Preparation of brain membranes containing a single type of opioid receptor highly selective for dynorphin.

Opioid receptors on guinea pig brain membranes were alkylated by the naltrexone analogue beta-chlornaltrexamine. Binding of the prototypical mu and kappa ligands, [3H]dihydromorphine and [3H]ethylketocyclazocine, was more readily affected by the reagent than was binding of the delta ligand, 3H-labeled [D-Ala2, D-Leu5]enkephalin. Treatment of membranes with beta-chlornaltrexamine in the presence of dynorphin resulted in significant protection of [3H]ethylketocyclazocine binding sites, without protection of [3H]dihydromorphine or 3H-labeled [D-Ala2, D-Leu5]enkephalin sites. Similarly, [D-Ala2, D-Leu5]enkephalin and sufentanil selectively protected binding sites for 3H-labeled [D-Ala2, D-Leu5]enkephalin and [3H]dihydromorphine, respectively. Scatchard analysis of [3H]ethylketocyclazocine binding to untreated membranes suggested two types of binding site with 40-fold difference in affinities. Membranes treated with beta-chlornaltrexamine in the presence of dynorphin retained about 40% of the high-affinity sites and lost the low-affinity sites. Selective protection of sites with high affinity for dynorphin and ethylketocyclazocine was confirmed in competition binding assays. These results strongly suggest that the three types of opioid receptor are not interconvertible and provide further evidence that the endogenous peptide dynorphin is a highly selective ligand of the kappa opioid receptor.     

Selective protection of stereospecific enkephalin and opiate binding against inactivation by N-ethylmaleimide: evidence for two classes of opiate receptors.

Stereospecific binding of 3H-labeled [D-Ala2,D-Leu5]enkephalin is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide. This inactivation, like that of opiate binding, exhibits pseudo-first-order kinetics with a half-inactivation time of 10-12 min. The presence of opiates or enkephalins during incubation with N-ethylmaleimide provides good protection. Ouantitative studies of protection demonstrate that naltrexone and morphine are 20 and 8 times, respectively, more effective in protecting the binding of [3H]naltrexone than that of [3H]enkephalin. [D-Ala2,Leu]Enkephalin and [D-Ala2,Met]enkephalin, however, are more effective (7 and 30 times, respectively) for the protection of 3H-labeled [D-Ala2,D-Leu5]enkephalin binding. These results provide strong evidence for the existence of two classes of opiate receptor in rat brain.     

Visualization of mu1 opiate receptors in rat brain by using a computerized autoradiographic subtraction technique.

We have developed a quantitative computerized subtraction technique to demonstrate in rat brain the regional distribution of mu1 sites, a common very-high-affinity binding site for both morphine and the enkephalins. Low concentrations of [D-Ala2, D-Leu5]enkephalin selectively inhibit the mu1 binding of [3H]dihydromorphine, leaving mu2 sites, while low morphine concentrations eliminate the mu1 binding of [3H][D-Ala2, D-Leu5]enkephalin, leaving delta sites. Thus, quantitative differences between images of sections incubated in the presence and absence of these low concentrations of unlabeled opioid represent mu1 binding sites. The regional distributions of mu1 sites labeled with [3H]dihydromorphine were quite similar to those determined by using [3H][D-Ala2, D-Leu5]enkephalin. High levels of mu1 binding were observed in the periaqueductal gray, medial thalamus, and median raphe, consistent with the previously described role of mu1 sites in analgesia. Other regions with high levels of mu1 binding include the nucleus accumbens, the clusters and subcallosal streak of the striatum, hypothalamus, medial habenula, and the medial septum/diagonal band region. The proportion of total specific binding corresponding to mu1 sites varied among the regions, ranging from 14% to 75% for [3H][D-Ala2, D-Leu5]enkephalin and 20% to 52% for [3H]dihydromorphine.     

Distinct high-affinity binding sites for benzomorphan drugs and enkephalin in a neuroblastoma--brain hybrid cell line.

