Enhancement Of Ca2 Research Articles

Abstract P1066: Monoamine Oxidase A And Organic Cation Transporter 3 Coordinate Intracellular Adrenergic Signaling To Calibrate Cardiac Function

Aims: We have recently identified a functional pool of intracellular β 1 adrenergic receptors (β 1 ARs) crucial for cardiac function. However, the systematic signaling network of intracellular β 1 ARs remains elusive. Here, we aim to uncover the integrative control of cardiac function, compartmentalized β 1 AR signaling, and potential interface between two critical modulators of catecholamine homeostasis, MAO-A and OCT3. Methods and Results: Cardiac deletion of MAO-A (MAO-A-CKO) elevated myocardium catecholamines, cAMP levels and cardiac ejection fraction by disrupting intracellular catecholamine degradation. Deletion and pharmacological inhibition of MAO-A both enhanced cardiac adrenergic response, with elevated ejection fraction and heart rate. Inhibiting OCT3 by corticosterone prevented the impacts of MAO-A inhibition on cardiac function. Using subcellular anchored Förster resonance energy transfer (FRET) biosensors, we then determined the regulation of compartmentalized β 1 AR/PKA signaling at the sarcoplasmic reticulum (SR) and plasma membrane (PM) microdomains. We also examined local PKA-substrates phosphorylation and myocyte excitation-contraction coupling. MAO-A deletion and inhibitor (MAOi) enhanced local PKA activity at the SR, phosphorylation of phospholamban, Ca 2+ cycling, and contractile response. Whereas overexpression of MAO-A impairs SR-localized PKA activity and phospholamban phosphorylation. In contrast, these modifying of MAO-A did not affect PKA activity at PM and L-type Ca 2+ channel phosphorylation. Inhibiting OCT3 blocked blocking catecholamine transport and diminished the efficacy of MAO-A inhibitor (MAO-A) and MAO-A-CKO to regulate SR-localized β 1 AR signaling. In parallel, OCT3 inhibition and deletion prevented the MAO-A-dependent enhancement of Ca 2+ cycling and cardiac contractile function. Conclusion: Together, we show that MAO-A and OCT3 act in a concert to fine-tune the intracellular catecholamines, activation of SR-localized β 1 AR-PKA signaling, and cardiac fight-or-flight response. Further, we reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of those drugs in cardiac implications.

Characterizing SERCA Function in Murine Skeletal Muscles after 35-37 Days of Spaceflight.

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35–37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA.

Investigations on phase coexistence and functional properties of BCZT lead-free piezoceramic

Large piezoelectric properties was observed in (1-x)BZT-(x)BCT where x=0.5 or Ba0.85Ca0.15Ti0.9Zr0.1O3 (denoted as BCZT), leading to a promising candidate for lead-free piezoelectric materials. However, phase formation of the BCZT is controversial and still unclear since various phase coexistences were identified in the literatures, for instances, the mixed phases of rhombohedral-tetragonal (R-T), ortho-rhombic-tetragonal (O-T) or rhombohedral-orthorhombic-tetragonal (R-O-T). Additionally, it is well known that the crystal structure plays a crucial role on the occurrence of polarization in the piezoceramics. Therefore, this work aims to investigate the coexistence of phase formation at room temperature for the BCZT powder and ceramic. Moreover, the electrical properties as a function of temperature, frequency and electric field were observed in order to evaluate the extrinsic contribution of piezoelectric response. It was found that, according to the results from temperature-dependent dielectric properties as well as Rietveld refinement of XRD profiles, the coexistence of O-T phase was observed in the BCZT powder and ceramic. Furthermore, the enhancement of Ca2+ substitution into Ba2+ site in BCZT ceramic caused the shrinkage of unit cell, leading to the shift of XRD profile and Raman spectra. In addition, it was found that the applications of frequency and electric field can influence on changes of domain-wall motion and micro-polar cluster in the BCZT piezoceramic.

Effects of TNFα on Dynamic Cytosolic Ca2 + and Force Responses to Muscarinic Stimulation in Airway Smooth Muscle.

