Restoration Of Ca2 Research Articles

Targeting alarmin release reverses Sjogren's syndrome phenotype by revitalizing Ca2+ signalling.

Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that is embodied by the loss of salivary gland function and immune cell infiltration, but the mechanism(s) are still unknown. The aim of this study was to understand the mechanisms and identify key factors that leads to the development and progression of pSS. Immunohistochemistry staining, FACS analysis and cytokine levels were used to detect immune cells infiltration and activation in salivary glands. RNA sequencing was performed toidentify the molecular mechanisms involved in the development of pSS. The function assays include in vivo saliva collection along with calcium imaging and electrophysiology on isolated salivary gland cells in mice models of pSS. Western blotting, real-time PCR, alarmin release, and immunohistochemistry was performed to identify the channels involved in salivary function in pSS. We provide evidence that loss of Ca2+ signaling precedes a decrease in saliva secretion and/or immune cell infiltration in IL14α, a mouse model for pSS. We also showed that Ca2+ homeostasis was mediated by transient receptor potential canonical-1 (TRPC1) channels and inhibition of TRPC1, resulting in the loss of salivary acinar cells, which promoted alarmin release essential for immune cell infiltration/release of pro-inflammatory cytokines. In addition, both IL14α and samples from human pSS patients showed a decrease in TRPC1 expression and increased acinar cell death. Finally, paquinimod treatment in IL14α restored Ca2+ homeostasis that inhibited alarmin release thereby reverting the pSS phenotype. These results indicate that loss of Ca2+ signaling is one of the initial factors, which induces loss of salivary gland function along with immune infiltration that exaggerates pSS. Importantly, restoration of Ca2+ signaling upon paquinimod treatment reversed the pSS phenotype thereby inhibiting the progressive development of pSS.

Tetramisole is a new IK1 channel agonist and exerts IK1 -dependent cardioprotective effects in rats.

Cardiac ischemia, hypoxia, arrhythmias, and heart failure share the common electrophysiological changes featured by the elevation of intracellular Ca2+ (Ca2+ overload) and inhibition of the inward rectifier potassium (IK1) channel. IK1 channel agonists have been considered a new type of anti‐arrhythmia and cardioprotective agents. We predicted using a drug repurposing strategy that tetramisole (Tet), a known anthelminthic agent, was a new IK1 channel agonist. The present study aimed to experimentally identify the above prediction and further demonstrate that Tet has cardioprotective effects. Results of the whole‐cell patch clamp technique showed that Tet at 1–100 μmol/L enhanced IK1 current, hyperpolarized resting potential (RP), and shortened action potential duration (APD) in isolated rat cardiomyocytes, while without effects on other ion channels or transporters. In adult Sprague–Dawley (SD) rats in vivo, Tet showed anti‐arrhythmia and anticardiac remodeling effects, respectively, in the coronary ligation‐induced myocardial infarction model and isoproterenol (Iso, i.p., 3 mg/kg/day, 10 days) infusion‐induced cardiac remodeling model. Tet also showed anticardiomyocyte remodeling effect in Iso (1 μmol/L) infused adult rat ventricular myocytes or cultured H9c2 (2‐1) cardiomyocytes. Tet at 0.54 mg/kg in vivo or 30 μmol/L in vitro showed promising protections on acute ischemic arrhythmias, myocardial hypertrophy, and fibrosis. Molecular docking was performed and identified the selective binding of Tet with Kir2.1. The cardioprotection of Tet was associated with the facilitation of IK1 channel forward trafficking, deactivation of PKA signaling, and inhibition of intracellular calcium overload. Enhancing IK1 may play dual roles in anti‐arrhythmia and antiventricular remodeling mediated by restoration of Ca2+ homeostasis.

