Rat Brain Membranes Research Articles

Characterization of a novel Ca2+-Activated potassium channel in rat brain rough endoplasmic reticulum

ObjectivesPotassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). MethodsRats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to −60 mV following incorporation of rat brain RER vesicles into planar bilayers. ResultsWe observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. ConclusionOur study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.

SYNTHESIS AND PROPERTIES OF NEW 1-HYDRAZINOCARBONYLMETHYL-7-BROMO-5-PHENYL-3-ARYLYDENE-1,2-DIHYDRO-3H-1,4-BENZODIAZEPIN-2-ONES

It is known that 1,4-benzodiazepines have neurotropic properties. Previously, we synthesized a series of 3-arylidene-1,2-dihydro-3H-1,4-benzdiazepin-2-ones 11-18 and showed that they exhibit significant analgesic activity. They also show significant affinity for central benzodiazepine receptors and TSPO receptors CNS. With this in mind, we have previously modified the series of 3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones by alkylation of 3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones with monobromoacetic acid methyl ester to the first position of the 1,4-diazepine ring to obtain 1-methoxycarbonylmethyl-7-bromo-5-phenyl-3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones 19-26, which showed pronounced analgesic activity. The aim of this work is to synthesize and study the affinity for CBR and TSPO receptors 1-hydrazinocarbonylmethyl-7-bromo-5-phenyl-3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones 27-31, which have not been previously described in the literature. Compounds 27-31 were synthesized as a result of the reaction of 1-methoxycarbonylmethyl-7-bromo-5-phenyl-3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones 19-26 with hydrazine hydrate while stirring the components in ethanol. The yields of the reaction products were 65-92%. It was shown that the hydrazinolysis technique used provides high yields of the target compounds. The structure of the synthesized compounds was confirmed by mass spectrometry and 1H NMR spectroscopy. The affinity of the synthesized compounds to the central benzodiazepine receptors CNS (CBR) and TSPO receptors CNS was studied. The affinity of compounds 27-31 was determined in vitro by radioligand analysis by their ability to displace the commercial radioligands [3H] flumazenil and [3H]PK11195 from their specific binding sites in the GABAA receptor complex and TSPO receptors of the synaptic and the mitochondrial fractions of the rat brain membranes, respectively. Compound 27 displaces the commercial [3H]flumazenil radioligand from its specific binding sites in the GABAA receptor complex by 80.1%, and simultaneously exhibits a very low affinity for TSPO receptors. Derivative 27 is the most potent CBR ligand among the investigated compounds and it is a promising compound for pharmacological research. It has been established that in the synthesized series of 3-fluorobenzylidene derivatives, varying the position of the fluorine atom in the benzylidene fragment leads to change affinity for TSPO receptors. It has been established that the para- position of the fluorine atom in the 3-benzylidene radical of the 1,4-benzodiazepin-2-one molecule is the most important descriptor that determines their affinity for the TSPO receptors. These data agree with the data available in the literature on the effect of the position of chlorine and bromine atoms in the 3-benzylidene radical of the 1,4-benzodiazepin-2-one molecule on the affinity for the TSPO receptors. It was found that the main structural fragment that determines the affinity of binding of 3-benzylidene derivatives to TSPO receptors is the presence of electronegative substituents in the para-position of the benzylidene fragment: para–Hal>>оrtho–Hal>meta–Hal (affinity for TSPO) (Hal= Br, F) Thus, the expediency of further study of this class of compounds is shown.

Alkoxy chain length governs the potency of 2-benzylbenzimidazole 'nitazene' opioids associated with human overdose.

