dl-Buthionine-S,R-sulfoximine affects intestinal alkaline phosphatase activity

The susceptibility of intestinal alkaline phosphatase to dl-buthionine-S,R-sulfoximine was investigated in chicks fed a commercial diet. The results show that dl-buthionine-S,R-sulfoximine produced inhibition of intestinal alkaline phosphatase activity. This effect showed dose- and time-dependency and it was caused by either in vivo dl-buthionine-S,R- sulfoximine administration or in vitro dl-buthionine-S,R-sulfoximine incubation with villus tip enterocytes. dl-Buthionine-S,R-sulfoximine did not act directly on intestinal alkaline phosphatase but it provoked glutathione depletion which led to changes in the redox state of the enterocyte as shown by the production of free hydroxyl radicals and an incremental increase in the carbonyl content of proteins. The reversibility of the buthionine sulfoximine effect on intestinal alkaline phosphatase was proved by addition of glutathione monoester to the duodenal loop.     

Sodium deoxycholate inhibits chick duodenal calcium absorption through oxidative stress and apoptosis

High concentrations of sodium deoxycholate (NaDOC) produce toxic effects. This study explores the effect of a single high concentration of NaDOC on the intestinal Ca(2+) absorption and the underlying mechanisms. Chicks were divided into two groups: 1) controls and 2) treated with different concentrations of NaDOC in the duodenal loop for variable times. Intestinal Ca(2+) absorption was measured as well as the gene and protein expressions of molecules involved in the Ca(2+) transcellular pathway. NaDOC inhibited the intestinal Ca(2+) absorption, which was concentration dependent. Ca(2+)-ATPase mRNA decreased by the bile salt and the same occurred with the protein expression of Ca(2+)-ATPase, calbindin D(28k) and Na(+)/Ca(2+) exchanger. NaDOC produced oxidative stress as judged by ROS generation, mitochondrial swelling and glutathione depletion. Furthermore, the antioxidant quercetin blocked the inhibitory effect of NaDOC on the intestinal Ca(2+) absorption. Apoptosis was also triggered by the bile salt, as indicated by the TUNEL staining and the cytochrome c release from the mitochondria. As a compensatory mechanism, enzyme activities of the antioxidant system were all increased. In conclusion, a single high concentration of NaDOC inhibits intestinal Ca(2+) absorption through downregulation of proteins involved in the transcellular pathway, as a consequence of oxidative stress and mitochondria mediated apoptosis.     

Oxidative stress studied in intact mammalian cells

Exposure of isolated rat hepatocytes to toxic doses of menadione (2-methyl-1,4-naphthoquinone) results in enhanced formation of active oxygen species, depletion of cellular glutathione and protein thiols, and perturbation of intracellular calcium ion homeostasis. An increase in cytosolic Ca2+ concentration, resulting from inhibition of the plasma membrane Ca2+ translocase by menadione metabolism, appears to be critically involved in the development of cytotoxicity.     

Mitochondrial dysfunction is responsible for the intestinal calcium absorption inhibition induced by menadione

Menadione (MEN) inhibits intestinal calcium absorption by a mechanism not completely understood. The aim of this work was to find out the role of mitochondria in this inhibitory mechanism. Hence, normal chicks treated with one i.p. dose of MEN were studied in comparison with controls. Intestinal calcium absorption was measured by the in situ ligated intestinal segment technique. GSH, oxidoreductase activities from the Krebs cycle and enzymes of the antioxidant system were measured in isolated mitochondria. Mitochondrial membrane potential was measured by a flow cytometer technique. DNA fragmentation and cytochrome c localization were determined by immunocytochemistry. Data indicate that in 30 min, MEN decreases intestinal Ca(2+) absorption, which returns to the control values after 10 h. GSH was only decreased for half an hour, while the activity of malate dehydrogenase and alpha-ketoglutarate dehydrogenase was diminished for 48 h. Mn(2+)-superoxide dismutase activity was increased in 30 min, whereas the activity of catalase and glutathione peroxidase remained unaltered. DNA fragmentation and cytochrome c release were maximal in 30 min, but were recovered after 15 h. In conclusion, MEN inhibits intestinal Ca(2+) absorption by mitochondrial dysfunction as revealed by GSH depletion and alteration of the permeability triggering the release of cytochrome c and DNA fragmentation.     

Lack of relationship between activity of intestinal alkaline phosphatase and calcium or phosphate absorption

The effects of vitamin D3 and the aqueous extract of Solanum malacoxylon on intestinal alkaline phosphatase and tissue phosphate content were studied on rachitic chicks treated with large doses of ethane-1-hydroxy-1,1 diphosphonate (EHDP). The EHDP treatment blocks the increase of intestinal calcium or phosphate absorption induced by the vitamin D3, while it has no effects on the rise of intestinal alkaline phosphatase activity or the increment in tissue phosphate content. The lack of correlation between the increment of alkaline phosphatase and that of Ca or phosphate absorption in vitamin D3 plus EHDP treated chicks excludes a participation of the alkaline phosphatase in the mechanism of Ca or P intestinal absorption. The Ca or phosphorus absorption are elicited specifically by 1,25-(OH)2-D3, while alkaline phosphatase activity and phosphate tissue concentration respond to a broader spectrum of stimuli.     

