assayed. It should be notedhowever, that the actual levels of steroid production were decreased in primary culturecompared to those seen in freshly isolated suspensions. This decreased response to acute- 83 -Ang II treatment (ie. 4 hr) is likely due to a partial de-differentiation that takes placein the homogeneous GLOM and FASC populations in primary culture (Fig 9). Such dedifferentiation can be partially overcome by chronic trophic hormone treatment.ii) Inositol Phosphate Accumulation.a lnsPl. lnsP2. lnsP3Ang II treatment for 3 mm. increased the production of 3H inositol labelledInsPi, lnsP2 and lnsP3 in both GLOM and FASC primary cultures (Fig 23a).Qualitatively similar results were obtained after a 10 mm. stimulation although bothcell types showed greater increases in InsPi production at this time point (Fig 23b).This indicates that Ang II induces inositol phosphate production in both cell types. It alsosuggests that the metabolism of the inositol phosphates after their production is similarin both cell types.b) lnsP(1.4.5)P3Utilizing an isomer specific radioligand binding assay we found that Ang Ilincreased the total mass of lns(1 ,4,5)P3 to similar extents in both GLOM and FASCprimary cultures (Fig 24a). This increase occured rapidly, within 15 seconds ofexposure to the ligand. Ang II also induced lns(1,4,5)P3 increases in freshly isolatedsuspensions of GLOM and FASC cells (Fig 24b). Therefore, the initiation of Ang IImediated signal transduction in FASC cells was not a culture artifact.FASC and GLOM cells in primary culture were also treated with the calciumionophore Br-A23187 in calcium containing HBSS (1.3 mM). This treatment resultedin an increase in lns(1 ,4,5)P3 in both cell types (Fig 24c).- 84 -Fig 21: Any Il-mediated steroidoyenesis in freshly isolated suspensions of GLOM andFASC cells.Freshly isolated suspensions of GLOM and FASC cells were treated with or without theindicated doses of Ang II for 4 hrs and the medium was assayed for steroid content by RIA.A-aldosterone (ALDO); E-corticosterone (CS). All Treatments: 11 Basal, 11 1 nM1OnM, •lOOnM Any II. Data represent steroid/b5 cells/4hrs., (n=3,means ± SEM, *p<05 **p<o1 vs. basal).-84a-16000)08000200)C10C-)0AGLOM FASCBGLOM FASC- 85 -Fig 22: Any Il-mediated steroidogenesis of GLOM and FASC cells in primary culture.Primary cultures of GLOM and FASC cells were treated without or with the indicateddoses of Ang II for 4 hrs and the medium was assayed for steroid content by RIA.A-aldosterone (ALDO); .-corticosterone (CS). All Treatments: 111Basal, IJ 1 nM,lOnM, • lOOnM Ang II. Data represent steroid/105 cells/4hrs. (n=3,means ± SEM, *p<05 **p<o1 vs. basal).CS(ng)ALDO(pg)N)C-0)00I’)0C’) 0ODC,) 0- 86 -Fig 23: Ang II -mediated inositol phosphate production& Primary cultures of GLOM and FASC cells were labelled with 3H myo-inositol andtreated with Ang II (lOOnM). InsPi, lnsP2, lnsP3 accumulations were assayed by ionexchange chromatography. 11 Basal, • + Ang II, 3mm. (n=4, means ± SEM, *p<05**p<O1 vs basal).The % increase in inositol phosphates in GLOM and FASC cells after 100 nM Ang IItreatment for either 3 min.(1I)or 10 mm (B) are shown. Unstimulated basal levelsrepresent 0% increase (n=4 for each, means).-86a-U)U)>ci)U)ci)0Acv)b0InsP 1 InsP2 InsP3 InsP 1 InsP2 InsP38.06.04.02.00.05004003002001000BIP1 1P2 1P3 Ipi IF 1P3- 87Fig 24: Ang Il-mediated lns(1.4.5)Pp production.& GLOM and FASC cells in primary culture were treated with Ang II (lOOnM) for theindicated times, and Ins(1 ,4,5)P3 accumulations were assayed using an isomer specificradioligand binding assay. D Osec, l5sec, • 3osec Ang II (Data represent pmoleslns(1,4,5)P3/ 06cells, n=8, means ±SEM, * p<.05, **p.<.o1 vs. basal).j GLOM and FASC cells in suspension prior to culture were treated with Ang 11(100nM) and lns(1,4,5)P3 levels were determined (n=4, means ±SEM, * p<.05, vs.basal).j GLOM and FASC cells in suspension were treated with the calcium ionophore BrA23187 (25 jim) and lns(1,4,5)P3 levels were determined (n=4, means ±SEM,*p<05 vs. basal).Ins(1,4,5)IIns(1,4,5)’Ins(1,4,5)-L0N.)4S0)0)000F’)4G)0’r0N)- 88 -iii) [Ca2]i in cell groups.[Ca2]i was assayed in GLOM and FASC primary cultures (day 4-12) usingFura-2 microfluorimetry. At rest, small groups of 10-30 GLOM and FASC cells hadmean [Ca2]i levels of 74 ± 7 nM and 80 ±20 nM (n=7) respectively. Transient AngII treatment (< 1 mm) resulted in rapid (<30 sec.) increases in [Ca2]i in GLOMpopulations (Fig 25). The timing of the onset of the [Ca2]i response correlated withthe rapid rise in Ins(1 ,4,5)P3. The magnitude of the [Ca2]i response was dosedependent in these GLOM cell populations. A representative graph for a single GLOMpopulation is shown in Fig 26a.In FASC populations, transient Ang II treatment resulted in much smaller[Ca2]i increases than those seen in GLOM populations (Fig. 25). At high Ang II doses,the increase in FASC populations was approximately one fourth that of GLOM populations.A representative graph of one FASC population is shown in Fig 26b.Thapsigargin, which acts to release calcium from intracellular stores in anlns(1,4,5)P3 independent manner (Thastrup et al., 1990), induced a stable [Ca2]iincrease in both GLOM and FASC populations. These [Ca2]i increases were stable incalcium containing medium and transient in calcium-free medium (Fig 27).- 89 -Fig 25: Any Il-mediated changes in [Ca2]i in groups of FASC and GLOM cells inprimary culture.GLOM and FASC cells were cultured on glass coverslips and groups of cells (10-30)were assayed for [Ca2]i using Fura-2 microfluorimetry after transient (< 1 mm)treatment with the indicated doses of Ang II.El1nM 1OnM, •lOOnM Ang II. (n=7, means ±SEM, **p<o1 vs mM Ang IItreatment).[Ca2]iincrease(nM)LC)CCC0000C,) C)cz cc 3- 90 -Fig 26: Examples of [Ca2ji changes in GLOM and FASC cell groups.GLOM and FASC cells were cultured on glass coverslips and groups of cells (10-30)were assayed for [Ca2ji using Fura-2 microfluorimetry after transient (< 1 mm.)treatment with Ang II at the indicated dose (labelled arrows).A GLOM: the population responds to Ang II treatment with an increase in [Ca2]i , themagnitude of which is Ang II dose dependent (representative example taken from asample size, n=7).B FASC: the population responds very little to Ang II at any dose (representativeexample taken from a sample size, n=7).-90a-+c..JcA+c’Jctsq C 0050040030020010000500400300200100’02010Time (mm)Bq 00 10 20Time (mm)- 91Fig 27: Comparison of Any II and Thapsigargin induced [Ca 2+ changes in cell groups.A GLOM: Ang II and thapsigargin both induce [Ca2]i increases in the population(representative example taken from a sample size, n=4).B FASC: Only thapsigargin induces a significant [Ca21i increase in the population(representative example taken from a sample size, n=5).Single Arrows: transient treatment with Ang II (lOOnM). Horizontal Bars: duration ofstatic treatment with thapsigargin (200nM).-91a-+c..Jct+c..JA0015Time (mm)500-400-30020010000500400-300-200-100•0I • I -10 15B0010 10 20Time (mm)30- 92 -iv) [Ca2]i in single cells.As experiments with the small groups of cells suggested a differential regulationof Ang Il-mediated [Ca2]i increases in GLOM and FASC cells, we assayed single cells todetermine whether similar differences could be observed at this level. In culture, allGLOM cells assayed were characteristically flattened and contained only small amounts ofcytoplasmic lipid. All FASC cells assayed were laden with cytoplasmic lipid. None of thecells examined had the morphologic characteristics of adrenal medullary cells.At rest, individual GLOM and FASC cells had mean [Ca2ji levels of 109 ± 7 nM(n=58) and 126 ± lOnM (n=39), respectively. Individual GLOM cells responded totransient Ang II treatment with rapid increases in [Ca2]i over a dose range of 0.1 tolOOnM. Once a threshold Ang II dose was reached in an individual GLOM cell themagnitude of the [Ca2+li increase appeared to be near maximal, independent ofincreasing dose (Fig. 28a). Ang Il-mediated [Ca2]i increases in single GLOM cells alsooccured in Ca2+ free medium and in the presence of the calcium channel blockerverapamil, indicating that the source of the calcium was an intracellular pool (Fig 29).The majority of individual FASC cells did not respond to Ang II with [Ca2]iincreases, although they did respond to the non-fluorescent calcium ionophore Br A-23187 (Fig 28b). The response to the calcium ionophore indicates that the lack ofresponse by FASC cells to Ang II was not due to a Fura-2 loading artifact.In a comparison of individual cells, lOnM Ang II stimulated [Ca2ji increases of>5OnM in 90% (53/58) of GLOM cells but in only 28% (11/39) of FASC cells. Onlycells that exhibited increases of >5OnM were considered responsive as this ensured thatthe [Ca2+]i changes observed were not due to baseline variation. A frequencydistribution indicates that the magnitude of [Ca2]i increases exhibited by responsiveGLOM cells was highly variable (Fig 30). The mean [Ca2]i increases were341 ±6OnM for responsive GLOM cells and 309 ±55nM for all GLOM cells tested. In- 93 -contrast, the mean [Ca2]i increases werel 84 ± 48nM in responsive FASC cells and70 ± 1 7nM for all FASC cells tested. Therefore, much fewer individual FASC cellsresponded to transient Ang II treatment, and those that did exhibited a smaller increasein [Ca2]i compared to responsive GLOM cells.All the above experiments were carried out using transient (<1 mm) Ang Iltreatment. Under these conditions there was little or no evidence of sustained [Ca2+]ioscillations. However, if GLOM cells were perfused with Ang II an initial peak followedby lesser oscillatory peaks occurred (Fig 31a).As is the case in rat FASC cells, Ang II also increases inositol phosphateproduction in Yl mouse adrenocortical tumour cells without a resultant increase insteroidogenesis (Begeot et al., 1987). Therefore, we assayed [Ca2ii levels in thesecells in conjunction with Ang II treatment. We found that transient Ang II treatment, atdoses up to 1 aiM, did not increase [Ca2]i in 4 individual Yl cells assayed. Arespresentative example is shown in Fig 31b.v) Calcium lonophore-Mediated SteroidoyenesisThe data presented above suggest a disconnection between Ang Il-mediatedincreases in lns(1 ,4,5)P3 production and intracellular calcium release in FASC cells.To determine whether there was a direct link between this observation and thedifferences in steroidogenesis between the zones, we compared steroidogenesis in GLOMand FASC cultures after [Ca2]i was raised by treatment with calcium ionophore. Incalcium containing medium, the ionophore induced dose dependent increases insteroidogenesis in both GLOM and FASC cells (Fig 32). Therefore, like GLOM cells, FASCcells have retained the intrinsic capacity to respond to elevated [Ca2ji levels byincreasing steroidogenesis.- 94 -Fig 28: Examples of [Ca2ji changes in individual GLOM and FASC cells.GLOM and FASC cells were cultured on glass coverslips and individual cells were assayedfor [Ca2]i using Fura-2 microfluorimetry after transient (< 1 mm.) treatment withAng II or calcium ionophore at the indicated dose (labelled arrows).A GLOM: the cell responds to Ang II treatment with an increase in [Ca2]i that is nearmaximal once a threshold of 1.0 nM Ang II is reached (representative example takenfrom a sample size, n=58 from 11 cultures).B FASC: the cell responds very little to Ang II but does respond to calcium ionophoretreatment, indicating proper Fura-2 loading (representative example taken from asample size, n=39 from 8 cultures).Single arrows: transient Ang II treatment with indicated dose. Double arrow: transientnon-fluorescent calcium ionophore Br-A23187 (IP) treatment (25 iiM).-94a-A‘-: q 00 ‘-I I0 10 20500-400300200100•0600500400300200100-0+c’Jct$+c’JcTime (mm)B‘— c 00 0d11I0 10 20 30Time (mm)- 95 -Fig 29: Ana Il-mediated [Ca2]i chanaes in individual GLOM cells in the absence ofextracellular calcium (A) and in the presence of verapamil (B).& An individual GLOM cell was transiently treated with 100 nM Ang II (arrows) in thepresence of extracellular calcium (1 .8mM) and in the absence of extracellular calcium(under horizontal bar). Notice that the first Ang II treatment in the absence of calciuminduced an increase in [Ca2]i comparable to that seen before the removal of calcium.However, the second Ang II treatment did not increase [Ca2+]i presumably due to anexhaustion of the intracellular calcium pool (Kojima et al., 1987). This pool refilledafter restoration of extracellular calcium as a further Ang II treatment resulted inincreased [Ca2+]i (a representative example taken from a sample size, n=8)An individual cell in calcium containing medium was transiently treated with 100 nMAng II (arrows) in the absence or presence of the calcium channel blocker verapamil(100 nM, under the horizontal bar). The [Ca2+]i increases were similar in the absenceor presence of verapamil (a representative example taken from a sample size, n=3).-95a-+c’JasAC+c..Jas0 20 40 606005004003002001000600500400300200100-0Time (mm)80BI— I0 10 20 30 40Time (mm)- 96 -Fig 30: Frequency distribution of increases in [Ca2]i in single GLOM and FASC cellsafter lOnM Any II treatment.Each [Ca2+]i interval represents 5OnM increases and the % frequency represents thepercentage of the total number of FASC or GLOM cells assayed which responded withinthat interval. Cells which had increases in [Ca2]i of 50 nM were considered nonresponsive. 10% of GLOM cells and 72% of FASC cells were non-responsive. Nonresponsive cells are not included in the figure (ie.10% GLOM cells, 72% FASC cells).