Non-selective Agonist Research Articles

Hypotonicity-Induced Increase in Duodenal Mucosal Permeability Is Regulated by Cholinergic Receptors in Rats

BackgroundThe role of cholinergic receptors in the regulation of duodenal mucosal permeability in vivo is currently not fully described.AimsTo elucidate the impact of nicotinic and muscarinic acetylcholine receptor signaling in response to luminal hypotonicity (50 mM NaCl) in the proximal small intestine of rat.MethodsThe effect on duodenal blood-to-lumen clearance of 51Cr-EDTA (i.e., mucosal permeability) and motility was studied in the absence and presence of nicotinic and muscarinic receptor agonists and antagonists, a sodium channel blocker (tetrodotoxin), and after bilateral cervical vagotomy.ResultsRats with duodenal contractions responded to luminal hypotonicity by substantial increase in intestinal permeability. This response was absent in animals given a non-selective nicotinic receptor antagonist (mecamylamine) or agonist (epibatidine). Pretreatment with tetrodotoxin reduced the increase in mucosal permeability in response to luminal hypotonicity. Further, the non-selective muscarinic receptor antagonist (atropine) and agonist (bethanechol) reduced the hypotonicity-induced increase in mucosal permeability, while vagotomy was without an effect, suggesting that local enteric reflexes dominate. Finally, neither stimulating nor blocking the α7-nicotinic receptor had any significant effects on duodenal permeability in response to luminal hypotonicity, suggesting that this receptor is not involved in regulation of duodenal permeability. The effect of the different drugs on mucosal permeability was similar to the effect observed for duodenal motility.ConclusionsA complex enteric intramural excitatory neural reflex involving both nicotinic and muscarinic receptor subtypes mediates an increase in mucosal permeability induced by luminal hypotonicity.

Resveratrol enhances A1 and hinders A2A adenosine receptors signaling in both HeLa and SH-SY5Y cells: Potential mechanism of its antitumoral action.

Despite great efforts, effective treatment against cancer has not yet been found. However, natural compounds such as the polyphenol resveratrol have emerged as promising preventive agent in cancer therapy. The mode of action of resveratrol is still poorly understood, but it can modulate many signaling pathways related to the initiation and progression of cancer. Adenosinergic signaling may be involved in the antitumoral action of resveratrol since resveratrol binds to the orthosteric binding site of adenosine A2A receptors and acts as a non-selective agonist for adenosine receptors. In the present study, we measured the impact of resveratrol treatment on different adenosinergic pathway components (i.e. adenosine receptors levels, 5'-nucleotidase, adenosine deaminase, and adenylyl cyclase activities, protein kinase A levels, intracellular adenosine and other related metabolites levels) and cell viability and proliferation in HeLa and SH-SY5Y cell lines. Results revealed changes leading to turning off cAMP signaling such as decreased levels of A2A receptors and reduced adenylyl cyclase activation, increased levels of A1 receptors and increased adenylyl cyclase inhibition, and lower levels of PKA. All these changes could contribute to the antitumoral action of resveratrol. Interestingly, these effects were almost identical in HeLa and SH-SY5Y cells suggesting that resveratrol enhances A1 and hinders A2A adenosine receptors signaling as part of a potential mechanism of antitumoral action.

Efficacy and safety of pemafibrate versus bezafibrate in coronary artery disease patients receiving statin treatment: a randomized, open-label, cross-over study

