Thromboxane A2 increases endothelial permeability through upregulation of interleukin-8

Thromboxane A₂ (TXA₂), a major prostanoid formed from prostaglandin H₂ by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA₂ mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-κB (NF-κB). U46619 induced the activation of NF-κB through IκB kinase (IKK) activation, IκB phosphorylation and NF-κB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-κB activation in endothelial cells.     

Thromboxane A2 stimulates mitogen activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A₂ mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of ³²P to a specific substrate for MAPK. U46619 also liberated [³H)arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [³H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca²⁺ ions. This is a first demonstration that TXA₂ activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

MeJA诱导的拟南芥保卫细胞H2O2积累与质膜H+-ATPase的关系

茉莉酸类物质(JAs)作为与昆虫啃噬及损伤相关的植物激素和信号分子在植物防御反应中起重要作用,但是茉莉酸引起的早期防御反应的机理仍不清楚。该研究以拟南芥叶片保卫细胞为材料,结合非损伤微测(NMT)及激光共聚焦技术探讨了茉莉酸诱导的保卫细胞中质膜H+-ATPase与H2O2积累的调控关系。结果表明:茉莉酸甲酯(MeJA)处理导致H+迅速跨膜外排和H2O2积累,H+外排和H2O2积累能够被钒酸钠抑制,而二苯基碘(DPI)处理则对MeJA诱导的H+跨膜外排无显著影响。研究结果证明,在MeJA诱导的早期信号事件中,质膜H+-ATPase的激活先于H2O2的产生。     

Role of phospholemman and the 70 kDa inhibitor protein in regulating Na/K ATPase activity in pulmonary artery smooth muscle cells under U46619 stimulation

Treatment of bovine pulmonary smooth muscle cells with U46619 inhibited the Na⁺/K⁺ ATPase activity in two parallel pathways: one of which is mediated via glutathionylation of the pump and the other by augmenting the inhibitory activity of the 70 kDa inhibitor protein of Na⁺/K⁺ ATPase. Although phospholemman deglutathionylates the pump leading to its activation, the inhibitor is responsible for irreversible inhibition of Na⁺/K⁺ ATPase in an isoform specific manner during treatment of the cells with U46619.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.     

Mechanism of the gas phase reactions of the C5H5Ni and C5H5Co ions with substituted pyridines. A combined experimental and theoretical study

Different products have been observed in the reactions of C₅H₅Co⁺ and C₅H₅Ni⁺ ions with halogen-substituted pyridines (XPy) that have been studied by ion trap mass spectrometry (ITMS) techniques. In particular, an addition product C₅H₅M(XPy)⁺ and a product ion C₅H₄M(Py)⁺ corresponding to a loss of a HX molecule (X = F, Cl, Br) have been detected. The relative yield of these products is determined by the nature of the metal and by the nature and position of the halogen on the pyridine ring. A computational study at the DFT level on model-systems formed by 2-fluoro and 2-bromopyridine reacting either with the C₅H₅Ni⁺ or the C₅H₅Co⁺ ion has been carried out. This study shows the existence of a general mechanistic pattern. The rate-determining step of this mechanism is the migration of the halogen from the pyridine ring to the metal. A final hydrogen abstraction step carried out by the halogen leads to the expulsion of a HX molecule. The existence of avoided crossings between surfaces of different multiplicities (ground and first excited state) allows the system to follow lower energy reaction pathways. The barrier determined for the reactions involving 2-bromopyridine is significantly lower than that found for 2-fluoropyridine. This is mainly due to the poor migrating/leaving character and low polarizability of fluorine compared to that of bromine.     

The association of thromboxane A2 receptor with lipid rafts is a determinant for platelet functional responses

We have investigated the presence of thromboxane A₂ (TXA₂) receptor associated with lipid rafts in human platelets and the regulation of platelet function in response to TXA₂ receptor agonists when lipid rafts are disrupted by cholesterol extraction. Platelet aggregation with TXA₂ analogs U46619 and IBOP was almost blunted in cholesterol-depleted platelets, as well as α_IIbβ₃ integrin activation and P-selectin exposure. Raft disruption also inhibited TXA₂-induced cytosolic calcium increase and nucleotide release, ruling out an implication of P2Y₁₂ receptor. An important proportion of TXA₂ receptor (40%) was colocalized at lipid rafts. The presence of the TXA₂ receptor associated with lipid rafts in platelets is important for functional platelet responses to TXA₂.     

