Protein kinase C
Luminol enhanced chemoluminescence
Murine peritoneal neutrophils
Springer Online Journal Archives 1860-2000
Summary In binding competition assays using a protein kinase C preparation from mouse brain (particulate fraction)3H-labelled 12-O-tetradecanoylphorbol-13-acetate (TPA), for a series of new diterpene esters (DTE) the relative binding affinity [rba=K i a (TPA)/K i a (DTE)] in relation to TPA was determined. A wide range of values was noticed, some of the DTE binding more strongly than TPA (rba 〉1), others binding less strongly than TPA (rba 〈1) In comparative terms, competition for specific binding sites appears to correlate better with irritant than with promoting acitvity of the DTE. Using mouse peritoneal neutrophils, binding of [3H]-TPA was determined by a modification of the “cold-acetone filter assay”; saturation of high-affinity sites (K d a =0.2 nM) was obtained at concentrations ≦ 1 nM, but there was also evidence for specific binding at “low-affinity” sites (K d a =26 nM). Induction of chemoluminescence in the presence of luminol in mouse peritoneal neutrophils with a set of DTE usually elecited two peaks; at concentrations ≧10 nM DTE a short-lived, “spike-like” response lasting only from 0 to about 5 min (phase A) ist followed by a “plateau” response from about 5–120 min (phase B). This latter phase of chemoluminescence stimulation with luminol correlated well with theirritant potential of the DTE used. The sequence of the two phases can be inverted partially by using first TPA at 2,5 nM followed by a quick concentration increase to 100 nM; this indicates two different concentration-dependent events. As regards the intensity of the chemoluminescent response, quantitative but not qualitative differences between DTE were observed, which show some correlation with strong and weak tumour-promoting activity. Inhibition studies suggest the involvement of the myeloperoxidase/H2O2/Cl− system in the luminogenic response; it is suggested that the release of hypochlorite or a closely related oxidant may be instrumental in tumour promotion.
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