Inhibition Of Voltage-gated Calcium Channels Research Articles

Distribution and calcium signaling function of somatostatin receptor subtypes in rat pituitary

The somatostatin (SST) receptor family controls pituitary hormone secretion, but the distribution and specific roles of these receptors on the excitability and voltage-gated calcium signaling of hormone producing pituitary cells have not been fully characterized. Here we show that the rat pituitary gland expressed Sstr1, Sstr2, Sstr3, and Sstr5 receptor genes in a cell type-specific manner: Sstr1 and Sstr2 in thyrotrophs, Sstr3 in gonadotrophs and lactotrophs, Sstr2, Sstr3, and Sstr5 in somatotrophs, and none in corticotrophs and melanotrophs. Most gonadotrophs and thyrotrophs spontaneously fired high-amplitude single action potentials, which were silenced by SST without affecting intracellular calcium concentrations. In contrast, lactotrophs and somatotrophs spontaneously fired low-amplitude plateau-bursting action potentials in conjunction with calcium transients, both of which were silenced by SST. Moreover, SST inhibited GPCR-induced voltage-gated calcium signaling and hormone secretion in all cell types expressing SST receptors, but the inhibition was more pronounced in somatotrophs. The pattern of inhibition of electrical activity and calcium signaling was consistent with both direct and indirect inhibition of voltage-gated calcium channels, the latter being driven by cell type-specific hyperpolarization. These results indicate that the action of SST in somatotrophs is enhanced by the expression of several types of SST receptors and their slow desensitization, that SST may play a role in the electrical resynchronization of gonadotrophs, thyrotrophs, and lactotrophs, and that the lack of SST receptors in corticotrophs and melanotrophs keeps them excitable and ready to responses to stress.

Total Synthesis of Ganoapplanin Enabled by a Radical Addition/Aldol Reaction Cascade.

The total synthesis of the Ganoderma meroterpenoid ganoapplanin, an inhibitor of T-type voltage-gated calcium channels, is reported. Our synthetic approach is based on the convergent coupling of a readily available aromatic polyketide scaffold with a bicyclic terpenoid fragment. The three contiguous stereocenters of the terpenoid fragment, two of which are quaternary, were constructed by a diastereoselective, titanium-mediated iodolactonization. For the fusion of the two fragments and to simultaneously install the crucial biaryl bond, we devised a highly effective two-component coupling strategy. This event involves an intramolecular 6-exo-trig radical addition of a quinone monoacetal followed by an intermolecular aldol reaction. A strategic late-stage oxidation sequence allowed the selective installation of the remaining oxygen functionalities and the introduction of the characteristic spiro bisacetal structure of ganoapplanin.

Cortical Tagged Synaptic Long-Term Depression in the Anterior Cingulate Cortex of Adult Mice.

The anterior cingulate cortex (ACC) is a key cortical region for pain perception and emotion. Different forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), have been reported in the ACC. Synaptic tagging of LTP plays an important role in hippocampus-related associative memory. In this study, we demonstrate that synaptic tagging of LTD is detected in the ACC of adult male and female mice. This form of tagged LTD requires the activation of metabotropic glutamate receptor subtype 1 (mGluR1). The induction of tagged LTD is time-related with the strongest tagged LTD appearing when the interval between two independent stimuli is 30 min. Inhibitors of mGluR1 blocked the induction of tagged LTD; however, blocking N-methyl-d-aspartate receptors did not affect the induction of tagged LTD. Nimodipine, an inhibitor of L-type voltage-gated calcium channels, also blocked tagged LTD. In an animal model of amputation, we found that tagged LTD was either reduced or completely blocked. Together with our previous report of tagged LTP in the ACC, this study strongly suggests that excitatory synapses in the adult ACC are highly plastic. The biphasic tagging of synaptic transmission provides a new form of heterosynaptic plasticity in the ACC which has functional and pathophysiological significance in phantom pain.

Lactate ameliorates palmitate-induced impairment of differentiative capacity in C2C12 cells through the activation of voltage-gated calcium channels.

