Pharmacological Characterization of the Zebrafish (Danio Rerio) Histamine H1 Receptor Reveals the Involvement of the Second Extracellular Loop in the Binding of Histamine.

The zebrafish (Danio rerio) histamine H1 receptor gene (zfH1R) was cloned in 2007 and reported to be involved in fish locomotion. Yet, no detailed characterization of its pharmacology and signaling properties have so far been reported. In this study, we pharmacologically characterized the zfH1R expressed in HEK-293T cells by means of [3H]-mepyramine binding and G protein-signaling assays. The zfH1R [dissociation constant (KD), 0.7 nM] displayed similar affinity for the antagonist [3H]-mepyramine as the human histamine H1 receptor (hH1R) (KD, 1.5 nM), whereas the affinity for histamine is 100-fold higher than for the human H1R. The zfH1R couples to Gαq/11 proteins and activates several reporter genes, i.e., NFAT, NFÏ°B, CRE, VEGF, COX-2, SRE, and AP-1, and zfH1R-mediated signaling is prevented by the Gαq/11 inhibitor YM-254890 and the antagonist mepyramine. Molecular modeling of the zfH1R and human H1R shows that the binding pockets are identical, implying that variations along the ligand binding pathway could underly the differences in histamine affinity instead. Targeting differentially charged residues in extracellular loop 2 (ECL2) using site-directed mutagenesis revealed that Arg21045x55 is most likely involved in the binding process of histamine in zfH1R. This study aids the understanding of the pharmacological differences between H1R orthologs and the role of ECL2 in histamine binding and provides fundamental information for the understanding of the histaminergic system in the zebrafish. SIGNIFICANCE STATEMENT: The use of the zebrafish as in vivo models in neuroscience is growing exponentially, which asks for detailed characterization of the aminergic neurotransmitter systems in this model. This study is the first to pharmacologically characterize the zebrafish histamine H1 receptor after expression in HEK-293T cells. The results show a high pharmacological and functional resemblance with the human ortholog but also reveal interesting structural differences and unveils an important role of the second extracellular loop in histamine binding.

Effect of subchronic exposure to ambient fine and ultrafine particles on rat motor activity and ex vivo striatal dopaminergic transmission.

Alterations in dopaminergic transmission are associated with neurological disorders, such as depression, autism, and Parkinson's disease. Exposure of rats to ambient fine (FP) or ultrafine (UFP) particles induces oxidative and inflammatory responses in the striatum, a neuronal nucleus with dense dopaminergic innervation and critically involved in the control of motor activity.Objectives: We used an ex vivo system to evaluate the effect of in vivo inhalation exposure to FP and UFP on motor activity and dopaminergic transmission.Materials and Methods: Male adult Wistar rats were exposed to FP, UFP, or filtered air for 8 weeks (subchronic exposure; 5 h/day, 5 days/week) in a particle concentrator. Motor activity was evaluated using the open-field test. Uptake and release of [3H]-dopamine were assessed in striatal synaptosomes, and dopamine D2 receptor (D2R) affinity for dopamine was evaluated by the displacement of [3H]-spiperone binding to striatal membranes.Results: Exposure to FP or UFP significantly reduced spontaneous motor activity (ambulatory distance: FP -25%, UFP -32%; ambulatory time: FP -24%, UFP -22%; ambulatory episodes: FP -22%, UFP -30%), decreased [3H]-dopamine uptake (FP -18%, UFP -24%), and increased, although not significantly, [3H]-dopamine release (113.3 ± 16.3 and 138.6 ± 17.3%). Neither FP nor UFP exposure affected D2R density or affinity for dopamine.Conclusions: These results indicate that exposure to ambient particulate matter reduces locomotion in rats, which could be related to altered striatal dopaminergic transmission: UFP was more potent than FP. Our results contribute to the evidence linking environmental factors to changes in brain function that could turn into neurological and psychiatric disorders.HIGHLIGHTSYoung adult rats were exposed to fine (FP) or ultrafine (UFP) particles for 40 days.Exposure to FP or UFP reduced motor activity.Exposure to FP or UFP reduced dopamine uptake by striatal synaptosomes.Neither D2R density or affinity for dopamine was affected by FP or UFP.UFP was more potent than FP to exert the effects reported.

