Induction Of Ca2 Research Articles

Routes of Ca2+ Shuttling during Ca2+ Oscillations: FOCUS ON THE ROLE OF MITOCHONDRIAL Ca2+ HANDLING AND CYTOSOLIC Ca2+ BUFFERS

In some cell types, Ca(2+) oscillations are strictly dependent on Ca(2+) influx across the plasma membrane, whereas in others, oscillations also persist in the absence of Ca(2+) influx. We observed that, in primary mesothelial cells, the plasmalemmal Ca(2+) influx played a pivotal role. However, when the Ca(2+) transport across the plasma membrane by the "lanthanum insulation method" was blocked prior to the induction of the serum-induced Ca(2+) oscillations, mitochondrial Ca(2+) transport was found to be able to substitute for the plasmalemmal Ca(2+) exchange function, thus rendering the oscillations independent of extracellular Ca(2+). However, in a physiological situation, the Ca(2+)-buffering capacity of mitochondria was found not to be essential for Ca(2+) oscillations. Moreover, brief spontaneous Ca(2+) changes were observed in the mitochondrial Ca(2+) concentration without apparent changes in the cytosolic Ca(2+) concentration, indicating the presence of a mitochondrial autonomous Ca(2+) signaling mechanism. In the presence of calretinin, a Ca(2+)-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased, and the amount of Ca(2+) ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to proapoptotic/pronecrotic signals. We identified and characterized (experimentally and by modeling) three Ca(2+) shuttling pathways in primary mesothelial cells during Ca(2+) oscillations: Ca(2+) shuttled between (i) the endoplasmic reticulum (ER) and mitochondria, (ii) the ER and the extracellular space, and (iii) the ER and cytoplasmic Ca(2+) buffers.

Abstract 186: Increased STIM1 Expression Results in Altered Calcium Handling and Heart Failure

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor that partners with Orai1, resulting in store-operated Ca2+ entry (SOCE) that is important for maintaining endoplasmic reticulum (ER) Ca2+ homeostasis. STIM1 is expressed in the heart and upregulated during disease, but its role in disease progression is unclear. In this study we used transgenic mice with STIM1 overexpression in the heart to model the known increase of this protein in response to cardiac disease. We found that STIM1 transgenic myocytes showed elevated Ca2+ entry following store depletion and STIM1 co-localized with the type 2 ryanodine receptor (RyR2) in the sarcoplasmic reticulum (SR). In addition, STIM1 transgenic mice exhibited sudden cardiac death as early as 6 weeks of age, while mice that survived past 12 weeks developed cardiac hypertrophy that progressed to heart failure, pulmonary edema, activation of the fetal gene program, alterations in mitochondrial structure, and reduced ventricular functional performance. When pre-symptomatic STIM1 transgenic mice were subjected to disease stimuli including pressure overload stimulation or neurohumoral agonist infusion, they showed greater pathology compared to control mice. STIM1 elevation also disrupted normal Ca2+ handling in cardiac myocytes, which showed spontaneous Ca2+ transients that could be inhibited by the SOCE blocker SKF-96265, as well as increased diastolic Ca2+ levels and elevated Ca2+ spark frequency. In keeping with this increase in Ca2+ cycling we also found that STIM1 elevation resulted in an increased baseline activity of cardiac nuclear factor of activated T-cells (NFAT) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). This increased CaMKII activity did not, however, translate into additional RyR2 phosphorylation, suggesting that the augmented Ca2+ spark frequency observed was likely due to an elevation in SR Ca2+ load. Our results suggest that increased STIM1 expression elicits augmented Ca2+ entry, SR Ca2+ load and Ca2+ spark frequency, that leads to mitochondrial pathology and the induction of Ca2+ sensitive hypertrophic signaling pathways that contribute to cardiac disease.

The Mechanism of Ca(2+) Movement in the Involvement of Baicalein-Induced Cytotoxicity in ZR-75-1 Human Breast Cancer Cells.

