Inhibition Of Calcium Influx Research Articles

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.

The effects of bisphenols on the cardiovascular system ex vivo and in vivo

The human population is regularly exposed to bisphenols. The first compound of this class, bisphenol A, is burdened by numerous reports of its potential toxicity and has been hence replaced by its analogues, so-called next generation bisphenols. Their widespread use has made them pervasive throughout the environment. These endocrine disrupting chemicals can affect the cardiovascular system, and hence the aim of this study was to test 14 bisphenols (A, AF, AP, B, BP, C, E, F, G, M, P, PH, S and Z), and compare their effects in vitro (human and rat cell lines), ex vivo (isolated rat aorta) and in vivo (Wistar Han rats, acutely or chronically exposed to low environmental and high toxic doses). The majority of the tested bisphenols relaxed rat aorta, but their potency varied markedly. The most potent compound, bisphenol AF, had an EC50 of 57 μM. The mechanism of action was likely based on the inhibition of calcium influx via L-type calcium channels. The cytotoxicity of bisphenols towards 4 human and rat cell lines (H9c2, A-10, MCF7/S0.5 and MCF7/182R-6) showed variable potencies ranging from units of micromolar to millimolar concentrations. Based on these data, an effect on arterial blood pressure and possible cardiotoxicity was expected. Contrarily, the in vivo acute effects of three doses (0.005, 0.05 and 2.5 mg/kg) of bisphenol AF and 3 other analogues (A, S and F) on the cardiovascular system were rather biologically negligible. The most potent bisphenol, AF, was also administered chronically at a dose of 2.5 mg/kg for 4 weeks to rats, but had no impact on arterial blood pressure. Our results showed that bisphenols can relax vascular smooth muscles, but the effective concentrations are too high to produce clear cardiovascular effects in relation to common biological exposure as was confirmed with the most potent bisphenol AF.

Chitosan triggers actin remodelling and activation of defence genes that is repressed by calcium influx in grapevine cells

Defence to pathogens must be specific. In the past, we have dissected early signalling deployed by bacterial elicitors in a grapevine cell system. In the current work, we asked, how defence of fungi differs. Fungal diseases of grapevine pose great challenges for global viticulture and require massive plant protection measures. Plant cells are able to sense chitin, a central component of fungal cell walls and respond by activation of basal defence. We, therefore mapped early defence responses evoked by chitosan, a chitin fragment able to bind to chitin receptors. We found an activation of calcium influx, monitored by extracellular alkalinisation due to a co-transport of protons, remodelling of actin (but not of microtubules), and the activation of transcripts for phytoalexin synthesis, jasmonate-signalling, salicylate signalling, and chitinase. Interestingly, Gadolinium, an inhibitor of calcium influx, can inhibit extracellular alkalinisation in response to chitosan, while the induction of the phytoalexin synthesis transcripts was specifically promoted. In contrast, both DMSO and benzyl alcohol, compounds known to modulate membrane fluidity, partially inhibited the transcript responses to chitosan. We discuss these data with a model, where chitosan deploys signalling culminating in activation of defence related transcripts, but at the same time activates calcium influx that negatively feeds back on the same signal chain, which might be a mechanism to achieve a temporal signature that is rapid, but transient.

Relaxation mechanisms of chloroform root extracts of Prangos heyniae and Prangos uechtritzii on mouse corpus cavernosum.

