DESCRIPCIÓN DE LA METODOLOGÍA DE FORMACIÓN EN EL PROGRAMA DE INGENIERÍA

2.4.1: PREPARATION OF MEMBRANES FOR BINDING STUDIES:

The human lung fibroblast cell line CCD19Lu was cultured in plOO (55cm^) plates until confluent. Cells were washed in the dish twice with 5ml of PBS solution containing 1 protease inhibitor tablet (Boehringer Mannheim) per 100ml, then incubated with 3-4ml of trypsin-EDTA (porcine, 0.5g/l trypsin, 0.2g/l EDTA in HBSS w/o

calcium and magnesium, Gibco) at 37°C for at least 5 mins until the cells had detached from the culture plate. This was checked using a microscope.

The cells were spun in the trypsin at 1500rpm at 4°C for 5min. The supernatant was aspirated, and the pellet resuspended in 5ml cold PBS/PI. The cells were spun again, and this procedure was repeated.

The pellet was then resuspended in 200|il of cold PBS/PI. 2ml of cold water was added, and the cells were left to swell on ice for lOmins. The cells were initially sucked up and down a syringe, on ice, 20 times using a 2 5 0 needle, then transferred to a Bounce homogeniser and further lysed.

The lysate was transferred to an SS34 rotor, and spun in the Sorvall for 60mins at 4°C, 17000 rpm.

The pellet was resuspended in 1ml of protease inhibited PBS and homogenised on ice using a Bounce homogeniser. Any membrane to be stored was immediately transferred to -80°C.

2.4 2lSPECIFIC BINDING OF '^^I-ET-I TO LUNG FIBROBLAST MEMBRANES:

An initial experiment was conducted to measure specific binding of ET-1. 50p,g of membrane were incubated with 50pM ^^^I-ET-1, alone (NC), or with competition from lOOnM of unlabelled ET-1(C). Samples were prepared as below, and run in triplicate. A total radioactivity control was also included, which was added directly to the

scintillation vials and measured the total amount of radioactivity added to each tube at the start.

Group Membrane (pg) '“ l-E T -l (M) ET-1 (M)

NC 50 50 X 10'^ -

C 50 50 X 10'^ 100 X 10'^

The tubes were incubated at RT for 2 hours. They were then spun for lOmins at 14,000 rpm. The supernatant was carefully aspirated, and 100p,l of buffer was added taking care not to disturb the pellet. This was also aspirated. The pellets were then resuspended in PBS/PI containing 1% SDS. They were transferred to scintillation vials containing 5ml of scintillation fluid (Packard (Meriden, CT, USA)), and radioactivity was measured in the counter (Packard) in dpm

Results are displayed in fig. 3.2.

2.4.3: SPE C inC BINDING OF ^H-BO-123 TO DIFFERENT CONCENTRATIONS OF MEMBRANE PROTEIN:

This experiment was a preliminary study to investigate what concentration of membrane protein was necessary to obtain specific binding using tritiated BQ-123, an ETaR antagonist, and if that was reasonable given the expected yields from membrane preparation. Results are displayed in fig. 3.3.

Stock solutions of 50mM BQ-123 and 50nM ^H-BQ-123 were prepared. The membrane stock was bOSpg/ml. Samples were prepared as follows in siliconised tubes and made up to 200|xl with PBS/PI:

Group Membrane (pg) ^H-BQ-123(M) BQ-123 (M) 1 NC 50 2.5 X 10'" - 1 C 50 2.5 X 10'^ 2.5 X 10 " 2N C 30 2.5 X 10'^ - 2 C 30 2.5 X 10^ 2.5 X 10" 3N C - 2.5 X 10 ^ - 3 C - 2.5 X 10 '' 2.5 X 10 " 4 - 2.5 X 10 '' -

Each group contained 3 tubes. NC - no competitor, C - competed.

Group 1 had 90p,l of membrane, approximately 55p.g, and group 2 had 50pl, approximately 30pg. Group 3 was the no-membrane control, to measure how much radioactivity bound to the tubes. Group 4 was the total radioactivity control, which was added directly to the scintillation vials and measured the total amount of radioactivity added to each tube at the start.

The tubes were vortexed briefly and incubated at RT for 2 hours. They were then spun for lOmins at 14000rpm. The supernatant was carefully aspirated, and 100|Lil of PBS/PI was added taking care not to disturb the pellet. This was also aspirated.

The pellets were resuspended in lOOpl of PBS/PI containing 1% SDS, and transferred to scintillation vials containing 5ml of scintillation fluid. Radioactivity bound was measured in dpm using a scintillation counter.