Previously, we reported that in airway smooth muscle (ASM), the cytosolic Ca2+ ([Ca2+]cyt) and force response induced by acetyl choline (ACh) are increased by exposure to the pro-inflammatory cytokine tumor necrosis factor α (TNFα). The increase in ASM force induced by TNFα was not associated with an increase in regulatory myosin light chain (rMLC20) phosphorylation but was associated with an increase in contractile protein (actin and myosin) concentration and an enhancement of Ca2+ dependent actin polymerization. The sensitivity of ASM force generation to elevated [Ca2+]cyt (Ca2+ sensitivity) is dynamic involving both the shorter-term canonical calmodulin-myosin light chain kinase (MLCK) signaling cascade that regulates rMLC20 phosphorylation and cross-bridge recruitment as well as the longer-term regulation of actin polymerization that regulates contractile unit recruitment and actin tethering to the cortical cytoskeleton. In this study, we simultaneously measured [Ca2+]cyt and force responses to ACh and explored the impact of 24-h TNFα on the dynamic relationship between [Ca2+]cyt and force responses. The temporal delay between the onset of [Ca2+]cyt and force responses was not affected by TNFα. Similarly, the rates of rise of [Ca2+]cyt and force responses were not affected by TNFα. The absence of an impact of TNFα on the short delay relationships between [Ca2+]cyt and force was consistent with the absence of an effect of [Ca2+]cyt and force on rMLC20 phosphorylation. However, the integral of the phase-loop plot of [Ca2+]cyt and force increased with TNFα, consistent with an impact on actin polymerization and, contractile unit recruitment and actin tethering to the cortical cytoskeleton.

Enhanced Ca2+ influx in mechanically distorted erythrocytes measured with 19F nuclear magnetic resonance spectroscopy

We present the first direct nuclear magnetic resonance (NMR) evidence of enhanced entry of Ca2+ ions into human erythrocytes (red blood cells; RBCs), when these cells are mechanically distorted. For this we loaded the RBCs with the fluorinated Ca2+ chelator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA), and recorded 19F NMR spectra. The RBCs were suspended in gelatin gel in a special stretching/compression apparatus. The 5FBAPTA was loaded into the cells as the tetraacetoxymethyl ester; and 13C NMR spectroscopy with [1,6-13C]d-glucose as substrate showed active glycolysis albeit at a reduced rate in cell suspensions and gels. The enhancement of Ca2+ influx is concluded to be via the mechanosensitive cation channel Piezo1. The increased rate of influx brought about by the activator of Piezo1, 2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1) supported this conclusion; while the specificity of the cation-sensing by 5FBAPTA was confirmed by using the Ca2+ ionophore, A23187.

Age-Induced Differential Changes in the Central and Colonic Human Circadian Oscillators.

Aging modifies not only multiple cellular and homeostatic systems, but also biological rhythms. The circadian system is driven by a central hypothalamic oscillator which entrains peripheral oscillators, in both cases underlain by circadian genes. Our aim was to characterize the effect of aging in the circadian expression of clock genes in the human colon. Ambulatory recordings of the circadian rhythms of skin wrist temperature, motor activity and the integrated variable TAP (temperature, activity and position) were dampened by aging, especially beyond 74 years of age. On the contrary, quantitative analysis of genes expression in the muscle layer of colonic explants during 24 h revealed that the circadian expression of Bmal1, Per1 and Clock genes, was larger beyond that age. In vitro experiments showed that aging induced a parallel increase in the myogenic contractility of the circular colonic muscle. This effect was not accompanied by enhancement of Ca2+ signals. In conclusion, we describe here for the first time the presence of a molecular oscillator in the human colon. Aging has a differential effect on the systemic circadian rhythms, that are impaired by aging, and the colonic oscillator, that is strengthened in parallel with the myogenic contractility.

