Characterization of opioid binding sites in rat spinal cord.
Binding sites were characterized in rat whole spinal cord crude membrane preparations using selective labelling techniques with multiple methods of mathematical analysis of experimental curves. Mathematical analysis of single [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (DAGO) saturation curves suggested binding of the [3H]-ligand at one site, while displacement curves of low concentrations of [3H]-DAGO with selective mu-ligands indicated the presence of high- and low-affinity sites. All the [3H]-DAGO curves processed simultaneously by LIGAND analysis showed the presence of high (27%) and low (73%) affinity components, with a total Bmax of 3.19 pmol/g tissue. Eighty percent of [3H]-[D-Ala2,D-Leu5] enkephalin (DADLE) binding was displaced by DAGO with high affinity, indicating that a high percentage of [3H]-DADLE binding was at mu-sites. Saturation curves of [3H]-DADLE after inhibition of mu-sites by unlabelled DAGO (delta-sites) were monophasic with non-linear fitting analysis and the Bmax was 0.90 pmol/g tissue. Most mathematical analysis of single saturation curves of [3H]-(-)-bremazocine indicated binding at more than one site. DAGO, DADLE, U-69,593 and PD 117302 displaced 0.15 nM of [3H]-(-)-bremazocine biphasically: the percentages of displacement calculated with the non-linear fitting program were respectively 50 (mu-sites), 64 ((mu + delta)-sites), 18 and 25 (kappa-sites). Haloperidol displaced [3H]-(-)-bremazocine only at microM concentrations. suggesting no binding at sigma-sites. In the presence of 225 nM of DAGO, DADLE displaced only 21% of [3H]-(-)-bremazocine 0.15 nM binding (delta-sites). Most mathematical analysis of saturation curves of [3H]-(-)-bremazocine, after inhibition of binding at mu- and delta-sites by DAGO and DADLE, still indicated binding at more than one site and the selective kappa-ligands U-69,593 and PD 117302 displaced [3H]-(-)-bremazocine in these experimental conditions. LIGAND analysis of saturation and inhibition curves of [3H]-(-)-bremazocine by U-69,593 and PD 117302 showed the presence of high (43%) and low (57%) affinity components, with a total Bmax of 2,73 pmol/g tissue. Thus in rat spinal cord there are at least two mu-sites bound by [3H]-DAGO which amount together to approximately 47% of total opioid sites, delta-sites bound by [3H]-DADLE amounting to approximately 13%, kappa-sites and other unknown sites (possibly a kappa-subtype) bound by [3H]-(-)-bremazocine, which together are approximately 40% of total opioid sites.
Petrillo
P
P
Istituto di Richerche Farmacologiche Mario Negri, Milano, Italy.
Kowalski
J
J
Sbacchi
M
M
Tavani
A
A
D000818:Animals; D001575:Benzomorphans / Q000378:metabolism; D001665:Binding Sites; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D051381:Rats; D011957:Receptors, Opioid / Q000032:analysis*; D013116:Spinal Cord / Q000737:chemistry*
D001575:Benzomorphans; D004745:Enkephalins; D011957:Receptors, Opioid; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine; C027248:bremazocine