Thymopoietin, a potent antagonist at nicotinic receptors in C2 muscle cell cultures

Recent work has shown that thymopoietin, a polypeptide with actions in the immune and nervous systems, potently binds to the alpha-bungarotoxin (alpha-BGT) receptor. The present study was done to characterize the interaction of thymopoietin at the nicotinic alpha-BGT binding site in cultured muscle cells and to correlate these findings with the effects of the polypeptide on nicotinic receptor-mediated function. Inhibition studies showed that thymopoietin potently inhibited 125I-alpha-BGT binding in C2 muscle cells in culture, with an IC50 of 1.1 nM, a value similar to that for alpha-BGT. Thymopoietin bound to the alpha-BGT receptor in the cells in culture relatively slowly; at 10(-8) M thymopoietin, maximal inhibition occurred after 45 to 75 min of exposure to the polypeptide. Dissociation of thymopoietin from the receptor exhibited a much longer time course; recovery of alpha-BGT binding to control values after exposure to 10(-8) M thymopoietin occurred approximately 16 hr after removal of the polypeptide. The effects of thymopoietin on 125I-alpha-BGT binding correlated well with those on nicotinic function. Thymopoietin potently inhibited nicotinic receptor-mediated 22Na uptake in muscle cells in culture, with an IC50 of 2 nM. This effect was dependent on the length of the preincubation period with thymopoietin, with maximal inhibition occurring after 60 min of exposure to the polypeptide. Recovery of the functional response after thymopoietin (10(-8) M) exposure required about 16 hr. The mode of inhibition of receptor-mediated ion flux by thymopoietin was similar to that observed with alpha-BGT but distinct from that obtained with d-tubocurarine and gallamine. To conclude, thymopoietin, a thymic polypeptide associated with the immune system, potently inhibited both 125I-alpha-BGT binding and nicotinic receptor-mediated function in C2 muscle cells. These findings may have implications for myasthenia gravis and/or other neuromuscular disorders.     

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.     

Carbamoylcholine homologs: novel and potent agonists at neuronal nicotinic acetylcholine receptors

The classic muscarinic acetylcholine receptor (mAChR) agonist carbamoylcholine (carbachol) does not seem to be the most obvious lead for the development of selective ligands at nicotinic acetylcholine receptors (nAChRs). In the past, however, N-methylations of carbachol have provided N-methylcarbamoylcholine and N,N-dimethylcarbamoylcholine (DMCC), which predominantly display nicotinic activity. In this study, 12 homologous analogs of DMCC and its corresponding tertiary amine, N,N-dimethylcarbamoyl-N,N-dimethylaminoethanol, were synthesized and their binding affinities to native mAChR and nAChR sites estimated. One of the compounds in the series, 3-N,N-dimethylaminobutyl-N,N-dimethylcarbamate (7), displayed low nanomolar binding affinity to nAChRs and a 400-fold selectivity for nAChRs over mAChRs. Hence, a new series of compounds was synthesized in which alkyl and aryl groups and different ring systems were introduced in the carbamate moiety of 7. In a [3H]epibatidine binding assay, the Ki values of 7 and its analogs at rat alpha2beta2, alpha4beta2, alpha2beta4, alpha3beta4, and alpha4beta4 nAChRs, stably expressed in mammalian cell lines, ranged from low nanomolar to midmicromolar concentrations, whereas all of the compounds displayed weak binding to an alpha7/5-HT3 chimera and to native mAChRs. Compound 7 and its analogs were determined to be agonists at the alpha3beta4 nAChR subtype. This series includes the most potent and selective nicotinic agonists structurally derived from ACh to date. Furthermore, the compounds are tertiary amines, implying some advantages in terms of bioavailability pertinent to future in vivo pharmacological studies. Finally, observations made in the study hold promising perspectives for future development of ligands selective for specific nAChR subtypes.     

Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.     

Blockade of nicotinic currents in hippocampal neurons defines methyllycaconitine as a potent and specific receptor antagonist.

