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TRPV channels are a category of nonselective cation channels that activated by heat and ligands, and permeate monovalent and divalent ions. The mechanism of Ca2+ transfer through TRPV2 channel is not well known. Here, we investigated the reaction coordination and energy fluctuation of Ca2+ transition in TRPV2 channel by steered molecular dynamics (SMD) simulations and potential of mean force (PMF) calculation. Results showed that electrostatic interactions between Ca2+ and residues of the first and second gates had main roles in ions transfer through the channel, and also, we recognized important amino acids in this path. Moreover, results indicated that enter and exit of calcium ions needed to overcome barrier energies in first and second gates.

The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+sensor R-CEPIA1er was used to measure the concentration of Ca2+in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+release channel ryanodine receptor (RyR2) created a Ca2+release/reuptake system that mimicked aspects of cardiac myocyte Ca2+handling. SERCA2a function was quantified from the rate of [Ca2+]ERrefilling after ER Ca2+depletion; then, ER Ca2+leak was measured after SERCA inhibition. ER Ca2+uptake and leak were analyzed as a function of [Ca2+]ERto determine maximum ER Ca2+uptake rate and maximum ER Ca2+load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function.NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+sensor R-CEPIA1er was used to successfully analyze Ca2+uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+pump regulation.

The processes of regulation of smooth muscle contractile function depend greatly on the efficiency of maintaining Са2+-homeostasis by systems of active ionic transport of the plasma membrane (calcium pump and Na+,Ca2+-exchanger) and intracellular stores (sarcoplasmic reticulum and mitochondria). In this work, the effect of calixarene C-716 (macrocyclic compound capable of blocking functioning of the plasma membrane calcium pump) on the kinetics of relaxing K+-induced contraction of rat myomet­rium smooth muscles, while inhibiting the systems of active transport of calcium ions of intracellular Са2+-stores, were investigated. It was established that the application of 100 M calixarene at the maximum of the phase component of the K+-induced contractile response did not change the kinetic parameters of the relaxation process (the normalized maximal velocity of relaxation phase Vnr and the half-maximal time of relaxation τ1/2). The tonic component of K+-induced contraction remained without statistically significant changes. Blocking of Са2+-accumulating function of mitochondria by the protonophore СССР caused a significant increase in both the parameter Vnr and the tonic component of K+-induced contraction, while the τ1/2 index remained at the control level. Blocking calcium pump of sarcoplasmatic reticulum with thapsigargin (TG) caused a conside­rable increase in the τ1/2 ,Vnr parameters and the tonic component of the K+-induced contraction. Combination of TG and СССР induce further increase in the Vnr parameter and the tonic component of K+-induced contraction. The action of calixarene С-716 (100 M) at the background of the impact of TG and СССР was accompanied by further increase in the tonic contraction, Vnr remained at the increased level, and the τ1/2 index decreased. Possible mechanisms for changing the mechanokinetic parameters of relaxing K+-induced contraction of myometrium is discussed. Analysis of the results of our experimental data and the data of other researchers allow predicting that an increase in the rate of relaxation of myometrium smooth muscles at the action of calixarene C-716 under the conditions of blockage of intracellular Ca2+ depots is associated with a significant activation of NO synthesis by constitutive forms of NO-synthases, whereas an increase in the tonic component of K+-contraction under these conditions is probably due to an elevated concentration of Ca2+ ions in the myocytes.

Much contemporary evidence underscores the pathophysiological importance of Ca2+ handling by acidic organelles such as lysosomes. Whereas our knowledge of how Ca2+ is released from these acidic Ca2+ stores (the ‘outs’) is advancing, we know relatively little about how Ca2+ uptake is effected (the ‘ins’). Here I highlight new work identifying animal Ca2+/H+ (CAX) exchangers that localize to acidic organelles, mediate Ca2+ uptake and regulate cell migration in vivo. Continued molecular definition of the acidic Ca2+ store toolkit provides new insight into Ca2+-dependent function.

