Multiple forms of phosphoinositide-specific phospholipase C of different relative molecular masses in animal tissues. Evidence for modification of the platelet enzyme by Ca2+-dependent proteinase.

The Mr distribution of phosphoinositide-specific phospholipase C in the supernatants isolated from a variety of animal tissues was analysed by high-performance gel-filtration chromatography. In most tissues, at least four peaks of activity were resolved. However, different tissues showed quite marked differences in the distribution of activity between these peaks. In rat heart, lung and kidney, the predominant form had Mr approx. 90000, whereas the predominant form in brain had Mr approx. 290000. In liver, the Mr-90000 form predominated, but this tissue also contained relatively large amounts of a form of Mr approx. 150000. Phospholipase C in these tissues from other animal species gave similar distributions of activity between the peaks. In supernatants prepared from platelets sonicated in the presence of leupeptin (0.5 mM) or EGTA (20 mM), the Mr-290000 form predominated. However, when leupeptin or EGTA (inhibitors of Ca2+-dependent proteinase) was omitted from the sonication buffer, the Mr-290000 form appeared to be replaced by a form of Mr 100000. Similar changes in Mr were not demonstrated with the other tissues. These results may be relevant to the intracellular regulation of phospholipase C, since Ca2+-dependent proteolysis has been reported to occur during platelet activation.     

Isolation and characterization of a phospholipase A2 from an inflammatory exudate.

Sterile peritoneal exudates produced in rabbits injected with 1% glycogen contain a phospholipase A activity in a cell-free supernatant fraction that hydrolyzed a synthetic phospholipid (1,2-diacyl-sn-glycero-3-phospho-ethanolamine) and phospholipids of autoclaved Escherichia coli. This phospholipase activity (phosphatidylacylhydrolase EC 3.1.1.4) exhibited an apparent bimodal pH optimum (pH 6.0 and pH 7.5) and was Ca(2+)-dependent; Mg(2+) and monovalent cations (Na(+) and K(+)) did not substitute for Ca(2+) in the reaction; EDTA was a potent inhibitor. The phospholipase hydrolyzed 1-[1-(14)C]palmitoyl-2-acyl-sn-glycero-3-phosphoethanolamine to form only radio-active lysophosphatidylethanolamine as the product, indicating that the enzyme had phospholipase A(2) specificity. The phospholipase A(2) was purified 302-fold by two successive chromatographic steps on carboxymethyl Sephadex. Gel filtration (Sephadex G75) of the purified enzyme resulted in a single peak of biological activity with a molecular weight of approximately 14,800. The same estimate of molecular weight was obtained by SDS-polyacrylamide gel electrophoresis, which yielded a single band. Polyacrylamide gel electrophoresis of this fraction at pH 4.3 revealed a single protein band migrating beyond lysozyme, with the dye front, suggesting that this protein was more basic than lysozyme (pI 10.5). The enzymatic and physical-chemical characteristics of this soluble enzyme were remarkably similar to a recently described phospholipase A(2) of rabbit polymorphonuclear leukocytes derived from glycogen-induced peritoneal exudates. The possible origin and physiological role of this soluble enzyme are discussed.     

Protective mechanisms of Mg-gluconate against oxidative endothelial cytotoxicity.

The potential anti-radical properties and cytoprotective effects of Mg-gluconate were studied. When microsomal membranes were peroxidized by a *O2- driven, Fe-catalyzed oxy-radical system (R* = dihydroxyfumarate + Fe2+), Mg-gluconate inhibited lipid peroxidation (TBARS formation) in a concentration-dependent manner with IC50 being 2.3 mM. For the entire range of .25-2 mM, MgSO4 or MgCl2 were < or = 20% effective compared to Mg-gluconate. When cultured bovine aortic endothelial cells were incubated with the R* for 50 min. at 37 degrees C, 56% loss of total glutathione occurred. Pre-treatment (10 min.) of the cells with 0.25-4 mM Mg-gluconate before R* exposure significantly (p<0.05) prevented the GSH loss to varying degrees; the EC50 was 1.1 mM. In separate experiments, with 30 min. of free radical incubation of endothelial monolayers (approximately 65% confluent), cell survival/proliferation determined by the tetrazolium salt MTT assay, decreased to 38% of control at 24 hrs; Mg-gluconate concentration-dependently attenuated the lost cell survival with EC50 of approximately 1.3 mM. For comparison, the effects provided by MgSO4 or MgCl2 were significantly lower and were < or = 1/3 as potent as that produced by Mg-gluconate. In a Fenton-reaction system consisting of Fe(II)+ H2O2, Mg-gluconate but not other Mg-salts, significantly inhibited the formation of OH radicals as determined by the ESR DMPO-OH signal intensity. Mg-gluconate also dose-dependently inhibited the 'Fe-catalyzed' deoxyribose degradation suggesting that Mg-gluconate could displace Fe from 'catalytic sites' of oxidative damage. These data suggest that Mg-gluconate may serve as a more advantageous Mg-salt for clinical use due to its additional anti-radical and cytoprotective activities.     

Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium

Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids.     

Organ specificity and transcriptional control of metabolic routes revealed by expression QTL profiling of source-sink tissues in a segregating potato population

Background  

With the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization.     

The EGFR tyrosine kinase inhibitor tyrphostin AG-1478 causes hypomagnesemia and cardiac dysfunction

We determined whether the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) N-?(3-?chlorophenyl)-?6,?7-?dimethoxy-?4-?quinazolinamine (tyrphostin AG-1478) causes hypomagnesemia and cardiac dysfunction in rats. Tyrphostin was administered (3 times per week, intraperitoneal injection, to achieve 21.4 mg·(kg body mass)(-1)·day(-1)) to normomagnesemic rats for 5 weeks. Levels of magnesium in the plasma of the tyrphostin-treated rats decreased significantly by the following amount: 17% at week 1, 27% at week 2, and 26%-35% between weeks 3 to 5. Levels of the plasma lipid peroxidation marker 8-isoprostane rose significantly: by 58% at week 1, 168% at week 3, and 113% at week 5. At week 5, blood neutrophils from the tyrphostin-treated group displayed a 2.26-fold higher basal level of O(2)(·-) generation; the ratio of oxidized glutathione (glutathione disulfide; GSSG) to reduced glutathione (GSH) in the red blood cells increased 2.5-fold. At week 5, echocardiography revealed that TKI treatment resulted in significant cardiac systolic dysfunction, with impaired diastolic function and dilated cardiomyopathy. Since hypomagnesemia alone can trigger oxidative stress and cardiac injury, we suggest that inhibition of EGFR-TK caused magnesium wasting, which partly contributed to decreased cardiac contractility.     

Rapid agonist-induced decrease of 125I-pindolol binding to beta-adrenergic receptors. Relationship to desensitization of cyclic AMP accumulation in intact heart cells

Cyclic AMP accumulation in embryonic chick heart cells and binding of the beta-adrenergic antagonist 125I-pindolol to intact cells has been examined during the first 30 min of (-)-isoproterenol-induced desensitization. Myocardial beta-adrenergic receptors exist in two states which bind agonists with high (KD congruent to 10 nM) and low (KD congruent to 10 microM) affinities. Both activation and desensitization of cyclic AMP accumulation were mediated by (-)-isoproterenol binding to high affinity receptors. (-)-Isoproterenol-induced desensitization of cyclic AMP accumulation occurred with a t1/2 of 3.8 min. Desensitization was accompanied by a decrease in the number of 125I-pindolol binding sites assessed by equilibrium radioligand binding assays conducted at 4 degrees C or short (80 s) binding assays conducted at 37 degrees C. There was an excellent temporal correlation between loss of binding and loss of (-)-isoproterenol-stimulated cyclic AMP accumulation. After (-)-isoproterenol-induced desensitization, most of the remaining receptors assayed at 4 degrees C bound (-)-isoproterenol with high affinity. A rapid (-)-isoproterenol-induced decrease in the number of 125I-pindolol binding sites also occurred in adult canine heart cells and rat adipocytes. The data suggest that agonists do not cause uncoupling of surface receptors. Receptors may be uncoupled as a consequence of rapid sequestration into a hydrophobic compartment.