Modulation of rat brain opioid receptors by cannabinoids

The interaction of delta 9-tetrahydrocannabinol (delta 9-THC) and related cannabinoids with opioid receptors of neuronal membranes has been investigated. Treatment of membranes with delta 9-THC consistently decreased specific in vitro binding of [3H]dihydromorphine (mu opioid) in a dose-dependent fashion. Similar dose-dependent changes were elicited by cannabidiol and (+/-)-hexahydrocannabinol. Equilibrium binding studies in which brain membranes were titrated with [3H]dihydromorphine in the presence of delta 9-THC demonstrated that the decrease in [3H]dihydromorphine binding is due to a reduction in the number of binding sites, with no significant alteration in receptor affinity. This result suggests that the interaction of delta 9-THC with opioid receptors is a noncompetitive one. Delta 9-THC also inhibited the binding of the delta opioid [3H]D-Pen2, D-Pen5-enkephalin and the opioid antagonist [3H]naloxone (Ki = 16 and 19 microM, respectively) but failed to inhibit the binding of the kappa opioid [3H]ethylketocyclazocine (after suppression of mu and delta receptor binding), the phencyclidine analog [3H]N-(1-[2-theinyl]cyclohexyl)piperidine, the dopamine antagonist [3H]spiroperidol or the muscarinic antagonist [3H]quinuclidinyl benzilate. Moreover, delta 9-THC inhibited the binding of [3H]etorphine (potent opioid agonist) to solubilized, partially purified opioid receptors with a Ki value similar to that observed for the membrane-bound receptors. This finding indicates that the allosteric modulation of the opioid receptor by delta 9-THC is the result of a direct interaction with the receptor protein or with a specific protein-lipid complex and not merely the result of a perturbation of the lipid bilayer of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2,D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Central and peripheral cocaine receptors

High-affinity binding sites for [3H]cocaine (levo-[benzoyl-3,4-3H(N)]) were detected in membrane preparations from rat brain and liver. Analysis of binding to rat whole brain membranes revealed a high-affinity site (Kd = 16 nM) present at 0.65 pmol/mg of protein and a lower affinity site of (Kd = 660 nM) present at 5.1 pmol/mg of protein. The striatum had a much higher density of [3H]cocaine binding sites than either the frontal or occipital cortices. Also, the ratio of high/low affinity sites was highest for striatum, lower for cortices and lowest for whole brain. Scatchard analysis of cocaine binding to striatum and cortex suggested that, in addition to the high affinity binding (Kd = 16 nM), there were two or more lower affinity sites. Liver membranes bound [3H]cocaine with a single high affinity (Kd = 1.7 nM) present at high concentrations (Bmax = 66 pmol/mg of protein). Binding of cocaine to both brain and liver membranes was sensitive to proteases, reduced by high Na+ concentrations (30-100 mM), eliminated by denaturing temperatures (i.e., 95 degrees C for 5 min) and optimal at pH 7.4. Binding of [3H]cocaine to the striatal and cortical synaptic membranes was inhibited by cocaine and analogs in the following decreasing rank order: (-)-cocaine greater than (-)-norcocaine greater than (-)-cinnamoylcocaine greater than (+)-pseudococaine but (-)-ecgonine had no effect. The effective analogs also inhibited [3H]norepinephrine (levo[7-3H(N)]) and [3H]dopamine (dihydroxyphenylethylamine, 3,4-[7-3H]) uptake into cortical and striatal synaptosomes, respectively, with similar rank order.(ABSTRACT TRUNCATED AT 250 WORDS)     

D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin: a selective high affinity radioligand for delta opioid receptors with exceptional specific activity.

[D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin ([125I]DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. [125I]DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 +/- 67 pM and a receptor density (Bmax) value of 36.4 +/- 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM [125I] DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 +/- 0.88 X 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM [3H DPDPE and 0.8 nM [3H] [D-Pen2,4'-Cl-Phe4, D-Pen5]enkephalin, respectively. The dissociation rate of [125I]DPDPE (0.917 +/- 0.117 X 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of [125I]DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes [125I]DPDPE a valuable new radioligand for studies of delta opioid receptors.     

