The thermodynamics of agonist and antagonist binding to dopamine D-2 receptors

The ability of dopamine agonists and antagonists to compete with [3H]spiperone binding to rat striatal membrane preparations at 4, 15, 26, and 37 degrees varied markedly with temperature. Dopamine and the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (ADTN) were more potent at lower temperatures. The ability of the dopamine antagonists, haloperidol, cis-flupenthixol, cis-N-(1-benzyl-1-methypyrrolidin-3-yl)-5-chloro-2-methoxy-9- methylaminobenzamide (YM 09151-2), raclopride, and clozapine, and of the agonists apomorphine and pergolide, to compete with [3H]spiperone binding was little altered by temperature. (+)-Butaclamol was more potent at higher temperatures. In contrast, the antagonists sulpiride, metoclopramide, clebopride, sultopride, tiapride, piquindone, and zetidoline were more potent at lower temperatures. The interaction of the agonists dopamine and ADTN was driven by a decrease in enthalpy, allowing an energetically unfavorable decrease in entropy. The binding of the antagonists, haloperidol, cis-flupenthixol, YM 09151-2, raclopride, (+)-butaclamol, and clozapine, and also of the agonists, apomorphine and pergolide, was entropy driven. The interaction of the antagonists sulpiride, metoclopramide, clebopride, alizapride, sultopride, tiapride, piquindone, and zetidoline differed from that of other antagonists in being enthalpy driven. The observed entropy changes correlated with the lipophilicity of the displacing drugs and not with their intrinsic activity.     

Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4-dihydropyridine receptors.

The kinetic and equilibrium binding properties of the 1,4-dihydropyridine activator [3H](-)-S-Bay K 8644 and the antagonist [3H](+)-PN 200-110 were determined in rat heart membrane particulate preparations at temperatures between 4 and 37 degrees C. The binding of [3H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with KD = 3.57 nM and Bmax = 330 fmol/mg.protein at 25 degrees C. The association and dissociation rate constants were 3.4 x 10(7) min-1 M-1 and 0.095 min-1 respectively at 25 degrees C and decreased slightly at lower temperatures. In contrast, [3H](+)-PN 200-110 bound to high (KD(H) = 0.032 nM, Bmax(H) = 316 fmol/mg.protein) and low affinity sites (KD(L) = 27.6 nM and Bmax(L) = 6432 fmol/mg.protein) at 25 degrees C in rat heart preparation. A similar two-site binding of [3H](+)-PN 200-110 was found in rat brain preparation, but only a single binding site was detected in rat skeletal muscle. Binding of [3H](+)-PN 200-110 to the high and low affinity sites in cardiac membranes was sensitive and insensitive respectively to temperature. Association and dissociation rates of [3H](+)-PN 200-110 at the high affinity binding sites were best fitted as mono-exponential functions. Association and dissociation rates of [3H](+)-PN 200-110 were 3.94 x 10(8) min-1 M-1 and 7.86 x 10(-3) min-1 at 25 degrees C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4 degrees C. Thermodynamic analysis of equilibrium binding showed that the binding of activator was enthalpy driven, whereas the binding of antagonist to the high affinity site was both entropy- and enthalpy-driven and to the low affinity site was totally entropy-driven.     

Regulation of agonist binding to rat ET(B) receptors by cations and GTPgammaS

Extensive use for disulfiram (DSF) has been found in the aversion therapy treatment of recovering alcoholics. Although it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (ALDH), the specific mechanism of in vivo inhibition of the enzyme by the drug has not been determined yet. We have demonstrated in this report a novel, but simple and rapid method for structurally characterizing in vivo derived protein-drug adducts by linking on-line sample processing to HPLC-electrospray ionization mass spectrometry (HPLC-MS) and HPLC-tandem mass spectrometry (HPLC-MS/MS). Employing this approach, rats were administered DSF, and their liver mitochondria were isolated and solubilized. Both native and in vivo DSF-treated mitochondrial ALDH (mALDH) were purified in one step with an affinity cartridge. The in vivo DSF-treated mALDH showed 77% inhibition in enzyme activity as compared with that of the control. Subsequently, the control and DSF-inhibited mALDH were both subjected to HPLC-MS analyses. We were able to detect two adducts on DSF-inhibited mALDH, as indicated by the mass increases of approximately 71 and approximately 100 Da. To unequivocally determine the site and structure of these adducts, on-line pepsin digestion-HPLC-MS and HPLC-MS/MS were performed. We observed two new peptides at MH(+) = 973.7 and MH(+) = 1001.8 in the pepsin digestion of DSF-inhibited enzyme. These two peptides were subsequently subjected to HPLC-MS/MS for sequence determination. Both peptides possessed the sequence FNQGQC(301)C(302)C(303), derived from the enzyme active site region, and were modified at Cys(302) by N-ethylcarbamoyl (+71 Da) and N-diethylcarbamoyl (+99 Da) adducts. These findings indicated that N-dealkylation may be an important step in DSF metabolism, and that the inhibition of ALDH occurred by carbamoylation caused by one of the DSF metabolites, most likely S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO). Finally, there was no evidence of the presence of an intramolecule disulfide bridge modification on the peptide FNQGQCCC.     

