Sergio Sánchez-Armass

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2.

BACKGROUND Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation. METHODS J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases. RESULTS ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP. CONCLUSIONS Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin. GENERAL SIGNIFICANCE ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.

Effect of coexposure to DDT and manganese on freshwater invertebrates: Pore water from contaminated rivers and laboratory studies

An environmental survey of several rivers of the southern Huasteca area of Mexico revealed high concentrations of manganese (Mn) and the presence of DDT in the sediments and pore water. Therefore, acute (48-h) toxicities of Mn and DDT were assessed both independently and as a combination on 24-h-old neonates of Daphnia magna Strauss and Lecane quadridentata Ehrenberg. Daphnia magna showed high sensitivity to both toxicants, whereas L. quadridentata was highly resistant to DDT and less susceptible to Mn. For D. magna, the Mn and DDT coexposure was significantly more toxic than any of the singly tested compounds. When D. magna was exposed to sediment pore water, no association was found between the Mn content in the samples and the observed toxicity. Preliminary particle analysis of pore water showed different compounds of Mn, which apparently were not in bioavailable form.

Protein kinase C-mediated inhibition of recombinant T-type Cav3.2 channels by neurokinin 1 receptors.

The voltage-activated T-type calcium channel (CaV3.2) and the G protein-coupled neurokinin 1 (NK1) receptor are expressed in peripheral tissues and in central neurons, in which they participate in diverse physiological processes, including neurogenic inflammation and nociception. In the present report, we demonstrate that recombinant CaV3.2 channels are reversibly inhibited by NK1 receptors when both proteins are transiently coexpressed in human embryonic kidney 293 cells. We found that the voltage-dependent macroscopic properties of CaV3.2 currents were unaffected during NK1 receptor-mediated inhibition. However, inhibition was attenuated in cells coexpressing either the dominant-negative Gαq Q209L/D277N or the regulator of G protein signaling (RGS) proteins 2 (RGS2) and 3T (RGS3T), which are effective antagonists of Gαq/11. By contrast, inhibition was unaffected in cells coexpressing human rod transducin (Gαt), which buffers Gβγ. Channel inhibition was blocked by 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and bisindolylmaleimide I, selective inhibitors of phospholipase Cβ and protein kinase C (PKC), respectively. Inhibition was occluded by application of the PKC activator phorbol-12-myristate-13-acetate. Altogether, these data indicate that NK1 receptors inhibit CaV3.2 channels through a voltage-independent signaling pathway that involves Gαq/11, phospholipase Cβ, and PKC. Our results provide novel evidence regarding the mechanisms underlying T-type calcium channel modulation by G protein-coupled receptors. Functional coupling between CaV3.2 channels and NK1 receptors may be relevant in neurogenic inflammation, neuronal rhythmogenesis, nociception, and other physiological processes.

[Ca2+]i changes in guinea pig tracheal smooth muscle cells in culture: effects of Na+ and ouabain

The objective of this work was to confirm that the contractile effects of ouabain and Na+-free solutions in guinea pig tracheal rings are associated with increments in the cytosolic free Ca2+ concentration ([Ca2+]i) in cultured tracheal smooth muscle (TSM) cells. Cultured cells were α-actin positive. Histamine (50 μM) and Na+-free solution elicited a transient increase in [Ca2+]i, while the responses to thapsigargin (1 μM) and ouabain (1 mM) were long lasting. However, carbachol (10, 200, and 500 mM) and high K+-solution produced no effect on [Ca2+]i, suggesting that cultured guinea pig TSM cells display a phenotype change but maintain some of the tracheal rings physiological properties. The transient rise in [Ca2+]i in response to the absence of extracellular Na+ and the effect of ouabain may indicate the participation of the Na+/Ca2+ exchanger (NCX) in the regulation of [Ca2+]i.ResumenEl objectivo del presente trabajo es el de confirmar que los efectos contráctiles producidos por la ouabaína y la ausencia de Na+ en la solución en anillos de traquea de cobayo, se asocian a incrementos en la concentración libre de Ca2+ citosólico ([Ca2+]i) en células cultivadas de músculo liso traqueal de cobayo. Las células usadas en los experimentos eran positivas para α-actina de músculo liso. La histamina (50 μM) y las soluciones carentes de Na+ producen un incremento transitorio en la [Ca2+]i mientras que la thapsigargina (1 μM) y la ouabaína (1 nM) dan lugar a un incremento de larga duración. Sin embargo, el carbacol (10, 200 y 500 μM) y soluciones con alta concentración de K+ no producen cambios en la [Ca2+]i. Estos resultados sugieren que, en nuestras condiciones experimentales, las células cultivadas de músculo liso traqueal presentan cambios en su fenotipo, pero conservan algunas de las propiedades fisiológicas observadas en los anillos traqueales. El incremento transitorio de la [Ca2+]i producido por la ausencia de Na+ extracellar y el efecto de la ouabaína sugieren la participación del intercambiador Na+/Ca2+ (NCX) en la regulación de la [Ca2+]i.

