[Deposition of exogenous and endogenously generated unsaturated fatty acids in lipid droplets triacylglycerol as a mechanism of its sequestration in epithelial cells].

Neutral lipids are deposited in intracellular compartments called lipid droplets, which are known to be critically implicated in regulation of cellular lipid metabolism. These organelles consist of a core of neutral lipids, mainly triacylglycerol (TAG) and cholesteryl esters, surrounded by phospholipid monolayer. Using Nile red lipid staining and [3H]-arachidonic and [3H]-oleic acids as precursors for lipid biosynthesis, we have evaluated the mechanisms of lipid body induction elicited by exogenous fatty acids within primary cultured epithelial cells from the frog urinary bladder. It was found that arachidonic and oleic acids at concentrations 10-50 tM stimulated lipid droplets formation accompanied by accumulation of TAG and by the significant increase of incorporation of fatty acids into TAG indicating an enhanced TAG biosynthesis. No changes of cholesteryl esters content were observed under these conditions. In cells, prelabelled with [3H]-oleic acids, etomoxir, an inhibitor of O-carnitine palmitroyltansferase 1, decreased oxidation of oleic acid and increased its incorporation into TAG leading to intracellular TAG accumulation. In cells, prelabelled with [3H]-arachidonic acid, diclofenac, an inhibitor of cyclooxygenase 1 and 2, led to significant decrease in cellular PGE2 production and to reesterification of free arachidonic acid to TAG but not to phospholipids. Taking together, these data evidence that in isolated frog urinary bladder epithelial cells, reacylation of unsaturated free fatty acids into TAG is a main route of their metabolic conversion under the conditions of the increased cytosolic level of free fatty acids.

[Gangliosides GM1 and GD1a modulate inflammatory effect of bacterial lipopolysaccharide in epithelial cells].

It is known that exogenous gangliosides (GL) inhibit acute inflammatory signals in different cells induced by Escherichia coli lipopolysaccharide (LPS). Until now the mechanisms underlying their effect are unknown. We hypothesize that the anti-inflammatory effect of GL is caused by their ability to modify TLR4 translocation into the lipid rafts. To test this hypothesis, we studied the effect of exogenous GL on LPS-induced inflammatory reactions associated with increased nitric oxide and prostaglandin E2 (PGE2) production in epithelial cells isolated from the frog Rana temporia urinary bladder. It was shown that preincubation of cells with GM1 and GD1a in the concentration range from 100 nm to 50 µM reduced the effect of 25 µg/ml LPS E. coli on the increase of NO and PGE2 production. The effect of LPS was also eliminated in the presence of polymyxin B, capable to interact with lipid A in LPS molecule, which makes it inaccessible for binding to TLR4. The subcellular fractionation of epithelial cells in the sucrose density gradient in combination with immunoblotting revealed that LPS stimulates translocation of TLR4 into the lipid rafts in the cytoplasmic membrane. Preincubation of cells with GM1 or GD1a at concentration 20 µM completely eliminated the effect of LPS. A similar effect was revealed with 1 mM methyl-ß-cyclodextrin, a classical destructor of the lipid rafts. The results indicate the existence of a previously unknown mechanism of the anti-inflammatory effect of exogenous GL associated with their ability to interfere with LPS-induced translocation of TLR4 into the lipid rafts preventing LPS signal transduction. It is assumed that the observed effect of GL is based on their incorporation into cytoplasmic membrane and modification of the lipid rafts organization.

[Regulatory interconnections of cyclooxigenase and inducible no-synthase in urinary bladder epithelial cells of the frog Rana temporaria under action of bacterial stimuli].

