Serotonin metabolism in rat mesangial cells: involvement of a serotonin transporter and monoamine oxidase A.
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BACKGROUND: Serotonin is one of the factors regulating mesangial cell proliferation, and convergent evidence supports its involvement in the development of glomerulonephritis. In this study, we identified a serotonin transporter and the amine-degrading enzyme monoamine oxidases (MAOs) in mesangial cells, and we studied their involvement in serotonin degradation. METHODS: MAOs were characterized in membrane preparations and intact mesangial cells by enzyme assay using [14C]5-hydroxytryptamine and [14C]beta-phenylethylamine as specific substrates for MAO-A and MAO-B, respectively, and by Western blot analysis. The expression of a serotonin transporter was determined by [14C]5-hydroxytryptamine uptake experiments and Western blot. Mesangial cell proliferation was measured by BrdU incorporation. RESULTS: Quantitation of the MAO isoforms by enzyme assay and Western blot analysis showed that MAO-A was largely predominant in mesangial cells, accounting for approximately 90% of the total enzyme population. The MAO substrate [14C]serotonin was transported into mesangial cells by a saturable uptake system (Vmax 310 +/- 36 pmol/30 min/mg protein; Km 5.9 +/- 1.4 microM) displaying the pharmacological properties of a serotonin transporter. The expression of a serotonin transporter was confirmed by Western blot analysis. MAO activity measured in intact cells showed that after accumulation into mesangial cells, [14C]serotonin was metabolized by MAO-A. Finally, serotonin-mediated mesangial cell proliferation was significantly increased after irreversible MAO inhibition. CONCLUSIONS: Our results suggest that serotonin concentration and function in glomeruli may be regulated in part by its transport into mesangial cells and degradation by MAO-A. (+info)
32P-postlabelling of propylene oxide 1- and N(6)-substituted adenine and 3-substituted cytosine/uracil: formation and persistence in vitro and in vivo.
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Propylene oxide, a widely used monofunctional alkylating agent, has been shown to be genotoxic in in vitro test systems and induces tumors in the nasal tissues of experimental animals. Propylene oxide, like related alkylating agents, forms several different adducts with DNA bases, but predominantly at the 7-position of guanine. We have previously described the in vitro and in vivo formation and stability of this major adduct. The aim of the present study was to perform a similar investigation of other adducts of propylene oxide. 1-(2-Hydroxypropyl)adenine (1-HP-adenine) and 3-(2-hydroxypropyl)cytosine (3-HP-cytosine), as well as their rearrangement products to N(6)-(2-hydroxypropyl)adenine (N(6)-HP-adenine) and 3-(2-hydroxypropyl)uracil (3-HP-uracil), respectively, were analysed by a very sensitive (32)P-postlabelling method involving nuclease P1 enhancement and radioisotope detector-coupled HPLC separation. All four adducts could be detected in DNA treated in vitro with propylene oxide. The sum of the levels of 1- and N(6)-HP-adenine amounted to 3.5% and the sum of 3-HP-cytosine and 3-HP-uracil to 1.7%, respectively, of 7-(2-hydroxypropyl)guanine (7-HP-guanine). In male Fischer 344 rats exposed to 500 p.p.m. propylene oxide by inhalation for 20 days, 1-HP-adenine was detected in all analysed tissues, including nasal epithelium, lung and lymphocytes, whereas N(6)-HP-adenine was only found in the tissues of the nasal cavities. The highest level of 1-HP-adenine (2.0 mol/10(6) mol of normal nucleotides, i.e. 2% of 7-HP-guanine) was found in the respiratory nasal epithelium, which also represents the major target for tumour induction in the rat following inhalation of propylene oxide. The levels of this adduct in the lung and in the lymphocytes were considerably lower, amounting to 15 and 9%, respectively, of that of the respiratory nasal epithelium. In rats killed 3 days after cessation of exposure, practically no decrease in 1-HP-adenine was observed, indicating no or very slow repair. 3-HP-uracil could only be detected in the respiratory nasal epithelia of propylene-exposed rats and its concentration was as low as 0.02 mol/10(6) mol of normal nucleotides (0.02% of 7-HP-guanine). Since 3-HP-uracil was chemically much more stable than the latter, the obtained animal data suggest repair of the cytosine and/or uracil adducts. Incubation of propylene oxide-reacted DNA with a protein extract from mammalian cells indicated that an enzymatic repair mechanism exists for removal of 3-HP-cytosine, but not for 3-HP-uracil or 1- and N(6)-HP-adenine. Another finding was that uracil glycosylase is probably not involved. The level of 1-HP-adenine in the propylene oxide-exposed rats was approximately 50 times lower than that of 7-HP-guanine. Nevertheless, this adduct is conveniently analysed and has high chemical stability and recovery, which results in high sensitivity (detection limit 0.3 mol/10(9) mol of normal nucleotides using 10 microgram DNA). 1-HP-adenine might, therefore, be considered as an alternative to 7-HP-guanine for monitoring exposure to propylene oxide. (+info)
Use of the [(14)C]leucine incorporation technique to measure bacterial production in river sediments and the epiphyton.
