Study of NO-synthase activity and detection of NO-dependent antimicrobial effects in primary culture of frog urinary bladder epithelial cells

We have shown previously that endogenous nitric oxide (NO) in frog urinary bladders modulates the influence of arginine-vasotocin on the increase in the osmotic water permeability of the bladder epithelium. The goal of the present work was to obtain a primary culture of epithelial cells from a frog urinary bladder in order to study in vitro the cellular activity of NO-synthase (NOS) and its regulation. The best conditions for cultivating the cells turned out to be with the use of a modified L-15 medium containing 10% fetal bovine serum and gentamicin (40 μg/ml) at room temperature. Under these conditions, at least 50% of the cells preserved their viability for 8 days. The NOS activity was estimated from the accumulation of nitrites (NO ₂ ^? ) in the cultivation medium; the amount of NO ₂ ^? in the presence of nitro-L-arginine methyl ester (L-NAME), an inhibitor of all types of NOS, was considered unspecific and was subtracted from each value. The NO ₂ ^? accumulation was linear in time for three days of cultivation and was inhibited by 1400W, an inducible NOS (iNOS) inhibitor, or by 7-nitroindazole, an inhibitor of constitutive NOSs, at concentrations of 5–50 and 10–200 μM, respectively. One-day incubation of the cells in the medium with low doses of gentamicin (1 or 2 μg/ml) led to a marked increase in the amount of bacteria in the cultivation m5 mM edium identified as E. coliand Acinetobacter sp. An addition of 5 mM L-NAME to the cultivation medium increased the amount of the bacteria by 1.5-and 2.5-fold in the presence of 2 and 1 μg/ml gentamicin/ml, respectively. Thus, the epithelial cells of frog urinary bladders have the NO-dependent antibacterial effect, which seems to be the result of enhanced iNOS expression. The obtained data indicate that the primary culture of the epithelial cells from frog urinary bladders is a prospective model for studying the role of NO and the regulation of NOS activity, which is of particular interest for studying protective NO effects in epithelial tissues.