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1.
Recent studies focusing on the consequences of feeding for ion and water balance in freshwater fish have revealed the need for similar comparative studies in seawater fish. A detailed time course sampling of gastrointestinal (GI) tract contents following the ingestion of a single meal of a commercial diet revealed the assimilation of both water and dietary ions (Na+, Cl?, K+, Ca2+, Mg2+) along the GI tract of seawater-acclimated rainbow trout (Oncorhynchus mykiss) which had been fasted for 1 week. Consumption of the meal did not change the drinking rate. There was a large secretion of fluid into the anterior intestine and caecae (presumably bile and/or pancreatic secretions). As a result, net assimilation (63%) of the ingested water along the GI tract was lower than generally reported for fasted trout. Mg2+ was neither secreted into nor absorbed from the GI tract on a net basis. Only K+ (93% assimilated) and Ca2+ (43% assimilated) were absorbed in amounts in excess of those provided by ingested seawater, suggesting that dietary sources of K+ and Ca2+ may be important to seawater teleosts. The oesophagus–stomach served as a major site of absorption for Na+, Cl?, K+, Ca2+, and Mg2+, and the anterior intestine and caecae as a major site of net secretion for all of these ions, except Cl?. Despite large absorptive fluxes of these ions, the ionic composition of the plasma was maintained during the digestion of the meal. The results of the present study were compared with previous work on freshwater-acclimated rainbow trout, highlighting some important differences, but also several similarities on the assimilation of water and ions along the gastrointestinal tract during digestion. This study highlights the complicated array of ion and water transport that occurs in the intestine during digestion while revealing the importance of dietary K+ and Ca2+ to seawater-acclimated rainbow trout. Additionally, this study reveals that digestion in seawater-acclimated rainbow trout appears to compromise intestinal water absorption.  相似文献   

2.
The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca2+-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channels closed conformations. Vacuolar Ca2+ and Mg2+ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a permeable blocker. Vacuolar Ca2+—with a much higher affinity than Mg2+—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca2+-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channels closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca2+ at the vacuolar side. The specificity for Ca2+ compared to Mg2+ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca2+ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.Abbreviation SV Slow vacuolar  相似文献   

3.
Annexin A5 (AnxA5) binds to negatively charged phospholipid membranes in a Ca2+ dependent manner. Several studies already demonstrate that Mg2+ ions cannot induce the binding. In this paper, quartz crystal microbalance with dissipation monitoring (QCM-D), Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) and molecular dynamics (MD) were performed to elucidate the high specificity of Ca2+ versus Mg2+ on AnxA5 binding to membrane models. In the presence of Ca2+, AnxA5 showed a strong interaction with lipids, the protein is adsorbed mainly in α-helix under the DMPS monolayer, with an orientation of the α-helices axes slightly tilted with respect to the normal of the phospholipid monolayer as revealed by PMIRRAS. The Ca2+ ions interact strongly with the phosphate group of the phospholipid monolayer. In the presence of Mg2+, instead of Ca2+, no interaction of AnxA5 with lipids was detected. Molecular dynamics simulations allow us to explain the high specificity of calcium. Ca2+ ions are well exposed and surrounded by labile water molecules at the surface of the protein, which then favour their binding to the phosphate group of the membrane, explaining their specificity. To the contrary, Mg2+ ions are embedded in the protein structure, with a smaller number of water molecules strongly bound. We conclude that the embedded Mg2+ ions inside the AnxA5 structure are not able to link the protein to the phosphate group of the phospholipids for this reason.  相似文献   

4.
Ryanodine receptors (RyRs) are the Ca2+ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca2+ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca2+. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca2+ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca2+ at these sites to inhibit RyRs and this plays an important role in modulating their Ca2+-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca2+ and Mg2+ and how these mechanisms control Ca2+ release in excitation-contraction coupling and cardiac pacemaking.  相似文献   

5.
Calcium (Ca2+) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca2+ is crucial for the control of energy production and cellular responses to metabolic stress. Ca2+ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca2+ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca2+ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca2+ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca2+. In view of the fundamental importance of Ca2+ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca2+ entry. We review the experimental evidence for the existence of alternative Ca2+ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca2+ signalling.  相似文献   