Abstract Background Fibrates activate peroxisome proliferator-activated receptor (PPAR)-α which is associated with lipid metabolism. Bezafibrate is a non-selective PPAR-α agonist, whereas pemafibrate has been developed as a higher selective PPAR-α agonist. Objective The efficacy and safety of pemafibrate for 24-week in patients with dyslipidemia was examined in comparison with bezafibrate. Methods Sixty patients with hypertriglyceridemia (fasting triglyceride (TG) level of ≥150 mg/dL) were treated with pemafibrate of 0.2 mg/day or bezafibrate of 400 mg/day for 24-week in a randomized cross-over study. Percent change from baseline in TG levels was the primary endpoint, and that in HDL-C and apolipoprotein A-I (Apo A-I) levels was the secondary endpoints. Results A significantly greater reduction in TG percent change was observed in pemafibrate than in bezafibrate (−46.1% vs. −34.7%, p<0.001). There was no significant difference in HDL-C percent change between pemafibrate and bezafibrate (18.4% vs. 14.0%, p=0.067), whereas Apo A-I percent change was significantly greater in pemafibrate than in bezafibrate (9.2% vs. 5.7%, p=0.018). Pemafibrate and bezafibrate significantly decreased alanine aminotransferase (ALT) and gamma-glutamyltransferase (γ-GT) levels, and pemafibrate showed a greater reduction than bezafibrate (ALT: −21.9% vs. −10.6%, p=0.048; γ-GT: −43.5% vs. −33.1%, p=0.025). Creatinine levels significantly increased in both treatments (both p<0.001), however, creatinine percent change was significantly smaller in pemafibrate than in bezafibrate (5.72% vs. 15.5%, p<0.001). There was no difference in frequency of adverse event (AE) or serious AE between two treatments, but frequency of creatinine elevation (≥0.5 mg/d and/or 25%) was significantly higher in bezafibrate than in pemafibrate (16/60 vs. 3/60, p=0.004). Conclusion As compared with bezafibrate, pemafibrate is more effective to reduce TG levels and to elevate Apo A-I levels, and it is safer in terms of liver and renal function. Funding Acknowledgement Type of funding sources: None.

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aβ1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aβ1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aβ1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD.Graphical Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.

OX2R-selective orexin agonism is sufficient to ameliorate cataplexy and sleep/wake fragmentation without inducing drug-seeking behavior in mouse model of narcolepsy.

Acquired loss of hypothalamic orexin (hypocretin)-producing neurons causes the chronic sleep disorder narcolepsy-cataplexy. Orexin replacement therapy using orexin receptor agonists is expected as a mechanistic treatment for narcolepsy. Orexins act on two receptor subtypes, OX1R and OX2R, the latter being more strongly implicated in sleep/wake regulation. However, it has been unclear whether the activation of only OX2R, or both OX1R and OX2R, is required to replace the endogenous orexin functions in the brain. In the present study, we examined whether the selective activation of OX2R is sufficient to rescue the phenotype of cataplexy and sleep/wake fragmentation in orexin knockout mice. Intracerebroventricular [Ala11, D-Leu15]-orexin-B, a peptidic OX2R-selective agonist, selectively activated OX2R-expressing histaminergic neurons in vivo, whereas intracerebroventricular orexin-A, an OX1R/OX2R non-selective agonist, additionally activated OX1R-positive noradrenergic neurons in vivo. Administration of [Ala11, D-Leu15]-orexin-B extended wake time, reduced state transition frequency between wake and NREM sleep, and reduced the number of cataplexy-like episodes, to the same degree as compared with orexin-A. Furthermore, intracerebroventricular orexin-A but not [Ala11, D-Leu15]-orexin-B induced drug-seeking behaviors in a dose-dependent manner in wild-type mice, suggesting that OX2R-selective agonism has a lower propensity for reinforcing/drug-seeking effects. Collectively, these findings provide a proof-of-concept for safer mechanistic treatment of narcolepsy-cataplexy through OX2R-selective agonism.

Involvement of isoproterenol-induced intracellular Zn2+ dynamics in the basolateral amygdala in conditioned fear memory.

