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This study investigated the acute toxicity of cadmium (Cd) to the freshwater mussel Anodonta woodiana. The freshwater mussels were exposed to five concentrations of Cd (0 mg/L, 8.43 mg/L, 16.86 mg/L, 33.72 mg/L, and 67.45 mg/L) for up to 96 h. The 24-h, 48-h, 72-h, and 96-h LC50 values for Cd were estimated as 562.3 mg/L, 331.1 mg/L, 182.0 mg/L, and 134.9 mg/L, respectively. Caspase-3, caspase-8, caspase-9, and Ca-ATPase activities; protein and H2O2 levels; DNA fragmentation; and ultrastructure of the gill were also investigated. The activities of caspase-3 and caspase-9 in mussels were increased by Cd in a dose-dependent manner, where higher doses of Cd (33.72 mg/L and 67.45 mg/L) significantly increased the enzyme activities compared to the controls (P < 0.05). The caspase-8 activity was significantly depressed by a low dose of Cd (8.43 mg/L) but was clearly induced by higher doses of Cd (16.86 mg/L, 33.72 mg/L, and 67.45 mg/L) (P < 0.05). The Ca-ATPase activity and H2O2 levels were elevated and reached maximum values under the medium dose of Cd (16.86 mg/L). However, protein levels were decreased by Cd in an inverse dose–dependent manner. In the gills of the mussels, Cd treatment induced DNA fragmentation as demonstrated by DNA ladders observed via agarose gel electrophoresis. Moreover, ultrastructural alterations in gill cells of mussels treated with Cd (16.86 mg/L and 67.45 mg/L) for 96 h were observed by electronic microscopy. The ultrastructure abnormalities were characterized by the following features: (1) a disordered arrangement and breaking off of microvilli of epithelial cells; (2) chromatin condensed near the nuclear membrane and the appearances of extremely irregular nuclei, some with a fingerlike shape and an unclear, swollen, invaginated, or ruptured nuclear membrane and apoptotic bodies; (3) swollen and vacuolating mitochondria, some with disintegrated or missing cristae; (4) a disintegrated rough endoplasmic reticulum containing different sizes of vesicles; and (5) shrinking and deformation of Golgi bodies with decreased vesicle numbers. Our results demonstrated that Cd could activate caspase-3, caspase-8, caspase-9, and Ca-ATPase; cause ultrastructural changes; and produce DNA fragmentation in the mussels investigated. Based on the information obtained through this study, it is reasonable to conclude that Cd can induce apoptosis in the gills of the mussels, eventually leading to tissue damage. [ABSTRACT FROM AUTHOR]

Given that the cadmium (Cd) toxicity could be due to its interference with the calcium (Ca) homeostasis, the aim of this work was to study the effect of Cd over the presence, distribution and volume density (Vv) of Ca and Ca-ATPase in the secretory cells of the pars preconvoluta (PPC) and the pars convoluta (pc) in Rhinella arenarum. The severe effect of the xenobiotic (CdCl2 2.5 mg/kg) in sexually matured females was evaluated. Co-localization, as well as a marked reduction of Ca and Ca-ATPase, was observed in treated animals, in the areas analyzed, compared to control. Low calcium deposits were found in the secreting granules (SG) of the epithelial (ESC) and glandular secretory cells (GSC), while an increase in their cytoplasm and intracellular space was observed. The Ca-ATPase in treated and control animals was detected at the SG and the plasmatic membrane of the ESC and GSC. In relation to the Vv estimates, a substantial reduction of Ca deposits and Ca-ATPase activity was observed in the treated group, with respect to the control. Both amounts of Vv of Ca and Ca-ATPase activity were higher in PPC than in pc, and, higher in ESC than in GSC. These results were associated with the Cd concentration in the oviductal PC, determining that it is a bioaccumulator organ. Thus, this work demonstrated that the Cd interacted with Ca-ATPase, leading to an increase of cytosolic Ca, which is responsible for the possible disruptions in cellular metabolism. [ABSTRACT FROM AUTHOR]

Highlights • 76% of the interactions between reduced seawater pH and increasing copper concentrations were antagonistic. • The interaction of stressors did not result in deleterious effects on endosymbiont´s photosynthetic metabolism or Ca-ATPase activity. • Low copper concentrations had a consistent positive effect on Ca-ATPase activity in corals facing reduced seawater pH conditions. • Potential deleterious effects on the acid-base balance of corals were intensified by the combination of stressors. • Toxic effects of copper in future ocean acidification scenarios can be less severe than previously suggested. Abstract Coral reefs are threatened by global and local impacts, such as ocean acidification (OA) and metal contamination. Toxicity of metals, such as copper (Cu), is expected to be enhanced with OA. However, the interaction between these environmental stressors is still poorly evaluated. In the present study, the interactive effects of seawater acidification and increasing Cu concentrations were evaluated in a zooxanthellate scleractinian coral (Mussismilia harttii), using biochemical biomarkers involved in the coral calcification process and the photosynthetic metabolism of endosymbionts. Corals were kept under control conditions (no seawater acidification and no Cu addition in seawater) or exposed to combined treatments of reduced seawater pH (8.1, 7.8, 7.5 and 7.2) and environmentally relevant concentrations of dissolved Cu (measured: 1.0, 1.6, 2.3 and 3.2 μg/L) in a mesocosm system. After 15- and 35-days exposure, corals were analyzed for photochemical efficiency (F v/ F m), chlorophyll a content, Ca-ATPase and carbonic anhydrase (CA) activity. Results showed that 76% of the interactions between reduced seawater pH and increasing Cu concentrations were antagonistic. Only 24% of these interactions were additive or synergistic. In general, the combination of stressors had no significant deleterious effects in the photosynthetic metabolism of endosymbionts or Ca-ATPase activity. In fact, the lowest dissolved Cu concentration tested had a consistent positive effect on Ca-ATPase activity in corals facing any of the reduced seawater pH conditions tested. In turn, potentially deleterious effects on acid-base balance in M. harttii , associated with changes in CA activity, were intensified by the combination of stressors. Findings reported here indicate that Cu toxicity in future OA scenarios can be less severe than previously suggested in this coral holobiont. [ABSTRACT FROM AUTHOR]

