Increase In Extracellular Ca2 Research Articles

Prolonged Ca2+ release refractoriness and T-tubule disruption as determinants of increased propensity to cardiac alternans in the hypertensive heart disease.

Cardiac alternans is a dynamical phenomenon linked to the genesis of severe arrhythmias and sudden cardiac death. It has been proposed that alternans is caused by alterations in Ca2+ handling by the sarcoplasmic reticulum (SR), in both the SR Ca2+ uptake and release processes. The hypertrophic myocardium is particularly prone to alternans, but the precise mechanisms underlying its increased vulnerability are not known. Mechanical alternans (intact hearts) and Ca2+ alternans (cardiac myocytes) were studied in spontaneously hypertensive rats (SHR) during the first year of age after the onset of hypertension and compared with age-matched normotensive rats. Subcellular Ca2+ alternans, T-tubule organization, SR Ca2+ uptake, and Ca2+ release refractoriness were measured. The increased susceptibility of SHR to high-frequency-induced mechanical and Ca2+ alternans appeared when the hypertrophy developed, associated with an adverse remodeling of the T-tubule network (6 mo). At the subcellular level, Ca2+ discordant alternans was also observed. From 6 mo of age, SHR myocytes showed a prolongation of Ca2+ release refractoriness without alterations in the capacity of SR Ca2+ removal, measured by the frequency-dependent acceleration of relaxation. Sensitizing SR Ca2+ release channels (RyR2) by a low dose of caffeine or by an increase in extracellular Ca2+ concentration, shortened refractoriness of SR Ca2+ release, and reduced alternans in SHR hearts. The tuning of SR Ca2+ release refractoriness is a crucial target to prevent cardiac alternans in a hypertrophic myocardium with an adverse T-tubule remodeling.

Responsive Calcium (Ca<sup>2+</sup>) Alginate-Chitosan Based Hydrogel: A Promising Biomaterial for Spinal Cord Injury

Spinal cord injury is damage to the spinal cord which causes lesions in the spinal cord and leads to an increase in extracellular Ca2+. It results in additional neuronal loss which causes temporary/permanent disability or even death. The aim of this study was to determine characteristics and the best composition of alginate – chitosan hydrogel responsive to Calcium (Ca2+) for spinal cord injury. Hydrogel synthesis with its compositions, namely chitosan was dissolved in 0.4% acetic acid, neutralized in pH 7 with 0.5 M NaOH, added some 0.85% NaCl in it, and added 5 alginate variations which were dissolved in 0.85% NaCl, next will centrifugation method. Based on the FTIR test, hydrogel showed stretching vibrations of Chitosan’s O–H bonds appeared in 3415.93cm-1 wavenumber, while Na groups of alginate isomer appeared in 1413.82 cm-1 wavenumber. The results of the cytotoxicity test using the MTT Assay method showed live cell percentage from less than 50% to 52.61% in Sample B and 83.83% in Sample C. The results of the injectability test showed that all samples were injectable with the highest percentage of injectability at 98.283%. The results of the UV-Vis spectrophotometric test showed that all hydrogel samples were able to absorb Ca2+. Hydrogels can be degraded at more than 90% within 14 days. The results of the morphology test (SEM) obtained 84.7-99.6 μm pore sizes.

The increase of extracellular Ca2+ from physiological concentrations to hypercalcemia impairs sino-atrial automaticity