Beta-adrenergic receptors in the basolateral amygdala play an essential role in fear memory, while the physiological role of intracellular Zn2+ remains to be clarified. Intracellular Zn2+ level was decreased 5 min after local injection of 500μM isoproterenol (2μl), a nonselective beta-adrenergic receptor agonist into the basolateral amygdala, suggesting that intracellular Zn2+ dynamic is linked with beta-adrenergic receptor signaling in the basolateral amygdala. When isoproterenol was injected into the basolateral amygdala 20min prior to long-term potentiation (LTP) induction, LTP at perforant pathway-basolateral amygdala was enhanced and conditioned fear memory was also augmented, suggesting that isoproterenol leads to utilization of Zn2+ to consolidate fear memory followed by lowering intracellular Zn2+. We postulated that synaptic Zn2+ dynamics under conditioned fear experience regulates conditioned fear memory in cooperation with beta-adrenergic receptor signaling. When either intracellular Zn2+ chelator (ZnAF-2DA) or extracellular Zn2+ chelator (CaEDTA) was locally injected into the basolateral amygdala in the same manner, LTP was also enhanced. The local injection of ZnAF-2DA augmented fear memory. It is likely that the decrease in availability of intracellular Zn2+ by Zn2+ chelators under fear experience affects the function of Zn2+-required proteins followed by augmentation of fear memory and its related LTP. The present study suggests that beta-adrenergic receptor signaling is linked with intracellular Zn2+ signaling in the basolateral amygdala to consolidate conditioned fear memory. Because intracellular Zn2+ signaling is required for fear memory, the decrease in availability of intracellular Zn2+ may augment fear memory and its related LTP under non-physiological condition.

Effects of lysophosphatidic acid on sling and clasp fibers of the human lower esophageal sphincter

Background This study aims to explore the role of lysophosphatidic acid receptors in the regulation mechanisms of contraction and relaxation of human lower esophageal sphincter. Methods Between July 2015 and March 2016, muscle strips were collected from a total of 30 patients (19 males, 11 females; mean age: 62±9.9 years; range, 52 to 68 years) who underwent an esophagectomy for mid-third esophageal carcinomas. The specimens were maintained in oxygenated Krebs solution. Muscle tension measurement technique in vitro was used to examine the effects of non-selective lysophosphatidic acid receptors agonists and antagonists, as well as selective lysophosphatidic acid receptors agonists on the clasp and sling fibers of human lower esophageal sphincter. Results The non-selective dopamine receptor agonist lysophosphatidic acid induced the contraction of the clasp and sling fibers of the human lower esophageal sphincter. The response induced by nonselective lysophosphatidic acid receptor agonist was inhibited completely by non-selective lysophosphatidic acid receptor antagonist. The selective lysophosphatidic acid 1 and 2 receptor agonist and the selective lysophosphatidic acid 3 receptor agonist induced a concentration-dependent contractile response of the clasp and sling fibers of the human lower esophageal sphincter. There was no significant difference in contraction rates between the clasp and sling fibers (p>0.05). Conclusion This study indicates that lysophosphatidic acid regulates the lower esophageal sphincter is through its receptor; the lysophosphatidic acid receptors may be involved in the contractile response of the human lower esophageal sphincter.

Differential Effects of the G-Protein-Coupled Estrogen Receptor (GPER) on Rat Embryonic (E18) Hippocampal and Cortical Neurons.

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERβ. Studies related to the extranuclear, membrane-bound G-protein-coupled ER (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic day 18 (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and nonselective agonist, 17β-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca2+ rise in hippocampal neurons involves internal Ca2+ store release via activation of phospholipase C (PLC) and extracellular entry via Ca2+ channels. Immunocytochemistry results revealed no observable difference in GPER expression/localization in neurons, yet real-time qPCR (RT-qPCR) and Western blotting showed a higher GPER expression in the cortex than hippocampus, implying that GPER expression level may not fully account for its robust physiological effects in hippocampal neurons. We used RNA sequencing data to identify distinctly enriched pathways and significantly expressed genes in response to G-1 or E2 in cultured rat E18 hippocampal and cortical neurons. In summary, the identification of differential effects of GPER activation on hippocampal and cortical neurons in the brain and the determination of key genes and molecular pathways are instrumental toward an understanding of estrogen’s action in early neuronal development.