Antioxidant and antiapoptotic properties of melatonin restore intestinal calcium absorption altered by menadione

The intestinal Ca²⁺ absorption is inhibited by menadione (MEN) through oxidative stress and apoptosis. The aim of this study was to elucidate whether the antioxidant and antiapoptotic properties of melatonin (MEL) could protect the gut against the oxidant MEN. For this purpose, 4-week-old chicks were divided into four groups: (1) controls, (2) treated i.p. with MEN (2.5 μmol/kg of b.w.), (3) treated i.p. with MEL (10 mg/kg of b.w.), and (4) treated with 10 mg MEL/kg of b.w after 2.5 μmol MEN/kg of b.w. Oxidative stress was assessed by determination of glutathione (GSH) and protein carbonyl contents as well as antioxidant enzyme activities. Apoptosis was assayed by the TUNEL technique, protein expression, and activity of caspase 3. The data show that MEL restores the intestinal Ca²⁺ absorption altered by MEN. In addition, MEL reversed the effects caused by MEN such as decrease in GSH levels, increase in the carbonyl content, alteration in mitochondrial membrane permeability, and enhancement of superoxide dismutase and catalase activities. Apoptosis triggered by MEN in the intestinal cells was arrested by MEL, as indicated by normalization of the mitochondrial membrane permeability, caspase 3 activity, and DNA fragmentation. In conclusion, MEL reverses the inhibition of intestinal Ca ²⁺ absorption produced by MEN counteracting oxidative stress and apoptosis. These findings suggest that MEL could be a potential drug of choice for the reversal of impaired intestinal Ca ²⁺ absorption in certain gut disorders that occur with oxidative stress and apoptosis.     

Aluminium-induced impairment of transcellular calcium absorption in the small intestine: calcium uptake and glutathione influence

Aluminium (Al) has been recognised as a cause of bone tissue disorders. The aims of this work were to investigate: (i) whether Al affects calcium (Ca) entry into enterocyte, and (ii) the possibility that the Al effect upon calbindin-D-related Ca transport would be influenced by intestinal glutathione (GSH) levels. In isolated chicken duodenal enterocytes, 100 microM Al lactate produced a decrease in both, the maximum uptake rate and the affinity constant of 45Ca uptake (CaUPT). This effect of Al on CaUPT was concentration-dependent in the micromolar range, showing an inhibitory saturation type phenomenon which appeared to be higher at pH 6.5 than at pH 7.4, and was not modified by the Ca channel activators A23187 and capsaicin. The simultaneous administration of Al (50 mg elemental Al/kg body weight, as AlCl3) and GSH (10 mmol/kg body weight) to rats during 7 days, prevented the inhibitory effects of Al on Ca transport. The protective effect of GSH was accompanied by an increased duodenal calbindin-D9k expression. Experimental depletion of intestinal GSH by means of D,L-buthionin-[S,R] sulfoximine, a gamma-glutamylcystein-synthase inhibitor, given as a single i.p. dose of 2 mmol/kg body weight, enhanced the degree of reduction of Ca absorption ascribed to Al. Our results suggest that Al might interfere Ca uptake by enterocytes through a general effect on cell membrane, and that an oxidative stress state induced by Al would reduce intestine GSH level affecting calbindin-D function and/or synthesis, thus leading to a reduced transcellular Ca absorption in the small intestine.     

Store-operated calcium entry in human oocytes and sensitivity to oxidative stress

Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca(2+) influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca(2+) stores through inhibition of sarco(endo)plasmic Ca(2+)-ATPase with thapsigargin triggers Ca(2+) entry in resting human oocytes. Ba(2+) and Mn(2+) influx was also stimulated following inhibition, and Ca(2+) entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca(2+) entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca(2+)](i) that was dependent on the presence of extracellular Ca(2+). This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF.     

Mutual antagonism of calcium entry by capacitative and arachidonic acid-mediated calcium entry pathways

In nonexcitable cells, the predominant mechanism for regulated entry of Ca(2+) is capacitative calcium entry, whereby depletion of intracellular Ca(2+) stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca(2+) entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca(2+) pumps, depletion of Ca(2+) stores, or entry of Ca(2+) through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca(2+) overload.     