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): ESC FRM Lefoulon Delalande Aims To investigate whether extracellular hypercalcemia alters the conduction through L-type Ca2+ channels (LTCCs), impairing the pacemaker activity of the heart. Introduction In the sino-atrial node (SAN), membrane currents and the dynamics of intracellular Ca2+ ([Ca2+]i) generate the pacemaker activity of the heart. SAN dysfunctions (SNDs) harm heart automaticity and have been associated with abnormal dynamics of [Ca2+]i. The LTCCs, Cav1.2 and Cav1.3 carry the main Ca2+ influx of SAN cells, which is necessary to sustain [Ca2+]i dynamics. Modified extracellular Ca2+ ([Ca2+]o) could alter Ca2+ influx through these channels. For example, cancer and hyperparathyroidism can raise [Ca2+]o, causing an extracellular hypercalcemia that could alter [Ca2+]i dynamics and impair SAN activity and heart automaticity. Methods and results To test this hypothesis, we measured contractions, [Ca2+]i release and L-type Ca2+ current (ICa,L) in spontaneous cells of the murine SAN. Then, we recorded rate and propagation of the spontaneous action potentials (APs) generated by the SAN tissue ex-vivo. In spontaneously beating SAN cells, we observed that the modification of [Ca2+]o affected [Ca2+]i and cell contractility through changes of ICa,L. In particular, the increase of [Ca2+]o dysregulated pacemaker activity, likely through excessive Ca2+ influx mediated by Cav1.2. [Ca2+]o increase to hypercalcemia induced arrhythmia also in the intact SAN tissues, activating ectopic leading regions of pacemaking and impairing conduction towards the atria. Conclusions Hypercalcemia causes excessive Cav1.2-mediated Ca2+ influx, which alters [Ca2+]I leading to pacemaker impairment. Modulation of LTCC may reduce pacemaker dysfunctions, preventing SND progression.

Homer1 mediates CaSR-dependent activation of mTOR complex 2 and initiates a novel pathway for AKT-dependent β-catenin stabilization in osteoblasts

The calcium-sensing receptor (CaSR) is critical for skeletal development, but its mechanism of action in osteoblasts is not well-characterized. In the central nervous system (CNS), Homer scaffolding proteins form signaling complexes with two CaSR-related members of the G protein-coupled receptor (GPCR) family C, metabotropic glutamate receptor 1 (mGluR1) and mGluR5. Here, we show that CaSR and Homer1 are co-expressed in mineralized mouse bone and also co-localize in primary human osteoblasts. Co-immunoprecipitation experiments confirmed that Homer1 associates with CaSR in primary human osteoblasts. The CaSR-Homer1 protein complex, whose formation was increased in response to extracellular Ca2+, was bound to mechanistic target of rapamycin (mTOR) complex 2 (mTORC2), a protein kinase that phosphorylates and activates AKT Ser/Thr kinase (AKT) at Ser473 siRNA-based gene-silencing assays with primary osteoblasts revealed that both CaSR and Homer1 are required for extracellular Ca2+-stimulated AKT phosphorylation and thereby inhibit apoptosis and promote AKT-dependent β-catenin stabilization and cellular differentiation. To confirm the role of the CaSR-Homer1 complex in AKT initiation, we show that in HEK-293 cells, co-transfection with both Homer1c and CaSR, but neither with Homer1c nor CaSR alone, establishes sensitivity of AKT-Ser473 phosphorylation to increases in extracellular Ca2+ concentrations. These findings indicate that Homer1 mediates CaSR-dependent AKT activation via mTORC2 and thereby stabilizes β-catenin in osteoblasts.

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

An injectable, calcium responsive composite hydrogel for the treatment of acute spinal cord injury.

Immediately following spinal cord injury, further injury can occur through several secondary injury cascades. As a consequence of cell lysis, an increase in extracellular Ca2+ results in additional neuronal loss by inducing apoptosis. Thus, hydrogels that reduce extracellular Ca2+ concentration may reduce secondary injury severity. The goal of this study was to develop composite hydrogels consisting of alginate, chitosan, and genipin that interact with extracellular Ca2+ to enable in situ gelation while maintaining an elastic modulus similar to native spinal cord (∼1000 Pa). It was hypothesized that incorporation of genipin and chitosan would regulate hydrogel electrostatic characteristics and influence hydrogel porosity, degradation, and astrocyte behavior. Hydrogel composition was varied to create hydrogels with statistically similar mechanical properties (∼1000 Pa) that demonstrated tunable charge characteristics (6-fold range in free amine concentration) and degradation rate (complete degradation between 7 and 28 days; some blends persist after 28 days). Hydrogels demonstrate high sensitivity to Ca2+ concentration, as a 1 mM change during fabrication induced a significant change in elastic modulus. Additionally, hydrogels incubated in a Ca2+-containing solution exhibited an increased linear viscoelastic limit (LVE) and an increased elastic modulus above the LVE limit in a time dependent manner. An extension of the LVE limit implies a change in hydrogel cross-linking structure. Attachment assays demonstrated that addition of chitosan/genipin to alginate hydrogels induced up to a 4-fold increase in the number of attached astrocytes and facilitated astrocyte clustering on the hydrogel surface in a composition dependent manner. Furthermore, Western blots demonstrated tunable glial fibrillary acid protein (GFAP) expression in astrocytes cultured on hydrogel blends, with some hydrogel compositions demonstrating no significant increase in GFAP expression compared to astrocytes cultured on glass. Thus, alginate/chitosan/genipin hydrogel composites show promise as scaffolds that regulate astrocyte behavior and for the prevention of Ca2+-related secondary neuron damage during acute SCI.

