2.8.1 Preparation of arteries for radioligand binding assays
2nd and 3rd order branches of the intrapulmonary artery were identified and dissected free of the lung as described in Section 2.1. The arteries were then cut open lengthways and the endothelium was removed from the preparation by gentle rubbing of the luminal wall of the vessel with a cotton bud. Tissue was then placed in HBSS at 37oC (pH 7.4) for a period of equilibration (30 min).
2.8.2 Preparation of sea urchin egg homogenates
UnfertilizedLytechinus pictussea urchin eggs were collected during the gravid season between the months of May and September. Urchins were shipped from California and collection of gametes occurred immediately upon arrival. Eggs were obtained by stimulating ovulation of female sea urchins with an intracoelomic injection of KCl (0.5M; up to 0.5 ml total volume). Eggs were collected in artificial sea-water (ASW), of the following composition (mM): NaCl 435, MgSO4 15, CaCl2 11, KCl 10, NaHCO3 2.5 and EDTA 1.0 at pH
8.0.
Homogenates (50% v/v) of unfertilized sea urchin eggs were prepared in a similar manner to that described previously (Clapper, et al., 1987; Dargie, et al., 1990). Eggs were dejellied in ASW by filtering through 85-mm Nitex mesh (Plastok Associates Ltd, Merseyside, UK). Dejellied eggs were immediately washed by centrifugation at 800 g and 10oC in approximately 10 times their volume of AWS. Eggs were washed twice in Ca2+-free ASW with EGTA and twice in Ca2+-free ASW composed of (mM): NaCl 470, MgCl227,
Finally, eggs were washed with an ‘intracellular medium’ (IM), composed of (mM): potassium gluconate 250, N-methylglucamine 250, HEPES 20 and MgCl21. The pH was adjusted to 7.2 using acetic acid.
Eggs were then homogenised in IM plus 2 mM ATP, 20 U/ml creatine phosphokinase (CPK), 20 mM phosphocreatine, 50 g/ml leupeptin, 20 g/ml aprotinin and 100 g/ml SBTI (Soya bean trypsin inhibitor), using a dounce glass tissue homogeniser, size ‘A’ pestle. Cortical granules were removed and discarded by centrifugation at 13,000 g for 10 seconds at 4C. Homogenates were aliquoted (1 ml) into microcentrifuge tubes and stored at -80 C, until used for radioligand binding assays.
2.8.3 Preparation of tissue and acid extraction of endogenous NAADP from pulmonary artery smooth muscle
Tissue was dissected as described in section 2.8.1, and placed in HBSS at 37 oC (pH 7.4) for 30 minutes to equilibrate. Tissue was then placed into fresh HBSS at 37 oC as a control, or in HBSS containing test compound. After a given period of time tissue was removed from the HBSS, dried by quickly running the tissue over tissue paper, weighed and snap frozen in liquid nitrogen within 5 seconds of removal from HBSS and stored at -80oC until required for extraction of nucleotides.
The extraction of endogenous NAADP from tissue samples was carried out using a variation of a method previously described (Walseth, et al., 1991). Tissue samples were diluted 1:4 with ice cold perchloric acid (PCA; 1.5 M) in order to prevent any degradation of the nucleotides by enzymes in the samples and to precipitate proteins out of the solution. Tissues were then homogenized by sonicating the tissue five times for ten second bursts while on ice to prevent any overheating of the samples (Jencons Vibracell at amplitude 60). Samples were then left in an ice bath for twenty minutes in order to allow for full separation of the nucleotides from the protein sample. After this time samples were spun on a desktop centrifuge (13 000 g) for ten minutes in order to remove precipitated proteins. After centrifugation, the supernatant was
removed and the protein fraction was stored at -80oC until the level of protein within the samples could be determined as described in section 2.8.6.
The supernatant was then neutralized with a 1:1 addition of potassium bicarbonate (KHCO3, 2 M) and placed in an ice bath for 20 minutes. After this
time the samples were centrifuged at 13 000g for ten minutes to separate the nucleotide containing supernatant from the precipitated KClO4. The
supernatant was then removed and frozen at -80oC until required for binding.
2.8.4 Preparation of [32P]NAADP for use in radioligand binding assay
The synthesis of [32P]NAADP was carried out in a two-step reaction as described previously (Aarhus, et al., 1996b; Patel, et al., 2000a). Firstly, [32P]NADP was synthesised by incubating [32P]NAD with 0.5 U/ml human NAD kinase, 5 mM MgATP, and 100 mM HEPES for 1 hour. 100 mM nicotinic acid and 1 g/ml ADP-ribosyl cyclase were added to commence the second step, which was allowed to proceed for one further hour. The resulting mixture was pumped onto a high pressure liquid chromatography (HPLC) column. Separation was carried out on an anion-exchange resin (AGMP1, Biorad) using a concave upwards gradient of trifluoroacetic acid (TFA) as described previously (Aarhus, et al., 1996b; Billington and Genazzani, 2000a; Galione, et al., 2000; Patel, et al., 2000a). The NAADP fraction was then stored for use in the assay.
