Voltage-gated Calcium Channel Antagonist Research Articles

EXTH-41. COMBINATION TREATMENT OF CALCIUM CHANNEL ANTAGONIST MIBEFRADIL ENHANCES THE EFFECT OF HYDROXYUREA AT THERAPEUTIC RANGES IN HIGH-GRADE MENINGIOMA IN VITRO

Abstract High-grade meningiomas (MNs), WHO Grade II- III, are highly aggressive brain tumors that are difficult to treat. Standard chemotherapy agents are, at best, moderately effective in treating high-grade MNs. There is great interest in developing combinatorial drug treatment regimens to enhance the effects of standard chemotherapy. Many cancer types, including brain tumors, have an increase in the expression of voltage-gated calcium channels (VGCCs). These channels are involved in the regulation of cell cycle, proliferation, and apoptosis, among other functions. FDA-approved VGCC antagonists nimodipine (NIMO) and mibefradil (MIB) target two different VGCCs, L- and T-type channels, and have been of great interest for their use in combination therapy in other cancers. We sought to determine the efficacy of chemotherapy agents tested at clinically relevant concentrations for the treatment of high-grade MNs alone or in combination with NIMO or MIB. We cultured CH-157MN and IOMM-Lee MN cells and assessed their cell survival, apoptosis, and proliferation in the presence of temozolomide, octreotide, sunitinib, or hydroxyurea (HU). We confirmed that HU (150 µM) was the most efficacious chemotherapy agent, decreasing cell viability at therapeutic ranges. Monotherapy treatment with 2 or 4 µM MIB significantly decreased cell viability in both cell lines, whereas 1 µM NIMO only affected IOMM-Lee cells. The combination of HU and MIB further decreased cell viability but only in the most aggressive cell line, IOMM-Lee, whereas NIMO did not enhance HU’s ability to decrease cell survival. Remarkably, we detect an enhancement of HU’s effect by MIB when both agents are used within therapeutic ranges, as reported by pharmacokinetic studies. Here we demonstrate for the first time that T-type calcium channels play an important role in MN survival. The combination of HU and MIB suggests their use could be a promising treatment for high-grade MN.

Nimodipine augments cerebrovascular reactivity in aging but runs the risk of local perfusion reduction in acute cerebral ischemia.

The efficacy of cerebrovascular reactivity (CVR) is taken as an indicator of cerebrovascular health. We found that CVR tested with the inhalation of 10 % CO2 declined in the parietal cortex of 18-20-month-old rats. The CVR deficit in old rats was coincident with cerebrovascular smooth muscle cell and astrocyte senescence, revealed by the immuno-labeling of the cellular senescence marker p16 in these cells. In a next series of experiments, CVR was severely impaired in the acute phase of incomplete global forebrain ischemia produced by the bilateral occlusion of the common carotid arteries in young adult rats. In acute ischemia, CVR impairment often manifested as a perfusion drop rather than blood flow elevation in response to hypercapnia. Next, nimodipine, an L-type voltage-gated calcium channel antagonist was administered topically to rescue CVR in both aging, and cerebra ischemia. Nimodipine augmented CVR in the aged brain, but worsened CVR impairment in acute cerebral ischemia. A careful evaluation of benefits and side effects of nimodipine is recommended, especially in acute ischemic stroke.

Impact of the Voltage-Gated Calcium Channel Antagonist Nimodipine on the Development of Oligodendrocyte Precursor Cells

