Research ArticleCell Biology

Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions

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Sci. Signal.  30 Jan 2018:
Vol. 11, Issue 515, eaal2039
DOI: 10.1126/scisignal.aal2039
  • Fig. 1 Suppression of ouabain-sensitive 86Rb+ uptake by Ca2+ indicators in cultured mouse astrocytes.

    (A) Acetoxymethyl (AM) ester derivatives of Ca2+ indicators (Ca2+-insensitive and nonfluorescent), which cross cell membranes noninvasively, are cleaved by esterases in the cells. The ionized active form of Ca2+ indicators (which is Ca2+-sensitive) remains trapped within the cells. (B) Intracellular Fluo-4, Rhod-2, and BAPTA concentrations after the loading of various concentrations of AM indicators (n = 4 wells for each treatment). (C) Ouabain (1 mM)–sensitive 86Rb+ uptake without or with preincubation of Fluo-4 AM (1 to 9 μM), Rhod-2 AM (1 to 9 μM), or BAPTA AM (5 to 40 μM). The data were expressed as percent change from control and plotted against intracellular concentrations (n = 4 to 6 wells for each treatment). **P < 0.01, ****P < 0.0001 compared to vehicle control (0.2% DMSO). Means ± SEM are shown.

  • Fig. 2 Ca2+ indicators inhibited Na,K-ATPase in four different types of cultured cells.

    (A) Schematic diagram outlining the measurement of 86Rb+ uptake by cells and Na,K-ATPase activity in membrane preparation. (B to K) Ca2+ indicators (Fluo-4 AM, Rhod-2 AM, and Fura-2 AM) or BAPTA AM was preloaded for 30 min in cultured mouse astrocytes (B and G; n = 8 to 27 wells for each treatment), human astrocytes (C and H; n = 5 to 12 wells for each treatment), mouse cardiomyocytes (D and I; n = 6 to 12 wells for each treatment), human proximal tubule epithelial cells (E and J; n = 5 to 12 wells for each treatment), or mouse neurons (F and K; n = 5 wells for each treatment), and then 86Rb+ uptake (B to F) or Na,K-ATPase activity (G to K) was measured. Ouabain (1 mM) was used to identify the portion of 86Rb+ uptake or ATP hydrolysis mediated by the Na,K-ATPase. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to vehicle control (0.2% DMSO). Means ± SEM are shown.

  • Fig. 3 Ca2+ indicators directly inhibited Na,K-ATPase and diminished the GPCR-mediated enhancement of K+ uptake.

    (A) Schematic diagram outlining the ATPase assay in membrane preparations using salt and AM forms of BAPTA or Rhod-2. (B) Effect of BAPTA tetrapotassium salt (100 μM) to membrane preparations on Na,K-ATPase activity, compared to the preincubation of BAPTA AM (20 μM) in cultured mouse astrocytes (n = 8 to 14 wells for each treatment). (C) Effect of Rhod-2 tripotassium salt (10 μM) on ouabain-sensitive ATP hydrolysis in membrane preparation compared to the preincubation of Rhod-2 AM (2 μM) in cultured human astrocyte (n = 8 wells for each treatment). (D) Na,K-ATPase activity measurement in the absence or presence of BAPTA tetrapotassium salt (100 μM) or low–Ca2+ affinity 5,5′-dibromo BAPTA tetrapotassium salt (100 μM) in the zero Ca2+ assay buffer in cultured mouse astrocytes (n = 8 to 14 wells for each treatment). *P < 0.05, **P < 0.01, ****P < 0.0001 compared to vehicle control (B and D, 0.1% DMSO; C, 0.04% DMSO). Means ± SEM are shown.

  • Fig. 4 Ca2+ indicators inhibited Na,K-ATPase current in pyramidal neurons of hippocampal CA1 region.

    (A) Schematic diagram outlining the measurement of Na,K-ATPase current in a pyramidal neuron. (B) Na,K-ATPase current was measured by the application of 0.5 mM strophanthidin for 2 min. (C to G) Baseline recording of the holding current using vehicle without strophanthidin (B) (88). The membrane-impermeable Ca2+ indicators Fluo-4 (D), Rhod-2 (E), and Fura-2 (F) or the membrane-impermeable BAPTA (G) was included in the recording pipette to measure their effect on Na,K-ATPase current. Ten minutes after breaking into the neuron, Ca2+ indicator loading was detectable [inset in (D); scale bar, 10 μm]. (H) Na,K-ATPase current density was plotted and analyzed using one-way analysis of variance (ANOVA) (n = 4 to 5 mice; **P < 0.01, ***P < 0.001, ****P < 0.0001). Means ± SEM are shown.

  • Fig. 5 Ca2+ indicators induced swelling and cell death of mouse astrocytes.

