Caffeine-induced Calcium Release Research Articles

Investigating candidate modifier loci in malignant hyperthermia susceptibility using CRISPR/Cas gene editing

Previous studies indicate more than one gene may be involved in determining susceptibility to malignant hyperthermia (MH) in individuals.1 2 Thus a threshold model of MH, whereby several genes may contribute to phenotype through both major and modifier effects, may be more suitable than a classical single gene disorder model. We have previously found that variants in both PYGM and CPT2 are over-represented amongst individuals susceptible to MH. We aimed to examine whether loss of function in these genes has a modifying effect on caffeine-induced calcium release in mouse skeletal muscle cells modelling the RYR1 variant most commonly associated with MH in the UK.

Extracellular and Intracellular Angiotensin II Regulate the Automaticity of Developing Cardiomyocytes via Different Signaling Pathways.

Angiotensin II (Ang II) plays an important role in regulating various physiological processes. However, little is known about the existence of intracellular Ang II (iAng II), whether iAng II would regulate the automaticity of early differentiating cardiomyocytes, and the underlying mechanism involved. Here, iAng II was detected by immunocytochemistry and ultra-high performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry in mouse embryonic stem cell–derived cardiomyocytes (mESC-CMs) and neonatal rat ventricular myocytes. Expression of AT1R-YFP in mESC-CMs revealed that Ang II type 1 receptors were located on the surface membrane, while immunostaining of Ang II type 2 receptors (AT2R) revealed that AT2R were predominately located on the nucleus and the sarcoplasmic reticulum. While extracellular Ang II increased spontaneous action potentials (APs), dual patch clamping revealed that intracellular delivery of Ang II or AT2R activator C21 decreased spontaneous APs. Interestingly, iAng II was found to decrease the caffeine-induced increase in spontaneous APs and caffeine-induced calcium release, suggesting that iAng II decreased spontaneous APs via the AT2R- and ryanodine receptor–mediated pathways. This is the first study that provides evidence of the presence and function of iAng II in regulating the automaticity behavior of ESC-CMs and may therefore shed light on the role of iAng II in fate determination.

Phosphorylation of cardiac myosin–binding protein-C contributes to calcium homeostasis

Cardiac myosin-binding protein-C (cMyBP-C) is highly phosphorylated under basal conditions. However, its phosphorylation level is decreased in individuals with heart failure. The necessity of cMyBP-C phosphorylation for proper contractile function is well-established, but the physiological and pathological consequences of decreased cMyBP-C phosphorylation in the heart are not clear. Herein, using intact adult cardiomyocytes from mouse models expressing phospho-ablated (AAA) and phosphomimetic (DDD) cMyBP-C as well as controls, we found that cMyBP-C dephosphorylation is sufficient to reduce contractile parameters and calcium kinetics associated with prolonged decay time of the calcium transient and increased diastolic calcium levels. Isoproterenol stimulation reversed the depressive contractile and Ca2+-kinetic parameters. Moreover, caffeine-induced calcium release yielded no difference between AAA/DDD and controls in calcium content of the sarcoplasmic reticulum. On the other hand, sodium-calcium exchanger function and phosphorylation levels of calcium-handling proteins were significantly decreased in AAA hearts compared with controls. Stress conditions caused increases in both spontaneous aftercontractions in AAA cardiomyocytes and the incidence of arrhythmias in vivo compared with the controls. Treatment with omecamtiv mecarbil, a positive cardiac inotropic drug, rescued the contractile deficit in AAA cardiomyocytes, but not the calcium-handling abnormalities. These findings indicate a cascade effect whereby cMyBP-C dephosphorylation causes contractile defects, which then lead to calcium-cycling abnormalities, resulting in aftercontractions and increased incidence of cardiac arrhythmias under stress conditions. We conclude that improvement of contractile deficits alone without improving calcium handling may be insufficient for effective management of heart failure.

