Passive Ca2 Research Articles

Unexpected effects of old drugs – antihypoxic properties of lactulose

Faced with soaring costs of developing new drugs, researchers are looking for ways to repurpose old ones, that is «...the most fruitful basis for discovering a new drug is to start with an old drug». Lactulose is a well-known synthetic sugar used to treat constipation. It is not absorbed in the small intestine and is not broken down by human enzymes. Lactulose is metabolized in the colon by bacterial flora to short-chain fatty acids, monosaccharides, lactic and acetic acids, volatile fatty acids, hydrogen and methane. The problem is that all of this happens in the colon. Could something similar happen in Krebs solution under in vitro conditions?The aim of study – to test experimentally the hypothesis about the possible antihypoxic effect of the drug lactudose in vitro. However lactulose unexpectedly demonstrated a clear ability to restore spontaneous activity of the rat portal vein inhibited under hypoxia in vitro. Lactulose effectively restores the amplitude of vascular contractions caused by field electrical stimulation that was inhibited by hypoxia. When lactulose was added to the organ bath both before hypoxic exposure and electrical stimulation, stimulation caused a paradoxical increase in isometric tension. Preliminary data obtained suggest that the plasma membrane of vascular smooth muscle cells may primarily participate, and the mechanisms of energy supply to the contractile apparatus do not take such a large part in the response to hypoxia. The experiments showed, that one of the most vulnerablel inks in the excitation-contraction coupling chain in vascular tissues during hypoxia is the system of passive transmembrane Ca2+ transport. Probably, sugars that are not hydrolyzed by a living cell can have biological effects during hypoxia that are not related to their substrate influence. As an object of study, we focused on the disaccharide lactulose, which contains fructose and galactose. In the body, outside the intestines, there are no enzymes that could metabolize this bond and hydrolyze this compound. It seems that if lactulose has an antihypoxic effect, then this effect will depend exclusively on non-metabolic factors, which will make a significant contribution to the understanding of the fundamental mechanisms of the development of hypoxic conditions. The voltage-sensing domain of voltage-gated ion channels is a protein structure important for membrane voltage sensing that is characterized by specific, conserved, and charged amino acid residues. It is generally assumed that molecules are neutral and do not carry unpaired electrons. Charged molecules are called molecular ions, molecules with a multiplicity other than unity (i. e., with unpaired electrons and unsaturated valences). Thus, under certain changes in environmental conditions (for example, a change in pH), a previously neutral molecule can become electrically charged and thus acquire the ability to interact with a potential sensor in ion channels. Interestingly, even if a carbohydrate does not carry a formal charge, when bound it will change the dielectric environment, since the region previously filled with water (in the absence of significant conformational changes) will be occupied by an environment with a much lower dielectric constant. Thus, carbohydrates can act as universal electrostatic modulators of protein function, even if their binding causes little or no conformational change in the protein molecule. In conclusion, it should be noted, the fact that lactulose has antihypoxic properties is beyond doubt. From a clinician's point of view, this is already good. The compound is well known and completely harmless. But pharmacology is the science of the mechanisms of action of chemicals on living systems, and therefore we are obliged to propose such mechanisms. If not absolutely demonstrative, then at least theoretically acceptable. A possible metabolic shift towards glycolysis would be a good potential candidate for evidence, but is currently difficult to substantiate. It remains only to assume the possible interaction of the electrically modified (under conditions of hypoxia and associated acidosis) lactulose molecule with voltage-dependent calcium channels and the restoration of their activity, suppressed during hypoxia.

Sex-specific cardiovascular remodeling leads to a divergent sex-dependent development of heart failure in aged hypertensive rats

