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Transient Receptor Potential (TRP) ion channels like Vanilloid 1 (TRPV1) and Melastatin 3 (TRPM3) are nonselective cation channels expressed in primary sensory neurons and peripheral nerve endings, which are located in cholesterol- and sphingolipid-rich membrane lipid raft regions and have important roles in pain processing. Besides TRP ion channels a wide variety of voltage-gated ion channels were also described in the membrane raft regions of neuronal cells. Here we investigated the effects of lipid raft disruption by methyl-beta-cyclodextrin (MCD) and sphingomyelinase (SMase) on TRPV1, TRPM3 and voltage-gated L-type Ca2+ channel activation in cultured trigeminal neurons and sensory nerve terminals of the trachea. We also examined the mechanism of action of MCD by in silico modeling. Disruption of lipid rafts by MCD or SMase did not alter CIM0216-induced TRPM3 cation channel activation and the voltage-gated L-type Ca2+ channel activation by FPL 64126 or veratridine neither on trigeminal sensory neurons nor sensory nerve terminals. We provided the first structural explanation with in silico modeling that the activation of TRPV1, TRPM3 and voltage-gated L-type Ca2+ channels is affected differently by the cholesterol content surrounding them in the plasma membrane. It is concluded that modifying the hydrophobic interactions between lipid rafts and ion channels might provide a selective novel mechanism for peripheral analgesia. [ABSTRACT FROM AUTHOR]

The vegetal alkaloid toxin veratridine (VTD) is a selective voltage-gated Na+ (NaV) channel activator, widely used as a pharmacological tool in vascular physiology. We have previously shown that NaV 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 NaV 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 NaV channels in parasympathetic neurons and then vasorelaxation in resistance arteries, which could explain arterial hypotension after VTD intoxication. [ABSTRACT FROM AUTHOR]

【Objective】The field control effects of different biogenic pesticides on Empoasca onukii Matsuda (tea green leafhopper) was investigated, with an aim to provide a basis for the rational application of pesticides in tea gardens.【Method】Using a five-point sampling method, based on the investigation of the dynamic occurrence of E. onukii with yellow sticky traps in Zunyi of Guizhou Province, the field control effects of seven different biological pesticides biogenic pesticides spinetoram, azadirachtin and veratrine and their mixture with Metarhizium anisopliae CQMa421 (500-fold dilution) were evaluated, water was used as the control (CK).【Result】The results showed that E. onukii occurred in seven generations in Zunyi area from March to September 2021 (during tea picking period), with severe alternation between generations. And the number of E. onukii reached its peak of 899 in late June. The rise in temperature facilitated the initial reproduction of E. onukii, with the population gradually increasing with the temperature until it reached the peak period. The highest control effect single use of Metarhizium anisopliae CQMa421 against E. onukii was 63.22%, which was achieved 7 d after application. The highest control effects of single use of spinetoram, azadirachtin and veratridine against E. onukii was achieved 3 d after application and were 90.14%, 62.82% and 61.04%, respectively. The control effect of spinetoram, azadirachtin and veratridine mixed with M. anisopliae CQMa421 was higher than that of using CQMa421 only, with control effects at 3 d post-application being 87.12%, 71.41% and 72.86%, respectively. It was established the economic costs required for applying different combinations of pesticides in a 333 m2 tested tea garden, from lowest to highest, were as follows: spinetoram (20 yuan), veratrine (22 yuan), azadirachtin (24 yuan), spinetoram + CQMa421 (35 yuan), veratrine + CQMa421 (36 yuan), azadirachtin + CQMa421 (37 yuan), and CQMa421 (50 yuan).【Conclusion】The effects of single use of bio-pesticides against E. onukii ranked as: spinetoram > M. anisopliae CQMa421 > azadirachtin > veratrine; the effects of the combined use of bio-pesticides with M. anisopliae CQMa421 against E. onukii ranked as: spinetoram > veratrine > azadirachtin. In the actual application of pesticide in tea garden, spinetoram is recommended because of its fast, good and long-lasting control effect as well as low economic cost, and it is an excellent synergist of M. anisopliae CQMa421 to control E. onukii.

