Nitric oxide induces the expression of the monocarboxylate transporter MCT4 in cultured astrocytes by a cGMP-independent transcriptional activation.

The monocarboxylate transporter MCT4 is a proton-linked carrier particularly important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is exclusively expressed by astrocytes. Surprisingly, MCT4 expression in primary cultures of mouse cortical astrocytes is conspicuously low, suggesting that an external, nonastrocytic signal is necessary to obtain the observed pattern of expression in vivo. Here, we demonstrate that nitric oxide (NO), delivered by various NO donors, time- and dose-dependently induces MCT4 expression in cultured cortical astrocytes both at the mRNA and protein levels. In contrast, NO does not enhance the expression of MCT1, the other astrocytic monocarboxylate transporter. The transcriptional effect of NO is not mediated by a cGMP-dependent mechanism as shown by the absence of effect of a cGMP analog or of a selective guanylate cyclase inhibitor. NO causes an increase in astrocytic lactate transport capacity which requires the enhancement of MCT4 expression as both are prevented by the use of a specific siRNA against MCT4. In addition, cumulated lactate release by astrocytes over a period of 24 h was also enhanced by NO treatment. Our data suggest that NO represents a putative intercellular signal to control MCT4 expression in astrocytes and in doing so, to facilitate lactate transfer to other surrounding cell types in the central nervous system. © 2011 Wiley-Liss, Inc.

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation.

Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs.

Endothelium-derived nitric oxide (EDNO) plays a pivotal role in regulating pulmonary circulation. To determine whether there is a heterogeneity in EDNO-mediated responses of different sized pulmonary vessels, we studied small and large isolated pulmonary arteries of newborn lambs (diameter, 0.4-0.7 and 1.5-2.5 mm, respectively). The isometric tension of vessel rings were recorded while suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O2-5% CO2, 37 degrees C). In vessels preconstricted with norepinephrine, acetylcholine and bradykinin induced a greater relaxation of small pulmonary arteries than of large pulmonary arteries. Acetylcholine, bradykinin, and nitric oxide also induced a greater increase in cGMP content in small arteries than in large ones. The responses to acetylcholine and bradykinin were endothelium-dependent and inhibited by nitro-L-arginine, an inhibitor of nitric oxide synthase. In vessels without endothelium, the response to nitric oxide was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. The activity of soluble guanylyl cyclase of small arteries was greater than that of large arteries under basal conditions and after stimulation with S-nitroso-N-acetylpenicillamine, a nitric oxide donor. These results demonstrate that heterogeneity exists in EDNO-mediated relaxation of small and large pulmonary arteries in newborn lambs. A difference in the soluble guanylate cyclase activity of vascular smooth muscle may have contributed to this phenomenon.

Differential responses of newborn pulmonary arteries and veins to atrial and C-type natriuretic peptides.

Atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are important dilators of the pulmonary circulation during the perinatal period. We compared the responses of pulmonary arteries (PA) and veins (PV) of newborn lambs to these peptides. ANP caused a greater relaxation of PA than of PV, and CNP caused a greater relaxation of PV than of PA. RIA showed that ANP induced a greater increase in cGMP content of PA than CNP. In PV, ANP and CNP caused a similar moderate increase in cGMP content. Receptor binding study showed more specific binding sites for ANP than for CNP in PA and more for CNP than for ANP in PV. Relative quantitative RT-PCR for natriuretic peptide receptor A (NPR-A) and B (NPR-B) mRNAs show that, in PA, NPR-A mRNA is more prevalent than NPR-B mRNA, whereas, in PV, NPR-B mRNA is more prevalent than NPR-A mRNA. In conclusion, in the pulmonary circulation, arteries are the major site of action for ANP, and veins are the major site for CNP. Furthermore, the differences in receptor abundance and the involvement of a cGMP-independent mechanism may contribute to the heterogeneous effects of the natriuretic peptides in PA and PV of newborn lambs.

Congenital diaphragmatic hernia: current status and review of the literature.

Treatment of congenital diaphragmatic hernia (CDH) challenges obstetricians, pediatric surgeons, and neonatologists. Persistent pulmonary hypertension (PPHT) associated with lung hypoplasia in CDH leads to a high mortality rate at birth. PPHT is principally due to an increased muscularization of the arterioles. Management of CDH has been greatly improved by the introduction of prenatal surgical intervention with tracheal obstruction (TO) and by more appropriate postnatal care. TO appears to accelerate fetal lung growth and to increase the number of capillary vessels and alveoli. Improvement of postnatal care over the last years is mainly due to the avoidance of lung injury by applying low peak inflation pressure during ventilation. The benefits of other drugs or technical improvements such as the use of inhaled nitric oxide or extracorporeal membrane oxygenation (ECMO) are still being debated and no single strategy is accepted worldwide. Despite intensive clinical and experimental research, the treatment of newborn with CDH remains difficult.

Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma.

A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy.

Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy.

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.