IntroductionThe prevalence of heart failure with preserved ejection fraction (HFpEF) is continuously rising and predominantly affects older women often hypertensive and/or obese or diabetic. Indeed, there is evidence on sex differences in the development of HF. Hence, we studied cardiovascular performance dependent on sex and age as well as pathomechanisms on a cellular and molecular level.MethodsWe studied 15-week- and 1-year-old female and male hypertensive transgenic rats carrying the mouse Ren-2 renin gene (TG) and compared them to wild-type (WT) controls at the same age. We tracked blood pressure and cardiac function via echocardiography. After sacrificing the 1-year survivors we studied vascular smooth muscle and endothelial function. Isolated single skinned cardiomyocytes were used to determine passive stiffness and Ca2+-dependent force. In addition, Western blots were applied to analyse the phosphorylation status of sarcomeric regulatory proteins, titin and of protein kinases AMPK, PKG, CaMKII as well as their expression. Protein kinase activity assays were used to measure activities of CaMKII, PKG and angiotensin-converting enzyme (ACE).ResultsTG male rats showed significantly higher mortality at 1 year than females or WT male rats. Left ventricular (LV) ejection fraction was specifically reduced in male, but not in female TG rats, while LV diastolic dysfunction was evident in both TG sexes, but LV hypertrophy, increased LV ACE activity, and reduced AMPK activity as evident from AMPK hypophosphorylation were specific to male rats. Sex differences were also observed in vascular and cardiomyocyte function showing different response to acetylcholine and Ca2+-sensitivity of force production, respectively cardiomyocyte functional changes were associated with altered phosphorylation states of cardiac myosin binding protein C and cardiac troponin I phosphorylation in TG males only. Cardiomyocyte passive stiffness was increased in TG animals. On a molecular level titin phosphorylation pattern was altered, though alterations were sex-specific. Thus, also the reduction of PKG expression and activity was more pronounced in TG females. However, cardiomyocyte passive stiffness was restored by PKG and CaMKII treatments in both TG sexes.ConclusionHere we demonstrated divergent sex-specific cardiovascular adaptation to the over-activation of the renin-angiotensin system in the rat. Higher mortality of male TG rats in contrast to female TG rats was observed as well as reduced LV systolic function, whereas females mainly developed HFpEF. Though both sexes developed increased myocardial stiffness to which an impaired titin function contributes to a sex-specific molecular mechanism. The functional derangements of titin are due to a sex-specific divergent regulation of PKG and CaMKII systems.

SERCA-1 conformational change exerted by the Ca2+-channel blocker diltiazem affects mammalian skeletal muscle function

In skeletal muscle (SM), inward Ca2+-currents have no apparent role in excitation-contraction coupling (e-c coupling), however the Ca2+-channel blocker can affect twitch and tetanic muscle in mammalian SM. Experiments were conducted to study how diltiazem (DLZ) facilitates e-c coupling and inhibits contraction. 1) In complete Extensor Digitorum Longus (EDL) muscle and single intact fibres, 0.03 mM DLZ causes twitch potentiation and decreases force during tetanic activity, with increased fatigue. 2) In split open fibres isolated from EDL fibres, DLZ inhibits sarcoplasmic reticulum (SR) Ca2+-loading in a dose-dependent manner and has a potentiating effect on caffeine-induced SR Ca2+-release. 3) In isolated light SR (LSR) vesicles, SERCA1 hydrolytic activity is not affected by DLZ up to 0.2 mM. However, ATP-dependent Ca2+-uptake was inhibited in a dose-dependent manner at a concentration where e-c coupling is changed. 4) The passive Ca2+-efflux from LSR was reduced by half with 0.03 mM diltiazem, indicating that SR leaking does not account for the decreased Ca2+-uptake. 5) The denaturation profile of the SERCA Ca2+-binding domain has lower thermal stability in the presence of DLZ in a concentration-dependent manner, having no effect on the nucleotide-binding domain. We conclude that the effect of DLZ on SM is exerted by crossing the sarcolemma and interacting directly with the SERCA Ca2+-binding domain, affecting SR Ca2+-loading during relaxation, which has a consequence on SM contractility. Diltiazem effect on SM could be utilized as a tool to understand SM e-c coupling and muscle fatigue.

