Development of a multivariate statistical model to predict peroxisome proliferation in the rat, based on urinary 1H-NMR spectral patterns.

A previous report of this work (Ringeissen et al. 2003) described the use of nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical data analysis (MVDA) to identify novel biomarkers of peroxisome proliferation (PP) in Wistar Han rats. Two potential biomarkers of peroxisome proliferation in the rat were described, N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY). The inference from these results was that the tryptophan-nicotinamide adenine dinucleotide (NAD(+)) pathway was altered in correlation with peroxisome proliferation, a hypothesis subsequently confirmed by TaqMan analysis of the relevant genes encoding two key enzymes in the pathway, aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) and quinolinate phosphoribosyltransferase (EC 2.4.2.19). The objective of the present study was to investigate these data further and identify other metabolites in the NMR spectrum correlating equally with PP. MVDA Partial Least Squares (PLS) models were constructed that provided a better prediction of PP in Wistar Han rats than levels of 4PY and NMN alone. The resulting Wistar Han rat predictive models were then used to predict PP in a test group of Sprague Dawley rats following administration of fenofibrate. The models predicted the presence or absence of PP (above on arbitrary threshold of >2-fold mean control) in all Sprague Dawley rats in the test group.     

Development of a multivariate statistical model to predict peroxisome proliferation in the rat,based on urinary 1H-NMR spectral patterns

A previous report of this work (Ringeissen et al. 2003) described the use of nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical data analysis (MVDA) to identify novel biomarkers of peroxisome proliferation (PP) in Wistar Han rats. Two potential biomarkers of peroxisome proliferation in the rat were described, N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY). The inference from these results was that the tryptophan-nicotinamide adenine dinucleotide (NAD⁺) pathway was altered in correlation with peroxisome proliferation, a hypothesis subsequently confirmed by TaqMan® analysis of the relevant genes encoding two key enzymes in the pathway, aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) and quinolinate phosphoribosyltransferase (EC 2.4.2.19). The objective of the present study was to investigate these data further and identify other metabolites in the NMR spectrum correlating equally with PP. MVDA Partial Least Squares (PLS) models were constructed that provided a better prediction of PP in Wistar Han rats than levels of 4PY and NMN alone. The resulting Wistar Han rat predictive models were then used to predict PP in a test group of Sprague Dawley rats following administration of fenofibrate. The models predicted the presence or absence of PP (above on arbitrary threshold of >2-fold mean control) in all Sprague Dawley rats in the test group.     

Synthesis of thiostatins (major acute-phase alpha 1 proteins) in different strains of Rattus norvegicus

1. Two different thiostatin proteins were detected by crossed immunoelectrophoresis in plasma of the following inbred strains of Rattus norvegicus; Wistar, Sprague Dawley, Hooded Wistar, Lewis, Porton-Albino, ACI, Long Evans, and Katholiek, a mutant strain of the Brown Norway. 2. Only one thiostatin protein was detected using crossed immunoelectrophoresis in plasma from the Buffalo rat. 3. Comparison of partially purified thiostatins from Buffalo, Wistar, and Sprague Dawley rats in polyacrylamide gels showed that the thiostatin protein in Buffalo rat plasma corresponded to thiostatin 1 of the two thiostatins of the Wistar and Sprague Dawley rats. 4. Thiostatin 1 mRNA and thiostatin 2 mRNA, of approximately 1.7 kilobases, were both demonstrated in RNA from Buffalo rat liver.     

