Differential Binding Properties of Adenosine Receptor Agonists and Antagonists in Brain

The binding properties of N6-cyclohexyl [3H]adenosine ( [3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ( [3H]DPX) in rat forebrain membrane are compared. The kinetic parameters of binding for each ligand are quite distinct, with [3H]CHA displaying two populations of binding sites (KD = 0.4 +/- 0.05 nM and 4.2 +/- 0.3 nM; Bmax = 159 +/- 17 and 326 +/- 21 fmol/mg protein), whereas [3H]DPX yielded monophasic Scatchard plots (KD = 13.9 +/- 1.1 nM; Bmax = 634 +/- 27 fmol/mg protein). The metals copper, zinc, and cadmium are potent inhibitors of [3H]CHA binding, with respective IC50 concentrations of 36 microM, 250 microM, and 70 microM. Copper is a much less potent inhibitor of [3H]DPX binding (IC50 = 350 microM). The inhibitory effect of copper on both [3H]CHA and [3H]DPX binding is apparently irreversible, as membranes pretreated with copper cannot be washed free of its inhibitory effect. The inhibitory effect of both copper and zinc on [3H]CHA binding was reversed by the guanine nucleotide Gpp(NH)p. [3H]DPX binding is only partially inhibited by zinc and cadmium (60% of specific binding remains unaffected), suggesting that this adenosine receptor ligand binds to two separate sites. Guanine nucleotides had no effect on the inhibition of [3H]DPX binding by either copper or zinc. Differential thermal and proteolytic denaturation profiles are also observed for [3H]CHA and [3H]DPX binding, with the former ligand binding site being more labile in both cases. Stereospecificity is observed in the inhibition of both [3H]CHA and [3H]DPX binding, with L-N-phenylisopropyladenosine (PIA) being 50-fold more potent than D-PIA in both cases. Evidence is therefore provided that adenosine receptor agonists and antagonists have markedly different binding properties to brain adenosine receptors.     

Taurine Modulation of the Benzodiazepine-γ-Aminobutyric Acid Receptor Complex in Brain Membranes

In unwashed brain membranes taurine produced an inhibition of [3H]flunitrazepam [( 3H]FNZ) binding with IC50 ranging between 31.5 and 11.9 microM; the IC20 varied between 18 and 26 nM. This inhibitory effect was of a mixed type, with a reduction in Bmax and an increase in KD. Various precursors and metabolites of taurine have a less inhibitory effect. Taurine also has little inhibitory effect (IC50 above 500 microM) on the binding of [3H]ethyl-beta-carboline-3-carboxylate. In extensively washed membranes, 10(-5) M taurine produces a 16-21% increase in the binding of [3H]FNZ while 10(-5) M gamma-aminobutyric acid (GABA) increases it between 31 and 42%. However, if 10(-5) M GABA plus 10(-5) M taurine is included in the assay there is a dramatic inhibitory effect. Taurine causes an inhibition of the GABAergic enhancement of [3H]FNZ binding with an IC50 between 7.3 and 7.8 microM. Binding experiments with [3H]taurine done under different conditions failed to detect a Na+-independent and specific [3H]taurine receptor. These results suggest that endogenous taurine, the second most abundant free amino acid in brain, may play an important modulatory role in the GABA-benzodiazepine receptor complex.     

Opioid binding site in EL-4 thymoma cell line

Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of [3H] bremazocine indicated a single site with a KD = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10(6) cells (51 pmols/mg total cell proteins). To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of [3H] bremazocine with an IC50 value = 0.57 microM. The two stereoisomers levorphanol and dextrorphan showed the same affinity for this site (IC50 = 2.9 microM and 1.9 microM respectively). While morphine, [D-Pen2, D-Pen5] enkephalin and beta-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC50 = 60 microM, that was similar to naloxone (IC50 = 69 microM).     

