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PubMed2003

Characterization of antigenic properties of horseradish peroxidase with monoclonal antibodies.

Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO(1) and 36F(9) were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.

Amino Acid Sequence↗
PubMed2003

Identification of novel target proteins of cyclic GMP signaling pathways using chemical proteomics.

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the coprecipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5\'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.

Animals↗
PubMed2006

Pharmacological characterization of protease activated receptor-1 by a serum responsive element-dependent reporter gene assay: major role of calmodulin.

We studied the protease activated receptor-1 coupling to a serum response element (SRE)-dependent luciferase activity readout in transfected COS-7 cells. Thrombin, with a pEC50 of 10.5, was 3000-fold more potent than the peptide agonists SFLLR and its derived compound C721-40 in stimulating luciferase activity, although the three agonists exhibited similar efficacy at the maximal concentration tested. Interestingly, SFLLR- and C721-40-induced luciferase activity was biphasic, suggesting that at least two populations of G proteins couple to the receptor. Further pharmacological characterization of this system was performed using selective protease activated receptor-1 antagonists. SCH203099 and ER-112787 blocked SFLLR-induced luciferase activity with similar potencies (pK(B) of 7), slightly higher than that exhibited by an arylisoxazole derivative compound from Merck (pK(B) of 6.1). These values correlated with their affinities established by competition binding experiments using [3H]-C721-40 as radioligand for protease activated receptor-1. Transduction mechanisms of protease activated receptor-1 coupling to SRE-dependent luciferase activity were examined using specific inhibitors. The Ca2+ chelator BAPTA-AM, as well as the calmodulin inhibitors W-7 and ophiobolin A, robustly inhibited SFLLR-induced SRE activation. Overexpression of RGS2 and a dominant negative rhoA protein abolished the SFLLR signal in an additive manner, suggesting a major role of Gq and G12/13 proteins. Furthermore, inhibition of phospholipase C, MAP-kinases, phosphatidyl inositol-3 kinase, rho-kinase and Ca2+/calmodulin-dependent protein kinases, all downstream effectors of Gq and G12/13, partially blocked the SFLLR-induced luciferase signal. Taken together, this SRE-luciferase assay reveals a complex network of transduction pathways of protease activated receptor-1 in accordance with the pleiotrophic action of thrombin.

Animals↗
PubMed1998

Analysis of the ternary complex formation of human urokinase with the separated two domains of its receptor.

Human urokinase-type-plasminogen-activator receptor (uPAR) is a glycolipid-anchored membrane glycoprotein comprising three structurally similar domains. We have succeeded in direct observation of the ternary complex formation of single-chain urokinase (scuPA) or its N-terminal fragment (ATF) with the separated domain-1 (N-terminal domain) and domain-(2+3) (internal and C-terminal domain) of human uPAR, by means of gel-filtration HPLC analysis. This complex was found to consist of the three components in an equimolar ratio (thus referred to as the three-part complex). To determine the nature of the interaction between these components, cross-linking experiments involving various kinds of cross-linkers and competitive binding assay on ELISA were performed. These experiments have shown that each uPAR domain can bind directly to scuPA at low affinity, and that both these domains contribute to the high-affinity binding between scuPA and uPAR in a synergistic manner. It can be considered that the synergistic effect of domain-1 and domain-(2+3) on scuPA binding would result from a conformational change, and that this steric event might trigger the signal transduction reported for scuPA/uPAR binding.

Azides↗
PubMed1995

Interaction of histidine-proline-rich glycoprotein with plasminogen: effect of ligands, pH, ionic strength, and chemical modification.

The association of plasma histidine-proline-rich glycoprotein (HPRG) with plasminogen (PLG) was examined using a sucrose density gradient assay in order to evaluate the effects of several relevant conditions on complex formation. Addition of PLG shifts the S-value of 125I-labeled HPRG from 4.8S to 6.8S, providing the first direct evidence that HPRG associates with the zymogen form of plasmin in solution. Complex formation is not sensitive to pH in the range of pH 6.5-8.5, but is abolished at high ionic strength (1 M NaCl). No species differences were found, as either rabbit or human HPRG bound readily to rabbit or human PLG. Of the ligands of HPRG tested, mesoheme (20 microM) but not heparin (M(r) 10,000, 10 microM) inhibits the formation of the HPRG-PLG complex. Modification of lysine residues of HPRG or competitive binding by lysine and anti-fibrinolytic agents containing primary amino groups also inhibits association. Conversely, modification of arginine or histidine residues of HPRG has no effect on complex formation. These results indicate that HPRG has independent binding sites for heparin and PLG and confirm that one or more lysine residues of HPRG are involved in its recognition by PLG. The protein-protein interaction was also quantitatively characterized at thermodynamic equilibrium by analytical ultracentrifugation. The stoichiometry and dissociation constant (KD) of the complex were determined from the equilibrium distribution of fluorescein-isothiocyanate-labeled PLG in the presence of HPRG. The experimental data were analyzed by nonlinear least-squares curve fitting and indicated that a heterodimer is formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Modulation of endothelin-A receptor, Galpha subunit, and RGS2 expression during H9c2 cardiomyoblast differentiation.

