Effect of some analgesics on Paraoxonase-1 purified from human serum

The in vitro effects of the analgesic drugs, lornoxicam, indomethacin, tenoxicam, diclofenac sodium, ketoprofen and lincomycine, on the activity of purified human serum paraoxonase (hPON1) (EC 3.1.8.1.) were evaluated. hPON1 was purified from human serum with a final specific activity of 3840 U mg^?1 and a purity of 25.3 % using simple chromatographic methods, including DEAE-Sephadex anion exchange and Sepharose 4B-L-tyrozine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis indicated a single protein band corresponding to hPON1. The six analgesics dose-dependently decreased in vitro hPON1 activity, with IC₅₀ values for lornoxicam, indomethacin, tenoxicam, diclofenac sodium, ketoprofen and lincomycine of 0.136, 0.195, 0.340, 1.639, 6.23 and 9.638 mM, respectively. K_i constants were 0.009, 0.097, 0.306, 0.805, 13.010 and 11.116 mM, respectively. Analgesics showed different inhibition mechanisms: lornoxicam, diclofenac sodium and lincomycine were uncompetitive, indomethacin and tenoxicam were competitive, ketoprofen was noncompetitive. According to the results, inhibition potency was lornoxicam>indomethacin>tenoxicam> diclofenac sodium>ketoprofen> lincomycine.     

Agrobacterium-mediated transformation and assessment of factors influ-encing transgene expression in loblolly pine (Pinus taeda L.)

INTRODUCTIONRecombinant DNA technology is a powerful toolfor the introduction of foreign genes into longlivedperennials and fOr fundamelltal studies of gene expression. Using such techniques, we can overcomethe difficulties associated with the breeding of a long-lived perennial. At present, although considerablereseaxch effort has been devoted to the genetic en-gineering of fOrest trees, it has lagged behind ad-vances made in herbaceous crops due both to eco-nomics and the recalcitrant n…     

Lithium and Protein Kinase C Modulators Regulate Swelling-Activated K-Cl Cotransport and Reveal a Complete Phosphatidylinositol Cycle in Low K Sheep Erythrocytes

K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li _i ) but not external (Li _o ) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K _i of ∼7 mm. In Cl, Li _i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li _i reduced the V _max and increased the K _m for K efflux in Cl. Furthermore, Li _i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li _i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume. Received: 1 November 1999/Revised: 6 June 2000     

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Platelet-derived growth factor (PDGF), apotent serum mitogen for vascular smooth muscle cells (VSMCs), plays animportant role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, isinvolved in ion homeostasis. VSMCs possess K-Cl COT activity and theKCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-ClCOT activity and mRNA expression in primary cultures of rat VSMCs. K-ClCOT was determined as the Cl-dependent Rb influx and mRNA expression bysemiquantitative RT-PCR. Twenty four-hour serum deprivation inhibitedbasal K-Cl COT activity. Addition of PDGF increased total proteincontent and K-Cl COT activity in a time-dependent manner. PDGFactivated K-Cl COT in a dose-dependent manner, both acutely (10 min)and chronically (12 h). AG-1296, a selective inhibitor of the PDGFreceptor tyrosine kinase, abolished these effects. Actinomycin D andcycloheximide had no effect on the acute PDGF activation of K-Cl COT,suggesting posttranslational regulation by the drug. Furthermore, PDGFincreased KCC1 and decreased KCC3 mRNA expression in a time-dependentmanner. These results indicate that chronic activation of K-Cl COTactivity by PDGF may involve regulation of the two KCC mRNA isoforms,with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

     

KCl Cotransport Regulation and Protein Kinase G in Cultured Vascular Smooth Muscle Cells

K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.     

A likelihood method for the detection of selection and recombination using nucleotide sequences

Different regions along nucleotide sequences are often subject to different evolutionary forces. Recombination will result in regions having different evolutionary histories, while selection can cause regions to evolve at different rates. This paper presents a statistical method based on likelihood for detecting such processes by identifying the regions which do not fit with a single phylogenetic topology and nucleotide substitution process along the entire sequence. Subsequent reanalysis of these anomalous regions may then be possible. The method is tested using simulations, and its application is demonstrated using the primate psi eta-globin pseudogene, the V3 region of the envelope gene of HIV-1, and argF sequences from Neisseria bacteria. Reanalysis of anomalous regions is shown to reveal possible immune selection in HIV-1 and recombination in Neisseria. A computer program which implements the method is available.      