Baicalein (5,6,7-trihydroxyflavone) (1) has been found to be active against a wide variety of cancer cells. However, the molecular mechanism underlying the effects of 1 on the induction of Ca(2+) movement and cytotoxicity in human breast cancer cells is unknown. This study examined the relationship between 1-induced Ca(2+) signaling and cytotoxicity in ZR-75-1 human breast cancer cells. The in vitro investigations reported herein produced the following results: (i) Compound 1 increased intracellular Ca(2+) concentration ([Ca(2+)]i) in a concentration-dependent manner. The signal was decreased by approximately 50% by removal of extracellular Ca(2+). (ii) Compound 1-triggered [Ca(2+)]i increases were significantly suppressed by store-operated Ca(2+) channel blockers 2-aminoethoxydiphenyl borate (2-APB) and the PKC inhibitor GF109203X. (iii) In Ca(2+)-free medium, compound 1-induced [Ca(2+)]i increases were also inhibited by GF109203X. Furthermore, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-ditert-butylhydroquinone (BHQ) abolished 1-induced [Ca(2+)]i increases. Inhibition of phospholipase C (PLC) with U73122 abolished 1-induced [Ca(2+)]i increases. (iv) Compound 1 (20-40 μM) caused cytotoxicity, increased reactive oxygen species (ROS) production, and activated caspase-9/caspase-3. Furthermore, compound 1-induced apoptosis was significantly inhibited by prechelating cytosolic Ca(2+) with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester) or by decreasing ROS with the antioxidant NAC (N-acetylcysteine). Together, baicalein (1) induced a [Ca(2+)]i increase by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-dependent, 2-APB-sensitive store-operated Ca(2+) channels. Moreover, baicalein (1) induced Ca(2+)-associated apoptosis involved ROS production in ZR-75-1 cells.

Disruption of cytochrome c oxidase function induces the Warburg effect and metabolic reprogramming.

Defects in mitochondrial oxidative phosphorylation complexes, altered bioenergetics and metabolic shift are often seen in cancers. Here we show a role for the dysfunction of electron transport chain component, cytochrome c oxidase (CcO) in cancer progression. We show that genetic silencing of the CcO complex by shRNA expression and loss of CcO activity in multiple cell types from the mouse and human sources resulted in metabolic shift to glycolysis, loss of anchorage dependent growth and acquired invasive phenotypes. Disruption of CcO complex caused loss of transmembrane potential and induction of Ca2+/Calcineurin-mediated retrograde signaling. Propagation of this signaling, includes activation of PI3-kinase, IGF1R and Akt, Ca2+ sensitive transcription factors and also, TGFβ1, MMP16, periostin that are involved in oncogenic progression. Whole genome expression analysis showed up regulation of genes involved in cell signaling, extracellular matrix interactions, cell morphogenesis, cell motility and migration. The transcription profiles reveal extensive similarity to retrograde signaling initiated by partial mtDNA depletion, though distinct differences are observed in signaling induced by CcO dysfunction. The possible CcO dysfunction as a biomarker for cancer progression was supported by data showing that esophageal tumors from human patients show reduced CcO subunits IVi1 and Vb in regions that were previously shown to be hypoxic core of the tumors. Our results show that mitochondrial electron transport chain defect initiates a retrograde signaling. These results suggest that a defect in CcO complex can potentially induce tumor progression.

Biochemical characterization of the roles of glycines 24 and 27 and threonine 179 in tropomyosin from the fast skeletal trunk muscle of the atlantic salmon.

Atlantic salmon fast skeletal muscle is composed of an α-type tropomyosin that shares 20 substitutions with a mammalian homologue. Prominent isomorphisms considered to be potentially destabilizing include threonine 179 (core, Ala in rabbit) and a unique pair of glycines, residues 24 and 27. Bacterially expressed mutant tropomyosins were isolated without exposure to elevated temperature or organic solvent. The Thr179Ala mutant is more resistant to heat-induced denaturation (Tm > 40 °C) than the nonmutant control (Tm = 37 °C) as monitored by far-UV circular dichroism [0.1 M salt and 1 mM DTT (pH 7)]. Changing one glycine to alanine has no detectable effect on the Tm versus the control value. An increase of 1 °C is observed for the double mutant (Gly27Ala/Gly24Ala). The preferred site of chymotryptic cleavage of recombinant salmon tropomyosin is between Leu 11 and Lys 12. The rate of the subsequent cleavage at Leu 169, the preferred site in rabbit tropomyosin, is reduced via replacement of Thr 179 with Ala. Thin filaments reconstituted with this mutant display greater Ca(II) induction of the myosin steady-state MgATPase versus control. Glycine-induced local instability is demonstrated by Escherichia coli outer membrane protease T (Omp-T) cleavage between Lys 6 and Lys 7. The control is more susceptible to proteolysis at this site than the mutants, which are distinct from each other: control > Gly27Ala > Gly24Ala > double mutant (most resistant to Omp-T). Similarly, the double mutant is comparatively more resistant to cleavage at Leu 11. By inference, the mid- and amino-terminal sections of salmon tropomyosin are intrinsically less stable than those in mammals, suggesting greater flexibility. Neighboring glycines make up a new destabilization motif in tropomyosin.

