Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

Molecular Interactions between an Enzyme and Its Inhibitor for Selective Detection of Limonene.

Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer's drugs. However, application of enzyme inhibition to odorant sensors is less common because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limonene─the major odorant of citrus and an AChE inhibitor─using molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 µM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant-enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.

Concatemers to re-investigate the role of α5 in α4ß2 nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ion channels expressed in the central nervous systems. nAChRs containing the α4, ß2 and α5 subunits are specifically involved in addictive processes, but their functional architecture is poorly understood due to the intricacy of assembly of these subunits. Here we constrained the subunit assembly by designing fully concatenated human α4ß2 and α4ß2α5 receptors and characterized their properties by two-electrodes voltage-clamp electrophysiology in Xenopus oocytes. We found that α5-containing nAChRs are irreversibly blocked by methanethiosulfonate (MTS) reagents through a covalent reaction with a cysteine present only in α5. MTS-block experiments establish that the concatemers are expressed in intact form at the oocyte surface, but that reconstitution of nAChRs from loose subunits show inefficient and highly variable assembly of α5 with α4 and ß2. Mutational analysis shows that the concatemers assemble both in clockwise and anticlockwise orientations, and that α5 does not contribute to ACh binding from its principal (+) site. Reinvestigation of suspected α5-ligands such as galantamine show no specific effect on α5-containing concatemers. Analysis of the α5-D398N mutation that is linked to smoking and lung cancer shows no significant effect on the electrophysiological function, suggesting that its effect might arise from alteration of other cellular processes. The concatemeric strategy provides a well-characterized platform for mechanistic analysis and screening of human α5-specific ligands.

From Crystal Structures of RgIA4 in Complex with Ac-AChBP to Molecular Determinants of Its High Potency of α9α10 nAChR.

α9-containing nicotinic acetylcholine receptors (nAChRs) have been shown to play critical roles in neuropathic pain. The α-conotoxin (α-CTx) RgIA and its analog RgIA4 were identified as the most selective inhibitor of α9α10 nAChR. However, the mechanism of their selectivity toward α9α10 nAChR remains elusive. Here, we reported the co-crystal structure of RgIA and RgIA4 in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at resolution of 2.6 Å, respectively. Based on the structure of the complexes, together with molecular dynamic simulation (MD-simulation), we suggested the key residues of α9α10 nAChR in determining its high affinity for RgIA/RgIA4. This is the first time the complex between pain-related conotoxins and Ac-AChBP was reported and the complementary side of α9 subunit in binding of the antagonists shown. These results provide realistic template for the design of new therapeutic in neuropathic pain.

Improvement of the Solubilization and Extraction of Curcumin in an Edible Ternary Solvent Mixture.

A water-free, ternary solvent mixture consisting of a natural deep eutectic solvent (NADES), ethanol, and triacetin was investigated concerning its ability to dissolve and extract curcumin from Curcuma longa L. To this purpose, 11 NADES based on choline chloride, acetylcholine, and proline were screened using UV-vis measurements. A ternary phase diagram with a particularly promising NADES, based on choline chloride and levulinic acid was recorded and the solubility domains of the monophasic region were examined and correlated with the system's structuring via light scattering experiments. At the optimum composition, close to the critical point, the solubility of curcumin could be enhanced by a factor of >1.5 with respect to acetone. In extraction experiments, conducted at the points of highest solubility and evaluated via HPLC, a total yield of ~84% curcuminoids per rhizome could be reached. Through multiple extraction cycles, reusing the extraction solvent, an enrichment of curcuminoids could be achieved while altering the solution. When counteracting the solvent change, even higher concentrated extracts can be obtained.

The Effects of Structural Alterations in the Polyamine and Amino Acid Moieties of Philanthotoxins on Nicotinic Acetylcholine Receptor Inhibition in the Locust, Schistocerca gregaria.

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 µM at -75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 µM at -75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 µM at -75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at -75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 µM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Polymeric Membrane Electrodes Using Calix[4]pyrrole Bis/Tetra-Phosphonate Cavitands as Ionophores for Potentiometric Acetylcholine Sensing with High Selectivity.