2.4.4: COMPETITION BINDING STUDY - BINDING OF ^H-BO-123 TO LUNG

FIBROBLAST MEMBRANES IN THE PRESENCE OF INCREASING

CONCENTRATIONS OF UNLABELLED BO-123:

The aim of this experiment was to confirm the specificity of ligand binding. Solutions of 50nM ^H-BQ-123 and protease inhibited PBS (1 tablet per 100ml) were prepared (BQ-123 is a specific ETa receptor antagonist). Solutions of unlabelled BQ-123 were prepared as follows: Stock concentration (M). Final concentration in sample (M) 2 X 10" 1 X 10^ 2 X 1 0 ^ 1 X 1 0 " 2 X 10'^ 1 X 10'^ 2 X 10'^ 1 X 10" 2 X 1 0 " 1 X 10^ 2 X 10"" 1 X 10'" 2 X lO '" 1 X 10"

Samples were made up as shown below in siliconised tubes. All samples were made up to 200pl with PBS/PI.

Group Membrane (pg) ’H-BQ-123 (M) BQ-123 (M) 1 - 2.5 X 10'^ - 2 60 2.5 X 10^ - 3 60 2.5 X 10^ 1 X 10'’ 4 60 2.5 X 10 '' 1 X 10" 5 60 2.5 X lO " 1 X 1 0 ' 6 60 2.5 X lO " 1 X 10" 7 60 2.5 X 10" 1 X 10" 8 60 2.5 X 10 " 1 X 1 0 '" 9 60 2.5 X lO " 1 X 1 0 "

Each group contained 3 tubes. Group 1 was a control to check how much radioactivity bound to the tubes in the absence of membrane. Group 2 was a control for non-specific binding, showing the amount of radioactivity bound with no competitor. Groups 3-9 measured binding in the presence of decreasing concentrations of unlabelled ligand. A total radioactivity control was also included, which was added directly to the scintillation vials and measured the total amount of radioactivity added to each tube at the start.

The tubes were incubated at RT for 2 hours. They were then spun for lOmins at 14,000 rpm. The supernatant was carefully aspirated, and lOOp.1 of buffer was added taking care not to disturb the pellet. This was also aspirated. The pellets were then resuspended in PBS containing 1% SDS, and transferred to the scintillation counter.

2.4.5: SATURATION B E R IN G STUDY - BINDING OF INCREASING

CONCENTRATIONS OF ^H-BO-123 TO LUNG FIBROBLAST MEMBRANES:

The aim of this experiment was to determine the Kj (dissociation constant) of H-BQ- 123 binding in the fibroblast line CCD19Lu, and to determine the abundance of the receptor in these cells by finding Bmax (no. of binding sites). To this end, a saturation binding experiment was performed. Cell membranes were incubated with increasing concentrations of ^H-BQ-123 in the presence or absence of a constant concentration of unlabelled BQ-123 competitor.

Membranes were prepared as described in section 2.4.1. The membrane concentration was determined by BCA protein assay to be 2400pg/ml. And membrane volume for samples calculated accordingly An initial stock solution of 0.5p.M ^H-BQ-123 was prepared in PBS/PI and diluted to make stocks of 0.25 and 0.125|LiM, 62.5, 31.5 and 15.625nM. A competitor solution of lOOpM BQ-123 was also prepared, giving a final concentration in 50|xl total volume of 20pM BQ-123.

Samples were prepared as follows in siliconised tubes and made up to 50pl with protease inhibited PBS. They were vortexed briefly, and incubated at RT for 3 hours.

Group Membrane (Pg) ^H-BQ-123 (nM) BQ-123 (nM) 1 C 60 200 200 IN C 60 200 - 2 C 60 100 200 2N C 60 100 - 3 C 60 50 200 3N C 60 50 - 4 C 60 25 200 4NC 60 25 - 5 C 60 12.5 200 5N C 60 12.5 - 6 C 60 6.25 200 6N C 60 6.25 -

After incubation, the samples were spun down at 13,000 rpm, 4°C for 5 mins. The supernatant was aspirated, and the pellets were washed rapidly with lOOpl pro tease inhibited PBS, taking care not to disturb the pellet. The pellets were resuspended in lOOp.1 1% SDS in PBS and left standing at RT for 15 mins. The resuspension was repeated, the solution was transferred to scintillation vials containing 5ml of scintillation fluid, and the samples were left to count overnight.

