Role of thromboxane A2 in early BDL-induced portal hypertension

Although the mechanisms of cirrhosis-induced portal hypertension have been studied extensively, the role of thromboxane A(2) (TXA(2)) in the development of portal hypertension has never been explicitly explored. In the present study, we sought to determine the role of TXA(2) in bile duct ligation (BDL)-induced portal hypertension in Sprague-Dawley rats. After 1 wk of BDL or sham operation, the liver was isolated and perfused with Krebs-Henseleit bicarbonate buffer at a constant flow rate. After 30 min of nonrecirculating perfusion, the buffer was recirculated in a total volume of 100 ml. The perfusate was sampled for the enzyme immunoassay of thromboxane B(2) (TXB(2)), the stable metabolite of TXA(2). Although recirculation of the buffer caused no significant change in sham-operated rats, it resulted in a marked increase in portal pressure in BDL rats. The increase in portal pressure was found concomitantly with a significant increase of TXB(2) in the perfusate (sham vs. BDL after 30 min of recirculating perfusion: 1,420 +/- 803 vs. 10,210 +/- 2,950 pg/ml; P < 0.05). Perfusion with a buffer containing indomethacin or gadolinium chloride for inhibition of cyclooxygenase (COX) or Kupffer cells, respectively, substantially blocked the recirculation-induced increases in both portal pressure and TXB(2) release in BDL group. Hepatic detection of COX gene expression by RT-PCR revealed that COX-2 but not COX-1 was upregulated following BDL, and this upregulation was confirmed at the protein level by Western blot analysis. In conclusion, these results clearly demonstrate that increased hepatic TXA(2) release into the portal circulation contributes to the increased portal resistance in BDL-induced liver injury, suggesting a role of TXA(2) in liver fibrosis-induced portal hypertension. Furthermore, the Kupffer cell is likely the source of increased TXA(2), which is associated with upregulation of the COX-2 enzyme.     

Purification and characterization of chitinase from Alcaligenes xylosoxydans

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecularmass of chitinase was estimated to be 45 kDa and44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 °C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l^–1. Cu²⁺ and Na⁺ at 5 mM inhibited chitinase activity to 25% while Ca²⁺, Mg²⁺ and Ba²⁺ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.     

Effects of peroxynitrite on pig coronary artery smooth muscle

We examined the effects of peroxynitrite pretreatment of pig coronary arteries on their sarcoplasmic reticulum (SR) Ca(2+) pump function. Pretreating rings from de-endothelialized arteries with peroxynitrite, followed by a wash to remove this agent, led to a decrease in the force of contraction produced in response to the SR Ca(2+) pump inhibitor cyclopiazonic acid (CPA, IC(50) = 87 +/- 6 microM). Inclusion of catalase and superoxide dismutase with the peroxynitrite did not alter its effect indicating that the inhibition was produced by peroxynitrite. Contractions produced by 30 mM KCl were not affected by up to 250 microM peroxynitrite. Smooth muscle cells cultured from this artery gave a transient increase in cytosolic Ca(2+) in response to CPA. Treating the cells with peroxynitrite inhibited this increase. Treating the SR-enriched isolated subcellular membrane fraction with peroxynitrite produced an inhibition of the ATP-dependent azide-insensitive oxalate-stimulated Ca(2+) uptake. Thus, peroxynitrite damages the SR Ca(2+)pump in the coronary artery, and this inhibition appears to lead to an inability of the arteries to respond to CPA. Thus, peroxynitrite produced from superoxide and NO in the arteries may compromise regulation of coronary tone which requires mobilization of Ca(2+) from the SR.     

Role of platelet-derived growth factor in vascular remodeling during pulmonary hypertension in the ovine fetus

Platelet-derived growth factor (PDGF) is a potent smooth muscle cell mitogen that may contribute to smooth muscle hyperplasia during the development of chronic pulmonary hypertension (PH). We studied changes in PDGFalpha- and beta-receptor and ligand expression in lambs with chronic intrauterine PH induced by partial ligation of the ductus arteriosus (DA) at gestational age 124-128 days (term = 147 days). Western blot analysis performed on whole lung homogenates from PH animals after 8 days of DA ligation showed a twofold increase in PDGFalpha- and beta-receptor proteins compared with age-matched controls (P < 0.05). Lung PDGF-A and -B mRNA expression did not differ between PH and control animals. We treated PH animals with NX1975, an aptamer that selectively inhibits PDGF-B, by infusion into the left pulmonary artery for 7 days after DA ligation. NX1975 reduced the development of muscular thickening of small pulmonary arteries by 47% (P < 0.05) and right ventricular hypertrophy (RVH) by 66% (P < 0.02). Lung PDGFalpha- and beta-receptor expression is increased in perinatal PH, and NX1975 reduces the increase in wall thickness of small pulmonary arteries and RVH in this model. We speculate that PDGF signaling contributes to structural vascular remodeling in perinatal PH and that selective PDGF inhibition may provide a novel therapeutic strategy for the treatment of chronic PH.     

Osmosis: a macroscopic phenomenon,a microscopic view

At present, physical chemistry employs the tools of thermodynamics to treat osmosis across a semipermeable membrane. We propose a model in terms of momentum transfer, the inherent asymmetry of which leads quantitatively to the van't Hoff relationship; qualitatively, the solute molecules can be looked upon as micropumps that suck solvent through the pores in the membrane.     

Multiplexing nuclear receptors for agonist identification in a cell-based reporter gene high-throughput screen

High-throughput screening (HTS) has become an essential part of the drug discovery process. Due to the rising requirements for both data quality and quantity, along with increased screening cost and the demand to shorten the time for lead identification, increasing throughput and cost-effectiveness has become a necessity in the hit identification process. The authors present a multiplexed HTS for 2 nuclear receptors, the farnesoid X-activated receptor and the peroxisome proliferator-activated receptor delta in a viable cell-based reporter gene assay. The 2 nuclear receptors were individually transfected into human hepatoma cells, and the transient transfected cell lines were pooled for the multiplexed screen. Hits identified by the multiplexed screen are similar to those identified by the individual receptor screens. Furthermore, the multiplexed screen provides selectivity information if ligands selective for one and not the other receptor are one of the hit criteria. The data demonstrate that multiplexing nuclear receptors can be a simple, efficient, cost-effective, and reliable alternative to traditional HTS of individual targets without compromising data quality.     

Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase

Measurement of adenosine deaminase (ADA) activity using spectrophotometric method presents problem, regarding the quantitative estimation of the substrate degradation and product formation, due to the closely apposed lambda(max) of the substrates, product and the inhibitor. The feasibility of applying reverse-phase HPLC technique, for studying adenosine deaminase-catalyzed reaction product and inhibition study was examined. We have drawn a comparison between the HPLC-based method over the corresponding spectrophotometric method. A gradient elution pattern was used to separate substrate (adenosine and deoxyadenosine), product (inosine and deoxyinosine) and standard adenosine deaminase inhibitor (erythro-9-(3-nonyl-p-aminobenzyl)-adenine) in the HPLC method. The product formation was quantitated by monitoring the absorbance at 260 nm with the progress of time. The limit of detection as well as the limit of quantification of the respective enzymatic product were found to be in nano molar (nM) range in the HPLC method. This study was also extended to monitor adenosine deaminase activity in different cancer cells of hematological origin. The HPLC-based method is found to be suitable for the quantitative estimation of adenosine deaminase-catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC-based method has specific advantages over the spectrophotometric method. Moreover, the concentration of different nucleotides in cell lysate and body fluid can be measured using this HPLC method.