Agonist-induced degradation of phosphatidylcholine (PC) is of interest as this pathway of diacylglycerol (DG) generation may provide added opportunities for the regulation of protein kinase C (PKC). In REF52 cells pH]myristic acid is preferentially incorporated into PC; this, coupled with the use of pH]eholine, allows for quantitation of both the water-soluble and the lipid products generated when PC is degraded. In cells prelabeled with pH]choline, TPA stimulated a time-dependent release, into the medium, of choline and not phosphocholine or glycerophosphocholine. Treatment of pH]myristic acid-labeled cells with either phorbol diesters, sn-1,2-dioetanoyiglycerol, or vasopressin elicited the formation of labeled phosphatidate (PA) and DG. The temporal pattern of PC hydrolysis in cells treated with TPA is indicative of a precursor (PA)-product (DG) relationship for an enzymatic sequence initiated by phospholipase D. Adding propranolol, a phosphatidate phosphohydrolase inhibitor, eliminated TPA-induced DG formation, whereas PA generation was unaffected. From these data we conclude that TPA elicits DG formation from PC by the sequential actions of phospholipase D and phosphatidate phosphohydrolase.
By employing early-passaged rabbit kidney epithelial cells in tissue culture, we demonstrated that angiotensin II (AII) has unique mechanisms of signal transduction. First, unlike its action in other target tissues, micromolar concentrations of AII are required to induce small rises in cytosolic calcium, [Ca2+J, an action which is not accompanied by the release of inositol phosphates (IP). In contrast, nanomolar bradykinin (BK) mobilizes [Ca2", through activation of phospholipase C and release of IP. Neither of these stimulated calcium responses exhibits pertussis toxin (PTx) sensitivity. Secondly, AII and BK at 10' to 10'-M stimulate cAMP indirectly through PGE2 production in distal cells. AII-and BK-stimulated PGE2 release is PTx inhibitible, suggestive of the presence of a GTP binding protein mediating the response. By contrast, arginine vasopressin fails to elicit rises in ICa2+I, but exerts its primary effect on cAMP production in distal cells via direct coupling to a stimulatory GTP binding protein, as evidenced by uncoupling with cholera toxin. Regulation of PGE2 synthesis appears to occur via phospholipase A2, not C, by all three peptides.
Nephrotoxic cysteine conjugates derived from a variety of halogenated alkenes are enzymatically activated via the beta-lyase pathway to yield reactive sulfur-containing metabolites which bind covalently to cellular macromolecules. Mitochondria contain beta-lyase enzymes and are primary targets for binding and toxicity. Previously, mitochondrial protein and/or DNA have been considered as molecular targets for cysteine conjugate metabolite binding. We now report that metabolites of nephrotoxic cysteine conjugates form covalent adducts with rat kidney mitochondrial phospholipids. Rat kidney mitochondria were incubated with the 35S-labeled conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), S-(1,2-dichlorovinyl)-L-cysteine, and S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine. Quantitation of metabolite binding to whole mitochondria and to mitochondrial protein and lipid fractions revealed that as much as 42% of the 35S-label associated with the mitochondria was found in the lipid fraction. Total lipids were also extracted from 35S-treated mitochondria and separated by thin-layer chromatography. 35S-Containing metabolites were found in the lipid fractions from mitochondria treated with each of the conjugates. Lipids from both [35S]CTFC- and [35S]-TFEC-treated mitochondria contained major 35S-labeled lipid adducts which had similar mobility by thin-layer chromatography. Fatty acid analysis, 19F and 31P NMR spectroscopy, and mass spectrometric analyses confirmed that the major TFEC and CTFC adducts are thioamides of phosphatidylethanolamine.
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