Methyllycaconitine, a toxin isolated from the seeds of Delphinium brownii, inhibited acetylcholine- and anatoxin-induced whole-cell currents in cultured fetal rat hippocampal neurons, at picomolar concentrations. This antagonism was specific, concentration dependent, reversible, and voltage independent. Furthermore, methyllycaconitine inhibited 125I-alpha-bungarotoxin binding to adult rat hippocampal membranes, protected against the alpha-bungarotoxin-induced pseudoirreversible blockade of nicotinic currents, and shifted the concentration-response curve of acetylcholine to the right in fetal rat hippocampal neurons, suggesting a possible competitive mode of action for this toxin. Remarkably low concentrations of methyllycaconitine (1-1000 fM) decreased the frequency of anatoxin-induced single-channel openings, with no detectable decrease in the mean channel open time. These actions of methyllycaconitine commend this neurotoxin for the characterization of the alpha-bungarotoxin-sensitive subclass of neuronal nicotinic receptors, which has hitherto eluded functional demonstration.     

Nicotinic antagonist effects in the mediodorsal thalamic nucleus: Regional heterogeneity of nicotinic receptor involvement in cognitive function

Nicotine has been found in many studies to improve cognitive function. However, some studies have not found this effect and others have seen nicotine-induced impairments. Systemic administration bathes the brain with drugs. However, the brain is quite intricately organized with various regions playing very different roles in the bases of cognitive function. We have examined the role of nicotinic receptors in a variety of brain areas for memory. In the hippocampus and amygdala, local infusions of both α7 and α4β2 antagonists methyllyaconitine (MLA) and dihydro-β-erythroidine (DHβE) significantly impair memory. In the current studies we locally infused acute and chronic doses of MLA and DHβE into the mediodorsal thalamic nucleus and tested memory function on a 16-arm radial maze. The rats also received systemic nicotine to determine the impact of more generalized nicotine effects. Since nicotinic treatments are being developed for cognitive impairment of schizophrenia, interactions were studied with the antipsychotic drug clozapine. In the acute study, the 6.75 μg/side of DHβE improved working memory. Co-administration of MLA reversed the DHβE-induced improvement. Chronic DHβE infusions into the mediodorsal thalamic nucleus also improved working memory. Systemic nicotine reversed this effect. Clozapine had no significant interaction. Nicotinic α4β2 receptors in the mediodorsal thalamic nucleus appear to play an opposite role with regard to working memory than those in the hippocampus and amygdala. Heterogeneity in response to nicotinic drugs given systemically may be due to anatomically distinct nicotinic systems in the brain and their unique roles in the neural bases of cognitive function.     

Modeling the nicotinic receptor loss in dementia using the nicotinic antagonist mecamylamine: Effects on human cognitive functioning

The loss of central nicotinic receptors is a neurochemical hallmark of several degenerative brain disorders, notably Alzheimer's disease (AD) and Parkinson's disease (PD). However, uncertainty has remained about the significance of this loss for the cognitive symptomatology of these disorders. Symptoms of impaired acquisition of information and short-term storage, impaired memory consolidation, attention, visual perception, and speed may reflect nicotinic lesions. Administration of the nicotinic antagonist mecamylamine in young and elderly humans produces significant dose-related impairment of new learning, liberalization of response bias, and slowing of reaction time. These results suggest that mecamylamine may in part model the results of nicotinic receptor loss in AD and that nicotinic augmentation of some aspects of cognitive functioning may be a worthwhile strategy to pursue, particularly if agents can be developed that are more selective than nicotine itself. © 1994 Wiley-Liss, Inc.     

Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells

Nonylphenol (NP) is the most critical metabolite of alkylphenol polyethoxylate detergents. NP is known as an endocrine disruptor with estrogenic activities and as an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Estrogen has modulatory roles on ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). Ca(2+)-ATPase inhibitors can modulate the cytosolic calcium concentration ([Ca(2+)](c)]) and thus can affect the calcium signaling coupled with nAChRs. Therefore, NP is predicted to have complex effects on the Ca(2+) signaling and secretion coupled with nAChRs. This study investigated these effects using bovine adrenal chromaffin cells. The results show that NP suppressed the Ca(2+) signaling coupled with nAChRs and voltage-operated Ca(2+) channels in a dose-dependent manner, with IC(50)s of 1 and 5.9 microM, respectively. Estradiol exhibits similar suppression but much lower inhibitory potencies. NP alone induced a transient rise in [Ca(2+)](c) in the presence or absence of extracellular calcium. Thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, partially suppressed the [Ca(2+)](c) rise induced by NP, but NP totally blocked the [Ca(2+)](c) rise induced by thapsigargin. This illustrates that NP can cause Ca(2+) release from thapsigargin-insensitive pools. Thapsigargin suppressed the Ca(2+) signaling coupled with nAChRs but increased that coupled with voltage-operated Ca(2+) channels. We propose that three routes are responsible for the effects of NP on nAChRs: named receptor channels, voltage-gated Ca(2+) channels, and Ca(2+)-induced Ca(2+) release. Three routes are related to the characteristics of NP as steroid-like compounds and Ca(2+)-ATPase inhibitor.     