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.

Animals↗
PubMed1993

Modification of a competitive protein binding assay for determination of inositol 1,4,5-trisphosphate.

A modification of a competitive protein-binding method for the determination of inositol 1,4,5-triphosphate (Ins(1,4,5)P3), facilitated by the use of a cell harvester, is described. The method is based on the competition of [3H]Ins(1,4,5)P3 with Ins(1,4,5)P3 in the sample for binding to a binding protein prepared from bovine adrenal cortex. The assay is carried out in 96-well microtiter plates in a final volume of 100 microliters and free [3H]Ins(1,4,5)P3 is separated from bound by filtration using a cell harvester. This allows the rapid measurement of large numbers of samples, with high reproducibility. Ins(1,4,5)P3 bound to a single class of high-affinity receptors with a KD of 2.3 +/- 0.2 nM and a Bmax of 289 +/- 7 fmol/mg of protein. The binding was rapid, reaching equilibrium after 20 min, and reversible. The sensitivity of the assay allows the determination of amounts of Ins(1,4,5)P3 as small as 0.1 pmol, which corresponds to a concentration of 5 nM in the sample. The application of the method to measure the formation of Ins(1,4,5)P3 in DDT1 MF-2 smooth muscle cells activated with ATP or bradykinin is described. The modification is a rapid, sensitive, convenient, and relatively inexpensive method for the determination of Ins(1,4,5)P3 in large numbers of samples.

Adenosine Triphosphate↗
PubMed2006

Aplindore (DAB-452), a high affinity selective dopamine D2 receptor partial agonist.

The pharmacology of aplindore (DAB-452) was characterized in CHO-K1 cells stably transfected with the human dopamine D(2) receptor short isoform (CHO-D(2s)) and in a behavioral model for post-synaptic agonism in rats. In [(3)H]-spiperone competition binding studies, aplindore showed high affinity for dopamine D(2) and D(3) receptors and low affinity for the dopamine D(4), serotonin (5-HT)(1A), 5-HT(2) receptors and the alpha1-adrenoceptor. The high potency partial agonist activity of aplindore was demonstrated in [(35)S]guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding, extracellular signal-regulated kinase (ERK)-phosphorylation and intracellular calcium flux assay using fluorometric plate reader ([Ca(2+)](i)-FLIPR) format. The [Ca(2+)](i)-FLIPR assay was conducted with CHO-D(2S) receptor cells also stably expressing chimeric G(alphaq/o)-proteins. In all assay modalities, the potencies and intrinsic activities of aplindore were lower than dopamine and higher than aripiprazole. In contrast to the [(35)S]GTPgammaS binding and ERK-phosphorylation assays, the [Ca(2+)](i)-FLIPR assay was able to detect the low partial agonist activity of SDZ 208-912. In unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, aplindore induced contralateral turning, which was blocked by the dopamine D(2) receptor antagonist raclopride. The dopamine D(2) receptor selective partial agonist profile of aplindore suggests that it should be effective for the treatment of dopaminergic-based disorders, such as schizophrenia and Parkinson's disease.

Animals↗
PubMed2003

Functional approach to investigate Lp(a) in ischaemic heart and cerebral diseases.

BACKGROUND: Lp(a), a major cardiovascular risk factor, contains a specific apolipoprotein, apo(a), which by virtue of structural homology with plasminogen inhibits the formation of plasmin, the fibrinolytic enzyme. A number of clinical reports support the role of Lp(a) as a cardiovascular or cerebral risk factor, and experimental data suggest that it may contribute to atherothrombosis by inhibiting fibrinolysis. DESIGN: A well-characterized model of a fibrin surface and an apo(a)-specific monoclonal antibody were used to develop a functional approach to detect pathogenic Lp(a). The assay is based on the competitive binding of Lp(a) and plasminogen for fibrin, and quantifies fibrin-bound Lp(a). High Lp(a) binding to fibrin is correlated with decreased plasmin formation. In a transversal case-control study we studied 248 individuals: 105 had a history of ischaemic cardiopathy (IC), 52 had cerebro-vascular disease (CVD) of thrombotic origin, and 91 were controls. RESULTS: The remarkably high apo(a) fibrin-binding in CVD (0.268 +/- 0.15 nmol L-1) compared with IC (0.155 +/- 0.12 nmol L-1) suggests the existence of peculiar and poorly understood differences in pro- or anti-thrombotic mechanisms in either cerebral and/or coronary arteries. CONCLUSIONS: Our results demonstrated that Lp(a) fibrin-binding and small Apo(a) isoforms are associated with athero-thrombotic disease.

