Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Solubilization and Characterization of Striatal Dopamine Receptors

Abstract: Dopamine receptor binding proteins were sol-ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio - 2 - hydroxy - 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [³H]spiroperidol ([³H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO-solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga-tion at 100,000 ×g for 2 h. The CHAPSO-solubilized dopamine receptor proteins, as well as the prelabeled [³H]Spi-receptor protein complex, bind specifically to wheat germ agglutinin (WGA)-agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO-solubilized, prelabeled [³H]Spi-receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA-agarose-bound [³H]Spi-receptor protein complex with digitonin (CHAPSO-digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [³H]Spi-receptor complex in the CHAPSO-digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO-solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [³H]Spi to CHAPSO-solubilized dopamine receptor proteins are similar to those for membrane-bound proteins. Chronic treatment with haloperidol increases the B_max, and does not change the K_D for [³H]Spi in the CHAPSO-solubilized and in the membrane-bound preparations. Thus, the CHAPSO-solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane-bound dopamine receptors.     

High-and Low-Affinity States of Striatal D2 Receptors Are Not Affected by 6-Hydroxydopamine or Chronic Haloperidol Treatment

Specific D2 binding in rat striatum was characterized and then the effects of chronic disruption of dopaminergic activity on antagonist and agonist binding to these sites were studied. D2 receptors were defined as those sites capable of binding [3H]spiperone in the presence of cinanserin, a 5-HT2 antagonist, but not in the presence of (+)-butaclamol, a D2 and 5-HT2 blocker. Saturation, competition, and kinetic analyses suggested that D2 receptors are a homogeneous population exhibiting more complex interactions with agonists than antagonists. Antagonist binding was monophasic and guanine nucleotide-insensitive whereas agonist binding was biphasic and guanine nucleotide-sensitive. D2 receptor density was elevated by more than 40% following dopamine depletion by 6-hydroxydopamine or chronic receptor blockade by haloperidol. However neither treatment altered the affinities or magnitudes of the high- and low-affinity components associated with agonist binding to the D2 receptor.     

D2 Receptor Antagonists Do Not Modify Guanine Nucleotide-Sensitive Interactions Between Dopamine and D1 Dopamine Receptors Under In Vitro Conditions

Abstract: This study investigated possible D₁/D₂ interactions in rat and bovine striatal tissue by examining the effects of D₂ antagonists on the action of dopamine at D₁ dopamine receptors. In addition, the extent to which D₂ antagonists may induce an agonist low-affinity state of the D₁ receptor was evaluated in comparison with the effects of the guanine nucleotide analogue 5′-guanylylimidodiphosphate [Gpp(NH)p]. In saturation experiments dopamine caused a dose-dependent decrease in rat striatal and bovine caudate D₁ receptor density. This effect of dopamine, which has been shown to be sensitive to Gpp(NH)p, was not altered by pretreatment with either of the selective D₂ antagonists eticlopride (200 nM) or domperidone (200 nM). Results from displacement experiments show that the affinity of dopamine for D₁ receptors and the proportion of receptors in an agonist high-affinity state, are reduced by Gpp(NH)p (100 µM) but not by eticlopride. A molar excess of dopamine (100 µM) promotes the dissociation of (±)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol ([³H]SCH 23390) from rat striatal D₁ receptors at a rate that is significantly slower than when dissociation is initiated using 1 µM piflutixol. After pretreatment with Gpp(NH)p, [³H]SCH 23390 dissociation induced by dopamine occurred at an even slower rate. Pretreatment with eticlopride had no effect on the dopamine-induced rate of [³H]SCH 23390 dissociation. These results indicate that all experimental approaches detected dopamine effects at D₁ receptors that are Gpp(NH)p sensitive and D₂ antagonist insensitive and provide no evidence to support a D₁/D₂ link operating at the receptor level.     

