Determination of prostaglandin profiles in lipopolysaccharide-challenged guinea pig spleen.

We previously reported that splenic extract from lipopolysaccharide (LPS)-challenged guinea pigs inhibits the exaggerated febrile response of splenectomized guinea pigs, suggesting that the spleen generates an inhibitory factor. Earlier results indicate that the factor is a lipid. In an effort to identify this factor, lipid fractions, isolated from splenic extracts of control and LPS-challenged guinea pigs, were analyzed with emphasis on identifying and quantifying prostanoids, which according to current knowledge are the likely bioactive factors. Prostaglandins have been extensively implicated in central and peripheral thermoregulation, and thus these lipids were targeted for characterization in the spleen. Analysis was done on the splenic extracts using solid-phase extraction, analytical and preparative thin-layer chromatography (TLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Four prostaglandins (PGs, 6-keto-PGF(1α) , PGF(2α) , PGE(2) and PGD(2) ) were identified and quantified. Our data shows that these PG levels are doubled in LPS-treated guinea pig spleen compared with the control group. The methods used in this investigation to characterize PG in the spleen offer significant advantages over immunoassays previously used to identify and quantify PG in the spleen and other biological tissues. These methods will be utilized in further research needed to definitively characterize the role of splenic-derived PG in modulation of the febrile response induced by LPS.

Investigating metal-binding in proteins by nuclear magnetic resonance.

Metal ions play a key role for the function of many proteins. The interaction of the metal ion with the protein and its involvement in the function of the protein vary widely. In some proteins, the metal ion is bound tightly to the ligand residues and may be the key player in the function of the protein, as in the case of blue copper proteins. In other proteins, the metal ion is bound only temporarily and loosely to the protein, as in the case of some metalloenzymes and other proteins where the metal ion acts as a cofactor necessary for the function of the protein. Such proteins are often known as metal ion-activated proteins. The review focuses on recent nuclear magnetic resonance (NMR) studies of a series of metal-dependent proteins and the characterization of the metal-binding sites. In particular, we focus on NMR techniques for studying metal binding to proteins such as chemical shift mapping, paramagnetic NMR and changes in backbone dynamics upon metal binding.

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder.

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.

Effect of partial outflow obstruction on the distribution of free fatty acids and phospholipids in the rabbit bladder.

The urinary bladder is separated into two distinct components, the mucosal epithelium (urothelium) and the underlying detrusor smooth muscle. Specific bladder dysfunctions such as partial outlet obstruction may contribute to the breakdown and damage of cell membranes. The major component of cell membranes is phospholipids, and the release of free fatty acids (FFA) from membrane phospholipids is suggestive of degradative lipase activity. The current investigation is concerned with the effect of partial outlet obstruction on the subcellular distribution of free fatty acids and phospholipids (PL) in rabbit bladder muscle and mucosa. Partial outlet obstructions were surgically created in mature male New Zealand White rabbits by standard methodology. At 2 weeks following surgery, rabbits were euthanized and the bladders, removed and separated into smooth muscle and mucosa. Muscle and mucosa were homogenized and separated by differential centrifugation to obtain separate subcellular fractions including plasma membranes, mitochondria, microsomes, and cytosol. The homogenate and supernatant fraction, free of membranes, were also saved. The free-fatty-acid (FFA) and choline-containing phospholipid (PL) content and the rate of generation of FFA were quantitated using in vitro enzymatic colorimetric methods. Relative to controls there was a significant increase in the FFA content of the obstructed smooth muscle and an increase in the PL content of the obstructed mucosa. There was an increase in FFA content in the mitochondrial fraction and a decrease in the supernatant of the obstructed smooth muscle. The PL content was reduced in the obstructed smooth muscle microsomal and supernatant fractions and was increased in the supernatant fraction of the mucosa. Endogenous lipase activity among control bladders was more than 10-fold greater in mucosa than in muscle. The FFA generation of the smooth muscle was significantly reduced by partial outlet obstruction. In conclusion, partial outlet obstruction causes bladder dysfunction due to activation of enzymes that hydrolyze cellular and subcellular membranes. The increase in endogenous lipase activity and generation of FFA among obstructed bladders indicates that the pathological state affects the membrane structure needed for normal bladder function.

Fatty acid profiles in normal and obstructed rabbit bladder smooth muscle and mucosa.

