Glial cells exhibit distinct cellular domains, somata, and filopodia. Thus the cytoplasmic pH (pH(cyt)) and/or the behavior of the fluorescent ion indicator might be different in these cellular domains because of distinct microenvironments. To address these issues, we loaded C6 glial cells with carboxyseminaphthorhodafluor (SNARF)-1 and evaluated pH(cyt) using spectral imaging microscopy. This approach allowed us to study pH(cyt) in discrete cellular domains with high temporal, spatial, and spectral resolution. Because there are differences in the cell microenvironment that may affect the behavior of SNARF-1, we performed in situ titrations in discrete cellular regions of single cells encompassing the somata and filopodia. The in situ titration parameters apparent acid-base dissociation constant (pK'(a)), maximum ratio (R(max)), and minimum ratio (R(min)) had a mean coefficient of variation approximately six times greater than those measured in vitro. Therefore, the individual in situ titration parameters obtained from specific cellular domains were used to estimate the pH(cyt) of each region. These studies indicated that glial cells exhibit pH(cyt) heterogeneities and pH(cyt) oscillations in both the absence and presence of physiological HCO(3)(-). The amplitude and frequency of the pH(cyt) oscillations were affected by alkalosis, by acidosis, and by inhibitors of the ubiquitous Na(+)/H(+) exchanger- and HCO(3)(-)-based H(+)-transporting mechanisms. Optical imaging approaches used in conjunction with BCECF as a pH probe corroborated the existence of pH(cyt) oscillations in glial cells.
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 alpha-actin positive. Histamine (50 microM) and Na(+)-free solution elicited a transient increase in [Ca2+]i, while the responses to thapsigargin (1 microM) 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.
Stimulation of macrophages with extracellular ATP or Bz‐ATP induces Reactive Oxygen Species (ROS) production. We tested the idea that in J774 macrophages activation of P2X7 receptors (P2X7R) by ATP leads to NADPH oxidase assembly and ROS production via Ca2+ entry and PKC activation. We used whole‐cell voltage clamp to measure P2X7R‐mediated currents, Fura‐2 to determine [Ca2+]i, 2′,7‐dichlorodihidrofluorescein to assay ROS production, PCR and western blot for P2X7R expression and phospho‐specific antibodies to determine activation of PKC isoforms. Stimulating J774 cells with Bz‐ATP (100 μM) induced an inward current at −80 mV that was sensitive to A438079 (10 μM), a blocker specific for P2X7R. ROS production induced by Bz‐ATP was also inhibited by A438079, PKC inhibitors and by removing extracellular Ca2+. Similarly, the increase in [Ca2+]i seen with Bz‐ATP was inhibited by zero external Ca2+ and A438079. Bz‐ATP induced PKC α activation. Importantly, Bz‐ATP induced a transient activation of PKC δ, while PKC ζ displayed an oscillatory pattern and was inhibited by A438079, confirming the participation of P2X7R. In summary, we propose that purinergic stimulation in macrophages induced ROS production due to Ca2+ entry via P2X7R and subsequent PKC activation which may, in turn, induce NADPH oxidase assembly and activation.Supported by grants 79897 and 105457 to JA and P. P‐C
The Na(+)/H(+) exchanger has been the only unequivocally demonstrated H(+)-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl(-)-H(+) symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl(-)-H(+) symporter is NO(3)(-) > Br(-) > Cl(-) >> I(-) = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 muM inhibits the gluconate-dependent alkalinization by 30 +/- 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization.
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