Inhibition Of Voltage-gated Calcium Channels Research Articles

A Potent Voltage-gated Calcium Channel Inhibitor Engineered from a Nanobody Targeted to Auxiliary CaVβ Subunits

Inhibiting high-voltage-activated calcium channels (HVACCs; CaV1/CaV2) is therapeutic for myriad cardiovascular and neurological diseases. For particular applications, genetically-encoded HVACC blockers may enable channel inhibition with greater tissue-specificity and versatility than is achievable with small molecules. Here, we engineered a genetically-encoded HVACC inhibitor by first isolating an immunized llama nanobody (nb.F3) that binds auxiliary HVACC CaVβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to CaVβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (CaV-aβlator), an E3 ubiquitin ligase, ablated currents from diverse reconstituted HVACCs, and from endogenous CaV1/CaV2 channels in cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, CaV-aβlator redistributed CaV1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces CaV-aβlator as a potent genetically-encoded HVACC inhibitor, and describes a general approach that can be broadly adapted to generate versatile modulators for macro-molecular membrane protein complexes.

Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway

Nifedipine is a voltage-gated calcium channel inhibitor widely used in the treatment of hypertension. Nifedipine has been reported to have antioxidant and anti-apoptotic effects and promotes cell proliferation. However, the effects of nifedipine on oxidative stress and apoptosis in osteoarthritic (OA) chondrocytes are still unclear. In this study, we sought to investigate whether nifedipine alleviates oxidative stress and apoptosis in OA through nuclear factor erythroid-2-related factor 2 (Nrf2) activation. The cytotoxicity of nifedipine against human chondrocytes was detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, whereas mRNA and protein expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The oxidative stress level was analyzed by measuring reactive oxygen species (ROS), glutathione peroxidase (GSH-px), catalase (CAT) and superoxide dismutase (SOD) activities. The role of Nrf2 in the effect of nifedipine on OA was analyzed using an Nrf2 inhibitor brusatol (BR). The result showed that nifedipine inhibited the expression of matrix metalloprotein(MMP)-13, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, inducible nitric oxide (NO) synthase (iNOS), and prostaglandin E2 (PGE2), as well as reduced ROS production in human OA chondrocytes, which was partially reversed by BR. Nifedipine prevented cartilage degeneration and contributed to the expression of Nrf-2 in chondrocytes. These results indicate that nifedipine inhibited inflammation and oxidative stress in chondrocytes via activation of Nrf-2/HO-1 signaling.

Gabapentinoid prescription in oral medicine and oral surgery practice. Part I − Experience from a French Orofacial pain clinic

Gabapentinoids, pregabalin and gabapentin, are neuronal voltage-gated calcium channel inhibitors mainly prescribed for the treatment of partial epilepsy and neuropathic pain. Although their efficacy as first-line treatments for painful neuropathic conditions (and several non-neuropathic painful conditions) has been well established in general medical practice, their efficacy and prescription in Oral Medicine and Oral Surgery practice has received little attention so far. This didactic article, the first of a two-part series, aims to present the experience of a French tertiary orofacial pain clinic regarding the prescription of gabapentinoids in Orofacial pain conditions that fall within the scope of the oral surgeon's practice.

Gabapentinoid prescription in Oral Medicine and Oral Surgery practice. Part II − a systematic scoping review of the literature