Novel synthetic opioids (NSOs) are emerging in recreational drug markets worldwide. In particular, 2-benzylbenzimidazole 'nitazene' compounds are problematic NSOs associated with serious clinical consequences, including fatal respiratory depression. Evidence from in vitro studies shows that alkoxy chain length can influence the potency of nitazenes at the mu-opioid receptor (MOR). However, structure-activity relationships (SARs) of nitazenes for inducing opioid-like effects in animal models are not well understood compared to relevant opioids contributing to the ongoing opioid crisis (e.g., fentanyl). Here, we examined the in vitro and in vivo effects of nitazene analogues with varying alkoxy chain lengths (i.e., metonitazene, etonitazene, isotonitazene, protonitazene, and butonitazene) as compared to reference opioids (i.e., morphine and fentanyl). Nitazene analogues displayed nanomolar affinities for MOR in rat brain membranes and picomolar potencies to activate MOR in transfected cells. All compounds induced opioid-like effects on locomotor activity, hot plate latency, and body temperature in malemice, and alkoxy chain length markedly influenced potency. Etonitazene, with an ethoxy chain, was the most potent analogue in MOR functional assays (EC50 = 30pM, Emax = 103%) and across all in vivo endpoints (ED50 = 3-12μg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

Effect of moderate prenatal ethanol exposure on the differential expression of two histamine H3 receptor isoforms in different brain regions of adult rat offspring.

We have reported that prenatal alcohol exposure (PAE) elevates histamine H3 receptor (H3R) agonist-mediated inhibition of glutamatergic neurotransmission in the dentate gyrus. Here, we hypothesized that PAE alters the expression of two prominent H3R isoforms namely, the rH3A and rH3C isoforms, which have differing intrinsic activities for H3R agonists, in a manner that may contribute to heightened H3R function in PAE rats. In contrast to our predictions, we found different effects of sex and PAE in various brain regions with significant interactions between sex and PAE in dentate gyrus and entorhinal cortex for both isoforms. Subsequently, to confirm the PAE-and sex-induced differences on H3R isoform mRNA expression, we developed a polyclonal antibody selective for the rH3A inform. Western blots of rH3A mRNA-transfected HEK-293 cells identified a ~ 48 kDa band of binding consistent with the molecular weight of rH3A, thus confirming antibody sensitivity for rH3A protein. In parallel, we also established a pan-H3R knockout mice line to confirm antibody specificity in rodent brain membranes. Both qRT-PCR and H3R agonist-stimulated [35S]-GTPγS binding confirmed the absence of mH3A mRNA and H3 receptor-effector coupling in H3R knockout (KO) mice. Subsequent western blotting studies in both rat and mouse brain membranes were unable to detect rH3A antibody binding at ~48 kDa. Rather, the H3RA antibody bound to a ~ 55 kDa band in both rat and mouse membranes, including H3R KO mice, suggesting H3RA binding was not specific for H3Rs in rodent membranes. Subsequent LC/MS analysis of the ~55 kDa band in frontal cortical membranes identified the highly abundant beta subunit of ATPase in both WT and KO mice. Finally, LC/MS analysis of the ~48 kDa band from rH3A mRNA-transfected HEK-293 cell membranes was able to detect rH3A protein, but its presence was below the limits of quantitative reliability. We conclude that PAE alters rH3A and rH3C mRNA expression in some of the same brain regions where we have previously reported PAE-induced alterations in H3R-effector coupling. However, interpreting the functional consequences of altered H3R isoform expression was limited given the technical challenges of measuring the relatively low abundance of rH3A protein in native membrane preparations.

RO6807936 as a Novel PET Radiotracer for in Vitro and in Vivo Visualization and Quantification of BACE1 in the Rodent and Baboon Brain.

