Small Molecules as Modulators of Voltage-Gated Calcium Channels in Neurological Disorders: State of the Art and Perspectives.

Voltage-gated calcium channels (VGCCs) are widely expressed in the brain, heart and vessels, smooth and skeletal muscle, as well as in endocrine cells. VGCCs mediate gene transcription, synaptic and neuronal structural plasticity, muscle contraction, the release of hormones and neurotransmitters, and membrane excitability. Therefore, it is not surprising that VGCC dysfunction results in severe pathologies, such as cardiovascular conditions, neurological and psychiatric disorders, altered glycemic levels, and abnormal smooth muscle tone. The latest research findings and clinical evidence increasingly show the critical role played by VGCCs in autism spectrum disorders, Parkinson's disease, drug addiction, pain, and epilepsy. These findings outline the importance of developing selective calcium channel inhibitors and modulators to treat such prevailing conditions of the central nervous system. Several small molecules inhibiting calcium channels are currently used in clinical practice to successfully treat pain and cardiovascular conditions. However, the limited palette of molecules available and the emerging extent of VGCC pathophysiology require the development of additional drugs targeting these channels. Here, we provide an overview of the role of calcium channels in neurological disorders and discuss possible strategies to generate novel therapeutics.

Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity.

There remains an unmet need for reliable fully synthetic adjuvants that increase lasting protective immune responses from vaccines. We previously reported a high-throughput screening for small molecules that extended nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) activation after a Toll-like receptor 4 (TLR4) ligand, lipopolysaccharide (LPS), stimulation using a human myeloid reporter cell line. We identified compounds with a conserved aminothiazole scaffold including 2D216 [N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide], which increased murine antigen-specific antibody responses when used as a co-adjuvant with LPS. Here, we examined the mechanism of action in human cells. Although 2D216 activated the major mitogen-activated protein kinases, it did not interact with common kinases and phosphatases and did not stimulate many of the pattern recognition receptors (PRRs). Instead, the mechanism of action was linked to intracellular Ca2+ elevation via Ca2+ channel(s) at the plasma membrane and nuclear translocation of the nuclear factor of activated T-cells (NFAT) as supported by RNA-seq data, analysis by reporter cells, Ca2+ flux assays, and immunoblots. Interestingly, 2D216 had minimal, if any, activity on Jurkat T cells but induced cytokine production and surface expression of costimulatory molecules on cells with antigen-presenting functions. A small series of analogs of 2D216 were tested for the ability to enhance a TLR4 ligand-stimulated autologous mixed lymphocyte reaction (MLR). In the MLR, 2E151, N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide, was more potent than 2D216. These results indicate that a small molecule that is not a direct PRR agonist can act as a co-adjuvant to an approved adjuvant to enhance human immune responses via a complementary mechanism of action.

Marjoram extract prevents ischemia reperfusion-induced myocardial damage and exerts anti-contractile effects in aorta segments of male wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Marjoram (Origanum majorana L.) is an herb traditionally used as a medicine in different countries, as Morocco and Iran, because of its beneficial cardiovascular effects. Some studies suggest that these effects are due, at least in part, to the presence of phenolic compounds such as rosmarinic acid (RA) and luteolin. AIM OF THE STUDY: To analyze the possible cardiprotective effects of a marjoram extract (ME) reducing myocardial damage after coronary ischemia-reperfusion (IR) and its possible antihypertensive effects reducing the response of aorta segments to the vasoconstrictors noradrenaline (NA) and endothelin-1 (ET-1). MATERIALS AND METHODS: Male Wistar rats (300g) were used. After sacrifice, the heart was immediately removed and mounted in a perfusion system (Langendorff). The aorta was carefully dissected and cut in 2 mm segments to perform vascular reactivity experiments. RESULTS: In the heart, ME perfusion after IR reduced heart rate and prevented IR-induced decrease of cardiac contractility, possibly through vasodilation of coronary arteries and through the upregulation of antioxidant markers in the myocardium that led to reduced apoptosis of cardiomyocytes. In the aorta, ME decreased the vasoconstrictor response to NA and ET-1 and exerted a potent anti-inflammatory and antioxidant effect. Neither RA nor 6-hydroxi-luteolin-O-glucoside, major compounds of this ME, were effective in improving cardiac contractility after IR or attenuating vasoconstriction to NA and ET-1 in aorta segments. CONCLUSION: In conclusion, ME reduces the myocardial damage induced by IR and the contractile response to vasoconstrictors in the aorta. Thus, it may be useful for the treatment of cardiovascular diseases such as myocardial infarction and hypertension.