Erectile dysfunction (ED) is the inability to achieve/maintain an erection. Because of the side effects, interactions, or ineffectiveness of currently used drugs, novel drug discovery studies are ongoing. The roots of Turkish endemic plants Prangos uechtritzii and Prangos heyniae are traditionally used as aphrodisiacs in Anatolia and contain coumarin-like relaxant compounds. This study aims to reveal the relaxant effect mechanisms of chloroform root extracts of P. heyniae (Ph-CE) and P. uechtritzii (Pu-CE). Isolated organ bath experiments were performed on Swiss albino mouse corpus cavernosum by DMT strip myograph. Relaxant responses to extract (10-7 -10-4 g/ml) were obtained in the presence/absence of NO and H2 S synthesis inhibitors nitro-l-arginine methyl ester (l-NAME, 100 μM) and aminooxyacetic acid (AOAA, 10 mM) respectively. Sodium nitroprusside (SNP, 10-9 to 10-4 M) and Na2 S (10-6 to 3× 10-3 M)-induced relaxations and CaCl2 (10-6 to 10-4 M), KCl (10-2.1 to 10-0.9 M) and phenylephrine (3× 10-8 to 3× 10-5 M)-induced contractions were taken in the presence/absence of the extracts (10-4 g/ml). Relaxations induced by Ph-CE but not by Pu-CE were inhibited in the presence of l-NAME and AOAA. Ph-CE increased Na2 S- and SNP-induced relaxations. Ph-CE and Pu-CE decreased the contractions of KCl, phenylephrine, and CaCl2 . It was concluded that NO and H2 S synthesis/downstream mechanisms play roles in relaxations of Ph-CE but not in Pu-CE-induced relaxations. Inhibition of calcium influx appears to be involved in the relaxant effect of Ph-CE and Pu-CE. Since the extracts act directly by relaxing smooth muscle or through H2 S as well as NO, they may be a potential therapeutic agent in diseases such as ED where the bioavailability of NO is impaired.

Regulated extravascular microenvironment via reversible thermosensitive hydrogel for inhibiting calcium influx and vasospasm.

Arterial vasospasm after microsurgery can cause severe obstruction of blood flow manifested as low tissue temperature, leading to tissue necrosis. The timely discovery and synchronized treatment become pivotal. In this study, a reversible, intelligent, responsive thermosensitive hydrogel system is constructed employing both the gel–sol transition and the sol–gel transition. The “reversible thermosensitive (RTS)” hydrogel loaded with verapamil hydrochloride is designed to dynamically and continuously regulate the extravascular microenvironment by inhibiting extracellular calcium influx. After accurate implantation and following in situ gelation, the RTS hydrogel reverses to the sol state causing massive drug release to inhibit vasospasm when the tissue temperature drops to the predetermined transition temperature. Subsequent restoration of the blood supply alleviates further tissue injury. Before the temperature drops, the RTS hydrogel maintains the gel state as a sustained-release reservoir to prevent vasospasm. The inhibition of calcium influx and vasospasm in vitro and in vivo is demonstrated using vascular smooth muscle cells, mice mesenteric arterial rings, and vascular ultrasonic Doppler detection. Subsequent animal experiments demonstrate that RTS hydrogel can promote tissue survival and alleviate tissue injury responding to temperature change. Therefore, this RTS hydrogel holds therapeutic potential for diseases requiring timely detection of temperature change.

Dopamine receptor D2 regulates GLUA1-containing AMPA receptor trafficking and central sensitization through the PI3K signaling pathway in a male rat model of chronic migraine