Introduction: Gabapentinoids, pregabalin and gabapentin, neuronal voltage-gated calcium channel inhibitors are first-line treatments for painful neuropathic conditions (and several non-neuropathic painful conditions). Nevertheless, their efficacy and prescription in Oral Medicine and Oral Surgery practice has received little attention so far. A previous article, the first of a two-part series, presented the experience of a French tertiary orofacial pain clinic regarding the prescription of gabapentinoids in orofacial conditions. This second article aimed to explore the scientific literature on the subject. Material and methods: A systematic scoping review was conducted on multiple relevant databases (MEDLINE®, Cochrane®, Agence Nationale de Sécurité du Médicament et des produits de santé, Haute Autorité de Santé) and journal archives (JOMOS, JSOMFS) to assess the indications, non-indications and contraindications of gabapentinoids in an Oral Medicine/Oral Surgery context. Results: Out of 131 records selected during the initial screening, 34 matched the inclusion criteria and were used for subsequent analyses. Gabapentinoids were prescribed in three clinical contexts: orofacial pain management (32 studies), anxiolysis (1 study) and prevention of postoperative nausea/vomiting (1 study), with variable quality of evidence: high (6 studies), moderate (3 studies), low (5 studies) and very low (20 studies) quality studies (GRADE scale). Untoward effects of gabapentinoids were reported in 16 studies, mainly neurological (vertigo, drowsiness, sedation) and gastro-intestinal (nausea, vomiting, diarrhea, constipation). Gabapentinoids were ineffective in preemptive and postoperative analgesia and for the management of mucositis-related pain. Discussion: There is some evidence supporting the use of gabapentinoids in Oral Medicine/Oral Surgery in adherence with current practices observed in France and other countries (practices often extrapolated from their use in other non-orofacial painful conditions). The methodological quality of the studies included in this scoping review is often poor and publication bias is most probable in this field. Therefore, any conclusion drawn from such studies must be subject to circumspection. Conclusion: Data obtained from the present scoping review suggests the potential use of gabapentinoids as second-line treatments for anxiolysis, prevention of postoperative nausea/vomiting and the management of trigeminal neuralgia and masticatory myalgia. Other potential indications of gabapentinoids in Oral Medicine/Oral Surgery practice include cranial neuralgias, post-traumatic trigeminal neuropathies, first bite syndrome, burning mouth syndrome and migraine prophylaxis, when other treatment options are inefficient or unavailable.

L-type voltage-gated calcium channel regulation of in vitro human cortical neuronal networks

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here, we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days, inhibitory GABAergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that, although basal neuronal firing rate is unaffected, there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening.

A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary CaVβ subunits.

Inhibiting high-voltage-activated calcium channels (HVACCs; CaV1/CaV2) is therapeutic for myriad cardiovascular and neurological diseases. For particular applications, genetically-encoded HVACC blockers may enable channel inhibition with greater tissue-specificity and versatility than is achievable with small molecules. Here, we engineered a genetically-encoded HVACC inhibitor by first isolating an immunized llama nanobody (nb.F3) that binds auxiliary HVACC CaVβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to CaVβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (CaV-aβlator), an E3 ubiquitin ligase, ablated currents from diverse HVACCs reconstituted in HEK293 cells, and from endogenous CaV1/CaV2 channels in mammalian cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, CaV-aβlator redistributed CaV1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces CaV-aβlator as a potent genetically-encoded HVACC inhibitor, and describes a general approach that can be broadly adapted to generate versatile modulators for macro-molecular membrane protein complexes.

Retinoid receptor-based signaling plays a role in voltage-dependent inhibition of invertebrate voltage-gated Ca2+ channels

The retinoic acid receptor (RAR) and retinoid X receptor (RXR) mediate the cellular effects of retinoids (derivatives of vitamin A). Both RAR and RXR signaling events are implicated in hippocampal synaptic plasticity. Furthermore, retinoids can interact with calcium signaling during homeostatic plasticity. We recently provided evidence that retinoids attenuate calcium current (ICa) through neuronal voltage-gated calcium channels (VGCCs). We now examined the possibility that constitutive activity of neuronal RXR and/or RAR alters calcium influx via the VGCCs. We found that in neurons of the mollusk Lymnaea stagnalis, two different RXR antagonists (PA452 and HX531) had independent and opposing effects on ICa that were also time-dependent; whereas the RXR pan-antagonist PA452 enhanced ICa, HX531 reduced ICa Interestingly, this effect of HX531 occurred through voltage-dependent inhibition of VGCCs, a phenomenon known to influence neurotransmitter release from neurons. This inhibition appeared to be independent of G proteins and was largely restricted to Cav2 Ca2+ channels. Of note, an RAR pan-antagonist, LE540, also inhibited ICa but produced G protein-dependent, voltage-dependent inhibition of VGCCs. These findings provide evidence that retinoid receptors interact with G proteins in neurons and suggest mechanisms by which retinoids might affect synaptic calcium signaling.

