Endogenous Ca2 Research Articles

Tonantzitlolone is a nanomolar potency activator of transient receptor potential canonical 1/4/5 channels.

The diterpene ester tonantzitlolone (TZL) is a natural product, which displays cytotoxicity towards certain types of cancer cell such as renal cell carcinoma cells. The effect is similar to that of (-)-englerin A, and so, although it is chemically distinct, we investigated whether TZL also targets transient receptor potential canonical (TRPC) channels of the 1, 4 and 5 type (TRPC1/4/5 channels). The effects of TZL on renal cell carcinoma A498 cells natively expressing TRPC1 and TRPC4, modified HEK293 cells overexpressing TRPC4, TRPC5, TRPC4-TRPC1 or TRPC5-TRPC1 concatemer, TRPC3 or TRPM2, or CHO cells overexpressing TRPV4 were studied by determining changes in intracellular Ca2+ , or whole-cell or excised membrane patch-clamp electrophysiology. TZL induced an elevation of intracellular Ca2+ in A498 cells, similar to that evoked by englerin A. TZL activated overexpressed channels with EC50 values of 123nM (TRPC4), 83nM (TRPC5), 140nM (TRPC4-TRPC1) and 61nM (TRPC5-TRPC1). These effects of TZL were reversible on wash-out and potently inhibited by the TRPC1/4/5 inhibitor Pico145. TZL activated TRPC5 channels when bath-applied to excised outside-out but not inside-out patches. TZL failed to activate endogenous store-operated Ca2+ entry or overexpressed TRPC3, TRPV4 or TRPM2 channels in HEK 293 cells. TZL is a novel potent agonist for TRPC1/4/5 channels, which should be useful for testing the functionality of this type of ion channel and understanding how TRPC1/4/5 agonists achieve selective cytotoxicity against certain types of cancer cell.

Action potential counting at giant mossy fiber terminals gates information transfer in the hippocampus

Neuronal communication relies on action potential discharge, with the frequency and the temporal precision of action potentials encoding information. Hippocampal mossy fibers have long been recognized as conditional detonators owing to prominent short-term facilitation of glutamate release displayed during granule cell burst firing. However, the spiking patterns required to trigger action potential firing in CA3 pyramidal neurons remain poorly understood. Here, we show that glutamate release from mossy fiber terminals triggers action potential firing of the target CA3 pyramidal neurons independently of the average granule cell burst frequency, a phenomenon we term action potential counting. We find that action potential counting in mossy fibers gates glutamate release over a broad physiological range of frequencies and action potential numbers. Using rapid Ca2+ imaging we also show that the magnitude of evoked Ca2+ influx stays constant during action potential trains and that accumulated residual Ca2+ is gradually extruded on a time scale of several hundred milliseconds. Using experimentally constrained 3D model of presynaptic Ca2+ influx, buffering, and diffusion, and a Monte Carlo model of Ca2+-activated vesicle fusion, we argue that action potential counting at mossy fiber boutons can be explained by a unique interplay between Ca2+ dynamics and buffering at release sites. This is largely determined by the differential contribution of major endogenous Ca2+ buffers calbindin-D28K and calmodulin and by the loose coupling between presynaptic voltage-gated Ca2+ channels and release sensors and the relatively slow Ca2+ extrusion rate. Taken together, our results identify a previously unexplored information-coding mechanism in the brain.

Calcium-Mobilizing Behaviors of Neutral Cyclic ADP-Ribose Mimics that Integrate Modifications to the Nucleobase, Northern Ribose and Pyrophosphate.

Cyclic adenosine diphosphate ribose (cADPR) is an endogenous Ca2+ mobilizer involved in diverse cellular processes. Mimics of cADPR play a crucial role in investigating the molecular mechanism(s) of cADPR-mediated signaling. Here, compound 3, a mimic of cADPR in which a neutral triazole moiety and an ether linkage were introduced to substitute the pyrophosphate and "northern" ribose components, respectively, was synthesized for the first time. The pharmacological activities in Jurkat cells indicated that this mimic is capable of penetrating plasma membrane and inciting Ca2+ release from the endoplasmic reticulum (ER) through the action of ryanodine receptors (RyRs) and triggering Ca2+ influx. Furthermore, a uridine moiety was introduced in place of adenine and the new cADPR mimics 4 and 5 were synthesized. The results of biological investigation showed that these mimics also targeted RyRs and retained moderate Ca2+ agonistic activities. The results indicated that the neutral cADPR mimics had the same targets for inducing Ca2+ signaling.