Palmitic acid (PA), a saturated fatty acid enriched in high-fat diet, has been implicated in the development of skeletal muscle regeneration dysfunction. This study aimed to examine the effects and mechanisms of lactate (Lac) treatment on PA-induced impairment of C2C12 cell differentiation capacity. Furthermore, the involvement of voltage-gated calcium channels in this context was examined. In this study, Lac could improve the PA-induced impairment of differentiative capacity in C2C12 cells by affecting Myf5, MyoD and MyoG. In addition, Lac increases the inward flow of Ca2+, and promotes the depolarization of the cell membrane potential, thereby activating voltage-gated calcium channels during C2C12 cell differentiation. The enchancement of Lac on myoblast differentiative capacity was abolished after the addition of efonidipine (voltage-gated calcium channel inhibitors). Therefore, voltage-gated calcium channels play an important role in improving PA-induced skeletal muscle regeneration disorders by exercising blood Lac. Our study showed that Lac could rescue the PA-induced impairment of differentiative capacity in C2C12 cells by affecting Myf5, MyoD and MyoG through the activation of voltage-gated calcium channels.

LC-derived excitatory synaptic transmission to dorsal raphe serotonin neurons is inhibited by activation of alpha2-adrenergic receptors.

In the central nervous system, noradrenaline transmission controls the degree to which we are awake, alert, and attentive. Aberrant noradrenaline transmission is associated with pathological forms of hyper- and hypo-arousal that present in numerous neuropsychiatric disorders often associated with dysfunction in serotonin transmission. In vivo, noradrenaline regulates the release of serotonin because noradrenergic input drives the serotonin neurons to fire action potentials via activation of excitatory α1-adrenergic receptors (α1-AR). Despite the critical influence of noradrenaline on the activity of dorsal raphe serotonin neurons, the source of noradrenergic afferents has not been resolved and the presynaptic mechanisms that regulate noradrenaline-dependent synaptic transmission have not been described. Using an acute brain slice preparation from male and female mice and electrophysiological recordings from dorsal raphe serotonin neurons, we found that selective optogenetic activation of locus coeruleus terminals in the dorsal raphe was sufficient to produce an α1-AR-mediated excitatory postsynaptic current (α1-AR-EPSC). Activation of inhibitory α2-adrenergic receptors (α2-AR) with UK-14,304 eliminated the α1-AR-EPSC via presynaptic inhibition of noradrenaline release, likely via inhibition of voltage-gated calcium channels. In a subset of serotonin neurons, activation of postsynaptic α2-AR produced an outward current through activation of GIRK potassium conductance. Further, in vivo activation of α2-AR by systemic administration of clonidine reduced the expression of c-fos in the dorsal raphe serotonin neurons, indicating reduced neural activity. Thus, α2-AR are critical regulators of serotonin neuron excitability.

A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels.

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.

Cancer Stem Cells of Hepatocellular Carcinoma Are Suppressed by the Voltage-gated Calcium Channel Inhibitor Amlodipine.

The membrane transporters activated in cancer stem cells (CSCs) are the target of novel cancer therapies for hepatocellular carcinoma (HCC). The present investigation demonstrated the expression profiles of ion channels in CSCs of HCC. Cells that highly expressed aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were separated from HepG2 cells, a human HCC cell line, by fluorescence-activated cell sorting, and CSCs were identified based on the formation of tumorspheres. Gene expression profiles in CSCs were investigated using microarray analysis. Among HepG2 cells, ALDH1A1 messenger RNA level was higher in CSCs than in non-CSCs. Furthermore, CSCs exhibited resistance to cisplatin and had the capacity to redifferentiate. The results of the microarray analysis of CSCs showed the up-regulated expression of several genes related to ion channels, such as calcium voltage-gated channel auxiliary subunit gamma 4 (CACNG4). The cytotoxicity of the CACNG4 inhibitor amlodipine was higher at lower concentrations in CSCs than in non-CSCs, and markedly decreased the number of tumorspheres. The cell population among HepG2 cells that highly expressed ALDH1A1 was also significantly reduced by this inhibitor. CACNG4 plays a role in maintaining CSCs, and its inhibitor, amlodipine, could potentially be a targeted therapeutic agent against HCC.

Direct-Current Electrical Field Stimulation of Patient-Derived Colorectal Cancer Cells.

