Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Effects of Ca2+ on ethanolaminephosphotransferase and cholinephosphotransferase in rabbit platelets

The effects of Ca2+ on ethanolaminephosphotransferase [EC 2.7.8.1] and cholinephosphotransferase [EC 2.7.8.2] activities in rabbit platelet membranes were studied using endogenous diglyceride and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. Both transferases required Mn2+, Co2+, or Mg2+ as a metal cofactor and the optimal concentrations of the metals for both activities were about 5, 10, and 5 mM, respectively. When 5 mM Mg2+ was used as a cofactor, both transferase activities were inhibited by a low concentration of Ca2+ (half maximal inhibition at approx. 15 microM). In the presence of 5 mM Mn2+, however, approx. 5 mM Ca2+ was required to produce half maximal inhibition. The Ca2+-induced inhibition was reversible and the rate of the inhibition was not affected either by the concentrations of the CDP-compound or by exogenously added diacylglycerol. The relationship between Ca2+ and both Mg2+ and Mn2+ on the transferase activities was competitive. 45Ca2+ binding (and/or uptake) to the platelet membranes was inhibited by Mn2+, Mg2+, and Co2+, in a concentration-dependent manner. However, the inhibitory effects of the three metal ions on the total Ca2+ binding (and/or uptake) did not correlate with the activation of both transferase activities by the three metal ions in the presence of Ca2+. These results suggest that both transferase activities are regulated by low concentrations of Ca2+ in the presence of optimal concentrations of Mg2+, and that the inhibition is mediated directly by Ca2+, which interacts with a specific metal cofactor binding site(s) of the transferases.     

Effect of NO3- transport and reduction on intracellular pH: an in vivo NMR study in maize roots

The effect of NO3- uptake on cellular pH was studied in maize roots by an in vivo 31P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO3- uptake from those due to NO3- reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO3- uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO3- uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO3- uptake is an acidifying process and suggest a possible involvement of NO3- reduction in pH homeostasis. In the presence of NO3-, a transient depolarization of transmembrane electric potential difference (Em) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover Em was found. This was related to a higher activity of the plasma membrane (PM) H+-ATPase. When NO3- was administered as potassium salt, its uptake increased and a greater depolarization of Em took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K+ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H+-ATPase expression during nitrate exposure is suggested.     

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.     

Improved use of organic phosphate by Skeletonema costatum through regulation of Zn2 + concentrations

The maximum growth rate (1.4-2 x 10(5) cells ml(-1) d(-1)), cell final yields (2.6-5.2 x 10(5) cells ml(-1)) and extracellular alkaline phosphatase activity (2.4-10.6 microg phosphate released ml(-1) h(-1)) of the red tide alga, Skeletonema costatum, increased when Zn2+ was increased from 0 to 24 pM, but decreased with 66 pM Zn2+ in growth medium with glycerophosphate as the sole phosphorus source. Extracellular carbonic anhydrase activity and the affinity for HCO3- and CO2 uptake increased when Zn2+ was increased from 0 to 12 pM, but then decreased at higher concentrations. The results suggested that utilization of organic phosphate required more Zn2+ than the uptake of inorganic carbon did, while utilization of dissolved inorganic carbon by Skeletonema costatum was very sensitive to Zn2+ concentration variations.     

Capsaicin inhibits platelet-activating factor-induced cytosolic Ca2+ rise and superoxide production