To date, it has been established that the symbiosome membrane (SM), i.e., plant-derived membrane of symbiosomes, nitrogen-fixing compartments of legume root nodules, is equipped with Ca-ATPase transporting Ca ions through the SM from the cytosol of infected cells into the symbiosome space (SS). Earlier in the experiments on the SM vesicles isolated from broad bean root nodules some data indicating the action of the Ca-ATPase as ATP-driven Ca/H antiporter were obtained. In the present work performed on isolated symbiosomes from the same plant object, further evidence in favor of calcium-proton countertransport mechanism of the pump operation was obtained. These were expressed in vanadate-sensitive alkalinization of the SS coupled with Ca uptake by symbiosomes catalyzed by the SM Ca-ATPase, stimulation of the kinetics of the latter process in the response to artificial acidification of the SS and expectable modulation of ITP-hydrolyzing activity of this enzyme caused by the variation of pH within this compartment. The above findings are discussed in the framework of the model describing the mechanism of Ca-ATPase operation as an ATP-driven Ca/H exchanger and on this base allow us to put forward the hypothesis about the involvement of this enzyme in symbiosome signaling in a Ca- and pH-dependent manner. [ABSTRACT FROM AUTHOR]

Selenium (Se) can play a protective role against heavy metal toxicity. This experiment aims to evaluate the effect of Se supplementation at different doses on the chicken brains. Oxidative stress was induced in the chicken brains by chromium(VI). A total of 105 Hyland brown male chickens were randomly divided into seven groups, including the control group, poisoned group [6%LD KCrO body weight (B.W.)], and detoxification groups KCrO (6%LD) + Se (0.31, 0.63, 1.25, 2.50, and 5.00 NaSeO mg/kg B.W.) orally in water for 42 days. The chickens were detected by the activities of mitochondrial membrane potential, 2′-benzoyloxycinnamaldehyde, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and Ca-ATPase. Cr(VI) administration caused histopathological damage. In addition, changes in oxidative stress indicators were observed in the chicken's brains. Se supplement increased the levels of GSH, mitochondrial membrane potential (MMP), and Ca-ATPase and reduced MDA activity in the detoxification groups. However, the high-dose Se supplementation groups of 2.50 and 5.00 mg/kg reduced the activities of GSH, MMP, and Ca-ATPase; increased the brain-body ratio; and increased SOD activity. In conclusion, Cr(VI) exposure caused oxidative stress. Se exerted a remission effect on toxic responses in the chicken brains. However, a high Se concentration was synergistic to the toxic effect of Cr(VI). [ABSTRACT FROM AUTHOR]

Various organic compounds are applied upon cryopreservation and their adding into cell suspension causes modification of subcellular systems, providing cell survival during freeze-thawing. The aim of the study was to assess the modifying effect of cryoprotectant PEG-1500 and low temperatures on Ca-ATPase activity in saponin-permeabilized erythrocytes. PEG-1500 was revealed to inhibit erythrocyte Ca-ATPase activity despite the presence of endogenous effectors able to stimulate the enzyme function. Presumably, the Ca-ATPase modification was determined by the physicochemical properties of the polymer solution, since the removal of PEG-1500 out of the medium recovered the enzyme activity. Reversibility of Ca-ATPase inhibition was characteristic of erythrocytes both exposed to cryoprotectant without freezing and frozen-thawed in the PEG-1500 presence. The cell freeze-thawing without cryoprotectant had no effect on Ca-ATPase, suggesting that membrane form of enzyme is cryoresistent. Although the efficiency of erythrocyte cryopreservation with PEG-1500 depends on the incubation temperature before freezing stage, the functional indices of Ca-ATPase in erythrocytes exposed to PEG-1500 at 37 and 5-7°C had no significant distinctions if the subsequent ATP hydrolysis was conducted at 37°C. However, the enzyme activity was additionally slowed down when the temperature of enzymatic reaction was decreased to 5-7°C after erythrocyte preincubation with PEG-1500 under the same conditions. The identified changes in Ca-ATPase activity in erythrocytes in the PEG-1500 presence were most likely determined by a modifying effect of the cryoprotectant on the membrane structure; as a result, the Ca-ATPase endogenous effectors present in the medium could not overcome the restrictions imposed on the enzyme function by a modified membrane macroenvironment. [ABSTRACT FROM AUTHOR]