Dual action of amitriptyline on NMDA receptors: enhancement of Ca-dependent desensitization and trapping channel block

Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca2+-dependent desensitization and 2) trapping channel block. Inhibition of NMDARs by ATL was found to be dependent upon external Ca2+ concentration ([Ca2+]) in a voltage-independent manner, with an IC50 of 0.72 μM in 4 mM [Ca2+]. The ATL IC50 value increased exponentially with decreasing [Ca2+], with an e-fold change observed per 0.69 mM decrease in [Ca2+]. Loading neurons with BAPTA abolished Ca2+-dependent inhibition, suggesting that Ca2+ affects NMDARs from the cytosol. Since there is one known Ca2+-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca2+-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC50 of 220 µM at 0 mV. An e-fold change in ATL IC50 was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca2+]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca2+], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.

Inhibition of PI3Kinase-α is pro-arrhythmic and associated with enhanced late Na+ current, contractility, and Ca2+ release in murine hearts

BackgroundPhosphoinositide 3-kinase α (PI3Kα) is a proto-oncogene with high activity in the heart. BYL719 (BYL) is a PI3Kα-selective small molecule inhibitor and a prospective drug for advanced solid tumors. We investigated whether acute pharmacological inhibition of PI3Kα has pro-arrhythmic effects. Methods & ResultsIn isolated wild-type (WT) cardiomyocytes, pharmacological inhibition of PI3Kα (BYL719) increased contractility by 28%, Ca2+ release by 20%, and prolonged action potential (AP) repolarization by 10–15%. These effects of BYL719 were abolished by inhibition of reverse-mode Na+/Ca2+ exchanger (NCX) (KB-R7943) or by inhibition of late Na+ current (INa-L) (ranolazine). BYL719 had no effect on PI3Kα-deficient cardiomyocytes, suggesting BYL719 effects were PI3Kα-dependent and mediated via NCX and INa-L. INa-L was suppressed by activation of PI3Kα, application of exogenous intracellular PIP3, or ranolazine. Investigation of AP and Ca2+ release in whole heart preparations using epicardial optical mapping showed that inhibition of PI3Kα similarly led to prolongation of AP and enhancement of Ca2+ release. In hearts of PI3Kα-deficient mice, β-adrenergic stimulation in the presence of high Ca2+ concentrations and 12-Hz burst pacing led to delayed afterdepolarizations and ventricular fibrillation. In vivo, administration of BYL719 prolonged QT interval [QTcF (Fridericia) increased by 15%] in WT, but not in PI3Kα-deficient mice. ConclusionsPharmacological inhibition of PI3Kα is arrhythmogenic due to activation of INa-L leading to increased sarcoplasmic reticulum Ca2+ load and prolonged QT interval. Therefore, monitoring of cardiac electrical activity in patients receiving PI3K inhibitors may provide further insights into the arrhythmogenic potential of PI3Ka inhibition.

Mechanisms of Cannabinoid 2 receptor agonist JWH133-mediated modulations in cell calcium signals of mouse pancreatic acinar cells and its application for treatment of acute pancreatitis

Objective To evaluate the effect of JWH133 on acetylcholine (Ach)-induced Ca2+ oscillations, and to elucidate its mechanism and target. Methods Pancreatic acinar cells were isolated by enzymatic hydrolysis. Patch-clamp whole cell recording techniques were used to measure the effects of 10 μmol/L JWH133 on 10 nmol/L ACh-induced intracellular Ca2+ oscillations in pancreatic acinar cells acutely dissociated from wild-type (WT), and Cannabinoid 2 receptor (CB2R) knockout (KO) mice, and the pharmacological effects of JWH133 on the Ca2+ oscillations. The effect of 10 μmol/L JWH133 on the enhancement of Ca2+ oscillation induced by 10 mmol/L L-Arginine (L-Arg) was measured. The model of acute pancreatitis in mice was induced by L-Arg, and the histological structure, cell morphology and hemorrhagic necrosis were examined. Results The results showed that JWH133 could inhibit ACh-induced Ca2+ oscillations likely through CB2R. Patch-clamp recordings demonstrated that 10 μmol/L of JWH133 could reduce the 10 nmol/L ACh-induced Ca2+ oscillations current to (26.0±4.2)% (P 0.05, n=9). 10 μmol/L of JWH133 attenuated the enhancement of calcium oscillations current to (106.0±16.7)% (P<0.01, n=10) induced by 10 mmol/L L-Arg. In L-Arg acute pancreatitis model, JWH133 improved L-Arg -induced pancreatic pathological changes (P<0.05, n=5). Conclusion JWH133 inhibits intracellular calcium signals by selectively inhibiting extracellular CB2R. In L-Arg -induced acute mouse pancreatitis model, JWH133 significantly improves pathological changes. All these results suggest that JWH133 is a promising candidate for treatment of acute pancreatitis. Key words: Acute pancreatitis; Cannabinoid 2 receptor agonist; Calcium signals; Patch clamp