Earlier we have shown that in epithelial cells of the frog urinary bladder under action of bacterial lipopolysaccharides (LPS) there is activated expression of inducible NO-synthase (iNOS) and there is increased the NO production, which can play an important role in providing protective cell reactions from pathogens. The goal of the present work consisted in study of cyclooxigenase (cOG) products and mechanisms of their regulatory effect on expression of iNOS under action of LPS. In experiments on urinary bladder epithelial cells on the frog Rana temporaria it has been shown that incubation of the cells for 21 h with LPS leads to a rise in production of PGE2 and nitrites, stable NO metabolites. Inhibitor of iNOS 1400W decreased sharply production of nitrites, but did not affect the PGE2 level. Both the basal and the LPS-stimulated level of PGE2 and nitrites were inhibited in the presence of selective cOG inhibitors--SC-560 (cOG-1) and NS-398 (cOG-2). The IC50 value amounted to 90, 220, and 470 microM for NS-398, SC-560, and diclofenac (unspecific inhibitor of both isoforms), respectively. PGE2 and butaprost, the EP2-receptor agonist, but not agonists of EP1/EP3 or EP1 receptors, partially eliminated the inhibitory action of diclofenac on production of nitrites. Action of PGE2 was accompanied by an increase in the intracellular cAMP. Analysis of expression of iNOS mRNA in the epithelial cells incubated with LPS or LPS + inhibitor of cOG has shown the LPS-stimulated rise in expression of iNOS mRNA to decrease sharply in the presence of SC-560 or NS-398. Thus, the epithelial cells of the frog urinary bladder have the effectively functioning system of the congenital immune protection against bacterial pathogens, the most important component of this system being PGE2 and NO. Analysis of mechanisms of regulatory interactions of cOG and iNOS indicates that in this cell type the main regulators of iNOS expression and of the nitrogen oxide level are products of the cOG catalytic activity.

[Lipopolysaccharide E. coli inhibits the arginine-vasotocin-induced increase of osmotic water permeability in the frog urinary bladder].

Since Gram-negative bacteria are known to be present in the cavity of urinary bladders in amphibian species, it was interesting to study the effect of bacterial endotoxins on epithelial signaling network which provides the arginine-vasotocin-induced increase of osmotic water permeability (OWP). The effect of LPS E. coli on AVT-induced OWP was studied in isolated frog Rana temporaria L. urinary bladder incubated during 20-21 hours in modified L-15 culture medium in sterile conditions. The LPS (25 microg/ml) was added into the mucosal solution. It was shown that exposure to LPS caused a strong suppression of the increase of OWP under AVT (0.5 nM), forskolin (35 microM) or IBMX (200 microM). Moreover, LPS induced more than 2-folds decrease both ofbasal and AVT-stimulated content of cAMP in the bladder tissue. The inhibitory effect of LPS on AVT-induced increase of OWP was eliminated in the presence of ODQ, 20 microM, a cytosolic guanylate cyclase inhibitor. With the use of RT-PCR it was shown that the expression of mRNA iNOS was 10-fold increased in 6 hours after LPS administration. These findings demonstrate the ability of frog bladder mucosal epithelial cells to recognize bacterial LPS and initiate antipathogen immune response related to increased production of nitric oxide. The activation of signal transduction cascade mediated by the LPS-induced immune response leads to a decrease of intracellular cAMP and down-regulates AVT-stimulated OWP acting at least in part through NO/cGMP-dependent signaling pathway.

[No-synthase activity in primary cultured frog urinary bladder epithielial cells and no-dependent antimicrobial effect].

We have shown previously that endogenous NO modulates the effect of arginine-vasotocin on the increase in the osmotic water permeability of the frog urinary bladder epithelium. The aim of the present work was to develop a procedure of cultivation of epithelial cells from the frog urinary bladder as a primary culture in order to study in vitro the cellular production of NO and its regulation. Isolated cells were cultivated in modified L-15 medium with 10% FBS and gentamycin (40 microg/ml) at room temperature. Under these conditions, at least 50% cells kept their viability until 8 days of incubation. NO-synthase (NOS) activity was estimated as nitrite (NO2-) accumulation in culture medium; NO2- concentration in the presence of L-NAME, inhibitor of all NOS types, was considered as NOS-independent and was subtracted from each value. The nitrite accumulation was linear in time during 3 days of cultivation and was inhibited by 1400W, inducible NOS (iNOS) inhibitor, and 7-nitroindazole, constitutive NOS's inhibitor, at doses 5-50 and 10-200 microM, respectively. One-day incubation of he cells in the medium with low concentration of gentamycin (1 or 2 microg/ml) led to the significant increase in amount of bacterial in cultured fluid identified as E. coli and Acinetobacter sp. Addition of L-NAME (5 - 103 M) to the medium potentiated the bacteria growth 1.5- and 2.5-times in the presence of 2 and 1 microg gentamycin/ml, respectively. Thus, epithelial cells form the frog urinary bladder possess NO-dependent antibacterial effect which is probably provided by induction of iNOS expression. Taken together, these data demonstrate that the primary culture of the frog urinary bladder epithelial cells is a perspective experimental model for the study of regulation of NOS activity and NO production being of particular interest in relation to the defense effect of NO in epithelia.

[The presence of nitric oxide synthase in mucosal epithelial cells of the frog urinary bladder].