The high-affinity binding of benzomorphan drugs (ethylketocyclazocine and N-allylnorcyclazocine) and [DAla2,DLeu5] enkephalin was examined in a mouse neuroblastoma--Chinese hamster brain clonal hybrid cell line (NCB-20). Scatchard analysis of saturation binding isotherms indicated the presence of a single binding site for 3H-labeled [DAla2,DLeu5]enkephalin (Kd = 3 nM) and multiple binding sites for [3H]ethylketocyclazocine (Kd = 4 and 20 nM) and N-[3H]allylnorcyclazocine (Kd = 0.5 and 15 nM). Both ethylketocyclazocine and N-allylnorcyclazocine competed (Ki = 10 and 30 nM, respectively) with [3H][DAla2,DLeu5]enkephalin binding in NCB-20 cells but neither [DAla2,DLeu5]enkephalin nor morphine could completely inhibit the specific binding of [3H]ethylketocyclazocine (7 nM) or N-[3H]allylnorcyclazocine (3 nM). Furthermore, not all benzomorphan drugs (e.g., ethylketocyclazocine) were totally efficacious in displacing 3 nM N-[3H]allylnorcyclazocine binding in the presence or absence of high concentrations of [DAla2,DLeu5]enkephalin. The data presented suggest that benzomorphan drugs interact with three distinct high-affinity binding sites: (i) a site that binds enkephalin and morphine in addition to ethylketocyclazocine and N-allylnorcyclazocine; (ii) a site that binds both ethylketocyclazocine and N-allylnorcyclazocine but not enkephalin and morphine; and (iii) a site that binds N-allylnorcyclazocine but not enkephalin, morphine, or ethylketocyclazocine. The first of these sites was comparable to the delta opiate receptor expressed in NG108-15 and N4TG1 cell lines based on the potency series obtained for various opiates and benzomorphan drugs in competition studies with [3H][DAla2,DLeu5]-enkephalin. However, the specific high-affinity benzomorphan binding sites thus far are unique and may represent biochemical correlates of kappa and sigma opiate receptors which have been proposed to exist on the basis of physiological studies.     

Possible role of distinct morphine and enkephalin receptors in mediating actins of benzomorphan drugs (putative kappa and sigma agonists).

The binding of many opiates and enkephalins to enkephalin (delta) and morphine (mu) receptors was compared by using three different binding assays: (i) 125I-labeled[D-Ala2, D-Leu5]enkephalin or 125I-labeled[D-Ala2,N-Me-Phe4,Met(O)5ol]-enkephalin to brain membranes; (ii) [3H]ethylketocyclazocine to brain membranes; and (iii) [3H]diprenorphine and [3H]naloxone to neuroblastoma cell and brain membranes, respectively. According to their relative binding potencies and the effects of Na+ and GTP on the binding to these two receptors, opiates and enkephalins can be classified into seven classes: (i) morphine-type mu agonists; (ii) enkephalin-type delta agonists; (iii) mixed agonists-antagonists; (iv) putative kappa agonists; (v) putative sigma agonists; (vi) nalorphine-type antagonists; and (vii) opiate antagonists. Studies with [3H]ethylketocyclazocine do not reveal specific kappa receptors distinct from those already described that bind morphine and enkephalins. The benzomorphan analogs ketocyclazocine and ethylketocyclazocine (putative kappa agonists) and N-allylnormetazocine (putative sigma agonist) bind to morphine (mu) and enkephalin (delta) receptors with similarly high affinities. The potency of putative kappa agonists, measured by competition with binding of the 3H-labeled antagonist, is greatly reduced by the presence of Na+ and GTP; the "Na+ and GTP ratios" are similar to those of morphine and enkephalins. However, Na+ and GTP greatly decrease the potency of binding of putative sigma agonists to enkephalin receptors but only slightly decrease the binding to morphine receptors. These data suggest that putative kappa agonists have agonistic activity toward both receptors, whereas putative sigma agonists behave as agonists for enkephalin receptors but have antagonist activity for morphine receptors. Mixed agonist-antagonists also show smaller difference in affinity to both receptors. These findings may have important implications for understanding the differences in the pharmacological effects of these drugs.     

Enkephalins have a direct positive inotropic effect on cultured cardiac myocytes.