Inactivation Regulates RGK-Mediated Inhibition of Voltage-Gated Calcium Channels

Voltage-gated Ca2+ channels (VGCC) directly control muscle contraction and neurotransmitter release, and slower processes such as cell differentiation, migration, and death. They are potently inhibited by RGK GTP-ases (Rem1, Rem2, Rad and Gem/Kir), which decrease Ca2+ channel membrane expression, as well as directly inhibit membrane-resident channels. The mechanisms of membrane-resident channel inhibition are difficult to study because RGK-overexpression causes complete or near complete channel inhibition. Using titrated levels of RGK expression in Xenopus oocytes that inhibit WT P/Q-type calcium channels by ∼50%, we show that inhibition depends on channel inactivation. Interestingly, fast-inactivating channels, including Familial Hemiplegic Migraine mutants, are more potently inhibited than WT channels, while slow-inactivating channels, such as those expressed with the β2a auxiliary subunit, are spared. We found similar results in L-type channels, and, remarkably, an insensitivity of Timothy Syndrome mutants to RGK inhibition. Further results suggest that RGKs slow channel recovery from inactivation and identify RGKs as potential modulating factors in channelopathies. Finally, our results confirm a previously proposed immobilization of calcium channel voltage sensors by Rad, which we were now able to observe in electrophysiological experiments with titrated levels of Rad. Thus, RGK-mediated inhibition is a much subtler affair than previously thought, and physiological conditions can control the levels of inhibition.

(-)-Hardwickiic Acid and Hautriwaic Acid Induce Antinociception via Blockade of Tetrodotoxin-Sensitive Voltage-Dependent Sodium Channels.

For an affliction that debilitates an estimated 50 million adults in the United States, the current chronic pain management approaches are inadequate. The Centers for Disease Control and Prevention have called for a minimization in opioid prescription and use for chronic pain conditions, and thus, it is imperative to discover alternative non-opioid based strategies. For the realization of this call, a library of natural products was screened in search of pharmacological inhibitors of both voltage-gated calcium channels and voltage-gated sodium channels, which are excellent targets due to their well-established roles in nociceptive pathways. We discovered (-)-hardwickiic acid ((-)-HDA) and hautriwaic acid (HTA) isolated from plants, Croton californicus and Eremocarpus setigerus, respectively, inhibited tetrodotoxin-sensitive sodium, but not calcium or potassium, channels in small diameter, presumptively nociceptive, dorsal root ganglion (DRG) neurons. Failure to inhibit spontaneous postsynaptic excitatory currents indicated a preferential targeting of voltage-gated sodium channels over voltage-gated calcium channels by these extracts. Neither compound was a ligand at opioid receptors. Finally, we identified the potential of both (-)-HDA and HTA to reverse chronic pain behavior in preclinical rat models of HIV-sensory neuropathy, and for (-)-HDA specifically, in chemotherapy-induced peripheral neuropathy. Our results illustrate the therapeutic potential for (-)-HDA and HTA for chronic pain management and could represent a scaffold, that, if optimized by structure-activity relationship studies, may yield novel specific sodium channel antagonists for pain relief.

Doses of Pregabalin that Decrease Ethanol Intake Potentiate the Disruptive Effects of Ethanol on Learning in Outbred Rats

