Gated sodium-23 nuclear magnetic resonance images of an isolated perfused working rat heart.

Sodium-23 nuclear magnetic resonance images of phantoms and gated images of isolated perfused working rat hearts were obtained. By synchronizing the nuclear magnetic resonance process to the heartbeat, images were obtained at systole and at diastole.

Selective cytotoxicity of low-density lipoprotein to helper T cells of cutaneous T-cell lymphoma after photoperoxidation with 8-methoxypsoralen.

Lymphocyte-containing plasma subjected to photolysis in the presence of 8-methoxypsoralen (methoxsalen, 8-MOP) has previously been shown to be effective against cutaneous T-cell lymphoma and the AIDS-related complex. The mechanism of this effect was thought to involve photoreaction of 8-MOP with DNA, based on certain in vitro experiments. The results of this study suggest a different mechanism. Low-density lipoprotein (LDL) from fresh human plasma was photosensitized by addition of 8-MOP and exposure to UV light (mp-LDL), and the reactions of the LDL lipids and the chemical actions induced by these reactions were monitored. In a separate procedure, LDL was peroxidized with hydrogen peroxide and peroxidase (p-LDL). mp-LDL and p-LDL were then tested in cytotoxicity assays on HuT-78 helper T cells of cutaneous T-cell lymphoma. These results indicate that (a) LDL in plasma in the presence of very low concentrations of 8-MOP (200 ng/mL) can be peroxidized by UV light; (b) this photoperoxidized LDL is cytotoxic to helper T cells of cutaneous T-cell lymphoma in a dose-dependent manner; but (c) it does not kill normal lymphocytes under similar conditions. The findings also suggest alternative therapeutic strategies for treatment of cutaneous T-cell lymphoma, such as direct utilization of peroxidized LDL.

Cell death induced by peroxidized low-density lipoprotein: endopepsis.

Peroxidized low-density lipoprotein (p-LDL) has been previously demonstrated to be preferentially cytotoxic to certain malignant cells compared to normal cells of the same type. We present evidence that p-LDL is at least partially taken up through the LDL receptor and that it becomes localized in lysosomes. The integrity of lysosomes of p-LDL-treated cells is compromised, and leakage of their contents into the cytosol occurs. This leakage occurs early and precedes mitochondrial dysfunction. Brefeldin A inhibits this leakage, perhaps by interfering with the traffic between endosomes and lysosomes. Electron micrographs taken at various times suggest a mechanism of cell death which resembles certain aspects of the broad definition of apoptosis. However, we suggest that the cell death observed following p-LDL-induced release of lysosomal contents is essentially unique, with released lysosomal enzymes degrading the cell from within. We suggest that this process should be described as endopepsis.

Effect of the sodium/potassium ratio on glyceraldehyde 3-phosphate dehydrogenase interaction with red cell vesicles.

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the 31P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K (1977) Biochim. Biophys. Acta 464, 82--92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation changed induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na4 + K+)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew (1970) J. Physiol. London 207, 393--402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by 31P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na+ + K+)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.

Membrane mediated link between ion transport and metabolism in human red cells.

When 10(-6) M oubain is added to human red cell that have been incubated without glucose for two hours, there is a significant shift in the 31P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na+ + K+)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na+ + K+)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10(-6) M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na+ + K+)-ATPase and that the interaction is inhibited by 10(-6) M ouabain.

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape.

Na+ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na+ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the 31P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg2+/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.

Relation between red cell membrane (Na+ + K+)-ATPase and band 3 protein.