The beta-site amyloid precursor protein cleaving enzyme (BACE1) is responsible for initiating the generation of beta-amyloid, the major constituent of amyloid plaques in Alzheimer's disease (AD). The purpose of this study was to develop a specific BACE1 radioligand for visualization of the distribution pattern and quantification of the BACE1 protein in the rodent and monkey brain both in vitro by autoradiography and in vivo by positron emission tomography (PET). The BACE1 inhibitor RO6807936 originating from an in-house chemical drug optimization program was selected based on its PET tracer like physicochemical properties and a favorable pharmacokinetic profile. Saturation binding analysis of [3 H]RO6807936 revealed specific and high affinity binding (KD = 2.9 nM) and a low Bmax value (4.3 nM) of the BACE1 protein in native rat brain membranes. [3 H]RO6807936 binding showed a ubiquitous distribution on rat brain slices in vitro with higher levels in the CA3 8 cell layer and the granule cell layer of the hippocampus. In a next step, RO6807936 was successfully radiolabelled with carbon-11 and showed acceptable uptake in the baboon brain as well as a widespread and rather homogeneous distribution consistent with rodent data. In vivo blockade studies with a specific BACE1 inhibitor reduced uptake of the tracer to homogenous levels across brain regions and demonstrated specificity of the signal. Our data warrant further profiling of this PET tracer candidate in humans to investigate BACE1 expression in normal individuals and those with AD and as an imaging biomarker for target occupancy studies in clinical drug trials.

Agonistic properties of a series of psychotropic drugs at 5-HT1A receptors in rat and human brain membranes determined by [35S]GTPγS binding assay.

Many psychoactive compounds have been developed to have more beneficial clinical efficacy than conventional drugs by adding agonistic action at 5-HT1A receptors. The aim of the present study was to evaluate several psychotropic drugs that had been reported to behave as an agonist at 5-HT1A receptor (aripiprazole, brexpiprazole, asenapine, lurasidone, and vortioxetine) in both rat and postmortem human brain membranes. The [35S]GTPγS binding assay for Gi/o proteins coupled with 5-HT1A receptors was performed in rat brain membranes and postmortem human brain membranes. The specific binding was stimulated by brexpiprazole in rat hippocampus, human hippocampus, and human prefrontal cortex. Aripiprazole also behaved as an agonist in the same brain regions. Interestingly, its potency was much higher in rat hippocampal membranes than in human brain membranes, indicating the possibility of species differences. Although vortioxetine was an efficacious stimulator at high concentrations, its potency was undeterminable because of a lack of saturability. In addition to 5-HT1A receptor agonism, involvement of other components, e.g., 5-HT1B receptor agonism, was speculated by the biphasic inhibitory effects of the selective 5-HT1A receptor neutral antagonist. Negligible stimulatory effects were obtained as to lurasidone and asenapine. Our previous studies have raised the concept of a psychoactive drug group with a common pharmacological mechanism of action, i.e., 5-HT1A receptor agonism, consisting of perospirone, aripiprazole, ziprasidone, clozapine, quetiapine, nemonapride, and trazodone. The present study demonstrates the data indicating that brexpiprazole and probably vortioxetine are included in this drug group. Lurasidone and asenapine are excluded from this group.

Nano-LC-FTMS Analysis of the Primary Structure and Post-translational Modifications of the α1 Subunit of the GABAA Receptor

GABAAR is one of the most significant drug targets in the treatment of neuropsychiatric disorders such as epilepsy, insomnia, anxiety, as well as anesthesia in surgical operations. However, complete information on the regulation of GABAA receptor activity is lacking. Therefore, studies are needed on post-translational modifications of receptor subunits that exhibit different pharmacological and physiological properties. GABAAR has been immunopurified from rat brain membranes on protein A agarose beads immobilized with complex anti-GABAAR antibody. Primary structure and post-translational modifications of the α1 subunit of GABAAR have been characterized by nano-LC-FTMS peptide mapping by direct in situ–gel digestion on the one-dimensional gel. The primary structure of the α1 subunit of GABAAR has been identified with high sequence coverage (81%, 371 from 455 amino acids). The extracellular domains, N-terminal and the C-terminus, have been identified with extensive sequence coverage, 85 and 100 % (214 from 249 amino acids), respectively. Other soluble domains, including the M1-M2 linker, and the M3-M4 linker, have also been determined with 100 and 85 % (75 from 88 amino acids), respectively. Transmembrane domains, including M1, M2, and M4, were identified almost completely. Post-translational modifications of the α1 subunit peptides have been found, such as phosphorylation, methionine oxidation, and carbamidomethylation.