Copyright of Guangdong Agricultural Sciences is the property of South China Agricultural University, Guangdong Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Transient Receptor Potential (TRP) ion channels like Vanilloid 1 (TRPV1) and Melastatin 3 (TRPM3) are nonselective cation channels expressed in primary sensory neurons and peripheral nerve endings, which are located in cholesterol- and sphingolipid-rich membrane lipid raft regions and have important roles in pain processing. Besides TRP ion channels a wide variety of voltage-gated ion channels were also described in the membrane raft regions of neuronal cells. Here we investigated the effects of lipid raft disruption by methyl-beta-cyclodextrin (MCD) and sphingomyelinase (SMase) on TRPV1, TRPM3 and voltage-gated L-type Ca2+ channel activation in cultured trigeminal neurons and sensory nerve terminals of the trachea. We also examined the mechanism of action of MCD by in silico modeling. Disruption of lipid rafts by MCD or SMase did not alter CIM0216-induced TRPM3 cation channel activation and the voltage-gated L-type Ca2+ channel activation by FPL 64126 or veratridine neither on trigeminal sensory neurons nor sensory nerve terminals. We provided the first structural explanation with in silico modeling that the activation of TRPV1, TRPM3 and voltage-gated L-type Ca2+ channels is affected differently by the cholesterol content surrounding them in the plasma membrane. It is concluded that modifying the hydrophobic interactions between lipid rafts and ion channels might provide a selective novel mechanism for peripheral analgesia.

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na+ (NaV) channels. In arteries, NaV channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of NaV channels in vascular tone using TTX. We first characterized the expression of NaV channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular NaV channels consist of NaV1.2 channel subtype with NaVβ1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks NaV channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications. [ABSTRACT FROM AUTHOR]

Veratridine (VTD) is a plant neurotoxin that acts by blocking the voltage-gated sodium channels (VGSC) of cell membranes. Symptoms of VTD intoxication include intense nausea, hypotension, arrhythmia, and loss of consciousness. The treatment for the intoxication is mainly focused on treating the symptoms, meaning there is no specific antidote against VTD. In this pursuit, we were interested in studying the molecular interactions of VTD with cyclodextrins (CDs). CDs are supramolecular macrocycles with the ability to form host–guest inclusion complexes (ICs) inside their hydrophobic cavity. Since VTD is a lipid-soluble alkaloid, we hypothesized that it could form stable inclusion complexes with different types of CDs, resulting in changes to its physicochemical properties. In this investigation, we studied the interaction of VTD with β-CD, γ-CD and sulfobutyl ether β-CD (SBCD) by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. Docking and molecular dynamics studies confirmed the most stable configuration for the inclusion complexes. Finally, with an interest in understanding the effects of the VTD/CD molecular interactions, we performed cell-based assays (CBAs) on Neuro-2a cells. Our findings reveal that the use of different amounts of CDs has an antidote-like concentration-dependent effect on the cells, significantly increasing cell viability and thus opening opportunities for novel research on applications of CDs and VTD. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na+ (NaV) channels. In arteries, NaV channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of NaV channels in vascular tone using TTX. We first characterized the expression of NaV channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular NaV channels consist of NaV1.2 channel subtype with NaVβ1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks NaV channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.

Despite considerable advances to improve colorectal cancer (CRC) survival over the last decade, therapeutic challenges remain due to the rapid metastatic dissemination of primary tumors and screening limitations. Meanwhile, the rise of CRC in younger adults (Early-onset CRC), commonly diagnosed with a metastatic form of the disease, shows the pressing need to develop more effective targeted therapies to decrease the high mortality rates associated with metastatic disease. Hyperactivation of the Rictor-mTORC2-AKT signaling pathway drives key metastatic players in diverse malignant tumors, including early- and late-onset colorectal cancer. Selective mTORC2 inhibitors are becoming a potential treatment strategy for CRC due to the therapeutic limitations of mTORC1 inhibitors. Veratridine (VTD), a lipid-soluble alkaloid extracted from Liliaceae plants, can transcriptionally increase UBXN2A, which induces 26S proteasomal degradation of the Rictor protein, a key member in the mTORC2 complex. Destabilization of Rictor protein by VTD decreases Akt phosphorylation on Ser 473 , which is responsible for metastatic signaling downstream of the mTORC2 pathway in diverse malignant tumors. VTD decreases the population of metastatic colon cancer stem cells and functions as an angiogenesis inhibitor. VTD effectively reduces the spheroid growth rate and restricts cell migration. Live cell migration and invasion assays alongside biomechanical-force-based experiments revealed that VTD suppresses colon cancer cell invasiveness and the ensuing risk of tumor metastasis. A CRC mouse model that mimics the natural stages of human sporadic CRC revealed that VTD treatment significantly decreases tumor growth in a UBXN2A-dependent manner. This study showed a novel mechanistic connection between a ubiquitin-like protein and mTORC2-dependent migration and invasion in CRC tumors. This study revealed the therapeutic benefit of selective inhibition of Rictor in CRC, particularly in tumors with a hyperactive Rictor-mTORC2 signaling pathway. Finally, this study opened a new platform for repurposing VTD, a supplemental anti-hypertension molecule, into an effective targeted therapy in CRC tumors., Competing Interests: Declarations Competing Interests The authors declare no competing financial interests.