Enkephalins have peripheral vascular effects, and enkephalinergic innervation of the heart has been reported. To determine whether enkephalins have direct effects on myocardium, we studied the effects of [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin on amplitude of contraction (measured with an optical-video system) in spontaneously beating monolayer cultures of chicken embryo ventricular cells, a preparation devoid of intact neural elements. [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin as well as [Met5]- and [Leu5]enkephalin increased contractility in a concentration-dependent manner. The enkephalin-induced maximal contractile effects were 28% and 30% above control, with EC50 values of 0.53 and 0.17 microM for [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin, respectively. The positive inotropic effect was antagonized by naloxone but not by propranolol, phentolamine, diphenhydramine, or cimetidine. Naloxone alone had no effect on contractility at a concentration (0.1 microM) that blocked positive inotropic effects of [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin. To demonstrate the presence of opiate receptors, we studied [3H]naloxone binding in homogenates of cultured chicken embryo ventricular cells. Analysis of binding curves under equilibrium conditions indicated that [3H]naloxone bound specifically to membranes of cultured heart cells with KD = 18.5 +/- 5.4 nM and Bmax = 46.8 +/- 11.7 fmol/mg of protein. We conclude that enkephalins exert a direct positive inotropic effect on cultured heart cells, increasing contractile state via specific opiate receptors.     

beta-Endorphin: characterization of binding sites specific for the human hormone in human glioblastoma SF126 cells.

The established human glioblastoma cell line SF126 was found to bind tritiated human beta-endorphin (beta h-EP) in a saturable fashion. From displacement studies, the ED50 was estimated to be about 2.5 nM. The Kd was estimated as 1.9 X 10(-9) M and Scatchard analysis showed a biphasic pattern with a predominant low-affinity component. Binding reached a maximum at about 90 min at 22 degrees C and was instantaneously reversible. Tritiated [D-Ala2,D-Leu5]enkephalin and tritiated dihydromorphine did not bind to the cells. Sodium at a concentration of 150 mM decreased the specific binding by 80%. The interaction with the cellular binding site appeared to be mediated by the COOH-terminal segment of beta h-EP, as beta h-EP-(6-31) retained a high potency for displacing tritiated beta h-EP, and beta h-EP-(1-27) has no activity. Camel beta-EP was only about 1% as active as the human hormone.     

Novel opiate binding sites selective for benzomorphan drugs

The simultaneous addition of [D-Ala2, D-Leu5]-enkephalin and morphiceptin at concentrations at which 98% of enkephalin (delta) and morphine (mu) receptors are occupied only partially inhibits the binding of [3H]diprenorphine to rat brain membranes. These conditions, furthermore, do not affect the curves for displacement of [3H]diprenorphine binding by unlabeled diprenorphine. These data suggest that [3H]diprenorphine binds to a third subtype of opiate binding site, which has high affinity for diprenorphine but very low affinity for mu and delta agonists. The [3H]-diprenorphine binding observed in the presence of morphiceptin and [D-Ala2, D-Leu5]enkephalin exhibits high affinity for several benzomorphan drugs in the chemical family of 6,7-benzomorphan (e.g., cyclazocine, ethylketocyclazocine, SKF 10047, UM 1072, oxilorphan, etc). Because of its selectivity for most benzomorphan drugs, this putative receptor site is tentatively referred to as a benzomorphan binding site. Its regional distribution in rat brain is similar to that of morphine (mu) receptors but differs from that for enkephalin (delta) receptors. The content of benzomorphan binding sites in rat brain is only one-half to one-third that of morphine receptors. The relative affinities of various opioids to morphine enkephalin, and benzomorphan binding sites are also described.     

Kappa opiate receptors localized by autoradiography to deep layers of cerebral cortex: relation to sedative effects.

Kappa opiate drugs differ from other opiates in their unique sedative actions and lack of cross-tolerance. We have visualized kappa opiate receptors by in vitro autoradiography using the kappa drugs [3H]ethylketazocine ([3H]EKC) and [3H]bremazocine. Though these ligands also label mu and delta opiate receptors, their binding is rendered kappa specific by coincubation with morphine and [D-Ala2, D-Leu5]enkephalin (DADL-Enk) to displace mu and delta interactions, respectively. Labeling patterns with [3H]EKC and [3H]bremazocine are the same and differ markedly from localizations of mu and delta opiate receptors visualized with [3H]dihydromorphine and [3H]DADL-Enk, respectively. The highest density and most selective localization of putative kappa receptors occurs in layers V and VI of the cerebral cortex. In these layers cells are localized which project to the thalamus regulating sensory input to the cortex. Receptors in these layers could account for the unique sedative and possibly analgesic effects of kappa opiates.     