Nitric Oxide Induces the Expression of the Monocarboxylate Transporter MCT4 in Cultured Astrocytes by a cGMP-Independent Transcriptional Activation

The monocarboxylate transporter MCT4 is a proton-linked carrier particularly important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is exclusively expressed by astrocytes. Surprisingly, MCT4 expression in primary cultures of mouse cortical astrocytes is conspicuously low, suggesting that an external, nonastrocytic signal is necessary to obtain the observed pattern of expression in vivo. Here, we demonstrate that nitric oxide (NO), delivered by various NO donors, time- and dose-dependently induces MCT4 expression in cultured cortical astrocytes both at the mRNA and protein levels. In contrast, NO does not enhance the expression of MCT1, the other astrocytic monocarboxylate transporter. The transcriptional effect of NO is not mediated by a cGMP-dependent mechanism as shown by the absence of effect of a cGMP analog or of a selective guanylate cyclase inhibitor. NO causes an increase in astrocytic lactate transport capacity which requires the enhancement of MCT4 expression as both are prevented by the use of a specific siRNA against MCT4. In addition, cumulated lactate release by astrocytes over a period of 24 h was also enhanced by NO treatment. Our data suggest that NO represents a putative intercellular signal to control MCT4 expression in astrocytes and in doing so, to facilitate lactate transfer to other surrounding cell types in the central nervous system. (C) 2011 Wiley-Liss, Inc.

Phosphorylation of CARMA1 by HPK1 is critical for NF-kappaB activation in T cells.

Activation of the NF-kappaB pathway in T cells is required for induction of an adaptive immune response. Hematopoietic progenitor kinase (HPK1) is an important proximal mediator of T-cell receptor (TCR)-induced NF-kappaB activation. Knock-down of HPK1 abrogates TCR-induced IKKbeta and NF-kappaB activation, whereas active HPK1 leads to increased IKKbeta activity in T cells. Yet, the precise molecular mechanism of this process remains elusive. Here, we show that HPK1-mediated NF-kappaB activation is dependent on the adaptor protein CARMA1. HPK1 interacts with CARMA1 in a TCR stimulation-dependent manner and phosphorylates the linker region of CARMA1. Interestingly, the putative HPK1 phosphorylation sites in CARMA1 are different from known PKC consensus sites. Mutations of residues S549, S551, and S552 in CARMA1 abrogated phosphorylation of a CARMA1-linker construct by HPK1 in vitro. In addition, CARMA1 S551A or S5549A/S551A point mutants failed to restore HPK1-mediated and TCR-mediated NF-kappaB activation and IL-2 expression in CARMA1-deficient T cells. Thus, we identify HPK1 as a kinase specific for CARMA1 and suggest HPK1-mediated phosphorylation of CARMA1 as an additional regulatory mechanism tuning the NF-kappaB response upon TCR stimulation.

Altérations dans le cordon ombilical de nouveau-nés avec une restriction de la croissance intra-utérine

Introduction: Le retard de croissance intra-utérin (RCIU) est défini comme une incapacité du foetus à atteindre son plein potentiel de croissance. C'est une complication fréquente (affectant ~8% des grossesses), associée à un risque accru de mortalité et morbidité périnatales et de maladies chroniques à l'âge adulte, telles que les maladies coronariennes, l'hypertension, ou le diabète. Une croissance foetale adéquate est déterminée principalement par la disponibilité en oxygène et nutriments apportés au foetus par la circulation ombilico-placentaire. Chez l'homme, le tonus vasculaire ombilical est régulé majoritairement par la voie du monoxyde d'azote (NO)/GMPc. Nous avons émis l'hypothèse que le RCIU pourrait être associé à des altérations dans la régulation de la circulation ombilicale, en particulier dans la voie du NO/GMPc. Méthodes: Cette étude a été conçue pour identifier dans des cordons ombilicaux, les changements structurels, fonctionnels et moléculaires survenant en cas de RCIU, en particulier dans la veine om-bilicale. Résultats: De façon générale, le diamètre du cordon ombilical était significativement réduit chez les nouveau-nés avec RCIU par rapport aux contrôles. Les mesures histomorphométriques ont mis en évidence une diminution significative de la surface transversale totale ainsi que de muscle lisse dans la veine ombilicale en cas de RCIU. Les études pharmacologiques effectuées sur des anneaux vasculaires de veines ombilicales ont montré une diminution de la tension maximale induite par des vasoconstricteurs chez les garçons avec RCIU, et une réduction significative de la relaxation induite par le NO chez les filles avec RCIU. Cette altération de la relaxation s'accompagne de modifications de plusieurs composants de la voie du NO/GMPc au niveau du muscle lisse de la veine ombilicale des filles avec RCIU. Enfin l'addition d'un inhibiteur non-spécifique des phosphodiesterases (PDEs) a permis d'améliorer la réponse au NO dans tous les groupes et surtout de compenser la réduction de la relaxation induite par le NO chez les filles avec RCIU. Conclusion: Cette étude a permis de mettre en évidence des modifications structurelles dans le cordon ombilical de nouveau-nés présentant un RCIU, ainsi que des changements fonctionnels et moléculaires dans la veine ombilicale, en particulier dans la voie du NO/GMPc, qui pourraient contribuer au développement du RCIU. L'effet bénéfique de l'inhibition des PDEs sur la relaxation suggère qu'elles pourraient constituer des cibles thérapeutiques potentielles.

Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF(-)GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF(-)GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in 'equivalent-light'' scenarios.

Glutamatergic transmission in the nucleus tractus solitarii: from server to peripherals in the cardiovascular information superhighway

Afferent nerves carrying signals from mechanoreceptors in the aortic arch and carotid sinus terminate predominantly in the nucleus tractus solitarii (NTS). Signal transduction and neurotransmission in the NTS are critical for central cardiovascular reflex control, but little was known about either until the late 1970's. None of the numerous neuroactive chemicals found in the NTS had met strict criteria as a neurotransmitter in the baroreflex arc until data suggested that the excitatory amino acid L-glutamate (GLU) might be released from baroreceptor afferent terminals in the NTS. In anesthetized animals microinjection into the NTS of GLU, which can be demonstrated in terminals in the NTS, produces cardiovascular responses like those seen with activation of the baroreceptor reflex. Similar responses occur in awake animals if the chemoreceptor reflex is eliminated; otherwise, in conscious animals responses mimic those of chemoreceptor reflex activation. GLU is released in the NTS upon selective activation of the baroreceptor, and possibly the chemoreceptor, reflex. Responses to selective agonists as well as baroreflex responses are eliminated by GLU antagonists microinjected into the NTS. Non-NMDA (N-methyl-D-aspartic acid) receptors seem to predominate at primary baroreceptor synapses in the NTS while NMDA receptors may be involved at later synapses. Although inhibition of soluble guanylate cyclase attenuates responses to ionotropic glutamate agonists in the NTS, nitric oxide does not seem to play a role in glutamate transmission in the NTS. GLU may also participate in transmission at cardiovascular neurons beyond the NTS. For example, a role has been suggested for GLU in the ventrolateral medulla and spinal cord. Work continues concerning GLU signal transduction and mechanisms that modulate that transduction both at the NTS and at other cardiovascular nuclei

Reversal by methylene blue of tetanic fade induced in cats by nitric oxide

Previous data from our laboratory have indicated that nitric oxide (NO) acting at the presynaptic level increases the amplitude of muscular contraction (AMC) of the phrenic-diaphragm preparations isolated from indirectly stimulated rats, but, by acting at the postsynaptic level, it reduces the AMC when the preparations are directly stimulated. In the present study we investigated the effects induced by NO when tetanic frequencies of stimulation were applied to in vivo preparations (sciatic nerve-anterior tibial muscle of the cat). Intra-arterial injection of NO (0.75-1.5 mg/kg) induced a dose-dependent increase in the Wedensky inhibition produced by high frequencies of stimulation applied to the motor nerve. Intra-arterial administration of 7.2 µg/kg methylene blue did not produce any change in AMC at low frequencies of nerve stimulation (0.2 Hz), but antagonized the NO-induced Wedensky inhibition. The experimental data suggest that NO-induced Wedensky inhibition may be mediated by the guanylate cyclase-cGMP pathway

Relationship between the actions of atrial natriuretic peptide (ANP), guanylin and uroguanylin on the isolated kidney

Guanylin and uroguanylin are peptides that bind to and activate guanylate cyclase C and control salt and water transport in many epithelia in vertebrates, mimicking the action of several heat-stable bacteria enterotoxins. In the kidney, both of them have well-documented natriuretic and kaliuretic effects. Since atrial natriuretic peptide (ANP) also has a natriuretic effect mediated by cGMP, experiments were designed in the isolated perfused rat kidney to identify possible synergisms between ANP, guanylin and uroguanylin. Inulin was added to the perfusate and glomerular filtration rate (GFR) was determined at 10-min intervals. Sodium was also determined. Electrolyte dynamics were measured by the clearance formula. Guanylin (0.5 µg/ml, N = 12) or uroguanylin (0.5 µg/ml, N = 9) was added to the system after 30 min of perfusion with ANP (0.1 ng/ml). The data were compared at 30-min intervals to a control (N = 12) perfused with modified Krebs-Hanseleit solution and to experiments using guanylin and uroguanylin at the same dose (0.5 µg/ml). After previous introduction of ANP in the system, guanylin promoted a reduction in fractional sodium transport (%TNa+, P<0.05) (from 78.46 ± 0.86 to 64.62 ± 1.92, 120 min). In contrast, ANP blocked uroguanylin-induced increase in urine flow (from 0.21 ± 0.01 to 0.15 ± 0.007 ml g-1 min-1, 120 min, P<0.05) and the reduction in fractional sodium transport (from 72.04 ± 0.86 to 85.19 ± 1.48, %TNa+, at 120 min of perfusion, P<0.05). Thus, the synergism between ANP + guanylin and the antagonism between ANP + uroguanylin indicate the existence of different subtypes of receptors mediating the renal actions of guanylins.