Intracellular Ca Release Research Articles

Transcriptome profiling of DPP stimulated DPSCs identifies the role of autophagy in odontogenic differentiation

Dentin phosphophoryn (DPP), synthesized and processed predominantly by the odontoblasts, serves both a structural and signaling role in dentin. In the ECM, DPP functions as an avid calcium and collagen binding protein and it also plays a crucial role as a scaffold for cell attachment and survival. The signaling function of DPP was demonstrated when undifferentiated mesenchymal cells stimulated with DPP, mediated calcium signaling through release of intracellular Ca2+. The objective of this study was to identify potentially novel signaling mechanisms that mediate odontoblast differentiation. Therefore, transcriptomes of DPSCs (dental pulp stem cells) with or without DPP stimulation were compared by bulk RNA-seq. Analysis of the unbiased RNA-seq data were subjected to functional enrichment analysis using Gene Ontology (GO) and KEGG pathways. Results identified several upregulated genes which were associated with autophagy, that were subsequently validated by RT-PCR. Western blotting analysis confirmed the up regulation of several autophagy markers such as ATG5, BECN1 and LC3A/B at specific time points. Autophagosome formation was also observed with DPP treatment. Additionally, autophagy supported a role for odontoblast differentiation of DPSCs. These findings suggest that DPP mediated autophagy might be a potential mechanism for the survival and terminal differentiation of DPSCs.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, Aβ, Aδ, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

Nanoparticulate System Based on Calcium-Crosslinked Carbomer Retards Percutaneous Drug Permeation: New Insight Into Skin Barrier Functions.

The stratum corneum poses a formidable barrier for dermal and transdermal delivery of drugs. Besides the stratum corneum barrier, the viable epidermis poses another challenge to pharmaceutical formulators. A drug is probably transdermally permeable if it rapidly crosses the epidermal secondary barrier, while stimulation of lamellar body secretion from granular cells and intracellular release of Ca++ from endoplasmic reticulum (ER) result in retardation. To evaluate the skin permeability of lidocaine HCl loaded in nanoparticles made of carbomer calcified with calcium gluconate, while figuring out the physiological mechanism that regulates the Ca++ related skin barrier function. Lidocaine hydrochloride was loaded in a nanoparticulate system based on calcified carbomer, fabricated by using a water-in-oil microemulsion as a precursor. In vitro release and percutaneous permeation testing were carried out to compare between calcified and non-calcified nanoparticles. In addition, comparison was also made between calcified nanoparticles using carbomer gels prepared at two pH values and at two different ratios of Ca++/carbomer. A unique structure of the calcified nanoparticles has been proposed, in which the carbomer nanoparticles are partially coated by gluconate ions through hydrogen bonding and partially through ionic interactions with calcium ions. Although the in vitro release data showed no difference between non-calcified and calcified carbomer nanoparticles, a calcium-related phenomenon of skin retardation has been revealed. It has been proposed that stimulation of lamellar body secretion from granular cells and Ca++ release from ER, which is elicited by the calcium gluconate-coated nanoparticles, result in dermal retardation of lidocaine.

Synchronization of spatially discordant voltage and calcium alternans in cardiac tissue.