Integration of nicotinic acid adenine dinucleotide phosphate (NAADP)-dependent calcium signalling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is currently the most potent endogenous Ca2+ mobilizing second messenger. Upon specific extracellular stimulation, rapid production of NAADP has been observed in different cell types from sea urchin eggs to mammalian cells. More than 20years after the discovery of NAADP, there is still controversy surrounding its metabolism and target receptors/ion channels and organelles. This article briefly reviews recent developments in the NAADP field. Besides the metabolism of NAADP, this review focuses on assumed organelles and putative targets, e.g. ion channels, with special emphasis on ryanodine receptor type 1 (RyR1) and two-pore channels (TPCs). The role of NAADP as a Ca2+ trigger is also discussed and the importance of NAADP in the formation of initial Ca2+ microdomains is highlighted.

Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation.

Background and PurposeThe mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small‐molecule agonist, but the pharmacology of these channels is otherwise limited.Experimental ApproachYoda1 analogues were generated by synthetic chemistry. Intracellular Ca2+ and Tl+ measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta.Key ResultsModification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 μM Yoda1‐induced Ca2+‐entry with IC50s of 1.3 μM (HEK 293 cells) and 1.5 μM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP‐evoked Ca2+ elevation or store‐operated Ca2+ entry in HEK 293 cells or Ca2+ entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose‐dependent relaxation of aortic rings, which was mediated by an endothelium‐ and NO‐dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1.Conclusion and ImplicationsChemical antagonism of Yoda1‐evoked Piezo1 channel activity is possible, and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains.

Do not neglect calcium: a systematic review and meta-analysis (meta-regression) of its digestibility and utilisation in growing and finishing pigs.

Ca digestibility and utilisation in growing pigs are not well understood, and are usually neglected in diet formulation. This has implications not only for the accurate determination of its requirements but also for its interactions with other nutrients. A systematic review and meta-analysis (meta-regression) of published trials was carried out to quantify factors affecting Ca absorption and utilisation, and to derive an estimate of Ca endogenous excretion. The analysis was carried out on the data from forty studies, corresponding to 201 treatments performed on 1204 pigs. The results indicated that although Ca absorption and retention (g/kg of body weight per d) increased with increasing Ca intake (P<0·001), non-phytate-P intake (P<0·001) and exogenous phytase supplementation (P<0·001), these values decreased with increasing phytate-P intake (P<0·05). Interactions between exogenous phytase and Ca intake, indicating reduced efficacy of this enzyme (P<0·001), and between phytate-P intake and exogenous phytase, counteracting the direct negative effect of phytate-P (P<0·05) on Ca absorption and retention, were also detected. There were no effects of animal-related characteristics, such as pig genotype in Ca absorption and retention. The large amount of variance explained in Ca absorption (90 %) and retention (91 %) supported our choice of independent variables. Endogenous Ca losses obtained via linear regression were 239 mg/kg of DM intake (95 % CI 114, 364). These outcomes advance the current understanding of Ca digestibility and utilisation, and should contribute towards establishing requirements for digestible Ca. Consequently, pig diets will be more correctly formulated if digestible Ca values are used in estimating requirements for Ca.