Alcohol abuse is a serious public health concern, with over three million Americans suffering from an alcohol use disorder annually. Currently available therapeutics for treating alcohol use disorder have limited clinical effectiveness; as such, there is a pressing need for new treatments. Pregabalin (Lyrica®), a voltage‐gated calcium channel inhibitor, has garnered interest as a potential therapeutic to help reduce alcohol intake and maintain abstinence in patients with alcohol use disorder. However, the data supporting such an indication are lacking and there do not appear to be any published studies investigating whether pregabalin can alter the acute effects of alcohol on learning and memory. The purpose of this study was twofold: 1) determine doses of pregabalin that decrease ethanol‐ and food‐maintained responding, and 2) test the hypothesis that doses of pregabalin that decrease ethanol intake will also alter the disruptive effects of ethanol on complex acquisition and performance behavior. Two separate groups of adult Long‐Evans rats were used for these studies. The first group of rats (n=9) responded under a multiple fixed‐ratio 10, variable‐interval 80‐sec schedule of ethanol (32% v/v) and food (45‐mg pellets) reinforcement, respectively. The second group (n=12) responded under a multiple schedule of repeated acquisition and performance of response sequences in which responding in both components was maintained by food reinforcement under a second‐order fixed‐ratio 3 schedule. In the first group, 18 – 180 mg/kg (i.p.) of pregabalin dose‐dependently decreased both ethanol‐ and food‐maintained responding, as well as post‐session blood ethanol concentrations; however, food‐maintained responding was more potently decreased than ethanol‐maintained responding. This lack of selectivity of pregabalin for reducing ethanol intake was further complicated by the effects of pregabalin and ethanol on the acquisition and performance task. In the second group, ethanol alone (0.56 – 2.37 g/kg, i.p.) dose‐dependently decreased response rates and increased percent errors in both the acquisition and performance components, although acquisition was more sensitive to disruption than performance. Furthermore, when doses of pregabalin (18 – 100 mg/kg, i.p.) that had no effect alone under this baseline were administered prior to ethanol, the dose‐effect curves for both response rate and accuracy were shifted leftward (i.e., smaller doses of ethanol were needed to decrease response rates and increase percent errors). These findings indicate that pregabalin can decrease ethanol intake, but that these doses will likely decrease responding for other reinforcers and potentiate the disruptive effects of ethanol on complex acquisition and performance behaviors. As such, pregabalin might not be useful in treating patients with alcohol use disorder even if they are already abstinent from alcohol because there is an increased risk of negative health consequences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Attenuation of Vascular Contractility in Metastatic Breast Cancer Mice

Breast cancer (BC) is the one of crucial health burden among women worldwide. Even though the alteration of vascular function were observed by few researchers in metastatic BC, the changes in vascular reactivity and its underlying mechanism were not fully understood. Here, we investigate the physiological changes of vascular function from isolated mesenteric arteries in metastatic breast cancer mice model. In this study, 105 of 4T1 murine BC cells were injected into the subcutaneous or tail vein of female Balb‐c mice aged 8 weeks old. Mice were randomly divided into the following group: 1) control; n=10, 2) non‐metastatic model (4T1; n=10), 3) tumor‐bearing model (4T1M; n=10), and 4) tail vein injection model (4T1ML; n=10). Mesenteric arteries were removed after 4 weeks of tail vein injection group and 5 weeks of tumor‐bearing groups. Isometric tension was investigated in response to various vasoconstrictors such as phenylephrine (PhE), Serotonin (5‐HT), Angiotensin II (ANG II) and high potassium chloride (HK). Our results showed that PhE‐induced vasoconstriction was no significant differences in the non‐metastatic group compared to control. However, the attenuated vasoconstriction in response to PhE, 5‐HT, and High K was found in metastatic cancer groups. Application of L‐type voltage‐gated calcium channel inhibitor (Nifedipine) induced vascular relaxation with PhE‐induced contraction in each group, but there were no significant differences among groups. Interestingly, PhE‐induced contraction in metastatic groups was not inhibited by Rho kinase inhibitor (Y‐27632), but not in control. From above results, it is suggested that decreased Rho kinase activity might be attributable to attenuated vasoconstriction in mesenteric artery from advanced breast cancer mice.Support or Funding InformationThis study was supported by a National Research Foundation of Korea grant funded by Ministry of Science, ICT, & Future Planning (2015R1C1A1A01054038).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suramin-Induced Neurotoxicity: Preclinical Models and Neuroprotective Strategies.