Increasing evidence implicates Ca(2+) in the control of cell migration. However, the underlying mechanisms are incompletely understood. Acidic Ca(2+) stores are fast emerging as signaling centers. But how Ca(2+) is taken up by these organelles in metazoans and the physiological relevance for migration is unclear. Here, we identify a vertebrate Ca(2+)/H(+)exchanger (CAX) as part of a widespread family of homologues in animals. CAX is expressed in neural crest cells and required for their migration in vivo. It localizes to acidic organelles, tempers evoked Ca(2+) signals, and regulates cell-matrix adhesion during migration. Our data provide new molecular insight into how Ca(2+) is handled by acidic organelles and link this to migration, thereby underscoring the role of noncanonical Ca(2+) stores in the control of Ca(2+)-dependent function.

Literature data on the role of physiological prion in Ca2+-homeostasis regulation, violation promoting prion-induced neurodegenerations, are summarized. The electrophysiological neuronal disorders and synaptic dysfunction which occur during prion di­seases and are accompanied by changes in intracellular ionized Ca2+ concentration, are described. Short description of Ca2+-transport systems in neurons is presented. Special attention is paid to data obtained in animal models of prion infections and PrP-knocked animals, indicating possible role of PrPc in the process of Ca2+-transport in neurons. In particular, a PrPc mediated participation in functioning of calcium channels due to binding with NR2D-subunit of N-methyl-D-aspartate (NMDA)-receptors of Ca2+-channels, which provides its neuroprotective role, is described. Also, a PrPc participation in the synaptic structure formation and neurotransmission is characterized. The interaction of physiological prion with synapsin-1 and synaptophysin that regulates exo-endoendocytosis synaptic vesicles cycle, was studied. The violation of Ca2+-homeostasis as one of important factors during prion pathologies is characterised.

This teaching article uses the report by Kwan et al., “Effects of methacholine, thapsigargin, and La3+on plasmalemmal and intracellular Ca2+transport in lacrimal acinar cells,” where the effects of Ca2+-mobilizing agents in regulating Ca2+fluxes were examined under various conditions. Upper-level undergraduate and new graduate students in physiology are the targe audience. Teaching and learning points are put forth in this article to illustrate 1) the characteristics of methacholine- and thapsigargin-induced Ca2+responses, 2) the different endoplasmic reticulum Ca2+stores accessible to methacholine and thapsigargin, 3) the inhibitory effects of La3+on Ca2+extrusion and Ca2+influx, and 4) the facilitatory role of La3+on endoplasmic reticulum Ca2+recycling. Each of the above concepts is first explained with references to the figures adapted from the original article. A list of student learning questions then follows, where the answers are found in the teaching notes for the instructors. It is the objective of this article to make both teaching and learning Ca2+regulation a rewarding experience for all.

Ca2+ is a ubiquitous cellular signal. Altered expression of specific Ca2+ channels and pumps are characterizing features of some cancers. The ability of Ca2+ to regulate both cell death and proliferation, combined with the potential for pharmacological modulation, offers the opportunity for a set of new drug targets in cancer. However, the ubiquity of the Ca2+ signal is often mistakenly presumed to thwart the specific therapeutic targeting of proteins that transport Ca2+. This Review presents evidence to the contrary and addresses the question: which Ca2+ channels and pumps should be targeted?

Calix[4]arenes, functionalized at the wide rim with two or four N 2 -sulfonylamidine groups were prepared. In the crystalline state, the bowl shaped calix[4]arene-bis- N -sulfonyltrifluoromethylacetamidine 3b is associated through intermolecular hydrogen bonds, NH⋯O S, while the phenyl rings of the Ph–SO 2 -fragments are hosted in the cavities of the nearby molecules of 3b . Calixarene 4b influences Ca 2+ transport in Mg 2+ , ATP-dependent calcium pumps.

It is thought that a plasma membrane Ca 2+ -transport ATPase (PMCA) and a Na + /Ca 2+ -exchange (NCE) mechanism are involved in epithelial Ca 2+ transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca 2+ -extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens . Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca 2+ -transporting condition to the stages of CaCO 3 deposit formation and degradation. During Ca 2+ -transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.