Identification of a high-affinity peripheral-type benzodiazepine binding site in rat aortic smooth muscle membranes

The existence of a benzodiazepine binding site in rat aortic smooth muscle membranes was explored employing [3H]Ro5-4864 as radioligand. The binding site was concentrated in the mitochondrial fraction enriched with cytochrome c oxidase and semicarbazide-insensitive monoamine oxidase. [3H]Ro5-4864 binds to the membranes in the mitochondrial fraction with high affinity. The dissociation constant (KD) determined by saturation binding was 2.8 +/- 0.7 nM (n = 5). The association rate constant (k1) was 4.7 +/- 0.8 x 10(6) M1 min-1, and the dissociation rate constant (k-1) was 0.028 +/- 0.005 min-1 (n = 3). The kinetically determined KD was 6.0 +/- 0.8 nM (n = 3) at 0.5 nM [3H]Ro5-4864. The density of binding determined from saturation binding experiments was 14.0 +/- 1.2 pmol/mg protein (n = 5). The Hill coefficient of binding was 0.94 +/- 0.02 (n = 5) indicating that [3H] Ro5-4864 binds to a single site. The [3H]Ro5-4864 binding was inhibited by Ro5-4864 (Ki = 6.1 +/- 1.9 nM), PK 11195 (Ki = 8.9 +/- 1.8 nM), diazepam (Ki = 87.3 +/- 3.4 nM), flunitrazepam (Ki = 94.6 +/- 1.8 nM), clonazepam (Ki = 6.3 +/- 1.3 microM) and Ro15-1788 (Ki = 16.8 +/- 1.5 microM). The rank order of potency of the competitive inhibition of [3H]Ro5-4864 binding (Ro5-4864 = PK 11195 greater than diazepam = flunitrazepam much greater than clonazepam greater than Ro15-1788) is characteristic of the peripheral-type benzodiazepine binding site. The data indicate an abundant high affinity peripheral-type benzodiazepine binding site of unknown function in rat aortic smooth muscle cells.     

Ascorbic acid prevents nonreceptor "specific" binding of [3H]-5-hydroxytryptamine to bovine cerebral cortex membranes

[3H]-5-Hydroxytryptamine ([3H]-5-HT) decomposes rapidly when exposed to air in solution at physiological pH if antioxidants are not present. The decomposition products appear to bind to two saturable sites on brain membranes (apparent Kd values = 1-2 and 100-1000 nM). This binding mimics "specific" ligand/receptor binding in that it is inhibited by 10 microM unlabeled 5-HT. This inhibition is not competitive, but rather is due to the prevention of [3H]-5-HT breakdown by excess unlabeled 5-HT. Unlike genuine ligand/receptor binding, the binding of [3H]-5-HT breakdown products is essentially irreversible and does not display a tissue distribution consistent with binding to authentic 5-HT receptors. [3H]-5-HT decomposition can be eliminated by the inclusion of 0.05 to 5 mM ascorbic acid. At these concentrations ascorbic acid is not deleterious to reversible [3H]-5-HT binding. When [3H] 5-HT exposure to air occurs in the presence of brain membranes, the apparent antioxidant activity of brain membranes themselves affords protection against [3H]-5-HT degradation equal to ascorbic acid. This protection is effective below final [3H]-5-HT concentrations of 10 nM. Above 10 nM [3H]-5-HT, addition of ascorbic acid or other antioxidants is necessary to avoid the occurrence of additional low affinity (apparent Kd = 15-2000 nM) binding sites that are specific but nonetheless irreversible. When care is taken to limit [3H]-5-HT oxidation, the only reversible and saturable specific binding sites observed are of the 5-HT1 high affinity (Kd = 1-2 nM) type. Radioligand oxidation artifacts may be involved in previous reports of low affinity (Kd = 15-250 nM) [3H]-5-HT binding sites in brain membrane preparations.     

