Search Results (30)

Trimethylamine N-oxide (TMAO) is an endogenous osmolyte produced by enzymatic reactions starting in the human gut, where microbiota release trimethylamine (TMA) from foods, and ending in the liver, where TMA is oxidized to TMAO by flavin-containing monooxygenase 3 (FMO3). While physiological concentrations of TMAO help proteins preserve their folding, high levels of this metabolite are harmful and promote oxidative stress, inflammation, and atherosclerosis. In humans, elevated levels of circulating TMAO predispose individuals to cardiovascular diseases and chronic kidney disease and increase mortality risk, especially in the elderly. How TMAO exerts its negative effects has been only partially elucidated. In hypertensive rats, the eNOS substrate L-arginine and Taurisolo^®, a nutraceutical endowed with TMAO-reducing activity, act synergistically to reduce arterial blood pressure. Here, we investigate the molecular mechanisms underpinning this synergism and prove that TMAO, the target of Taurisolo^®, acts as direct inhibitor of endothelial nitric oxide synthase (eNOS) and competes with L-arginine at its catalytic site, ultimately inhibiting NO production and acetylcholine (Ach)-induced relaxation in murine aortas. Full article

Donepezil (DH), a selective acetylcholinesterase inhibitor, is widely used to manage symptoms of mild to moderate Alzheimer’s disease by enhancing cholinergic neurotransmission and preventing acetylcholine breakdown. Despite the effectiveness of oral formulations, extensive hepatic metabolism and low systemic bioavailability have driven the search for alternative delivery systems. This study focuses on nasal delivery as a non-parenteral substitute, utilizing hydroxypropyl methylcellulose (HPMC) for its mucoadhesive properties and methyl-β-cyclodextrin (Me-β-CD) for its ability to enhance permeability and form inclusion complexes with drugs. Prior studies demonstrated the potential of HPMC-based nasal films for nose-to-brain delivery of donepezil and highlighted Me-β-CD’s role in improving drug solubility. Building on this, transparent gel formulations containing DH, HPMC, and 2,6 Me-β-CD were developed to investigate molecular interactions within two- and three-component systems. This study utilized a combination of nuclear magnetic resonance (NMR) spectroscopy and density functional theory (DFT) to provide detailed insights into the interactions between DH, 2,6-Me-β-CD, and HPMC. The findings provide critical insights into drug–excipient interactions, aiding the optimization of stability, solubility, and controlled release. This advances the rational design of nanotechnology-based drug delivery systems for enhanced therapeutic efficacy. Full article

Botulinum neurotoxins are synthetized by anaerobic, spore-forming bacteria that inhibit acetylcholine release at the level of the neuromuscular and autonomic cholinergic junctions, thus inducing a series of symptoms, the most relevant of which is flaccid paralysis. At least seven serotypes and over 40 subtypes are known, and they are among the most poisonous natural substances. There are different forms of botulism according to the route of contamination, but the clinical manifestation of descending symmetric flaccid paralysis is consistent, regardless of the route of contamination. It is very severe and potentially lethal. The induced paralysis lasts as long as the toxin is active, with variable length, according to the serotype of the toxin. This transient activity, as well as the precise mechanism of action, are the basis for the rationale behind use of the toxin in therapy for several clinical conditions, particularly, spastic conditions, as well as chronic migraine and axillary hyperhidrosis. The toxin has also been approved for the reduction in facial wrinkles; all these clinical applications, coupled with the toxin’s risks, have earned botulinum the title of a two-faced Janus protein. No approved vaccines are currently available, andthe only approved antidotes are the human specific intravenous immunoglobulins for infant botulism and the heptavalent equine immunoglobulins/(F(ab’)² for adults. Nanobodies, which show great promise, may penetrate neuronal cells to inactivate the toxin within the cytoplasm, and Ebselen, a non-toxic, economic, small-molecule inhibitor, has the characteristic of inhibiting the toxin irrespective of the serotype. Full article