Histamine H3 Receptor Activation Modulates Glutamate Release in the Corticostriatal Synapse by Acting at CaV2.1 (P/Q-Type) Calcium Channels and GIRK (KIR3) Potassium Channels.

The striatum is innervated by histaminergic fibers and expresses a high density of histamine H3 receptors (H3Rs), present on medium spiny neurons (MSNs) and corticostriatal afferents. In this study, in sagittal slices from the rat dorsal striatum, excitatory postsynaptic potentials (EPSPs) were recorded in MSNs after electrical stimulation of corticostriatal axons. The effect of H3R activation and blockers of calcium and potassium channels was evaluated with the paired-pulse facilitation protocol. In the presence of the H3R antagonist/inverse agonist clobenpropit (1 µM), the H3R agonist immepip (1 µM) had no effect on the paired-pulse ratio (PPR), but in the absence of clobenpropit, immepip induced a significant increase in PPR, accompanied by a reduction in EPSP amplitude, suggesting presynaptic inhibition. The blockade of CaV2.1 (P/Q-type) channels with ω-agatoxin TK (400 nM) increased PPR and prevented the effect of immepip. The CaV2.2 (N-type) channel blocker ω-conotoxin GVIA (1 µM) also increased PPR, but did not occlude the immepip action. Functional KIR3 channels are present in corticostriatal terminals, and in experiments in which immepip increased PPR, the KIR3 blocker tertiapin-Q (30 nM) prevented the effect of the H3R agonist. These results indicate that the presynaptic modulation by H3Rs of corticostriatal synapses involves the inhibition of Cav2.1 calcium channels and the activation of KIR3 potassium channels.

Activation of α-adrenoceptors depresses synaptic transmission of myelinated afferents and inhibits pathways mediating primary afferent depolarization (PAD) in the in vitro mouse spinal cord.

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Cell-based assays and molecular dynamics analysis of a boron-containing agonist with different profiles of binding to human and guinea pig beta2 adrenoceptors.

The design of beta2 adrenoceptor (ß2AR) agonists is attractive because of their wide-ranging applications in medicine, and the details of agonist interactions with ß2AR are interesting because it is considered a prototype for G-protein coupled receptors. Preclinical studies for agonist development have involved biological assays with guinea pigs due to a similar physiology to humans. Boron-containing Albuterol derivatives (BCADs) designed as bronchodilators have improved potency and efficacy compared with their boron-free precursor on guinea pig ß2ARs (gpß2ARs), and two of the BCADs (BR-AEA and boronterol) conserve these features on cells expressing human ß2ARs (hß2ARs). The aim of this study was to test the BCAD Politerol on gpß2ARs and hß2ARs in vitro and in silico. Politerol displayed higher potency and efficacy on gpß2AR than on hß2AR in experimental assays, possible explanations are provided based on molecular modeling, and molecular dynamics simulations of about 0.25 µs were performed for the free and bound states adding up to 2 µs in total. There were slight differences, particularly in the role of the boron atom, in the interactions of Politerol with gpß2ARs and hß2ARs, affecting movements of transmembrane domains 5-7, known to be pivotal in receptor activation. These findings could be instrumental in the design of compounds selective for hß2ARs.

Adenosine A2A and histamine H3 receptors interact at the cAMP/PKA pathway to modulate depolarization-evoked [3H]-GABA release from rat striato-pallidal terminals.

