Histamine H1-Receptor in Heart: Unique Electrophoretic Mobility and Autoradiographic Localization

Histamine H1-receptors, visualized in the guinea pig heart by autoradiography using [125I]iodobolpyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1-receptor was shown to be present on a major 68-kDa and a less abundant 54- to 58-kDa protein. The 68-kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1-receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.     

Pharmacological Characterization and Autoradiographic Localization of Histamine H2 Receptors in Human Brain Identified with [125I]Iodoaminopotentidine

125I-Aminopotentidine (125I-APT), a reversible probe of high specific radioactivity and high affinity and selectivity for the H2 receptor, was used to characterize and localize this histamine receptor subtype in human brain samples obtained at autopsy. On membranes of human caudate nucleus, specific 125I-APT binding at equilibrium revealed a single component, with a dissociation constant of 0.3 nM and maximal capacity of about 100 fmol/mg of protein. At 0.2 nM, 125I-APT specific binding, as defined with tiotidine, an H2-receptor antagonist chemically unrelated to iodoaminopotentidine, represented 40-50% of the total. Specific 125I-APT binding was inhibited by a series of typical H2-receptor antagonists that displayed apparent dissociation constants closely similar to corresponding values at the reference biological system, i.e., guinea pig atrium. This indicates that the pharmacology of the H2 receptor is the same in the human brain as on this reference system. However, histamine was about 10-fold more potent in inhibiting 125I-APT binding to membranes of human brain than of guinea pig brain. 125I-APT binding was also inhibited by amitriptyline and mianserin, two antidepressant drugs, in micromolar concentrations corresponding to effective plasma concentrations of treated patients. The distribution of H2 receptors was established autoradiographically with 125I-APT on a series of coronal sections of human brain after assessing the pharmacological specificity of the labeling. The highest density of 125I-APT sites was found in the basal ganglia, various parts of the limbic system, e.g., hippocampus or amygdaloid complex, and the cerebral cortex. H2 receptors displayed a laminar distribution in cerebral cortex and hippocampal formation. A low density of sites was found in cerebellum as well as in hypothalamus, the brain area where all the perikarya and the largest number of axons of histaminergic neurons are found. The widespread distribution of H2 receptors in the human brain is consistent with the alleged modulatory role of histamine mediated by this subtype of receptor.     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.     

Specific Binding of [3H]Pyrilamine to Histamine H1 Receptors in Guinea Pig Brain In Vivo: Determination of Binding Parameters by a Kinetic Four-Compartment Model

The binding of [3H]pyrilamine, a selective ligand of histamine H1 receptors, to guinea pig brain in vivo was compared with its binding to a brain homogenate. The pharmacological properties (regional distribution, saturability, and stereoselectivity) of the [3H]pyrilamine binding in vivo were similar to those of the in vitro binding to brain homogenate. A dynamic four-compartment model was proposed for the analysis of the kinetics of [3H]pyrilamine binding in vivo. The receptor constants in vivo were determined by a computer-fitting method after correcting the radioactivity of arterial plasma and brain for the presence of radioactive metabolites. The in vivo association and dissociation were 213 and 42 times, respectively, slower than those of in vitro binding at 37 degrees C. A possible mechanism for slow association and dissociation in vivo is discussed.     

Labelling of 5-Hydroxytryptamine3 Receptors with a Novel 5-HT3 Receptor Ligand, [3H]RS-42358–197

Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine₃ (5-HT₃) receptor ligand [³H]quipazine in rat cerebral cortical membranes with an affinity (pK_i) of 9.8 ± 0.1, while having weak affinity (pK_i < 6.0) in 23 other receptor binding assays. [³H]RS-42358–197 was then utilized to label 5-HT₃ receptors in a variety of tissues. [³H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (K_D) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (B_max) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [³H]RS-42358–197 was similar to that labelled with [³H]GR 65630, but was significantly less than that found with [³H]-quipazine. The binding of [³H]RS-42358–197 had a pharmacological profile similar to that of [³H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT₃ receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [³H]RS-42358-197 binding. [³H]RS-42358–197 also labelled a population (B_max= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (K_D= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT₃ receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [³H]RS-42358–197 have detected differences in 5-HT₃ receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT₃ receptor.     

Histamine H1 Receptor Antagonists Produce Increases in Extracellular Acetylcholine in Rat Frontal Cortex and Hippocampus