The vegetal alkaloid toxin veratridine (VTD) is a selective voltage-gated Na⁺ (Na_V) channel activator, widely used as a pharmacological tool in vascular physiology. We have previously shown that Na_V channels, expressed in arteries, contribute to vascular tone in mouse mesenteric arteries (MAs). Here, we aimed to better characterize the mechanisms of action of VTD using mouse cecocolic arteries (CAs), a model of resistance artery. Using wire myography, we found that VTD induced vasorelaxation in mouse CAs. This VTD-induced relaxation was insensitive to prazosin, an α1-adrenergic receptor antagonist, but abolished by atropine, a muscarinic receptor antagonist. Indeed, VTD–vasorelaxant effect was totally inhibited by the Na_V channel blocker tetrodotoxin (0.3 µM), the NO synthase inhibitor L-NNA (20 µM), and low extracellular Na⁺ concentration (14.9 mM) and was partially blocked by the NCX1 antagonist SEA0400 (45.4% at 1 µM). Thus, we assumed that the VTD-induced vasorelaxation in CAs was due to acetylcholine release by parasympathetic neurons, which induced NO synthase activation mediated by the NCX1-Ca²⁺ entry mode in endothelial cells (ECs). We demonstrated NCX1 expression in ECs by RT-qPCR and immunohisto- and western immunolabelling. VTD did not induce an increase in intracellular Ca²⁺ ([Ca²⁺]i), while SEA0400 partially blocked acetylcholine-triggered [Ca²⁺]i elevations in Mile Sven 1 ECs. Altogether, these results illustrate that VTD activates Na_V channels in parasympathetic neurons and then vasorelaxation in resistance arteries, which could explain arterial hypotension after VTD intoxication. Full article

Purpose: This study aimed to compare the effects of the phytoestrogens resveratrol (RES) and genistein (GEN) on the contractility of isolated uterine smooth muscle from rats, focusing on both spontaneous and stimulated contractions, and to investigate the underlying mechanisms. Methods: Uterine strips were suspended vertically in perfusion chambers containing Kreb’s solution, various concentrations of RES and GEN were added to the ex vivo uterine strips, and contractions were measured before and after incubation with RES or GEN. Results: (1) Both RES and GEN inhibited K⁺-induced contractions in a dose-dependent manner; the β/β₂-adrenoceptor antagonist propranolol (PRO), ICI118551, the ATP-dependent K⁺ channel blocker glibenclamide (HB-419) and the NO synthase inhibitor N-nitro-L-arginine (L-NNA) diminished the inhibitory effects of RES and GEN on K⁺-induced contractions. (2) RES and GEN also dose-dependently inhibited PGF_2α-induced uterine contractions. (3) The inhibitory effects of RES and GEN were observed in spontaneous contractile activities as well; PRO, ICI118551, HB-419 and L-NNA attenuated the inhibitory effects of RES and GEN on the spontaneous contractions of isolated uterine muscle strips. (4) RES and GEN significantly decreased the cumulative concentration response of Ca²⁺ and shifted the Ca²⁺ cumulative concentration–response curves to the right in high-K⁺ Ca²⁺-free Kreb’s solution. (5) RES and GEN markedly reduced the first phasic contraction induced by oxytocin, acetylcholine, and prostaglandin F_2α but did not alter the second phasic contraction caused by CaCl₂ in Ca²⁺-free Kreb’s solution. Conclusions: RES and GEN can directly inhibit both spontaneous and activated contractions of isolated uterine smooth muscle. The mechanisms underlying the inhibitory effects of RES and GEN likely involve β adrenergic receptor activation, reduced Ca²⁺ influx and release, the activation of ATP-dependent K⁺ channels and increased NO production. Full article

In recent years, particular attention has been paid to the serotonin 4 receptor, which is well expressed in the brain, but also peripherally in various organs. The cerebral distribution of this receptor is well conserved across species, with high densities in the basal ganglia, where they are expressed by GABAergic neurons. The 5-HT₄ receptor is also present in the cerebral cortex, hippocampus, and amygdala, where they are carried by glutamatergic or cholinergic neurons. Outside the central nervous system, the 5-HT₄ receptor is notably expressed in the gastrointestinal tract. The wide distribution of the 5-HT₄ receptor undoubtedly contributes to its involvement in a plethora of functions. In addition, the modulation of this receptor influences the release of serotonin, but also the release of other neurotransmitters such as acetylcholine and dopamine. This is a considerable asset, as the modulation of the 5-HT₄ receptor can therefore play a direct or indirect beneficial role in various disorders. One of the main advantages of this receptor is that it mediates a much faster antidepressant and anxiolytic action than classical selective serotonin reuptake inhibitors. Another major benefit of the 5-HT₄ receptor is that its activation enhances cognitive performance, probably via the release of acetylcholine. The expression of the 5-HT₄ receptor is also altered in various eating disorders, and its activation by the 5-HT₄ agonist negatively regulates food intake. Additionally, although the cerebral expression of this receptor is modified in certain movement-related disorders, it is still yet to be determined whether this receptor plays a key role in their pathophysiology. Finally, there is no longer any need to demonstrate the value of 5-HT₄ receptor agonists in the pharmacological management of gastrointestinal disorders. Full article

