NO-SYNTHASE ACTIVITY IN PRIMARY CULTURED FROG URINARY BLADDER EPITHELIAL CELLS AND NO-DEPENDENT ANTIMICROBIAL EFFECT
E. M. Fock, V. T. Bachteeva, E. A. Lavrova, S. D. Nikolaeva, R. G. Parnova 1
I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, St. Petersburg;
1 e-mail: parnova@iephb.ru
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 μg/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
μM, respectively. One-day incubation of he cells in the medium with low concentration of gentamycin (1 or 2 μg/ml) led to the significant increase in
amount of bacterial in cultured fluid identified as E. coli and Acinetobacter sp. Addition of L-NAME (5·10-3 M) to the
medium potentiated the bacteria growth 1.5- and 2.5-times in the presence of 2 and 1 μg 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.
Key words: nitric oxide, NO-synthase, primary cell culture, frog urinary bladder epithelium, antibacterial effect
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