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Characterization, localization and solubilization of P2x-purinoceptors

Bo, Xuenong; (1993) Characterization, localization and solubilization of P2x-purinoceptors. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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A radioligand binding assay was established to study the biochemical characteristics of P2x-purinoceptors. α,β-Methylene ATP was tritiated, and it was found that the binding of [3H]α,β-methylene ATP ([3H]α,β-MeATP) to rat bladder membrane preparations was rapid, fully reversible, and saturable. Competitive displacement showed that the potency order of the unlabelled purinergic ligands in displacing [3H]α,β-MeATP binding was: α,β-methylene ATP > β,Y-methylene ATP > suramin > ATP > 2-methylthio ATP > ADP >> adenosine, which indicates the binding sites are, or are related, to P2X-purinoceptors. Saturation assays revealed high- and low-affinity binding state, with Kd values of approximally 6 and 80 nM respectively. The optimal pH value of the binding was around 7, and the specific binding sites could be completely denatured by heat. The binding was greatly influenced by Na+, Ca2+, and Mg2+ ions in the media. Autoradiographic localization showed that the [3H]α,β-MeATP binding sites were distributed only over the smooth muscle cells of the rat urinary bladder, vas deferens, and the rabbit ear artery, which indicates that the binding is tissue-specific. The basic criteria for the recognition of a ligand binding site as a receptor or a subunit were fulfilled. Further competitive displacement experiments with other nucleotides, base and polyphosphate suggest that the critical structure for the interaction of the ligand with P2X- purinoceptor is the polytriphosphate moiety. A comparative study carried out on the urinary bladder and urethra of rat, guinea-pig and rabbit showed that rat bladder contained the highest density of [3H]α,β-MeATP binding sites, followed by rabbit and guinea-pig. Semi-quantitation of the autoradiograms revealed a similar order of binding site densities. The urethra of the rat and guinea-pig, a tissue where the P2X-purinoceptor-mediated responses have been shown to be absent, lacked specific labelling. In human urinary bladder, P2X-purinoceptor-mediated neurogenic contractile responses were reported to be absent or to exist only in some individuals. Both radioligand binding assay and autoradiographic localization showed that specific binding sites of [3H]α,β-MeATP were observed only in about 38 - 42% of the bladder detrusor of old male subjects, and the density was much lower than those in rat, guinea-pig and rabbit bladder. In many blood vessels extracellular ATP has been reported to elicit contractions of smooth muscle. Radioligand binding assays showed that rabbit ear artery contained a high density of high-affinity [3H]α,β-MeATP binding sites. Autoradiographic localization demonstrated specific binding sites of [3H]α,β-MeATP in many blood vessels from rat, guinea-pig and rabbit. Generally, medium- and small-sized muscular arteries, such as rat saphenous, rat tail, and rabbit ear arteries, contained higher densities of binding sites than large elastic arteries like aorta. In some large arteries such as rabbit carotid, renal and hepatic arteries, the binding sites were denser over the outer region of the vascular wall than the inner region. Only sparse specific binding was observed in all veins other than the portal vein from all the three species. In the portal veins, the densities over the circular and longitudinal muscles were different. The high-affinity component of the specific [3H]α,β-MeATP binding sites were successfully solubilized from rat vas deferens with 2% digitonin. The solubilized [3H]α,β-MeATP binding sites still possessed the characteristics of the membrane-bound binding sites, i.e., rapid association and dissociation, reversibility, and saturability. The potency order of the unlabelled ligands in displacing the [3H]α,β-MeATP binding was similar to that obtained from membrane binding experiments. Sucrose density gradient ultracentrifugation showed that the sedimentation coefficient of receptor-detergent complex was 12.1 S. Upon UV irradiation [3H]α,β-MeATP was cross-linked to a molecule of 62,000 daltons in rat vas deferens membrane, which might be the functional receptor or the binding subunit.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Characterization, localization and solubilization of P2x-purinoceptors
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10098248
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