2.4.6: SPECIFIC BINDING OF ^^^I-ET-3 TO DIFFERENT CONCENTRATIONS OF MEMBRANE PROTEIN:

Stock solutions of 200nM BQ-123 and InM ^^^I-ET-3 were prepared. Samples were prepared as follows in siliconised tubes and made up to lOOpI with PBS/PI:

G roup M em brane (pg) *“ I-ET-3(M) E T -3(M ) BQ-123 (M)

IN C 60 1.0 X 10'' - 200 X 10" 1 C 60 1.0 X lO " 100 X 10" 200 X 10" 2N C 30 1.0 X lO " - 200 X 10" 2 C 30 1.0 X lO " 100 X 10" 200 X 10" 3N C - 1.0 X 10 * - 200 X 10" 3 C - 1.0 X icr^ 100 X 10" 200 X 10" 4 - 1.0 X lO " - -

Each group contained 3 tubes. NC - no competitor, C - competed.

Group 3 was the no-membrane control, to measure how much radioactivity bound to the tubes. Group 4 was the total radioactivity control, which was added directly to the scintillation vials and measured the total amount of radioactivity added to each tube at the start.

The tubes were vortexed briefly and incubated at RT for 2 hours. They were then spun for lOmins at 14000rpm. The supernatant was carefully aspirated, and lOOpl of PBS/PI was added taking care not to disturb the pellet. This was also aspirated.

The pellets were resuspended in lOOpl of PBS|/PI containing 1% SDS, and transferred to scintillation vials containing 5ml of scintillation fluid (Packard (Meriden, CT, USA)). Radioactivity bound was measured in dpm using a scintillation counter (Packard).

Results are displayed in fig. 3.4.

2.4.7: BINDING OF ^^^I-ET-3 IN THE PRESENCE OF SPECIFIC INHIBITORS OF ETa a n d ETn RECEPTOR:

Samples were prepared as follows:

Group Membrane (pg) ’“ I-ET-3(M) ET-3 (M) BQ-123 (M)

NS 30 60 X 10'-' 6 X 10" 4 X 10"

NS-hA 30 60 X 1 0'" 6 X 10" -

NS-hA-hB 30 60 X ICl'" - -

NS+B 30 60 X 1 0'" - 4 X 10"

NM - 60 X 1 0'" - -

NS is measuring non-specific binding (ie. Binding when both ETa and ETb receptor are blocked. NS+A is measuring non-specific plus ETa receptor binding, NS+B non­ specific plus ETb receptor binding, and NS-hA-hB total binding. NM is the no membrane control.

The tubes were vortexed briefly and incubated at RT for 2 hours. They were then spun for lOmins at 14000rpm. The supernatant was carefully aspirated, and lOOpl of PBS/PI was added taking care not to disturb the pellet. This was also aspirated.

The pellets were resuspended in lOOpf of PBS/PI containing 1% SDS, and transferred to scintillation vials containing 5ml of scintillation fluid (Packard (Meriden, CT, USA)). Radioactivity bound was measured in dpm using a scintillation counter (Packard). A sample to measure total radioactivity was added directly to scintillation fluid.

Results are displayed in fig.3.5.

2.4.8: SATURATION BINDING STUDY - BINDING OF INCREASING CONCENTRATIONS OF '^^I-ET-3 TO LUNG FIBROBLAST MEMBRANES:

The aim of this experiment was to determine the Ka (dissociation constant) of '^^I-ET- 3 binding in the fibroblast line CCD19Lu, and to determine the abundance of the receptor in these cells by finding Bmax (no. of binding sites). ET-3 is a naturally occurring agonist for both ETa and ETb receptors, but has a higher affinity for ETrR. BQ-123 was used to block ETa receptors prior to adding ET-3 in this experiment.

Membranes were prepared as described in section 2.4.1. The membrane concentration was determined by BCA protein assay to be 995pg/ml. An initial stock solution of 30nM ^^^I-ET3 was prepared in PBS/PI and diluted to give final concentrations as

shown below. Competitor solutions were ET-3 (final concentration lOOnM) and BQ- 123 (final concentration 200nM).

Samples were prepared as follows in siliconised tubes and made up to 50pl with protease inhibited PBS. They were vortexed briefly, and incubated at RT for 3 hours.

Group Membrane (Pg) i25l_ET-3 (pM) ET-3 (nM) BQ-123 (nM) 1 C 30 3000 100 200 IN C 30 3000 - 200 2 C 30 1000 100 200 2N C 30 1000 - 200 3 C 30 300 100 200 3N C 30 300 - 200 4 C 30 100 100 200 4NC 30 100 - 200 5 C 30 30 100 200 5N C 30 30 - 200

After incubation, the samples were spun down at 13,000 rpm, 4°C for 5 mins. The supernatant was aspirated, and the pellets were washed rapidly with lOOpl protease inhibited PBS, taking care not to disturb the pellet. The pellets were resuspended in lOOpl 1% SDS in PBS and left standing at RT for 15 mins. The resuspension was repeated, the solution was transferred to scintillation vials containing 5ml of scintillation fluid, and the samples were left to count overnight.