The birds were developed to be 16 groups (4 groups of TT and 4 groups of RR, with male and female lines, respectively). The mating was conducted between similar groups of population (TT vs. TT, and RR vs. RR) to obtain second generation. The change in metabolism such as Ca–ATPase activity and Nτ–methylhistidine (Nτ–MH), also selection response between generations were analyzed. Parameters of metabolism were subjected to statistical analysis of T-test to compare between productions characteristic (TT and RR), especially for second generation. Body weight was also statistically analyzed by the same method. Selection response in TT group (3.10% or 2.74%) was higher than that in RR group (1.20% or 1.13%). Metabolism aspect in the quails of second generation either rate of protein turnover or activity of bone Ca-ATPase enzyme showed the change toward the productivity specification. Rate of muscle protein synthesis was higher and enzyme activity of Ca-ATPase was lower in group of TT population than those in group of RR population.

Using fluorescent microscopy, we found decreased expression of the β1 subunit of Na/K-ATPase and subunits of Ca-ATPase, increased expression of the α1S subunit of the L-type Ca-channel, and no changes in the expression of the α2 subunit of Na/K-ATPase in rat postural muscle under the conditions of modeled hypogravity. In the phasic muscle, we observed decreased expression of the β1 subunit, which was similar to that found in the postural muscle, whereas the other studied parameters remained without alterations. However, a decrease in the fluorescence intensity of the β1 subunit was insignificant due to a high variability of data. Thus, hypogravity negatively influenced primarily those skeletal muscles that are responsible for static load. [ABSTRACT FROM AUTHOR]

Using vesicles of symbiosome membrane (SM), it was shown that the Ca-ATPase can function as an ATP-energized Ca/H antiporter. The initial rate of the acidic shift inside the vesicles, as well as the rate of the ITP-dependent alkalization of the medium inside them markedly increased in the presence of valinomycin. This process was rapidly stopped by eosin Y, a known inhibitor of the type IIB Ca-ATPase. ITP-dependent uptake of Ca was blocked after the addition to the reaction mixture of nigericin in the presence of K. Under these conditions, the alkaline shift of pH inside the vesicles occurred, leading to the inhibition of operation of the calcium pump in SM. Evaluation of the pH shifts inside the vesicles by using pH-indicator pyranine confirmed the ion-exchange mechanism of the Ca-ATPase functioning in the SM. [ABSTRACT FROM AUTHOR]

Aims: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the A T I receptor. Methods and results: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS and results of the isolated complexes showed that Atrap interacts with the cardiac Ca2+-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca2+ uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca2+]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap--/-- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap--/-- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap--/-- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap--/-- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap--/-- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap--/-- (725 ± 48 μL/s) compared with WT mice (1065 ± 122 μL/s; P = 0.01; n = 15). Conclusion: We identified Atrap as a novel regulatory protein of the cardiac Ca2+-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation. [ABSTRACT FROM AUTHOR]

Cd2+ has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd2+ on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca2+-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd2+ concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd2+ showed a significant inhibition of progressive motility, reaching values even lower at higher Cd2+ concentrations. In addition, it was found that the activity of the plasma membrane Ca2+-ATPase reached maximal inhibition at 50 nm free Cd2+, with a K50% inhibition of 18.3 nm free Cd2+. The dynein-ATPase activity was maximally inhibited by 25 nm free Cd2+ in the assay medium, with a K50% inhibition of 11.3 nm Cd2+. Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd2+. [ABSTRACT FROM AUTHOR]

Control of smooth muscle is vital for health. The major route to contraction is a rise in intracellular [Ca2+], determined by the entry and efflux of Ca2+ and release and re-uptake into the sarcoplasmic reticulum (SR). We review these processes in myometrium, to better understand excitation-contraction coupling and develop strategies for preventing problematic labours. The main mechanism of elevating [Ca2+] is voltage-gated L-type channels, due to pacemaker activity, which can be modulated by agonists. The rise of [Ca2+] produces Ca-calmodulin and activates MLCK. This phosphorylates myosin and force results. Without Ca2+ entry uterine contraction fails. The Na/Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) remove Ca2+, with contributions of 30% and 70% respectively. Studies with PMCA-4 knockout mice show that it contributes to reducing [Ca2+] and relaxation. The SR contributes to relaxation by vectorially releasing Ca2+ to the efflux pathways, and thereby increasing their rates. Agonists binding produces IP3 which can release Ca from the SR but inhibition of SR Ca2+ release increases contractions and Ca2+ transients. It is suggested that SR Ca2+ targets K+ channels on the surface membrane and thereby feedback to inhibit excitability and contraction.