Synthetic cannabinoids reduce the inflammatory activity of microglia and subsequently improve neuronal survival in vitro

Microglia are resident immune cells of the brain that survey the microenvironment, provide trophic support to neurons, and clear debris to maintain homeostasis and healthy brain function. Microglia are also drivers of neuroinflammation in several neurodegenerative diseases. Microglia produce endocannabinoids and express both cannabinoid receptor subtypes suggesting that this system is a target to suppress neuroinflammation. We tested whether cannabinoid type 1 (CB1) or type 2 (CB2) receptors could be targeted selectively or in combination to dampen the pro-inflammatory behavior of microglia, and whether this would have functional relevance to decrease secondary neuronal damage. We determined that components of the endocannabinoid system were altered when microglia are treated with lipopolysaccharide and interferon-gamma and shift to a pro-inflammatory phenotype. Furthermore, pro-inflammatory microglia released cytotoxic factors that induced cell death in cultured STHdhQ7/Q7 neurons. Treatment with synthetic cannabinoids that were selective for CB1 receptors (ACEA) or CB2 receptors (HU-308) dampened the release of nitric oxide (NO) and pro-inflammatory cytokines and decreased levels of mRNA for several pro-inflammatory markers. A nonselective agonist (CP 55,940) exhibited similar influence over NO release but to a lesser extent relative to ACEA or HU-308. All three classes of synthetic cannabinoids ultimately reduced the secondary damage to the cultured neurons. The mechanism for the observed neuroprotective effects appeared to be related to cannabinoid-mediated suppression of MAPK signaling in microglia. Taken together, the data indicate that activation of CB1 or CB2 receptors interfered with the pro-inflammatory activity of microglia in a manner that also reduced secondary damage to neurons.

REDUCTION OF HEART RATE VIA P2X4 RECEPTOR ACTIVATION AS A PROMISING TREATMENT STRATEGY IN PULMONARY ARTERIAL HYPERTENSION

Objective: Mounting evidence suggest that the ATP-sensitive ionotropic P2X4 receptor (P2X4R) improves cardiac performance and optimizes myocardial energy demand by reducing chronotropy. Here, we set to investigate whether the digitalis-like effect of the P2X4R in the sinus node was preserved in rats with right heart failure due to pulmonary arterial hypertension (PAH). Design and method: Male Wistar rats received a single subcutaneous injection of either monocrotaline (MCT; PAH group) or vehicle (CTRL group). Right heart failure associated to PAH normally occurs in Wistar rats 21 to 25 days after MCT administration. Myographic recordings were performed in spontaneously beating atria (chronotropic effect) isolated from both animal groups. Results: Baseline spontaneous atrial rate was 250 ± 5 (n = 26) bpm in CTRL and 237 ± 7 bpm (n = 23) in PAH group (p > 0.05). ATP (0.01–1 mM) concentration-dependently reduced the spontaneous atrial rate of both animal groups. The negative chronotropic effect of ATP (100 M) was smaller in PAH rats (-9 ± 2%; n = 14 vs. -22 ± 4%; n = 25, p < 0.05), but this divergence was attenuated upon increasing the concentration of ATP to 1 mM (CTRL: -54 ± 5%; n = 15 vs. MCT: -50 ± 7%; n = 8; p > 0.05). The P2X4R antagonist, 5-BDBD (10 M), attenuated the negative chronotropic action of ATP (100 M) in CTRL (from -19 ± 5% to -13 ± 3%; n = 5, p > 0.05) and PAH (from -8 ± 3% to 1 ± 8%; n = 4, p > 0.05) rats. The positive allosteric modulator of P2X4R, ivermectin (30 M), potentiated the negative chronotropic effect of ATP (100 M) in CTRL (from -31 ± 5% to -56 ± 14%; n = 5, p > 0.05) and PAH (from -12 ± 6% to -24 ± 9%; n = 4, p < 0.05). The non-selective P2XR agonist, CTP (1 mM), which contrary to ATP does not breakdown into adenosine, also decreased atrial chronotropy in CTRL (-24.31 ± 6.73%; n = 6) and PAH (-11.82 ± 1.98%; n = 6) rats. Conclusions: Data suggest that the negative chronotropic effect of P2X4R activation is preserved in atria of rats with heart failure associated to PAH, thus strengthening the hypothesis that heart rate reductions using this ATP-sensitive receptor may increase cardiac performance in patients with PAH-induced heart failure.

