Lead‐related effects on rat fibroblasts

Lead (Pb) is an environmental toxicant that can induce structural and functional abnormalities of multiple organ systems, including the central nervous and the immune systems. The aim of this study was to evaluate the effects of extracellular Pb supplementation on the cellular content of the metal and on the proliferation and the survival of normal rat fibroblasts.We found that the concentration of Pb in the culture medium was 0.060 M and the normal Pb concentration in rat fibroblasts was 3.1 ± 0.1 ng/10⁷ cells. Then we exposed the cells to increasing concentration of Pb (as Pb acetate) from 0.078–320 M. We observed a dosedependent inhibition of cell proliferation after 48 h, which was already apparent at a concentration of 0.312 M (p = 0.122) and became statistically significant for concentration higher than 0.625 M (p = 0.0003 at 5 M). Cell proliferation was completely compromised at 320 M Pb total inhibition of cell proliferation.To investigate the mechanisms of Pbmediated inhibition of cell proliferation, we evaluated the occurrence of apoptosis in the same cells and found that cytosolic DNA fragments, hallmark of apoptotic cell death, increased significantly at Pb concentrations from 2.5–10.0 M. The occurrence of apoptosis was also confirmed by FACS analysis which showed the appearance of a subdiploid peak at Pb concentrations from 5–20 M. The distribution of cells in the cell cycle showed a dosedependent accumulation of cells in the G₀/G₁ phase mainly compensated by a decrease in the percentage of cells in the S phase. In conclusion, our results demonstrate that induction of apoptosis contributes to the Pbinduced inhibition of cell proliferation in rat fibroblasts.     

Do rat cardiac myocytes release ATP on contraction?

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is 10-fold below the EC₅₀ values for most purinergic receptors expressed in the heart (1.5–20 µM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP. cardiomyocytes; luciferase     

Influence of a single γ-irradiation on rat microflora

Changes in leukocyte counts and in the gut microflora of laboratory rats irradiated with a single whole-body dose of γ rays (5.0 Gy) were determined. The number of leukocytes was lower especially 1 and 2 weeks after irradiation. A significant decrease in lymphocytes was observed 1 week and in monocytes 1 and 2 weeks after irradiation. In parallel with these changes, an increase in common microflora was observed; some microorganisms, which normally are not present in duodenum, liver and mouth cavity, were detected in these organs.     

Effects of β-phenylethylamine on polyphosphoinositide turnover in rat cerebral cortex

The effects of the trace amine, -phenylethylamine, on the hydrolysis of inositol phospholipids in rat cerebral cortical slices was studied using a direct assay involving prelabeling with [³H]inositol and then examining the production of [³H]inositol phosphates in the presence of lithium. Phenylethylamine exhibited two different effects. Millimolar concentrations of phenylethylamine stimulated the production of [³H]inositol phosphates to about 200% of control, while much smaller concentrations (micromolar) inhibited noradrenaline(NE)-stimulated [³H]inositol phosphate formation dose-dependently. The ₁-antagonist, prazosin, inhibited the increases in [³H]polyphosphoinositide turnover stimulated by phenylethylamine and by NE, though it inhibited phenylethylamine to a lesser extent than NE. It appears, therefore, that phenylethylamine affects [³H]inositol phosphate formation by acting as a partial ₁-agonist.     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

Effect of β-phenylethylamine on dopaminergic systems of the rat brain

The time course of changes in the dopamine concentration in dopaminergic neurons of the nigro-neostriatal and mesolimbic systems of the rat brain during 1 h after intraperitoneal injection of -phenylethylamine (100 mg/kg) was studied by quantitative fluorescence-histochemical analysis. The results showed that -phenylethylamine causes a marked fall in the dopamine level in neurons of dopaminergic systems of the brain. The dopamine level in the bodies of dopaminergic neurons changes more than in their axon terminals. The fall in the dopamine concentration in the dopaminergic systems of the brain during the first hour is irregular in character: in the terminals between 10 and 30 min and in the bodies between 30 and 45 min there is actually a temporary increase in the dopamine concentration. The rise in the dopamine concentration in the terminals coincides with a sharp fall in the dopamine level in the neuron bodies, and conversely, the fall in the dopamine concentration in the terminals after 30 min is accompanied by some increase in the dopamine concentration in the neuron bodies. The results suggest that the increase in motor activity described in the literature in animals after injection of -phenylethylamine is connected with its action on catecholaminergic, especially dopaminergic, brain systems.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 578–584, November–December, 1979.     