Gamma-hydroxybutyrate is a weak agonist at recombinant GABA(B) receptors

Gamma-hydroxybutyrate (GHB) is a neuromodulator with high affinity binding sites in the mammalian brain. However, the receptor for GHB has not yet been identified. There are indications that GHB and gamma-aminobutyric acid (GABA) mediate their effects via the same receptor. We tested this hypothesis using GABA(B)R1/R2 receptors co-expressed with Kir3 channels in Xenopus oocytes. GHB activated these receptors with an EC50 of approximately 5 mM and a maximal stimulation of 69% when compared to the GABA(B) receptor agonist L-baclofen. GHB and L-baclofen did not amplify each others effect nor did they stimulate the GABA(B) receptor in a linearly additive manner. CGP54626A, 2-OH saclofen and CGP35348, three competitive GABA(B) receptor antagonists, inhibited the GHB induced response completely. A concentration of 30 mM GHB displaced [125I]CGP64213 binding at GABA(B)R1 expressed in COS cells by 21%. These results indicate that GHB is a weak partial agonist at the GABA binding site of GABA(B)R1/R2.     

Incorporation of the pi subunit into functional gamma-aminobutyric Acid(A) receptors.

mRNA encoding the recently cloned gamma-aminobuytyric acid(A) receptor (GABAR) pi subunit is expressed in the hippocampus and in several non-neuronal tissues including the uterus and ovaries. Whereas native GABARs are pentamers composed primarily of alphabetagamma, alphabetadelta, or alphabetaepsilon subunits, it has not been demonstrated clearly that the pi subunit incorporates into functional GABARs to form alphabetapi receptors and, if so, with what properties. We provide electrophysiological evidence that the pi subunit can coassemble with either alpha5beta3 or alpha5beta3gamma3 subunits to produce recombinant GABARs with distinct pharmacological and biophysical properties. Compared with alpha5beta3 receptors, GABARs produced by coexpression of alpha5beta3pi subunits had a lower GABA EC(50) value, were enhanced to a lesser extent by loreclezole, had different IC(50) values for pregnenolone sulfate and lanthanum, and were insensitive to benzodiazepines. Incorporation of both pi and gamma3 subunits into an alpha5beta3gamma3pi isoform was suggested by reduced enhancement by diazepam and a high zinc IC(50) value. Current-voltage relations for the alpha5beta3pi subunit combination outwardly rectified more than currents from alpha5beta3gamma3 but less than alpha5beta3 combination GABARs. Single-channel alpha5beta3 GABAR currents had a main conductance state of 15.2 picoSeimens (pS). Coexpression of the pi subunit with alpha5beta3 subtypes increased the conductance level to 23.8 pS, similar to the conductance level of alpha5beta3gamma3 GABARs (26.9 pS). We conclude that the pi subunit coassembles with alpha, beta, and gamma subunits to form functional alphabetapi or alphabetagammapi GABARs and, thus, could have a significant impact on the function of native GABARs expressed in the brain or non-neuronal tissue.     