Abstract MP135: Cardiac Sarcoplasmic Reticulum Calcium Cycling is Regulated by Kinase Independent Function of Pi3kgamma

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular contractility. We investigated whether the lack of PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Isolated cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine induced calcium release indicating that PI3Kγ locally regulates the function of SR. The intracellular calcium remained at elevated levels in the cardiomyocytes of PI3Kγ -/- for a prolonged period after caffeine treatment. This could be due to changes in phosphorylation of SERCA2, Ryanodine receptor (RyR 2 ) or phospholamban (PLN). In fact, when we looked at phosphorylation of PLN in cardiac lysates, a major regulator of cardiac contractility and relaxation, PI3Kγ -/- mice showed significantly reduced PLN phosphorylation compared to littermate controls. Previous studies from our laboratory suggested that absence of PI3Kγ leads to increase in protein phosphatase (PP) activity which could be possible reason for rapid dephosphorylation of PLN, resulting in inhibition of SERCA2 pump. We observed increased SR associated PP activity and PLN associated PP activity in PI3Kγ -/- mice. We also observed increased association of PP-1 and PP2A with PLN in the absence of PI3Kγ. The altered calcium handling in the cardiomyocytes of PI3Kγ -/- mice could be restored to the level of WT controls by okadaic acid mediated inhibition of PP, suggesting that PI3Kγ plays a role in regulating PP activity associated with SR. To test whether PI3Kγ activity is required for PLN dephosphorylation and SR calcium cycling, we used mice with cardiac specific overexpression of kinase dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ). PI3Kγ inact /PI3Kγ -/- mice showed restored PLN phosphorylation, improved caffeine induced calcium reuptake, decreased SR and PLN associated PP activity. These studies show a novel regulation of PP and SR calcium regulation by kinase independent function of PI3Kγ. The underlying mechanism of PP regulation by PI3Kγ will be presented.

A novel homozygous mutation in the TRDN gene causes a severe form of pediatric malignant ventricular arrhythmia

Triadin is a protein expressed in cardiac and skeletal muscle that has an essential role in the structure and functional regulation of calcium release units and excitation-contraction coupling. Mutations in the triadin gene (TRDN) have been described in different forms of human arrhythmia syndromes with early onset and severe arrhythmogenic phenotype, including triadin knockout syndrome. The purpose of this study was to characterize the pathogenetic mechanism underlying a case of severe pediatric malignant arrhythmia associated with a defect in the TRDN gene. We used a trio whole exome sequencing approach to identify the genetic defect in a 2-year-old boy who had been resuscitated from sudden cardiac arrest and had frequent episodes of ventricular fibrillation and a family history positive for sudden death. We then performed invitro functional analysis to investigate possible pathogenic mechanisms underlying this severe phenotype. We identified a novel homozygous missense variant (p.L56P) in the TRDN gene in the proband that was inherited from the heterozygous unaffected parents. Expression of a green fluorescent protein (GFP)-tagged mutant human cardiac triadin isoform (TRISK32-L56P-GFP) in heterologous systems revealed that the mutation alters protein dynamics. Furthermore, when co-expressed with the type 2 ryanodine receptor, caffeine-induced calcium release from TRISK32-L56P-GFP was relatively lower compared to that observed with the wild-type construct. The results of this study allowed us to hypothesize a pathogenic mechanism underlying this rare arrhythmogenic recessive form, suggesting that the mutant protein potentially can trigger arrhythmias by altering calcium homeostasis.

Abstract 334: Cardiac Myosin Binding Protein C Phosphorylation Regulates Calcium Homeostasis