Characterization of opioid-binding sites in zebrafish brain

The pharmacological profile of opioid-binding sites in zebrafish brain homogenates has been studied using radiolabeled binding techniques. The nonselective antagonist [(3)H]diprenorphine binds with high affinity (K(D) = 0.27 +/- 0.08 nM and a B(max) = 212 +/- 14.3 fmol/mg protein), displaying two different binding sites with affinities of K(D1) = 0.08 +/- 0.02 nM and K(D2) = 17.8 +/- 9.18 nM. The nonselective agonist [(3)H]bremazocine also binds with high affinity to zebrafish brain membranes but only displays one single binding site with a K(D) = 1.1 +/- 0.09 nM and a B(max) = 705 +/- 19.3 fmol/mg protein. Competition binding assays using [(3)H]diprenorphine and several unlabeled ligands were performed. The synthetic selective agonists for mammalian opioid receptors DPDPE ([DPen(2),D-Pen(5)]-enkephalin), DAMGO ([D-Ala(2),NMe-Phe(4),Gly(5)-ol]-enkephalin), and U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] failed to effectively displace [(3)H]diprenorphine binding, whereas nonselective ligands and the endogenous opioid peptides such as dynorphin A showed good affinities in the nanomolar range, although several of the endogenous peptides only displaced approximately 50% of the specifically bound [(3)H]diprenorphine. Our results provide evidence that, although the selective synthetic compounds for mammalian receptors do not fully recognize the opioid-binding sites in zebrafish brain, the activity of the endogenous zebrafish opioid system might not significantly differ from that displayed by the mammalian opioid system. Hence, the study of zebrafish opioid activity may contribute to an understanding of endogenous opioid systems in higher vertebrates.     

A new, highly selective CCK-B receptor radioligand ([3H][N-methyl-Nle28,31]CCK26-33): evidence for CCK-B receptor heterogeneity

[N-methyl-Nle28,31]CCK26-33 (SNF 8702) is a nonsulfated cholecystokinin octapeptide analog that is highly selective for cholecystokinin-B (CCK-B) receptors. Inhibition studies using [125I] Bolton-Hunter-labeled CCK-8 show that SNF 8702 has over 4,000-fold greater affinity for CCK receptors in guinea pig cortex relative to those in guinea pig pancreas. SNF 8702 was tritium-labeled to a specific activity of 23.7 Ci/mmol and its binding properties characterized for guinea pig brain membrane preparations. [3H]SNF 8702 binds to a single site with high affinity (Kd = 0.69-0.90 nM) in guinea pig cortex, cerebellum, hippocampus and pons-medulla. Of these four tissues, the highest receptor density was measured in the cortex (86 fmol/mg of protein) and the lowest in the pons-medulla (22 fmol/mg of protein). In contrast to findings of single-site binding in some brain regions, evidence for CCK-B receptor heterogeneity is observed under other conditions. [3H]SNF 8702 binding to membranes prepared from whole guinea pig brain shows biphasic association kinetics at a concentration of 2.0 nM consistent with the presence of binding site heterogeneity. Binding site heterogeneity is consistently observed for [3H]SNF 8702 binding to guinea pig whole brain membranes in saturation studies where a high-affinity site (Kd = 0.31 nM) is distinguished from a low-affinity site (Kd = 3.3 nM). Binding site heterogeneity is also observed for the midbrain-thalamic region. CCK-B receptor heterogeneity is suggested by the effect of the guanyl nucleotide analogue, guanylyl-imidodiphosphate (Gpp(NH)p), on [3H]SNF 8702 binding to CCK-B receptors in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Naloxonazine effects on the interaction of enkephalin analogs with mu-1, mu and delta opioid binding sites in rat brain membranes