Novel PA/PVDF hollow fiber nanofiltration membrane with high permeability and Ca2+/antibiotics selectivity for drinking water purification

Emerging contaminants, including antibiotics, threaten water safety and public health. To remove these contaminants while retaining beneficial minerals in water, such as calcium (Ca), a novel thin-film composite nanofiltration (NF) membrane was manufactured through polymerization of a barrier layer composed of polypiperazine amide onto polyvinylidene fluoride (PVDF) hollow fiber (HF) substrate. The pore size of the PVDF surface was refined by introducing poly(vinylpyrrolidone) via a thermally induced phase separation method. Then piperazine (PIP) and trimesoyl chloride were selected to synthesize the NF membrane by interfacial polymerization with NaHCO3 as an additive. The influence of PIP concentration on the membrane morphology and separation performance was investigated. The optimized HF NF membrane (NF3) exhibited high water permeability (8.08 ​L/(m2 ​h ​bar)) due to its strong hydrophilicity. It also demonstrated a molecular weight cut-off of 378 ​Da and an enhanced negative surface charge (−43.96 ​mV), which was beneficial for the exclusion of antibiotics and passage of Ca2+. The high tetracycline rejection (98.9 ​%) enabled the NF3 membrane to achieve superior Ca2+/antibiotic selectivity (37.27) compared with most commercially available NF membranes. This study offers novel insights into tailoring the mineral/micropollutant selectivity of HF NF membranes for drinking water purification.

Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

NaHCO3 addition enhances water permeance and Ca/haloacetic acids selectivity of nanofiltration membranes for drinking water treatment

The existence of disinfection by-products such as haloacetic acids (HAAs) in drinking water severely threatens water safety and public health. Nanofiltration (NF) is a promising strategy to remove HAAs for clean water production. However, NF often possesses overhigh rejection of essential minerals such as calcium. Herein, we developed highly selective NF membranes with tailored surface charge and pore size for efficient rejection of HAAs and high passage of minerals. The NF membranes were fabricated through interfacial polymerization (IP) with NaHCO3 as an additive. The NaHCO3-tailored NF membranes exhibited high water permeance up to ∼24.0 L m − 2 h − 1 bar−1 (more than doubled compared with the control membrane) thanks to the formation of stripe-like features and enlarged pore size. Meanwhile, the tailored membranes showed enhanced negative charge, which benefitted their rejection of HAAs and passage of Ca and Mg. The higher rejection of HAAs (e.g., > 90%) with the lower rejection of minerals (e.g., < 30% for Ca) allowed the NF membranes to achieve higher minerals/HAAs selectivity, which was significantly higher than those of commercially available NF membranes. The simultaneously enhanced membrane performance and higher minerals/HAAs selectivity would greatly boost water production efficiency and water quality. Our findings provide a novel insight to tailor the minerals/micropollutants selectivity of NF membranes for highly selective separation in membrane-based water treatment.

A comprehensive overview of the complex world of the endo- and sarcoplasmic reticulum Ca2+-leak channels

Inside cells, the endoplasmic reticulum (ER) forms the largest Ca2+ store. Ca2+ is actively pumped by the SERCA pumps in the ER, where intraluminal Ca2+-binding proteins enable the accumulation of large amount of Ca2+. IP3 receptors and the ryanodine receptors mediate the release of Ca2+ in a controlled way, thereby evoking complex spatio-temporal signals in the cell. The steady state Ca2+ concentration in the ER of about 500 μM results from the balance between SERCA-mediated Ca2+ uptake and the passive leakage of Ca2+. The passive Ca2+ leak from the ER is often ignored, but can play an important physiological role, depending on the cellular context. Moreover, excessive Ca2+ leakage significantly lowers the amount of Ca2+ stored in the ER compared to normal conditions, thereby limiting the possibility to evoke Ca2+ signals and/or causing ER stress, leading to pathological consequences. The so-called Ca2+-leak channels responsible for Ca2+ leakage from the ER are however still not well understood, despite over 20 different proteins have been proposed to contribute to it. This review has the aim to critically evaluate the available evidence about the various channels potentially involved and to draw conclusions about their relative importance.

Multiscale Modeling of Cardiovascular Function Predicts That the End-Systolic Pressure Volume Relationship Can Be Targeted via Multiple Therapeutic Strategies.

