A partially purified, membrane-bound Na+-K+-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1 mM FeCl3, 1 mM ADP, and 0.1-100 mM H2O2 for either 15 or 30 min at 37 degrees C. The activity of ouabain-sensitive Na+-K+-ATPase was reduced proportionally to the concentration of H2O2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na+-K+-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na+-K+-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degrees C. Administration of 10 mM dithiothreitol alleviated the reductions in enzyme activity, in turnover rate, and in SH content without suppressing MDA formation. Addition of 2 mM butylated hydroxytoluene to the incubation mixture prevented the lipid peroxidation without totally normalizing the enzyme activity in the H2O2 experiment, whereas this antioxidant restored the ATPase activity to normal in the ultraviolet experiment. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na+-K+-ATPase activity, a reduced amount of SH groups, and an increased MDA.(ABSTRACT TRUNCATED AT 250 WORDS)

Sarcolemmal and mitochondrial phospholipids were extracted from normally perfused and ischemic regions of the dog heart and the composition of these extracts was analyzed. Relatively pure sarcolemmal fraction obtained from myocardium subjected to 3 h of ischemia exhibited a significantly lower concentration of phospholipids than that obtained from normal myocardium. In particular, phosphatidylcholine and phosphatidylethanolamine were reduced by approximately 33%. The fatty acid content in the sarcolemmal phospholipid fraction was also reduced by approximately 30% without any change in the relative composition. In the mitochondrial fraction, the relative phospholipid composition was also altered by ischemia; the major components (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) being reduced by approximately 15-20%. An attempt was made to correlate these biochemical changes with ultrastructural lesions observed electron microscopically. These observations revealed extensive regional variability and a wide heterogeneity in the extent of ultrastructural damage evident in the different organelles even in a single cell. This may suggest that ischemic damage, in the early stages, may advance at widely varying rates in different regions. Our findings demonstrate that significant biochemical and structural disorganization occurs during 3 h of ischemia in the myocardium and raise the possibility that one of the initiating events is the activation of sarcolemmal and mitochondrial phospholipases.

In this study we prepared sarcolemmal fractions from bovine and rat hearts; their Na+K+ ATPase activities, measured in the presence of saponin to unmask latent Na+K+ ATPase, were 59.4 and 48.8 mu mol Pi/mg protein.h, respectively. The rate of Na+ dependent Ca2+ uptake was linear for the first 10 s and a plateau was reached in 3 min. Oxidation by free radical generation either with H2O2, FeSO4 plus DTT or xanthine oxidase plus hypoxanthine stimulated Na+/Ca2+ exchange in a time-dependent manner. The stimulation was abolished by deferoxamine or o-phenanthroline. By contrast, oxidation by HOCl inhibited Na+/Ca2+ exchange in proportion to its concentration, and this inhibition was antagonized by DTT. DTT alone had no effect on the exchange. Insulin stimulated Na+/Ca2+ exchange, its maximal effect was attained after 30 min incubation with 100 mu units/ml. N-ethylmaleimide inhibited the exchange both in the presence and in the absence of insulin. Sarcolemmal fractions prepared from hearts of alloxan-treated, acutely diabetic rats showed a significant decrease in Na+/Ca2+ exchange. Addition of insulin in vitro significantly stimulated Na+/Ca2+ exchange of both diabetic and control groups. The results indicate that sarcolemmal Na+/Ca2+ exchange function is modulated by oxidation-reduction states and by the presence of insulin.

Ethanol in saline solution (15!b, v/v) was infused into anesthetized rabbits at a rate of 0.494 ml/min for the first 12 minutes and then at 0.247 ml/min for 108 minutes. Three hours after the infusion, heart triglyceride and lipoprotein lipase were assayed. Oxidation and esterification of fatty acids (palmitate-14 C as an indicator) were assessed by using either tissue homogenates or perfused hearts taken from the rabbits. Oxidation-reduction states of the perfused hearts were examined by measuring the tissue levels of dehydrogenase-linked substrates. The infusion of ethanol resulted in 180% increase in heart triglyceride content, but the infusion of norepinephrine (3 /Ltg/kg/min) did not change the content. No change in plasma free fatty acids and triglyceride or heart lipoprotein lipase activity was detected. Addition of ethanol had little effect on the distribution of palmitate-1 4C in the lipids of tissue slices and homogenates. On the other hand, prior infusion of ethanol resulted in depression of 14 COj production (70 and 50%) and enhanced fatty acid esterification into triglyceride (270 and 170$) both in homogenates and perfused hearts. Mitochondrial and cytoplasmic redox states were shifted to more oxidized states by ethanol infusion. It is postulated from these results that an accumulation of triglyceride in the rabbit heart in response to ethanol administration is a result of decreased fatty acid oxidation rather than of increased tnglyceride uptake or increased fatty acid synthesis.

Previous work from this laboratory has indicated that actomyosin bands prepared from heart muscle of dogs preserve undiminished contractility for one hour after the death of the animal (1). This suggested the possibility of investigating the contractile properties of actomyosin bands from human hearts obtained at autopsy. Such a study would make possible a comparison of actomyosin prepared from normal human heart muscle with that obtained from heart muscle of patients who had died of congestive heart failure. The method could also be used to obtain information on the effect of cardiac glycosides and other drugs on the contractile elements of normal and failing human heart muscle. To the present, only actomyosin preparations from hearts with artificially produced failure have been studied (2, 3). In these "models" obtained from dogs' hearts, the mechanism and nature of failure may vary from that encountered in man.The present report deals with a comparison of the contractile properties of actomyosin prepared from normal and failing human hearts and with the effect of calcium chloride and digoxin and the action of nicotine on these preparations. MATERIAL AND METHODSActomyosin solution was prepared in the cold (40 C.) as follows: The heart was minced with scissors and 10 Gm. supernatant fluid was again centrifuged at 4,000 rpm for 10 minutes. Following this, the supernatant fluid was diluted with a fivefold volume of triple glass distilled water; the pH was adjusted to 7.0 by careful addition of 1/10 N acetic acid. Usually, 17 ml. of the acid was sufficient. The solution was then centrifuged at 1,500 rpm for 30 minutes. The supernatant was discarded and the precipitate was suspended in about 20 ml. of triple glass distilled water and centrifuged again for 15 minutes. The precipitate was dissolved in 2 M KCl to a final concentration of 0.5 M KCl. One ml. of this solution was used for the formation of actomyosin bands. The solution was pipetted on the surface of a glass slide, from which it was transferred into a solution contained in a Langmuir trough. This trough solution contained 0.05 M KCl and 0.001 M magnesium chloride, and was buffered to a pH of 7.5 with 1/100 M barbital buffer. The temperature in the trough was kept at 240 C.The technique for the formation of bands has been previously described (4). However, since certain modifications were used, the method is briefly summarized at this point. The protein solution spread on the surface of the trough was compressed between two wooden sticks into bands, and then pushed gently into the contraction chamber containing 40 ml. of the trough solution (Figure 1). One end of the band was firmly fixed in position by a rigid connection with the bottom of the trough (Figure 1). The other end of the band was attached to the elongated arm of a Roller Smith torsion balance. The length of the band was approximately 2 cm., the width about 5 mm. Only afterloaded contractions were studied. The band was loaded by moving the weighing spring of the torsion balance by the desire...