The authors have characterized the opioid receptors of rat brain membranes using self- and cross-displacement studies with both tritiated and unlabeled [D-Ala2, D-Leu5]-enkephalin and [D-Ala2, MePhe4, Gly-ol5]-enkephalin. Mathematical modeling demonstrated the presence of three classes of binding sites, corresponding to mu, delta and the putative mu-1 classes of site. Unlabeled naloxonazine shows high affinity for all three classes of sites, with highest affinity for the mu-1 sites. Membranes were preincubated with 50 nM naloxonazine or with controls (50 nM naloxone or buffer) for 30 min. Preincubation of membranes with 50 nM naloxonazine resulted in a dramatic, nearly 2-fold reduction in the binding of [3H][D-Ala2, D-Leu5]-enkephalin and [3H][D-Ala2, MePhe4, Gly-ol5]-enkephalin relative to the controls. Quantitative analyses using mathematical modeling with program "LIGAND" suggested that this effect was primarily "competitive," i.e., attributable to changes in affinity, with no apparent or detectable noncompetitive or irreversible effects on binding capacities for the three classes of sites.     

3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain

Characterization of the adenosine A2 receptor has been limited due to the lack of available ligands which have high affinity and selectivity for this adenosine receptor subtype. In the present study, the binding of a highly A2-selective agonist radioligand, [3H]CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) is described. [3H]CGS 21680 specific binding to rat striatal membranes was saturable, reversible and dependent upon protein concentration. Saturation studies revealed that [3H]CGS 21680 bound with high affinity (Kd = 15.5 nM) and limited capacity (apparent Bmax = 375 fmol/mg of protein) to a single class of recognition sites. Estimates of ligand affinity (16 nM) determined from association and dissociation kinetic experiments were in close agreement with the results from the saturation studies. [3H]CGS 21680 binding was greatest in striatal membranes with negligible specific binding obtained in rat cortical membranes. Adenosine agonists ligands competed for the binding of 5 nM [3H]CGS 21680 to striatal membranes with the following order of activity; CGS 21680 = 5'-N-ethylcarboxamidoadenosine greater than 2-phenylaminoadenosine (CV-1808) = 5'-N-methylcarboxamidoadenosine = 2-chloroadenosine greater than R-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6cyclopentyltheophylline greater than S-phenylisopropyladenosine. The nonxanthine adenosine antagonist, CGS 15943A, was the most active compound in inhibiting the binding of [3H]CGS 21680. Other adenosine antagonists inhibited binding in the following order; xanthine amine congener = (1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 1,3-dipropyl-8-cyclopentylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than 8-phenyltheophylline greater than 8-cyclopentyltheophylline = xanthine carboxylic acid congener greater than 8-parasulfophenyltheophylline greater than theophylline greater than caffeine. The pharmacological profile of both adenosine agonist and antagonist compounds to compete for the binding of [3H]CGS 21680 was consistent with a selective interaction at the high affinity adenosine A2 receptor. A high positive correlation (r = 0.98, P less than .01) was observed between the pharmacological profile of adenosine ligands to inhibit the binding of [3H]CGS 21680 and the selective binding of [3H]NECA (+50 nM CPA) to high affinity A2 receptors. However, some differences between these assays were found for compounds which have moderate affinity and nonselective actions at both the A1 and A2 adenosine receptor subtypes. Unlike data obtained with nonselective adenosine ligands, the present results indicate that [3H]CGS 21680 directly labels the high affinity A2 receptor in rat brain without the need to block binding activity at the A1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of [Dmt1]DALDA and DAMGO in binding and G protein activation at mu, delta, and kappa opioid receptors

[Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine) binds with high affinity and selectivity to the mu opioid receptor and is a surprisingly potent and long-acting analgesic, especially after intrathecal administration. In an attempt to better understand the unique pharmacological profile of [Dmt1]DALDA, we have prepared [3H][Dmt1]DALDA and compared its binding properties with that of [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Kinetic studies revealed rapid association of [3H][Dmt1]DALDA when incubated with mouse brain membranes (K+1 = 0.155 nM(-1) min(-1)). Dissociation of [3H][Dmt1]DALDA was also rapid (K(-1) = 0.032 min(-1)) and indicated binding to a single site. [3H][Dmt1]DALDA binds with very high affinity to human mu opioid receptor (hMOR) (Kd = 0.199 nM), and Kd and Bmax were reduced by sodium but not Gpp(NH)p [guanosine 5'-(beta,gamma-imido)triphosphate]. Similar Kd values were obtained in brain and spinal cord tissues and SH-SY5Y cells. The hMOR:hDOR (human delta opioid receptor) selectivity of [Dmt1]DALDA ( approximately 10,000) is 8-fold higher than DAMGO. However, [Dmt1]DALDA is less selective than DAMGO against hKOR (human kappa opioid receptor) (26-versus 180-fold). The Ki values for a number of opioid ligands were generally higher when determined by competitive displacement binding against [3H][Dmt1]DALDA compared with [3H]DAMGO, with the exception of Dmt1-substituted peptide analogs. All Dmt1 analogs showed much higher affinity for the mu receptor than corresponding Tyr1 analogs. [35S]GTPgammaS (guanosine 5'-O -(3-[35S]thio)triphosphate) binding showed that [Dmt1]DALDA and DAMGO are full agonists at hMOR and hDOR but are only partial agonists at hKOR. The very high affinity and selectivity of [3H][Dmt1]DALDA for the mu receptor, together with its very low nonspecific binding (10-15%) and metabolic stability, make [3H][Dmt1]DALDA an ideal radioligand for labeling mu receptors.     

Selective labeling of serotonin uptake sites in rat brain by [3H]citalopram contrasted to labeling of multiple sites by [3H]imipramine

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes [3H]citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for [3H]citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of [3H]citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of [3H]citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of [3H]imipramine binding reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific [3H]imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of [3H]imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in [3H]citalopram and serotonin-sensitive [3H]imipramine binding with only a small effect on serotonin-insensitive [3H]imipramine binding. The dissociation rate of [3H]imipramine or [3H]citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive [3H]imipramine high affinity binding sites closely resembles that of [3H]citalopram binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]zacopride binding to 5-hydroxytryptamine3 sites on partially purified rabbit enteric neuronal membranes

5-Hydroxytryptamine3 (5-HT3) receptors are present in both central and peripheral neuronal tissues but radioligand binding studies have thus far been limited to crude membranes from brain and vagus nerve. The present studies describe the isolation and characterization from the rabbit small bowel of neuronal membranes enriched in binding sites for the potent 5-HT3 ligand, [3H]zacopride. The number of specific [3H]zacopride binding sites per milligram of protein was increased 6-fold in a 10,000 to 100,000 x g membrane fraction as compared to the homogenate. [3H]Zacopride bound to these membranes with high specificity (greater than 90%), exhibited high affinity for a homogeneous population of binding sites (Kd = 0.3 nM) and its binding was inhibited competitively by other 5-HT3 compounds with the following rank order of potency: ICS 205-930 greater than GR 38032F greater than or equal to quipazine greater than BRL 24924 approximately MDL 72222 much greater than metoclopramide greater than 2-CH3-5-HT3. On a discontinuous sucrose gradient, specific [3H]zacopride binding was increased an additional 3.5-fold and copurified with three plasma membrane markers. Fractionation on a continuous sucrose gradient demonstrated that specific [3H]zacopride binding was associated with the enteric neuronal plasma membranes. Comparative studies in rabbit vagus nerve also demonstrated a large number (maximum binding = 148 fmol/mg of protein) of high affinity [3H]zacopride binding sites (Kd = 0.4 nM), in membranes that exhibited a density and binding characteristics similar to those from enteric neurons. Thus, membranes enriched in 5-HT3 binding sites can be isolated from both enteric and vagus neurons and [3H]zacopride is a potent ligand useful for characterization of these sites.     