Cecropia pachystachya protection against preproIAPP cytotoxicity is independent of Ca2+ homeostasis: lessons learned using a novel yeast model of preproIAPP-induced Ca2+ intracellular dysregulation

Despite the progresses in therapeutics, type 2 diabetes is still an epidemic with alarming numbers worldwide. New strategies for prevention and treatments are imperative. A venue of research with potential is the aggregation of Islet Amyloid PolyPeptide (IAPP), a contributor to pancreatic β-cell dysfunction. To address the impact of IAPP on calcium (Ca2+) signalling, we developed a dual reporter yeast model independently expressing preproIAPP-GFP (ppIAPP) and encoding the lacZ gene under the control of Crz1-recognition elements. In this reporter system, ppIAPP induced Crz1 hyperactivation, a yeast transcription factor activated by the Ca2+/calmodulin/calcineurin pathway, which was followed by the increase in β-galactosidase activity. Encouraged by the reported healthy effects of Urticaceae plants against metabolic disturbances, we tested the protective potential of Cecropia pachystachya against IAPP-induced cytotoxicity using the newly designed yeast model. Although C. pachystachya extract exerted no beneficial effects towards the prevention of ppIAPP cytotoxicity, treatment with C. pachystachya enriched C-glycosyl flavonoid fraction (EFF-Cp) significantly improved viability of ppIAPP-expressing cells. Potential bioactivities of C. pachystachya extract and EFF-Cp towards the restoration of Ca2+ homeostasis disrupted by ppIAPP expression were also assessed. Neither prevented Crz1 hyperactivation, suggesting that the EFF-Cp-induced protection against ppIAPP toxicity was mediated by Ca2+-independent mechanisms. Keywords: Amylin, Calcium signaling; Cecropia pachystachya; Diabetes; Islet Amyloid Polypeptide (IAPP)

Local recovery of cardiac calcium-induced calcium release interrogated by ultra-effective, two-photon uncaging of calcium.

Key points In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery.Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias.We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two‐photon photolysis of a new ultra‐effective caged calcium compound.The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation.Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. In cardiac myocytes Ca2+‐induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca2+ release occurs via subcellular Ca2+ signalling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both SR refilling and restoration of Ca2+ sensitivity of the RyRs. We used two‐photon (2P) photolysis of the ultra‐effective caged Ca2+ compound BIST‐2EGTA and laser‐scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in the presence of cAMP or low‐dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST‐2EGTA and rhod‐2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca2+ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self‐learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca2+ sensitivity of CICR. This new 2P approach provides composite information of Ca2+ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.

The Interplay Between Depression and Parkinson´s Disease: Learning the Link Through Ca2+/cAMP Signaling.

Parkinson´s disease (PD) and depression have an interplay at multiple cellular levels, a phenomenon which is translated into clinical data showing that depressive patients presented an enhanced risk for developing PD. The pathogenesis of both diseases is under intensive debate as correlated to dysregulations related to Ca2+ signaling. Then, revealing this interplay between these diseases may provide novel insights into the pathogenesis of them. Publications involving Ca2+ signaling, PD and depression (alone or combined) were collected by searching PubMed and EMBASE. Not surprisingly, calcium (Ca2+) channel blockers (CCBs), classical antihypertensive medicines, have been demonstrated off-label effects, such as alleviating both PD and depression symptoms. A mechanism under debate for the antiparkinsonism and antidepressant effects associated to CCBs is focused on the restoration of Ca2+ signaling dysregulations. In addition, previous studies have observed that CCBs can affect Ca2+/cAMP signaling. Thus, this article discussed the role of Ca2+/cAMP signaling in the interplay between depression and PD, including the implications for the pharmacotherapy involving CCBs.

MLP-deficient human pluripotent stem cell derived cardiomyocytes develop hypertrophic cardiomyopathy and heart failure phenotypes due to abnormal calcium handling

Muscle LIM protein (MLP, CSRP3) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

Boron Deficiency Increases Cytosolic Ca2+ Levels Mainly via Ca2+ Influx from the Apoplast in Arabidopsis thaliana Roots.

Boron (B) is a micronutrient for plant development, and its deficiency alters many physiological processes. However, the current knowledge on how plants are able to sense the B-starvation signal is still very limited. Recently, it has been reported that B deprivation induces an increase in cytosolic calcium concentration ([Ca2+]cyt) in Arabidopsis thaliana roots. The aim of this work was to research in Arabidopsis whether [Ca2+]cyt is restored to initial levels when B is resupplied and elucidate whether apoplastic Ca2+ is the major source for B-deficiency-induced rise in [Ca2+]cyt. The use of chemical compounds affecting Ca2+ homeostasis showed that the rise in root [Ca2+]cyt induced by B deficiency was predominantly owed to Ca2+ influx from the apoplast through plasma membrane Ca2+ channels in an IP3-independent manner. Furthermore, B resupply restored the root [Ca2+]cyt. Interestingly, expression levels of genes encoding Ca2+ transporters (ACA10, plasma membrane PIIB-type Ca2+-ATPase; and CAX3, vacuolar cation/proton exchanger) were upregulated by ethylene glycol tetraacetic acid (EGTA) and abscisic acid (ABA). The results pointed out that ACA10, and especially CAX3, would play a major role in the restoration of Ca2+ homeostasis after 24 h of B deficiency.

