Cardioprotective effects of alantolactone on isoproterenol-induced cardiac injury and cobalt chloride-induced cardiomyocyte injury

Objectives Alantolactone (AL) is a compound extracted from the roots of Inula Racemosa that has shown beneficial effects in cardiovascular disease. However, the cardioprotective mechanism of AL against hypoxic/ischemic (H/I) injury is still unclear. This research aimed to determine AL’s ability to protect the heart against isoproterenol (ISO)-induced MI injury in vivo and cobalt chloride (CoCl2) induced H/I injury in vitro. Methods Electrocardiography (ECG), lactate dehydrogenase (LDH), creatine kinase (CK), and cardiac troponin I (cTnI) assays in addition to histological analysis of the myocardium were used to investigate the effects of AL in vivo. Influences of AL on L-type Ca2+ current (ICa-L) in isolated rat myocytes were observed by the patch-clamp technique. Furthermore, cell viability, apoptosis, oxidative stress injury, mitochondrial membrane potential, and intracellular Ca2+ concentration were examined in vitro. Results The results indicated that AL treatment ameliorated the morphological and ECG changes associated with MI, and decreased levels of LDH, CK, and cTnI. Furthermore, pretreatment with AL elevated antioxidant enzyme activity and suppressed ROS production. AL prevented H/I-induced apoptosis, mitochondria damage, and calcium overload while reducing ICa-L in a concentration and time dependent fashion. The 50% inhibiting concentration (IC50) and maximal inhibitory effect (Emax) of AL were 17.29 μmol/L and 57.73 ± 1.05%, respectively. Conclusion AL attenuated MI-related injury by reducing oxidative stress, apoptosis, calcium overload, and mitochondria damage. These cardioprotective effects may be related to the direct inhibition of ICa-L.

The Changes of KCNQ5 Expression and Potassium Microenvironment in the Retina of Myopic Guinea Pigs

KCNQ5 is suggestively associated with myopia, but its specific role in the myopic process has not been studied further. The aim of this study was to investigate the expression of potassium channel gene KCNQ5 and the changes of K+ microenvironment within the retina of form deprivation myopia (FDM) guinea pigs. A total of 60 guinea pigs were randomly divided into the normal control (NC) group, the self-control (SC) group, and the form-deprivation (FD) group for different treatments. Molecular assays and immunohistochemistry (IHC) were conducted to measure the expression and distribution of KCNQ5-related gene and protein in the retina. We determined the K+ concentration in the retina. In addition, the possible effects of form deprivation on potassium ionic currents and the pharmacological sensitivity of KCNQ5 activator Retigabine and inhibitor XE991 to the M-current in RPE cells were investigated using the patch-clamp technique. As a result, FD eyes exhibited more myopic refraction and longer AL. The mRNA and protein levels of KCNQ5 significantly decreased in the FD eyes, but the K+ concentration increased. In addition, the M-type K+ current [IK(M)] density decreased in FD RPE cells, and were activated or inhibited in a concentration-dependent manner due to the addition of Retigabine or XE991. Overall, KCNQ5 was significantly downregulated in the retina of FD guinea pigs, which may be associated with the increasing K+ concentration, decreasing IK(M) density, and elongating ocular axis. It suggested that KCNQ5 may play a role in the process of myopia, and the intervention of potassium channels may contribute to the prevention and control of myopia.

GPER/β-Alanine Positive Interaction in The Dorsal Root Ganglion Uncovers Potential Mechanisms: Mediating Continuous Neuronal Sensitisation and Neuroinflammation Responses in Neuropathic Pain

