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Extracellular pH (extracellular + ph)
Selected AbstractsPurinergic activation of a leak potassium current in freshly dissociated myocytes from mouse thoracic aortaACTA PHYSIOLOGICA, Issue 2 2009S. Hayoz Abstract Aim:, Exogenous ATP elicits a delayed calcium-independent K+ current on freshly isolated mouse thoracic aorta myocytes. We investigated the receptor, the intracellular pathway and the nature of this current. Methods:, The patch-clamp technique was used to record ATP-elicited delayed K+ current in freshly dissociated myocytes. Results:, ATP-elicited delayed K+ current was not inhibited by a ,cocktail' of K+ channel blockers (4-AP, TEA, apamin, charybdotoxin, glibenclamide). The amplitude of the delayed K+ current decreased after the reduction of extracellular pH from 7.4 to 6.5. These two characteristics suggest that this current could be carried by the TASK subfamily of ,twin-pore potassium channels' (K2P). Purinergic agonists including dATP, but not ADP, activated the delayed K+ current, indicating that P2Y11 is the likely receptor involved in its activation. The PKC activator phorbol ester 12,13-didecanoate stimulated this current. In addition, the PKC inhibitor Gö 6850 partially inhibited it. Real-time quantitative PCR showed that the genes encoding TASK-1 and TASK-2 are expressed. Conclusion:, Our results indicate that blocker cocktail-insensitive delayed K+ current in freshly dissociated aortic myocytes is probably carried by the TASK subfamily of twin-pore channels. [source] Gap junctional coupling between progenitor cells at the retinal margin of adult goldfishDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2001Fuminobu Tamalu Abstract We prepared living slice preparations of the peripheral retina of adult goldfish to examine electrical membrane properties of progenitor cells at the retinal margin. Cells were voltage-clamped near resting potential and then stepped to either hyperpolarizing or depolarizing test potentials using whole-cell voltage-clamp recordings. Electrophysiologically examined cells were morphologically identified by injecting both Lucifer Yellow (LY) and biocytin. All progenitor cells examined (n = 37) showed a large amount of passively flowing currents of either sign under suppression of the nonjunctional currents flowing through K+ and Ca2+ channels in the cell membrane. They did not exhibit any voltage-gated Na+ currents. Cells identified by LY fills were typically slender. As the difference between the test potential and the resting potential increased, 13 out of 37 cells exhibited symmetrically voltage- and time-dependent current decline on either sign at the resting potential. The symmetric current profile suggests that the current may be driven and modulated by the junctional potential difference between the clamping cell and its neighbors. The remaining 24 cells did not exhibit voltage dependency. A gap junction channel blocker, halothane, suppressed the currents. A decrease in extracellular pH reduced coupling currents and its increase enhanced them. Dopamine, cAMP, and retinoic acid did not influence coupling currents. Injection of biocytin into single progenitor cells revealed strong tracer coupling, which was restricted in the marginal region. Immature ganglion cells closely located to the retinal margin exhibited voltage-gated Na+ currents. They did not reveal apparent tracer coupling. These results demonstrate that the marginal progenitor cells couple with each other via gap junctions, and communicate biochemical molecules, which may subserve or interfere with cellular differentiation. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 204,214, 2001 [source] Sodium/bicarbonate cotransporter NBCn1/slc4a7 increases cytotoxicity in magnesium depletion in primary cultures of hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2009Deborah S. Cooper Abstract Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO3 transport provides protection against damage or injury in cardiac ischemia. In this study, we examined the contribution of the sodium/bicarbonate cotransporter NBCn1 (slc4a7) to cytotoxicity in cultured hippocampal neurons of rats. In neurons exposed to extracellular pH (pHo) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pHo 7.4. At pHo 6.5, the intracellular pH of neurons was ,1 unit lower than that at pH 7.4. Immunochemistry showed a marked increase in NBCn1 immunofluorescence in plasma membranes and cytosol of the soma as well as in dendrites, at pHo 6.5. NBCn1 expression also increased by 40% in a prolonged Mg2+ -free incubation at normal pHo. Knockdown of NBCn1 in neurons had negligible effect on cell viability. The effect of NBCn1 knockdown on cytotoxicity was then determined by exposing neurons to 0.5 mm glutamate for 10 min and measuring lactate dehydrogenase (LDH) release from neurons. Compared to normal incubation (pHo 7.2 for 6 h) after glutamate exposure, acidic incubation (pHo 6.3 for 6 h) reduced cytotoxicity by 75% for control neurons and 78% for NBCn1-knockdown neurons. Thus, both controls and knockdown neurons showed acidic protection from cytotoxicity. However, in Mg2+ -free incubation after glutamate exposure, NBCn1 knockdown progressively attenuated cytotoxicity. This attenuation was unaffected by acidic