A role for calcium in resistin transcriptional activation in diabetic hearts

The adipokine resistin has been proposed to link obesity, insulin resistance and diabetes. We have previously reported that diabetic hearts express high levels of resistin while overexpression of resistin in adult rat hearts gives rise to a phenotype resembling diabetic cardiomyopathy. The transcriptional regulation of resistin in diabetic cardiac tissue is currently unknown. This study investigated the mechanism of resistin upregulation and the role of Serca2a in its transcriptional suppression. We demonstrate that restoration of Ca2+ homeostasis in diabetic hearts, through normalization of Serca2a function genetically and pharmacologically, suppressed resistin expression via inhibition of NFATc. H9c2 myocytes stimulated with high-glucose concentration or Ca2+ time-dependently increased NFATc and resistin expression while addition of the Ca2+ chelator BAPTA-AM attenuated this effect. NFATc expression was enhanced in hearts from ob/ob diabetic and from cardiac-specific Serca2a−/− mice. Similarly, NFATc increased resistin expression in myocytes cultured in low glucose while the NFATc inhibitor VIVIT blocked glucose-induced resistin expression, suggesting that hyperglycemia/diabetes induces resistin expression possibly through NFATc activation. Interestingly, overexpression of Serca2a or VIVIT mitigated glucose-stimulated resistin and NFATc expression and enhanced AMPK activity, a downstream target of resistin signaling. NFATc direct activation of resistin was verified by resistin promoter luciferase activity and chromatin-immunoprecipitation analysis. Interestingly, activation of Serca2a by a novel agonist, CDN1163, mirrored the effects of AAV9-Serca2a gene transfer on resistin expression and its promoter activity and AMPK signaling in diabetic mice. These findings parse a role for Ca2+ in resistin transactivation and provide support that manipulation of Serca2a-NFATc-Resistin axis might be useful in hyper-resistinemic conditions.

Moderate weight loss attenuates chronic endoplasmic reticulum stress and mitochondrial dysfunction in human obesity

ObjectiveIn obese patients undergoing caloric restriction, there are several potential mechanisms involved in the improvement of metabolic outcomes. The present study further explores whether caloric restriction can modulate endoplasmic reticulum (ER) stress and mitochondrial function, as both are known to be mechanisms underlying inflammation and insulin resistance (IR) during obesity. MethodsA total of 64 obese patients with BMI ≥35 kg/m2 underwent a dietary program consisting of 6 weeks of a very-low-calorie diet followed by 18 weeks of low-calorie diet. We evaluated changes in the metabolic and inflammatory markers -TNFα, hsCRP, complement component 3 (C3c), and retinol binding protein 4 (RBP4)-, in the ER stress markers and modulators -eIF2α-P, sXBP1, ATF6, JNK-P, CHOP, GRP78, and SIRT1-, and in mitochondrial function parameters -mitochondrial reactive oxygen species (mROS), glutathione peroxidase 1 (GPX1), cytosolic Ca2+, and mitochondrial membrane potential. ResultsThe dietary intervention produced an 8.85% weight loss associated with enhanced insulin sensitivity, a less marked atherogenic lipid profile, and a decrease in systemic inflammation (TNFα, hsCRP) and adipokine levels (RBP4 and C3c). Chronic ER stress was significantly reduced (ATF6-CHOP, JNK-P) and expression levels of SIRT1 and GRP78 – a Ca2+-dependent chaperone – were increased and accompanied by the restoration of Ca2+ depots. Furthermore, mROS production and mitochondrial membrane potential improvement were associated with the up-regulation of the antioxidant enzyme GPX1. ConclusionsOur data provide evidence that moderate weight loss attenuates systemic inflammation and IR and promotes the amelioration of ER stress and mitochondrial dysfunction, increasing the expression of chaperones, SIRT1 and antioxidant GPX1.

