[No-synthase activity in primary cultured frog urinary bladder epithielial cells and no-dependent antimicrobial effect].
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.
Fok
E M
EM
Bakhteeva
V T
VT
Lavrova
E A
EA
Nikolaeva
S D
SD
Parnova
R G
RG
D000150:Acinetobacter / Q000254:growth & development; D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology* / Q000201:enzymology / Q000276:immunology / Q000382:microbiology; D004926:Escherichia coli / Q000254:growth & development; D007097:Imines / Q000494:pharmacology; D007191:Indazoles / Q000494:pharmacology; D008297:Male; D008826:Microbial Sensitivity Tests; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D011896:Rana temporaria / Q000378:metabolism*; D001743:Urinary Bladder / Q000166:cytology* / Q000201:enzymology / Q000276:immunology / Q000382:microbiology
D003470:Culture Media; D004791:Enzyme Inhibitors; D007097:Imines; D007191:Indazoles; C496401:N-((3-(aminomethyl)phenyl)methyl)ethanimidamide; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C080122:7-nitroindazole; D019331:NG-Nitroarginine Methyl Ester