Reversible and irreversible binding of beta-funaltrexamine to mu, delta and kappa opioid receptors in guinea pig brain membranes

The effect of beta-funaltrexamine (beta-FNA), an irreversible mu receptor blocker in isolated tissue bioassays, on mu, kappa and delta opioid receptor binding and the binding of beta-[3H]FNA were determined in guinea pig brain membranes. beta-FNA inhibited the binding of mu, kappa and delta opioid ligands to their receptors with Ki values of 2.2, 14 and 78 nM, respectively. Pretreatment of brain membranes with beta-FNA (less than 2 microM) followed by extensive washing inhibited mu binding and to a lesser degree delta binding, without changing kappa binding. The extent of the irreversible inhibition was dependent on the concentration of beta-FNA, and this inhibition on mu binding could be observed with as little as 1 nM beta-FNA. The irreversible inhibition of mu binding by beta-FNA pretreatment was due to a decrease in the number of binding sites with little change in Kd, and was more pronounced in the presence of increasing concentrations of NaCl. Specific binding of beta-[3H]FNA to opioid receptors was demonstrated. The rate of specific binding with 2 nM beta-[3H]FNA was rapid in the initial 10 min and did not reach maximum in 90 min. The dissociation of bound beta-[3H]FNA (5 nM added) by the addition of excess unlabeled naloxone reached maximum at 30 min with approximately 35% of specifically bound beta-[3H]FNA remaining. Mu opioids were most effective in preventing specific binding of beta-[3H]FNA when added before beta-[3H]FNA. Opioids added 1 hr after 2 nM beta-[3H]FNA could displace maximally only 70 to 75% of specific binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local anesthetics differentiate dihydropyridine calcium antagonist binding sites in rat brain and cardiac membranes

Local anesthetics were used to probe differences in the binding of [3H]nitrendipine to dihydropyridine calcium antagonist binding sites on rat brain and cardiac membranes. Local anesthetics inhibited [3H]nitrendipine binding to brain and cardiac membranes with the rank order of potency, dibucaine = proadifen much greater than tetracaine greater than meproadifen greater than RAC-109 (S) greater than RAC-109 (R) greater than benzocaine. Lidocaine, procaine, piperocaine and bupivacaine produced either a small potentiation or inhibition of [3H]nitrendipine binding. Dibucaine inhibited [3H]nitrendipine binding to brain membranes (IC50, 4.9 +/- 0.5 microM) by increasing the Kd, whereas in cardiac membranes (IC50, 8.5 +/- 0.9 microM) it both increased the Kd and decreased the maximum binding site capacity of [3H]nitrendipine. The potency of dibucaine to inhibit [3H]nitrendipine binding was reduced in both tissues by monovalent (Li+ greater than Na+ = K+ = Rb+; EC50, 40-50 mM) and divalent (Ca++, Mg++ and Mn++; EC50, 10-50 microM) cations. These cations reduced the effect of dibucaine on the Kd of [3H]nitrendipine in brain and on the maximum binding site capacity of [3H]nitrendipine in cardiac membranes. Inhibition of [3H]nitrendipine binding by dibucaine was best described by high (2 microM) and low (50 microM) affinity sites. The apparent affinities of these sites, but not the fractional occupancies, were similar in brain and cardiac membranes. Na+ modulated the occupancies of these sites in brain, but not in cardiac membranes, whereas Ca++ inhibited occupancy of the high affinity site in both tissues. The effects of Li+ were similar to those of Ca++. These findings indicate that brain and cardiac dihydropyridine calcium antagonist binding sites are coupled to different allosteric effectors or exist in a different membrane environment.     

Identification and characterization of delta opioid binding sites in the bovine pineal.

Bovine pineal membranes were shown to possess a single class of high-affinity binding sites for the opioid peptide, [125I]iodiotyrosyl27-beta-endorphin (beta E) (Kd = 47 pM, Bmax = 2.4 fmol/mg of tissue). The rank order of potency at this beta E site was deltorphin greater than [D-Ser2]-Leu-enkephalin-Thr greater than [D-Pen2,D-Pen5]enkephalin much greater than dermorphin greater than [D-Ala2,MePhe4,Gly5-ol]enkephalin much greater than (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl]] benzeneacetamide (U69593) greater than [des-Tyr1]beta E greater than beta E(6-31). These results suggest that beta E binds to delta opioid sites and excludes the possibility of significant binding to mu, kappa and epsilon sites. The presence of delta binding sites was confirmed by use of the delta selective ligand [3H][D-Pen2,D-Pen5]enkephalin (Kd = 1.5 nM). The Bmax observed using [D-Pen2,D-Pen5]enkephalin is similar to that obtained with [125I]beta E, confirming that essentially all pineal opioid sites are of the delta type. The virtual absence of mu opioid sites was confirmed using the mu-selective opioid ligand [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin. These results suggest that endogenous or circulating opioid peptides may modulate pineal function by interaction with delta opioid sites.     

