Acute and chronic hypokalemia sensitize the isolated heart to hypoxic injury.

We examined the effects of acute and/or chronic hypokalemia on responses to 30 min of hypoxia and recovery in the isolated, perfused heart model. We found that both acute hypokalemia and chronic hypokalemia impaired contractility [expressed as maximum slope of pressure increase over time (dP/dt): 501 +/- 49 and 529 +/- 48 vs. 1,302 +/- 118 mmHg/s, P < 0.01] and recovery of ATP concentrations (determined with 31P NMR spectroscopy: 30 +/- 6 and 40 +/- 10 vs. 67 +/- 5% initial, P < 0.05) at 30 min of recovery. Moreover, the combination of acute hypokalemia and chronic hypokalemia had additive effects (dP/dt 166 +/- 15 mmHg/s and ATP 21 +/- 7% initial, both P < 0.01). We also measured cytosolic calcium with surface fluorescence spectroscopy after indo 1 loading. Acute hypokalemia and acute hypokalemia + chronic hypokalemia increased cytosolic calcium (averaged throughout the cardiac cycle) during and after hypoxia (390- to 460-nm ratio at 30 min of recovery: 0.46 +/- 0.07 and 0.65 +/- 0.07 vs. 0.18 +/- 0.03, P < 0.01), whereas control and chronic hypokalemia hearts had only small changes with hypoxia and recovery. Finally, when we examined mitochondria isolated from hearts perfused under experimental conditions, we found that chronic hypokalemia-alone mitochondria and chronic hypokalemia + acute hypokalemia mitochondria had marked impairment of state 3 respiration compared with control hearts (52 +/- 13 and 50 +/- 9 vs. 128 +/- 10 natm.min-1.mg protein-1 with succinate as substrate, P < 0.01), whereas acute hypokalemia mitochondria demonstrated only subtle changes. These data suggest that both acute hypokalemia and chronic hypokalemia impair cardiac responses to hypoxia. The mechanism may involve impairment of calcium metabolism, but cytosolic calcium alterations do not explain all of the metabolic and functional effects of acute hypokalemia and chronic hypokalemia in the setting of hypoxia.

Effects of acetate on energy metabolism and function in the isolated perfused rat heart.

Impairment of cardiac contractile function is an important component of acetate associated hypotension during hemodialysis treatments. We examined the effect of acetate on cardiac energy metabolism using the isovolumic isolated perfused heart model. In this preparation, acetate (10 M) caused decreases in tissue ATP concentrations (12.3 +/- 0.8 vs. 15.6 +/- 1.0 micromol/g dry at 30 min, P < 0.05) as well as marked impairment of systolic function (dpdt = 863 +/- 135 vs. 1288 +/- 166 mm Hg/second at 30 min, P < 0.05). Although altering perfusate calcium concentrations (0.6, 1.2 and 2.4 mM) affected physiological responses to acetate (5 and 10 mM), the reductions in tissue ATP concentrations were similar. In isolated heart mitochondria, acetate (100 microM -10 mM) selectively impaired octanoate and palmityl carnitine supported State 3 respiration in a dose dependent fashion (P < 0.01), but did not affect respiration when succinate, pyruvate/malate or malate/glutamate was used as substrate. We suggest that high concentrations of acetate selectively impair fatty acid metabolism in heart issue. This in turn leads to decreases in ATP production and tissue ATP concentrations that ultimately result in impaired contractile function. As this occurs at relatively low concentrations of acetate, this finding may be relevant to other parenterally-administered acetate containing fluids.

Post-insult treatment with interleukin-1 receptor antagonist decreases oxidative lung injury in rats given intratracheal interleukin-1.