Partial bladder outlet obstruction results in progressive loss in contractile and specific cellular and subcellular membrane functions. There is evidence that ischemic activation of proteolytic and lipolytic enzymes play a major role in the etiology of bladder dysfunction secondary to partial outlet obstruction. The specific aims of the current study were to determine the fatty acid profiles in normal rabbit bladder smooth muscle and mucosa and to determine the effect of partial outlet obstruction on the distribution and content of free and total fatty acids. Fatty acids were isolated by extraction from obstructed and normal bladder smooth muscle and mucosal homogenates, and samples were analyzed by gas chromatography. All samples contained palmitic, stearic, oleic, linoleic, and arachidonic acids. A 100% increase in total fatty acid concentration was observed in the obstructed bladder muscle tissue relative to normal bladders, although the concentration of total arachidonic acid remained constant in the two groups. Significantly higher levels of free arachidonic acid were observed in the obstructed bladder muscle group compared to the normal group. No changes were observed in fatty acid concentrations or distributions in bladder mucosa. These data show that fatty acid composition is altered as a result of bladder obstruction and support the idea that obstruction increases the activity of lipase activity and/or decreases acyl transferase activity. Neurourol. Urodynam. 18:697-711, 1999.

Identification of components of Prunus africana extract that inhibit lipid peroxidation.

Extractive and chromatographic separations were performed on V-1326, a chloroform extract from the bark of Prunus africana (also referred to as Pygeum africanum), which is used to treat the symptoms associated with benign prostate hyperplasia (BPH). The relative amounts of eleven identified constituents in crude V-1326 and in separated fractions were determined using gas chromatographic analysis. The ability of V-1326 and its separated fractions to inhibit ferrous ion-induced stimulation of lipid peroxidation in microsomal preparations from rabbit livers was evaluated. The extract, V-1326, and fractions containing high levels of myristic acid potently inhibited lipid peroxidation.

Physiological effects of macrocycle 1 on the rabbit corpus cavernosum.

When the penis is in the flaccid state, the corpus cavernosum smooth muscle is under adrenergic control in order to maintain the sinusoids in a contracted condition (primarily via alpha-adrenergic stimulation). Penile erection is mediated by relaxation of corporal smooth muscle through a variety of direct and indirect mechanisms. Pharmacological agents that either inhibit alpha-adrenergic transmission or facilitate corporal smooth muscle relaxation will be beneficial for penile erection. Preliminary studies on macrocycle 1, a novel bioactive agent, demonstrated that this compound inhibits receptor-stimulated contraction without significantly altering field-stimulated contraction. The current study was designed to determine the physiological effects of macrocycle 1 on the response of the rabbit corpus cavernosum to various forms of stimulation. Sexually mature male New Zealand White rabbits were used in this study. The corpus cavernosum was dissected sharply from the removed penis and two longitudinal strips were prepared for isometric tension studies. Each strip was prestimulated with 200 mumol/l phenylephrine to produce a maximal contraction. The influences of macrocycle 1 (15.6, 62.5, 250, 1,000 mumol/l) on both the contractile response to phenylephrine and the relaxant effects of field stimulation, carbachol, ATP and nitroprusside were determined. The results demonstrated that although basal tension was not altered, the contractile response to phenylephrine was decreased by 5, 18, 47 and 57%, respectively, by the different concentrations of macrocycle 1. The degree of relaxation induced by field stimulation of various frequencies was significantly enhanced by macrocycle 1 in a concentration-dependent manner. Similarly, the degrees of relaxation induced by bethanechol, ATP and nitroprusside were all significantly enhanced in a concentration-dependent manner. The conclusion from this study is that macrocycle 1 significantly inhibits phenylephrine-stimulated contraction and significantly enhances field-stimulated and pharmacologically induced relaxation. Although the mechanisms of action are not clear, its physiological effects on the rabbit corpus cavernosum implicate a potential for the treatment of erectile dysfunction.

Physiological effects of a novel bioactive agent.