Agonist-induced regulation of inositol tetrakisphosphate isomers and inositol pentakisphosphate in adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II (AII) rapidly stimulates the formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and causes marked long-term changes in the levels of highly phosphorylated inositols. Glomerulosa cells prelabeled with [3H]inositol for 48 h and exposed to AII for 10 min showed prominent increases in inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and smaller increases in two additional tetrakisphosphates, Ins-1,3,4,6-P4 and another (Ins-3,4,5,6-P4) eluting in the position of Ins-3,4,5,6-P4 and its stereoisomer, Ins-1,4,5,6-P4, on anion exchange liquid chromatography. A concomitant decrease in InsP5 indicates that an increase in Ins-1,4,5,6-P4, the breakdown product of InsP5, is probably responsible for the initial rise in Ins-3,4,5,6-P4 during 10 min stimulation by AII. During prolonged stimulation by AII, Ins-1,3,4,5-P4 began to decline from its high, stimulated level after the first hour but the level of Ins-1,3,4,6-P4 remained elevated for several hours. There were also progressive increases in the levels of Ins-3,4,5,6-P4 and InsP5 during stimulation for up to 16 h with AII. Treatment of adrenal cells for 16 h with the cyclic AMP-mediated secretagogue, adrenocorticotropic hormone (ACTH), slightly increased basal levels of Ins-1,3,4,6-P4, Ins-3,4,5,6-P4, and InsP5, and enhanced the subsequent AII-stimulated increases in the two additional tetrakisphosphate isomers but not of inositol trisphosphates or Ins-1,3,4,5-P4. This change in the pattern of the higher inositol phosphate response to AII was manifested within 2 h after exposure to ACTH, and was mimicked by treatment with 8-bromo cyclic AMP or forskolin. Treatment with 50 microM cycloheximide abolished the ACTH-induced increases in inositol polyphosphate responses during AII stimulation, but had no effect on the responses of untreated cells to AII. The conversion of [3H]Ins-1,3,4-P3 to [3H]Ins-1,3,4,6-P4, a reaction linking the receptor-mediated InsP3 response to higher inositol phosphates, was enhanced in permeabilized cells that were pretreated for 16 h with either ACTH or AII. These results demonstrate that the reactions by which Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4 are formed and converted to InsP5 are influenced by agonist-stimulated regulatory processes that include both calcium-dependent and cyclic AMP-dependent mechanisms of target cell activation. They also reveal changes consistent with agonist-induced conversion of InsP5 to its dephosphorylated metabolite, Ins-1,4,5,6-P4, during short-term stimulation by AII.     

Metabolism of inositol 1,3,4-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells

Angiotensin stimulates rapid and prominent increases in inositol polyphosphates and their metabolites in bovine glomerulosa cells labeled with [3H]inositol. In addition to the early formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), as well as their intermediate product, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), delayed increases in two new InsP4 isomers were consistently observed by high resolution high performance liquid chromatography. Studies on the metabolism of purified Ins-1,3,4,5-P4 preparations, labeled with [3H]inositol and 32P to monitor sites of dephosphorylation, were performed in permeabilized glomerulosa cells. In addition to rapid degradation of Ins-1,3,4,5-P3 to Ins-1,3,4-P3 and then to Ins-3,4-P2, there was delayed formation of one of the putative InsP4 isomers observed during AII stimulation in intact cells. The kinetics of formation of the new InsP4 isomer, and the lack of phosphate in its 5 position based on isotope ratios, were consistent with its origin from Ins-1,3,4-P3. This was confirmed by the conversion of [3H]Ins-1,3,4-P3 to the new InsP4 isomer in permeabilized cells by a kinase distinct from that which phosphorylates Ins-1,4,5-P3. These results have demonstrated that the dephosphorylation sequence of Ins-1,4,5-P3 metabolism is accompanied by a complex cycle of higher phosphorylations with formation of new intermediates of potential significance in cellular regulation.     

