Targeting of c-kit+ haematopoietic progenitor cells prevents hypoxic pulmonary hypertension.

Haematopoietic c-kit+ progenitor cells may contribute to pulmonary vascular remodelling and pulmonary hypertension (PH). Stromal derived factor-1 (SDF-1/CXCL12) and its receptors CXCR4 and CXCR7 have been shown to be critical for homing and mobilisation of haematopoietic c-kit+ progenitor cells in the perivascular niche. We administered AMD3100, a CXCR4 antagonist, and CCX771, a CXCR7 antagonist, to chronic hypoxia exposed mice in order to study the role of c-kit+ progenitor cells in PH. CXCL12, CXCR4 and CXCR7 protein expression, haemodynamic parameters, right ventricular mass, extent of vascular remodelling and perivascular progenitor cell accumulation were studied. Chronic hypoxia-exposed mice showed increased total lung tissue expression of CXCR4, CXCR7 and CXCL12 after development of PH. This was associated with significantly increased right ventricular systolic pressure and evidence of right ventricular hypertrophy, vascular remodelling and perivascular c-kit+/sca-1+ progenitor cell accumulation. CCX771 administration did not abrogate these effects. In contrast, administration of AMD3100, whether alone or combined with CCX771, prevented vascular remodelling, PH and perivascular accumulation of c-kit+/sca-1+ progenitor cells, with a synergistic effect of these agents. This study offers important pathophysiological insights into the role of haematopoietic c-kit+ progenitors in hypoxia-induced vascular remodelling and may have therapeutic implications for PH.

Sodium channel and sodium pump in normal and pathological muscles from patients with myotonic muscular dystrophy and lower motor neuron impairment.

TWO SODIUM TRANSPORT SYSTEMS HAVE BEEN ANALYZED IN THIS WORK: the voltage-sensitive sodium channel and the (Na(+), K(+)) ATPase pump. The sodium channel has been studied using a tritiated derivative of tetrodotoxin; the sodium pump has been studied using tritiated ouabain. Properties of interaction of tritiated tetrodotoxin and of tritiated ouabain with their respective receptors were observed in normal human skeletal muscle and in muscles of patients with myotonic muscular dystrophy and with lower motor neuron impairment. Levels of sodium pump and of sodium channels were measured at different stages of membrane purification. Microsomal fractions of normal human muscle have maximal binding capacities for tetrodotoxin of 230 fmol/mg of protein and of 7.4 pmol/mg of protein for ouabain. Dissociation constant for the complexes formed by the tetrodotoxin derivative and by ouabain with their respective receptors were 0.52 nM and 0.55 muM, respectively. In muscles from patients with myotonic muscular dystrophy, the maximal binding capacity for tetrodotoxin, i.e., the number of Na(+) channels was found to be very similar to that found for normal muscle. The maximal binding capacity for ouabain, i.e., the number of Na(+) pumps was three- to sixfold lower than in normal muscle. Dissociation constants for the complexes formed with the tetrodotoxin derivative and with ouabain were the same as for normal muscle. In muscles from patients with lower motor nerve impairment, the maximal binding capacities for tetrodotoxin and for ouabain were twice as high as in normal muscle. Again, dissociation constants for the complexes formed with the tetrodotoxin derivative and with ouabain were nearly unchanged as compared with normal muscle. These results suggest that sodium transport systems involved in the generation of action potentials and/or in the regulation of the resting potential are altered both in myotonic muscular dystrophy and in lower motor neuron impairment.

Synthesis and properties of new photoactivable derivatives of tetrodotoxin.

The Pfitzner-Moffatt oxidation procedure has been used to prepare two new photoactivable derivatives of tetrodotoxin that have been synthesized with high specific radioactivities (17.5 Ci/mmol and 30 Ci/mmol). They specifically bind to axonal membranes with affinities of 5.2-14.2 nM. They dissociate from their membrane complex with half-lives of 10.8 and 20 min. In the dark, these compounds give a reversible block of the sodium channels. After ultraviolet irradiation, they induce an irreversible blockade of the nerve channels.

Biochemical characterization of plasma membrane isolated from human skeletal muscle.

Specific components of ion translocation systems were studied in excitable plasma membranes isolated from normal human muscle. Na+-K+ ATPase and ouabain-sensitive K+ phosphatase activities were 8.9 +/- 1 mumol Pi/h per mg protein and 96 +/- 9 nmol/min per mg protein, respectively. Scatchard analysis of equilibrium binding assays with [3H]ouabain showed non-linear curves consistent with high- and low-affinity sites (estimated Kd 3 nM and 0.22 microM). Two families of receptors with different affinities for a tritiated TTX derivative (estimated Kd 0.4 and 4 nM) were also identified suggesting the existence in human muscle of at least two classes of voltage-dependent Na+ channels. In addition (+)-[methyl-3H]PN200-110, a potent Ca2+ antagonist used for labeling voltage-dependent Ca2+ channels, was observed to bind to a homogeneous population of receptors in the plasma membrane (Kd = 0.2 nM).

Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na+ channel.

Binding studies indicate that ciguatoxin and brevetoxin allosterically enhance in a very similar way the binding of [3H]batrachotoxinin A 20-alpha-benzoate to the neuronal Na+ channel protein. Moreover ciguatoxin competitively inhibits the binding of [3H]brevetoxin-3 to rat brain membranes. The affinity of ciguatoxin for the Na+ channel is at least 20-50-times higher than that of brevetoxin. These results indicate that ciguatoxin and brevetoxins act at the same binding site on the sodium channel.

Tibolone actions on normal and breast cancer cells.

Tibolone and its main derivatives were studied in an original model of cultures of normal human epithelial breast (HBE) cells on proliferation, differentiation and apoptosis, the three mechanisms responsible for breast homeostasis. Tibolone and its Delta4 isomer were antiproliferative, both in the absence and presence of oestradiol (E2). The oestrogenic 3alpha and 3beta hydroxy derivatives did not display any mitogenic activities in HBE cells. Moreover, at 1 microM, they were antiproliferative. Tibolone and its Delta isomer increased the 17beta hydroxysteroid dehydrogenase activity similarly to that observed with progestins [1]. Apoptosis was increased in HBE cells to a similar range as with the pure pregnane progestin, Org2058. We also studied the extent of apoptosis in hormone-dependent breast cancer cell lines. Tibolone and its Delta4 isomer also increased apoptosis, especially in ZR75-1 cells containing progesterone and androgen receptors [2]. We could demonstrate that these pro-apoptotic actions of tibolone and its Delta4 isomer were mediated at least partially through the bcl-2-family of proteins. Moreover, the antiproliferative and pro-apoptotic activities of tibolone, as well as Org2058, were mediated by increasing catalase activities in breast cancer cells. Thus, in breast cells, tibolone slows down the proliferation rate, increases differentiation and apoptosis. These actions seem to be optimal on breast tissue.

Characterization and visualization of [125I] stromal cell-derived factor-1alpha binding to CXCR4 receptors in rat brain and human neuroblastoma cells.

Stromal cell-Derived Factor-1 (SDF-1alpha), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and modulates cell migration, differentiation, and proliferation. CXCR4 has been reported to be expressed in various tissues including brain. Moreover, CXCR4 has recently been shown to be one of the coreceptors for HIV-1 infection which could be implicated in HIV encephalitis. In the present study, the binding properties and autoradiographic distribution of [125I]SDF-1alpha binding to CXCR4 were characterized in the adult rat brain. SDF-1alpha binding and CXCR4 coupling system were also studied in human neuroblastoma cell line SK-N-SH. The binding of [125I]SDF-1alpha on rat brain sections was specific, time-dependent and reversible. The highest densities of CXCR4 were detected in the choroid plexus of the lateral and the dorsal third ventricle. Lower densities of [125I]SDF-1alpha binding sites were observed in various brain regions including cerebral cortex, anterior olfactory nuclei, hippocampal formation, thalamic nuclei, blood vessels and pituitary gland. In the choroid plexus, the IC(50) and K(d) of [125I]SDF-1alpha binding were respectively 0.6 nM and 0. 36 nM. Similar IC(50) values were obtained in other brain structures. A CXCR4 antagonist, bicyclam, competed with SDF-1alpha binding (30% inhibition at 10(-6) M). In SK-N-SH cells, [125I]SDF-1alpha bound to CXCR4 with a K(d) of 5.0 nM and a maximal binding capacity of 460 fmol/mg of protein. SDF-1alpha induced a rapid and transient intracellular calcium increase in SK-N-SH cells. These findings suggest that CXCR4 is highly expressed in some brain structures and have a regulatory role in the nervous system. The significance of this expression in the brain parenchyma and more specifically in the choroid plexus remains to be clarified in the normal as well as in the infected brain.

Cryopreservation and culture of human corneal keratocytes.