MgCl2和Gpp（NH）p对腺苷A1受体激动剂和拮抗剂结合的作用比较

AIM: To investigate modulation of antagonist and agonist binding to adenosine A1 receptors by MgCI2 and 5'-guanylimidodiphosphate (Gpp(NH)p) using rat brain membranes and the A1 antagonist [^3H]-8-cyclopentyl-1,3-dipropylxanthine ([^3H]DPCPX) and the A1 agonist [^3H]-2-chloro-N^6-cyclopentyladenosine ([^3H]CCPA). METHODS:Parallel saturation and inhibition studies were performed using well-characterised radioligand binding assays and aBrandel Cell Harvester. RESULTS: MgCI2 produced a concentration-dependent decrease (44%), whereasGpp(NH)p increased [^3H]DPCPX binding (19%). In [^3H]DPCPX competition studies, agonist affinity was 1.5-14.6-fold higher and 4.6-10-fold lower in the presence of l0 mmol/L MgCl2 and l0μmol/L Gpp(NH)p respectively;antagonist affinity was unaffected. The decrease in agonist affinity with increasing Gpp(NH)p concentrations was due to a reduction in the proportion of binding to the high affinity receptor state. In contrast to [^3H]DPCPX, MgCl2produced a concentration-dependent increase (72%) and Gpp(NH)p a decrease (85%) in [^3H]CCPA binding.Using [^3H]CCPA, agonist affinities were 5-17-fold higher than those for [^3H]DPCPX, consistent with binding onlyto the high affinity receptor state. Agonist affinity was 1.3-10.5-fold higher and 2.4-4.7-fold lower on addingMgCl2 or Gpp(NH)p respectively; antagonist affinities were as for [^3H]DPCPX. CONCLUSION: The inconsistencies surrounding the effects of MgCl2 and guanine nucleotides on radioligand binding to adenosine A1 receptorswere systematically examined. The effects of MgCl2 and Gpp(NH)p on agonist binding to A1 receptors are consistent with their roles in stimulating GTP-hydrolysis at the G-protein α-subunit and in blocking formation of the highaffinity agonist-receptor-G protein complex.     

Differential allosteric modulation by amiloride analogues of agonist and antagonist binding at A(1) and A(3) adenosine receptors

The diuretic drug amiloride and its analogues were found previously to be allosteric modulators of antagonist binding to A(2A) adenosine receptors. In this study, the possibility of the allosteric modulation by amiloride analogues of antagonist binding at A(1) and A(3) receptors, as well as agonist binding at A(1), A(2A), and A(3) receptors, was explored. Amiloride analogues increased the dissociation rates of two antagonist radioligands, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one ([3H]PSB-11), from A(1) and A(3) receptors, respectively. Amiloride and 5-(N,N-dimethyl)amiloride (DMA) were more potent at A(1) receptors than at A(3) receptors, while 5-(N,N-hexamethylene)amiloride (HMA) was more potent at A(3) receptors. Thus, amiloride analogues are allosteric inhibitors of antagonist binding at A(1), A(2A), and A(3) adenosine receptor subtypes. In contrast to their effects on antagonist-occupied receptors, amiloride analogues did not affect the dissociation rates of the A(1) agonist [3H]N(6)-[(R)-phenylisopropyl]adenosine ([3H]R-PIA) from A(1) receptors or the A(2A) agonist [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) from A(2A) receptors. The dissociation rate of the A(3) agonist radioligand [125I]N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]I-AB-MECA) from A(3) receptors was decreased significantly by amiloride analogues. The binding modes of amiloride analogues at agonist-occupied and antagonist-occupied receptors differed markedly, which was demonstrated in all three subtypes of adenosine receptors tested in this study. The effects of the amiloride analogues on the action of the A(3) receptor agonist were explored further using a cyclic AMP functional assay in intact CHO cells expressing the human A(3) receptor. Both binding and functional assays support the allosteric interactions of amiloride analogues with A(3) receptors.     

SCH-202676: An allosteric modulator of both agonist and antagonist binding to G protein-coupled receptors