Molecular heterogeneity of leukotriene receptors: correlation of smooth muscle contraction and radioligand binding in guinea-pig lung

The [3H]leukotriene C4 ([3H]LTC4) and [3H]leukotriene D4 ([3H] LTD4) specific binding sites in guinea-pig lung membranes were characterized and correlated with smooth muscle contractile activities of a series of LTC-, D- and E-type analogs. [3H]LTC4 bound to the specific sites with high affinity (dissociation constant Kd = 15 +/- 5 nM), saturable capacity (maximum binding = 68 +/- 15 pmol/mg of membrane protein), stereoselectivity and specificity. The [3H]LTC4 specific binding sites were detected in the membranes isolated from leukotriene sensitive (e.g., lung and heart) or insensitive (e.g., brain and red blood cells) tissues. [3H] LTD4 also bound to specific sites with high affinity (Kd = 0.20 +/- 0.05 nM), low capacity (maximum binding = 1.1 +/- 0.2 pmol/mg of membrane protein) stereoselectivity and specificity. The [3H] LTD4 specific binding sites were detected in the membranes isolated from lung and trachea. [3H]LTC4 specific binding was inhibited by treatment of the membranes with the sulfhydryl alkylating agent N-ethylmaleimide. [3H]LTD4 specific binding was more sensitive to heat treatment and p-hydroxymercuribenzoate than the [3H]LTC4 specific binding. Radioligand competition activities of the LTD- and LTE-type analogs correlated well with the agonist and antagonist smooth muscle contractile activities. In contrast, the radioligand competition activity of the LTC-type analogs did not correlate with smooth muscle contractile activities. These results indicate that the [3H]LTC4 and [3H]LTD4 specific binding sites in guinea-pig lung membranes are chemically and physically distinct. The [3H]LTD4 specific binding sites represent physiologically and pharmacologically important receptors, and the smooth muscle contraction induced by LTD-, and possible LTE-, type analogs are mediated through the LTD4 receptors.     

Guanyl-5'-yl-lmidodiphosphate regulation of ligand binding to LTD4 receptors on guinea pig lung membranes

We investigated the mechanism of guanyl-5'-yl-imidodiphosphate (GppNHp) regulation of peptidoleukotrienes (LTs) and LT-antagonists binding to LTD4 receptors on guinea pig lung membranes (GPLMs). In saturation experiments, [3H]LTD4 saturable (maximum binding = 943 +/- 39 fmol/mg of protein) binding to GPLM was significantly (P less than .01) inhibited by GppNHp (60 nM, maximum binding = 446 +/- 113 fmol/mg of protein) in a concentration-dependent manner. No significant change in the affinity (Kd = 0.29 +/- 0.02 nM vs. 0.43 +/- 0.12 nM for control and treated GPLM, respectively) for [3H]LTD4 was observed. The binding affinity for the selective LTD4 antagonist ICI 198,615 (Ki = 0.13 +/- 0.04 nM) as determined by competition against [3H]LTD4, was not changed by GppNHp. Saturation analysis of [3H]ICI 198,615 binding confirmed that GppNHp did not change the apparent affinity or site-density for this ligand. In competition experiments against [3H]-ICI 198,615, GppNHp (1 microM) caused a significant (P less than .01) rightward shift of the inhibition by agonists (94-, 50- and 8-fold shifts for LTD4, LTE4 and YM-17690, respectively). In contrast, inhibition of [3H]ICI 198,615 by four LTD4 antagonists (ICI 198,615, 4-[5-cyclopentylcarbonylamino-1-[3-cyanobenzyl] indol-3-yl-methyl]3-methoxybenzoic acid, 4-[5-cyclopentylcarbonylamino-3-chloroindol-1-y-methyl]3-met hoxybenzoic acid and FPL55712) was not affected by GppNHp. Taken together the data suggest that LTD4 receptors are coupled to a G-protein that modulates the affinity of agonists but not antagonists binding.     

