Calcium Rapidly Down-Regulates Human Renal Epithelial Sodium Channels Via a W-7-Sensitive Mechanism

Increases in intracellular calcium (Ca²⁺) inhibit renal sodium (Na⁺) absorption in cortical collecting ducts, but the precise mechanism is unclear. We, therefore, studied the effects of raising intracellular Ca²⁺ (using 10 µmol/L A23187, a Ca²⁺ ionophore) on wild-type and Liddle-mutated human epithelial Na⁺ channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. A23187 decreased amiloride-sensitive Na⁺ current by 55 % in oocytes expressing wild-type hENaC, an effect prevented by co-exposure to 50 μmol/L W-7 (to inhibit the Ca²⁺/calmodulin complex). By contrast, co-exposure to 50 μmol/L calphostin (to inhibit protein kinase C) or 5 μmol/L KN-62 (to inhibit Ca²⁺/calmodulin-dependent protein kinase II) had no effect on the decrease in amiloride-sensitive Na⁺ current elicited by A23187 alone. Whereas A23187 reduced amiloride-sensitive Na⁺ current in oocytes expressing wild-type hENaC, it had no similar effect in those expressing Liddle-mutated hENaCs, suggesting that the activity of individual Na⁺ channels in situ was unchanged by the rise in intracellular Ca²⁺. These data suggest that the A23187-induced rise in intracellular Ca²⁺ inhibited wild-type hENaC through a W-7-sensitive mechanism, which likely reflected enhanced removal of Na⁺ channels from the cell membrane by endocytosis. We, therefore, propose that Na⁺ absorption in cortical collecting duct cells is inhibited by Ca²⁺, possibly when complexed with calmodulin.     

Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP

Ca²⁺ changes induced by nitric oxide (NO^·) were investigated in cultured human endothelial cells. Sodium nitroprusside (SNP) (1–100 μmol/L) and S-Nitroso-N-acetylpenicillamine (SNAP) (100 μmol/L) were used as NO^· donors. The cytoplasmatic Ca²⁺ concentration was calculated using ratiometric FURA2 fluorescence measurements. Both NO^· donors caused transient oscillatory Ca²⁺ changes, which were not detectable in the presence of oxyhemoglobin (50 μmol/L). Digital ratio imaging revealed initiation sites within cells where Ca²⁺ increases started spreading, which indicates that nonuniformly distributed targets might be involved in these reactions. Calcium was released from intracellular stores as indicated by experiments performed in Ca²⁺-free buffer. L-type Ca²⁺-channel blocker diltiazem (100 μmol/L) was not able to block these responses. NO^·-induced Ca²⁺ release from intracellular stores caused capacitative Ca²⁺ entry. Both thapsigargin (1 μmol/L) and cyclopiazonic acid (10 μmol/L) inhibited the SNP response completely, whereas neither ryanodine (up to 100 μmol/L) nor dantrolene (100 μmol/L) was able to inhibit Ca²⁺ changes induced by SNP, indicating that primarily inositol 1,4,5-triphosphate (IP₃)-dependent stores are released upon stimulation with NO^·. A small inhibitory effect of ATP- and SNP-induced peak [Ca²⁺]_i increase was measured in the presence of both caffeine (20 mmol/L) and procaine (1 mmol/L). Evidence is presented that cGMP is not involved in NO^·-induced Ca²⁺ signals, as neither inhibitors of guanylate cyclase (methylene blue and LY (83583) nor cell permeant analogues of cGMP altered or simulated [Ca²⁺]_i changes. An inhibitor of cGMP-dependent protein kinase was also ineffective. We therefore propose that endothelial cells have specific targets proximal or at IP₃ receptors to induce Ca²⁺ changes in endothelial cells stimulated with NO^·. J. Cell. Physiol. 172:296–305, 1997. © 1997 Wiley-Liss, Inc.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP₃) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully. No effect of Ca²⁺ concentration on [³H]-InsP₃ binding to unreconstituted InsP₃ receptor could be observed either at 4°C or at 25°C, whereas the effect of [Ca²⁺] on reconstituted InsP₃ receptor depended on the temperature. The Ca²⁺ concentration outside the proteolipsome ([Ca²⁺]_o) had no detectable effect on InsP₃ binding to InsP₃ receptor at 4°C. In contrast, with increase of [Ca²⁺]_o from 0 to 100 nmol/L at 25°C, the InsP₃ binding activity increased gradually. Then the InsP₃ binding activity was decreased drastically at higher [Ca²⁺]_o and inhibited entirely at 50 μmol/L [Ca²⁺]_o. Conformational studies on intrinsic fluorescence of the reconstituted InsP₃ receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP₃ receptor could not be affected by [Ca²⁺]_o at 4°C. While at 25°C, the effects of 10 μmol/L [Ca²⁺]_o on global, membrane and cytoplasmic conformation of the reconstituted InsP₃ receptor were different significantly from that of 100 nmol/L [Ca²⁺]_o.     