A novel thiadiazole compound, SCH-202676 (N-(2,3-diphenyl-1,2, 4-thiadiazol-5-(2H)-ylidene)methanamine), has been identified as an inhibitor of both agonist and antagonist binding to G protein-coupled receptors (GPCRs). SCH-202676 inhibited radioligand binding to a number of structurally distinct, heterologously expressed GPCRs, including the human mu-, delta-, and kappa-opioid, alpha- and beta-adrenergic, muscarinic M1 and M2, and dopaminergic D1 and D2 receptors, but not to the tyrosine kinase epidermal growth factor receptor. SCH-202676 had no direct effect on G protein activity as assessed by [35S]guanosine-5'-O-(gamma-thio)triphosphate binding to purified recombinant G(oalpha)- or G(betagamma)-stimulated ADP-ribosylation of G(oalpha) by pertussis toxin. In addition, SCH-202676 inhibited antagonist binding to the beta2-adrenergic receptor expressed in Escherichia coli, a system devoid of classical heterotrimeric G proteins. SCH-202676 inhibited radiolabeled agonist and antagonist binding to the alpha2a-adrenergic receptor with an IC50 value of 0.5 microM, decreased the Bmax value of the binding sites with a slight increase in the KD value, and inhibited agonist-induced activation of the receptor. The effects of SCH-202676 were reversible. Incubation of plasma membranes with 10 microM SCH-202676 did not alter subsequent radioligand binding to the alpha2a-adrenergic receptor and the dopaminergic D1 receptor. Taken together, our data suggest that SCH-202676 has the unique ability to allosterically regulate agonist and antagonist binding to GPCRs in a manner that is both selective and reversible. The scope of the data presented suggests this occurs by direct interaction with a structural motif common to a large number of GPCRs or by activation/inhibition of an unidentified accessory protein that regulates GPCR function.     

Chronic theophylline exposure increases agonist and antagonist binding to A1 adenosine receptors in rat brain

Chronic treatment of rats with theophylline (75mg/kg twice-daily for 21 consecutive days) significantly increased the specific binding of [3H]CHA and [3H]DPCPX in cerebral cortical membranes. The absolute increase in the number of binding sites following theophylline treatment was approximately the same for each ligand, although this number represents a larger percentage of the total sites available to [3H]CHA. Saturation analysis of [3H]DPCPX binding indicated that theophylline treatment increased the maximum number of binding sites from 799 +/- 13 to 920 +/- 22 fmol/mg protein, while the affinity of [3H]DPCPX for A1 receptors was unaltered. These results suggest that chronic theophylline exposure produces both an increase in the number of A1 adenosine receptors and an enhancement of coupling of these receptors to G proteins.     

Sertindole is a serotonin 5-HT2c inverse agonist and decreases agonist but not antagonist binding to 5-HT2c receptors after chronic treatment

RATIONALE: Sertindole is a novel antipsychotic drug with high affinity for dopamine D2, alpha-1-adrenoceptors and serotonin 5-HT2A and 5-HT2c receptors. The 5-HT2c receptor component of sertindole may be clinically relevant as this receptor subtype is implicated in regulation of anxiety, cognition/memory and brain plasticity. OBJECTIVE: To characterise the interaction of sertindole with the 5-HT2C receptor using rat choroid plexus as a physiological receptor source. RESULTS: Sertindole had nanomolar affinity for the 5-HT2c receptor in vitro. Sertindole antagonised 5-HT-stimulated phosphoinositide (PI) hydrolysis and, like clozapine, also inhibited basal PI hydrolysis suggesting that sertindole is a 5-HT2C receptor inverse agonist. The effect of repeated sertindole dosing on 5-HT2C receptors was studied in rats treated for 21 days with sertindole (20, 300 and 1250 microg/kg/day). Clozapine (25 mg/kg/day) was used as a comparison drug. 5-HT2C receptor binding in the choroid plexus was measured with antagonist and agonist ligands ([3H]mesulergine and [125I]DOI) using quantitative autoradiography 8 days after withdrawal. Clozapine decreased 5-HT2C receptor antagonist and agonist binding sites equally by 36% and 32%, respectively. Sertindole did not induce significant changes in the total number of 5-HT2C receptors, but the highest dose of sertindole lowered the affinity of [3H]mesulergine for 5-HT2C receptors. This was most likely due to residual sertindole levels in the brain which was supported by direct concentration measurements. In contrast, sertindole induced a highly significant and dose-related decrease in 5-HT2C agonist binding (up to 77%). Neither drug affected striatal D2 receptor binding. CONCLUSIONS: Sertindole, like clozapine, was found to be a serotonin 5-HT2C receptor inverse agonist. The preferential downregulation of 5-HT2C receptor agonist (G-protein-coupled) sites by chronic administration seemed to differentiate sertindole from clozapine at these dose regimens. The 5-HT2c receptor downregulation during repeated dosing may contribute to therapeutic efficacy and/or side effects of sertindole treatment.     