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is heavily phosphorylated to regulate normal cardiac function under basal conditions. However, its phosphorylation level is significantly decreased in patients with heart failure and atrial fibrillation. Furthermore, decreased cMyBP-C phosphorylation causes reduced myofilament contractility and decreased calcium sensitivity. The impact of such decreases in cMyBP-C phosphorylation and myofilament calcium sensitivity on the overall calcium handling and potential induction of arrhythmogenesis is not known. Objective: To determine the necessity and sufficiency of cMyBP-C phosphorylation to regulate calcium cycling and contractility at the isolated cardiomyocyte level. Methods and Results: Contractile properties and calcium kinetics were measured in intact cardiomyocytes isolated from 3-month-old, mixed sex, mice expressing phospho-ablated (S273A/S282A/S302A) cMyBP-C (AAA) or phospho-mimetic (S273D/S282D/S302D) cMyBP-C (DDD) and nontransgenic (NTG) control mice. AAA cells displayed a significant decrease in fractional shortening compared to NTG and DDD cells (9.4% vs 12.8% in NTG, and 12.3% in DDD, p<0.0001). Similarly, AAA myocytes demonstrated abnormal calcium kinetics with increased diastolic calcium levels (20%, p<0.001 vs . NTG and DDD) and prolonged decay time of the calcium transient (26%, p<0.01 vs NTG and DDD) when compared with NTG and DDD myocytes. Caffeine-induced calcium release in AAA myocytes indicated no change in SR calcium content, while sodium-calcium exchanger function, assessed as the time constant (τau) of calcium decline, was increased (89%, p<0.01 vs NTG). However, these depressive effects in AAA myocytes were relieved by isoproterenol (100 nmol/L) stimulation. Furthermore, stress conditions (2 Hz + ISO) increased after-contractions in AAA cardiomyocytes (60% in AAA vs 13% in NTG, p<0.001 and 15% in DDD, p<0.001). In addition, preliminary data indicate increased susceptibility of the AAA mice to arrhythmias under stress conditions in vivo. Conclusion: Dephosphorylation of cMyBP-C is sufficient to reduce sarcomere contractility and impair calcium cycling resulting in spontaneous after-contractions and arrhythmias under stress conditions.

Assessing the pathogenicity of RYR1 variants in malignant hyperthermia

Background.Missense variants in the ryanodine receptor 1 gene (RYR1) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. MethodsWe identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. ResultsWe identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. ConclusionsSegregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.

The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle.

BackgroundThe SH3 and cysteine-rich domain 3 (Stac3) gene is specifically expressed in the skeletal muscle. Stac3 knockout mice die perinatally. In this study, we determined the potential role of Stac3 in postnatal skeletal muscle growth, fiber composition, and contraction by generating conditional Stac3 knockout mice.MethodsWe disrupted the Stac3 gene in 4-week-old male mice using the Flp-FRT and tamoxifen-inducible Cre-loxP systems.ResultsRT-qPCR and western blotting analyses of the limb muscles of target mice indicated that nearly all Stac3 mRNA and more than 70 % of STAC3 protein were deleted 4 weeks after tamoxifen injection. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei, with no effect on the total myofiber number. Grip strength and grip time tests indicated that postnatal Stac3 deletion decreased limb muscle strength in mice. Muscle contractile tests revealed that postnatal Stac3 deletion reduced electrostimulation-induced but not the ryanodine receptor agonist caffeine-induced maximal force output in the limb muscles. Calcium imaging analysis of single flexor digitorum brevis myofibers indicated that postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum.ConclusionsThis study demonstrates that STAC3 is important to myofiber hypertrophy, myofiber-type composition, contraction, and excitation-induced calcium release from the sarcoplasmic reticulum in the postnatal skeletal muscle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0088-4) contains supplementary material, which is available to authorized users.

0346 : Characterization of the calcium deregulation in cardiomyocytes from mdx mice, the main rodent model of the Duchenne muscular dystrophy

The Duchenne muscular dystrophy (DMD) is caused by an absence or mutation of the protein dystrophin, which leads to smooth, skeletal and cardiac muscles degeneration. Intracellular calcium deregulation, notably abnormally high cytosolic calcium concentration, is thought to contribute to muscle necrosis and to cardiomyopathy development. Despite its importance in cell necrosis, the mechanisms involved in this calcium deregulation are not fully understood. In our study, we have investigated several parameters of the calcium homeostasis in cardiomyocytes from mdx mice, the main rodent model of the DMD. All the parameters have been measured by using confocal microscopy calcium imaging experiments in cardiomyocytes loaded with the calcium indicator Fluo-3AM. Our results show an increase of the occurrence of spontaneous calcium events such as a higher frequency of calcium sparks and waves in the mdx cardiomyocytes compared with wild type mice. We also found that the calcium sensitivity of the ryanodine receptors (RyRs) is increased in cardiomyocytes from mdx mice. Finally, the sarcoplasmic reticulum (SR) calcium content has been evaluated by caffeine-induced calcium release. We found reduced calcium content of the SR in the mdx mice, which could be explained, at least in part, by an excessive spontaneous calcium activity due to the enhanced RyRs sensitivity. Our data suggest that targeting this SR calcium “leak” with pharmacological tools could be beneficial for the DMD patients. The author hereby declares no conflict of interest

Caffeine-Induced Suppression of GABAergic Inhibition and Calcium-Independent Metaplasticity.