2.4.9: COMPETITION BINDING STUDY - BINDING OF ^^^I-ET-3 TO LUNG FIBROBLAST MEMBRANES IN THE PRESENCE OF INCREASING

CONCENTRATIONS OF TINT ABFTI.FD BO-123:

The aim of this experiment was to confirm the specificity of ligand binding. Solutions of 2pM ^^^I-ET-3 and protease inhibited PBS (1 tablet per 100ml) were prepared. Samples were made up as shown below in siliconised tubes. All samples were made up to 200pl with PBS/PI. G roup Membrane (Pg) 125I.ET-3 (pM) ET-3 (M) BQ-123 (nM) 1 30 0 . 2 1 0 X i c r * 2 0 0 2 30 0 . 2 1 X 10'^ 2 0 0 3 30 0 . 2 0.1 X 10^ 2 0 0 4 30 0 . 2 10 X ICfi^ 2 0 0 5 30 0 . 2 1 X 10'" 2 0 0 6 30 0 . 2 0.1 X 10 12 1 0 0

Each group contained 3 tubes. Binding was measured in the presence of decreasing concentrations of unlabelled ligand. A total radioactivity control was also included, which was added directly to the scintillation vials and measured the total amount of radioactivity added to each tube at the start.

The tubes were incubated at RT for 2 hours. They were then spun for lOmins at 14,000 rpm. The supernatant was carefully aspirated, and lOOpl of buffer was added taking care

not to disturb the pellet. This was also aspirated. The results of this experiment are not shown as no binding curve was achieved.

2.4.10: INTF.RNAT.TSATION OF ‘^^I-ET-1 IN LUNG FIBROBLAST CF.T.T.S:

This method was based on that of Cramer et al [Cramer H. (1997)] who showed internalisation of ^^^I-ET-1 in transfected CHO cells.

Sixteen lOcm^ dishes of CCD19Lu were washed three times with 5ml of PBS warmed to 37°C. Cells were habituated to serum-free medium for 1 hour at 37°C, 5% CO2, then

the medium was aspirated and replaced with serum-free medium at 4°C containing the stimulus. Groups were as follows (four dishes in each group, final volume 3ml):

G roup Final cone. ^^^I-ET-1

(pM)

Final conc. ET-1 (nM)

No competitor (0 mins) 50 -

Competed (0 mins) 50 50

No competitor (20 mins) 50 -

Competed (20 mins) 50 50

Dishes were incubated at 4°C for 3 hours to allow passive binding to occur. The medium was then collected to measure unbound radioactivity and the dishes were washed three times with ice-cold PBS/PI. Control dishes were returned immediately to the fridge in 3ml of 50mM glycine, pH 2.5. After 15 mins the glycine was collected for measurement of surface bound radioactivity and replaced with 3ml of 1% SDS in IN NaOH which had been warmed to 50°C. The dishes were left at RT for 15 mins and the cells were scraped in the detergent.

Stimulated dishes were incubated at 37°C, 5% CO2 for 20 mins in fresh serum-free

medium. The medium was then discarded, and the cells were treated with acid and detergent in the same way as the controls.

Each 3ml sample (medium/acid/detergent from each dish) was divided between two scintillation vials containing 8ml of scintillation fluid, and radioactivity was measured in a liquid scintillation counter.

2.5: ISOLATION OF ENDOTHELIN RECEPTO RS AND ANALYSIS OF POST- TRANSLATIONAL MODIFICATIONS:

2.5.1: SYNTHESIS OF ((dA W 5’-S-N-(e-

MALEIMIDOCAPROYLOXYISUCCINIMIDE ENDOTHELIN:

Having established that the receptor was expressed in the fibroblast line CCD19Lu, the next step was to be able to isolate the receptor in sufficient quantity and purity to analyse its post-translational modifications. To this end, a synthetic, endothelin-based ligand was synthesised. This comprised a poly-A oligonucleotide attached to endothelin via a heterobifunctional cross-linker, N-(8-Maleimidocaproyloxy)succinimide (EMCS). Two methods were used. The initial synthesis was carried out according to the procedure of Roos et al [Roos M. (1998)]. The subsequent synthesis was carried out according to a modified procedure.

i) Synthesis of ((dA)^-5’-S-N-(£-Maleimidocaprovloxv)succinimide endothelin (I):

L2mg of EMCS was dissolved in 200|li1 of acetonitrile. It was added to the ET-1 solution in 5 portions over the course of 1 hour, vortexing vigorously between additions, to give a final EMC concentration of 4pM, a 10-fold excess. The reaction mixture was then incubated at RT for 2.5 hours, then adjusted to pH 6 with 140mM KH2PO4 and stored at -20°C.