This study is designed to examine the participation of the major red cell membrane protein, band 3 protein, in the chain which transmits information from the cardiac glycoside site on the external face of the cell (Na+ + K+)-ATPase to the megadalton glycolytic enzyme complex within the cell. The experiments show that the anion transport inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, affects the resonance of 2,3-diphosphoglycerate, as does the cardiac glycoside cation transport inhibitor, ouabain. Resonance shifts induced by the cardiac glycoside alone are modulated by addition of the anion transport inhibitor which indicates that there is coupling in the red cell between the (Na+ + K+)-ATPase and band 3 protein. Band 3 protein was separated from the membrane and partially purified following the technique of Yu and Steck ((1975) J. Biol. Chem. 250, 9170-9175). When glyceraldehyde-3-phosphate dehydrogenase was added to the separated band 3 protein preparation, addition of cardiac glycosides caused shifts in the 31P resonance of glyceraldehyde 3-phosphate. These experiments indicate that there is coupling between the (Na+ + K+)-ATPase and band 3 protein in the separated preparation and suggest that the anion and cation transport systems may be closely related spatially and functionally in the intact red cell.

Ouabain-sensitive interaction between human red cell membrane and glycolytic enzyme complex in cytosol.

Binding of 2,3-diphosphoglycerate to monophosphoglycerate mutase, of which it is an obligatory cofactor, causes changes in the resonance positions of the 31P nuclear magnetic resonance spectra of both phosphate groups. It has previously been shown that these resonances shift when other glycolytic enzymes, such as phosphoglycerate kinase, are added to form the 2,3-diphosphoglycerate . monophosphoglycerate mutase . phosphoglycerate kinase complex. In view of this association, we have examined the set of glycolytic enzymes from aldolase to pyruvate kinase and found evidence of direct communication between all of these enzymes. A multi-enzyme complex of 1--2 . 10(6) daltons has been separated from broken cell ghosts by Biogel column filtration and evidence has been presented to show that this complex exhibits aldolase, glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase activity. The glycolytic multi-enzyme complex interacts with the outer face of inside-out vesicles prepared from human red cells and the interaction is suppressed by application of 10(-6) M ouabain to the inner face of these vesicles. These studies show that the conformation of the enzymes comprising the megadalton complex are responsive to the application of ouabain to the outer red cell membrane surface.

Interaction of fluorinated ether anesthetics with artificial membranes.

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T1) measurements are consistent with substantial immobilization of the lipid-bound anesethetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.

Alteration of aliphatic lipid proton NMR linewidths by malignant tumors in guinea pigs.

Water-suppressed proton nuclear magnetic resonance spectroscopy was used to observe plasma lipoprotein lipid methyl and methylene resonances from guinea pigs which had been injected with viable or heat-killed line 1 or line 10 tumor cells or sterile oil. It was shown that the widths of these resonances became significantly sharper as the number of tumor cells grew. Plasma from tumor-free control animals showed no change in the NMR linewidths. It is concluded that the changes observed reflect a specific host response to viable tumor cells, and in these models there is a reciprocal relationship between the number of viable tumor cells and the linewidths of plasma lipoprotein methyl and methylene resonances.

Nuclear magnetic resonance imaging: potential cardiac applications.

During the past several years, the production of high resolution images of organs in intact animals and human beings using nuclear magnetic resonance (nmr) has generated much interest and raised the possibility that the technique could be usefully applied to clinical problems. Because the images are derived from biochemical as well as structural information, valuable data relating to the metabolic status of the tissues and organs may be obtained. Furthermore, nuclear magnetic resonance imaging involves no potentially hazardous ionizing radiation. The technology of the technique is complex and much work remains to be done defining the biochemical and physiologic basis of such images, but the potential rewards of defining the metabolic state of organs such as heart and brain in the intact animal and human justify continued research.

Effects of reduced coronary flow on thallium-201 accumulation and release in an in vitro rat heart preparation.