Most patients who develop heart failure are unable to elevate their cardiac output on demand due to impaired contractility and/or reduced ventricular filling. Despite decades of research, few effective therapies for heart failure have been developed. In part, this may reflect the difficulty of predicting how perturbations to molecular-level mechanisms that are induced by drugs will scale up to modulate system-level properties such as blood pressure. Computer modeling might help with this process and thereby accelerate the development of better therapies for heart failure. This manuscript presents a new multiscale model that uses a single contractile element to drive an idealized ventricle that pumps blood around a closed circulation. The contractile element was formed by linking an existing model of dynamically coupled myofilaments with a well-established model of myocyte electrophysiology. The resulting framework spans from molecular-level events (including opening of ion channels and transitions between different myosin states) to properties such as ejection fraction that can be measured in patients. Initial calculations showed that the model reproduces many aspects of normal cardiovascular physiology including, for example, pressure-volume loops. Subsequent sensitivity tests then quantified how each model parameter influenced a range of system level properties. The first key finding was that the End Systolic Pressure Volume Relationship, a classic index of cardiac contractility, was ∼50% more sensitive to parameter changes than any other system-level property. The second important result was that parameters that primarily affect ventricular filling, such as passive stiffness and Ca2+ reuptake via sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), also have a major impact on systolic properties including stroke work, myosin ATPase, and maximum ventricular pressure. These results reinforce the impact of diastolic function on ventricular performance and identify the End Systolic Pressure Volume Relationship as a particularly sensitive system-level property that can be targeted using multiple therapeutic strategies.

Cardiomyocyte-Specific Deletion of Orai1 Reveals Its Protective Role in Angiotensin-II-Induced Pathological Cardiac Remodeling.

Pathological cardiac remodeling correlates with chronic neurohumoral stimulation and abnormal Ca2+ signaling in cardiomyocytes. Store-operated calcium entry (SOCE) has been described in adult and neonatal murine cardiomyocytes, and Orai1 proteins act as crucial ion-conducting constituents of this calcium entry pathway that can be engaged not only by passive Ca2+ store depletion but also by neurohumoral stimuli such as angiotensin-II. In this study, we, therefore, analyzed the consequences of Orai1 deletion for cardiomyocyte hypertrophy in neonatal and adult cardiomyocytes as well as for other features of pathological cardiac remodeling including cardiac contractile function in vivo. Cellular hypertrophy induced by angiotensin-II in embryonic cardiomyocytes from Orai1-deficient mice was blunted in comparison to cells from litter-matched control mice. Due to lethality of mice with ubiquitous Orai1 deficiency and to selectively analyze the role of Orai1 in adult cardiomyocytes, we generated a cardiomyocyte-specific and temporally inducible Orai1 knockout mouse line (Orai1CM–KO). Analysis of cardiac contractility by pressure-volume loops under basal conditions and of cardiac histology did not reveal differences between Orai1CM–KO mice and controls. Moreover, deletion of Orai1 in cardiomyocytes in adult mice did not protect them from angiotensin-II-induced cardiac remodeling, but cardiomyocyte cross-sectional area and cardiac fibrosis were enhanced. These alterations in the absence of Orai1 go along with blunted angiotensin-II-induced upregulation of the expression of Myoz2 and a lack of rise in angiotensin-II-induced STIM1 and Orai3 expression. In contrast to embryonic cardiomyocytes, where Orai1 contributes to the development of cellular hypertrophy, the results obtained from deletion of Orai1 in the adult myocardium reveal a protective function of Orai1 against the development of angiotensin-II-induced cardiac remodeling, possibly involving signaling via Orai3/STIM1-calcineurin-NFAT related pathways.

Intercellular Bridge Mediates Ca2+ Signals between Micropatterned Cells via IP3 and Ca2+ Diffusion

Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca2+ communication between postmitotic cells. In this work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca2+ signal transfer over the intercellular bridges. With the use of such micropatterns, considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca2+ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca2+ diffusion and endogenous Ca2+ response in the intercellular-bridge-connected cell, indicating two different pathways of the Ca2+ communication. Extracellular Ca2+ and the paracrine pathway have practically no effect on the endogenous Ca2+ response, demonstrated by application of Ca2+-free medium, exogenous ATP, and P2Y13 receptor antagonist. In contrast, the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and inositol trisphosphate (IP3) receptor blocker 2-aminoethyl diphenylborate significantly inhibit the endogenous Ca2+ increase, which signifies involvement of IP3-sensitive calcium store release. Notably, passive Ca2+ diffusion into the connected cell can clearly be detected when IP3-sensitive calcium store release is abolished by 2-aminoethyl diphenylborate. Those observations prove that both passive Ca2+ diffusion and IP3-mediated endogenous Ca2+ response contribute to the Ca2+ increase in intercellular-bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.