Discovery of a polysubstituted phenyl containing novel N-phenylpyrazole scaffold as potent ryanodine receptor activator.

To develop the novel ryanodine receptors (RyRs) insecticides, encouraged by our previous research work, a series of novel N-phenylpyrazole derivatives containing a polysubstituted phenyl ring scaffold were designed and synthesized. The bioassays results indicated that some title compounds exhibited excellent insecticidal activity. For oriental armyworm (Mythimna separata), compounds 7f, 7g, 7i and 7o at 0.5 mg L-1 displayed 100% larvicidal activity, and even at 0.1 mg L-1, 7o was 30% larvicidal activity, comparable to chlorantraniliprole (30%) and better than cyantraniliprole (10%). Compounds 7f and 7o had the median lethal concentrations (LC50) of 8.83 × 10-2 and 7.12 × 10-2 mg L-1, respectively, close to chlorantraniliprole (6.79 × 10-2 mg L-1). Additionally, for diamondback moth (Plutella xylostella), the larvicidal activity of compounds 7f and 7i were 90% and 70% at 0.01 mg L-1, respectively, better than chlorantraniliprole (50%) and cyantraniliprole (40%). More impressively, the LC50 value of 7f was 4.2 × 10-3 mg L-1, slightly lower than that of chlorantraniliprole (5.0 × 10-3 mg L-1). The molecular docking between compound 7f and RyRs of diamondback moth validated our molecular designation. Furthermore, the calcium imaging experiment explored the influence of compound 7o on the calcium homeostasis in the central neurons of the third larvae of oriental armyworm. The results of this study indicated that 7o is a potent novel lead targeting at RyRs.

Congenetic Hybrids Derived from Dearomatized Isoprenylated Acylphloroglucinol with Opposite Effects on Cav3.1 Low Voltage-Gated Ca2+ Channel.

A hybrid of dearomatized isoprenylated acylphloroglucinol (DIAP) and monoterpenoid, hypatone A (1), together with its biosynthetic analogues 2-4 is characterized from Hypericum patulum. Structurally, 1 possesses an unprecedented spiro[bicyclo[3.2.1]octane-6,1'-cyclohexan]-2',4',6'-trione core as elucidated by extensive spectroscopic and X-ray crystallographic analyses. Biological studies reveal that compounds 1 and 2-4 produce opposite effects on Cav3.1 low voltage-gated Ca2+ channel, with 1 and 4, respectively, being the most potent Cav3.1 agonist and antagonist from natural products. Further studies suggest that compound 1 and its biogenetical precursor, 2, have the same binding site on Cav3.1 and that the rigid cagelike moiety at C-5 and C-6 is a key structural feature responsible for 1 being an agonist. Furthermore, 1 can normalize the pathological gating of a mutant Cav3.1 channel found in spinocerebellar ataxia 42 (SCA42), a hereditary neurodegenerative disorder with no available therapy. Collectively, our findings provide valuable tools for future studies on Cav3.1 physiology and pathophysiology, as well as afford possible leads for developing new drugs against SCA42, epilepsy, and pain.

Anticancer Activity of Euplotin C, Isolated from the Marine Ciliate Euplotes crassus, Against Human Melanoma Cells.

Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.

1,25-Dihydroxyvitamin-D3 induces brain proteomic changes in cuprizone mice during remyelination involving calcium proteins.

Dietary supplementation of vitamin D is commonly recommended to patients with multiple sclerosis. We recently found that high-dose of the hormonally active 1,25-dihydroxyvitamin-D3 (1,25D) promotes myelin repair in the cuprizone model for de- and remyelination. In the present study, we quantified 5062 proteins, of which 125 were differentially regulated in brain tissue from 1,25D treated mice during remyelination, compared to placebo. Proteins upregulated in the early remyelination phase were involved in calcium binding, e.g. calretinin (>1.3 fold, p < 0.005), S10A5 and secretagogin, and involved in mitochondrial function, e.g. NADH-ubiquinone oxidoreductase chain 3, and acyl-coenzyme A synthetase. Calretinin, S10A5 and secretagogin expression levels were characterized using immunohistochemistry. Calretinin immunoreactivity was significantly increased (>3 fold, p = 0.016) in the medial septal nuclei of 1,25D treated mice in the early remyelination phase. Our results indicate that vitamin D may influence remyelination by mechanisms involving an increase in calretinin expression and potentially other calcium binding proteins.

EBIO Does Not Induce Cardiomyogenesis in Human Pluripotent Stem Cells but Modulates Cardiac Subtype Enrichment by Lineage-Selective Survival.

Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.

Antinociceptive effect of a novel armed spider peptide Tx3-5 in pathological pain models in mice.