BackgroundThe pathogenesis of chronic migraine remains unresolved. Recent studies have affirmed the contribution of GLUA1-containing AMPA receptors to chronic migraine. The dopamine D2 receptor, a member of G protein-coupled receptor superfamily, has been proven to have an analgesic effect on pathological headaches. The present work investigated the exact role of the dopamine D2 receptor in chronic migraine and its effect on GLUA1-containing AMPA receptor trafficking.MethodsA chronic migraine model was established by repeated inflammatory soup stimulation. Mechanical, periorbital, and thermal pain thresholds were assessed by the application of von Frey filaments and radiant heat. The mRNA and protein expression levels of the dopamine D2 receptor were analyzed by qRT‒PCR and western blotting. Colocalization of the dopamine D2 receptor and the GLUA1-containing AMPAR was observed by immunofluorescence. A dopamine D2 receptor agonist (quinpirole) and antagonist (sulpiride), a PI3K inhibitor (LY294002), a PI3K pathway agonist (740YP), and a GLUA1-containing AMPAR antagonist (NASPM) were administered to confirm the effects of the dopamine D2 receptor, the PI3K pathway and GULA1 on central sensitization and the GLUA1-containing AMPAR trafficking. Transmission electron microscopy and Golgi-Cox staining were applied to assess the impact of the dopamine D2 receptor and PI3K pathway on synaptic morphology. Fluo-4-AM was used to clarify the role of the dopamine D2 receptor and PI3K signaling on neuronal calcium influx. The Src family kinase (SFK) inhibitor PP2 was used to explore the effect of Src kinase on GLUA1-containing AMPAR trafficking and the PI3K signaling pathway.ResultsInflammatory soup stimulation significantly reduced pain thresholds in rats, accompanied by an increase in PI3K-P110β subunit expression, loss of dopamine receptor D2 expression, and enhanced GLUA1-containing AMPA receptor trafficking in the trigeminal nucleus caudalis (TNC). The dopamine D2 receptor colocalized with the GLUA1-containing AMPA receptor in the TNC; quinpirole, LY294002, and NASPM alleviated pain hypersensitivity and reduced GLUA1-containing AMPA receptor trafficking in chronic migraine rats. Sulpiride aggravated pain hypersensitivity and enhanced GLUA1 trafficking in CM rats. Importantly, the anti-injury and central sensitization-mitigating effects of quinpirole were reversed by 740YP. Both quinpirole and LY294002 inhibited calcium influx to neurons and modulated the synaptic morphology in the TNC. Additional results suggested that DRD2 may regulate PI3K signaling through Src family kinases.ConclusionModulation of GLUA1-containing AMPA receptor trafficking and central sensitization by the dopamine D2 receptor via the PI3K signaling pathway may contribute to the pathogenesis of chronic migraine in rats, and the dopamine D2 receptor could be a valuable candidate for chronic migraine treatment.

Safranal Induces Vasorelaxation by Inhibiting Ca2+ Influx and Na+/Ca2+ Exchanger in Isolated Rat Aortic Rings.

Introduction: Safranal, which endows saffron its unique aroma, causes vasodilatation and has a hypotensive effect in animal studies, but the mechanisms of these effects are unknown. In this study, we investigated the mechanisms of safranal vasodilation. Methods: Isolated rat endothelium-intact or -denuded aortic rings were precontracted with phenylephrine and then relaxed with safranal. To further assess the involvement of nitric oxide, prostaglandins, guanylate cyclase, and phospholipase A2 in safranal-induced vasodilation, aortic rings were preincubated with L-NAME, indomethacin, methylene blue, or quinacrine, respectively, then precontracted with phenylephrine, and safranal concentration–response curves were established. To explore the effects of safranal on Ca2+ influx, phenylephrine and CaCl2 concentration–response curves were established in the presence of safranal. Furthermore, the effect of safranal on aortic rings in the presence of ouabain, a Na+-K+ ATPase inhibitor, was studied to explore the contribution of Na+/Ca2+ exchanger to this vasodilation. Results: Safranal caused vasodilation in endothelium-intact and endothelium-denuded aortic rings. The vasodilation was not eliminated by pretreatment with L-NAME, indomethacin, methylene blue, or quinacrine, indicating the lack of a role for NO/cGMP. Safranal significantly inhibited the maximum contractions induced by phenylephrine, or by CaCl2 in Ca2+-free depolarizing buffer. Safranal also relaxed contractions induced by ouabain, but pretreatment with safranal totally abolished the development of ouabain contractions. Discussion/Conclusion: Inhibition of Na+-K+ ATPase by ouabain leads to the accumulation of Na+ intracellularly, forcing the Na+/Ca2+ exchanger to work in reverse mode, thus causing a contraction. Inhibition of the development of this contraction by preincubation with safranal indicates that safranal inhibited the Na+/Ca2+ exchanger. We conclude that safranal vasodilation is mediated by the inhibition of calcium influx from extracellular space through L-type Ca2+ channels and by the inhibition of the Na+/Ca2+ exchanger.