Flip-Flopping Retinal in Microbial Rhodopsins as a Template for a Farnesyl/Prenyl Flip-Flop Model in Eukaryote GPCRs.

Thirty years after the first description and modeling of G protein coupled receptors (GPCRs), information about their mode of action is still limited. One of the questions that is hard to answer is: how do the allosteric changes in the GPCR induced by, e.g., ligand binding in the end activate a G protein-dependent intracellular pathway (e.g., via the cAMP or the phosphatidylinositol signal pathways). Another question relates to the role of prenylation of G proteins. Today’s “consensus model” states that protein prenylation is required for the assembly of GPCR-G protein complexes. Although it is well-known that protein prenylation is the covalent addition of a farnesyl- or geranylgeranyl moiety to the C terminus of specific proteins, e.g., α or γ G protein, the reason for this strong covalent binding remains enigmatic. The arguments for a fundamental role for prenylation of G proteins other than just being a hydrophobic linker, are gradually accumulating. We uncovered a dilemma that at first glance may be considered physiologically irrelevant, however, it may cause a true change in paradigm. The consensus model suggests that the only functional role of prenylation is to link the G protein to the receptor. Does the isoprenoid nature of the prenyl group and its exact site of attachment somehow matter? Or, are there valid arguments favoring the alternative possibility that a key role of the G protein is to guide the covalently attached prenyl group to – and it hold it in – a very specific location in between specific helices of the receptor? Our model says that the farnesyl/prenyl group – aided by its covalent attachment to a G protein -might function in GPCRs as a horseshoe-shaped flexible (and perhaps flip-flopping) hydrophobic valve for restricting (though not fully inhibiting) the untimely passage of Ca2+, like retinal does for the passage of H+ in microbial rhodopsins that are ancestral to many GPCRs.

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promiseto revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues withchamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to8months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Cryo-EM reveals ligand induced allostery underlying InsP3R channel gating

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are cation channels that mobilize Ca2+ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP3R activation is the coupled interplay between binding of InsP3 and Ca2+ that switches the ion conduction pathway between closed and open states to enable the passage of Ca2+ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here, we present the electron cryo-microscopy structure of InsP3R1 from rat cerebellum determined to 4.1 Å resolution in the presence of activating concentrations of Ca2+ and adenophostin A (AdA), a structural mimetic of InsP3 and the most potent known agonist of the channel. Comparison with the 3.9 Å-resolution structure of InsP3R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP3R channel.

The mitochondrial calcium uniporter regulates procoagulant platelet formation

Essentials Mitochondrial hyperpolarization enhances the conversion of platelets to a procoagulant phenotype. Mitochondrial calcium uniporter (MCU) function is essential in procoagulant platelet formation. Mitochondrial calcium uniporter deletion does not impact other aspects of platelet activation. Ablation of MCU results in the emergence of a permeability transition pore-independent pathway. SUMMARY: Background Procoagulant platelets comprise a phenotypically distinct subpopulation of activated platelets with high-level phosphatidylserine externalization. When initiated by co-stimulation with thrombin and a glycoprotein VI (GPVI) agonist, the transition to the procoagulant phenotype is mediated by extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation. Objectives The intracellular mechanisms coordinating these distinct cytoplasmic and mitochondrial processes remain unclear. The mitochondrial calcium uniporter (MCU) protein is a central component of the transmembrane ion channel that allows the passage of Ca2+ from the cytosol into the mitochondrial matrix. Here we investigate the role of the MCU in the regulation of procoagulant platelet formation. Results Procoagulant platelet formation was directly correlated with pre-stimulatory mitochondrial transmembrane potential, a key determinant of calcium flux from the cytoplasm to the mitochondria. The role of MCU in the regulation of procoagulant platelet formation was investigated using MCU null platelets. Procoagulant platelet formation in MCU null platelets was significantly decreased coincident with decreased mPTP formation. In contrast, neither granule release nor initial integrin activation was altered in response to stimulation. In the genomic absence of MCU, developmental induction of an alternative intracellular pathway partially rescued procoagulant platelet formation. Conclusion These results identify a key role for the mitochondrial calcium uptake channel in the regulation of mPTP-mediated procoagulant platelet formation and suggest a novel pharmacologic target for procoagulant-platelet-related pathologies.