Background: The pathogenesis of neuropathic pain and the reasons for the prolonged unhealing are still unknown. Increasing evidence suggests that estrogen sex differences play a role in pain sensitivity, but few studies focused on the role of estrogen receptor which maybe an important molecular component contributing to peripheral pain transduction. We aimed to investigate the impact of oestrogen receptors in nociceptive neuronal response in the dorsal root ganglion (DRG) and spinal dorsal horn using a spared nerve injury (SNI) rat model of chronic pain. Methods: We used a class of estrogen receptors antagonists and agonists intrathecal (i.t.) administrated to male rats with SNI or normal rats to identify the main receptor. Moreover, we applied genes identified through genomic metabolic analysis to determine the key metabolism point and elucidate potential mechanisms mediating continuous neuronal sensitisation and neuroinflammation responses in neuropathic pain. The excitability of DRG neurons was detected using the patch clamp technique. Immunohistochemistry, Western blotting, qPCR and behavioral testing were used to assess the expressions, cellular distributions, and actions of main receptor and its related signaling molecules.Results: Increasing the expression and function of G protein-coupled estrogen receptor (GPER), but not estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), in the DRG, but not the dorsal spinal cord, contributed to SNI-induced neuronal sensitisation. Inhibiting GPER expression in the DRG alleviated SNI-induced pain behaviours and neuroinflammation by downregulating IL-1β and IL-6 expression as well as restoring GABAα2 expression simultaneously. Additionally, the positive interaction between GPER and β-alanine, β-alanine accumulation enhances pain sensation and promotes chronic pain development. Conclusion: GPER activation in the DRG causes a positive interaction of β-alanine with IL-1β and IL-6 expression and represses GABAα2 involved in post-SNI neuropathic pain development. Blocking GPER and eliminating β-alanine in the DRG may prevent neuropathic pain development.

A Comparison of the Effect of Lead (Pb) on the Slow Vacuolar (SV) and Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.

Cyclodextrins Exert a Ligand-like Current Inhibitory Effect on the KV1.3 Ion Channel Independent of Membrane Cholesterol Extraction

Cyclodextrins (CDs) are cyclic oligosaccharides capable of forming water-soluble complexes with a variety of otherwise poorly soluble molecules including cholesterol and different drugs. Consistently, CDs are widely used in research and clinical practice to deplete cholesterol from cellular membranes or to increase solubility and bioavailability of different pharmaceuticals at local concentrations in the millimolar range. Effects of CDs exerted on cellular functions are generally thought to originate from reductions in cholesterol levels. Potential direct, ligand-like CD effects are largely neglected in spite of several recent studies reporting direct interaction between CDs and proteins including AMP-activated protein kinase, β-amyloid peptides, and α-synuclein. In this study, by using patch-clamp technique, time-resolved quantitation of cholesterol levels and biophysical parameters and applying cholesterol-extracting and non-cholesterol-extracting CDs at 1 and 5 mM concentrations, we provide evidence for a previously unexplored ligand-like, cholesterol-independent current inhibitory effect of CDs on KV1.3, a prototypical voltage-gated potassium channel with pathophysiological relevance in various autoimmune and neurodegenerative disorders. Our findings propose that potential direct CD effects on KV channels should be taken into consideration when interpreting functional consequences of CD treatments in both research and clinical practice. Furthermore, current-blocking effects of CDs on KV channels at therapeutically relevant concentrations might contribute to additional beneficial or adverse effects during their therapeutic applications.

Investigation of Novel Small Molecular TRPM4 Inhibitors in Colorectal Cancer Cells

(1) Background: Transient receptor potential melastatin (TRPM4) ion channel aberrant expression or malfunction contributes to different types of cancer, including colorectal cancer (CRC). However, TRPM4 still needs to be validated as a potential target in anti-cancer therapy. Currently, the lack of potent and selective TRPM4 inhibitors limits further studies on TRPM4 in cancer disease models. In this study, we validated novel TRPM4 inhibitors, CBA, NBA, and LBA, in CRC cells. (2) Methods: The potency to inhibit TRPM4 conductivity in CRC cells was assessed with the whole-cell patch clamp technique. Furthermore, the impact of TRPM4 inhibitors on cellular functions, such as viability, proliferation, and cell cycle, were assessed in cellular assays. (3) Results: We show that in CRC cells, novel TRPM4 inhibitors irreversibly block TRPM4 currents in a low micromolar range. NBA decreases proliferation and alters the cell cycle in HCT116 cells. Furthermore, NBA reduces the viability of the Colo205 cell line, which highly expresses TRPM4. (4) Conclusions: NBA is a promising new TRPM4 inhibitor candidate, which could be used to study the role of TRPM4 in cancer disease models and other diseases.