Selenium and Zinc against Aβ<Subscript>25–35</Subscript>-Induced Cytotoxicity and Tau Phosphorylation in PC12 Cells and Inhibits γ-cleavage of APP

Amyloid beta (Aβ) is the main component of the amyloid plaques that accumulate in the brains of Alzheimer patients. The present study was conducted to investigate whether the combined treatment with selenium (Se) and zinc (Zn) offers more beneficial effects than that provided by either of them alone in reversing Aβ_25–35-induced neurotoxicity in PC12 cells. Cells were pretreated with 0.1 μmol/L of Se and Zn for 4 h, after treated with 10 mmol/L Aβ_25–35 for 24 h. Cells were divided into control and five treated groups, and received either 10 mmol/L Aβ_25–35,10 mmol/L Aβ_25–35 + 0.1 μmol/L Se, 10 mmol/L Aβ_25–35 + 0.1 μmol/L Zn, 10 mmol/LAβ_25–35 + 0.1 μmol/L Se + 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. The result showed that cell viability was decreased in MTT metabolic rate; LDH release and MDA, H₂O₂, and NO levels were increased and the GSK-3β and phosphorylated tau protein level were increased in Aβ_25–35-treated group (P < 0.05 or P < 0.01), which whole changes were attenuated by Se and Zn and Se combined Zn. In order to evaluate whether the Se and Zn have an effect on processing pathway of amyloid precursor protein (APP), we examined the activity of γ-secretase in primary cultured cortical neuron cells. ELISA analysis showed that Se and Zn could inhibit the activity of γ-secretase. Then we also investigated the effect of Se and Zn on the Aβ_1–40 concentration and APP-N-terminal fragment expression from APP695 stably transfected Chinese hamster ovary (CHO) cells. APP695 stably transfected CHO cells were treated with 0.1 μmol/L Se and Zn; cells were divided into control and four treated groups, which received either 0.5 M DAPT, 0.1 μmol/L Se, 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. Se and Zn could decrease Aβ_1–40 production and increase the APP-N-terminal fragment protein expression. These experiments indicate that Se and Zn have a protective effect on AD pathology that a possible mechanism is inhibiting the activity of γ-secretase to decreasing Aβ_1–40 production further influencing the APP processing. Altogether, our findings may provide a novel therapeutic target to treat AD sufferers.     

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.     

Fed-Batch Cultivation of Arthrospira platensis Using Carbon Dioxide from Alcoholic Fermentation and Urea as Carbon and Nitrogen Sources

To reduce CO₂ emissions from alcoholic fermentation, Arthrospira platensis was cultivated in tubular photobioreactor using either urea or nitrate as nitrogen sources at different light intensities (60 μmol m^?2 s^?1?≤?I?≤?240 μmol m^?2 s^?1). The type of carbon source (pure CO₂ or CO₂ from fermentation) did not show any appreciable influence on the main cultivation parameters, whereas substitution of nitrate for urea increased the nitrogen-to-cell conversion factor (Y _X/N ), and the maximum cell concentration (X _m ) and productivity (P _X ) increased with I. As a result, the best performance using gaseous emissions from alcoholic fermentation (X _m ?=?2,960?±?35 g m^?3, P _X ?=?425?±?5.9 g m^?3 day^?1 and Y _X/N ?=?15?±?0.2 g g^?1) was obtained at I?=?120 μmol m^?2 s^?1 using urea as nitrogen source. The results obtained in this work demonstrate that the combined use of effluents rich in urea and carbon dioxide could be exploited in large-scale cyanobacteria cultivations to reduce not only the production costs of these photosynthetic microorganisms but also the environmental impact associated to the release of greenhouse emissions.     