Urinary bladder smooth muscle contraction can be evaluated using field stimulation (neurohumoral transmission), carbachol (muscarinic stimulation), and KCl (direct membrane depolarization). We recently evaluated the activity of a novel organic chemical, macrocycle-1, on the contractile responses of the bladder to field stimulation, carbachol, and KCl. Isolated strips of rabbit bladder were mounted in individual baths containing 7.5 ml Tyrode's solution. The response to FS (1-32 Hz), carbachol (1 mumol/l), and KCl (120 mmol/l) were determined in the presence and absence of 3 different concentrations of macrocycle-1. Maximal tension, the rate of tension generation, the time to maximal tension, and the rate of decay following maximal tension were determined. The results can be summarized as follows: (1) In the absence of macrocycle-1, maximal tension and the maximal and mean rate of tension generation increased with frequency, whereas the time to maximal tension was constant. The rate of decay of tension following maximum tension was greater for 8, 16 and 32 Hz as compared to 1 or 2 Hz. (2) The maximal response to KCl was lower than either FS or carbachol. The maximal rates of tension generation for carbachol and KCl were lower than that of FS; and the rate of tension generation for KCl was lower than that of carbachol. The time to maximal stimulation for KCl was greater than that of either carbachol or FS. (3) Macrocycle-1 had a greater inhibitory effect on KCl stimulation than on carbachol stimulation; and a greater inhibitory effect on KCl and carbachol stimulation than on FS. (4) The rate of tension generation was more sensitive to macrocycle-1 inhibition than was the maximal tension responses to all methods of stimulation. Our current hypothesis is that macrocycle-1 is acting as an intracellular calcium buffer whose affinity constant and association rate does not interfere with rapid intracellular release mechanisms (FS) while it inhibits slow intracellular calcium release mechanisms (carbachol and KCl).

Endotoxin increases lung Cu,Zn superoxide dismutase mRNA: O2 raises enzyme synthesis.

Administration of endotoxin to adult rats increases lung Cu,Zn superoxide activity after 72 h of exposure to greater than 95% O2. The increased activity is brought about mainly by a faster rate of Cu,Zn superoxide dismutase synthesis; rats treated with endotoxin but not exposed to hyperoxia do not exhibit these findings (Hass, Frank, and Massaro, J. Biol. Chem. 257: 9379-9383, 1982). We now report that 48 h after treatment of adult rats with endotoxin there was a decreased rate of Cu,Zn superoxide dismutase synthesis by lung slices from air- and O2- exposed rats, although, in both groups, the lung concentration of Cu,Zn superoxide dismutase mRNA was increased approximately 45%. Exposure of endotoxin-treated rats to greater than 95% O2 or air for an additional 24 h (72 h all told) resulted in continued elevation of Cu,Zn superoxide dismutase mRNA only in lungs of O2- exposed rats. In vitro exposure of lung slices from air-breathing saline- or endotoxin-treated rats to 95% O2 for 6 h led to an increased rate of Cu,Zn superoxide dismutase synthesis only in slices from endotoxin-treated rats. We conclude that endotoxin treatment leads to an increased concentration of Cu,Zn superoxide dismutase mRNA in rat lungs, but a sustained elevation of the mRNA, and its translation into an increased rate of Cu,Zn superoxide dismutase synthesis requires exposure of the lung to hyperoxia.

Rat lung Cu,Zn superoxide dismutase. Isolation and sequence of a full-length cDNA and studies of enzyme induction.

The synthesis of Cu,Zn SOD by rat lung increases spontaneously in the fetus in late gestation and during exposure of neonatal and adult rats to greater than 95% O2. To explore the regulation of these increases, we measured rat lung Cu,Zn SOD synthesis and activity. We also cloned and sequenced a rat lung Cu,Zn SOD cDNA that was used to measure Cu,Zn SOD mRNA concentration. We found that (a) under normal gestational and postgestational conditions the synthesis of this enzyme was regulated pretranslationally; (b) the increased synthesis that occurs under hyperoxia (greater than 95% O2), was pretranslationally mediated in otherwise unmanipulated neonatal rats but translationally controlled in hyperoxic adult rats; and (c) in lungs of rats made tolerant to greater than 95% O2 by allowing 24 h rest in air after an initial 48 h in greater than 95% O2, the increased Cu,Zn SOD synthesis that occurred during the second period of hyperoxia was regulated pretranslationally. We conclude Cu,Zn SOD gene expression in the lung is developmentally regulated under normal conditions and in response to an oxidant challenge. Tolerance, whether endogenous or induced, appears to require the accumulation of increased amounts of Cu,Zn SOD mRNA.

Regulation of the synthesis of superoxide dismutases in rat lungs during oxidant and hyperthermic stresses.