Melanocortin-1 Receptor Positively Regulates Human Artery Endothelial Cell Migration.

Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²⁺ mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²⁺ signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.

Endothelial Upregulation of Mechanosensitive Channel Piezo1 in Pulmonary Hypertension

RationaleThe Piezo channel family (Piezo1 and Piezo2), one of the most important family of mechanosensing ion channels. Surprisingly, there are no studies on Piezo in the pulmonary hypertension. We focus on Piezo1 because of its imprtance in vascular deveopment, especially in endothelial cells. Our working hypothesis is that upregulated Piezo1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PAEC proliferation or apoptosis in patients with IPAH.MethodsRNA levels of Piezo1 and Piezo2 was quantified by RT‐PCR Analysis. Protein levels of Piezo1, bax, bcl‐2, Calpain‐1 and Calpain‐2 was quantified by Western blot analysis. adult Male C57BL/6 mice (6–8 weeks) were exposed to persistent chronic hypoxia (10% O2) for 21 days to establish hypoxia‐induced PH. For Sugen 5416/hypoxia (SuHx) induced‐PH rat model, SD rats (age, 6–8 weeks old) were either injected s.c. with 20 mg·kg−1 SU5416 (Sigma‐Aldrich, St. Louis, MO, USA) or vehicle, exposed to hypoxic conditions for 3 weeks (10% O2) and returned to normoxic condition (21% O2) for 2 weeks. In the MCT group, a single intraperitoneal injection of MCT (60 mg/kg, Sigma Aldrich) was given at day 1 to establish the MCT‐induced PH.ResultsPiezo1 and Piezo2 have high expression in rat lung tissue and human PAEC, and also Piezo1 has significantly higher mRNA levels in both human PASMC and PAEC than Piezo2 (n=5, p&lt;0.01). Upregulated Piezo1 in whole‐lung tissue (mainly containing ECs) from MCT‐mediated PH (MCT‐PH) compared to normal control rats (1.00±0.07; 4.74±0.41, n&gt;5, p&lt;0.001). Compared to controls, Piezo1 was increased in whole‐lung tissues in hypoxic mice (1.00±0.07; 4.74±0.41, n&gt;5, p&lt;0.01) and SuHx rats (1.00±0.15; 1.99±0.31, n&gt;5, p&lt;0.01). Also, compared to normoxia condition, increased Piezo1 was associated with a increased Calpain‐1 and Calpain‐2 treated with hypoxia (3% O2 for 72 hours) (1.00±0.16; 4.14±0.75; 1.00±0.14, 2.32±0.35; 1.00±0.07; 1.74±0.14, n&gt;5, p&lt;0.01). Moreover, PAECs from IPAH patients exhibited significant elevation of Piezo1 compared to normal control subjects (1.00±0.09; 6.20±1.78, n=5, p&lt;0.01).ConclusionsThese results are the first to implicate that Piezo1 as a mechanosensor may play an important role in the development of pulmonary hypertension via, at least in part, induction of PAEC apoptosis and enhancement of Ca2+ in PAECs. Piezo1 may be involved in the transcriptional regulation under hypoxia and play critical roles in the development of PH. Therefore, further studies are needed to identify the potential contribution of Piezo1 in PH.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Brain cholesterol metabolite 24-hydroxycholesterol modulates inotropic responses to β-adrenoceptor stimulation: The role of NO and phosphodiesterase