Dynamics of body calcium and net calcium requirements for maintenance of Saanen goats

AbstractThe objectives of the current study were to investigate the dynamics of body calcium (Ca) and to estimate the net Ca maintenance requirements (NCam) of Saanen goats, using45Ca as a radiotracer. Eighteen castrated male Saanen goats (25 ± 2.3 kg body weight (BW)) received a basal diet (ground ear maize, ground maize and vitamin–mineral premix). The treatments consisted of adding limestone to the basal diet to provide Ca content of 0.6, 1.7 and 3.0 g/kg dry matter (DM). The experiment lasted 45 days (i.e. 36 d of adaptation and 9 days of measurements). On day 38, 0.5 ml of 7.4 MBq45Ca solution was administrated before feeding. From days 39 to 45, samples of faeces, blood and urine were collected, and Ca concentration determined. The Ca intake, Ca in faeces, Ca in urine, faecal endogenous Ca and true absorbed Ca increased linearly as Ca content in the diets increased, while retained Ca increased at a decreasing rate. Dry matter intake decreased at an increasing rate with increased Ca content in the diets. In contrast, Ca content in the diets did not affect biological availability of Ca, or Ca in plasma. The true biological availability of Ca from limestone in Saanen goats was 0.72. The daily NCamwas 11.6 (±1.3) mg/kg BW. The current results might help to understand Ca dynamics in goats and enhance the formulation of balanced diets to best meet Ca requirements of Saanen goats.

Variations in Ca2+ Influx Can Alter Chelator-Based Estimates of Ca2+ Channel-Synaptic Vesicle Coupling Distance.

The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca2+ channels (VGCCs) and Ca2+ sensors for exocytosis. This VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca2+ chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca2+ reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca2+ buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca2+ influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca2+ sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca2+ entry durations are not considered when estimating VGCC-sensor coupling distances.SIGNIFICANT STATEMENT The coupling distance between voltage-gated Ca2+ channels and Ca2+ sensors for exocytosis critically determines the timing and probability of neurotransmitter release. Perfusion of presynaptic terminals with the exogenous Ca2+ chelator EGTA has been widely used for both qualitative and quantitative estimates of this distance. However, other presynaptic terminal parameters such as the amplitude and duration of Ca2+ entry can also influence EGTA inhibition of exocytosis, thus confounding conclusions based on EGTA alone. Here, we performed reaction-diffusion simulations of Ca2+-driven synaptic vesicle fusion, which delineate the critical parameters influencing an accurate prediction of coupling distance. Our study provides guidelines for characterizing and understanding how variability in coupling distance across chemical synapses could be estimated accurately.

Crosstalk between calcium and melatonin affects postharvest physiological deterioration and quality loss in cassava

Rapid postharvest physiological deterioration largely reduces the quality and marketability of cassava. The molecular mechanism underlying cassava postharvest physiological deterioration and quality loss is largely unknown. The present study aimed to investigate the role of calcium and its relationship with melatonin in cassava postharvest physiological deterioration. Transcriptomic analyses indicate that most of the calcium ion (Ca2+) sensor genes are upregulated in cassava tuberous roots at different postharvest stages. Exogenous CaCl2 reduces postharvest physiological deterioration, increases the endogenous levels of Ca2+ and melatonin, reduces the degradation of ascorbic acid and starch, and induces the expression of genes related to melatonin biosynthesis after harvest. These effects are reversed by the exogenous application of a Ca2+ chelator (EGTA). Exogenous melatonin also increases endogenous melatonin levels and reduces ascorbic acid and starch degradation during postharvest physiological deterioration but do not affect endogenous Ca2+ content. Together, these findings demonstrate that calcium-induced activation of melatonin biosynthesis plays a role in reducing postharvest physiological deterioration and quality loss in cassava. Additionally, pretreatment with EGTA arrests the melatonin-induced reduction of postharvest physiological deterioration, suggesting the possible crosstalk between melatonin and calcium during postharvest physiological deterioration.

Calmodulin antagonist affects peroxisomal functionality by disrupting both peroxisomal Ca2+ and protein import.