Inhibitory action of carvedilol, a novel alpha, beta-adrenoceptor antagonist, on catecholamine secretion and calcium influx in cultured bovine adrenal chromaffin cells

The effect of carvediolol on the secretory function was studied using cultured bovine adrenal chromaffin cells. Carvedilol caused the concentration-dependent inhibition of catecholamine secretion evoked by carbamylcholine, high K+ or veratridine. The drug also caused the inhibition of radioactive calcium uptake stimulated by these secretagogues into the cells, and the inhibition of calcium uptake was observed in parallel with that of catecholamine secretion. The inhibitory action of carvedilol on catecholamine secretion was shown to be similar to that caused by a classical beta-adrenoceptor antagonist, propranolol. Furthermore, although the level of carbamylcholine-stimulated catecholamine secretion inhibited by diltiazem, a potent calcium channel antagonist, was significantly raised by elevating the calcium concentration in the reaction mixture, increasing the concentration of calcium ions in the mixture failed to induce any substantial influence on the secretion inhibited by carvedilol, as well as propranolol, under the same experimental conditions. These results seem to indicate that carvedilol may cause the inhibition of catecholamine secretion through its blocking action on calcium influx into the cells, and suggest the possibility that the inhibitory action of carvedilol on calcium influx is presumably based on its stabilizing action on the plasma membranes rather than its blocking action on the calcium channels in the chromaffin cell.     

Amphetamine enhances Ca2+ entry and catecholamine release via nicotinic receptor activation in bovine adrenal chromaffin cells

Amphetamine, a psychostimulant, has been shown to act as a channel blocker of muscle nicotinic receptors and to induce a Ca(2+)-dependent secretion from adrenal chromaffin cells. In this study, the relationship between amphetamine and nicotinic receptors was studied using bovine adrenal chromaffin cells as a model system. Our results show that D-amphetamine sulfate alone induced an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [3H]norepinephrine release in a dose-dependent and extracellular Ca(2+)-dependent manner. Two common nicotinic receptor antagonists, hexamethonium and mecamylamine, suppressed the D-amphetamine sulfate-induced [Ca(2+)](c) rise and [3H]norepinephrine release. In addition, D-amphetamine sulfate inhibited the 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP)-induced [Ca(2+)](c) rise and [3H]norepinephrine release, but not the high K(+)- or veratridine-induced [Ca(2+)](c) increase and [3H]norepinephrine release. Antagonists, including alpha-bungarotoxin and choline, that are more specific for alpha7 nicotinic receptors were capable of inhibiting the D-amphetamine sulfate-induced [Ca(2+)](c) rise, while D-amphetamine sulfate was found to be capable of inhibiting the [Ca(2+)](c) rise induced by the alpha7-nicotinic receptor agonists, epibatidine and choline. Moreover, D-amphetamine sulfate dose-dependently suppressed [3H]nicotine binding to chromaffin cells. We, therefore, conclude that D-amphetamine sulfate acts as a nicotinic receptor agonist to induce [Ca(2+)](c) increase and [3H]norepinephrine release in bovine adrenal chromaffin cells.     

LF 16.0335, a novel potent and selective nonpeptide antagonist of the human bradykinin B2 receptor