The heart is an excitable medium which is excited by membrane potential depolarization and propagation. Membrane potential depolarization brings in calcium (Ca) through the Ca channels to trigger intracellular Ca release for contraction of the heart. Ca also affects voltage via Ca-dependent ionic currents, and thus, voltage and Ca are bidirectionally coupled. It has been shown that the voltage subsystem or the Ca subsystem can generate its own dynamical instabilities which are affected by their bidirectional couplings, leading to complex dynamics of action potential and Ca cycling. Moreover, the dynamics become spatiotemporal in tissue in which cells are diffusively coupled through voltage. A widely investigated spatiotemporal dynamics is spatially discordant alternans (SDA) in which action potential duration (APD) or Ca amplitude exhibits temporally period-2 and spatially out-of-phase patterns, i.e., APD-SDA and Ca-SDA patterns, respectively. However, the mechanisms of formation, stability, and synchronization of APD-SDA and Ca-SDA patterns remain incompletely understood. In this paper, we use cardiac tissue models described by an amplitude equation, coupled iterated maps, and reaction-diffusion equations with detailed physiology (the ionic model) to perform analytical and computational investigations. We show that, when the Ca subsystem is stable, the Ca-SDA pattern always follows the APD-SDA pattern, and thus, they are always synchronized. When the Ca subsystem is unstable, synchronization of APD-SDA and Ca-SDA patterns depends on the stabilities of both subsystems, their coupling strengths, and the spatial scales of the initial Ca-SDA patterns. Spontaneous (initial condition-independent) synchronization is promoted by enhancing APD instability and reducing Ca instability as well as stronger Ca-to-APD and APD-to-Ca coupling, a pattern formation caused by dynamical instabilities. When Ca is more unstable and APD is less unstable or APD-to-Ca coupling is weak, synchronization of APD-SDA and Ca-SDA patterns is promoted by larger initially synchronized Ca-SDA clusters, i.e., initial condition-dependent synchronization. The synchronized APD-SDA and Ca-SDA patterns can be locked in-phase, antiphase, or quasiperiodic depending on the coupling relationship between APD and Ca. These theoretical and simulation results provide mechanistic insights into the APD-SDA and Ca-SDA dynamics observed in experimental studies.

Regulatory Effects of Damaged Renal Epithelial Cells After Repair by Porphyra yezoensis Polysaccharides with Different Sulfation Degree on the Calcium Oxalate Crystal-Cell Interaction.

BackgroundThe interaction between urinary microcrystals and renal epithelial cells is closely related to kidney stone formation. However, the mechanism of cell state changes that affect crystal–cell interaction remains unclear.MethodsThis study investigated the relationship between the sulfate group (–OSO3−) content in Porphyra yezoensis polysaccharide (PYP) and the ability to repair damaged cells, as well as the changes in cell adhesion and endocytosis of nano-calcium oxalate monohydrate (COM) crystals before and after PYP repair of damaged renal tubular epithelial cells. The sulfur trioxide–pyridine method was used to sulfate PYP (–OSO3− content of 14.14%), and two kinds of sulfated PYPs with –OSO3− content of 20.28% (SPYP1) and 27.14% (SPYP2) were obtained. The above three PYPs were used to repair oxalate-damaged human proximal tubular epithelial cells (HK-2), and the changes in the biochemical indicators of the cells before and after the repair and the changes in cell adhesion and endocytosis of nano-COM crystals were detected.ResultsAfter repair by PYPs, the cell viability increased, the number of reactive oxygen species decreased, and the reduction of mitochondrial membrane potential and the release of intracellular Ca2+ were suppressed. The cells repaired by PYPs inhibited the adhesion of nano-COM crystals while promoting the endocytosis of the adhered crystals. The endocytosed crystals mainly accumulated in the lysosome. The ability of PYPs to repair cell damage, inhibit crystal adhesion, and promote crystal endocytosis was enhanced when the –OSO3− content increased. Among them, SPYP2 with the highest –OSO3− content showed the best biological activity.ConclusionSPYP2 showed the best ability to repair damaged cells, followed by SPYP1 and PYP. SPYP2 may become a potential green drug that inhibits the formation and recurrence of calcium oxalate stones.

SR-Mitochondria Crosstalk Shapes Ca Signalling to Impact Pathophenotype in Disease Models Marked by Dysregulated Intracellular Ca Release.