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While most of the current treatment strategies focus on immune cell regulation, except for the drug siponimod, there is no therapeutic intervention that primarily aims at neuroprotection and remyelination. Recently, nimodipine showed a beneficial and remyelinating effect in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Nimodipine also positively affected astrocytes, neurons, and mature oligodendrocytes. Here we investigated the effects of nimodipine, an L-type voltage-gated calcium channel antagonist, on the expression profile of myelin genes and proteins in the oligodendrocyte precursor cell (OPC) line Oli-Neu and in primary OPCs. Our data indicate that nimodipine does not have any effect on myelin-related gene and protein expression. Furthermore, nimodipine treatment did not result in any morphological changes in these cells. However, RNA sequencing and bioinformatic analyses identified potential micro (mi)RNA that could support myelination after nimodipine treatment compared to a dimethyl sulfoxide (DMSO) control. Additionally, we treated zebrafish with nimodipine and observed a significant increase in the number of mature oligodendrocytes (* p≤ 0.05). Taken together, nimodipine seems to have different positive effects on OPCs and mature oligodendrocytes.

Assays for L-type voltage gated calcium channels

Voltage gated calcium channels (VGCCs) are pursued as drug targets for neurodegenerative and cardiovascular diseases. High throughput drug screening targeting VGCCs depends on patch-clamp electrophysiology or fluorophore-based calcium imaging that requires powerful equipment and specialized expertise thus leading to cost escalation. Moreover, VGCC needs to be transfected into cell lines such as HEK-293. We report the presence of L-type VGCC (L-VGCC) subunit proteins, Cav1.2, α2δ and β in HEK-293 cells and the application of simple methods for its assay. Endogenous expression of the channel in HEK-293 cells overcomes the need for transfection. L-VGCC in HEK-293 cells was activated either by the agonist, BayK8644 or by KCl-mediated depolarization. Activity was detected using the calcium sensing probe, GCaMP6m by live imaging. L-VGCC activity induced enhancement in GCaMP6m fluorescence returned to baseline corresponding to channel-closure. Activity was also shown using a methodology involving end-point detection of the calcium dependent interaction of α-CaMKII with NMDA receptor subunit GluN2B sequence. This methodology further simplifies the assay as it eliminates the need for real time imaging. Activation was blocked by the specific L-type VGCC antagonist, nifedipine. Finding the protein and activity of L-VGCC in HEK-293 cells offers commercially viable assays for drug screening.

Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca2+) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Cav blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca2+]cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca2+ pools, and Ca2+ influx by ORAI channels have been associated with the increase in [Ca2+]cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil’s effect on [Ca2+]cyt. To address these questions, we monitored changes in [Ca2+]cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca2+]cyt at concentrations above 10 μM (IC50 around 50 μM) and a fast Ca2+ increase capacity at 100 μM. We found that inhibiting IP3 receptors, depleting the ER-Ca2+ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca2+]cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca2+ influx by store-operated Ca2+ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP3R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

CACNA1S haploinsufficiency confers resistance to New World arenavirus infection

Understanding the genetics of susceptibility to infectious agents is of great importance to our ability to combat disease. Here, we show that voltage-gated calcium channels (VGCCs) are critical for cellular binding and entry of the New World arenaviruses Junín and Tacaribe virus, suggesting that zoonosis via these receptors could occur. Moreover, we demonstrate that α1s haploinsufficiency renders cells and mice more resistant to infection by these viruses. In addition to being more resistant to infection, haploinsufficient cells and mice required a lower dosage of VGCC antagonists to block infection. These studies underscore the importance of genetic variation in susceptibility to both viruses and pharmaceutics.

Chitosan nanoparticles release nimodipine in response to tissue acidosis to attenuate spreading depolarization evoked during forebrain ischemia