Several cues for a directional migration of colorectal cancer cells were identified as being crucial in tumor progression. However, galvanotaxis, the directional migration in direct-current electrical fields, has not been investigated so far. Therefore, we asked whether direct-current electrical fields could be used to mobilize colorectal cancer cells along field vectors. For this purpose, five patient-derived low-passage cell lines were exposed to field strengths of 150-250 V/m in vitro, and migration along the field vectors was investigated. To further study the role of voltage-gated calcium channels on galvanotaxis and intracellular signaling pathways that are associated with migration of colorectal cancer cells, the cultures were exposed to selective inhibitors. In three out of five colorectal cancer cell lines, we found a preferred cathodal migration. The cellular integrity of the cells was not impaired by exposure of the cells to the selected field strengths. Galvanotaxis was sensitive to inhibition of voltage-gated calcium channels. Furthermore, signaling pathways such as AKT and MEK, but not STAT3, were also found to contribute to galvanotaxis in our in vitro model system. Overall, we identify electrical fields as an important contributor to the directional migration of colorectal cancer cells.

A Chemoenzymatic Approach To Produce a Cyclic Analogue of the Analgesic Drug MVIIA (Ziconotide).

Ziconotide (ω-conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)-mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six- to nine-residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage-gated calcium channels (CaV 2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins.

Betulinic acid analogs inhibit N- and T-type voltage-gated calcium channels to attenuate nerve-injury associated neuropathic and formalin models of pain

Over the past three decades, there has been a significant growth in the use of natural products, with approximately 80% of individuals using them for some aspect of primary healthcare. Our laboratories have identified and studied natural compounds with analgesic effects from dry land plants or their associated fungus during the past ten years. Here, we isolated and characterized thirteen betulin analogs and fifteen betulinic acid analogs for their capacity to prevent calcium influx brought on by depolarization in sensory neurons. The in vitro inhibition of voltage-gated calcium channels by the top drugs was then assessed using whole cell patch clamp electrophysiology. In vivo experiments, conducted at two sites, evaluated the best compound in acute and tonic, neuropathic, inflammatory, post-operative and visceral models of pain. We found that the betulinic acid analog 8 inhibited calcium influx in rat dorsal root ganglion neurons by inhibiting N- (CaV2.2) and T- (CaV3) type voltage-gated calcium channels. Moreover, intrathecal delivery of analog 8 had analgesic activity in both spared nerve injury model of neuropathic pain and acute and tonic pain induced by formalin. The results presented herein highlight the potential antinociceptive properties of betulinic acid analog 8 and set the stage for the development of novel non-opioid pain therapeutics based on the triterpenoid scaffold of betulinic acid.

Pharmacological basis of bergapten in gastrointestinal diseases focusing on H+/K+ ATPase and voltage-gated calcium channel inhibition: A toxicological evaluation on vital organs.

Aim and objectives: This study aimed to establish a pharmacological basis for evaluating the effects of bergapten (5-methoxypsoralen) in gastrointestinal diseases and assessment of its toxicological profile. Methods: The pharmacokinetic profile was evaluated using the SwissADME tool. AUTODOCK and PyRx were used for evaluating the binding affinities. The obtained results were further investigated for a post-dock analysis using Discovery Studio Visualizer 2016. The Desmond software package was used to conduct molecular dynamic simulations of best bound poses. Bergapten was further investigated for antidiarrheal, anti-secretory, charcoal meal transit time, anti-ulcer, anti-H. pylori activity. Results: Bergapten at a dose of 50, 100, and 200mg/kg was proved effective in reducing diarrheal secretions, intestinal secretions, and distance moved by charcoal meal. Bergapten at the aforementioned doses acts as a gastroprotective agent in the ethanol-induced ulcer model that can be attributed to its effectiveness against H. pylori. Bergapten shows concentration-dependent relaxation of both spontaneous and K+ (80mM)-induced contractions in the isolated rabbit jejunum model; the Ca2+ concentration-response curves (CRCs) were shifted to the right showing potentiating effect similar to papaverine. For molecular investigation, the H+/K+ ATPase inhibitory assay indicated inhibition of the pump comparable to omeprazole. Oxidative stress markers GST, GSH, and catalase showed increased expression, whereas the expression of LPO (lipid peroxidation) was reduced. Histopathological examination indicated marked improvement in cellular morphology. ELISA and western blot confirmed the reduction in inflammatory mediator expression. RT-PCR reduced the mRNA expression level of H+/K+ ATPase, confirming inhibition of the pump. The toxicological profile of bergapten was evaluated by an acute toxicity assay and evaluated for behavioral analysis, and the vital organs were used to analyze biochemical, hematological, and histopathological examination. Conclusion: Bergapten at the tested doses proved to be an antioxidant, anti-inflammatory, anti-ulcer, and antidiarrheal agent and relatively safe in acute toxicity assay.