1. In the present paper, we describe the in vitro pharmacological properties of LF 16.0335 (1-[[3-[(2,4-dimethylquinolin-8-yl)oxymethyl]-2,4-dichloro-phenyl]sulphonyl] -2(S) - [[4 -[4-(aminoiminomethyl)phenylcarbonyl]piperazin-1-yl]carbonyl]pyrrolidine), a novel and potent nonpeptide antagonist of the human bradykinin (BK) B₂ receptor.
2. LF 16.0335 displaced [³H]-BK binding to membrane preparations from CHO cells expressing the cloned human B₂ receptor, INT 407 cells and human umbilical vein with K_i values of 0.84±0.39 nM, 1.26±0.68 nM and 2.34±0.36 nM, respectively.
3. In saturation binding studies performed in INT 407 cell membranes in the presence or absence of LF 16.0335, B_max values of [³H]-BK were not significantly changed suggesting that LF 16.0335 behaves as a competitive antagonist.
4. LF 16.0335 had no affinity for the cloned human kinin B₁ receptor stably expressed in 293 cells. In addition, this compound at 1 μM did not significantly bind to a range of 40 different membrane receptors and eight ion channels except muscarinic M₂ and M₁ receptors for which an IC₅₀ value of 0.9 and 1 μM was obtained.
5. BK stimulates in a concentration-dependent manner phosphoinositosides (IPs) production in cultured INT 407 cells. Concentration-response-curves to BK were shifted to the right in the presence of LF 16.0335 (0.1 μM) without reduction of the maximum. LF 16.0335 inhibited the concentration-contraction curve to BK in the human umbilical vein giving a pA₂ value of 8.30±0.30 with a Schild plot slope that was not different from unity.
6. These results demonstrate that LF 16.0335 is a potent, selective and competitive antagonist of the human bradykinin B₂ receptor.

     

Neuroprotective effects of RPR 104632, a novel antagonist at the glycine site of the NMDA receptor, in vitro

The NMDA antagonist and neuroprotective effects of RPR 104632 (2H-1,2,4-benzothiadiazine-1-dioxide-3-carboxylic acid), a new benzothiadiazine derivative, with affinity for the glycine site of the NMDA receptor-channel complex are described. RPR 104632 antagonized the binding of [³H]5,7-dichlorokynurenic acid to the rat cerebral cortex, with a K_i of 4.9 nM. This effect was stereospecific, since the (-)-isomer was 500-fold more potent than the (+)-isomer. The potent affinity of RPR 104632 for the glycine site was confirmed by the observation that RPR 104632 inhibited [³H]N-(1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) binding in the presence of N-methyl- -aspartate (NMDA) (IC₅₀ = 55 nM), whereas it had no effect on the competitive NMDA site or on the dissociative anaesthetic site. RPR 104632 inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (IC₅₀ = 890 nM) in a non-competitive manner and markedly reduced NMDA-induced neurotoxicity in rat hippocampal slices and in cortical primary cell cultures. These results suggest that RPR 104632 is a high-affinity specific antagonist of the glycine site coupled to the NMDA receptor channel with potent neuroprotective properties in vitro.     

Study of the novel non-xanthine heterocyclic compound GU285 as a potent non-selective adenosine receptor antagonist in the rat

The novel pyrazolo[3,4-d]pyrimidine compound GU285 (4-amino-6-alpha-carbamoylethylthio-1- phenylpyrazolo[3,4-d]pyrimidine, CAS 134896-40-5) was examined for its ability (1) to inhibit binding of adenosine (ADO) receptor ligands in rat brain membranes, (2) to antagonise functional responses to ADO agonists in rat right and left atria and coronary resistance vessels, and (3) to reduce the fall in heart rate and arterial blood pressure produced by the ADO A1 agonist N6-cyclopentyladenosine (CPA) in the intact, anaesthetized rat. GU285 competitively inhibited binding of the ADO A1 agonist [3H]-R-N6-phenylisopropyladenosine (R-PIA) yielding a Ki value of 11 (7-18) nmol.l-1 (geometric mean +/- 95% Cl). When assayed against the ADO A2A selective agonist [3H]-2-[p-(2-carboxyethyl)- phenethylamino]-5'-N-ethylcarboxamidoadenosine, (CGS21680), a Ki of 15 (10-24) nmol.l-1 was obtained. In spontaneously beating right atria, GU285 competitively antagonized negative chronotropic effects of R-PIA with a pA2 of 8.7 +/- 0.3 and in electrically paced left atria, GU285 competitively antagonized negative inotropic effects of R-PIA with a pA2 of 9.0 +/- 0.1. In the potassium-arrested, perfused rat heart GU285 (1 mumol.l-1) antagonized only the high sensitivity, ADO A2B mediated component of the biphasic relaxation of the coronary vasculature produced by NECA. The low sensitivity component was unchanged. GU285 (1 mumol.kg-1) antagonized the negative chronotropic and hypotensive effects of the adenosine A1 agonist CPA in anaesthetized rats, producing a 10-fold rightward shift in the dose-response relationship. These data demonstrate that in the rat, GU285 is a potent, non-selective adenosine receptor antagonist that maintains its activity in vivo.     

