Stimulation of the dopamine 1 receptor increases lung edema clearance.

We previously reported that lung edema clearance was stimulated by dopamine (DA). The purpose of this study was to determine whether the DA-mediated stimulation of edema clearance occurs via an adrenergic or dopaminergic regulation of alveolar epithelial Na, K-ATPase. When isolated perfused rat lungs were coinstilled with DA and SCH 23390 (a specific D(1) receptor antagonist), there was a dose-dependent attenuation of the stimulatory effects of DA. Coinstillation with S-sulpiride (a specific D(2) receptor antagonist) or propranolol (a beta-adrenergic antagonist) did not alter DA-stimulated clearance. Similarly, the specific dopaminergic D(1) agonist fenoldopam increased lung edema clearance, but quinpirole (a specific dopaminergic D(2) agonist) did not. (125)I-SCH 23982 binding studies suggested that D(1) receptors are expressed on alveolar type II (ATII) cells with an apparent dissociation constant (K(d)) of 4.4 nM and binding maximum (Bmax) 9.8 pmol/mg. Consistent with these results, the D(1) receptor messenger RNA (mRNA) and protein were detected in ATII cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. These data demonstrate a novel mechanism involving the activation of dopaminergic D(1) receptors which mediates DA-stimulated edema removal from rat lungs.

Modulation of lung liquid clearance by isoproterenol in rat lungs.

beta-Adrenergic agonists have been reported to increase lung liquid clearance by stimulating active Na+ transport across the alveolar epithelium. We studied mechanisms by which beta-adrenergic isoproterenol (Iso) increases lung liquid clearance in isolated perfused fluid-filled rat lungs. Iso perfused through the pulmonary circulation at concentrations of 10(-4) to 10(-8) M increased lung liquid clearance compared with that of control lungs (P < 0.01). The increase in lung liquid clearance was inhibited by the beta-antagonist propranolol (10(-5) M), the Na(+)-channel blocker amiloride (10(-4) M), and the antagonist of Na-K-ATPase, ouabain (5 x 10(-4) M). Colchicine, which inhibits cell microtubular transport of ion-transporting proteins to the plasma membrane, blocked the stimulatory effects of Iso on active Na+ transport, whereas the isomer lumicolchicine, which does not affect cell microtubular transport, did not inhibit Na+ transport. In parallel with these changes, the Na-K-ATPase alpha 1-subunit protein abundance and activity increased in alveolar type II cells stimulated by 10(-6) M Iso. Colchicine blocked the stimulatory effect of Iso and the recruitment of Na-K-ATPase alpha 1-protein to the basolateral membrane of alveolar type II cells. Accordingly, Iso increased active Na+ transport and lung liquid clearance by stimulation of beta-adrenergic receptors and probably by upregulation of apical Na+ channels and basolateral Na-K-ATPase mechanisms. Recruitment from intracellular pools and microtubular transport of Na+ pumps to the plasma membrane participate in beta-adrenergic stimulation of lung liquid clearance in rat lungs.

The role of magnetic resonance spectroscopy in the assessment of kidney viability.

Renal transplant programmes are seriously limited by the continuing shortage of donor organs. Kidneys from marginal and non-heart-beating donors are increasingly being used, but their viability may be compromised. There is currently no rapid yet accurate method for assessing donor organ viability which can be applied within the window of opportunity between harvesting and implantation. Magnetic resonance spectroscopy (MRS) is a non-invasive technique which is being increasingly applied to delineate biochemical changes in vivo. Studies in animal models and humans now suggest that phosphorus-31 MRS may be useful in the non-invasive assessment of isolated donor kidney viability.

Liver transplantation: current and potential applications of magnetic resonance spectroscopy.

Magnetic resonance spectroscopy (MRS) allows the noninvasive measurement of whole organ metabolism due to the presence of the MR-sensitive nucleus phosphorus 31 in adenosine triphosphate (ATP), its precursors, and break-down products. In small animal liver transplant studies it has been used to analyze the metabolic effects of cold and warm ischemia, hypothermic reperfusion, and the relative efficacy of different organ preservation solutions. In recent large animal studies MRS has been developed to provide continuous dynamic information on ATP metabolism during graft reperfusion and the bioenergetic consequences of altering preservation solutions. These basic experimental data need to be critically evaluated in human liver transplantation. Encouraging preliminary data on many possible clinical applications have already been obtained, such as the assessment of human donor liver viability and posttransplant graft function. At present, the cost and technically demanding nature of MRS may restrict its application to research units.

