Kinin-Destroying (Kininase) Activity of Cultured Rodent Fibroblasts L-929 1 2

Extracts of a cultured cell line of rodent fibroblasts, L-929, were reported to have kinin-forming activity when incubated at acid pH with kininogen substrate from rat plasma or Murphy-Sturm lymphosarcoma tissue (1). The kinin-forming acid protease was purified and further characterized with regard to pH optimum, cellular localization, and molecular weight (2). While the fibro-blast preparation did not contain kininogen substrate, it did have kinin-destroying activity that was inactivated at acid pH as well as by 1,10-phenanthroline (1). In view of the possible involvement of the vasopeptide kinin system in the growth and development of transplanted tumors containing considerable fibroblast networks (3), the nature of this kinin-destroying activity of fibroblasts was studied further and forms the basis of this report.

Methods. Cell culture and cell extract preparation. Mouse fibroblasts L-929, obtained from Microbiological Associates (Be-thesda, Maryland), were grown for 4 days in roller bottles in minimum Eagle's medium containing 10% fetal calf serum as described previously (1). The cells were scraped from the bottle wall at the time of study, suspended in minimum Eagle's medium, the cell number was determined by microscopic examination, and the cell suspension was centrifuged in a refrigerated centrifuge at 3500 rpm. The cells then were resuspended in physiologic saline and disrupted by freeze-thaw technique repeated 10 times. Aliquot volumes of 0.2-0.5 ml of the cell extract, approximating 6 × 10⁶ cells, were used for the assays.

Kinin-destroying activity. Fibroblast suspensions (0.2-0.5 ml) were incubated at 37° with 0.5 ml of synthetic bradykinin (1 × 10^-6 g, Sandoz, Hanover, New Jersey) dissolved in 0.05 M phosphate buffer, pH 7.4. Additional buffer was added to 1 ml when indicated, and at 1-, 3-, and 5-min intervals, 1 mg of 1,10-phenanthroline was added to individual incubation mixtures to terminate the reaction.