To study the relation between myocardial thallium-201 (TI-201) uptake, TI-201 release, and reduced coronary flow, isolated Langendorff rat hearts (n = 8) were perfused for 3 hours at constant flows ranging from physiologic (12 ml/min) to severely ischemic (1.5 ml/min); thallium activity was monitored with a scintillation probe. Each heart was perfused for 1 hour with thallium buffer, followed by 2 hours with thallium-free buffer at the same flow rate. Accumulation curves for all 4 flows were monoexponential. However, release curves during the 2 hours of washout with thallium-free buffer demonstrated a biexponential configuration. The early fast release component decreased with reductions in coronary flow, and the later slow release component did not vary significantly with flow. These data show that thallium clearance has at least 2 components: a rapid (possibly extracellular) component related to coronary flow and a slow (possibly intracellular) component independent of coronary flow. These findings should be useful in providing a better understanding of thallium redistribution observed clinically.

Limitations of potassium cardioplegia during cardiac ischemic arrest: a phosphorus 31 nuclear magnetic resonance study.

Cold K+ cardioplegia is commonly used to preserve the myocardium during surgical ischemia. Since the K+-induced membrane depolarization could cause a Ca2+-mediated breakdown of adenosine triphosphate, this study compared the influence of different electrolytes on high-energy phosphate metabolism during cardioplegic arrest phosphate metabolism during cardioplegic arrest and subsequent recovery of mechanical function. An isolated working heart was subjected to hypothermic ischemia for one hour. Metabolic studies were assessed on phosphorus 31 nuclear magnetic resonance (NMR). Results show that (1) K+ cardioplegia is harmful when the Ca2+ content is equal to 2 mEq/I; (2) deleterious effects of K+ are markedly reduced by lowering the Ca2+ content; (3) the most adequate preservation is provided by a Mg2+-rich-Ca2+-poor perfusate; (4) this protection is not enhanced by addition of K+. Finally, 31P NMR appears particularly appropriate for evaluating myocardial protection techniques since it allows noninvasive serial monitoring of high-energy phosphate content and subsequent correlation with functional recovery after ischemia.

The NMR blood test for cancer: current status.

Our laboratory has developed nuclear magnetic resonance (NMR) techniques for detecting cancer. Using water-suppressed proton (H-1) NMR spectroscopy, we observed that the linewidths of the resonances of methyl and methylene moieties in lipoprotein lipids were consistently narrower in plasma samples from cancer patients than in those from controls. These findings have been corroborated by a number of independent laboratories, but other investigators have been unable to reproduce our results. One reason for the variability of results obtained with H-1 NMR may be that hypertriglyceridemia also induces linewidth narrowing of lipoprotein lipid methyl and methylene resonances, and can cause false positive results. We show that this ambiguity can be circumvented by using a second test based on the carbon-13 (C-13) NMR spectrum of plasma. Here we postulate that the cancer-associated changes seen in H-1 and C-13 NMR spectra are caused by peroxidation of lipoprotein lipids, an effect that may be induced by tumor necrosis factor-alpha released during malignancy.

Nuclear magnetic resonance studies of intracellular ions in perfused frog heart.

Intracellular sodium, potassium, and lithium were observed in a perfused frog heart by nuclear magnetic resonance (NMR) spectroscopy. A perfusate buffer containing the shift reagent, dysprosium tripolyphosphate, was used in combination with mathematical filtering or presaturation of the extracellular resonance to separate the intra- and extracellular sodium NMR signals. Addition of 10 microM ouabain to the perfusate, perfusion with a zero potassium, low-calcium buffer, and replacement of 66% of the perfusate sodium with lithium resulted in the following percent changes in the intracellular sodium levels (mean +/- SD): ouabain + 460 +/- 60% (n = 6), zero potassium + 300 +/- 30% (n = 3), and lithium - 51 +/- 6% (n = 3). An increase of 45% in the intracellular sodium was observed when changing the pacing rate from 0 to 60 beats/min (with proportional changes for intermediate pacing rates). The ratio of intracellular potassium to sodium concentration was determined to be 2.3 by NMR, indicating that a substantial amount of the intracellular potassium is undetectable with these NMR methods. In addition, intracellular lithium was observed during perfusion with a lithium-containing perfusate.

Intracellular sodium and lithium NMR relaxation times in the perfused frog heart.