Dopamine stimulates sodium transport and liquid clearance in rat lung epithelium.

Pulmonary edema clearance is driven primarily by active sodium transport out of the alveoli, mediated predominantly by apical sodium channels and the basolateral NA,K-ATPase. We postulated that dopamine, analogous to its effects in other transporting epithelia, could regulate these sodium transport mechanisms and affect lung liquid clearance. We therefore studied the effects of dopamine on sodium transport and liquid clearance in isolated perfused rat lungs. Instillation of dopamine into the airways caused a dose-dependent increase in liquid clearance from isolated rat lungs of up to 33% above control values at 10(-8) to 10(-4) M concentrations. 10(-6) M amiloride, which selectively inhibits apical sodium channels, decreased basal liquid clearance by 34% but did not inhibit the dopamine-mediated stimulation of lung liquid clearance. Instillation of 10(-4) M amiloride into rat airways, which inhibits other sodium transport mechanisms non-selectively, decreased basal lung liquid clearance by 49% and inhibited the dopamine-mediated stimulation of lung liquid clearance. Perfusion of rat lungs with 5 x 10(-4) M ouabain to specifically inhibit Na,K-ATPase reduced both basal clearance (by 55%) and the dopamine-stimulated increase in lung fluid clearance. Conceivably, the stimulation of lung liquid clearance by dopamine is due to a modulation of Na,K-ATPase in the pulmonary epithelium.

In vivo assessment of metabolic perturbations following alanine and glucagon administration using 31P-MRS in the rat.

This study set out to validate the use of 31P-NMR spectroscopy together with alanine +/- glucagon infusions to assess hepatic gluconeogenic flux in vivo. Bolus infusions of alanine (2.8 or 5.6 mmol/kg) +/- glucagon (250 microg/kg) were used. Maximal changes in the phosphomonoesters (PME), inorganic phosphate (Pi) and beta-NTP occurred 40 mins post infusion. PME increased 13.1% (p < 0.02) and 20.8% (P < 0.01) at 2.8 mmol/kg + glucagon and 5.6 mmol/kg +/- glucagon, respectively. Pi was unaltered at 2.8 mmol/kg but increased by 28.8% (P < 0.01) at 5.6 mmol/kg alanine + glucagon. beta-NTP decreased by 14.4% (P < 0.02) and 16.1% (P < 0.02) at 5.6 mmol/kg -/+ glucagon, respectively. This latter infusion showed slower recovery rates of NTP which remained 12.3% (P < 0.05) lower 70 min post infusion compared with pre-infusion values. 31 P-NMR analysis of liver extracts revealed that PME increases were partly due to 3-phosphoglycerate and corroborated reductions in beta-NTP and gamma-NTP: beta-NDP ratio upon infusion of 5.6 mmol/kg alanine +/- glucagon. Hepatic glucose output from perfused liver experiments showed no difference between alanine concentrations indicating maximal glucose output at the lower concentration. This study has shown that in vivo 31P-NMR in combination with alanine infusion, can be used to determine metabolic changes associated with gluconeogenesis.

Evidence for distinct behaviour of phosphatidylcholine and sphingomyelin at the low density lipoprotein surface.

This study demonstrates that the use of high field 1H NMR spectroscopy permits individual detection of phosphatidylcholine and sphingomyelin molecules at the surface of native low density lipoprotein (LDL) particles. Distinct behaviour was observed for the choline head group -N(CH3)3 resonances of these different phospholipids revealing preferential immobilisation for phosphatidylcholine. This suggests the existence of reversible and irreversible phosphatidylcholine-apolipoprotein B interactions and is consistent with microdomain formation at the surface monolayer of LDL. The novel resonance assignment and results show that 1H NMR can provide efficient and practical means for future studies on the structure and dynamics at the LDL surface.

Role of nitric oxide and superoxide anion in spontaneous lung chemiluminescence.