Functional expression of voltage-gated Na(+) channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug‐induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin‐2 (mot‐2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot‐2 enhances tumor cell invasion and migration. Thus, mot‐2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin‐like protein called UBXN2A in the regulation of mot‐2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull‐down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70‐interacting protein (CHIP)‐dependent ubiquitination of mot‐2. We subsequently showed that UBXN2A increases proteasomal degradation of mot‐2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot‐2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot‐2 in cancer cells. Consistent with the in vitro results, UBXN2A+/− mice exhibited selective elevation of mot‐2 in colon tissues. An in vitro Anti‐K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot‐2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A‐mot‐2 complex in tumors in an azoxymethane/dextran sulfate sodium‐induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot‐2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot‐2‐enriched tumors. This finding validates the UBXN2A‐CHIP axis as a novel and potential therapeutic target in CRC.

Alleviating chronic pain is challenging, due to lack of drugs that effectively inhibit nociceptors without off-target effects on motor or central neurons. Dorsal root ganglia (DRG) contain nociceptive and non-nociceptive neurons. Drug screening on cultured DRG neurons, rather than cell lines, allows for the identification of drugs most potent on nociceptors with no effects on non-nociceptors (as a proxy for unwanted side effects on central nervous system and motor neurons). However, screening using DRG neurons is currently a low-throughput process, and there is a need for assays to speed this process for analgesic drug discovery. We previously showed that veratridine elicits distinct response profiles in sensory neurons. Here, we show evidence that a veratridine-based calcium assay allows for an unbiased and efficient assessment of a drug effect on nociceptors (targeted neurons) and non-nociceptors (nontargeted neurons). We confirmed the link between the oscillatory profile and nociceptors, and the slow-decay profile and non-nociceptors using 3 transgenic mouse lines of known pain phenotypes. We used the assay to show that blockers for Nav1.7 and Nav1.8 channels, which are validated targets for analgesics, affect non-nociceptors at concentrations needed to effectively inhibit nociceptors. However, a combination of low doses of both blockers had an additive effect on nociceptors without a significant effect on non-nociceptors, indicating that the assay can also be used to screen for combinations of existing or novel drugs for the greatest selective inhibition of nociceptors. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms involved in the biogenesis of N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) and N-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca(2+)-activated D-type phosphodiesterase activity. We report here that the de novo biosynthesis of NAPE is enhanced in a Ca(2+)-dependent manner when rat cortical neurons are stimulated with the Ca(2+)-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronal N-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimetic N-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.

Copyright of Xinjiang Agricultural Sciences is the property of Xinjiang Agricultural Sciences Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Graphical abstract Abstract The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Na v 1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Na v 1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Na v 1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel Na v Rh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Na v 1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6. [ABSTRACT FROM AUTHOR]

Purpose/Aim of the study: the retinal relaxing factor (RRF) is an unidentified paracrine factor, which is continuously released from retinal tissue and causes smooth muscle cell relaxation. This study tried to identify the cellular source of the RRF. Furthermore, the possible RRF release by voltage-dependent sodium channel activation and the calcium-dependency of the RRF release were investigated. Materials and methods: mouse femoral arteries were mounted in myograph baths for in vitro isometric tension measurements. The vasorelaxing effect of chicken retinas, which contain no vascular cells, and of solutions incubated with MIO-M1 or primary Müller cell cultures were evaluated. The RRF release of other retinal cells was investigated by using cell type inhibitors. Concentration-response curves of veratridine, a voltage-dependent sodium channel activator, were constructed in the presence or absence of mouse retinal tissue to evaluate the RRF release. The calcium-dependency of the RRF release was investigated by evaluating the vasorelaxing effect of RRF-containing solutions made out of chicken retinas in the absence or presence of calcium. Results: Chicken retinas induced vasorelaxation, whereas solutions incubated with Müller cell cultures did not. Moreover, the gliotoxin DL-α-aminoadipic acid, the microglia inhibitor minocycline, and the tetrodotoxin-resistant voltage-dependent sodium channel 1.8 inhibitor A-803467 could not reduce the RRF-induced relaxation. Concentration-response curves of veratridine were not enlarged in the presence of retinal tissue, and RRF-containing solutions made in the absence of calcium induced a substantial, but reduced vasorelaxation. Conclusions: the RRF is not released from vascular cells and probably neither from glial cells. The retinal cell type that does release the RRF remains unclear. Veratridine does not stimulate the RRF release in mice, and the RRF release in chickens is calcium-dependent as well as calcium-independent. [ABSTRACT FROM AUTHOR]