Botulinum toxin type A (BONT-A) has shown promise in improving the mood-related symptoms of psychiatric disorders by targeting muscles linked to the expression of negative emotions. We conducted a systematic review of past and ongoing efficacy trials of BONT-A therapy for psychiatric disorders to identify relevant trends in the field and discuss the refinement of therapeutic techniques. A comprehensive search for published clinical trials using BONT-A injections for psychiatric disorders was performed on 4 May 2023 through OVID databases (MEDLINE, Embase, APA PsycINFO). Unpublished clinical trials were searched through the ClinicalTrials.gov and International Clinical Trial Registry Platform public registries. The risk of bias was assessed using the JBI Critical Appraisal tools for use in systematic reviews. We identified 21 studies (17 published, 4 unpublished clinical trials) involving 471 patients. The studies focused on evaluating the efficacy of BONT-A for major depressive, borderline personality, social anxiety, and bipolar disorders. BONT-A was most commonly injected into the glabellar area, with an average dose ranging between 37.75 U and 44.5 U in published studies and between 32.7 U and 41.3 U in unpublished trials. The results indicated significant symptom reductions across all the studied psychiatric conditions, with mild adverse effects. Thus, BONT-A appears to be safe and well-tolerated for psychiatric disorders of negative affectivity. However, despite the clinical focus, there was a noted shortage of biomarker-related assessments. Future studies should focus on pursuing mechanistic explorations of BONT-A effects at the neurobiological level. Full article

The cholinergic system, relying on the neurotransmitter acetylcholine (ACh), plays a significant role in muscle contraction, cognition, and autonomic nervous system regulation. The enzymes acetylcholinesterase, AChE, and butyrylcholinesterase, BChE, responsible for hydrolyzing ACh, can fine-tune the cholinergic system’s activity and are, therefore, excellent pharmacological targets to address a range of medical conditions. We designed, synthesized, and profiled 14 N-alkyl quaternary quinuclidines as inhibitors of human AChE and BChE and analyzed their impact on cell viability to assess their safety in the context of application as potential therapeutics. Our results showed that all of the 14 tested quinuclidines inhibited both AChE and BChE in the micromolar range (K_i = 0.26 − 156.2 μM). The highest inhibition potency was observed for two bisquaternary derivatives, 7 (1,1′-(decano)bis(3-hydroxyquinuclidinium bromide)) and 14 (1,1′-(decano)bis(3-hydroxyiminoquinuclidinium bromide)). The cytotoxic effect within 7–200 μM was observed only for monoquaternary quinuclidine derivatives, especially those with the C12–C16 alkyl chain. Further analysis revealed a time-independent mechanism of action, significant LDH release, and a decrease in the cells’ mitochondrial membrane potential. Taking all results into consideration, we can confirm that a quinuclidine core presents a good scaffold for cholinesterase binding and that two bisquaternary quinuclidine derivatives could be considered as candidates worth further investigations as drugs acting in the cholinergic system. On the other hand, specific cell-related effects probably triggered by the free long alkyl chain in monoquaternary quinuclidine derivatives should not be neglected in future N-alkyl quaternary quinuclidine derivative structure refinements. Such an effect and their potential to interact with other specific targets, as indicated by a pharmacophore model, open up a new perspective for future investigations of these compounds’ scaffold in the treatment of specific conditions and diseases other than cholinergic system-linked disorders. Full article

ATP, being a well-known universal high-energy compound, plays an important role as a signaling molecule and together with its metabolite adenosine they both attenuate the release of acetylcholine in the neuro-muscular synapse acting through membrane P2 and P1 receptors, respectively. In this work, using a mechanomyographic method, we analyzed the presynaptic mechanisms by which ATP and adenosine can modulate the transduction in the rat m. soleus and m. extensor digitorum longus. N-ethylmaleimide, a G-protein antagonist, prevents the modulating effects of both ATP and adenosine. The action of ATP is abolished by chelerythrin, a specific phospholipase C inhibitor, while the inhibitory effect of adenosine is slightly increased by Rp-cAMPS, an inhibitor of protein kinase A, and by nitrendipine, a blocker of L-type Ca²⁺ channels. The addition of DPCPX, an A₁ receptor antagonist, fully prevents the inhibitory action of adenosine in both muscles. Our data indicate that the inhibitory action of ATP involves metabotropic P2Y receptors and is mediated by phospholipase C dependent processes in rat motor neuron terminals. We suggest that the presynaptic effect of adenosine consists of negative and positive actions. The negative action occurs by stimulation of adenosine A₁ receptors while the positive action is associated with the stimulation of adenosine A_2A receptors, activation of protein kinase A and opening of L-type calcium channels. The combined mechanism of the modulating action of ATP and adenosine provides fine tuning of the synapse to fast changing conditions in the skeletal muscles. Full article