Opiate antagonistic properties of an octapeptide somatostatin analog.

The somatostatin analog D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-NH-CH(CH2OH)CHOHCH3 (SMS 201-995) displaces [3h[naloxone from its binding sites (IC50, 38 +/- 60 nM), being more than 200 times more potent than somatostatin. As measured by the difference between [3H]dihydromorphine, [3H][D-Ala2,D-Leu5]enkephalin, and (-)-[3H]bremazocine binding, SMS 201-995 appears to be highly specific for the opiate mu binding site. Electrophysiological data from hippocampal cultures and results from animal studies (tail flick, mydriasis) demonstrate the opiate antagonistic properties of SMS 201-995. SMS 201-995 is an opiate mu antagonist with a peptide structure. That this property is displayed by a somatostatin analog is somewhat unexpected.     

Dynorphin 1-8 binds to opiate kappa receptors in the neurohypophysis

In order to clarify the effects of endogenous opiate peptides on the vasopressin system, we have investigated the presence of different opiate receptor subtypes in the neurohypophysis by radioreceptor assay and autoradiography. [3H]-etorphine binding to membrane preparations revealed the presence of high- and low-affinity binding sites (KD, 1.2 nM and 8.1 nM). Displacement of [3H]-etorphine by opiate receptor subtype-specific ligands gave the following results: the preferential mu agonists DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-oL) and the tetrapeptide morphiceptin did not displace etorphine; the preferential sigma receptor agonists DADLE (D-Ala2,D-Leu5-enkephalin) or DSTLE (D-Ser2,Leu5,Thr6-enkephalin) and beta-endorphin, a preferential agonist of the epsilon receptor, displaced [3H]-etorphine from its low-affinity site only, and dynorphin 1-8, a preferential kappa agonist, displaced [3H]-etorphine from its high-affinity binding site. Film autoradiography of neurohypophyseal sections incubated with [3H]-etorphine showed a displacement of 30% of the labeled ligand by unlabeled dynorphin 1-8. Exposure of rat neurointermediate lobes in organ culture to dynorphin 1-8 caused a small but significant stimulation of vasopressin release. These results demonstrate the existence of dynorphin 1-8 sensitive opiate receptors of the kappa subtype in the neurohypophysis and their possible involvement in vasopressin release.     

Characterization of [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin binding to delta opiate receptors in the rat brain and neuroblastoma--glioma hybrid cell line (NG 108-15).

Specific binding properties of the tritium-labeled delta opiate receptor agonist [3H][2-D-penicillamine, 5-D-penicillamine]enkephalin [( 3H][D-Pen2, D-Pen5]enkephalin) were characterized in the rat brain and in a mouse neuroblastoma-rat glioma hybrid cell line (NG 108-15). Saturation isotherms of [3H][D-Pen2, D-Pen5]enkephalin binding to rat brain and NG 108-15 membranes gave apparent Kd values of 1-6 nM. These values are in good agreement with the Kd value obtained from the kinetic studies. The Bmax value in NG 108-15 membranes was 235.3 fmol/mg of protein. An apparent regional distribution of [3H][D-Pen2, D-Pen5]enkephalin binding was observed in the rat brain. A number of enkephalin analogues inhibited [3H][D-Pen2, D-Pen5]enkephalin binding with high affinity (IC50 values of 0.5-5.0 nM) in both NG 108-15 and rat brain membranes. However, putative mu receptor-selective ligands such as morphine, [D-Ala2, MePhe4, Gly5-ol]enkephalin, [MePhe3, D-Pro4]morphiceptin, and naloxone were less effective inhibitors of [3H][D-Pen2, D-Pen5]enkephalin binding in both systems tested. These data suggest that [3H][D-Pen2, D-Pen5]enkephalin is a potent and selective ligand for the delta opioid receptor.     

Conformationally restricted analogs of somatostatin with high mu-opiate receptor specificity.