Adult↗
PubMed2002

Interaction between human serum albumin and the felbamate metabolites 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one and 2-phenylpropenal.

Felbamate is an anti-epileptic drug associated with hepatotoxicity and aplastic anemia. These toxicities are believed to be mediated by the formation of the reactive species 2-phenylpropenal. 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one is a metabolic precursor for 2-phenylpropenal. 4-Hydroxy-5-phenyl-[1,3]oxazinan-2-one exists in equilibrium with 3-oxo-2-phenylpropyl carbamate, which can undergo beta-elimination to form 2-phenylpropenal. The work presented here investigates the interaction between 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one and human serum albumin (HSA). HSA (40 mg/mL) was found to decrease the half-life of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one from 4.57 +/- 0.44 h to 1.07 +/- 0.10 h at pH 7.4. This decrease in the half-life of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one was due to increased beta-elimination of 3-oxo-2-phenylpropyl carbamate, presumably through HSA-mediated general base catalysis. The k(cat) for HSA-catalyzed decomposition of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one was determined to be 12.04 min(-)(1) M(-)(1). Competitive binding assays using warfarin and ibuprofen showed that HSA-catalyzed decomposition of 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one is dependent on the subdomain IIA binding site of HSA. LC/MS/MS analyses of trypsin digests of HSA incubations with either 4-hydroxy-5-phenyl-[1,3]oxazinan-2-one or 2-phenylpropenal identified HSA-2-phenylpropenal adducts formed specifically at residues His-242 and His-247. These HSA-2-phenylpropenal adducts were found to be slowly reversible, with a decrease in alkylation of 74.0 +/- 0.6% after extensive dialysis. Interestingly, only the bis-adduct (His-242 and His-247) could be identified after dialysis. These results demonstrate the first direct example of 2-phenylpropenal conjugation to a human protein in vitro and suggest the possibility that HSA may be involved in the development of felbamate toxicity either by antigen formation or as a route of detoxification of 2-phenylpropenal.

Aldehydes↗
PubMed1997

Kappa2 opioid receptors in limbic areas of the human brain are upregulated by cocaine in fatal overdose victims.

Cocaine is thought to be addictive because chronic use leads to molecular adaptations within the mesolimbic dopamine (DA) circuitry that affect motivated behavior and emotion. Although the reinforcing effects of cocaine are mediated primarily by blocking DA reuptake into the presynaptic nerve terminal, reciprocal signaling between DA and endogenous opioids has important implications for cocaine dependence. The present study used the opioid antagonist 6 beta-[125iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([125I]IOXY) after pretreatment with the site-directed acylating agents 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid iazole -HCl (mu-selective) and N-phenyl-N-[1-(2-(4-isothiocyanato)-phenethyl)-4-piperidinyl]-p ropana mide-HCl (delta-selective) to examine the effect of cocaine exposure on the distribution and density of kappa2 receptors in autopsy studies of human cocaine fatalities. The selective labeling of the kappa2 receptor subtype was demonstrated by competition binding studies, which gave a pharmacological signature (IOXY >/= (+)-bremazocine >> U50,488 >/= U69,593) distinct from either the kappa1 or kappa3 receptor subtypes. Visualization of [125I]IOXY labeling revealed that kappa2 receptors localize to mesocortical and subcortical limbic areas, including the cingulate, entorhinal, insular, and orbitofrontal cortices and the nucleus accumbens and amygdala. The number of kappa2 receptors in the nucleus accumbens and other limbic brain regions from cocaine fatalities was increased twofold as compared with age-matched and drug-free control subjects. Cocaine overdose victims, who experienced paranoia and marked agitation before death, also had elevated densities of kappa2 receptors in the amygdala. These findings demonstrate for the first time that kappa2 receptor numbers are upregulated by cocaine exposure. The molecular adaptation of kappa2 receptor numbers may play a role in the motivational incentive associated with episodes of binge cocaine use and in the dysphoria that follows abrupt cocaine withdrawal.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1989

[Clinical significance of competitive protein binding assay of 25-hydroxyvitamin D concentration].