Suramin is a trypan blue analogon originally developed to treat protozoan infections, which was found to have diverse antitumor effects. One of the most severe side effects in clinical trials was the development of a peripheral sensory-motor polyneuropathy. In this study, we aimed to investigate suramin-induced neuropathy with a focus on calcium (Ca2+) homeostasis as a potential pathomechanism. Adult C57Bl/6 mice treated with a single injection of 250 mg/kg bodyweight suramin developed locomotor and sensory deficits, which were confirmed by electrophysiological measurements showing a predominantly sensory axonal-demyelinating polyneuropathy. In a next step, we used cultured dorsal root ganglia neurons (DRGN) as an in vitro cell model to further investigate underlying pathomechanisms. Cell viability of DRGN was significantly decreased after 24-hour suramin treatment with a calculated IC50 of 283 µM. We detected a suramin-induced Ca2+ influx into DRGN from the extracellular space, which could be reduced with the voltage-gated calcium channel (VGCC) inhibitor nimodipine. Co-incubation of suramin and nimodipine partially improved cell viability of DRGN after suramin exposure. In summary, we describe suramin-induced neurotoxic effects on DRGN as well as potentially neuroprotective agents targeting intracellular Ca2+ dyshomeostasis.

A Phase I, Randomized, Double-Blind, Laser-Evoked Potential Study to Evaluate the Analgesic/Antihyperalgesic Effect of ASP9226, a State-Dependent N-Type Voltage-Gated Calcium Channel Inhibitor, in Healthy Male Subjects.

ObjectiveEvaluate the analgesic/antihyperalgesic effects of ASP9226, a state-dependent N-type voltage-gated calcium channel inhibitor, in healthy male subjects.DesignRandomized, double-blind, double-dummy, placebo- and active comparator–controlled crossover study.SettingHPR Dr. Schaffler GmbH, Munich, Germany.SubjectHealthy male subjects aged 18–55 years.MethodsTwenty-four eligible subjects were randomly assigned to one of four treatment sequences and received single doses of ASP9226 (30 mg or 50 mg), pregabalin (150 mg), or placebo during four treatment periods. Laser-evoked potentials (LEP) and postlaser pain visual analog scales (VAS) on capsaicin-treated skin were assessed during main assessment days (the first day of each study period). Primary and secondary end points were the differences in LEP N2-P2 peak-to-peak (PtP) amplitudes and VAS score, respectively, in all subjects.ResultsOverall, treatment with pregabalin resulted in a significantly lower LEP N2-P2 PtP amplitude vs placebo (–3.30 μV, P < 0.0001). There were no clinically relevant differences in N2-P2 PtP amplitudes between placebo and either ASP9226 dose (–0.31 μV and –0.27 μV). Furthermore, subjects reported significantly lower VAS pain scores with pregabalin vs placebo (–9.90%, P < 0.0001) in contrast to ASP9226 30 mg (–2.1%) and ASP9226 50 mg (1.2%) vs placebo. Subgroup analysis of LEP and VAS pain in participants with positive prestudy capsaicin response (n = 13) were in keeping with results in all subjects.ConclusionsASP9226 was well tolerated; however, there was no improvement in LEP and VAS pain scores with ASP9226 at either dose vs placebo.

Essential oil of lavender in anxiety disorders: Ready for prime time?

Anxiety disorders are some of the most common psychiatric disorders, with potentially debilitating consequences on individual function. Existing pharmacotherapies for anxiety disorders are limited by delay to therapeutic effect, dependence, tolerance, withdrawal, and abuse potential. Therefore, safe and evidence-based complementary or alternative therapies may be important allies in the care of patients with anxiety disorders. Essential oils are lipophilic and concentrated botanical extracts that exhibit many properties of drugs, although they are not Food and Drug Administration approved and have limitations characteristic of herbal preparations. Lavender essential oil has an extensive anecdotal history of anxiolytic benefit that has recently been supported by clinical efficacy studies. The 2 primary terpenoid constituents of lavender essential oil, linalool and linalyl acetate, may produce an anxiolytic effect in combination via inhibition of voltage-gated calcium channels, reduction of 5HT1A receptor activity, and increased parasympathetic tone. The objectives of this article are to provide a brief overview of lavender oil in aromatherapy, explore variability in the constituents of lavender oil, summarize its pharmacology and safety profile, as well as describe its body of research that has been conducted for anxiety.