PURPOSE: To assess the effects of two different concentrations of albumin in a cryoprotective solution and two freezing methods on human corneal keratocyte ctyopreservation. METHODS: Isolated keratocytes were used for cryopreservation. Solutions of 10% dimethylsulfoxide with either 2% or 10% human albumin were used as cryoprotective agents. Cells either were transferred directly into a -80 degrees C freezer (freezing rate, 2 degrees C/min) or were cooled in a programmed freezer (1 degrees C/min until -40 degrees C and then 10 degrees C/min), which resulted in four different cryopreservation protocols. Cells were stored at -80 degrees C, then were thawed at 37 degrees C, and subsequently were cultured. Keratocytes were studied by means of trypan blue staining, growth assay, apoptosis assays, transmission electron microscopy, and immunochemistry. RESULTS: The percentage of cells that were alive after thawing ranged from 80% to 99% by trypan blue staining and from 45% to 60% by flow cytometry. The ratio of the number of living cells at the end of primary culture after cryopreservation to that before cryopreservation was significantly (P=0.04) higher after direct transfer into the -80 degrees C freezer than after controlled-rate freezing, whereas the albumin concentration had no significant influence on this ratio (P=0.45). The percentage of apoptotic cells was significantly higher after cryopreservation than in the control group of noncryopreserved cells; more than 5% 24 hours after thawing. Cryopreservation did not modify the keratocyte ultrastructure. Fibroblast growth factor dramatically decreased the serum-induced cell expression of alpha smooth muscle actin, whereas cryopreservation had no influence on this cell expression. CONCLUSIONS: A freeze-thaw trauma, which was related to cryopreservation-induced cell apoptosis, was revealed during primary culture after thawing. Direct transfer into the -80 degrees C freezer resulted in better postcryopreservation growth in the culture than controlled-rate freezing. A change in albumin concentration from 2% to 10% did not affect the results.

In vitro effects of dexamethasone on human corneal keratocytes.

PURPOSE: To investigate whether cultured human keratocytes express the glucocorticoid receptor (GR) and to assess the influence of dexamethasone (DEX) on these cells. METHODS: Human keratocytes were cultured in medium supplemented with various concentrations of DEX (ranging from 10(-10) to 10(-4) M). Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-s ulfophenyl)-2H-tetrazolium inner salt (MTS) assay at 2, 4, and 6 days of culture. Some experiments were performed in the presence of mifepristone (RU38486), an antiglucocorticoid molecule. The early phase of apoptosis was studied by means of keratocyte staining with a fluorescein conjugate of annexin V and propidium iodide, and cells were analyzed by flow cytometry. Glucocorticoid receptor mRNA was detected in keratocytes by means of reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemical staining of the cells was performed with a monoclonal anti-human GR. RESULTS: RT-PCR and immunocytochemistry showed the expression of GR (mRNA and protein) in cultured keratocytes. Dexamethasone significantly increased keratocyte proliferation with concentrations ranging from 10(-9) to 10(-5) M, with a maximum effect at 10(-7) M (P < 0.005). Dexamethasone's proproliferative effect was inhibited by RU38486. However, DEX also induced apoptosis of cultured keratocytes at any concentration used. CONCLUSIONS: These results indicate that cultured human keratocytes express the GR and proliferate in response to DEX stimulation (10(-9)-10(-5) M), which also induces keratocyte apoptosis.

Multidrug-resistance circumvention and inhibition of [h-3] azidopine photolabeling of p-glycoprotein by new dihydropyridine derivatives displaying a low-affinity for calcium channels.

This study was aimed to characterize the reversing activity of S16209 and S16317, two new dihydropyridines with low affinity for calcium channels. In vivo, S16209 (75 mg/kg) and S16317 (25 mg/kg) potentiate the antitumor activity of vincristine (VCR) in VCR-resistant leukemia bearing mice. In vitro, a complete sensitization to adriamycin (ADR) or VCR is obtained with 2.5 muM of S16209 in S1/tMDR and KB-A1 cells and with 2.5 muM of S16317 in S1/tMDR and P388/ADR-10 cells. These two compounds are also more potent than verapamil and cyclosporin A in increasing actinomycin-D cytotoxicity in DC-3F/AD cells. In the presence of ADR or VCR, a 4 h co-incubation followed by a post-incubation of 20 h with 2.5 muM S16209 is sufficient to completely overcome the resistance of human KB-A1 and S1/tMDR cells to these cytotoxic drugs. S16209 and S16317 increase ADR accumulation in resistant cells, and completely inhibit the photolabeling of P-gp by [H-3]azidopine at 100 and 10 muM, respectively, suggesting that the reversing activity of these two compounds is mainly due to a specific inhibition of the P-gp mediated efflux of cytotoxic drugs.

Proapoptotic effects of antiestrogens, progestins and androgen in breast cancer cells.