Transcript dynamics at early stages of molecular interactions of MYMIV with resistant and susceptible genotypes of the leguminous host, Vigna mungo.

Initial phases of the MYMIV- Vigna mungo interaction is crucial in determining the infection phenotype upon challenging with the virus. During incompatible interaction, the plant deploys multiple stratagems that include extensive transcriptional alterations defying the virulence factors of the pathogen. Such molecular events are not frequently addressed by genomic tools. In order to obtain a critical insight to unravel how V. mungo respond to Mungbean yellow mosaic India virus (MYMIV), we have employed the PCR based suppression subtractive hybridization technique to identify genes that exhibit altered expressions. Dynamics of 345 candidate genes are illustrated that differentially expressed either in compatible or incompatible reactions and their possible biological and cellular functions are predicted. The MYMIV-induced physiological aspects of the resistant host include reactive oxygen species generation, induction of Ca2+ mediated signaling, enhanced expression of transcripts involved in phenylpropanoid and ubiquitin-proteasomal pathways; all these together confer resistance against the invader. Elicitation of genes implicated in salicylic acid (SA) pathway suggests that immune response is under the regulation of SA signaling. A significant fraction of modulated transcripts are of unknown function indicating participation of novel candidate genes in restricting this viral pathogen. Susceptibility on the other hand, as exhibited by V. mungo Cv. T9 is perhaps due to the poor execution of these transcript modulation exhibiting remarkable repression of photosynthesis related genes resulting in chlorosis of leaves followed by penalty in crop yield. Thus, the present findings revealed an insight on the molecular warfare during host-virus interaction suggesting plausible signaling mechanisms and key biochemical pathways overriding MYMIV invasion in resistant genotype of V. mungo. In addition to inflate the existing knowledge base, the genomic resources identified in this orphan crop would be useful for integrating MYMIV-tolerance trait in susceptible cultivars of V. mungo.

The acidic transformed nano-VO2 causes macrophage cell death by the induction of lysosomal membrane permeabilization and Ca2+ efflux

Because of its outstanding thermochromic characteristics and metal-insulator transition (MIT) property, nano-vanadium dioxide (abbreviated as nano-VO2 or nVO2) has been applied widely in electrical/optical devices and design of intelligent window. However, the biological effect of nVO2 is not well understood, especially when affected by environmental factors or living organisms. For VO2 is an amphoteric oxide, we simulated pH's influence to nVO2’s physicochemical properties by exposure nVO2 in water of different pH values. We found that nVO2 transformed to a new product after exposure in acidic water for two weeks, as revealed by physicochemical characterization such as SEM, TEM, XRD, and DLS. This transformation product formed in acidic water was referred as (acidic) transformed nVO2). Both pristine/untransformed and transformed nVO2 displayed no obvious toxicity to common epithelial cells; however, the acidic transformed nVO2 rapidly induced macrophage cell death. Further investigation demonstrated that transformed nVO2 caused macrophage apoptosis by the induction of Ca2+ efflux and the following mitochondrial membrane permeabilization (MMP) process. And a more detailed time course study indicated that transformed nVO2 caused lysosomal membrane permeabilization (LMP) at the earlier stage, indicating LMP could be chosen as an earlier and sensitive end point for nanotoxicological study. We conclude that although nVO2 displays no acute toxicity, its acidic transformation product induces macrophage apoptosis by the induction of LMP and Ca2+ efflux. This report suggests that the interplay with environmental factors or living organisms can results in physicochemical transformation of nanomaterials and the ensuing distinctive biological effects.