6beta-Acyloxy(nor)tropanes: affinities for antagonist/agonist binding sites on transfected and native muscarinic receptors

A series of esters of 6beta-hydroxynortropane and the N-methyl analogue 6beta-tropanol were synthesized and screened versus binding of an antagonist (quinuclidinyl benzilate) and an agonist (oxotremorine-M) at sites on human m(1)-, m(2)-, m(3)-, and m(4)-muscarinic receptors in transfected cell membranes and on native M(1)-muscarinic receptors in rat brain membranes and native M(2)-muscarinic receptors in rat heart membranes. Most 6beta-acyloxy(nor)tropanes had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at transfected m(1)- and native M(1)-receptors, indicative of agonist activity. 6beta-Acetoxynortropane had K(i) values versus oxotremorine-M binding at m(1)-, m(2)-, and m(4)-receptors ranging from 4 to 7 nM. N-Methylation reduced affinity greatly as did increasing the size of the acyl moiety. The affinity of 6beta-benzoyloxynortropane and other analogues with larger acyl moieties was little affected by N-methylation or in some cases was increased. 6beta-Acyloxy(nor)tropanes and classical muscarinic agonists, such as muscarine and oxotremorine, had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at native M(2)-muscarinic receptors of heart, but not at transfected m(2)-muscarinic receptors. Antagonist/agonist binding ratios were not obtained for transfected m(3)-receptors, since significant oxotremorine-M binding could not be detected. 6beta-Acyloxy(nor)tropane, two other (nor)tropanes, and the classical muscarinic agonists had higher affinity versus agonist binding compared to antagonist binding for transfected m(4)-receptors. The antagonist/agonist binding ratio method is clearly not always reliable for predicting agonist activity at muscarinic receptors.     

The melatonergic agonist and clinically active antidepressant, agomelatine, is a neutral antagonist at 5-HT(2C) receptors

The novel antidepressant, agomelatine, behaves as an agonist at melatonergic receptors, and as an antagonist at edited, human serotonin2C(VSV) receptors [h5-HT2C(VSV)Rs]. However, its actions at constitutively active 5-HT2CRs have yet to be characterized, an issue addressed herein. At unedited h5-HT2C(INI)Rs expressed in HEK-293 cells, 5-HT enhanced [35S]GTPγS binding to Gαq, whereas the inverse agonists SB206,553 and S32006 inhibited binding and, by analogy to the neutral antagonist, SB242,084, agomelatine exerted no effect alone. Mirroring these observations, 5-HT stimulated, whereas SB206,553 and S32006 inhibited, [3H]inositol phosphate formation. Both the agonist actions of 5-HT and the inverse agonist actions of SB206,553 and S32006 were abolished by agomelatine and SB242,084. As demonstrated by bioluminescence resonance energy transfer, 5-HT enhanced, whereas SB206,553 and S32006 decreased, association of 'h5-HT2C(INI)-Rluc-tagged' receptors with yellow-fluorescence-protein-coupled β-arrestin2. These actions of 5-HT, SB206,553 and S32006 were prevented by agomelatine and SB242,084 were ineffective alone. As shown by ELISA and confocal microscopy, prolonged (18 h) exposure to SB206,553 or S32006 enhanced cell surface expression of N-terminal Flag-tagged h5-HT2C(INI)Rs: these effects were blocked by agomelatine and SB242,084, which were inactive alone. Finally, following pre-exposure to SB206,553 or S32006 for 18 h, 5-HT triggered 5-HT2CR-mediated elevations in cytosolic Ca2+ in primary cultures of mice cortical neurons. Agomelatine and SB242,084, inactive alone, prevented these actions of SB206,553 and S32006. In conclusion, agomelatine behaves as a neutral antagonist at constitutively active h5-HT2C(INI)Rs and native, cortical 5-HT2CRs. It will be of interest to determine whether the neutral antagonist properties of agomelatine are related to its favourable clinical profile of antidepressant properties with few side-effects and no discontinuation syndrome.     