Organophosphate insecticides disturb neuronal network development and function via non-AChE mediated mechanisms

Exposure to organophosphate (OP) insecticides has been related to several adverse health effects, including neurotoxicity. The primary insecticidal mode of action of OP insecticides relies on (irreversible) binding to acetylcholine esterase (AChE), with –oxon metabolites having a much higher potency for AChE inhibition than the parent compounds. However, OP insecticides can also have non-AChE-mediated effects, including changes in gene expression, neuroendocrine effects, disruption of neurite outgrowth and disturbance of the intracellular calcium (Ca2+) homeostasis. Since Ca2+ is involved in neurotransmission and neuronal development, our research aimed to assess the effects of two widely used OP insecticides, chlorpyrifos (CPF) and diazinon (DZ) and their respective -oxon metabolites, on intracellular Ca2+ homeostasis in human SH-SY5Y cells and rat primary cortical cultures. Furthermore, we assessed the acute and chronic effects of exposure to these compounds on neuronal network maturation and function in rat primary cortical cultures using microelectrode array (MEA) recordings. While inhibition of AChE appears to be the primary mode of action of oxon-metabolites, our data indicate that both parent OP insecticides (CPF and DZ) inhibit depolarization-evoked Ca2+ influx and neuronal activity at concentrations far below their sensitivity for AChE inhibition, indicating that inhibition of voltage-gated calcium channels is a common mode of action of OP insecticides. Notably, parent compounds were more potent than their oxon metabolites, with exposure to diazinon-oxon (DZO) having no effect on both neuronal activity and Ca2+ influx. Human SH-SY5Y cells were more sensitive to OP-induced inhibition of depolarization-evoked Ca2+ influx than rat cortical cells. Acute exposure to OP insecticides had more potent effects on neuronal activity than on Ca2+ influx, suggesting that neuronal activity parameters are especially sensitive to OP exposure. Interestingly, the effects of DZ and chlorpyrifos-oxon (CPO) on neuronal activity lessened after 48 h of exposure, while the potency of CPF did not differ over time. This suggests that neurotoxicity after exposure to different OPs has different effects over time and occurs at levels that are close to human exposure levels. In line with these results, chronic exposure to CPF during 10 days impaired neuronal network development, illustrating the need to investigate possible links between early-life OP exposure and neurodevelopmental disorders in children and highlighting the importance of non-AChE mediated mechanisms of neurotoxicity after OP exposure.

Zinc drives vasorelaxation by acting in sensory nerves, endothelium and smooth muscle

Zinc, an abundant transition metal, serves as a signalling molecule in several biological systems. Zinc transporters are genetically associated with cardiovascular diseases but the function of zinc in vascular tone regulation is unknown. We found that elevating cytoplasmic zinc using ionophores relaxed rat and human isolated blood vessels and caused hyperpolarization of smooth muscle membrane. Furthermore, zinc ionophores lowered blood pressure in anaesthetized rats and increased blood flow without affecting heart rate. Conversely, intracellular zinc chelation induced contraction of selected vessels from rats and humans and depolarized vascular smooth muscle membrane potential. We demonstrate three mechanisms for zinc-induced vasorelaxation: (1) activation of transient receptor potential ankyrin 1 to increase calcitonin gene-related peptide signalling from perivascular sensory nerves; (2) enhancement of cyclooxygenase-sensitive vasodilatory prostanoid signalling in the endothelium; and (3) inhibition of voltage-gated calcium channels in the smooth muscle. These data introduce zinc as a new target for vascular therapeutics.