Ca(2+) /calmodulin-dependent protein kinase II equally induces sarcoplasmic reticulum Ca(2+) leak in human ischaemic and dilated cardiomyopathy.

The sarcoplasmic reticulum (SR) Ca(2+) leak is an important pathomechanism in heart failure (HF). It has been suggested that Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is only relevant for the induction of the SR Ca(2+) leak in non-ischaemic but not in ischaemic HF. Therefore, we investigated CaMKII and its targets as well as the functional effects of CaMKII inhibition in human ischaemic cardiomyopathy (ICM, n = 37) and dilated cardiomyopathy (DCM, n = 40). Western blots showed a significantly increased expression (by 54 ± 9%) and autophosphorylation at Thr286 (by 129 ± 29%, P < 0.05 each) of CaMKII in HF compared with healthy myocardium. However, no significant difference could be detected in ICM compared with DCM as to the expression and autophosphorylation of CaMKII nor the phosphorylation of the target sites ryanodine receptor 2 (RyR2)-S2809, RyR2-S2815, and phospholamban-Thr17. Isolated human cardiomyocytes (CMs) of patients with DCM and ICM showed a similar frequency of diastolic Ca(2+) sparks (confocal microscopy) as well as of major arrhythmic events (Ca(2+) waves, spontaneous Ca(2+) transients). Despite a slightly smaller size of Ca(2+) sparks in DCM (P < 0.01), the calculated SR Ca(2+) leak [Ca(2+) spark frequecy (CaSpF) × amplitude × width × duration] did not differ between CMs of ICM vs. DCM. Importantly, CaMKII inhibition by autocamide-2-related inhibitory peptide (AIP, 1 µmol/L) reduced the SR Ca(2+) leak by ∼80% in both aetiologies (P < 0.05 each) and effectively decreased the ratio of arrhythmic cells (P < 0.05). Functional and molecular measures of the SR Ca(2+) leak are comparable in human ICM and DCM. CaMKII is equally responsible for the induction of the 'RyR2 leakiness' in both pathologies. Thus, CaMKII inhibition as a therapeutic measure may not be restricted to patients suffering from DCM but rather may be beneficial for the majority of HF patients.

Induction of Ca2+-dependent cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength.

In liver mitochondria loaded with Ca2+ or Sr(2+), α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca2+-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca2+ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca2+ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca2+ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca2+ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca2+ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca2+ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca2+ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.

CaV3.2 knockout mice display enhanced myogenic tone due to reduced BKCa‐mediated feedback (1077.3)

In resistance arteries, there is an emerging view that CaV3.2 channel restrains vascular constriction through a feedback response that involves ryanodine receptors (RyR), Ca2+ spark generation and the modulation of large‐conductance Ca2+ activated K+ channel (BKCa). In the present study, we tested this hypothesis by examining the vasomotor, electrophysiological and Ca2+ responses of mesenteric arterial smooth muscle isolated from wild type (WT) and CaV3.2 knockout (KO) mice. Using vessel myography, we first observed enhanced myogenic tone in KO arteries, a finding consistent with the loss of feedback response. Control myography and electrophysiological experiments subsequently confirmed the inability of Ni2+ (CaV3.2 blocker) to either constrict arteries or suppress T‐type currents in KO tissues. Line scan analysis, performed on opened arteries loaded with fluo‐4, illustrated the presence of Ca2+ sparks in all arteries with the subsequent application of Ni2+ only affecting WT arteries. Consistent with Ca2+ imaging, the frequency of BKCa‐induced spontaneous transient outward currents (STOC) dropped in WT but not KO cells upon the pharmacological suppression of CaV3.2 channel. Overall, our findings support the view that CaV3.2 channels drive a local Ca2+‐induced Ca2+ release‐like process that restrains arterial constriction through the induction of Ca2+ sparks and the activation of BKCa channels.

