SYNTHESIS OF KININOGEN AND DEGRADATION OF BRADYKININ BY PC12 CELLS

1 In this study, the abilities of PC12 cells to synthesize and degrade kinins were investigated. Kinin formation was assessed as kinin and k ininogen content of cells and supernatants in serum-free incubations b y use of a bradykinin-specific radioimmunoassay. Expression of kininog en mRNA was demonstrated by reverse-transcriptase PCR. Kinin degradati on pathways of intact PC12 cells were characterized by identification of the kinin fragments generated from tritiated bradykinin either in t he absence or presence of the angiotensin I-converting enzyme inhibito r ramiprilat. 2 Kinin immunoreactivity in the supernatant of PC12 cell cultures accumulated in a time-dependent fashion during incubations i n serum-free media. This effect was solely due to de novo synthesis an d release of kininogen (35 pg bradykinin h(-1) mg(-1) protein) since i t could be suppressed by cycloheximide. Continuous synthesis of kinino gen was a specific property of PC12 cells, as it was not observed in c ultured macro-or microvascular endothelial cells. PC12 cells contained only minor amounts of stored kininogen. The rate of kininogen synthes is was not affected by ramiprilat, bacterial lipopolysaccharide, nerve growth factor or dexamethasone, but was stimulated 1.4 fold when cell s were pretreated for 1 day with 1 mu M desoxycorticosterone. 3 By use of cDNA probes specific for kininogen subtype mRNAs, expression of lo w-molecular-weight kininogen and T-kininogen in PC12 cells was confirm ed. Expression of high molecular weight kininogen mRNA was also shown, though only at the lowest limit of detection of the assay. 4 Degradat ion of tritiated bradykinin by PC12 cells occurred with a half-life of 48 min resulting in the main fragments [1-7]- and [1-5]-bradykinin. T he degradation rate of bradykinin decreased to 15% in the presence of ramiprilat (250 nM). Apart from angiotensin I-converting enzyme direct cleavage of bradykinin to [1-7]- and [1-5]-bradykinin still occurred under this condition as a result of additional kininase activities. 5 Along with previous findings of B-2-receptor-mediated catecholamine re lease, these results now confirm the hypothesis that a cellular kinin system is expressed in PC12 cells. The presence of such a system may r eflect a role of kinins as local neuromodulatory mediators in the peri pheral sympathetic system.