DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

A benzo[b]thiophene-based selective type 4 S1P receptor agonist

S1P receptors (S1PR1-5) are a group of GPCRs activated by a high affinity binding with S1P that have important roles in the regulation of the immune system. A potent S1PR agonist FTY720 is an immunomodulator used to treat multiple sclerosis and several ‘second generation’ drugs are under clinical development. Subtype-selective agonists have been reported for each S1PR isotype, some of which are used as pharmacological tools for functional studies. Here we report the discovery and initial characterization of compound 5c, a benzo[b]thiophene amino carboxylate which exhibits potent and selective agonist activity for S1PR4. Compound 5c has an EC₅₀ = 200 nM as an agonist in GTPγ³⁵S binding assay for S1PR4 and exhibits no activity against S1PR1,2,3,5. We confirmed its potent activity and decent S1PR subtype selectivity using biochemical and cellular assays.     

A Very Large Number of GABAergic Neurons Are Activated in the Tuberal Hypothalamus during Paradoxical (REM) Sleep Hypersomnia

We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD₆₇ mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD₆₇ in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD⁺, Fos-ir/MCH⁺, and GAD⁺/MCH⁺ double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD⁺ neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis.     

ANTAGONIST AND AGONIST ACTIVITIES OF THE MOUSE AGOUTI PROTEIN FRAGMENT (91–131) AT THE MELANOCORTIN-1 RECEPTOR

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91–131) (AP_91–131) at the melanocortin type-1 receptor (MC1-R) were assessed using B16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP_91–131 was about 3-fold less potent than the natural agonist α-melanocyte-stimulating hormone (α-MSH) in displacing the radioligand [¹²⁵I]-[Nle⁴, D-Phe⁷]-α-MSH (K_i 6.5±0.8 nmol/l). α-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP_91–131; the IC₅₀ values for AP_91–131 in the two assay systems were 91±22 nM and 95±15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP_91–131 with IC₅₀ values of 9.6±1.8 nM and 5.0±2.4 nM, respectively. This indicates inverse agonist activity of AP_91–131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP_91–131 in the adenylate cyclase and melanin assays. On the other hand, AP_91–131 inhibited cell growth similar to α-MSH (IC₅₀ 11.0±2.1 nM; maximal inhibition 1.8-fold higher than that of α-MSH). Furthermore, MC1-R was down-regulated by AP_91–131 with about the same potency and time-course as with α-MSH. These results demonstrate that AP_91–131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different α-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.     

Melanotropin receptors in the cartilaginous fish,Potamotrygon reticulatus and in the lungfish,Lepidosiren paradoxa

1. P. reticulatus skins in vitro exhibit punctate melanophores, and darken in response to the melanotropins in a dose-related manner.2. The relative potencies (Ac-Nle⁴-alpha-MSH_4–13-NH₂< alpha-MSH = Ac-Cys⁴,Cys¹⁰-alpha-MSH_4–13-NH₂) suggest that the supression of the amino-terminal tripeptide may affect the ideal peptide conformation for interaction with the receptor, which is restored with cyclization of the decapeptide fragment.3. Our data suggest that hormonal control of P. reticulatus pigment cells is mainly exerted by alpha-MSH, as none of the other agonists (isoproterenol, norepinephrine, MCH and melatonin) exhibited pigment dispersing or aggregating activities.4. L. paradoxa scales possess non-innervated epidermal and dermal melanophores, which exhibit stellate shape in vitro.5. Norepinephrine is a weak partial agonist in L. paradoxa epidermal melanophores, in the presence or in the absence of the beta-adrenoceptor blocker propranolol, suggesting that catecholamines do not play a role in the control of pigment cells.6. Both epidermal and dermal melanophore responses to MCH and to its analogue MCH_5–15, and to melatonin were also slow, partial and obtained with high doses of the hormones.7. The relative insensitivity of L. paradoxa melanophores is in accordance with the animal cryptic behavior, since it lives in muddy dark waters, and does not seem to depend on chromatic adaptation for camouflage.     

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [³H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 M) or THIP plus the antagonist bicuculline methobromide (150 M of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (K _D) of 7.9±0.4 nM and aB _max of 3.42±0.08 pmol×mg^–1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA withK _D-values of 6.8±0.9 nM and 476±311 nM, respectively. The correspondingB _max values were 4.41±0.42 pmol×mg^–1 and 5.81±1.20 pmol×mg^–1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. TheK _D value (14.3±1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.     

Synthesis and preliminary evaluation of [H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors

The selective antagonist radioligand [³H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([³H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74 Ci/mmol. In preliminary saturation binding studies, [³H]PSB-0413 showed high affinity for platelet P2Y₁₂ receptors with a K_D value of 4.57 nM. Human platelets had a high density of P2Y₁₂ receptors exhibiting a B_max value of 7.66 pmol/mg of protein.     