Effects of pH on β-hydroxybutyrate transport in rat erythrocytes and thymocytes

Entry of β-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or α-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [β-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ from 29 to 16 to 10 mM, entry $\text{V}$ being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external β-hydroxybutyrate enhanced efflux slightly. At high external pH, external β-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal β-hydroxybutyrate (βHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled βHB^?/OH^? exchange, βHB^? and OH^? competing for influx, C : OH^? moving faster than C : βHB^?, empty C being immobile. They are also compatible with coupled βHB^?/H⁺ copermeation, empty C moving inward faster than H⁺ : C : βHB^?, H⁺ : C being immobile, and C : βHB^? (without H⁺) being so unstable as not to be formed in significant amounts (relative to C, H⁺ : C, and H⁺ : C : βHB^?).     

Metabolic effects of tumour necrosis factor-α on rat brown adipose tissue

Intravenous administration of a single dose (100 g/kg bw) of recombinant tumour necrosis factor- (TNF, cachectin) to rats increased the rate ofin vitro fatty acid synthesis in interscapular brown adipose tissue (IBAT) from both glucose and alanine, without changes in the oxidation of these substrates to¹⁴CO₂. Lactate production and glycerol release were also unaffected by treatment with the cytokine. Additionally, the presence of TNF in the incubation media did not affect fatty acid synthesis, suggesting an indirect effect of the cytokine. The activities of different enzymes of glucose and alanine metabolism such as hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase and alanine transaminase, did not suffer changes as a consequence of TNF administration. The same applied to the enzymatic activities involved in fatty acid synthesis such as fatty acid synthase, acetyl-CoA carboxylase and ATP-citrate lyase. Conversely, citrate levels in IBAT were increased in animals treated with TNF, suggesting that it could be the cause for the increased fatty acid synthesis in this tissue.     

Studies on the binding of d-α-tocopherol to rat liver nuclei

The present study describes the nature and characteristics of the intranuclear binding sites of [³H]d-α-tocopherol in rat liver. When radioactively labeled d-α-tocopherol was intravenously administered to rats, approximately 55% of the nuclear radioactivity was associated with an intranuclear nucleoprotein complex. This complex, which was extractable by high concentrations of NaCl, was characterized by equilibrium density ultracentrifugation on a 30 to 60% linear sucrose gradient. About 50% of the high-salt-extracted radioactivity was coprecipitable with macromolecules by 10% ice-cold trichloroacetic acid (TCA). This TCA-precipitable radioactivity was completely ethanol soluble. Alkaline conditions favored the solubilization of the vitamin-receptor complex. Among various enzymes tested, only Pronase and trypsin were capable of dissociating the vitamin-receptor complex. Both ionic (sodium dodecyl sulfate) and nonionic (Triton X-100) detergents solubilized α-tocopherol from the nuclei and concomitantly released some of the associated macromolecules. In addition, treatment of nuclei with low concentrations of Triton X-100 showed that about 30% of the nuclear bound α-tocopherol is associated with inner core sites in the nucleoprotein complex with very high affinity for the vitamin. Dissociation of the nucleoprotein complex (chromatin) by high-salt solubilization and subsequent partial reassociation of the components by salting out procedures revealed the high affinity association of α-tocopherol with the reconstituted DNA-protein complex. Subfractionation of this complex further revealed that α-tocopherol is predominantly associated with the fraction containing phenol-soluble nonhistone proteins having a high affinity for DNA. In vitro binding studies also showed that there are specific saturable binding sites for d-α-tocopherol in rat liver nuclei.     

Protective effects of exogenous β-hydroxybutyrate on paraquat toxicity in rat kidney

In this study, we demonstrated the protective effects of β-hydroxybutyrate (β-HB) against paraquat (PQ)-induced kidney injury and elucidated the underlying molecular mechanisms. By histological examination and renal dysfunction specific markers (serum BUN and creatinine) assay, β-HB could protect the PQ-induced kidney injury in rat. PQ-induced kidney injury is associated with oxidative stress, which was measured by increased lipid peroxidation (MDA) and decreased intracellular anti-oxidative abilities (SOD, CAT and GSH). β-HB pretreatment significantly attenuated that. Caspase-mediated apoptosis pathway contributed importantly to PQ toxicity, as revealed by the activation of caspase-9/-3, cleavage of PARP, and regulation of Bcl-2 and Bax, which were also effectively blocked by β-HB. Moreover, treatment of PQ strongly decreased the nuclear Nrf2 levels. However, pre-treatment with β-HB effectively suppressed this action of PQ. This may imply the important role of β-HB on Nrf2 pathway. Taken together, this study provides a novel finding that β-HB has a renoprotective ability against paraquat-induced kidney injury.     