Physiological studies have established the role of plant high-capacity vacuolar H + /Ca 2+ exchange activity in ion homeostasis and signal transduction. The molecular characterization and structure–function analyses of these transporters are just beginning to emerge. In yeast, Ca 2+ signaling molecules regulate vacuolar H + /Ca 2+ exchange. Recently, some of the Ca 2+ dependent ‘molecular relay' molecules have been characterized in plants; however, the regulation of plant vacuolar H + /Ca 2+ exchange remains an open question.

To investigate the hypothesis that prolonged partial ischemia would result in a depression in homogenate sarcoplasmic reticulum (SR) Ca2+-sequestering and mechanical properties in muscle, a cuff was placed around the hindlimb of 8 adult Sprague–Dawley rats (267 ± 5.8 g; × ± S.E.) and partially inflated (315 mm Hg) for 2 h. Following occlusion, the EDL was sampled both from the ischemic (I) and contralateral control (C) leg and SR properties compared with the EDL muscles extracted from rats (n = 8) immediately following anaesthetization (CC). Ischemia was indicated by a lower (p < 0.05) concentration (mmol.kg dry wt–1) of ATP (19.0 ± 0.7 vs. 16.7 ± 0.7) and phosphocreatine (58.1 ± 5.7 vs. 35.0 ± 4.6) in I compared to C. Although Ca2+-ATPase activity (μmol·g protein–1.sec–1 ), both maximal and submaximal, was not different between C and I (19.7 ± 0.4 vs. 18.5 ± 1.3), reductions (p < 0.05) in Ca2+-uptake (mmol·g protein–1.sec–1 ) of between 18.2 and 24.7% across a range of submaximal free Ca2+-levels were observed in I compared to C. Lower submaximal Ca2+-ATPase activity and Ca2+-uptake were also observed in the EDL in C compared to CC animals. Time dependent reductions (p < 0.05) were found in peak twitch and maximal tetanic tension in EDL from I but not C. It is concluded that partial ischemia, resulting in modest reductions in energy state in EDL, induces a reduction in Ca2+-uptake independent of changes in Ca2+-ATPase activity. These changes reduce the coupling ratio and the efficiency of Ca2+-transport by SR.

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100–130% when membrane vesicles are incubated (15–20 s at 37 °C) with 1 mm MgATP and 1 µm Ca2+ (b) in the presence of 100 µm Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 µm Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.

α-Crystallin, an endogenous low-molecular-weight protein with chaperone activity, exerted protective effects on membrane systems of Ca2+ transport into the sarcoplasmic reticulum of skeletal muscles. Protective action of α-crystallin depended on the body state. This effect was not observed in the control and after adaptation to stress, while after stress, especially against the background of adaptation, α-crystallin increased the rate of Ca2+ transport into the sarcoplasmic reticulum and thermal resistance of Ca2+ pump. The mechanisms of α-crystallin activation during stress are discussed.

This study evaluated whether annexins I, II and VI possess Ca2+ transport activity in phospholipid membranes by the burst method, and the activity of each was compared with that of annexin V. Briefly, in the presence of 400 microM Ca2+, each annexin at 50 nM was added to large unilamellar vesicles (LUV) which were then burst in fura-2 solution with 0.2% Triton X-100, followed by examination of Ca2+ signals. Annexins I, II, V and VI were all shown to express, each to a different degree, Ca2+ activity toward phosphatidylserine/phosphatidyl- ethanolamine-LUV. Ca2+ signal intensity increased dependent on annexin concentration, and the Ca2+ transport activity of annexin V and VI was higher than that of annexin I and II. However, none of annexin I, II, V and VI expressed Ca2+ transport activity in LUV produced using phosphatidylcholine. Ca(2+)-incorporated LUV with no annexin showed signals whose intensity was proportional to Ca2+ concentration. The Ca2+ transport activity of the annexins could be effectively measured by the burst method. Ca2+ signal intensity would thus appear to be unique for each of the annexins and to be determined by the particular function and specificity of each of those considered in this study.