Obesity Preserves Myocardial Function During Blockade of the Glycolytic Pathway

BackgroundObesity is defined by excessive accumulation of body fat relative to lean tissue. Studies during the last few years indicate that cardiac function in obese animals may be preserved, increased or diminished.ObjectiveStudy the energy balance of the myocardium with the hypothesis that the increase in fatty acid oxidation and reduced glucose leads to cardiac dysfunction in obesity.Methods30-day-old male Wistar rats were fed standard and hypercaloric diet for 30 weeks. Cardiac function and morphology were assessed. In this paper was viewed the general characteristics and comorbities associated to obesity. The structure cardiac was determined by weights of the heart and left ventricle (LV). Myocardial function was evaluated by studying isolated papillary muscles from the LV, under the baseline condition and after inotropic and lusitropic maneuvers: myocardial stiffness; postrest contraction; increase in extracellular Ca2+ concentration; change in heart rate and inhibitor of glycolytic pathway.ResultsCompared with control group, the obese rats had increased body fat and co-morbities associated with obesity. Functional assessment after blocking iodoacetate shows no difference in the linear regression of DT, however, the RT showed a statistically significant difference in behavior between the control and the obese group, most notable being the slope in group C.ConclusionThe energy imbalance on obesity did not cause cardiac dysfunction. On the contrary, the prioritization of fatty acids utilization provides protection to cardiac muscle during the inhibition of glycolysis, suggesting that this pathway is fewer used by obese cardiac muscle.

Ca(2+) efflux is involved in cinnamaldehyde-induced growth inhibition of Phytophthora capsici.

As a destructive fungus-like plant pathogen, the oomycete Phytophthoracapsici is unable to synthesize its own ergosterol as the potential target of fungicide cinnamaldehyde (CA). In this study, CA exerted efficient inhibitory effects on both mycelial growth (EC50=0.75 mM) and zoospore germination (MIC=0.4 mM) of P . capsici . CA-induced immediate Ca2+ efflux from zoospores could be confirmed by the rapid decrease in intracellular Ca2+ content determined by using Fluo-3 AM and the increase in extracellular Ca2+ concentration determined by using ICP-AES (inductively coupled plasma atomic emission spectrometry). Blocking Ca2+ influx with ruthenium red and verapamil led to a higher level of CA-induced Ca2+ efflux, suggesting the simultaneous occurrence of Ca2+ influx along with the Ca2+ efflux under CA exposure. Further results showed that EGTA-induced decrease in intracellular Ca2+ gave rise to the impaired vitality of P . capsici while the addition of exogenous Ca2+ could suppress the growth inhibitory effect of CA. These results suggested that Ca2+ efflux played an important role in CA-induced growth inhibition of P . capsici . The application of 3-phenyl-1-propanal, a CA analog without α,β- unsaturated bond, resulted in a marked Ca2+ influx in zoospores but did not show any growth inhibitory effects. In addition, exogenous cysteine, an antagonist against the Michael addition (the nucleophilic addition of a carbanion or another nucleophile) between CA and its targets, could attenuate CA-induced growth inhibition of P . capsici by suppressing Ca2+ efflux. Our results suggest that CA inhibits the growth of P . capsici by stimulating a transient Ca2+ efflux via Michael addition, which provides important new insights into the antimicrobial action of CA.

Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg(2+) concentrations. Changes in Mg(2+) concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca(2+)/Mg(2+) ratio facilitated Ca(2+) influx in both control and prostate cancer cells, a significantly higher Ca(2+) influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca(2+)/Mg(2+) ratio per se. Additionally, an increase in the extracellular Ca(2+)/Mg(2+) ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca(2+)/Mg(2+) ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca(2+)/Mg(2+) ratio could be essential for the initiation/progression of prostate cancer.

A set of SNARE proteins in the contractile vacuole complex of Paramecium regulates cellular calcium tolerance and also contributes to organelle biogenesis

The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca2+. No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca2+ after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca2+ stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca2+ in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca2+ tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis.

Acute Activation of the Renal Betaine/GABA Transporter in Response to a Decrease in Extracellular Calcium

The betaine/GABA transporter (BGT1) is important for osmoprotection in kidney medullary cells. We previously reported an acute (30 min) increase in extracellular Ca2+caused dose dependent inhibition of BGT-1 in renal MDCK cells. To determine if extracellular Ca2+might be a local regulator of BGT-1, we have tested the response to low Ca2+serum-free growth medium (LCM, 0.05 mM Ca2+). Chronic treatment (8–24 h) of MDCK cell monolayers completely blocked hypertonic adaptation of BGT1 and disrupted tight junctions. In contrast, acute treatment activated BGT1 transport within 30 min in MDCK cells previously adapted to hypertonic growth medium containing normal Ca2+(1.6 mM). Activation was significant after 60–90 min and was independent of medium osmolarity. Peak transport was increased 50% in isotonic LCM and 100% in hypertonic (500 mOsm) LCM over controls. The activation was reversed by restoration of normal Ca2+. Perfusion of Fura-2-loaded MDCK cells with LCM decreased intracellular Ca2+by 31% within 6-7 min. Inclusion of staurosporine (0.6 μM), a protein kinase C inhibitor, potentiated the action of LCM. We suggest that activation of BGT1 by LCM may be due in part to inhibition of protein kinase C.

Involvement of cationic channels in proliferation and migration of human mesenchymal stem cells

Human mesenchymal stem cells (HMSCs) have been applied in various clinic settings. Ion channels play an important role in cellular physiology. However, the potential role of cationic channels in regulating the proliferation and migration properties of hMSCs remains to be determined. In the present study, the functional expression of ion channels in hMSCs was investigated by patch clamp. MTT assay and BrdU stainings were used to assess the proliferation of hMSCs. hMSC migration was evaluated by Transwell migration assays. The results show that sodium-, L-type calcium, potassium currents have been identified in hMSCs. TEA (K+ channel blocker), nifedipine (Ca2+ channel blocker) can inhibit both proliferation and migration of hMSCs. The increase of extracellular Ca2+ concentration promoted both proliferation and migration of hMSCs. TTX, a Na+ channel blocker, promoted cell proliferation but inhibited cell migration. Our data suggest that cationic channels (sodium, L-type calcium, potassium channels) play important roles in regulating proliferation and migration of hMSCs.

Peripheral Nerve Injury Increases Glutamate-Evoked Calcium Mobilization in Adult Spinal Cord Neurons