Ca2+ is a second messenger in many physiological and phytopathological processes. Peroxisomes are subcellular compartments with an active oxidative and nitrosative metabolism. Previous studies have demonstrated that peroxisomal nitric oxide (NO) generation is dependent on Ca2+ and calmodulin (CaM). Here, we used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) containing a type 1 peroxisomal-targeting signal motif (PTS1; CFP-PTS1), which enables peroxisomes to be visualized in vivo, and also used a cell-permeable fluorescent probe for Ca2+ Analysis by confocal laser-scanning microscopy (CLSM) enabled us to visualize the presence of endogenous Ca2+ in the peroxisomes of both roots and guard cells. The presence of Ca2+ in peroxisomes and the import of CFP-PTS1 are drastically disrupted by both CaM antagonist and glutathione (GSH). Furthermore, the activity of three peroxisomal enzymes (catalase, glycolate oxidase and hydroxypyruvate reductase) containing PTS1 was clearly affected in these conditions, with a decrease of between 41 and 51%. In summary, data show that Ca2+ and CaM are strictly necessary for protein import and normal functionality of peroxisomal enzymes, including antioxidant and photorespiratory enzymes, as well as for NO production.

Measurement of the true ileal calcium digestibility of some feed ingredients for broiler chickens

Results from three experiments relating to the measurement of ileal calcium (Ca) digestibility in feed ingredients for broiler chickens are presented herein. The first experiment was conducted to determine the true ileal Ca digestibility of dicalcium phosphate (DCP), monocalcium phosphate (MCP), poultry by-product meal (PBPM), fish meal (FM) and canola meal (CM). Five semi-purified diets containing DCP, MCP, PBPM and FM with a dietary Ca concentration of 9 g/kg, and CM with a dietary Ca concentration of 5.71 g/kg were generated. A Ca- and phosphorus (P)-free diet was used to determine the basal ileal endogenous Ca losses. Titanium dioxide was incorporated in all diets as an indigestible marker. Each diet was randomly allotted to six replicate cages (eight birds per cage) and fed from 21 to 24 days post-hatch. True Ca digestibility coefficients of DCP, MCP, PBPM, FM and CM were determined to be 0.28, 0.33, 0.29, 0.24 and 0.31, respectively. These lower digestibility coefficients were unexpected and it was speculated that this finding may be due to the length of adaptation to assay diets and/or assay methodology. To investigate these possibilities, two more experiments were conducted. In the second experiment, the influence of dietary adaptation length was examined with DCP and MCP. The assay diets with DCP and MCP, and the Ca- and P-free diet were similar to those used in Experiment 1. Each diet was randomly allocated to four replicate cages (15 birds per cage) and fed from 21 to 24 days post-hatch. Digesta samples of five birds from each replicate were collected after 24, 48 and 72 h of feeding. Dietary adaptation length had no influence (P > 0.05) on the true Ca digestibility of MCP, but increased that of DCP at 24 h. True ileal Ca digestibility of DCP and MCP after 24, 48 and 72 h of adaptation were 0.45, 0.36, and 0.35, and 0.30, 0.32 and 0.34, respectively. The third experiment was conducted to determine the effect of assay methodology (direct, regression and difference methods) and Ca:non-phytate P ratio (2:1 and 1.16:1) on the Ca digestibility of DCP. The true Ca digestibility coefficients of DCP determined by the direct, difference and regression methods with Ca:non-phytate P ratio of 1.16:1 were 0.34, 0.21 and 0.13, respectively. The true Ca digestibility of DCP determined by the direct method was higher (P < 0.05) than those determined by difference and regression methods The true Ca digestibility coefficient of DCP with Ca:non-phytate P ratios of 2:1 and 1.16:1 were 0.25 and 0.21, respectively, and was not influenced (P > 0.05) by the Ca to non-phytate P ratio.

An improved measurement of the Ca2+-binding affinity of fluorescent Ca2+ indicators

