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.     

Identification of spinal 5-HT1C binding sites in the rat: characterization of [3H]mesulergine binding.

5-Hydroxytryptamine1C (5-HT1C) recognition sites were characterized in rat spinal cord using [3H]mesulergine. In competition experiments with different 5-HT receptor agonists and antagonists, the rank order of drug potencies was consistent with drug affinities for the 5-HT1C receptor: mesulergine, mianserin, 5-HT greater than ketanserin, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane greater than 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole, spiperone greater than 8-hydroxy-2-(di-n-propylamino)tetralin, pindolol. Bmax was 3.7 +/- 0.3 pmol/g and Kd 1.7 +/- 0.1 nM, with sites found in cervical, thoracic and lumbosacral cord. Inclusion of 20 nM spiperone to block potential 5-HT2 sites did not significantly alter drug affinities. There was a high correlation between drug affinities for [3H]mesulergine-labeled 5-HT1C sites in spinal cord and those reported in pig cortex (r = 0.94) and choroid plexus (r = 0.93), but poor correlation with 5-HT2 (high or low affinity states) or other 5-HT sites. Unlike [3H]mesulergine, the specific binding of [3H]5-HT, [3H]mianserin and [3H]ketanserin was low and no saturation studies could be performed with [3H]1-4-bromo-2-5-dimethoxy phenylisopropylamine. Limited competition studies suggest that [3H]5-HT labels 5-HT1C sites, [3H]ketanserin labels spinal 5-HT2 sites and [3H]mianserin labels both sites under the assay conditions studied, but the population of spinal 5-HT2 is small and not well characterized by these [3H]radioligands. In contrast, the population of spinal 5-HT1C receptors is substantial and [3H]mesulergine is the most useful [3H]radioligand for studies of spinal 5-HT1C sites.     

Characterization of [3H]leukotriene D4 binding sites in guinea-pig ventricular myocardium

[3H]Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High-performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 3% of membrane-bound [3H]LTD4 was converted to [3H]LTC4 or [3H]LTE4 at 30 degrees C. The specific [3H] LTD4 binding, assayed in the presence of 80 mM L-serine-borate, reached a stable steady state within 45 min at 30 degrees C (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded an apparent dissociation constant (Kd) of 3.4 +/- 2.1 nM and a maximum number of binding sites of 850 +/- 91 fmol/mg of protein. Competition binding studies with [3H]LTD4, synthetic 5S, 6R-LTD4 (LTD4) and its diastereoisomer 5R,6S-LTD4, LTE4, LTC4 and the putative LT antagonists FPL 55712, 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid (2-nor-LTD1) and SKF 88046 demonstrated a potency order of LTD4 greater than LTE4 greater than LTC4 greater than 5R,6S-LTD4 much greater than 2-nor-LTD1. FPL 55712 and SKF 88046 were ineffective in displacing the specific [3H]LTD4 binding. Pretreatment of the heart membranes with the sulfhydryl reducing reagent dithiothreitol decreased the specific [3H]LTD4 binding in a concentration-dependent manner. Scatchard analyses of saturation isotherms indicated that 0.3 mM dithiothreitol pretreatment of heart membranes decreased the maximum number of binding sites of the [3H]LTD4 binding to 368 +/- 61 fmol/mg of protein with minimal effects on the apparent Kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two distinct populations of [3H]prazosin and [3H]yohimbine binding sites in the plasma membranes of rat mesenteric artery