Stroke is an important cause of mortality and disability. Treatment options are limited, therefore the progress in this regard is urgently needed. Nimodipine, an L-type voltage-gated calcium channel antagonist dilates cerebral arterioles, but its systemic administration may cause potential side effects. We have previously constructed chitosan nanoparticles as drug carriers, which release nimodipine in response to decreasing pH typical of cerebral ischemia. Here we have set out to evaluate this nanomedical approach to deliver nimodipine selectively to acidic ischemic brain tissue.After washing a nanoparticle suspension with or without nimodipine (100 μM) on the exposed brain surface of anesthetized rats (n = 18), both common carotid arteries were occluded to create forebrain ischemia. Spreading depolarizations (SDs) were elicited by 1M KCl to deepen the ischemic insult. Local field potential, cerebral blood flow (CBF) and tissue pH were recorded from the cerebral cortex. Microglia activation and neuronal survival were evaluated in brain sections by immunocytochemistry.Ischemia-induced tissue acidosis initiated nimodipine release from nanoparticles, confirmed by the significant elevation of baseline CBF (47.8 ± 23.7 vs. 29.3 ± 6.96%). Nimodipine shortened the duration of both SD itself (48.07 ± 23.29 vs. 76.25 ± 17.2 s), and the associated tissue acidosis (65.46 ± 20.2 vs. 138.3 ± 66.07 s), moreover it enhanced the SD-related hyperemia (48.15 ± 42.04 vs. 17.29 ± 11.03%). Chitosan nanoparticles did not activate microglia.The data support the concept that tissue acidosis linked to cerebral ischemia can be employed as a trigger for targeted drug delivery. Nimodipine-mediated vasodilation and SD inhibition can be achieved by pH-responsive chitosan nanoparticles applied directly to the brain surface.

1,4-Dihydropyridine: A Dependable Heterocyclic Ring with the Promising and the Most Anticipable Therapeutic Effects.

Nowadays, heterocyclic compounds act as a scaffold and are the backbone of medicinal chemistry. Among all of the heterocyclic scaffolds, 1,4-Dihydropyridine (1,4-DHP) is one of the most important heterocyclic rings that possess prominent therapeutic effects in a very versatile manner and plays an important role in synthetic, medicinal, and bioorganic chemistry. The main aim of the study is to review and encompass relevant studies related to 1,4-DHP and excellent therapeutic benefits of its derivatives. An extensive review of Pubmed-Medline, Embase and Lancet's published articles was done to find all relevant studies on the activity of 1,4-DHP and its derivatives. 1,4-DHP is a potent Voltage-Gated Calcium Channel (VGCC) antagonist derivative which acts as an anti-hypertensive, anti- anginal, anti-tumor, anti-inflammatory, anti-tubercular, anti-cancer, anti-hyperplasia, anti-mutagenic, anti-dyslipidemic, and anti-ulcer agent. From the inferences of the study, it can be concluded that the basic nucleus, 1,4-DHP which is a voltage-gated calcium ion channel blocker, acts as a base for its derivatives that possess different important therapeutic effects. There is a need of further research of this basic nucleus as it is a multifunctional moiety, on which addition of different groups can yield a better drug for its other activities such as anti-convulsant, anti-oxidant, anti-mutagenic, and anti-microbial. This review would be significant for further researches in the development of several kinds of drugs by representing successful matrix for the medicinal agents.

Using L-type Voltage-Gated Calcium Channels (LVGCC) Antagonists to Treat Withdrawal Symptoms of Chronic Alcohol and Drug Abuse

L-type Voltage-Gated Calcium Channels (LVGCC) are among ion-channels which their functions would be altered during some abused drugs withdrawal and alcohol withdrawal [1-3]. During alcohol consumption, the levels of L-type Voltage-Gated Calcium Channels would be increased in some parts of the brain including hippocampus and striatum.

Protective effects of voltage-gated calcium channel antagonists against zinc toxicity in SN56 neuroblastoma cholinergic cells.

One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well.

T-type Calcium Channels Determine the Vulnerability of Dopaminergic Neurons to Mitochondrial Stress in Familial Parkinson Disease.

SummaryParkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.

T-Type voltage gated calcium channels: a target in breast cancer?