Frog sympathetic ganglion neurons exhibit a novel Ca2+ uptake mechanism, release-activated calcium transport or RACT, which is manifest in both cytosolic and store [Ca2+] signals as greatly accelerated Ca2+ uptake after Ca2+ release from internal stores. RACT is activated by Ca2+ release but not by Ca2+ entry and serves to selectively refill Ca2+ stores after release. RACT lowers cytosolic [Ca2+] with a rate constant about 1.6 times that of the SERCA pump with empty ER. RACT is thapsigargin-insensitive, was eliminated by ryanodine, but was not affected by blocking mitochondrial or plasma membrane Ca2+ transport. A Ca2+ flux model with RACT in the ER membrane reproduced the cytosolic and store [Ca2+] responses to all stimuli.

Anthropogenic carbon dioxide (CO2) emissions from fossil carbon storages to atmosphere are causing major impacts on the Earth s climate. The increase of atmospheric temperatures due to a strengthening of the natural greenhouse effect is a well known phenomenon. Moreover, a large fraction of the atmospheric CO2 that has been released by mankind enters the ocean and induces, besides other effects, the acidification of surface waters ( Ocean Acidification ). The effects of increasing partial pressures of atmospheric CO2 on parameters that describe the abiotic environment have been examined extensively. It is now of particular interest to gain a detailed comprehension of the complex interactions between the changing environment and the metabolism of marine organisms. This knowledge is essential when aiming to understand the ongoing changes in their entirety. In the spotlight of this thesis are coccolithophores, unicellular algae that significantly impact the marine carbon cycle. Coccolithophores perform photosynthesis and calcification. Both processes rely on different carbon species and thus have a different effect on the carbonate system of the surface ocean. In connection with the formation of calcite particles, they further impact the sinking rates of particulate carbon from the photic zone into the ocean. The ratio of photosynthesis to calcite precipitation rate varies with the composition of the carbonate system. In order to understand the dependencies of both individual processes on the complex cabonate system, a detailed understanding of cellular carbon fluxes is essential. Until now, it is impossible to measure these fluxes directly. Therefore, mathematical models are used in this thesis to examine these fluxes. All presented models describe Emiliania huxleyi, one of the globally most abundant and important coccolithophores. The first part of this PhD thesis summarises current knowledge concerning the intracellular formation of coccoliths, i.e. the calcite platelets that surround the cells. Former propositions about the provision of carbon and calcium ions (Ca2 ) towards the site of calcite precipitation are reviewed and discussed. Furthermore, abiotic prerequisites on initiation and continuation of calcite precipitation, as well as the biological control of precipitation onset and crystal growth are of particular interest. Gaps of knowledge are discussed. In the second part, different hypotheses concerning the pathway of Ca2 are discussed on basis of analytical calculations. It turns out that a continuous trans- port across the membrane of the compartment, in which calcite precipitation takes place, i.e. the coccolith vesicle, is more likely than is a vesicle-based transport. The latter possibility constitutes the hypothesis currently favoured in literature. A transport via molecules that exhibit high Ca2 binding capacities, as found in other coccolithophores, is thought to be unlikely for E. huxleyi, because there is currently no evidence hinting at comparable molecules. Based on the assumption that Ca2 is transported continuously into the coccolith vesicle, a numerical model is developed, which examines the influence of different membrane transporters on the precipitation rate of calcite. These transporters convey varying substrate stoichiometries and are inserted into the in silico membrane of the coccolith vesicle. The influence of these transporters on the calcite precipitation rate is examined. Since the precipitation rate is known from literature, it is possible to exclude some of the tested transport stoichiometries. Two potential substrate stoichiometries are detected that serve as basic assumptions in the next step of modelling calcite precipitation. In the third part, different numeric models are developed that examine carbon fluxes through cytosol, coccolith vesicle, and chloroplast. Based on current knowledge of external carbon sources for organic matter production and calcite precipitation, it is concluded that both processes are supplied independently of each other with carbon under replete conditions. When external CO2 becomes limiting, how- ever, external bicarbonate ions that usually constitute the substrate for calcite precipitation are used by photosynthesis also. Moreover, an energy-efficient possibility is proposed how CO2 could be accumulated around Ribulose-1,5-bisphosphat (RubisCO), the CO2 fixing enzyme, without being transported against concentration gradients. The general discussion summarises the findings of this thesis and picks up gaps that were not discussed in detail previously. Moreover, measurements and models are proposed that might fill gaps of knowledge. Finally, implications of this work on physiological, ecological, and biogeochemical aspects of coccolithophore research are given.