Postsynaptic alpha adrenoceptor subtypes have been investigated by radioligand binding studies in plasma membrane vesicles prepared from rat mesenteric arteries using [3H]prazosin and [3H]yohimbine. Both the radioligands displayed monophasic saturation in binding with a single component on Scatchard analysis. In the estrogenized female rat mesenteric artery, the specific binding of [3H]prazosin was rapid, saturable, reversible and of high affinity (0.65 +/- 0.05 nM) with a maximum binding capacity (Bmax) of 177 +/- 14 fmol/mg of protein. The maximum number of [3H]yohimbine binding sites were 427 +/- 31 fmol/mg of protein with the Kd equal to 34.5 +/- 3.8 nM. There was no evidence of cooperativity in the binding of both the ligands. The Kd values of [3H]prazosin and [3H]yohimbine, calculated from their respective kinetic analyses of binding, were in good agreement with the Kd values estimated from Scatchard plots. Prazosin was 15,000 times more potent in competing at the [3H]prazosin binding sites than at the [3H]yohimbine sites. In contrast, unlabeled yohimbine was 100-fold more potent in competing at the [3H]yohimbine binding sites than at the [3H]prazosin sites. The affinity of BE 2254 was 10,500 times higher for the [3H]prazosin binding sites than its affinity for the [3H]yohimbine binding sites. Non-alpha adrenoceptor antagonists competed poorly for both the radioligand binding sites. The Kd and Bmax of [3H]prazosin and [3H]yohimbine binding in the membranes of male rat mesenteric arteries were not significantly different from the corresponding values in the membranes of estrogenized female rat mesenteric artery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of high affinity [3H]pirenzepine and (-)-[3H] quinuclidinyl benzilate binding to muscarinic cholinergic receptors in rabbit peripheral lung

We have characterized the binding of the selective muscarinic antagonist [3H]pirenzepine ([3H])PZ) and the classical muscarinic antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) to muscarinic cholinergic sites in rabbit peripheral lung membranes. For both radioligands, high affinity binding with pharmacologic specificity was demonstrated. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 4.5 nM and the Kd for (-)-[3H]QNB binding was 6.2 pM. Comparison of the total binding capacity values determined by saturation experiments with [3H] PZ and (-)-[3H]QNB demonstrates that approximately 78% of the total muscarinic binding sites in rabbit peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (-)-[3H]QNB binding by the muscarinic agonist carbachol in peripheral lung membranes. If the pulmonary muscarinic receptor with high affinity for PZ proves to have an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.     

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)     

3H]Mianserin: differential labeling of serotonin and histamine receptors in rat brain

Mianserin, a tetracyclic antidepressant, is a potent serotonin (5-HT) and histamine H1 antagonist in peripheral smooth muscle systems. Mianserin was found to possess high affinity for 5-HT2 and histamine H1 receptor binding sites in brain membranes. By using [3H]mianserin, both 5-HT2 and histamine H1 receptors can be specifically labeled in rat cerebral cortex membranes. Simultaneous incubation of brain membranes with 300 nM triprolidine or 30 nM spiroperidol enables the selective labeling of 5-HT2 or histamine H1 receptors, respectively. In the guinea-pig cerebellum, [3H]mianserin exclusively labels histamine H1 receptors, since 5-HT2 sites are virtually absent in this area.     

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.     

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)     

Identification and characterization of [3H]nitrendipine binding sites in rat spinal cord

The present study reports the existence of high-affinity [3H]nitrendipine ([3H]NIT) binding sites in rat spinal cord. Characterization studies revealed [3H]NIT binding to synaptosomes to be specific, rapid and saturable, occurring at a single population of sites. The Bmax was 51 fmol/mg of protein and Kd 0.22 nM with a Hill slope of 0.96. Studies with nifedipine and verapamil demonstrated that the latter binds to a site allosterically linked to the 1,4-dihydropyridine binding sites in spinal cord. The Ca++ channel agonist methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5 carboxylate acted competitively at the 1,4-dihydropyridine binding site and inhibited specific [3H]NIT binding completely. Organic Ca++ antagonists inhibited binding to various degrees. Treatment with EDTA reduced specific [3H]NIT binding in spinal cord by 83%. This was restored by externally added Ca++. The effect of various mono-, di- and trivalent cations on specific [3H]NIT binding as well as its restoration in EDTA-treated preparations was tested. Na+, K+, Li+, Ca++, Mg++, Mn++ and Ba++ were found to have no significant effect. Other cations inhibited binding of [3H]NIT in the sequence La greater than Cd++ greater than Cu++ greater than Co++. Regional studies in rat spinal cord demonstrated 3-fold higher specific [3H]NIT binding sites in the dorsal cord compared to the ventral cord. Moreover, further variations were also found in cervical, thoracic and lumbar regions of the spinal cord. The results demonstrate that binding of the labeled calcium antagonist in spinal cord membranes is of high affinity and completely reversible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cocaine and related drugs in nonhuman primates. I. [3H]cocaine binding sites in caudate-putamen