Fluorescent Ca2+ indicators are widely used to measure the intracellular Ca2+ concentration ([Ca2+]i) in living cells, including neurons. By calibrating an indicator in solutions that mimic the main ionic constituents of the actual cytoplasm, [Ca2+]i can be determined from the measured fluorescence intensity. However, different studies have reported considerably different Ca2+-binding affinities (Kd) for the same indicator, even though they used calibrating solutions with similar compositions. In this paper, we present a method to accurately determine the Kd values of non-ratiometric Ca2+ indicators in solutions that mimicked a standard patch-clamp internal solution. The free Ca2+ concentration ([Ca2+]) in these solutions, which was set by either EGTA or HEDTA, was measured with a Ca2+-selective macroelectrode. We found that such a measurement was critical for an accurate calibration of the Ca2+ indicators. The Kd values of OGB-1, OGB-6F, fluo-5F, and fluo-4FF were 0.26 ± 0.01, 8.7 ± 0.4, 1.00 ± 0.05, and 23.0 ± 0.7 μM, respectively. Calculating [Ca2+] with Maxchelator, a widely used computer program, led to a significant underestimation of the Kd values of OGB-6F, fluo-5F, and fluo-4FF. This is because the purity of EGTA was considerably less than that advertised by the manufacturer. In addition, the Kd value of HEDTA was overestimated by Maxchelator. Therefore, besides batch-to-batch variations, the fact that [Ca2+] in the calibrating solutions of many studies was estimated with Maxchelator is very likely a reason for the different published values of Kd of Ca2+ indicators. Using a reaction-diffusion model to reproduce Ca2+ rises in a nerve terminal, we further showed that incorrect calibration of fluorescent Ca2+ indicators can underlie the large variation of the endogenous Ca2+ binding ratio between different types of excitatory synapses.

Effects of Ca2+ on nitric oxide-induced adventitious rooting in cucumber under drought stress

Cucumber (Cucumis sativus L. 'Xinchun 4') was used to explore the relationship between nitric oxide (NO) and calcium (Ca2+) during adventitious rooting under drought stress. Rooting parameters, endogenous Ca2+ fluorescent intensity and the antioxidant enzymes activity (SOD, CAT and APX) in cucumber explants under drought stress were investigated. The results showed that treatment with 200 μmol·L-1 CaCl2 and 0.05% PEG significantly improved the number and length of adventitious root in cucumber explants under drought stress, while the application of Ca2+ chelating agent (EGTA) and channel inhibitor (BAPTA/AM) significantly decreased NO-induced number and length of adventitious root under drought stress. Under drought stress, the fluorescence intensity of Ca2+ in hypocotyls treated with NO and CaCl2 was improved, however, the Ca2+ fluorescence intensity in the hypocotyls treated with NO scavenger (cPTIO) was significantly lower than that in the hypocotyls treated with NO. Under drought stress, the activities of antioxidant enzymes in the cucumber explants were significantly promoted by the treatments with NO and CaCl2, however, Ca2+ chelating agent and channel inhibitor significantly decreased the activity of antioxidant enzymes induced by NO. In conclusion, Ca2+ might be involved in the process of NO-adjusted antioxidant enzymes activity during adventitious rooting under drought stress, which alleviated the negative effects of drought on the adventitious rooting and promoted the formation of adventitious roots.

Different sources of calcium for starter pig diets

Abstract The study was conducted to determine the true Ca absorption coefficients and growth performance of piglets fed diets containing different sources of Ca. In the metabolism study, 30 piglets uncastrated, with initial weight averages of 20.52 ± 1.84 kg, were assigned to 5 treatments with 6 piglets per treatment in a randomized complete block design. A basal diet was formulated to meet the piglets nutritional requirements, except to Ca (0.09%), and was supplemented with different source of Ca (limestone, monodicalcium phosphate, calcined bone meal, and oyster meal) to provide 0.64% total Ca. A low-Ca diet (0.018%) and 6 additional piglets were used to estimate the endogenous Ca excretion in the feces. Total feces and urine were collected to determine the apparent and true absorption of Ca. After the adaptation period (7 d), the excreted feces were collected twice daily, weighed, stored in plastic bags in freezer (−18 °C) until the end of the collection period (5 d). The volume of the urine collected for 24 h was measured and a 20% aliquot was stored in glass containers in refrigerator (3 °C) until the end of the collection period (5 d). Experimental diets, feces and urine were analyzed for Ca. To determine the effect of different Ca sources on the growth performance, 160 piglets were assigned, to 2 sexes (males intact and females) and 4 sources of Ca (the same ones used in the metabolism study), in a 2 × 4 factorial arrangement and randomized complete block design with 4 pens per treatment and 5 intact males or 5 females per pen. At the end of the study (22 d), blood samples were collected of the all piglets and 5 piglets per treatment were selected randomly regardless of sex to collect samples of organs. All samples were stored at −18 °C. Response criteria were: weight gain, feed intake, feed efficiency, Ca deposition in organs, bone strength, weight and size of the third metatarsal bone, and serum in Ca. The sources of Ca and direct or indirect collection method did not affect the absorption coefficients. There was no interaction among the sources of Ca and sex of the piglets for the performance variables. The piglets growth performance was similar among treatments. Calcined bone meal and oyster meal resulted in a greater content of mineral matter in the kidney (P = 0.013). The bone meal resulted in a greater Ca concentration in the piglets hearts (P