24R,25-Dihydroxyvitamin D3Promotes the Osteoblastic Differentiation of Human Mesenchymal Stem Cells

Although 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is considered the most biologically active vitamin D3 metabolite, the vitamin D3 prohormone, 25-hydroxyvitamin D3 [25(OH)D3], is metabolized into other forms, including 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. Herein we show that 24R,25(OH)2D3 is fundamental for osteoblastic differentiation of human mesenchymal stem cells (hMSCs). Our approach involved analyses of cell proliferation, alkaline phosphatase activity, and pro-osteogenic genes (collagen 1A1, osteocalcin, vitamin D receptor [VDR], vitamin D3-hydroxylating enzymes [cytochrome P450 hydroxylases: CYP2R1, CYP27A1, CYP27B1 and CYP24A1]) and assessment of Ca(2+) mineralization of extracellular matrix. 24R,25(OH)2D3 inhibited hMSC proliferation, decreased 1α-hydroxylase (CYP27B) expression, thereby reducing the ability of hMSCs to convert 25(OH)D3 to 1α,25(OH)2D3, and promoted osteoblastic differentiation through increased alkaline phosphatase activity and Ca(2+) mineralization. 24R,25(OH)2D3 decreased expression of the 1α,25(OH)2D3 receptor, VDR. 24R,25(OH)2D3 but not 1α,25(OH)2D3 induced Ca(2+) mineralization dependent on the absence of the glucocorticoid analog, dexamethasone. To elucidate the mechanism(s) for dexamethasone-independent 1α,25(OH)2D3 inhibition/24R,25(OH)2D3 induction of Ca(2+) mineralization, we demonstrated that 1α,25(OH)2D3 increased whereas 24R,25(OH)2D3 decreased reactive oxygen species (ROS) production. 25(OH)D3 also decreased ROS production, potentially by conversion to 24R,25(OH)2D3. Upon inhibition of the vitamin D3-metabolizing enzymes (cytochrome P450s), 25(OH)D3 increased ROS production, potentially due to its known (low) affinity for VDR. We hypothesize that vitamin D3 actions on osteoblastic differentiation involve a regulatory relationship between 24R,25(OH)2D3 and 1α,25(OH)2D3. These results implicate 24R,25(OH)2D3 as a key player during hMSC maturation and bone development and support the concept that 24R,25(OH)2D3 has a bioactive role in the vitamin D3 endocrine system.

Cross-talk between reactive oxygen species and polyamines in regulation of ion transport across the plasma membrane: implications for plant adaptive responses

Many stresses are associated with increased accumulation of reactive oxygen species (ROS) and polyamines (PAs). PAs act as ROS scavengers, but export of putrescine and/or PAs to the apoplast and their catabolization by amine oxidases gives rise to H2O2 and other ROS, including hydroxyl radicals ((•)OH). PA catabolization-based signalling in apoplast is implemented in plant development and programmed cell death and in plant responses to a variety of biotic and abiotic stresses. Central to ROS signalling is the induction of Ca(2+) influx across the plasma membrane. Different ion conductances may be activated, depending on ROS, plant species, and tissue. Both H2O2 and (•)OH can activate hyperpolarization-activated Ca(2+)-permeable channels. (•)OH is also able to activate both outward K(+) current and weakly voltage-dependent conductance (ROSIC), with a variable cation-to-anion selectivity and sensitive to a variety of cation and anion channel blockers. Unexpectedly, PAs potentiated (•)OH-induced K(+) efflux in vivo, as well as ROSIC in isolated protoplasts. This synergistic effect is restricted to the mature root zone and is more pronounced in salt-sensitive cultivars compared with salt-tolerant ones. ROS and PAs suppress the activity of some constitutively expressed K(+) and non-selective cation channels. In addition, both (•)OH and PAs activate plasma membrane Ca(2+)-ATPase and affect H(+) pumping. Overall, (•)OH and PAs may provoke a substantial remodelling of cation and anion conductance at the plasma membrane and affect Ca(2+) signalling.