A handful of bis/tetra-phosphonate calix[4]pyrroles with recognition sites embedding in hydrophobic cavitands were evaluated for the first time as ionophores for polymeric membrane Ach+-selective electrodes. Highly selective potentiometric Ach+ could be achieved over its analogues, especially for Ch+, which differs only by an acetate tail from Ach+. The superior performance of the proposed ISEs might be ascribed to a dual-site binding mode, in which the trimethylammonium head and acetate tail were accommodated by the phosphonate group-bridged aryl walls and the bowl-shaped aromatic cavity, through cation-π/charge-dipole interaction and the convergent four N-H···O hydrogen bonds, respectively. To gain more insight into the performance of the proposed ISEs, the cation-ionophore complex constants in the membrane phase were determined, and the binding affinity trend correlates well with the selectivity pattern. These results suggest that conformational preorganization of the ionophores and complementary weak interactions do change the selectivity of the ionophores. Studies on the influence of the sample solution pH demonstrated that the developed ISEs can be employed in a wide pH range of 4.0-9.6 with a fast response (<60 s), good reversibility, and long lifetime. Optimizing the membrane components, such as ionophores, lipophilic additives, and plasticizers, yielded ISEs, showing Nernstian responses to Ach+ with improved linear ranges and detection limits (a slope of -59.5 mV/dec in the linear range of 1 × 10-6-1 × 10-2 M with a detection limit of 3 × 10-7 M), which led to the success of the determination of Ach+ in spiked urine and milk samples.

Quaternary-Ammonium-Modulated Surface-Enhanced Raman Spectroscopy Effect: Discovery, Mechanism, and Application for Highly Sensitive In Vitro Sensing of Acetylcholine.

Quaternary ammonium (QA) plays multiple roles in biological functions, whose dysregulation may result in multiple diseases. However, how to efficiently detect QA-based materials such as acetylcholine (ACh) still remains a great challenge, especially in complex biological environments. Here, a new effect [called quaternary-ammonium-modulated surface-enhanced Raman spectroscopy (QAM-SERS) effect] is discovered, showing that the existence of QA will modulate the intensity of SERS signals in a concentration-dependent manner. When the QAM-SERS effect is used, a new method is easily developed for in vitro detection of ACh with an extremely high sensitivity and an ultrawide dynamic range. Particularly, the linear dynamic range can be freely tuned to adapt for various physiological samples. As a proof-of-concept experiment, the time-dependent secretion of ACh from PC12 cells was successfully monitored using the QAM-SERS method, which were under either the stimulation of potassium ions or the incubation of drugs. The discovery of the QAM-SERS effect provides an easy and universal strategy for detecting ACh as well as other QA-contained molecules, which can also inspire new insights into the roles that QA could play in biology and chemistry.

Colorimetric Differentiation of Flavonoids Based on Effective Reactivation of Acetylcholinesterase Induced by Different Affnities between Flavonoids and Metal Ions.

Flavonoids are closely related to human health, and the distinguishiment of flavonoids is an important but difficult issue. We herein unveil a novel colorimetric sensor array for the rapid identification of 7 flavonoids (e.g., gallocatechin (GC), morin hydrate (MH), puerarin (Pu), epigallocatechin gallate (EGCG), catechin (C), rac Naringenin (rN), and Flavone (Fla)) for the first time. The colorimetric performances of gold nanoparticles (AuNPs) are characteristically correlated with thiocholine, which is issued from the enzymatic hydrolysis of acetylcholine (AcCh). Therefore, as a proof-of-concept design, three sensors (Cu2+/acetylcholinesterase (AcChE)/AcCh/AuNPs, Zn2+/AcChE/AcCh/AuNPs, and Mn2+/AcChE/AcCh/AuNPs) were constructed to form our sensor array. The distinct affinities between flavonoids and metal ions would cause varying degrees of effective reactivation of AcChE, leading to unique colorimetric response patterns upon being challenged with the seven flavonoids for their pattern recognition, enabling an excellent identification of the seven flavonoids at a concentration of 20 nM and different concentrations of individual flavonoids, as well as mixtures of them.

Paracetamol inhibits Ca2+ permeant ion channels and Ca2+ sensitization resulting in relaxation of precontracted airway smooth muscle.

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator.

Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4).

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MßCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MßCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

A Density-Functional Study of the Conformational Preference of Acetylcholine in the Neutral Hydrolysis.

