Oxidative effects of chronic ethanol consumption on the functions of heart and kidney: folic acid supplementation.

AIMS: The principal aim of this study was to investigate the oxidative effects of chronic ethanol consumption on the functions of the heart and the kidney and the possible modification of this effect by folic acid supplementation. Moreover, in order to find whether this oxidative profile affects cardiovascular function, parameters such as heart rate and glomerular filtration rate were also assessed. METHODS: Four experimental groups of rats were used: control, ethanol-exposed, control supplemented with folic acid and ethanol-exposed plus folic acid. Ethanol-exposed rats were subjected to a chronic ethanol treatment (2 months), in which the level of alcohol reaches 30% v/v. Diet and ethanol solution were provided ad libitum, and folic acid supplementation was 8 vs. 2 ppm. Energy intake, creatinine clearance and heart rate were determined. Antioxidant enzyme activity and lipid and protein peroxidation of the kidney and the heart were measured by the spectrophotometric method. RESULTS: Ethanol increases heart size and catalase (CAT) activity and decreases lipid peroxidation in heart without changing heart rate. However, in the kidney, ethanol decreases CAT activity, increases lipid peroxidation and decreases glomerular filtration rate. Folic acid supplementation avoids these situations; it does not, however, improve glomerular function. CONCLUSION: Chronic ethanol consumption has many effects on the antioxidant enzymatic activity of the heart and the kidney, leading to increased renal lipid peroxidation prevented by folic acid supplementation.

The effects of ethanol upon hydric balance and arterial pressure in rats: folic acid as a possible hypotensor.

AIMS: Chronic alcohol intake is related to hypertension. There are, however, few studies concerning the effect of ethanol upon hydric balance in relation to arterial pressure. Folic acid intake has beneficial effects upon the cardiovascular system decreasing hyperhomocysteinemia, however, more studies imply that it is related with other mechanisms. Therefore, we have studied the effects of chronic alcohol intake (30% v/v) upon hydric-saline balance and hypertension and have found that dietary supplementation with folic acid (8 mg/kg) improves the above parameters. MAIN METHODS: Our study used four experimental groups of rats: control, alcohol, alcohol with folic acid and control with folic acid. In all cases we measured the clearance of Na(+), K(+) and aldosterone; osmolarity in urine, liquid and solid ingestion; homocysteine levels in serum; cardiac frequency and arterial blood pressure. KEY FINDINGS: The alcohol intake increases serum aldosterone and homocysteine, which is reflected in an increase in arterial blood pressure. In addition, we have found that alcohol intake reduces both liquid and solid ingestion (causing a malnourishment status), the clearance of creatinine, aldosterone, Na(+) and K(+), and the ratio ClNa(+)/ClCr; it also increases urine osmolarity. Folic acid supplementation increases the clearance of Na(+) and the ratio ClNa(+)/ClCr. SIGNIFICANCE: Folic acid intake improves the hypertension provoked by alcohol by increasing the aldosterone clearance, drastically reducing the serum levels of this hormone and thus its hypertensor effect.

Dietary selenium plus folic acid as an antioxidant therapy for ethanol-exposed pups.

BACKGROUND: Nutrients such as folic acid and selenium are decreased in dams exposed to ethanol during gestation and lactation, affecting their metabolism, antioxidant balance, and the future health of their progeny. We will study whether the supplementation of the maternal diet with folate and selenium can prevent ethanol-induced oxidative liver disorders in the offspring. METHODS: Dams were randomised into four groups: control, alcohol, alcohol+folic acid+Se, and control+folic acid+Se. We determined selenium by graphite-furnace atomic absorption and antioxidant enzyme activities, lipid peroxidation, and protein carbonyl by spectrophotometry in the offspring. RESULTS: Alcohol increased serum Se levels and glutathione peroxidase (GPx) activity. However, in the liver of pups from ethanol-exposed dams a decrease in selenium was provoked and GPx activity increased with the double supplementation. Glutathione reductase (GR) and catalase (CAT) activities increased with ethanol, while double supplementation significantly decreased the GR activity. The supplemented diet reduced the protein peroxidation found in ethanol pups. CONCLUSIONS: These results suggest that folic acid+Se could be effective in neutralising the damage of ethanol consumption in pups since it prevents peroxidation protein products.

Stimulation by thimerosal of histamine-induced Ca(2+) release in intact HeLa cells seen with aequorin targeted to the endoplasmic reticulum.