    (A) Assessment of viable cell numbers by counting. Cultured mouse astrocytes were treated with increasing concentrations of ouabain (0 to 5 mM), DMSO (0 to 0.2%), Ca2+ indicators (1 to 10 μM in 0.2% DMSO), and BAPTA AM (5 to 40 μM in 0.2% DMSO) for 30 min, and cell viability was examined at 24 hours (n = 8 wells for each treatment). *P < 0.05, **P < 0.01 compared to no-treatment control. (B) Same as in (A), except the loading time was extended to 2 hours (n = 5 to 6 wells for each treatment). *P < 0.05, ***P < 0.001 compared to no-treatment control. (C) LDH release was examined immediately after 2 hours of treatment with the indicators (n = 6 wells for each treatment). *P < 0.05, **P < 0.001, ****P < 0.0001 compared to no-treatment control. (D) Relative increases in the diameter of viable cultured mouse astrocytes at 24 hours induced by treatment of various concentrations of ouabain, DMSO, Ca2+ indicators, and BAPTA AM for 2 hours (n = 4 wells for each treatment). (E) Effect of loading of BAPTA AM (20 μM) on the ability of TFLLR-NH2 (30 μM) and FMRF (15 μM) to enhance 86Rb+ uptake in cultured wild-type or MrgA1+/− mouse astrocytes, respectively (n = 4 to 38 wells for each treatment). n.s. (not significant), *P < 0.05, **P < 0.01 compared to (−) BAPTA AM of control group (0.1% DMSO). (F) Effect of preloading Rhod-2 AM (4.5 μM) and BAPTA AM (20 μM) on the ability of ATP (100 μM) to increase 86Rb+ uptake in cultured mouse astrocytes (n = 4 to 38 wells for each treatment). n.s., *P < 0.05 compared to (−) ATP of each group, #P < 0.05 compared to (+) ATP of control group (0.1% DMSO). Means ± SEM are shown.

  • Fig. 6 Ca2+ indicators altered glucose uptake and lactate release of cultured mouse astrocytes and neurons.

    (A) Effect of loading Fluo-4 AM, Fura-2 AM, and BAPTA AM on [3H]-2-deoxyglucose ([3H]-2-DG) uptake by mouse astrocyte cultures and neuronal cultures (n = 4 to 12 wells for each treatment). (B) Effect of Rhod-2 AM on [3H]-2-DG uptake by mouse astrocyte and neuronal cultures (n = 4 to 8 wells for each treatment). *P < 0.05, **P < 0.01, ***P < 0.001 compared to vehicle control (0.2% DMSO; A and B). (C) Effect of mitochondrial oxidative metabolism inhibition with sodium cyanide (NaCN; 100 μM; n = 8 wells for each treatment) or glycolysis inhibition with iodoacetate (IAA; 300 μM; n = 8 to 16 wells for each treatment) on [3H]-2-DG uptake by cultured mouse astrocytes and neurons in the presence or absence of Rhod-2 AM (2 μM). (D) Effect of inhibition of mitochondrial oxidative metabolism by Rhod-2 AM (2 μM) and NaCN (100 μM) or IAA (300 μM) on lactate release by cultured mouse astrocytes (n = 4 to 8 wells for each treatment). *P < 0.05, **P < 0.01, ****P < 0.001 compared to vehicle control (0.04% DMSO); #P < 0.01, ####P < 0.01 compared to (+) Rhod-2 AM of control group (C and D). (E) Images of astrocytes loaded with MitoTracker (20 nM) (top) and Rhod-2 AM (2 and 10 μM) (bottom). Scale bar, 20 μm. White arrows correspond to mitochondrial morphological changes. Means ± SEM are shown.

  • Fig. 7 Ca2+ indicator disrupts astrocyte K+ uptake and tissue environments in vivo.

    (A) Schematic diagram outlining the application of Ca2+ indicator and sample collection using microdialysis in freely moving mice. After overnight probe equilibration, samples were collected every 15 min before, during, and after infusion of artificial cerebrospinal fluid (aCSF) containing Fluo-4 AM, Rhod-2 AM, or vehicle. (B) Image of acutely prepared coronal brain section confirming the delivery of Rhod-2 AM into tissue. Dashed circle indicates position of microdialysis probe. Scale bar, 500 μm. (C) Comparison of extracellular K+ measurements with Fluo-4 AM, Rhod-2 AM, or control application in vivo (n = 6 to 8 mice for each treatment; *P < 0.05, **P < 0.01 compared to control group). BL, baseline. (D) Comparison of ATP and glycerol measurements with Fluo-4 AM, Rhod-2 AM, or control application in vivo (n = 6 to 8 mice for each treatment; n.s., *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group). Means ± SEM are shown.

  • Fig. 8 Expression of GCaMP3 does not affect K+ uptake of mouse astrocytes.