We estimated the influence of plasmids with DNA carrying the neurotrophin-3 (NT-3) gene on apoptosis in the gastrocnemius muscle and content of Ca-ATPase in the latter after sciatic nerve injury. Sixty adult Wistar rats were randomly divided into the saline (control) and NT-3 groups. The related indices, such as expression of caspase-3 protein, the rate of apoptosis in the muscle evaluated using a TUNEL technique, and the level of Ca-ATPase estimated using Western blot, were observed. Expression of caspase-3 protein was elevated at different post-operative times after peripheral nerve injury; NT-3 expression and the rate of muscle cell apoptosis decreased, whereas the level of Ca-ATPase in the sarcoplasmic reticulum increased; significant differences were observed compared with the saline group ( P < 0.05). The mitigation mechanism of NT-3 on muscle atrophy after peripheral nerve injury is expressed as inhibition of caspase-3 gene expression, increase in the Ca-ATPase level, and reduction in the rate of muscle apoptosis. [ABSTRACT FROM AUTHOR]

In a previous study, we reported lower sarcoplasmic reticulum (SR) Ca(2+) pump ionophore ratios in rat soleus compared to red and white gastrocnemius (RG, WG) muscles which may be indicative of greater SR Ca(2+) permeability in soleus. Here we assessed the lipid composition of the SR membranes obtained from these muscles to determine if SR docosahexaenoic acid (DHA) content and fatty acid unsaturation could help to explain the previously observed differences in SR Ca(2+) permeability. Since we have shown previously that sarcolipin may also influence SR Ca(2+) permeability, we also examined the levels of sarcolipin in rat muscle. We found that SR membrane DHA content was significantly higher in soleus (5.3 ± 0.2 %) compared to RG (4.2 ± 0.2 %) and WG (3.3 ± 0.2 %). Likewise, total SR membrane unsaturation and unsaturation index (UI) were significantly higher in soleus (% unsaturation: 59.1 ± 2.4; UI: 362.9 ± 0.8) compared to RG (% unsaturation: 55.3 ± 1.0; UI: 320.9 ± 2.5) and WG (% unsaturation: 52.6 ± 1.1; UI: 310. ± 2.2). Sarcolipin protein was 17-fold more abundant in rat soleus compared to RG and was not detected in WG; however, comparisons between soleus, RG, and WG in sarcolipin-null mice revealed that, in the absence of sarcolipin, ionophore ratios are still lowest in soleus and highest in WG. Overall, our results suggest that SR membrane DHA content and unsaturation, and, in part, sarcolipin expression may contribute to SR Ca(2+) permeability and, in turn, may have implications in muscle-based metabolism and diet-induced obesity. [ABSTRACT FROM AUTHOR]

The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle. [ABSTRACT FROM AUTHOR]

Migraine headaches are considered to be associated with increased mitochondrial energy metabolism. Mitochondrial oxidative stress is also important in migraine headache pathophysiology although riboflavin and selenium (Se) induced a modulator role on mitochondrial oxidative stress in the brain. The current study aimed to determine the effects of Se with/without riboflavin on the microsomal membrane Ca-ATPase (MMCA), lipid peroxidation, antioxidant, and electroencephalography (EEG) values in glyceryl trinitrate (GTN)-induced brain injury rats. Thirty-two rats were randomly divided into four groups. The first group was used as the control, and the second group was the GTN group. Se and Se plus oral riboflavin were administered to rats constituting the third and fourth groups for 10 days prior to GTN administration. The second, third, and fourth groups received GTN to induce headache. Ten hours after the administration of GTN, the EEG records and brain cortex samples were obtained for all groups. Brain cortex microsomes were obtained from the brain samples. The brain and microsomal lipid peroxidation levels were higher in the GTN group compared to the control group, whereas they were decreased by selenium and selenium + riboflavin treatments. Vitamin A, vitamin C, vitamin E, and reduced glutathione (GSH) concentrations of the brain and MMCA, GSH and glutathione peroxidase values of microsomes were decreased by the GTN administration, although the values and β-carotene concentrations were increased by Se and Se + riboflavin treatments. There was no significant change in EEG records of the four groups. In conclusion, Se with/without riboflavin administration protected against GTN-induced brain oxidative toxicity by inhibiting free radicals and the modulation of MMCA activity and supporting the antioxidant redox system. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the immune adherence function of erythrocytes and erythrocyte induced by dietary nickel chloride (NiCl) in broilers fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg NiCl for 42 days. Blood samples were collected from five broilers in each group at 14, 28, and 42 days of age. Changes of erythrocyte parameters showed that total erythrocyte count (TEC), hemoglobin (Hb) contents, and packed cell volume (PCV) were significantly lower ( p < 0.05 or p < 0.01) and erythrocyte osmotic fragility (EOF) was higher ( p < 0.05 or p < 0.01) in the 600 and 900 mg/kg groups at 28 and 42 days of age than those in the control group, and the sodium-potassium adenosine triphosphatase (Na/K-ATPase) and calcium adenosine triphosphatase (Ca-ATPase) activities were significantly decreased ( p < 0.05 or p < 0.01) in the NiCl-treated groups. The results of erythrocyte immune adherence function indicated that erythrocyte C receptor rosette rate (E-CRR) was significantly decreased ( p < 0.05 or p < 0.01) in the 600 and 900 mg/kg groups and in the 300 mg/kg group at 42 days of age, whereas the erythrocyte immune complex rosette rate (E-ICRR) was markedly increased ( p < 0.05 or p < 0.01) in the 300, 600, and 900 mg/kg groups at 28 and 42 days of age. It was concluded that dietary NiCl in excess of 300 mg/kg caused anemia and impaired the erythrocytic integrity, erythrocytic ability to transport oxygen, and erythrocyte immune adherence function in broilers. Impairment of the erythrocytes and erythrocyte immune adherence function was one of main effect mechanisms of NiCl on the blood function. [ABSTRACT FROM AUTHOR]