Drug-Induced Up-Regulation of Dopamine D2 Receptors on Cultured Cells

Abstract: Ligand-induced up-regulation of recombinant dopamine D2 receptors was assessed using C₆ glioma cells stably expressing the short (415-amino-acid; D2_S) and long (444-amino-acid; D2_L) forms of the receptor. Overnight treatment of C6-D2_L cells with N-propylnorapomorphine (NPA) caused a time- and concentration-dependent increase in the density of receptors, as assessed by the binding of radioligand to membranes prepared from the cells, with no change in the affinity of the receptors for the radioligand. The effect of 10 µM NPA was maximal after 10 h, at which time the density of D2_L receptors was more than doubled. The agonists dopamine and quinpirole also increased the density of D2_L receptors. The receptor up-regulation was not specific for agonists, because the antagonists epidepride, sulpiride, and domperidone caused smaller (30–60%) increases in receptor density. Prolonged treatment with 10 µM NPA desensitized D2_L receptors, as evidenced by a reduced ability of dopamine to inhibit adenylyl cyclase, whereas treatment with sulpiride was associated with an enhanced responsiveness to dopamine. The magnitude of NPA-induced receptor up-regulation in each of four clonal lines of C6-D2_L cells (mean increase, 80%) was greater than in all four lines of C6-D2_S cells (33%). Inactivation of pertussis toxin-sensitive G proteins had no effect on the basal density of D2_L receptors or on the NPA-induced receptor up-regulation. Treatment with 5 µg/ml of cycloheximide, on the other hand, decreased the basal density of receptors and attenuated, but did not prevent, the NPA-induced increase. Chimeric D1/D2 receptors were used to identify structural determinants of dopamine receptor regulation. Treatment with the D1/D2 agonist NPA decreased the density of D1 and chimeric CH4 and CH3 receptors. The latter two receptors have D1 sequence from the amino-terminus to the amino-terminal end of transmembrane region (TM) VII and VI, respectively. CH2, with D1 sequence up to the amino-terminal end of TM V, and thus the third cytoplasmic loop of the D2 receptor, was up-regulated by NPA or the D2-selective agonist quinpirole. Quinpirole treatment decreased the density of CH3 and had no effect on CH4 or D1 receptors. The different responses of CH2 and CH3 to agonist treatment suggest a role for TM V and the third cytoplasmic loop in the direction of receptor regulation.     

pH Selectivity of N-Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

In Human Brain Two Subtypes of D1 Dopamine Receptors Can Be Distinguished on the Basis of Differences in Guanine Nucleotide Effect on Agonist Binding

D1 dopamine receptors were identified in membranes of human nucleus caudatus, nucleus accumbens, amygdala, and globus pallidus, by the specific binding of [3H](+)-R-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7 -ol [( 3H]SCH 23390). In these four brain regions, dopamine/[3H]SCH 23390 competition binding curves were computer-analyzed to a two-site model, distinguishing a high- (RH) and low- (RL) affinity site for dopamine. The ability of guanine nucleotides (0.4 mM GTP or 0.1 mM 5'-guanylylimidodiphosphate) to provoke a conversion of RH into RL was different between these brain regions. In amygdala, a complete conversion was seen, whereas there was no guanine nucleotide-effect on RH in globus pallidus. In nucleus caudatus and nucleus accumbens, guanine nucleotides provoked only a partial conversion of RH into RL, suggesting that these brain regions may contain guanine nucleotide-sensitive and -insensitive receptors. Heating of the membranes at 60 degrees C for 5 min had the same effect as guanine nucleotides. The pharmacological profiles of the guanine nucleotide-sensitive and -insensitive D1 receptors were similar, suggesting that D1 receptors in human brain are heterogeneous only with respect to their effector-coupling mechanism: guanine nucleotide-sensitive receptors, which are capable of undergoing functional coupling with Gs, and guanine nucleotide-insensitive receptors, which are not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contributions of Conserved Serine Residues to the Interactions of Ligands with Dopamine D2 Receptors

Four dopamine D2 receptor mutants were constructed, in each of which an alanine residue was substituted for one of four conserved serine residues, i.e., Ser-193, Ser-194, Ser-197, and Ser-391. Wild-type and mutant receptors were expressed transiently in COS-7 cells and stably in C6 glioma cells for analysis of ligand-receptor interactions. In radioligand binding assays, the affinity of D2 receptors for dopamine was decreased 50-fold by substitution of alanine for Ser-193, implicating this residue in the binding of dopamine. Each mutant had smaller decreases in affinity for one or more of the ligands tested, with no apparent relationship between the class of ligand and the pattern of mutation-induced changes in affinity, except that the potency of agonists was decreased by substitution for Ser-193. The potency of dopamine for inhibition of adenylyl cyclase was reduced substantially by substitution of alanine for Ser-193 or Ser-197. Mutation of Ser-194 led to a complete loss of efficacy for dopamine and p-tyramine, which would be consistent with an interaction between Ser-194 and the p-hydroxyl substituent of dopamine that is necessary for activation of the receptors to occur. Because mutation of the corresponding residues of beta 2-adrenergic receptors has very different consequences, we conclude that although the position of these serine residues is highly conserved among catecholamine receptors, and the residues as a group are important in ligand binding and activation of receptors by agonists, the function of each of the residues considered separately varies among catecholamine receptors.     

