Production of lysophospholipids and free fatty acids by a sarcolemmal fraction from canine myocardium.

The potential for injury of myocardial sarcolemma by endogenous lipases was studied. The sarcolemmal fraction was incubated for 30 min under conditions found optimal for hydrolysis of exogenous phosphatidylethanolamine (5 mM calcium, pH 7.0, 37°C). Incubation of the sarcolemmal fraction increased significantly the level of total free fatty acids (14.1 to 31.1 nmoles/mg protein, P < 0.001); in addition, production of arachidonic acid was increased significantly (P < 0.01). Lysophosphatidylcholine was increased significantly (P < 0.01) but the content of lysophosphatidylethanolamine was unchanged. A large proportion of the above free fatty acids (10.2 nmoles/mg protein) was derived from the hydrolysis of triacylglycerols. These data demonstrate that the sarcolemmal fraction preferentially hydrolyses endogenous membrane phosphatidylcholine at neutral pH in the presence of calcium with the formation of lysophosphatidylcholine and free fatty acids including arachidonic acid.     

Inhibition of tumor necrosis factor-alpha by thalidomide in magnesium deficiency

The effect of thalidomide on circulating cytokines and myocardial lesion formation was investigated in Mg-deficient rats. After two weeks on a Mg-deficient diet, rats show an increase in circulating levels of tumor necrosis factor-alpha and interleukin 1. Thalidomide (1 mg/day) caused a complete inhibition of the increase in circulating tumor necrosis factor-alpha levels, without having an effect on interleukin 1. However, a marked increase in cardiomyopathic lesion formation was observed in Mg-deficient animals treated with thalidomide; possible mechanisms for thalidomide's enhancement of myocardial injury are discussed. (Mol Cell Biochem129: 195–200, 1993)     

Is the ATP — dependent protection of lysosomes against osmotic lysis a function of the lysosomal proton pump

Summary Rat liver lysosomes have been used to characterize further the effects of ATP on lysosomal stability during incubation at 37°C at hypo-osmolarity. As previously reported, when the osmotically-supporting solute is the salt of a strong base (K⁺), ATP protects against lysis during incubation. However, if the osmotically-supporting solute is the salt of a weak base, e.g. Tris HCl or NH₄Cl, ATP actually promotes lysis during incubation. Thus, ATP can exert destabilizing as well as protective effects on lysosomes. The destabilizing effect is eliminated by protonophores. The protective effect in the presence of potassium salts is not eliminated by protonophores. Moreover, when incubation is in the presence of a salt of a weak base, protonophores actually cause an ATP-dependent protective effect to be established. The destabilizing effect occurs at 37°C, but not at 0°C. The Mg^–+-dependence of the destabilizing effect was found to be similar to that found earlier for the ATP-dependent protective effect, insofar as only 1 mM MgCl₂ in the presence of 1 mM EDTA is sufficient for nearly maximal stimulation of both effects. The destabilizing effect may result from a H^– ion gradient across the lysosomal membrane which is maintained by the lysosomal ATP-dependent proton pump. The protective effect, on the other hand, does not depend on such a gradient being maintained; on the contrary, protonophores appear to act as enablers of the protective effect. The question that remains to be answered is: does the protective effect derive in some way from the same ATP-driven mechanism which constitutes the proton pump? Some possible answers to this question are considered.Abbreviations Mops 2-(N-morpholine)-propanesulfonic acid - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-Dinitrophenol - EDTA Ethylenediaminetetracetic acid     

Spin Trapping of Free Radicals Formed During Peroxidation of Sarcolemmal Membranes (SLM)

The generation of free radicals in a superoxide (O₂-)driven Fe⁺³ catalysed reactions with isolated myocytic sarcolemma using electron spin resonance was investigated. Incubation of highly purified canine myocytic sarcolemma in the presence of the spin trap, 2-methyl-2-nitrosopropane (MNP). followed by the addition of dihydroxyfurmarate (DHF) and Fe⁺³-ADP resulted in the generation and detection of radical adducts of this spin trap. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl radical adduct following exposure to DHF/Fe⁺³-ADP. With sarcolemma and the alkyl nitroso compound, the only radical product trapped was the methyl radical formed by β-scission of alkoxyl radical. The participation of hydroperoxide-derived radicals in this system verified that the decomposition of unsaturated hydroperoxy fatty acid does proceed via a free radical mechanism.     

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.     

A calcium-stimulated, ouabain-inhibited ATPase in a myocardial fraction enriched with sarcolemma

An active intracellular to extracellular Ca²⁺ efflux has been proposed in heart muscle. A myocardial sarcolemmal ATPase stimulated by an intracellular pCa and serving as a Ca²⁺ pump has been postulated. A recently developed myocardial sarcolemmal preparation has not permitter a search for such an enzymatic activity. In these studies, an ATPase has been demonstrated in the sarcolemma which is activated by Mg²⁺, stimulated as the Ca²⁺ is raised to a pCa of 6, and is inhibited by ouabain. These findings suggest a mechanism by which Ca²⁺ within the myocardium may be modulated and thus how the force of contraction may be altered by cardiac glycosides.     

Formation of superoxide in the reaction of photolytically altered nifedipine--a nitroso compound--with unsaturated membrane lipids.

Nifedipine, which is unstable at light, is photolytically converted to the corresponding 4-[2'-nitrosophenyl]-pyridine (NTP). We reported earlier that NTP react with unsaturated lipids in a pseudo Diels-Alder reaction, thus forming stable nitroxide radicals. In this paper we report that superoxide is being generated in the latter reaction. Superoxide formation was evidenced by SOD-inhibitable cytochrome c reduction in the reaction of NTP with egg phosphatidylcholine at molar ratio 1:1, and 1:3. In this reaction an ESR-observable nitroxide radical was formed. Maximum nitroxide formation was observed after 90 min; the addition of SOD (93 units/ml) increased the concentration of nitroxide. This effect of SOD was reversed by catalase, indicating involvement of hydrogen peroxide in this effect. The nitroxide radical formation appears to be metal-independent, since neither iron salts, nor an iron chelator, desferal, influenced the nitroxide formation. Although production of superoxide in our system was only observed at high concentrations of NTP and of unsaturated lipids, this reaction may be of potential cytotoxic significance due to redox cycling of the nitroxide/hydroxylamine couple in cellular systems.