A remarkable Activity of Steroid Biosynthesis in Captopril Preserved Leydig Cells of Mice Implicated Leukotriene B4 and Gonadotropin Releasing Hormones in vitro

In this study, investigated whether captopril inhibited steroidogenesis and components of the Leukotriene B4 pathways are involved in GnRH agonist (GnRH)-induced testis steroidogenesis in mice Leydig cells. Primary cultures of mice Leydig cells were established. Purified Leydig cells from adult albino mice were incubated with gradual various concentrations of GnRH with and without Captopril, Luteinizing hormone (LH); LTB4, steroidogenic (testosterone) activity and LTB4 concentration were measured after various time intervals and Leydig cell viability. The maneuvers of Leydig cells treated media was covered the singular and dual actions of antisteroidogenic of captopril and the reversible activity by GnRH-LTB4 as well as contribution of LTB4 in Leydig cells testosterone production endpoint. The different treatment media are Medium alone; Medium plus captopril 60 μM, 65 μM, 70 μM, 75 μM and 80 μM 100 μM; Medium plus 2.5mU/ml leukotriene B4; Medium plus 0.1 mM LH; Medium plus 0.1 μM GnRH; Medium plus 65 μM captopril plus 2.5 mU/ml leukotriene B4;Medium plus 65 μM captopril plus 0.1 mM LH; Medium plus 65 μM captopril plus 0.1 mM GnRH; Medium plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4 and Medium plus 65 μM captopril plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4. Basal testosterone levels were maximal at 0.1 μM GnRH concentration and superior testosterone yield in Leydig cells incubated 0.1 μM GnRH media than without GnRH media, and the activity profile LTB4 flow up. That comparable result led to highly correlated approved the contribution of LTB4 in GnRH stimulated Leydig cell steroidogenic end point. Furthermore captopril had an abolishment effect partially of testosterone yield and recovered and improved by GnRH and LTB4. The Leydig cells viability results suggest that the major effect of GnRH is probably beyond the LTB4. The entire key; GnRH induced testosterone production and upregulated LTB4 Levels at both the captopril inhibitory LTB4-testesteron Leydig cells culture media and captopril abolished LTB4 levels; it also activated endogenous LTB4, but not LH motivated testosterone pathway. Our data show that GnRH positively regulates steroidogenesis via LTB4 signaling in mice Leydig cells. LTB4 activation by GnRH may be responsible for the induction of Ca++ signaling indirect. Possibility improve the captopril steroidogenic disruption in Leydig cells via LTB4 and/or GnRH induction of endogenous LTB4, likewise the positive maintenance of Leydig cells viability matched induce testosterone synthesis. The LTB4 production, which may ultimately modulate steroidogenesis in mice Leydig cells, and promise new antidotal and preventative of captopril adverse effects.

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

ity, inhibited mitochondrial swelling induced by Ca 2+ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca 2+ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca 2+ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDAinduced highamplitude swell� ing of mitochondria by reducing free Ca 2+ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate cal� cium ions. Polycation spermine, which is known as an inhibitor of the classical CsAsensitive pore, at the physiological concentration of 1 mM inhibited CsAi nsensitive swelling of liver mitochondria induced by sequential addition of Ca 2+ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca 2+ �dependent pore, inhibited HDA� induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω�dioic acids weaker than their monocarboxylic analogues.

Carbonic anhydrase induction in euryhaline crustaceans is rate-limited at the post-transcriptional level

The transfer of euryhaline crustaceans from full-strength seawater to low salinity results in both a rapid up-regulation of carbonic anhydrase (CA; EC 4.2.1.1) mRNA and a slow induction of CA activity. There is a delay of several days between the two processes, which is attributed to the time required to synthesize new enzyme. These delays may also be due to limitations in the cellular uptake of Zn, which is a required post-translational active site modification to CA. To investigate these processes, the euryhaline crabs, Callinectes sapidus and Carcinus maenas, were acclimated to salinities below their isosmotic points (22.5 and 25ppt, respectively) for 7days to activate the physiological and molecular mechanisms of osmoregulation. CA mRNA increased 90-fold in C. sapidus and 2-fold in C. maenas within 6h; whereas it took 48h for the initial increases in CA activity (120% and 31%), and 4 to 7days for new acclimated levels (300% and 100%, respectively). Crabs were then transferred to lower salinities (10 and 15ppt) to induce further CA activity and to determine if previous increases in CA mRNA reduced the time required for subsequent CA induction. Additionally, the expression of the Zn transporter ZIP1 was examined in C. sapidus at 35 and 22.5ppt. In both species, prior CA mRNA elevation failed to accelerate the rate of CA induction. Levels of CA mRNA did not change in either crab following transfer from intermediate to low salinity. Taken together, these results show that the timecourse of CA induction at low salinity is not limited by the expression of CA mRNA, but by the synthesis of new enzyme from an existing pool of mRNA. No increases in ZIP1 expression occurred at low salinity, therefore these delays may be due to the limits of cellular Zn uptake.