Local Recovery of CICR Probed by Ultra-Effective Calcium Uncaging

In cardiac myocytes a small amount of Ca2+ enters the cytoplasm upon depolarization and initiates Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). Ca2+ release occurs via subcellular local Ca2+ signaling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both, SR refilling and restoration of Ca2+ sensitivity of the RyRs. In this work we used two photon (2P) photolysis of the ultra effective caged Ca2+ compound BIST-2EGTA [1] and laser-scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in presence of a low dose of caffeine (to mimic sensitisation of RyRs) or cyclopiazonic acid (CPA, SERCA inhibitor to slow SR refilling). Whole-cell patch-clamped cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (5 ms, 810 nm) with different intra pulse intervals were applied to test Ca2+ release amplitude recovery. Data recorded in the presence of CPA showed slower recovery with a time constant of ∼76 ms compared to control condition (∼56 ms). In the presence of a low dose of caffeine the recovery was faster than control (∼31 ms). We obtained comparable results with the well established “ryanodine method” [2], where a low dose of ryanodine is used to trigger repetitive sparks from single cluster of RyRs. This new 2P approach provides composite information of Ca2+ release amplitude recovery reflecting both SR refilling and restoration of Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias. 1. Agarwal, H. K., Janicek, R., Chi, S.-H., Perry, J. W., Niggli, E., and Ellis-Davies, G. C. R. (2016), Calcium uncaging with visible light. JACS 138, 3687-3693. 2. Sobie, E. A., Song, L.-S. and Lederer, W. J. (2005), Local recovery of Ca2+ release in rat ventricular myocytes. J. Physiol, 565, 441-447.

Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides.

The membrane protein complex between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN’s inhibitory effects on SERCA, increasing the ATPase’s apparent Ca2+ affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca2+ flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults.

P53 orchestrates calcium signaling in vivo

Intracellular calcium (Ca2+) governs several aspects of the homeostasis of living organisms. The levels of Ca2+ in the cytosol are maintained at a concentration of about 0.1 μM by the continuous action of ATP-powered Ca2+ pumps, which transport Ca2+ ions across the plasma membrane to the outside of the cell or into the lumen of the endoplasmic reticulum (ER) and other vesicles.1 In response to a variety of stimuli, Ca2+ is released from these storage sites leading to the activation of several Ca2+ responsive proteins. Intracellular Ca2+ fluxes play fundamental roles in regulating a wide variety of cellular responses, among which programmed cell death has been one of the most studied biological process in the last decade. Several reports have demonstrated that the Ca2+ content within the ER determines cell sensitivity to apoptotic stress and that changes in the Ca2+ flux from the ER to mitochondria represent a signal capable of triggering the rupture of the mitochondrial membrane as well as the consequent release of several pro-apoptotic factors into the cytosol.1 This pathway is inactivated or impaired in cancer progression, with important consequences. Indeed, several tumor suppressor genes, such as PML, PTEN and TP53, have been unexpectedly found enriched at the ER where they regulate the Ca2+ crosstalk between the ER and mitochondria thus favoring Ca2+-dependent apoptosis after stress stimuli.2 Particularly intriguing is the role of p53, a stress induced tumor suppressor, able to orchestrate mitochondrial apoptosis acting both in the nucleus via transcriptional regulation of several proteins (e.g. Bcl-xL, Bcl-2, PUMA and others) as well as in the cytoplasm.3 Despite its biological importance, the mechanism of Ca2+ flux between the ER and mitochondria and its regulation by tumor suppressors is not completely understood. Contributing in filling this gap, Pinton and colleagues found that p53 localizes at ER and at mitochondria-associated membranes (MAMs), and upon activation by a variety of stimuli, is able to bind the sarco/ER Ca2+-ATPase (SERCA) pump, leading to an increase of Ca2+ release from the ER and a consequent mitochondrial Ca2+ overload that profoundly impacts on the ability of the cells to undergo apoptosis.4 The same group has also recently overcome the technical limitations that slowered so far the acquisition of evidences on the in vivo impact of this process, by using a novel strategy that, for the first time, allows the direct monitoring of intracellular Ca2+ fluxes within a tumor mass.5,6 For the first time Giorgi and colleagues, starting from the techniques already used to measure Ca2+ in vivo,7 undoubtedly demonstrated a role for p53 in modulating Ca2+ homeostasis in tumors. The authors injected murine p53−/− embryo fibroblasts (MEF) transduced with the oncogene H-RASV12 alone or with wild type (wt) p53 into a skinfold chamber, that had been implanted in the dorsal skin of female athymic mice. Upon tumor growth, the masses originated from these cells have been treated with phthalocyanine, a light-activatable agent used in cancer photodynamic therapy. Photo-activation of the drug with a LED light caused ROS formation in the ER and consequent Ca2+ release. The monitoring of pro-apoptotic Ca2+ signal with the ratiometric Ca2+ indicator Fura-2 by spinning disc confocal microscopy unveiled a massive release of Ca2+ ions into the cytoplasm of p53+/+ tumors after photo-activation. Instead, Ca2+ release was almost absent in the p53–/– counterpart. Of note, the higher Ca2+ response evoked by p53 was instrumental for an efficient induction of apoptosis and reduction of tumor mass. More interestingly, restoration of Ca2+ fluxes in p53–/– cells by overexpression of the mitochondrial Ca2+ uniporter (MCU) or of the sarco/ER Ca2+-ATPase (SERCA) pump, was able to rescue the apoptotic sensitivity of the cells to activated phthalocyanine. On the other hand, pharmacological manipulation of Ca2+ signaling in p53+/+ tumors efficiently prevented apoptosis in vitro and in vivo, meaning that a functional p53 is required to trigger an efficient stress-induced Ca2+ release able to sustain mitochondrial swelling and apoptosis (Fig. 1). Figure 1. Intravital imaging of ER-mitochondrial Ca2+ transfer and its regulation by p53. Ca2+ is transferred to the ER by the SERCA pumps and is released by inositol 1,4,5-triphosphate (IP3)-gated channels (IP3R). p53 binds and activates SERCA pumps thus increasing ... The potential impact of this new technique is wide opening the possibility of studying protein and RNA-based modulators of the Ca2+-mediated apoptotic cascade in vivo and also allowing the screening of molecules that could change cancer cell sensitivity to apoptotic signals.