The purpose of this review article is to discuss the potential of T-type voltage gated calcium channels (VGCCs) as drug targets in breast cancer. Breast cancer, attributable to the different molecular subtypes, has a crucial need for therapeutic strategies to counter the mortality rate. VGCCs play an important role in regulating cytosolic free calcium levels which regulate cellular processes like tumorigenesis and cancer progression. In the last decade, T-type VGCCs have been investigated in breast cancer proliferation. Calcium channel blockers, in general, have shown ananti-proliferative and cytotoxic effects. T-type VGCC antagonists have shown growth inhibition owing to the inhibition of CaV3.2 isoform. CaV3.1 isoform has been indicated as a tumour-suppressor gene candidate and is reported to support anti-proliferative and apoptotic activity in breast cancer. The distribution of T-type VGCC isoforms in different breast cancer molecular subtypes is diverse and needs to be further investigated. The role of T-type VGCCs in breast cancer migration, metastasis and more importantly in epithelial to mesenchymal transition (EMT) is yet to be elucidated. In addition, interlaced therapy, using a combination of chemotherapy drugs and T-type VGCC blockers, presents a promising therapeutic approach for breast cancer but more validation and clinical trials are needed. Also, for investigating the potential of T-type VGCC blocker therapy, there is a need for isoform-specific agonists/antagonists to define and discover roles of T-type VGCC specific isoforms. Our article provides a review of the role of T-type VGCCs in breast cancer and also discusses future of the research in this area so that it can be ascertained whether there is any potential of T-type VGCCs as drug targets in breast cancer.

Vascular wall regulator of G-protein signalling-1 (RGS-1) is required for angiotensin II–mediated blood pressure control

G-Protein coupled receptors (GPCRs) activate intracellular signalling pathways by coupling to heterotrimeric G-proteins that control many physiological processes including blood pressure homeostasis. The Regulator of G-Protein Signalling-1 (RGS1) controls the magnitude and duration of downstream GPCR signalling by acting as a GTPase-activating protein for specific Gα-proteins. RGS1 has contrasting roles in haematopoietic and non-haematopoietic cells. Rgs1−/−ApoE−/− mice are protected from Angiotensin II (Ang II)-induced aortic aneurysm rupture. Conversely, Ang II treatment increases systolic blood pressure to a greater extent in Rgs1−/−ApoE−/− mice than ApoE−/− mice, independent of its role in myeloid cells. However the precise role of RGS1 in hypertension and vascular-derived cells remains unknown. We determined the effects of Rgs1 deletion on vascular function in ApoE−/− mice. Rgs1 deletion led to enhanced vasoconstriction in aortas and mesenteric arteries from ApoE−/− mice in response to phenylephrine (PE) and U46619 respectively. Rgs1 was shown to have a role in the vasculature, with endothelium-dependent vasodilation being impaired, and endothelium-independent dilatation to SNP being enhanced in Rgs1−/−ApoE−/− mesenteric arteries. To address the downstream signalling pathways in vascular smooth muscle cells (VSMCs) in response to Ang II-stimulation, we assessed pErk1/2, pJNK and pp38 MAPK activation in VSMCs transiently transfected with Rgs1. pErk1/2 signalling but not pJNK and pp38 signalling was impaired in the presence of Rgs1. Furthermore, we demonstrated that the enhanced contractile response to PE in Rgs1−/−ApoE−/− aortas was reduced by a MAPK/Erk (MEK) inhibitor and an L-type voltage gated calcium channel antagonist, suggesting that Erk1/2 signalling and calcium influx are major effectors of Rgs1-mediated vascular contractile responses, respectively. These findings indicate RGS1 is a novel regulator of blood pressure homeostasis and highlight RGS1-controlled signalling pathways in the vasculature that may be new drug development targets for hypertension.

Mechanisms of calcium sequestration by isolated Malpighian tubules of the house cricket Acheta domesticus.

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca2+ within internal calcium stores (Ca-rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion-selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca2+ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca2+ transport was specific to midtubule segments, where 97% of the Ca2+ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage-gated (L-type) calcium channels decreased Ca2+ influx ≥fivefold in adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated tubules, suggesting basolateral Ca2+ influx is facilitated by voltage-gated Ca2+ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca2+ had opposite effects on tubule Ca2+ transport. The adenylyl cyclase-cAMP-PKA pathway promotes Ca2+ sequestration whereas both 5-hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca2+ sequestration through stimulatory (cAMP) and inhibitory (Ca2+ ) regulatory pathways.