GABAergic inhibition plays a critical role in the regulation of neuron excitability; thus, it is subject to modulations by many factors. Recent evidence suggests the elevation of intracellular calcium ([Ca2+]i) and calcium-dependent signaling molecules underlie the modulations. Caffeine induces a release of calcium from intracellular stores. We tested whether caffeine modulated GABAergic transmission by increasing [Ca2+]i. A brief local puff-application of caffeine to hippocampal CA1 pyramidal cells transiently suppressed GABAergic inhibitory postsynaptic currents (IPSCs) by 73.2 ± 6.98%. Time course of suppression and the subsequent recovery of IPSCs resembled DSI (depolarization-induced suppression of inhibition), mediated by endogenous cannabinoids that require a [Ca2+]i rise. However, unlike DSI, caffeine-induced suppression of IPSCs (CSI) persisted in the absence of a [Ca2+]i rise. Intracellular applications of BAPTA and ryanodine (which blocks caffeine-induced calcium release from intracellular stores) failed to prevent the generation of CSI. Surprisingly, ruthenium red, an inhibitor of multiple calcium permeable/release channels including those of stores, induced metaplasticity by amplifying the magnitude of CSI independently of calcium. This metaplasticity was accompanied with the generation of a large inward current. Although ionic basis of this inward current is undetermined, the present result demonstrates that caffeine has a robust Ca2+-independent inhibitory action on GABAergic inhibition and causes metaplasticity by opening plasma membrane channels.

Mitochondrial Hyperfusion during Oxidative Stress Is Coupled to a Dysregulation in Calcium Handling within a C2C12 Cell Model

Atrial Fibrillation is the most common sustained cardiac arrhythmia worldwide harming millions of people every year. Atrial Fibrillation (AF) abruptly induces rapid conduction between atrial myocytes which is associated with oxidative stress and abnormal calcium handling. Unfortunately this new equilibrium promotes perpetuation of the arrhythmia. Recently, in addition to being the major source of oxidative stress within cells, mitochondria have been observed to fuse, forming mitochondrial networks and attach to intracellular calcium stores in response to cellular stress. We sought to identify a potential role for rapid stimulation, oxidative stress and mitochondrial hyperfusion in acute changes to myocyte calcium handling. In addition we hoped to link altered calcium handling to increased sarcoplasmic reticulum (SR)-mitochondrial contacts, the so-called mitochondrial associated membrane (MAM). We selected the C2C12 murine myotube model as it has previously been successfully used to investigate mitochondrial dynamics and has a myofibrillar system similar to atrial myocytes. We observed that rapid stimulation of C2C12 cells resulted in mitochondrial hyperfusion and increased mitochondrial colocalisation with calcium stores. Inhibition of mitochondrial fission by transfection of mutant DRP1K38E resulted in similar effects on mitochondrial fusion, SR colocalisation and altered calcium handling. Interestingly the effects of ‘forced fusion’ were reversed by co-incubation with the reducing agent N-Acetyl cysteine (NAC). Subsequently we demonstrated that oxidative stress resulted in similar reversible increases in mitochondrial fusion, SR-colocalisation and altered calcium handling. Finally, we believe we have identified that myocyte calcium handling is reliant on baseline levels of reactive oxygen species as co-incubation with NAC both reversed and retarded myocyte response to caffeine induced calcium release and re-uptake. Based on these results we conclude that the coordinate regulation of mitochondrial fusion and MAM contacts may form a point source for stress-induced arrhythmogenesis. We believe that the MAM merits further investigation as a therapeutic target in AF-induced remodelling.

