Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO.

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.

Phorbol esters and chemotactic factor induce distinct changes in cytoplasmic Ca2+ and pH in granulocytic like HL60 cells.

Differentiation of HL60 cells into neutrophil-like cells after exposure to dimethylsulfoxide is accompanied by an increase in intracellular pH (pHi) which results from an increased activity of the Na+/H+ antiporter at physiological pHi value, but not at acidic pHi values. The functional responses of differentiated HL60 cells to the chemotactic peptide, N-formylmethionylleucylphenylalanine (fMLP), and to phorbol myristate acetate (PMA) were studied. In differentiated cells fMLP produced a large increase in cytosolic Ca2+ levels and a small biphasic change in pHi, whereas PMA produced cellular acidification, which was potentiated by ethylisopropylamiloride and no change in [Ca2+]i. In undifferentiated HL60 cells, PMA produced the opposite effect on pHi, i.e., a cellular alkalinization. The extent of the acidification produced by PMA in differentiated HL60 cells correlated with the production of reactive oxygen species.

Activation of cardiac endothelium as a compensatory component in endotoxin-induced cardiomyopathy: role of endothelin, prostaglandins, and nitric oxide.

BACKGROUND: In view of growing evidence of an important endothelial paracrine regulation of cardiac function, the present study investigated the role of cardiac endothelium-derived endothelin-1 (ET-1), prostaglandins, and nitric oxide (NO) during endotoxin-induced cardiomyopathy in rabbits. METHODS AND RESULTS: Immunohistochemical studies showed a marked transient coinduction of the inducible isoforms of NO synthase (NOS-2) and cyclooxygenase (COX-2) in endocardial endothelium and coronary arteriolar endothelium of hearts 12 hours after intravenous administration of lipopolysaccharide (LPS+12h); staining for both isoforms was much weaker 24 hours later (LPS+36h). Nitrotyrosine localization was similar to that of NOS-2, suggesting a NOS-2-related endothelial formation of peroxynitrite in septic hearts. Contractile performance of papillary muscles was depressed in both LPS-treated groups. In the LPS+12h group, however, isometric twitches were significantly prolonged (482+/-14 versus 420+/-14 ms in the saline-treated group, P<0.005). This twitch prolongation was completely reversed by simultaneous administration of BQ-123 and indomethacin to block endogenous ET-1 and prostaglandins, respectively. In addition, in the LPS+12h group, myocardial inotropic responsiveness to exogenous ET-1 was enhanced (P<0.01). CONCLUSIONS: Cardiac endothelial activation and myocardial sensitization to endothelium-derived mediators may be part of an adaptive response in the early (12 hours) stages of septic cardiomyopathy.

Why are circulating concentrations of endothelin-1 so low?

Physiological and pathophysiological roles of endothelins are still unclear. One reason is that circulating endothelin levels in normal and pathological states are much lower than the concentrations necessary to elicit contractions in vitro. It is usually assumed that endothelin accumulates in diseased tissues and that, because of its degradation, only a small fraction of it reaches the systemic circulation. Such a hypothesis does not fit with recent observations showing that low circulating endothelin levels may be active. We show here that most of the current inferences about the actions of endothelin assume that the peptide acts in the vessel wall under conditions known as non-stoichiometric binding conditions, that is, under conditions in which the receptor concentration in tissues ([Ro]) is smaller than the equilibrium dissociation constant of endothelin receptor complexes (Kd). Under stoichiometric binding conditions (defined by the condition [Ro] > Kd), most ligand molecules are bound to receptors and cannot be present in a free form. Estimates of [Ro] and Kd from the literature suggests that in vivo endothelin probably binds stoichiometrically to its receptors. Under this condition, most of tissue endothelin is probably bound to receptors. It is therefore suggested that plasma endothelin levels are low probably because tissue free endothelin levels are low, and this is not inconsistent with the presence of high tissue levels of active (that is, bound) endothelin. When the topology of the vessels with respect to the site of production (or of delivery) of endothelin is considered, stoichiometric binding may also account for the higher sensitivity to Et-1 of in vivo preparations. It also suggests that autocrine and paracrine actions of Et-1 are favoured at low and high secretory rates respectively, thus providing an explanation for the dual (vasodilator and vasoconstricting) actions of endothelin. Finally, the stoichiometric binding model predicts that functional receptors also act as clearance receptors and provides an explanation for the observation that antagonists of endothelin receptors are also clearance antagonists.