6.
To test the effects of ketamine on metal ion balance in the spinal cord tissues after ischemic reperfusion (I/R), 24 white adult Japanese rabbits were randomly assigned to sham operation group, I/R group or ketamine-treated I/R group. Spinal cord injuries in I/R group and ketamine-treated I/R group were induced by aortic occlusions. Rabbits in ketamine-treated I/R group were intravenously infused 10 mg/kg ketamine twice: once at 10 min before aortic clamping and once at the onset of reperfusion. Post-operative neurological functions and concentrations of ions Ca2+, Mg2+, Cu2+ and Zn2+ in the spinal cord were assessed. Compared with the sham operation group, rabbits in the I/R group showed significantly worsened neurological functions as scored with the modified Tarlov criteria and altered concentrations of ions Ca2+, Mg2+, Cu2+ and Zn2+. These unfavorable changes were significantly reversed in the ketamine-treated I/R group, suggesting that the potent protective effects of ketamine against the I/R-induced spinal cord injuries may be due to its ability to maintain ion balance in the I/R affected tissues.  相似文献   

7.
Two new bismacrocyclic Gd3+ chelates containing a specific Ca2+ binding site were synthesized as potential MRI contrast agents for the detection of Ca2+ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca2+-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L1) or by a propylene (L2) unit [H4BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H3DO3A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd3+ complexes towards Ca2+ and Mg2+ was studied by 1H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca2+ binding to Gd2L1 and Gd2L2, respectively, with a distinct selectivity of Gd2L1 towards Ca2+ compared with Mg2+. For Ca2+ binding, association constants of log K = 1.9 (Gd2L1) and log K = 2.7 (Gd2L2) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu3+ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca2+ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca2+ addition. A 1H nuclear magnetic relaxation dispersion and 17O NMR study on Gd2L1 in the absence and in the presence of Ca2+ was performed to assess the microscopic parameters influencing relaxivity. On Ca2+ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

8.
Cation/H+ exchangers (CAXs) are membrane proteins that transport Ca2+ and other cations using the H+ gradient generated by H+-ATPase or H+-pyrophosphatase. This study reports the characterization of CAX2 from Puccinellia tenuiflora with respect to molecular and functional properties. PutCAX2 was cloned from a cDNA library of P. tenuiflora seedlings. The expression of PutCAX2 in shoots and roots was induced by Ca2+ and Ba2+ treatments. A green fluorescent protein (GFP) marker revealed that PutCAX2 was located on the endoplasmic reticulum (ER) membrane. Four yeast transformants were created using GFP fusion PutCAX2 and truncated PutCAX2s, and their growth in the presence of various cations (Fe3+, Al3+, Mn2+, Cu2+, Co2+, Ni2+, Mg2+, Zn2+, Na+, Li+, Ca2+, and Ba2+) was analyzed. The N-terminally truncated PutCAX2 (GFP-ΔNPutCAX2) and the N and C-terminally truncated PutCAX2 (GFP-ΔNCPutCAX2) transformants grew well in the presence of 100 and 150 mM Ca2+ or 8 and 20 mM Ba2+, whereas the GFP-PutCAX2 and C-terminally truncated PutCAX2 (GFP-ΔCPutCAX2) transformants did not show any tolerance to Ca2+ or Ba2+. The Ba2+ content in whole yeast cells expressing GFP-ΔNPutCAX2 or GFP-ΔNCPutCAX2 was lower than that in other yeast transformants. Moreover, the efflux experiment showed that the Ba2+ efflux rate of yeast cells expressing GFP-ΔNPutCAX2 and GFP-ΔNCPutCAX2 was higher than that of other yeast cells. To our knowledge, this is the first report on the molecular and functional characterization of a novel ER-localized CAX protein from a wild halophyte plant; the results suggest that the N-terminus of PutCAX2 acts as an auto-inhibitory domain, which affects the Ca2+ and Ba2+ tolerance of yeast.  相似文献   