Aims24-Hydroxycholesterol (24HC) is the main brain cholesterol metabolite, which level in the circulation is significantly changed under physiological and pathological conditions. Here, we have studied the effect of 24HC on the inotropic responses to β-adrenoceptor (AR) stimulation. Main methodsElectrical stimulation-evoked contractions were recorded in isolated atria from mice. Fluorescent dyes, Fluo-4 and DAF-FM, were used for estimation of Ca2+ transient and NO production, respectively. Key findingsWe revealed that 24HC in the submicromolar range attenuated β-AR-induced positive inotropy in isolated atria. This was accompanied by a decrease in Ca2+ transient and unchanged nitric oxide (NO) production. However, β1-AR-induced positive inotropy and enhancement of Ca2+ transient were increased by 24HC due to suppression of NO production. Only β2-AR-dependent inotropy and enhancement of Ca2+ transient were decreased by 24HC in a NO-independent manner. Inhibition of phosphodiesterase (PDE) suppressed effect of 24HC on β2-AR-dependent contractility as well as on non-subtype specific β-AR activation. Moreover, 24HC counteracted positive inopropic action of PDE inhibitors, IBMX and rolipam. Thus, 24HC modulates the effects of β1- and β2-AR stimulation via different mechanisms linked with change in activity of NO synthase or PDE, respectively. Under conditions of non-selective activation of β-ARs, the depressant effect of 24HC related with β2-AR-dependent signaling dominates. SignificanceWe suggest that 24HC could serve as a modulator of atrial β-AR signaling, contributing to regulation of contractility.

Revealing the role of calcium codoping on optical and scintillation homogeneity in Lu2SiO5:Ce single crystals

The presence of impurities and dopants (or codopants) can influence solute transport at the solid-liquid interface and its stability during bulk crystal growth, which ultimately affect the crystal uniformity. Codoping with Ca2+ can significantly improve the performance of Lu2SiO5:Ce single crystals, well-known scintillators for nuclear medical imaging, albeit at the expense of growth stability due to surface tension reduction. However, an understanding of the correlation between crystal growth properties and radial performance uniformity in Czochralski-grown LSO:Ce, Ca single crystals is still lacking. In this work, we reveal this essential correlation by studying the roles of Ca2+ concentration on the radial distribution of stable Ce3+ ions and oxygen vacancy (VO) related defects as well as solute transport at the solid-liquid interface. Through mapping of optical and scintillation properties across cross-sections of 0.1 at% and 0.4 at% Ca codoped boules and the defect formation energies derived from density functional theory (DFT) calculations, a direct link between Ca2+ ions, stable Ce3+ or Ce4+ ions, and {CaLu + VO} complex defects is established. The light yield enhancement in Ca2+ codoped LSO:Ce single crystals is attributed to the dissociation of spatially correlated Ce ions and oxygen vacancies. Based on the quantitative comparison of photoluminescence (PL) and radioluminescence (RL) emissions from Ce1 (seven-coordinated) and Ce2 (six-coordinated) sites, the preferential occupation of stable Ce4+ is believed to depend on the concentration of stable Ce4+. The underlying causes of symmetry and variation in the radial distribution of optical and scintillation properties, as consequences of Ca2+ distribution, are discussed from the perspective of crystal growth.

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca2+]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ETA- or ETB-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca2+ transients in adult rat ventricular myocytes. The negative inotropic phase was ETB receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca2+ transients required ETA receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP3) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca2+ transient amplitude induced by ET-1 were independent of InsP3 signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca2+ transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP3 and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca2+ transients following ET-1 stimulation.

Decreased Caffeine-Induced Locomotor Activity via Microinjection of CART Peptide into the Nucleus Accumbens Is Linked to Inhibition of the pCaMKIIa-D3R Interaction.