The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 µg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.

Calcitriol attenuates cardiac remodeling and dysfunction in a murine model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex reproductive and metabolic disorder affecting 10 % of reproductive-aged women, and is well associated with an increased prevalence of cardiovascular risk factors. However, there are few data concerning the direct association of PCOS with cardiac pathologies. The present study aims to investigate the changes in cardiac structure, function, and cardiomyocyte survival in a PCOS model, and explore the possible effect of calcitriol administration on these changes. PCOS was induced in C57BL/6J female mice by chronic dihydrotestosterone administration, as evidenced by irregular estrous cycles, obesity and dyslipidemia. PCOS mice progressively developed cardiac abnormalities including cardiac hypertrophy, interstitial fibrosis, myocardial apoptosis, and cardiac dysfunction. Conversely, concomitant administration of calcitriol significantly attenuated cardiac remodeling and cardiomyocyte apoptosis, and improved cardiac function. Molecular analysis revealed that the beneficial effect of calcitriol was associated with normalized autophagy function by increasing phosphorylation levels of AMP-activated protein kinase and inhibiting phosphorylation levels of mammalian target of rapamycin complex. Our findings provide the first evidence for the presence of cardiac remodeling in a PCOS model, and vitamin D supplementation may be a potential therapeutic strategy for the prevention and treatment of PCOS-related cardiac remodeling.

Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D3 (VD3)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

Magnesium sulfate provides neuroprotection in lipopolysaccharide-activated primary microglia by inhibiting NF-κB pathway.

BACKGROUND: Magnesium sulfate has been used as an anticonvulsant in severe preeclamptic or eclamptic women prior to surgical trauma, but its effects on neuroinflammation is not well defined. In the present study, we investigated the neuroprotective effects of magnesium sulfate in lipopolysaccharide (LPS)-induced microglia and explored the underlying mechanism. MATERIALS AND METHODS: Microglia was incubated with LPS in the presence or absence of various concentrations of magnesium sulfate, or L-type calcium channel activator BAY-K8644. The levels of inflammatory mediators, such as nitric oxide, prostaglandin E2, interleukin 1ß, and tumor necrosis factor α, were measured using enzyme-linked immunosorbent assay. The expression of inducible nitric oxide synthase mRNA was detected by reverse-transcription polymerase chain reaction. Nuclear factor κB (NF-κB) activity in the nuclear extract of microglia was detected by NF-κB p50/p65 transcription factor assay kit. RESULTS: Magnesium sulfate at 5 and 10 mmol/L significantly inhibited the release of nitric oxide, prostaglandin E2, interleukin 1ß, and tumor necrosis factor α, and the expression of inducible nitric oxide synthase mRNA in LPS-activated microglia. Furthermore, magnesium sulfate inhibited the translocation of NF-κB from the cytoplasm to the nucleus in a dose-dependent manner. Notably, these effects were significantly reversed by L-type calcium channel activator BAY-K8644. CONCLUSIONS: Magnesium sulfate protects microglia against LPS-induced release of inflammatory mediators, and these effects may be mediated by inhibiting L-type calcium channels and NF-κB signaling.

The slow afterhyperpolarization: a target of ß1-adrenergic signaling in hippocampus-dependent memory retrieval.

In rodents, adrenergic signaling by norepinephrine (NE) in the hippocampus is required for the retrieval of intermediate-term memory. NE promotes retrieval via the stimulation of ß1-adrenergic receptors, the production of cAMP, and the activation of both protein kinase A (PKA) and the exchange protein activated by cAMP. However, a final effector for this signaling pathway has not been identified. Among the many targets of adrenergic signaling in the hippocampus, the slow afterhyperpolarization (sAHP) is an appealing candidate because its reduction by ß1 signaling enhances excitatory neurotransmission. Here we report that reducing the sAHP is critical for the facilitation of retrieval by NE. Direct blockers of the sAHP, as well as blockers of the L-type voltage-dependent calcium influx that activates the sAHP, rescue retrieval in mutant mice lacking either NE or the ß1 receptor. Complementary to this, a facilitator of L-type calcium influx impairs retrieval in wild-type mice. In addition, we examined the role of NE in the learning-related reduction of the sAHP observed ex vivo in hippocampal slices. We find that this reduction in the sAHP depends on the induction of persistent PKA activity specifically in conditioned slices. Interestingly, this persistent PKA activity is induced by NE/ß1 signaling during slice preparation rather than during learning. These observations suggest that the reduction in the sAHP may not be present autonomously in vivo, but is likely induced by neuromodulatory input, which is consistent with the idea that NE is required in vivo for reduction of the sAHP during memory retrieval.