-96a-FASCa)U)C”a)0+C’.’C”0>750700-749650-699600-649550-599500-549450-499400-449350-399300-349250-299200-249150-199100-14950-99CC)U)C”a)C.)C+C’.’C”0>750700-749650-699600-649550-599500-549450-499400-449350-399300-349250-299200-249150-1 99100-1 4950-990 10 20 0 10 20% frequency % frequency- 97 -Fig 31: Any Il-mediated [Ca2]i responses in an individual GLOM cell (A) and a Ylmouse adrenal cortical tumour cell (B).An individual GLOM cell was treated either trasiently (arrow) or perfused (under thehorizontal bar) with 100 nM Ang II. Notice that the Ang II perfusion caused anoscillatory pattern after the initial [Ca2ji peak increase. Oscillatory patterns wereseen with dual wavelength fluoresence as shown here (representative example takenfrom a sample size, n=3), or by direct chart recording of fluctuations in single(350nm) wavelength fluorescence (data not shown, n=4).An individual Yl mouse adrenocortical tumour cell was treated with the indicateddoses of Ang Il (single arrows) or 25 tm of calcium ionophore Br-A23187 (IP, doublearrow). Ang II did not increase [Ca2]i significantly while the ionophore did(representative example taken from a sample size, n=4).-97a-+c..JcA400300C2005 7 9 11 13Time (mm)15B10007006005004003002001000CC‘ifCC0‘if:i‘if2 4 6 8 10 12 14 16Time (mm)- 98 -Fig 32: Calcium ionophore-mediated steroidogenesis of GLOM and FASC cells in primaryculture.Primary cultures of GLOM and FASC cells were treated without or with the indicateddoses of the calcium ionophore A-Br 23187 for 4 hrs and the medium was assayed forsteroid content by RIA.A-aIdosterone (ALDO); E.-corticosterone (CS). All treatments: C Basal, 1 0.1 i.LM,1iiM, •1OItM Br A-23187. Data represent steroid/b5 cells/4hrs (n=3,means ± SEM, **p 1(I) C)(I) C)- 99 -5. ras Oncoprotein Induced Transformation and Differentiation of FASC and GLOM Cells.FASC and GLOM cells were infected with high titres of Kirsten murine sarcomavirus (KiMSV) in primary culture. This retrovirus contains the v-Ki-ras oncogene(Shih et al., 1982). Morphological alterations, transformation parameters andsteroidogenic differentiation were then monitored in the KiMSV infected cells.i) KiMSV Infection and Selection of Morphologically Altered CellsIn 3 separate experiments involving 3 or more cultures in each, KiMSV infectionhad no discernable effects on FASC cells. The cells remained epithelial, monolayered andstationary. In addition, very few FASC cells survived passaging with or without viralinfection. Therefore, FASC cells will not be considered further here.After infection of GLOM cells in primary culture with KiMSV, discrete foci didnot appear. However, heterogeneity within the culture dish did arise. About one weekafter viral infection, diffuse areas of multilayering developed among the flat fibroblasticGLOM monolayer. Within these multilayers rounded cells emerged (Fig 33). In infectedGLOM cultures, the multilayered areas were often transient, reverting back to amonolayer form after a further 1-2 weeks in primary culture or after enzymaticpassaging. The rounded cells that sat atop the infected GLOM multilayers were looselyadherent and could be easily detached by gentle agitation. Once detached, these cells werecollected, washed by centrifugation and replated into secondary culture. Thus, we wereable to select cells which exhibited the strongest morphological response to KiMSVinfection. We have designated these cells KGLOM.100 -Fig 33: Morphology of KiMSV infected GLOM cells.a) Uninfected GLOM cells Day 9. primary culture: The cells are flat, fibroblastic andmonolayered.b) KiMSV infected GLOM cells Day 10. primary culture: An area of diffuse cellularmultilayering is shown.c) KiMSV infected GLOM cells at day 16 in primary culture: An area of intense cellularmultilayering with detaching and loosely adherent rounding cells is shown.d) K1MSV infected GLOM cells in secondary culture: Rounded cells were removed fromprimary culture multilayers by gentle agitation and replated into secondary culturewere they exhibit a fusiform, transformed morphology.Phase microscopy; bar=5Ojim./00&- 101ii) Transformation Associated Parameters of KGLOM (KiMSV infected ylomerulosa) Cellsa Transformation in vitroIn secondary culture, KGLOM cells took on a retractile, spindle shaped‘transformed’ morphology (Fig 33d). There were very few (less than 1%) flat,fibroblastic cells in these post-selection KGLOM secondary cultures. Thus, the selectionprocess efficiently separated the morphologically altered KGLOM cells from surroundingmorphologically normal glomerulosa cells. Immunotluorescence staining indicated thatthe concentration of p21 ras was greater in KGLOM cells than in uninfected GLOM cells(Fig 34,35). The ras p21 protein observed by immunotluorescence was at least in partmembrane associated as it could be extracted from membrane ruffles as well as cellbodies by treatment with the non-ionic detergent triton X-100 (Fig 36).Immunoprecipitation indicated that p21 ras was present in KGLOM cells at levelssimilar to that of the original KNRK producer line (Fig 37a). In addition, the viral formof the protein was expressed in KGLOM but not in unirifected GLOM cells. This wasillustrated by the second slightly more slowly migrating p21 species in KGLOM cells(Fig 37b). This species arises due to phospholylation of the mutant protein at amino acidposition 59 (Shih et al., 1982). Interestingly, in unifected GLOM cells an additional 27kd protein (p27) that was specifically immunoprecipitated by the anti-ras antibody(Fig 37b). Tryptic mapping of p27 isolated from uninfected explant derived adrenalfibroblasts suggests that p27 may be a cellular ras homologue that is not expressed intransformed cells (MacAuley, 1987).KGLOM cells grew rapidly and continued to divide after reaching confluence. Inpassage 5, KGLOM cells grew to a saturation density of approximately 3 X cells/cm2which was approximately lOX greater than that of uninfected glomerulosa cells in thesame passage. Also in passage 5, KGLOM cells grew rapidly in both 10% andl % serum- 102 -(FBS), indicating that they were not dependent upon high concentrations of serumderived factors for growth. (Fig 38). In contrast, in the same passage, uninfected GLOMcells remained strictly serum dependent.b) Tumorigenicity in vivoPassage 10 KGLOM cells were injected into various sites in immunosuppressedmale rats. Palpable tumours formed rapidly, within 9-12 days (Table 4). Thesetumours were all >1 cm3 in size, contained areas of neo-vascularization, were oftencentrally necrotic, and were usually locally invasive. Most tumours were pleomorphic.Those that arose in connective tissue (le. sub-cutaneous, suprarenal fatpad) werepredominantly sarcomatous, consisting mostly of whorls of bipolar, attenuated cells(Fig 39a). In contrast, when KGLOM cells were injected into the adrenal gland or invadedthe kidney, the tumours formed were usually more carcinomatous with cells in clumpsand chords. In one instance in the adrenal, the tumour was almost almost entirelycarcinomatous and undifferentiated, consisting of disorganized cell clumps and chords(Fig 39b).- 103Fig 34: p21 ras expression in KGLOM cells (p2).Immunofluorescence staining for p21 ras. The cell bodies and processes arepositive.No first antibody control.a,c) Fluorescence microscopy; b,d) phase microscopy; bar=20 j.tm.103- 104Fig 35: p21 ras expression in uninfected GLOM cells (p2).abi Immunofluorescence staining for p21 ras. The cells are slightly positive.çJj. No first antibody control.a,c) Fluorescence microscopy; b,d) phase microscopy; bar=20 tim.104L-a’--..a‘f;:.4..9---- 105Fig 36: p1 ras extraction in KGLOM cells (p5).j)j Immunofluorescence staining for p21 ras. The cell bodies, processes andmembrane ruffles are positive..d)j Immunofluorescence staining for p21 ras after a 2 minute treatment with 1%triton X-1 00. Most of the positive staining has been extracted..J)j No first antibody control in the absence of triton extraction.a,c)Fluorescence microscopy; b,d) phase microscopy; bar=20 jim.- 106 -Fig 37: p21 ras immunoprecipitation in GLOM and KGLOM cells (p5).Cells were labelled with 355 methionine, lysed and p21 ras was immunoprecipitatedusing the monoclonal antibody Y13-259. Equal amounts of TCA precipitable c.p.m persample were then loaded onto 12/5% SDS gels and separated by electrophoresis.Immunoprecipitated 35S labelled proteins were then visualized by autoradiography... p21 ras expression in GLOM and KGLOM cells was compared to NRK (v-Ki rasnegative control) and KNRK cells (v-Ki ras positive control). Lane 1, NRK; Lane 2,KNRK; Lane 3, KGLOM; Lane 4, GLOM. Notice the great increase in p21 in the KNRK andKGLOM cells as compared to their normal counterparts.B. Specificity of the immunoprecipitation. Lane 1, GLOM; Lane 2 KGLOM (no 1stantibody); Lane 3 KGLOM. The 21 kd protein is indeed p21 ras as it is absent in the nofirst antibody condition (Lane 2). This autoradiogram was underexposed to show theviral p21 doublet in KGLOM cells (lane 3). The slightly more slowly migrating p21 rasspecies is formed by phosphorylation of the viral protein (Shih et aL, 1982). In GLOMcells (lane 1) there was a second immunoprecipitated peptide p27 (kD) that was notpresent in KGLOM cells (lane 3).Ib Oh.nplAP50—33—2 8—1 8—aC.IBIap2l rasIthI27j4P21 rasa4,18—107 -Fig 38: Proliferation of GLOM and KGLOM cells (p5).GLOM and KGLOM cells were plated at 2 x io per 16 mm well and cultured with 1%C) or 10% () fetal bovine serum. At the indicated times, the cells were trypsindissociated and the cell numbers were estimated by DNA assay. Data represent means+1- SEM; each in quadrupilcate.II—II•I•oo0000(0L()cJC’)C’Jc’J0-coC>%,c’J0cx>0I•I•I•(0(0C)CJ(oix)#iieo‘cJ00#iieo- 108 -TABLE 4KGLOM Tumour HistopathologyInjection Site Tumour Sizea HistopathologybSarcomatous/CarcinomatousSubcutaneous +Suprarenal Fatpad ++ +++/+Intraperitoneal ++•Intradrenal/kidneyc + +/++In an effort to determine if KGLOM tumour histopathology wasaffected by the site of origin, cells (passage 8) were injected into anumber of different anatomical sites. Each site was injectedseparately in 3 different animals. Palpable tumours, some verylarge, formed within 9-12 days at all sites injected.a Tumour volume was estimated by measurement after excision. This was very difficultin the intraperitoneal and fatpad tumours as they were usually multifocal and locallyinvasive (+) 1-2cm3; (++) >2cm3.b Usually tumours were pleiomorphic except for one intradrenal tumour that wasentirely carcinomatous. Areas consisting of whorls of attenuated, bipolar cells werecharacterized as sarcomatous. Areas consisting of clumps and chords of cells werecharacterized as carcinomatous. The ratio of the number of symbols in each catagoryrepresents an estimation of the relative proportion of sarcomatous vs. carcinomatousareas per tumour).C Cells were injected into the adrenal in 3 animals and into the kidney once. Thesetumours were always more restricted in size than those produced in the connectivetissue sites. In addition, overall they were more carcinomatous than tumours thatarose in the connective tissue sites.- 109 -FiQ 39: KGLOM tumour histopathology.1 X 106 KGLOM cells (plo) were injected into either the suprarenal fat pad or directlyinto the adrenal gland of immunosuppressed rats. Two weeks later the animals weresacrificed and the tumours removed and processed.a) Suprarenal fatpad tumour: Cells are attenuated and align themselves in whorls andparallel arrays, suggesting that the tumour is sarcomatous. At the bottom of the figure isthe outer region of the uninjected normal adrenal cortex. This was the predominatinghistopathology of the tumours that formed in connective tissue (see Table 4).b) Intradrenal tumour: The normal organization of the adrenal cortex is disrupted butthe cells are arranged in clumps and plates suggesting that the tumour is anundifferentiated carcinoma. The normal suprarenal fat pad is seen at the top of thefigure. It should be noted that a mixture of sarcomatous and carcinomatous areas wereseen in the other intradrenal tumours (see Table 4).Hematoxylin and eosin staining; light microscopy; bar=100 urn.e- 110 -iii) Steroidogenic Differentiation of KGLOM cellsKGLOM cells with a transformed morphology expressed P-45Oscc and retractedfrom the substratum after treatment with cAMP (Fig 40), Both of these traits aremarkers of steroidogenic differentiation. Thus, the cells selected from KiMSV infectedGLOM cultures were not non-steroidogenic stromal fibroblasts.More than 90% of KGLOM cells expressed P-45Oscc and also overexpressed p21ras. We were unable to show definitively that this combination occurred within the sameKGLOM cells as a double label immunofluorescence could not be carried out. This was thecase because treatment with a detergent such as triton X-100, which is necessary toexpose the P-45Oscc protein in the inner mitochondrial membrane, extracts p21 rasfrom the cells (Fig 36). Clearly however, morphologically transformed KGLOM cells,which overexpressed p21 ras , also expressed P-45Oscc (compare Fig 34 and Fig 40).KGLOM cells in passage 2 produced corticosterone basally and responded to bothACTH and cAMP with increased corticosterone production (Fig 41). It should be notedthat the amount of corticosterone produced by KGLOM cells in passage 2 wasconsiderably less than that produced in primary cultures of uninfected GLOM cells (seeFig 9). Interestingly however, uninfected GLOM cells in passage 2 had completely dedifferentiated and no longer produced corticosterone under any conditions. Importantly,this de-differentiation indicated that the corticosterone produced by KGLOM cells was notderived from normal GLOM cells that may have been carried over during the originalselection/passaging process.— 111Fig 40: P-45Oscc expression in KGLOM cells.KGLOM cells (p2) were treated for 24 hrs with cAMP and then stained byimmunofluorescence for P-45Oscc.P-45Oscc immunofluorescence. The cell bodies and cell extensions are positivewith the staining confined to the mitochondria.