preincubation before glutamate exposure. We conclude that NBCn1 has a dynamic upregulation in low pHo and Mg2+ depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg2+ -free conditions. [source] Intracellular pH homeostasis in the filamentous fungus Aspergillus nigerFEBS JOURNAL, Issue 14 2002Stephan J. A. Hesse Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ,pH over the cytoplasmic membrane of 6.6,6.7 was reached (pHex 0.7,0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ,pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ,pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m -chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ,pH. Nonlethal levels of the metabolic inhibitor azide (N3,, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively). [source] OCTN3: A Na+ -independent L -carnitine transporter in enterocytes basolateral membraneJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005J.M. Durán L -carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+ -free conditions chicken enterocytes take up L -carnitine until the cell to medium L -carnitine ratio is 1. This uptake was inhibited by L -carnitine, D -carnitine, ,-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L - 3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+ -independent, (ii) trans-stimulated by intravesicular L -carnitine, and (iii) cis-inhibited by TEA and cold L -carnitine. L - 3H-carnitine efflux from L - 3H-carnitine preloaded enterocytes was also Na+ -independent, and trans-stimulated by L -carnitine, D -carnitine, ,-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L -carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl- L -carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L -carnitine transport activity measured in the three experimental conditions. © 2004 Wiley-Liss, Inc. [source] Epidermal growth factor stimulates proton efflux from chondrocytic cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Kevin E.H. Lui Proton efflux from chondrocytes alters the extracellular pH and ionic composition of cartilage, and influences the synthesis and degradation of extracellular matrix. Epidermal growth factor (EGF) promotes chondrocyte proliferation during skeletal development and accumulates in the synovial fluid in rheumatoid arthritis. The purpose of this study was to investigate the effect of EGF on proton efflux from chondrocytes. When monitored using a Cytosensor microphysiometer, EGF was found to rapidly activate proton efflux from CFK2 chondrocytic cells and rat articular chondrocytes. The actions of EGF were concentration-dependent with half-maximal effects at 0.3,0.7 ng/ml. Partial desensitization and time-dependent recovery of the response were observed following repeated exposures to EGF. EGF-induced proton efflux was dependent on extracellular glucose, and inhibitors of Na+/H+ exchange (NHE) markedly attenuated the initial increase in proton efflux. The response was diminished by inhibitors of phosphatidylinositol 3-kinase and phospholipase C, but not by inhibitors of MEK (MAPK/ERK kinase) or protein kinase A or C. Thus, EGF-induced proton efflux involves glucose metabolism and NHE, and is regulated by a discrete subset of EGF-activated signaling pathways. In vivo, proton efflux induced by EGF may lead to an acidic environment, enhancing turnover of cartilage matrix during development and in rheumatoid arthritis. © 2002 Wiley-Liss, Inc. [source] Respiration of steelhead trout sperm: sensitivity to pH and carbon dioxideJOURNAL OF FISH BIOLOGY, Issue 1 2003R. L. Ingermann Steelhead trout Oncorhynchus mykiss sperm held in seminal plasma or sperm-immobilizing buffer (pH 8·6) at 10° C consumed O2 at the rate of c. 2 nmol O2 min,1 10,9 sperm; the rate of O2 consumption was not different in sperm held for 4 or 24 h. Decreasing the extracellular pH from 8·5 to 7·5 either by diluting semen with buffer titrated with HCl or by increasing the partial pressure of CO2 in the incubation atmosphere resulted in c. a 40% decrease in the rate of sperm respiration. The data did not, however, support the hypothesis that the precipitous reduction in the capacity for sperm motility that occurs as external pH is reduced is a result of a decrease in cellular metabolism. The rate of O2 consumption of freshly collected semen from different males was not correlated to cellular ATP content or to the proportion of sperm that were motile upon activation; the initial ATP content and sperm motility were positively correlated. The rate of O2 consumption was not significantly increased following sperm activation or by the addition of an uncoupler of oxidative phosphorylation, carbonyl cyanide p -trifluoromethoxyphenylhydrazone, suggesting that these sperm have little, if any, capacity for increased oxidative metabolism. [source] Glial connexins and gap junctions in CNS inflammation and diseaseJOURNAL OF NEUROCHEMISTRY, Issue 3 2008Tammy Kielian Abstract Gap junctions facilitate direct cytoplasmic communication between neighboring cells, facilitating the transfer of small molecular weight molecules involved in cell signaling and metabolism. Gap junction channels are formed by the joining of two hemichannels from adjacent cells, each composed of six oligomeric protein subunits called connexins. Of paramount importance to CNS homeostasis are astrocyte