The level of serum 25-hydroxyvitamin D (25-OH-D3) was determined in 94 cases of healthy volunteers and 98 patients with various diseases. The mean level of the controls was 16.2 +/- 4.6 ng/ml (x /- S). The patients were divided into 5 groups, 1) Osteomalacia and rickets 42 cases, showing typical changes of bone in X-ray films. In 12 of them the disease was caused by vitamin D deficiency. The mean value of 25-OH-D3 was 3.3 +/- 2.0 ng/ml, being much lower than that of controls (P less than 0.001). In 14 cases of hypophosphatemia, 8 cases of renal tubular acidosis and 8 cases of renal insufficiency all complicated with osteomalacia the mean value of 25-OH-D3 was 17.9 +/- 11.4, 18.2 +/- 9.6 and 19.2 +/- 5.6 ng/ml (P greater than 0.05) respectively. 2) Intestinal malabsorption 18 cases. The mean level of 25-OH-D3 was 4.5 +/- 3.2 ng/ml (P less than 0.001). Some of them had hypocalcemia and/or secondary hyperparathyroidism. 2 showed osteoporosis and 1 osteomalacia. 3) Chronic liver disease 24 cases, the mean value of 25-OH-D3 was 6.2 +/- 5.6 ng/ml, being much lower than that of the controls (P less than 0.001). 4) 10 cases after taking anti-epileptic drugs had a mean level of 25-OH-C3 6.9 +/- 5.8 ng/ml (P less than 0.001). 1 had X-ray evidence of osteomalacia. 5) 4 cases of overdosage of vitamin D had a mean value of 110.3 +/- 22.5 ng/ml, being much higher than that of the controls (P less than 0.001). Competitive protein binding assay for measuring 25-OH-D3 is a simple and economic method. It is sensitive and specific as it provides distinct discrimination between healthy controls and patients with vitamin D deficiency or overdosage.

Adult↗
PubMed2003

Interaction of DNA polymerase I (Klenow fragment) with the single-stranded template beyond the site of synthesis.

Cocrystal structures of DNA polymerases from the Pol I (or A) family have provided only limited information about the location of the single-stranded template beyond the site of nucleotide incorporation, revealing contacts with the templating position and its immediate 5' neighbor. No structural information exists for template residues more remote from the polymerase active site. Using a competition binding assay, we have established that Klenow fragment contacts at least the first four unpaired template nucleotides, though the quantitative contribution of any single contact is relatively small. Photochemical cross-linking indicated that the first unpaired template base beyond the primer terminus is close to Y766, as expected, and the two following template bases are close to F771 on the surface of the fingers subdomain. We have constructed point mutations in the region of the fingers subdomain implicated by these experiments. Cocrystal structures of family A DNA polymerases predict contacts between the template strand and S769, F771, and R841, and our DNA binding assays provide evidence for the functional importance of these contacts. Overall, the data are most consistent with the template strand following a path over the fingers subdomain, close to the side chain of R836 and a neighboring cluster of positively charged residues.

Base Sequence↗
PubMed2004

Estrogen receptor ligands. Part 4: The SAR of the syn-dihydrobenzoxathiin SERAMs.

A series of estrogen receptor ligands based on a dihydrobenzoxathiin scaffold is described and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The most active analogue, 22, was found to be 40-fold ERalpha selective in a competitive binding assay, and 22 demonstrated very potent in vivo antagonism of estradiol driven proliferation in an immature rat uterine weight gain assay.

Animals↗
PubMed1984

Enzymatic function in crystals of delta 5-3-ketosteroid isomerase. Catalytic activity and binding of competitive inhibitors.

Crystals of the steroid-metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni, exhibit many enzymatic properties. Each enzyme subunit in the lattice binds a competitive inhibitor, progesterone, with the same stoichiometry (1:1) and affinity (KD = 6 X 10(-6) M) as the enzyme in solution. Another competitive inhibitor, 19-nortestosterone, competes with progesterone for the same binding sites in the crystal. The enzyme crystals catalyze the conversion of delta 5- to delta 4-ketosteroids, but because the enzyme is so efficient, and substrate diffusion into the crystal is so slow, substrate cannot penetrate deeply into the crystal before being converted to product. A general theoretical formulation is presented to account for the effects of substrate diffusion into enzyme crystals of different shapes and sizes. The dependence of apparent mean enzyme activity in steroid isomerase crystals as a function of crystal size is shown to be consistent with this theoretical formulation. These inhibitor binding and catalytic properties suggest that the enzyme is in an active conformation within these crystals.