Excess of glucocorticoid induces myocardial remodeling and alteration of calcium signaling in cardiomyocytes

Ventricular dysfunction is one of the important side effects of the anti-inflammatory agent, glucocorticoid (GC). The present study was undertaken to examine whether abnormal calcium signaling is responsible for cardiac dysfunction due to an excess of GC hormone. The synthetic GC drug, dexamethasone (DEX), significantly (P<0.001, n=20) increased heart weight to body weight ratio, left ventricular remodeling, and fibrosis. The microarray analysis showed altered expression of several genes encoding calcium cycling/ion channel proteins in DEX-treated rat heart. The altered expression of some of the genes was validated by real-time PCR and western blotting analyses. The expression of the L-type calcium channels and calsequestrin was increased, whereas sarcoendoplasmic reticulum calcium transport ATPase 2a (SERCA2a) and junctin mRNAs were significantly reduced in DEX-treated rat left ventricular tissues. In neonatal rat ventricular cardiomyocytes, DEX also increased the level of mRNAs of atrial- and brain natriuretic peptides, L-type calcium channels, and calsequestrin after 24 h of treatment, which were mostly restored by mifepristone. The caffeine-induced calcium release was prolonged by DEX compared to the sharp release in control cardiomyocytes. Taken together, these data show that impaired calcium kinetics may be responsible for cardiac malfunction by DEX. The results are important in understanding the pathophysiology of the heart in patients treated with excess GC.

Participation of inositol trisphosphate and ryanodine receptors inBufo arenarumoocyte activation

Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.

Imbalance between CaM kinase II and calcineurin activities impairs caffeine-induced calcium release in hypertrophic cardiomyocytes

Cardiac hypertrophy impairs Ca 2+ handling in the sarcoplasmic reticulum, thereby impairing cardiac contraction. To identify the mechanisms underlying impaired Ca 2+ release from the sarcoplasmic reticulum in hypertrophic cardiomyocytes, we assessed Ca 2+-dependent signaling and the phosphorylation of phospholamban, which regulates Ca 2+ uptake during myocardial relaxation and is in turn regulated by Ca 2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin. In cultured rat cardiomyocytes, treatment with endothelin-1, angiotensin II, and phenylephrine-induced hypertrophy and increased CaMKII autophosphorylation and calcineurin expression. The calcineurin level reached its maximum at 72 h and remained elevated for at least 96 h after endothelin-1 or angiotensin II treatment. By contrast, CaMKII autophosphorylation, phospholamban phosphorylation, and caffeine-induced Ca 2+ mobilization all peaked 48 h after these treatments. By 96 h after treatment, CaMKII autophosphorylation and phospholamban phosphorylation had returned to baseline, and caffeine-induced Ca 2+ mobilization was impaired relative to baseline. A similar biphasic change was observed in dystrophin levels in endothelin-1-induced hypertrophic cardiomyocytes, and treatment with the novel CaM antagonists DY-9760e and DY-9836 significantly inhibited the hypertrophy-induced dystrophin breakdown. Taken together, the abnormal Ca 2+ regulation in cardiomyocytes following hypertrophy is in part mediated by an imbalance in calcineurin and CaMKII activities, which leads to abnormal phospholamban activity.

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca2+ inside the ER ([Ca2+]ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca2+]ER (EC50) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca2+]ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 µM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 µM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca2+. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland.

Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture

Crude decoction, aqueous and ethanolic extracts of two medicinal plants (Psidium guajava and Diospyros mespiliformis), widely used in the central plateau of Burkina Faso to treat many diseases were evaluated for their antagonistic effects on caffeine induced calcium release from sarcoplasmic reticulum of rat skeletal muscle cells. These different extracts showed a decrease of caffeine induced calcium release in a dose dependent manner. Comparison of the results showed that Psidium guajava leaf extracts are more active than extracts of Diospyros mespiliformis and that crude decoctions show better inhibitory activity. The observed results could explain their use as antihypertensive and antidiarrhoeal agents in traditional medicine, by inhibiting intracellular calcium release.

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.

