Many recent reports present evidence that bradykinin-related oligopeptides, collectively called kinins, play important role in the central nervous system (CNS) upon injury, disease, infection and inflammation. The presence of kinin receptors of both constitutive B2 type and inflammation-inducible B1 type in CNS is well documented. All components of the tissue kallikrein-dependent kinin-generating system have been identified in the brain. We hypothesize that a significant proportion of total kinin in CNS may be produced by a surface adsorption-dependent cascade similar to the contact-phase system operating in plasma and composed of high molecular weight kininogen (HK), plasma prekallikrein and factor XII. In this mechanism, a primary event is the binding of HK to negatively charged surfaces of some cells.

In an attempt to present the first rationale for that hypothesis, we hereby characterize the binding of kininogens by model human neuroblastoma cell lines IRM–32 and BE(2) C. These cells were incubated with increasing concentrations of biotin-labeled HK or LK (low molecular weight kininogen), in the presence of zinc ions, at 4^oC or 37^oC, for 1 hour. The amount of bound kininogen was determined by enzymatic reaction after incubation with streptavidin-conjugaded horseradish peroxidase. From the saturation binding plots obtained, the binding parameters were estimated. For HK binding to IRM-32 cells, the dissociation constant (K_d) varied from 10 to 25 nM and the maximal amount of HK bound was up to 0.2 pmoles per million cells. The binding significantly increased at lower temperature. The BE(2) C cells showed lower affinity to HK than IRM-32 cells. LK bound to both IRM-32 and BE(2) C cells with the affinity lower by at least one order of magnitude. Moreover, the LK binding was essentially independent of the presence zinc ions. The binding of HK to the neuroblastoma cells was determined both in their resting state as well as after their differentiation into neuronal cells. Using fluorescein-labeled HK we were able to visualize kininogen molecules bound on the neuron surface. Hundred-fold excess of unlabeled HK (but not LK) completely displaced fluorescein-labeled HK from the cell surface, suggesting that the binding was relatively specific. No significant proteolytic degradation of kininogens upon 1-hr incubation with neuroblastoma cells was observed. Hence, a secondary adsorption of (pre)kallikrein, or some other kininogenase(s), to the cell-surface adsorbed HK is necessary for triggering the kinin-formation cascade. Further studies are carried out in our laboratory to specify the full mechanism of neuron surface-dependent kinin release and to suggest its possible contribution to some pathophysiologic processes in nervous tissue.

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