Propofol pretreatment alleviates mast cell degranulation by inhibiting SOC to protect the myocardium from ischemia-reperfusion injury.

Propofol (PPF) has a protective effect on myocardial ischemia-reperfusion (I/R) injury (MIRI). The purpose of this study was to investigate whether the myocardial protective effect of propofol is related to the inhibition of mast cell degranulation and explore the possible mechanisms involved. Our in vivo results showed that compared with the sham group, cardiac function, infarct size, histopathological damage, apoptosis, and markers of myocardial necrosis were significantly increased in the ischemia-reperfusion group, and propofol pretreatment alleviated these effects. In the coculture system, propofol-treated mast cells reduced their tryptase activity, resulting in cardiomyocyte protective effects, such as decreased apoptosis of cardiomyocytes and decreased expression of myocardial necrosis markers. Finally, experimental results in vitro revealed that thapsigargin (TG) can increase mast cell degranulation, tryptase release, calcium ion concentration, and the expression of STIM1 and Orai1 induced by H/R, but propofol pretreatment can partially reverse the above effects. These results suggested that the cardioprotective effect of propofol is achieved in part by inhibiting calcium influx through store-operated Ca2+ channels (SOCs) and thus alleviating mast cell degranulation.

Schwarzinicine A inhibits transient receptor potential canonical channels and exhibits overt vasorelaxation effects.

This study investigated the vasorelaxant effects of schwarzinicine A, an alkaloid recently reported from Ficus schwarzii Koord. Regulation of calcium homeostasis in vascular smooth muscle cells (VSMC) is viewed as one of the main mechanisms for controlling blood pressure. L‐type voltage‐gated calcium channel (VGCC) blockers are commonly used for controlling hypertension. Recently, the transient receptor potential canonical (TRPC) channels were found in blood vessels of different animal species with evidence of their roles in the regulation of vascular contractility. In this study, we studied the mechanism of actions of schwarzinicine A focusing on its regulation of L‐type VGCC and TRPC channels. Schwarzinicine A exhibited the highest vasorelaxant effect (123.1%) compared to other calcium channel blockers. It also overtly attenuated calcium‐induced contractions of the rat isolated aortae in a calcium‐free environment showing its mechanism to inhibit calcium influx. Fluorometric intracellular calcium recordings confirmed its inhibition of hTRPC3‐, hTRPC4‐, hTRPC5‐ and hTRPC6‐mediated calcium influx into HEK cells with IC50 values of 3, 17, 19 and 7 μM, respectively. The evidence gathered in this study suggests that schwarzinicine A blocks multiple TRPC channels and L‐type VGCC to exert a significant vascular relaxation response.

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor.

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 104 cells/3 μL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,β-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,β-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,β-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.

Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycota), Cold-Water Extract Relaxes the Rat Isolated Bladder

Ophiocordyceps sinensis is a popular medicinal mushroom used for various health conditions, including alleviation of frequent urination, which is a major symptom of overactive bladder (OAB) syndrome. This study aimed to investigate the effect of O. sinensis (OCS02 cultivar) cold-water extract (CWE) against bladder contractility using the organ bath technique. The bladder was removed from male Sprague-Dawley rats and cut into longitudinal strips of 2 mm × 8 mm. In some experiments, the urothelium was removed to study its role in CWE-induced responses. CWE elicited a biphasic response consisting of an immediate, transient contraction that was followed by a sustained relaxation in bladder strips precontracted with carbachol, a muscarinic agonist. Removal of urothelium did not alter the magnitude of the contractile response but significantly attenuated the relaxation response. In the presence of L-NAME (nitric oxide synthase inhibitor) and sodium nitroprusside (nitric oxide donor), CWE-induced transient contraction was enhanced, whereas the relaxation response was significantly reduced. Following preincubation with CWE, the amplitude and the frequency of the spontaneous myogenic contractions induced by carbachol, as well as the contractile response toward calcium, were significantly suppressed. Findings from this study show that the urothelium plays a role in the relaxant effect of CWE. Its mechanisms of action include the regulation of nitric oxide and inhibition of calcium influx.