ç)j No first antibody control. There is no mitochondrial staining.a,c) fluorescence microscopy; b,d) phase microscopy; bar=20 jim.III orT- 112 -Fig 41: Corticosterone production by GLOM and KGLOM cells (p2).Subconfluent passage 2 cultures in 16 mm wells were either not treated (D), treatedwith 10 nM ACTH () or treated with 0.5 mM cAMP () for 72 hours.Corticosterone in the media was determined by RIA. Data represent means +1- SEM eachin triplicate; corticosterone/105cells/24 hrs; ** p<.01 vs untreated.CS(ng)-1 I’30F\).o•)0III0 6- 113 -iv) Inhibition of p21 ras Function in KGLOM cellsIn order to determine whether v-Ki-ras p21 expression was directlyresponsible for the maintainence of steroidogenic differentiation in ras transformedKGLOM cells we treated the cells with lovastatin, a pharmacologic inhibitor of p21function.Lovastatin acts as a competitive inhibitor of HMG CoA reductase activity (Albertset al., 1980). Therefore, it inhibits p21 ras function by preventing the addition of aprenyl group (C15-farnesyl) to the carboxy terminus. This covalent modification ofp21 is critical for the sub-plasmalemmal localization and biological function of theprotein (Schafer et al., 1989). As well as interrupting p21 ras function, lovastatin alsointerferes with cholesterol biosynthesis. Therefore, pregnenolone (100 ng/ml) wasadded to the culture medium in these experiments in an attempt to prevent theinterruption of steroidogenesis due to an inadequate production of cholesterol. This dosewas chosen as it was low enough to avoid cross-reactivity with corticosterone during theradioimmunoassay.GLOM cells early in primary culture (Day 4) were treated with increasing dosesof lovastatin for up to 26 hrs in the presence or absence of ACTH/cAMP. Lovastatin didnot alter basal CS production but it did slightly reduce the CS increase induced by eitheracute (8hr) or chronic (26 hr) ACTH/cAMP treatment (Fig 42) . As has already beenshown, GLOM cells de-differentiate with increasing time in primary culture. By day 28,both basal and ACTH/cAMP induced CS production were considerably less than that seenat day 4 (compare Figs 42 and 43). In addition, at day 28, lovastatin did not decreaseeither basal or ACTH/cAMP induced steroidogenesis (Fig 43). These data suggest thatlovastatin did not inhibit steroidogenesis per Se. However, when large amounts of steroidare being produced, as occurred at day 4, lovastatin treatment may cause a cholesteroldeficit that cannot be completely overcome by the addition of 100 ng/ml pregnenelone.- 114 -Lovastatin did not decrease basal steroidogenesis in KGLOM cells. However,lovastatin did inhibit ACTH/cAMP induced corticosterone production in a dose dependentmanner (Fig 44). This inhibition was more readily apparent in chronically treated cells(26 hrs), as acute trophic hormone treatment (8 hrs) increased KGLOM steroidogenesisto only a small degree. Also, this inhibition was considerably greater than that observedin day 4 GLOM cells (compare Figs 42 and 44).During the first 12 hours of lovastatin treatment KGLOM cells retracted from thesubstratum and rounded up. However, by 24 hours many of the cells began to flatten andrevert somewhat to an untransformed morphology (Fig 45). In addition, in lovastatintreated cells p21 ras staining was diffuse throughout the cytoplasm and there was nolonger any localized staining along the plasma membrane (Fig 45).- 115 -Fig 42: Corticosterone production in lovastatin treated early primary GLOM cultures.GLOM cells at day 4 early in primary culture were treated with the indicated doses oflovastatin for 8 or 26 hrs in the presence of 100 ng/ml pregnenolone. Concurrently,either no further treatment was added (D), 10 nM ACTH was added (u), or 0.5 mM8Br-cAMP () was added. Culture medium was then assayed for corticosterone byRIA. Data represent means +1- SEM each in triplicate; corticosterone/105cells/timeperiod specified, *p<05 **p<01 vs same treatment (no addition, ACTH or cAMP) whenno lovastatin added.-115a-0)CCl)C-)0)CCl)C)1200800400400GLOM (Day 4) 8 hr0.0 0.5 5.0 50.00• -1200800LOVASTATIN iMGLOM (Day 4) 26 hrn00.0 0.5 5.0 50.0LOVASTATIN .tM- 116Fig 43: Corticosterone production in lovastatin treated late primary GLOM cultures.GLOM cells at day 28 late in primary culture were treated with the indicated doses oflovastatin for 8 or 26 hrs in the presence of 100 ng/ml pregnenolone. Concurrently,either no further treatment was added (III), 10 nM ACTH was added (), or 0.5 mM8Br-cAMP (•) was added. Culture medium was then assayed for corticosterone byRIA. Data represent means +1- SEM each in triplicate; corticosterone/105cells/timeperiod specified.-116a-0)Cl)00)Cl)0GLOM (Day 28) 8 hrrJ0.0 0.5100500100500I—50.0LOVASTATIN jtMGLOM (Day 28) 26 hr0.0 0.5 50.0LOVASTATIN iiM- 117 -Fig 44: Corticosterone production in lovastatin treated KGLOM cultures.KGLOM cells (p3) were treated with the indicated doses of lovastatin for 8 or 26 hrs inthe presence of 100 ng/ml pregnenolone. Concurrently, either no further treatment wasadded ( III), 10 nM ACTH was added (), or 0.5 mM 8Br-cAMP ( ) was added. Theculture medium was then assayed for corticosterone by RIA. Data represent means +1-SEM each in triplicate; corticosterone/1 cells/time period specified, *p<05**p<ol vs same treatment (no addition, ACTH or cAMP) when no lovastatin added.-117a-0)CC.’)C-)20100KGLOM 8 hrr100200.0 0.5 5.0 50.0LOVASTATIN p.MKGLOM 26 hr0)CC/)C-)0.0 0.5 5.0 50.0LOVASTATIN p.M- 118 -Fig 45: p21 localization in lovastatin treated KGLOM cells.KGLOM cells (p2) were treated for 26 hrs with 5 im lovastatin and then fixed andstained for p21 ras by immunofluoresecence.a,b) Lovastatin treated: The cells are somewhat flattened and p21 ras is found diffuselythroughout the cytoplasm.c,d) Untreated controls (no lovastatin): The cells are spindle shaped and there areconcentrations of p21 ras along the plasma membrane.a,c flourescence microscopy; b,d phase microscopy; bar=20 jim.118 o.- 119 -6. Parenchymal Differentiation of a Mixed Parenchymal-Stromal (MIX) Cell Populationin CultureAs already mentioned in Sec. 3. II. c., (FASC vs GLOM), homogeneous FASC andGLOM parenchymal cell populations de-differentiated with increasing time in culture.This was reflected by the decreased steroidogenic response to ACTH treatment late inprimary culture (see Fig 9, 42 vs. 43), and by the almost total non-responsivenessafter passaging (see Fig 10). This de-differentiaton was associated with a decrease in theactivity of the steroidogenic pathway enzyme A5,313-HSD in FASC and GLOM cells withincreasing time in culture. However, in a separate density-isolated adrenocorticalfraction which contained a mixture of stromal, endothelial and parenchymal cells(designated MIX), i5,313-HSD activity was not reduced but became enhanced with time.In addition, steroidogenic differentiation was maintained in long term primary cultureand after passaging. This phenomenom in MIX cultures will be described in some detailhere.i) CharacterizationMIX populations originated from Band 5 (1.057 g/ml) on the density gradient(Table 2). These populations contained only 60% A5,313-HSD positive cells at the timeof isolation. In addition, the cells in this population were heterogeneous in both size andlipid content and they tended to form cellular aggregates while they were within thedensity gradient (Fig 6c).- 120 -ii Primary Culture MorpholoQyAs described previously, FASC cells formed homogeneous epithelial monolayers,retained their original epithelial morphology, and underwent little proliferation inprimary culture. GLOM cells were initially epithelial but after the first week in culturethey formed monolayers of flat, fibroblastic cells that began to proliferate (Fig 11).Unlike the two parenchymal populations, the MIX populations immediatelyexhibited heterogeneity in culture. After 4 days, fibroblasts and endothellal cell colonieswere distinguishable from the lipid-containing parenchymal cells (Fig 46a). A few dayslater MIX cultures contained epithelial islands surrounded by areas of bipolarfibroblastic cells. After 1 week, cellular multilayers began to form as proliferationcontinued (Fig 47). Initially these multilayers were predominantly fibroblastic, butduring the second week in culture refractile and rounded cells appeared (Fig 46b). Thismultilayering morphology was reminiscent of that seen in KiMSV infected GLOM cultures(see Fig 33). Monitoring by time lapse indicated that in the MIX multilayers flattenedcells gradually rounded, acquired cytoplasmic lipid and proliferated (Fig 48).Occasionally large colonies of these rounded, lipid filled cells would detatch from themultilayer and float in the medium (Fig 46c).121Fig 46: Morphology of MIX adrencocortical cell population in primary culture.a) Day 4: A variety of cell types are present including epithelial adrenocorticalparenchymal cells, fibroblastic cells and endothelial cells.b) Day 9: A multilayered culture is shown. Rounded, lipid containing containing cells arelocated apically on an underlying bed of fibroblastic cells.C) Day 16: A large colony of rounded cells that have emerged from a multilayer similarto that shown in (b) above.Phase microscopy; bar=50 jim.- 122 -Fig 47: Growth of FASC. GLOM and MIX cells in primary culture.1 X MIX, FASC or GLOM cells were seeded in 16mm wells. At the times indicated,triplicate cultures were trypsinized and counted. Data represent means, all SEM < 10%,each time point is in triplicate. The results are representative of 3 experiments.-122a-Cci)C-)128400 10 20 30Days in Culture- 123 -Fig 48: Time lapse microscopy of proliferatino MIX cells in primary culture.Frames from a time lapse microscopic analysis of a 10 day old multilayered culture ofMIX cells. This shows rounded cells in the same field that emerge from the multilayerand undergo cell division over a 72 hour period, a) 0 hr; b) 20 hr; C) 28 hr; d) 45 hr;e) 48 hr; f) 72 hr.The small arrow designates a rounded cell which begins to divide in c); completesdivision in d); and shows the daughter cells moving apart in d).The large arrow shows a rounded cell which begins to divide in c); completes division ine); and shows the daughter cells moving apart in f).Phase microscopy; bar= 50 jim.1i3&- 124 -iii) Steroidogenic Differentiation in Primary Culture.When assayed after 4 days in primary culture, all 3 density-isolated populations(FASC, GLOM, MIX), were steroidogenic and produced corticosterone in the absence oftrophic hormone treatment. Interestingly, on a per cell basis, MIX cultures produced themost corticosterone (Fig 49) even though a significant proportion of this population wasnot parenchymal. ACTH treatment (24 hr) resulted in dramatic increases incorticosterone production in all 3 populations. FASC cultures produced the mostcorticosterone in response to ACTH, which was to be expected given that the zonafasciculata is the major site of glucocorticoid production in 3jQ.When assayed after 16 days in primary culture, all 3 populations producedfluorogenic steroid and responded to ACTH treatment with increased steroidogenesis (Fig50a). At this time point, FASC cultures produced the most fluorogenic steroid inresponse to ACTH. However, after 56 days in primary culture, the ACTH responsivenessof FASC and GLOM cultures was greatly reduced (Fig 50b). On the other hand, thesteroidogenic response to ACTH was greatly increased in 56 day old MIX culturescompared to day 16.The increased responsiveness to ACTH treatment suggested that a greater degreeof cyto-differentiation took place in MIX cultures compared to that which occurred in theFASC and GLOM cultures. A second possibility is that there is a proliferating,differentiated cell population in the MIX cultures. To provide a quantitative measure ofcyto-differentiation in the absence of trophic hormone treatment, we estimated thepercentage of A5,313-HSD positive cells at various time points in primary culture. Aswas expected (Ryback and Ramachandran, 1981b), the percentage of z5,3I3-HSDpositive FASC and GLOM cells decreased gradually during the first 21 days in culture(Fig 51 a). In contrast, the percentage of 5,313-HSD positive cells decreased very littlein MIX cultures. This finding, coupled with the high rate of proliferation, indicated a- 125greater than 10 fold increase in the number of A5,313-HSD positive cells in the MIXcultures after 21 days in culture (Fig 51b). When MIX multilayers were stained ini1LL, it was the rounded cells that contained lipid and A5,36-HSD activity (Fig 52a,b).These cells also expressed P-45Oscc, in the absence of ACTH treatment (Fig 52c-f).Interestingly, under these conditions the underlying fibroblastic cells in the multilayerwere P-45Oscc negative.- 126 -Fig 49: Corticosterone production by FASC. GLOM and MIX cells in primary culture.FASC ( III), GLOM (E1) and MIX () cells were seeded at approximately 1 X iO per16 mm well. At Day 6 the cells were left untreated (A) or were treated with lOnM ACTH(B) for 24 hrs. Medium was then collected and assayed by RIA for corticosterone. Datarepresent means +1- SEM each in triplicate; corticosterone/1 cells/24hrs.-126a-200C)a)010006000a)04000200080000- 127Fig 50: Fluoroyenic steroid production by FASC. GLOM and MIX cells in primaryculture.Cells were seeded at approximately 1 X 1 per 35 mm dish. Fluorogenic steroidproduction was determined at either day 16 or day 56 in primary culture in the absence( EJ) or presence of (•) 10 nM ACTH for 4 hours. Data represent means +1- SEMeach in triplicate; fluorogenic steroid production/dish/4 hrs.FluorogenicSteroid(ng)010010010010000000000000000000FluorogenicSteroid(ng)p’.I (Ji 0)Cl) C)><-lI\3—a><- 128 -Fig 51: A5.3f3-Hydroxysteroid dehydroQenase (5.3B-HSD activity of FASC. GLOMand MIX cells in primary culture.A) % A5.313-HSD positive cells. Cells were seeded at 1 X cells per 16 mm well. Atthe indicated time in culture the cells were dissociated, plated on coverslips and stainedfor A5,313-HSD activity. The percentage of positive cells was then determined bymicroscopic examination. Data represent means of at least 10 fields of> 50 cells each.All SEM < 15%.B) Number of A5.313-HSD positive cells. Values were determined by multiplyingpercent 5,3B-HSD positive in (A) with the total number of cells at indicated timepoints shown in Fig 47.