Triadin (Trisk 95) Overexpression Blocks Excitation-Contraction Coupling in Rat Skeletal Myotubes

To identify the function of triadin in skeletal muscle, adenovirus-mediated overexpression of Trisk 95 or Trisk 51, the two major skeletal muscle isoforms, was induced in rat skeletal muscle primary cultures, and the physiological behavior of the modified cells was analyzed. Overexpression did not modify the expression level of their protein partners ryanodine receptor, dihydropyridine receptor, and the other triadin. Caffeine-induced calcium release was also unaffected by triadin overexpression. Nevertheless, in the absence of extracellular calcium, depolarization-induced calcium release was almost abolished in Trisk 95 overexpressing myotubes (T95 myotubes), and not modified in Trisk 51 overexpressing myotubes (T51 myotubes). This was not because of a modification of dihydropyridine receptors, as depolarization in presence of external calcium still induced a calcium release, and the activation curve of dihydropyridine receptor was unchanged, in both T95 and T51 myotubes. The calcium release complex was also maintained in T95 myotubes as Trisk 95, ryanodine receptor, dihydropyridine receptor, and Trisk 51 were still co-localized. The effect of Trisk 95 overexpression on depolarization-induced calcium release was reversed by a simultaneous infection with an antisense Trisk 95 adenovirus, indicating the specificity of this effect. Thus, the level of Trisk 95 and not Trisk 51 is important on regulating the calcium release complex, and an excess of this protein can lead to an inhibition of the physiological function of the complex.

Apoptotic effect of ethanol is potentiated by caffeine-induced calcium release in rat astrocytes

In this study, we investigated agents that increased intracellular calcium levels and their correlation with apoptotic cell death induction. We used rat astrocytes to investigate the increase in cytosolic Ca 2+ (Ca c 2+) and apoptosis induction by drugs that mobilize Ca 2+ from different sources. We observed that thapsigargin (Thap), caffeine (Caff) and FCCP which caused similar increases in Ca c 2+ levels (30–40%), also induced similar apoptotic rates (30–35%). On the other hand, antimycin (Anti), staurosporine (STS) and ethanol (Eth) promoted higher increases in Ca c 2+ (55–65 %) and higher apoptotic rates (55–85%). Eth induced cell death in a concentration- and time-dependent manner. After treatment with Eth plus Caff for 6, 12 and 24 h, these effects were strongly potentiated. Results suggest that there might be a correlation between Ca c 2+ increase and the rate of apoptosis. It is possible that Eth induces cell death by activation of more than one pathway and Ca 2+ might be one of the elements involved. The present work indicates that Ca 2+ can potentiate death by ethanol in rat astrocytes.

Caffeine inhibits cytokine expression in lymphocytes

Caffeine alters intracellular calcium signalling patterns in lymphocytes which are important for the specific regulation of activation and effector function in lymphocytes. The effect of caffeine on calcium signalling is probably mediated via a ryanodine receptor type 3 dependent intracellular calcium store which releases calcium after exposure to caffeine. Also, caffeine decreases lymphocyte cytotoxicity against allogenic myocyte. Which cytotoxic mechanisms are actually altered by caffeine is unknown. In mouse splenocyte cultures containing about 87% lymphocytes we show that concanavalin A (ConA, 5 μg/ml) stimulated cells increase the expression of TNF-α, IL-2 and IFN-γ (ELISA) significantly. Caffeine (3.75 mM) inhibits cytokine expression of ConA stimulated cells almost completely. Ryanodine (1 μM) specifically blocks ryanodine receptors and thereby prevents caffeine induced calcium release. In our experiments, however, ryanodine has no effect on ConA stimulated IL-2 and IFN-γ expression and only suppresses TNF-α expression by 20%. Furthermore, ryanodine does not prevent the inhibitory effect of caffeine on TNF-α, IL-2 and IFN-γ expression in stimulated effector cells. We postulate that caffeine suppresses cytokine expression and thereby contributes to decreased cytotoxicity of lymphocytes against allogenic myocytes. The ryanodine receptor dependent intracellular calcium store does not seem to play a significant role in this process. Possibly, the blockade of IP3 receptors by caffeine is more important for cytokine suppression.