Effect of recombinant erythropoietin on ischemia–reperfusion-induced apoptosis in rat liver

Ischemia–reperfusion (I/R) cannot be avoided in liver transplantation procedures, and apoptosis is a central mechanism of cell death after liver reperfusion. Protective effect of recombinant erythropoietin (rhEPO) on liver apoptosis has not been clearly investigated. This work investigated intraportal (IP) rhEPO-protective effect in a rat model of hepatic I/R-induced apoptosis and its appropriated time and dose of administration. Eight groups were included (n = 10/group): sham-operated, I/R (45 min ischemia and 2 h reperfusion), preconditioned rhEPO I/R (24 h or 30 min before ischemia), and postconditioned rhEPO I/R (before reperfusion) using two different rhEPO doses (1,000 and 5,000 IU/kg). When compared with the sham-operated group, the I/R group showed significant increase of serum levels of aspartate and alanine aminotransferases (AST, ALT), hepatic caspase-9 activity(894.99 ± 176.90 relative fluorescence units (RFU)/mg/min versus 458.48 ± 82.96 RFU/mg/min), and Fas ligand (FasL) expression, histopathological damages, and significant decrease in the antiapoptotic Bcl-xL/apoptotic Bax ratio(0.38 ± 0.21 versus 3.35 ± 0.77) rhEPO-improved ALT and AST but failed to reduce FasL expression in all groups compared with the I/R group. Thirty minutes and 24 h preconditioning with rhEPO (1,000 IU/kg) increased Bcl-xL/Bax ratio and reduced caspase-9 activity, and the same effect was observed when higher dose was given 24 h before ischemia. Preconditioning was more effective than postconditioning in improving caspase-9 activity, and no dose-dependent effect was observed. In conclusion, single IP rhEPO injection 30 min before ischemia has an advantage over rhEPO postconditioning in improving post-hepatic I/R-induced apoptosis with no additional time- and dose-dependent effects which may provide potentially useful guide in liver transplantation procedures.     

Central effects of TNFα on thermogenesis and fever in the rat

Intracerebroventricular (icv) injection of purified recombinant human tumour necrosis factor (TNF , 4–8g) in conscious rats, produced increases in colonic temperature (1.0°C) and resting oxygen consumption (VO₂, 14%) which were maximal after 80–90 minutes. Pretreatment with propranolol (10mg/kg s.c) significantly inhibited the rise in VO₂, and prevented the increase in body temperature. Icv injection of an antagonist to corticotropin releasing factor (-helical CRF 9-41, 25 g), which prevents the pyrogenic and thermogenic actions of interleukin-1, did not influence the effects of TNF on temperature or VO₂. Injection of a fragment of TNF (113–130 amino acid sequence) did not affect body temperature or VO₂. TNF injection (icv) significantly increased brown adipose tissue (BAT)in vitro mitochondrial GDP binding, and this effect was slightly inhibited, but not prevented, by surgical denervation of the tissue, and was unaffected by pretreatment with -helical CRF 9-41. These data indicate that TNF can stimulate thermogenesis by a direct central action. The effects are largely, but not totally, dependent on the sympathetic nervous system but, unlike the thermogenic actions of interleukin they do not require release of CRF.     

The effect of exogenous δ-aminolaevulinate on rat liver haem and cytochromes

The activity of delta-aminolaevulinate synthetase is generally regarded as rate-limiting for hepatic haem biosynthesis. It has been suggested that cytochrome synthesis may also be regulated by changes in delta-aminolaevulinate synthetase activity. This hypothesis was studied by injecting product, delta-aminolaevulinate, into adult rats over a 4-240h period. The concentrations of hepatic mitochondrial cytochromes a, b, c and c(1) were unchanged by treatment with delta-aminolaevulinate, allylisopropylacetamide or phenobarbital. In control animals, total microsomal haem content equalled the sum of cytochromes b(5) plus P-450. After delta-aminolaevulinate administration the total amount of microsomal haem, measured as the pyridine haemochromogen, exceeded these components, indicating the formation of a ;free' haem pool. Haem synthesis does not appear rate-limiting for hepatic cytochrome synthesis in the adult rat.     

Effect of phenobarbital on the oligosaccharide structure of rat α-acid glycoprotein

We have recently shown that the administration of phenobarbital to rats leads t an increased serum α₁-acid glycoprotein content with alterations in the relative proportion of the sugar moiety. Therefore, α₁-acid glycoprotein was purified from normal (α₁-acid glycoprotein_N) and phenobarbital-treated rats (α₁-acid glycoprotein_PB). Glycans were separated by AX-10 chromatography and analysed by gas chromatography. It appears that, compared to α₁-acid glycoprotein_N, α₁-acid glycoprotein_PB had a higher carbohydrate content (31.7% compared to 26%) and a non-negligible amount of neutral oligosaccharide (12.2% compared to 1.3%). No tetrasialyl oligosaccharides in α₁-acid glycoprotein_PB were detected, whereas their relative proportion in α₁-acid glycoprotein_N was 27%.     