9.
10.
Here we show that positive modulators (CyPPA and NS309) of Ca2+-activated K+ channels of small (SK) and intermediate (IK) conductances in cerebellar neurons decrease glutamate-evoked Ca2+ entry into neurons independently on the presence of Mg2+ in extracellular media. An analysis of neuronal viability after long-term (240 min) glutamate treatments demonstrated neuroprotective action of CyPPA and NS309. Extracellular Mg2+ did not protect neurons from apoptosis during prolonged treatment with glutamate. Activation of SK and IK channels results in local membrane hyperpolarization, which enhances Mg2+ block of NMDA receptors and reduces activation of voltage-dependent Ca2+ channels, which can explain neuroprotection caused by CyPPA or NS309. The obtained results reveal an important role Ca2+-activated K+ channels of small and intermediate conductance in the regulation of Ca2+ entry into cerebellar neurons via NMDA receptors and voltage-gated Ca2+ channels.  相似文献   

11.
Effects of intracellular Mg2+ on a native Ca2+-and voltage-sensitive large-conductance K+ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca2+ ([Ca2+]i) of 10−5–10−4 M, addition of 1–10 mM Mg2+ increased the open probability (Po) of the channel, which shifted the Po –membrane potential (Vm) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg2+-induced increase in Po was suppressed at a relatively low [Ca2+]i (10−5.5–10−6 M). Dwell-time histograms have revealed that addition of Mg2+ mainly increased Po by extending open times at 10−5 M Ca2+ and extending both open and closed times simultaneously at 10−5.5 M Ca2+. Since our data showed that raising the [Ca2+]i from 10−5 to 10−4 M increased Po mainly by shortening the closed time, extension of the closed time at 10−5.5 M Ca2+ would result from the Mg2+-inhibited Ca2+-dependent activation. At a constant Vm, adding Mg2+ enhanced the sigmoidicity of the Po–[Ca2+]i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg2+ on this channel is to elevate Po by lengthening the open time, while extension of the closed time at a relatively low [Ca2+]i results from a lowering of the sensitivity to Ca2+ of the channel by Mg2+, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.  相似文献   

12.
Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca2+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca2+. We show that hyperphosphorylation by exogenous PKA increases open probability (P o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca2+; lowering [Ca2+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca2+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg2+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca2+. We show that the endogenous kinase is both Ca2+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca2+-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca2+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca2+ regulation.  相似文献   

13.

Background  

E-NTPase/E-NTPDase is activated by millimolar concentrations of Ca2+ or Mg2+ with a pH optimum of 7.5 for the hydrolysis of extracellular NTP and NDP. It has been generally accepted that E-NTPase/E-NTPDase plays regulatory role in purinergic signalling, but other functions may yet be discovered.  相似文献   

14.
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 Ca2+-ATPase transporting Ca2+ 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 Ca2+-ATPase as ATP-driven Ca2+/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 Ca2+ uptake by symbiosomes catalyzed by the SM Ca2+-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 Ca2+-ATPase operation as an ATP-driven Ca2+/H+ exchanger and on this base allow us to put forward the hypothesis about the involvement of this enzyme in symbiosome signaling in a Ca2+- and pH-dependent manner.  相似文献   