Vacuolar Ca-transporters could play an important role for salt tolerance in rice ( Oryza sativa L.) root. Here, we compared the expression profiles of putative vacuolar cation/H exchanger (CAX) and calmodulin-regulated autoinhibited Ca-ATPase (ACA) in rice roots of salt tolerant cv. Pokkali and salt sensitive cv. IR29. In addition to five putative vacuolar CAX genes in the rice genome, a new CAX gene ( OsCAX4) has been annotated. In the present study, we isolated the OsCAX4 gene and showed that its encoded protein possesses a unique transmembrane structure and is potentially involved in transporting not only Ca but also Mn and Cu. These six OsCAX genes differed in their mRNA expression pattern in roots of tolerant versus sensitive rice cultivars exposed to salt stress. For example, OsCAX4 showed abundant expression in IR29 (sensitive) upon prolonged salt stress. The mRNA expression profile of four putative vacuolar Ca-ATPases ( OsACA4- 7) was also examined. Under control conditions, the mRNA levels of OsACA4, OsACA5, and OsACA7 were relatively high and similar among IR29 and Pokkali. Upon salt stress, only OsACA4 showed first a decrease in its expression in Pokkali (tolerant), followed by a significant increase. Based on these results, a role of vacuolar Ca transporter for salt tolerance in rice root was discussed. [ABSTRACT FROM AUTHOR]

Myotoxic effects related to intracellular Ca disturbances have been reported for local anesthetics. Such effects might derive from Ca-ATPase dysfunction. The aim of this work was to describe the effect of lidocaine and bupivacaine on the sarcoplasmic reticulum (SR) Ca-ATPase from fast-twitch skeletal muscle and to identify the affected steps of the enzyme's cycle. SR sealed vesicles were isolated from rabbit fast-twitch muscles by ultracentrifugation. The effect of the anesthetics on Ca-ATPase activity was assessed with a colorimetric method and Ca binding, uptake, phosphorylation of the enzyme by ATP, Ca dissociation kinetics and phosphoenzyme formation and decomposition levels were tested with radioisotopic methods. Lidocaine and bupivacaine inhibited Ca-ATPase activity with half-maximal inhibitory concentrations (K) of 25.3 and 31.4 mM, respectively, and the steady-state Ca transport ability with K values of 33.6 and 46.5 mM, decreasing the maximal transport rate without modification of the Ca or ATP affinity for the enzyme. This is consistent with an absence of competition for the transport and catalytic sites. The anesthetics did not inhibit Ca binding but inhibited the phosphorylation partial reactions. Ca dissociation kinetics was not affected, but the phosphoenzyme levels were decreased, and the decomposition rate of the phosphoenzyme became faster in the presence of the anesthetics. It is concluded that lidocaine and bupivacaine at concentrations available in pharmaceutical formulations for clinical medical and dental uses inhibit the SR Ca-ATPase through inhibition of key phosphorylation steps of the enzymatic cycle. [ABSTRACT FROM AUTHOR]

Key message: Arabidopsis Ca -ATPase ACA8 plays a role in sucrose signalling during early seedling development by integrating developmental signals with carbon source availability. Abstract: Calcium (Ca) is an essential signal transduction element in eukaryotic organisms. Changes in the levels of intracellular Ca affect multiple developmental processes in plants, including cell division, polar growth, and organogenesis. Here, we report that the plasma-membrane-localised Arabidopsis Ca-ATPase ACA8 plays a role in sucrose signalling during early seedling development. Disruption of the ACA8 gene elevated the expression of genes that encode transporters for Ca efflux. The seedlings that carried a T-DNA insertion mutation in ACA8 experienced water stress during early development. This response was unrelated to inadequate osmoregulatory responses and was most likely caused by disruption of cell membrane integrity and severe ion leakage. In addition, aca8- 1 seedlings displayed a significant decline in photosynthetic performance and arrested root growth after removal of sucrose from the growth medium. The two phenomena resulted from impaired photosynthesis, reduced cell proliferation in the root meristem and the sucrose control of cell-cycle events. All of the stress-response phenotypes were rescued when expression of ACA8 was restored in aca8-1 mutant. Taken together, our results indicate that ACA8-mediated Ca signalling contributes to modulate early seedling development and coordinates root development with nutrient availability. [ABSTRACT FROM AUTHOR]

We investigated the effect of chitooligosaccharides on the denaturation of weever (Lateolabrax japonicus) myofibrillar protein during frozen storage at −18°C for 90 days. Chitooligosaccharides (2.5–10g dry weight) were added to 100g of myofibrillar protein, and the changes in the Ca-adenylpyrophosphatase (ATPase) activity, unfrozen water content, solubility, and sulfhydryl content of the myofibrillar protein were examined during frozen storage. We observed that the Ca-ATPase activity and solubility of the myofibrillar protein decreased gradually during frozen storage at −18°C following the addition of chitooligosaccharides. In contrast, the Ca-ATPase activity and solubility of the myofibrillar protein of the control group decreased markedly during the first 45 days of storage and then further decreased gradually for up to 90 days of storage, indicating a biphasic denaturation pattern. The addition of chitooligosaccharides resulted in a significant increase in unfrozen water and sulfhydryl contents of the myofibrillar protein of the treatment group compared with that of the control group (p <0.05) during frozen storage at −18°C. [ABSTRACT FROM AUTHOR]