A Facile Approach to Bis(isoxazoles), Promising Ligands of the AMPA Receptor

A convenient synthetic approach to novel functionalized bis(isoxazoles), the promising bivalent ligands of the AMPA receptor, was elaborated. It was based on the heterocyclization reactions of readily available electrophilic alkenes with the tetranitromethane-triethylamine complex. The structural diversity of the synthesized compounds was demonstrated. In the electrophysiological experiments using the patch clamp technique on Purkinje neurons, the compound 1,4-phenylenedi(methylene)bis(5-aminoisoxazole-3-carboxylate) was shown to be highly potent positive modulator of the AMPA receptor, potentiating kainate-induced currents up to 70% at 10−11 M.

Formation and function of bacterial outer membrane proteins- incorporated liposomes using an in vitro translation system

Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a β-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from E. coli.. Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes, with various lipid compositions, was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.

Block of Voltage-Gated Sodium Channels as a Potential Novel Anti-cancer Mechanism of TIC10

Background: Tumor therapeutics are aimed to affect tumor cells selectively while sparing healthy ones. For this purpose, a huge variety of different drugs are in use. Recently, also blockers of voltage-gated sodium channels (VGSCs) have been recognized to possess potentially beneficial effects in tumor therapy. As these channels are a frequent target of numerous drugs, we hypothesized that currently used tumor therapeutics might have the potential to block VGSCs in addition to their classical anti-cancer activity. In the present work, we have analyzed the imipridone TIC10, which belongs to a novel class of anti-cancer compounds, for its potency to interact with VGSCs.Methods: Electrophysiological experiments were performed by means of the patch-clamp technique using heterologously expressed human heart muscle sodium channels (hNav1.5), which are among the most common subtypes of VGSCs occurring in tumor cells.Results: TIC10 angular inhibited the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state was weak with an extrapolated Kr of about 600 μM. TIC10 most probably did not interact with fast inactivation. In protocols for slow inactivation, a half-maximal inhibition occurred around 2 µM. This observation was confirmed by kinetic studies indicating that the interaction occurred with a slow time constant. Furthermore, TIC10 also interacted with the open channel with an affinity of approximately 4 µM. The binding site for local anesthetics or a closely related site is suggested as a possible target as the affinity for the well-characterized F1760K mutant was reduced more than 20-fold compared to wild type. Among the analyzed derivatives, ONC212 was similarly effective as TIC10 angular, while TIC10 linear more selectively interacted with the different states.Conclusion: The inhibition of VGSCs at low micromolar concentrations might add to the anti-tumor properties of TIC10.

Molecular and functional characterization of the mouse intracardiac nervous system

Background: The intracardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate to the neuronal regulation of cardiac functions and are involved in the initiation of cardiac arrhythmias. Therefore, deciphering the role of the ICNS in cardiac physiology and physiopathology is mandatory. Whereas transgenic mouse models represent powerful tools to reach this goal, the mouse ICNS is still poorly characterized. Objective: The objective of the present study was to provide a phenotypic, electrophysiological and pharmacological characterization of the mouse ICNS. Methods: Global cardiac innervation and phenotypic diversity was investigated by performing immunohistochemistry on cleared murine heart and on tissue sections. Patch clamp technique was used for electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons. Results: We identified the expression of 7 distinct neuronal markers within mouse intracardiac neurons demonstrating the neurochemical diversity of this network. Of note, we described for the first time in mouse, the existence of neuron expressing the calcium binding protein calbindin, the neuropeptide Y (NPY) and the cocain and amphetamine regulated transcript (CART) peptide. Electrophysiological studies also revealed the existence of two different neuronal population based on their electrical behavior. Finally, we demonstrated that these neurons can be modulated by several neuromodulators. Conclusion: This study demonstrated that mouse ICNS shares similar molecular and functional complexity to that of other species and therefore is a suitable model to decipher the role of individual neuronal subtypes in the modulation of cardiac function and in the initiation of cardiac arrhythmias.