Cross state-dependent memory retrieval between cannabinoid CB1 and serotonergic 5-HT1A receptor agonists in the mouse dorsal hippocampus

Understanding the neurobiological mechanisms of drug-related learning and memory formation may help the treatment of cognitive disorders. Dysfunction of the cannabinoid and serotonergic systems has been demonstrated in learning and memory disorders.The present paper investigates the phenomenon called state-dependent memory (SDM) induced by ACPA (a selective cannabinoid CB1 receptor agonist) and 8-OH-DPAT (a nonselective 5-HT1A receptor agonist) with special focus on the role of the 5-HT1A receptor in the effects of both ACPA and 8-OH-DPAT SDM and cross state-dependent memory retrieval between ACPA and 8-OH-DPAT in a step-down inhibitory avoidance task.The dorsal hippocampal CA1 regions of adult male NMRI mice were bilaterally cannulated, and all drugs were microinjected into the intended injection sites. A single-trial step-down inhibitory avoidance task was used to assess memory retrieval and state-dependence.Post-training and/or pre-test microinjections of ACPA (1 and 2 ng/mouse) and 8-OH-DPAT (0.5 and 1 μg/mouse) dose-dependently induced amnesia. Pre-test administration of the same doses of ACPA and 8-OH-DPAT reversed the post-training ACPA- and 8-OH-DPAT-induced amnesia, respectively. This phenomenon has been named SDM. 8-OH-DPAT (1 μg/mouse) reversed the amnesia induced by ACPA (0.5, 1, and 2 ng/mouse) and induced ACPA SDM. ACPA (2 ng/mouse) reversed the amnesia induced by 8-OH-DPAT (0.25, 0.5, and 1 μg/mouse) and induced 8-OH-DPAT SDM. Pre-test administration of a 5-HT1A receptor antagonist, (S)-WAY 100,135 (0.25 and 0.5 μg/mouse), 5 min before ACPA and 8-OH-DPAT dose-dependently inhibited ACPA- and 8-OH-DPAT-induced SDM, respectively.The present study results demonstrated ACPA- and 8-OH-DPAT- induced SDM. Overall, the data revealed that dorsal hippocampal 5-HT1A receptor mechanisms play a pivotal role in modulating cross state-dependent memory retrieval between ACPA and 8-OH-DPAT.

Modulation of Morphine Analgesia, Antinociceptive Tolerance, and Mu-Opioid Receptor Binding by the Cannabinoid CB2 Receptor Agonist O-1966.

Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.

Cryo-EM structures of the β3 adrenergic receptor bound to solabegron and isoproterenol

The β3-adrenergic receptor (β3AR) is the most essential drug target for overactive bladder and has therapeutic potentials for the treatments of type 2 diabetes and obesity. Here, we report the cryo-electron microscopy structures of the β3AR-Gs signaling complexes with the selective agonist, solabegron and the nonselective agonist, isoproterenol. Comparison of the isoproterenol-, mirabegron-, and solabegron-bound β3AR structures revealed that the extracellular loop 2 changes its conformation depending on the bound agonist and plays an essential role in solabegron binding. Moreover, β3AR has an intrinsically narrow exosite, regardless of the agonist type. This structural feature clearly explains why β3AR prefers mirabegron and solabegron, as the narrow exosite is suitable for binding with agonists with elongated shapes. Our study deepens the understanding of the binding characteristics of β3AR agonists and may pave the way for developing β3AR-selective drugs.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Dopaminergic signaling regulates zebrafish larvae's response to electricity.

