Change in soluble epoxide hydrolase (sEH) during cisplatin-induced acute renal failure in mice.

Cisplatin is one of the most effective chemotherapeutic agents against various types of cancers; however, it is also associated with nephrotoxicity. Recently, it was reported that inflammatory mechanisms play a key role in the development of nephrotoxicity. Epoxyeicosatrienoic acids (EETs) have an anti-inflammatory effect and are metabolized by soluble epoxide hydrolase (sEH: encoded by EPHX2 gene). Here, we determined the change in sEH activity and EPHX2 expression in renal tissue associated with the development of cisplatin-induced nephrotoxicity. Cisplatin administration decreased hydrolase activity accompanied by down-regulation of sEH and EPHX2 expression. The down-regulation occurred prior to the elevation of blood urea nitrogen (BUN) and tumor necrosis factor-α (TNF-α) gene expression or at treatment with low dose cisplatin. In addition, a negative correlation was found between EPHX2 expression and renal thiobarbituric acid reactive substance (TBARS), and edaravone, a radical scavenger, administration did not down-regulate expression of this gene. The results of this study suggest that cisplatin decreased sEH activity through the down-regulation of sEH and EPHX2 expression, and this down-regulation was involved in a negative feedback loop to protect renal tissue from further damage. Thus, sEH is a potential therapeutic target of cisplatin-induced nephrotoxicity.

Fluorometric determination of nitrite with 2,3-diaminonaphthalene by reverse phase HPLC under alkaline conditions.

INTRODUCTION: In this study, a simple and sensitive fluorometric HPLC method was described for the determination of nitrite, an indicator of nitric oxide production in biological samples. METHODS: Nitrite was reacted with 2,3-diaminonaphthalene (DAN) to form fluorescent 2,3-naphthotriazole (NAT). Because NAT has higher fluorescence intensity at alkaline conditions, a reverse phase HPLC method employing a polymer-based column was applied to separate NAT using an alkaline mobile phase system. RESULTS: NAT was well separated from DAN and other fluorescent substances with increased detection sensitivity under alkaline conditions. A linear relationship (r=0.999) between the fluorescence intensity of NAT and the concentration of nitrite was observed in the range from 0 to 800 nM with the detection limit of about 25 nM. DISCUSSION: The present HPLC method appears suitable for routine analysis of nitrite in crude biological samples, since the polymer-based column can withstand rigorous cleaning with either acid or base.

Nitrogen oxide air pollutants interfere with the measurement of nitric oxide using 2,3-diaminonaphthalene: reduction of background interference.

Nitrite, a stable metabolite of nitric oxide (NO), is measured fluorometrically as an indicator of NO production using 2,3-diaminonaphthalene. In cultured cells, it has been believed that a longer period of incubation improves the detection sensitivity because of the accumulation of nitrite formed from NO in culture media. However, here we show that nitrite formed from nitrogen oxide air pollutants accumulates continuously in culture media during the incubation and interferes with the measurement of NO as nitrite. Thus, a proper period of incubation is important to allow maximum nitrite signals from NO with minimum background nitrite from the air.

Marked increase in the histamine content of neointima after stent implantation of pig coronary artery and growth-promoting effects of histamine in cultured smooth muscle cells.

After coronary stent implantation, the unfavorable in-stent restenosis often occurs by the formation of neointima due to the proliferation of smooth muscle cells. Platelet-derived growth factor (PDGF) and other peptide growth factors contribute to this process, but little is known about the role of non-peptide factors in this process. In the present study, the role of histamine, a non-peptide factor, in the formation of neointima was investigated using a pig coronary model of in-stent restenosis and a culture system of coronary smooth muscle cells. A Palmaz-Schatz stent was implanted in the left anterior descending coronary artery of male pigs. At 1, 2 and 4 weeks after stenting, the histamine content of neointima was determined to be 326 +/- 82, 1427 +/- 280 and 440 +/- 69 pmol/mg protein, respectively, by HPLC fluorometry. In contrast, the histamine content of arterial media from the untreated control arteries was only 15.3 +/- 1.6 pmol/mg protein. These results demonstrate that the histamine content of neointima is about 20 to 90-fold that of the normal media. In vitro, histamine by itself did not stimulate the proliferation of cultured smooth muscle cells, but potentiated the PDGF-stimulated proliferation of the cultured cells via a mechanism independent of H1 and H2 histamine receptors. Thus, histamine may be an important non-peptide factor in the pathogenesis of in-stent restenosis.

Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin.

Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis. Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials. In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100 micrograms/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin. Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100 micrograms/ml heparin. HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100 micrograms/ml of HRG was co-added with 100 micrograms/ml heparin. Interestingly, micromolar concentrations of the zinc ion (0-10 microM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG. Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media. These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials.

Role of Rho-associated protein kinase and histamine in lysophosphatidic acid-induced airway hyperresponsiveness in guinea pigs.

Inhalation of oleoyl lysophosphatidic acid (LPA) induced airway hyperresponsiveness to acetylcholine (ACh). In contrast, palmitoyl and stearoyl LPA exerted minimal effects. Airway hyperresponsiveness was inhibited by inhalation of Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). Mepyramine, an H1 histamine receptor antagonist and ketotifen, an inhibitor of histamine release and H1 histamine receptor antagonist, also inhibited airway hyperresponsiveness induced by LPA; however, aspirin failed to attenuate this response. The incubation of lung fragments with LPA gave rise to releases in histamine. On the other hand, LPA produced no significant changes on the smooth muscle contraction evoked by ACh. These findings suggest that LPA-induced airway hyperresponsiveness is attributable to activation of the Rho/ROCK-mediated pathway via endothelial cell differentiation gene (EDG) receptors, probably EDG 7. Moreover, histamine release may be involved.