Regional binding of [3H]‐MK801 to NMDA receptors in young and aged rat brain membranes

Norepinephrine (NE) and dopamine (DA) are essential neuromodulators in the central nervous system. They regulate several crucial physiological processes, including arousal/attention, focus, neuroendocrine functions, and the sleep‐wake cycle; they also play complex roles in regulating cognition and affect; therefore, dysfunctions in catecholaminergic neurotransmission are associated with various neurological consequences. Previously, we confirmed that in brain slices from aged Fischer 344 rats, there is an age‐related decrease in glutamate‐stimulated NE release in the cerebral cortex and hippocampus (Gonzales et al., 1991 & Dezfuli et al., 2019) and a regionally specific decline in glutamate‐stimulated DA release, with a deficit in the striatum but not in the olfactory tubercle (Aljohani et al., 2021a). Importantly, our studies demonstrated that NMDA receptors mediate glutamate‐stimulated NE and DA release. We also have shown that the alpha‐2 adrenergic receptors agonist clonidine decreases this glutamate‐stimulated NE release in a concentration‐dependent manner; while in contrast, blockade of these receptors with phentolamine, an alpha‐1 and alpha‐2 adrenergic receptor blocker, or with idazoxan, a selective alpha‐2 receptor blocker, markedly potentiates glutamate‐stimulated NE release in cortical brain slices (Aljohani et al., 2021b). Since aging has heterogeneous effects on NMDA receptors in different brain areas, we have now begun to examine [3H]‐MK801 binding to NMDA receptors to compare changes in receptor density and responsiveness during aging in the cortex, hippocampus, striatum, and olfactory tubercle, as a possible mechanistic explanation for the changes in NE and DA release. The data from cortical membrane preparations suggest that young and old rats have similar NMDA receptor densities but may differ in their response to stimulation with glutamate/glycine.

Pharmacokinetics and pharmacodynamics of the synthetic cannabinoid, 5F-MDMB-PICA, in male rats

5F-MDMB-PICA is a popular synthetic cannabinoid associated with analytically confirmed intoxications. In vitro studies show 5F-MDMB-PICA is a potent cannabinoid-1 receptor (CB1) agonist, but little information is available about in vivo pharmacokinetics and pharmacodynamics. To this end, the present study had three aims: 1) to develop a validated method for detection of 5F-MDMB-PICA and its metabolites in rat plasma, 2) to utilize the method for investigating pharmacokinetics of 5F-MDMB-PICA in rats, and 3) to relate 5F-MDMB-PICA pharmacokinetics to pharmacodynamic effects. 5F-MDMB-PICA and its metabolites were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) and method validation followed forensic standards. Male Sprague-Dawley rats bearing surgically implanted jugular catheters and subcutaneous (s.c.) temperature transponders received 5F-MDMB-PICA (50, 100, or 200 μg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, 240, and 480 min post-injection, and plasma was assayed using LC-MS/MS. At each blood draw, body temperature, and catalepsy scores were recorded. Maximum plasma concentrations (Cmax) of 5F-MDMB-PICA rose linearly with increasing dose (1.72–6.20 ng/mL), and plasma half-life (t1/2) ranged from 400 to 1000 min 5F-MDMB-PICA-3,3-dimethylbutanoic acid and 5OH-MDMB-PICA were the only metabolites detected, and plasma concentrations were much lower than the parent drug. 5F-MDMB-PICA induced robust hypothermia and catalepsy-like symptoms that were significantly correlated with concentrations of 5F-MDMB-PICA. Radioligand binding in rat brain membranes revealed 5F-MDMB-PICA displays high affinity for CB1 (IC50 = 2 nM) while metabolites do not. In summary, 5F-MDMB-PICA is a potent CB1 agonist in rats whose pharmacodynamic effects are related to circulating concentrations of the parent drug and not its metabolites.

In silico Screening and Behavioral Validation of a Novel Peptide, LCGA-17, With Anxiolytic-Like Properties.