networks formed by gap junctions, which play a critical role in maintaining the homeostatic regulation of extracellular pH, K+, and glutamate levels. Inflammation is a hallmark of several diseases afflicting the CNS. Within the past several years, the number of publications reporting effects of cytokines and pathogenic stimuli on glial gap junction communication has increased dramatically. The purpose of this review is to discuss recent observations characterizing the consequences of inflammatory stimuli on homocellular gap junction coupling in astrocytes and microglia as well as changes in connexin expression during various CNS inflammatory conditions. [source] Resistance to acidic and alkaline environments in the endodontic pathogen Enterococcus faecalisMOLECULAR ORAL MICROBIOLOGY, Issue 5 2006K. Nakajo Background/aims:, This study aimed to investigate the biochemical mechanisms employed by the endodontic pathogen Enterococcus faecalis to confer acid- and alkali-resistance and to compare these with the mechanisms of representative oral streptococci. Methods:,E. faecalis JCM8728, Streptococcus mutans NCTC10449 and Streptococcus sanguinis ATCC10556 were used to assess both acid- and alkali-resistance by examining: (i) growth in complex media; (ii) stability of intracellular pH (pHin); (iii) cell durability to leakage of preloaded BCECF (2,,7,-bis-(2-carboxyethyl)-5,6-carboxy-fluorescein); and (iv) cell permeability to SYTOX-Green. Results:, Growth was initiated by E. faecalis at pH 4.0,11.0, by S. mutans at pH 4.0,9.0 and by S. sanguinis at pH 5.0,9.0. The pHin was similar to the extracellular pH in S. mutans and S. sanguinis at pH 5,10, while the pHin of E. faecalis was maintained at approximately 7.5,8.5 when extracellular pH was 7.5,10 and was maintained at levels equivalent to the extracellular pH when pH < 7.5. Cell membranes of E. faecalis were resistant to BCECF leakage when extracellular pH was 2.5,12 and to SYTOX-Green permeability at pH 4,10. The cell membrane durability to extracellular pH in E. faecalis was higher than that observed in the Streptococcus strains. Conclusion:, Compared to S. mutans, E. faecalis was found to be equally resistant to acid and more resistant to alkalis. The results suggest that pH-resistance in E. faecalis is attributed to membrane durability against acid and alkali, in addition to cell membrane-bound proton-transport systems. These characteristics may account for why E. faecalis is frequently isolated from acidic caries lesions and from persistently infected root canals where calcium hydroxide medication is ineffective. [source] Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifoliaNEW PHYTOLOGIST, Issue 3 2007Hai-Yong Qu Summary ,,The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca2+]cyt) is essential for pollen-tube growth. Local Ca2+ influx mediated by Ca2+ -permeable channels plays a key role in maintaining this [Ca2+]cyt gradient. ,,Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. ,,We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba2+) , Ca2+ > magnesium (Mg2+) > strontium (Sr2+) > manganese (Mn2+). This channel conductance was selective for Ca2+ over chlorine (Cl,) (relative permeability PCa/PCl = 14 in 10 mm extracellular Ca2+). We also showed that the channel was inhibited by the Ca2+ channel blockers lanthanum (La3+) and gadolinium (Gd3+). Furthermore, channel activity depended on extracellular pH and pollen viability. ,,We propose that the Ca2+ -permeable channel is likely to play a role in mediating Ca2+ influx into the growing pollen tubes to maintain the [Ca2+]cyt gradient. [source] pH-Dependent Spectral Properties of HpIX, TPPS2a, mTHPP and mTHPC,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2001Beata, underlíková ABSTRACT Lower extracellular pH in tumors as compared to normal tissues has been proposed to be a factor contributing to the tumor selective uptake of several photosensitizers. Therefore, the pH dependence of absorption and fluorescence spectral properties of four different drugs relevant for photodynamic therapy (hematoporphyrin IX [HpIX], disulfonated meso -tetraphenylporphine [TPPS2a], meso -tetra(3-hydroxyphenyl)porphine [mTHPP] and meso -tetra(3-hydroxyphenyl)chlorin [mTHPC]) has been examined. Spectral analysis of the dyes dissolved in phosphate buffered saline (PBS) indicates pH-dependent modification in the physiologically important region (6.0,8.0) only in the case of HpIX. This modification is probably related to the protonation of carboxylic groups. Spectral changes of HpIX in PBS observed at acidic pH values <5, as well as those of the rest of the drugs (inflection points of titration curves occurred at about 5.1, 3.8 and 2.4 for TPPS2a, mTHPP and mTHPC, respectively), are likely to be due to the protonation of imino nitrogens. The tumor localizing properties of mTHPP and mTHPC reported in the literature appear to be due to factors other than pH-dependent changes in the lipophilicity of the drugs. [source] Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protonsTHE JOURNAL OF PHYSIOLOGY, Issue 7 2009María Isabel Niemeyer The ClC transport protein family comprises both Cl, ion channel and H+/Cl, and H+/NO3, exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl, passage and in addition serves as a staging post for H+ exchange. This