Attenuation of heat shock-induced cardioprotection by treatment with the opiate receptor antagonist naloxone

Whole body hyperthermia induces heat shock proteins (HSPs), which confer cardioprotection. Several opioid receptor subtypes are expressed in the heart and are linked to cardioprotection; however, no one has attempted to link the protection elicited by heat stress (HS) to opioids. Therefore, we investigated the effect of an opiate receptor antagonist, naloxone, on HS-induced cardioprotection. Anesthetized Sprague-Dawley rats were subjected to HS (42 degrees C for 20 min) with and without naloxone pretreatment and were allowed to recover for 48 h. They then underwent 30 min of ischemia followed by 2 h of reperfusion. An acute HS group was given an intravenous bolus of naloxone (3 mg/kg) 10 min before index ischemia. Infarct size (IS), expressed as a percentage of the area at risk (IS/AAR), was determined. The right heart was excised for analysis of HSP content by Western blot. Heat-shocked rats showed significant reductions in IS/AAR versus control (16 +/- 3 vs. 58 +/- 4%, P < 0.001). Pretreatment with naloxone before HS attenuated the protective effects in a dose-dependent fashion, with significant attenuation of protection occurring at 15 mg/kg naloxone versus heat shock (42 +/- 6 vs. 16 +/- 3%, P < 0.001). Acute treatment with naloxone (3 mg/kg) 48 h after recovery from HS also significantly attenuated the delayed protective effect (47 +/- 4 vs. 16 +/- 3%, P < 0.001). No difference was seen in the level of HSP70 induced in the different groups. We conclude that heat shock-induced cardioprotection can be attenuated by naloxone, an opiate receptor antagonist, without reducing the levels of certain HSPs. These results suggest there may be a link between the endogenous release of opioids and HS that mediates cardioprotection.     

Decrease in the number of high-affinity opiate binding sites during the aging process inMytilus edulis (Bivalvia)

As Mytilus edulis(Bivalvia) ages, the number of stereospecific opiate binding sites in the visceral ganglia significantly decreases. The number of high-affinity etorphine sites decreased by 30%; the number of naloxone sites decreased by 23%. The affinity of the ligands did not vary with age. In addition, the augmentation by opioids of dopamine levels in the visceral ganglia was less in older animals and was accompanied by a reduced sensitivity of the ganglia to etorphine and DAMA (d-Ala ²,Met⁵-enkephalinamide).     

On the specificity of naloxone as an opiate antagonist.

Since the discovery of endogenous opioid peptides in brain (68,69,97,113, 128) and the pituitary gland (26,81,105,125) there has been considerable interest in their possible roles in a variety of physiological and pharmacological processes. Many studies have used antagonism by naloxone as a criterion for implicating endogenous opiates in a process, assuming that naloxene has no pharmacological actions other than those related to blockade of opiate receptors. The doses of naloxene used are often higher than those required to antagonize the analgesic and other effects of morphine. However, multiple forms of opiate receptors are present in nervous tissue and higher concentrations of naloxene are required to antagonize effects mediated by some of these receptors (83). Although the earlier literature supports the assumption that the effects of naloxene are due to the blockade of opiate receptors (87), there are an increasing number of reports which indicate that naloxene may have pharmacological actions unrelated to opiate receptor blockade. The subsequent review serves to emphasize that antagonism by naloxene is a necessary but not sufficient criterion for invoking the mediation of a response by an endogenous opiate (61). Additional lines of evidence which serve to strengthen the conclusion that endogenous opiates mediate a process will be considered.     

Effect of the opiate antagonist naloxone on the electrical activity of identified neurons in the edible snail

The opiate antagonist naloxone modifies the electric activity of some identified neurons of the Helix lucorum which have not been preliminary exposed to the effect of exogenous opioids. Some neurons are excited while others are inhibited by naloxone, and in both cases the reaction may have both a short and long latent period. The reactions depend on naloxone dose and become less expressed or are blocked when naloxone is administered together with the agonists of opiate receptor (morphine, D-Ala2, D-Leu5-enkephalin, bremazocine and beta-endorphin). Opioids alone do not produce any effect on neurons. The effect of naloxone on neurons is assumed to be a result of the elimination by the opiate antagonist of the tonic effect of endogenous opioids by their replacing on opiate receptors which are constantly stimulated by endogenous ligands.     