The oxidizing thiol reagent, thimerosal, has been shown to activate reversibly the inositol 1,4,5-trisphosphate (InsP(3)) receptor in several cell types. We have studied here the effects of thimerosal by monitoring the [Ca(2+)] inside the endoplasmic reticulum (ER) of intact HeLa cells with targeted aequorin. We show that thimerosal produced little effects on the ER-Ca(2+)-pump and only slightly increased the ER-Ca(2+)-leak in intact cells. Instead, thimerosal increased the sensitivity to histamine of ER-Ca(2+)-release by about two orders of magnitude, made the response much more prolonged at saturating histamine concentrations and enhanced both cytosolic and mitochondrial [Ca(2+)] responses to histamine. Moreover, inhibition of ER-Ca(2+)release by cytosolic [Ca(2+)] microdomains was fully preserved and sensitive to BAPTA-loading, and histamine-induced Ca(2+) release remained quantal in the presence of both thimerosal and intracellular BAPTA. The effects of thimerosal were reversible in the presence of dithiotreitol, suggesting the possible presence of a physiological redox regulatory mechanism. However, in permeabilized cells thimerosal potentiated InsP(3)-induced Ca(2+) release but oxidized glutathione had no effect. In addition, thimerosal increased the [Ca(2+)](ER) steady-state level in permeabilized cells. Thimerosal partially inhibited also plasma membrane Ca(2+)extrusion and increased Ca(2+)(Mn(2+)) entry through the plasma membrane, both phenomena contributing to increase the steady-state cytosolic [Ca(2+)]. Thimerosal-induced Ca(2+) entry was additive to that induced by emptying of the ER, suggesting that store-operated Ca(2+) channels may not be involved. These results provide new insights on the mechanisms of activation and inactivation of InsP(3) receptors.

Low activity of mitochondrial HMG-CoA synthase in liver of starved piglets is due to low levels of protein despite high mRNA levels.

The unusually low hepatic ketogenic capacity of piglets has been correlated with lack of expression of the mitochondrial HMG-CoA synthase gene. However, we have shown that starvation of 2-week-old piglets increased the mRNA levels of mitochondrial HMG-CoA synthase to a level similar to that observed in starved rats (S. H. Adams, C. S. Alho, G. Asins, F. G. Hegardt, and P. F. Marrero, 1997, Biochem. J. 324, 65-73). We now report that antibodies against pig mitochondrial HMG-CoA synthase detected the pig enzyme in mitochondria of 2-week-old starved piglets and that the pig mitochondrial HMG-CoA synthase cDNA encodes an active enzyme in the eukaryotic cell line Mev-1, with catalytic behavior similar to that of the rat enzyme when expressed in the same system. We also show that low activity of pig mitochondrial HMG-CoA synthase correlates with low expression of the pig enzyme. The discrepancy in mitochondrial HMG-CoA synthase gene expression between the high levels of mRNA and low levels of enzyme was not associated with differences in transcript maturation, which suggests that an attenuated translation of the pig mRNA is responsible for the diminished ketogenic capacity of pig mitochondria.

Regulation of the rat liver carnitine palmitoyltransferase I gene transcription by thyroid hormone.

L-CPT I isotype is the main locus of control for liver LCFA oxidation. T3 levels have been described as controlling L-CPT I gene expression, and in this paper we demonstrate that rat liver CPT I promoter responds to T3. Using deleted reporter constructs we located the thyroid hormone-responsive element between -2935 and -2918, consisting of a DR4. This response is mediated by the binding of the thyroid to this sequence as a monomer, homodimer, or heterodimer with RXR.

Lysophosphatidic acid inhibits Ca2+ signaling in response to epidermal growth factor receptor stimulation in human astrocytoma cells by a mechanism involving phospholipase C(gamma) and a G(alphai) protein.

The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca(2+) concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca(2+) transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase Cgamma (PLCgamma)-1, and G(alphai) protein. LPA was found to induce coupling of PLCgamma-1 to the EGFR by a mechanism involving a G(alphai) protein, in the absence of tyrosine phosphorylation of both PLCgamma and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G(alphai)-protein-mediated translocation of PLCgamma-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins.

Secretory phospholipase A2 induces phospholipase Cgamma-1 activation and Ca2+ mobilization in the human astrocytoma cell line 1321N1 by a mechanism independent of its catalytic activity.

The effect of secretory phospholipase A2 (sPLA2) on intracellular Ca2+ signaling in human astrocytoma cells was studied. sPLA2 increased cytosolic [Ca2+] ([Ca2+]c) in both Ca2+-containing and Ca2+-free medium, thus suggesting Ca2+ release from intracellular stores. The activation by sPLA2 of arachidonate release via cytosolic PLA2 (cPLA2) was also independent of extracellular Ca2+. As sPLA2 requires Ca2+ for activity, these results indicate that both Ca2+ mobilization and cPLA2 activation induced by sPLA2 are unrelated to phospholipase activity but dependent on signaling mechanisms. The sPLA2-induced [Ca2+]c peak was sensitive to Bordetella pertussis toxin and inhibited by caffeine, suggesting its mediation by inositol 1,4,5-trisphosphate (IP3). sPLA2 induced tyrosine phosphorylation and membrane targeting of phospholipase Cgamma-1 (PLCgamma-1). Moreover, the Ca2+ peak was sensitive to protein tyrosine kinase inhibitors. sPLA2 activates two signaling pathways: one leading to the activation of the MAP kinase/cPLA2 cascade and another leading to PLCgamma activation and Ca2+ release.

Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin.

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin.

Changes in the free calcium concentration of the endoplasmic reticulum ([Ca2+]er) play a central role controlling cellular functions like contraction, secretion or neuronal signaling. We recently reported that recombinant aequorin targeted to the endoplasmic reticulum (ER) [Montero M., Brini M., Marsault R. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 1995; 14: 5467-5475, Montero M., Barrero M.J., Alvarez J. [Ca2+] microdomains control agonist-induced Ca2+ release in intact cells. FASEB J 1997; 11: 881-886] can be used to monitor selectively [Ca2+]er in intact HeLa cells. Here we have used a herpes simplex virus type 1 (HSV-1) based system to deliver targeted aequorin into a number of different cell types including both postmitotic primary cells (anterior pituitary cells, chromaffin cells and cerebellar neurons) and cell lines (HeLa, NIH3T3, GH3 and PC12 cells). Functional studies showed that the steady state lumenal [Ca2+]er ranged from around 300 microM in granule cells to 800 microM in GH3 cells. InsP3-coupled receptor stimulation with agonists like histamine (in HeLa, NIH3T3 and chromaffin cells), UTP and bradykinin (in PC12 cells) or thyrotropin-releasing hormone (TRH, in GH3 cells) produced a very rapid decrease in lumenal [Ca2+]er. Caffeine caused a rapid Ca2+ depletion of the ER in chromaffin cells, but not in the other cell types. Depolarization by high K+ produced an immediate and reversible increase of [Ca2+]er in all the excitable cells (anterior pituitary, GH3, chromaffin cells and granule neurons). We conclude that delivery of recombinant aequorin to the ER using HSV amplicon provides the first direct quantitative and dynamic measurements of [Ca2+]er in several primary non-dividing cells.

Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells. A comparative study.

We have measured the [Ca2+] in the endoplasmic reticulum ([Ca2+]er) of intact HeLa cells at both 22 degrees C and 37 degrees C using endoplamsic reticulum-targeted, low Ca2+ affinity aequorin reconstituted with coelenterazine n. Aequorin consumption was much slower at 22 degrees C, and this allowed performing a much longer study of the dynamics of [Ca2+]er. The steady-state [Ca2+]er (500-600 microM) was not modified by the temperature, although both the rates of pumping and leak were decreased at 22 degrees C. The behavior of both [Ca2+]er and cytoplasmic [Ca2+] ([Ca2+]c) after the addition of increasing concentrations of agonists and/or Ca2+-ATPase inhibitors, or following incubation in Ca2+-free medium were compared. We show that agonists induce a fast but relatively small decrease in [Ca2+]er, which is enough to produce a sharp increase in [Ca2+]c. Termination of Ca2+ release is controlled by feedback inhibition of the inositol 1,4,5-trisphosphate receptors by [Ca2+]c, a mechanism that appears to be designed to release the minimum amount of Ca2+ necessary to produced the required [Ca2+]c signal. We also show that Ca2+ release is inhibited progressively when [Ca2+]er decreases below a threshold of about 150 microM, even in the absence of Ca2+ pumping or -Ca2+-c increase. This effect is consistent with a regulation of the inositol 1,4,5-trisphosphate-gated channels by [Ca2+]er.

[Ca2+] microdomains control agonist-induced Ca2+ release in intact HeLa cells.

We have monitored specifically the [Ca2+] in the lumen of the endoplasmic reticulum (ER) of intact HeLa cells using an ER-targeted low-Ca2+-affinity aequorin. The steady-state [Ca2+] in the ER was around 600 microM. Histamine induced a concentration-dependent decrease in lumenal [Ca2+], whose rate increased near one order of magnitude and became "quantal" when cytosolic [Ca2+] ([Ca2+]c) was clamped with the Ca2+ chelator BAPTA. This effect was not due to decreased Ca2+ pumping because simultaneous addition of a SERCA inhibitor produced only additive effects. Given that inhibition by [Ca2+]c of the inositol 1,4,5-trisphosphate-gated channels requires a [Ca2+]c much higher than that observed in the bulk cytosol after histamine addition, we conclude that local [Ca2+]c microdomains at the site of release strongly inhibit agonist-induced Ca2+ mobilization in intact cells. This effect should play a key role in the mechanism controlling cytosolic [Ca2+] oscillations and waves, and therefore in the generation of spatio-temporal Ca2+ patterns.