EMC-ET was purified on a micro-FPLC (Amersham-Pharmacia SMART system) using a Sephasil C-18 column. A gradient of 2-70% acetonitrile in 0.1% TEA was used. Solvent A was 0.1% TEA in H2O. Solvent B was 0.1% TEA in acetonitrile. After a test

run using a steady gradient from 2-70% acetonitrile over 30 mins at a flow of 200pl/min and recording at wavelength 280nm, it was established that major fractions eluted at approximately 36, 42 and 46% acetonitrile. Eor all subsequent runs, 20pl of reaction product was loaded on the column and the flow rate was 200pl/min. The gradient was run from 0-30% acetonitrile in 5 mins, from 30-50% in 20 mins, and from 50-70% in 5 mins. Purified fractions were stored at -20°C.

Samples from all likely fractions from each run were analysed using MALDI-TOE mass spectrometry without any further processing, and fractions containing purified EMC-ET were identified. The mass of EMC-ET is 2684.0, so the expected mass on mass spectrometry (MH^) was 2685.0.

Deprotection o f (dA)30:

330nmol (3.23mg) of (dA)30-5’-SS-R-l-O-dimethoxytrityl hexamethylendisulphide was dissolved in 45pl of lOOmM triethylammonium acetate. lOpl of a solution of ImM DTT in 200mM Tris-HCl, pH 8.0, was added, the mix was vortexed, and incubated at RT for 3 hours.

Meanwhile, the previously synthesised EMC-ET was dried in a speed-vac, and adjusted to pH 6 using a solution of 200mM MES, to a final volume of 150pl.

Reaction with EMC-ET:

The oligo/DTT reaction mixture was diluted 1:10 and run on a fast desalting column (Pharmacia PC 3.2/10) eluting with 50mM MES, pH 6, 5mM EDTA. The flow rate was 300pl/min, and the chromatogram was recorded at 280nm. Only the first half of the major peak was collected to reduce the risk of unreacted DTT getting into the final reaction mixture. The fraction collected was added to the EMC-ET immediately it came off the column, and the mixture was incubated at RT overnight under nitrogen to allow the reaction to take place without dimérisation of the oligonucleotides. The reaction product was stored at -20°C.

Purification o f the synthetic ligand:

The reaction product was further purified on the Sephasil C-18 column using a gradient of 2-70% acetonitrile. First, two test chromatograms were taken to check for coincident peaks when measuring at 280nm and 260nm. This indicated that oligonucleotide and protein were present in the same peak, thus denoting the desired product. Once this peak had been identified, the remaining sample was purified.

Solvent A was 2% acetonitrile in lOOmM TE A A, 2mM BU4NHSO3. Solvent B was 70% acetonitrile in the same buffer. The gradient was run in a number of different ways, but the best separation was achieved running from 0-50% B in 15 min at 200pl/min, and from 50-100% B in 30 mins at 50pl/min. All major peaks were collected and stored at - 20°C.

n) Svnthesis of (tdA)3n-5’-S-N-l£-Maleimidocaprovloxv)succinimide endothelin (2):

0.5g of oligo(dT)cellulose powder was added to 1ml of O.IM NaOH and packed in a column, which was rinsed with water until the column effluent had a pH of < 8. The column was equilibrated with 5 column volumes of loading buffer (50mM sodium citrate, pH 7.5, 0.5M LiCl, ImM EDTA, 0.1% (w/v) SDS) and transferred to a 15ml tube. 140.6 OD (3.02mg) of DMT-hexyl-SS-(dA)3o (BioTeZ, Berlin-Buch GmbH) was

dissolved in 4ml of loading buffer, and mixed with the oligo(dT)cellulose. The mixture was shaken at room temperature for 2hrs. 1600pl of IM DTT and 80|xl of 200mM tributyl phosphine (TBP) in DMF were added, and shaking continued for 3hrs at RT. The suspension was then transferred to a column, repacked, and washed with 10 column

volumes of reaction buffer (50mM sodium citrate, pH 7.0, 0.5M LiCl, ImM EDTA,