Structures and metabolism of inositol tetrakisphosphates and inositol pentakisphosphate in bovine adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II stimulates rapid increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), followed by slower increases in two additional inositol tetrakisphosphate (InsP4) isomers. One of these InsP4 isomers was previously identified as Ins-1,3,4,6-P4 and shown to be a precursor of inositol pentakisphosphate (InsP5). Analysis of the third InsP4 isomer, purified from cultured bovine adrenal cells labeled with [3H]inositol and stimulated by angiotensin II, revealed that the polyol produced by periodate oxidation, borohydrate reduction, and dephosphorylation was [3H]iditol. This finding is consistent with precursor structures of either Ins-1,4,5,6-P4 or Ins-3,4,5,6-P4 (= L-Ins-1,4,5,6-P4) for the third InsP4 isomer. The [3H]iditol was readily converted to [3H]sorbose by the stereospecific enzyme, L-iditol dehydrogenase, indicating that it originated from Ins-3,4,5,6-P4. Chicken erythrocytes labeled with [3H]inositol also contained high levels of Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4, as well as InsP5, but only small amounts of Ins-1,3,4,5-P4. Both [3H]Ins-1,3,4,6-P4 and [3H]Ins-3,4,5,6-P4, but not [3H]Ins-1,3,4,5-P4, were phosphorylated to form InsP5 in permeabilized bovine glomerulosa cells. In addition, InsP5 itself was slowly dephosphorylated to Ins-1,4,5,6-P4, indicating that its structure is Ins-1,3,4,5,6-P5. These results demonstrate that the higher inositol phosphates are metabolically interrelated and are linked to the receptor-regulated InsP3 response by the conversion of Ins-1,3,4-P3 through Ins-1,3,4,6-P4 to Ins-1,3,4,5,6-P5. The source of Ins-3,4,5,6-P4, the other precursor of InsP5, is not yet known but its elevation in angiotensin II-stimulated glomerulosa cells suggests that its formation is also influenced by agonist-regulated processes.     

Ca2+ regulates the inositol tris/tetrakisphosphate pathway in intact and broken preparations of insulin-secreting RINm5F cells

The two-step isomerization of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to Ins-1,3,4-P3 via the intermediate inositol 1,3,4,5-tetrakisphosphate (Ins-P4) was studied in intact RINm5F cells and in subcellular fractions. Muscarinic stimulation with carbamylcholine leads to a rapid (2 s) rise in both Ins-1,4,5-P3 and Ins-P4, whereas Ins-1,3,4-P3 was produced only after a lag of at least 5 s. In cells with depleted Ca2+ stores, the rise in Ins-1,4,5-P3 was nearly tripled, and that of Ins-1,3,4-P3 markedly diminished as compared to control cells. Raising the free Ca2+ concentration from 10(-7) to 10(-5) M increased inositol 1,4,5-triphosphate-3-kinase activity in cytosolic fractions by 2 1/2-fold (EC50 for Ca2+ approximately 0.8 microM) but had no effect on the activity of inositol 1,4,5-triphosphate-5-phosphomonoesterase. At 10(-7) M Ca2+ these two enzymes displayed comparable activity when assayed at concentrations of Ins-1,4,5-P3 occurring in stimulated cells; however, at 10(-5) M Ca2+, kinase activity predominates. These results suggest that Ins-1,4,5-P3 counter-regulates its own levels through the activity of inositol 1,4,5-trisphosphate 3-kinase and that the increase in [Ca2+]i may account for the transience of the rise in Ins-1,4,5-P3 seen during muscarinic stimulation of RINm5F cells.     

Ca2+-mediated generation of inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets. Studies with K+, glucose, and carbamylcholine

The role of Ca2+ in the generation of inositol phosphates was investigated using rat pancreatic islets after steady state labeling with myo-[2-3H]inositol. Depolarizing K+ concentrations (24 mM) evoked early (2 s) increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) as measured by high performance anion-exchange chromatography. The increase in Ins-1,4,5-P3 was transient and was followed by a more pronounced rise in Ins-1,3,4-P3. These effects were dependent on the presence of extracellular Ca2+ but were not secondary to release of either neurotransmitters or metabolites of arachidonic acid. K+ also promoted the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) and of the other phosphoinositides. Glucose (16.7 mM) was less marked in its effects but still promoted rapid increases in Ins-1,3,4,5-P4 (2 s) and Ins-1,4,5-P3 (10 s) and a slower rise in Ins-1,3,4-P3 (30 s). The levels of all three metabolites rose steadily over 10 min stimulation. These responses to glucose could be largely, although not entirely, inhibited by depletion of extracellular Ca2+ or by Ca2+ channel blockade with verapamil (20 microM). Carbamylcholine (0.5 mM) was the most potent stimulus used evoking early rises in Ins-1,4,5-P3 and Ins-1,3,4,5-P4 (2 s) followed by Ins-1,3,4-P3 (10 s), effects which were only partially dependent on extracellular Ca2+. The results suggest that a Ca2+-mediated PtdIns-4,5-P2 hydrolysis accounts for most of the Ins-1,4,5-P3 generated in response to glucose but not carbamylcholine. In addition, glucose may exert effects on inositol phosphate metabolism which are Ca2+ independent.     