Stimulation of human TRPA1 channels by clinical concentrations of the antirheumatic drug auranofin.

Gold compounds, which were widely used to treat rheumatoid arthritis, have been recently used as experimental agents for tumor treatment. Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1) is a Ca(2+)-permeable ion channel that senses acute and inflammatory pain signals. Electrophilic compounds such as mustard oil and cinnamaldehyde activate TRPA1 by interacting with TRPA1 cysteine residues. Here we investigate the effects of the gold compound auranofin (AUR) on TRPA1 channels. Intracellular Ca(2+) and whole cell patch-clamp recordings were performed on human embryonic kidney cells transiently expressed with TRPA1, TRP melastatin 8 (TRPM8), and vanilloid type TRP (TRPV1-4) channels. AUR stimulated TRPA1 in a concentration-dependent manner with a half-maximum potency of around 1.0 µM. The AUR-induced response was effectively blocked by HC030031, a TRPA1 antagonist. On the other hand, AUR failed to activate TRPM8 and TRPV1-4 channels, which are highly expressed in sensory neurons as nociceptors. The stimulatory effect on TRPA1 channels depended on the C414, C421, C621, and C633 cysteine residues and not on the inhibition of thioredoxin reductase by AUR. Moreover, AUR effectively activated TRPA1 channels expressed in human differentiated neuroblastoma cell lines. The study shows that AUR is a potent stimulator of TRPA1 channels.

Astemizole synergizes calcitriol antiproliferative activity by inhibiting CYP24A1 and upregulating VDR: a novel approach for breast cancer therapy.

BACKGROUND: Calcitriol antiproliferative effects include inhibition of the oncogenic ether-à-go-go-1 potassium channel (Eag1) expression, which is necessary for cell cycle progression and tumorigenesis. Astemizole, a new promising antineoplastic drug, targets Eag1 by blocking ion currents. Herein, we characterized the interaction between calcitriol and astemizole as well as their conjoint antiproliferative action in SUM-229PE, T-47D and primary tumor-derived breast cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Molecular markers were studied by immunocytochemistry, Western blot and real time PCR. Inhibitory concentrations were determined by dose-response curves and metabolic activity assays. At clinically achievable drug concentrations, synergistic antiproliferative interaction was observed between calcitriol and astemizole, as calculated by combination index analysis (CI <1). Astemizole significantly enhanced calcitriol's growth-inhibitory effects (3-11 folds, P<0.01). Mean IC(20) values were 1.82 ± 2.41 nM and 1.62 ± 0.75 µM; for calcitriol (in estrogen receptor negative cells) and astemizole, respectively. Real time PCR showed that both drugs alone downregulated, while simultaneous treatment further reduced Ki-67 and Eag1 gene expression (P<0.05). Astemizole inhibited basal and calcitriol-induced CYP24A1 and CYP3A4 mRNA expression (cytochromes involved in calcitriol and astemizole degradation) in breast and hepatoma cancer cells, respectively, while upregulated vitamin D receptor (VDR) expression. CONCLUSIONS/SIGNIFICANCE: Astemizole synergized calcitriol antiproliferative effects by downregulating CYP24A1, upregulating VDR and targeting Eag1. This study provides insight into the molecular mechanisms involved in astemizole-calcitriol combined antineoplastic effect, offering scientific support to test both compounds in combination in further preclinical and clinical studies of neoplasms expressing VDR and Eag1. VDR-negative tumors might also be sensitized to calcitriol antineoplastic effects by the use of astemizole. Herein we suggest a novel combined adjuvant therapy for the management of VDR/Eag1-expressing breast cancer tumors. Since astemizole improves calcitriol bioavailability and activity, decreased calcitriol dosing is advised for conjoint administration.

Application of human stem cell-derived cardiomyocytes in safety pharmacology requires caution beyond hERG.