Differences and Similarities in Muscarinic Receptors of Rat Heart and Retina: Effects of Agonists, Guanine Nucleotides, and N-Ethylmaleimide

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.     

Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)     

D2 Dopamine Receptors in Calf Globus Pallidus: Agonist High- and Low-Affinity Sites Not Regulated by Guanine Nucleotide

By use of the radioligand [3H]spiroperidol, D2 3,4-dihydroxyphenylethylamine (dopamine) receptor binding characteristics were studied in calf globus pallidus and compared with those of neostriatum. Antagonist competition curves were monophasic and revealed similar affinities for neostriatum and globus pallidus, suggesting a uniform receptor population with one affinity state for antagonists. In both regions, competition curves with the agonist dopamine were biphasic, distinguishing a high- and low-agonist-affinity state. In neostriatum and globus pallidus, respectively, 45% and 19% of [3H]spiroperidol binding was displaced with high affinity and the remainder with low affinity. In neostriatum, the addition of 0.4 mM GTP resulted in a partial conversion from high- to low-affinity state with a remaining high-affinity component of 15%. In globus pallidus, dopamine binding was not altered by GTP. The capability of GTP to modulate agonist binding to D2 receptors appears to be dependent on their neuroanatomical localization.     

Dopamine D2-receptors mediate hypothermia in mice: ICV and IP effects of agonists and antagonists

The effect of centrally and peripherally administered dopamine D1 and D2 specific compounds on core body temperature in mice was investigated. Quinpirole (LY-17155), a D2 agonist, induced a dose-dependent fall in body temperature (2.4–11.6%; p<0.003) when injected intraperitoneally (ip, 0.3–3.0 mg/kg) and intracerebroventricularly (icv, 0.1 mg/kg). This quinpirole-induced (1.0 mg/kg, ip) hypothermia was reversed by the central and peripheral administration of the D2 antagonists S-(–)-sulpiride (3.0–30.0 mg/kg, ip; 0.1–3.0 mg/kg, icv) and spiperone (0.03 and 0.1 mg/kg, ip; 0.03–3.0 mg/kg, icv). Domperidone, a D2 antagonist which does not cross the blood brain barrier, had no effect on quinpirole-induced hypothermia (1.0–10.0 mg/kg, ip). Domperidone partially reversed quinpirole-induced hypothermia at 0.1–30.0 mg/kg, icv. The D1 agonist, SKF-38393 at a high dose of 10.0 mg/kg, ip mildly attenuated quinpirole-induced hypothermia (a 1.8% increase in temperature). SKF-38393 at 10.0 mg/kg, icv potentiated quinpirole-induced hypothermia. SCH-23390 (0.1–3.0 mg/kg, ip), a D1 antagonist, had no effect on quinpirole-induced hypothermia and potentiated the hypothermia when administered icv. An ineffective icv dose of spiperone (0.01 mg/kg) in reversing quinpirole-induced hypothermia was rendered effective by prior administration of SCH-23390 (0.1–3.0 mg/kg, icv) but not by SKF-38393 (1.0–10.0 mg/kg, icv). These data suggest a central D2 receptor mechanism mediating hypothermia in mice which is capable of being modulated by the D1 receptor.     

A simple novel approach for the purification of pertussis toxin

Abstract Pertussis toxin (PT) was purified from concentrated culture supernatants of Bordetella pertussis by a single step based on affinity chromatography on heparin-Sepharose 6B with a recovery of 36% in two fractions. The fraction of highest specific activity (0.91 mg of toxin per mg protein) constituted 15.3% of toxin content in crude material and appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibited the five bands corresponding to toxin subunits. The purified fraction induced clustering of Chinese hamster ovary cells at as little as 0.06 ng per ml. PT solution gave a single precipitation line with rabbit immune serum raised against crude Bordetella pertussis supernatant.     