Heat shock proteins are induced at normal temperatures by oxidants and during reoxygenation following hypoxia. We now report cyanide-resistant O2 consumption increased 30-50% in rat lungs exposed to heat shock or reoxygenation following hypoxia. The synthesis of Cu,Zn superoxide dismutase, but not Mn superoxide dismutase, was increased in rat lung slices by in vivo hyperthermia (39 degrees C), by in vitro heat shock (41 degrees C), and during incubation of lung slices with the Cu chelator diethyldithiocarbamate, which decreased the activity of Cu,Zn superoxide dismutase. The heat shock-induced increase in Cu,Zn superoxide dismutase developed 2 h later than the induction of heat shock proteins and was not blocked by actinomycin D. The rates of synthesis of both superoxide dismutases were decreased 50% by hypoxia and failed to increase during reoxygenation. During hypoxia the activity of Cu,Zn superoxide dismutase decreased about 50%, but the activity of Mn superoxide dismutase remained unchanged. We conclude that hyperthermia increases the synthesis of Cu,Zn superoxide dismutase, the synthesis of Cu,Zn superoxide dismutase and Mn superoxide dismutase are not coordinately regulated by hyperthermia or by the oxidant stress produced by lowering the activity of Cu,Zn superoxide dismutase, and the synthesis of heat shock proteins and Cu,Zn superoxide dismutase are regulated at different levels of gene expression.

Mitogenic response of rat lung to endotoxin exposure.

Adult rats exposed to hyperoxia are protected from lung injury by treatment with bacterial endotoxin. Experiments were undertaken to determine whether endotoxin treatment produces a mitogenic effect on the lung. Endotoxin treatment caused a significant (P less than .05) loss of body weight (8%) in rats exposed to either air or greater than 95% O2 for 24 hr. Therefore, experiments were also undertaken in which both saline- and endotoxin-treated rats were starved for the duration of the experiments to make equal any nutritional imbalance. The rate of DNA synthesis in lung slices from fed rats treated with endotoxin did not differ from that of saline-treated controls. In contrast, lung DNA synthesis in starved rats treated with endotoxin increased 50%. The effect of endotoxin treatment was similar in rats breathing air or greater than 95% O2, and lung protein synthesis generally paralleled lung DNA synthesis. These results indicate that endotoxin does exert a mitogenic effect on the lung and this effect can be masked by the nutritional imbalance resulting from endotoxin administration.

Developmental regulation of rat lung Cu,Zn-superoxide dismutase.

In the present investigation we found that lung Cu,Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu,Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu,Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu,Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu,Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu,Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu,Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.

Differences in CuZn superoxide dismutase induction in lungs of neonatal and adult rats.

The failure of adult rats to survive prolonged exposure to greater than 95% O2 is generally ascribed to the inability of their lungs to increase antioxidant enzyme synthesis in response to the oxidant challenge. We studied the synthesis rate of the antioxidant enzyme CuZn superoxide dismutase (CuZn SOD) in lungs of adult and neonatal rats exposed to conditions that alter the lung's oxidant-to-antioxidant balance. Lung CuZn SOD synthesis in the adult was significantly increased after 24 h of hyperoxia but fell to control levels after further exposure, whereas in neonatal lungs an increased rate of synthesis of CuZn SOD was found only after 72 h of hyperoxia. The adult lung responded to two in vitro oxidant stresses, [diethyldithiocarbamate exposure and heat (42 degrees C)] with increases in CuZn SOD synthesis twice the magnitude of those in the neonatal lung. These data indicate that the adult lung is at least as capable as the neonatal lung of increasing its synthesis of CuZn SOD in response to an oxidative stress. However, the inability of the adult lung to maintain an increased rate of CuZn SOD synthesis during in vivo hyperoxia may contribute to the poor tolerance of the adult lung to greater than 95% O2.

PO2-dexamethasone interactions in fibroblast growth and antioxidant enzyme activity.