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca2+ in an in vitro experimental model in which Ca2+ stores had been depleted and their refilling had been blocked by thapsigargin. 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100 µmol/L SKF‐96365 and 100 µmol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca2+ channels CaV1.2 channels, namely 1 µmol/L nifedipine and 10 µmol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20 µmol/L niflumic acid, a non‐selective inhibitor of Ca2+‐dependent Cl− channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca2+‐activated K+ (BK) channels. 3 Furthermore, contraction was significantly inhibited when Na+ in the bathing solution was replaced by N‐methyl–d‐glucamine (NMDG+) to 39.9 ± 7.2% of control, but not when it was replaced by Li+ (114.5 ± 24.4% of control). In addition, when Na+ had been replaced by NMDG+, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na+ had been replaced by Li+ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control). 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and CaV1.2 channels in the contractions induced by the re‐addition of Ca2+ to the solution bathing guinea‐pig tracheal rings under conditions of Ca2+‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na+, suggesting a role for SOCC in mediating the Na+ influx.

Regulation of Kv7.2/Kv7.3 channels by cholesterol: Relevance of an optimum plasma membrane cholesterol content

Kv7.2/Kv7.3 channels are the molecular correlate of the M-current, which stabilizes the membrane potential and controls neuronal excitability. Previous studies have shown the relevance of plasma membrane lipids on both M-currents and Kv7.2/Kv7.3 channels. Here, we report the sensitive modulation of Kv7.2/Kv7.3 channels by membrane cholesterol level. Kv7.2/Kv7.3 channels transiently expressed in HEK-293 cells were significantly inhibited by decreasing the cholesterol level in the plasma membrane by three different pharmacological strategies: methyl-β-cyclodextrin (MβCD), Filipin III, and cholesterol oxidase treatment. Surprisingly, Kv7.2/Kv7.3 channels were also inhibited by membrane cholesterol loading with the MβCD/cholesterol complex. Depletion or enrichment of plasma membrane cholesterol differentially affected the biophysical parameters of the macroscopic Kv7.2/Kv7.3 currents. These results indicate a complex mechanism of Kv7.2/Kv7.3 channels modulation by membrane cholesterol. We propose that inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol depletion involves a loss of a direct cholesterol-channel interaction. However, the inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol enrichment could include an additional direct cholesterol-channel interaction, or changes in the physical properties of the plasma membrane. In summary, our results indicate that an optimum cholesterol level in the plasma membrane is required for the proper functioning of Kv7.2/Kv7.3 channels.

Inhibition of CaV2.3 channels by NK1 receptors is sensitive to membrane cholesterol but insensitive to caveolin-1

Voltage-gated, CaV2.3 calcium channels and neurokinin-1 (NK1) receptors are both present in nuclei of the central nervous system. When transiently coexpressed in human embryonic kidney (HEK) 293 cells, CaV2.3 is primarily inhibited during strong, agonist-dependent activation of NK1 receptors. NK1 receptors localize to plasma membrane rafts, and their modulation by Gq/11 protein-coupled signaling is sensitive to plasma membrane cholesterol. Here, we show that inhibition of CaV2.3 by NK1 receptors is attenuated following methyl-β-cyclodextrin (MBCD)-mediated depletion of membrane cholesterol. By contrast, inhibition of CaV2.3 was unaffected by intracellular diffusion of caveolin-1 scaffolding peptide or by overexpression of caveolin-1. Interestingly, MΒCD treatment had no effect on the macroscopic biophysical properties of CaV2.3, though it significantly decreased whole-cell membrane capacitance. Our data indicate that (1) cholesterol supports at least one component of the NK1 receptor-linked signaling pathway that inhibits CaV2.3 and (2) caveolin-1 is dispensable within this pathway. Our findings suggest that NK1 receptors reside within non-caveolar membrane rafts and that CaV2.3 resides nearby but outside the rafts. Raft-dependent modulation of CaV2.3 could be important in the physiological and pathophysiological processes in which these channels participate, including neuronal excitability, synaptic plasticity, epilepsy, and chronic pain.

Cytosolic pH gradients in cultured neuronal cell lines studied by laser scanning confocal microscopy, real-time confocal microscopy, and spectral imaging microscopy

Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.