The purpose of this study was to characterize the inhibitory modulation of cocaine- and amphetamine-regulated transcript (CART) peptides, particularly with respect to the function of the D3 dopamine receptor (D3R), which is activated by its interaction with phosphorylated CaMKIIα (pCaMKIIα) in the nucleus accumbens (NAc). After repeated oral administration of caffeine (30 mg/kg) for five days, microinjection of CART peptide (0.08 μM/0.5 μl/hemisphere) into the NAc affected locomotor behavior. The pCaMKIIα-D3R interaction, D3R phosphorylation and cAMP/PKA/phosphorylated CREB (pCREB) signaling pathway activity were measured in NAc tissues, and Ca2+ influx and pCaMKIIα levels were measured in cultured NAc neurons. We found that CART attenuated the caffeine-mediated enhancement of depolarization-induced Ca2+ influx and CaMKIIα phosphorylation in cultured NAc neurons. Repeated microinjection of CART peptides into the NAc decreased the caffeine-induced enhancement of Ca2+ channels activity, pCaMKIIα levels, the pCaMKIIα-D3R interaction, D3R phosphorylation, cAMP levels, PKA activity and pCREB levels in the NAc. Furthermore, behavioral sensitization was observed in rats that received five-day administration of caffeine following microinjection of saline but not in rats that were treated with caffeine following microinjection of CART peptide. These results suggest that caffeine-induced CREB phosphorylation in the NAc was ameliorated by CART peptide due to its inhibition of D3R phosphorylation. These effects of CART peptides may play a compensatory role by inhibiting locomotor behavior in rats.

Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca(2+) oscillations in mouse pancreatic acinar cells.

Emerging evidence demonstrates that the blockade of intracellular Ca2+ signals may protect pancreatic acinar cells against Ca2+ overload, intracellular protease activation, and necrosis. The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca2+ signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca2+ oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca2+ oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca2+ oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca2+ oscillations and L-arginine-induced enhancement of Ca2+ signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis.

Shear Stress Enhances Ca2+ Spark Occurrence in Rat Ventricular Myocytes Via Mitochondrial NADPH Oxidase-Reactive Oxygen Species Signaling

It has been reported that shear stress enhances Ca2+-induced Ca2+ release during depolarization in ventricular myocytes. To know molecular basis for the increase in global Ca2+ releases we assessed the effects of shear stress (∼16 dyn/cm2) on local Ca2+ release events (Ca2+ sparks) and examined cellular mechanisms for the shear-mediated Ca2+ spark regulation using confocal Ca2+ imaging in rat ventricular myocytes. The frequency of Ca2+ sparks was immediately (within 1 s) increased by shear stress to about 1.5-fold, and further increased to about 2-fold by prolonged (20 s) shear exposure. Inhibition of nitric oxide synthetase and interference of cytoskeletal integrity using L-NAME (1 mM) and colchicine (10 μM), respectively, did not affect the shear-mediated enhancement in spark frequency. Blockers for Na+-Ca2+ exchanger and L-type Ca2+ channel, KB-R7943 (5 μM) or Ni2+ (5 mM), did not alter the shear effect on spark occurrence. Inhibition of NADPH oxidase (NOX) and mitochondrial uncoupling using diphenyleneiodonium (3 μM) and carbonyl cyanide 3-chlorophenylhydrazone (plus oligomycin, 1 μg/ml), respectively, reduced shear mediated spark enhancements. Pretreatment of reducing agent dithiothreitol (2 mM) also significantly reduced the shear-mediated spark enhancement. Measurement of intracellular reactive oxygen species (ROS) using its specific dye 2′,7′-dichlorofluorescein revealed increase in ROS level by shear stress. Sarcoplasmic reticulum (SR) Ca2+ content was not altered immediately after the application of shear stress, but significantly increased after 20-s long shear exposure. Our data suggest that shear stress enhances the frequency of Ca2+ sparks partly by producing ROS via mitochondrial NOX, and that prolonged enhancement in spark frequency by shear stress may be also mediated by increased SR Ca2+ loading. These mechanisms may partly explain the shear-mediated enhancement in Ca2+ transient in ventricular myocytes.