A series of cyclic, conformationally restricted analogs of somatostatin have been prepared and tested for their ability to inhibit the binding of [3H]naloxone and [D-Ala2, D-Leu5] [3H]enkephalin to rat brain membranes. The most potent analog, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 where Pen is penicillamine in [D-Phe5, Cys6, Tyr7, D-Trp8, Pen11]somatostatin-(5-12)-octapeptide amide, exhibited high affinity for mu-opiate receptors (IC50 value of [3H]naloxone = 3.5 nM), being 7800 times more potent than somatostatin. The cyclic octapeptide also displayed high mu-opiate receptor selectivity with an IC50 [( D-Ala2,D-Leu5]enkephalin)/IC50 (naloxone) ratio of 271. The high affinity and selectivity of the somatostatin analog for mu-opiate receptors may be of use in examining the physiological role(s) of the mu-opiate receptor.     

Thiazide diuretic drug receptors in rat kidney: identification with [3H]metolazone.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. We report here that [3H]metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. [3H]Metolazone bound with high affinity (Kd = 4.27 nM) to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or no binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated with their clinical potency. Halide anions (Cl-, Br-, and I-) specifically inhibited high-affinity binding of [3H]metolazone to this site. [3H]Metolazone also bound with lower affinity (Kd = 289 nM) to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had Ki values of 0.6-10 microM for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity [3H]metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.     

Multiple opiate receptors: [3H]ethylketocyclazocine receptor binding and ketocyclazocine analgesia.

The receptor binding of the kappa agonist [3H]ethylketocyclazocine to brain homogenates in vitro and ketocyclazocine (kappa) analgesia in vivo has been investigated and compared to morphine, a mu agonist. Saturation analysis of [3H]ethylketocyclazocine binding in both mice and rats yielded biphasic Scatchard plots similar to those of opiate mu agonists, antagonists, enkephalins, and endorphins. Treatment of brain membranes with monovalent and divalent cation, chelating agents, protein-modifying reagents, and enzymes affected [3H]ethylketocyclazocine binding in a manner similar to that of [3H]morphine. Naloxazone, a long-acting antagonist that selectively abolished high-affinity [3H-DAla2,Met5]enkephalinamide binding in vivo, also selectively blocked high-affinity [3H]ethylketocyclazocine binding. Evaluation of analgesia with writhing and tail-flick assays in animals whose high-affinity binding sites were blocked by naloxazone demonstrated a 6- to 7-fold increase in median effective dose (ED50) values of ketocyclazocine. This decrease in analgesic potency was comparable to morphine's decreased potency in similarly treated mice. These biochemical and pharmacological results suggest that the analgesic properties of both kappa and mu agonists may be mediated through the same subpopulation of receptors, the high-affinity binding sites.     

Interconverting mu and delta forms of the opiate receptor in rat striatal patches.

The binding of a radiolabeled "mu receptor" prototype opiate, dihydromorphine (H2morphine), and the binding of a "delta receptor" prototype, [D-Ala2,D-Leu5]enkephalin (D-Enk), to slide-mounted rat caudate slices were simultaneously compared quantitatively and visualized by autoradiography. Generally, D-Enk bound to opiate receptors distributed evenly throughout the entire striatum (type 2 pattern), whereas H2morphine labeled discrete islands or patches of receptors (type 1 pattern). In the presence of Mn2+ (3 mM) or other divalent cations, however, Na+ and GTP at 25 degrees C caused an increase in D-Enk binding at the expense of H2morphine binding at striatal opiate receptor patches. Thus, these conditions shifted D-Enk binding from an even pattern to one that included both an even and patchy distribution. These incubation conditions not only promoted D-Enk binding to striatal patches but also enabled the opiate receptor to regulate adenylate cyclase with the same (P less than 0.01) ligand selectivity pattern as that obtained by the displacement of D-Enk binding. The relative affinity of opiate receptors in striatal patches for opiate peptides, naloxone, and morphine appears to be a function of incubation conditions and coupling to adenylate cyclase and is not indicative of distinctly different opiate receptors. We postulate a three-state allosteric model consisting of mu agonist-, mu antagonists-, and adenylate cyclase-coupled delta-agonist-preferring states, whose equilibrium may be regulated by a sulfhydryl group mechanism.     

Morphine and opioid peptides reduce inhibitory synaptic potentials in hippocampal pyramidal cells in vitro without alteration of membrane potential.