Diastolic Ca release (DCR) from sarcoplasmic reticulum (SR) Ca release channel ryanodine receptor (RyR2) has been linked to multiple cardiac pathologies, but its exact role in shaping divergent cardiac pathologies remains unclear. We hypothesize that the SR-mitochondria interplay contributes to disease phenotypes by shaping Ca signalling. A genetic model of catecholaminergic polymorphic ventricular tachycardia (CPVT2 model of CASQ2 knockout) and a pre-diabetic cardiomyopathy model of fructose-fed mice (FFD), both marked by DCR, are employed in this study. Mitochondria Ca (mCa) is modulated by pharmacologically targeting mitochondria Ca uniporter (MCU) or permeability transition pore (mPTP), mCa uptake, and extrusion mechanisms, respectively. An MCU activator abolished Ca waves in CPVT2 but exacerbated waves in FFD cells. Mechanistically this is ascribed to mitochondria's function as a Ca buffer or source of reactive oxygen species (mtROS) to exacerbate RyR2 functionality, respectively. Enhancing mCa uptake reduced and elevated mtROS production in CPVT2 and FFD, respectively. In CPVT2, mitochondria took up more Ca in permeabilized cells, and had higher level of mCa content in intact cells vs. FFD. Conditional ablation of MCU in the CPVT2 model caused lethality and cardiac remodelling, but reduced arrhythmias in the FFD model. In parallel, CPVT2 mitochondria also employ up-regulated mPTP-mediated Ca efflux to avoid mCa overload, as seen by elevated incidence of MitoWinks (an indicator of mPTP-mediated Ca efflux) vs. FFD. Both pharmacological and genetic inhibition of mPTP promoted mtROS production and exacerbation of myocyte Ca handling in CPVT2. Further, genetic inhibition of mPTP exacerbated arrhythmias in CPVT2. In contrast to FFD, which is more susceptible to mtROS-dependent RyR2 leak, in CPVT2 mitochondria buffer SR-derived DCR to mitigate Ca-dependent pathological remodelling and rely on mPTP-mediated Ca efflux to avoid mCa overload. SR-mitochondria interplay contributes to the divergent pathologies by disparately shaping intracellular Ca signalling.

The effects of acetylcholine on intracellular calcium fluorescence in smooth muscle cells of human esophagogastric junction cultured in vitro.

Most esophageal motility studies are based on animals. It is necessary to explore smooth muscle motility in the human esophagus. This study was undertaken to explore the feasibility of in vitro culture of smooth muscle cells (SMCs) from human esophagogastric junction (EGJ) and to determine changes of intracellular calcium (Ca2+ ) fluorescence ([Ca2+ ]i ) in SMCs stimulated by acetylcholine (ACh). Primary cells of EGJ (Clasp, Sling, esophageal circular muscle (ECM), and longitudinal muscle (ELM)) were obtained by enzymatic digestion (ED) and explant culture with tissues (EC-T) from 9 upper esophageal carcinoma patients. Cells were cultured in smooth muscle cell medium (SMCM) and DMEM/F-12medium containing 10% newborn bovine serum (10%-F12), respectively, and then identified by α-SMA staining. After incubation with 5μM Fluo-3/am, the effect of 10-6 mM ACh on [Ca2+ ]i in Ca2+ -containing and Ca2+ -free buffers was evaluated by confocal microscopy. Cultured cells from ED and EC-T were identified as SMCs by α-SMA with spindle surface and "hills and valleys" morphology. Cells cultured in 10%-F12showed better morphology. The main characteristic of [Ca2+ ]i in Clasp-, Sling- and ECM-SMCs was the release of intracellular Ca2+ stores; the main characteristic in ELM-SMCs was extracellular Ca2+ influx. However, these cells seemed not to rely on a unique Ca2+ activity, instead combining the two activities to maintain [Ca2+ ]i . It was feasible to culture human EGJ SMCs in vitro; moreover, Ach-induced changes of [Ca2+ ]i in EGJ SMCs represent a complex interaction of intracellular Ca2+ release and extracellular Ca2+ influx.

PTPN5 promotes follicle‐stimulating hormone secretion through regulating intracellular calcium homeostasis

Protein tyrosine phosphatase non-receptor type 5 (PTPN5), also called striatal-enriched protein tyrosine phosphatase (STEP), is highly expressed in neurons of the basal ganglia, hippocampus, cortex, and related structures, also in the pituitary. Gonadotropins are the key regulator of the reproduction in mammals. In this study, PTPN5 is detected to express in murine pituitary in a developmental manner. Moreover, the expression of PTPN5 in the pituitary is heavily reduced after ovary removal. Follicle-stimulating hormone (FSH) secretion in gonadotropes is regulated by PTPN5 via binding GnRH to GnRH-R. Two parallel signaling pathways, Gs-protein kinase A (PKA)-PTPN5 and Gq-phospholipases C (PLC)-p38 MAPK-PTPN5, cooperatively regulate GnRH-induced FSH secretion. We also show that influx of Ca2+ activates the Ca2+ -dependent phosphatase calcineurin, leading to the phosphorylation and activation of PTPN5. The intracellular release of Ca2+ is reduced via TC2153. In conclusion, blocking or knocking out of PTPN5 reduces the release of FSH in whole pituitary. Mechanically, PTPN5 regulates gonadotropes' function through regulating intracellular calcium homeostasis.

Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca2+) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Cav blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca2+]cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca2+ pools, and Ca2+ influx by ORAI channels have been associated with the increase in [Ca2+]cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil’s effect on [Ca2+]cyt. To address these questions, we monitored changes in [Ca2+]cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca2+]cyt at concentrations above 10 μM (IC50 around 50 μM) and a fast Ca2+ increase capacity at 100 μM. We found that inhibiting IP3 receptors, depleting the ER-Ca2+ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca2+]cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca2+ influx by store-operated Ca2+ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP3R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

Microcystin-leucine arginine induced the apoptosis of GnRH neurons by activating the endoplasmic reticulum stress resulting in a decrease of serum testosterone level in mice

Microcystin-leucine arginine (MC-LR) is a kind of toxin produced by cyanobacterial, resulting in decrease of testosterone levels in serum and leading to impaired spermatogenesis. Gonadotropin-releasing hormone (GnRH) neurons play crucial roles in the regulation of testosterone release. Meanwhile, it has been demonstrated that MC-LR is capable of entering the GnRH neurons and inducing apoptosis. Nevertheless, the molecular mechanism of MC-LR induced apoptosis of GnRH neurons remains elusive. In present study, we found that MC-LR inhibited the cell viability of GT1–7 cells. In addition, we discovered apoptosis of GnRH neurons and GT1–7 cells treated with MC-LR. And increased intracellular ROS production and the release of intracellular Ca2+ were all observed following exposure to MC-LR. Furthermore, we also found the endoplasmic reticulum stress (ERs) and autophagy were activated by MC-LR. Additionally, pretreatment of the ERs inhibitor (4-Phenyl butyric acid) reduced the apoptotic rate of GT1–7 cells comparing with MC-LR exposure alone. Comparing with MC-LR treatment alone, apoptotic cell death was increased by pretreatment of GT1–7 cells with an autophagy inhibitor (3-methyladenine). Together, our data implicated that the treatment of MC-LR induced the apoptosis of GnRH neurons by activating the ERs resulting in a decrease of serum testosterone level in mice. Autophagy is a protective cellular process which was activated by ER stress and thus protected cells from apoptosis upon MC-LR exposure.

Cellular contribution to left and right atrial dysfunction in chronic arterial hypertension in pigs.

AimsAtrial contractile dysfunction contributes to worse prognosis in hypertensive heart disease (HHD), but the role of cardiomyocyte dysfunction in atrial remodelling in HHD is not well understood. We investigated and compared cellular mechanisms of left (LA) and right atrial (RA) contractile dysfunction in pigs with HHD.Methods and results In vivo electrophysiological and magnetic resonance imaging studies were performed in control and pigs treated with 11‐deoxycorticosterone acetate (DOCA)/high‐salt/glucose diet (12 weeks) to induce HHD. HHD leads to significant atrial remodelling and loss of contractile function in LA and a similar trend in RA (magnetic resonance imaging). Atrial remodelling was associated with a higher inducibility of atrial fibrillation but unrelated to changes in atrial refractory period or fibrosis (histology). Reduced atrial function in DOCA pigs was related to reduced contraction amplitude of isolated LA (already at baseline) and RA myocytes (at higher frequencies) due to reduced intracellular Ca release (Fura 2‐AM, field stimulation). However, Ca regulation differed in LA and RA cardiomyocytes: LA cardiomyocytes showed reduced sarcoplasmic reticulum (SR) [Ca], whereas in RA, SR [Ca] was unchanged and SR Ca2+‐ATPase activity was increased. Sodium–calcium exchanger (NCX) activity was not significantly altered. We used ORM‐10103 (3 μM), a specific NCX inhibitor to improve Ca availability in LA and RA cardiomyocytes from DOCA pigs. Partial inhibition of NCX increased Ca2+ transient amplitude and SR Ca in LA, but not RA cells.ConclusionsIn this large animal model of HHD, atrial remodelling in sinus rhythm in vivo was related to differential LA and RA cardiomyocyte dysfunction and Ca signalling. Selective acute inhibition of NCX improved Ca release in diseased LA cardiomyocytes, suggesting a potential therapeutic approach to improve atrial inotropy in HHD.