Voltage-Activated Elementary Calcium Release Events in Isolated Mouse Skeletal Muscle Fibers

The elementary Ca²⁺-release events underlying voltage-activated myoplasmic Ca²⁺ transients in mammalian muscle remain elusive. Here, we looked for such events in confocal line-scan (x,t) images of fluo-3 fluorescence taken from isolated adult mouse skeletal muscle fibers held under voltage-clamp conditions. In response to step depolarizations, spatially segregated fluorescence signals could be detected that were riding on a global increase in fluorescence. These discrete signals were separated using digital filtering in the spatial domain; mean values for their spatial half-width and amplitude were 1.99 ± 0.09 μm and 0.16 ± 0.005 ΔF/F ₀ (n = 151), respectively. Under control conditions, the duration of the events was limited by the pulse duration. In contrast, in the presence of maurocalcine, a scorpion toxin suspected to disrupt the process of repolarization-induced ryanodine receptor (RyR) closure, events uninterrupted by the end of the pulse were readily detected. Overall results establish these voltage-activated low-amplitude local Ca²⁺ signals as inherent components of the physiological Ca²⁺-release process of mammalian muscle and suggest that they result from the opening of either one RyR or a coherently operating group of RyRs, under the control of the plasma membrane polarization.     

Stimulation of primary osteoblasts with ATP induces transient vinculin clustering at sites of high intracellular traction force

Adenosine 5′-triphosphate (ATP), released in response to mechanical and inflammatory stimuli, induces the dynamic and asynchronous protrusion and subsequent retraction of local membrane structures in osteoblasts. The molecular mechanisms involved in the ligand-stimulated herniation of the plasma membrane are largely unknown, which prompted us to investigate whether the focal-adhesion protein vinculin is engaged in the cytoskeletal alterations that underlie the ATP-induced membrane blebbing. Using time-lapse fluorescence microscopy of primary bovine osteoblast-like cells expressing green fluorescent protein-tagged vinculin, we found that stimulation of cells with 100 μM ATP resulted in the transient and rapid clustering of recombinant vinculin in the cell periphery, starting approximately 100 s after addition of the nucleotide. The ephemeral nature of the vinculin clusters was made evident by the brevity of their mean assembly and disassembly times (66.7 ± 13.3 s and 99.0 ± 6.6 s, respectively). Traction force vector maps demonstrated that the vinculin-rich clusters were localized predominantly at sites of high traction force. Intracellular calcium measurements showed that the ligand-induced increase in [Ca²⁺]_i clearly preceded the clustering of vinculin, since [Ca²⁺]_i levels returned to normal within 30 s of exposure to ATP, indicating that intracellular calcium transients trigger a cascade of signalling events that ultimately result in the incorporation of vinculin into membrane-associated focal aggregates.     

Aluminum resistance in wheat involves maintenance of leaf Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> content,decreased lipid peroxidation and Al accumulation,and low photosystem II excitation pressure

The phytotoxic aluminum species (Al³⁺) is considered as the primary factor limiting crop productivity in over 40 % of world’s arable land that is acidic. We evaluated the responses of two wheat cultivars (Triticum aestivum L.) with differential Al resistance, cv. Yecora E (Al-resistant) and cv. Dio (Al-sensitive), exposed to 0, 37, 74 and 148 μM Al for 14 days in hydroponic culture at pH 4.5. With increasing Al concentration, leaf Ca²⁺ and Mg²⁺ content decreased, as well as the effective quantum yield of photosystem II (PSII) photochemistry (Φ _PSII ), while a gradual increase in leaf membrane lipid peroxidation, Al accumulation, photoinhibition (estimated as F _v /F _m ), and PSII excitation pressure (1 ? q _p ) occurred. However, the Al-resistant cultivar with lower Al accumulation, retained larger concentrations of Ca²⁺ and Mg²⁺ in the leaves and kept a larger fraction of the PSII reaction centres (RCs) in an open configuration, i.e. a higher ratio of oxidized to reduced quinone A (Q_A), than plants of the Al-sensitive cultivar. Four times higher Al concentration in the nutrient solution was required for Al-resistant plants (148 μM Al) than for Al-sensitive (37 μM Al), in order to establish the same closed RCs. Yet, the decline in photosynthetic efficiency in the cultivar Dio was not only due to closure of PSII RCs but also to a decrease in the quantum yield of the open RCs. We suggest that Al³⁺ toxicity may be mediated by nutrient deficiency and oxidative stress, and that Al-resistance of the wheat cultivar Yecora E, may be due at least partially, from the decreased Al accumulation that resulted to decreased reactive oxygen species (ROS) formation. However, under equal internal Al accumulation (exposure Al concentration: Dio 74 μM, Yecora E 148 μM) that resulted to the same oxidative stress, the reduced PSII excitation pressure and the better PSII functioning of the Al-resistant cultivar was probably due to the larger concentrations of Ca²⁺ and Mg²⁺ in the leaves. We propose that the different sensitivities of wheat cultivars to Al³⁺ toxicity can be correlated to differences in the redox state of Q_A. Thus, chlorophyll fluorescence measurements can be a promising tool for rapid screening of Al resistance in wheat cultivars.     