Piezo1 initiates platelet hyperreactivity and accelerates thrombosis in hypertension

BackgroundThrombosis is the pathological basis of cardiovascular and cerebrovascular diseases, which seriously threaten human life and health. Among them, nearly half of cardiovascular disease patients suffer from severe hypertension complications. Hypertension is thought to cause abnormal platelet activation and increases the risk of thrombosis, but the related mechanism is still vague. ObjectivesThis study hypothesized that the abnormal hemodynamics of blood under hypertension might affect platelet function and accelerate thrombosis by activating mechanoreceptor Piezo1. MethodsTo assess the activation effect of hypertension on mechanoreceptor Piezo1, we injected Piezo1 agonist Yoda1 and antagonist GsMTx‐4 through the tail vein, then examined the platelet activation status and thrombosis. ResultsOur results displayed that antagonist GsMTx‐4 effectively inhibited calcium influx caused by hypertension and agonist Yoda1. Antithrombotic studies proved that the inhibition of Piezo1 effectively inhibited arterial thrombosis and reduced the infarct size of stroke in hypertensive mice. ConclusionsOur study explains the activation of mechanoreceptor Piezo1 under hypertension is the key to abnormal platelet activation and thrombosis while providing novel platelet intervention strategies to prevent thrombosis.

CSIG-24. TARGETED INHIBITION OF CDK5-MEDIATED REGULATION OF HUMAN ENDOGENOUS RETROVIRUS K (HML-2) ENVELOPE PROTEIN IN ATYPICAL TERATOID RHABDOID TUMOR

Abstract Atypical teratoid rhabdoid tumor (ATRT) is a pediatric brain tumor with a high mortality rate characterized by mutations in/ deletions of SWI/SNF matrix-associated actin-dependent regulator of chromatin sub-family B member 1 (SMARCB1). We previously showed that loss of SMARCB1 causes up-regulation and release of HML-2 subfamily of human endogenous retrovirus K envelope protein (HML-2 ENV), resulting in maintenance of pluripotency. Here, we investigated intracellular trafficking and release of HML-2 ENV. Further, we demonstrate two potential therapeutic strategies to decrease intracellular HML-2 ENV: 1) inhibition of calcium influx by ouabain, a cardiac glycoside toxic to neural stem cells, and 2) targeted inhibition of cyclin-dependent kinase 5 (CDK5), which is restricted to neurons by p35, its activator protein, by TP5. ATRT cell lines and tumor tissue obtained from patients were confirmed for SMARCB1 loss and increased HML-2 ENV. Cell viability and intracellular HML-2 ENV concentration were measured after treatment with ouabain and TP5 (CDK5 antagonist). We evaluated the calcium-mediated effect of ouabain on HML-2 intracellular concentration by treating the cells with ouabain, the calcium chelators calcimycin and EGTA, and calpeptin, a calpain inhibitor, which activates CDK5, and measuring HML-2 ENV and p35. We evaluated HML-2 ENV for a CDK5 consensus phosphorylation site and performed co-immunoprecipitation to evaluate direct interaction. We evaluated activity of CDK5 in ATRT cell lines by autoradiography. Both Ouabain and TP5 caused a decrease in cell viability in a dose-dependent manner. Further, ouabain treatment decreased HML-2 ENV intracellular concentration. We found that HML-2 ENV contains a consensus phosphorylation site for CDK5. We discovered that HML-2 ENV was bound to CDK5. We established that ATRT cell lines had hyperactive CDK5. Finally, we established that the effect of ouabain on HML-2 ENV was due to indirect inhibition of calcium-mediated activation of calpain and thus CDK5.