A molecular basis for agonist and antagonist actions at GABA(C) receptors

We modelled the N-terminal ligand-binding domain of the rho1 GABA(C) receptor based on the Lymnaea stagnalis acetylcholine-binding protein (L-AChBP) crystal structure using comparative modelling and validated using flexible docking guided by known mutagenesis studies. A range of known rho1 GABA(C) receptor ligands comprising seven full agonists, 10 partial agonists, 43 antagonists and 12 inactive molecules were used to evaluate and validate the models. Of the 50 models identified, six models that allowed flexible ligand docking in accordance with the experimental data were selected and used to study detailed receptor-ligand interactions. The most refined model to accommodate all known active ligands featured a cavity comprising of a volume of 488 A(3). A detailed analysis of the interaction between the rho1 GABA(C) receptor model and the docked ligands revealed possible H-bonds and cation-pi interactions between the different ligands and binding site residues. Based on quantum mechanical/molecular mechanical (QM/MM) calculations, the model showed distinctive conformations of loop C that provided a molecular basis for agonist and antagonist actions. Agonists elicit loop C closure, while a more open loop C was observed upon antagonist binding. The model differentiates the role for key residues known to be involved in either binding and/or gating.     

Bremazocine is an agonist at kappa-opioid receptors and an antagonist at mu-opioid receptors in the guinea-pig myenteric plexus.

The agonist and antagonist activity of bremazocine at opioid receptors in the guinea-pig myenteric plexus preparation was determined in untreated tissues and in tissues in which either mu-9 or kappa-opioid receptors were blocked preferentially. After pretreatment of the tissue with beta-funaltrexamine for 90 min followed by washing out, the IC50 value of the selective mu-ligand [D-Ala2,MePhe4,Gly-ol5]enkephalin was increased 67 fold whereas the IC50 values of the selective kappa-ligand U-69,593 and of the non-selective kappa-ligand bremazocine were not significantly changed. In this experimental design bremazocine acted only on kappa-receptors. After pretreatment of the tissue with beta-chlornaltrexamine and 10 microM of the mu-ligand for 30 min followed by washout, the IC50 value of the mu-ligand was increased 2 fold whereas the IC50 value of the selective kappa-ligand was increased 32 fold and that of bremazocine 62 fold. Under these experimental conditions, it was shown that bremazocine is an antagonist against [D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-receptor (Ke = 1.6 nM). The residual agonist activity of bremazocine is at the kappa-receptor. In naive myenteric plexus preparations the mu-antagonist activity of bremazocine cannot be demonstrated because its potency at the kappa-receptor is very high. This dual action may be of importance for the responses of bremazocine in other peripheral and central tissues.     

International Union of Pharmacology. XXXIII. Mammalian gamma-aminobutyric acid(B) receptors: structure and function

The gamma-aminobutyric acid(B) (GABA(B)) receptor was first demonstrated on presynaptic terminals where it serves as an autoreceptor and also as a heteroreceptor to influence transmitter release by suppressing neuronal Ca(2+) conductance. Subsequent studies showed the presence of the receptor on postsynaptic neurones where activation produces an increase in membrane K(+) conductance and associated neuronal hyperpolarization. (-)-Baclofen is a highly selective agonist for GABA(B) receptors, whereas the established GABA(A) receptor antagonists, bicuculline and picrotoxin, do not block GABA(B) receptors. The receptor is G(i)/G(o) protein-coupled with mixed effects on adenylate cyclase activity. The receptor comprises a heterodimer with similar subunits currently designated 1 and 2. These subunits are coupled via coiled-coil domains at their C termini. The evidence for splice variants is critically reviewed. Thus far, no unique pharmacological or functional properties have been assigned to either subunit or the variants. The emergence of high-affinity antagonists for GABA(B) receptors has enabled a synaptic role to be established. However, the antagonists have generally failed to establish the existence of pharmacologically distinct receptor types within the GABA(B) receptor class. The advent of GABA(B1) knockout mice has also failed to provide support for multiple receptor types.     

烟碱对大脑皮层毒蕈碱型受体与其激动剂和拮抗剂结合的调节

烟碱对大脑皮层毒蕈碱型受体与其激动剂和拮抗剂结合的调节汪海，崔文玉，刘传缋（军事医学科学院毒物药物研究所，北京１００８５０，中国）关键词烟碱；烟碱受体；毒蕈碱受体；大脑皮质；氧颤莫林；奎纽定二苯羟乙酸目的：研究烟碱对脑Ｍ受体结合性能的调节作用．方法：...     

Thermodynamic analysis of agonist and antagonist binding to the chicken brain melatonin receptor.