Systemic administration of recombinant human interleukin-1 receptor antagonist (IL-1Ra) caused a rapid and sustained elevation of plasma IL-1Ra levels and decreased the leak of intravascularly injected 125I-labeled albumin into lungs of rats given human recombinant interleukin-1 intratracheally. IL-1Ra treatment decreased leak when given 0.5 h before, 1.25 h after, or 2.5 h after IL-1 administration. Similarly, IL-1Ra treatment decreased lavage leukocytes and neutrophils when given 0.5 h before, 1.25 h after, or 2.5 h after IL-1 administration. Pretreatment with IL-1Ra also decreased lung myeloperoxidase activity and breath H2O2 concentration increases in rats given IL-1 intratracheally. Our results suggest that IL-1Ra treatment may decrease acute lung injury and neutrophil influx even when given after an IL-1 inciting insult.

Toxic effects of dimethylthiourea in rats.

Dimethylthiourea (DMTU) is a small, highly diffusible molecule that effectively scavenges toxic oxygen metabolites in vitro and reduces oxidative injury in many biologic systems. Nonetheless, for unknown reasons, DMTU has occasionally failed to decrease damage in some systems where injury is presumed to be mediated by oxygen metabolites. We hypothesized that the inconsistent pattern of protection might partially reflect a direct toxicity of DMTU. Our results supported this premise. We found that rats treated with commonly used doses of highly purified DMTU had increased lung accumulation of intravenously injected iodine 125-labeled albumin (4 hours after DMTU treatment) and decreased blood glutathione levels (24 hours after DMTU treatment) when compared with saline-injected control rats. In contrast, rats treated with dimethylurea, a analog of DMTU, did not develop increased accumulation of labeled albumin in the lungs or decreased blood glutathione levels. We conclude that DMTU has intrinsically toxic effects in rats and that DMTU toxicity may at times obscure its protective action.

A novel system for the culture of human lung: lung development and the response to injury.

Existing methods of fetal lung organ culture are complicated and require special skills. With the use of a polyester-based plastic sheet, we have developed a simpler human fetal lung organ culture that is viable for 6 wk. This novel method permits the study of growth and differentiation, pulmonary surfactant secretion, and the response of human lung tissue to injury in vitro. Lung tissues, obtained from human fetuses ranging in gestational age from 14 to 18 wk, were cultured on the polyester sheet in Dulbecco's modified Eagle medium supplemented with 15% fetal calf serum and gentamicin. Microscopic study of the fetal lung before culturing revealed round epithelial tubules, lined by glycogen-rich columnar cells and a thick cellular interstitium. After 1 wk in culture, morphological examination showed the development and expansion of alveolar saccules and thinning of the interstitium; type I and II pneumocytes as well as fibroblasts and myofibroblasts were present. Lipid analysis of the tissues, 2 wk after the initiation of the culture, demonstrated a high percentage of dipalmitoyl phosphatidylcholine characteristic of pulmonary surfactant. Treatment of the organ culture with asbestos fibers induced type II cell hyperplasia, increased numbers of collagen fiber bundles within the interstitium, and the accumulation of multi-lamellated surfactant material within the alveolar lumens. We conclude that this organ culture system is suitable for studying lung growth, development, and injury in human tissue.

Mechanism of impaired energy metabolism during acidosis: role of oxidative metabolism.

Isolated perfused rat hearts were used to study the effects of metabolic acidosis on energy metabolism. Hearts perfused with different substrates (glucose, pyruvate, and succinate) were subjected to metabolic acidosis. With all substrates, there were comparable decrements in oxygen consumption (approximately 35%), cardiac function (decrease in first derivative of pressure of 65%), and similar changes in high-energy phosphates (approximately 150% increases in inorganic phosphate and 25% decreases in phosphocreatine concentrations) with metabolic acidosis. To further investigate the metabolic effects of acidosis, isolated cardiac mitochondria were exposed to different incubation media pH conditions and given simple metabolites (glutamate/malate, succinate, or pyruvate) or fatty acids (octanoate). Reduction of incubation media pH to 6.0 did not significantly affect either coupled respiration rate or the respiratory control ratio (RCR) with any substrate. These data suggest that metabolic acidosis induces decreases in energy production in the isolated perfused heart by inhibiting mitochondrial substrate utilization and not by impairing glycolysis. However, this impairment of mitochondrial function is not a direct effect of acidosis itself but appears to occur secondarily to some other effects of acidosis which are, as yet, incompletely understood.