Biochemical studies on the mechanism of difference in the renal toxicity of 5-hydroxy-L-tryptophan between Sprague Dawley and Wistar rats.

The biochemical mechanisms of the renal toxicity of 5-hydroxy-L-tryptophan to rats were studied using Wistar and Sprague Dawley rats, which had different LD50 values. When the amino acid was injected intraperitoneally, Wistar rats, which had a low LD50 value of 5-hydroxy-L-tryptophan, excreted larger amonts of serotonin and smaller amounts of 5-hydroxyindole acetic acid into the urine than Spraque Dawley rats, which had a high LD50 value. The activity of renal aromatic L-amino acid decarboxylase was higher in Wistar rats than in Sprague Dawley rats, while the activity of renal aromatic amino acid transaminase was in an opposite relationship. The activity of renal monoamine oxidase was almost the same in both strains and the activity of renal UDP glucuronyltransferase in Wistar rats was higher than in Sprague Dawley rats. Since the renal damage caused in rats by 5-hydroxy-L-tryptophan was very similar to that caused by serotonin, the amine formed from the administered amino acid was thought to be an important factor for the renal necroses, and difference in serotonin formation from the administered precursor amino acid may be one of the important factors leading to the difference in LD50 values in the two strains of rats.     

Calcium-calmodulin stimulates inositol 1,4,5-trisphosphate kinase activity from insulin-secreting RINm5F cells

In a cytosolic fraction derived from insulin-secreting RINm5F cells, the rate of conversion of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) was half-maximally stimulated by 0.8 microM Ca2+ (Biden, T. J., and Wollheim, C. B. (1986) J. Biol. Chem. 261, 11931-11934). In the present study we show that after initial purification by anion exchange chromatography, the Ins-1,4,5-P3 kinase activity responsible for that conversion is stimulated by Ca2+-calmodulin, but not by Ca2+ alone. This is almost certainly due to a specific interaction of the enzyme and its activator since kinase activity was retained on a calmodulin-linked Sepharose 6B column in the presence of Ca2+ but eluted upon chelation of the cation. After this two-step purification, Ins-1,4,5-P3 kinase activity was maximally stimulated 5-fold by 10 microM calmodulin in the presence of 10(-5) M Ca2+, and 2 1/2-fold at 10(-6) M Ca2+. Under these conditions the minimum concentrations of calmodulin needed to stimulate activity were in the 10-50 nM range. At 10(-7) M Ca2+, calmodulin (up to 30 microM) was without effect. Stimulated Ins-1,4,5-P3 kinase activity was inhibited in a dose-dependent fashion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) although the calmodulin antagonist had no effect on the residual activity seen at 10(-7) M Ca2+. These results strongly support our previous suggestion that alterations in cytosolic free Ca2+ concentrations play an important role in regulating the levels of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 during cellular stimulation.     

Epidermal growth factor (EGF) stimulates inositol trisphosphate formation in cells which overexpress the EGF receptor

EGF is a low molecular weight polypeptide hormone which acts as a regulator of cell growth and differentiation. The A-431 cell line has been used frequently to examine receptor-mediated biochemical effects of EGF, since this cell line has an increased (20-50 fold) level of EGF receptors. We have utilized A-431 cells to examine the influence of EGF on formation of an intracellular second messenger, inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3), and other inositol phosphates. The results show that EGF induces rapid formation of Ins-1,4,5-P3 as well as Ins-1,3,4-P3 and Ins-1,3,4,5-P4. There is a concurrent decrease in the level of the lipid precursor for Ins-1,4,5-P3, phosphatidylinositol 4,5-biphosphate (PIP2). Furthermore, we have examined five other cell lines that overexpress the EGF receptor and find that EGF treatment induces formation of inositol polyphosphates in those cell lines also.     

Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, we describe the partial purification of the 3-kinase [approximately 400-fold purified, specific activity = 0.12 mumol/(min.mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, we propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, an intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [gamma-32P]ATP by thin-layer chromatography. Using this new assay method, we evaluated kinetic parameters (Km for ATP = 40 microM, Km for Ins-1,4,5-P3 = 0.7 microM, Ki for ADP = 12 microM) and divalent cation specificity (Mg2+ much greater than Mn2+ greater than Ca2+) for the 3-kinase. Studies with various inositol polyphosphates indicate that the substrate-binding site is quite specific to Ins-1,4,5-P3. Nevertheless, Ins-2,4,5-P3 could be phosphorylated at a velocity approximately 1/20-1/30 that of Ins-1,4,5-P3.     

Metabolism of inositol-1,3,4,6-tetrakisphosphate to inositol pentakisphosphate in adrenal glomerulosa cells

Angiotensin II stimulates rapid formation of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) in bovine adrenal glomerulosa cells. In addition to being rapidly metabolized to lower inositol phosphates, Ins-1,4,5-P3 is converted to Ins-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and Ins-1,3,4-P3 which is in turn phosphorylated to a further Ins-P4 isomer, namely Ins-1,3,4,6-P4. In bovine adrenocortical cytosol [3H]Ins-1,3,4,5-P4 and [3H]Ins-1,3,4-P3 were converted to Ins-1,3,4,6-P4 and inositol pentakisphosphate (Ins-P5) in a metabolic sequence suggesting that unlike Ins-1,3,4,5-P4, Ins-1,3,4,6-P4 is a direct precursor of Ins-P5. Consistent with this assumption, [3H]Ins-1,3,4,6-P4 was converted to Ins-P5 in electropermeabilized adrenal glomerulosa cells. These findings demonstrate that Ins-1,3,4,6-P4 is an intermediate link between InsP3 metabolism and the higher inositol phosphates detected in several tissues.     

Orexin-Immunopositive Structures in the Thalamic Reticular Nucleus

Journal of Evolutionary Biochemistry and Physiology - Orexin-A and orexin-B were immunolocalized in laboratory rodents (adult Wistar and Sprague–Dawley rats, C57Bl/6J mice) in neurons of...     

Hippocampal 8-[3H]Hydroxy-2-(Di-n-Propylamino)Tetralin Binding Site Densities, Serotonin Receptor (5-HT1A) Messenger Ribonucleic Acid Abundance, and Serotonin Levels Parallel the Activity of the Hypothalamopituitary-Adrenal Axis in Rat

We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared with the relatively resistant Fischer F344/N rat, is related to a hyporesponsive hypothalamopituitary-adrenal axis to inflammatory and other stress mediators. Because serotonin (5-HT) and the 5-HT1A receptor are important stimulators of this axis, we have investigated the levels of 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites, 5-HT1A mRNA, 5-HT, and 5-hydroxyindoleacetic acid in various brain regions of Lewis, outbred Harlan Sprague Dawley, and Fischer F344/N rats. Lewis rats expressed significantly fewer hippocampal and frontal cortical 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites and less 5-HT1A mRNA than Harlan Sprague Dawley and Fischer F344/N rats. Adrenalectomy increased the number of 8-[3H]hydroxy-2,3-(di-n-propylamino)tetralin binding sites and 5-HT1A mRNA expression in the hippocampus of all three strains. Levels of hippocampal 5-HT in Fischer F344/N rats were significantly greater than levels detected in the same regions from Lewis and Harlan Sprague Dawley rats. Hypothalamic 5-HT and 5-hydroxyindoleacetic acid levels in Harlan Sprague Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5-hydroxyindoleacetic acid in the hypothalamus of all three strains. We conclude that hippocampal 5-HT1A receptor densities and 5-HT levels in the rat parallel the activity and responsiveness of the hypothalamopituitary-adrenal axis.     