Salidroside Attenuates Hypoxia-Induced Expression of Connexin 43 in Corpus Cavernosum Smooth Muscle Cells

Introduction: Connexin 43 (Cx43) is the major component of gap junction in corpus cavernosum smooth muscle, which allows rapid intercellular communication. Cx43 coordinates corpus cavernosum smooth muscle cells and ensures erectile function. The role of hypoxia in Cx43 dysfunction resulting in erectile dysfunction has not been well studied, and salidroside has shown cell protective effects under hypoxia. Objective: We aimed to investigate the protective role of salidroside and the underlying mechanisms in hypoxia-induced dysfunction of Cx43. Methods: Corpus cavernosum smooth muscle cells prepared from young male Sprague-Dawley rats were pretreated with or without salidroside and exposed to hypoxic condition for 48 h. The cell viability, expression of hypoxia-inducible factor-1α (HIF-1α) and Cx43, and Ca²⁺ signals were investigated. Results: Pretreatment with salidroside attenuated loss of hypoxia-induced cell viability markedly and could downregulate the HIF-1α protein expression under hypoxia. Moreover, the expression of Cx43 was significantly increased by hypoxia but was decreased with salidroside pretreatment. The salidroside pretreated group exhibited enhanced release of intracellular Ca²⁺ in corpus cavernosum smooth muscle cells compared with the hypoxia group after stimulation. Conclusion: Salidroside has a protective effect against hypoxia-induced damage to corpus cavernosum smooth muscle cells.

Cryptochromes Mediate Intrinsic Photomechanical Transduction in Avian Iris and Somatic Striated Muscle.

Irises isolated from the eyes of diverse species constrict when exposed to light. Depending on species this intrinsic photomechanical transduction response (PMTR) requires either melanopsin or cryptochrome (CRY) photopigment proteins, generated by their respective association with retinoid or flavin adenine dinucleotide (FAD) chromophores. Although developmentally relevant circadian rhythms are also synchronized and reset by these same proteins, the cell type, mechanism, and specificity of photomechanical transduction (PMT) and its relationship to circadian processes remain poorly understood. Here we show that PMTRs consistent with CRY activation by 430 nm blue light occur in developing chicken iris striated muscle, identify relevant mechanisms, and demonstrate that similar PMTRs occur in striated iris and pectoral muscle fibers, prevented in both cases by knocking down CRY gene transcript levels. Supporting CRY activation, iris PMTRs were reduced by inhibiting flavin reductase, but unaffected by melanopsin antagonism. The largest iris PMTRs paralleled the developmental predominance of striated over smooth muscle fibers, and shared their requirement for extracellular Ca2+ influx and release of intracellular Ca2+. Photo-stimulation of identified striated myotubes maintained in dissociated culture revealed the cellular and molecular bases of PMT. Myotubes in iris cell cultures responded to 435 nm light with increased intracellular Ca2+ and contractions, mimicking iris PMTRs and their spectral sensitivity. Interestingly PMTRs featuring contractions and requiring extracellular Ca2+ influx and release of intracellular Ca2+ were also displayed by striated myotubes derived from pectoral muscle. Consistent with these findings, cytosolic CRY1 and CRY2 proteins were detected in both iris and pectoral myotubes, and knocking down myotube CRY1/CRY2 gene transcript levels specifically blocked PMTRs in both cases. Thus CRY-mediated PMT is not unique to iris, but instead reflects a more general feature of developing striated muscle fibers. Because CRYs are core timing components of circadian clocks and CRY2 is critical for circadian regulation of myogenic differentiation CRY-mediated PMT may interact with cell autonomous clocks to influence the progression of striated muscle development.

