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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 form pathogens. The goal of the present work consisted in study of cyclooxygenase (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 μM 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 cPG 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: liver > kidney >> brain > urinary bladder (epithelium) > heart > testis. 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 (p < 0.05). 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.