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

Abstract

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 (NO2−) in the cultivation medium; the amount of NO2− 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 NO2− 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.