Selective Somatostatin 5 (SST5) and Somatostatin 2 (SST2) Nonpeptide Agonists Potently Suppress Glucose- and Tolbutamide-Stimulated Dynamic Insulin Secretion From Isolated Human Islets

Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in newborns and infants and arises from dysregulated insulin secretion. Rapid recognition and treatment are vital to prevent seizures, permanent developmental delays, coma, or even death. Very few medical options exist to treat congenital HI patients: the KATP channel activator diazoxide, the injectable somatostatin receptor peptide agonists octreotide and lanreotide, or chronic glucose infusions. However, side effects and/or limited efficacy render these therapies inadequate for many patients.Somatostatin is a 14-amino acid peptide hormone with a broad spectrum of biological actions, which are regulated through five somatostatin receptor subtypes (SST1-SST5). Somatostatin’s common physiological role is to down-regulate secretion of other hormones in various tissues. Its role in the maintenance of euglycemia is to regulate insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Somatostatin regulates insulin secretion by decreasing the intracellular levels of cAMP, inhibition of voltage-gated calcium channels (VGCC), activation of the G protein-activated inward rectifier K+ channel (GIRK), and direct inhibition of insulin exocytosis.Several studies have evaluated the effect of somatostatin, somatostatin peptide analogs, and a limited number of nonpeptide somatostatin receptor agonists on insulin secretion in static assays using isolated human islets. However, the lack of highly selective agonists has made the interpretation of the contribution of SST receptor sub-types difficult to discern. Our programs for the treatment of hyperinsulinism, acromegaly, and other indications have led to the development of selective nonpeptide SST2, SST3, SST4, and SST5 agonists, possessing EC50s < 1 nM in cell-based assays of receptor activation and selectivity > 130 times over the other members of the family. The ability of these selective nonpeptide agonists to regulate glucose- and tolbutamide-stimulated dynamic insulin secretion from human islets was evaluated using a perifusion system (Biorep, FL).We found that selective SST2 and SST5 agonists potently suppressed dynamic insulin secretion in contrast to SST3 or SST4 selective agonists. Importantly, SST5 agonists were shown to have a greater effect than selective SST2 agonists or diazoxide, demonstrating their potential utility in human conditions such as congenital HI. In addition, SST5 activation is also known to have a smaller effect on glucagon secretion and is also less prone to agonist-driven desensitization than SST2 activation. Taken together, these studies support our program to identify, characterize, and develop potent, nonpeptide, orally-bioavailable, selective SST5 agonists with appropriate pharmaceutical and safety characteristics for the treatment of congenital HI.

Repositioning Trimebutine Maleate as a Cancer Treatment Targeting Ovarian Cancer Stem Cells.

The overall five-year survival rate for late-stage patients of ovarian cancer is below 29% due to disease recurrence and drug resistance. Cancer stem cells (CSCs) are known as a major contributor to drug resistance and recurrence. Accordingly, therapies targeting ovarian CSCs are needed to overcome the limitations of present treatments. This study evaluated the effect of trimebutine maleate (TM) targeting ovarian CSCs, using A2780-SP cells acquired by a sphere culture of A2780 epithelial ovarian cancer cells. TM is indicated as a gastrointestinal motility modulator and is known to as a peripheral opioid receptor agonist and a blocker for various channels. The GI50 of TM was approximately 0.4 µM in A2780-SP cells but over 100 µM in A2780 cells, demonstrating CSCs specific growth inhibition. TM induced G0/G1 arrest and increased the AV+/PI+ dead cell population in the A2780-SP samples. Furthermore, TM treatment significantly reduced tumor growth in A2780-SP xenograft mice. Voltage gated calcium channels (VGCC) and calcium-activated potassium channels (BKCa) were overexpressed on ovarian CSCs and targeted by TM; inhibition of both channels reduced A2780-SP cells viability. TM reduced stemness-related protein expression; this tendency was reproduced by the simultaneous inhibition of VGCC and BKCa compared to single channel inhibition. In addition, TM suppressed the Wnt/β-catenin, Notch, and Hedgehog pathways which contribute to many CSCs characteristics. Specifically, further suppression of the Wnt/β-catenin pathway by simultaneous inhibition of BKCa and VGCC is necessary for the effective and selective action of TM. Taken together, TM is a potential therapeutic drug for preventing ovarian cancer recurrence and drug resistance.

Activation of GABAB receptors results in excitatory modulation of calyx terminals in rat semicircular canal cristae.