Abstract Taurine–magnesium coordination compound (TMCC) exhibits antiarrhythmic effects in cesium–chloride-and ouabain-induced arrhythmias; however, the mechanism underlying these effects on arrhythmia remains poorly understood. Here, we investigated the effects of TMCC on aconitine-induced arrhythmia in vivo and the electrophysiological effects of this compound in rat ventricular myocytes in vitro. Aconitine was used to induce arrhythmias in rats, and the dosages required to produce ventricular premature contraction (VPC), ventricular tachycardia (VT), ventricular fibrillation (VF), and cardiac arrest (CA) were recorded. Additionally, the sodium current (I Na ) and L-type calcium current (I Ca,L ) were analyzed in normal and aconitine-treated ventricular myocytes using whole-cell patch-clamp recording. In vivo, intravenous administration of TMCC produced marked antiarrhythmic effects, as indicated by the increased dose of aconitine required to induce VPC, VT, VF, and CA. Moreover, this effect was abolished by administration of sodium channel opener veratridine and calcium channel agonist Bay K8644. In vitro, TMCC inhibited aconitine-induced increases in I Na and I Ca,L. These results revealed that TMCC inhibited aconitine-induced arrhythmias through effects on I Na and I Ca,L. Highlights • TMCC inhibits aconitine-induced arrhythmias in vivo. • TMCC restores aconitine-induced I Na and I Ca,L abnormalities in ventricular myocytes. • Activators of I Na and/or I Ca, L abolish TMCC-induced antiarrhythmic effects. [ABSTRACT FROM AUTHOR]

Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug‐induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin‐2 (mot‐2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot‐2 enhances tumor cell invasion and migration. Thus, mot‐2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin‐like protein called UBXN2A in the regulation of mot‐2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull‐down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70‐interacting protein (CHIP)‐dependent ubiquitination of mot‐2. We subsequently showed that UBXN2A increases proteasomal degradation of mot‐2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot‐2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot‐2 in cancer cells. Consistent with the in vitro results, UBXN2A+/− mice exhibited selective elevation of mot‐2 in colon tissues. An in vitro Anti‐K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot‐2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A‐mot‐2 complex in tumors in an azoxymethane/dextran sulfate sodium‐induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot‐2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot‐2‐enriched tumors. This finding validates the UBXN2A‐CHIP axis as a novel and potential therapeutic target in CRC. [ABSTRACT FROM AUTHOR]

Aims The sperm plasma membrane contains specific ion channels and transporters that initiate changes in Ca 2+ , Na + , K + and H + ions in the sperm cytoplasm. Ion channels are key regulators of the sperm membrane potential, cytoplasmic Ca 2+ and intracellular pH (pH i ), which leads to regulate motility, capacitation, acrosome reaction and other physiological processes crucial for successful fertilization. Expression of epithelial sodium channels (ENaC) and voltage-gated sodium channels (Na v ) in human spermatozoa has been reported, but the role of Na + fluxes sodium channels in the regulation of sperm cell function remains poorly understood. In this context, we aimed to analyze the physiological role of Na v channels in human sperm. Main methods Motility and hyperactivation analysis was conducted by CASA analysis. Flow cytometry and spectrophotometry approaches were carried out to measure Capacitation, Acrosome reaction, immunohistochemistry for Tyr-residues phosporylation, [Ca 2+ ] i levels and membrane potential. Key findings Functional studies showed that veratridine, a voltage-gated sodium channel activator, increased sperm progressive motility without producing hyperactivation while the Na v antagonist lidocaine did induce hyperactivated motility. Veratridine increased protein tyrosine phosphorylation, an event occurring during capacitation, and its effects were inhibited in the presence of lidocaine and tetrodotoxin. Veratridine had no effect on the acrosome reaction by itself, but was able to block the progesterone-induced acrosome reaction. Moreover, veratridine caused a membrane depolarization and modified the effect of progesterone on [Ca 2+ ] i and sperm membrane potential. Significance Our results suggest that veratridine-sensitive Na v channels are involved on human sperm fertility acquisition regulating motility, capacitation and the progesterone-induced acrosome reaction in human sperm. [ABSTRACT FROM AUTHOR]

Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridineevoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF- 05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non-pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery. [ABSTRACT FROM AUTHOR]

Purpose: Disturbances in retinal blood flow are a prominent feature of vision threatening retinal diseases. The regulation of tone in retinal resistance vessels involves the perivascular retinal tissue, but it is unknown to what extent neurons or glial cells contribute to the effect. Therefore, the purpose of the present study was to study the contribution of neurons in the perivascular retina to vascular tone during activation of voltage-gated sodium channels with veratridine and electrical field stimulation (EFS). Methods: Porcine retinal arterioles with and without perivascular tissue were mounted in an isometric myograph system for studying the effects of the voltage-gated sodium channel opener veratridine and EFS on retinal vascular tone. Results: Veratridine induced concentration-dependent relaxation of retinal arterioles which was more pronounced in arterioles with preserved perivascular retinal tissue than in isolated vessels. In the presence of this tissue, veratridine-induced relaxation was inhibited by the voltage-gated sodium channel blocker tetrodotoxin and the nitric oxide synthase inhibitor, Nω-Nitro-L-arginine methyl ester (L-NAME), but was unaffected by the inhibition of the cyclo-oxygenase inbitior ibuprofen and by blocking of adenosine receptors with 8-(p-Sulfophenyl)theophylline hydrate (8-PSPT). Electrical field stimulation induced no changes in retinal vascular tone. Conclusions: Sodium channels of neuronal origin are likely to be involved in the regulation of retinal vascular tone. The lack of effect of EFS on retinal vascular tone may be due to the lack of autonomic nerves in the retina. [ABSTRACT FROM PUBLISHER]

Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patchclamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (τ, ∼2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (τ ∼15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (τ >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when cytoplasmic Na rises and suppresses pump activity when cytoplasmic Na declines. [ABSTRACT FROM AUTHOR]

Key points The role of enteric glial cell activity in the acute regulation of epithelial barrier and secretomotor functions of the intestines under physiological conditions is not clear., We used transgenic mice to modify glial activity and found that enteric glia significantly contribute to the neurogenic ion transport while glial activity does not appear to play a major role in the acute regulation of barrier function., The selective activation of glial activity evoked electrogenic ion transport primarily through neural pathways and was sufficient to drive electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport., These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine and might provide novel therapeutic avenues for functional diarrheal diseases., Abstract Enteric glial cells are often implicated in the regulation of epithelial barrier and secretomotor functions of the intestines. But whether glial cell activity regulates these functions acutely under physiological conditions is not clear. We addressed this issue by using transgenic animal models to modify the activity of enteric glia, either reducing glial expression of connexin 43 in Sox10::CreERT2+/−/Cx43f/f mice or activating glial calcium responses in GFAP::hM3Dq mice, and tested the effects on colonic barrier function and electrogenic ion transport in Ussing chambers. We assessed neuronal-dependent and -independent contributions by activating or inhibiting neurogenic activity with veratridine and tetrodotoxin, respectively. Our results show that the reduction of glial Cx43 expression in Sox10::CreERT2+/−/Cx43f/f mice significantly reduced neurogenic ion transport. The selective glial activation in tissues from GFAP::hM3Dq mice evoked electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport with veratridine and glial driven responses consisted of both tetrodotoxin-sensitive and -insensitive components. The selective glial stimulation did not affect transmural ion conductance or cell-impermeant dye flux but the baseline ion conductance was more variable in Sox10::CreERT2+/−/Cx43f/f tissues. Together, our findings show that glial activity contributes to the regulation of electrogenic ion transport in the intestine through effects on neurons and possibly direct effects on epithelial cells. However, glial activity does not appear to play a major role in the acute regulation of barrier function. These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine. [ABSTRACT FROM AUTHOR]