The preventive effect of Se-methylselenocysteine on γ-radiation-induced oxidative stress in rat lungs

We investigated the preventive effect of Se-methylselenocysteine (MSC) administration on γ-radiation (whole body irradiation, single 10-Gy dose)-induced oxidative damage in rat lungs. Rats were pretreated with MSC (0.75 mg/rat/day) for 1 week before γ-irradiation. The MSC pretreatment prevented the irradiation-induced increase in lipid peroxidation and the concomitant decrease in cellular glutathione content. The prevention of irradiation-induced oxidative damage in MSC-pretreated rat lungs appeared to be associated with increased antioxidant capacity, particularly in the glutathione system. The 1-week MSC treatment resulted in an increase in glutathione peroxidase, glutathione reductase, and glucose 6-phosphate dehydrogenase activities, which are involved in glutathione redox cycling. An increase in catalase activity was also observed in the rat lungs. Additionally, a significantly increased level of nuclear factor erythroid 2-related factor 2 (Nrf2) was exhibited in the MSC-treated rat lungs. Heme oxygenase 1, glutathione S-transferase pi, and peroxiredoxin 1, which are known target proteins of Nrf2, were also increased in MSC-treated lungs. These results implicate Nrf2 signaling in the MSC-induced activation of the antioxidant system.     

Effect of α-p-chlorophenoxyisobutyrate on the metabolism of isoprenoid compounds in the rat

1. Feeding of alpha-p-chlorophenoxyisobutyrate (CPIB) to rats increased ubiquinone concentration in the liver but not in other tissues. The increase was progressive with the time of feeding and related to the concentration of CPIB in the diet. 2. Incorporation of [1-(14)C]acetate, but not of [2-(14)C]mevalonate, into sterols in the liver in vivo or by liver slices in vitro was decreased on feeding the rats with CPIB. However, incorporation of mevalonate into ubiquinone increased. 3. CPIB, when added in low concentrations to liver slices, had no effect on isoprene synthesis from acetate; higher concentrations, however, were inhibitory. 4. No activation of ubiquinone synthesis from mevalonate was observed when CPIB was added to the liver slices synthesizing ubiquinone. 5. The increase in ubiquinone in CPIB-fed animals appears to be due to increased synthesis in the initial stages and to decreased catabolism in the later stages. 6. An inverse relationship was found between the concentration of ubiquinone in the liver and the serum sterol concentration in CPIB-fed rats.     

Action of ω-conotoxin on calcium currents of rat pituitary GH3 cells

The effect of -conotoxin (-CgTX) on calcium currents of rat pituitary GH3 cells was studied by the voltage clamp method on a whole cell, under tight junction conditions. Two different components of the inward calcium current were observed in a solution containing 15 mM Ca²⁺. The first was activated with a holding potential of –80 mV and by testing pulses more positive than –55 mV. A shift of holding potential to –40 mV led to steady-state inactivation of this low-threshold component of the current. -CgTX at the initial moment after its application had an activating action on both components of the calcium current: low-threshold and high-threshold, but the increase in the first was much greater. In the present experiments the currents increased as early as 30 sec after replacement of the external solution; later the drop of current took place with temporal parameters characteristic of the spontaneous current drop in the control solution during cell dialysis. Incubation of the cells in growth medium containing 5 µM -CgTX for 2 h led to an increase in the density of both types of calcium currents in the GH3 cells, which was reduced after incubation for 2 h in the same medium. Thus -CgTX was found to have an activating action on calcium currents of GH3 cells at the initial moment after application of the toxin. The absence of a marked blocking action of -CgTX on the calcium currents of the test cells confirms the high tissue specificity of action of the toxin as a blocker of high-threshold calcium channels in the nerve cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 2, pp. 199–205, March–April, 1991.     

Effects of succinylacetone on methyl α-d-glucoside uptake by the rat renal tubule

Succinylacetone, a catabolic end-product of tyrosine, is excreted in large quantities in urine from individuals with hereditary tyrosinemia and the Fanconi syndrome. Succinylacetone inhibits rat renal tubular concentrative uptake of the glucose transport analogue, methyl α-d-glucoside, in a noncompetitive and reversible fashion. This compound also depresses oxygen consumption by the rat renal tubule without fine structural damage to mitochondria. It is concluded that succinylacetone may be a useful probe in elucidation of the biochemical mechanism underlying the human Fanconi syndrome.