Antitubercular activity of trifluoperazine, a calmodulin antagonist

Trifluoperazine, a calmodulin antagonist, completely inhibited the growth of mycobacteria. The minimum inhibitory concentrations in shake cultures in a synthetic medium containing 0.2% Tween 80 were 5 and 8 micrograms/ml, respectively, for the human pathogenic strain Mycobacterium tuberculosis H37Rv and M. tuberculosis resistant to isoniazid. When added to a growing culture of M. tuberculosis H37Rv on the 10th day (mid exponential phase), trifluoperazine 50 micrograms/ml further arrested growth of this organism. It is suggested that trifluoperazine or similar calmodulin antagonists might be useful as antitubercular drugs.     

Circadian pattern of motor activity in adults with attention-deficit/hyperactivity disorder

This study aims to describe the 24-hour activity rhythm in adults with attention-deficit/hyperactivity disorder (ADHD). A total of 18 ADHD patients and 37 healthy controls (HCs) wore an actigraph for 7 days. ADHD patients showed higher motor activity than HCs at 4:00, 6:00, 15:00 and 16:00 hour. Within the theoretical framework of the two-process model of sleep regulation, the observed data may be explained by lower homeostatic sleep pressure in ADHD. This could lead to an increase in motor activity in the second half of the night, when sleep need decreases more rapidly, and in the first half of the afternoon, when patients do not experience the typical post-lunch dip.     

The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist

Abstract Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae . Different growth conditions of the producer strain affect microcin activity. The production of a microcin antagonist is responsible for the changes in microcin activity. The microcin antagonist is induced when cells are iron-deprived, resulting in a low microcin activity. The microcin antagonist was purified using a procedure developed for the isolation of a catechol-type siderophore, and its activity was titrated using purified microcin. The inhibitory effect of the microcin antagonist is not observed when this compound is forming a complex with iron. The same inhibitory effect on microcin activity was obtained using purified enterochelin from Escherichia coli . The microcin antagonist was identified as enterochelin through thin-layer chromatography.     

A physicochemical study of the interaction of phosphatidylinositol with buprenorphine and naloxone

The interactions of two opioid molecules (buprenorphine and naloxone) with phosphatidylinositol and phosphatidylcholine were studied in lipid monolayers at the air-water interface. The influence of Na⁺, Ca²⁺, and Mn²⁺ ions in these interactions has also been determined. Neither buprenorphine nor naloxone influence the ordered state of phosphatidylcholine monolayers. On the contrary, both opioid molecules interact specifically with phosphatidylinositol monolayers. The area/molecule of phosphatidylinositol spread on buprenorphine containing subphases is highly affected by this molecule and also by ions. The phosphatidylinositol/naloxone interactions are rather weak and less affected by ions.Abbreviations PI phosphatidylinositol - PC phosphatidylcholine - Bup Buprenorphine - Nx naloxone - ODS octadecyl silica - k capacity factor - logP partition coefficient between octanol and water     

Attenuation of cocaine-induced conditioned place preference by Polygala tenuifolia root extract

A recent investigation indicated that Polygala tenuifolia Willdenow extract (PTE) possesses a potential antipsychotic effect. In this study, we examined the effects of PTE on the cocaine-induced changes in locomotor activity, conditioned place preference (CPP), fos-related antigen-immunoreactivity (FRA-IR), and activator protein (AP)-1 DNA binding activity. Cocaine-induced behavioral effects (hyperlocomotion and CPP) occurred in parallel with increases in FRA-IR and AP-1 DNA binding activity in the nucleus accumbens. These responses induced by cocaine were consistently attenuated by concurrent treatment with PTE (25 mg or 50 mg/kg/day, i.p. x 7). The adenosine A2A receptor antagonist, 1,3,7-trimethyl-8-(3-chlorostyrl)xanthine (0.5 or 1.0 mg/kg, i.p.), reversed the PTE-mediated pharmacological action in a dose related manner; neither the adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (0.5 or 1.0 mg/kg, i.p.) nor the A2B receptor antagonist, alloxazine (1.5 or 3.0 mg/kg, i.p.) significantly affected this pharmacological action. Our results suggest that PTE prevents cocaine-induced behavioral effects, at least in part, via the activation of the adenosine A2A receptor.     