We previously reported that the activation of histamine H3 receptors (H3Rs) selectively counteracts the facilitatory action of adenosine A2A receptors (A2ARs) on GABA release from rat globus pallidus (GP) isolated nerve terminals (synaptosomes). In this work, we examined the mechanisms likely to underlie this functional interaction. Three possibilities were explored: (a) changes in receptor affinity for agonists induced by physical A2AR/H3R interaction, (b) opposite actions of A2ARs and H3Rs on depolarization-induced Ca2+ entry, and (c) an A2AR/H3R interaction at the level of adenosine 3',5'-cyclic monophosphate (cAMP) formation. In GP synaptosomal membranes, H3R activation with immepip reduced A2AR affinity for the agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680) (Ki control 4.53 nM; + immepip 9.32 nM), whereas A2AR activation increased H3R affinity for immepip (Ki control 0.63 nM; + CGS-21680 0.26 nM). Neither A2AR activation nor H3R stimulation modified calcium entry through voltage-gated calcium channels in GP synaptosomes, as evaluated by microfluorometry. A2AR-mediated facilitation of depolarization-evoked [2,3-3H]-γ-aminobutyric acid ([3H]-GABA) release from GP synaptosomes (130.4 ± 3.6% of control values) was prevented by the PKA inhibitor H-89 and mimicked by the adenylyl cyclase activator forskolin or by 8-Bromo-cAMP, a membrane permeant cAMP analogue (169.5 ± 17.3 and 149.5 ± 14.5% of controls). H3R activation failed to reduce the facilitation of [3H]-GABA release induced by 8-Bromo-cAMP. In GP slices, A2AR activation stimulated cAMP accumulation (290% of basal) and this effect was reduced (- 75%) by H3R activation. These results indicate that in striato-pallidal nerve terminals, A2ARs and H3Rs interact at the level of cAMP formation to modulate PKA activity and thus GABA release.

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells.

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H3 receptor (hH3R445 and hH3R365) on [35S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) and depolarization-evoked [3 H]-dopamine release. Maximal specific binding (Bmax) of [3 H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells, respectively, with similar dissociation constants (Kd, 0.86 nM and 0.81 nM). The mRNA of the hH3R365 isoform was 40.9 ± 7.9% of the hH3R445 isoform. No differences in receptor affinity were found for the H3R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [35S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH3R445 cells ([35S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH3R365 cells; cAMP accumulation, -74.0 ± 4.9% versus -43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [3 H]-dopamine release evoked by 100 mM K+ (-18.9 ± 3.0% and -20.5 ± 3.3%, for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells), or the inhibition of depolarization-induced increase in [Ca2+]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH3R445 cells 0.639 ± 0.049, SH-SY5Y-hH3R365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH3R445 and hH3R365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.

Functional histamine H3 and adenosine A2A receptor heteromers in recombinant cells and rat striatum.

In the striatum, histamine H3 receptors (H3Rs) are co-expressed with adenosine A2A receptors (A2ARs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows H3Rs and A2ARs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors can physically interact has not yet been assessed. To test this hypothesis, a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric A2AR302-Gαqi4 and wild-type H3Rs in transfected HEK-293T cells. H3R activation with the agonist RAMH resulted in Ca2+ mobilization (pEC50 7.31 ±â€¯0.23; maximal stimulation, Emax 449 ±â€¯25% of basal) indicative of receptor heterodimerization. Functional H3R-A2AR heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cells. In membranes from rat striatal synaptosomes, H3R activation decreased A2AR affinity for the agonist CGS-21680 (pKi values 8.10 ±â€¯0.04 and 7.70 ±â€¯0.04). Moreover, H3Rs and A2ARs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a H3R-A2AR heteromer with possible physiological implications for the modulation of the intra-striatal transmission.

pVHL suppresses Akt/ß-catenin-mediated cell proliferation by inhibiting 14-3-3ζ expression.