BackgroundCentral sensitization in the spinal cord requires glutamate receptor activation and intracellular Ca2+ mobilization. We used Fura-2 AM bulk loading of mouse slices together with wide-field Ca2+ imaging to measure glutamate-evoked increases in extracellular Ca2+ to test the hypotheses that: 1. Exogenous application of glutamate causes Ca2+ mobilization in a preponderance of dorsal horn neurons within spinal cord slices taken from adult mice; 2. Glutamate-evoked Ca2+ mobilization is associated with spontaneous and/or evoked action potentials; 3. Glutamate acts at glutamate receptor subtypes to evoked Ca2+ transients; and 4. The magnitude of glutamate-evoked Ca2+ responses increases in the setting of peripheral neuropathic pain.ResultsBath-applied glutamate robustly increased [Ca2+]i in 14.4 ± 2.6 cells per dorsal horn within a 440 x 330 um field-of-view, with an average time-to-peak of 27 s and decay of 112 s. Repeated application produced sequential responses of similar magnitude, indicating the absence of sensitization, desensitization or tachyphylaxis. Ca2+ transients were glutamate concentration-dependent with a Kd = 0.64 mM. Ca2+ responses predominantly occurred on neurons since: 1) Over 95% of glutamate-responsive cells did not label with the astrocyte marker, SR-101; 2) 62% of fura-2 AM loaded cells exhibited spontaneous action potentials; 3) 75% of cells that responded to locally-applied glutamate with a rise in [Ca2+]i also showed a significant increase in AP frequency upon a subsequent glutamate exposure; 4) In experiments using simultaneous on-cell recordings and Ca2+ imaging, glutamate elicited a Ca2+ response and an increase in AP frequency. AMPA/kainate (CNQX)- and AMPA (GYKI 52466)-selective receptor antagonists significantly attenuated glutamate-evoked increases in [Ca2+]i, while NMDA (AP-5), kainate (UBP-301) and class I mGluRs (AIDA) did not. Compared to sham controls, peripheral nerve injury significantly decreased mechanical paw withdrawal threshold and increased glutamate-evoked Ca2+ signals.ConclusionsBulk-loading fura-2 AM into spinal cord slices is a successful means for determining glutamate-evoked Ca2+ mobilization in naïve adult dorsal horn neurons. AMPA receptors mediate the majority of these responses. Peripheral neuropathic injury potentiates Ca2+ signaling in dorsal horn.

Stanniocalcin 2 Is a Negative Modulator of Store-Operated Calcium Entry

The regulation of cellular Ca(2+) homeostasis is essential for innumerable physiological and pathological processes. Stanniocalcin 1, a secreted glycoprotein hormone originally described in fish, is a well-established endocrine regulator of gill Ca(2+) uptake during hypercalcemia. While there are two mammalian Stanniocalcin homologs (STC1 and STC2), their precise molecular functions remain unknown. Notably, STC2 is a prosurvival component of the unfolded protein response. Here, we demonstrate a cell-intrinsic role for STC2 in the regulation of store-operated Ca(2+) entry (SOCE). Fibroblasts cultured from Stc2 knockout mice accumulate higher levels of cytosolic Ca(2+) following endoplasmic reticulum (ER) Ca(2+) store depletion, specifically due to an increase in extracellular Ca(2+) influx through store-operated Ca(2+) channels (SOC). The knockdown of STC2 expression in a hippocampal cell line also potentiates SOCE, and the overexpression of STC2 attenuates SOCE. Moreover, STC2 interacts with the ER Ca(2+) sensor STIM1, which activates SOCs following ER store depletion. These results define a novel molecular function for STC2 as a negative modulator of SOCE and provide the first direct evidence for the regulation of Ca(2+) homeostasis by mammalian STC2. Furthermore, our findings implicate the modulation of SOCE through STC2 expression as one of the prosurvival measures of the unfolded protein response.

The TRPV4 Channel Contributes to Intercellular Junction Formation in Keratinocytes

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca(2+) dynamics. We found that TRPV4 interacts with beta-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca(2+)-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca(2+) increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.

Acute atrial arrhythmogenesis in murine hearts following enhanced extracellular Ca2+ entry depends on intracellular Ca2+ stores

AimTo investigate the effect of increases in extracellular Ca2+ entry produced by the L-type Ca2+ channel agonist FPL-64176 (FPL) upon acute atrial arrhythmogenesis in intact Langendorff-perfused mouse hearts and its dependence upon diastolic Ca2+ release from sarcoplasmic reticular Ca2+ stores.MethodsConfocal microscope studies of Fluo-3 fluorescence in isolated atrial myocytes were performed in parallel with electrophysiological examination of Langendorff-perfused mouse hearts.ResultsAtrial myocytes stimulated at 1 Hz and exposed to FPL (0.1 μm) initially showed (<10 min) frequent, often multiple, diastolic peaks following the evoked Ca2+ transients whose amplitudes remained close to control values. With continued pacing (>10 min) this reverted to a regular pattern of evoked transients with increased amplitudes but in which diastolic peaks were absent. Higher FPL concentrations (1.0 μm) produced sustained and irregular patterns of cytosolic Ca2+ activity, independent of pacing. Nifedipine (0.5 μm), and caffeine (1.0 mm) and cyclopiazonic acid (CPA) (0.15 μm) pre-treatments respectively produced immediate and gradual reductions in the F/F0 peaks. Such nifedipine and caffeine, or CPA pre-treatments, abolished, or reduced, the effects of 0.1 and 1.0 μm FPL on cytosolic Ca2+ signals. FPL (1.0 μm) increased the incidence of atrial tachycardia and fibrillation in intact Langendorff-perfused hearts without altering atrial effective refractory periods. These effects were inhibited by nifedipine and caffeine, and reduced by CPA.ConclusionEnhanced extracellular Ca2+ entry exerts acute atrial arrhythmogenic effects that is nevertheless dependent upon diastolic Ca2+ release. These findings complement reports that associate established, chronic, atrial arrhythmogenesis with decreased overall inward Ca2+ current.