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Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [(14)C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 microM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 microM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined. (+info)
Sterol synthesis is up-regulated in cholesterol-loaded pigeon macrophages during induction of cholesterol efflux.
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The extent to which cholesterol synthesis is modulated in macrophage foam cells by changes in cholesterol influx and efflux was determined using thioglycollate-elicited peritoneal macrophages from normal and cholesterol-fed White Carneau (WC) and Show Racer (SR) pigeons. In peritoneal macrophages from normocholesterolemic pigeons, sterol synthesis from [(14)C]-acetate was down-regulated by more than 90% following incubation in vitro with beta-VLDL. Sterol synthesis was increased when the cellular free cholesterol concentration was decreased in response to stimulation of cholesterol efflux with apoHDL/phosphatidylcholine vesicles and cyclodextrin. Peritoneal macrophages isolated from hypercholesterolemic pigeons were loaded with cholesterol to levels similar to foam cells from atherosclerotic plaques (375-614 microg/mg cell protein), and had an extremely low rate of sterol synthesis. When cholesterol efflux was stimulated in these cells, sterol synthesis increased 8 to 10-fold, even though the cells remained grossly loaded with cholesterol. Cholesterol efflux also stimulated HMG-CoA reductase activity and LDL receptor expression. This suggests that only a small portion of the total cholesterol pool in macrophage foam cells was responsible for regulation of sterol synthesis, and that cholesterol generated by hydrolysis of cholesteryl esters was directed away from the regulatory pool by efflux from the cells. When the increase in sterol synthesis was blocked with the HMG-CoA reductase inhibitor mevinolin, there was no difference in the cholesterol content of the cells, or in the mass efflux of cholesterol into the culture medium.Thus, under these conditions, the increase in cholesterol synthesis during stimulation of cholesterol efflux does not appear to contribute significantly to the mass of cholesterol in these macrophage foam cells. Whether a similar situation exists in vivo is unknown. (+info)
Rapid hepatic metabolism of 7-ketocholesterol in vivo: implications for dietary oxysterols.
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7-Ketocholesterol is a major dietary oxysterol and the predominant non-enzymically formed oxysterol in human atherosclerotic plaque. We tested the hypothesis that 7-ketocholesterol is preferentially retained by tissues relative to cholesterol in vivo. To ensure rapid tissue uptake, acetylated low density lipoprotein, labeled with esters of [(14)C]-7-ketocholesterol and [(3)H]cholesterol, was injected into rats via a jugular catheter. At timed intervals (2 min to 24 h) rats (n = 48 total) were exsanguinated and tissues were dissected and assayed for radioactivity. In two experiments the majority of both radiolabels appeared in the liver after 2 min. In all tissues, (14)C appeared transiently and did not accumulate. Rather, it was metabolized in the liver and excreted into the intestine mainly as aqueous-soluble metabolites (presumably bile acids). By 9 h, (14)C in the liver had decreased to 10% of the injected dose while 36% was present in the intestine. In contrast, at 9 h 38% of (3)H was evident in the liver while only 5% was found in the intestine. Unlike [(3)H]cholesterol, little (14)C was found to re-enter the circulation, indicating that enterohepatic recycling of 7-ketocholesterol was negligible. This is the first report of the distribution of an oxysterol relative to cholesterol, administered simultaneously, in a whole animal model. The finding that [(14)C]-7-ketocholesterol is rapidly metabolized and excreted by the liver suggests that diet may not be a major source of oxysterols in atherosclerotic plaque, and that perhaps dietary oxysterols make little or no contribution to atherogenesis. (+info)
Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages.