Plant auto-inhibited Ca-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues-highly conserved in other ACA isoforms-localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation. [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize the sarcoplasmic-endoplasmic reticulum Ca- ATPase ( SERCA) isoforms in rabbit masticatory muscles compared with those in fast-twitch muscle. It was hypothesized that combined expression of the SERCA isoforms in fast- and slow-twitch muscles accounts for lower Ca- ATPase activity. SERCA was isolated by differential centrifugation, the isoforms were determined by ELISA, and the activity of each isoform was measured using a colorimetric method. Activity was tested for significance by anova, and the distribution of isoforms was assessed using the chi-square test ( P < 0.05) and correlated to SERCA activity using Spearman's rank correlation. SERCA1 was predominant (90.5%) in fast-twitch muscle, whereas a mixture of SERCA isoforms was found in masticatory muscles: 62-78% was SERCA2, 20-37% was SERCA1, and the SERCA3 content was negligible. Depressor muscles showed a significantly higher content (77.8%) of SERCA2, and elevator muscles showed a higher content (35.4%) of SERCA1. Elevator muscles showed higher expression of SERCA2a (58%), and depressor muscles showed higher expression of SERCA2b (20%). The SERCA1 content was mainly SERCA1a and significantly higher for elevator muscles (33%), whereas depressor muscles showed a higher content of SERCA1b (4%). The SERCA1 content of fast-twitch muscle was mainly SERCA1a (88.5%). It is concluded that the mixture of different SERCA isoforms, along with a substantial content of SERCA2b, in masticatory muscles would support lower Ca- ATPase activity and calcium transport. [ABSTRACT FROM AUTHOR]

We studied the effect of iron nitrosyl complexes, NO donors, of various structural types on the activity of Ca-ATPase of sarcoplasmic reticulum (SR) and phosphodiesterase (PDE) of cyclic guanosine monophosphate (cGMP). It was established that iron nitrosyl complexes with organic ligands modulate functions of both enzymes. They effectively inhibited the hydrolytic and transport functions of Ca-ATPase SR at concentrations 0.1 - 0.01 mM and decoupled ATP hydrolysis and active Ca transport at concentrations 0.01 - 0.0001 mM, thus disrupting the Ca balance in cells. This influenced thrombogenesis and adhesion of metastatic cells to capillary endothelium. The compound [Fe(SC(NH))(NO)][Fe(SO)(NO)] produced non-competitive and reversible inhibition of Ca-ATPase SR functioning with K = 0.70∙10 M. All studied iron nitrosyl complexes inhibited the activity of PDE-cGMP, which led to accumulation of cGMP, which is a secondary messenger influencing the in vivo anti-aggregation effect. The obtained results suggested that the studied iron nitrosyl complexes could be considered as potential drugs. [ABSTRACT FROM AUTHOR]

The effect of the local anesthetics procaine and tetracaine on sarcoplasmic reticulum membranes isolated from two masticatory muscles, masseter and medial pterygoid, was tested and compared to fast-twitch muscles. The effects of the anesthetics on Ca-ATPase activity, calcium binding, uptake, and phosphorylation of the enzyme by inorganic phosphate (P i) were tested with radioisotopic methods. Calcium binding to the Ca-ATPase was non-competitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner. The inhibition of the activity depended on pH, calcium concentration, the presence of the calcium ionophore calcimycin, and the membrane protein concentration. Unlike fast-twitch membranes, the pre-exposure of the masseter and medial pterygoid membranes to the anesthetics enhanced the enzymatic activity in the absence of calcimycin, supporting their permeabilizing effect. Procaine and tetracaine also interfered with the calcium transport capability, decreasing the maximal uptake without modification of the calcium affinity for the ATPase. Besides, the anesthetics inhibited the phosphorylation of the enzyme by P i in a competitive manner. Tetracaine revealed a higher inhibitory potency on Ca-ATPase compared to procaine, and the inhibitory concentrations were lower than usual clinical doses. It is concluded that procaine and tetracaine not only affect key steps of the Ca-ATPase enzymatic cycle but also exert an indirect effect on membrane permeability to calcium and suggest that the consequent myoplasmic calcium increase induced by the anesthetics might account for myotoxic effects, such as sustained contraction and eventual rigidity of both fast-twitch and masticatory muscles. [ABSTRACT FROM AUTHOR]

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS) mice hearts that Na/K- and Ca-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS mice. Na/K-ATPase activity from crude preparations of adult male eNOS mice hearts and kidneys was reduced compared with wild-type animals (32 %, p < 0.05 and 16 %, p < 0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na/K-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p < 0.0001). In addition, although cardiac Ca-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca-ATPase 2 protein in eNOS mice was very high (290 % compared with wild-type animals, p < 0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p < 0.01 and 35 %, p < 0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases. [ABSTRACT FROM AUTHOR]

We analyse a kinetic cycle of the Ca-ATPase molecular pump using mesoscopic non-equilibrium thermodynamics. The pump is known to generate heat, and by analysing the operation on the mesoscopic level, we are able to introduce a temperature difference and the corresponding heat flux in the description. Integration over the internal coordinates then results in non-linear flux-force relations describing the operation of the pump on the macroscopic level. Specifically, we obtain an expression for the heat flux associated with the active transport and the coupling of heat effects to the transport of ions and the rate of the ATP-hydrolysis. [ABSTRACT FROM AUTHOR]

