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1.
Free Radic Res ; 48(10): 1190-9, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24985354

ABSTRACT

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was ∼ 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was ∼ 1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only ∼ 1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface.


Subject(s)
Eosinophils/metabolism , Ion Channels/metabolism , Neutrophils/metabolism , Respiratory Burst/physiology , Fluorescent Antibody Technique , Humans , Immunoblotting , Membrane Glycoproteins/metabolism , Microscopy, Confocal , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Oxygen Consumption/physiology , Reactive Oxygen Species/metabolism
2.
J Physiol ; 535(Pt 1): 95-106, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11507160

ABSTRACT

1. Cardiorespiratory response to acidosis is initiated by the carotid body. 2. The direct effect of extracellular pH (pH(o)) on the chloride currents of isolated chemoreceptor cells of the rat carotid body was investigated using the whole-cell patch-clamp technique. 3. On applying intra- and extracellular solutions with a symmetrical high-Cl(-) content and with the monovalent cations replaced with membrane-impermeant ones, an inwardly rectifying Cl(-) current was found. 4. The current activated slowly and did not display any time-dependent inactivation. Current activation was present at membrane potentials negative to 0 mV (pH(o) = 7.0). 5. The current was activated by extracellular acidosis and inhibited by alkalosis in the physiologically relevant pH range of 7.0-7.8. 6. The current was reduced by 0.1 mM Cd2+ to the level of the leak current and by 1 mM anthracene-9-carboxylic acid (9-AC) to about 40 %, while 0.1 mM Ba2+ had no effect. 7. Application of 1 mM 9-AC caused a slow but statistically significant increase in the resting pH(i) (from a mean of 7.29 to 7.37 in 5 min) in clusters of chemoreceptor cells in CO(2)/HCO3(-)-buffered media as measured with carboxy-SNARF-1. 8. When membrane potential changes were estimated in the cell-attached mode, 1 mM 9-AC hyperpolarized three out of five tested cells (by 14 mV in average) incubated in CO(2)/HCO3(-)-buffered media. 9. In summary, chemoreceptor cells express an inwardly rectifying Cl(-) current, which is directly regulated by pH(o). The current may participate in intracellular acidification and membrane depolarization during acidic challenge.


Subject(s)
Carotid Body/physiology , Chemoreceptor Cells/physiology , Chloride Channels/physiology , Hydrogen/metabolism , Animals , Anthracenes/pharmacology , Cations, Divalent/pharmacology , Chloride Channels/antagonists & inhibitors , Electric Conductivity , Electrophysiology , Hydrogen-Ion Concentration , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Ions , Membrane Potentials/drug effects , Rats , Rats, Wistar
3.
Am J Physiol Cell Physiol ; 281(2): C700-8, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11443069

ABSTRACT

The two-pore-domain K(+) channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca(2+)-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M(1) muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5'-O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca(2+) concentration, and diacylglycerol) do not mediate the inhibition. Unlike the G(q)-coupled receptors, stimulation of the G(i)-activating M(2) muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-beta(2) (which is responsive also to G(i) beta gamma-subunits) renders M(2) receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.


Subject(s)
Nerve Tissue Proteins , Potassium Channel Blockers , Potassium Channels, Tandem Pore Domain , Type C Phospholipases/physiology , Androstadienes/pharmacology , Animals , Calcium/physiology , Electric Conductivity , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Female , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/physiology , Inositol 1,4,5-Trisphosphate/physiology , Oocytes/metabolism , Potassium Channels/physiology , Protein Kinase C/physiology , Protein-Tyrosine Kinases/physiology , Receptors, Cell Surface/physiology , Type C Phospholipases/metabolism , Wortmannin , Xenopus laevis
4.
Glia ; 34(1): 52-8, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11284019

ABSTRACT

The effect of pH(o) on plasma membrane chloride current of cultured rat cortical astrocytes was investigated using the whole-cell patch-clamp technique. In the presence of intra- and extracellular solutions with symmetrical high Cl(-) content and K(+) channel inhibitors, the cells exhibited an inwardly rectifying current. The current activated slowly at potentials negative to -40 mV and did not display time-dependent inactivation. The current was inhibited by 0.1 mM Cd(2+), 0.1 mM Zn(2+), 1 mM 9-anthracene-carboxylic acid, and 0.2 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not by 10 mM Ba(2+) or 3 mM Cs(+). Reversal potential of the current followed the chloride equilibrium potential and was not influenced by changes in K(+) or Na(+) concentration. The inwardly rectifying chloride current was augmented by extracellular acidosis and reduced by alkalosis. The pH sensitivity was most pronounced in the physiologically relevant pH(o) range of 6.9--7.9. Lowering pH to 6.4 induced no additional increase in steady-state current amplitude compared with pH(o) 6.9, but it substantially slowed the activation kinetics. According to its kinetic and pharmacological properties this chloride current is similar to that found in cultured rat astrocytes after long-term treatment with dibutyryl-cAMP, however, in our cultures it was consistently expressed without any treatment with the drug. Considering that astrocytes possess carbonic anhydrase and Cl(-)/HCO3(-) antiporter, this current may participate in the regulation of the interstitial and astrocyte pH.