Specific binding sites for [3H]cocaine were identified in caudate-putamen membranes prepared from nonhuman primate brains (Macaca fascicularis and Saimiri sciureus). Saturation of the sites was determined in competition studies using a fixed concentration of [3H]cocaine (2.7 nM) and increasing concentrations of unlabeled cocaine (1 pM-100 microM). Computer resolution of the shallow displacement curve (nH, 0.58) revealed that a two-component binding model [Kd1, 19.2 nM, maximum binding1 (Bmax1), 28.3 pmol/g of tissue; Kd2, 1120 nM, Bmax2, 431 pmol/g of tissue] was statistically preferred over a one-component model (K.50, 283 nM, Bmax, 471 pmol/g of tissue). Binding of [3H]cocaine was NaCl-dependent, with specific binding reduced by 72% when NaCl (100 mM) was omitted from the incubation medium. [3H]Cocaine was displaced stereoselectively by the enantiomers of cocaine and by the diastereoisomers of cocaine and its phenyltropane analog. Cocaine congeners displaced specifically bound [3H]cocaine with IC50 values ranging from 17 nM to over 100 microM in the following rank order of potency: WIN 35,428 greater than WIN 35,065-2 greater than (-)-cocaine greater than WIN 35,981 greater than (-)-norcocaine greater than WIN 35,140 greater than (+)-cocaine, (+)-pseudococaine greater than 3 alpha-tropanyl-1H-indole-carboxylic acid ester greater than 1 alpha H-3 alpha-5 alpha H-tropan-3-yl-3,5-dichlorobenzoate greater than benzoylecgonine, benzoylnorecgonine and (-)-pseudococaine. Several monoamine uptake inhibitors structurally unrelated to cocaine also displaced [3H]cocaine with IC50 values ranging from 1.6 nM to 50 microM. The rank order of potency was: ( +/- )-trans-3-(3',4'-dichlorophenyl)-N-methyl-1-indanamine greater than mazindol greater than nomifensine greater than methylphenidate 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]- 4-(3-phenylpropyl)piperazine, N-[1-(2- benzo(b)thiophenyl)cyclohexyl]piperidine greater than (-)-cocaine greater than 1-amino-4-phenylbicyclo-[2,2,2]-octane greater than bupropion, nisoxetine greater than desipramine, talsupram greater than citalopram. Other drugs, including the dopamine releasing agent (+)-amphetamine and the dopamine receptor agonists (-)-apomorphine, (+)-4-propyl-9-hydroxy-naphthoxazine, quinpirole and SKF 38393 were weak displacers of [3H]cocaine. Monoamine neurotransmitters also were relatively weak, but dopamine was considerably more potent than either norepinephrine or serotonin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [3H]acetylcholine release from rabbit retina labeled in vitro with [3H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [3H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [3H]acetylcholine with the following order of potency: apomorphine greater than or equal to SKF(R)82526 greater than SKF 85174 greater than SKF(R)38393 greater than or equal to pergolide greater than or equal to dopamine (EC50 = 4.5 microM) greater than SKF(S)82526 greater than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of [3H]acetylcholine: SCH 23390 (IC50 = 1 nM) greater than (+)-butaclamol greater than or equal to cis-flupenthixol greater than fluphenazine greater than perphenazine greater than trans-flupenthixol greater than R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating [3H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [3H]SCH 23390, or as determined by adenylate cyclase activity. [3H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [3H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [3H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [3H]SCH 23390 binding sites (r = 0.755, P greater than .05, n = 8). The potencies of antagonists to inhibit dopamine-evoked [3H]acetylcholine release were correlated with their potencies to inhibit the dopamine-stimulated adenylate cyclase (r = 0.759, P less than .05, n = 5) and with their affinities at [3H]SCH 23390 binding sites (r = 0.998, P less than .01, n = 7). We conclude that in rabbit retina dopamine evokes calcium-dependent [3H]acetylcholine release through activation of a site with the pharmacological characteristics of a D-1 dopamine receptor.     

