Cytosolic pH measurements in single cardiac myocytes using carboxy-seminaphthorhodafluor-1

Blank, Paul S.; Silverman, Howard S.; Chung, Oliver; Hogue, Barbara A.; Stern, Michael D.; Hansford, Richard G.; Lakatta, Edward G.; Capogrossi, M. C.

doi:10.1152/ajpheart.1992.263.1.h276

Cited by 43 publications

(53 citation statements)

References 11 publications

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“…Using the 568-nm excitation line of a kryptonargon laser confocal microscope (MRC-1024, BioRad), the fluorescence emitted from the ROIs at wavelengths of 570/40 nm and 640/40 nm was collected by two independent photodetectors (255-gray levels), every 1 min in oocytes and every 10 s in N2A cells. After background subtraction, the mean fluorescence intensities at the two emissions were used to obtain the 640/570 ratios that were converted to pH values against the intracellular calibration pH (43). The accuracy of the pH dependence of the emission ratios was preserved in the pH i range of 5.5 to 8.5.…”

Section: Methodsmentioning

confidence: 99%

Regulation of neuronal connexin-36 channels by pH

González‐Nieto

Gómez‐Hernández

Larrosa

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

104

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Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the Cand N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pH i since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions. alkalosis ͉ electrical synapses ͉ intracellular pH ͉ pH gating ͉ gap junction

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Section: Methodsmentioning

confidence: 99%

Regulation of neuronal connexin-36 channels by pH

González‐Nieto

Gómez‐Hernández

Larrosa

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

104

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“…Serum deprivation alone induces apoptosis only after 24 h, and the dose of ultraviolet (UV) irradiation that is being used does not induce apoptosis in the presence of serum, whereas the combination induces apoptosis after 5 h (Kennedy et al 1999). Thus, following serum deprivation/UV-exposure, the cells were trypsinized, loaded with the pH-sensitive fluorophore SNARF1, and analyzed by fluorescence-activated cell sorter (FACS; Blank et al 1992). The results (Fig.…”

Section: Akt/pkb Prevents the Cytosolic Acidification And Mitochondrimentioning

confidence: 99%

“…For measuring the pH i , cells were trypsinized and loaded with 2 µM SNARF1-acetate (Molecular Probes) for 8 min at room temperature and subjected to FACS analysis. Calibration for each cell line was performed by adding 10 µM nigericin to equilibrate the pH i with controlled pH of the extracellular medium (40 mM HEPES, 115 mM KCl, 1 mM MgCl 2 , pH 6.0-8.0; Blank et al 1992).…”

Section: Facs Analysismentioning

confidence: 99%

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

Gottlob¹,

Majewski²,

Kennedy³

et al. 2001

Genes Dev.

866

704

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The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival. Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a PT pore component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release. However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to its metabolism. Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism. We provide evidence that such coupling serves as a downstream effector function for Akt. First, Akt increases mitochondria-associated hexokinase activity. Second, the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase. Finally, ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis. We therefore propose that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane. Extrinsic signals emanating from cell-surface growth factor and cytokine receptors are major determinants of mammalian cell survival. The transduction of these signals oppose both basal intrinsic proapoptotic activity, as well as external proapoptotic stimuli (Raff 1992;Raff et al. 1993). Analysis of downstream signaling pathways has shown that activation of the PI-3 kinase/Akt(PKB) pathway plays a major role in cell survival induced by cell surface receptors. Following the initial demonstration that activation of the serine/threonine kinase Akt promotes cell survival (Dudek et al. 1997; KauffmannZeh et al. 1997;Kennedy et al. 1997), mounting reports established Akt as a major determinant of cell survival. Akt has been reported to mediate cell survival by various growth factors and cytokines in a variety of cell types and blocks apoptosis induced by multiple apoptotic stimuli (for review, see Datta et al. 1999;Kandel and Hay 1999). Various specific targets of Akt have been proposed to mediate the antiapoptotic activity of Akt (for review, see Datta et al. 1999;Kandel and Hay 1999). However, because growth factors promote cell survival via maintenance of the metabolic function of mitochondria , it is likely that Akt exerts its effect through similar mechanisms, which may be more fundamental and generally conserved.In mammalian cells, apoptosis has been described as a multistep process that can be initiated by a variety of stimuli. Mitochondria play a major role in this process through the release of cytochrome c and other proapoptotic proteins that normally reside in the intermembrane space between the inner and outer mitochondrial membranes (for review, see Gross et al. 1999;Desagher and Martinou 2000). Cytochrome c release is considered an ea...

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“…Intracellular pH was measured by using a modification of the method of Blank et al (3). Cells were loaded with 20 M carboxyseminapthorhodofluor (SNARF) by incubation for 15 min at 37°C in medium supplemented with 10 mM HEPES (pH 7.4).…”

Section: Cellular Hmentioning

confidence: 99%

Beneficial effect of taurine depletion on osmotic sodium and calcium loading during chemical hypoxia

Schaffer

Solodushko

Kakhniashvili

2002

American Journal of Physiology-Cell Physiology

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Cellular sodium excess is cytotoxic because it increases both the intracellular osmotic load and intracellular calcium concentration ([Ca2+]i). Because sodium levels rise during hypoxia, it is thought to contribute to hypoxic injury. Thus the present study tested the hypothesis that taurine-linked reductions in [Na+]i reduce hypoxia-induced cell injury. Taurine depletion was achieved by exposing isolated neonatal cardiomyocytes to medium containing the taurine analog β-Alanine. As predicted, the β-Alanine-treated cell exhibited less hypoxia-induced necrosis and apoptosis than the control, as evidenced by less swelling, shrinkage, TdT-mediated dUTP nick end labeling staining, and accumulation of trypan blue. After 1 h of chemical hypoxia, [Na+]i was 3.5-fold greater in the control than the taurine-deficient cell. Although more taurine was lost from the control cell than from the β-Alanine-treated cell during hypoxia, the combined taurine and sodium osmotic load was lower in the β-Alanine-treated cell. Taurine deficiency also reduced the degree of hypoxia-induced calcium overload. Thus the observed resistance against hypoxia-induced necrosis and apoptosis is probably related to an improvement in sodium and calcium handling.

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Cytosolic pH measurements in single cardiac myocytes using carboxy-seminaphthorhodafluor-1

Cited by 43 publications

References 11 publications

Regulation of neuronal connexin-36 channels by pH

Regulation of neuronal connexin-36 channels by pH

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

Beneficial effect of taurine depletion on osmotic sodium and calcium loading during chemical hypoxia

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