Neutrophils accumulate and contribute to skeletal muscle dysfunction after ischemia-reperfusion.

Skeletal muscles subjected to ischemia and then reperfusion develop contractile dysfunction for reasons that are unclear. We found that rats pretreated with vinblastine 4 days before study had decreased numbers of blood neutrophils and increased gastrocnemius muscle function after ischemia (3h) and reperfusion (4 h) compared with untreated rats or rats treated 4 days before study with saline. By comparison, rats pretreated with vinblastine or saline 1 day before study had increased blood neutrophils and decreased gastrocnemius muscle contractile function after ischemia-reperfusion compared with untreated rats. In addition, numbers of neutrophils in gastrocnemius muscles paralleled numbers of blood neutrophils and correlated with gastrocnemius muscle edema and contractile function after ischemia and reperfusion. The results indicate that neutrophils accumulate and may play an important role in the genesis of skeletal muscle contractile dysfunction after ischemia-reperfusion.

Xanthine oxidase-derived H2O2 contributes to reperfusion injury of ischemic skeletal muscle.

We hypothesized that xanthine oxidase (XO)-derived hydrogen peroxide (H2O2) contributes to ischemic skeletal muscle injury during reperfusion. We found that after ischemia (3 h) and then reperfusion (4 h) rat gastrocnemius muscles had decreased contractile function following direct stimulation. Three lines of investigation suggested that XO-derived H2O2 contributes to reperfusion injury of ischemic skeletal muscle. First, treatment with dimethylthiurea (DMTU), a highly permeant O2 metabolite scavenger, but not urea, just before reperfusion improved muscle function in legs subjected to ischemia and then reperfusion. Second, gastrocnemius muscles from rats fed tungsten or allopurinol had negligible XO activities and increased muscle function after ischemia and reperfusion. Third, as assessed by measurement of skeletal muscle catalase activity in the presence of aminotriazole, H2O2 was measured during reperfusion of ischemic muscles from untreated or urea-treated rats but not during reperfusion of muscles from rats treated with DMTU, tungsten, or allopurinol.

Formation of lung surfactant films from intact lamellar bodies.

Lamellar bodies, an intracellular source of lung alveolar surfactant, were isolated from rat lung homogenates and studied in the Langmuir-Adams surface balance. By layering intact lamellar bodies on the surface of a more dense sucrose subphase, we studied the factors affecting film formation from surface tension-vs-time data and determined surface tension-surface area isotherms by compression and expansion of the resulting films. We found that films with properties representative of the alveolar surfactant are formed in the presence of Ca2+ or Mg2+ alone, or either plus Na+; that film formation is incomplete with Na+ alone or on ion-free subphases; and that Ca2+-induced film formation is blocked by chelation with EGTA but is unaffected by diisopropylfluorophosphate. The results suggest that divalent cations induce film formation by interactions at sites within the lamellar bodies and may be responsible for the binding of membrane lipids to membrane proteins in lung surfactant.

Characteristics of pulmonary surfactant in adult respiratory distress syndrome associated with trauma and shock.

Broncho-alveolar lavage fluid was obtained from a 24-year-old man who developed the adult respiratory distress syndrome one day after massive trauma and hemorrhagic shock. The lungs were available 3 days later when organ transplantation was performed. When the various fractions of the lavage material obtained by centrifugation, including the purified surface-active lipid-protein aggregates, were examined on the film balance, they revealed the usual minimal surface tension of 16 to 18 dyne per cm at 37 degrees C, but the compressibility of the films from the lungs with adult respiratory distress syndrome was 5 to 10 times higher than the normal range. This suggests that surfactant films in the adult respiratory distress syndrome are less responsive to stress, and that as a result, a loss of film elasticity may contribute to the abnormal pressure-volume relationships observed with the intact lung. Changes in the lipid-to-protein ratios of the purified lipid-protein aggregates were also found, as indicated by the recovery of 3 lipid-protein aggregates with different isopycnic densities from the lung with adult respiratory distress syndrome; only one major aggregate could be recovered from the lavages of normal lungs.