2.8.5 Radioligand binding assay to determine levels of NAADP within pulmonary artery smooth muscle
Endogenous levels of NAADP within the tissue were determined using a variation of a technique previously described (Masgrau, et al., 2003). Sea urchin egg homogenate was made up to a final concentration of 1.25% in IM. [32P]NAADP (50 000 CPM) and known concentrations of unlabelled NAADP (0.001 – 300 nM) or unknown samples were added to 200l of homogenate to make a final volume of 250 l in the reaction vessel. Due to the irreversible binding of NAADP to its target in sea urchin egg (Aarhus, et al., 1996a;
Billington and Genazzani, 2000a; Patel, et al., 2000a) it is important that the homogenate is exposed to both the radiolabelled and unlabelled NAADP simultaneously otherwise one will not displace the other. Once mixed the samples were left for 15 minutes at room temperature (22 oC) to allow binding to reach equilibrium. Binding was terminated by rapid filtration through glass microfibre (GF/B) filters (Whatman, US) using a Brandell cell harvester. Filters were washed immediately prior to binding with ice-cold HEPES-EDTA solution of the following composition (mM): HEPES 10, EDTA 1, pH 7.2. Following filtration, filters were washed four times with 4 – 5 ml of HEPES- EDTA solution. Filters were then removed and retained [32P]NAADP was determined using either Cerenkov Spectrometry or storage phosphor screen autoradiography.
In storage phosphor screen autoradiography, filters are exposed to a storage phosphor screen (General purpose storage phosphor screen, 35 x 43 cm, Amersham Bioscience, UK) within an exposure cassette (Amersham Bioscience, UK) for thirty minutes. During this time an imprint of the retained radiation within the filters is created on the screen. The phosphor screen is composed of fine crystal of BaFBR:Eu2+ contained within an organic binder. The radiation from the filters causes the oxidation of Eu2+ to Eu3+ and the reduction of BaFBr to BaFBr-. These ions remain oxidized and reduced after removal of the screen from the filters, and thereby retains the energy of the ionizing radiation. The release of the stored energy occurs when the storage phosphor is stimulated by light of an appropriate wavelength. The screen is stimulated by red light (wavelength: 633 nm) which is absorbed by the BaFBr– complex. This frees electrons and reduces the Eu3+to Eu2+*(a Eu2+ion with an excited electron). As this excited electron falls to the ground state it releases energy in the form of blue light (emission maxima: 390 nm). The Typhoon scanner system (Amersham Biosciences, UK) uses a band- pass filter, allowing light around 390 nm to pass through to the detector. The light is then collected and measured by the detector. Therefore the emitted light intensity is proportional to the radioactivity. An image of the filter is then displayed on the computer screen with the detected light depicted in a grey scale. Therefore, the more light detected the darker the image. The image is then quantified using
densitometry analysis (ImageQuant software, Amersham Biosciences, UK) and analyzed using Prism software as described below.
Total binding of [32P]NAADP to sea urchin egg homogenate was determined in the absence of unlabelled NAADP and unknown sample. The amount of [32P]NAADP retained on the filters after filtration decreased as the concentration of unlabelled NAADP increased as they compete for the same binding site. This allowed for the construction of a dissociation curve for [32P]NAADP binding to sea urchin egg homogenate. Non-specific binding was determined as being the amount of radiation (CPM) retained in the presence of 1 M NAADP. This value was subtracted from the values obtained for the known concentrations of NAADP or unknown samples in order to determine specific binding. By obtaining the values for the retained radiation against various concentrations of NAADP and plotting them as a graph of CPM versus concentration of NAADP it was possible to fit a curve to the data using non- linear regression and fitting a sigmoidal concentration displacement curve with
Fig. 2.15Example of dissociation curve used to determine the concentration of NAADP levels within pulmonary arteries.
variable slope fitted using Equation 2.3 (Section 2.6.7) using Prism analysis software (Fig. 2.15; Graphpad, CA, USA). Using this curve it was possible to extrapolate the concentration of NAADP in the unknown samples by determining their position on the dissociation curve. Binding of NAADP to its receptor has been shown to be affected by the presence, or absence of K+ ions
(Dickinson and Patel, 2003). In order to minimize any effects caused by a differing concentration of K+ions in the reaction mixture the samples of known NAADP concentration were subjected to the same acid extraction protocol as the unknown samples.
2.8.6 BCA protein assay and determination of concentrations of NAADP within artery smooth muscle
The protein concentrations of the tissues from which NAADP were extracted for the radioligand binding were determined spectrophotometrically using the bicinchonic acid (BCA) assay according to the manufacturers instructions (Sigma-Aldrich, Dorset, UK). This assay relies on the alkaline reduction of Cu2+ to Cu+ by proteins and on the fact that the bicinchonic acid:Cu+ complex has absorption maxima of 562 nm. Samples containing 10- 50g protein in a volume of 50l were incubated with 1 ml BCA solution and 0.08 % copper sulphate pentahydrate for 30 minutes at 37oC. Incubations were then given five more minutes at room temperature (20 oC), after which their absorbance was measured at 563 nm. Standard protein concentrations of BSA were used to construct a standard curve using Prism analysis software (Graphpad, CA, USA), from which protein concentrations from tissue samples could be determined by interpolation. Following this, concentrations of NAADP were corrected for the dilutions during acid extraction and using these data the concentration of NAADP were expressed as pmol per mg of protein.