We have used a combination of a shift reagent and mathematical filtering or presaturation of the extracellular sodium resonance for the quantitative investigation of the intracellular sodium and lithium relaxation times in the perfused frog heart. While the T1 of the intracellular sodium was found to consist of a single-exponential time constant (approximately 23 ms), the T2 was better fit as a double-exponential decay with time constants of approximately 2 and 17 ms. However, the relative amplitudes of the two time constants in the T2 decay were found to be inconsistent with those which would be expected from a homogeneous pool of nuclei undergoing quadrupolar interactions. The relaxation times were not changed by a fivefold increase in the intracellular sodium level (due to perfusion with a ouabain-containing buffer). The T1 and T2 of the intracellular lithium (after perfusion with lithium-containing buffer) were both well fit by single exponentials (700- and 31-ms time constants, respectively).

Observation of intracellular potassium and sodium in the heart by NMR: a major fraction of potassium is "invisible".

Using 39K and 23Na NMR in conjunction with extracellularly localized shift reagents, we have determined the intracellular concentrations of NMR visible sodium and potassium in isolated, perfused rat hearts. We find this concentration to be 9.9 mM/kg cell water for sodium and 31 mM/kg cell water for potassium. Values of activity determined by ion-sensitive microelectrodes are in good agreement with our sodium value but do not agree with our potassium value. Our results mean that a major pool of intracellular potassium is, on average, significantly immobilized and that the mobile NMR visible fraction (31 mM/kg) is not in exchange with the NMR invisible pool (114 mM/kg). The immobilized fraction is characterized by T2 values which are too short to be observed by our conventional spectrometer. This fraction is, therefore, said to be "invisible" under our experimental conditions.

A synthetic functional metabolic compartment. The role of propinquity in a linked pair of immobilized enzymes.

A system was created to model the influence of microcompartments on linked enzymatic reactions. Creatine kinase and hexokinase were covalently attached to Sepharose beads. The gel could be perfused in a specially constructed chamber inside a 360-MHz NMR spectrometer at different flow rates with solutions containing various concentrations of substrates. 31P NMR studies were carried out on the linked enzymatic reaction, creatine phosphate + glucose----creatine + glucose 6-phosphate in two enzyme gels differing in only one aspect, the average distance between hexokinase and creatine kinase. At a distance on the order of 0.1 mm between the enzymes, the average bulk concentrations of substrates and products in the perfusate determined the overall function of the linked system. At an average distance of the order of 10 nm, flux through the linked pair was much higher and much less dependent on the concentration of the intermediate substrate/product ADP/ATP. Even at adenine nucleotide concentrations far below the Km of hexokinase, substantial amounts of glucose 6-phosphate were produced when the enzymes were near but not when they were distant. From saturation transfer measurements and turnover calculations, the lifetime of ATP in the system is estimated to be 0.14-0.5 s when the enzymes are near. This compares to 6 s for distant enzymes. From this it appears that the pair of linked enzymes comprise a functional compartment supported by propinquity in which hexokinase has preferential access to ATP produced by creatine kinase, and creatine kinase to ADP from the hexokinase reaction.

Complete inhibition of creatine kinase in isolated perfused rat hearts.

Transient exposure of an isolated isovolumic perfused rat heart to low concentrations (0.5 mM) of perfusate-born iodoacetamide resulted in complete inhibition of creatine kinase and partial inhibition of glyceraldehyde-3-phosphate dehydrogenase in the heart. At low levels of developed pressure, hearts maintained mechanical function, ATP, and creatine phosphate levels at control values. However, iodoacetamide-inhibited hearts were unable to maintain control values of end diastolic pressure or peak systolic pressure as work load increased. Global ischemia resulted in loss of all ATP without loss of creatine phosphate, indicating lack of active creatine kinase. These results indicate that isovolumic perfused rat hearts are able to maintain normal function and normal levels of high-energy phosphates without active creatine kinase at low levels of developed pressure.