Previous studies have found GABA in vestibular end organs. However, existence of GABA receptors or possible GABAergic effects on vestibular nerve afferents has not been investigated. The current study was conducted to determine whether activation of GABAB receptors affects calyx afferent terminals in the central region of the cristae of semicircular canals. We used patch-clamp recording in postnatal day 13-18 (P13-P18) Sprague-Dawley rats of either sex. Application of GABAB receptor agonist baclofen inhibited voltage-sensitive potassium currents. This effect was blocked by selective GABAB receptor antagonist CGP 35348. Application of antagonists of small (SK)- and large-conductance potassium (BK) channels almost completely blocked the effects of baclofen. The remaining baclofen effect was blocked by cadmium chloride, suggesting that it could be due to inhibition of voltage-gated calcium channels. Furthermore, baclofen had no effect in the absence of calcium in the extracellular fluid. Inhibition of potassium currents by GABAB activation resulted in an excitatory effect on calyx terminal action potential firing. While in the control condition calyces could only fire a single action potential during step depolarizations, in the presence of baclofen they fired continuously during steps and a few even showed repetitive discharge. We also found a decrease in threshold for action potential generation and a decrease in first-spike latency during step depolarization. These results provide the first evidence for the presence of GABAB receptors on calyx terminals, showing that their activation results in an excitatory effect and that GABA inputs could be used to modulate calyx response properties.NEW & NOTEWORTHY Using in vitro whole cell patch-clamp recordings from calyx terminals in the vestibular end organs, we show that activation of GABAB receptors result in an excitatory effect, with decreased spike-frequency adaptation and shortened first-spike latencies. Our results suggest that these effects are mediated through inhibition of calcium-sensitive potassium channels.

Insoluble tau aggregates induce neuronal death through modification of membrane ion conductance, activation of voltage-gated calcium channels and NADPH oxidase.

Most neurodegenerative disorders are associated with aggregation and accumulation of misfolded proteins. One of these proteins, tau, is involved in a number of pathologies including Alzheimer's disease and frontotemporal dementia. Aggregation and phosphorylation of tau have been shown to be a trigger for abnormal signal transduction and disruption of cellular homeostasis. Here, we have studied the effect of extracellular tau at different stages of aggregation in cortical co-cultures of neurons and astrocytes, to understand how this process affects tau pathogenicity. We found that the species formed after prolonged invitro aggregation of tau (longer than 1day) are able to stimulate reactive oxygen species (ROS) production through the activation of NADPH oxidase without decreasing the level of the endogenous antioxidant glutathione. The same late insoluble aggregates of tau induced calcium signals in neurons and a gradual increase in the ionic current of artificial membranes. Both tau-induced calcium signals and ROS production in NADPH oxidase were reduced in the presence of the inhibitor of voltage-gated calcium channels (VGCC) nifedipine. This suggests that insoluble aggregates of tau incorporate into the membrane and modify ionic currents, changing plasma membrane potential and activating VGCCs, which induces a calcium influx that triggers ROS production in NADPH oxidase. The combination of all these effects likely leads to toxicity, as only the same insoluble tau aggregates which demonstrated membrane-active properties produced neuronal cell death.

Analgesic effects of the CTK 01512-2 toxin in different models of orofacial pain in rats

Orofacial pain is clinically challenging, having therapeutic failures and side effects. This study evaluated the antinociceptive activities of the CTK 01512-2 toxin, the TRPA1 channel antagonist, and the selective inhibitor of the N-type voltage-gated calcium channels (N-type VGCC), in different pain models. The trigeminal ganglia were stimulated in vitro with capsaicin. The in vivo models received subcutaneous (sc) injections of formalin into the upper lip of the rats, Freund's Complete Adjuvant (FCA) into the temporomandibular joint (TMJ), and infraorbital nerve constrictions (IONC). CTK 01512-2 at concentrations of 30, 100, and 300pmol/site, intrathecally (ith), and MVIIA at 10, 30, and 100pmol/site in the formalin test, guided the doses for the models. The glutamate levels in the CSF of the rats that were submitted to IONC were analyzed. CTK 01512-2 decreased the nociceptive behavior in the inflammatory phase of the formalin test (65.94 ± 7.35%) and MVIIA in the neurogenic phase (81.23 ± 3.36%). CTK 01512-2 reduced facial grooming with FCA in the TMJ (96.7 ± 1.6%), and in the IONC neuropathy model, it decreased heat hyperalgesia (100%) and cold hyperalgesia (81.61 ± 9.02%). The levels of glutamate in the trigeminal ganglia in vitro (81.40 ± 8.59%) and in the CSF in vivo (70.0 ± 9.2%) were reduced. The roles of TRPA1 in pain transduction and the performance of CTK 01512-2 in the inhibition of the N-type VGCCs were reinforced. This dual activity may represent an advantage in clinical treatments.

