| Abstract: |
Kappa receptor multiplicity is a complex area. We now present evidence from binding studies suggesting the existence of four kappa receptor subtypes. The guinea pig cerebellum contains high levels of U50,488-sensitive, or κ1, receptors. Kappa opiates (U50,488, tifluadom, Mr2034, Mr2266 and Win44,441) complete [3H]ethylketocyclazocine binding to κ1 receptors with K(i) values under 10 nM and Hill coefficients of approximately one, as does dynorphin A (K(i), 0.27 ± 0.05 nM; Hill coefficient, 0.83 ± 0.20, n = 4). However, competition studies with dynorphin B yield a Hill coefficient of 0.46 ± 0.03 (n = 5) and nonlinear regression analysis of the competition curve is best fit by two sites. α-Neoendorphin competition curves (Hill coefficient, 0.46 ± 0.07; n = 3) also were best fit with two components. Competition studies with both α-neoendorphin and dynorphin B together suggest that both compounds label the same site with high affinity. Similar results were obtained using [3H]U69,593. Dynorphin B and α-neoendorphin competed binding with Hill coefficients of 0.45 ± 0.04 (n = 3) and 0.59 ± 0.09 (n = 3), respectively. These data suggest two subtypes of κ1 receptors in the guinea pig cerebellum κ(1a) and κ(1b). Classical kappa opiates and dynorphin A have high affinity for both subtypes whereas dynorphin B and α-neoendorphin label κ(1b) over 50-fold more potently than κ(1a) sites. [3H]Naloxone benzoylhydrazone ([3H]NalBzoH) labels a novel, U50,488-insensitive kappa receptor subtype, κ3, in membranes from calf striatum, rat and mouse brain. We now have developed a relatively selective assay in calf striatum. EDTA greatly decreases the ability of [3H]NalBzoH to label mu receptors and in its presence greater than 90% of specific binding corresponds to κ3 sites. [3H]NalBzoH labels κ3 sites with high affinity (K(D), 0.74 ± 0.04 nM; n = 4). The density of κ3 receptors in calf striatal membranes was quite high (B(max), 14.6 ± 0.69 fmol/mg wet weight of tissue; n = 4), approximately twice the levels of mu receptors and greater than the level of κ1 receptors labeled with [3HU69,593. Competition studies with selective mu and delta ligands indicated that this site correlated with neither mu nor delta receptors. In contrast, a large number of kappa ligands competed binding quite potently with K(i) values under 10 nM. However, U50,488 at concentrations up to 1 μM did not significantly inhibit [3H]NalBzoH binding to κ3 sites in calf striatal membranes despite its potent inhibition of [3H]NalBzoH binding to κ1 receptors in the guinea pig cerebellum (K(i), 6.1 nM). Similarly, nor-Binaltorphimine, a selective kappa antagonist, competed κ1 (U50,488-sensitive) binding (K(i), 3.5 nM) far more potently than κ3 binding (K(i), 103 nM). Metkephamid, on the other hand, competed κ3 binding (K(I) 4.3 nM) far more effectively than κ1 binding (K(i), 187 nM). Although morphine lowered both κ1 and κ3 binding with similar potencies (K(i) values, 32.8 and 49 nM, respectively), [D-Ala2,MePhe4,Gly(ol)5]enkephalin inhibited κ3 binding (K(i) 8.2 nM) much more potently than κ1 binding (K(i) > 350 nM). κ3 binding was highly stereospecific and a series of nonopioid neuroactive compounds did not significantly lower binding. |
| Keywords: |
unclassified drug; nonhuman; animal cell; animal; cerebellum; brain; binding sites; cattle; radioisotope; morphine; ligand binding; receptors, opioid, mu; in vitro; naloxone; 3,4 dichloro n methyl n [2 (1 pyrrolidinyl)cyclohexyl]benzeneacetamide; pyrrolidines; receptor subtype; cyclazocine; n allylnormetazocine; pentazocine; drug concentration; dynorphin a; ethylketazocine; kappa opiate receptor; guinea pig; edetic acid; levallorphan; guinea pigs; receptors, opioid; norbinaltorphimine; alpha neoendorphin; priority journal; article; receptors, opioid, kappa; enkephalin derivative; support, u.s. gov't, p.h.s.; 3,4-dichloro-n-methyl-n-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-isomer; dynorphin b; quadazocine; ethylketocyclazocine; 5,9 diethyl 2 (3 furylmethyl) 2' hydroxy 6,7 benzomorphan; n (tetrahydrofurfuryl)normetazocine; n allyl 6 deoxy 6 benzoylhydrazido 14 hydroxydihydronormorphinone; tifluadom
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