-128a-0U)ci0>4-,U)0010080604020U)ci0G)>4-,U)0000 7 14 21Day64200 10 20Days in Culture- 129 -Fig 52: Differentiation marker expression of multilayered MIX primary cultures in1LThese cultures were not treated with ACTH or cAMP.a) Oil Red 0: Day 12 in culture. Notice that the rounded cells emerging from themultilayer are positive for lipid. Underlying fibroblastic cells (out of focus) are for themost part negative.b) z\5.313-HSD: Day 11 in culture. Notice that rounded cells are most A5,36-HSDpositive.a,b) light microscopy; bar=20 jim.c.d) P-45Oscc Day 7: (same field) Rounded cells are just beginning to emerge from themultilayer, some of which are P-45Oscc positive. The underlying fibroblastic cells arenegative.e.f) P-45Oscc Day 14: (same field) The rounded cells are now all P-45Oscc positive.c,e) fluorescence microscopy; d,f) phase microscopy; bar=20 jim.lzqLLIt2S.‘1 ••-.-9-9.•.90• I. •S..•v-f?4 -Vt—S-S.•S5 4-•- •C.4C- 130 -iv) Secondary Culture MorphologyRounded MIX cells, either detached or slightly adherent to the underlyingfibroblastic cells, were selectively passaged by gentle agitation using a pasteur pipettein a fashion similar to that used for passaging KiMSV infected GLOM cells. Secondarycultures of these cells were initially homogeneous (Fig 53a). The cells were elongated,branched, refractile and contained cytoplasmic lipid. After a few days, a second,morphologically distinct cell type appeared in these cultures (Fig 53b). Initially thesecells were rare, and they were large, flat, and fibroblastic. These fibroblastic cellsserved as a substratum for the predominating retractile cells which selectively adheredto the fibroblastic cells.v) Steroidogenic Differentiation in Secondary CultureIn secondary culture, the initially homogeneous MIX cells were P-45Osccpositive (Fig 54). When heterogeneous secondary MIX cultures were treated with ACTH,or its second messenger cAMP, the retractile cells rounded up, while the underlyingfibroblastic cells did not (Fig 55a,b). As cAMP-induced rounding is considered to be acharacteristic of many steroidogenic cell types, this suggested that the fibroblastic cellsare stromally derived. However, after CAMP treatment both the refractile cells and theunderlying fibroblasic cells expressed P-45Oscc (Fig 55c,d), indicating a commonparenchymal origin for both cell types.Using time lapse videomicroscopy we were unable to determine if the fibroblasticcells arose by modulation from the refractile cells or if they represented a separate celltype carried over from primary cultures. The retractile cells remained adherent andcontinued to replicate in secondary culture only if they were in contact with the flattenedfibroblastic cells, suggesting a requirement for heterologous cell interactions.- 131Upon reaching confluence these heterogeneous secondary cultures once againformed predominantly fibroblastic multilayers from which refractile and rounded cellsonce again emerged in the absence of trophic hormone treatment (Fig 53c). Afteranother round of selective passaging, MIX cells in passage 2 continued to producefluorogenic steroid and responded to ACTH treatment with increased steroidogenesis (Fig56a). After non-enzymatic, dissociation GLOM cells in passage 2 produced negligibleamounts of fluorogenic steroid either basally or after ACTH treatment. FASC cells werenot assayed in these experiments as they did not survive mechanical passaging. MIX cellsin passage 2 also produced corticosterone basally and responded to ACTH and cAMP,although at reduced levels compared to primary cultures (Fig 56b). GLOM cells inpassage 2 produced negligible amounts of corticosterone either basally or after ACTH orcAMP treatment.- 132 -Fig 53: Morphology of MIX adrencocortical cell population in secondary culture.Loosely adherent and detaching rounded cells from multilayering primary MIX cultureswere removed by gentle agitation and passaged into secondary culture (p1).a) Day 2: Cultures were relatively homogeneous. Cells contained lipid and often hadbranching processes. Some cells did not spread and remained rounded (not shown here,but see Fig 54).b) Day 10: Cultures became heterogeneous. Small, refractile cells utilized large, flatfibrobastic cells as an underlying substratum.c) Day 21: Multilayers once again formed with emerging rounded cells that containedlipid.Phase microscopy; bar=50 lim.‘sq4- 133 -Fig 54: P-45Oscc expression in MIX (p1) cells.Two days after selective passaging MIX cells were stained for P-45Oscc expression inthe absence of ACTH or cAMP treatment. The cells are P-45Oscc positive and individualmitochondria are visible in flattened cells.a) phase microcopy; b) fluorescence microscopy; bar=20 urn.133 LFe- 134 -Fig 55: Response to cAMP and P-45Oscc expression in heterogeneous MIX (p1)cultures.Heterogeneous MIX (p1) cultures were treated with 0.5 mM 8Br-cAMP for 24 hours.a) no cAMP: Refractile lipid containing cells adhering to underlying fibroblastic cell.b) cAMP treated (morphology): The majority of the retractile cells responded to cAMPby rounding up. Some cells detached from the underlying fibroblastic cell. The latterremained flat. a,b) phase microscopy; bar=50 jim.c.d) cAMP treated (P-45Oscc expresion): Both the rounded and fibroblastic cells are P45Oscc positive. C) fluorescence microscopy; d) phase microscopy; bar=20 jim.I3LIOL.- 135 -Fig 56: Steroidogenesis of passage 2 GLOM and MIX cultures.GLOM cultures were passaged mechanically by scraping to avoid enzymatic dissociation.The latter method has already been shown to result in loss of steroidogenicdifferentiation (see Fig 10). MIX multilayered cultures were selectively passaged byremoving detached and loosely adherent rounded cells.Fluorogenic steroid production: Subconfluent GLOM and MIX cultures in 35 mm disheswere treated without () or with () 10 nM ACTH for 24 hours. Data representmeans ÷1- SEM; each in triplicate; fluorogenic steroid/dish/24 hours; ** p <.01 vsuntreated.Corticosterone production: Subconfluent GLOM and MIX cultures in 16 mm Wellswere not treated (LII), treated with 10 nM ACTH () or treated with 0.5 mM cAMP( •) for 72 hours. Corticosterone in the media was determined by RIA. Data representmeans +1- SEM each in triplicate; corticosterone/105cells/24 hrs; ** p<.O1 vs.untreated.001CS(ng)FluorometricSteroid(ng)-I C)C.;,00101 000100- 136 -IV. DISCUSSIONThe initial attempts at adrenocortical parenchymal cell purification were notwholly successful. However, density gradient centrifugation produced 2 homogeneouspopulations, one derived from the zona glomerulosa (GLOM) and the other derived fromthe zona fasciculata (FASC). In primary culture, these cells exhibited phenotypicdifferences associated with their stage of adrenocortical development. For example,GLOM cells responded to Ang II while FASC cells did not. An interruption of Ang II-mediated signal transduction at the level of intracellular calcium release appeared tocontribute to this phenotype in FASC cells. Infection with KiMSV, which contains the rasoncogene, was associated with oncogenic transformation and steroidogenic differentiationin GLOM but not FASC cells. This difference may be representative of the state of thesignal transduction pathways in the two cell types. Both GLOM and FASC cells dedifferentiated with time and/or passaging in culture. In contrast, in a mixedparenchymal/stromal cell population (MIX) steroidogen ic differentiation occurredspontaneously over the longterm. This phenomenom was associated with a cellularmultilayering that may mimic some aspects of the histological organization presentduring adrenocortical development In yjyn.1. Preliminary Attempts at Parenchymal Cell SeparationA major objective of this study was to produce homogeneous populations ofadrenocortical parenchymal cells. A number of different separation techniques were usedin an attempt to isolate such populations from crude, whole gland preparations. Longterm treatment of whole gland cultures with cAMP caused the majority of theparenchymal cells to retract from the substratum and round up. This morphologicalresponse is typical of many steroidogenic cell types in culture (Gill et al., 1980;- 137Amsterdam et al., 1989; Hornsby et al., 1989). The rounded cells were selectivelyremoved from the culture dish, thereby leaving behind a bed of non-responsivefibroblastic cells. However, the detached cells did not reattach to the substratum whenpassaged to new dishes. Differential substratum adhesion removed large numbers offibroblasts and factor VIII positive endothelial cells from whole gland suspensions priorto culture. However, a small percentage of contaminating cells were also carried overwith the lipid containing parenchymal cells during serial transferring. Therefore, thismethod enriched the parenchymal cell component but it did not purify it. Chemical antistromal cells agents such as d-valine (Ryback and Ramachandran, 1981 a) were not usedto try and eliminate contaminants from these populations as there was a concern thatthey would also be detrimental to the growth and/or function of the mesodermallyderived parenchymal cells. Also, we did not attempt to clone the lipid containingepithelial cells because rat adrenocortical cells have a short pre-senescent lifespan andrapidly de-differentiate in monolayer culture (O’Hare and Neville, 1973b; Hornsby etal., 1974, Ryback and Ramachandran, 1981b).Removal of the capsule by dissection is often used to separate zones of the ratadrenal cortex (Haning et al., 1970, Hornsby et al., 1974; Ryback and Ramachandran,1981a,b; Kojima et al., 1984,1985; Payet et al., 1984; Woodcock 1990). This processproduces two fractions, a capsular adherent zona glomerulosa and the de-capsulatedzonae fasciculata/reticularis. While this method is efficient in producing populationsthat have the overall characteristics of a particular zona, it does not remove the stromalcells associated with the connective tissue framework of the gland or the endothelial cellsthat are derived from the extensive the capillary network that courses throughout thecortex. In explant culture, adrenocortical capsules, but not de-capsulated glandsproduced large numbers of migrating fibroblastic cells. While these explant derivedfibroblasts resemble myofibroblasts ultrastucturally and produce large amounts of anextracellular matrix that contains collagen, they also exhibit limited parenchymal- 138 -characteristics under some culture conditions, or after ras-mediated transformation(Slavinski et al., 1974, 1976; Slavinski-Turley and Auersperg, 1978; Auersperg etal., 1981,1990). The connective tissue capsule can be dissected away from the zonaglomerulosa prior to collagenase dissociation (Hornsby et al., 1974). This procedureremoves most myofibroblastic cells from the capsular fraction but it does not addressthe presence of stromal and/or endothelial cells within the zona glomerulosa itself.When de-capsulated glands were collagenase dissociated and seeded into culture,the great majority of the cells contained large cytoplasmic lipid inclusions. During thefirst week in culture, these cells formed epithelial monolayers except for small areascontaining fibroblastic cells. Over a period of weeks the latter have been observed toovergrow the epithelial monolayer (Neville and O’Hare, 1973a; Ryback andRamachandran, 1981b). Thus, decapsulation provided a population that was enrichedwith, but not homogeneous for, parenchymal epithelial cells.2. Percoll Density Gradient Parenchymal Cell SeparationThe parenchymal cells of the zona fasciculata and glomerulosa have a number ofdifferent physical properties. There are large differences in cell size, nuclear tocytoplasmic ratio, lipid content and cell density (Idleman, 1970; Tait et al., 1974;Nickerson, 1976; Nussdorfer 1986). We attempted to obtain homogeneous parenchymalcell populations by exploiting the extreme differences in cell density in these two celltypes.Serum albumin gradients, used previously to estimate the density of rat zonafasciculata and zona glomerulosa cells, do not separate the cells efficiently because theyare composed of both density and viscosity components (Tait et al., 1974). This dualgradient produces a sedimentation rate that is dependent on both cell density and size.Thus, the larger the cell and the greater its density the faster the sedimentation rate. As- 139 -zona fasciculata cells from decapsulated glands are large (18 j.tm diameter) and of lowdensity (1.040 g/ml), they tended to travel through.the albumin gradients at a ratesimilar to capsular zona glomerulosa cells which were small (12 jim diameter) and ofhigh density (1.072 g/ml). Due to the large difference in density between the 2 celltypes, separation on the basis of density alone was predicted to be much more efficient.Percoll, whose density differs with its concentration, is useful in this regard because itsviscosity is similar to that of tissue culture media. Thus the viscosity of percoll does notvary with changes in concentration/density. Contaminating zona fasciculata cells havebeen. removed from capsular glomerulosa cell preparations by sedimenting the cells incontinuous percoll gradients (Chu and Hyatt, 1986). We went one step further andconstructed discontinuous density gradients. This has two advantages. Firstly, the densityinterfaces could be set at our choosing. Secondly, the cells settle out at the densityinterface closest to their own density (isopycnic point). Therefore, sedimentation ratesare not a variable that has to be controlled for between experiments. In its final form, an8 step discontinuous gradient was utilized. Density interfaces that were similar to thosedetermined previously for fasciculata and glomerulosa cells on albumin gradients werepositioned at opposite ends of the gradient. These were separated by numerousintervening interfaces in an effort to achieve maximal parenchymal cell separation.To initially characterize the constituents of each cell band obtained from thedensity gradient, we used the presence of cytoplasmic lipid inclusions and A5,313-HSDactivity as markers of steroidogenic differentiation. The presence of the microsomalenzyme A5,38-HSD is not an exclusive marker for endocrine derived steroidogeniccells. For example, it is found in low amounts in some some peripheral tissues includingskin (Labrie, 1990). However, all adrenocortical parenchymal cells contain largeamounts of this enzyme when they are producing endpoint steroids (Simpson et al.,1990).