The aim of the study was to develop better anxiolytics and antidepressants. We focused on GABAA receptors and the α2δ auxiliary subunit of V-gated Ca2+ channels as putative targets because they are established as mediators of efficacious anxiolytics, antidepressants, and anticonvulsants. We further focused on short peptides as candidate ligands because of their high safety and tolerability profiles. We employed a structural bioinformatics approach to develop novel tetrapeptides with predicted affinity to GABAA receptors and α2δ. In silico docking studies of one of these peptides, LCGA-17, showed a high binding score for both GABAA receptors and α2δ, combined with anxiolytic-like properties in a Danio rerio behavioral screen. LCGA-17 showed anxiolytic-like effects in the novel tank test, the light–dark box, and the social preference test, with efficacy comparable to fluvoxamine and diazepam. In binding assays using rat brain membranes, [3H]-LCGA-17 was competed more effectively by gabapentinoid ligands of α2δ than ligands of GABAA receptors, suggesting that α2δ represents a likely target for LCGA-17. [3H]-LCGA-17 binding to brain lysates was unaffected by competition with ligands for GABAB, glutamate, dopamine, serotonin, and other receptors, suggesting specific interaction with α2δ. Dose-finding studies in mice using acute administration of LCGA-17 (i.p.) demonstrated anxiolytic-like effects in the open field test, elevated plus maze, and marble burying tests, as well as antidepressant-like properties in the forced swim test. The anxiolytic effects were effectively blocked by bicuculline. Therefore, LCGA-17 is a novel candidate anxiolytic and antidepressant that may act through α2δ, with possible synergism by GABAA receptors.

Kigelia africana (Lam.) Benth leaf extract inhibits rat brain and liver mitochondrial membrane permeability transition pore opening

The effect of Kigelia africana on mitochondrial membrane permeability transition has not been explored. In this study, the effect of a solvent fraction of Kigelia africana leaf extract on mitochondrial membrane permeability transition of rat brain and liver was evaluated. A methanol extract of K. africana leaves was fractionated into different solvents by vacuum liquid chromatography and following preliminary screening, the dichloromethane:ethylacetate (1:1) fraction was selected for further assays. Constituent phytochemicals in the fraction were revealed by thin-layer chromatography and further purification was carried out to characterize the compounds. Brain and liver mitochondria were isolated and used for mitochondrial membrane permeability transition and adenosine triphosphatase assays. Exogenous Ca2+ and Al3+ were used to trigger the mitochondrial membrane permeability transition opening. Physicochemical properties revealed by thin-layer chromatography showed that the isolated compounds were flavonoids. The extract inhibited mitochondrial membrane permeability transition opening in the presence and absence of triggering agents in brain and liver mitochondria. It also inhibited mitochondrial lipid peroxidation and adenosine triphosphatase activity. These results suggest that the extract can limit the rate of apoptosis via inhibition of mitochondrial membrane permeability transition which is pivotal to the mitochondrial apoptotic pathway and is an important therapeutic target in some pathological conditions.

Humulone Modulation of GABAA Receptors and Its Role in Hops Sleep-Promoting Activity.