The aim of our study was to characterize the voltage gated sodium channel Na v 1.8 in bull spermatozoa. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the nonsignificant variations between different ejaculates. Functional characterization was undertaken using A-803467, a selective blocker of Na v 1.8, and veratridine as an opener of the voltage gated sodium channels while molecular characterization was done using western blotting and indirect immunofluorescence assays. In vitro capacitation was induced using heparin, and to study the functional involvement of Na v 1.8 in regulation of capacitation induced hyper sperm motility, A-803467 was used. Selective blocking of Na V 1.8 by A-803467 at 6 and 8 μM concentration significantly (P < 0.05) decreased the forward progressive sperm motility in a time-dependent manner, while, blocking at higher concentrations (10 and 15 μM) resulted in fast forward motility in spermatozoa after 2 h of incubation and it was observed up to 3 h. Treatment of sperm cells with veratridine (6, 8, 10, 15, 20 μM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 4 h. However, hyperactive motility was induced by veratridine at higher concentrations (10 and 15 μM) after 2 h of incubation. In vitro capacitated spermatozoa treated with A-803467 revealed significant (P < 0.05) reduction in forward progressive motility after 2 h of incubation. Both A-803467 and veratridine altered the percentage of spermatozoa showing high mitochondrial transmembrane potential in concentration- and time-dependent manner. High concentrations (10 and 15 μM) of A-803467 and veratridine resulted in bent neck condition in spermatozoa along with significant (P < 0.05) reduction in membrane integrity (HOST negative). Immunoblot revealed the presence of a single protein band of 260 kDa molecular weight along with positive immunoreactivity (IR) in head, neck, middle piece and tail of the spermatozoa. Strongest IR was observed in the neck and middle piece whereas weak IR was observed in tail and acrosomal region of the spermatozoa. Results of our present study evidently revealed the presence of voltage gated sodium channel Na v 1.8 in bull spermatozoa and its functional involvement in regulation of spermatozoa dynamics in terms of motility, membrane integrity, acrosome integrity, capacitation and mitochondrial transmembrane potential. Further studies are warranted to unravel their mechanistic pathways and/or their interaction with other ion channels in regulating sperm dynamics. [ABSTRACT FROM AUTHOR]

Crambescin B carboxylic acid, a synthetic analog of crambescin B, was recently found to inhibit the voltage-sensitive sodium channels (VSSC) in a cell-based assay using neuroblastoma Neuro 2A cells. In the present study, whole-cell patch-clamp recordings were conducted with three heterologously expressed VSSC subtypes, Na v 1.2, Na v 1.6 and Na v 1.7, in a human embryonic kidney cell line HEK293T to further characterize the inhibition of VSSC by crambescin B carboxylic acid. Contrary to the previous observation, crambescin B carboxylic acid did not inhibit peak current evoked by depolarization from the holding potential of −100 mV to the test potential of −10 mV in the absence or presence of veratridine (VTD). In the presence of VTD, however, crambescin B carboxylic acid diminished VTD-induced sustained and tail currents through the three VSSC subtypes in a dose-dependent manner, whereas TTX inhibited both the peak current and the VTD-induced sustained and tail currents through all subtypes of VSSC tested. We thus concluded that crambescin B carboxylic acid does not block VSSC in a similar manner to TTX but modulate the action of VTD, thereby causing an apparent block of VSSC in the cell-based assay. [ABSTRACT FROM AUTHOR]

Carotid bodies (CBs) are secondary sensory receptors in which the sensing elements, chemoreceptor cells, are activated by decreases in arterial PO2 (hypoxic hypoxia). Upon activation, chemoreceptor cells (also known as Type I and glomus cells) increase their rate of release of neurotransmitters that drive the sensory activity in the carotid sinus nerve (CSN) which ends in the brain stem where reflex responses are coordinated. When challenged with hypoxic hypoxia, the physiopathologically most relevant stimulus to the CBs, they are activated and initiate ventilatory and cardiocirculatory reflexes. Reflex increase in minute volume ventilation promotes CO2 removal from alveoli and a decrease in alveolar PCO2 ensues. Reduced alveolar PCO2 makes possible alveolar and arterial PO2 to increase minimizing the intensity of hypoxia. The ventilatory effect, in conjunction the cardiocirculatory components of the CB chemoreflex, tend to maintain an adequate supply of oxygen to the tissues. The CB has been the focus of attention since the discovery of its nature as a sensory organ by de Castro (1928) and the discovery of its function as the origin of ventilatory reflexes by Heymans group (1930). A great deal of effort has been focused on the study of the mechanisms involved in O2 detection. This review is devoted to this topic, mechanisms of oxygen sensing. Starting from a summary of the main theories evolving through the years, we will emphasize the nature and significance of the findings obtained with veratridine and tetrodotoxin (TTX) in the genesis of current models of O2-sensing.