- 140 -The constituents of the lowest density cell band (#1) were morphologicallyhomogeneous. The cells were large and they contained large cytoplasmic lipid inclusionsthat obscured the nucleus. However, only about half of these cells were steroidogenic.The rest were probably capsular-adherent adipocytes, as the great majority of cellsfrom the adrenal fat pad settled at this density interface.Bands obtained from the middle of the gradient had a heterogenous make-up, bothin cell size and lipid content. In addition, in these bands (#4-6) the cells tended to formlarge (<50 cells) aggregates during the percoll separation. These cells were alsoheterogeneous for steroidogenic differentiation. For example, Band 5 (1 .057 g/ml)contained 60% A5,3B-HSD positive cells. On the basis of the absence of lipid and theirmorphology in primary culture it is believed that the majority of the i5,3B-HSDnegative cells in band 5 consisted of both stromal and endothelial cells. Therefore, thisband was designated MIX (see below).In bands #2 and #7 the great majority of the cells were A5,3B-HSD positive.Within each of these bands the cells were morphologically homogeneous. However,comparatively, the cells of each band were very different from each other. Band #2 cellswere large and filled with cytoplasmic lipid inclusions that obscured the nucleus. Band#7 cells were small and contained only scanty amounts of cytoplasmic lipid. in yi,the zona fasciculata contains columns of large, lipid laden cells, while the zonaglomerulosa contains clustered nests of small relatively lipid poor cells (Nussdorfer1986). Therefore, the physical characteristics of the cells suggested that Band #2arose predominantly from the zona fasciculata and Band #7 from the zona glomerulosa.In addition, the densities of Band #2 (1 .034 g/mI) and Band #7 (1 .069 g/mI) weresimilar to those determined for zona fasciculata and zona glomerulosa cells on serumalbumin gradients (Tait et al., 1974; Bell et al., 1978). Therefore Band #2 wasdesignated FASC (fasciculata) and Band #7 was designated as GLOM (glomerulosa).Fractionation of adrenal capsules (glomerulosa) from inner glands- 141 -(fasciculata/reticularis), followed by density separation of the cells from each fractionsupported these conclusions. It should be noted at this point that neither of these bandscontained ]! the cells of a particular zona. Thus, other density bands likely containedcells from these zonae that had slightly different physical characteristics.3. in vitro Characterization of FASC and GLOM CellsDue to their intra-population homogeneity and different parenchymal origins,FASC and GLOM cells were characterized in depth. In suspension FASC and GLOM cellsexhibited the steroidogenic characteristics of fasciculata and glomerulosa cellsrespectively (Haning at al., 1970; Tait et aI., 1974; Douglas et al., 1978, Braley et al.,1986). In primary culture ACTH, but not Ang II, dramatically increased CS productionin FASC cells. This indicated that a fasciculata phenotype was maintained. Both ACTH andAng II increased Aldo production in GLOM cultures, which indicated that a glomerulosacell phenotype was maintained.After one week in primary culture, FASC cells remained homogeneous, epithelialand responded to a 48 hr treatment with cAMP by retracting from the substratum.During the same period, GLOM cells rapidly lost lipid, spread extensively, and assumedfibroblastic shapes that changed little after a 48 hour cAMP treatment. cAMP inducedretraction by adrenocortical cells in culture has long been used as an indicator ofparenchymal origin (Gill et al., 1980), although there are culture conditions that canreduce this morphologic response (Hornsby et al., 1989). Regardless, the expression ofthe steroidogenic enzyme P-45Oscc in the fibroblastic GLOM cells indicated that theywere of parenchymal rather than stromal origin. Therefore, cell morphology andcAMP-induced rounding are not sufficient in delineating certain adrenocorticalparenchymal cells from stromal fibroblasts. Also, cAMP-mediated increases in steroidproduction without GLOM cell rounding/retraction indicated that the induction of- 142 -steroidogenesis is not dependent on this morphological response as has been suggested byothers (Zor, 1983; Hall, 1984; Almahabohi and Hall, 1991).Ultrastructurally, FASC cells maintained many of the characteristics of fasciculatacells in JyQ. These included large cytoplasmic lipid inclusions and mitochoridria withtubulovescicular cristae. GLOM cells that had modulated to a fibroblastic morphologycontinued to also showed exhibit ultrastructural characteristics steroidogenicdifferentiation. These included a well developed smooth endoplasmic reticulum andmitochondria with tubular cristae. In addition, while fibroblastic GLOM cells did notcontain large lipid inclusions they did not exhibit many of the myofibroblasticultrastructural characteristics observed in explant derived adrenocortical fibroblasts(Slavinski-Turley and Auersperg, 1978).Parenchymal cells of glandular epithelia usually express cytokeratin intermediatefilaments (Steinert and Liem, 1990). However, there was no evidence of cytokeratinexpression in FASC or GLOM cells. The steroidogenic parenchymal cells of the ratovarian follicle (ie. granulosa cells) also do not express cytokeratiris (Amsterdam et aL,1989). The absence of cytokeratins in steroidogenic rat cells may be species specific.For example, keratins are expressed in human adrenocortical cells (Henzen-Logmans etal., 1988).The culture characteristics of FASC and GLOM cells may reflect the developmentalmechansims and the mesodermal origin of the rat adrenal cortex. Adrenocorticalcytogenesis appears to take place along a continuum as as an adrenocyte migrates inwardfrom the fibroblastic capsule through the zones of the gland before degenerating in thezona reticularis (Gottschau, 1883; Ingle and Higgins, 1938; Wright and Voncina, 1977;Taki and Nickerson 1985; lannacone and Weinberg, 1987). The rapid culturemodulation of GLOM cells to a fibroblastic morphology suggests that while they areparenchymal in origin they are not far removed from a stromal cell phenotype. Insupport of this suggestion, GLOM cells expressed the intermediate filament protein- 143vimentin after fibroblastic modulation. Vimentin is often expressed in connectivetissue/stromal cells (Steinert and Liem, 1990). It should be noted that the fibroblasticmodulation in GLOM cells is not an example of complete epithelial/mesenchymaltransformation (Greenberg and Hay, 1988). For example, fibroblastic GLOM cellsexpress both P-45Oscc and vimentin. Thus, GLOM cells are able to simultaneouslyexpress parenchymal and fibroblastic characteristics. This is in contrast to explantderived adrenocortical fibroblasts which are bipotential, but take on an epithelialmorphology and reduce their production of ECM when they express steroidogeniccharacteristics (Turley 1980).The fibroblastic modulation of GLOM cells is a phenomenon which is likelyenhanced in monolayer culture, but it is not trivial as FASC cells exhibit a morerestricted parenchymal phenotype under the same culture conditions. Therefore, theability of GLOM cells to express some characteristics of fibroblasts while remainingsteroidogenic suggests that these cells may be man intermediate position in theadrenocortical developmental pathway, located somewhere between a bipotentialstromal/parenchymal capsular cell (explant derived fibroblast) and a fully committedparenchymal cell (FASC). These conclusions are in agreement with the centripitalmigration hypothesis (Gottschau, 1883; lannacone and Weinberg, 1 987) which suggeststhat ‘adrenocyte’ development takes place along a single clonal continuum from thecapsule to the medulla.4. Any ii- Mediated Signal Transduction in FASC and GLOM CellsThe centripital migration hypothesis states that zona glomerulosa cellsdifferentiate into zona fasciculata cells as they move inwards during normaladrenocortical development. There is ample experimental evidence to support thistheory. Presumably this process is gradual, both temporally and spatially. The temporal- 144 -component can be followed by examining labelled cells as they move through the cortex.The spatial component may be delineated by a non-descript “intermediate zone” that isfound between the two zonae (Nickerson, 1976). Regardless, as the migrating cells takeon a fasciculata phenotype they lose their ability to respond steroidogenically to Ang II(Haning et al., 1970; Tait et al., 1974; Douglas et al., 1978; Braley et al., 1986). Thistrophic hormone acts primarily by altering intracellular calcium [Ca2]i metabolismand activating protein kinase C (Barrett et al., 1989). It does this by intially activatingphosphatidyl inositol specific phospholipase C which produces the second messengersdiacylgicerol (DAG) and lns(1 ,4,5)P3. We have demonstrated a loss in the capacity ofAng II to trigger an elevation in [Ca2]i in rat fasciculata (FASC) cells despite the factthat lns(1 ,4,5)P3 levels were initially increased. Interestingly, when [Ca2]i waselevated independently in these cells, steroidogenesis was stimulated. Therefore, aninterruption of signal transduction at the level of increased [Ca2+]i may be one of thefactors that prevents Ang II from eliciting a steroidogenic response in FASC cells. Incontrast, the transduction pathway was intact in GLOM cells.Rat fasciculata cells have much fewer Ang II receptors than their glomerulosacell counterparts (Douglas et al.,1978; Healy et al., 1984; Maurer and Reubi, 1986;Husain et al., 1987). This difference between the zones has led to the speculation thatthe lack of a steroidogenic response by rat fasciculata cells may be accounted for entirelyby the inability of the hormone to initiate signal transduction in these cells.Nevertheless, in rat fasciculatalreticularis cell preparations isolated by dissectionprior to cell dissociation, Ang II increased phospholipid turnover and overall inositolphosphate production (Whitley et al., 1984,1987). However, because of the possiblepresence of contaminating cells in such preparations it has been difficult to preciselyidentify the cell type which responded to Ang II in these studies. In addition it has notbeen determined if production of the lns(1 ,4,5)P3 isomer is increased. Consequently, inthis study, the homogeneous sub-populations of rat glomerulosa (GLOM) and fasciculata- 145 -(FASC) cells were utilized to carry out a detailed comparison of Ang II mediated signaltransduction. As has been disscussed above, these sub-populations did not appear to becontaminated by stromal, endothelial or adrenal medullary cells. The absence ofmedullary cells which was determined by cell density, morphology, and the lack ofcatecholamine staining is an important consideration as these cells have also been shownto contain Ang II receptors in the rat (Healy et al., 1984; Maurer and Reubi, 1986).We have confirmed that Ang II treatment increases the production of inositolphosphates in FASC cells. We have also shown that Ang II specifically triggers theincreased production of lns(1,4,5)P3 in FASC as well as GLOM cells. Therefore, in spiteof the drastically reduced receptor number (Douglas et al., 1978; Healy et al., 1984;Maurer and Reubi, 1986; Husain et al., 1987), Ang II initiates signal transduction inrat fasciculata cells. Such inconsistency between receptor number and a functionalresponse is not unknown. For example, Ang II induces hypertrophy of rat fasciculatacells jfly.j (Nussdorfer et al., 1981; Mazzocchi and Nussdorfer, 1986), increaseslnsP3 prQduction in mouse Y-1 adrenocortical tumour cells (Langlois et al., 1990) andinitiates signal transduction in bovine adrenal medullary cells (Zimlichman et al.,1987; Stauderman and Pruss, 1989) even though few hormone receptors are detected inany of these cell types (Healy et al., 1984; Maurer and Reubi, 1986; Husain et al.,1987; Begeot et al., 1987).In zona glomerulosa cells, Ang Il-mediated lns(1,4,5)P3 production elicits arelease of calcium from intracellular stores (Barrett et al., 1989). This produces anintial elevation in [Ca2]i that falls within minutes. This [Ca2]i peak is essential forthe initiation of a steroidogenic response. Over a longer period, extending up to hours,Ang II elicits a secondary increase in [Ca2]i which is associated with an influx ofextracellular calcium (Kojima et al., 1985). This secondary increase in [Ca 2]i isessential for the maintenance of the steroidogenic response to Ang II and is strictlydependent on the initial intracellular calcium release (Alkon and Rasmussen, 1988). At- 146 -the cell population level, this secondary response results in a raised plateau of [Ca2’iiwhich is