High- and low-affinity binding sites for [3H]-alpha, beta-methylene ATP in rat urinary bladder membranes.
1. The characteristics of [3H]-alpha, beta-methylene adenosine 5'triphosphate ([3H]-alpha, beta-MeATP) binding to membrane preparations of rat urinary bladder detrusor were studied. 2. The rat bladder membrane preparation was obtained by multiple centrifugation. [3H]-quinuclidinyl benzilate [( 3H]-QNB) binding to this preparation demonstrated that the muscarinic receptor density was 4.32 times higher than that in the homogenate. [3H]-alpha, beta-MeATP binding was increased 3.88 times. 3. Saturation analysis revealed that the rat bladder membrane contained a high density of [3H]-alpha, beta-MeATP binding sites, which could be divided into a high-affinity component (Kd = 8.1-8.9 nM) and a low-affinity component (Kd = 67.0-119.8 nM). 4. Magnesium ions inhibited the maximum binding in a concentration-dependent manner. The maximum high-affinity binding was reduced from 10.32 pmol mg-1 protein in magnesium-free buffer to 4.62 pmol mg-1 protein with 25 mM MgCl2, while the maximum low-affinity binding was reduced from 58.84 pmol mg-1 protein to 14.24 pmol mg-1 protein. Kd values were not greatly affected. 5. The binding was a rapid reversible process. The association rate constants were 7.64 x 10(7) M-1 min-1 for high-affinity binding, and 7.31 x 10(6) M-1 min-1 for low-affinity binding. The dissociation rate constants were 0.2896 min-1 for high-affinity binding, and 0.6348 min-1 for the low-affinity binding. 6. Displacement experiments with unlabelled purinoceptor ligands confirmed that [3H]-alpha, beta-MeATP mainly binds to P2X-purinoceptors. The potency order was: a,/i-methylene ATP > fy-methylene ATP > suramin > ATP > ADP > 2-methylthio ATP > adenosine. 7. The results indicate that [3H]-a,,B-MeATP is a radioligand for the P2x-purinoceptor, which satisfies the basic criteria for use in radioligand binding assay.
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Department of Anatomy and Developmental Biology, University College London.
Burnstock
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000378:metabolism; D011983:Receptors, Purinergic / Q000378:metabolism*; D014316:Tritium; D001743:Urinary Bladder / Q000378:metabolism*
D011976:Receptors, Muscarinic; D011983:Receptors, Purinergic; D014316:Tritium; D011813:Quinuclidinyl Benzilate; D000255:Adenosine Triphosphate; D008274:Magnesium; C002630:alpha,beta-methyleneadenosine 5'-triphosphate