Electrical stimulation of brain or muscle activities has gained attention for studying the molecular and cellular mechanisms involved in electric-induced responses. We recently showed zebrafish's response to electricity. Here, we hypothesized that this response is affected by the dopaminergic signaling pathways. The effects of multiple dopamine agonists and antagonists on the electric response of 6 days-postfertilization zebrafish larvae were investigated using a microfluidic device with enhanced control of experimentation and throughput. All dopamine antagonists decreased locomotor activities, while dopamine agonists did not induce similar behaviors. The D2-selective dopamine agonist quinpirole enhanced the movement. Exposure to nonselective and D1-selective dopamine agonists apomorphine and SKF-81297 caused no significant change in the electric response. Exposing larvae that were pretreated with nonselective and D2-selective dopamine antagonists butaclamol and haloperidol to apomorphine and quinpirole, respectively, restored the electric locomotion. These results reveal a correlation between electric response and dopamine signaling pathway. Furthermore, they demonstrate that electric-induced zebrafish larvae locomotion can be conditioned by modulating dopamine receptor functions. Our electrofluidic assay has profound application potential for fundamental electric-induced response research and brain disorder studies especially those related to the dopamine imbalance and as a chemical screening method when investigating biological pathways and behaviors.

Evidence for Bell-Shaped Dose-Response Emetic Effects of Temsirolimus and Analogs: The Broad-Spectrum Antiemetic Efficacy of a Large Dose of Temsirolimus Against Diverse Emetogens in the Least Shrew (Cryptotis parva).

Temsirolimus is a prodrug form of sirolimus (rapamycin). With its analogs (everolimus, ridaforolimus, and rapamycin), it forms a group of anticancer agents that block the activity of one of the two mammalian targets of rapamycin (mTOR) complexes, mTORC1. We investigated the emetic potential of varying doses (0, 0.5, 1, 2.5, 5, 10, 20, and 40 mg/kg, i.p.) of temsirolimus in the least shrew. Temsirolimus caused a bell-shaped and dose-dependent increase in both the mean vomit frequency and the number of shrews vomiting with maximal efficacy at 10 mg/kg (p < 0.05 and p < 0.02, respectively). Its larger doses (20 or 40 mg/kg) had no significant emetic effect. We also evaluated the emetic potential of its analogs (5, 10, and 20 mg/kg, i.p.), all of which exhibited a similar emetic profile. Our observational studies indicated that temsirolimus can reduce the shrew motor activity at 40 mg/kg, and subsequently, we examined the motor effects of its lower doses. At 10 and 20 mg/kg, it did not affect the spontaneous locomotor activity (distance moved) but attenuated the mean rearing frequency in a U-shaped manner at 10 mg/kg (p < 0.05). We then determined the broad-spectrum antiemetic potential of a 20 mg/kg (i.p.) dose of temsirolimus against diverse emetogens, including selective and nonselective agonists of 1) dopaminergic D2/3 receptors (apomorphine and quinpirole); 2) serotonergic 5-HT3 receptors [5-HT (serotonin) and 2-methyl-5-HT]; 3) cholinergic M1 receptors (pilocarpine and McN-A-343); 4) substance P neurokinin NK1 receptors (GR73632); 5) the L-type calcium (Ca2+) channel (LTCC) (FPL64176); 6) the sarcoplasmic endoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin; 7) the CB1 receptor inverse agonist/antagonist, SR141716A; and 8) the chemotherapeutic cisplatin. Temsirolimus prevented vomiting evoked by the aforementioned emetogens with varying degrees. The mechanisms underlying the pro- and antiemetic effects of temsirolimus evaluated by immunochemistry for c-fos expression demonstrated a c-fos induction in the AP and NTS, but not DMNX with the 10 mg/kg emetic dose of temsirolimus, whereas its larger antiemetic dose (20 mg/kg) had no significant effect. Our study is the first to provide preclinical evidence demonstrating the promising antiemetic potential of high doses of temsirolimus and possibly its analogs in least shrews.