In experiments on frog Rana temporaria L. urinary bladder, we investigated localization of NO-synthase (NOS) in urinary bladder slices and measured NOS activity in the suspension of mucosal epithelial cells. Intensive NADPH-diaphorase staining which is widely used as an indicator of NOS activity was found in mucosal epithelium. Almost all mucosal epithelial cells isolated in Ca2+ -free conditions demonstrated positive NADPH-diaphorase reactivity. Direct measurement of NOS activity in suspension of mucosal cells determined by the rate of conversion of L-arginine to L-citrullin showed that the enzyme activity was reduced in absence of external Ca2+ and was inhibited by L-NAME: non-specific NOS inhibitor, and 1400 W: a highly selective iNOS inhibitor (control: 754 +/- 184; L-NAME, 1 mM 329 +/- 87; 1400 W, 20 mM: 547 +/- 25; Ca2+ -free/EDTA: 490 +/- 184 cpm [3H]-citrullin/10(6) cells per 45 min, p < 0.05, n = 7-8). The data obtained demonstrate that frog urinary bladder mucosa epithelial cells provided antidiuretic hormone-induced increase of osmotic water permeability contain nitric oxide synthase. The presence of inducible (iNOS) as well as constitutive isoform(s) revealed in these cells allows to suggest involvement of NOS in intracellular signaling pathways regulated water transport across the epithelium.

[Atrial natriuretic factor stimulates the frog urinary bladder osmotic permeability in presence of a cyclic nucleotide phosphodiesterase inhibitor]. / Atrial'nyi natriureticheskii faktor stimuliruet osmoticheskuiu pronitsaemost' mochevogo puzyria liagushki v prisutstvii ingibitora fosfodiésteraz tsiklicheskikh nukleotidov.

The role of atrial natriuretic factor (ANF) in regulation of osmotic water permeability was studied in isolated frog Rana temporaria L. urinary bladder. It was found that ANF (rANF, 1-28) added to the serosal solution at concentrations 5 x 10(-8) M and higher dosedependently stimulated the arginine-vasotocin (AVT)-induced increase of osmotic water permeability. The effect of ANF was revealed only in presence of 3-isobuthyl-1-methylxantine (180 microM) and was accompanied by significant elevation of cGMP level in urinary bladder homogenate and isolated mucosal epithelial cells. C-ANF (des[Gln18, Ser19, Gly20, Leu21, Gly22]-ANF-(4-23)-NH2), a specific agonist of NPR-C receptor, exerted no effect on osmotic water permeability. ANF induced a significant increase of cAMP in urinary bladder homogenates (AVT, 5 x 10(-11) M: 52.3 +/- 10.6; AVT + ANF, 10(-7) M: 114.2 +/- 26.9 pmol/mg protein, n = 5, p < 0.05). The activity of adenylate cyclase in crude plasmatic membrane fraction was not changed. Milrinone, a specific inhibitor of phosphodiesterase 3, at concentrations from 25 to 80 microM, enhanced both the hydroosmotic response to AVT and AVT-stimulated cAMP production. Altogether these data demonstrate that, in the frog urinary bladder, ANF stimulates the AVT-induced increase of osmotic water permeability acting probably through NPR-A receptor-coupled mobilization of cGMP and cGMP-dependent inhibition of phosphodiesterase 3.

[Effect of cGMP-dependent signaling system in regulation of osmotic permeability in the frog urinary bladder]. / Rol' tsGMF-zavisimoi signal'noi sistemy v reguliatsii osmoticheskoi pronitsaemosti mochevogo puzyria liagushki.

In frogs' isolated urinary bladders, contribution of cytosolic guanylate cyclase and cGMP-dependent protein kinase to regulation of osmotic permeability was studied. ODQ (25-100 microM), an inhibitor of cytosolic guanylate cyclase induced an increase of vasotocin-activated osmotic permeability but had no effect on the hormone-activated transepithelial urea transport. In isolated mucosal epithelial cells ODQ (50 microM) decreased the concentration of intracellular cGMP. In these cells L-NAME (0.5 nM), an inhibitor of NO synthase, also decreased the level of cGMP whereas cAMP was significantly increased. 8-pCPT-cGMP (25 and 50 microM), a permeable cGMP analogue which selectively activates protein kinase G, inhibited vasotocin-induced increase of water transport along osmotic gradient indicating that protein kinase G is involved in regulation of water reabsorption. The data obtained show that NO/cGMP signalling system in the frog urinary bladder appears to be a negative modulator of vasotocin-activated increase of osmotic permeability.