Quantum dots modulate intracellular Ca2+ level in lung epithelial cells.

While adverse effects of nanoparticles on lung health have previously been proposed, few studies have addressed the direct effects of nanoparticle exposure on the airway epithelium. In this work, we examine the response of the pulmonary airway to nanoparticles by measuring intracellular Ca2+ concentration ([Ca2+]i) in the Calu-3 epithelial layer stimulated by 3-mercaptopropionic-acid (3MPA) coated CdSe-CdS/ZnS core-multishell quantum dots (QDs). Simultaneous transient transepithelial electrical resistance (TEER) decrease and global [Ca2+]i increase in Calu-3 epithelial layer, accompanied by cell displacements, contraction, and expansion, were observed under QD deposition. This suggests that a QD-induced global [Ca2+]i increase in the Calu-3 epithelial layer caused the transient TEER decrease. The [Ca2+]i increase was marked and rapid in the apical region, while [Ca2+]i decreased in the basolateral region of the epithelial layer. TEER transient response and extracellular Ca2+ entry induced by QD deposition were completely inhibited in cells treated with stretched-activated (SA) inhibitor GdCl3 and store-operated calcium entry (SOCE) inhibitor BTP2 and in cells immersed in Ca2+-free medium. The voltage-gated calcium channel (VGCC) inhibitor nifedipine decreased, stabilized, and suppressed the TEER response, but did not affect the [Ca2+]i increase, due to QD deposition. This demonstrates that the Ca2+ influx activated by QDs’ mechanical stretch occurs through activation of both SA and SOCE channels. QD-induced [Ca2+]i increase occurred in the Calu-3 epithelial layer after culturing for 15 days, while significant TEER drop only occurred after 23 days. This work provides a new perspective from which to study direct interactions between airway epithelium and nanoparticles and may help to reveal the pathologies of pulmonary disease.

Pre-clinical evaluation of voltage-gated calcium channel blockers derived from the spider P. nigriventer in glioma progression

This study investigated the effects of P/Q- and N-type voltage-gated calcium channel (VGCC) blockers derived from P. nigriventer in glioma progression, by means of in vitro and in vivo experiments. Glioma cells M059J, U-138MG and U-251MG were used to evaluate the antiproliferative effects of P/Q- and N-type VGCC inhibitors PhTx3-3 and Phα1β from P. nigriventer (0.3–100 pM), in comparison to MVIIC and MVIIA from C. magus (0.3–100 pM), respectively. The toxins were also analyzed in a glioma model induced by implantation of GL261 mouse cells. PhTx3-3, Phα1β and MVIIA displayed significant inhibitory effects on the proliferation and viability of all tested glioma cell lines, and evoked cell death mainly with apoptosis characteristics, as indicated by Annexin V/propidium iodide (PI) positivity. The antiproliferative effects of toxins were confirmed by flow cytometry using Ki67 staining. None of the tested toxins altered the proliferation rates of the N9 non-tumor glial cell line. Noteworthy, the administration of the preferential N-type VGCC inhibitors, Phα1β (50 pmol/site; i.c.v.), its recombinant form CTK 01512-2 (50 pmol/site; i.c.v. and i.t.), or MVIIA (10 pmol/site; i.c.v.) caused significant reductions of tumor areas in vivo. N-type VGCC inhibition by Phα1β, CTK 01512-2, and MVIIA led to a marked increase of GFAP-activated astrocytes, and Iba-1-positive microglia, in the peritumoral region, which might explain, at least in part, the inhibitory effects of the toxins in tumor development. This study provides novel evidence on the potential effects of P. nigriventer-derived P/Q-, and mainly, N-type VGCC inhibitors, in glioma progression.

Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed.The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs).Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells.To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner.The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive.Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle [3x/day, 2x], imatinib [100 mg/kg, 2x/day], TET [150 mg/kg, 1x/day] or imatinib + TET [3x/day: 2x with imatinib, 1x with TET]. The results of this study demonstrated that TET given orally once a day is superior to imatinib given twice a day in inhibiting the development of both circulating leukemic cells and leukemic stem cells while the combination of TET with imatinib further improves efficacy (See Figure).To determine whether TET has efficacy on human CML stem-like cells, a colony forming cell (CFC) assay with bone marrow cells from a de-novo CML patient was performed. The bone marrow cells were treated with 10 µM (IC50) TET for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine BCR-ABL or ABL only cells. Replating efficiency of TET treated cells was 54% compared to 67% of solvent controls. In primary colonies, 95% of colonies from solvent control cultures were BCR-ABL positive compared to 70% of colonies treated with ES-3000. In secondary colonies (representing stem-ness), the TET treatment group was negative for BCR-ABL colonies while 79% of solvent control colonies still tested positive for BCR-ABL, indicating efficacy of TET in CML stem-like cells (See Table).We conclude that TET reduces leukemic stem cells in both a murine model of CML and a CFC assay which demonstrates its potential for development as an adjuvant therapy for CML patients demonstrating a lack of optimal response to TKIs, alone. [Display omitted] [Display omitted] DisclosuresMichaels:Escend Pharmaceuticals, Inc.: Equity Ownership. Bates:Escend Pharmaceuticals, Inc.: Equity Ownership.

Calcium modulation of doxorubicin cytotoxicity in yeast and human cells.

Doxorubicin is a widely used chemotherapeutic agent, but its utility is limited by cellular resistance and off-target effects. To understand the molecular mechanisms regulating chemotherapeutic responses to doxorubicin, we previously carried out a genomewide search of doxorubicin-resistance genes in Schizosaccharomyces pombe fission yeast and showed that these genes are organized into networks that counteract doxorubicin cytotoxicity. Here, we describe the identification of a subgroup of doxorubicin-resistance genes that, when disrupted, leads to reduced tolerance to exogenous calcium. Unexpectedly, we observed a suppressive effect of calcium on doxorubicin cytotoxicity, where concurrent calcium and doxorubicin treatment resulted in significantly higher cell survival compared with cells treated with doxorubicin alone. Conversely, inhibitors of voltage-gated calcium channels enhanced doxorubicin cytotoxicity in the mutants. Consistent with these observations in fission yeast, calcium also suppressed doxorubicin cytotoxicity in human breast cancer cells. Further epistasis analyses in yeast showed that this suppression of doxorubicin toxicity by calcium was synergistically dependent on Rav1 and Vph2, two regulators of vacuolar-ATPase assembly; this suggests potential modulation of the calcium-doxorubicin interaction by fluctuating proton concentrations within the cellular environment. Thus, the modulatory effects of drugs or diet on calcium concentrations should be considered in doxorubicin treatment regimes.

Regulation of electrical activity and neuronal excitability in Helisoma trivolvis by carbon monoxide

Carbon monoxide (CO), like other gaseous neuromodulators, has a dual nature as both a toxic gas and a physiologically relevant signaling molecule. In the nervous system, high concentrations of CO can lead to neuronal injury while lower concentrations are found to be neuroprotective. The number of cellular targets affected by physiological concentrations of CO is rapidly growing and includes ion channels in various cell types. The modulation of ion channels by CO in neurons, however, and the effect it has on neural activity are incompletely understood. Here, the well-characterized buccal neurons, B5 and B19, of the freshwater snail, Helisoma trivolvis, were used to investigate the role that CO plays in regulating spontaneous firing activity and neuronal excitability. Neurons were studied in single-cell culture, thereby removing other signals normally present in the intact nervous system and allowing for the optimal characterization of physiological effects of CO. We found that the CO donor molecule, carbon monoxide releasing molecule-2 (CORM-2), hyperpolarized the resting membrane potential of B5 neurons and silenced their spontaneous firing activity. These effects were mediated through the inhibition of a persistent sodium current. CORM-2 also inhibited neuronal excitability. This effect was mediated by the inhibition of voltage-gated calcium channels by CO. The general findings of CO acting as a hyperpolarizing signal and an inhibitor of neuronal excitability extended to B19 neurons. Taken together, these findings suggest that CO is a potent modulator of ion channels with broad implications for the modulation of neural activity in a wide range of neuron-types.

Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells

TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner.