Human neutrophil protein kinase C: calcium-induced changes in the solubility of the enzyme do not always correlate with enzymatic activity

Abstract

We hypothesized that calcium and 1,2-diacylglycerols stimulated human neutrophil (PMN) protein kinase C (EC 2.7.1.37) in a two-step mechanism. The proposed mechanism entails (1) increased insoluble protein kinase C activity and (2) endogenous protein phosphorylation, events which have not been biochemically dissociated. PMN which were treated with 100 nM ionomycin shifted protein kinase C activity from being mostly soluble to insoluble. Concentrations of ionomycin greater than 300 nM stimulated a doubling of total cellular (soluble + insoluble) protein kinase activity and stimulated increased endogenous phosphorylation of PMN proteins. Intracellular calcium (measured with fura-2) increased from 65 nM (basal) to 680 nM using 500 nM ionomycin; calcium increases were dose-dependent. The anti-inflammatory agents acetylsalicylic acid and sodium salicylate (but not ibuprophen, indomethacin or acetaminophen) inhibited ionomycin-induced protein kinase C activation and protein phosphorylation in a dose-dependent manner by inhibiting the production of diacylglycerols. 1-Oleoyl-2-acetylglycerol reversed the inhibitory effect of salicylates. In contrast to the effect of acetylsalicylates on protein kinase C functional activity the distribution of phorbol receptors was unaffected in acetylsalicylate-treated, ionomycin-stimulated PMN using a phorbol-binding assay. Our results show that ionomycin increased intracellular diacylglycerol levels 3.5-fold over those present in control PMN, while acetylsalicylate decreased diacylglycerol production in ionomycin-stimulated PMN below baseline values. These results support the hypothesis that increased intracellular calcium activated protein kinase C leading to protein phosphorylation in two distinct dissociable events: (1) increased intracellular calcium; and (2) increased 1,2-diacylglycerol levels.

Neutrophil superoxide and hydrogen peroxide production in patients with acute liver failure

“…Although previous in vitro studies on neutrophil function from healthy control subjects has shown that paracetamol (even at toxic concentrations) does not affect chemotaxis or superoxide production [25][26][27], it is possible that an in vivo overdose could inhibit neutrophil function. Although previous in vitro studies on neutrophil function from healthy control subjects has shown that paracetamol (even at toxic concentrations) does not affect chemotaxis or superoxide production [25][26][27], it is possible that an in vivo overdose could inhibit neutrophil function.…”

“…The cause of ALF in this series of patients was paracetamol overdose. Although previous in vitro studies on neutrophil function from healthy control subjects has shown that paracetamol (even at toxic concentrations) does not affect chemotaxis or superoxide production [25][26][27], it is possible that an in vivo overdose could inhibit neutrophil function. In this study, all patients assessed were receiving an infusion of N-acetylcysteine when the assays were performed.…”