Effects of altitude on myoglobin and mitochondrial protein in canine skeletal muscle.

Myoglobin and mitochondrial protein contents were measured spectrophotometrically in the sternothyroid muscle of 8 dogs, first in Dnver, at a PB of 635 mm Hg, and after a 3-week exposure to a PB of 435 mm Hg, using a hypobaric chamber. The PaO2 at a PB of 635 mm Hg was 81.9 mm Hg and at a PB of 435 mm Hg, it was 54.6 mm Hg. Myoglobin concentration increased from 3.67 +/- 1.07 to 5.47 +/- 0.90 mg/g fresh muscle while mitochondrial protein content increased from 3.04 +/- 0.80 to 4.96 +/- 2.59 mg N2/g fresh muscle upon exposure to simulated altitude. It is believed that these changes in the biochemistry of the muscle are related to alterations in muscle fiber type composition, which seem to be adaptive in nature.

Metabolism of glycerol monoethers in cultured liver cells and implications for monoglyceride pathways.

A comparative study has been made of the assimilation and metabolism of rac-1 and 2-[9, 10(-3)H]-octadec-9-enylglycerol in a clone of epithelial-like cells isolated from rabbit liver. Based on cell protein content, the free glycerol ether isomers attained equal cellular concentrations. As shown by isolation and degradation experiments, however, the incorporation of radioactive 1-monether was appreciably higher than that of radioactive 2-monoether in both the triacylglycerol and phospholipid fractions. The 1-monoether, unlike the 2-monether, was also a significant source of esterified fatty acids in both lipid fractions. In addition, the 1-monoether, but not the 2-monoether, was an active precursor of plasmalogens, particularly ethanolamine plasmalogen. In contrast to the 1-monoether, the 2-monoether was a more active precursor of triacylglycerols than it was of phospholipids. The results indicate that in the rabbit liver cells the pathway of complex lipid synthesis from 1-monoether was via 1-alkyl-sn-glycerol-3-phosphoric acid and from 2-monoether via 1-alkyl-2-acyl-sn-glycerol.

Isolation of pulmonary pressor substances from bovine amniotic fluid.

The pulmonary pressor activity of bovine amniotic fluid resides in substances that appear to be non-polar lipids as suggested by the various chemical and chromatographic procedures. In preliminary experiments, it was found that the pressor activities of the hexane extracts were blocked by prior administration of aspirin to the calf (1 mg/kg), suggesting that pressor effect was mediated via the prostaglandin system. The amniotic fluid lipids, however, differed from the known prostaglandins in being less polar as indicated by thin layer chromatography and in possessing greater pressor activity. It is possible, of course, that the lipids with pulmonary pressor activity participate in the metabolism of the prostaglandins.

Isolation and characterization of lamellar bodies and tubular myelin from rat lung homogenates.

Three surface-active fractions which differ in their morphology have been isolated from rat lung homogenates by ultracentrifugation in a discontinuous sucrose density gradient. In order of increasing density, the fractions consisted, as shown by electron microscopy, primarily of common myelin figures, lamellar bodies, and tubular myelin figures. The lipid of all three fractions contained approximately 94% polar lipids and 2% cholesterol. In the case of the common myelin figures and the lamellar bodies, the polar lipids consisted of 73% phosphatidylcholines, 9% phosphatidylserines and inositols, and 8% phosphatidylethanolamines. In the case of the tubular myelin figures, the respective percentages were 58, 19, and 5. Over 90% of the fatty acids of the lecithins of all three fractions were saturated. Electrophoresis of the proteins of the fractions in sodium dodecyl sulfate or Triton X-100 revealed that the lamellar bodies and the tubular myelin figures differed in the mobilities of their proteins. The common myelin figures, however, contained proteins from both of the other fractions. These data indicate that, whereas the lipids of the extracellular, alveolar surfactant(s) originate in the lamellar bodies, the proteins arise from another source. It is further postulated that the tubular myelin figures represent a liquid crystalline state of the alveolar surface-active lipoproteins.