Platelet-activating factor (PAF) is an important participant in the inflammatory process. We studied the regulation of PAF activity by capsaicin in human promyelocytic leukemia HL-60 cells. Capsaicin inhibited PAF-induced superoxide production in a concentration-dependent manner. In addition to PAF, the fMLP- and extracellular ATP-induced superoxide productions were inhibited by capsaicin, whereas PMA-induced superoxide production was not affected. In the PAF-stimulated cytosolic Ca2+ increase, capsaicin inhibited in particular the sustained portion of the raised Ca2+ level without attenuation of the peak height. In the absence of extracellular Ca2+, the PAF-induced Ca2+ elevation was not inhibited by capsaicin because capsaicin only inhibited the Ca2+ influx from the extracellular space. In addition, capsaicin did not affect PAF-induced inositol 1,4,5-trisphosphate production, suggesting that phospholipase C activation by PAF is not affected by capsaicin. Store-operated Ca2+ entry (SOCE) induced by thapsigargin was inhibited by capsaicin in a concentration-dependent manner. This capsaicin effect was also observed on thapsigargin-induced Ba2+ and Mn2+ influx. Furthermore, capsaicin's inhibitory effect on the thapsigargin-induced Ca2+ rise overlapped with that of SK&F96365, an inhibitor of SOCE. Both capsaicin and SK&F96365 also inhibited PAF-induced cytosolic superoxide generation in HL-60 cells differentiated by all-trans-retinoic acid. Our data suggest that capsaicin exerts its anti-inflammatory effect by inhibiting SOCE elicited via PLC activation, which occurs upon PAF activation and results in the subsequent superoxide production.     

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.     

Functional expression of the human hZIP2 zinc transporter

Zinc is an essential nutrient for humans, yet we know little about how this metal ion is taken up by mammalian cells. In this report, we describe the characterization of hZip2, a human zinc transporter identified by its similarity to zinc transporters recently characterized in fungi and plants. hZip2 is a member of the ZIP family of eukaryotic metal ion transporters that includes two other human genes, hZIP1 and hZIP3, and genes in mice and rats. To test whether hZip2 is a zinc transporter, we examined (65)Zn uptake activity in transfected K562 erythroleukemia cells expressing hZip2 from the CMV promoter. hZip2-expressing cells accumulated more zinc than control cells because of an increased initial zinc uptake rate. This activity was time-, temperature-, and concentration-dependent and saturable with an apparent K(m) of 3 microM. hZip2 zinc uptake activity was inhibited by several other transition metals, suggesting that this protein may transport other substrates as well. hZip2 activity was not energy-dependent, nor did it require K(+) or Na(+) gradients. Zinc uptake by hZip2 was stimulated by HCO(3)(-) treatment, suggesting a Zn(2+)-HCO(3)(-) cotransport mechanism. Finally, hZip2 was exclusively localized in the plasma membrane. These results indicate that hZip2 is a zinc transporter, and its identification provides one of the first molecular tools to study zinc uptake in mammalian cells.     

Nitrate Reductase from the Marine Diatom Skeletonema costatum (Biochemical and Immunological Characterization)

Assimilatory nitrate reductase (NR) was purified from the marine diatom Skeletonema costatum (clone Skel) using Cibacron blue-Sepharose affinity chromatography. The single-step purification scheme yielded a 103-fold purification of specific activity with an overall recovery of 40.8%. Only NADH-dependent NR activity (form EC 1.6.6.1) was observed in this species. Kinetic analysis revealed that this form had apparent Michaelis constants of 3.6 [mu]M for NADH and 295 [mu]M for NO3- when purified from cells grown in NO3--enriched seawater. The S. costatum NR exhibits a pH optimum of 7.4, a temperature optimum of 14[deg]C, and enzyme activity not sensitive to Mg2+ inhibition. The strong temperature dependence of NR activity in S. costatum may contribute to the seasonal and latitudinal distributions and abundances of this bloom-forming species. Chromatographically isolated NR was further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single polypeptide with an apparent molecular mass of 110 kD. The 110-kD polypeptide was used to generate polyclonal antibodies. The antiserum recognized a single 110-kD polypeptide in western blots of total proteins from S. costatum, as well as the native enzyme. Western blot analysis also revealed an antigenic similarity of NR from two additional diatom species, whereas no cross-reactivity was observed with NR from other phytoplankton taxa, including prymnesiophytes, dinoflagellate, cyanobacterium, and green alga. This result suggests a structural diversity of NR in phytoplankton and identifies the potential for development of taxon-specific NR antisera for ecological studies.     