Non-selective cation channels mediate chloroquine-induced relaxation in precontracted mouse airway smooth muscle.

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca2+ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca2+ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca2+-free conditions, and, following a restoration of Ca2+, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca2+ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.

ІР3-Чутливі Са2+-канали ендоплазматичного ретикулума секреторних клітин зовнішньоорбітальних сльозових залоз щура

The role of inositol-1,4,5-trisphosphate of (IP3)-sensitive Ca2+ channels in Ca2+ homeostasis maintenance under activation of M-cholinergic receptors and P2Y receptors in the secretory cells of the rat lacrimal gland was investigated. The study was carried out on intact and permeabilized secretory cells of exorbital lacrimal glands of rats. The cells were isolated using the modified Herzog, Sides, Miller method (1976) and permeabilized with digitonin (50 mg per 0.5 million cells). The functioning of the Ca(2+)-transport systems was estimated by changes of Ca2+ content in the studied cells, which was determined by the spectrophotometric method using arsenazo III. It was shown that IP3-sensitive Ca2+ channels (IP3Rs) of investigated cells are directly inhibited by 2-APB (10 microM/l). On the other hand, the channels are activated by IP3, cholinomimetic (carbacholine) and purine receptor agonist (ATP). When both M-cholinergic receptors and P2Y receptors were activated Ca2+ was released from the same IP3-sensitive store because the effects of ATP and carbacholine at high concentrations (1mM/l and 10 microM/l, respectively) on the Ca2+ content were non-additive. The presence of the store-operated Ca(2+)-channels in secretory cells of the lacrimal gland is confirmed by the observed increase of cellular Ca2+ content as a result of Ca2+ mobilization from the store by carbacholine or thapsigargin and following restoration of Ca2+ concentration in the extracellular solution.