Binding Sites↗
PubMed2001

Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1.

Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.

3T3 Cells↗
PubMed1984

Somatomedin/insulin-like growth factor tissue receptors.

There are two types of Sm/IGF receptors based on results of competitive binding experiments and investigations of receptor structure. The type I receptor preferentially interacts with IGF I rather than IGF II and interacts weakly with insulin. This receptor has a binding subunit of Mr = 130 000 which is disulphide bonded to form larger structures of Mr greater than 300 000. The type II receptor prefers IGF II to IGF I and does not interact with insulin. Its binding subunit is not linked by disulphide bonds to other membrane components (Mr = 260 000 with reduction, 220 000 without reduction). Subunit organization of the type I receptor appears to be similar to that of the insulin receptor but it is unlikely that the insulin and Sm/IGF binding sites are on a common alpha subunit. The type I receptor is down-regulated by IGFs and insulin. A rapid increase in ligand binding to the type II receptor by insulin has been described in intact rat adipocytes. The original idea that an IGF receptor mediates the growth-promoting action of both IGFs and insulin while acute metabolic effects of insulin and IGFs are mediated by the insulin receptor is an oversimplification . There now are clear examples of insulin stimulating growth by acting through the insulin receptor and, conversely, instances of IGF stimulating glucose transport by acting through an IGF receptor. Radioreceptor assays which measure IGF I in preference to IGF II (human placental membrane) and which measure IGF II in preference to IGF I (rat liver and rat placental membranes) have been utilized for clinical measurements of Sm/IGF levels, but are less specific than the respective radioimmunoassays. With the demonstration of Sm/IGF receptors on circulating human mononuclear cells and human skin fibroblasts, it is expected that these systems will be useful for investigations of patients with possible end-organ resistance to Sm/IGF.

Animals↗
PubMed1997

Agonist activity of antimigraine drugs at recombinant human 5-HT1A receptors: potential implications for prophylactic and acute therapy.

The actions of several serotonergic ligands in use or under development for the treatment of migraine headaches were examined at recombinant human 5-HT1A receptors stably expressed in Chinese Hamster Ovary cells. Affinities (K(i)s) at this site were determined in competition binding experiments with [3H]-8-OH-DPAT ([3H](+/-)8-hydroxy-N,N-dipropylaminotetralin), whilst agonist efficacy was measured by stimulation of [35S]-GTP gamma S (guanylyl-5'-[gamma[35S]thio]-triphosphate) binding. Of the prophylactic antimigraine drugs tested, methysergide and lisuride behaved as efficacious agonists (Emax > or = 90% relative to 5-HT) whereas pitozifen and (-)propranolol acted as a partial agonist (60%) and an antagonist, respectively. This suggests that there is no correlation between agonism at 5-HT1A receptors and prophylactic antimigraine action. In contrast, serotonin, dihydroergotamine, sumatriptan, naratriptan and alniditan, which are effective in acute interruption of migraine attacks, each displayed high efficacy (Emax = 100, 100, 92.6, 79.3, 79.1% respectively) and marked affinity (Ki = 18.7, 0.6, 127, 26.4 and 3.0 nM respectively) at 5-HT1A receptors. EC50 values for agonist stimulation of [35S]-GTP gamma S binding correlated with respective Ki values at 5-HT1A receptors (r = 0.93) and the stimulation of [35S]-GTP gamma S binding by these compounds was antagonised by the selective 5-HT1A antagonist WAY 100,635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo-hexanecarboxamide; 100 nM). These data suggest that agonism at 5-HT1A receptors may be involved in some actions of drugs used in acute antimigraine therapy. In comparison with the above compounds, novel ligands targeted at 5-HT1B/1D receptors, such as GR125,743 (N-[4-methoxy-3-(4-methyl-piperazin-1-yl)phenyl] -3-methyl-4-(4-pyridyl)benzamide) and GR 127,935 (N-[4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-2'-methyl-4'-(5-m ethyl-1, 2,4-oxadiazol-3-yl)-biphenyl-4-carboxamide), only weakly activated [35S]-GTP gamma S binding (32.4 and 32.1% efficacy) and displayed moderate affinity at 5-HT1A receptors (Kis 53.1 and 49.8 nM) suggesting that they constitute useful tools to differentiate 5-HT1A and 5-HT1B/1D receptor-mediated actions. In conclusion, the present data indicates that several antimigraine agents exhibit marked 5-HT1A receptor activity and that although this is unlikely to be important for prophylactic action it may be relevant to the ancilliary properties of drugs used for acute migraine treatment.

8-Hydroxy-2-(di-n-propylamino)tetralin↗