Actions of lipoxygenase metabolites in isolated rat lungs

Leukotrienes constrict smooth muscle and could be important for the regulation of the pulmonary circulation. We examined the production and action of lipoxygenase metabolites in isolated lungs, where we controlled the perfusing fluid used. Arachidonate injected into isolated rat lungs perfused with cell- and protein-free physiological salt solution caused a transient pressor response. Following indomethacin, arachidonate caused a delayed slow pressure rise followed by edema. The lung effluent contracted the guinea pig ileum. High-pressure liquid chromatography (HPLC) analysis of the perfusate demonstrated the presence of leukotrienes (LTC4 and LTD4). Diethylcarbamazine, a leukotriene synthesis inhibitor, prevented the slow pressure rise and edema seen after indomethacin plus arachidonate. In lungs perfused with cell- and protein-free physiological salt solution, LTC4, but not LTD4, caused a transient pressure rise followed by a sustained pressure rise. The sustained rise was abolished by a leukotriene-receptor blocker (FPL 55712) but not by indomethacin. In blood-perfused lungs, LTC4 caused only the transient pressure rise that was not blocked by FPL 55712. In lungs perfused with physiological salt solution containing albumin, LTC4 had no effect. We concluded that 1) perfused nonsensitized rat lungs produced LTC4 and LTD4; 2) LTC4 may be a major pulmonary vasoconstrictor; and 3) albumin binding limits the pressor effect of LTC4.     

Direct airway injury results in elevated levels of sulfidopeptide leukotrienes, detectable in airway secretions.

Sulfidopeptide leukotrienes (LTC4/D4/E4) are suspected to be important lipid mediators in inflammatory responses in the lung. Previous investigations have provided evidence to support enhanced synthesis and secretion of these eicosanoids into bronchoalveolar lavage fluid in patients with Adult Respiratory Distress Syndrome (ARDS). We have prospectively examined the relationship between sulfidopeptide leukotriene levels in tracheal aspirates of 14 intubated and mechanically ventilated patients. When compared with the aspirate from one patient who required ventilation because of respiratory muscle weakness, the tracheal aspirates from eight ARDS patients had elevated leukotriene levels (range 2020-2052 pg/aspirate). However, the aspirates from four of the five patients with direct airway injury [inhalational burn (n = 3) and massive aspiration of gastric contents (n = 2)] contained significantly higher amounts of sulfidopeptide leukotrienes (range 10309-52244 pg/aspirate). Three of the five patients with direct airway injury did not develop ARDS. We conclude that simple aspiration of tracheal secretions can be used to monitor airway leukotriene biosynthesis in patients with lung injury and that elevated airway leukotriene levels may reflect airway epithelial damage, but may not predict the development of ARDS.     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

Water Flow in Wheat Seedlings after Small Water Deficits

Cultures of Scenedesmus obliquus when grown heterotrophically for 10 or 30 days without addition of fresh medium showed 85 and 98% loss of their photosynthetic capacity respectively. This loss in photosynthetic capacity was accompanied by an increase in quantum requirement. No major changes in the pigment amounts or types were detected which would explain the decay in photosynthetic capacity. Partial reactions mediated by photosystem II or I showed a more or less constant decay over a period of 30 days. Photosystem II reactions appeared less stable than those of photosystem I, decaying by 95% as compared with 70%, over this time period. The results of comparative studies on aged cells for their potential of cytochrome f photooxidation, fluorescence kinetics, 520 nm absorbance change and the variable influence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on the photosynthetic capacity of such cells, suggest that it is the inherent ability of the cells to photooxidize plastohydroquinone which is affected primarily. In addition, secondary changes were noted in the activity of reactions on the water-splitting side of photosystem II and in the P700 — plastocyanin — cytochrome f complex.     

Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin E2 in rabbits bearing the VX2 carcinoma: Effects of hydrocortisone and indomethacin

In rabbits bearing the prostaglandin-producing VX₂ carcinoma, the plasma concentration of 13,14-dihydro-15-keto-PGE₂ (PGE₂-M) was elevated within one week after tumor implantation and preceded the development of hypercalcemia. Both the rate of rise and magnitude of the increase were greater for the metabolite than for PGE₂; at the time of peak hypercalcemia (about 4 to 5 weeks after tumor implantation), the increase over basal in plasma PGE₂-M was about 75 fold whereas it was previously shown that the increase in PGE₂ was less than 2 fold. Indomethacin, which inhibits PGE₂ synthesis in VX₂ cells in culture, lowered in parallel plasma calcium and PGE₂-M in tumor-bearing rabbits. Administration of hydrocortisone to rabbits bearing the VX₂ tumor prevented the development of hypercalcemia when given at the time of tumor implantation and reversed the elevated plasma calcium in previously untreated animals; the steroid hormone also lowered plasma concentrations of PGE₂-M. These findings are consistent with our hypothesis that the hypercalcemic syndrome in VX₂ tumor-bearing rabbits is due to the secretion of PGE₂ by the tumor.     

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbecco's phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Ham's F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (相似文献   

Electrical and adaptive properties of rod photoreceptors in bufo marinus. I. Effects of altered extracellular Ca(2+) levels

The effects of altering extracellular Ca(2+) levels on the electrical and adaptive properties of toad rods have been examined. The retina was continually superfused in control (1.6 mM Ca(2+)) or test ringer’s solutions, and rod electrical activity was recorded intracellularly. Low-calcium ringer’s (10(-9)M Ca(2+)) superfused for up to 6 min caused a substantial depolarization of the resting membrane potential, an increase in light-evoked response amplitudes, and a change in the waveform of the light-evoked responses. High Ca(2+) ringer’s (3.2 mM) hyperpolarized the cell membrane and decreased response amplitudes. However, under conditions of either low or high Ca(2+) superfusion for up to 6 min, in both dark-adapted and partially light-adapted states, receptor sensitivity was virtually unaffected; i.e., the V-log I curve for the receptor potential was always located on the intensity scale at a position predicted by the prevailing light level, not by Ca(2+) concentration. Thus, we speculate that cytosol Ca(2+) concentration is capable of regulating membrane potential levels and light-evoked response amplitudes, but not the major component of rod sensitivity. Low Ca(2+) ringer’s also shortened the period of receptor response saturation after a bright but nonbleaching light flash, hence accelerating the onset of both membrane potential and sensitivity recovery during dark adaptation.

Exposure of the retina to low Ca(2+) (10(-9)M) ringer’s for long periods (7-15 min) caused dark-adapted rods to lose responsiveness. Response amplitudes gradually decreased, and the rods became desensitized. These severe conditions of low Ca(2+) caused changes in the dark-adapted rod that mimic those observed in rods during light adaptation. We suggest that loss of receptor sensitivity during prolonged exposure to low Ca(2+) ringer’s results from a decrease of intracellular (intradisk) stores of Ca(2+); i.e., less Ca(2+) is thereby released per quantum catch.

     

Treatment of severe pulmonary hypertension: a bradykinin receptor 2 agonist B9972 causes reduction of pulmonary artery pressure and right ventricular hypertrophy

Bradykinin is an important modulator of endothelial cell function and has also a powerful cardioprotective effect. Here we report that treatment of severely pulmonary hypertensive rats (that recapitulate several of the physiological and pathological characteristics of the human pulmonary vascular disease, including dramatic right ventricular hypertrophy, pericardial effusion and death) with a newly synthesized long-acting bradykinin B2 receptor agonist B9972 caused reduction of the pulmonary artery pressure (PAP=51+/-2.0 versus 68+/-2.8 of untreated animals) and of right ventricular hypertrophy (Rv/Lv+S=0.55+/-0.02 versus 0.73+/-0.03 of untreated rats) and activation of Akt. Long-term stimulation with B9972 in our animal model of SPH resulted in decreased expression of the B2 receptor in lung vasculature. Treatment with B9972 decreased the number of plexiform lesions in the lungs by inducing cell apoptosis in the obliterated vessels and by restoring caveolin-1 expression. B9972 also promoted eNOS activation. In vitro B9972 caused activation of caspase-3 as well as Erk and induction of prostacyclin production in rat pulmonary microvascular EC. Taken together our data suggest that a stable bradykinin B2 agonist B9972 demonstrates the capacity to reduce severe pulmonary hypertension, right ventricular hypertrophy and induce apoptosis of hyperproliferative cells in pre-capillary pulmonary arterioles.