The mechanisms controlling degradation of cytosolic ß-catenin are important for regulating ß-catenin co-transcriptional activity. Loss of von Hippel-Lindau protein (pVHL) has been shown to stabilize ß-catenin, increasing ß-catenin transactivation and ß-catenin-mediated cell proliferation. However, the role of phosphoinositide 3-kinase (PI3K)/Akt in the regulation of ß-catenin signaling downstream from pVHL has never been addressed. Here, we report that hyperactivation of PI3K/Akt in cells lacking pVHL contributes to the stabilization and nuclear accumulation of active ß-catenin. PI3K/Akt hyperactivation is facilitated by the up-regulation of 14-3-3ζ and the down-regulation of 14-3-3ε, 14-3-3η and 14-3-3θ. Up-regulation of 14-3-3ζ in response to pVHL is important for the recruitment of PI3K to the cell membrane and for stabilization of soluble ß-catenin. In contrast, 14-3-3ε and 14-3-3η enhanced PI3K/Akt signaling by inhibiting PI3K and PDK1, respectively. Thus, our results demonstrated that 14-3-3 family members enhance PI3K/Akt/ß-catenin signaling in order to increase proliferation. Inhibition of Akt activation and/or 14-3-3 function strongly reduces ß-catenin signaling and decreases cell proliferation. Thus, inhibition of Akt and 14-3-3 function efficiently reduces cell proliferation in 786-0 cells characterized by hyperactivation of ß-catenin signaling due to pVHL loss.

Dopamine D1 receptor activation maintains motor coordination and balance in rats.

Dopamine (DA) modulates motor coordination, and its depletion, as in Parkinson's disease, produces motor impairment. The basal ganglia, cerebellum and cerebral cortex are interconnected, have functional roles in motor coordination, and possess dopamine D1 receptors (D1Rs), which are expressed at a particularly high density in the basal ganglia. In this study, we examined whether the activation of D1Rs modulates motor coordination and balance in the rat using a beam-walking test that has previously been used to detect motor coordination deficits. The systemic administration of the D1R agonist SKF-38393 at 2, 3, or 4 mg/kg did not alter the beam-walking scores, but the subsequent administration of the D1R antagonist SCH-23390 at 1 mg/kg did produce deficits in motor coordination, which were reversed by the full agonist SKF-82958. The co-administration of SKF-38393 and SCH-23390 did not alter the beam-walking scores compared with the control group, but significantly prevented the increase in beam-walking scores induced by SCH-23390. The effect of the D1R agonist to prevent and reverse the effect of the D1R antagonist in beam-walking scores is an indicator that the function of D1Rs is necessary to maintain motor coordination and balance in rats. Our results support that D1Rs mediate the SCH-23390-induced deficit in motor coordination.

The Histamine H3 Receptor: Structure, Pharmacology, and Function.

Among the four G protein-coupled receptors (H1-H4) identified as mediators of the biologic effects of histamine, the H3 receptor (H3R) is distinguished for its almost exclusive expression in the nervous system and the large variety of isoforms generated by alternative splicing of the corresponding mRNA. Additionally, it exhibits dual functionality as autoreceptor and heteroreceptor, and this enables H3Rs to modulate the histaminergic and other neurotransmitter systems. The cloning of the H3R cDNA in 1999 by Lovenberg et al. allowed for detailed studies of its molecular aspects. In this work, we review the characteristics of the H3R, namely, its structure, constitutive activity, isoforms, signal transduction pathways, regional differences in expression and localization, selective agonists, antagonists and inverse agonists, dimerization with other neurotransmitter receptors, and the main presynaptic and postsynaptic effects resulting from its activation. The H3R has attracted interest as a potential drug target for the treatment of several important neurologic and psychiatric disorders, such as Alzheimer and Parkinson diseases, Gilles de la Tourette syndrome, and addiction.

Pharmacoresistant temporal lobe epilepsy modifies histamine turnover and H3 receptor function in the human hippocampus and temporal neocortex.