Humulus lupulus L. (hops) is a major constituent of beer. It exhibits neuroactive properties that make it useful as a sleeping aid. These effects are hypothesized to be mediated by an increase in GABAA receptor function. In the quest to uncover the constituents responsible for the sedative and hypnotic properties of hops, recent evidence revealed that humulone, a prenylated phloroglucinol derivative comprising 35–70% of hops alpha acids, may act as a positive modulator of GABAA receptors at low micromolar concentrations. This raises the question whether humulone plays a key role in hops pharmacological activity and potentially interacts with other modulators such as ethanol, bringing further enhancement in GABAA receptor-mediated effects of beer. Here we assessed electrophysiologically the positive modulatory activity of humulone on recombinant GABAA receptors expressed in HEK293 cells. We then examined humulone interactions with other active hops compounds and ethanol on GABA-induced displacement of [3H]EBOB binding to native GABAA receptors in rat brain membranes. Using BALB/c mice, we assessed humulone’s hypnotic behavior with pentobarbital- and ethanol-induced sleep as well as sedation in spontaneous locomotion with open field test. We demonstrated for the first time that humulone potentiates GABA-induced currents in α1β3γ2 receptors. In radioligand binding to native GABAA receptors, the inclusion of ethanol enhanced humulone modulation of GABA-induced displacement of [3H]EBOB binding in rat forebrain and cerebellum as it produced a leftward shift in [3H]EBOB displacement curves. Moreover, the additive modulatory effects between humulone, isoxanthohumol and 6-prenylnaringenin were evident and corresponded to the sum of [3H]EBOB displacement by each compound individually. In behavioral tests, humulone shortened sleep onset and increased the duration of sleep induced by pentobarbital and decreased the spontaneous locomotion in open field at 20 mg/kg (i.p.). Despite the absence of humulone effects on ethanol-induced sleep onset, sleep duration was increased dose-dependently down to 10 mg/kg (i.p.). Our findings confirmed humulone’s positive allosteric modulation of GABAA receptor function and displayed its sedative and hypnotic behavior. Humulone modulation can be potentially enhanced by ethanol and hops modulators suggesting a probable enhancement in the intoxicating effects of ethanol in hops-enriched beer.

Characterization of the N6-etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism.

The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66h) metabolized N6-etheno-ATP. Applied N6-etheno-ATP was recovered in the medium as N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and surprisingly N6-etheno-adenine; intracellular N6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N6-etheno-ATP, N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and N6-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors (3H-α,β-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (35S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N6-etheno-adenosine was partially converted to N6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N6-etheno-ATP was quickly metabolized, with N6-etheno-adenine being the main product in naïve rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N6-etheno-adenosine to N6-etheno-adenine.

Tritiated JNJ‐63514230 is a novel GluN2B‐selective radioligand with no affinity to sigma receptors

IntroductionCommonly used GluN2B radioligands exhibit limited selectivity towards GluN2B receptors as they interact with sigma and other off‐target receptors. Here we describe development and characterization of [3H] JNJ‐63514230, a novel radioligand selective for GluN2B‐containing NMDA receptors.MethodsThe selectivity JNJ 63514230 was determined using functional assays and CEREP panel. Saturation binding and competitive radioligand binding experiments were used to characterize [3H]‐JNJ 63514230 properties. We have also tested utility of [3H]‐JNJ 63514230 as a tracer for ex‐vivo autoradiography in rat brain.ResultsWe confirmed that widely used tracers [3H] Ifenprodil and [3H] Ro 25‐6981 have substantial affinity to sigma receptors as measured in radioligand competition experiments (CEREP). In contrast, new tracer [3H] JNJ‐63514230 exhibits no affinity to sigma 1 and sigma 2 receptors in CEREP assays. Saturation binding in rat brain membranes yielded a Kd value of 2.53 nM and Bmax of 2531 fmol/mg. Association constant Kon, dissociation constant Koff, and calculated equilibrium binding constant (Kon/Koff) were 0.0067±0.001 min−1 nM‐1, 0.021±0.0002 min−1, and 3.28±0.61 nM, respectively (average of 3 individual experiments each ± SD). Enhanced selectivity for GluN2B‐containing receptors translates to reduced non‐specific binding and better signal to noise ratio in both in vitro radioligand binding assays and ex vivo autoradiography.ConclusionsWe showed that new radioligand [3H] JNJ 63514230 is superior to previous previously described GluN2B tracers as it permits accurate measurement of affinity to GluN2B‐containing receptors (Ki) without using additional blockers to mask native sigma receptors.Support or Funding InformationAuthors are full time employees of Janssen Research & Development, LLC. The research was funded by the company.