The relationship between the cold-induced increase in sarcoplasmic reticulum (SR) Ca2+ transport proteins and the development of muscular nonshivering theermogenesis (NST) was investigated by determining the time course of expression of the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA), Ca2+ release channel, and calsequestrin in control and cold-acclimating ducklings. 45Ca2+ uptake and [3H]ryanodine binding measurements with skeletal muscle homogenates showed that a cold acclimation period of approximately 4 wk was required to observe a substantial increase in Ca(2+)-ATPase activity and Ca2+ release channel content, which correlates well with NST development Immunoblot analysis of muscle homogenates showed no differences in calsequestrin levels but revealed that the decrease in SERCA2a content was delayed in cold-acclimating birds and that the SERCA1 level was increased after 4 wk of cold acclimation. The persistence of SERCA2a may be related to shivering thermogenesis occurring preferentially in slow-twitch fibers. SERCA1 may account for most of the cold-induced increase in 45Ca2+ uptake activity, suggesting the preferential occurrence of a Ca(2+)-dependent NST in fast-twitch fibers.

The relationship between the level of accumulation of lipid peroxidation products and the status of the Ca2+-transporting system in the sarcoplasmic reticulum of the rat myocardium is studied against the background of two cardioprotective factors, namely adaptation to periodic hypoxia and a diet enriched in polyunsaturated fatty acids of the ω-3 class. It is shown that the diet leads to an increase of level of lipid peroxidation products by 1.8 times in the heart and by 19 times in the liver, whereas a adaptation has no effect on the level of lipid peroxidation products in either of these organs. At the same time, the combined action of both factors considerably enhances the resistance of the myocardial Ca2+-transporting system to free radical-induced oxidation. Inin vitro experiments it is shown that adaptation to periodic hypoxia results in a more than twofold deceleration of Ca2+ transport inhibition during the oxidation induction by the Fe2+/ascorbate system; the diet causes a 3.5-fold deceleration of such inhibition. The results show that the accumulation of a high level of lipid peroxidation products is not always followed by damage to the Ca2+-transporting system in the myocardial sarcoplasmic reticulum.

The effect of citrinin on Ca2+ transport was studied in isolated kidney cortex and liver mitochondria, and baby hamster kidney cultured cells. The mycotoxin significantly inhibited the activity of 2-oxoglutarate and pyruvate dehydrogenases in both kidney cortex and liver mitochondria. Citrinin promoted a decrease in the velocity and in the total capacity of Ca2+ uptake, in both mitochondria. Apparently, citrinin acts by a mechanism similar to ruthenium red. In intact cultured cells, citrinin also had a preferential effect on mitochondrial Ca2+ fluxes. Citrinin promoted a marked decrease in the Ca2+ level in the mitochondrial matrix, whereas that of the extramitochondiral fraction became less affected. All the observed effects were dependent on the citrinin concentration.

The rate of Ca2+ accumulation in the myocardial sarcoplasmic reticulum is studied in experiments on rats under conditions of hind paw functional off-loading by suspending animals by the tail for 1 to 40 days, as well as during a 2-week period of readaptation after a 40-day load relief. The rate of Ca2+ transport in the myocardial sarcoplasmic reticulum reliably drops (by 33%) after 40 days of suspension. At earlier stages of off-loading Ca-pump activity in the sarcoplasmic reticulum does not change reliably. After resumption of the loads in animals suspended for 40 days, the transporting function of the myocardial sarcoplasmic reticulum rapidly reverts to the control level, which is indicative of a reversible pattern of load relief-induced changes in the rate of Ca2+ transport.

