Effect of apoptosis-related compounds on Ca2+ transport system in isolated rat liver nuclei

The effect of various inhibitors of DNA topoisomerase II, which has been shown to induce apoptotic cell death, on Ca2+ transport in isolated rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. The presence of aurintricarboxylic acid (ATA; 10-6 to 10-4 M), etoposide (10-4 M), genistein (10-5 and 10-4 M) or amsacrine (10-4 M) in the reaction mixture caused a significant increase in Ca2+ release from the nuclei. Also, these compounds (10-4 M) significantly inhibited Ca2+ uptake by the nuclei. However, the presence of ATA (10-5 and 10-4 M) in the enzyme reaction mixture did not significantly inhibit Ca2+-ATPase activity, which is involved in the nuclear Ca2+ uptake, in the liver nuclei, while etoposide (10-4 M), genistein (10-4 M) and amsacrine (10-4 M) appreciably decreased the enzyme activity. Meanwhile, addition of Ca2+ clearly activated DNA fragmentation in the liver nuclei. The Ca2+ activated DNA fragmentation was significantly prevented by the presence of etoposide, genistein and amsacrine with the concentrations of 10-5 and 10-4 M in the reaction mixture, although ATA (10-5 and 10-4 M) had no effect. The present study demonstrates that some apoptosis inducible compounds used can influence on Ca2+ transport system in isolated rat liver nuclei, suggesting a decrease of nuclear Ca2+ level involved in nuclear functions. (Mol Cell Biochem 166: 183-189, 1997)     

Influence of vanadate on glycolysis,intracellular sodium,and pH in perfused rat hearts

Vanadium compounds have been shown to cause a variety of biological and metabolic effects including inhibition of certain enzymes, alteration of contractile function, and as an insulin like regulator of glucose metabolism. However, the influence of vanadium on metabolic and ionic changes in hearts remains to be understood. In this study we have examined the influence of vanadate on glucose metabolism and sodium transport in isolated perfused rat hearts. Hearts were perfused with 10 mM glucose and varying vanadate concentrations (0.7100 M) while changes in high energy phosphates (ATP and phosphocreatine (PCr)), intracellular pH, and intracellular sodium were monitored using 31P and 23Na NMR spectroscopy. Tissue lactate, glycogen, and (Na+, K+)-ATPase activity were also measured using biochemical assays. Under baseline conditions, vanadate increased tissue glycogen levels two fold and reduced (Na+, K+)-ATPase activity. Significant decreases in ATP and PCr were observed in the presence of vanadate, with little change in intracellular pH. These changes under baseline conditions were less severe when the hearts were perfused with glucose, palmitate and b-hydroxybutyrate. During ischemia vanadate did not limit the rise in intracellular sodium, but slowed sodium recovery on reperfusion. The presence of vanadate during ischemia resulted in attenuation of acidosis, and reduced lactate accumulation. Reperfusion in the presence of vanadate resulted in a slower ATP recovery, while intracellular pH and PCr recovery was not affected. These results indicate that vanadate alters glucose utilization and (Na+, K+)-ATPase activity and thereby influences the response of the myocardium to an ischemic insult.     

Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Heart FoF1-ATPase changes during the acute phase of Trypanosoma cruzi infection in rats

The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K_0.5 values, and a decreased total V_max. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100 - solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels.     

Brain-corticosteroid hormone dialogue: Slow and persistent

Summary 1. The stress response system is shaped by genetic factors and life experiences, of which the effect of a neonatal life event is among the most persistent. Here we report studies focused on the nature-nurture question using rat lines genetically selected for extreme differences in dopamine phenotype as well as rats exposed as infants to the traumatic experience of maternal deprivation.2. As key to the endocrine and behavioural adaptations occurring in these two animal models the hormone corticosterone (CORT) is considered. The stress hormone exerts slow and persistent genomic control over neuronal activity underlying the stress response system via high affinity mineralocorticoid (MR) and glucocorticoid receptors (GR). This action is exerted in a coordinate manner and involves after stress due to the rising CORT levels progressive activation of both receptor types.3. Short periods of maternal separation (neonatal handling) trigger subsequently enhanced maternal care and sensory stimulation. However, a prolonged period (24h) of depriving the infant of maternal care disrupts the stress hyporesponsive period (SHRP) and causes an inappropriate rise in CORT. During development exposure to CORT and to sensory stimulation has long-lasting consequences for organization of the stress response system.4. We find that these factors embodied by mother-pup interaction are critical for dopamine phenotype, CORT receptor dynamics and neuroendocrine regulation in adult life. The findings provide a conceptual framework to study dopamine-related psychopathology against a background of genetic predisposition, early life events, stress hormones and brain development.     