Inflammation alters AMPA-stimulated calcium responses in dorsal striatal D2 but not D1 spiny projection neurons.

Neuroinflammation precedes neuronal loss in striatal neurodegenerative diseases and can be exacerbated by the release of proinflammatory molecules by microglia. These molecules can affect trafficking of AMPARs. The preferential trafficking of calcium-permeable versus impermeable AMPARs can result in disruptions of [Ca2+ ]i and alter cellular functions. In striatal neurodegenerative diseases, changes in [Ca2+ ]i and L-type voltage-gated calcium channels (VGCCs) have been reported. Therefore, this study sought to determine whether a proinflammatory environment alters AMPA-stimulated [Ca2+ ]i through calcium-permeable AMPARs and/or L-type VGCCs in dopamine-2- and dopamine-1-expressing striatal spiny projection neurons (D2 and D1 SPNs) in the dorsal striatum. Mice expressing the calcium indicator protein, GCaMP in D2 or D1 SPNs, were utilized for calcium imaging. Microglial activation was assessed by morphology analyses. To induce inflammation, acute mouse striatal slices were incubated with lipopolysaccharide (LPS). Here we report that LPS treatment potentiated AMPA responses only in D2 SPNs. When a nonspecific VGCC blocker was included, we observed a decrease of AMPA-stimulated calcium fluorescence in D2 but not D1 SPNs. The remaining agonist-induced [Ca2+ ]i was mediated by calcium-permeable AMPARs because the responses were completely blocked by a selective calcium-permeable AMPAR antagonist. We used isradipine, the highly selective L-type VGCC antagonist to determine the role of L-type VGCCs in SPNs treated with LPS. Isradipine decreased AMPA-stimulated responses selectively in D2 SPNs after LPS treatment. Our findings suggest that dorsal striatal D2 SPNs are specifically targeted in proinflammatory conditions and that L-type VGCCs and calcium-permeable AMPARs are important mediators of this effect.

High-frequency voltage oscillations in cultured astrocytes.

Because of their close interaction with neuronal physiology, astrocytes can modulate brain function in multiple ways. Here, we demonstrate a yet unknown astrocytic phenomenon: Astrocytes cultured on microelectrode arrays (MEAs) exhibited extracellular voltage fluctuations in a broad frequency spectrum (100–600 Hz) after electrical stimulation. These aperiodic high-frequency oscillations (HFOs) could last several seconds and did not spread across the MEA. The voltage-gated calcium channel antagonist cilnidipine dose-dependently decreased the power of the oscillations. While intracellular calcium was pivotal, incubation with bafilomycin A1 showed that vesicular release of transmitters played only a minor role in the emergence of HFOs. Gap junctions and volume-regulated anionic channels had just as little functional impact, which was demonstrated by the addition of carbenoxolone (100 μmol/L) and NPPB (100 μmol/L). Hyperpolarization with low potassium in the extracellular solution (2 mmol/L) dramatically raised oscillation power. A similar effect was seen when we added extra sodium (+50 mmol/L) or if we replaced it with NMDG+ (50 mmol/L). The purinergic receptor antagonist PPADS suppressed the oscillation power, while the agonist ATP (100 μmol/L) had only an increasing effect when the bath solution pH was slightly lowered to pH 7.2. From these observations, we conclude that astrocytic voltage oscillations are triggered by activation of voltage-gated calcium channels and driven by a downstream influx of cations through channels that are permeable for large ions such as NMDG+. Most likely candidates are subtypes of pore-forming P2X channels with a low affinity for ATP.