Effect of miconazole on intracellular Ca2+ levels and proliferation in human osteosarcoma cells

The effect of miconazole, an anti-fungal drug, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in human osteosarcoma cells (MG63) was explored by using the Ca2+-sensitive dye fura-2. Miconazole acted in a concentration-dependent manner with an EC50 of 75 microM. The Ca2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca2+ reduced 50% of the signal. In Ca2+-free medium, the [Ca2+]i rise induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) was completely inhibited by pretreatment with 20 microM miconazole. Pretreatment with thapsigargin partly inhibited miconazole-induced Ca2+ release. The miconazole-induced Ca2+ release was not changed by inhibition of phospholipase C with 2 microM U73122. By using tetrazolium as a fluorescent probe, it was shown that 10-100 microM miconazole decreased cell proliferation rate in a concentration-dependent manner. Collectively, this study shows that miconazole induces [Ca2+]i rises in human osteosarcoma cells via releasing Ca2+ mainly from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. Furthermore, miconazole may be cytotoxic to the cells at higher concentrations.     

Nordihydroguaiaretic acid-induced Ca2+ handling and cytotoxicity in human prostate cancer cells

The effect of nordihydroguaiaretic acid (NDGA), a compound commonly used as a lipoxygenases inhibitor, on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells was investigated. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. NDGA increased [Ca2+]i in a concentration-dependent manner with an EC50 of 30 microM. The Ca2+ signal comprised a gradual and sustained increase. Removal of extracellular Ca2+ partly decreased the NDGA-induced [Ca2+]i increase, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and intracellular Ca2+ release. NDGA-induced Ca2+ influx was independently confirmed by measuring NDGA-induced Mn2+ -coupled quench of fura-2 fluorescence. The NDGA-induced Ca2+ influx was not affected by L-type Ca2+ channel blockers. In Ca2+ -free medium, the NDGA-induced [Ca2+]i increase was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with NDGA abolished thapsigargin-induced [Ca2+]i increase. NDGA-induced intracellular Ca2+ release was not altered by inhibition of phospholipase C. Overnight treatment with 20-50 microM NDGA inhibited cell proliferation rate in a concentration-dependent manner. Several other lipoxygenases inhibitors did not alter [Ca2+]i. Collectively, this study shows that in prostate cells, NDGA induced a [Ca2+]i increase via releasing stored Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. NDGA also caused cytotoxicity at higher concentrations.     

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory effect of eugenol on Cu2+-catalyzed lipid peroxidation in human erythrocyte membranes

1. The effects of eugenol on lipid peroxidation catalyzed by hydrogen peroxide (H2O2) or benzoyl peroxide (BPO) in the presence of copper ions were studied in human erythrocyte membranes. 2. The production of hydroxyl radicals was suggested in the peroxidation system catalyzed by H2O2/Cu2+. 3. H2O2/Cu2+-dependent peroxidation was inhibited by eugenol in a concentration-dependent manner; peroxidation was inhibited 62% by 200 microM eugenol. 4. In the presence of eugenol, the peroxidation catalyzed by BPO/Cu2+ was inhibited in a concentration-dependent manner, and more than 100 microM eugenol completely inhibited peroxidation. 5. The inhibitory effect of eugenol was non-competitive against Cu2+ in H2O2/Cu2+- and BPO/Cu2+-dependent peroxidation. 6. It is suggested that eugenol inhibits formation of hydroxyl radicals.     

Decreased sensitivity of atopic mononuclear cells to prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2)