lower in magnitude than the initial peak. At the single cell level, the effect ofthis calcium influx is unclear, although it may play a role in producing [Ca2]ioscillations (Quinn et al., 1988; Johnson et al., 1989). Recently, it has been shownthat in addition to initiating the opening of calcium channels at the plasma membrane,continued occupancy of Ang II receptors also helps trigger the secondary influx ofcalcium into the cell via receptor-mediated endocytosis (Hunjady et al., 1991). Thus,rather than acting to inhibit the generation of lnsP(1 ,4,5)P3, the reduced number ofAng II receptors in rat fasciculata cells may inhibit the secondary influx of extracellularcalcium. If this is indeed the case, an interruption in signal transduction distal toreceptor-mediated lns(1 ,4,5)P3 generation must occur in order to prevent theabsoulute increase in steroidogenesis observed in these cells.In GLOM cells, transient Ang II treatment (< 1 mm) produced sharp [Ca2]ipeaks that returned to baseline within minutes. These responses continued to occur inthe presence of the calcium channel blocker verapamil and in the absence ofextracellular calcium. A second Ang II treatment in calcium free medium did not increase[Ca2]i suggesting that the intracellular calcium pool was exhausted after an initialrelease (Kojima et al., 1987). There was no evidence of maintained [Ca2+]i plateaus oroscillations in the GLOM cells unless Ang II treatment was continued for longer periods(ie. 10 mm). Taken together, these results indicated that transient Ang II treatmentinitiated an intracellular calcium release only.Ninety percent of individual GLOM cells assayed responded to Ang II treatmentwith increased [Ca2]i. This further attests to the parenchymal purity of the GLOMpopulation. The magnitude of the [Ca2hii increase was dependent on the dose of Ang IIadded. In single GLOM cells however, the peaks were near maximal once a threshold dosewas reached. This direct comparison suggests that the dose-dependent increases in[Ca2]i peaks observed in GLOM populations reflected an increased recruitment of- 147responding single cells. This observation has been noted in other cell types where it hasbeen suggested that each individual cell has its own constant threshold for intracellularcalcium release under a particular set of conditions (Berridge and Irvine 1989).Transient Ang II treatment of FASC populations resulted in [Ca2]i increasesthat were much lower in magnitude than those seen in GLOM populations. Further, mostof the individual FASC cells assayed (72%) did not respond to Ang II at all, and those thatdid exhibited mean [Ca2+]i increases that were only about one half of those observed inresponding GLOM cells. Therefore, it appeared that the initial Ang Il-mediatedproduction of lns(1 ,4,5)P3 was not efficiently translated into an intracellular calciumrelease in rat fasciculata cells.Yl mouse adrenocortical tumour cells respond steroidogenically to ACTHtreatment but not to Ang II, although the latter does increase the generation oflns(1,4,5)P3 (Begeot et al., 1987; Langlois et al., 1990). However, in a small sampleassayed in this study, Ang II treatment did not induce an intracellular calcium release.Thus, as was seen in FASC cells, Ang Il-mediated signal transduction may be initiated butnot completed in steroidogenically refractory Yl cells. Interestingly, rat fasciculata andYl cells have very similar Ang II receptor numbers, approximately 10,000 per cell(Douglas et al., 1978; Begeot et al., 1987).If the reduced ability of Ang II to increase [Ca2]i is an important negativeregulatory control mechanism in the rat zona fasciculata, then an artificial [Ca2jiincrease should overcome this control and stimulate steroidogenesis in FASC cells. Thiswas indeed the case. Calcium ionophore treatment, which increased [Ca2]i, alsoelicited a dose dependent increase in steroidogenesis in both GLOM and FASC cells.Calcium ionophore treatment alone, without an additional stimulation of PKC, has beenreported to be a weak stimulator of steroidogenesis in glomerulosa cells (Kojima et al.,1984, Capponi et al., 1988). The observation that such a weak stimulator is sufficientto increase steroidogenesis in FASC cells suggests that the portion of the Ang Il-mediated- 148 -signal transduction pathway distal to intracellular calcium release is maintained inthese cells. This is in contrast to ovine fasciculata cells where Ang II treatment elicits a[Ca2+]i increase but is unable to increase steroidogenesis, presumably because of apathway interruption further downstream (Viard et al., 1990).Interestingly, calcium ionophore treatment also increased lns(1 ,4,5)P3production in GLOM and FASC cells. This suggests that extracellular calcium influx maybe important in maintaining protein kinase C activation (via DAG production) as well asre-initiating a secondary lns(1 ,4,5)P3-mediated intracellular calcium release after aninitial exhaustion and re-filling of the intracellular calcium pool. As has been discussedabove, refilling of the intracellular calcium pool is also dependent upon an extracellularcalcium influx. Thus, such a cycling of intracellular release and extracellular calciuminflux may be self-perpetuating, thereby leading to the [Ca2]i oscillations observedduring long term Ang II treatment. In support of this suggestion, it has recently beendetermined that extracellular calcium is required for the maintained stimulation ofPIP2-specific PLC during Iongterm Ang II treatment of rat glomerulosa cells (Foster etal., 1991). Also, growth factor induced [Ca2]i oscillations in fibroblast lines aredependent upon the continued, intermittant generation of InsP3 (Harootunian et al.,1991).There are a number of possible explanations for the interruption oflns(1 ,4,5)P3-mediated intracellular calcium release in FASC cells. One could involvequalitative differences among the Ang II receptors present in GLOM and FASC cells as 2different receptor sub-types have been identified, each with different signaltransduction capabilities (Johnson and Aguilera, 1991). Another explanation couldinvolve the absence of an intracellular calcium pool in FASC cells. This does not appearto be the case as thapsigargin, which releases intracellular calcium in an lns(1 ,4,5)P3independent manner (Takemura et al., 1989; Thastrup et al., 1990), increased [Ca2]iin both GLOM and FASC cells. However, this increase was smaller in magnitude and- 149 -slower in onset in FASC cells. Thus, it is possible that the intracellular calcium pool isreduced or restricted in FASC cells.Differential InsP3 metabolism could also inhibit [Ca2]i increases in FASCcells. For example, a rapid conversion of lnS(1,4,5)P3 into InS(1,3,4)P3 couldimpede intracellular calcium release (Berridge and Irvine, 1989). In fact, this type ofdifferential lnsP3 metabolism appears to be responsible for the inability of the peptidehormone endothelin to increase intracellular calcium or steroidogenesis in capsulederived rat glomerulosa cells (Woodcock et al., 1990). If it occured in Ang II treatedFASC cells, this lnsP3 conversion would also produce inositol 1,3,4,5-tetrakisphosphate (lnsP4) as an intermediate (Berridge and Irvine, 1989). lnsP4 hasbeen shown to further prevent [Ca2+]i increases in other cell types by causing asequestration of intracellular calcium (Hill et al., 1988). However, in a crudecomparison of InsPi, lnsP2 and lnsP3 production after 3 and 10 minute Ang IItreatments no large differences in inositol phosphate metabolism were evident in GLOMand FASC cells. For example, in both cell types lnsPi production increased similarlyafter 10 minutes due to inositol phosphate phosphatase activity.The status of intracellular Ins(1 ,4,5)P3 receptors within the cell could also bea factor in the decreased [Ca2]i release in FASC cells. There is some evidence ofheterogeneity among these receptors in the adrenal cortex (Enyedi and Williams, 1988;Challiss et al., 1990). Also, signal pathway cross-talk can modify the function of thesereceptors. For example, cAMP-mediated activation of protein kinase A induces thephosphorylation of lns(1 ,4,5)P3 receptors in the rat cerebellum, thereby decreasingtheir ability to release intracellular calcium by up to 90% (Supattapone et al., 1988).in yitrQ, chronic ACTH treatment of rat glomerulosa cells results in the appearance of afasciculata-like phenotype (Hornsby et al., 1.974; Roskelley and Auersperg, 1990), andACTH plays a major role in the emergence of the fasciculata phenotype inyJ2(Nussdorfer, 1986). As the major second messenger of ACTH action is cAMP, it is- 150 -possible that the ability of the Ins(1 ,4,5)P3 receptor to induce calcium fluxes acrossintracellular membranes is decreased in rat fasciculata cells. Therefore, heterogeneityamong lns(1 ,4,5)P3 receptors or alterations to receptor phosphorylation states couldcontribute to the differences observed in Ang Il-mediated [Ca2]i responses in GLOMand FASC cells.Locally acting paracrine or autocrine factors also modulate steroidogenicfunction. For example, TGF-13, which is expressed in the bovine adrenal cortex, stronglyinhibits Ang II mediated steroidogenesis in bovine adrenocortical cells in yJljQ. (Hottaand Baird, 1986; Feige et al., 1991). Thus, a zone specific expression of TGF-13 in therat could help to facilitate the differential Ang II responses of FASC and GLOM cells. Toour knowledge however, levels of TGF-13 expression in the different zones of the ratadrenal cortex have as yet not been determined.5. Expression of the ras Oncopene in GLOM CellsAfter infection with KiMSV, primary GLOM cultures produced morphologicallyaltered cells that could be selectively passaged into secondary culture. These cells(designated KGLOM) contained viral p21 ras and rapidly became oncogenicallytransformed. Therefore, expression of the v-Ki ras oncogene and its effect on signaltransduction is compatible with both steroidogenic differentiation and oncogenictransformation in pre-senescent rat zona glomerulosa cells.Both clinical and experimental evidence strongly suggest thatcarcinogenesis is a multistep process (Bishop, 1987). Initial assays utilizing DNAisolated from tumour tissues identified activated ras genes on the basis of their ability totransform immortal fibroblast lines (Barbacid, 1987). However, it has becomeapparent that ras alone is inefficient in fully transforming numerous pre-senescent celltypes in i1r. (Weinberg, 1985). Therefore, it has been hypothesized that along with- 151 -the expression of activated ras, further genetic or epigenetic changes are required tofully transform pre-senescent cells. In certain experimental situations activation ofsecondary co-operating oncogenes, or a loss of a tumour suppressor can act as theadditional genetic component (Land et al., 1983; Hunter, 1991). In others, tumourpromoters or escape from the control of surrounding normal cells can act as theadditional epigenetic factors (Dotto et al., 1988; Missero et al., 1990). In presenescent rat adrenocortical fibroblasts derived from tissue explants, v-Ki rasexpression eventually produces cells that are tumorigenic (Auersperg et al., 1977,1981; Auersperg, 1978). However, in mass cultures complete oncogenictransformation occurs very slowly over multiple passages and tends to be completed atthe time when surrounding untransformed cells senesce (Auersperg and Calderwood,1984; Auersperg et aI., 1986). This process takes place independently of the levels ofras expression and it can be accelerated if the ras and src oncogenes are co-expressed(MacAuley et al., 1986; MacAuley and Pawson, 1988). These results suggest thatexpression of the ras oncogene alone was not sufficient to fully transform adrenocorticalfibroblasts in mass culture. Thus, at first glance it was surprising that GLOM cells wereso rapidly transformed by v-Ki ras in this study. However, it should be noted that weselected morphologically altered GLOM cells after K1MSV infection in primary culture.In this way we isolated those cells that were most susceptible to ras-mediatedmorphological changes and we removed them from the influence of surrounding normalcells. Previously, simple genetic selection of pre-senescent rat embryo fibroblasts thatexpress activated ras p21 has been shown to abrogate the need for a second co-operatingoncogene to induce transformation (Land et al., 1 986). Therefore, it was notunreasonable to find that a more phenotypically rigorous method of selecting v-rasexpressing cells (ie. morphologic alteration) resulted in the rapid transformation ofpre-senescent GLOM cells.