Steroidal Antagonists of Progesterone- and Prostaglandin E1-Induced Activation of the Cation Channel of Sperm.

The cation channel of sperm (CatSper) is the principal entry point for calcium in human spermatozoa and its proper function is essential for successful fertilization. As CatSper is potently activated by progesterone, we evaluated a range of steroids to define the structure-activity relationships for channel activation and found that CatSper is activated by a broad range of steroids with diverse structural modifications. By testing steroids that failed to elicit calcium influx as inhibitors of channel activation, we discovered that medroxyprogesterone acetate, levonorgestrel, and aldosterone inhibited calcium influx produced by progesterone, prostaglandin E1, and the fungal natural product l-sirenin, but these steroidal inhibitors failed to prevent calcium influx in response to elevated K+ and pH. In contrast to these steroid antagonists, we demonstrated for the first time that the T-type calcium channel blocker ML218 acts similarly to mibefradil, blocking CatSper channels activated by both ligands and alkalinization/depolarization. These T-type calcium channel blockers produced an insurmountable blockade of CatSper, whereas the three steroids produced antagonism that was surmountable by increasing concentrations of each activator, indicating that the steroids selectively antagonize ligand-induced activation of CatSper rather than blocking channel function. Both the channel blockers and the steroid antagonists markedly reduced hyperactivated motility of human sperm assessed by computer-aided sperm analysis, consistent with inhibition of CatSper activation. Unlike the channel blockers mibefradil and ML218, which reduced total and progressive motility, medroxyprogesterone acetate, levonorgestrel, and aldosterone had little effect on these motility parameters, indicating that these steroids are selective inhibitors of hyperactivated sperm motility.SIGNIFICANCE STATEMENTThe steroids medroxyprogesterone acetate, levonorgestrel, and aldosterone selectively antagonize progesterone- and prostaglandin E1-induced calcium influx through the CatSper cation channel in human sperm. In contrast to T-type calcium channel blockers that prevent all modes of CatSper activation, these steroid CatSper antagonists preferentially reduce hyperactivated sperm motility, which is required for fertilization. The discovery of competitive antagonists of ligand-induced CatSper activation provides starting points for future discovery of male contraceptive agents acting by this unique mechanism.

Ethanol and opioids do not act synergistically to depress excitation in carotid body type I cells.

ObjectiveThe combination of opioids and ethanol can synergistically depress breathing and the acute ventilatory response to hypoxia. Multiple studies have shown that the underlying mechanisms for this may involve calcium channel inhibition in central neurons. But we have previously identified opioid receptors in the carotid bodies and shown that their activation inhibits calcium influx into the chemosensitive cells. Given that the carotid bodies contribute to the drive to breathe and underpin the acute hypoxic ventilatory response, we hypothesized that ethanol and opioids may act synergistically in these peripheral sensory organs to further inhibit calcium influx and therefore inhibit ventilation.MethodsCarotid bodies were removed from 56 Sprague–Dawley rats (1021 days old) and then enzymatically dissociated to allow calcium imaging of isolated chemosensitive type I cells. Cells were stimulated with high K+ in the presence and absence of the µ-opioid agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) (10 µM), a maximal sublethal concentration of ethanol (3 g L-1, 65.1 mM) or a combination of both.ResultsDAMGO alone significantly inhibited Ca2+ influx but this effect was not potentiated by the high concentration of ethanol.ConclusionThese results indicate for the first time that while opioids may suppress breathing via an action at the level of the carotid bodies, ethanol is unlikely to potentiate inhibition via this pathway. Thus, the synergistic effects of ethanol and opioids on ventilatory parameters are likely mediated by central rather than peripheral actions.

Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development.

Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identif ied these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might inf luence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium inf lux inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1–3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative inf luence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the f luorophores such as red f luorescent protein (RFP) and yellow f luorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by inf iltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modif ication or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

Targeting regulator of G protein signaling 1 in tumor-specific T cells enhances their trafficking to breast cancer.

Reduced infiltration of anti-tumor lymphocytes remains a major cause of tumor immune evasion and is correlated with poor cancer survival. Here, we found that upregulation of regulator of G protein signaling (RGS)1 in helper TH1 cells and cytotoxic T lymphocytes (CTLs) reduced their trafficking to and survival in tumors and was associated with shorter survival of patients with breast and lung cancer. RGS1 was upregulated by type II interferon (IFN)-signal transducer and activator of transcription (STAT)1 signaling and impaired trafficking of circulating T cells to tumors by inhibiting calcium influx and suppressing activation of the kinases ERK and AKT. RGS1 knockdown in adoptively transferred tumor-specific CTLs significantly increased their infiltration and survival in breast and lung tumor grafts and effectively inhibited tumor growth in vivo, which was further improved when combined with programmed death ligand (PD-L)1 checkpoint inhibition. Our findings reveal RGS1 is important for tumor immune evasion and suggest that targeting RGS1 may provide a new strategy for tumor immunotherapy.

P008 Store-operated calcium entry controls immune cell function and activation in Inflammatory Bowel Disease

Abstract Background Inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) and Crohn’s disease (CD), both representing major clinical challenges in need of new treatment modalities to improve patient care. Store-operated calcium entry (SOCE) is the predominant calcium influx pathway in immune cells regulating many of their functional properties. Conditional knockout mice, in which SOCE signaling components are deleted in T cells, have revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, it is currently unknown whether the pharmacologic inhibition of SOCE is a suitable drug target in IBD and it remains elusive, which immune cell subsets are affected by the pharmaceutical blockade of SOCE. Therefore, we here aimed to investigate the effects of the SOCE inhibitor BTP-2 on the function and activation of lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes (LPMC) were isolated from UC and CD patients undergoing colon resection. Cells were ex -vivo stimulated with PMA/Ionomycin in the presence or absence of increasing concentrations of BTP-2, fixed and stored at -80°C until acquisition by mass cytometry. LPMCs were stained using 37 lineage and functional markers targeting B, T, NK or myeloid cells and the resulting flow cytometry standard (FCS) files were analyzed by using cytobank and the R/Bioconductor 9 packages. Additionally, Ca2+ influx measurements were performed in order to assess the impact of SOCE inhibition on the calcium homeostasis of various immune cell subsets. Results Data on B, T, NK, and myeloid cells revealed that treatment with BTP-2 causes a dose-dependent inhibition of calcium influx, demonstrating that BTP-2 is a potent inhibitor of SOCE in human LPMCs and PBMCs. By applying mass cytometry, we investigated how the gradual pharmacologic inhibition of SOCE affects the activation and pro-inflammatory functions of various immune cell subsets obtained from colon specimens of patients with UC and CD, which were characterized by the accumulation of CD4+ effector T cells, IFNγ-producing CD8+ T cells and IL-17-producing innate lymphoid cells (ILCs) compared to non-inflamed specimens. Remarkably, inhibition of SOCE attenuated the production of cytokines including IL-2, IL-4, IL-6, IL-17, TNF-α and IFNγ by T cells and ILCs, reduced the activation of B cells and decreased the production of IFNγ by myeloid cells without affecting the viability of primary human epithelial cells. Conclusion Our data reveal for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis.

Oxoglaucine mediates Ca2+ influx and activates autophagy to alleviate osteoarthritis through the TRPV5/calmodulin/CAMK-II pathway.