Nalbuphine: an autoradiographic opioid receptor binding profile in the central nervous system of an agonist/antagonist analgesic

Nalbuphine is a potent agonist/antagonist analgesic with a low side effect profile and low abuse potential. Previous studies have shown that nalbuphine produces predominantly agonist (analgesic) effects at kappa receptors and antagonist (morphine-reversal) effects at mu receptors in vivo. The present study was designed to localize the sites of nalbuphine binding to mu, delta and kappa opioid receptors in the central nervous system (CNS) using in vitro labeling light microscopic autoradiography. Mu, delta and kappa opioid receptors were labeled selectively using [3H]dihydromorphine, D-[3H]Ala2-D-Leu5-enkephalin and (-)-[3H]ethylketocyclazocine, respectively. In displacement studies in rat brain homogenates, nalbuphine had the highest affinity (Ki) for mu receptors (0.5 nM) with progressively lower affinities for kappa (29 nM) and delta (60 nM) opioid receptors. In autoradiographic studies in slide-mounted sections of guinea pig brain and monkey spinal cord, nalbuphine (300 nM) displaced completely the binding at mu and kappa receptors without significantly altering the binding at delta receptors. The binding of [3H]nalbuphine in slide-mounted sections of guinea pig forebrain was saturable and showed a curvilinear profile indicating the presence of two binding sites with apparent dissociation constant (Kd) values of 0.5 and 12 nM. Morphine and U-50,488H, which have high affinities for mu and kappa opioid receptors, respectively, inhibited [3H]nalbuphine binding with IC50 values of 0.9 and 10 nM, respectively. In saturation studies, morphine (50 nM) and U-50,488H (100 nM) selectively blocked the high and low affinity components of [3H]nalbuphine binding, respectively. The autoradiographic distribution of [3H]nalbuphine binding sites in the CNS corresponds well to the distribution of mu and kappa opioid receptors. In addition, CNS areas (deep layers of the cerebral cortex, laminae I and II of the spinal cord, substantia gelatinosa of the trigeminal nerve, periaqueductal gray and thalamic nuclei) that mediate analgesia contain high concentrations of [3H]nalbuphine binding sites. In summary, these data demonstrate that nalbuphine acts on mu and kappa opioid receptors and identify anatomical loci in the CNS in which nalbuphine may produce its actions.     

Putative selective 5-HT-2 antagonists block serotonin 5-HT-1c receptors in the choroid plexus

The binding of [3H]mianserin to rat choroid plexus was characterized and compared with two other radioligands that label the 5-HT (serotonin)-1c receptor ([3H]mesulergine and [125I] lysergic acid diethylamide). [3H]Mianserin binding to a crude membrane preparation of choroid plexus from rat brain was rapid, saturable and of high affinity (Kd = 1 nM). The density of sites labeled by [3H]mianserin and [3H]mesulergine was equal. Furthermore, an excellent correlation was found between the potencies of drugs in competing for [3H]mianserin binding and for [125]lysergic acid diethylamide binding. Based on these data, it was concluded that [3H]mianserin labels the 5-HT-1c binding site. Using this ligand, the binding of the putative selective 5-HT-2 antagonist ritanserin to the 5-HT-1c site was evaluated. Ritanserin was a potent inhibitor of [3H]mianserin binding with a Ki value of 0.2 nM. Functional studies of 5-HT-stimulated phosphoinositide hydrolysis, the transmembrane signaling pathway for the 5-HT-1c receptor, showed that ritanserin blocks the effect of 5-HT and that it functions as a competitive antagonist of the 5-HT-1c receptor in intact choroid plexus. The potencies of ritanserin and several other drugs, including other 5-HT-2 antagonists, at the 5-HT-1c binding site correlated with their potencies at blocking 5-HT-stimulated phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)