Calcium and Calmodulin Are Involved in Nitric Oxide-Induced Adventitious Rooting of Cucumber under Simulated Osmotic Stress.

Osmotic stress is a major form of abiotic stress that adversely affects growth and development of plants and subsequently reduces yield and quality of crops. In this study, the effect of nitric oxide (NO) and calcium (Ca2+) on the process of adventitious rooting in cucumber (Cucumis sativus L.) under simulated osmotic stress was investigated. The results revealed that the effect of exogenous NO and Ca2+ in promoting the development of adventitious roots in cucumber seedlings under simulated osmotic stress was dose-dependent, with a maximal biological response at 10 μM NO donor nitroprusside (SNP) or 200 μM Ca2+. The application of Ca2+ chelators or channel inhibitors and calmodulin (CaM) antagonists significantly reversed NO-induced adventitious rooting, implying that endogenous Ca2+/CaM might be involved in NO-induced adventitious rooting under osmotic stress. Moreover, intracellular Ca amount was also increased by NO in cucumber hypocotyls during the development of adventitious roots under osmotic stress. This increase of endogenous Ca2+ was inhibited by NO specific scavenger 2-(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), nitrate reductase inhibitors tungstate (Na2WO4) and sodium azide (NaN3). This gives an indication that Ca2+ might be a downstream signaling molecule in the adventitious root development by NO under osmotic condition. The results also show that NO or Ca2+ play a positive role in improving plant water status and photosynthetic system by increasing chlorophyll content and photochemical activity in leaves. Furthermore, NO and Ca2+ treatment might alleviate the negative effects of osmotic stress by decreasing membrane damage and reactive oxygen species (ROS) production by enhancing the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Therefore, Ca2+/CaM may act as a downstream signaling molecule in NO-induced development of adventitious root under simulated osmotic stress through improving the photosynthetic performance of leaves and activating antioxidative system in plants.

Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores.

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.

C1q/tumor necrosis factor-related protein-3 enhances the contractility of cardiomyocyte by increasing calcium sensitivity

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine that protects against myocardial infarction-induced cardiac dysfunction through its pro-angiogenic, anti-apoptotic, and anti-fibrotic effects. However, whether CTRP3 can directly affect the systolic and diastolic function of cardiomyocytes remains unknown. Adult rat cardiomyocytes were isolated and loaded with Fura-2AM. The contraction and Ca2+ transient data was collected and analyzed by IonOptix system. 1 and 2μg/ml CTRP3 significantly increased the contraction of cardiomyocytes. However, CTRP3 did not alter the diastolic Ca2+ content, systolic Ca2+ content, Ca2+ transient amplitude, and L-type Ca2+ channel current. To reveal whether CTRP3 affects the Ca2+ sensitivity of cardiomyocytes, the typical phase-plane diagrams of sarcomere length vs. Fura-2 ratio was performed. We observed a left-ward shifting of the late relaxation trajectory after CTRP3 perfusion, as quantified by decreased Ca2+ content at 50% sarcomere relaxation, and increased mean gradient (μm/Fura-2 ratio) during 500–600ms (-0.163 vs. −0.279), 500–700ms (-0.159 vs. −0.248), and 500–800ms (-0.148 vs. −0.243). Consistently, the phosphorylation level of cardiac troponin I at Ser23/24 was reduced by CTRP3, which could be eliminated by preincubation of okadaic acid, a type 2A protein phosphatase inhibitor. In summary, CTRP3 increases the contraction of cardiomyocytes by increasing the myofilament Ca2+ sensitivity. CTRP3 might be a potential endogenous Ca2+ sensitizer that modulates the contractility of cardiomyocytes.