[Various effects of prostaglandin E2 on reabsorption of water and urea in the amphibia osmosis-regulating epithelium]. / Razlichie vliianii prostaglandina E2 na reabsorbtsiiu vody i mocheviny v osmoreguliruiushchem épitelii amfibii.

Principal similarities between molecular pathways providing the enhancement of water and urea reabsorption under the action of argininvasotocin (AVT) in amphibian urinary bladder suggest that prostaglandin E2 (PGE2) could be a negative regulator of urea transport. To analyse this hypothesis, the role of PGE2 in regulation of urea transport was studied in isolated frog (Rana temporaria L.) urinary bladder. The urea permeability (Pu) was determined from the rate of efflux of (14) Curea from mucosal to serosal solution in isoosmotic conditions. The water permeability was measured in separate experiments in presence of an osmotic gradient. In contrast to water permeability, we were unable to demonstrate any inhibitory effect of 10-1000 nM PGE2 on AVT-stimulated urea transport using a variety of protocols. It was found that basolateral PGE2 exposure (10 nM-1 microM) caused an increase in Pu with no effect on osmotic water flow. The PGE2 effect was markedly inhibited by phloretin, a specific inhibitor of urea transporter. Sulprostone, an EP1/EP3 prostaglandin E2 receptor agonist, had no effect on Pu suggesting the contribution of EP2/EP4 receptor subtypes. In presence of osmotic water flow, the AVT-induced urea transport was significantly higher. This water flow-dependent urea permeability was inhibited by PGE2 although the inhibitory effect was less pronounced in comparison to the action of PGE2 on osmotic water flow. On the basis of these results we can make a conclusion that PGE2 has different role in regulation of water and urea transport in the frog urinary bladder. PGE2 could be considered as a stimulator of urea transport and an inhibitor of osmotic water flow activated by the AVT. The ability of PGE2 to regulate various types of cAMP-dependent transport by different mechanisms seems to be based on the presence of multiple basolateral PGE2 receptor subtypes in amphibian osmosis-regulatory epithelium.

[Comparative study of the action of antineoplastic platinum compounds with varying nephrotoxic effects]. / Sravnitel'noe issledovanie deistviia protivoopukholevykh soedinenii platiny, obladaiushchikh razlichnym nefrotoksichnym éffektom.

Studying the action of the two antitumour platinum compounds--cisplatin capable of exerting a nephrotoxic action and cycloplatam which has no damaging effect on the kidney, it was found that 3 h after the administration of cycloplatam the content of platinum in the kidney was 2 times lower than in the cfse of cisplatin. Due to different dynamics of the excretion of platinum compounds from the kidney 5 lays after their addition the content of platinum in the kidney was the same in both cases. The content of platinum in the nuclei, mitochondria and supernatant with respect to a total content in the kidney cortex was almost equal for both compounds. Inhibition of nephrotoxic effect of cisplatin after the animals were pretreated with choline chloride or paraaminohippurate is not connected with a decrease of platinum in the kidney either 3 h, or 5 days after the injection of these preparations. The mechanisms of nephrotoxic action of cisplatin and its prevention are discussed.

Renal failure and nephrotoxic drug-induced disturbances in rat kidney tissue.

The injection to rats of glycerol, cisplatin, uranyl acetate, sodium dichromate, and mercuric chloride is followed on the third day by acute renal failure. A new approach for quantitative estimation of disturbance of excretory renal function is presented. The decrease in renal function due to uranyl acetate was 77%; sodium dichromate, 71%; mercuric chloride, 52%; cisplatin, 25%; and glycerol, 10%. The kidneys still maintained serum ion concentration close to normal values. Injection of nephrotoxic drugs increased kidney wet weight by 24-57%. This was caused by swelling of renal tissue and increases in dry weight of the kidneys. The sodium content increased in the renal cortex and decreased in the papilla. The potassium content of the renal cortex is increased. The effect of some nephrotoxic drugs is suggested to depend on an increased number of cells in the renal cortex (probably due to hemostasis and inflammation) and a decrease of renal medulla function. The above drugs induce disturbance of kidney tissue but have no effect on the ion and water content in liver and m. gastrocnemius.

[The use of prednisolone for the attenuation of the nephrotoxic effect of cisplatin]. / Ispol'zovanie prednizolona dlia oslableniia nefrotoksicheskogo éffekta tsisplatina.