1. The binding of 2-[125I]-iodomelatonin to chicken brain membranes, and the inhibition of binding by melatonin, N-acetyltryptamine and luzindole, were examined at temperatures between 4 degrees C and 37 degrees C. 2. At all temperatures studied, the binding affinity (Kd or Ki) for 2-[125I]-iodomelatonin, melatonin (both agonists) and, to a lesser extent, N-acetyltryptamine (a partial agonist) was reduced by inclusion of guanosine triphosphate (GTP, 1 mM) in the assay. GTP did not affect the Ki for luzindole, a melatonin receptor antagonist. 3. The maximal density of binding sites (Bmax) was not affected by temperature but the Kd showed a peak at 21 degrees C with lower values at both higher and lower temperatures giving curvilinear van't Hoff plots (lnKA vs l/temperature). 4. Derived changes in entropy (delta S degree) and enthalpy (delta H degree) of binding for all of the melatonin ligands decreased as temperature increased. 5. The affinity, and thus the free energy of binding, delta G degree, of these ligands at the melatonin receptor have identical values at several temperatures yet at these temperatures delta S degree and delta H degree were very different, implying that more than one intermolecular force must be involved in the binding of ligand and receptor. 6. Conceivably, the large positive delta S degree observed at low temperatures, perhaps as a result of hydrophobic interactions, is compensated by a corresponding, but opposite, change in enthalpy at higher temperatures. However, it is not clear what type of binding force(s) would show such a temperature-dependence. 7. These studies suggest that caution must be exercised in the molecular interpretation of derived measures of delta S degree and delta H degree obtained from direct measurements of delta G degree.     

A N-terminal PTHrP peptide fragment void of a PTH/PTHrP-receptor binding domain activates cardiac ET(A) receptors

1. Adult ventricular cardiomyocytes show an unusual structure-function relationship for cyclic AMP-dependent effects of PTHrP. We investigated whether PTHrP(1 - 16), void of biological activity on classical PTHrP target cells, is able to mimic the positive contractile effect of PTHrP(1 - 34), a fully biological agonist on cardiomyocytes. 2. Adult ventricular cardiomyocytes were paced at a constant frequency of 0.5 Hz and cell contraction was monitored using a cell-edge-detection system. Twitch amplitudes, expressed as per cent cell shortening of the diastolic cell length, and rate constants for maximal contraction and relaxation velocity were analysed. 3. PTHrP(1 - 16) (1 micromol l(-1)) mimicked the contractile effects of PTHrP(1 - 34) (1 micromol l(-1)). It increased the twitch amplitude from 5.33+/-0.72 to 8.95+/-1.10 (% dl l(-1)) without changing the kinetic of contraction. 4. PTH(1 - 34) (10 micromol l(-1)) affected the positive contractile effect of PTHrP(1 - 34), but not that of PTHrP(1 - 16). 5. RpcAMPS (10 micromol l(-1)) inhibited the positive contractile effect of PTHrP(1 - 34), but not that of PTHrP(1 - 16). 6. The positive contractile effect of PTHrP(1 - 16) was antagonized by the ET(A) receptor antagonist BQ123. 7. Sarafotoxin 6b and PTHrP(1 - 16), but not PTHrP(1 - 34), replaced (3)H-BQ123 from cardiac binding sites. 8. We conclude that N-terminal PTHrP peptides void of a PTH/PTHrP-receptor binding domain are able to bind to, and activate cardiac ET(A) receptors.     

CGP 36216 is a selective antagonist at GABA(B) presynaptic receptors in rat brain

In rat neocortical preparations maintained in Mg(2+)-free Krebs medium, baclofen depressed the frequency of spontaneous discharges in a concentration-dependent manner (EC(50) = 6 microM), sensitive to (3-aminopropyl)ethylphosphinic acid (CGP 36216) (100, 300 and 500 microM) (pA(2) = 3.9 +/- 0.1). By contrast, CGP 36216, up to 1 mM, was ineffective in antagonising baclofen-induced hyperpolarisations, mediated through gamma-aminobutyric acid(B) (GABA(B)) postsynaptic receptors. In electrically stimulated brain slices preloaded with [3H]GABA, CGP 36216 increased [3H]GABA release (IC(50) = 43 microM), which was reversed by baclofen (20 microM). While CGP 36216 is ineffective at GABA(B) postsynaptic receptors, it is appreciably more active at presynaptic receptors.