Abstract: Lesions of the neuronal histaminergic system or pharmacological blockade of histamine receptors, e.g., with histamine H₁ receptor antagonists, can enhance the performance of rats in several tests of learning and memory. The underlying neuronal systems that mediate these behavioral effects are not known. Here, we examined the effects of treatment with histamine H₁ antagonists on extracellular levels of acetylcholine (ACh) in adult rats anesthetized with urethane (1.25 g/kg). ACh was quantified using in vivo microdialysis and HPLC with electrochemical detection. Basal levels of ACh in the frontal cortex and hippocampus were in the range of 0.54 ± 0.13 and 0.96 ± 0.17 pmol/20 min, respectively. Injection (intraperitoneally) of saline did not produce significant increases in ACh levels, even though there was a slight and gradual increase in cortical ACh levels throughout the course of the experiments (up to 4 h after an injection). Administration of the H₁ receptor antagonist chlorpheniramine (intraperitoneally) produced a dose-dependent increase of cortical ACh levels to a maximum of 260, 280, and 570% of baseline values after doses of 5, 10, and 20 mg/kg, respectively. In the hippocampus, ACh content increased to a maximum of ～600% of baseline levels after chlorpheniramine administration (20 mg/kg, i.p.). Administration of the H₁ antagonist pyrilamine (intraperitoneally) increased cortical ACh content to a maximum of 300 and 500%, whereas hippocampal ACh levels increased to 215 and 280% after doses of 10 and 20 mg/kg, respectively. In an additional experiment using nonanesthetized, freely moving rats, cortical ACh content showed a moderate increase (to 190%) after saline injections (intraperitoneally) and a much higher increase (to 370%) after chlorpheniramine treatment (20 mg/kg, i.p.). These data suggest that cortical and hippocampal levels of ACh can be effectively modulated by systemic treatment with histamine H₁ antagonists. The increases in ACh levels produced by H₁ antagonists may suggest that some histaminergic receptors exert an inhibitory influence over central ACh levels. The enhanced availability of ACh in the forebrain may contribute to the behavioral effects observed with H₁ antagonist treatment.     

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.     

Ontogeny of Receptor Binding Sites for [3H]Glutamic Acid and [3H]Kainic Acid in the Rat Cerebellum

The development of the specific binding sites for L-[3H]glutamic acid (KD = 370 nM) and for [3H]kainic acid (KD = 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.     

Effect of Histamine H2 Receptor Antagonists on the Secretion of Cerebrospinal Fluid in the Cat

Following a recent report that epithelial cells of the choroid plexus possess histamine H2 receptors, the effect of cimetidine and ranitidine, histamine H2 receptor antagonists, on the secretion and electrolyte content of CSF was examined. Fifty cats were divided into one control (n = 6) and six experimental groups. CSF was collected by puncture of the cisterna magna following pentobarbital anesthesia, and its volume, concentrations of Na+, K+, Cl-, and pH were determined. Cimetidine or ranitidine (50, 20, or 10 mg/kg) was injected intravenously 2 h after the start of the test, and their concentrations were measured in hourly blood samples and in 30-min aliquots of CSF in the 50 mg/kg experimental groups. Whereas the secretion of CSF did not change over 6 h in the control group, it decreased significantly by 30-60 min after injection of cimetidine or ranitidine and remained low for the following 6 1/2 h in all experimental groups except the 10-mg ranitidine group. Peak cimetidine and ranitidine concentrations in CSF in the 50-mg experimental groups were noted 60 and 90 min, respectively, after intravenous injection. CSF electrolyte concentrations and pH did not change during the test in any group. We conclude that intravenous cimetidine or ranitidine can significantly reduce CSF secretion in the cat, possibly by competitive inhibition of the histamine effect on H2 receptors located on the choroid plexus epithelial cell, or by a direct effect on the capillaries of the choroid plexus.     

Focal Stimulation of the Mossy Fibers Releases Endogenous Dynorphins That Bind K1-Opioid Receptors in Guinea Pig Hippocampus

Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a kappa 1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid peptide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous kappa ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1-8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin- and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of kappa 1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that prodynorphin-derived opioids released under physiological conditions from the mossy fibers act at kappa 1 receptors in the guinea pig dentate gyrus.     

Comparison of 125I-SCH 23982 and [3H]SCH 23390 as Ligands for the D-1 Dopamine Receptor

125I-SCH 23982, an antagonist with high affinity and selectivity for the D-1 subtype of dopamine receptors, has recently been synthesized. Densities of D-1 receptors in rat brain obtained from autoradiographic studies using this iodinated ligand are 5- to 10-fold less than densities reported with tritiated analogues such as [3H]SCH 23390. A direct comparison of these two ligands using striatal homogenates confirmed this discrepancy. One explanation for this difference is that 125I-SCH 23982 labels a subset of the sites labeled by [3H]SCH 23390. However, the distributions of sites labeled by the ligands in autoradiograms of horizontal sections of rat brain were virtually identical. Furthermore, 127I-SCH 23982 displaced 100% of the specifically bound [3H]SCH 23390 in striatal homogenates with a Hill coefficient of approximately 1. These results are not consistent with the existence of a subset of receptors recognized by 125I-SCH 23982 and suggest that both ligands label the same population of receptors. An alternative explanation for the discrepancy in Bmax values is that an unlabeled inhibitor is present in commercial preparations of 125I-SCH 23982. When all of the solvent (including any volatile inhibitors) was removed from commercial preparations of 125I-SCH 23982 prior to use in radioligand binding experiments, the discrepancy in Bmax values was eliminated.     