Effects of prednisolone, estradiol, and testosterone on the transport of Ca2+ and the respiration induced by it in the heart and liver mitochondria of rats were studied. Prednisolone and testosterone were found to reduce the Ca-accumulating capacity of the mitochondria, the rates of ion entry and exit, and the rate of Ca2+-induced respiration. Estradiol, while inhibiting Ca2+ transport across mitochondrial membrane, did not influence the respiration in the phase of Ca2+ absorption, but accelerated it in the phase of ion exit. These data suggest that due to their lipophilic properties, the steroids become incorporated in the mitochondrial membrane, thereby changing its viscosity and permeability and limiting the mobility of transmitter proteins.

The rate of Ca2+ accumulation in the sarcoplasmic reticulum of the skeletal muscle (m. gastrocnemius lateralis, m. vastus medialis, andm. soleus) is studied in rats under conditions of functional off-loading of the hind paws (suspending animals by the tail). The rate of Ca2+ transport in the sarcoplasmic reticulum is shown to be stepped up in all these muscles. In the sarcoplasmic reticulum ofm. gastrocnemius lateralis andm. vastus medialis the Ca2+ transport rate reliably drops, which does not occur inm. soleus. During a 2-week period of readaptation of animals suspended for 40 days, the Ca2+-transporting function of them. soleus sarcoplasmic reticulum gradually recovers to reach the control values, whereas the time course of recovery of Ca2+-pump activity in the sarcoplasmic reticulum ofm. gastrocnemius lateralis andm. vastus medialis has a phasic pattern.

In this work, we compared the effect of K+ on vesicles derived from the longitudinal (LSR) and terminal cisternae (HSR) of rabbit white muscle. In HSR, K+ was found to inhibit both the Ca2+ accumulation and the heat released during ATP hydrolysis by the Ca2+-ATPase (SERCA1). This was not observed in LSR. Valinomycin abolished the HSR Ca2+-uptake inhibition promoted by physiological K+ concentrations, but it did not modify the thermogenic activity of the Ca2+ pump. The results with HSR are difficult to interpret, assuming that a single K+ is binding to either the ryanodine channel or to the Ca2+-ATPase. It is suggested that an increase of K+ in the assay medium alters the interactions among the various proteins found in HSR, thus modifying the properties of both the ryanodine channel and SERCA1.

Article de synthese sur les relations entre la structure moleculaire de l'ATPase et son fonctionnement dans le transport du calcium Ca ++ , chez les mammiferes. Synthese sur la cinetique du transport, sur la liaison et sur la translocation du Ca ++ par l'ATPase. Etude spectroscopique de la structure de l'ATPase, modification de cette structure par mutagenese et hypothese d'un mecanisme d'action moleculaire

It is known that stress applied to pigs can be of influence on meat quality combined with disturbed regulation of the intracellular Ca 2+ concentration. However, the effect on meat tenderness is not sufficiently known. In an experiment with pigs two kinds of stress (immobilisation by a nose snare (nose) and the use of an electrical goad (goad)) for 5 min just before slaughter were used and the results compared with minimum stress pigs (control). To quantify the level of stress, the heart rate during stress application was determined and it was found that the stress effect was highest in the goad pigs and surprisingly decreasing during the use of a nose snare. The meat quality did not differ between nose and control pigs, but was inferior in the goad group. Also, the goad stress significantly reduced the Ca 2+ transport of the sarcoplasmic reticulum. The principal hypothesis, that a disturbed Ca 2+ transport will affect the tenderness by activating the calpain system, could not be verified. Tenderness did not differ between experimental groups using 24 h post-mortem samples, neither was there a difference in tenderness after storing the meat samples for 6 days.

Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase.