Identification of lectin binding sites in the rat brain

Lectins belong to a class of proteins or glycoproteins able to bind carbohydrates. The study reported here describes the identification of lectin-binding sites in the adult rat brain. The results indicate that among the 31 lectins utilized, eight show a specific positive reaction with neurons. Staining was also observed with other cerebral structures such as myelin, leptomeninges, choroid plexus and capillaries. Lectins are, therefore, an important histochemical tool and can be easily and reliably used for the identification of cells and cerebral structures in the adult rat brain.Abbreviations Gal galactose - Fuc fucose - Man mannose - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - NeuNAc sialic acid     

The role of magnesium in regulating CCK-8-evoked secretory responses in the exocrine rat pancreas

This study investigates the effect of magnesium (Mg²⁺) on the secretory responses and the mobilization of calcium (Ca²⁺) and Mg²⁺ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10^–10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg²⁺ [Mg²⁺]_o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg²⁺]_o Similar effects of perturbation of [Mg²⁺]_o on amylase secretion and ⁴⁵Ca²⁺ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca²⁺ concentration [Ca²⁺]_i in zero and normal [Mg²⁺]_o compared to a much reduced response in elevated [Mg²⁺]_o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB₂ cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca²⁺]_i. In magfura-2-loaded acinar cells CCK-8 (10^–8 M) stimulated an initial transient rise in intracellular free Mg²⁺ concentration [Mg²⁺]_i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na⁺ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg²⁺ resulted in an elevation in [Mg²⁺]_i. Upon stimulation with CCK-8, [Mg²⁺]_i. decreased only slightly compared with the response obtained in normal [Mg²⁺]_o. CCK-8 caused a net efflux of Mg²⁺ in pancreatic segments; this effect was abolished when extracellular sodium [Na⁺]_o was replaced with either NMDG or choline. The results indicate that Mg²⁺ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca²⁺ mobilization. Moreover, the movement of Mg²⁺ in pancreatic acinar cells is dependent upon extracellular Na⁺.     

Donors of nitric oxide mimic effects of ischaemic preconditioning on reperfusion induced arrhythmias in isolated rat heart

NO has been implicated in the mechanism of ischaemic preconditioning. To verify this hypothesis further we have attempted to reproduce effects of ischaemic preconditioning by nitric oxide donors administration prior to the ischaemia. The effect of glyceryl trinitrate (GTN) and 3-morpholino-sydnonimine-hydrochloride (SIN- 1), NO donors, on reperfusion induced ventricular tachycardia (VT) and ventricular fibrillation (VF) in Langendorff perfused rat hearts subjected to 10 min regional ischaemia followed by 10 min reperfusion were examined. Results: GTN, 500 M and SIN-1, 10 M, administered for 5 min and washed for another 5 min prior to ischaemia (to mimic ischaemic preconditioning), almost completely abolished reperfusion induced VF. GTN and SIN-1, administered at the time of reperfusion, increased the incidence of sustained VF and the duration of VT and VF. When given 5 min before the ischaemia and throughout the ischaemia and the reperfusion, SIN-1 abolished VF. Adenosine, 10 M, applied according to the above three protocols, did not affect reperfusion arrhythmias, although adenosine induced changes in coronary flow and post-ischaemic reflow were similar to those produced by the NO donors. In conclusions: (1) NO is able to mimic the effect of ischaemic preconditioning on reperfusion arrhythmias in rat heart, supporting the view that NO may be one of the endogenous substances triggering ischaemic preconditioning; (2) In crystalloid-perfused heart, NO may be deleterious when its administration is restricted to the reperfusion period.     

Dietary linolenic acid-mediated increase in vascular prostacyclin formation

To define vascular effects of an enhanced dietary -linolenic acid intake, 28 spontaneously hypertensive rats were fed a 3% sunflowerseed oil (44% linoleic acid) diet; in 3 groups (7 rats each), the diet was supplemented with 1, 2.5 or 5% linseed oil containing 62% -linolenic acid. -Linolenic acid was incorporated up to 12% in the aorta of the 5% linseed oil group. The eicosapentaenoic acid content was not significantly increased. The content of arachidonic acid and docosatetraenoic acid was moderately reduced in rats fed 5% linseed oil. The generation of 6-keto-PGF₁ (degradation product of prostacyclin) assessed by HPLC/electrochemical detection was, however, markedly increased (p < 0.05) in rats fed 2.5 and 5% linseed oil. The minor prostanoids TXB₂, PGE₂ and PGF₂ were not significantly altered. The high systolic and diastolic blood pressure of SHR monitored by radio telemetry was more effectively reduced (p < 0.05) in the light, i.e. sleep, cycle. An increased prostacyclin formation and lowered vascular arachidonic acid content associated with enhanced dietary -linolenic acid intake would thus be expected to prove beneficial in the prevention of vascular disorders.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.