Temperature-dependent 5-hydroxytryptamine (5-HT)-sensitive [3H]spiperone binding to rat cortical membranes: regulation by guanine nucleotide and antidepressant treatment

5-Hydroxytryptamine (5-HT)-sensitive binding of [3H]spiperone to rat cortical membranes was examined at 22 degrees C and 37 degrees C. At the lower temperature a significantly higher density of specific sites with identical affinity for [3H]spiperone (0.15 nM) was observed. Furthermore, a proportion of sites at 22 degrees C, although not at 37 degrees C, possessed high affinity for 5-HT and these were apparently converted to low affinity sites by GTP (100 microM). Examination of the apparent affinities of the 5-HT derivatives 5-methoxytryptamine, 5,6-dihydroxytryptamine and tryptamine revealed differences in the potencies of these analogs that were particularly evident for the high affinity proportion of sites observed at 22 degrees C. Subchronic treatment of rats with the antidepressants mianserin and iprindole reduced the density of [3H] spiperone binding sites when assays were performed at either temperature. However, antidepressant treatment appeared to preferentially reduce the number of sites that possess high affinity for 5-HT.     

Identification and characterization of two sigma-like binding sites in the mouse neuroblastoma x rat glioma hybrid cell line NG108-15.

NG108-15 cells were shown to possess high affinity binding sites for 1,3-di(2-[5-3H]tolyl)guanidine ([3H]DTG), a selective sigma ligand (Kd = 23 nM; maximal number of binding sites = 15.6 pmol/mg). The rank order of potency of drugs at this site was DTG greater than haloperidol greater than pentazocine greater than 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] greater than phencyclidine (PCP) greater than metaphit greater than (-)-3-PPP greater than (-)-N-allylnormetazocine [(-)-SKF 10,047] greater than (-)-butaclamol greater than (+)-butaclamol greater than (+)-SKF 10,047 greater than dizolcipine (MK 801 [5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine- maleate]) greater than ketamine. Both, Kd value and pattern of ligand selectivity suggest a close relationship to sigma sites in rodent brain. However, in comparison to sigma sites in brain stereoselectivity for the benzomorphan, SKF 10,047 was reversed and the affinities for benzomorphans were only moderate to low. Thus, sigma binding sites in NG108-15 cells seem to correspond to recently detected sigma sites in a pheochromocytoma cell line (PC12). [3H]-1-(2-thienyl)-cyclohexyl]piperidine ([3H]TCP), a PCP receptor-selective ligand binds to NG108-15 cells with moderate affinity (Kd = 139 nM; maximal number of binding sites = 4.7 pmol/mg of protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of vasoactive intestinal polypeptide (VIP) binding in circular muscle and characterization of VIP binding in canine small intestinal mucosa.

The present study examined the localization and characterization of [125I]vasoactive intestinal polypeptide (VIP) binding to synaptosomes and enterocyte membranes using preparations made from homogenized canine intestinal mucosa and compared it to [3H]saxitoxin binding and VIP-immunoreactive content (markers for synaptosomes). The highest [125I]VIP binding was located in the P2 fraction and was correlated with the locations of maximal [3H]saxitoxin binding and VIP-immunoreactive content. This correlation indicates that VIP receptors are present on synaptosomes of canine small intestinal mucosa. A fraction enriched in synaptosomes contained a high density of saturable VIP receptors (352 +/- 26.40 fmol/mg) having high affinity (Kd, 0.23 nM) for [125I]VIP. Studies of association and dissociation of [125I]VIP to this site revealed that binding was fully reversible and yielded a Kd value similar to that from equilibrium binding. Competition binding experiments suggested the presence of two binding sites, a high and a low affinity binding site. The order of competition potency was VIP greater than peptide histidine isoleucine greater than secretin greater than peptide histidine methionine greater than or equal to [D-Ala4]VIP greater than or equal to [Phe1]VIP greater than VIP10-28 greater than [4-Cl-D-Phe6-Leu17]VIP. All these competitors displaced all specifically bound VIP. VIP, peptide histidine isoleucine and secretin interacted differentially with each of the two binding sites. Peptide histidine methionine, [D-Ala4]VIP, [Phe1]VIP, VIP10-28 and [4-Cl-D-Phe6-Leu17]VIP interacted with a single low affinity at all binding sites. Other VIP binding sites were sought in circular muscle and submucosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]SCH 23390 labels both dopamine-1 and 5-hydroxytryptamine1c receptors in the choroid plexus