Exosomes as carriers transporting long non‑coding RNAs: Molecular characteristics and their function in cancer (Review).

Long non-coding RNAs (lncRNAs) comprise a sizeable class of non-coding RNAs with a length of over 200 base pairs. Little is known about their biological function, although over 20,000 lncRNAs have been annotated in the human genome. Through a diverse range of mechanisms, their primary function is in the regulation of the transcription of protein-coding genes. lncRNA transcriptional activation can result from a group of nucleus-retained and chromatin-associated lncRNAs, which function as scaffolds in the cis/trans recruitment of transcription factors, co-activators or chromatin remodelers, and/or promoter enhancers. Exosomes are released as extracellular vesicles and they are produced by endocytic pathways. Their synthesis is initiated by various processes including ceramide synthesis, release of intracellular Ca2+ or acid-base balance disorders. Prior to vesicle creation, selective cargo loading occurs in the Endosomal Sorting Complex Required for Transport. Participation of endosomal sorting proteins such as tetraspanins or specific sumoylated proteins required for transport has been indicated in research. The endosomal-sorting complex consists of four components, these induce the formation of multivesicular bodies and the induction of membrane deformation to form exosomes. Nanovesicles could be formed inside multivesicular bodies to allow transport outside the cell or digestion in lysosomes. The molecular content of exosomes is more heterogenic than its synthesis process, with different cargoes being examined inside vesicles with regard to the type or stage of cancers. This paper will review the importance of lncRNAs as crucial molecular content of exosomes, indicating its involvement in tumour suppression, pro-tumorigenic events and the development of novel therapeutic approaches in the near future. Further studies of their mechanisms of function are essential, as well as overcoming several challenges to gain a clearer insight to the approaches for the best clinical application.

Altered Intracellular Calcium Homeostasis and Arrhythmogenesis in the Aged Heart.

Aging of the heart is associated with a blunted response to sympathetic stimulation, reduced contractility, and increased propensity for arrhythmias, with the risk of sudden cardiac death significantly increased in the elderly population. The altered cardiac structural and functional phenotype, as well as age-associated prevalent comorbidities including hypertension and atherosclerosis, predispose the heart to atrial fibrillation, heart failure, and ventricular tachyarrhythmias. At the cellular level, perturbations in mitochondrial function, excitation-contraction coupling, and calcium homeostasis contribute to this electrical and contractile dysfunction. Major determinants of cardiac contractility are the intracellular release of Ca2+ from the sarcoplasmic reticulum by the ryanodine receptors (RyR2), and the following sequestration of Ca2+ by the sarco/endoplasmic Ca2+-ATPase (SERCa2a). Activity of RyR2 and SERCa2a in myocytes is not only dependent on expression levels and interacting accessory proteins, but on fine-tuned regulation via post-translational modifications. In this paper, we review how aberrant changes in intracellular Ca2+ cycling via these proteins contributes to arrhythmogenesis in the aged heart.

Membrane hyperpolarization and depolarization of rat thymocytes by azoxystrobin, a post harvest fungicide

Azoxystrobin, a broad-spectrum fungicide, has been increasingly used in the agricultural industry. In Japan in 2018, azoxystrobin at five times the normal limit was detected in a shipment of Australian barley that had been used in food products. Therefore, the effects of azoxystrobin need to be carefully examined to predict potential adverse reactions in humans. In this study, the effects of azoxystrobin on the membrane potential and intracellular Ca2+ levels of thymocytes have been photochemically examined using flow cytometry. Azoxystrobin hyperpolarized plasma membrane potential. This hyperpolarization appeared to be due to the activation of Ca2+-dependent K+ channels, as both the removal of extracellular Ca2+ and addition of charybdotoxin attenuated the observed hyperpolarization. In the presence of quinine, an anti-malarial drug that blocks Ca2+-dependent K+ channels, azoxystrobin depolarized the membranes instead. Azoxystrobin increased intracellular Ca2+ levels in a concentration-dependent manner through the influx of extracellular Ca2+ and intracellular release of Ca2+, as confirmed by reduction in azoxystrobin-induced response in the absence of extracellular Ca2+. It appears likely that azoxystrobin at micromolar concentrations modifies membrane ion permeability in thymocytes. Since changes in membrane potential and intracellular Ca2+ levels occur during typical physiological lymphocyte responses, azoxystrobin may disturb lymphocyte function.