Lithium-induced decrease of brain inositol and increase of brain inositol-1-phosphate is transient

The effects of a single does of LiCl (2.5 or 10 mEq/kg) on brain inositol and inositol-1-phosphate (Ins1P), intermediates of brain phosphoinositude (PI) turnover, were determinated in male Han: Wistar rats. There was a remarkable, 36–58 fold elevation of brain Li⁺ as the single does of LiCl was increased 4-fold. Moreover, the accumulation of brain lithium was slow during repeated administration of LiCl. Brain lithium did not correlate with changes in brain PI turnover either after a single or repeated doses. Thus, after a single does of LiCl the increases in brain Ins1P were much less than the decreases in brain inositol. Also, brain inositol was significantly decreased only with the high dose of LiCl whereas brain Ins1P accumulation was more prominent with the lower dose. Moreover, repeated daily doses of LiCl only transiently increased brain Ins1P at 1 and 7 d whereas inositol remained at control levels throughout the 14 d observation period. Lithium probably caused the transient decrease in brain inositol by inhibiting several enzymes, in addition to the inhibition of myo-inositol mono-phosphates, in the PI cycle. Moreover, a slow dampening down of PI turnover by lithium, possible via an inhibitory action on G-protein-coupling, may also explain the present findings.     

Comparison of the urinary excretion time courses of pyrene-1,6-dione,pyrene-1,8-dione and 1-hydroxypyrene in rats intravenously exposed to pyrene

Abstract

The urinary excretion time courses of pyrene-1,6-dione (P16D), pyrene-1,8-dione (P18D) and 1-hydroxypyrene (1-OHP) were compared in Sprague–Dawley and Wistar rats. Groups of five male rats, of about 200 g of body weight, were injected intravenously with 0.05, 0.5, 5 and 50 µmol pyrene kg^?1 of body weight. Urine was collected at 2, 4, 6, 8, 10, 12, 18, 24, 30, 42 and 48 h post-dosing. Pyrene metabolites were measured by high-performance liquid chromatography (HPLC)/fluorescence after enzymatic hydrolysis of the glucurono- and sulfo-conjugates, extraction on Sep-Pak C18 cartridges and, for the analysis of dione metabolites, derivatization to stable diacetoxypyrene molecules. Over the 48-h sampling period, on average 17.4–25.6% of the injected pyrene was excreted overall as P16D, 6.4–8.8% as P18D and 0.6–0.8% as 1-OHP in the urine of Sprague–Dawley rats. By comparison, on average 10.3–14.7% of the intravenous pyrene dose was recovered as P16D, 4.8–6.4% as P18D and 0.3–0.4% as 1-OHP in the urine of Wistar rats. In both strains of rats there was no clear effect of the dose on the 0–48-h cumulative urinary excretion of P18D and 1-OHP over the entire dose range, while the percentage of dose recovered overall as P16D in urine at the highest dose (50 µmol kg^?1) was statistically lower than at the other doses. The 0–48-h cumulative percentage of pyrene dose excreted as metabolites in the urine of Sprague–Dawley rats was also significantly higher than in Wistar rats (p<0.01) exposed under identical conditions. As for the urinary excretion-time courses of the different metabolites, for a given dose and strain of rats, excretion curves of P16D, P18D and 1-OHP generally evolved in parallel. There was also no clear effect of the dose on the excretion rate, thus half-life, of pyrene metabolites, except for P16D in Sprague–Dawley rats at the highest dose where elimination tended to be slower compared with the other doses (p<0.01). The average first-order elimination half-life of P16D, P18D and 1-OHP was 4.0, 5.7 and 4.1 h, respectively, in Sprague–Dawley rats, and 5.1, 6.1 and 5.1 h, respectively, in Wistar rats (all doses combined but excluding the highest dose for P16D). This study showed the relative importance of metabolic pathways leading to diones compared with 1-OHP. These dioxygenated metabolites appear to be interesting biomarkers of pyrene exposure at environmentally and occupationally relevant doses. Their adequacy as biomarkers of human exposure has yet to be confirmed.     

