The biochemical mechanisms of action of tigliane and phorbol esters

Initial studies adding TPA and Sap A to Human Mononuclear cell (HMNC) cultures found that the mitogenic action of Sap A was abolished if cells were incubated for 3 days prior to phorbol ester addition. Previously the phorbol ester receptor has been found to be a family of protein kinases (PKC's). The relationship between biochemical action and biological effect was investigated using rat brain as the enzyme source. Phorbol ester-stimulated kinase activities in rat brain hydroxylapatite FPLC fractions were investigated using a modified PS/Triton micellar assay. Proteins in the elution profile were separated by SDS-PAGE and immuno-blotted with antisera specific for PKC α, β1, γ, δ and ε to identify the PKC isotypes present. These investigations were coordinated with phorbol ester activation studies on PKC Isotypes (α, β1, γ, δ and ε) purified from bovine brain. This methodology was then applied to screen eluted proteins from subpopulations of HMNC as well as Daudi cells and mouse macrophages. These approaches revealed several phorbol ester-sensitive protein kinase activities, which could not be identified as PKC α, β1, γ, δ and ε Isotypes by elution or immunological definition, in crudely fractionated brain and cell extracts. Some of these activities demonstrated tissue specificity when elution positions characteristic of brain were compared to those seen for cultured cells. Preparations of HMNC's were also found to contain an entirely novel kinase activity which was stimulatable by Resiniferatoxin (Rx, a daphnane ester) only in the absence of added calcium (termed Rx-Kinase). A similar activity was also isolated, in greater quantity, from starch-elicited mouse peritoneal macrophages under identical conditions. Fractions of mouse Rx-Kinase activity were found to potently activate the reconstituted mouse NADPH oxidase system to generate superoxide in vitro In the presence of Rx and the absence of calcium (i.e. conditions corresponding to its histone kinase requirements in vitro).This suggests major differences between this putative phorbol ester receptor and the PKC family of isotypes.