Autoradiographic Localization of [125I-Tyr0]Bradykinin Binding Sites in Brains of Wistar-Kyoto and Spontaneously Hypertensive Rats

1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).2. Serial sections of brains were cut from adult WKY and SHR and specific [¹²⁵I-Tyr⁰]bradykinin ([¹²⁵I-Tyr⁰]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques.3. Specific binding of [¹²⁵I Tyr⁰]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85–90% of total binding) was of high affinity and saturable with K _D values in the range of 100 pM and a B _max of 0.75 fmol per mg tissue equivalent in the NTS–X–AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg⁹-BK. The B₂ receptor antagonist icatibant inhibited [¹²⁵I-Tyr⁰]BK binding with a K _i value of 0.63 ± 0.19 nM in WKY and 0.91 ± 0.73 nM in SHR, while K _i values for the B> ₁ receptor agonist DesArg⁹-BK were 1475 ± 1055 and 806 ± 362 nM in WKY and SHR, respectively.4. Our finding of specific high-affinity [¹²⁵I-Tyr⁰]BK B₂ binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B₂ receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.     

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Potent and selective adenosine A₁ receptor (A₁AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a K_i value of 0.96 nM for PQ-69 in cloned hA₁ receptor, which was 217-fold more selective compared with hA_2A receptors and >1,000-fold selectivity for hA₁ over hA₃ receptor. The results obtained from [³⁵S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A₁AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A₁AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t_1/2) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A₁AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A₁AR function, and it could be developed as a potential therapeutic agent.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9424-5) contains supplementary material, which is available to authorized users.     

4-Aminopiperidine ureas as potent selective agonists of the human β3-Adrenergic receptor

The preparation and structure–activity relationships (SARs) of potent agonists of the human β₃-adrenergic receptor (AR) derived from a 4-aminopiperidine scaffold are described. Examples combine human β₃-AR potency with selectivity over human β₁-AR and/or human β₂-AR agonism. Compound 29s was identified as a potent (EC₅₀=1 nM) and selective (greater than 400-fold over β₁- with no β₂-AR agonism) full β₃-AR agonist with in vivo activity in a transgenic mouse model of thermogenesis.     

[3H]Serotonin binding sites in goldfish retinal membranes

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [³H]5HT was sensitive to pre-treatment of the membranes at 40° or 60° C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [³H]5HT binding to retinal membranes. The 5HT₂ agonist, 1-(-naphtyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodopheny1-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT_1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B_max 2.45 pmol/mg of protein, k_d 6.86 nM), and a low affinity component (B_max 53.46 pmol/mg of protein, K_d 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (K_d 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existance of 5HT₁ receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a suptype of receptor expressed in glial cells.Abbreviations used B _max maximum binding capacity - CPP, 1 (3 chlorophenyl)piperazine - CLN clonidine - DMI desimipramine - DMT 5-methoxy-N,N-dimethyltryptamine - DOI (+)-1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane - DPAT (+)-8-hydroxy-2-(D1-N-propylamino)tetralin - GppNHp 5-guanylylimidodiphosphate - HAL haloperidol - 5HT serotonin - IC₅₀ concentration of drug producing 50% inhibition of binding - IMI imioramine - K_d equilibrium dissociation constant - MIAN mianserin - NOM nomifensin - NP 1-(1-napthyl)piperazine - PRP propranolol In memory of Dr. Boris Druian who died on Dec. 24, 1991.     

Biochemistry and pharmacokinetics of potent non-steroidal cytochrome P45017α inhibitors

Two potent non-steroidal inhibitors (CB7645 and CB7661) of human cytochrome P450_17α were tested for in vivo activity in WHT mice. There were no signs of toxicity, but there was no effect on the androgen-dependent organs. The pharmacokinetics and biochemistry of the compounds in mice were investigated. Following i.p. administration of 0.5 mmol/kg of CB7645 and CB7661, peak plasma levels of 13.4 and 3.4 μM, respectively, occurred after 2–4 h, both compounds were cleared rapidly (terminal half-lives 2.7 and 3.3 h, respectively) and neither was detectable at 24 h. CB7645 produced some decrease in plasma testosterone at 4 h, but this was not sustained. When tested in vitro against the WHT testicular enzyme, the CB7645 and CB7661 were competitive inhibitors with K_i values of 10 and 13 nM, respectively. However, the K_m for the substrate progesterone was lower at 4.3 nM. These data indicate that, for effective and continuous inhibition of the murine cytochrome P450_17α enzyme, higher peak levels of the compounds would be required, and these levels would need to be maintained throughout the treatment period.     

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.     

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.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.     

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-endorphin fragment 12–19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [³H]Octarphin was found to bind to macrophages with high affinity (K _d = 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met⁵]enkephalin (K _i > 10 μM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immuno-competent cells in vitro and in vivo: at concentration of 1–10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 μg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.