- 152 -Interestingly, rapidly transforming KGLOM celisdid not express a 27 kD protein(p27) that was immunoprecipitated from normal GLOM cells using the anti-rasantibody. p27 is also absent from slowly transforming adrenocortical fibroblasts, butnot from their normal counterparts. Tryptic mapping of p27 suggests that it is a rashomologue (MacAuley, 1987). Thus p27 may be an example of a ras related protein thatacts in the manner of a tumour suppressor, as it is no longer expressed in transformedcells.KGLOM cells formed tumours in immunosuppressed rats. Palpable (>1 cm3)tumours formed rapidly, in less than two weeks, regardless the site of injection. Ingeneral, the tumours were sarcomatous, although there were occasional carcinomatousareas and rare carcinomas. The latter were composed of undifferentiated epithelialclumps and cords and they were found where the tumours had formed within, or hadinvaded into, the kidney or the adrenal gland. The ability of KGLOM cells to form bothsarcomas and carcinomas is consistant with the dual parenchymal/stromal nature ofthese cells as discussed above. This same duality has been observed in ras transformedexplant-derived fibroblasts (Auersperg et al., 1981).Due to its biochemical function, sub-plasmalemmal location, and transformingpotential, it has long been assumed that ras p21 is involved in the transduction ofproliferative signals (Macara, 1991). However, jnyjy, cellular ras p21 is expressedin a number of non-proliferating, differentiating tissues (Chesa et al., 1987; Furth etal., 1987). While the oncogenic transformation elicited by the expression of activatedras p21 is usually associated with increased proliferation and a loss of differentiation,there are cell types in which the opposite occurs. For example neuronal differentiationis induced in adrenomedullary PC-12 tumour cells (Noda et al., 1985; Kremer et al.,1991) and adipocyte differentiation occurs in 3T3-L1 pre-adipocytes (Benito et al.,1991). Neuronal cell differentiation in PC-12 cells is ras specific as it can be blocked- 153 -by lovastatin, an inhibitor of ras function (Mendola and Barker, 1990). In PC-12 cellsp21 ras appears to impinge upon a pathway that stimulates protein kinase C, while in3T3-L1 cells it may transduce signals normally initiated activation of the insulinreceptor which is a tyrosine kinase. Therefore, the effect of activated p21 rasexpression on signal transduction, proliferation or differentiation appears to be celltype specific.v-Ki ras mediated transformation of GLOM cells was associated with both anincreased proliferative potential and the maintenance of steroidogenic differentiation.This suggested that v-Ki ras expression was associated with greater degree ofproliferative and steroidogenic autonomy. Such autonomy is a hallmark of malignantcells. KGLOM cells produced corticosterone constitufively and responded to ACTH andcAMP treatment with increased steroidogenesis. However, this steroidogenic increasewas the greatest after after a chronic (ie. >24 hr) trophic hormone treatment. Acute (ie.8 hr) trophic hormone treatment resulted in only a small increase in corticosteroneproduction.Acute trophic hormone treatment of adrenocortical cells activates signaltransduction pathways leading to substrate delivery to steroidogenic enzyme systems.For example, acute ACTH treatment induces steroidogenesis in a cAMP dependent mannerby increasing the association of cholesterol with P-45Oscc (Jefcoate et al., 1987). Incontrast, chronic ACTH treatment acts by inducing steroidogenic enzyme gene expression(Simpson et al., 1990). Thus, it is probable that steroidogenic enzyme expression isrequired prior to the maximal ACTH or cAMP-mediated steroidogenic increases inKGLOM cells. This also occurs in ras transformed ovarian granulosa cells which respondto chronic cAMP treatment with increased progesterone production (Amsterdam et al.,1988; Pan et al., 1991). Importantly, after passaging, uninfected GLOM cells were nolonger steroidogenic under any conditions. Therefore, the maintenance of steroidogenesis- 154 -and ACTH/cAMP responsivenss observed in KGLOM cells was not due to the carry over ofnormal GLOM cells during the selection process.Lovastatin, which acts as a pharmacological inhibitor of p21 ras function bypreventing membrane localization of the protein (Schafer et al., 1989; Chen et al.,1991), caused some reversion to a flattened fibroblastic phenotype in KGLOM cells after24 hrs. This occured after an initial rounding of the cells during the first 12 hrs aftertreatment. In addition, lovastatin inhibited steroidogenesis in KGLOM cells to a muchgreater degree than it did in primary cultures of GLOM cells. Therefore, the maintenanceof differentiation appeared to depend on v-ras expression rather than on other aspects ofthe transformed phenotype.Expression of activated ras genes has been associated with both thetransformation and steroidogenic differentiation of other cell types as well. For example,v-Ki ras transformation enhances the limited steroidogenic potential of adrenocorticalfibroblasts (Auersperg et al., 1990) and it is associated with the maintenance ofprogesterone production in ovarian granulosa cells (Pan et al., 1991). Specifically,granulosa cells immortalized by transfection of a complete SV-40 genome are notsteroidogenic but they can be induced to produce progesterone in response to cAMP afterexpression of an activated Ha-ras oncogene (Amsterdam et al., 1988). Activated Ki-rasp21 expression also induces non-steroidogenic rat ovarian surface epithelial cells toproduce progesterone (Pan et al., 1991). Therefore, it appears that in general,steroidogenic differentiation is positively influenced by ras p21 expression. Themechanism responsible for this differentiative effect is unknown, but presumably itinvolves the stimulation of steroidogenic, “template” specific signal transductionpathways which are impinged upon by the ras oncogene. It must be noted thatsteroidogenic differentiation can be associated with the expression of oncogenes otherthan ras. For example, it has recently been reported that early gene regions of the SV- 155 -40 virus alone are able to produce immortalized ovarian granulosa cells that respond tocAMP by producing progesterone (Rao et al., 1991).Steroidogenic cells respond to the presence of numerous trophic hormones. Whilethey always stimulate steroidogenesis, these trophic hormones can have opposing effectson cell proliferation depending on the signal transduction pathway that is activated. Forexample, the activation of adenylate cyclase by ACTH increases steroidogenesis andinhibits ceilular proliferation in adrenocortical cells (Ramachandran and Suyama,1975). Adrenocortical cells also respond to angiotensin II, which activates thephosphatidyl inositol specific PLC pathway (Barrett et al., 1989). The resultingproduction of DAG and lns(1 ,4,5)P3 increases both steroidogenesis and cellproliferation, presumably by stimulation of PKC (Gill et al., 1980). As we have shown,expression of viral p21 ras is also associated with a steroidogenic and proliferativephenotype in zona glomerulosa (GLOM) cells. Thus, p21 ras could act by impinging uponthe PLC mediated pathway in these cells. If this were the case it could explain theinability of K1MSV to transform zona fasciculata (FASC) cells. As discussed above, thereis an interruption in the Ang Il-mediated pathway at a point distal to PLC stimulation butproximal to PKC stimulation in these cells. Therefore, the differentiation specificalteration in the signal transduction template of FASC cells may make them non-responsive to the transforming effect of the ras oncogene. This is further supported bythe finding that stimulation of PKC directly with phorbol esters increases proliferationof fasciculata/reticularis parenchymal cells (McAllister and Hornsby, 1987). It shouldbe noted however, that in our experiments other possible explanations for the nonresponsiveness of FASC cells to viral ras transformation have not been ruled out. Theseinclude possible differences in KiMSV infectivity, and the low rate of proliferation ofFASC cells in primary culture which may suppress pro-virus integration and resultingviral gene expression (Shih et al., 1982). Oncogenic ras p21 could also act to enhancesteroidogenic differentiation by autonomously activating tyrosine kinase-mediated- 156 -pathways. For example, IGF-1, whose receptor is a transmembrane tyrosine kinase,enhances the cAMP-induced expression of steroid hydroxylase genes (Naseerudin andHornsby 1990).6. Parenchymal Cell Proliferation and Differentiation in MIX CulturesHomogeneous FASC and GLOM cells did not retain their steroidogenicdifferentiation in long term primary culture or after passaging. This decline has beennoted previously in rat fasciculata cells obtained by de-capsulation (Ryback andRamachandran, 1981b). Obviously, the rather primitive culture conditions used inthese studies may have played a part in this loss of differentiation. For example,culturing adrenocortical cells on extracellular matrices or in atmospheres with a low02 tension enhances long term differentiation by maintaining the expression andpreventing the oxidative degradation of steroidogenic enzymes (Gill et al., 1980;Hornsby, 1980; Georgiu et at., 1987). However, in the present study, an adrenocorticalpopulation which consists of a mixture of parenchymal, endothelial and stromal cells(designated MIX) has been identified that both maintains and produces large numbers ofdifferentiated steroidogenic cells in long term primary culture. This differentiationoccured in the absence of trophic hormone treatment, and it was associated with a highrate of proliferation and a cellular multilayering that did not occur in the homogeneousFASC and GLOM cultures.During embryogeriesis, the adrenal cortex arises from a condensation ofmesodermal cells that proliferate rapidly and initiate steroidogenic differentiation priorto the production of ACTH by the pituitary (Idleman, 1970; Jost, 1975; Nussdorfer,1986). With the appearance of ACTH, adrenocortical growth and differentiation aregreatly accelerated and brought to completion. In the adult, the adrenal cortex retains aregenerative potential as evidenced by the the re-appearance of a complete gland after- 157 -enucleation removes all but a sub-capsular layer of parenchymal cells (lngle andHiggins, 1938; Taki and Nickerson 1985). Therefore, the adult adrenal cortex is alsocapable of both proliferation and steroidogenic differentiation. It appears that MIXcultures contain the cells and the conditions that allow for similar, comcomitantproliferation and differentiation.When adrenocortical parenchymal cells are treated with ACTH or cAMP,steroidogenesis is increased and cells often morphologically round up after retractingfrom the underlying substratum (Hall, 1984; Hornsby et al., 1989). Long term MIXcultures produced increasing numbers of rounded cells that emerged from an underlyingbed of fibroblastic cells. This process occurred in the absence of ACTH or cAMPtreatment. The rounded MIX cells were positive for markers of steroidogenicdifferentiation including: cytoplasmic lipid; t5,313-HSD activity; P-45Osccexpression; and the production of fluorogenic steroid and corticosterone. Therefore, inMIX cultures parenchymal cell differentiation occurred independently of trophichormone treatment.In multilayered MIX cultures, the underlying fibroblastic cells may act as afeeder layer that is conducive to the differentiation of an associated population of preexisting parenchymal stem cells. In a number of jnyJiio systems, feeder layers act tosupport proliferation rather than differentiation (Watt, 1991). However, the formationof MIX multilayers was associated with sustained proliferation after confluence. This ledto high saturation densities and extreme cell crowding that, in turn, may have beenconducive to the induction of proliferation and differentiation. The heterogeneouscellular multilayering in MIX cultures (ie. fibroblastic cells on one side of themultilayer and spherical, parenchymal cells on the other) may represent an iny.Ltrnrecapitulation of the normal in yJy.Q adrenocortical histoarchitecture whereproliferating parenchymal cells associate with subcapsular fibroblast-like cells priorto differentiating and moving inwards within the cortex. In support of this suggestion, a- 158 -similar cellular multilayering occurred after KiMSV (ras) infection of GLOM cultures.This also led to the production of proliferating, steroidogenic cells.As discussed above, it is likely that parenchymal cells arise from multipotentsub-capsular fibroblasts that differentiate into steroidogenic cells as they move into thezonae of the adrenal cortex (lannacone and Weinberg, 1987). Thus, it is reasonable tospeculate that the fibroblastic cells themselves could have given rise to the steroidogeniccells that emerged from the MIX culture multilayers. However, the experimentalevidence does not exclude the possibility of another alternative, that the flattened cellswhich undergo differentiation in MIX cultures represent parenchymal cells that haveinitially modulated from an epithelial to a fibroblastic morphology, as was seen innormal GLOM cultures.The production of soluble factors which act in a paracrine fashion may also play arole in the differentiative process that takes place in MIX cultures. For example, longterm bone marrow cultures, which contain a mixture of adherent stromal cells,hematopoietic stem cells and detaching colonies of differentiating hematopoietic cells,have been utilized to identify a number of the critical cytokines that act in a paracrinefashion during stem cell renewal and differentiation (Dexter et al., 1979; Humphries etal., 1981; Metcalf, 1989; Eaves et al., 1991). As MIX cultures also producedifferentiated cells that can be removed from an underlying stroma, they could proveuseful in identifying those soluble factors that are important in the renewal anddifferentiation of cells derived from steroidogenic tissues. One factor that could beinvolved is IGF-1 which is produced in the adrenal cortex kuthi2. (Han et al., 1987) andenhances both the proliferation (Horiba et al., 1987; van Dijk et al., 1988) andsteroidogenesis (Nasserrudin and Hornsby, 1990) of adrenocortical cells in yJfl.Extracellular matrix (ECM) production in MIX cultures may also effectsteroidogenic differentiation. It has been shown that fibronectin and Matrigel, abasement membrane derived ECM, will enhance steroidogenic enzyme expression in- 159 -bovine adrenocortical cells (Flasch et al., 1990; Hornsby, 1991). In addition,adrenocortical fibroblasts derived from explant cultures have been found to producelarge amounts of an heterogeneous ECM, the production of which varies depending on thedifferentiation state of the cells (Turley, 1980; Auersperg et al., 1990). Therefore, thefibroblastic cells in MIX cultures may produce an ECM that is conducive to steroidogenicdifferentiation of a parenchymal population. This