Role of Calcium Signaling in Prostate Cancer Progression: Effects on Cancer Hallmarks and Bone Metastatic Mechanisms.

Advanced prostate cancers that progress to tumor metastases are often considered incurable or difficult to treat. The etiology of prostate cancers is multi-factorial. Among other factors, de-regulation of calcium signals in prostate tumor cells mediates several pathological dysfunctions associated with tumor progression. Calcium plays a relevant role on tumor cell death, proliferation, motility-invasion and tumor metastasis. Calcium controls molecular factors and signaling pathways involved in the development of prostate cancer and its progression. Such factors and pathways include calcium channels and calcium-binding proteins. Nevertheless, the involvement of calcium signaling on prostate cancer predisposition for bone tropism has been relatively unexplored. In this regard, a diversity of mechanisms triggers transient accumulation of intracellular calcium in prostate cancer cells, potentially favoring bone metastases development. New therapies for the treatment of prostate cancer include compounds characterized by potent and specific actions that target calcium channels/transporters or pumps. These novel drugs for prostate cancer treatment encompass calcium-ATPase inhibitors, voltage-gated calcium channel inhibitors, transient receptor potential (TRP) channel regulators or Orai inhibitors. This review details the latest results that have evaluated the relationship between calcium signaling and progression of prostate cancer, as well as potential therapies aiming to modulate calcium signaling in prostate tumor progression.

The cytotoxic role of the inhibition of PI‐3‐kinase, and acto‐myosin cross‐bridge formation in human metastatic breast cancer cells.

IntroductionExternally added survival/growth factors are essential for the survival of healthy cells, however cancer cells are believed to be self‐sufficient in supporting their auto‐proliferation without the need of exogenously added growth factors. The Phosphatidyl‐Inositol‐3‐kinase (PI‐3‐kinase) pathway is crucial in cell survival. Also, cancer cells exhibit enhanced potential to metastasize via increased acto‐myosin cross bridges formation which requires increased Ca2+ influx via cell surface voltage‐gated Ca2+ channels leading to activation of myosin light chain kinase (MLCK). The MLCK then phosphorylates myosin and forms acto‐myosin cross‐bridges. The role of MLCK, PI‐3‐kinase, and calcium channel in cancer cell growth has not been extensively tested. In this study, we tested the individual effects of blocking cell survival pathway (PI‐3 kinase), voltage‐gated Ca2+ channels and MLCK on MCF‐7 cell (a human metastatic breast cancer cell) survival using pharmacological inhibitors.HypothesisWe hypothesized that all three drugs would inhibit cell proliferation.AimsOur aim was to firstly test if these drugs had any effect at all on cancer cell growth. Secondly, it was to test each drug at different concentrations and see which concentration had the most effect on cell death.MethodsPI‐3‐kianse inhibitor (Wortmannin); MLCK inhibitor (ML‐7), and lastly, a voltage‐gated calcium channel inhibitor (nifedipine) were used. 1% Bitter melon extract (BME) was also tested as this was found to inhibit MCF‐cell growth in our lab by earlier investigators. Cells were thawed, mixed with a medium that contained all essential nutrients for cell growth (DMEM + fetal bovine serum), and then were added onto 96‐well plates and allowed to grow for 48 hours. We incorporated control plates (no drug), plates with drugs at different concentrations, a plate with the compound the drug was originally dissolved in (DMSO or ethanol), and a plate with 1% BME (bitter melon extract). The plates were analyzed under a microscope and cell viability was measured using MTT assays. Images of each plate were taken and correlated with the MTT assay results. Each experiment was repeated at least 4 times in duplicate or triplicate to plot graphs and draw statistical inferences.Results and DiscussionAll three drugs independently inhibited cell growth in a dose‐dependent manner. The observable differences between the different drugs were the concentrations at which they were effective. The results imply that Ca2+ influx and its downstream pathway leading to acto‐myosin interaction is essential for the MCF‐7 cell attachment to the plate and hence its survival.Support or Funding InformationSummer research stipend to medical students at MUCOMDepartmental faculty research support to faculties at MUCOM