Effects of guanine nucleotides on cns neuropeptide receptors

The effect of nucleotides on central nervous system neuropeptide receptor binding was investigated. The guanine nucleotides, guanosine-5′-triphosphate and guanylyl-5′-imidodiphosphate, significantly inhibited the binding of radiolabeled vasoactive intestinal polypeptide but not that of [Tyr⁴]bombesin to rat brain membranes. Vasoactive intestinal polypeptide binding was inhibited by guanine nucleotides in a dose-dependent manner. Using a 20 μM dose, 60% of the specific vasoactive intestinal polypeptide binding was inhibited by guanylyl-5′-imidodiphosphate, which was more potent than guanosine-5′-triphosphate, whereas other nucleotides were not effective. This reduction in binding was a consequence of lower affinity of the receptor for vasoactive intestinal polypeptide, which in turn resulted from an increased rate of dissociation.     

D1 and D2 Dopamine Receptors in Caudate-Putamen of Nonhuman Primates (Macaca fascicularis)

D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 +/- 0.027 nM) with a density (Bmax) of 35.7 +/- 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 +/- 0.007 nM and the density was 25.7 +/- 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151-2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.     

Dopamine D2 Receptors in the Anterior Pituitary: A Single Population Without Reciprocal Antagonist/Agonist States

Although dopamine agonists can recognize two states of the D2 dopamine receptor in the anterior pituitary (D2high and D2low), we examined whether the dopamine antagonists such as [3H]spiperone could recognize these two sites with different affinities. Using up to 30 concentrations of [3H]spiperone, however, we could only detect a single population of binding sites (porcine anterior pituitary homogenates) with a dissociation constant (KD) of 130 pM. When specific [3H]spiperone binding was defined by a low concentration of (+)-butaclamol (100 nM), the apparent density was low. When defined by a high concentration of (+)-butaclamol (10 microM), nonspecific sites became detectable, thus revealing two apparent populations of sites for [3H]spiperone, only one of which was specific for dopamine. Sodium chloride reduced the KD of the single population of specific D2 sites to 64 pM. Guanine nucleotide by itself had no effect on the KD, but enhanced the density by 25%. Since the density-enhancement could be eliminated by extensive washing of membranes, and could be restored by preincubation with dopamine, the nucleotide-induced elevation of D2 density appeared to be a result of the release of tightly bound endogenous dopamine. Thus, monovalent cations and guanine nucleotides appear to have separate regulatory effects on the anterior pituitary D2 receptor that modulate antagonist-receptor interactions. Several maneuvers were used to test whether [3H]spiperone could differentiate between the two agonist-detected subpopulations of sites. Twentyfold different concentrations of [3H]spiperone (47 pM and 1000 pM) were found to label identical proportions of receptors in the D2high and D2low states as detected by the agonist 6,7-dihydroxyaminotetralin (ADTN), suggesting that spiperone labelled equal proportions of D2high and D2low sites without differential affinity for them. In addition, competition of spiperone for D2high sites selectively labelled by the agonist [3H]n-propylnorapomorphine (NPA) had a virtually identical KD for spiperone as did the total D2 receptor population as determined by direct binding studies (75 pM versus 64 pM). [3H]Spiperone also bound to a uniform population of D2low sites induced by preincubation with guanine nucleotide with identical affinity as to the total D2 population. Thus, these data do not support a "reciprocal model" for the D2 receptor (i.e., antagonist having low affinity for D2high and high affinity for D2low in a manner reciprocal to agonists).(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and reconstitution of the N-formylpeptide receptor from HL-60 derived neutrophils

A multifunctional receptor for N-formylpeptides exists on the membranes of neutrophils. This receptor has now been isolated from neutrophils derived from HL-60 promyelocytic leukemia cells. After solubilization by Nonidet-P40 and purification by affinity chromatography and HPLC the isolated receptor was reconstituted into egg phosphatidylcholine vesicles by SM-2 Bio-Bead removal of the Nonidet-P40. Analysis of the affinity and selectivity of the receptor was done by direct binding of two high-affinity ligands, formyl-Met-Leu-[3H]Phe-OH and formyl-Nle-Leu-Phe-[3H]Tyr-OH. The data suggest that the receptor can be isolated and reconstituted without apparent alteration of its binding affinity and selectivity, and that there appear to be no co-factors or subunits upon which these binding characteristics are dependent.