The apical dendrite of a cortical projection neuron (CPN) is generated from the leading process of the migrating neuron as the neuron completes migration. This transformation occurs in the cortical marginal zone (MZ), a layer that contains the Cajal-Retzius neurons and their axonal projections. Cajal-Retzius neurons (CRNs) are well known for their critical role in secreting Reelin, a glycoprotein that controls dendritogenesis and cell positioning in many regions of the developing brain. In this study, we examine the possibility that CRNs in the MZ may provide additional signals to arriving CPNs, that may promote the maturation of CPNs and thus shape the development of the cortex. We use whole embryonic hemisphere explants and multiphoton microscopy to confirm that CRNs display intracellular calcium transients of <1-min duration and high amplitude during early corticogenesis. In contrast, developing CPNs do not show high-amplitude calcium transients, but instead show a steady increase in intracellular calcium that begins at the time of dendritic initiation, when the leading process of the migrating CPN is encountering the MZ. The possible existence of CRN to CPN communication was revealed by the application of veratridine, a sodium channel activator, which has been shown to preferentially stimulate more mature cells in the MZ at an early developmental time. Surprisingly, veratridine application also triggers large calcium transients in CPNs, which can be partially blocked by a cocktail of antagonists that block glutamate and glycine receptor activation. These findings outline a model in which CRN spontaneous activity triggers the release of glutamate and glycine, neurotransmitters that can trigger intracellular calcium elevations in CPNs. These elevations begin as CPNs initiate dendritogenesis and continue as waves in the post-migratory cells. Moreover, we show that the pharmacological blockade of glutamatergic signaling disrupts migration, while forced expression of a bacterial voltage-gated calcium channel (CavMr) in the migrating neurons promotes dendritic growth and migration arrest. The identification of CRN to CPN signaling during early development provides insight into the observation that many autism-linked genes encode synaptic proteins that, paradoxically, are expressed in the developing cortex well before the appearance of synapses and the establishment of functional circuits.

Therapeutic options for amyotrophic lateral sclerosis (ALS) are scarce and controversial. Although the aetiology of neuronal vulnerability is unknown, growing evidence supports a complex network in which multiple toxicity pathways, rather than a single mechanism, are involved in the pathogenesis of ALS. However, most cellular models only explain single pathogenic mechanisms. The present study proposes the two main cytotoxic mechanisms: (1) veratridine (VTD), which induced Na + and Ca 2+ overload; and (2) the TARD DNA-binding protein 43 (TDP-43) in NSC-34 cell line as an in vitro model of ALS. The study was carried out by MTT as an indirect measurement of cell viability and by flow cytometry to determine cell death stages. The impact of Ca 2+ overload combined with TDP-43 overexpression increased early apoptosis of NSC-34 cells. Furthermore, we found that ITH33/IQM9.21 (ITH33) exerted a neuroprotective effect in this model by reducing activation of the apoptotic pathway. Therefore, treatment with VTD in TDP-43 overexpressing NSC-34 cells is a good in vitro ALS model that makes it possible to test new neuroprotective compounds such as ITH33. [ABSTRACT FROM AUTHOR]

Alternatives for the treatment of amyotrophic lateral sclerosis (ALS) are scarce and controversial. The etiology of neuronal vulnerability in ALS is being studied in motor neuron-like NSC-34 cells to determine the underlying mechanisms leading to selective loss of motor neurons. One such mechanism is associated with mitochondrial oxidative stress, Ca overload, and low expression of Ca-buffering proteins. Therefore, in order to elicit neuronal death in ALS, NSC-34 cells were exposed to the following cytotoxic agents: (1) a mixture of oligomycin 10 µM and rotenone 30 µM (O/R), or (2) phenylarsine oxide 1 µM (PAO) (to mimic excess free radical production during mitochondrial dysfunction), and (3) veratridine 100 µM (VTD) (to induce overload of Na and Ca and to alter distribution of Ca-buffering proteins [parvalbumin and calbindin-D28k]). Thus, the aim of the study was to test the novel neuroprotective compound ITH33/IQM9.21 (ITH33) and to compare it with riluzole on in vitro models of neurotoxicity. Cell viability measured with MTT showed that only ITH33 protected against O/R at 3 μM and PAO at 10 μM, but not riluzole. ITH33 and riluzole were neuroprotective against VTD, blocked the maximum peak and the number of [Ca] oscillations per cell, and restored the effect on parvalbumin. However, only riluzole reversed the effect on calbindin-D28k levels. Therefore, ITH33 was neuroprotective against oxidative stress and Na/Ca overload, both of which are involved in ALS. Graphical Abstract: [ABSTRACT FROM AUTHOR]

The in vivo antidote activity of 72 diterpenoid alkaloids isolated from plants of the genera Aconitum, Delphinium, and Consolida and their derivatives was tested against poisoning of mice by a lethal dose of aconitine. The structure-activity relationship was analyzed. The use of diterpenoid alkaloids as versatile antidotes against poisoning of animals and humans by aconitine-, veratridine-, and batrachotoxin-type neurotoxins was justified. [ABSTRACT FROM AUTHOR]