15.
Recent studies from our laboratory have shown that in the mouse and rat nephron Ca2+ and Mg2+ are not reabsorbed in the medullary part of the thick ascending limb (mTAL) of Henle's loop. The aim of the present study was to investigate whether the absence of transepithelial Ca2+ and Mg2+ transport in the mouse mTAL is due to its relative low permeability to divalent cations. For this purpose, transepithelial ion net fluxes were measured by electron probe analysis in isolated perfused mouse mTAL segments, when the transepithelial potential difference (PDte.) was varied by chemical voltage clamp, during active NaCl transport inhibition by luminal furosemide. The results show that transepithelial Ca2+ and Mg2+ net fluxes in the mTAL are not driven by the transepithelial PDte. At zero voltage, a small but significant net secretion of Ca2+ into the tubular lumen was observed. With a high lumen-positive PDte generated by creating a transepithelial bath-to-lumen NaCl concentration gradient, no Ca2+ and Mg2+ reabsorption was noted; instead significant and sustained Ca2+ and Mg2+ net secretion occurred. When a lumen-positive PDte was generated in the absence of apical furosemide, but in the presence of a transepithelial bath-to-lumen NaCl concentration gradient, a huge Ca2+ net secretion and a lesser Mg2+ net secretion, not modified by ADH, were observed. Replacement of Na+ by K+ in the lumen perfusate induced, in the absence of PDte changes, important but reversible net secretions of Ca2+ and Mg2+. In conclusion, our results indicate that the passive permeability of the mouse mTAL to divalent cations is very low and not influenced by ADH. This nephron segment can secrete Ca2+ and Mg2+ into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. Given the impermeability of this epithelium to Ca2+ and Mg2+, the secretory processes would appear to be of cellular origin. Received: 30 January 1996/Revised: 24 April 1996  相似文献   

16.
Of various metal ions (Ca2+, Cr3+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+ and Zn2+) added to the culture medium of Agrobacterium tumefaciens at 1 mM, only Ca2+ increased Coenzyme Q10 (CoQ10) content in cells without the inhibition of cell growth. In a pH-stat fed-batch culture, supplementation with 40 mM of CaCO3 increased the specific CoQ10 content and oxidative stress by 22.4 and 48%, respectively. Also, the effect of Ca2+ on the increase of CoQ10 content was successfully verified in a pilot-scale (300 L) fermentor. In this study, the increased oxidative stress in A. tumefaciens culture by the supplementation of Ca2+ is hypothesized to stimulate the increase of specific CoQ10 content in order to protect the membrane against lipid peroxidation. Our results improve the understanding of Ca2+ effect on CoQ10 biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ10 by biological processes.  相似文献   

17.
Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Cav1 and Cav2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Cav3 family) is less documented. We have studied the blocking effect of Cd2+, Co2+ and Ni2+ on T-currents expressed by human Cav3 channels: Cav3.1, Cav3.2, and Cav3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca2+ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd2+ and Co2+ block was 2- to 3-fold more potent for Cav3.2 channels (EC50 = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co2+ and Ni2+ shift the voltage dependence of Cav3.1 and Cav3.3 channels activation to more positive potentials. Interestingly, block of those two Cav3 channels by Co2+ and Ni2+ was drastically increased at extreme negative voltages; in contrast, block due to Cd2+ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd2+ on Cav3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.  相似文献   

18.
Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca2+ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca2+, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca2+ and the swelling of mitochondria. Inhibitors of the Ca2+ uniporter, ruthenium red and La3+, as well as EGTA added in 10 min after the Pal/Ca2+-activated pore opening, prevent the release of Ca2+ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr2+], which leads to the activation of phospholipase A2 and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca2+ cycle, in which Ca2+ uptake is mediated by the Ca2+ uniporter and Ca2+ efflux occurs via a short-living Pal/Ca2+-activated pore.  相似文献   

19.
Supplementation with CaCl2·2H2O (50 mg l−1) or CuSO4·5H2O (10 mg l−1) improved mannitol production by Candida magnoliae by 14.5 and 18.6% (25 and 32 g/L), respectively. When used in combination, they acted synergistically: Ca2+ decreased the intracellular concentration of mannitol 30%, whereas Cu2+ increased the intracellular activity of mannitol dehydrogenase 1.6-times more than control. Ca2+ probably works by altering the permeability of cells to mannitol, whereas, Cu2+ increases the activity of an enzyme responsible for mannitol biosynthesis.  相似文献   

20.
Relevant Ca2+ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca2+-mobilizing agents. Vasopressin produced a cytoplasmic Ca2+ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca2+ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca2+. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca2+ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca2+ pool was not. The vasopressin-induced cytoplasmic Ca2+ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca2+ pool, was sensed as a mitochondrial Ca2+ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca2+ peak was affected by cyclosporin A when the Ca2+ signal was induced by irreversible discharge of the intracellular Ca2+ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca2+ signals generated in the reticular store.  相似文献   

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