Inhibition of hepatocyte plasma membrane Ca2+-ATPase activity by menadione metabolism and its restoration by thiols

Incubation of isolated rat hepatocytes with cytotoxic concentrations of menadione resulted in inhibition of plasma membrane Ca2+-ATPase activity. This could be restored by subsequent treatment with either dithiothreitol or reduced glutathione, suggesting that the inhibition by menadione was due to oxidation of sulfhydryl groups critical for Ca2+-ATPase activity.     

Oxidative stress in Ca2+‐induced membrane permeability transition in brain mitochondria

Mitochondrial permeability transition (PT) is a non-selective inner membrane permeabilization, typically promoted by the accumulation of excessive quantities of Ca(2+) ions in the mitochondrial matrix. This phenomenon may contribute to neuronal cell death under some circumstances, such as following brain trauma and hypoglycemia. In this report, we show that Ca(2+)-induced brain mitochondrial PT was stimulated by Na(+) (10 mM) and totally prevented by the combination of ADP and cyclosporin A. Removal of Ca(2+) from the mitochondrial suspension by EGTA or inhibition of Ca(2+) uptake by ruthenium red partially reverted the dissipation of the membrane potential associated with PT. Ca(2+)-induced brain mitochondrial PT was significantly inhibited by the antioxidant catalase, indicating the participation of reactive oxygen species in this process. An increased detection of reactive oxygen species, measured through dichlorodihydrofluorescein oxidation, was observed after mitochondrial Ca(2+) uptake. Ca(2+)-induced dichlorodihydrofluorescein oxidation was enhanced by Na(+) and prevented by ADP and cyclosporin A, indicating that PT enhances mitochondrial oxidative stress. This could be at least in part a consequence of the extensive depletion in NAD(P)H that accompanied this Ca(2+)-induced mitochondrial PT. NADPH is known to maintain the antioxidant function of the glutathione reductase/peroxidase and thioredoxin reductase/peroxidase systems. In addition, the occurrence of mitochondrial PT was associated with membrane lipid peroxidation. We conclude that PT further increases Ca(2+)-induced oxidative stress in brain mitochondria leading to secondary damage such as lipid peroxidation.     

The relations between intestinal alkaline phosphatase and carbohydrates with regard to calcium absorption.

The effect of carbohydrates on calcium absorption were studied in situ following the injection of a solution containing CaCl2 (+45Ca) into the ileal loop. The increase in Ca absorption was proportional to the concentration of carbohydrates injected and could be attributed to a progressive increase in the duration of absorption. In the ileal loop, sorbitol was much more effective than L-arabinose at equal concentrations in activating absorption. Such differences in the action of these carbohydrates were also observed in vitro with alkaline phosphatase extracted from the ileum. The transphosphorylating effect of the enzyme was much more pronounced in the case of sorbitol. Since the carbohydrate is a phosphate acceptor, it might influence the duration of absorption by reducing the inhibition exerted by phosphate upon a transfer mechanism which involves phosphatase, another possibility is that carbohydrate could postpone calcium insolubility through the formation of a phosphocarbohydrate complex.     

Menadione increases hepatic tight-junctional permeability. Its effect can be decreased by butylated hydroxytoluene and verapamil.

Infusion of menadione at two different doses [2.7 mg and 5.5 mg in 100 microliters of dimethyl sulphoxide (DMSO)] into perfused rat livers for 30 min caused no or a 6-fold increase respectively in junctional permeability to horseradish peroxidase as compared with controls receiving 100 microliters of DMSO alone. The total glutathione (GSH) contents in these livers measured at the end of the experiments were 115% and 53%, compared with the controls. The free-radical scavenger butylated hydroxytoluene (BHT) (final concn. 5 microM) protected against the GSH depletion caused by the higher dose of menadione and partially decreased the menadione-induced increase in junctional permeability. Verapamil, a Ca2(+)-channel blocker which was added into the perfusion medium (final concn. 40 microM) 10 min before the infusion of 5.5 mg of menadione, completely abolished the effect of menadione on junctional permeability. Menadione exposure therefore increases tight-junctional permeability in the liver; this may involve a depletion of GSH and a subsequent increase in intracellular Ca2+.     

Modulation of intestinal vitamin D receptor availability and calcium ATPase activity by essential fatty acids

The physiological mechanisms by which essential fatty acids (EFAs) affect calcium (Ca(2+)) retention is not clear, but suggestions have included changes in membrane fluidity, receptor modulation and induction of second messengers. The vitamin D receptor (VDR) is essential for the functioning of 1,25(OH)(2)D(3)which increases Ca(2+)absorption. Activity of the intestinal basolateral membrane (BLM) Ca(2+)ATPase correlates with the degree of Ca(2+)absorption. Therefore, changes in ATPase activity and VDR availability due to EFAs may influence calcium retention. We have investigated the effect of long-term dietary supplementation with EFAs on Ca(2+)ATPase activity (measured colourimetrically) and VDR availability (measured with the ELISA technique) after the loss of oestrogen induced by ovariectomy (OVX) in female Sprague Dawley rats. Control animals underwent anaesthesia and a surgical procedure but the ovaries were left intact (sham). Ca(2+)ATPase activity was significantly lower in OVX animals than in the intact animals (P<0.05) and following supplementation with EFAs, was significantly higher than in sham controls (P<0.05). A higher number of VDR was measured after OVX and declined due to EFA supplementation; these differences in activity of the ATPase and number of receptors could be ascribed to membrane changes due to EFA supplementation, feedback control by serum calcium or the direct influence of the EFAs.     

Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells

The effects of cholesterol depletion from the plasma membrane with methyl-beta-cyclodextrin (MbetaCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MbetaCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MbetaCD on exocytotic membrane fusion and mobilization of Ca(2+) and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MbetaCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MbetaCD affected a process downstream of Ca(2+) influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MbetaCD inhibited the Ca(2+) increase induced by antigen. Furthermore, we found that MbetaCD significantly inhibited Ca(2+) influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca(2+) release from the intracellular Ca(2+) store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MbetaCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MbetaCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells.     

Electromechanical effects of menadione on isolated rat heart in relation to oxidative stress

Although Ca2+ overloading has been observed in hepatocytes and in the isolated liver treated with 0.2 mM menadione, it has not been determined if menadione has similar effects on cardiac tissue and, if so, whether Ca2+ overloading leads to cardiac contracture, and if such an event results from plasma membrane peroxidation initiated by oxidative stress. The present study reveals that when the isolated heart is perfused with 0.2 mM menadione for 30 min, it shows Ca2+ overloading, which can not be reversed even after 30 min of drug-free perfusion. The time courses of glutathione, ethane, and LDH release from the hearts do not show a parallel pattern of abnormality between 30 and 60 min, indicating that contractile failure precedes the development of lipid peroxidation or plasma membrane disintegration. The evidence that the plasma membrane of menadione-treated rat cardiac tissue remains intact is supported by the observation that the resting membrane potential of the atrium remains virtually unchanged during the 30 min of drug exposure and then gradually falls (-67 +/- 3.1 vs. -76 +/- 2 mv) only during the last 10 min of the drug washout. Interestingly, even after the atria are treated with menadione for 30 min and followed by washout of 30 min, and have shown calcium overloading, as evidenced by contracture, they are still capable of generating action potentials in response to electrical field stimulation.     

Enhancement of rat intestinal calcium absorption by vanadate.

Vanadate alters intestinal transport and may have a role in regulating cell function. To determine whether it influences calcium absorption, we tested the effects of acute and chronic vanadate administration on calcium absorption using single-pass perfusion of jejunal and ileal segments of the in vivo rat intestine. Acute vanadate administration increased the lumen-to-mucosa and net fluxes of calcium in both the jejunum and ileum. The increase was largely due to an enhancement of the saturable fluxes of calcium and was observed at 10(-4) M concentration of vanadate, but not at higher or lower concentrations of the oxyanion, except at the highest concentration used, 10(-2) M, where calcium absorption was inhibited. Chronic vanadate administration caused, on the other hand, no changes in calcium absorption. We have demonstrated previously that rat intestinal (Na+ + K+)-ATPase is inhibited by vanadate, an effect that could raise cell sodium and increase the efflux of sodium across the brush border membrane. The results suggest that the vanadate enhancement of calcium absorption may be related to an increased entry of calcium into the mucosa, possibly as a result of an augmented exchange through the Na+/Ca+ antiport system. Alternatively, vanadate may influence access to a calcium channel in the mucosal membrane of the intestinal epithelium, leading to the observed increase in absorption.     

Differentiation of the changes in alkaline phosphatase from calcium ion-activated adenosine triphosphatase activities associated with increased calcium absorption in chick intestine.

The pattern of response of the intestinal enzymes Ca2+-activated adenosine triphosphatase and alkaline phosphatase in the chick to 1,25-dihydroxycholecalciferol is consistent with a role for the former but not the latter enzyme in the vitamin D-dependent absorption of calcium.     

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca(2+)-ATPase (PMCA) is an ATP-driven pump that is critical for the maintenance of low resting [Ca(2+)](i) in all eukaryotic cells. Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism, has the capacity to cause ATP depletion and thus inhibit PMCA activity. This has potentially fatal consequences, particularly for non-excitable cells in which the PMCA is the major Ca(2+) efflux pathway. This is because inhibition of the PMCA inevitably leads to cytosolic Ca(2+) overload and the consequent cell death. However, the relationship between metabolic stress, ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted. There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion. In particular, there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function. Moreover, membrane phospholipids, mitochondrial membrane potential, caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA. The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca(2+) overload and cytotoxicity.     

Calcium signalling and pancreatic cell death: apoptosis or necrosis?

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.