Nickel-Induced Structural and Functional Alterations in Porcine Granulosa Cells In Vitro

The present study was aimed at investigating the effect of nickel chloride (NiCl₂) on secretion of progesterone (P), ultrastructure and apoptosis in porcine granulosa cells. NiCl₂ was added to the cells to achieve a Ni²⁺ concentration of 62.5, 125, 250, 500 and 1,000 μmol/L. A control group contained no NiCl₂ addition. Quantification of P was performed directly from aliquots of the media from control and treated porcine granulosa cells after 48 h of culture using radioimmunoassay. Quantification of apoptotic cells was performed using terminal deoxynucleotidyl transferase dUTP nick end labelling assay, and ultrastructural changes were analyzed using transmission electron microscopy. A concentration-dependent depletion of P production was observed significantly for 1,000 μmol/L NiCl₂. The percentage of apoptotic cells was increased in all experimental groups significantly only after addition of 1,000 μmol/L NiCl₂. After addition of ≥250 μmol/L NiCl₂, a higher incidence of euchromatin was observed. Also, lipid droplets and vacuoles in the cytoplasm increased after addition of ≥250 μmol/L NiCl₂. NiCl₂ induced the decrease in numbers of mitochondria and smooth endoplasmic reticulum after treatment with ≥500 μmol/L NiCl₂. Our findings suggest a negative effect of NiCl₂ on steroidogenesis and apoptosis as well as ultrastructure of porcine granulosa cells.     

Plant Impact on CO<Subscript>2</Subscript> Consumption by Silicate Weathering: The Role of Bamboo

CO₂ consumption by silicate weathering has exerted a major control on atmospheric CO₂ over geologic time. In order to assess plant impact on this process, the study compared water geochemistry and CO₂ consumption rates by silicate weathering in watersheds covered by bamboos and other forests. Our study showed that SiO₂ concentrations (80?~?150 μmol/L, average 105 μmol/L) in water from pure bamboo forest watersheds were higher than that (15?~?85 μmol/L, average 60 μmol/L) from other watersheds. Si/(Na_silicate?+?K_silicate) ratios in water draining from bamboo watersheds (2.0?~?4.0, average 2.9) were higher than that from other watersheds ?>(0.7?~?2.7, average 2.2). CO₂ consumption rates by silicate weathering in bamboo watersheds (1.8?~?3.4 10⁵ mol/km²/yr, average 2.5 10⁵ mol/km²/yr) were higher than that in other watersheds (1.5?~?2.6 10⁵ mol/km²/yr, average 2.0 10⁵ mol/km²/yr). Therefore, bamboo-enhanced silicate weathering is a potential biogeochemical remediation approach for atmospheric CO₂.     

Oxidative Stress and Ca2+ Signals Involved on Cadmium-Induced Apoptosis in Rat Hepatocyte

Cadmium (Cd) is an important industrial and environmental pollutant. In animals, the liver is the major target organ of Cd toxicity. In this study, rat hepatocytes were treated with 2.5～10 μM Cd for various durations. Studies on nuclear morphology, chromatin condensation, and apoptotic cells demonstrate that Cd concentrations ranging within 2.5～10 μM induced apoptosis. The early-stage marker of apoptosis, i.e., decreased mitochondrial membrane potential, was observed as early as 1.5 h at 5 μM Cd. Significant (P?P?2+ concentration ([Ca²⁺] _i ) of Cd-exposed cells significantly increased (P?2+] _i may play an important role in apoptosis. Overall, these results showed that oxidative stress and Ca²⁺ signaling were critical mediators of the Cd-induced apoptosis of rat hepatocytes.     