Experiments were designed to evaluate the tissue content of tele-methylhistamine (t-MeHA) and histamine as well as H3 receptor (H3 Rs) binding and activation of the heterotrimeric guanine nucleotide binding αi/o proteins (Gαi/o) coupled to these receptors in the hippocampus and temporal neocortex of patients (n = 10) with pharmacoresistant mesial temporal lobe epilepsy (MTLE). Patients with MTLE showed elevated tissue content of t-MeHA in the hippocampus. Analyses revealed that a younger age at seizure onset was correlated with a higher tissue content of t-MeHA, lower H3 R binding, and lower efficacy of Gαi/o protein activation in the hippocampus. We conclude that the hippocampus shows a reduction in the H3 R function associated with enhanced histamine. In contrast, the temporal neocortex displayed a high efficacy of H3 Rs Gαi/o protein activation that was associated with low tissue contents of histamine and t-MeHA. These results indicate an overactivation of H3 Rs leading to decreased histamine in the temporal neocortex. However, this situation was lessened in circumstances such as a longer duration of epilepsy or higher seizure frequency. It is concluded that decrease in H3 Rs function and enhanced levels of histamine may contribute to the epileptic activity in the hippocampus and temporal neocortex of patients with pharmacoresistant MTLE.

Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 µM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

Inhibition of Uterine Contractility by Thalidomide Analogs via Phosphodiesterase-4 Inhibition and Calcium Entry Blockade.

Uterine relaxation is crucial during preterm labor. Phosphodiesterase-4 (PDE-4) inhibitors have been proposed as tocolytics. Some thalidomide analogs are PDE-4 inhibitors. The aim of this study was to assess the uterus-relaxant properties of two thalidomide analogs, methyl 3-(4-nitrophthalimido)-3-(3,4-dimethoxyphenyl)-propanoate (4NO2PDPMe) and methyl 3-(4-aminophthalimido)-3-(3,4-dimethoxyphenyl)-propanoate (4APDPMe) and were compared to rolipram in functional studies of spontaneous phasic, K⁺-induced tonic, and Ca2+-induced contractions in isolated pregnant human myometrial tissues. The accumulation of cAMP was quantified in HeLa cells. The presence of PDE-4B2 and phosphorylated myosin light-chain (pMLC), in addition to the effect of thalidomide analogs on oxytocin-induced pMLC, were assessed in human uterine myometrial cells (UtSMCs). Thalidomide analogs had concentration-dependent inhibitory effects on spontaneous and tonic contractions and inhibited Ca2+-induced responses. Tonic contraction was equipotently inhibited by 4APDPMe and rolipram (IC50 = 125 ± 13.72 and 98.45 ± 8.86 µM, respectively). Rolipram and the thalidomide analogs inhibited spontaneous and tonic contractions equieffectively. Both analogs increased cAMP accumulation in a concentration-dependent manner (p < 0.05) and induced changes in the subcellular localization of oxytocin-induced pMLC in UtSMCs. The inhibitory effects of thalidomide analogs on the contractions of pregnant human myometrium tissue may be due to their PDE-4 inhibitory effect and novel mechanism as calcium-channel blockers.

[Isoforms of the human histamine H3 receptor: Generation, expression in the central nervous system and functional implications]. / Isoformas del receptor a histamina H3 humano: generación, expresión en el sistema nervioso central (SNC) e implicaciones funcionales.

Histamine plays a significant role as a neuromodulator in the human central nervous system. Histamine-releasing neurons are exclusively located in the tuberomammillary nucleus of the hypothalamus, project to all major areas of the brain, and participate in functions such as the regulation of sleep/wakefulness, locomotor activity, feeding and drinking, analgesia, learning, and memory. The functional effects of histamine are exerted through the activation of four G protein-coupled receptors (H1, H2, H3 and H4), and in the central nervous system the first three receptors are widely expressed. The H3 receptor (H3R) is found exclusively in neuronal cells, where it functions as auto- and hetero-receptor. One remarkable characteristic of the H3R is the existence of isoforms, generated by alternative splicing of the messenger RNA. For the human H3R, 20 isoforms have been reported; although a significant number lack those regions required for agonist binding or receptor signaling, at least five isoforms appear functional upon heterologous expression. In this work we review the evidence for the generation of human H3R isoforms, their expression, and the available information regarding the functionality of such receptors.