Ischemia/reperfusion (I/R)-induced endothelial dysfunction occurs in various cardiovascular disorders. I/R injury is partially driven by the release of cytokines. Known for its use in senotherapy, the JAK inhibitor ruxolitinib is able to block the release of cytokines. We investigated the effect of ruxolitinib on the cytokine release and endothelial-dependent vasorelaxation in an in vitro model of I/R. Aortic segments of C57BL/6J mice (N = 12/group) were divided into three groups: control, in vitro I/R (I/R group), and in vitro I/R with ruxolitinib during ischemic incubation (I/R+Ruxo group). We determined cytokine expression. In organ bath chambers, we investigated the maximal endothelial-dependent relaxation to acetylcholine (R_maxACh) and maximal endothelial-independent relaxation to sodium-nitroprusside (R_maxSNP). R_maxACh was decreased in I/R compared to the control (83.6 ± 2.4 vs. 48.6 ± 3.4%; p < 0.05) and I/R+Ruxo (74.4 ± 2.6 vs. 48.6 ± 3.4%; p < 0.05). R_maxSNP was comparable between all groups. IL-10 was detectable only in I/R+Ruxo. CXCL5, CCL2, CCL3, CCL8, CCL11, ICAM-1, IL-1α, IL-7, TNF-α, and G-CSF were decreased or not detectable in I/R+Ruxo. In I/R+Ruxo, ICAM-1 was reduced in rings only from male mice. Treatment of the aorta from mice during in vitro ischemia with the senomorphic agent ruxolitinib reduces cytokine release and protects the endothelium from I/R-mediated dysfunction. Full article

STAT3 plays a protective role against ischemic brain injury; however, it is not clear which brain cell type mediates this effect, and by which mechanism. We tested the hypothesis that endothelial STAT3 contributes to protection from cerebral ischemia, by preserving cerebrovascular endothelial function and blood–brain barrier (BBB) integrity. The objective of this study was to determine the role of STAT3 in cerebrovascular endothelial cell (EC) survival and function, and its role in tissue outcome after cerebral ischemia. We found that in primary mouse brain microvascular ECs, STAT3 was constitutively active, and its phosphorylation was reduced by oxygen-glucose deprivation (OGD), recovering after re-oxygenation. STAT3 inhibition, using two mechanistically different pharmacological inhibitors, increased EC injury after OGD. The sub-lethal inhibition of STAT3 caused endothelial dysfunction, demonstrated by reduced nitric oxide release in response to acetylcholine and reduced barrier function of the endothelial monolayer. Finally, mice with reduced endothelial STAT3 (Tie2-Cre; STAT3^flox/wt) sustained larger brain infarcts after middle cerebral artery occlusion (MCAO) compared to wild-type (WT) littermates. We conclude that STAT3 is vital to maintaining cerebrovascular integrity, playing a role in EC survival and function, and protection against cerebral ischemia. Endothelial STAT3 may serve as a potential target in preventing endothelial dysfunction after stroke. Full article

Botulinum neurotoxins (BoNTs) are potent inhibitors of synaptic vesicle fusion and transmitter release. The natural target of BoNTs is the peripheral neuromuscular junction (NMJ) where, by blocking the release of acetylcholine (ACh), they functionally denervate muscles and alter muscle tone. This leads them to be an excellent drug for the therapy of muscle hyperactivity disorders, such as dystonia, spasticity, and many other movement disorders. BoNTs are also effective in inhibiting both the release of ACh at sites other than NMJ and the release of neurotransmitters other than ACh. Furthermore, much evidence shows that BoNTs can act not only on the peripheral nervous system (PNS), but also on the central nervous system (CNS). Under this view, central changes may result either from sensory input from the PNS, from retrograde transport of BoNTs, or from direct injection of BoNTs into the CNS. The aim of this review is to give an update on available data, both from animal models or human studies, which suggest or confirm central alterations induced by peripheral or central BoNTs treatment. The data will be discussed with particular attention to the possible therapeutic applications to pathological conditions and degenerative diseases of the CNS. Full article