Transient receptor potential channel 6 knockdown prevents apoptosis of renal tubular epithelial cells upon oxidative stress via autophagy activation

Reactive oxygen species (ROS) are generated under various pathological conditions such as renal ischemia/reperfusion (I/R) injury and provoke damage to multiple cellular organelles and processes. Overproduction of ROS causes oxidative stress and contributes to damages of renal proximal tubular cells (PTC), which are the main cause of the pathogenesis of renal I/R injury. Autophagy is a dynamic process that removes long-lived proteins and damaged organelles via lysosome-mediated degradation, which has an antioxidant effect that relieves oxidative stress. The canonical transient receptor potential channel 6 (TRPC6), a nonselective cation channel that allows passage of Ca2+, plays an important role in renal disease. Yet, the relationship between TRPC6 and autophagy, as well as their functions in renal oxidative stress injury, remains unclear. In this study, we found that oxidative stress triggered TRPC6-dependent Ca2+ influx in PTC to inhibit autophagy, thereby rendering cells more susceptible to death. We also demonstrated that TRPC6 knockout (TRPC6-/-) or inhibition by SAR7334, a TRPC6-selective inhibitor, increased autophagic flux and mitigated oxidative stress-induced apoptosis of PTC. The protective effects of TRPC6 ablation were prevented by autophagy inhibitors Chloroquine and Bafilomycin A1. Moreover, this study also shows that TRPC6 blockage promotes autophagic flux via inhibiting the PI3K/Akt/mTOR and ERK1/2 signaling pathways. This is the first evidence showing that TRPC6-mediated Ca2+ influx plays a novel role in suppressing cytoprotective autophagy triggered by oxidative stress in PTC, and it may become a novel therapeutic target for the treatment of renal oxidative stress injury in the future.

BH3 mimetic-elicited Ca2+ signals in pancreatic acinar cells are dependent on Bax and can be reduced by Ca2+-like peptides

BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of anti- and pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death. These compounds have been developed as anti-cancer agents to counteract increased levels of Bcl-2 proteins often present in cancer cells. Application of a chemotherapeutic drug supported with a BH3 mimetic has the potential to overcome drug resistance in cancers overexpressing anti-apoptotic Bcl-2 proteins and thus increase the success rate of the treatment. We have previously shown that the BH3 mimetics, BH3I-2′ and HA14-1, induce Ca2+ release from intracellular stores followed by a sustained elevation of the cytosolic Ca2+ concentration. Here we demonstrate that loss of Bax, but not Bcl-2 or Bak, inhibits this sustained Ca2+ elevation. What is more, in the absence of Bax, thapsigargin-elicited responses were decreased; and in two-photon-permeabilised bax−/− cells, Ca2+ loss from the ER was reduced compared to WT cells. The Ca2+-like peptides, CALP-1 and CALP-3, which activate EF hand motifs of Ca2+-binding proteins, significantly reduced excessive Ca2+ signals and necrosis caused by two BH3 mimetics: BH3I-2′ and gossypol. In the presence of CALP-1, cell death was shifted from necrotic towards apoptotic, whereas CALP-3 increased the proportion of live cells. Importantly, neither of the CALPs markedly affected physiological Ca2+ signals elicited by ACh, or cholecystokinin. In conclusion, the reduction in passive ER Ca2+ leak in bax−/− cells as well as the fact that BH3 mimetics trigger substantial Ca2+ signals by liberating Bax, indicate that Bax may regulate Ca2+ leak channels in the ER. This study also demonstrates proof-of-principle that pre-activation of EF hand Ca2+-binding sites by CALPs can be used to ameliorate excessive Ca2+ signals caused by BH3 mimetics and shift necrotic death towards apoptosis.

Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling.

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+ signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+ signaling, including most prominently an inhibition of the passive ER Ca2+ leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+ leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+ signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+ signaling machinery are different.

Mitochondrial Calcium Entry Is Essential in Agonist-Induced Procoagulant Platelet Formation

Procoagulant platelets are a subpopulation of activated platelets with high-level phosphatidylserine (PSer) externalization. When initiated by co-stimulation with thrombin (THR) and a GPVI agonist, such as convulxin (CVX) or collagen, both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant platelet phenotype. The intracellular mechanisms coordinating these processes remain unclear. Here we investigated the role of the mitochondrial calcium uniporter in the regulation of procoagulant platelet formation.The flux of calcium from the cytosol to the mitochondrial matrix is limited by the ion-impermeable inner mitochondrial membrane. Initially, a pharmacologic approach was utilized to investigate the importance of mitochondrial calcium entry and elevation. Washed human or murine platelets were stimulated with THR and/or CVX in physiologic buffer with 2 mM CaCl2. Mitochondrial transmembrane potential was evaluated using 0.5 μM tetramethylrhodamine methylester (TMRM). Limitation of mitochondrial calcium entry either through alteration of pre-stimulatory mitochondrial membrane potential (i.e. inhibition of F0F1 ATP-ase, mitochondrial uncoupling etc.) or using the ruthenium red analog (Ru360) abrogated both mPTP and procoagulant platelet formation consistent with the hypothesis that elevated calcium levels within the mitochondrial matrix drives these processes.The molecular pathway mediating this rapid mechanism of calcium entry was investigated using mitochondrial calcium uniporter (MCU) null platelets. MCU is a transmembrane ion channel that allows the passage of Ca2+ from the cytosol into the mitochondrial matrix. Neither platelet aggregatory ability nor granule release was altered in response to single or dual-agonist stimulation. In contrast, procoagulant platelet formation in MCU null platelets was significantly decreased coincident with decreased mPTP formation as measured by loss of mitochondrial transmembrane potential. Whereas the number of annexin V positive platelets decreased from 60% in WT to 32% in MCU null (p<0.05, n=4). Thus, these results identify a key role of mitochondrial calcium uptake channel in the regulation of strong agonist-initiated procoagulant platelet formation and suggest a novel pharmacologic target that could be used the treatment of procoagulant-platelet related pathologies. DisclosuresNo relevant conflicts of interest to declare.

Ca2+ transport and signalling in enamel cells.

Dental enamel is one of the most remarkable examples of matrix-mediated biomineralization. Enamel crystals form de novo in a rich extracellular environment in a stage-dependent manner producing complex microstructural patterns that are visually stunning. This process is orchestrated by specialized epithelial cells known as ameloblasts which themselves undergo striking morphological changes, switching function from a secretory role to a cell primarily engaged in ionic transport. Ameloblasts are supported by a host of cell types which combined represent the enamel organ. Fully mineralized enamel is the hardest tissue found in vertebrates owing its properties partly to the unique mixture of ionic species represented and their highly organized assembly in the crystal lattice. Among the main elements found in enamel, Ca2+ is the most abundant ion, yet how ameloblasts modulate Ca2+ dynamics remains poorly known. This review describes previously proposed models for passive and active Ca2+ transport, the intracellular Ca2+ buffering systems expressed in ameloblasts and provides an up-dated view of current models concerning Ca2+ influx and extrusion mechanisms, where most of the recent advances have been made. We also advance a new model for Ca2+ transport by the enamel organ.

Segment-specific Ca2+ transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca2+ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca2+ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca2+ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca2+ regulation. Basolateral Ca2+ transport across distal tubules of Drosophila varied throughout the life cycle; Ca2+ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾168h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca2+ uptake and Ca2+ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca2+ transport is bidirectional. Ca2+ uptake across the distal segment of tubules of young adults and Ca2+ release across the distal segment of tubules of older adults was also suggestive of reversible Ca2+ storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca2+ sequestration during periods of high dietary calcium intake and passive Ca2+ release during periods of calcium deficiency.