Acute and subchronic exposure to air particulate matter induces expression of angiotensin and bradykinin-related genes in the lungs and heart: Angiotensin-II type-I receptor as a molecular target of particulate matter exposure.

BACKGROUND: Particulate matter (PM) adverse effects on health include lung and heart damage. The renin-angiotensin-aldosterone (RAAS) and kallikrein-kinin (KKS) endocrine systems are involved in the pathophysiology of cardiovascular diseases and have been found to impact lung diseases. The aim of the present study was to evaluate whether PM exposure regulates elements of RAAS and KKS. METHODS: Sprague-Dawley rats were acutely (3 days) and subchronically (8 weeks) exposed to coarse (CP), fine (FP) or ultrafine (UFP) particulates using a particulate concentrator, and a control group exposed to filtered air (FA). We evaluated the mRNA of the RAAS components At1, At2r and Ace, and of the KKS components B1r, B2r and Klk-1 by RT-PCR in the lungs and heart. The ACE and AT1R protein were evaluated by Western blot, as were HO-1 and γGCSc as indicators of the antioxidant response and IL-6 levels as an inflammation marker. We performed a binding assay to determinate AT1R density in the lung, also the subcellular AT1R distribution in the lungs was evaluated. Finally, we performed a histological analysis of intramyocardial coronary arteries and the expression of markers of heart gene reprogramming (Acta1 and Col3a1). RESULTS: The PM fractions induced the expression of RAAS and KKS elements in the lungs and heart in a time-dependent manner. CP exposure induced Ace mRNA expression and regulated its protein in the lungs. Acute and subchronic exposure to FP and UFP induced the expression of At1r in the lungs and heart. All PM fractions increased the AT1R protein in a size-dependent manner in the lungs and heart after subchronic exposure. The AT1R lung protein showed a time-dependent change in subcellular distribution. In addition, the presence of AT1R in the heart was accompanied by a decrease in HO-1, which was concomitant with the induction of Acta1 and Col3a1 and the increment of IL-6. Moreover, exposure to all PM fractions increased coronary artery wall thickness. CONCLUSION: We demonstrate that exposure to PM induces the expression of RAAS and KKS elements, including AT1R, which was the main target in the lungs and the heart.

Pharmacological characterization of the zebrafish Hrh2a histamine H2 receptor.

The zebrafish, Danio rerio, is a widely adopted in vivo model that conserves organs such as the liver, kidney, stomach, and brain, being, therefore, suitable for studying human diseases, drug discovery and toxicology. The brain aminergic systems are also conserved and the histamine H1, H2 and H3 receptors were previously cloned and identified in the zebrafish brain. Genome studies identified another putative H2 receptor (Hrh2) with ∼50% sequence identity with H2 receptor orthologs. In this study, we recombinantly expressed both zebrafish H2 receptor paralogs (hrh2a and hrh2b) and compared their pharmacology with the human H2 receptor ortholog. Our results showed that both zebrafish receptors conserve all the class A GPCR motifs. However, in contrast with the Hrh2a paralog, the Hrh2b does not possess all the amino acid residues shown to participate in histamine binding. The zebrafish Hrh2a receptor displays high affinity for [3H]-tiotidine with a binding profile for H2 receptor ligands similar to that of the human H2 receptor. The zebrafish Hrh2a receptor couples to GαS and Gαq/11 proteins, resulting in cAMP accumulation and activation of several reporter genes linked to the Gαq/11 pathway. Additionally, this receptor shows high constitutive activity, with histamine potency in the low nanomolar range for cAMP accumulation and the micromolar range for the activation of the NFAT response element. Moreover, dimaprit and amthamine seem to preferentially activate GαS over Gαq/11 proteins via the zebrafish Hrh2a receptor. These results can contribute to clarifying the functional roles of the H2 receptor in zebrafish.

In silico identification of a biarylamine acting as agonist at human ß3 adrenoceptors and exerting BRL37344-like effects on mouse metabolism.