STIM1 and STIM2 differently regulate endogenous Ca2+ entry and promote TGF-β-induced EMT in breast cancer cells

The Ca2+ sensor proteins STIM1 and STIM2 are crucial elements of store-operated calcium entry (SOCE) in breast cancer cells. Increased SOCE activity may contribute to epithelial–mesenchymal transitions (EMT) and increase cell migration and invasion. However, the roles of STIM1 and STIM2 in TGF-β-induced EMT are still unclear. In this study, we demonstrate roles of STIMs in TGF-β-induced EMT in breast cancer cells. In particular, STIM1 and STIM2 expression affected TGF-β-induced EMT by mediating SOCE in MDA-MB-231 and MCF-7 breast cancer cells. The specific SOCE inhibitor YM58483 blocked TGF-β-induced EMT, and differing effects of STIM1 and STIM2 on TGF-β-induced EMT correlated with differing roles in SOCE. Finally, we showed that STIM2 is associated with non-store-operated calcium entry (non-SOCE) during TGF-β-induced EMT, whereas STIM1 is not. What's more, non-SOCE have a large possibility to be ROCE. In conclusion, STIM1 and STIM2 proteins play important roles in TGF-β-induced EMT and these effects are related to both SOCE and non-SOCE.

Blockade of RyRs in the ER Attenuates 6-OHDA-Induced Calcium Overload, Cellular Hypo-Excitability and Apoptosis in Dopaminergic Neurons.

Calcium (Ca2+) dyshomeostasis induced by endoplasmic reticulum (ER) stress is an important molecular mechanism of selective dopaminergic (DA) neuron loss in Parkinson’s disease (PD). Inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), which are located on the ER surface, are the main endogenous Ca2+ release channels and play crucial roles in regulating Ca2+ homeostasis. However, the roles of these endogenous Ca2+ release channels in PD and their effects on the function and survival of DA neurons remain unknown. In this study, using a 6-hydroxydopamine (6-OHDA)-induced in vitro PD model (SN4741 Cell line), we found that 6-OHDA significantly increased cytoplasmic Ca2+ levels ([Ca2+]i), which was attenuated by pretreatment with 4-phenyl butyric acid (4-PBA; an ER stress inhibitor) or ryanodine (a RyRs blocker). In addition, in acute midbrain slices of male Sprague-Dawley rats, we found that 6-OHDA reduced the spike number and rheobase of DA neurons, which were also reversed by pretreatment with 4-PBA and ryanodine. TUNEL staining and MTT assays also showed that 4-PBA and ryanodine obviously alleviated 6-OHDA-induced cell apoptosis and devitalization. Interestingly, a IP3Rs blocker had little effect on the above 6-OHDA-induced neurotoxicity in DA neurons. In conclusion, our findings provide evidence of the different roles of IP3Rs and RyRs in the regulation of endogenous Ca2+ homeostasis, neuronal excitability, and viability in DA neurons, and suggest a potential therapeutic strategy for PD by inhibiting the RyRs Ca2+ channels in the ER.

Dynamics of volume-averaged intracellular Ca2+ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations.

The intracellular concentration of free calcium ions ([Ca2+ ]i ) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro- or nanodomain of highly elevated [Ca2+ ]i in the vicinity of open voltage-gated Ca2+ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca2+ ]i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca2+ ]i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance. These data allow us to predict changes in [Ca2+ ]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short-term plasticity of glutamatergic synapses. Many aspects of short-term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+ ]i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+ ]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+ ]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity-dependent dynamic changes in global [Ca2+ ]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch-clamp and microfluorimetry. We use low concentrations of a low-affinity Ca2+ indicator dye (100μm Fura-6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500μm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+ ]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD =543±51nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38±0.20 s-1 ), which we need to postulate in order to model the measured presynaptic [Ca2+ ]i transients.