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10^-6 mol·l^-1) induces a 30- to 40-fold activation of Na⁺/H⁺ exchange (the ethylisopropylamiloride-inhibited component of the ²²Na influx) and a fourfold stimulation of the Na⁺, K⁺ pump (ouabain-inhibited component of ⁸⁶Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (-phorbol 12-myristate, 13-acetate) increases Na⁺/H⁺ exchange by 10 times and decreases the Na⁺, K⁺ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na⁺, K⁺ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na⁺/H⁺ exchange-independent mechanism of the Na⁺, K⁺ pump regulation by -adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na⁺/H⁺ exchange, whereas hypotonic swelling induces an increase in the rate of ⁸⁶Rb⁺ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH₀ recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na⁺/H⁺ exchange, Na⁺, K⁺ pump and a non-identified system providing for K⁺ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.Abbreviations cAMP cyclic adenosine monophosphate - CCCP carbonylcyamide m-chlorophenylhydrazone - DMSO dimethylsulphoxide - EIPA ethylisopropylamiloride - NA noradrenaline - PMA -phorbol 12-myristate, 13-acetate - RVD regulatory volume decrease - RVI regulatory volume increase     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

Na+/H+ exchange in ehrlich ascites tumor cells: Activation by cytoplasmic acidification and by treatment with cupric sulphate

Summary Exposure of Ehrlich cells to isotonic Na⁺-propionate medium induces a rapid cell swelling. This treatment is likely to impose an acid load on the cells. Cell swelling is absent in K⁺-propionate medium but may be induced by the ionophore nigericin, which mediates K⁺/H⁺ exchange. Cell swelling in Na⁺-propionate medium is blocked by amiloride, but an alternative pathway is introduced by addition of the ionophore monensin, which mediates Na⁺/H⁺ exchange. Consequently, swelling of Ehrlich cells in Na⁺-propionate medium is due to the operation of an amiloride-sensitive, Na⁺-specific mechanism. It is concluded that this mechanism is a Na⁺/H⁺ exchange system, activated by cytoplasmic acidification. We have previously demonstrated that the heavy metal salt CuSO₄ in micromolar concentrations inhibits regulatory volume decrease (RVD) of Ehrlich cells following hypotonic swelling. The present work shows that CuSO₄ inhibits RVD as a result of a net uptake of sodium, of which the major part is sensitive to amiloride. Measurements of intracellular pH show that CuSO₄ causes significant cytoplasmic alkalinization, which is abolished by amiloride. Concomitantly, CuSO₄ causes an amiloride-sensitive net proton efflux from the cells. The combined results confirm that a Na⁺/H⁺ exchange system exists in Ehrlich cells and demonstrate that the heavy metal salt CuSO₄ activates this Na⁺/H⁺ exchange system.     

Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na/Ca exchanger

Phosphatidylinositol biphosphate (PtdIns-4,5P₂) plays a key role in the regulation of the mammalian heart Na⁺/Ca²⁺ exchanger (NCX1) by protecting the intracellular Ca²⁺ regulatory site against H⁺_i and (H⁺_i + Na⁺_i) synergic inhibition. MgATP and MgATP-γ-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P₂ incubated in the absence of MgATP and at normal [Na⁺]_i, alkalinization increases the affinity for Ca²⁺_i to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca²⁺_i any further. On the other hand, prevention of Na⁺_i inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P₂. Experiments on NCX1–PtdIns-4,5P₂ cross-coimmunoprecipitation show that the relevant PtdIns-4,5P₂ is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca²⁺_i regulatory site is seen in the deprotonated and PtdIns-4,5P₂-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

Binding of a thromboxane A2/prostaglandin H2 agonist [H]U46619 to washed human platelets