Inhibition of nitric oxide (.NO) synthase by nitro-L-arginine (NLA) decreased baseline chemiluminescence in a dose-dependent fashion up to 78% at 300 microM NLA. This inhibition was prevented by pretreatment with 1 mM arginine. Similarly, addition of superoxide dismutase (SOD; 200 U/ml) to the perfusion buffer inhibited spontaneous light emission by 57%. Addition of NLA after SOD or vice versa did not inhibit light emission any further, suggesting that both .NO and O2.- were precursors of the same oxidant. Production of additional extracellular O2.- by neutrophils activated with phorbol 12-myristate 13-acetate increased light emission by >200%, but this increase was insensitive to NLA. Increasing the intracellular steady-state O2.- concentration by perfusion of control lungs with the Cu and Zn-containing SOD inhibitor diethyldithiocarbamate (1 mM) stimulated light emission up to fourfold, but this spontaneous chemiluminescence was also insensitive to NLA. In experiments using cultured endothelial cells supplemented with extracellular bovine serum albumin (BSA), 5 microM of the Ca2+ ionophore A-23187 (a stimulant of .NO synthase) stimulated chemiluminescence by 40%. This increase was again SOD and NLA sensitive. Addition of NLA after SOD or vice versa did not change light emission. These results suggest that the background chemiluminescence of isolated-perfused intact lungs may result from the constant release of small amounts of O2.- and .NO by endothelial cells into the capillary lumen, which in turn react with BSA in the perfusion buffer.

Changes in adipose tissue composition in malnourished patients before and after liver transplantation: a carbon-13 magnetic resonance spectroscopy and gas-liquid chromatography study.

We investigated adipose tissue fatty acid composition in 22 moderately to severely malnourished patients with cirrhosis and in 22 healthy volunteers by in vivo carbon-13 magnetic resonance spectroscopy (MRS). Gas-liquid chromatography (GLC) of adipose tissue samples was also performed in 11 of the patients and in 4 volunteers. In vivo 13C magnetic resonance spectra were obtained from the subcutaneous adipose tissue before and after eight weeks following orthotopic liver transplantation (OLT). Adipose tissue biopsy samples were obtained for GLC analysis at the time of transplantation in the patients and at inguinal hernia repair in the 4 volunteers. No significant differences were found in the subcutaneous adipose tissue total-saturated, -polyunsaturated or -monounsaturated fatty acid composition between patients and healthy volunteers by in vivo 13C MRS. GLC analysis of adipose tissue samples confirmed that total levels of saturated, poly-, and monounsaturated fatty acids remained the same but revealed significant differences in levels of individual fatty acids, particularly n-3 fatty acids (total n-3, cirrhotics: .84% +/- .07% vs. controls: 1.36% +/- .13%, P < .01). Eight weeks following transplantation, recipients showed a considerable increase in body mass (pretransplantation: 59.3 +/- 3.2 vs. posttransplantation: 63.2 +/- 3 kg, P < .01). 13C MRS revealed a significant increase in saturated fatty acids (pretransplantation: 21.6 +/- 2.8 vs. posttransplantation: 25.5% +/- 1.2%, P < .05) and a significant decrease in unsaturated fatty acids. The application of noninvasive MRS techniques may be important to identify the differential uptake of fats, examining both specific fatty acids and different body fat compartments. In the future, this may be useful in optimizing the dietary management of severely malnourished patients with chronic liver disease before liver transplantation.

Characterisation of secondary metabolites associated with neutrophil apoptosis.

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis. '

Effects of n-3 fatty acids on the NMR profile of plasma lipoproteins.

The effects of fish oil supplementation (14.5 g n-3 fatty acids/day) on plasma lipoprotein particles in healthy volunteers were assessed by high resolution 13C and 1H nuclear magnetic resonance (NMR) spectroscopy. Resonances not previously observed in the 13C and 1H spectra of plasma and isolated lipoproteins were detected after fish oil ingestion. The 13C resonances, centered at 14.3, 127.1, and 131.6 ppm, have been assigned to specific carbon groups (CH3-CH2-CH = CH-, CH3-CH2-CH = CH-CH2-, CH3-CH2-CH = CH-CH2-, respectively) in eicosapentaenoic acid (C20:5n-3) and docosahexaenoic (C22:6n-3) DHA. The new lipid resonance observed in the 1H spectra of plasma (0.941 ppm) is consistent with the incorporation of these n-3 fatty acids into lipoprotein particles. The presence of increased EPA and DHA in plasma lipids was confirmed by gas-liquid chromatography. A marked reduction in the intensity of the methylene signal from very low density lipoproteins (VLDL) was also observed with fish oil. This reduction arises from a decrease in plasma triglyceride concentration (ca. 18%) and a reduction in the number of VLDL particles. Transverse relaxation studies of isolated VLDL and low density lipoprotein (LDL) showed significant elevation in the T2 of the -(CH2)n- and CH3- signals from non-n-3 fatty acids. The relaxation characteristics and signal intensity of the novel 1H peak (0.941 ppm) point to the existence of n-3 enriched microenvironments within lipoprotein particles. These findings suggest that incorporation of EPA and DHA into VLDL and LDL, after fish oil ingestion, leads to significant alteration in the molecular architecture of lipoprotein particles.

Development of a rapid and efficient magnetic resonance imaging technique for analysis of body fat distribution.

Fast scan magnetic resonance imaging techniques for adipose tissue (AT) quantification were compared to a conventional T1-weighted spin-echo (SE) sequence (TR = 500 ms, TE = 20 ms), imaging a mid-abdominal slice. A rapid T1-weighted SE sequence (TR = 36 ms, TE = 14 ms) was optimal, with minimal distortion (field, motion, flow artefact). Tissue contrast was higher and visceral AT was clearly differentiated. Quantification of all AT compartments (total, subcutaneous, internal, visceral) showed close agreement with the T1-weighted SE sequence and reproducibility was high (coefficient of variation < 4.7%). For AT quantification in a whole subject, this fast technique allows each image to be acquired serially at the magnet isocenter, as the subject is moved through the scanner (serial isocenter scanning, SIS). This method provides minimal image distortion and allows rapid coverage of the whole body.

An in vivo 13C magnetic resonance spectroscopic study of the relationship between diet and adipose tissue composition.

13C magnetic resonance spectroscopy (MRS) is a noninvasive technique used in the study of lipids. We applied 13C MRS to assess the effects of long-term dietary variation on adipose tissue composition in humans. In vivo 13C MRS was used to analyze the fatty acid composition of adipose tissue in 88 healthy volunteers with significantly different diets (38 vegans, 11 vegetarians, and 39 omnivores) assessed by analysis of dietary records. Results were compared with the serum lipid profile. 13C MRS revealed clear differences in the adipose tissue composition of vegans, which contained more unsaturated (P < 0.01) and fewer saturated fatty acids (P < 0.01) compared with omnivores and vegetarians. The vegan subjects had a significantly lower intake of saturated fatty acids and higher intake of polyunsaturated fatty acids than either the omnivore or the vegetarian groups (P < 0.01). These findings were associated with significantly lower levels of serum total cholesterol and low density lipoprotein-cholesterol in the vegan group compared with the omnivores. Our results demonstrate the use of 13C MRS for the noninvasive study of adipose tissue composition and its application to the study of the interaction between long-term dietary and metabolic risk factors in humans.

NMR investigation of the futile cycling of ethanol in chronic alcoholic rats.

Weight gain efficiency differences previously reported between alcohol-fed rats and their controls were investigated. Additionally, the futile cycling of ethanol proposed to explain such differences was studied by NMR spectroscopy. Male Sprague-Dawley rats were fed a nutritionally adequate diet containing 36% of the calories as alcohol, and their paired controls were fed an isocaloric diet for 11 weeks to establish conditions of chronic alcohol feeding. Normalized metabolic efficiencies varied significantly during the initial 2-week period (6.86 +/- 0.51 vs. 2.83 +/- 0.18 g/kcal x 10(-2) for control and alcohol-fed groups, respectively, and to a lesser extent over the entire feeding period (6.41 +/- 0.78 vs. 4.60 +/- 0.27 g/kcal x 10(-2) for control and alcohol-fed groups, respectively. Alcohol-induced weight gain inefficiency in metabolism has previously been studied and explained by a variety of different biochemical and physiological mechanisms. One possible pathway of energy wastage may occur due to ethanol futile cycling from ethanol to acetaldehyde through the microsomal ethanol oxidation system pathway, and simultaneously from acetaldehyde to ethanol via the ADH pathway. This futile cycle represents a net loss of 6 ATP/cycle, corresponding to the loss of two reducing equivalents (NADH and NADPH). 1H NMR spectroscopy was used to test for this cycling in blood extracts after administration of 1,1-2H2 ethanol. No futile cycling was detected either during the initial 2 weeks of feeding or after the entire feeding period.