Electrophysiological characterization of a novel potent and orally active NMDA receptor antagonist: CGP 37849 and its ethylester CGP 39551

The selectivity and potency of the novel competitive N-methyl-D-aspartate (NMDA) receptor antagonists, CGP 37849 and CGP 39551, were investigated in vitro and in vivo using electrophysiological approaches. Like the reference blocker DL-AP5, both compounds acted in vitro (hippocampus, substantia nigra, spinal cord) to antagonize the excitatory actions of exogenously administered NMDA as well as the synaptically elicited, physiological NMDA receptor responses in hippocampus and spinal cord. In all isolated preparations CGP 37849 was more potent than CGP 39551, and 5- to 10-fold more potent than DL-AP5. Neither compound showed any marked effect on responses evoked by quisqualate and kainate. NMDA excited dopaminergic cells in the pars compacta region of the substantia nigra in a concentration-dependent manner. This effect also could be selectively antagonized by CGP 37849 and CGP 39551. In the anaesthetized rat, excitatory responses of hippocampal pyramidal cells evoked by iontophoretic application of NMDA were antagonized by CGP 37849 and CGP 39551 following their oral administration without reducing quisqualate or kainate responses. In contrast to the in vitro situation, CGP 39551 was more potent than CGP 37849 in vivo. Effective doses were 30 mg/kg p.o. for CGP 39551 and 100 mg/kg p.o. for CGP 37849. In conclusion, it is demonstrated that CGP 37849 and CGP 39551 selectively antagonize NMDA evoked neuronal responses in vivo and in vitro and that the drugs are centrally active following their oral administration.     

ACET is a highly potent and specific kainate receptor antagonist: characterisation and effects on hippocampal mossy fibre function

Kainate receptors (KARs) are involved in both NMDA receptor-independent long-term potentiation (LTP) and synaptic facilitation at mossy fibre synapses in the CA3 region of the hippocampus. However, the identity of the KAR subtypes involved remains controversial. Here we used a highly potent and selective GluK1 (formerly GluR5) antagonist (ACET) to elucidate roles of GluK1-containing KARs in these synaptic processes. We confirmed that ACET is an extremely potent GluK1 antagonist, with a K_b value of 1.4 ± 0.2 nM. In contrast, ACET was ineffective at GluK2 (formerly GluR6) receptors at all concentrations tested (up to 100 μM) and had no effect at GluK3 (formerly GluR7) when tested at 1 μM. The X-ray crystal structure of ACET bound to the ligand binding core of GluK1 was similar to the UBP310-GluK1 complex. In the CA1 region of hippocampal slices, ACET was effective at blocking the depression of both fEPSPs and monosynaptically evoked GABAergic transmission induced by ATPA, a GluK1 selective agonist. In the CA3 region of the hippocampus, ACET blocked the induction of NMDA receptor-independent mossy fibre LTP. To directly investigate the role of pre-synaptic GluK1-containing KARs we combined patch-clamp electrophysiology and 2-photon microscopy to image Ca²⁺ dynamics in individual giant mossy fibre boutons. ACET consistently reduced short-term facilitation of pre-synaptic calcium transients induced by 5 action potentials evoked at 20-25 Hz. Taken together our data provide further evidence for a physiological role of GluK1-containing KARs in synaptic facilitation and LTP induction at mossy fibre-CA3 synapses.     

Pharmacological profile of GR117289 in vitro: a novel, potent and specific non-peptide angiotensin AT1 receptor antagonist.