Immunolabeling of Central Serotonin 5-HT1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti-Peptide Antiserum

Abstract: A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT_1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT_1B/1Dβ receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin- O -carboxymethylglycyl[¹²⁵I]iodotyrosinamide ([¹²⁵I]GTI; a selective 5-HT_1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT_1B receptors. The remaining 40% of [¹²⁵I]GTI binding sites were shown to be 5-HT_1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT_1B/1Dβ, but not the 5-HT_1D/1Dα, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT_1Dβ receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [¹²⁵I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT_1Dβ component of [¹²⁵I]GTI specific receptor binding sites.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Effects of Nucleotides on [3H]Bradykinin Binding in Guinea Pig: Further Evidence for Multiple B2 Receptor Subtypes

Abstract: We have suggested recently the existence of three subtypes of B₂ bradykinin receptors in tissues of guinea pigs. We have classified these B₂ bradykinin receptors into B_2a, B_2b, and B_2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [³H]bradykinin to the B₂ subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B_2a and B_2b subtypes, specific [³H]bradykinin binding was reduced with GDP (100 μM) and the nonmetabolized analogue of GTP, guanyl-5′-yl-imidodiphosphate (GppNHp; 100 μM). Competition studies with bradykinin and with [Hyp³]-bradykinin, which shows approximately 20-fold greater selectivity for the B_2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 μM). The competition experiments demonstrated that binding to the B_2a subtype, which has higher affinity for [Hyp³]-bradykinin and bradykinin than the B_2b subtype, was lost in the presence of GppNHp, whereas binding to the B_2b subtype was unaffected. In contrast, GppNHp (100 μM) and GDP (100 μM) failed to alter specific [³H]bradykinin binding to B_2b and B_2c subtypes in lung. [³H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 μM each). Based on this evidence, we suggest that the B_2a bradykinin subtype is coupled to G proteins. The B_2b and B_2c subtypes are either not coupled to G proteins, or may be coupled to the G^o-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides. These data provide further evidence for three subtypes of B₂-type bradykinin receptors in guinea pig.     

Guinea Pig Histamine H1 Receptor. II. Stable Expression in Chinese Hamster Ovary Cells Reveals the Interaction with Three Major Signal Transduction Pathways

Abstract: A cDNA encoding a guinea pig histamine H₁ receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H₁), the H₁ receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a G_i/G_o protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca²⁺ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca²⁺ level of which only the second sustained phase was dependent on the extracellular Ca²⁺ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca²⁺. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca²⁺- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A₂, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H₁ receptor, the histamine-induced arachidonic acid release was also Ca²⁺- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H₁) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca²⁺, and PMA. These various observations show that stimulation of a single receptor subtype, the guinea pig H₁ receptor, can trigger four major intracellular signals through coupling to several G proteins that are variously modulated by extracellular Ca²⁺ and protein kinase C activation.     

Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D2 Receptor Recognize Native Dopamine Receptor Protein in Rat Striatum

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Inhibitory Effect of Trifluoromethylphenylpiperazine on [3H]Acetylcholine Release in Guinea Pig Hippocampal Synaptosomes Is Mediated by a 5-Hydroxytryptamine1 Receptor Distinct from 1A, 1B, and 1C Subtypes

The effect of the serotonergic receptor agonist 1-(m-trifluoromethylphenyl)piperazine (TFMPP) was studied on the K(+)-evoked [3H]acetylcholine [( 3H]ACh) release from guinea pig hippocampal synaptosomes loaded with [3H]choline. TFMPP (5-1,000 microM) inhibited the evoked ACh release in a dose-dependent manner (IC50 = 81.8 microM). The inhibitory effect of TFMPP was mimicked by CGS-12066B (10, 30, and 100 microM), a 5-hydroxytryptamine1B (5-HT1B)/5-HT1D receptor agonist; 1-(m-chlorophenyl)piperazine (100 microM), a 5-HT1C/5-HT1B receptor agonist; and 5-carboxamidotryptamine (10 microM), a nonselective 5-HT1 receptor agonist. 8-Hydroxy-2-(di-n-propylamino)tetralin (10 and 100 microM), a 5-HT1A receptor agonist, and quipazine (10 and 100 microM), a 5-HT2 receptor agonist, did not have any significant effect. Serotonergic antagonists, such as dihydroergotamine (0.1 and 1 microM), metergoline (0.1 microM), methysergide (0.5 and 1 microM), or yohimbine (1 and 10 microM), blocked the TFMPP effect dose-dependently. In contrast, methiotepine (0.3 and 1 microM), propranolol (1 microM), ketanserin (0.1 microM), mesulergine (0.1 microM), ICS 205930 (0.1 and 1 microM), and spiroperidol (1 and 7 microM) did not affect the TFMPP-induced inhibition of the evoked ACh release. These data suggest that, in guinea pig hippocampus, the K(+)-evoked ACh release is modulated by a 5-HT1 receptor distinct from the 5-HT1A, 5-HT1B, and 5-HT1C subtypes.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.