Mitigation of oxidant injury to lung microvasculature by intratracheal instillation of antioxidant enzymes.

We quantitated the ability of intratracheally administered liposome-encapsulated antioxidant enzymes to reduce reactive oxygen species injury to the pulmonary microvasculature. Cationic liposomes containing 3,500 U of Cu,Zn superoxide dismutase (Cu,Zn SOD) and 3,124 U of catalase were instilled into rabbits. The animals were killed 2-72 h later and their lungs were removed and perfused with Krebs Ringer with 5% wt/vol of fat-free bovine serum albumin. The pulmonary filtration co-efficient (Kf,c) was measured before and after adding 500 microM xanthine and 5 mU/ml xanthine oxidase (XO) into the lung perfusate. Two hours after a single intratracheal instillation of liposome-entrapped Cu,Zn SOD and catalase, lung antioxidant enzyme activities were 34 and 125% higher than the corresponding control values, remained virtually unchanged for up to 8 h post-instillation, and then decreased, reaching baseline values between 24 and 72 h. Addition of xanthine and XO into the lung perfusate of un-instilled rabbits, or rabbits that received liposomes with inactivated enzymes, caused a 100% increase in Kf,c (control value: 2 +/- 0.12 ml.min-1 x cmH2O-1 per 100 g dry lung weight). On the other hand, Kf,c values of rabbits lungs instilled with liposome-encapsulated active Cu,Zn SOD and catalase and challenged with xanthine and XO 8-24 h later remained at baseline levels. Instillation of liposomes containing either enzyme was equally effective in preventing the increase in Kf,c, indicating that both superoxide anions and hydrogen peroxide were necessary for the initiation of injury. We concluded that intratracheal instillation of liposome-encapsulated antioxidant enzymes caused a transient increase of lung antioxidant enzyme levels which protects the pulmonary microvasculature from free radical-initiated injury.

Activated polymorphonuclear leukocytes increase low-level chemiluminescence of isolated perfused rat lungs.

Low-level chemiluminescence was measured in isolated perfused rat lungs subjected to different types of oxidative stress: perfusion with tert-butyl hydroperoxide (t-BOOH) or stimulation of polymorphonuclear cells (PMN). The time required for t-BOOH-dependent lung chemiluminescence to return to background levels was proportional to the concentration of t-BOOH. From the half times of the decay at different t-BOOH concentrations, we estimated that the lungs metabolize organic peroxides at a rate of 0.045 mM/min. Use of a high dose of t-BOOH (3 mM)or pretreatment of lungs with 1,3-bis(2-chloroethyl)-nitrosourea (100 micrograms/ml) to inhibit glutathione reductase produced chemiluminescence that was much greater and did not decay. Stimulation of 5 x 10(7) PMN with 1 micrograms of phorbol myristate acetate resulted in significant increases in chemiluminescence that occurred in the absence of a significant lung weight gain or measurable lipid peroxidation. Perfusion of isolated lungs with superoxide dismutase (100 U/ml) completely inhibited the chemiluminescence response to PMN activation, whereas treatment with 100 microM U-74389F, a lipid-soluble antioxidant, also significantly decreased PMN-dependent chemiluminescence. Neither catalase (2,000 U/ml) nor 100 microM U-78518F, a water-soluble antioxidant, decreased chemiluminescence after PMN activation. These results indicate that low-level chemiluminescence is a sensitive indicator of oxidative stress in the isolated perfused rat lung and provides a tool for devising and characterizing the effectiveness of antioxidant interventions.