Rats experiments have indicated that prednisolone significantly decreases the nephrotoxic effect of the antitumor drug cisplatin. A single injection of the agent in a dose of 5 mg/kg body weight was followed by acute renal failure, which led to an increase in serum levels of creatinine and urea, to a rise in kidney weight, changes in renal water and ion content. The injection of prednisolone (5 mg/kg body weight) 3 hours prior to cisplatin administration resulted in a two-fold decrease in the concentration of nitrogen metabolic end products, without any effects on renal tissue. The protective effect of the hormone is not related to the decrease in renal cisplatin accumulation.

[The stimulation of organic acid secretion in the kidneys of the frog Rana temporaria with the repeated administration of para-aminohippurate and triiodothyronine]. / Stimuliatsiia sekretsii organicheskikh kislot v pochkakh liagushki Rana temporaria pri neodnokratnom vvedenii paraaminogippurata i triiodtironina.

Investigating possible ways of the increase in the rate of organic acid transport in the kidney of frogs, it has been demonstrated that animals which passed hypobiosis exhibit the increase in maximum capacity of the kidney to secretion of paraaminohippurate during substrate stimulation evoked by the injection of this salt twice a day within three days, as well as by the injection of triiodthyronine once a day within three days. The effect produced is similar to kidney reaction in adult mammals.

[The water-salt metabolic state of guinea pigs after a prolonged stay at different temperatures of a hyperbaric environment]. / Sostoianie vodno-solevogo obmena u morskikh svinok posle dlitel'nogo prebyvaniia pri razlichnykh temperaturakh giperbaricheskoi sredy.

Guinea-pigs were exposed for 6-10 days to a helio-oxygen mixture with the pressure 6 x 10(5) and 36 x 10(5) Pa and temperature 30.5 degrees C to 34.5 degrees C. The concentration of cations was studied in the blood serum and cortex, outer medulla and papilla of the kidney. At the optimal temperature of the helio-oxygen mixture, the concentrations of sodium, potassium, calcium, magnesium and osmotically active substances did not differ from the control in the blood serum. In these conditions, the renal ability to create osmotic gradient does not change, i.e. the systems responsible for the permanence of the cation composition of internal milieu work in a sufficiently effective way and maintain a stable concentration of ions in the blood serum.

[The action of cisplatin on the developing meso- and metanephros of chick embryos]. / Osobennosti deistviia tsis-platina na razvivaiushchiisia mezo- i metanefros kurinykh émbrionov.

Injection of various doses of cisplatin to 2-14-day chick embryos showed that within 2-8 days of incubation cisplatin produces total toxic effect, the number of dead embryos being dependent on a dose of the drug. Within 9-16 days of incubation, i.e. a period when both the mature mesonephros and the developing metanephros are in action, no significant changes were observed in the content of urea and uric acid, the weight of the meso- and metanephros, their water content, and ion content of the blood. Electron microscopic studies revealed no structural changes in the renal tubules. The data obtained suggest that cisplatin does not produce any nephrotoxic effect in chick embryos irrespectively of their age.

Cisplatin: nephrotoxic action in vertebrates and its prevention.

1. The kidney of frog and black sculpin appeared to be much less sensitive to the toxic action of CP in comparison with rat and pigeon. 2. The impairment of renal function after CP administration resulted in increased serum urea in rat, uric acid in pigeon and magnesium in black sculpin. 3. Kidney swelling is important feature of CP nephrotoxicity in rat and pigeon but not in frog and fish. 4. Pretreatment with choline chloride, PAH, furosemide and ethacrynic acid reduced the nephrotoxic action of CP in rat but did not prevent the accumulation of platinum in renal tissue which appeared to be a function of the dose injected to investigated animals.

[Morphofunctional correlates of the nephrotoxic action of cis-platin]. / Morfofunktsional'nye korreliaty nefrotoksicheskogo deistviia tsis-platiny.

A single injection of 5 mg/kg cisplatin causes (in 2 days) focal changes in rat renal parenchyma. These changes correlate well with the decrease in glomerular filtration rate, increase in blood urea concentration, decrease in water load excretion. Cellular dystrophy was found in the convoluted and straight proximal renal tubules, in line with a marked thickening of basal membrane in damaged cells, that appears to account for the impairment of paraaminohippurate secretion, the increase of kidney mass and its swelling. On day 5 after injection the renal parenchyma exhibited signs of reparation, while renal function was abnormal.