[3H]SCH 23390 has been used to label the D-1 subtype of dopamine receptors. Quantitative autoradiographic studies with [3H]SCH 23390 have revealed high levels of binding in many regions of rat brain including the caudate-putamen, nucleus accumbens, substantia nigra and choroid plexus. The selectivity of the binding of [3H]SCH 23390 was characterized further in studies using homogenates of canine choroid plexus. Scatchard analysis of the binding of increasing concentrations of [3H]SCH 23390 resulted in a curvilinear plot when (+)-butaclamol was used to define specific binding. Nonlinear regression analysis of untransformed data was consistent with the presence of two distinct classes of binding sites. The high-affinity site (Kd, 0.4 nM) was present at low density (maximum binding sites, 30 fmol/mg of protein) and had the pharmacological properties expected of a D-1 receptor. The low-affinity site (Kd, 24 nM) was present at a much greater density (maximum binding sites, 350 fmol/mg of protein) and had the pharmacological properties expected of a 5-hydroxytryptamine1c receptor. Quantitative autoradiographic studies of the binding of [3H]SCH 23390 to sections of rat brain also suggested that 5-hydroxytroptamine1c receptors in the choroid plexus are labeled by [3H]SCH 23390. It is possible that [3H]SCH 23390 labels 5-hydroxytryptamine1c receptors in other brain regions as well.     

In vivo labeling of 5-hydroxytryptamine uptake sites in mouse brain with [3H]-6-nitroquipazine

6-Nitroquipazine (DU 24565; 6-nitro 2-piperazinylquinoline) is a very potent 5-hydroxytryptamine (5-HT; serotonin) uptake inhibitor. It has been demonstrated very recently that [3H]-6-nitroquipazine is a suitable radioligand for studying 5-HT uptake sites. The present study evaluates [3H]6-nitroquipazine as a radioligand for in vivo labeling of 5-HT uptake sites in mouse brain. Very high uptake of radioactivity in the brain after i.v. administration of [3H]-6-nitroquipazine was shown. Regional distribution of the radioactivity in mouse brain 3 hr after injection of [3H]-6-nitroquipazine was in the order (highest to lowest) hypothalamus greater than midbrain greater than striatum greater than hippocampus greater than cerebral cortex greater than medulla oblongata greater than cerebellum. The regional distribution of in vivo [3H]-6-nitroquipazine binding in mouse brain was highly correlated with that in rat brain obtained from previous in vitro binding studies. Coadministration of carrier 6-nitroquipazine (5 mg/kg) significantly decreased the radioactivity in the hypothalamus, whereas that in the cerebellum and cerebral cortex was increased. Because the cerebellum has very low density of [3H]-6-nitroquipazine binding sites, the radioactivity in the cerebellum could, therefore, reflect the amount on nonspecific binding and free ligand. Kinetic studies showed highest in vivo specific binding 1 hr after injection of [3H]-6-nitroquipazine and slow clearance of specific binding. Specific binding in the hypothalamus was inhibited in a stereoselective manner by the stereoisomers of norzimelidine. Furthermore, specific binding in the hypothalamus was reduced by several 5-HT uptake inhibitors, in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of 5-hydroxytryptamine1B receptors in rat spinal cord via [125I]iodocyanopindolol binding and inhibition of [3H]-5-hydroxytryptamine release.