We used intracellular recording in the hippocampal slice in vitro to characterize further the mechanisms behind the unusual excitatory action of opiates and opioid peptides on hippocampal pyramidal cells in vivo. No significant effect on resting membrane potential, input resistance, or action potential size in cortical area 1 (CA1) pyramidal cells was observed with morphine sulfate, beta-endorphin, [Met5]enkephalin, or [D-Ala2, D-Leu5]enkephalin at 1-50 microM. However, in all cells studied, these agents markedly reduced the size of inhibitory postsynaptic potentials generated by stimulation of the stratum radiatum or alveus. Excitatory postsynaptic potentials were also diminished in many of these cells. The effects of the opioids were antagonized by naloxone. These results are consistent with excitation of pyramidal neurons by a disinhibitory mechanism.     

Reconstitution of high-affinity opioid agonist binding in brain membranes.

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.     

Opiate receptor subtypes in the rat hypothalamus and neurointermediate lobe

The potent opiate radioligands [3H]etorphine, [3H]ethylketocyclazocine (EKC), and [3H]naloxone, bound specifically and saturably to a single class of membrane-binding sites in rat neurointermediate lobe (NIL), with Kd values of 3.7, 24, and 51 nM, respectively. In the hypothalamus (Ht), [3H]etorphine bound to specific and saturable sites with a Kd of 2.9 nM. Binding-inhibition studies with [3H]etorphine and unlabeled etorphine-HCl as well as [3H]EKC and unlabeled EKC, revealed high and low affinity binding sites in rat Ht and NIL as well as in the neural lobe of the bovine pituitary gland. [3H]naloxone also bound specifically to two classes of sites in Ht membranes, but to only a single class of low affinity sites in NIL membranes. Specific binding represented 80-90% of total [3H]etorphine binding, about 75% of total [3H]EKC binding, and 45-55% of total [3H]naloxone binding at 22 C in NIL and Ht, respectively. Relative binding potencies derived from Ki values for binding-inhibition studies of [3H]etorphine with opioid peptides and opiates were: NIL, etorphine-HCl greater than dynorphin A greater than naloxone-HCl greater than dynorphin-(1-9) greater than beta-endorphin much greater than alpha-neoendorphin approximately (Leu5)enkephalin approximately DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol); Ht, etorphine HCl greater than naloxone-HCl greater than beta-endorphin greater than dynorphin A much greater than DAGO greater than morphiceptin much greater than (Leu5)enkephalin. Specific [3H]etorphine binding was also demonstrable after preincubation of NIL membranes with DAGO and (Leu5)enkephalin and after preincubation of Ht membranes with morphiceptin and (Leu5)enkephalin; such binding could be displaced by nonradioactive dynorphin A. In addition, [3H]etorphine binding to bovine neural lobe was displaceable by naloxone-HCl, with an ED50 of 43 nM. Specific ligands for sigma-opiate receptors, such as (+)SKF 10,047 (N-allylnorcyclazocine), phencyclidine (PCP), and (-)cyclazocine, displaced specifically bound [3H]etorphine and [3H]EKC from NIL membranes only at high (micromolar) concentrations. However, specific [3H]PCP sites were of higher affinity in NIL and Ht membranes, with similar Kd values of 102 and 190 nM respectively, and different concentrations (0.15 and 1.32 pmol/mg protein, respectively). These data have revealed several differences in the opiate-binding properties of rat Ht and NIL membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties and localization of beta-endorphin receptor in rat brain.

Stereospecific high-affinity binding sites for beta h-[3H]endorphin could be demonstrated in the P2 pellet of rat brain homogenate. Scatchard analysis of the binding data revealed binding sites with Kd values of 0.81 and 6.8 nM and density of 120 and 240 fmol/mg of protein. Distribution of beta h-[3H]endorphin binding in various brain regions parallels that of opiate receptor:striatum greater than thalamus greater than amygdala greater than hypothalamus, septum greater than cortex greater than midbrain, brainstem. Similar to their effect on 3H-labeled agonist binding, Na+ and other monovalent cations, GTP, trypsin, chymotrypsin, phospholipase A2, and N-ethylmaleimide all inhibited the specific binding of beta h-[3H]endorphin. In contrast to their action on alkaloid and enkephalin binding, Ca2+, Mg2+, and Mn2+ also inhibited beta h-[3H]endorphin binding. These data suggest a difference between beta h-endorphin and alkaloid/enkephalin binding sites.