Rhizobial infection does not require cortical expression of upstream common symbiosis genes responsible for the induction of Ca(2+) spiking.

For the establishment of an effective root nodule symbiosis, a coordinated regulation of the infection processes between the epidermis and cortex is required. However, it remains unclear whether the symbiotic genes identified so far are involved in epidermal and/or cortical infection, e.g. epidermal and cortical infection thread formation or cortical cell division. To analyze the symbiotic gene requirements of the infection process, we have developed an epidermis-specific expression system (pEpi expression system) and examined the symbiotic genes NFR1, NFR5, NUP85, NUP133, CASTOR, POLLUX, CCaMK, CYCLOPS, NSP1 and NSP2 for involvement in the infection process in the epidermis and cortex. Our study shows that expression of the upstream common symbiosis genes CASTOR, POLLUX, NUP85 and NUP133 in the epidermis is sufficient to induce formation of infection threads and cortical cell division, leading to the development of fully effective nodules. Our system also shows a requirement of CCaMK, CYCLOPS, NSP1 and NSP2 for the entire nodulation process, and the different contributions of NFR1 and NFR5 to cortical infection thread formation. Based on these analyses using the pEpi expression system, we propose a functional model of symbiotic genes for epidermal and cortical infection.

SERCA2a gene therapy in heart failure: an anti-arrhythmic positive inotrope.

Therapeutic options that directly enhance cardiomyocyte contractility in chronic heart failure (HF) therapy are currently limited and do not improve prognosis. In fact, most positive inotropic agents, such as β-adrenoreceptor agonists and PDE inhibitors, which have been assessed in HF patients, cause increased mortality as a result of arrhythmia and sudden cardiac death. Cardiac sarcoplasmic reticulum Ca(2)(+) -ATPase2a (SERCA2a) is a key protein involved in sequestration of Ca(2)(+) into the sarcoplasmic reticulum (SR) during diastole. There is a reduction of SERCA2a protein level and function in HF, which has been successfully targeted via viral transfection of the SERCA2a gene into cardiac tissue in vivo. This has enhanced cardiac contractility and reduced mortality in several preclinical models of HF. Theoretical concerns have been raised regarding the possibility of arrhythmogenic adverse effects of SERCA2a gene therapy due to enhanced SR Ca(2)(+) load and induction of SR Ca(2)(+) leak as a result. Contrary to these concerns, SERCA2a gene therapy in a wide variety of preclinical models, including acute ischaemia/reperfusion, chronic pressure overload and chronic myocardial infarction, has resulted in a reduction in ventricular arrhythmias. The potential mechanisms for this unexpected beneficial effect, as well as mechanisms of enhancement of cardiac contractile function, are reviewed in this article.