Effects of 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4-Dihydroxyphenylethylamine and [3H]5-Hydroxytryptamine in Neostriatal Synaptosomal Preparations

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4-dihydroxyphenylethylamine-([3H]DA), [3H]5-hydroxytryptamine ([3H]5-HT), and [3H]gamma-aminobutyric acid [( 3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5-HT uptake (IC50 value 0.14 microM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 microM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 microM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5-HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4-electron oxidation product of MPTP, i.e., 1-methyl-4-phenyl-pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 microM) and of [3H]5-HT uptake (IC50 value 0.78 microM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Na(+)- and Cl(-)-dependent [3H]GABA binding to catfish brain particles

The uptake of [3H]GABA by homogenates of catfish brain was previously shown to be temperature-sensitive and sodium-dependent, and to display saturation kinetics. The present study is a continuation of this work and was undertaken to characterize the initial binding of [3H]GABA to its transport system. [3H]GABA binding to catfish brain particles at 4 degrees C displayed saturability and was totally dependent on both Na+ and Cl-, the optimum concentrations of which were 150 mM and 75 mM, respectively. The effects of a number of drugs on binding were established. Unlabelled GABA was the most potent inhibitor (IC50 = 3.2 microM). The structural analogues nipecotic acid and guvacine were also strongly inhibitory. Interestingly, verapamil, a Ca2+ channel blocker, also inhibited [3H]GABA binding (IC50 = 38 microM). Harmaline, known to compete for Na+ binding in other transport systems, did not appear to influence Na+ binding but was effective at displacing [3H]GABA. These results suggest that the interaction of GABA with its carrier is similar to that found in the mammalian nervous system and is further evidence that GABA is involved in neurotransmission in catfish brain.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

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 kd/slope factor values of 9.6 +/- 1.4 microM/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 (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 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 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl- aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX- B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX- B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.     

Stereoselectivity for the (R)-Enantiomer of HA-966 (l-Hydroxy-3-Aminopyrrolidone-2) at the Glycine Site of the N-Methyl-d-Aspartate Receptor Complex

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.     

The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid.

The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM-propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics.     

The cannabinoid receptor agonist CP-55,940 and ethyl arachidonate interfere with [(3)H]batrachotoxinin A 20 alpha-benzoate binding to sodium channels and inhibit sodium channel function

Recent investigations in our laboratory showed that voltage-gated sodium channels (VGSCs) in brain are sensitive to inhibition by various synthetic cannabinoids and endocannabinoids. The present experiments examined the effects of the cannabinoid-1 (CB1) receptor agonist CP-55,940 and ethyl arachidonate on [(3)H]batrachotoxinin A 20 alpha-benzoate ([(3)H]BTX-B]) binding and VGSC-dependent depolarization of the nerve membrane in synaptoneurosomes isolated from mouse whole brain. CP-55,940 acted as a full inhibitor of [(3)H]BTX-B binding and its IC(50) was established at 22.3 microM. At its maximum effect concentration, ethyl arachidonate achieved partial (approximately 70%) inhibition and was less effective than CP-55,940 as an inhibitor of binding (IC(50)=262.7 microM). The potent CB1 receptor antagonist AM251 (2 microM) had no significant effect on the displacement of [(3)H]BTX-B by either compound (P>0.05). Scatchard analyses showed that CP-55,940 and ethyl arachidonate reduce the binding of [(3)H]BTX-B by lowering its B(max) but ethyl arachidonate also increased the K(d) of radioligand binding. In kinetic experiments, CP-55,940 and ethyl arachidonate were found to boost the dissociation of [(3)H]BTX-B from VGSCs to rates that exceed the maximum velocity achievable by veratridine, indicating they operate as allosteric inhibitors of [(3)H]BTX-B binding. Neither compound was effective at changing the initial rate of association of [(3)H]BTX-B with sodium channels. CP-55,940 and ethyl arachidonate inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes with IC(50)s of 3.2 and 50.1 microM respectively. These inhibitory effects were again not influenced by 2 microM AM251. Our data demonstrate that the potent cannabinoid receptor agonist CP-55,940 and the ethyl ester of arachidonic acid have the ability to associate with VGSCs and inhibit their function independently of effects on CB1 receptors. Binding data comparisons using mouse brain preparations indicate CP-55,940 is approximately 10,000 times more potent as a CB1 receptor ligand than a sodium channel ligand while ethyl arachidonate shows a much smaller differential. Ethyl arachidonate has been shown previously to be the principal metabolite of ethanol in the brains of intoxicated individuals and effects of this ester on VGSCs and CB1 receptors may contribute to the depressant effects of alcohol.     