Human embryonic stem cell-derived cardiomyocytes (hESC-CM) have been proposed as a new model for safety pharmacology. So far, a thorough description of their basic electrophysiology and extensive testing, and mechanistic explanations, of their overall pro-arrhythmic ability is lacking. Under standardized conditions, we have evaluated the sensitivity of hESC-CM to proarrhythmic provocations by blockade of hERG and other channels. Using voltage patch clamp, some ion current densities (pA/pF) in hESC-CM were comparable to adult CM: I(Kr) (-12.5 ± 6.9), I(Ks) (0.65 ± 0.12), I(Na,peak) (-72 ± 21), I(Na,late) (-1.10 ± 0.36), and I(Ca,L) (-4.3 ± 0.6). I(f) density was larger (-10 ± 1.1) and I(K1) not existent or very small (-2.67 ± 0.3). The low I(K1) density was corroborated by low KCNJ2 mRNA levels. Effects of pro-arrhythmic compounds on action potential (AP) parameters and provocation of early afterdepolarizations (EADs) revealed that Chromanol293B (100 µmol/l) and Bay K8644 (1 µmol/l) both significantly prolonged APD(90). ATX-II (<1 µmol/l ) and BaCl(2) (10 µmol/l ) had no effect on APD. The only compound that triggered EADs was hERG blocker Cisapride. Computer simulations and AP clamp showed that the immature AP of hESC-CM prevents proper functioning of I(Na)-channels, and result in lower peak/maximal currents of several other channels, compared to the adult situation. Lack of functional I(K1) channels and shifted I(Na) channel activation cause a rather immature electrophysiological phenotype in hESC-CM, and thereby limits the potential of this model to respond accurately to pro-arrhythmic triggers other than hERG block. Maturation of the electrical phenotype is a prerequiste for future implementation of the model in arrhythmogenic safety testing.

Thalamic ryanodine receptors are involved in controlling the tonic firing of thalamocortical neurons and inflammatory pain signal processing.

Ryanodine receptors (RyRs) are highly conductive intracellular Ca(2+) release channels which are widely expressed in the CNS. They rapidly increase the intracellular Ca(2+) concentrations in neuronal cells in response to Ca(2+) influx through voltage-gated Ca(2+) channels. A previous study reported that RyRs were expressed in thalamocortical (TC) neurons, but their physiological function has remained elusive. Here, we show that the activation of RyRs in TC neurons in mice decreases their tonic firing rate while blocking them induces the opposite response. Furthermore, activation of RyRs in ventroposteriomedial/ventroposteriolateral nuclei reduces the behavioral responses to inflammatory pain and blocking them increases the responses. This study highlights the importance of the intracellular Ca(2+) release via RyRs in controlling the excitability of TC neurons and in inflammatory pain signal processing in the thalamus.

Titanium nitride oxide coating on rough titanium stimulates the proliferation of human primary osteoblasts.

OBJECTIVES: Titanium is widely used in contemporary endosseous implantology and there is considerable thrust to further promote osseointegration by implant surface modifications. The aim of this study was to evaluate the effect of a titanium-nitride-oxide (TiNOx) coating on commercially pure microroughened titanium by assessing the proliferation and differentiation of human primary osteoblasts. MATERIALS AND METHODS: Cell proliferation, gene expression, alkaline phosphatase activity, osteoprotegerin and osteocalcin secretion were analyzed for a time course of 3 weeks, with or without additional stimulation by 1.25(OH)(2) vitamin D(3) 100 nM. RESULTS: A 1.5-fold increase in the proliferation rate of cells grown on TiNOx-coated titanium as compared with uncoated surfaces was observed. SEM views indicated that the cells' normal morphology with their numerous extensions was maintained. The differentiation process on the TiNOx surface was only affected to a minor degree and translated into a slight delay in osteoblast maturation when compared to uncoated titanium. CONCLUSION: Pending confirmation of these results in vivo, TiNOx coatings could potentially accelerate and enhance osseointegration.

Calcitriol (1,25-dihydroxycholecalciferol) enhances mast cell tumour chemotherapy and receptor tyrosine kinase inhibitor activity in vitro and has single-agent activity against spontaneously occurring canine mast cell tumours.

Calcitriol potentiates the effect of multiple chemotherapy agents in a variety of tumour models. In this study, we examine whether calcitriol increases chemotherapy or tyrosine kinase inhibitor in vitro cytotoxicity in canine mastocytoma C2 cells. We also evaluate the in vivo effect of DN101, a highly concentrated oral formulation of calcitriol designed specifically for cancer therapy, as a single-agent therapy in dogs with mast cell tumours (MCTs). Calcitriol exhibits synergistic, antiproliferative activity when used in combination with CCNU, vinblastine, imatinib or toceranib in vitro. The concentrations required for 50% growth inhibition were generally two- to six-fold lower when the drugs were used in combination than when used individually. High-dose oral calcitriol induced remission in 4 of 10 dogs (one complete remission, three partial remissions), although the majority experienced toxicity, necessitating discontinuation of the trial. Further evaluation of calcitriol in combination therapy for dogs with MCTs is warranted.

A selective small molecule glucagon-like peptide-1 secretagogue acting via depolarization-coupled Ca(2+) influx.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca(2+) release from intracellular stores, but to extracellular Ca(2+) influx. The response was inhibited by the presence of either L-type Ca(2+) channel blockers or K(+) ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Galphas- nor Galphaq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca(2+) influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.