Stimulation of calcium influx and suppression of autophagy play important roles in the pathogenesis of osteoarthritis (OA). In this study, we used a novel inhibitor of TRPV5 cation channels - oxoglaucine to attenuate progression of deterioration and pathological changes in OA patient-derived chondrocytes and OA animal model, by activating autophagy. Inhibition by oxoglaucine of calcium influx was assessed in cells.. Analyses were also carried out to investigate the effect of oxoglaucine on OA by detection of anti-inflammatory response, TRPV5/CAMK-II/calmodulin pathway, autophagy, and cartilage protection both in vitro and in vivo. demonstrated by macroscopic evaluation and histological findings. Oxoglaucine suppressed expression of proinflammatory and apoptosis-related proteins, including TNF-α, IL-6, IL-1β, MMP-13, CASP-3, and BAX, and prevented matrix degradation in OA chondrocytes. It also successfully blocked Ca2+ influx, activating autophagy dose-dependently asshown by up-regulated expression of LC-3II/I, Beclin-1, ATG5, ATG7, higher autophagic influx and formation of autophagic vesicles. It also decreased expression of mRNA and protein of TRPV5, CAMK-II, and calmodulin. Conversely, 1,25-dihydroxyvitamin D3, anagonist of TRPV5 channels, reversed the oxoglaucine-induced calcium influx inhibition and autophagy activation, demonstrating the association of oxoglaucine with TRPV5. Further, oxoglaucine prevented the apoptosis and matrix degradation of articular cartilage in a rat model of OA. Oxoglaucine protects against cartilage damage by blocking the TRPV5/CAMK-II/calmodulin pathway to inhibit Ca2+ influx and activate autophagy. Our results indicate that oxoglaucine has the potential to become a candidate drug for treatment of OA.

Visualization of Ca Channel Blocker on Human Adrenal Tissue by Mass Spectrometry Imaging ~Its Predominant Distribution at Aldosterone-Producing Cells ~

Primary aldosteronism (PA) is the main cause of secondary hypertension, accounting for approximately 5–10% of all hypertension. Amlodipine, a third-generation calcium channel blocker, is one of the most frequently administered pharmaceuticals medications of hypertension, binds specifically to Cav1.2, a calcium channel primarily localized in the cardiovascular system, and exerts antihypertensive effects through inhibiting calcium influx into the vascular smooth muscle cells. In addition, calcium influx also plays important roles in aldosterone production and amlodipine was also reported to influence in vitro functions of Cav1.3, a calcium channel involved in aldosterone secretion. Ca channel blockers were also reported to reduce plasma aldosterone concentration by some clinical studies although with mild degrees. However, in vivo effects of amlodipine to aldosterone secretion has remained virtually unknown. A novel technique “Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI)” has been recently developed, which did make it possible to visualize non-labeled small molecules on tissue sections. Therefore, in this study, we firstly applied MALDI-MSI to visualize amlodipine on human adrenal glands including aldosterone producing adenoma (APA). We performed selective imaging of amlodipine using MALDI-MSI on the resected adrenal tissues from APA patients. Frozen sections containing whole representative tumor area were coated with a matrix called CHCA (α-Cyano-4-hydroxycinnamic acid) by deposition as a pretreatment. We subsequently analyzed and detected a precursor ion with MS at m/z 407.1 and then an amlodipine-specific ion with MS/MS at m/z 318.1. We also examined the concordance of amlodipine distribution obtained by this method with immunohistochemistry. Human resected adrenal tissues obtained from the patients APAs treated with and without amlodipine before adrenalectomy were examined. Periadrenal adipose tissues were also analyzed as a control tissue of non-aldosterone-producing tissues. Amlodipine was specifically detected and visualized only in the administered cases. Amlodipine was more abundantly detected in adrenal tissues than periadrenal adipose tissues. On the other hand, significant different was not detected between tumors and adjacent adrenal glands by semi-quantification using MALDI-MSI. In this study, we firstly visualized amlodipine directly in human tissue sections using MALDI-MSI. Increased accumulation of amlodipine in APAs treated with amlodipine did indicate direct effects of amlodipine on aldosterone production but further investigations are required for clarification between neoplastic and non-neoplastic aldosterone producing cells.