Effects of Cadmium Stress on Leaf Chlorophyll Fluorescence and Photosynthesis of Elsholtzia argyi—A Cadmium Accumulating Plant

A hydroponic experiment was conducted to investigate the effects of cadmium (Cd) on chlorophyll fluorescence and photosynthetic parameters on a Cd accumulating plant of Elsholtzia argyi. Four weeks-seedlings of E. argyi were treated with 0 (CK) 5, 10, 15, 20, 25, 30, 40, 50 and 100 μmol L^?1 Cd for 21days. Fv/Fo, Fv/Fm, qP, ΦPSП, ETR and Fv′/Fm′ were significantly increased under low Cd (5–15 μmol L^?1 for Fv/Fo, Fv/Fm and qP, 5–10 μmol L^?1 for ΦPSП, ETR and Fv′/Fm′) stress, and these parameters were similar to control under Cd ≤ 50μmol L^?1. All above parameters were significantly decreased at 100 μmol L^?1 Cd. Compared with control, Pn was significantly (P < 0.05) increased under 5–30 μmol L^?1 Cd. However, 50 and 100 μmol L^?1 Cd significantly (P < 0.05) reduced it. Gs and Tr were substantially decreased at 50–100 and 40–100 μmol L^?1 Cd, respectively. Ci was significantly increased at 50 and 100 μmol L^?1 Cd. High Cd-induced decrease of Pn is not only connected to stomatal limitation but also to the inhibition of Fv/Fo, Fv/Fm, ΦPSП, qP, ETR and increase of NPQ. Maintain chlorophyll fluorescence and photosynthesis parameters under its Cd tolerance threshold were one of tolerance mechanisms in E. argyi.     

Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na⁺ reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 μmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na⁺ current by 52 % and increased the membrane density of Na⁺ channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 μmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na⁺ reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage.     

N-acetylcysteine-induced vasodilation involves voltage-gated potassium channels in rat aorta

AimsN-acetylcysteine (NAC) has a protective effect against vascular dysfunction by decreasing the level of reactive oxygen species (ROS) in experimental and human hypertension. This study was designed to examine whether NAC would relax vascular rings in vitro via nitric oxide–cyclic guanosine monophosphate (NO–cGMP) pathway, extracellular Ca²⁺ and/or K⁺ channels.Main methodsRat aortic arteries were mounted in an organ bath, contracted with 0.1, 0.5 or 1 µmol/L phenylephrine to plateau, and the vasodilatory effect of NAC was examined in the absence or presence of ROS scavengers, inhibitors of NO–cGMP pathway or K⁺ channels. Vascular smooth muscle cells (VSMCs) were loaded with a calcium sensitive fluorescent dye fluo-3 AM, and [Ca²⁺]_i was determined with laser-scanning confocal microscopy.Key findingsNAC (0.1–4 mmol/L) dose-dependently relaxed rat aorta pre-contracted with phenylephrine. Endothelium removal, endothelial nitric oxide synthase inhibitor N^ω-Nitro-l-arginine (L-NNA) (100 µmol/L) or soluble guanylyl cyclase (sGC) inhibitor (ODQ) (10 µmol/L) did not affect NAC-induced vasodilation. In contrast, NAC-induced vasodilation was blunted after extracellular calcium was removed and calcium imaging showed that 4 mmol/L NAC quickly decreased [Ca²⁺]_i in fluo-3 AM loaded VSMCs. NAC-induced vasodilation was significantly reduced in the presence of voltage-gated K⁺ channels (Kv) inhibitor 4-aminopyridine (4-AP).SignificanceThe vasodilatory effect of NAC may be explained at least partly by activation of voltage-gated K⁺ channels.     

Biochemical characteristics of the Ca2+ pumping ATPase in the peribacteroid membrane from broad bean root nodules

Ca²⁺-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 Ca²⁺ through the PBM by the Ca²⁺-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP?>?ITP???GTP???UTP???CTP, the K _m 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 Ca²⁺-ATPase for Ca²⁺ of about 0.1 μM. The transport activity of Ca²⁺-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 Ca²⁺-ATPases which are present in other plant membranes.     