Subcellular Structures and Function of Myocytes Impaired During Heart Failure Are Restored by Cardiac Resynchronization Therapy

Cardiac resynchronization therapy (CRT) is an established treatment for patients with chronic heart failure. However, CRT-associated structural and functional remodeling at cellular and subcellular levels is only partly understood. To investigate the effects of CRT on subcellular structures and protein distributions associated with excitation-contraction coupling of ventricular cardiomyocytes. Our studies revealed remodeling of the transverse tubular system (t-system) and the spatial association of ryanodine receptor (RyR) clusters in a canine model of dyssynchronous heart failure (DHF). We did not find this remodeling in a synchronous heart failure model based on atrial tachypacing. Remodeling in DHF ranged from minor alterations in anterior left ventricular myocytes to nearly complete loss of the t-system and dissociation of RyRs from sarcolemmal structures in lateral cells. After CRT, we found a remarkable and almost complete reverse remodeling of these structures despite persistent left ventricular dysfunction. Studies of whole-cell Ca(2+) transients showed that the structural remodeling and restoration were accompanied with remodeling and restoration of Ca(2+) signaling. DHF is associated with regional remodeling of the t-system. Myocytes undergo substantial structural and functional restoration after only 3 weeks of CRT. The finding suggests that t-system status can provide an early marker of the success of this therapy. The results could also guide us to an understanding of the loss and remodeling of proteins associated with the t-system. The steep relationship between free Ca(2+) and contraction suggests that some restoration of Ca(2+) release units will have a disproportionately large effect on contractility.

Decrease in phosphatidylinositol 4,5‐bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels

The activity of the cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels diminishes over time in the presence of extracellular Ca(2+), a phenomenon referred to as desensitization or adaptation. Here we show that activation of TRPM8 by cold or menthol evokes a decrease in cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] levels. The decrease in PtdIns(4,5)P(2) levels was accompanied by increased inositol 1,4,5 trisphosphate (InsP(3)) production, and was inhibited by loading the cells with the Ca(2+) chelator BAPTA-AM, showing that it was the consequence of the activation of phospholipase C (PLC) by increased intracellular Ca(2+) concentrations. PtdIns(4,5)P(2) hydrolysis showed excellent temporal correlation with current desensitization in simultaneous patch clamp and fluorescence-based PtdIns(4,5)P(2) level measurements. Intracellular dialysis of PtdIns(4,5)P(2) inhibited desensitization both in native neuronal and recombinant TRPM8 channels. PtdIns(4)P, the precursor of PtdIns(4,5)P(2), did not inhibit desensitization, consistent with its minimal effect in excised patches. Omission of MgATP from the intracellular solution accelerated desensitization, and MgATP reactivated TRPM8 channels in excised patches in a phosphatidylinositol 4-kinase (PI4K)-dependent manner. PLC-independent depletion of PtdIns(4,5)P(2) using a voltage-sensitive phosphatase (ci-VSP) inhibited TRPM8 currents, and omission of ATP from the intracellular solution inhibited recovery from this inhibition. Inhibitors of PKC had no effect on the kinetics of desensitization. We conclude that Ca(2+) influx through TRPM8 activates a Ca(2+)-sensitive PLC isoform, and the resulting depletion of PtdIns(4,5)P(2) plays a major role in desensitization of both cold and menthol responses.

SV2 Acts via Presynaptic Calcium to Regulate Neurotransmitter Release

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

Cch1 Restores Intracellular Ca2+ in Fungal Cells during Endoplasmic Reticulum Stress

Pathogens endure and proliferate during infection by exquisitely coping with the many stresses imposed by the host to prevent pathogen survival. Recent evidence has shown that fungal pathogens and yeast respond to insults to the endoplasmic reticulum (ER) by initiating Ca(2+) influx across their plasma membrane. Although the high affinity Ca(2+) channel, Cch1, and its subunit Mid1, have been suggested as the protein complex responsible for mediating Ca(2+) influx, a direct demonstration of the gating mechanism of the Cch1 channel remains elusive. In this first mechanistic study of Cch1 channel activity we show that the Cch1 channel from the model human fungal pathogen, Cryptococcus neoformans, is directly activated by the depletion of intracellular Ca(2+) stores. Electrophysiological analysis revealed that agents that enable ER Ca(2+) store depletion promote the development of whole cell inward Ca(2+) currents through Cch1 that are effectively blocked by La(3+) and dependent on the presence of Mid1. Cch1 is permeable to both Ca(2+) and Ba(2+); however, unexpectedly, in contrast to Ca(2+) currents, Ba(2+) currents are steeply voltage-dependent. Cch1 maintains a strong Ca(2+) selectivity even in the presence of high concentrations of monovalent ions. Single channel analysis indicated that Cch1 channel conductance is small, similar to that reported for the Ca(2+) current I(CRAC). This study demonstrates that Cch1 functions as a store-operated Ca(2+)-selective channel that is gated by intracellular Ca(2+) depletion. The inability of cryptococcal cells that lacked the Cch1-Mid1 channel to survive ER stress suggests that Cch1 and its co-regulator, Mid1, are critical players in the restoration of Ca(2+) homeostasis.