The binding characteristics of [³H]U46619 to washed human platelets were studied. [³H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a K_d of 11 ± 4 nM (n=4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a K_d of 20 ± 7nM and a B_max of 9.1 ± 2.3 fmole/10⁷ platelets (550 ± 141 binding sites per platelet) (n=4). A number of compounds that act as either agonists or antagonists of the TXA₂/PGH₂ receptor were tested for their ability to inhibit the binding of [³H]U46619 to washed human platelets. The K_ds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [³H]U46619 binds to the putative human platelet TXA₂/PGH₂ receptor.     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

Mechanism and significance of P4 ATPase-catalyzed lipid transport: Lessons from a Na/K-pump

Members of the P₄ subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanism that appears to be conserved throughout the family. A challenging problem is to understand how this mechanism is adapted in P₄ ATPases to flip phospholipids. P₄ ATPases form oligomeric complexes with members of the CDC50 protein family. While formation of these complexes is required for P₄ ATPase export from the endoplasmic reticulum, little is known about the functional role of the CDC50 subunits. The Na⁺/K⁺-ATPase and closely-related H⁺/K⁺-ATPase are the only other P-type pumps that are oligomeric, comprising mandatory β-subunits that are strikingly reminiscent of CDC50 proteins. Besides serving a role in the functional maturation of the catalytic α-subunit, the β-subunit also contributes specifically to intrinsic transport properties of the Na⁺/K⁺ pump. As β-subunits and CDC50 proteins likely adopted similar structures to accomplish analogous tasks, current knowledge of the Na⁺/K⁺-ATPase provides a useful guide for understanding the inner workings of the P₄ ATPase class of lipid pumps.     

Na+/H+-exchange in A6 cells: Polarity and vasopressin regulation

Summary We have analyzed the mechanism of Na⁺-dependent pH_i; recovery from an acid load in A6 cells (an amphibian distal nephron cell line) by using the intracellular pH indicator 27-bis(2-carboxyethyl)5, 6 carboxyfluorescein (BCECF) and single cell microspectrofluorometry. A6 cells were found to express Na⁺/H⁺-exchange activity only on the basolateral membrane: Na⁺/H⁺-exchange activity follows simple saturation kinetics with an apparent K _mfor Na⁺ of approximately 11 mm; it is inhibited in a competitive manner by ethylisopropylamiloride (EIPA). This Na⁺/H⁺-exchange activity is inhibited by pharmacological activation of protein kinase A (PKA) as well as of protein kinase C (PKC). Addition of arginine vasopressin (AVP) either at low (subnanomolar) or at high (micromolar) concentrations inhibits Na⁺/H⁺-exchange activity; AVP stimulates IP₃ production at low concentrations, whereas much higher concentrations are required to stimualte cAMP formation. These findings suggest that in A6 cells (i) Na⁺/H⁺-exchange is located in the basolateral membrane and (ii) PKC activation (heralded by IP₃ turnover) is likely to be the mediator of AVP action at low AVP concentrations.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation, and the Geigy Jubiläumsstiftung. Prof. Dr. V. Casavola and Dr. R. Guerra were supported by a research grant, No. 91.02470.CT14 of the Consiglio Nazionale della Ricerche (C.N.R.) We are grateful to Prof. Dr. B.C. Rossier of the Institute of Pharmacology of Lausanne (Switzerland) for the gift of the A6 cells, to H.P. Gaeggeler for the supply of the necessary culture media and to Jutka Forgo for her excellent help in the day-to-day culturing of the A6 cells. The secretarial assistance of D. Rossi is gratefully acknowledged.     

猪心线粒体Fo的纯化、重建及其质子转运功能

比较了猪心线粒体F_oF₁-ATPase膜部分F_o的四种纯化方法.结果表明,用NaBr从亚线粒体除去F_oF₁-ATPase的水溶性部分F₁-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的F_o.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的F_o含有b、OSCP（寡霉素敏感授予蛋白）、d、a、e、F₆、IF₁、A6L和c等9种亚基.用去污剂稀释法将纯化的F_o在脂质体上重建后,重建F_o表现较高的被动转运质子活性.这为在体外深入研究F_o的活性、构象与膜脂的关系,以及F_o与F₁-ATPase的组装等提供了很好的实验模型.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased