Search

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.

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate, arginase can affect the NO synthesis. In the present work, the properties of arginase from the frog Rana temporaria L. urinary bladder epithelial cells possessing the NO-synthase activity were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme had the temperature optimum in the range of 55-60 degrees C, K(m) for arginine 23 mM, and V(max) about 10 nmol urea/mg protein/min, and its activity was effictively inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10(-6) to 10(-4) M. The comparison of arginase activity in various frog tissues revealed the following pattern: liverkidneybrainurinary bladder (epithelium)hearttestis. The arginase activity in the isolated urinary bladder epithelial cells was 3 times higher than that in the intact urinary bladder. To evaluate the role of arginase in the regulation of NO production, epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO2-, the stable NO metabolite, was determined in the culture fluid after 18-20 h of cells incubation. The vast majority of the produced nitrites are associated with the NOS activity, as L-NAME, the NOS-inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in 199 medium. BEC (10(-4) M) increased the nitrite production by 18.0 % +/- 2.7 in the L-15 medium and by 24.2 +/- 3.5 in the 199 medium (p0.05). The obtained data indicate a relatively high arginase activity in the frog urinary bladder epithelium and its involvement in regulation of NO production by epithelial cells.

Na, K, Ca and Mg concentrations have been determined in the otoliths and otoconia of the lamprey, skate, guppy and hen. Lamprey otoliths sharply differ from the otoliths and otoconia of other animals with respect to their K and Ca content. In all the animals investigated excluding lamprey, only traces of K were found. Na concentration in the otilithic apparatus of the investigated animals varies within 0.475-1.04 mM/g. Na presumably is bound to proteins of the organic matrix and plays an important role in the growth of otoliths and otoconia. Concentration of Ca in the otoliths and otoconia of all animals excluding the lamprey was equal to 9.09-9.63 mM/g: organic substances constitute 4-10 per cent of their mass. In the lamprey, Ca concentration in the otoliths was found to be 5.33 mM/g, the content of organic matter amounting to more than 40 per cent. In constantly growing otoliths of fish, concentration of Mg was significantly lower than in otoconia of fish and other animals. This is presumably due to the fact that Mg inhibits crystalline growth of calcium salts.

Frogs acclimated to 4 degrees C were transported to a medium with temperature 20 degrees C, which caused polyuria; recovery of normal diuresis took about 24 h. During this period, hypernatremia was observed together with the increase in natriuresis, the rate of renal excretion of potassium ions with urea remaining constant. Water content of skeletal muscles decreased. Transportation of frogs acclimated to 20 degrees C into a medium with a temperature 4 degrees C decreased their diuresis. Renal excretion of sodium, calcium and magnesium ions remained unchanged, whereas that of potassium ions significantly decreased. The content of potassium and magnesium in the blood serum increased, that of sodium--decreased. Hydratation of muscled and kidneys was accompanied by the decrease of calcium, potassium and magnesium ions calculated per wet weight of the tissues, the level of sodium remaining unaffected. The data obtained indicate significant changes in the pattern of water and salt metabolism in frogs during temperature hysteresis.

Concentration of osmotically active substances in the blood serum of the sturgeon A. guldenstaedti and sevruga A. stellatus from the central part of the Caspian Sea is practically equal to that in the sea water. In the blood serum of the sturgeon and the sevruga, concentration of Na is higher, whereas concentration of K and Mg is lower than in the sea water. Excretion of the urine with relatively low Mg concentration presumably indicated that being isoosmotic to the medium, the fishes studied in the Caspian Sea do not swallow the sea water. Electronmicroscopic studies indicate that with respect to poor development of membranous structures at the base of cells in the proximal tubuli, the kidney of the sturgeon and sevruga stands closer to freshwater than marine teleosts. These data show specific pattern of the osmotic and ionic regulation in the sturgeon, i.e. its isoosmotic type during migration to the Caspian Sea.

To investigate the role of neurohumoral factors in acclimation of mussel muscle to a lowered salinity, studies have been made on the reaction of the intact mussel muscle and that of isolated muscle to change in the salinity from 26% to 10%. The general pattern of swelling of the adductor and the dynamics of its electrolyte composition in intact molluscs are similar to those in the isolated muscle. In the intact mussel this reaction of the adductor is slower. Acclimation of adductor cells is of the autonomic nature and does not depend on neurohumoral influences.

In animals in which sodium is a predominant cation of the blood serum or haemolymph (rat, newt Triturus vulgaris, sturgeon Acipenser güldenstädti, herring Clupea pallasi, ascidian Goniocarpa rustica), its content in the body amounts up to a half of the total bulk of electrolytes; in insects with potassium type of the haemolymph, the share of sodium is equal to 6.4%. In animals of the same species, total electrolyte content (sodium, potassium, calcium, magnesium) and the relationship between separate ions were found to be remarkably constant. During ontogenetic development of the sturgeon and herring, as well as in rat embryos beginning from the 15th day of gestation, the ratio between separate cations exhibits significant conservatism.

From the 19th day of embryonic period, within 4 days, i. e. up to the beginning of postnatal life, the weight of the heart in rats increases by 3.3 times, that of the liver--by 2.2 times, and kidneys--by 5.7 times, whereas the content of water (calculated per dry weight of the organs) decreases. Changes in the content of sodium, potassium, calcium and magnesium in these organs are the lowest provided calculations are made per wet weight; during organogenesis, gradual decrease in the content of electrolytes (calculated per dry weight of the tissue) takes place. The value of Na/K ratio decreases during organogenesis. The content of iron and copper in these organs is higher in adult animals, than in younger ones.

Intra-abdominal injections of platinum, chromium and cadmium salts to Myoxocephalus scorpius produce nephrotoxic effect which includes the disturbances in magnesium secretion. Basic ultrastructural changes in the nephron cells of the fish after the injection of nephrotoxic substances are similar to those in mammals. Cis-platinum induces significant damage in the terminal part of the proximal tubules. One day after the injection of chromium compounds, total damage of the proximal tubule is observed, whereas cadmium salt affects cells within the whole nephron. After 5 days of administration of cadmium and chromium salts, partial recovery was found with respect to both functional and ultrastructural properties of nephron cells. Administration of nephrotoxic substances which selectively injure different parts of the nephron enabled to perform more exact differentiation of the nephron elements in marine teleosts.