The promoting action of E2 in breast cancer cells has been, until now, mainly linked to its action on prolifieration. Because of the importance of an increase in apoptosis in breast cancer prevention, we have studied the possible effects of various antiestrogens, progestins and an androgen on its occurrence in three hormone-dependent breast cancer cell lines. The antiestrogens were, a triphenylethylene derivative, 4 hydroxytamoxifen (4OHTAM) and two steroidal antiestrogens, IC1182780 and RU58668. The progestins were Org2058, a pregnane derivative, tibolone (OrgOD14), a normethyltestosterone derivative and OrgOM38 (the delta4 isomer of OrgOD14) and the androgen dihydrotestosterone (DHT). Apoptosis was studied in MCF-7, ZR75-1 and T47-D cells using morphological approaches and flow cytometry. The antiestrogens, the progestins and DHT were proapoptotic but to different potencies according to the cell line studied. Indeed, the 'pure' steroidal antiestrogens were more efficient than 4OHTam in increasing apoptosis. We have also studied the level of expression of some of the proteins involved in the regulation of apoptosis. Bcl-2 and bcxL, two antiapoptotic members of the bcl-2 family proteins, were inhibited by the progestins and the antiestrogens. In contrast, the proapoptotic proteins, bax and bak seemed to be constitutively expressed. Thus, since the ratio of proapoptotic and antiapoptotic proteins determines apoptosis or cell survival, the hormone effects are operating by modulating the antiapoptotic regulators of the balance. These data demonstrate that antiestrogens, progestins, and androgens can promote apoptosis in breast cancer cells, an effect which could be of importance in the therapeutic prevention of breast cancer.

Biochemical studies of the structure, mechanism and differentiation of the voltage-sensitive sodium channel.

This paper describes how neurotoxins specific of the Na(+) channel are used to study its function, its structure and its differentiation in a variety of excitable and non-impulsive cells.

Interaction of insecticides of the pyrethroid family with specific binding sites on the voltage-dependent sodium channel from mammalian brain.

Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-alpha-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemonia sulcata toxin 2 binding, 125I-Tityus serrulatus toxin gamma binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-alpha-benzoate.

Distribution of voltage-dependent Na+ channels identified by high-affinity receptors for tetrodotoxin and saxitoxin in rat and human brains: quantitative autoradiographic analysis.

The localization of a putative voltage-dependent Na+ channel in adult rat and human brain was studied by light microscopic quantitative autoradiography using a tritiated derivative of tetrodotoxin ([3H]enTTX) and tritiated saxitoxin [( 3H]STX). Equilibrium binding experiments in the whole rat brain gave dissociation constants of 7.0 nM ([3H]enTTX) and 5.0 nM ([3H]STX). The dissociation constant for the binding of [3H]STX in the different human brain regions was near 1.5 nM. Autoradiograms demonstrated a heterogeneous distribution of toxin binding sites in the brain with a very good correlation of the mapping of tetrodotoxin and saxitoxin receptors. With the exception of a few regions, the same type of cartography was observed for human and rat brain structures. If toxin receptors were present in all brain regions, their density was particularly important in cerebral cortex, hippocampus, lateral septum and molecular layer of cerebellar cortex. Conversely, the medulla oblongata contained only low amounts of binding sites.

Axonal transport of the voltage-dependent Na+ channel protein identified by its tetrodotoxin binding site in rat sciatic nerves.

Na+ channels levels were measured in different segments of rat vagus and sciatic nerves by in vitro binding using a tritiated ethylene-diamine tetrodotoxin derivative ([3H]en-TTX). Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve indicating an anterograde and retrograde axoplasmic transport of Na+ channels. Accumulation of Na+ channels at the ligature was time-dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of a ligature was also visualized by autoradiographic studies in longitudinal sections of sciatic nerves using [3H]en-TTX.

Identification in mammalian brain of an endogenous substance with Na+ channel blocking activities similar to those of tetrodotoxin.

A substance with Na+ channel blocking activities has been isolated from pig brain after extraction and purification on sulfopropyl-Sephadex C-25, reversed-phase and carboxymethyl Synchropak high pressure liquid chromatography columns. The peptidic material i) displaces [3H]ethylenediamine tetrodotoxin ([3H]en-TTX) from its binding sites on rat brain membranes, (ii) it blocks 22Na+ influx induced by veratridine and sea anemone toxin on neuroblastoma and embryonic chick heart cells in culture, (iii) it specifically decreases the height of the action potential generated in frog sciatic nerve, and (iv) it blocks the fast Na+ current in voltage-clamped neuroblastoma cells. These properties are similar to those of tetrodotoxin while the endogenous factor is a peptide that is destroyed by proteases. These results suggest the presence in pig brain of a potent Na+ channel modulation activity.

Differential effects of non-peptidic tachykinin receptor antagonists on Ca2+ channels.

The effects of a range of non-peptidic neurokinin receptor antagonists on dihydropyridine binding at voltage-dependent Ca2+ channels from rat skeletal muscle were studied. As previously reported, the binding studies on dihydropyridine binding sites revealed a temperature-dependency effect of the tachykinin NK1 receptor antagonist (+/-)-CP 96345 ((2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl) methyl)-1-azabicyclo-[2.2.2.]-octan-3-amine) similar to d-cis-diltiazem. Its related homologue CP 99994 ((+)-2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperid ine) was devoid of such activity. However, RP 67580 (perhydroisoindol-4-one-(3aR,7aR)-7,7-diphenyl- 2[1-imino-2-(2-methoxyphenyl)ethyl]) and SR 48968 ((S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichloro phenyl) butyl]benzamide) (tachykinin NK1 and NK2 receptor antagonists) were also potent inhibitors of [3H]PN 200-110 (Isradipine) binding without temperature dependency, indicating that actions on ion channels may contribute to their pharmacological effects. Furthermore, all the compounds had affinity for the D888 ((-)-devapamil) phenylalkylamine site, indicating that many neurokinin antagonists may have affinity for Ca2+ channels.

Ca2+ channel inhibition by a new dihydropyridine derivative, S11568, and its enantiomers S12967 and S12968.

Biochemical and electrophysiological techniques were used to describe the Ca2+ channel blocking properties of a new dihydropyridine derivative, S11568 (+/-)- ([(amino-2-ethoxy)-2-ethoxy]methyl)-2-(dichloro-2',3'-phenyl)-4- ethoxy-carbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyridine and its enantiomers S12967 ((+)-S11568) and S12968 ((-)-S11568). In binding studies, S11568 and S12968 displaced specifically bound [3H]PN 200-110 from cardiac and vascular smooth muscle preparations with potencies of 5.6-51 nM, respectively. S12967 was 6- to 18-fold less potent than S12968. A good correlation was found between the IC50 value for the inhibition of 45Ca2+ uptake by A7r5 aortic smooth muscle cells and binding data. Whole-cell patch clamp studies in both guinea-pig ventricular myocytes and A7r5 cells yielded similar results. At holding potential (VH) -50 mV, S12968 inhibited L-type Ca2+ current with an IC50 value near 70 nM, 2- to 3-fold more potently than S11568 and 30-fold more potently than S12967. With VH -100 mV, all three compounds were less potent, with IC50 values ranging from 500 nM to 3 microM. These results demonstrate conclusively that S12968 is the more active enantiomer. Furthermore, the pronounced voltage dependence of its actions in vitro suggests that in vivo it could exhibit good selectivity for vascular smooth muscle over cardiac muscle.

Mechanism of inhibition of tumor necrosis factor production by chlorpromazine and its derivatives in mice.

In previous work, we reported that chlorpromazine inhibits tumor necrosis factor (TNF) production in endotoxin lipopolysaccharide-treated mice, and protects against lipopolysaccharide toxicity. Chlorpromazine is used as an antipsychotic and has several effects on the central nervous system. It acts on different neurotransmitter receptors and has other biochemical activities some of which, like inhibition of phospholipase A2, might be responsible for the inhibitory effect on TNF production. To investigate the role of these actions in the inhibition of TNF production by chlorpromazine, we have synthesized some chlorpromazine derivatives that do not have central activities. Some of these analogs have lost their affinity for various receptors and their phospholipase A2 inhibitory activity, but still inhibit TNF production. No correlation was found between TNF inhibition and the ability to inhibit nitric oxide (NO) synthase, whereas a good correlation was evident between TNF inhibition and antioxidant activity.

Autoradiographic localization of tetrodotoxin-sensitive Na+ channels in rat brain.

The localization of tetrodotoxin-sensitive Na+ channels in rat brain was studied using a tritiated derivative of tetrodotoxin. The autoradiographic distribution of a tritiated ethylenediamine derivative of tetrodotoxin [( 3H]en-TTX) binding showed a high concentration of sites in cortical layers, hippocampus, globus pallidus, substantia nigra and the molecular layer of the cerebellar cortex. Lower levels were found principally in the striatum and median forebrain bundle. The white matter was not labelled. The characteristic distribution of tetrodotoxin-sensitive Na+ channels was compared to that of nitrendipine-sensitive Ca2+ channels and to that of apamin-sensitive Ca2+-dependent K+ channels.