Human ß3-adrenoceptor (ß3AR) agonists were considered potential agents for the treatment of metabolic disorders. However, compounds tested as ß3AR ligands have shown marked differences in pharmacological profile in rodent and human species, although these compounds remain attractive as they were successfully repurposed for the therapy of urinary incontinence. In this work, some biarylamine compounds were designed and tested in silico as potential ß3AR agonists on 3-D models of mouse or human ß3ARs. Based on the theoretical results, we identified, synthesized and tested a biarylamine compound (polibegron). In CHO-K1 cells expressing the human ß3AR, polibegron and the ß3AR agonist BRL 37344 were partial agonists for stimulating cAMP accumulation (50 and 57% of the response to isoproterenol, respectively). The potency of polibegron was 1.71- and 4.5-fold higher than that of isoproterenol and BRL37344, respectively. These results indicate that polibegron acts as a potent, but partial, agonist at human ß3ARs. In C57BL/6N mice with obesity induced by a high-fat diet, similar effects of the equimolar intraperitoneal administration of polibegron and BRL37344 were observed on weight, visceral fat and plasma levels of glucose, cholesterol and triglycerides. Similarities and differences between species related to ligand-receptor interactions can be useful for drug designing.

Chronic H3R activation reduces L-Dopa-induced dyskinesia, normalizes cortical GABA and glutamate levels, and increases striatal dopamine D1R mRNA expression in 6-hydroxydopamine-lesioned male rats.

RATIONALE: Dyskinesias induced by L-3,4-dihydroxyphenylalanine, L-Dopa (LIDs), are the major complication in the pharmacological treatment of Parkinson's disease. LIDs induce overactivity of the glutamatergic cortico-striatal projections, and drugs that reduce glutamatergic overactivity exert antidyskinetic actions. Chronic administration of immepip, agonist at histamine H3 receptors (H3R), reduces LIDs and diminishes GABA and glutamate content in striatal dialysates (Avila-Luna et al., Psychopharmacology 236: 1937-1948, 2019). OBJECTIVES AND METHODS: In rats unilaterally lesioned with 6-hydroxydopamine in the substantia nigra pars compacta (SNc), we examined whether the chronic administration of immepip and their withdrawal modify LIDs, the effect of L-Dopa on glutamate and GABA content, and mRNA levels of dopamine D1 receptors (D1Rs) and H3Rs in the cerebral cortex and striatum. RESULTS: The administration of L-Dopa for 21 days induced LIDs. This effect was accompanied by increased GABA and glutamate levels in the cerebral cortex ipsi and contralateral to the lesioned SNc, and immepip administration prevented (GABA) or reduced (glutamate) these actions. In the striatum, GABA content increased in the ipsilateral nucleus, an effect prevented by immepip. L-Dopa administration had no significant effects on striatal glutamate levels. In lesioned and L-Dopa-treated animals, D1R mRNA decreased in the ipsilateral striatum, an effect prevented by immepip administration. CONCLUSIONS: Our results indicate that chronic H3R activation reduces LIDs and the overactivity of glutamatergic cortico-striatal projections, providing further evidence for an interaction between D1Rs and H3Rs in the cortex and striatum under normal and pathological conditions.

Histamine in brain development.

The function of histamine in the adult central nervous system has been extensively studied, but data on its actions upon the developing nervous system are still scarce. Herein, we review the available information regarding the possible role for histamine in brain development. Some relevant findings are the existence of a transient histaminergic neuronal system during brain development, which includes serotonergic neurons in the midbrain and the rhombencephalon that coexpress histamine; the high levels of histamine found in several areas of the embryo nervous system at the neurogenic stage; the presence of histaminergic fibers and the expression of histamine receptors in various areas of the developing brain; and the neurogenic and proliferative effects on neural stem cells following histamine H(1) - and H(2) -receptor activation, respectively. Altogether, the reviewed information supports a significant role for histamine in brain development and the need for further research in this field.