The Flavone Luteolin, an Endocrine Disruptor, Relaxed Male Guinea Pig Gallbladder Strips.

BackgroundLuteolin (3',4',5,7-tetrahydroxyflavone) is a flavone with a yellow crystalline appearance present in numerous plants such as broccoli, green chili, and carrot. Luteolin is considered to be an endocrine disruptor with potent estrogen agonist activity and potent progesterone antagonist activity. Luteolin has effects on smooth muscle. Luteolin relaxed guinea pig trachea smooth muscle as it inhibited both phosphodiesterase and reduced intracellular Ca2+. Luteolin also caused vasorelaxation in rat thoracic aorta smooth muscle by inhibiting intracellular Ca2+ release, inhibition of sarcolemmal Ca2+ channels, and activation of K+ channels. Luteolin or its glycosides from artichoke extracts may have an ameliorating effect on irritable bowel syndrome. The purpose of this study was to determine if luteolin had an effect on gallbladder motility.MethodsAn in vitro pharmacologic technique was utilized. Either cholecystokinin octapeptide (CCK) or KCl were used to induce tension in male guinea pig gallbladder strips maintained in Sawyer-Bartlestone chambers. Luteolin relaxed either the CCK- or KCl-induced tension in a concentration dependent manner. Various blockers were added to the chambers to determine which second messenger system(s) mediated the observed relaxation. Paired t-tests were used for statistical analysis. Differences between mean values of P < 0.05 were considered significant.ResultsTreatment of the gallbladder strips with luteolin prior to either KCl or CCK significantly (P < 0.001) decreased the amount of either KCl- or cholecystokinin-induced tension. The 2-aminoethoxydiphenylborane was used to ascertain if the release of intracellular Ca2+ mediated the luteolin-induced relaxation. It significantly (P < 0.001) decreased the amount of luteolin-induced relaxation. To ascertain if PKA mediated the luteolin-induced relaxation, PKA inhibitor 14-22 amide myristolated was used. It significantly (P < 0.01) reduced the amount of luteolin-induced relaxation. Neither KT5823, NG-methyl-L-arginine acetate salt, genistein, tetraethylammonium, nor fulvestrant had a significant effect. To ascertain if PKC mediated the luteolin-induced relaxation, the PKC inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together. They had no significant effect.ConclusionsLuteolin relaxed cholecystokinin- or KCl-induced tension by blocking extracellular Ca2+ entry as well as intracellular Ca2+ release. In addition, the actions of PKA are also involved in mediating the luteolin effect.

Action potential and calcium dependence of tonic somatodendritic dopamine release in the Substantia Nigra pars compacta.

Despite the importance of somatodendritic dopamine (DA) release in the Substantia Nigra pars compacta (SNc), its mechanism remains poorly understood. Using a novel approach combining fast-scan controlled-adsorption voltammetry (FSCAV) and single-unit electrophysiology, we have investigated the mechanism of somatodendritic release by directly correlating basal (non-stimulated) extracellular DA concentration ([DA]out ), with pharmacologically-induced changes of firing of nigral dopaminergic neurons in rat brain slices. FSCAV measurements indicated that basal [DA]out in the SNc was 40.7±2.0nM (at 34±0.5°C), which was enhanced by amphetamine, cocaine, and L-DOPA, and reduced by VMAT2 inhibitor, Ro4-1284. Complete inhibition of firing by TTX decreased basal [DA]out , but this reduction was smaller than the effect of D2 receptor agonist, quinpirole. Despite similar effects on neuronal firing, the larger decrease in [DA]out evoked by quinpirole was attributed to cell membrane hyperpolarization and greater reduction in cytosolic free Ca2+ ([Ca2+ ]in ). Decreasing extracellular Ca2+ also reduced basal [DA]out , despite increasing firing frequency. Furthermore, inhibiting L-type Ca2+ channels decreased basal [DA]out , although specific Cav 1.3 channel inhibition did not affect firing rate. Inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase (SERCA) also decreased [DA]out , demonstrating the importance of intracellular Ca2+ stores for somatodendritic release. Finally, invivo FSCAV measurements showed that basal [DA]out in the SNc was 79.8±10.9nM in urethane-anesthetized rats, which was enhanced by amphetamine. Overall, our findings indicate that although tonic somatodendritic DA release is largely independent of action potentials, basal [DA]out is strongly regulated by voltage-dependent Ca2+ influx and release of intracellular Ca2+ . OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Tetrahydroxystilbene glucoside-induced relaxation of the superior mesenteric artery via both endothelium-dependent and endothelium-independent mechanisms