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.     

Chemical modification-mediated optimisation of bronchodilatory activity of mepenzolate,a muscarinic receptor antagonist with anti-inflammatory activity

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(–)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM₃R). Compared to 1 and 5, (R)-(–)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(–)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(–)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.     

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.     

Reversible,long-term inhibition of ovulation with a gonadotropin-releasing hormone antagonist in the marmoset monkey (Callithrix jacchus)

The effects of weekly injections of a gonadotropin-releasing hormone (GnRH) antagonist (GnRHa) ([N-acetyl-DβNal1-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8D-Ala10] NH2 GnRH) on pituitary and ovarian function were examined in the marmoset monkey, Callithrix jacchus. In experiment 1, five cyclic females were given weekly injections of vehicle (50% propylene glycol in saline) for 6 weeks followed by GnRHa for 20 weeks, animals receiving either 200 μg GnRHa/injection (n = 2) or 67 μg GnRHa/injection (n = 3) for 10 weeks, after which the treatment was reversed. Bioactive luteinizing hormone (LH) and progesterone (Po) were measured in blood samples (0.2–0.4 ml) collected twice weekly until at least 8 weeks after the last GnRHa injection. GnRHa treatment, timed to begin in the midluteal phase, caused a rapid decline in LH and Po and luteal regression after a single injection (both doses). Po levels were consistently low (<10 ng/ml), and ovulation was inhibited throughout 200 μg treatment in all animals. Short periods of elevated Po (>10 ng/ml) were, however, occasionally seen during 67 μg treatment, indicating incomplete ovarian suppression. Mean LH levels were significantly lower during GnRHa treatment compared with the period of vehicle injection (all animals 200 μg; three animals 67 μg), and there were significant differences in LH levels between GnRHa treatments (200 μg vs. 67 μg) in four animals. Four animals resumed normal ovarian cycles after the end of GnRHa treatment (15/16 days, three animals; 59 days, one animal); the fifth animal died of unknown causes 32 days after the last GnRHa injection. In a second experiment, pituitary responsiveness to exogenous GnRH was tested 1 day after a single injection of vehicle or antagonist (200 or 67 μg). Measurement of bioactive LH indicated that pituitary response to 200 ng native GnRH was significantly suppressed in animals receiving the antagonist, the degree of suppression being dose related. A third experiment examined the effect of four weekly injections of 200 μg GnRHa on follicular size and granulosa cell responsiveness to human follicle-stimulating hormone (hFSH) in vitro. Follicular development beyond 1 mm was inhibited by GnRHa treatment (preovulatory follicles normally 2-4 mm) although granulosa cell responsiveness to FSH during 48 hr of culture was not impaired. These results suggest that the GnRHa-induced suppression of follicular development and ovulation was mediated primarily by an inhibition of pituitary gonadotropin secretion and not by a direct action at the level of the ovary.     

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

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

Electronic supplementary material

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

Determination of ZD9583, a thromboxane receptor antagonist, in human plasma and urine by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

A liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI-MS–MS) method is described for the determination of a thromboxane receptor antagonist (4Z)-6-((2S,4S,5R)-2-(1-(2-cyano-4-methylphenoxy)-1-methylethyl)-4-(3-pyridyl)-3-dioxan-5-yl)hex-4-enoic acid (ZD9583, I) in human plasma and urine. Proteins in plasma and urine samples are precipitated using acidified acetonitrile. The resulting supernatant is chromatographed on a C₈ reversed-phase chromatography column. Following the diversion of the solvent front from the mass spectrometer by a switching valve, the column eluate is passed on to the mass spectrometer via a heated nebulizer interface where the analyte is detected by multiple reaction monitoring (MRM). The method has a chromatographic run time of less than 2 min, a linear calibration curve with a range of 1–500 ng ml⁻¹ and intra- and inter-day precision estimates of less than 10% over the calibration range.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

Tryptophanol-derived oxazolopiperidone lactams: Identification of a hit compound as NMDA receptor antagonist

N-Methyl-d-aspartate receptors (NMDAR) exacerbated activation leads to neuron death through a phenomenon called excitotoxicity. These receptors are implicated in several neurological diseases (e.g., Alzheimer and Parkinson) and thus represent an important therapeutic target. We herein describe the study of enantiopure tryptophanol-derived oxazolopiperidone lactams as NMDA receptor antagonists. The most active hit exhibited an IC₅₀ of 63.4 μM in cultured rat cerebellar granule neurons thus being 1.5 fold more active than clinically approved NMDA antagonist amantadine (IC₅₀ = 92 μM).     