    (A) Representative images of astrocytes loaded with Rhod-2 AM (4.5 μM) or prepared from GFAP-Cre:GCaMP3fl transgenic mice (GFAP-GCaMP3). Scale bar, 100 μm. (B) Ouabain-sensitive 86Rb+ uptake in mouse astrocytes expressing GFAP-GCaMP3 or in astrocytes from GCaMP3fl transgenic mice induced to express GCaMP3 by Cre recombinase adenovirus in the presence or absence of Rhod-2 AM (4 μM). n = 6 to 23 wells for each treatment; *P < 0.05, **P < 0.01, ****P < 0.0001 compared to (−) Rhod-2 AM of control group (0.1% DMSO). (C) A number of spontaneous Ca2+ events in cultured mouse astrocytes were detected by Fluo-4 AM (2 and 4 μM), Rhod-2 AM (2.25 and 4.5 μM), or GCaMP3. In groups combining Ca2+ imaging of GCaMP3 with Rhod-2 AM, the number of Ca2+ events detected with Rhod-2 (left, orange channel) and GCaMP3 (right, green channel) was counted. n = 4 to 6 wells for each treatment; ***P < 0.001 compared to GFAP-GCaMP3 control group (0.1% DMSO). (D) Histogram comparing the number of spontaneous Ca2+ events in Adv-GCaMP3–expressing mouse astrocytes in the presence and absence of 5,5′-dibromo BAPTA [P = 0.0238, unpaired t test (Mann-Whitney test); n = 4 to 8 wells for each treatment]. Means ± SEM are shown.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/515/eaal2039/DC1

    Fig. S1. Structures and characters of Ca2+ indicator derivatives used in publications listed in PubMed.

    Fig. S2. DMSO does not affect 86Rb+ uptake by astrocytes.

    Fig. S3. Ca2+ indicators do not affect ouabain-insensitive 86Rb+ uptake.

    Fig. S4. Ca2+ indicators inhibit Na,K-ATPase–mediated ATP hydrolysis and 86Rb+ uptake in rat astrocyte cultures.

    Fig. S5. Ca2+ indicators induce lactate release in neurons and increase cell volume in neurons and astrocytes.

    Fig. S6. Quantification of spontaneous and pharmacologically evoked Ca2+ signals and 86Rb+ uptake in GCaMP3-expressing astrocytes.

    Movie S1. Beating cardiomyocytes in culture prepared from mouse (2× frame rate).

    Movie S2. ATP-induced Ca2+ response in astrocytes expressing GFAP-GCaMP3.

    Movie S3. ATP-induced Ca2+ response in astrocytes expressing AdV-GCaMP3.

    Movie S4. ATP-induced Ca2+ response in astrocytes loaded with Rhod-2 AM.

    Movie S5. Spontaneous Ca2+ response in astrocytes expressing GFAP-GCaMP3.

  • Supplementary Materials for:

    Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions

    Nathan A. Smith, Benjamin T. Kress, Yuan Lu, Devin Chandler-Militello, Abdellatif Benraiss, Maiken Nedergaard*

    *Corresponding author. Email: nedergaard{at}urmc.rochester.edu

    This PDF file includes:

    • Fig. S1. Structures and characters of Ca2+ indicator derivatives used in publications listed in PubMed.
    • Fig. S2. DMSO does not affect 86Rb+ uptake by astrocytes.
    • Fig. S3. Ca2+ indicators do not affect ouabain-insensitive 86Rb+ uptake.
    • Fig. S4. Ca2+ indicators inhibit Na,K-ATPase–mediated ATP hydrolysis and 86Rb+ uptake in rat astrocyte cultures.
    • Fig. S5. Ca2+ indicators induce lactate release in neurons and increase cell volume in neurons and astrocytes.
    • Fig. S6. Quantification of spontaneous and pharmacologically evoked Ca2+ signals and 86Rb+ uptake in GCaMP3-expressing astrocytes.
    • Legends for movies S1 to S5

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.25 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Beating cardiomyocytes in culture prepared from mouse (2× frame rate).
    • Movie S2 (.avi format). ATP-induced Ca2+ response in astrocytes expressing GFAP-GCaMP3.
    • Movie S3 (.avi format). ATP-induced Ca2+ response in astrocytes expressing AdV-GCaMP3.
    • Movie S4 (.avi format). ATP-induced Ca2+ response in astrocytes loaded with Rhod-2 AM.
    • Movie S5 (.avi format). Spontaneous Ca2+ response in astrocytes expressing GFAP-GCaMP3.

    Citation: N. A. Smith, B. T. Kress, Y. Lu, D. Chandler-Militello, A. Benraiss, M. Nedergaard, Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions. Sci. Signal. 11, eaal2039 (2018).

    © 2018 American Association for the Advancement of Science

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