Acetylcholine, which is associated with Alzheimer's disease, is widely known to have conformers. The preference of each conformer to undergo neutral hydrolysis is yet to be considered. In this study, we employed density-functional calculations to build the conformers and investigated their preference in one-step neutral hydrolysis. The results showed the preference in ten possible hydrolysis pathways involving seven acetylcholine conformers (reactant), four transition state structures, and two choline conformers (product). Three out of the seven acetylcholine conformers predicted from the results confirmed experimental findings on the conformers stability. We suggested that two out of ten possible pathways were observed in the experimental results based on agreement in reaction energy. Eventually, this study will emphasize the importance of considering acetylcholine conformers in its hydrolysis study.

Kinetics of Chemical Processes in the Human Brain. The Cholinergic Synapse-Mechanisms of Functioning and Control Methods.

In the framework of the kinetic model, the functioning of the cholinergic synapse is considered. The results of mathematical modeling of changes in the level of acetylcholine, induced pH impulse, the influence of the frequency of impulse transmission and inhibition of acetylcholinesterase are presented. Physicochemical explanation for a number of important physiological phenomena, such as neuromuscular paralysis, the molecular mechanism of neurological memory, and actions of nerve poisons and toxins, is given.

An allosteric binding site of the α7 nicotinic acetylcholine receptor revealed in a humanized acetylcholine-binding protein.

Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine-binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the ß8-ß9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel-mediated disorders.

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors.

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT3A receptors (5-HT3ARs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT3AR can be exchanged between the homomeric 5-HT3AR and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT3AR affect the affinity of nicotine, a competitive antagonist of 5-HT3ARs, in a cooperative manner. Using mutant cycle analyses between the 5-HT3A triad, loop A residues Asn-101 and Glu-102, ß9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

Nicotinic receptors in mouse prefrontal cortex modulate ultraslow fluctuations related to conscious processing.

The prefrontal cortex (PFC) plays an important role in cognitive processes, including access to consciousness. The PFC receives significant cholinergic innervation and nicotinic acetylcholine receptors (nAChRs) contribute greatly to the effects of acetylcholine signaling. Using in vivo two-photon imaging of both awake and anesthetized mice, we recorded spontaneous, ongoing neuronal activity in layer II/III in the PFC of WT mice and mice deleted for different nAChR subunits. As in humans, this activity is characterized by synchronous ultraslow fluctuations and neuronal synchronicity is disrupted by light general anesthesia. Both the α7 and ß2 nAChR subunits play an important role in the generation of ultraslow fluctuations that occur to a different extent during quiet wakefulness and light general anesthesia. The ß2 subunit is specifically required for synchronized activity patterns. Furthermore, chronic application of mecamylamine, an antagonist of nAChRs, disrupts the generation of ultraslow fluctuations. Our findings provide new insight into the ongoing spontaneous activity in the awake and anesthetized state, and the role of cholinergic neurotransmission in the orchestration of cognitive functions.

Telmisartan Inhibits Nitric Oxide Production and Vessel Relaxation via Protein Phosphatase 2A-mediated Endothelial NO Synthase-Ser1179 Dephosphorylation.

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser1179 levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser1179 levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser1179 levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser1179 dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.

Synthesis of Novel Nicotinic Ligands with Multimodal Action: Targeting Acetylcholine α4ß2, Dopamine and Serotonin Transporters.

Nicotinic acetylcholine receptors (nAChRs), serotonin transporters (SERT) and dopamine transporters (DAT) represent targets for the development of novel nicotinic derivatives acting as multiligands associated with different health conditions, such as depressive, anxiety and addiction disorders. In the present work, a series of functionalized esters structurally related to acetylcholine and nicotine were synthesized and pharmacologically assayed with respect to these targets. The synthesized compounds were studied in radioligand binding assays at α4ß2 nAChR, h-SERT and h-DAT. SERT experiments showed not radioligand [3H]-paroxetine displacement, but rather an increase in the radioligand binding percentage at the central binding site was observed. Compound 20 showed Ki values of 1.008 ± 0.230 µM for h-DAT and 0.031 ± 0.006 µM for α4ß2 nAChR, and [3H]-paroxetine binding of 191.50% in h-SERT displacement studies, being the only compound displaying triple affinity. Compound 21 displayed Ki values of 0.113 ± 0.037 µM for α4ß2 nAChR and 0.075 ± 0.009 µM for h-DAT acting as a dual ligand. Molecular docking studies on homology models of α4ß2 nAChR, h-DAT and h-SERT suggested potential interactions among the compounds and agonist binding site at the α4/ß2 subunit interfaces of α4ß2 nAChR, central binding site of h-DAT and allosteric modulator effect in h-SERT.