Adenosine A3 receptor-mediated cardioprotection against doxorubicin-induced mitochondrial damage

Cardiotoxicity associated with doxorubicin (DOX) treatment limits the therapeutic efficiency of this drug against cancer. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA), a selective agonist of A3 adenosine receptor (A3R), reduces DOX toxicity in newborn rat cultured cardiomyocytes. The study's aim was to determine whether the protection demonstrated by Cl-IB-MECA attenuates cardiac depression in vivo. In addition, we wished to examine whether this protective pathway affects the sarcoplasmic reticulum (SR) calcium uptake and release, as well as intramitochondrial Ca2+ accumulation induced by DOX.Rats were injected every alternate day (6 times) with (1) saline, (2) 2.5mg/kg i.p. DOX, (3) 33μg/kg i.v. Cl-IB-MECA, (4) DOX+Cl-IB-MECA. Left ventricular functions were assessed by invasive (pressure) and non-invasive (echocardiography) techniques at the end of the injection period and 4 weeks later. Cytosolic and intramitochondrial calcium levels were measured with indo-1 and rhod-2 probes. SR Ca2+ content was determined by exposing cultured rat cardiomyocytes to caffeine.Echocardiography data demonstrate left ventricular wall thinning (23%), an increase in the end systolic dimension (170%) and decreased fractional shortening (35±5% vs. 54±5%, p<0.01) in DOX-treated animals, compared to the control group. DOX increased Ca2+ levels in the cytosol and in mitochondria by diminishing the SR Ca2+ uptake. Pretreatment with Cl-IB-MECA attenuated left ventricular dysfunction, improved SR calcium storage capacity and prevented mitochondrial Ca2+ overload.We conclude that the adenosine A3 receptor agonist is effective in vivo against DOX cardiotoxicity via the restoration of Ca2+ homeostasis and prevention of mitochondrial damage that occurs as a result of Ca2+ overload.

Cannabidiol Targets Mitochondria to Regulate Intracellular Ca2+Levels

Cannabinoids and the endocannabinoid system have attracted considerable interest for therapeutic applications. Nevertheless, the mechanism of action of one of the main nonpsychoactive phytocannabinoids, cannabidiol (CBD), remains elusive despite potentially beneficial properties as an anti-convulsant and neuroprotectant. Here, we characterize the mechanisms by which CBD regulates Ca(2+) homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal cultures using fura-2 AM suggested that CBD-mediated Ca(2+) regulation is bidirectional, depending on the excitability of cells. Under physiological K(+)/Ca(2+) levels, CBD caused a subtle rise in [Ca(2+)](i), whereas CBD reduced [Ca(2+)](i) and prevented Ca(2+) oscillations under high-excitability conditions (high K(+) or exposure to the K(+) channel antagonist 4AP). Regulation of [Ca(2+)](i) was not primarily mediated by interactions with ryanodine or IP(3) receptors of the endoplasmic reticulum. Instead, dual-calcium imaging experiments with a cytosolic (fura-2 AM) and a mitochondrial (Rhod-FF, AM) fluorophore implied that mitochondria act as sinks and sources for CBD's [Ca(2+)](i) regulation. Application of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na(+)/Ca(2+) exchange inhibitor, CGP 37157, but not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBD-induced Ca(2+) responses. In established human neuroblastoma cell lines (SH-SY5Y) treated with mitochondrial toxins, CBD (0.1 and 1 microm) was neuroprotective against the uncoupler FCCP (53% protection), and modestly protective against hydrogen peroxide- (16%) and oligomycin- (15%) mediated cell death, a pattern also confirmed in cultured hippocampal neurons. Thus, under pathological conditions involving mitochondrial dysfunction and Ca(2+) dysregulation, CBD may prove beneficial in preventing apoptotic signaling via a restoration of Ca(2+) homeostasis.