Alkaline pH and Internal Calcium Increase Na+ and K+ Effluxes in LK Sheep Red Blood Cells in Cl−-free Solutions

We examined the effects of pH, internal ionized Ca (Ca²⁺ _i ), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K⁺ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K⁺ phenotypes. In LK SRBCs the K⁺ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K⁺ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K⁺ efflux was activated (100%) when Ca²⁺ _i was increased (+A23187, +Ca²⁺ _o ) into the μm range. However, in comparison to human red blood cells, the Ca²⁺ _i -induced increase in K⁺ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na⁺ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca²⁺ _i . In contrast, in HK SRBCs the K⁺ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca²⁺ _i . These studies suggest the presence in LK SRBCs, of at least two pathways for Cl⁻-independent K⁺ and Na⁺ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca²⁺ _i . Received: 23 May 1996/Revised: 6 December 1996     

Effect of SH-group reagents on net water transport in frog urinary bladder

The basal rate of water reabsorption and its acceleration by oxytocin, cyclic AMP (cAMP) or serosal hypertonicity in frog urinary bladders were monitored before and after exposure of the mucosal surface to sulfhydryl (SH) reactive reagents. The following observations were made: 1. N-ethylmaleimide (NEM, 10(-5)M) did not modify the basal water flux, but did potentiate the hydrosmotic response to oxytocin. At higher NEM concentrations, an increase in the basal flux was observed, while the oxytocin-induced water flux was strongly inhibited, if not, nullified. 2. Iodoacetamide (IAM, 10(-3)M) did not modify the basal water flux but did inhibit the oxytocin-, cAMP-, and serosal hypertonicity-induced increase in water permeability. Furthermore, the time course of the hydrosmotic response to oxytocin was significantly increased. 3. 5,5' dithio-bis-(2-nitrobenzoic acid) (DTNB, 10(-3)M) modified neither the basal nor the oxytocin-induced water flux when incubated at pH 8.1, but potentiated the inhibitory effect of NEM. However, at a mucosal pH of 6.5, DTNB inhibited the response to oxytocin by 30%. These results suggest that: (1) the three SH reagents affect differently the basal and the oxytocin-induced water pathways; and that (2) each of the changes in the oxytocin-induced paths occurs at a step following the hormonally-induced increase in intracellular cAMP concentration.     

Cation transport in vascular endothelial cells and aging

Summary To understand the generation and maintenance of Na and K gradients in cultured vascular endothelial cells, net Na and K movements were studied. Ouabain-sensitive (OS) net Na gain and K loss were estimated as the difference between the cation content in the presence of ouabain and that in the control. Ouabain-and furosemide-resistant (OFR) fluxes were determined in the presence of the two inhibitors. When the normal medium bicarbonate and phosphate buffers were replaced by N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid both the OS ans OFR fluxes decreased more than 50%. Ouabain-sensitive and ouabain-and furosemide-resistant fluxes decreased with increasing cellular age (passage number) an effect not observed when the cation movements were studied in the absence of bicarbonate and phosphate. These results suggest that cultured vascular endothelial cells possess bicarbonate-and phosphate-dependent Na and K pathways which account for a significant portion of their passive movements. Furthermore, the behavior of cation permeabilities with passage number suggests that these modulations may be related to the cellular aging process.     

Quinine and quinidine inhibit and reveal heterogeneity of K-Cl cotransport in low K sheep erythrocytes

Low K (LK) sheep red blood cells (SRBCs) serve as a model to study K-Cl cotransport which plays an important role in cellular dehydration in human erythrocytes homozygous for hemoglobin S. Cinchona bark derivatives, such as quinine (Q) and quinidine (QD), are effectively used in the treatment of malaria. In the present study, we investigated in LK SRBCs, the effect of various concentrations of Q and QD on Cl-dependent K efflux and Rb influx (K(Rb)-Cl flux), activated by either swelling in hyposmotic media, thiol alkylation with N-ethylmaleimide (NEM), or by cellular Mg (Mg _i ) removal through A23187 in the presence of external chelators. K efflux or Rb influx were determined in Cl and NO₃ medium and K(Rb)-Cl flux was defined as the Cl-dependent (Cl minus NO₃) component. K(Rb)-Cl flux stimulated by all three interventions was inhibited by both Q and QD in a dose-dependent manner. Maximum inhibition of K(Rb)-Cl flux occurred at Q and QD concentrations ?1 mm. The inhibitory effect of Q was manifested in Cl, but not in NO₃, whereas QD reduced K and Rb fluxes both in Cl and NO₃ media. The mean 50% inhibitory concentration (IC⁵⁰) of Q and QD to inhibit K(Rb)-Cl flux varied between 0.23 and 2.24 mm. From determinations of the percentages of inhibition of the different components of K and Rb fluxes, we found that SRBCs possess a Cl-dependent QD-sensitive and a Cl-dependent QD-insensitive K efflux and Rb influx. These two components vary in magnitude depending on the manipulation and directional flux, but in average they are about 50% of the total Cl-dependent flux. This study raises the possibility that, in SRBCs, the Cl-dependent K(Rb) fluxes are heterogeneous. This work was supported by a grant from the National Institutes of Health (NIH DK5RO1 37,160).