Evidence for receptor bound endothelin in renal but not in cardiac tissues from normal rats.

OBJECTIVES: The stoichiometric binding model (Frelin C, Guedin D. Cardiovasc Res 1994;28:1613-1622) implies that most endothelin in tissues is bound onto receptors rather than in a free state. The objective of this study was to assay receptor bound endothelins in normal rat tissues. METHODS: We first defined acidic conditions that promoted a mild and reversible denaturation of ETA and ETB receptors and that allowed dissociation of bound [125I]endothelin-1. The action of an acid wash on [125I]endothelin-1 binding to cell or tissue homogenates was then investigated. RESULTS: Acid washing of homogenates prepared from rat brain capillary endothelial cells that express prepro endothelin-1 mRNAs unmasked receptor sites. Acid washing of cardiac, lung, brain or liver homogenates did not increase [125I]endothelin-1 binding. An acid wash of kidney homogenates increased 2.2 fold [125I]endothelin-1 binding. Experiments using BQ-123 further indicated that the acid treatment of renal homogenates mainly unmasked ETA receptors. Masked renal ET receptors were mainly localized in the medulla. Treatment of rats with phosphoramidon decreased the density of masked ET receptors in kidney homogenates. CONCLUSION: As much as 50% of endothelin receptors in renal tissues are masked by endogenous endothelins. Most cardiac receptors are free of bound endothelins. These suggest that endothelins act as local rather than systemic mediators.

Cardiac capillary cells release biologically active nitric oxide at an early stage of in vitro development.

OBJECTIVE: Coronary microvascular endothelial cells (EC) may regulate the myocardial contractile function by releasing cardioactive agents such as nitric oxide (NO). However, understanding of these regulatory mechanisms is complicated by the fact that EC exhibit marked phenotypic changes, such as the loss of endothelial NO synthase (eNOS), when they are placed into culture. Recently, it has been shown that eNOS gene expression is regulated by specific cell-cell interactions with mural cells depending on vascular beds. Since EC and pericytes (PL) are closely associated in capillaries, we have enzymatically isolated these cells from rat hearts to develop a primary culture of capillary cells favoring the re-establishment of cell interactions in vitro. METHODS: Expression of transcripts for both eNOS and the inducible isoform (iNOS), was evaluated by using reverse transcription, polymerase chain reaction and Southern blot analysis. Expression of NOS proteins was detected with specific rhodamine-labeled antibodies. Production of NO was assessed (i) from nitrite measurements in culture supernatants by the Griess reaction, and (ii) from its antiproliferative action on cardiac fibroblasts (FIB) in non-contacted cocultures (reporter-cell bioassay) compared to that of sodium nitroprusside in homotypic FIB cultures. Fura-2 fluorescence was used to measure agonist-induced changes in cytosolic free calcium levels. RESULTS: In our heterotypic cultures, EC firstly proliferated to form spots of monolayers (i.e. first phase) before to be covered by PL on the following days (i.e. second phase). The data from RT-PCR analysis demonstrate the presence of mRNAs of both eNOS and iNOS at all developmental stages of the culture. However, eNOS protein was only detected and restricted to EC. During the first phase of cell growth (5-8 days), cells released nitrite and a labile factor, clearly identified as NO, that inhibited the FIB proliferation in reporter-cell bioassay. These effects, not observed during the second phase of cell growth (15-20 days), were prevented by hemoglobin (50 microM) and by N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM). At the two periods of culture, EC increased rapidly their cytosolic Ca(2+) concentration in response to bradykinin (10 nM). However, this calcium response was associated with an increase in nitrite production only in older cultures. CONCLUSIONS: Our data indicate that heterotypic cultures of native capillary cells preserve the eNOS expression by EC. This enzyme is basally active at an early stage of in vitro development, and then becomes activatable by a Ca(2+)-mobilizing agonist. NO released by growing EC downregulates the proliferation of cardiac FIB, an effect which could be important in the cardiovascular plasticity.

Endothelin-1 as a mediator of endothelial cell-pericyte interactions in bovine brain capillaries.

Endothelial cells and pericytes are closely associated in brain capillaries. Together with astrocytic foot processes, they form the blood-brain barrier. Capillaries were isolated from bovine brain cortex. Pure populations of endothelial cells and pericytes were isolated and cultured in vitro. Polarized monolayers of endothelial cells preferentially secreted immunoreactive endothelin-1 (Et-1) at their abluminal (brain-facing) membrane. They did not express receptors for Et-1. Pericytes expressed BQ-123-sensitive ETA receptors for endothelins as evidenced by 125I-Et-1 binding experiments. These receptors were coupled to phospholipase C as demonstrated by intracellular calcium measurements using indo-1-loaded cells. Addition of Et-1 to pericytes induced marked changes in the cell morphology that were associated with a reorganization of F-actin and intermediate filaments. It is concluded that Et-1 is a paracrine mediator at the bovine blood-brain barrier and that capillary pericytes are target cells for endothelium-derived Et-1.

The inotropic effect of endothelin-1 on rat atria involves hydrolysis of phosphatidylinositol.

Endothelin-1 induces a positive inotropic response in isolated left atria of the rat with an IC50 value of 20 nM. The contractile effect of endothelin is larger than that of other inotropic hormones such as phenylephrine and epinephrine and smaller than that of Bay K8644. In the spontaneously active right atria, endothelin induces a positive inotropic effect with no chronotropic effect. Endothelin does not modify intracellular levels of cAMP under basal conditions or after stimulation with isoproterenol but stimulates the formation of inositol phosphates. Mobilization of inositol phospholipids is observed in the same range of concentrations as for the contractile action of endothelin. The contractile action of endothelin is not mediated by protein kinase C. It is antagonized by blockers of L-type Ca2+ channels, low external Ca2+ concentrations and drugs such as caffeine and ryanodine that interfere with Ca2+ release by the sarcoplasmic reticulum.

Palytoxin acidifies chick cardiac cells and activates the Na+/H+ antiporter.

The cardiotoxic action of palytoxin was investigated using embryonic chick ventricular cells. Under normal ionic conditions, palytoxin produced an intracellular acidification which is partially compensated for by the Na+/H+ antiporter thereby leading to an increased rate of ethylisopropylamiloride-sensitive 22Na+ uptake. Under depolarizing membrane conditions, palytoxin produced a cellular acidification, a cellular alkalinization or no change in intracellular pH depending on the value of the extracellular pH. We propose that palytoxin acidifies cardiac cells by opening preexisting H+ conducting pathways in the plasma membrane.

Differential regulation of cardiac heme oxygenase-1 and vascular endothelial growth factor mRNA expressions by hemin, heavy metals, heat shock and anoxia.

Increasing attention has been paid to the effects of hypoxia, heavy metals and heat shocks on gene expression and to the similarities in their actions. This paper compares mRNA levels of two putative hypoxia, heavy metal and heat shock sensitive genes: heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) in myocyte-enriched cultures of neonatal rat heart cells. HO-1 mRNA expression is stimulated by hemin, Cd2+, Co2+ and heat shocks but not by Ni2+ or Mn2+. It is stimulated by long (13h) but not by short (4h) periods of anoxia. Conversely, VEGF mRNA expression is stimulated by short as well as long periods of anoxia, by Cd2+, Co2+, Ni2+ and Mn2+ but not by hemin or heat shocks. The results suggest that heavy metals, anoxia and heat shocks exert their effects on VEGF and HO-1 mRNA expression through separate though potentially overlapping mechanisms. Increased expressions of HO-1 and VEGF may be both cardioprotective under hypoxic/ischemic conditions.

An increase in intracellular pH is a general response of promonocytic cells to differentiating agents.

Intracellular pH (pHi) was measured in HL60 and U937 cells before and after differentiation into monocyte-macrophage like cells. 12-O-Tetradecanoyl phorbol-13-acetate (PMA), butyrate, interferon, retinoic acid and 1,25-dihydroxyvitamin D3 all increased pHi. The increases elicited were rapid with PMA, much slower with retinoic acid and interferon and still slower with 1,25-dihydroxyvitamin D3. Increases in pHi are due to an activation of the Na+/H+ exchange system. High pHi values are unlikely to serve as an early intracellular signal for initiating monocytic differentiation.

The activity of the Na+/H+ antiporter in cultured cardiac cells is dependent on the culture conditions used.