same conserved glutamate acts as a gate to regulate Cl, flow in ClC channels. The activity of ClC-2, a genuine Cl, channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than ,7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl, acts as a voltage-independent modulator, as though regulating the pKa of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl, efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts. [source] Effect of pH on the development of acrosomal responsiveness of human spermANDROLOGIA, Issue 2 2007N. L. Cross Summary Human sperm incubated in vitro gradually become responsive to inducers of the acrosome reaction. The roles of constituents of the incubation medium are not well understood. These experiments tested the effect of the extracellular pH on sperm acrosomal responsiveness. Sperm were incubated 24 h in media with pH varying between 6.7 and 7.6 and then exposed to progesterone to determine the number of sperm that had become acrosomally responsive. The number of responsive sperm was very low following incubation at pH 6.7,7.0, and increased with the pH over the range 7.0,7.6. Sperm incubated at low pH were not damaged as assessed by motility or viability, and if they were transferred to medium of high pH for 8 h, the inhibition of acrosomal responsiveness was reversed. Inhibition of acrosomal responsiveness at low pH was not due to impaired loss of sperm cholesterol, but was correlated with a reduced intracellular pH. The inhibition of acrosomal responsiveness by media of low pH may result from preventing the normal capacitation-related rise in intracellular pH. [source] Control of Cell Volume in Skeletal MuscleBIOLOGICAL REVIEWS, Issue 1 2009Juliet A. Usher-Smith Abstract Regulation of cell volume is a fundamental property of all animal cells and is of particular importance in skeletal muscle where exercise is associated with a wide range of cellular changes that would be expected to influence cell volume. These complex electrical, metabolic and osmotic changes, however, make rigorous study of the consequences of individual factors on muscle volume difficult despite their likely importance during exercise. Recent charge-difference modelling of cell volume distinguishes three major aspects to processes underlying cell volume control: (i) determination by intracellular impermeant solute; (ii) maintenance by metabolically dependent processes directly balancing passive solute and water fluxes that would otherwise cause cell swelling under the influence of intracellular membrane-impermeant solutes; and (iii) volume regulation often involving reversible short-term transmembrane solute transport processes correcting cell volumes towards their normal baselines in response to imposed discrete perturbations. This review covers, in turn, the main predictions from such quantitative analysis and the experimental consequences of comparable alterations in extracellular pH, lactate concentration, membrane potential and extracellular tonicity. The effects of such alterations in the extracellular environment in resting amphibian muscles are then used to reproduce the intracellular changes that occur in each case in exercising muscle. The relative contributions of these various factors to the control of cell volume in resting and exercising skeletal muscle are thus described. [source] Multiple P2X receptors on guinea-pig pelvic ganglion neurons exhibit novel pharmacological propertiesBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2001Yu Zhong Application of ATP and ,,,-methylene ATP (,,meATP) to voltage-clamped guinea-pig pelvic neurons produced three types of inward currents. A fast-desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly-desensitizing currents, and the remainder had biphasic responses. Slowly-desensitizing responses were characterized pharmacologically. The response to ,,meATP 100 ,M was 46±27% (range 0 , 100%) of that evoked by ATP 100 ,M in the same cell. Cross-desensitization indicated the presence of ,,meATP-sensitive and -insensitive receptors. The concentration-response curve for ,,meATP had an EC50 of 55 ,M, and a Hill coefficient of 0.99, while at the ,,meATP-insensitive receptor, ATP had an EC50 of 73 ,M, with a Hill coefficient of 1.78. The response to ,,meATP was blocked by pyridoxalphosphate-6-azophenyl-2,,4,-disulphonic acid (PPADS), suramin and Cibacron blue. However, the ,,meATP-insensitive receptor was inhibited by PPADS, but not by the other two antagonists. 2,- (or 3,-) O -trinitrophenyl-ATP was 10 times more potent in inhibiting responses to ,,meATP than to ATP (at the ,,meATP-insensitive receptor). Lowering extracellular pH potentiated responses to ,,meATP and ATP, while raising pH attenuated them. Co-application of Zn2+ (3 , 300 ,M) inhibited the responses to ,,meATP and ATP, with IC50 values of 286 and 60 ,M, respectively. In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X2 homomers, at least three distinct P2X receptors are present in guinea-pig pelvic neurons, probably homomeric P2X2, P2X3 and heteromeric P2X2/3 receptors. However, some of the novel pharmacological properties observed suggest that the guinea-pig P2X receptor subtypes may differ from their rat orthologues. British Journal of Pharmacology (2001) 132, 221,233; doi:10.1038/sj.bjp.0703778 [source] | |||||||||||||