The effect of PGE2 and PGD2 on several lymphocyte functions in vitro was evaluated in nonatopic and atopic subjects. Both PGE2 and PGD2 inhibited phytohemagglutinin-induced protein synthesis ([3H] leucine uptake) by nonatopic mononuclear cells and T cells in a dose-dependent manner (10(-6) to 10(-12) M). Protein synthesis by atopic mononuclear cells was not significantly suppressed by the above concentration of PGE2. Although PGD2 effectively suppressed protein synthesis by atopic mononuclear cells and T cells at 10(-6) M, lower concentrations were ineffective. Kinetic studies revealed significant differences in the suppressive effects of PGE2 and PGD2 on atopic and nonatopic mononuclear cells at 24 and 48 h, but not at 72 or 96 hr. Protein synthesis by T helper-enriched populations (suppressor cell depletion by anti-Leu-2b + complement) obtained from nonatopics was significantly reduced by PGE2 and PGD2, suggesting that these mediators may be directly inhibiting the responding population. By contrast, protein synthesis by T suppressor-enriched populations (helper cell depletion by OKT4 + complement) obtained from nonatopics was enhanced by PGE2 and PGD2, suggesting that the PG were activating these cells. Atopic T helper and T suppressor cells exhibited decreased responsiveness to PGE2 and PGD2 compared with nonatopic cells. PGE2 and PGD2 inhibited the phytohemagglutinin-stimulated proliferative response ([3H]thymidine uptake) by both atopic and nonatopic mononuclear cells in a dose-dependent manner and to the same extent. However, although PGE2 and PGD2 generated functional suppressor activity (when using a coculture technique) in nonatopic mononuclear cells, these mediators failed to activate atopic suppressor cells. These results suggest that reduced responses by atopic T cells to signals provided by PGE2 and PGD2 are not solely restricted to suppressor cell function, and could indicate an impaired ability to regulate immune and/or inflammatory reactions.     

Inositol trisphosphate accumulation by high K+ stimulation in cultured adrenal chromaffin cells

Stimulation with high K+ (KCl, 56 mM) of myo-[3H]inositol-prelabelled cells increased Ca2+ uptake and [3H]inositol trisphosphate (IP3) accumulation in a concentration-dependent manner. Nifedipine, a Ca2+ channel antagonist, inhibited high K+-induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Furthermore, ionomycin (1 microM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation. These results indicate the existence of the Ca2+ uptake-triggered mechanism of IP3 formation in cultured adrenal chromaffin cells.     

Inhibition of red cell Ca2+-dependent K+ channels by snake venoms

We have investigated the effects of several snake venoms on the Ca2+-dependent K+ channels of human red cells. A heat-resistant component of the venom of the snake Notechis scutatus irreversibly inhibited Ca2+-dependent K+ transport with a Ki value of 0.1-0.2 micrograms/ml. Metabolic changes of the cells modified the maximal effect of the venom. Binding of the venom required extracellular Ca2+ and was quick, but development of full inhibition required additional time. The effects of the venoms from Notechis scutatus and Leiurus quinquestriatus were additive, suggesting that both venoms act through different mechanisms. Venoms of the snakes Vipera russelli russelli and Oxyuranus scutellatus also inhibited Ca2+-dependent K+ transport with the same characteristics as the Notechis scutatus venom.     

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.     

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.     

Effects of peroxynitrite on sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle

Peroxynitritegenerated in arteries from superoxide and NO may damageCa²⁺ pumps. Here, we report the effects of peroxynitrite onATP-dependent azide-insensitive uptake of Ca²⁺ into pigcoronary artery vesicular membrane fractions F2 [enriched in plasmamembrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)].Membranes were pretreated with peroxynitrite and then with DTT toquench this agent. This pretreatment inhibited Ca²⁺ uptakein a peroxynitrite concentration-dependent manner, but the effect wasmore severe in F3 than in F2. The inhibition was thus not overcome byexcess DTT used to quench peroxynitrite and was not affected ifcatalase, SOD, or mannitol was added along with peroxynitrite. Suchdamage to the pump protein would be difficult to repair if producedduring ischemia-reperfusion. The acylphosphates formed with ATPin F3 corresponded mainly to the SR Ca²⁺ pump (110 kDa),but in F2 both PM (140 kDa) and 110-kDa bands were observed.Peroxynitrite treatment of F2 inhibited only the 110-kDa band.Inhibition of Ca²⁺ uptake and acylphosphate formation fromATP correlated well in peroxynitrite-treated F3 samples. However,inhibition of acylphosphates from orthophosphate (reverse reaction ofthe pump) was slightly poorer. Peroxynitrite treatment also covalentlycross-linked the pump protein, yielding no dimers but only largeroligomers. In contrast, cross-linking of the SR Ca²⁺ pumpin skeletal and cardiac muscles gives dimers as the first oligomers.Therefore, we speculate that SERCA2 has a different quaternarystructure in the coronary artery smooth muscle.