Alterations in peroxidase activity and phenylpropanoid metabolism induced by Nacobbus aberrans Thorne and Allen, 1944 in chilli (Capsicum annuum L.) CM334 resistant to Phytophthora capsici Leo.

Chilli CM334 (Capsicum annuum L.) is resistant to Phytophthora capsici Leonian (Pc), but Nacobbus aberrans Thorne and Allen, 1944 (Na) broke down its resistance in plants previously infected by the nematode. Peroxidase (POD) and L-phenylalanine ammonia-lyase (PAL) activity, total soluble phenols (TSP) and chlorogenic acid concentration in CM334 plants inoculated with either or both pathogens (Na-Pc) were compared; also, the toxic effect of some phenolic acids on Na was tested in vitro. The highest POD activity (5.3 μM tetraguaiacol mg^?1 protein min^?1) was registered in plants inoculated only with Pc, while those inoculated only with Na showed the lowest (3.3 μM) (P?≤?0.05). PAL activity was 39.9 nM trans-cinnamic acid μg^?1 protein min^?1 in plants inoculated only with Pc, and it was lower (19.3 nM) and similar in non-inoculated plants or those with Na and with Na-Pc (P?≤?0.05). Usually, plants inoculated with Pc alone had higher contents of TSP (P?≤?0.05) (1.9 mg tannic acid g^?1 dry matter) and plants inoculated with Na or Na-Pc had lower levels (0.8 and 0.9 mg) than those non-inoculated (1.3 mg). CM334 plants inoculated with Na showed a significant reduction (10–37% and 12–17%, in roots and leaves) in the concentration of chlorogenic acid as compared to the non-inoculated. Vanillic, trans-cinnamic, p-coumaric and syringic acids had greater nematicidal effects (P?≤?0.05) than chlorogenic acid in vitro. Apparently Na modified the defence responses in CM334 plants as POD and PAL activities and TSP and chlorogenic acid concentrations were reduced.     

Role of pertussis toxin-sensitive G-protein, K+ channels, and voltage-gated Ca2+ channels in the antinociceptive effect of inosine

Inosine is the first metabolite of adenosine. It exerts an antinociceptive effect by activating the adenosine A₁ and A_2A receptors. We have previously demonstrated that inosine exhibits antinociceptive properties in acute and chronic mice models of nociception. The aim of this study was to investigate the involvement of pertussis toxin-sensitive G-protein-coupled receptors, as well as K⁺ and Ca²⁺ channels, in the antinociception promoted by inosine in the formalin test. Mice were pretreated with pertussis toxin (2.5 μg/site, i.t., an inactivator of G_i/0 protein); after 7 days, they received inosine (10 mg/kg, i.p.) or morphine (2.5 mg/kg, s.c., used as positive control) immediately before the formalin test. Another group of animals received tetraethylammonium (TEA) or 4-aminopyridine (4-AP) (1 μg/site, i.t., a non-specific voltage-gated K⁺ channel blockers), apamin (50 ng/site, i.t., a small conductance Ca²⁺-activated K⁺ channel blocker), charybdotoxin (250 pg/site, i.t., a large-conductance Ca²⁺-activated K⁺ channel blocker), glibenclamide (100 μg/site, i.t., an ATP-sensitive K⁺ channel blocker) or CaCl₂ (200 nmol/site, i.t.). Afterwards, the mice received inosine (10 mg/kg, i.p.), diclofenac (10 mg/kg, i.p., a positive control), or morphine (2.5 mg/kg, s.c., a positive control) immediately before the formalin test. The antinociceptive effect of inosine was reversed by the pre-administration of pertussis toxin (2.5 μg/site, i.t.), TEA, 4-aminopyridine, charybdotoxin, glibenclamide, and CaCl₂, but not apamin. Further, all K⁺ channel blockers and CaCl₂ reversed the antinociception induced by diclofenac and morphine, respectively. Taken together, these data suggest that the antinociceptive effect of inosine is mediated, in part, by pertussis toxin-sensitive G-protein coupled receptors and the subsequent activation of voltage gated K⁺ channel, large conductance Ca²⁺-activated and ATP-sensitive K⁺ channels or inactivation of voltage-gated Ca²⁺ channels. Finally, small conductance Ca²⁺-activated K⁺ channels are not involved in the antinociceptive effect of inosine.     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.