Aging is a biological process characterized by a progressive functional impairment which is associated with increased susceptibility to a variety of diseases. The main purpose of this study is to understand the gender-based relationship between human aging and activities of two erythrocyte membranes bound enzymes, Ca-ATPase and Na/K-ATPase. Ca-ATPase and Na/K-ATPase activities were determined as per the previous reports. Statistical differences were analyzed with Student's t test. Our results show a significant ( p < 0.0001) decrease in the Ca-ATPase and Na/K-ATPase activities in males and females as a function of age. We also correlate the activities of ATPases with total antioxidant capacity of the plasma in term of ferric reducing ability of plasma values. The Ca-ATPase and Na/K-ATPase activities positively correlated with ferric reducing ability of plasma value. No significant differences in the ATPase activity between males and females were observed. Decreased activity of Ca-ATPase and Na/K-ATPase during human aging may be due to increased free radical generation which leads to oxidative stress and alter the erythrocyte membrane transport function and other activities. Our results emphasize the need to establish age-dependent reference values for membrane bound enzymes in studies involving its role in different disease conditions. [ABSTRACT FROM AUTHOR]

Ca-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH 7.0. Translocation of Ca through the PBM by the Ca-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP > ITP ≅ GTP ≅ UTP ≅ CTP, the K of the enzyme for MgATP being about 100 μM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca-ATPase for Ca of about 0.1 μM. The transport activity of Ca-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca-ATPases which are present in other plant membranes. [ABSTRACT FROM AUTHOR]

To determine the effect of denaturants [guanidine hydrochloride (GdnHCl) and urea] and polyols [with various molecular masses (62.1-600)] on calcium binding at the two hypothesized conformers (A and B forms) of the chemically equivalent sarcoplasmic reticulum Ca-ATPase, which bind two calcium ions in different manners, we examined the effect of these reagents on the calcium dependence of ATP-supported phosphorylation of the ATPase molecules and of their calcium-activated, acetyl phosphatate hydrolytic activity. (1) GdnHCl (~0.05 M) and urea (~0.5 M) increased the apparent calcium affinity ( K) of 2-6 μM of noncooperative binding [Hill coefficient ( n) ~ 1] of the A form to 10-40 μM. (2) The employed polyols transformed the binding of the A form into cooperative binding ( n ~ 2), accompanying the approach of its K value to that ( K = 0.04-0.2 μM) of the cooperative binding ( n ~ 2) of the B form; the transition concentration (0.025-2 M) of the polyols, above which such transformation occurs, was in inverse relation to their molecular mass. (3) The binding of the B form was resistant to these denaturants and polyols. Based on these data, a structural model of the two forms, calcium-binding domains of which are loosely and compactly folded, is presented. [ABSTRACT FROM AUTHOR]

A large number of plant Ca/H exchangers have been identified in endomembranes, but far fewer have been studied for Ca/H exchange in plasma membrane so far. To investigate the Ca/H exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca/H exchange in plasma membrane vesicles was measured by Ca-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca/H exchange was optimal at pH 7.0, and displayed transport specificity for Ca with saturation kinetics at K = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca-dependent transport of H out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca/H exchange increased comparing to control without valinomycin effect, suggesting that the Ca/H exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H:Ca. Eosin Y, a Ca-ATPase inhibitor, drastically inhibited Ca/H exchange in plasma membrane as it does for the purified Ca-ATPase in proteoliposomes, indicating that measured Ca/H exchange activity is mainly due to a plasma membrane Ca pump. These suggest that calcium (Ca) is transported out of Arabidopsis cells mainly through a Ca-ATPase-mediated Ca/H exchange system that is driven by the proton-motive force from the plasma membrane H-ATPase. [ABSTRACT FROM AUTHOR]