A review of the drug pregabalin

Pregabalin (PGB) is a well-established anticonvulsant and analgesic agent. The stydy reviewed the mechanism of action, pharmacokinetics, adverse drug reactions, contraindications, and various uses of PGB. Literature search was done to identify the relevant studies. PGB is an antagonist of voltage-gated calcium channels and specifically binds to α2-δ subunit to produce antiepileptic and analgesic activity. It has less protein binding activity and lacks hepatic metabolism. It is unlikely to cause pharmacokinetic drug-drug interactions. It has a wide safety margin and does not require serum drug monitoring. The above-mentioned favorable pharmacological benefits of PGB makes it a first-line or adjunctive therapy in various conditions like diabetic peripheral neuropathy, post-herpetic neuralgia, in partial seizures and generalized anxiety disorders.

The role of voltage‐gated calcium channels in neurotransmitter phenotype specification: Coexpression and functional analysis in Xenopus laevis

Calcium activity has been implicated in many neurodevelopmental events, including the specification of neurotransmitter phenotypes. Higher levels of calcium activity lead to an increased number of inhibitory neural phenotypes, whereas lower levels of calcium activity lead to excitatory neural phenotypes. Voltage-gated calcium channels (VGCCs) allow for rapid calcium entry and are expressed during early neural stages, making them likely regulators of activity-dependent neurotransmitter phenotype specification. To test this hypothesis, multiplex fluorescent in situ hybridization was used to characterize the coexpression of eight VGCC α1 subunits with the excitatory and inhibitory neural markers xVGlut1 and xVIAAT in Xenopus laevis embryos. VGCC coexpression was higher with xVGlut1 than xVIAAT, especially in the hindbrain, spinal cord, and cranial nerves. Calcium activity was also analyzed on a single-cell level, and spike frequency was correlated with the expression of VGCC α1 subunits in cell culture. Cells expressing Cav2.1 and Cav2.2 displayed increased calcium spiking compared with cells not expressing this marker. The VGCC antagonist diltiazem and agonist (−)BayK 8644 were used to manipulate calcium activity. Diltiazem exposure increased the number of glutamatergic cells and decreased the number of γ-aminobutyric acid (GABA)ergic cells, whereas (−)BayK 8644 exposure decreased the number of glutamatergic cells without having an effect on the number of GABAergic cells. Given that the expression and functional manipulation of VGCCs are correlated with neurotransmitter phenotype in some, but not all, experiments, VGCCs likely act in combination with a variety of other signaling factors to determine neuronal phenotype specification. J. Comp. Neurol. 522:2518–2531, 2014.

Quantitative Structure-Activity Relationship and Virtual Screening of ω-Conotoxins

ω-Conotoxins are active peptides composed of 24-31 amino acids isolated from venomous marine predatory cone snails. ω-Conotoxins selectively inhibit voltage-gated calcium channels (VGCCs) in nociceptors, so are considered attractive molecules for drug design. In this study, based on a set of new amino acid structure descriptors (c-scales) and genetic partial least squares (G/PLS) regression method, quantitative structure- activity relationship (QSAR) models for N-type and P/Q-type VGCC antagonists of ω-conotoxins were developed. Two virtual polypeptide libraries with 2244 peptides were designed and established for N-type and P/Q-type VGCC antagonists, respectively. Then, based on the biological activities predicted from the constructed QSAR models and chemical similarities to the probes MVIIAand MVIIC, the polypeptide libraries were virtually screened. As a result, the established QSAR models had good predictability (cross-validated correlation coefficient CV-r 2 >0.89). The structural diversity of the libraries was validated using principal component analysis (PCA) and hierarchical cluster analysis (HCA) approaches. Six N-type and nineteen P/Q-type VGCC antagonists with high selectivity and activity were identified by virtual screening. The results of this study will be valuable for finding highly active polypeptide and non-peptide mimetics. Furthermore, the established polypeptide QSAR models and virtual screening strategy can also be applied to other peptide systems.

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd(2+) , the removal of extracellular Ca(2+) and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.