The aim of the present study in rat spinal cord synaptosomes was to compare the pharmacological characteristics of the serotonin (5-HT)1B receptor defined by [125I]iodocyanopindolol [( 125I] ICYP) binding and the 5-HT autoreceptor defined by inhibition of [3H]-5-HT release. In Percoll gradient Fractions 3 and 4 of spinal cord synaptosomes, a single saturable binding site for [125I]ICYP with a maximum binding of 70 and 134 fmol/mg, respectively, was demonstrated in the presence of 30 microM isoproterenol. The Kd of 0.16 nM did not vary between fractions. Competition for [125I]ICYP binding by various 5-HT agonists and antagonists also indicated a single site model based on a Hill coefficient of approximately 1.0. The most potent compounds at displacing [125I]ICYP binding were RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole), 5-carboxyamidotryptamine HCl, 5-methoxytryptamine, 5-HT and CGS 12066B (7-trifluoromethyl-4(4 methyl-1-pyrolo[1,2-a]-quinoxaline malate). [125I]ICYP binding was not altered by compounds with activity at 5-HT1A, 5-HT1C, 5-HT2, 5-HT3 or alpha-2 receptor sites. Similar to the pharmacological characteristics of the 5HT1B site defined by [125I]ICYP, compounds most active at inhibiting 15 mM K(+)-stimulated release of [3H]-5-HT were RU24969 = 5-carboxyamidotryptamine HCl = CGS 12066B greater than 5-methoxytryptamine greater than 5-HT. Compounds with activity at 5-HT1A, 5-HT1C, 5-HT2 or 5-HT3 sites were inactive. A correlation analysis of selective 5-HT1B compounds comparing the pKD for displacement of [125I]ICYP vs. the IC50 for inhibition of [3H]-5-HT release demonstrated the pharmacological similarity of the presynaptic inhibitory 5-HT autoreceptor and the 5-HT receptor site defined by [125I]ICYP binding in spinal cord synaptosomes (r = 0.791, P = .0193). Although [125I]ICYP binding was unaltered, alpha-2 agonists such as clonidine, norepinephrine and UK 14304 [5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) as well as the alpha-2 antagonists rauwolscine and yohimbine also decreased the K(+)-stimulated release of [3H]-5-HT and phentolamine, an alpha-2 antagonist increased release. The action of these alpha-2 compounds to alter [3H]-5-HT release suggests the presence of heteroreceptors localized on 5-HT terminals in the spinal cord. These results point out that [125I]ICYP identifies the 5-HT1B receptor, and affinity of compounds for this site predicts action at the 5-HT1B autoreceptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of alpha-1 adrenergic receptors in the heart using [3H]WB4101: effect of 6-hydroxydopamine treatment

To identify and characterize the cardiac alpha-adrenoceptors, a radioreceptor binding assay using the potent alpha adrenergic antagonist, [3H]WB4101 was performed in rat hearts. Specific [3H]WB4101 binding to rat left ventricular homogenates was saturable, reversible and of high affinity (Kd = 0.18 nM) with a Bmax of 2.57 fmol/mg of tissue (27.7 fmol/mg of protein). Adrenergic agonists competed for specific [3H]WB4101 binding in the order: (-)-epinephrine > (-)-norepinephrine greater than (-)-isoproterenol. Stereospecificity of the [3H]WB4101 binding sites was also demonstrated with (-)-epinephrine, (Ki = 90) nM being 270 times as potent as (+)-epinephrine, (K1 = 24 microM). Adrenergic antagonists competed for the binding in the order: WB4101 = prazosin greater than yohimbine greater than (-)-propranolol. WB4101 and prazosin exhibited a markedly greater (2000 times) affinity for [3H]WB4101 binding sites than yohimbine. The affinities (pKi) of alpha agonists and antagonists for [3H]WB4101 binding sites in the rat heart closely correlated with their pharmacological potencies in the heart. Scatchard analysis for [3H]WB4101 binding, performed in five regions from control and 6-hydroxydopamine-treated rat hearts, revealed specific [3H]WB4101 binding (Bmax) significantly greater in the ventricles and intraventricular septae than in atria. At 1 week after 6-hydroxydopamine treatment, there was a significant increase (40%) in the Bmax for [3H]WB4101 binding to ventricles and intraventricular septae without a change in Kd. We conclude: 1) [3H]Wb4101 selectively labels postsynaptic alpha-1 adrenoceptors in the rat heart; 2) there is a definite regional variation for cardiac alpha-1 adrenoceptors; and 3) 6-hydroxydopamine treatment caused a significant increase in the density of alpha-1 adrenoceptors in ventricles and intraventricular septae, compatible with a postsynaptic localization of the [3H]WB4101 binding site.     

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.