Inositol polyphosphate production and regulation of cytosolic calcium during the biphasic activation of adrenal glomerulosa cells by angiotensin II

Stimulation of aldosterone production by angiotensin II in the adrenal glomerulosa cell is mediated by increased phosphoinositide turnover and elevation of intracellular Ca2+ concentration. In cultured bovine glomerulosa cells, angiotensin II caused rapid increases in inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) levels and cytosolic Ca2+ during the first minute of stimulation, when both responses peaked between 5 and 10 s and subsequently declined to above-baseline levels. In addition to this temporal correlation, the dose-response relationships of the angiotensin-induced peak increases in cytosolic Ca2+ concentrations and Ins-1,4,5-P3 levels measured at 10 s were closely similar. However, at later times (greater than 1 min) there was a secondary elevation of Ins-1,4,5-P3, paralleled by increased formation of inositol 1,3,4,5-tetrakisphosphate that was associated with cytosolic Ca2+ concentrations only slightly above the resting value. These results are consistent with the primary role of Ins-1,4,5-P3 in calcium mobilization during activation of the glomerulosa cell by angiotensin II. They also suggest that Ins-1,4,5-P3 participates in the later phase of the target-cell response, possibly by acting alone or in conjunction with its phosphorylated metabolites to promote calcium entry and elevation of cytosolic Ca2+ during the sustained phase of aldosterone secretion.     

Effects of Chronic Nitric Oxide Inhibition on the Renal Excretory Response to Leptin

Objective: Previous investigations have demonstrated that leptin promotes natriuresis with a renal tubular effect. However, the mechanisms involved in this response are unclear. The present study was designed to examine the hypothesis that the natriuretic response to leptin in normotensive Sprague‐Dawley rats is regulated by nitric oxide (NO). Research Methods and Procedures: The hemodynamic and renal excretory effects of intravenous bolus administration of pharmacological doses of synthetic murine leptin were examined in groups of control Sprague‐Dawley rats (n = 8), Sprague‐Dawley rats treated for 4 days with the NO synthase inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME) (n = 8), and Sprague‐Dawley rats treated for 4 days with l‐NAME followed by acute treatment with sodium nitroprusside (n = 8). Results: In the control group (n = 8), an intravenous bolus of leptin, 400 μg/kg body weight, increased urinary sodium excretion 4‐ to 6‐fold. In the Sprague‐Dawley rats chronically administered l‐NAME (n = 8), an intravenous bolus of 400 μg/kg of leptin did not increase sodium excretion. Acute sodium nitroprusside infusion to Sprague‐Dawley rats chronically treated with l‐NAME (n = 8) was associated with partial restoration of the sodium excretory response to leptin administration. Discussion: Collectively, these results are interpreted to suggest that the natriuretic and diuretic responses to leptin observed in the Sprague‐Dawley rat require a functional NO system.     

Does the inositol tris/tetrakisphosphate pathway exist in rat heart?

Appearance of two isomers of inositol trisphosphate (InsP3) was observed when [3H]inositol prelabelled rat heart ventricles were stimulated for 10 and 30 s with noradrenaline. In contrast, inositol tetrakisphosphate (InsP4) could not be detected. However the existence of the inositol tris/tetrakisphosphate pathway was demonstrated by studying [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) metabolism in a soluble fraction of rat heart. There, [3H]Ins-1,4,5-P3 was phosphorylated to form [3H]Ins-1,3,4,5-P4. Raising [Ca2+] from 1 nM to 1 microM increased InsP3 kinase activity by 2-fold (EC50 for Ca2+ approx. 56 nM). This effect appeared to be due to an increase of the apparent Vmax of the enzyme while the apparent Km was unchanged.