Enkephalin is a competitive antagonist of cholecystokinin in the gastrointestinal tract, as predicted from prior conformational analysis

Prior calculations based on ECEPP (Empirical Conformational Energies for Peptides Program) of the low energy minima for cholecystokinin (CCK) and Met-enkephalin have demonstrated that significant structural features of these two peptides are identical. This result suggested the possibility that Met-enkephalin, as well as other enkephalin analogues of similar structure, could associate with receptors for CCK. To test this theoretical result, we examined the ability of Met-enkephalin and its analogues to bind to peripheral CCK receptors in the rat gastrointestinal tract; in particular, we measured the ability of the opiate peptide to inhibit the effects of CCK in a physiological assay system which we have previously characterized: CCK-induced contraction of the isolated rat pyloric sphincter. We find that Met-enkephalin is an antagonist of the CCK-8-induced contraction, with a IC₅₀ of 110 nM. Furthermore, antibodies against CCK were found to cross-react with Met-enkephalin and its analogues in a manner which suggests a distinct structure-activity relationship. These experimental results strongly support the theoretical results of conformational analysis showing structural similarity between enkephalin and CCK. They further suggest that enkephalins could modulate the response of CCK systems under physiological conditions.     

Isolation of a heptapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln (valorphin) with some opiate activity.

Bovine hypothalamic tissue was extracted and purified by solid phase extraction and several reversed-phase HPLC steps. The amino acid sequence of the purified peptide was determined by Edman degradation to be Val-Val-Tyr-Pro-Trp-Thr-Gln. This was confirmed by comparison of its chromatographic behavior with that of the synthetic peptide, and mass spectrometric analysis resulted in a mass identical to the calculated mass for this peptide. This heptapeptide shows homology with residues 32-38 of the beta-chain of bovine hemoglobin. The peptide inhibited the electrically induced contractions of the guinea pig ileum muscle preparation; this inhibition was reversible by naloxone. It also inhibited the binding of 125I-DAMGO (selective for mu receptors) to rat brain with an IC50 of 10 microM and the binding of 3H-DPDPE (selective for sigma receptors) with an IC50 of 185 microM. With two valines at the N-terminus and some opiate activity, valorphin seems a suitable name for this newly isolated peptide.     

Association of the glutamate receptor subunit gene GRIN2B with attention-deficit/hyperactivity disorder

The glutamatergic signaling pathway represents an ideal candidate susceptibility system for attention-deficit/hyperactivity disorder (ADHD). Disruption of specific N-methyl-D-aspartate-type glutamate receptor subunit genes (GRIN1, 2A-D) in mice leads to significant alterations in cognitive and/or locomotor behavior including impairments in latent learning, spatial memory tasks and hyperactivity. Here, we tested for association of GRIN2B variants with ADHD, by genotyping nine single nucleotide polymorphisms (SNPs) in 205 nuclear families identified through probands with ADHD. Transmission of alleles from heterozygous parents to affected offspring was examined using the transmission/disequilibrium test. Quantitative trait analyses for the ADHD symptom dimensions [inattentive (IA) and hyperactive/impulsive (HI)] and cognitive measures of verbal working memory and verbal short-term memory were performed using the fbat program. Three SNPs showed significantly biased transmission (P < 0.05), with the strongest evidence of association found for rs2,284,411 (chi(2)= 7.903, 1 degree of freedom, P= 0.005). Quantitative trait analyses showed associations of these markers with both the IA and the HI symptom dimensions of ADHD but not with the cognitive measures of verbal short-term memory or verbal working memory. Our data suggest an association between variations in the GRIN2B subunit gene and ADHD as measured categorically or as a quantitatively distributed trait.