Subject(s)
Astrocytes/physiology , Cerebral Cortex/physiology , Potassium Channels, Inwardly Rectifying , Potassium Channels/physiology , Animals , Anthracenes/pharmacology , Antiporters/drug effects , Antiporters/physiology , Astrocytes/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Chloride-Bicarbonate Antiporters , Chlorides/pharmacology , Hydrogen-Ion Concentration , Membrane Potentials/drug effects , Patch-Clamp Techniques , Potassium Channel Blockers , Potassium Channels/drug effects , Rats
5.
Endocrinology ; 141(5): 1705-10, 2000 May.
Article in English | MEDLINE | ID: mdl-10803580

ABSTRACT

The effect of osmotic changes on aldosterone production, [Ca2+]i and voltage-gated Ca2+ currents, was studied in cultured rat glomerulosa cells. Alteration of osmolarity by sucrose addition in the 250-330 mosM range did not influence aldosterone production per se, but it substantially affected K+-stimulated aldosterone production. Hyposmosis markedly increased the hormone response evoked by raising [K+] from 3.6 to 5 mM, whereas hyperosmosis had a mild decreasing effect. Cytoplasmic [Ca2+]i, measured in single glomerulosa cells, did not show detectable change in response to either hyposmotic or hyperosmotic exposure, but the [Ca2+]i signal evoked by elevation of [K+] to 5 mM was augmented in hyposmotic solution. The osmosensitivity of the transient (T)-type and long-lasting (L)-type voltage-gated Ca2+ currents was studied using the nystatin-perforated voltage-clamp technique. Lowering osmolarity to 250 mosM significantly increased the amplitude of the T-type current, and it had a transient augmenting effect on L-type current amplitude. Hyperosmotic solution (330 mosM) reduced L-type current amplitude but did not evoke significant change in T-type current. These results indicate that the responsiveness of rat glomerulosa cells to physiological elevation of [K+] is remarkably influenced by changes in osmolarity by means of modulating the function of voltage-gated Ca2+ channels.


Subject(s)
Aldosterone/biosynthesis , Zona Glomerulosa/metabolism , Animals , Calcium/metabolism , Calcium Channels/metabolism , Osmolar Concentration , Potassium Channels/metabolism , Rats , Rats, Wistar
6.
Neuropharmacology ; 39(6): 1029-36, 2000 Apr 03.
Article in English | MEDLINE | ID: mdl-10727713

ABSTRACT

Fluoxetine, an antidepressant which is used world-wide, is a prominent member of the class of selective serotonin re-uptake inhibitors. Recently, inhibition of voltage-gated Na(+) and K(+) channels by fluoxetine has also been reported. We examined the effect of fluoxetine on voltage-gated calcium channels using the patch-clamp technique in the whole-cell configuration. In hippocampal pyramidal cells, fluoxetine inhibited the low-voltage-activated (T-type) calcium current with an IC(50) of 6.8 microM. Fluoxetine decreased the high-voltage-activated (HVA) calcium current with an IC(50) between 1 and 2 microM. Nifedipine and omega-conotoxin GVIA inhibited the HVA current by 24% and 43%, respectively. Fluoxetine (3 microM), applied in addition to nifedipine or omega-conotoxin, further reduced the current. When fluoxetine (3 microM) was applied first neither nifedipine nor omega-conotoxin attenuated the remaining component of the HVA current. This observation indicates that fluoxetine inhibits both L- and N-type currents. In addition, fluoxetine inhibited the HVA calcium current in carotid body type I chemoreceptor cells and pyramidal neurons prepared from prefrontal cortex. In hippocampal pyramidal cells high K(+)-induced seizure-like activity was inhibited by 1 microM fluoxetine; the mean burst duration was shortened by an average of 44%. These results provide evidence for inhibition of T-, N- and L-type voltage-gated calcium channels by fluoxetine at therapeutically relevant concentrations.


Subject(s)
Antidepressive Agents, Second-Generation/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Fluoxetine/pharmacology , Hippocampus/drug effects , Ion Channel Gating , Selective Serotonin Reuptake Inhibitors/pharmacology , Action Potentials , Animals , Anticonvulsants/pharmacology , Carotid Body/cytology , Carotid Body/drug effects , Carotid Body/physiology , Cells, Cultured , Chemoreceptor Cells/cytology , Chemoreceptor Cells/drug effects , Chemoreceptor Cells/physiology , Embryo, Mammalian , Epilepsy/chemically induced , Epilepsy/physiopathology , Hippocampus/cytology , Hippocampus/physiology , Nerve Net/physiopathology , Potassium , Prefrontal Cortex/cytology , Prefrontal Cortex/drug effects , Prefrontal Cortex/physiology , Pyramidal Cells/drug effects , Pyramidal Cells/physiology , Rats
7.
Pflugers Arch ; 435(3): 429-31, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9426301

ABSTRACT

Elevation of extracellular potassium concentration by as little as some tenth of mM activates rat adrenal glomerulosa cells. In the present study some factors responsible for this high K+ sensitivity were examined. Using whole-cell voltage-clamp technique we found that both T-type and L-type voltage-dependent Ca2+ channels have very low threshold potential (-71 and -58 mV, resp.). By means of patch-clamp technique combined with single-cell fluorimetry we also provided evidence that the activation of Igl, a K+-activated inward rectifying current is associated with Ca2+ influx. Both the low activation threshold of voltage-dependent Ca2+ channels and the function of Igl contribute to the exceptional K+ sensitivity of the glomerulosa cells.


Subject(s)
Calcium Channels/physiology , Potassium/pharmacology , Zona Glomerulosa/drug effects , Zona Glomerulosa/physiology , Animals , Calcium/metabolism , Calcium Channels/drug effects , Cells, Cultured , Cytoplasm/metabolism , Electric Conductivity , Electrophysiology , Fluorometry , Membrane Potentials , Patch-Clamp Techniques , Potassium/administration & dosage , Rats , Rats, Wistar , Signal Transduction
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