Assessing of tolerance to metallic and saline stresses in the halophyte Suaeda fruticosa: The indicator role of antioxidative enzymes

Many areas are simultaneously affected by high concentrations of salts and trace metal elements (TME), the latter constituting a serious threat to human health. In the present study, we determined the combined effect of high salinity and toxic levels of trace elements on physiological behavior of the halophytic species Suaeda fruticosa. Plants were cultivated for three months with an irrigation solution supplemented separately with different concentrations of Pb2+ and Zn2+ (0, 200, 400 and 600μM) with and without 200mM NaCl. Growth, total chlorophyll, water status and ion nutrition were quantified and antioxidant enzyme activities [ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and catalase (CAT)] were studied. Our results revealed that S. fruticosa has a strong ability to tolerate lead and zinc. This halophyte accumulated higher concentrations of TME in their roots. Growth parameters of S. fruticosa were not significantly affected by TME. An enhancement of Ca2+ concentration accompanied by a decrease of Mg2+ content was observed under Pb2+ or Zn2+ treatments whereas K+ content was not affected by TME. Of the antioxidant enzymes, the activity of CAT and APX was increased by metal stress. However, the activity of GPX was diminished by increasing TME concentrations. It was concluded that NaCl 200mM had a positive impact on the response of S. fruticosa to Zn2+ toxicity, acting through a decrease in Zn absorption.

The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position

Mutations in cardiac myosin binding protein C (cMyBP-C), a thick filament protein that modulates contraction of the heart, are a leading cause of hypertrophic cardiomyopathy (HCM). Electron microscopy and 3D reconstruction of thin filaments decorated with cMyBP-C N-terminal fragments suggest that one mechanism of this modulation involves the interaction of cMyBP-C's N-terminal domains with thin filaments to enhance their Ca2+-sensitivity by displacement of tropomyosin from its blocked (low Ca2+) to its closed (high Ca2+) position. The extent of this tropomyosin shift is reduced when cMyBP-C N-terminal domains are phosphorylated. In the current study, we have examined L348P, a sequence variant of cMyBP-C first identified in a screen of patients with HCM. In L348P, leucine 348 is replaced by proline in cMyBP-C's regulatory M-domain, resulting in an increase in cMyBP-C's ability to enhance thin filament Ca2+-sensitization. Our goal here was to determine the structural basis for this enhancement by carrying out 3D reconstruction of thin filaments decorated with L348P-mutant cMyBP-C. When thin filaments were decorated with wild type N-terminal domains at low Ca2+, tropomyosin moved from the blocked to the closed position, as found previously. In contrast, the L348P mutant caused a significantly larger tropomyosin shift, to approximately the open position, consistent with its enhancement of Ca2+-sensitization. Phosphorylated wild type fragments showed a smaller shift than unphosphorylated fragments, whereas the shift induced by the L348P mutant was not affected by phosphorylation. We conclude that the L348P mutation causes a gain of function by enhancing tropomyosin displacement on the thin filament in a phosphorylation-independent way.

Elevated Homocysteine Levels Result in Carbonyl Formation on RyR2 and Enhanced Sensitivity of Sarcoplasmic Reticulum to Activation by Calcium

Elevated levels in blood serum of homocysteine (>10µmol/L) is strongly correlated with the incidence of heart failure (HF) in humans. We demonstrate that the cyclic thioester, homocysteine thiolactone (HTL), a metabolic product of homocysteine, at a concentration of 100 nM, results in the formation of carbonyls on the ryanodine receptor from sheep cardiac muscle (RyR2), and an enhancement of Ca2+ dependent activation of ryanodine binding from 129.7 ± 3.4 nM to 102.0 ± 2.7 nM. While both improper Ca2+ handling and elevated homocysteine levels have been considered bio-markers in HF, a direct connection between the two has not previously been made. We propose that HTL reacts with lysine residues on RyR2, generating an Ne-homocysteine-protein, which results in carbonyl formation and an enhancement in the Ca2+ sensitivity of RyR2. This is a new molecular mechanism linking elevated levels of Homocysteine, post-translational modification of RyR2, improper Ca2+ handling and heart failure. This work was supported by NIH 1 R41 HL105063-01 to J. Abramson and R. Strongin.