Fetal rat lung fibroblasts were cultured in a gas phase of 20% O2, 5% CO2 (PO2 measured, 150 Torr) or 2% oxygen, 5% CO2 (PO2 measured, 25 Torr) with or without 100 nM dexamethasone (Dex). Superoxide dismutase (SOD) activity per cell increased spontaneously during 4 days of incubation at both PO2, but catalase (CAT) activity tended to fall during this time and glutathione peroxidase (GPx) activity showed no consistent trend during this interval. Cells cultured at a low PO2 had a lower protein content and SOD activity compared with air controls. Dex inhibited cell proliferation and enhanced intracellular accumulation of protein at the low PO2 but prevented the increase in protein content without affecting cell multiplication at a PO2 of 150 Torr. SOD activity per cell was enhanced by Dex at a low PO2 but reduced in 20% O2, 5% CO2. An increase in CAT and GPx activity per cell resulted on exposing fibroblasts to Dex in the presence of low PO2. These results show that Dex affects the growth and antioxidant enzyme activity of fetal lung fibroblasts, and this action of Dex can be modulated by changing the ambient PO2.

The effect of bacterial endotoxin on synthesis of (Cu,Zn)superoxide dismutase in lungs of oxygen-exposed rats.

Administration of bacterial endotoxin to rats exposed to greater than 95% O2 results in increased lung superoxide dismutase activity, decreased O2-induced lung damage, and a 3- to 4-fold improvement in survival rate (Frank, L., Yam, J., and Roberts, R. J. (1978) J. Clin. Invest, 61, 269-275). Antibodies to rat liver (Cu,Zn) superoxide dismutase were prepared and utilized to investigate the mechanism by which endotoxin treatment leads to increased lung superoxide dismutase activity. Assay of enzyme activity and of immunodetectable enzyme showed that the increased activity is due to an increase in the number of enzyme molecules rather than activation of existing enzyme. Compared to air controls, lung slices from rats exposed to greater than 95% O2 and treated with endotoxin have elevated rats of synthesis of (Cu,Zn)superoxide dismutase (51%) and of total protein (100%). Lung slices from untreated rats exposed to greater than 95% O2 have no such elevations. Endotoxin treatment thus appears to stimulate lung protein synthesis, leading to greater (Cu,Zn)superoxide dismutase activity due to an increased number of enzyme molecules.

Regulation of lung surfactant cholesterol metabolism by serum lipopoteins.

The isolated perfused rat lung was used as an experimental model in the study of the lipoprotein regulation of surfactant cholesterol metabolism. Addition of low density lipoproteins (LDL) to the perfusion medium at a cholesterol concentration of 0.5 micrometer had no inhibitory effect on [1-14C]-acetate incorporation into cholesterol of either the surfactant or residual fractions. Increasing the concentration of cholesterol in the medium to 2.5 micrometer resulted in significant inhibition of incorporation into cholesterol of both fractions. A similar inhibition resulted when lungs were perfused with 2.5 micrometer cholesterol in the form of high density lipoproteins (HDL). No inhibition of fatty acid synthesis, measured as incorporation into cholesteryl esters, was observed. The rate of uptake by perfused lung of cholesterol from both high and low density lipoproteins was similar. Competitive binding studies with 125I-labeled lipoproteins indicated the existence of lung receptors for both classes of lipoprotein. The rate of uptake of the apoprotein moiety of low density lipoproteins was significantly greater than that of high density lipoproteins. These data suggest that lung cholesterol metabolism may be subject to regulation by both low and high density serum lipoproteins.

Surfactant cholesterol metabolism of the isolated perfused rat lung.

1. The isolated perfused rat lung was used as the principal model to study surfactant cholesterol metabolism. 2. Cholesterol was found to represent over 50% of the neutral lipid of both the total surfactant and the lamellar body fractions. 3. De novo synthesis of cholesterol from [1-14C]acetate accounted for only 1% of the surfactant cholesterol, the remainder being derived from exogenous cholesterol supplied as serum lipoproteins. 4. Lipoprotein [1,2-3H2]cholesterol was incorporated into the lamellar body and extracellular surfactant fractions. The increase in the cholesterol specific activities of these fractions with time was consistent with a precursor-product relationship between the lamellar body cholesterol and that of the extracellular surfactant. 5. Incorporation of [methyl-14C]choline and [1,2-3H2]cholesterol indicated that the metabolism and secretion of lamellar body and extracellular surfactant cholesterol parallels that of phosphatidylcholine and suggests that most if not all extracellular surfactant cholesterol is derived from the lamellar body. 6. Comparison of the relative specific activities of incorporated [1,2-3H2]-cholesterol indicate that 59% of the total surfactant cholesterol is located extracellularly.