Objective : To investigate the change of myocardial mitochondrial Ca 2+ transport and its mechanism in the early stage after burns. Methods: Forty-eight Wistar rats were randomized into a normal control group ( n =8) and a burns group ( n =40). The rats of the burns group were given a 30%TBSA full-thickness. Myocardial mitochondria were isolated from normal and scalded rats which were sacrificed at the 1st, 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca 2+ transport velocity, membrane potential (MP), ATP content and cytosolic Ca 2+ concentration [Ca 2+ ] c were determined. The effects of exogenous ATP on mitochondrial Ca 2+ transport velocity were also investigated. Results : Mitochondrial Ca 2+ uptake velocity of the 1st hour post-burn was higher than that of the control, and Ca 2+ release velocity did not change significantly, but mitochondrial Ca 2+ transport velocity, MP and ATP content were all decreased at the 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca 2+ uptake velocity was positively correlated with MP after burn, and Ca 2+ release velocity with mitochondrial ATP content. [Ca 2+ ] c was increased at the 3rd, 6th, 12th and 24th hour post-burn. Exogenous ATP increased myocardial mitochondrial Ca 2+ uptake velocity of rats at the 3rd and 6th hour post-burn and Ca 2+ release velocity at the 3rd, 6th and 12th hour post-burn. Conclusions : Increase of [Ca 2+ ] c led to reinforcement of mitochondrial Ca 2+ uptake at the beginning of the post-burns period. ATP depletion and MP collapse cause myocardial mitochondrial Ca 2+ transport disorder in the following stages.

Ca2+ permeability mediated by voltage-dependent anion-selective channel protein 1 (VDAC1) can be tested by reconstitution of purified VDAC1 into liposomes. Here, we describe a setup for this membranal system, which has been used to study the transport activity of various transporters, including VDAC1, and allows detection of the passage of molecules across the lipid bilayer. Despite the disadvantage of needing radiolabeled molecules, this system is highly desirable when the transport properties of noncharged molecules and/or active transporters are studied.

Stress before slaughter affects the meat quality of pigs and a disorder in the Ca(2+) transport of the sarcoplasmic reticulum (SR) results in inferior meat quality. The object of this work was to determine the impact of stress on SR Ca(2+) transport in pig muscle in animals with no mutation in the Ca(2+) release channel. At about 80 kg live weight, pigs were stressed either by running for 4 min (n=9) or by a 5 min application of a nose snare (n=7). Immediately before and after the application of the stress, as well as 4 h after the stress, biopsy samples of the longissimus muscle were taken and the Ca(2+) uptake of the homogenised muscle samples determined. The nose snare stress reduced the rate of Ca(2+) uptake by 9% (P=0.02) and running by 18% (P=0.06). Uptake in samples taken 4 h after the stress was at initial levels. The pigs were slaughtered at an average live weight of 109 kg. Half the pigs were stressed just before slaughter for 5 min by the use of a nose snare. Immediately after exsanguination longissimus muscle samples were taken, Ca(2+) uptake measured, and later standard meat quality parameters were determined. Although the stress reduced the Ca(2+) uptake rate (61.3 vs. 46.4 nM/min per mg protein for control and stressed pigs; P=0.04), the meat quality was not significantly affected.

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40showed bands corresponding, respectively, to full-length RyR-1 (∼500 kDa) and IP3R-1 (∼250 kDa). Band intensity was reduced by just ∼20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+via Ca2+-ATPase activation (1 mM ATP and ∼100 nM Ca2+). Adequate Ca2+loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3or cADP ribose resulted in a rapid and sustained [Ca2+]npelevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+pathway.

Oxidative stress as a potential mechanism of Ca2+ disregulation in skeletal and cardiac muscles

pH-dependent Ca2+ and Na+ transport via Na-Ca+K exchange was measured in isolated and purified bovine retinal rod outer segments (ROS). Ca2+ transport was measured (1) as 45Ca uptake via reverse Na-Ca+K exchange or via Ca-Ca exchange and (2) as a Ca(2+)-induced rise in cytosolic free Ca2+ concentration via reverse Na-Ca+K exchange measured with the fluorescent Ca(2+)-indicating dye fluo-3. Na+ transport was inferred from a Na(+)-induced lowering of cytosolic free [Ca2+] measured with fluo-3. Both Na+ and Ca2+ influx into ROS mediated by the Na-Ca+K exchanger showed a steep (40-100-fold increase in influx rate when the pH was increased from 6.0 to 8.8) pH dependency when measured at low occupancy of the external transport site with Na+ or Ca2+, respectively. High occupancy of the transport site with either Na+ or Mg2+ caused a dramatic decrease in the pH dependence of either Ca2+ or Na+ influx. These observations corroborate the proposed presence of a common Na+ and Ca2+ transport site of the ROS Na-Ca+K exchanger.