Role of transport systems in cortisol release from human adrenal cells Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Abdul Rahman Asif aus Gujrat, Pakistan Göttingen 2004 D7 Referent: Prof. Dr. R. Hardeland Korreferent: Prof. Dr. D. Gradmann Tag der mündlichen Prüfung: To My parents & in loving memory of my grandma! She could not wait! CONTENTS I ABSTRACT ................................................................................................... IV LIST OF ABBREVIATIONS .......................................................................... VI 1 INTRODUCTION........................................................................................1 1.1 THE ADRENAL GLAND ANATOMY ...............................................................................................1 1.2 ADRENAL GLAND HORMONES....................................................................................................2 1.2.1 Biosynthesis of the steroid hormones...........................................................................................2 1.2.2 Regulation of adrenal glands........................................................................................................6 1.2.3 Actions of adrenal steroids ...........................................................................................................7 1.3 HUMAN ADRENOCORTICAL CELLS ............................................................................................8 1.4 RELEASE OF STERIODS THROUGH THE PLASMA MEMBRANE ............................................10 1.4.1 Organic anion transporter (OAT) family......................................................................................11 1.4.2 Organic anion transporter polypeptide (OATP) family................................................................13 1.4.3 P-glycoprotein (Pgp) family ........................................................................................................14 1.5 THE AIMS OF THE WORK ...........................................................................................................15 2 MATERIALS ............................................................................................16 2.1 CHEMICALS .................................................................................................................................16 2.2 RADIOCHEMICALS......................................................................................................................16 2.3 OLIGONUCLEOTIDES .................................................................................................................16 2.4 CELL LINES..................................................................................................................................19 2.5 CELL CULTURE MEDIA AND SUPPLEMENTS...........................................................................19 2.6 MISCELLANEOUS........................................................................................................................19 2.7 BUFFERS .....................................................................................................................................19 2.8 SCIENTIFIC SOFTWARES...........................................................................................................20 2.9 EQUIPMENTS ..............................................................................................................................21 3 METHODS ...............................................................................................23 3.1 AMPLIFICATION AND QUANTIFICATION OF GENE OF INTEREST BY PCR ...........................23 3.1.1 Isolation of total RNA from cultured cells....................................................................................23 3.1.2 Isolation of total RNA from human adrenal tissues.....................................................................23 3.1.3 Reverse transcription of mRNA..................................................................................................24 3.1.4 Polymerase chain reaction (PCR) ..............................................................................................24 3.1.5 Agarose gel electrophoresis.......................................................................................................24 3.1.6 Purification of PCR product from agarose gel ............................................................................25 3.1.7 Sequencing of the PCR product (non-radioactive dye terminated sequencing of DNA).............25 3.2 CULTIVATION OF HUMAN ADRENOCORTICAL CARCINOMA CELLS (NCI-H295R) ...............26 3.2.1 Culture media.............................................................................................................................26 3.2.2 Dissociation of cells from culture flasks......................................................................................26 3.2.3 Cryopreservation........................................................................................................................26 3.2.4 Thawing of cryopreserved cells..................................................................................................27 3.3 CORTISOL RELEASE FROM NCI-H295R CELLS AND ITS INHIBITION....................................27 3.3.1 Determination of cortisol by radioimmunoassay (RIA)................................................................27 3.4 UPTAKE OF RADIOACTIVE SUBSTANCES INTO THE NCI-H295R CELLS ..............................28 3.5 CULTIVATION OF HEK-293 CELLS.............................................................................................28 3.5.1 Culture media.............................................................................................................................29 3.5.2 Uptake of radiolabeled substances into HEK-293 cells ..............................................................29 3.6 CULTIVATION AND TRANSIENT TRANSFECTION IN COS-7 CELLS .......................................29 3.6.1 Culture media.............................................................................................................................29 3.6.2 Transfection of COS-7 cells by electroporation ..........................................................................30 3.7 EXPRESSION OF TRANSPORTER PROTEIN IN Xenopus laevis OOCYTES............................30 3.7.1 cRNA sycthesis ..........................................................................................................................30 3.7.2 cRNA injection into Xenopus laevis oocytes ..............................................................................31 3.7.3 Uptake of radiolabeled substance by transporter expressing Xenopus laevis oocytes ..............31 CONTENTS II 3.8 IMMUNOSTAINING OF NCI-H295R CELLS AND ADRENAL TISSUES......................................32 3.8.1 Preparation of cells for immunostaining .....................................................................................32 3.8.2 Immunostaining of cells..............................................................................................................32 3.8.3 Immunostaining of paraffin embedded adrenal tissue sections..................................................33 3.9 PREPARATION OF CYTOSOL AND MEMBRANE FRACTIONS OF NCI-H295R CELLS...........34 3.10 WESTERN BLOT ANALYSIS .....................................................................................................34 3.11 TWO-DIMENSIONAL GEL ELECTROPHORESIS OF PROTEINS ............................................35 3.11.1 First dimension electrophoresis................................................................................................35 3.11.2 Sample preparation for first dimension.....................................................................................35 3.11.3 Isoelectric focusing of proteins .................................................................................................36 3.11.4 Equilibration of proteins for SDS-PAGE ...................................................................................37 3.11.5 Second dimension....................................................................................................................37 3.11.6 Coomassie Brilliant Blue (CBB) staining ..................................................................................37 3.11.7 Silver staining...........................................................................................................................37 3.12 PROTEIN IDENTIFICATION BY MALDI-TOF MS.......................................................................38 3.12.1 In-gel digestion and preparation of proteins and proteolytic fragments for MALDI-TOF...........40 3.12.2 Matrix Solution Preparation ......................................................................................................40 3.12.3 Sample-Matrix Crystallization...................................................................................................41 3.12.4 MALDI-TOF Mass Spectrometry ..............................................................................................41 3.13 SELDI-TOF MASS SPECTROMETRY........................................................................................41 3.13.1 H-50 ProteinChip® preparation and analysis by SELDI-TOF...................................................42 4 RESULTS.................................................................................................44