Fingolimod (FTY720), a Sphinogosine-1-Phosphate Receptor Agonist, Mitigates Choroidal Endothelial Proangiogenic Properties and Choroidal Neovascularization.

Neovascular or wet age-related macular degeneration (nAMD) causes vision loss due to inflammatory and vascular endothelial growth factor (VEGF)-driven neovascularization processes in the choroid. Due to the excess in VEGF levels associated with nAMD, anti-VEGF therapies are utilized for treatment. Unfortunately, not all patients have a sufficient response to such therapies, leaving few if any other treatment options for these patients. Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator found in endothelial cells that participates in modulating barrier function, angiogenesis, and inflammation. S1P, through its receptor (S1PR1) in endothelial cells, prevents illegitimate sprouting angiogenesis during vascular development. In the present paper, we show that, in choroidal endothelial cells, S1PR1 is the most abundantly expressed S1P receptor and agonism of S1PR1-prevented choroidal endothelial cell capillary morphogenesis in culture. Given that nAMD pathogenesis draws from enhanced inflammation and angiogenesis as well as a loss of barrier function, we assessed the impact of S1PR agonism on choroidal neovascularization in vivo. Using laser photocoagulation rupture of Bruch’s membrane to induce choroidal neovascularization, we show that S1PR non-selective (FTY720) and S1PR1 selective (CYM5442) agonists significantly inhibit choroidal neovascularization in this model. Thus, utilizing S1PR agonists to temper choroidal neovascularization presents an additional novel use for these agonists presently in clinical use for multiple sclerosis as well as other inflammatory diseases.

Detection of pentylenetetrazol-induced seizure activity in the 19-21 Hz beta range using a magnetic coil induction method.

A force transducer or automatic scoring system is not sufficient to detect small or fine seizure activity. To improve previous assessments of epileptic behavior, a novel coil method was developed to detect an early behavioral marker for epileptic seizures. The present study used the γ-aminobutyric acid (GABA) receptor antagonist pentylenetetrazol (PTZ) to induce seizure activity and epileptic behavior in mice. A coil method was used to detect motor seizures consisting of small amplitude 19-21 Hz muscle contractions. Seizure activity in the 19-21 Hz range detected by the coil method was positively correlated with generalized clonic seizures with a kangaroo posture after PTZ administration. GABA receptor agonist valproic acid and ethosuximide decreased PTZ-induced 19-21 Hz seizure activity. The pattern of the amplitude ratio (%) of 19-21 Hz seizure activity after administration of the GABAA/C receptor antagonist picrotoxin was similar to the group that was treated with PTZ but different from the group that was treated with the nonselective muscarinic receptor agonist pilocarpine. The coil method detected 19-21 Hz seizure activity after PTZ administration. However, the force transducer method did not detect 19-21 Hz seizure activity. The coil method was more sensitive than the force transducer method for detecting epileptic behaviors. The findings may indicate a novel behavioral marker that can be detected by the coil method to reveal epileptic seizures, thus improving our understanding of the brain mechanisms of action and specific brain waves that are associated with PTZ-induced 19-21 Hz seizure activity.