1. This paper describes the effects of GR117289 (1-[[3-bromo-2-[2-(1H-tetrazol-5-yl)phenyl]-5-benzo-furanyl]methyl ]-2-butyl-4-chloro-1H-imidazole-5-carboxylic acid) at angiotensin receptors and binding sites in rabbit aorta, rat liver and bovine cerebellum preparations in vitro. 2. In rabbit isolated aortic strips, GR117289 (0.3, 1 and 3 nM) caused a concentration-related, insurmountable suppression of the concentration-response curve to angiotensin II (AII). When the contact time was increased, a greater degree of antagonism of AII was observed, suggesting that GR117289 is slow to reach equilibrium. A pKB of 9.8 +/- 0.1 was calculated for GR117289 after 3 h incubation. GR117289 (1 microM) did not affect contractile responses to phenylephrine or 5-hydroxytryptamine (5-HT) in the rabbit aorta. 3. GR117289 (1 nM) alone caused a marked suppression and a slight rightward displacement of the AII concentration-response curve. Co-incubation with the competitive, surmountable AT1 receptor antagonist, losartan (10 nM, 100 nM and 1 microM), resulted in a concentration-related upward and rightward displacement of the concentration-response curve to subsequently administered AII. In separate experiments in which preparations were pre-incubated with GR117289 (1 nM), subsequent addition of losartan (1 microM) for 2, 15 or 45 min caused a further, but similar, rightward displacement of the concentration-response curve to subsequently administered AII with a time-dependent increase in the maximum response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel potent ligands for the central nicotinic acetylcholine receptor: synthesis, receptor binding, and 3D-QSAR analysis

In the past few years the focus on central acetylcholine receptors has shifted from compounds with affinity for muscarinic acetylcholine receptors (mAChR) to compounds with affinity for nicotinic acetylcholine receptors (nAChR). The therapeutic potential includes treatment of a variety of diseases, e.g., Alzheimer's disease, Parkinson's disease, and Tourette's syndrome. This work describes the synthesis of six novel series of potent ligands with nanomolar affinity for the alpha4beta2 nAChR subtype. Structure-activity relationship (SAR) was evaluated by the calculation of a 3D-QSAR model. 3D-QSAR analysis of the compounds using the GRID/GOLPE methodology resulted in a model of high quality (R(2) = 0.97, Q(2) = 0.81). The coefficient plots reveal that the steric interactions between the target and our compounds are of major importance for the affinity. Bulky substituents in the 6-position of the pyridine ring will reduce the affinity of the compounds, whereas bulky ring systems including a sp(3)-nitrogen will increase the affinity of the compounds.     

Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2 (CysLT2) receptor

Background and purpose:

Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT₁ and CysLT₂ receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT₂ receptor antagonist.

Experimental approach:

Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT₁ and CysLT₂ receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts.

Key results:

In a CysLT₂ receptor reporter cell line, HAMI3379 antagonized leukotriene D₄- (LTD₄-) and leukotriene C₄- (LTC₄-) induced intracellular calcium mobilization with IC₅₀ values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT₁ receptor cell line (IC₅₀ > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT₂ and CysLT₁ receptor cell lines, HAMI3379 inhibited [³H]-LTD₄ binding with IC₅₀ values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC₄-induced perfusion pressure increase and contractility decrease. The selective CysLT₁ receptor antagonist zafirlukast was found to be inactive in this experimental setting.

Conclusions and implications:

HAMI3379 was identified as a potent and selective CysLT₂ receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT₂ receptor.     

Short chain bombesin pseudopeptides with potent bombesin receptor antagonist activity in rat and guinea pig pancreatic acinar cells

A series of potent bombesin antagonists based on the reduced C-terminal peptide bond modification which in the past resulted in the first really potent antagonists are compared for effects on bombesin-stimulated amylase release from and binding to rat and guinea pig pancreatic acini. It was found that the original member of this series, [Leu13 psi (CH2NH)Leu14] bombesin, displayed partial agonist activity with 11% efficacy in the rat. More recent analogues of this type which were found previously to be even more potent pure antagonists in the guinea pig pancreas or 3T3 cells, exhibited similarly higher binding affinity for rat acini but displayed even higher residual partial agonist activity in the rat. For instance, [D-Phe6,Leu13 psi (CH2NH)Phe14]bombesin-(6-14) was one of the most potent bombesin antagonists known in the guinea pig and 3T3 cell systems but has 40% partial agonist activity in the rat. Several structural modification strategies were developed to remove rat partial agonist properties with retention of high antagonist potency in all systems tested. The most effective of these was the substitution of a Cl on the aromatic ring of the Phe residue (p-Cl-Phe, Cpa) in position 14 to give [D-Phe6,Leu13 psi (CH2NH)Cpa14]bombesin-(6-14). This had higher binding affinities for both rat and guinea pig pancreatic acini and was a pure antagonist on both cell types. Another effective method was alteration of the stereochemistry of the position 14 amino acid in [D-Phe6,Leu13 psi (CH2ND)D-Phe14]bombesin-(6-14) which had somewhat lowered binding affinities but pure antagonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)