Neurodegenerative diseases, e.g., Alzheimer’s disease (AD), are a key health problem in the aging population. The lack of effective therapy and diagnostics does not help to improve this situation. It is thought that ligands influencing multiple but interconnected targets can contribute to a desired pharmacological effect in these complex illnesses. Histamine H₃ receptors (H₃Rs) play an important role in the brain, influencing the release of important neurotransmitters, such as acetylcholine. Compounds blocking their activity can increase the level of these neurotransmitters. Cholinesterases (acetyl- and butyrylcholinesterase) are responsible for the hydrolysis of acetylcholine and inactivation of the neurotransmitter. Increased activity of these enzymes, especially butyrylcholinesterase (BuChE), is observed in neurodegenerative diseases. Currently, cholinesterase inhibitors: donepezil, rivastigmine and galantamine are used in the symptomatic treatment of AD. Thus, compounds simultaneously blocking H₃R and inhibiting cholinesterases could be a promising treatment for AD. Herein, we describe the BuChE inhibitory activity of H₃R ligands. Most of these compounds show high affinity for human H₃R (K_i < 150 nM) and submicromolar inhibition of BuChE (IC₅₀ < 1 µM). Among all the tested compounds, 19 (E153, 1-(5-([1,1′-biphenyl]-4-yloxy)pentyl)azepane) exhibited the most promising in vitro affinity for human H₃R, with a K_i value of 33.9 nM, and for equine serum BuChE, with an IC₅₀ of 590 nM. Moreover, 19 (E153) showed inhibitory activity towards human MAO B with an IC₅₀ of 243 nM. Furthermore, in vivo studies using the Passive Avoidance Task showed that compound 19 (E153) effectively alleviated memory deficits caused by scopolamine. Taken together, these findings suggest that compound 19 can be a lead structure for developing new anti-AD agents. Full article

The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca²⁺-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca²⁺-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K⁺-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K⁺-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release. Full article

The authors in the current work suggested the potential repurposing of omarigliptin (OMR) for neurodegenerative diseases based on three new findings that support the preliminary finding of crossing BBB after a single dose study in the literature. The first finding is the positive results of the docking study with the crystal structures of A_2A adenosine (A2AAR) and acetylcholine esterase (AChE) receptors. A2AAR is a member of non-dopaminergic GPCR superfamily receptor proteins and has essential role in regulation of glutamate and dopamine release in Parkinson’s disease while AChE plays a major role in Alzheimer’s disease as the primary enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine into choline and acetate. Docking showed that OMR perfectly fits into A2AAR binding pocket forming a distinctive hydrogen bond with Threonine 256. Besides other non-polar interactions inside the pocket suggesting the future of the marketed anti-diabetic drug (that cross BBB) as a potential antiparkinsonian agent while OMR showed perfect fit inside AChE receptor binding site smoothly because of its optimum length and the two fluorine atoms that enables quite lean fitting. Moreover, a computational comparative study of OMR docking, other 12 DPP-4 inhibitors and 11 SGLT-2 inhibitors was carried out. Secondly, glucagon-like peptide-1 (GLP-1) concentration in rats’ brain tissue was determined by the authors using sandwich GLP-1 ELISA kit bio-analysis to ensure the effect of OMR after the multiple doses’ study. Brain GLP-1 concentration was elevated by 1.9-fold following oral multiple doses of OMR (5 mg/kg/day, p.o. for 28 days) as compared to the control group. The third finding is the enhanced BBB crossing of OMR after 28 days of multiple doses that had been studied using LC-MS/MS method with enhanced liquid–liquid extraction. A modified LC-MS/MS method was established for bioassay of OMR in rats’ plasma (10–3100 ng/mL) and rats’ brain tissue (15–2900 ng/mL) using liquid–liquid extraction. Alogliptin (ALP) was chosen as an internal standard (IS) due to its LogP value of 1.1, which is very close to the LogP of OMR. Extraction of OMR from samples of both rats’ plasma and rats’ brain tissue was effectively achieved with ethyl acetate as the extracting solvent after adding 1N sodium carbonate to enhance the drug migration, while choosing acetonitrile to be the diluent solvent for the IS to effectively decrease any emulsion between the layers in the stated method of extraction. Validation results were all pleasing including good stability studies with bias of value below 20%. Concentration of OMR in rats’ plasma were determined after 2 h of the latest dose from 28 days multiple doses, p.o, 5 mg/kg/day. It was found to be 1295.66 ± 684.63 ng/mL estimated from the bio-analysis regression equation. OMR passed through the BBB following oral administration and exhibited concentration of 543.56 ± 344.15 ng/g in brain tissue, taking in consideration the dilution factor of 10. The brain/plasma concentration ratio of 0.42 (543.56/1295.66) was used to illustrate the penetration power through the BBB after the multiple doses for 28 days. Results showed that OMR passed through the BBB more effectively in the multiple dose study as compared to the previously published single dose study by the authors. Thus, the present study suggests potential repositioning of OMR as antiparkinsonian agent that will be of interest for researchers interested in neurodegenerative diseases. Full article