MELATONİN AGONİSTİ OLAN RAMELTEONUN METOTREKSAT KAYNAKLI KEMİK TOKSİSİTESİNE KARŞI KORUYUCU ETKİSİ

Objective&#x0D; Methotrexate (MTX) used anti-metabolite, causes&#x0D; bone morbidity, including growth arrest and reduced&#x0D; bone mineral density. Melatonin, produced by the&#x0D; pineal gland, has also multiple positive effects in human&#x0D; bone cells, and positive effects on bone. Ramelteon&#x0D; (RMT) is a non-selective melatonin receptor agonist.&#x0D; In this study, we investigated whether ramelteon, a&#x0D; melatonin agonist, has a protective effect on MTXinduced&#x0D; bone toxicity.&#x0D; Material and Methods&#x0D; The rats divided into 4 groups, including Group 1&#x0D; control group; Group 2 MTX group (20 mg/kg); Group&#x0D; 3 MTX+RMT (20 mg/kg + 10 mg/kg); Group 4 RMT (10&#x0D; mg/kg). Oral ramelteon and intraperitoneal mtx were&#x0D; applied to the rats on the second day according to&#x0D; the groups. After 7 days, long bones were evaluated&#x0D; histologically with hematoxylin-eosin (HE) staining and&#x0D; immunohistochemically with Catepsin K and RUN X2&#x0D; staining. For statistical analysis immunohistochemical&#x0D; scores of the groups were compared between the&#x0D; groups for this purpose, the Oneway ANOVA Duncan&#x0D; test was used by SPSS-22.00 package program.&#x0D; Results&#x0D; There was no significant difference between the&#x0D; control group (group I) and the experimental groups&#x0D; (group II-group III-group IV) in H&amp;E staining of bone&#x0D; tissue sections (p&gt;0.05). No positive staining was&#x0D; observed in any of the groups in CAT-K and RUN-X&#x0D; immunostaining (p&gt;0.05).&#x0D; Conclusion&#x0D; It was showed that ramelteon has no anabolic&#x0D; function in bone turnover, histopathological and&#x0D; immunohistochemical, in bone toxicity induced by&#x0D; high-dose methotrexate on intact bone tissue.

Ankk1 Loss of Function Disrupts Dopaminergic Pathways in Zebrafish.

Ankyrin repeat and kinase domain containing 1 (ANKK1) is a member of the receptor-interacting protein serine/threonine kinase family, known to be involved in cell proliferation, differentiation and activation of transcription factors. Genetic variation within the ANKK1 locus is suggested to play a role in vulnerability to addictions. However, ANKK1 mechanism of action is still poorly understood. It has been suggested that ANKK1 may affect the development and/or functioning of dopaminergic pathways. To test this hypothesis, we generated a CRISPR-Cas9 loss of function ankk1 zebrafish line causing a 27 bp insertion that disrupts the ankk1 sequence introducing an early stop codon. We found that ankk1 transcript levels were significantly lower in ankk1 mutant (ankk127ins) fish compared to their wild type (ankk1+/+) siblings. In ankk1+/+ adult zebrafish brain, ankk1 protein was detected in isocortex, hippocampus, basolateral amygdala, mesencephalon, and cerebellum, resembling the mammalian distribution pattern. In contrast, ankk1 protein was reduced in the brain of ankk127ins/27ins fish. Quantitative polymerase chain reaction analysis revealed an increase in expression of drd2b mRNA in ankk127ins at both larval and adult stages. In ankk1+/+ adult zebrafish brain, drd2 protein was detected in cerebral cortex, cerebellum, hippocampus, and caudate homolog regions, resembling the pattern in humans. In contrast, drd2 expression was reduced in cortical regions of ankk127ins/27ins being predominantly found in the hindbrain. No differences in the number of cell bodies or axonal projections detected by anti-tyrosine hydroxylase immunostaining on 3 days post fertilization (dpf) larvae were found. Behavioral analysis revealed altered sensitivity to effects of both amisulpride and apomorphine on locomotion and startle habituation, consistent with a broad loss of both pre and post synaptic receptors. Ankk127ins mutants showed reduced sensitivity to the effect of the selective dopamine receptor antagonist amisulpride on locomotor responses to acoustic startle and were differentially sensitive to the effects of the non-selective dopamine agonist apomorphine on both locomotion and habituation. Taken together, our findings strengthen the hypothesis of a functional relationship between ANKK1 and DRD2, supporting a role for ANKK1 in the maintenance and/or functioning of dopaminergic pathways. Further work is needed to disentangle ANKK1’s role at different developmental stages.