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

Extracellular Ca2+ regulates the stimulus-elicited ATP release from urothelium

Accumulating evidence shows that the epithelial cells in urinary bladder (urothelium) serve as a sensory organ in micturition and/or in nociception pathway by releasing ATP in response to mechanical and/or chemical stimuli. Here, we compared the effects of capsaicin, acetylcholine, and prostaglandin E2 receptor EP1 agonist (ONO-DI-004) on the urothelial ATP release in primary cultured mouse urothelial cells in low Ca2+ medium. All of these chemicals induced a gradual ATP release from urothelium, implying that the downstream Ca2+ release from endoplasmic reticulum could trigger the ATP release. Consistent with this suggestion, blockade of inositol 1,4,5-triphosphate receptor reduced the distention-induced ATP release from urothelial tissues. The distention-induced ATP release was not affected by tetrodotoxin. However, an increase in extracellular Ca2+ diminished both chemical- and distention-induced ATP release from urothelium. Thus raising the extracellular Ca2+ concentration was found to inhibit stimulation-evoked ATP urothelial release.

Fluid Pressure-Gated Cation Channel in Atrial Myocytes

Regurgitant jets of blood in patients with mitral valve incompetence are known to predispose to atrial fibrillation. To understand cellular basis for the fibrillation induced by the fluid jet, we examined ionic currents induced by a fluid pressure (FP) in rat atrial myocytes. FP was applied by pressurized rapid (∼15 dyne/cm2) puffing of bathing solutions onto whole-cell clamped single atrial myocytes. Puffing (1-s long) of normal external solution produced inward current (IFP) at a resting membrane potential, which was inactivated independently of FP. The current-voltage relationship of IFP showed inward rectification with a reversal potential of ≈-18 mV. Ca2+-free extracellular solution enhanced IFP by ≈7-fold and eliminated the inactivation of IFP. IFP was decreased by extracellular divalent cations with the strongest suppression by Ca2+ (Ca2+ > Cd2+ > Ni2+). Removal of extracellular K+ or Na+ decreased IFP by ≈46% or ≈35%, respectively. IFP was almost completely suppressed in K+- and Na+-free extracellular solution. Increase of extracellular Ca2+ concentration to 75 mM enhanced IFP, indicating contribution of Ca2+ to IFP. IFP was resistant to the blockade of the stretch-activated channel or Na+-Ca2+ exchanger. Intracellular Ca2+ buffering with 4 mM EGTA did not alter the magnitude and inactivation of IFP. IFP was increased to ≈200% immediately after a depletion of Ca2+ in the sarcoplasmic reticulum using 10 mM caffeine. Our data provide functional evidence for a novel inwardly rectifying nonselective cation channel in rat atrial myocytes that is gated by fluid pressure. This channel appears to be inactivated by external Ca2+-dependent mechanism and accelerated by depletion of the Ca2+ store. The FP-dependent cation influx at resting potential may be a possible mechanism for the blood-jet induced atrial fibrillation in mitral valve incompetence.

Combined Proteomic and Cytological Analysis of Ca2+-Calmodulin Regulation inPicea meyeriPollen Tube Growth

Ca2+-calmodulin (Ca2+-CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca2+-CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca2+-CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca2+-CaM signaling rapidly induced an increase in extracellular Ca2+ influx, resulting in dramatically increased cytosolic Ca2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca2+-CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca2+-CaM in the regulation of tip growth machinery.