Sharma, Pawan; (1995) Stimulation of phagocytic cells by phorbol esters. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Initial studies using phorbol ester stimulation of superoxide anion (O2-) production from mouse peritoneal macrophages revealed a degree of selectivity. The action of a daphnane diterpene, Resiniferatoxin (Rx) was synergised by the presence of particulate zymosan and this effect only occurred from starch-elicited derived cell populations but not in resident macrophages. Previously the phorbol ester receptor has been found to be a family of protein kinases (PKC's). More recently a novel kinase activity which was stimulatable by Rx only in the absence of added calcium was discovered (termed Rx-kinase). A similar activity was also isolated in greater quantity from starch-elicited mouse peritoneal macrophages under identical conditions. To further understand the nature of this enzyme extensive studies of co-factor requirements, phorbol dibutyrate binding and, tissue distribution were investigated on Rx-kinase derived from hydroxyapatite FPLC from various sources. Interestingly Rx-kinase activity was discovered to be enhanced by priming of cells such as macrophages prior to purification. Proteins from elution fractions were purified on SDS-PAGE and when immunoblotted with specific antisera for PKC revealed a novel enzyme unrelated to previously known isotypes of PKC. A comparison of macrophage Rx-kinase and pooled rat brain PKC stimulation of the reconstituted membrane NADPH-oxidase system to generate superoxide anion in-vitro was investigated in a cell free assay system. Rx-kinase was found to potently activate the physiological substrate in-vitro in the presence of Rx and absence of calcium (i.e. conditions corresponding to its histone kinase assay requirements in-vitro.) To further evaluate the signalling pathways leading to the activation of oxidase, involvement of calcium ions in modulation of phagocytic cells and pure PKC isotypes stimulation of the oxidase itself was studied. Phorbol esters inhibited fMLP stimulated divalent cation influx but synergised with intracellular calcium to produce superoxide anion in neutrophils. Unstimulated PKC activity showed that calcium independent PKC's were more potent than the calcium dependent isotypes and corresponding to the action of Rx-kinase. Additionally the ability of daphnane derivative Thymeleatoxin A (TxA) and Rx to stimulate superoxide anion from neutrophils and monocytes were investigated. TxA was more potent in stimulating superoxide from neutrophils compared to monocytes. The relationship between biological response and biochemical mechanism involved in this system was investigated by studying PKC isotype profiles of neutrophils via hydroxyapatite chromatography.

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