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

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.