Synthesis and biological evaluation of a novel class of rofecoxib analogues as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs)

A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substituents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure-activity relationships identified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of COX-2 (COX-2 IC50 = 1.4 microM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC50 = 0.28 microM; 15-LOX IC50 = 0.32 microM), inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a) possesses dual COX-2 (IC50 = 2.7 microM) and 5-LOX (IC50 = 0.30 microM) inhibitor actions. Further in vivo studies employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA), but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of the COX and LOX enzymes.     

Discovery of piperazinylimidazo[1,2-a]pyridines as novel S4 binding elements for orally active factor Xa inhibitors

We have recently reported the discovery of orally active sulfonylalkylamide Factor Xa (FXa) inhibitors, as typified by compound 1 (FXa IC(50)=0.061 microM). Since the pyridylpiperidine moiety was not investigated in our previous study, we conducted detailed structure-activity relationship studies on this S4 binding element. This investigation led to the discovery of piperazinylimidazo[1,2-a]pyridine 2b as a novel and potent FXa inhibitor (FXa IC(50)=0.021 microM). Further modification resulted in the discovery of 2-hydroxymethylimidazo[1,2-a]pyridine 2e (FXa IC(50)=0.0090 microM), which was found to be a selective and orally bioavailable FXa inhibitor with reduced CYP3A4 inhibition.     

Direct evidence for the interaction between papaverine and alpha 2-adrenergic receptors in canine platelets

The effects of papaverine on specific [3H]-yohimbine binding to canine platelet alpha 2-adrenergic receptors and on the platelet aggregation were assessed and compared with those of verapamil. Both compounds concentration-dependently inhibited [3H]-yohimbine binding with KI values for respective compounds of 0.39 +/- 0.05 microM (n = 3) and 15 +/- 0.19 microM (n = 3). In the presence of either compound KD values in Scatchard analysis of the equilibrium ligand binding increased in concentration-dependent manner, whereas Bmax did not change, indicating competitive inhibition of the ligand binding by these compounds. (-)-Epinephrine (3 microM) potentiation of adenosine diphosphate (ADP, 0.1 microM) aggregation was inhibited by papaverine with IC50 of 11 +/- 3.6 microM (n = 4). In the same experiments verapamil inhibited the platelet aggregation with lower IC50 (3.1 +/- 0.87 microM, n = 4) in comparison with that for papaverine. These results suggest that papaverine, like verapamil, inhibits physiological response of canine platelets through alpha-adrenergic receptor stimulation by direct interaction with the receptors.     

Agonist-Dependent Internalization of γ-Aminobutyric AcidA/Benzodiazepine Receptors in Chick Cortical Neurons

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-(4-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50 = 9.8 +/- 2.9 nM) for displacement of [3H]flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50 = 4.0 +/- 0.3 nM). However, at concentrations from 0.1 to 10 microM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton X-100, a result suggesting that approximately 7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1-4 h at 37 degrees C) of neurons with 100 microM gamma-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H]flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154-176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.     

Effects of the sulfhydryl reagentN-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486

A steroid binding protein (Mr = 110,000) has previously been identified in the plasma membrane of Xenopus laevis oocytes by photoaffinity labeling with [3H]R5020. In order to further characterize this steroid receptor, the photoaffinity labeled receptor protein was solubilized with 0.1% Brij 35. The solubilized labeled receptor yielded an approximate mol. wt of 102,000 +/- 2,000 by sucrose density gradient centrifugation, suggesting that the solubilized receptor exists as a monomer. RU 486, a synthetic progestin antagonist for mammalian cytosolic receptor systems, inhibited up to 70% of [3H] R5020 photoaffinity binding to the 110,000-Dalton receptor with an IC50 of 5 microM and induced germinal vesicle breakdown (GVBD) with an EC50 of 9.0 +/- 0.6 microM. GVBD induced by RU 486 was slower than with progesterone, and RU 486 was less powerful than progesterone. Micromolar concentrations of RU 486 also potentiated GVBD induced by sub-optimal concentrations of progesterone or R5020. Furthermore, RU 486 inhibited oocyte plasma membrane adenylate cyclase with an apparent IC50 of 7.5 +/- 2.5 microM. The close correlation of the EC50 value for RU 486 induction of GVBD with the IC50 values for inhibition of [3H]R5020 photoaffinity labeling of the 110,000-Dalton receptor and inhibition of adenylate cyclase activity further supports the physiological significance of the oocyte plasma membrane steroid receptor.     

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)