could be tested by treating MIXcultures with ECM production inhibitors such as cis-hydroxy-L-proline andsubsequently determining the number of differentiating A5,313-HSD positive cells.Treatment of bone marrow cultures with this inhibitor prevents the stromal cell-dependent growth and differentiation of hematopoietic stem cells (Zuckerman et al.,1985).After selective passaging, rounded MIX cells re-formed multilayers whereinrefractile, lipid containing cells adhered to large, flattened fibroblasts. Although onlythe refractile cells responded morphologically to cAMP treatment, both cell typesexpressed P-45Oscc. At this point it cannot be stated with certainty that the appearanceof differentiated steroidogenic cells in MIX cultures was not due to the presence of anautonomously differentiating cell population that was not present in FASC or GLOMcultures. However, the progressive production of these steroidogenic cells in longtermprimary culture, and the intimate relationship between refractile and fibroblastic cellsin secondary culture suggests that heterologous cell-cell interactions are involved.7. ConclusionsUtilizing discontinuous density gradients we have separated adrenocorticalparenchymal cells into homogeneous populations which have the characteristics of zonafasciculata (FASC) and zona glomerulosa (GLOM) cells respectively. This methodprovides a quick and easy alternative to decapsulation for separating adrenocortical- 160 -parenchymal cell types, and results in preparations of superior purity with minimalstromal or endothelial cell contamination.Both FASC and GLOM cells were steroidogenic in primary culture. However, theyexhibited very different morphologies. FASC cells formed epithelial monolayers whileGLOM cells modulated to a fibroblastic form and expressed vimentin. This suggests thatGLOM cells may have both parenchymal and stromal characteristics, while FASC cellshave a more restricted parenchymal phenotype.In the course of differentiation, cells arising from a common precursor oftenacquire similar but subtly different phenotypes. The cells of the different adrenocorticalzones have a common origin and appear to differentiate along a spatial and developmentalcontinuum. It appears that a mesodermally derived sub-capsular stem cell gives rise tozona glomerulosa cells which in turn give rise to zona fasciculata cells (Wright andVoncina, 1977; Ueberberg et al., 1982; Farcnik and Auersperg, 1984; Taki andNickerson, 1985; Zajicek et al., 1986; lannaccone and Weinberg, 1987). The dualstromal and parenchymal charateristics exhibited by GLOM cells in primary culture areconsistant with them being in an intermediate developmental position, located betweenthe mesodermal stem cells and the parenchymal zona fasciculata (FASC) cells.In the rat adrenal cortex, a specific phenotypic consequence of the zonalinterconversion from a glomerulosa to a fasciculata cell type is the loss of steroidogenicresponsiveness to the trophic hormone Ang II (Haning et al., 1970; Tait et al., 1974;Douglas et al., 1978; Braley et al., 1986; Whitley et al., 1987). Our findings suggestthat this lost responsiveness arises, at least in part, due to a modification within the AngIl-mediated signal transduction pathway. That this modification takes place precisely atthe level of increased intracellular calcium indicates that developmental decisions can beenacted via alterations within signal transduction pathways distal to initial receptormediated events and proximal to downstream effector events.- 161The phenotypic effects of activated p21 ras expression are often associated withalterations to signal transduction pathways (Macara, 1991). It appears that thesealterations are cell-type, and thus transduction-template, specific (Auersperg andRoskelley, 1991). In GLOM cells, activated ras expression was associated with bothoncogenic transformation and the enhanced maintenance of steroidogenic differentiation.Therefore, ras may lead to the autonomous activation of PLC, or tyrosine kinasemediated, signal transduction pathways in GLOM cells as these pathways are normallyassociated with differentiation and proliferation in steroidogenic cells (Gill et al., 1980;Barrett et al; 1989; van Dijk et al., 1988, Nasseerudin and Hornsby 1990).While FASC and GLOM cells de-differentiated in long term primary culture, amixed parenchymal/stromal (MIX) population did not. Instead, MIX cultures producedlarge numbers of differentiated cells that could be selectively passaged. Thesedifferentiated cells emerged from highly proliferative cellular multilayers that weremorphologically similar to ACTH/cAMP treated cells or to those cells that appeared inKiMSV (ras) infected GLOM cultures. Thus, cellular multilayers may produce amicroenvironment that is conducive to the steroidogeriic differentiation of adrenocorticalstem-like cells. The specific characteristics associated with this microenvironment thatare crucial for its differentiative effect are as yet unknown. Some possibilites include:the establishment of heterologous cell-cell interactions; production of extracellularmatrices; and production of soluble growth factors that act in a paracrine fashion.Further examination and manipulation of this novel adrenocortical culture systemshould provide clues as to the specific factors which are crucial to the modulation of thesteroidogenic phenotype in a differentiative setting.- 162 -V. 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In a discontinuous, pre-made gradient thecells settle at interfaces that have a density nearest their own. Discontinuous gradientsare advantageous because they can be custom made and they are highly reproducible. Thegradient used here consited of 8 steps with a total of7 density interfaces between thesteps. Each step consisted of a particular percentage of SIP diluted with 1X DME/F12.Step # %SIP SIP (mfl ix DME/F12 (ml)1 15% 1.5 8.52 25% 2.5 7.53 30% 3.0 7.04 35% 3.5 6.55 40% 4.0 6.06 45% 4.5 5.57 50% 5.0 5.08 65% 6.5 3.5Gradient PreparationThe gradients were prepared in clear plastic, round bottom centrifuge tubes(15m1; 13 x 100 mm; Nalgene). Before using, each % SIP step was thoroughly mixedby pipetting. One ml of each step was then layered carefully into the tube from heaviest178 -to lightest (ie. step #8 first, step #1 last). This was done with an automatic pipettor toreduce turbulence that can occur when using manual bulbs. Each step was slowlyreleased onto the step below it ( ie. #7 on top of #8) at a rate of approximately 30sec/mi. As this was done, the pipette tip was kept just above the meniscus and fluid wasreleased along the wall of the tube to minimize turbulence or mixing with the step below.Upon completion, the interfaces between % SIP steps could be seen as sharp refractory,horizontal planes within the gradient. The gradients were stable for several hours.Usually, they were prepared the morning of the experiments, prior to cell dissociation.They were not constructed the night before, as the interfaces became slightly fuzzywhich reduced the efficiency of the cell separation.- 179 -Appendix B: lns(1.4.5)PlDeterminationThe mass of lns(1 ,4,5)P3 in Ang II or calcium ionophore treated cells was determinedusing a commercially available, competitive, isomer-specific radioligand binding assay(Amersham). The assay was carried out essentially as to manufacturers specificationswith slight modifications (see Palmer et al., 1989).lns(1 .4.5)Pp ExtractionCells in suspension or in culture were incubated with Ang II or calcium ionophore ABr231 87 in 125 tl of HBSS. At timed inteivals the incubations were terminated by theaddition of 25 il of cold 10% (v:v) percioric acid. Inositol phosphates were thenextracted into the medium during a 20 mm period on ice. The acidified samples werethen centrifuged 12,000 Xg for 2 mm (4°C) to remove cellular debris. Supernatantswere neutralized to pH 9.0 by the addition of 12.5 tl of 1.5M KOH with 60mM HEPES.100 j.tl aliquots of the neutralized samples were then used for assay.Assay ProcedureReagents-Assay buffer: 0.1 M Trix, 4mM EDTA, 4mg/mi BSA, pH 9.03H lns(1 ,4,5)P3 tracer: Enough tracer was used to give 4-6000 cpm per lOOjil inwater.-Ligand binding protein: Isolated from bovine adrenocortical microsomes a slurry wasdiluted in 10Oil assay buffer to give 25-40% binding of thelabelled tracer.-lns(1 ,4,5)P3 standards: From a stock solution of 4iiM, standards were diluted to give0.19, 0.38, 0.76, 1.5, 3.1, 12.5 and 25 pmol per 100111 iflwater. For non-specific binding, 400pmol of standard wasused to fully compete out the tracer.- 180 -ProtocolReagents were added to 10 X 55 mm polypropylene tubes for each of the followingcategories as summarized below (all volumes in ill):Total Counts Non-Specific Blank Standards Samples(TC) Binding (NSB) (Bo) (lñP’). (Extract)Buffer 100 100 100 100 100ddH2O 200---Extracted medium-- 1 00Standard- 1 00 1 00-Sample--- 100Tracer 100 100 100 100 100Binding Protein- 1 00 1 00 1 00 1 00The reagents were added in the order listed from top to bottom in the above summary. Alloperations were carried out in a walk in fridge (4°C). Tubes were thoroughly vortexedand left to stand for 15 minutes on ice. To separate free and bound tracer the tubes werecentfrifuged at 2000 Xg for 10 mm followed by decanting of the supernatant. The bindingprotein pellet, with bound tracer, was then resuspended in 200 jil dH2O by thoroughvortexing. The suspension was then placed into scintillation vials with 3 ml scintillationfluid (ACS, Amersham) and counted for 5 mm.Calculation of Results1) Percent of tracer bound in the absence of standard/sample (% Bo) was calculatedusing the following equation (25-40% was considered to be acceptable):%Bo = (Bo cpm-NSB cpm) X100(TC cpm)- 181 -C)a-C)0E00)02) Normalized percent bound (%B/Bo) for each standard/sample was calculated usingthe following equation:%B/Bo = (Standard or Sample cpm-NSB cpm) X 100(Bo cpm-NSB cpm)A standard curve was generated by plotting %B/Bo as a function of the logo lnsP3 pertube. Amounts of lnsP3 per sample were then extrapolated from this curve. Mediumblanks were then subtracted from all samples (these were negligable).What follows is a representative standard curve from one experiment.20log pmole lnsP3 = 1.65 - 0.0243 %B/Bo R = 1.00(each standard in duplicate)0 20 4Ô 60 80 100%B/Bo- 182 -Appendix C: p21 ras lmmunoprecipitationGLOM or KGLOM cells in passage 5 were grown to subconfluence in 60mm dishesand then labelled with 35S methionine prior to lysis and immunoprecipitation.Protocol1)CelI Labelling-Rinse 2 times with HBSS.-Pre-incubate 1 hour with methionine free DMEM medium (Sigma) supplemented with5% dialyzed FBS.-Incubate in 2m1 of the methionine free medium with 200 j.tCi 35S methionine (35STrans label, ICN) for 24 hours.-Chase for 1 hour with unlabelled methionine containing medium and 10% FBS.2) Cell Lysis-Rinse cells with cold HBSS and stand on ice.-Add 500 p.1 radiolabelled immunoprecipitation assay (RIPA) buffer with proteaseinhibitors. -Swirl RIPA buffer over surface of cells evenly for 2 minutes beingcareful to keep on ice to minimize proteolysis.-Scrape lysed cells from dish and wash and scrape again with 500 jil RIPA buffer.-Clear lysates by centrifuging 25,000 Xg for 90 mm at 4°C.-Calculate volume of cleared lysate required to yield 1 X 108 TCA precipitable c.p.m. byscintillation counting.3) Immunoprecipitation-On ice, dilute 108 c.p.m. aliquots of cell lysate to 250 p.1 with RIPA buffer.-Add 70 p.1 of protein A sepharose (PAS) linked to 2nd antibody with (experimental) orwithout. (control) 1 St antibody.-Incubate and rotate (—20 r.p.m.) for 4 hours at 4°C.-Wash immunoprecipitates by centrifuging 16,000 Xg 5 mm. and resuspending in 500jii fresh RIPA buffer 4X.-Release PAS bound proteins by boiling in 50 p.1 sample buffer for 5 mm.- 183 --Separate immunoprecipitated proteins by SDS-PAGE on 12.5% acrylamide gels.-Visualize immunoprecipitated proteins by autoradiography by placing the dried gelagainst X-ray film for 3-6 days (XAR-5 film, Kodak).Reagents1) Radiolabelled Immunoprecipitation Assay (RIPA) BufferCompound [Stock] Volume [Final]Sodium phosphate buffer 1 M 1.0 ml 10 mMNa2HPO4 adusted to pH7.2NaCI 5M 3.1 ml 154mMTriton X-100 (Sigma) 100% 1.0 ml 1% v:vSodium deoxycholate 5% w:v 10 ml 0.5%w:v(Fischer)Sodium dodecylsulfate 10% w:v 1 .0 ml 0.1 %w :v(Sigma)ddH2O N/A 84 ml N/ATOTAL 100 ml- 184 -2) Protease InhibitorsJust before lysis the following protease inhibitors are added to 4.85 ml RIPA bufferInhibitor [Stock] Volume [Final]Aprotinin (Sigma) 10 mg/mI 50 jil 100.tg/mlLeupeptin (Sigma) 100 mM 50 tl 1 mMPhenylmeghylsulfonyl 100 mM 50 jil 1 mMflouride (PMSF, Sigma)3) Protein A Sepharose (PAS)-Suspend 100 mg of PAS in 1 ml of 0.1 M glacin (pH 2.5) and rotate 20 mm at 4°C.-Wash with by centrifuging 10,000 Xg and resuspending in PBS (pH 7.2) 5 times;resuspend final time in 250 jtl PBS with 1 mg/mI BSA.- Add 30 ml of 2nd antibody (Rabbit anti rat lgG, Cappel, manufacturers optimaldilution); rotate 4°C overnight.- Wash 2X with 280 jil PBS containing 1 mM PMSF.-Add 8 jil anti ras 1st antibody (monoclonal Y13-259, 1.2 mg/mI); rotate 4°C for 2hours.-Wash 2 times with 280 .tl PBS/PMSF.-Add 70 j.tl to each aliquot of 250 tl cleared lysate.- 185 -41 Sample BufferCompound [Stock] Volume [Final]glycerol (Sigma) 1 00% 0.8 ml 8%w :v2-mercaptoethanol 1 00% 0.5 ml 5% v:v(Sigma)Sodium dodecylsulfate 1 0% 2.3 ml 2.3%Tris (Sigma) 1.0 M 625 LtI .0625MddH2O N/A 5.775 ml N/ATOTAL 10.0 ml
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Anatomy, Cell Biology and Physiology
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Rat adrenocortical cell differentiation : effects of signal transduction alterations, ras oncogene expression, and parenchymal/stromal interactions
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