Background Early life adversity ( ELA) is a risk factor for the later-life onset of gastrointestinal ( GI) diseases such as irritable bowel syndrome ( IBS); however, the mechanisms are poorly understood. Here, we utilized a porcine model of ELA, early weaning stress ( EWS), to investigate the influence of ELA on the development and function of the enteric nervous system ( ENS). Methods Female and castrated male (Male-C) piglets were weaned from their sow either at 15 days of age ( EWS) or 28 days of age (late weaning control, LWC). At 60 and 170 days of age, ileal mucosa-submucosa preparations were mounted in Ussing chambers and veratridine- and corticotropin releasing factor (CRF)-releasing factor-evoked short circuit current ( Isc) responses were recorded as indices of secretomotor neuron function. Enteric neuron numbers and the expression of select neurotransmitters and their receptors were also measured. Key Results Compared with LWC pigs, female, but not Male-C EWS, pigs exhibited heightened veratridine-induced I sc responses at 60 and 170 days of age that were inhibited with tetrodotoxin and atropine. Ileum from EWS pigs had higher numbers of enteric neurons that were choline acetyltransferase positive. Markers of increased cholinergic signaling (increased acetylcholinesterase) and downregulated mucosal muscarinic receptor 3 gene expression were also observed in EWS pigs. Conclusions & Inferences This study demonstrated that EWS in pigs induces lasting and sex-specific hypersensitivity of secretomotor neuron function and upregulation of the cholinergic ENS. These findings may represent a mechanistic link between ELA and lifelong susceptibility to GI diseases such as IBS. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the utility of cultured Anopheles gambiae Sua1B cells for insecticide screening applications without genetic engineering or other treatments. Sua1B cells were exposed to the known insecticidal compounds lindane and DIDS, which inhibited cell growth at micromolar concentrations. In patch clamp studies, DIDS produced partial inhibition (69%) of chloride current amplitudes, and an IC 50 of 5.1 μM was determined for Sua1B cells. A sub-set of chloride currents showed no response to DIDS; however, inhibition (64%) of these currents was achieved using a low chloride saline solution, confirming their identity as chloride channels. In contrast, lindane increased chloride current amplitude (EC 50 = 116 nM), which was reversed when cells were bathed in calcium-free extracellular solution. Voltage-sensitive chloride channels were also inhibited by the presence of fenvalerate, a type 2 pyrethroid, but not significantly blocked by type 1 allethrin, an effect not previously shown in insects. Although no evidence of fast inward currents typical of sodium channels was observed, studies with fenvalerate in combination with veratridine, a sodium channel activator, revealed complete inhibition of cell growth that was best fit by a two-site binding model. The high potency effect was completely inhibited in the presence of tetrodotoxin, a specific sodium channel blocker, suggesting the presence of some type of sodium channel. Thus, Sua1B cells express native insect ion channels with potential utility for insecticide screening. [ABSTRACT FROM AUTHOR]

The cardiac late Na + current is generated by a small fraction of voltage-dependent Na + channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na + component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na + current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.

We compare experimental and computational results for the actions of the cardioactive drugs Lidocaine, Verapamil, Veratridine, and Bay K 8644 on a tissue monolayer consisting of mainly fibroblasts and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSc-CM). The choice of the computational models is justified and literature data is collected to model drug action as accurately as possible. The focus of this work is to evaluate the validity and capability of existing models for native human cells with respect to the simulation of pharmaceutical treatment of monolayers and hiPSc-CM. From the comparison of experimental and computational results, we derive suggestions for model improvements which are intended to computationally support the interpretation of experimental results obtained for hiPSc-CM. [ABSTRACT FROM AUTHOR]

Primary cultures of bovine chromaffin cells are considered a good model to evaluate potential neuroprotective compounds for two major reasons: (i) they share many common features to neurons as they synthesize, store, and release neurotransmitters; they are excitable cells that express voltage-dependent calcium, potassium, and sodium channels; they express different neuronal receptor subtypes; and (ii) they can be easily cultured in high quantities from adult animals; as adult para-neurons, they can be used to reproduce different neurodegenerative-like cytotoxicity models. In this chapter, we describe protocols to mimic calcium overload (veratridine and thapsigargin) and oxidative stress (rotenone plus oligomycin-A and 6-hydroxydopamine) to evaluate potential neuroprotective compounds., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)