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Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4n-6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine (N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of (18)O from H2 (18)O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids. (+info)
Responses of contralateral SI and SII in cat to same-site cutaneous flutter versus vibration.
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The methods of (14)C-2-deoxyglucose ((14)C-2DG) metabolic mapping and optical intrinsic signal (OIS) imaging were used to evaluate the response evoked in the contralateral primary somatosensory receiving areas (SI and SII) of anesthetized cats by either 25 Hz ("flutter") or 200 Hz ("vibration") sinusoidal vertical skin displacement stimulation of the central pad on the distal forepaw. Unilateral 25-Hz stimulation consistently evoked a localized region of elevated (14)C-2DG uptake in both SI and SII in the contralateral hemisphere. In contrast, 200-Hz stimulation did not evoke elevated (14)C-2DG uptake in the contralateral SI but evoked a prominent, localized region of increased (14)C-2DG uptake in the contralateral SII. Experiments in which the OIS was recorded yielded results that complemented and extended the findings obtained with the 2DG method. First, 25-Hz central-pad stimulation evoked an increase in absorbance in a region in the contralateral SI and SII that corresponded closely to the region in which a similar stimulus evoked increased (14)C-2DG uptake. Second, 200-Hz stimulation of the central pad consistently evoked a substantial increase in absorbance in the contralateral SII but very little or no increase in absorbance in the contralateral SI. And third, 200-Hz central-pad stimulation usually evoked a decrease in absorbance in the same contralateral SI region that underwent an increase in absorbance during same-site 25-Hz stimulation. Experiments in which the OIS responses of both SI and SII were recorded simultaneously demonstrated that continuous (>1 s) 25-Hz central-pad stimulation evokes a prominent increase in absorbance in both SI and SII in the contralateral hemisphere, whereas only SII undergoes a sustained prominent increase in absorbance in response to 200-Hz stimulation to the same central-pad site. SI exhibits an initial, transient increase in absorbance in response to 200-Hz stimulation and at durations of stimulation >1 s, undergoes a decrease in absorbance. It was found that the stimulus-evoked absorbance changes in the contralateral SI and SII are correlated significantly during vibrotactile stimulation of the central pad-positively with 25-Hz stimulation and negatively with 200-Hz stimulation. The findings are interpreted to indicate that 25-Hz central-pad stimulation of the central pad evokes spatially localized and vigorous neuronal activation within both SI and SII in the contralateral hemisphere and that although 200-Hz stimulation evokes vigorous and well maintained neuronal activation within the contralateral SII, the principal effect on the contralateral SI of a 200-Hz stimulus lasting >1 s is inhibitory. (+info)
Cerebrovascular reactivity to CO(2) and hypotension after mild cortical impact injury.
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Cerebrovascular reactivity to CO(2) or hypotension was studied in vivo and in vitro [pressurized arteries ( approximately 82 micrometer) and arterioles ( approximately 30 micrometer)] at 1 h after mild controlled cortical impact (CCI) injury in rats. The cortical perfusion response [assessed using laser-Doppler flowmetry (LDF)] to altered CO(2) was diminished (up to 81%) after mild CCI injury. The responses to CO(2) alterations in arteries and arterioles isolated from the injured cortex were similar to responses in vessels isolated from sham-injured animals. After mild CCI injury, the autoregulatory response to hypotension (measured using LDF) was maintained or even enhanced, depending on the method used to measure the response. Vessels isolated from the injury site showed a response to changes in pressure similar to that in vessels isolated from sham-injured rats. We conclude that mild CCI injury produces complicated alterations in cerebrovascular control. Whereas the autoregulatory response to hypotension was maintained or even enhanced, the in vivo vascular response to CO(2) was severely compromised. The altered response to CO(2) was not caused by an intrinsic vascular perturbation but rather an altered milieu after mild CCI injury. (+info)