Tetrahydroxystilbene glucoside (TSG) is the main water-soluble component in Polygonum multiflorum Thunb, and it has many cardioprotective effects. Although TSG is able to relax blood vessels, its relaxation of rat superior mesenteric arteries and the underlying mechanism of this process are not clearly understood. The aim of the present study was to use in vivo and in vitro models to investigate the arterial relaxation effect of TSG on rat superior mesenteric arteries and the mechanisms involved. We found that TSG concentration-dependently relaxed the superior mesenteric artery with or without endothelium. The vasorelaxation induced by TSG is not related to the vasodilator derived factor NO but is rather by the inhibition of COX-2 activity and decreased TXA2. We also found that the vasorelaxation induced by TSG was attenuated by 4‑AP. Moreover, TSG also inhibited the contraction induced by an increase in external calcium concentration in Ca2+-free medium plus KCl (60 mM). These results suggest that TSG induces relaxation in mesenteric arterial rings through an endothelium-dependent pathway that involves the inhibition of COX-2 activity and decreased in TXA2 and through an endothelium-independent pathway via opening of a voltage-dependent K+ channel, blockade of Ca2+ influx and release of intracellular Ca2+.

Lipopeptide PAM3CYS4 Synergizes N-Formyl-Met-Leu-Phe (fMLP)-Induced Calcium Transients in Mouse Neutrophils.

N-Formyl-Met-Leu-Phe (fMLP), a mimic of N-formyl oligopeptides that are released from bacteria, is a potent leukocyte chemotactic factor. It induces intracellular calcium ([Ca]i) transient that is important for various neutrophil biological functions, e.g., adhesion, ROS, and cytokine productions. Toll-like receptors (TLRs), an essential part of host innate immunity, regulate neutrophil activities, but their role in [Ca]i signaling is less clear. In the present study, we examined the effect of several TLR ligands, including Pam3Cys4 (TLR1/2), lipopolysaccharide (LPS, TLR4), and lipoteichoic acid (LTA, TLR2/6), on calcium signaling and on the fMLP-induced [Ca]i transients in mouse neutrophils loaded with Fura-2/AM. We found that unlike fMLP, the three TLR ligands tested did not elicit any detectable Ca flux. However, Pam3Cys4, but not LPS or LTA, markedly synergized the fMLP-induced [Ca]i transients, and had no effect on the host component keratinocyte-derived cytokine (KC)- or C5a-induced calcium flux. The effect of Pam3Cys4 on the fMLP-induced [Ca]i transients is by enhancing extracellular Ca influx, not intracellular Ca release. Surprisingly, deletion of TLR2 or MyD88 in neutrophils had no impact on the Pam3Cys4's effect, suggesting a TLR2-MyD88-independent mechanism. Finally, using the pan PKC activator and inhibitor, we demonstrated that PKC negatively regulated fMLP-induced [Ca]i transients and that inhibition of PKC did not prohibit Pam3Cys4's synergistic effect on the fMLP-induced calcium influx. In conclusion, the present study identified a novel synergistic effect of Pam3Cys4 on fMLP-induced [Ca]i transients, a process important for many neutrophil biological functions.