Nafamostat and sepimostat identified as novel neuroprotective agents via NR2B N-methyl-D-aspartate receptor antagonism using a rat retinal excitotoxicity model

In addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [3H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

A New Tellurium- and Selenoorganic Compound as an Inhibitor of Acetylcholinesterase in Brain.

We studied the effect of a new cyanine dye containing selenium and tellurium on acetylcholinesterase activity in synaptic membrane in rat brain. The cyanine dye dose-dependently inhibits activity of this enzyme, and the concentration of half-maximal inhibition of acetylcholinesterase activity was 20.46 μM. The cyanine dye instantly inhibits the enzyme; the degree of inhibition depends on acetylthiocholine concentration: the lower is acetylthiocholine concentration, the higher is the degree of inhibition. On the Lineweaver-Burk plot, the concentration dependence curves of acetylcholinesterase with and without cyanine dye intersect in one point on the abscissa axis. In this case, the cyanine dye reduces the maximum inhibition rate (Vmax) and does not affect Michaelis constant (Km). The calculated inhibition constant Ki for the cyanine dye is 7.74 μM. Thus, the cyanine dye is a non-competitive inhibitor of acetylcholinesterase.

Discovery of Orexant and Anorexant Agents with Indazole Scaffold Endowed with Peripheral Antiedema Activity.

The endocannabinoid system represents an integrated neuronal network involved in the control of several organisms’ functions, such as feeding behavior. A series of hybrids of 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (mimonabant), a well-known inverse agonist of the type-1 cannabinoid receptor (CB1), once used as an antiobesity drug, and the N-(2S)-substitutes of 1-[(4-fluorophenyl)methyl]indazole-3-carboxamide with 1-amino-3-methyl-1-oxobutane (AB-Fubinaca), 1-amino-3,3-dimethyl-1-oxobutane (ADB-Fubinaca), and 3-methylbutanoate (AMB-Fubinaca), endowed with potent agonistic activity towards cannabinoid receptors CB1 and CB2 were in solution as C-terminal amides, acids, methyl esters and N-methyl amides. These compounds have been studied by binding assays to cannabinoid receptors and by functional receptor assays, using rat brain membranes in vitro. The most active among them as an agonist, (S)-1-(2,4-dichlorobenzyl)-N-(3,3-dimethyl-1-(methylamino)-1-oxobutan-2-yl)-1H-indazole-3-carboxamide (LONI11), and an antagonist, (S)-2-(1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamido)-3-methylbutanoic acid (LONI4), were tested in vivo in mic, to evaluate their ability to stimulate or suppress feeding behavior after intraperitoneal (i.p.) administration. For a LONI11 formalin test and a tail flick test after an administration by the subcutaneous (s.c.) and intracerebroventricular (i.c.v.) routes, respectively, were also carried out in vivo in mice to investigate the antinociceptive property at the central and peripheral levesl. We observed a significant orexant effect for LONI11 and an intense anorexant effect for (S)-methyl 2-(1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (LONI2) and LONI4. In zymosan-induced edema and hyperalgesia, LONI11 reduced the percent of paw volume increase and paw latency after s.c. administration, also suggesting a possible peripheral anti-inflammatory activity.

The Presence of Calcitonin Gene-Related Peptide and Its Receptors in Rat, Pig and Human Brain: Species Differences in Calcitonin Gene-Related Peptide Pharmacology