The use of a microsomal preparation from skeletal muscle revealed that both Ca transport and Ca-dependent ATP hydrolysis linked to Sarco-Endoplasmic Reticulum Ca-ATPase are inhibited by epigallocatechin-3-gallate (EGCG). A half-maximal effect was achieved at approx. 12 μM. The presence of the galloyl group was essential for the inhibitory effect of the catechin. The relative inhibition of the Ca-ATPase activity decreased when the Ca concentration was raised but not when the ATP concentration was elevated. Data on the catalytic cycle indicated inhibition of maximal Ca binding and a decrease in Ca binding affinity when measured in the absence of ATP. Moreover, the addition of ATP to samples in the presence of EGCG and Ca led to an early increase in phosphoenzyme followed by a time-dependent decay that was faster when the drug concentration was raised. However, phosphorylation following the addition of ATP plus Ca led to a slow rate of phosphoenzyme accumulation that was also dependent on EGCG concentration. The results are consistent with retention of the transporter conformation in the Ca-free state, thus impeding Ca binding and therefore the subsequent steps when ATP is added to trigger the Ca transport process. Furthermore, phosphorylation by inorganic phosphate in the absence of Ca was partially inhibited by EGCG, suggesting alteration of the native Ca-free conformation at the catalytic site. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The aim of this work was to isolate the sarcoplasmic reticulum (SR) Ca-ATPase from rabbit temporalis muscle and to determine the optimal conditions for calcium transport and enzymatic activity. Design: SR vesicles were isolated from rabbit temporalis muscle by differential centrifugation, the protein composition analyzed by electrophoresis and compared to fast-twitch muscle membrane suspensions. ELISA was used to determine the sarcoendoplasmic reticulum Ca-ATPase (SERCA) isoform. Ca-ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca-ATPase, calcium uptake, calcium efflux and phosphorylation by Pi were determined with radioisotopic techniques. Results: Sixty five percent of the total protein concentration of SR membranes suspensions from rabbit temporalis corresponded to SERCA. Of the total SERCA protein, 64% was SERCA 2, 35% was SERCA 1 and less than 1% was SERCA 3. The optimal conditions of the SERCA isolated from rabbit temporalis muscle were: pH 7.2, 5μM Ca2+, 100μM EGTA, 90μM Mg2+, 3mM ATP and 100mM KCl and did not differ from fast-twitch skeletal muscle. The temporalis maximal calcium uptake and Ca-ATPase activity were lower but the sensitivity to the specific Ca-ATPase inhibitor thapsigargin was higher. Calcium-binding to the enzyme and calcium efflux were similar while the phosphorylation of the enzyme by Pi was lower. Conclusion: The lower enzymatic activity and calcium transport capability of the Ca-ATPase isolated from rabbit temporalis, and the higher sensitivity to inhibitory drugs are consistent with the presence of a substantial proportion of SERCA 2, which can be expected in other rabbit masticatory muscles. [Copyright &y& Elsevier]

Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of β-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca-sensitive fluorescent dye Fura-2, to examine the passive Ca permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca leak from Ca-loaded SR vesicles. This enhanced rate of Ca leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca leak pathway in cardiac SR membranes in situ. [ABSTRACT FROM AUTHOR]

Abstract: Protein hydrolysates (SH, TH) were prepared from eel head by enzymatic treatment using protease. The eel head hydrolysates were used as a natural food preservative by adding to grass carp surimi at the 10% ranging. We compared the effect of protein hydrolysates of eel head with traditional antifreeze on the denatration, gel strength and contents of the salt soluble protein in grass carp surimi during frozen storage at -20°C for 80 days. The addition of eel head hydrolysate markedly decreased the activation rate of the Ca-ATPase. The contents of the salt soluble protein in the grass carp surimi containing eel head hydrolysates were higher than those without eel head hydrolysates (control).Therefore, the gel strength of grass carp surimi media with eel head hydrolysate lower than the others. The result suggests that eel head hydrolysate could suppress the denaturation of grass carp surimi during frozen storage. [Copyright &y& Elsevier]

We analyse the operation of the Ca-ATPase ion pump using a kinetic cycle diagram. Using the methodology of Hill, we obtain the cycle fluxes, entropy production and efficiency of the pump. We compare these results with a mesoscopic non-equilibrium description of the pump and show that the kinetic and mesoscopic pictures are in accordance with each other. This gives further support to the mesoscopic theory, which is less restricted and also can include the heat flux as a variable. We also show how motors can be characterised in terms of unidirectional backward fluxes. We proceed to show how the mesoscopic approach can be used to identify fast and slow steps of the model in terms of activation energies, and how this can be used to simplify the kinetic diagram. [ABSTRACT FROM AUTHOR]

Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats. [Copyright &y& Elsevier]

Abstract: The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca2+ flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T′ state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA. [Copyright &y& Elsevier]

Diabetes mellitus is known to impair glucose metabolism. The fundamental mechanism underlying hyperglycaemia in diabetes mellitus involves decreased utilization of glucose by the brain. However, mechanisms responsible for progressive failure of glycaemic regulation in type I (IDDM) diabetes need extensive and proper understanding. Hence the present study was initiated. Type I diabetes was induced in albino rat models with alloxan monohydrate (40 mg/Kg iv). Cerebral cortex and medulla oblongata were studied 48 h after alloxanisation. Diabetes caused an elevation in glucose, glutamate, aspartate, GABA and taurine levels and a decline in the glutamine synthetase activity. The activities of brain lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) exhibited significant decrease during diabetes. Ammonia content increased ( P < 0.01) as a function of diabetes. Na-K ATPase showed an elevation ( P < 0.01) and Ca-ATPase activity decreased ( P < 0.01). Calcium content enhanced ( P < 0.05) in the brain of diabetic rats. A General increase in the brain AMP, ADP and ATP was found on inducing diabetes. Impaired cerebral glucose metabolism accounts for the failure of cerebral glucose homeostasis. The impairment in the glycaemic control leads to disturbances in cerebral glutamate content (resulting in calcium overload and excitotoxic injury) and brain energy metabolism as reflected by alterations occurring in adenine nucleotide and the ATPases. The failure in the maintenance of normal energy metabolism during diabetes might affect glucose homeostasis leading to gross cerebral dysfunction during diabetes. [ABSTRACT FROM AUTHOR]

Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn detoxification by excretion into bile. Although many pathways of cellular Mn uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn detoxification by the Secretory Pathway Ca, Mn-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn. Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn toxicity. Taken together, our findings suggest a role for SPCA1 in Mn detoxification in liver. [ABSTRACT FROM AUTHOR]