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Introduction: Near-death experiences (NDE), reported as extremely vivid imagery, have long fascinated scientists and theologians. Recent quantitative electroencephalogram (EEG) studies suggest that the brain transiently becomes highly active during the immediate post-cardiac arrest (CA) period despite raw EEG showing progressive suppression. Additionally, the occipital lobes, harboring the visual cortices, appear to become the most active during NDE. Methods: Fifteen adult Wistar Rats (Male, 300-350gms) were implanted with EEG leads (L/R frontal lobes and L/R occipital lobes). One week later, rats underwent 7-minute asphyxial CA while undergoing continuous EEG. EEG sub-band analysis was done using Information Quantity (IQ), a well-established entropy based nonlinear calculation of the statistical variability and information content of EEG. Results: During CA induction, we find that EEG IQ in the R occipital lobe is significantly resistant to suppression at the EEG alpha sub-bands (8-12Hz; slope of EEG decrement = -0.086 IQ/min ±0.0028 [Std Dev]) and slow gamma sub-bands (30-50Hz; -0.0085 ± 0.0027 IQ/min) in comparison to either L frontal lobe (-0.0203 ± 0.0040 [alpha], -0.0261 ± 0.0044 [slow gamma]), R frontal lobe (-0.0193 ± 0.0043 [alpha], -0.0236 ± 0.0054[slow gamma]) or the L occipital lobe (-0.0198 ± 0.0045 [alpha], -0.0227 ± 0.0055 [slow gamma]). All p-values<0.001. Conclusions: In a rodent model of asphyxial CA, these data show that the R occipital lobe has profound resistance to loss of EEG entropy, most robustly in the alpha and slow gamma EEG sub-bands. In humans, gamma range EEG and its coherence with slower bands are associated with waking consciousness and higher cortical processing. While the R occipital region’s role and connectivity with surrounding temporo-parietal regions is established in imagery, our data is the first to demonstrate a critical role for the R occipital lobe during death. Upon further validation in humans, these data may better elucidate mechanisms of NDE-related vivid imagery.

Predictability of Peripheral Lymphocyte Reduction of Novel S1P1 Agonists by In Vitro GPCR Signaling Profile

Surrogate readouts of G-protein–coupled receptor signaling pathways using highly engineered systems are often employed in the drug discovery process. However, accumulating data have demonstrated the importance of selecting relevant biological activity rather than technically facile assays to support high-throughout screening and subsequent structure-activity relationship studies. Here we report a case study using sphingosine-1-phosphate receptor 1 (S1P1) as the model system to compare compound activity in six different in vitro assays with their ability to predict in vivo efficacy. S1P1 has long been validated as a therapeutic target for autoimmune diseases. In this article, in vivo and in vitro studies on 19 S1P1 agonists are reported. In vitro activities of these S1P1 agonists, together with S1P and FTY720p, on Ca2+ mobilization, adenylyl cyclase inhibition, extracellular signal-related kinase (ERK) phosphorylation, β-arrestin recruitment, and receptor internalization, were determined. The in vitro potency of these compounds was correlated with their ability to induce peripheral lymphocyte reduction. The results revealed that inhibition of adenylyl cyclase and induction of β-arrestin recruitment and receptor internalization are good indicators to predict in vivo efficacy, whereas induction of Ca2+ mobilization through Gqi/5 coupling and ERK phosphorylation is irrelevant. This study demonstrated the importance of identifying an appropriate in vitro assay to predict in vivo activity based on the biological relevance in the drug discovery setting.

Effect of diazoxide on AS-30D rat ascites hepatoma cells treated by Cd2+

For the last years, in several investigations, including ours, it was shown that oxidative stress and mitochondrial dysfunction mediated by disturbance of the mitochondrial respiratory chain and by induction of Ca2+ -dependent nonselective high-conductance pore of the inner mitochondrial membrane are involved in mechanism(s) of cytotoxic action of heavy metals. At the same time, possible interaction of heavy metals with other channels, in particular, with selective potassium channels, such as ATP-dependent potassium channels, which are generally considered to be protective for the cells, have not been studied. The aim of the present work was to examine the effect of diazoxide, an activator of mitochondrial ATP-dependent potassium channels, on mitochondrial physiology and cell survival in the presence of Cd2+. As a model system, we used AS-30D rat ascites hepatoma cells and isolated rat liver mitochondria. We found that diazoxide enhanced intracellular production of reactive oxygen species and induced significant stimulation of the resting respiration rate of the cells. Besides, diazoxide had a protective effect on AS-30D cells by increasing their survival that was substantially decreased in the presence of the tested concentrations of Cd2+ (50 and 100 microM). The protective effect of diazoxide was completely suppressed by increasing duration of incubation of the cells with Cd2+, and partially by addition to the assay medium of 5-hydroxyde- canoic acid (100 or 300 microM), a blocker of mitochondrial ATP-dependent potassium channels. In isolated rat liver mitochondria we found that diazoxide did not prevent the toxic action of Cd2+, since it produced no significant effects on the mitochondrial swelling and the respiration changes evoked by the heavy metal in the KCl assay media. Possible molecular mechanisms of the cytoprotective action of diazoxide are discussed.