Aim: The aim of present study is to investigate the binding characteristics of non-peptide calcitonin gene-related peptide (CGRP) receptor antagonists (i.e., gepants) in the brain membranes of rat, pig and human. Methods: The interaction of available gepants with the CGRP receptor was studied in the brain membranes of 3 different species using a radioligand competitive binding assay. In addition, the distribution of CGRP and its receptor component receptor activity modifying protein 1 (RAMP1) in rat cerebellum and cortex was explored using immunohistochemistry. Results: All gepants, except SB268262, displaced 100% of the radioligand specific binding in the brain tissue of all 3 species and showed highest affinity for CGRP receptors in human brain as compared to rat and pig brain membranes. Furthermore, radioligand binding studies revealed the presence of higher CGRP receptor density in human cerebellum compared to human cortex. The morphology, size and density of CGRP immunoreactive cells suggest that all cerebral cortical neurons were positive for CGRP. Slender receptor immunoreactive fibres were found spanning through the entire cortex. CGRP immunoreactivity was displayed in the cell soma of cerebellar Purkinje cells and in large neurons in the medial cerebellar nucleus. RAMP1 was found on the surface of the Purkinje cells and in parallel fibres, indicating presence in the granule cell axons. Conclusion: Cerebellum and cerebral cortex are rich in CGRP and CGRP receptors, which can be antagonized by gepants. However, all gepants display higher affinity for human CGRP receptors as compared to rat and pig CGRP receptors. Furthermore, human cerebellum seems to express higher density of CGRP receptors.

Isolation and Biological Activity of 8- Epitetrodotoxin and the Structure of a Possible Biosynthetic Shunt Product of Tetrodotoxin, Cep-226A, from the Newt Cynops ensicauda popei.

Tetrodotoxin (TTX, 1), a potent neurotoxin, has been found in various animal species in both marine and terrestrial environments. In this study, a new TTX analogue, 8- epiTTX (2), and a possible biosynthetic shunt compound of TTX, Cep-226A (3), were isolated from the newt Cynops ensicauda popei. The voltage-gated sodium ion channel (Nav) blocking activity of 2 and 6- epiTTX (4), a known analogue, were investigated by a colorimetric cell-based assay and compared with that of 1. The EC50 values for 2 and 4 were determined to be 110 ± 40 and 33 ± 11 nM, respectively, which were larger than that of 1 (1.9 ± 0.7 nM). The results indicated that the equatorial hydroxy group at C-8 in TTX significantly contributes to its Nav blocking activity, whereas the 6-epimer of TTX retains substantial activity, consistent with its previously reported toxicity in mice and binding affinity to rat brain membrane preparations. The presence of these epimers of TTX (2 and 4) and Cep-226A (3) in newts supports our hypothesis that TTX is derived from a monoterpene in terrestrial environments.

Functional activation of Gαq/11 protein via α1 -adrenoceptor in rat cerebral cortical membranes.

Although it is recognized that α1 -adrenoceptors are coupled to diverse intracellular signalling pathways, its primary transduction mechanisms are evoked by activating phospholipase C in the cell membrane through Gαq/11 , resulting in production of inositol 1,4,5-trisphosphate and diacylglycerol. However, there have been few studies that indicate directly the involvement of Gαq/11 proteins in this signalling pathway in the central nervous system. In the current study, we tried to pharmacologically characterize (-)-adrenaline-stimulated [35 S]GTPγS binding to Gαq/11 in rat brain membranes. Functional activation of Gαq/11 coupled to α1 -adrenoceptor was investigated by using [35 S]GTPγS binding/immunoprecipitation assay in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. The specific [35 S]GTPγS binding to Gαq/11 was stimulated by (-)-adrenaline in a concentration-dependent and saturable manner in rat cerebral cortical membranes. In hippocampal or striatal membranes, the stimulatory effects of (-)-adrenaline were scarce. The effect of (-)-adrenaline was potently inhibited by prazosin, a potent and selective α1 -adrenoceptor antagonist, but not by yohimbine, a selective α2 -adrenoceptor antagonist. The response was mimicked by cirazoline, but not by R(-)-phenylephrine. Although oxymetazoline also stimulated the specific [35 S]GTPγS binding to Gαq/11 as an apparent "super-agonist", detailed pharmacological characterization revealed that its agonistic properties in this experimental system were derived from off-target effects on 5-HT2A receptors, but not via α1 -adrenoceptors. In conclusion, functional coupling of α1 -adrenoceptors to Gαq/11 proteins are detectable in rat brain membranes by means of [35 S]GTPγS binding/immunoprecipitation assay. It is necessary to interpret the experimental data with caution when oxymetazoline is included as an agonist at α1 -adrenoceptors.