When chick cardiac cells are maintained in serum-free NCTC-135 medium, the pHi dependence of the Na+/H+ exchanger is much more acidic (pK = 7.0) than when a serum-free M199 medium is used (pK greater than 7.5). Phorbol esters activate the Na+/H+ exchanger and produce a cell alkalinization in cells maintained in NCTC-135 medium by shifting the pHi dependence of the system to more alkaline values. They have no action on cells maintained in M199 medium. The alkaline pHi dependence of the Na+/H+ exchanger and the loss of responsiveness of phorbol ester action in M199 medium correlate with increased rates of phosphatidylinositol turnover.

Expression of vascular endothelial growth factor-b in human astrocytoma.

Growth of human malignant gliomas is stringently dependent on an angiogenic process that probably involves vascular endothelial growth factor (VEGF). Expressions of mRNA coding for the different forms of VEGF were analyzed in surgical specimens from human astrocytomas. Low levels of placental growth factor (PGF) and VEGFC mRNA were observed in polymerase chain reaction, but not in Northern blot experiments. VEGF mRNA was found in some but not all grade and grade IV astrocytomas. VEGFB mRNA was observed in all tissue samples analyzed irrespective of the tumor grade. A new splice variant of VEGFB (VEGFB155) that lacks exons 5 and 6 is described. Expressions of VEGF mRNA in cultured glioblastomas cells were upregulated by hypoxia, but the sensitivity of the cells to hypoxia was reduced as compared with normal rat astrocytes. VEGF expression was depressed by dexamethasone. Expressions of VEGFB mRNA were affected neither by hypoxia nor by dexamethasone. The results indicate a coexpression of VEGF mRNA and VEGFB mRNA in human astrocytomas. Expression of VEGFB is markedly different from that of VEGF. Possible roles of VEGFB as a cofactor for hypoxia-induced angiogenesis in human astrocytomas are discussed.

The growth of heart cells in culture. Evidences for a multiple activation of the pleiotypic program.

Each medium renewal of confluent primary heart cell cultures derived from new born rats induces a pleiotypic response which leads to active proliferation. The presence of serum in the culture medium is essential for this activation of growth. Nutrient starvation prior to the activation decreases the response of the cells to serum. Serum starvation prior to the activation increases the serum dependence of the incorporation of labelled leucine but leaves the serum dependence of DNA synthesis unchanged. Ageing in culture decreases the serum dependence of the incorporation of labelled thymidine and amino acids but maintains it for alpha amino isobutyric acid transport. Several active components in human serum were distinguished by fractionated dialysis. A single dialyzable component stimulates both thymidine and amino acid incorporations. The transport of 2 deoxy-D-glucose is activated by another rapidly dialyzing component. The activation of alpha amino isobutyric acid transport may result from several components that are distanct from the previous ones. These results imply that a multiplicity of controls underly the pleiotypic activation of heart cell cultures by medium changes.

Pleiotypic response of rat heart cells in culture to serum stimulation.

The pleiotypic effects of medium replacement were studied in rat heart cell cultures. After each medium change alpha-aminoisobutyric acid and glucose transport are increased, RNA and protein syntheses are activated. DNA synthesis did not begin before 12 hours and was followed by a wave of mitoses. This sequence of events suggests that the stimulated cells were in early G 1 phase. DNA synthesis, following the shift to a fresh medium, is linearly related to the amount of serum used as is protein synthesis. However when serum concentrations higher than 20 percent were used no increased protein synthesis could be observed suggesting the existence of another limiting factor, which was probably the isoleucine content of the medium. The serum stimulating factor is heat stable, dialysable and was found in both human and fetal calf sera.

[The Na+/H+ exchanger in eukaryotic cells: biochemical and pharmacological properties and physiological role]. / L'échangeur Na+/H+ des cellules eucaryotes: propriétés biochimiques et pharmacologiques, rôle physiologique.

A membrane mechanism that catalyses the electroneutral exchange of Na+ for H+ has recently been characterized in a variety of eukaryotic cells. This exchanger is inhibited by amiloride, a potent diuretic drug. It has been implicated in a number of important physiological processes such as the regulation of the intracellular pH, the reabsorption of Na+ by the renal proximal tubule, the regulation of the cell volume and the fertilization of the sea urchin egg. The Na+/H+ exchanger seems able to mediate the action of growth factors. The biochemical and pharmacological properties of the Na+/H+ exchange system are reviewed. They are very similar in the different cell types that have been studied. Yet the Na+/H+ exchange system can fulfil different functions in different cell types depending i) on its properties of interaction with intracellular H+, ii) on the presence of other membrane structures that are involved in the maintenance of transmembrane Na+ and H+ gradients and iii) on the presence of extracellular messages that modify its catalytic properties and, among them, its interaction with internal H+.

Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchanger.

Amiloride analogs inhibit a number of transmembrane Na+ transport systems: 1) the epithelium Na+ channel, 2) the Na+/H+ exchange system and 3) the Na+/Ca2+ exchange system. Structure--activity relationships using amiloride derivatives with selected modification of each of the functional groups of the molecule indicate that the 3 Na+ transporting systems have distinct pharmacological profiles. 5-N Disubstituted derivatives of amiloride, such as ethylisopropylamiloride are the most potent inhibitors of the Na+/H+ exchange system. Conversely, amiloride derivatives that are substituted on the guanidino moiety, such as phenamil, are potent inhibitors of the epithelium Na+ channel. It is thus possible, by using selected amiloride derivatives to inhibit selectively one or another of the Na+ transport systems.

The Na+/H+ antiport of eukaryotic cells: relationship between the kinetic properties of the system and its physiological function.

The Na+/H+ antiport is present in the plasma membrane of virtually all vertebrate cells and it plays a central role in cell homeostasis. The pharmacological properties and the characteristics of the interaction of extracellular Na+, Li+, H+ and of intracellular H+ with the Na+/H+ antiport are reviewed herein. The kinetic properties of the system are shown to be essential for defining its four main physiological functions: transepithelial ion transport, control of the pHi, control of the intracellular Na+ concentration, and control of the cell volume. The activity of the Na+/H+ antiport can be modulated by a large number of effectors which are thought to act via protein kinases. At least three mechanisms of activation of the Na+/H+ exchanger are defined from the analysis of the kinetic properties of the system. Activation of the Na+/H+ antiport leads to very different consequences, depending upon the activity of other ion transporting systems in the membrane.

3,4 dichlorobenzamil-sensitive, monovalent cation channel induced by palytoxin in cultured aortic myocytes.

1. Smooth muscle cells were dispersed from rat aorta and then cultured. The action of palytoxin on rat aortic myocytes was analysed by measurement of 22Na+ uptake and single channel recording techniques. 2. Palytoxin induced an increase in 22Na+ uptake, with a concentration of 50 nM producing half-maximal activation. The action of palytoxin was inhibited by amiloride derivatives and by ouabain. The concentrations of inhibitor producing half-maximal inhibition were 10 microM for 3,4 dichlorobenzamil, 30 microM for benzamil, 100 microM for phenamil and 1 mM for ouabain. 3. In outside-out patches, palytoxin induced single channel currents that reversed near 0 mV with NaCl or KCl in the extracellular solution, but were outward with N-methyl-D-glucamine chloride or CaCl2 (110 mM), indicating that palytoxin induced a cation channel permeable to Na+ and K+ (PK/PNa = 1.2) but not to Ca2+ (PK/PCa > 30) or to N-methyl-D-glucamine (NMDG) (PK/PNMDG > 11) The unit channel conductance was 11-14 pS. 4. A high (> 0.1 mM) extracellular concentration of Ca2+ was necessary to observe channel activation by palytoxin. A high (150 mM) extracellular concentration of K+ partially prevented and reversed channel activation by palytoxin. 5. The channel activity was fully blocked by 3,4 dichlorobenzamil (20 microM) and partially blocked by phenamil (50 microM). It was not reduced by ouabain (200 microM).

Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y1 receptors.

1. ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio. 2. Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities. 3. ATP did not raise intracellular Ca2+ in B10 cells. Addition of apyrases III or VII (1 u ml(-1)) to ATP treated cells induced large intracellular Ca2+ transients. Apyrases had no action in the absence of ATP. 4. A 1 u ml(-1) apyrase III solution generated 20 microM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y1 receptors. 5. ATP was a full agonist of P2Y1 receptors in the presence of 1 u ml(-1) apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml(-1) apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s. 6. Apyrase VII (1 u ml(-1)) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve. 7. Results suggest that ATPDases prevent P2Y1 receptor activation by degrading ADP but may contribute to P2Y1 receptor activation by generating ADP from ATP.