QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 December 2007
doi: 10.1242/jcs.014522

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chalmers, S. Articles by McCarron, J. G. Search for Related Content PubMed PubMed Citation Articles by Chalmers, S. Articles by McCarron, J. G.

The mitochondrial membrane potential and Ca²⁺ oscillations in smooth muscle

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow, G4 0NR, UK

View larger version (15K): [in this window] [in a new window]   Fig. 1. Mitochondrial depolarization slowed the rate of [Ca2+]c decline following ICa activation and decreased the amplitude of the Ins(1,4,5)P3-induced [Ca2+]c rise. Plasmalemmal depolarization (–70 mV to +10 mV, 500 ms, A), local photolysis of caged Ins(1,4,5)P3 (UV-flash release at , B) and carbachol (CCh, 100 µM by pressure ejection, C) each transiently increased [Ca2+]c, as indicated by an increase in Fluo-4 fluorescence (controls, thin line). Inhibition of mitochondrial Ca2+ uptake by m depolarization with CCCP plus oligomycin (1 µM and 6 µM, respectively, A), increased the time for [Ca2+]c to decline to resting values (thick line), compared with vehicle-treated control (0.2% DMSO, thin line). The peak [Ca2+]c rise evoked by Ins(1,4,5)P3 release was decreased by m depolarization (B, thick line), as was the peak [Ca2+]c evoked by CCh (C, thick line), each compared with vehicle-treated control (0.2% DMSO, thin lines). By contrast, m depolarization did not alter the amplitude or rate of decline of [Ca2+]c increases evoked by caffeine (CAF; 10 mM by pressure ejection, D).

View larger version (63K): [in this window] [in a new window]   Fig. 2. Dual loading of colonic myocytes with Fluo-4 and TMRE allows simultaneous observation of [Ca2+]c and m, leaving shorter wavelengths to be used for UV-photolysis of caged compounds. (A) In spite of overlapping excitation and emission profiles (i), cross-talk between Fluo-4 and TMRE fluorescence signals was minimized using a custom-made dichroic mirror (black line, ii), dual-bandpass excitation (dark-blue line, ii) and emission (light-blue line, ii) filters and exciting at 475 and 560 nm (ii). (B) Virtual absence of overlap between Fluo-4 and TMRE channels in cells loaded with either Fluo-4 alone (10 µM, i) or, in another cell, with TMRE alone (10 nM, ii). Each cell was excited at 475 nm (left-hand panel) and 560 nm (right-hand panel). Pseudocolored images represent emission intensity from a 12-bit range (calibration in top-left corners). (C) Mitochondrial localization of TMRE (10 nM, i) was confirmed by its co-localization with the mitochondria-specific dye MitoTracker Green (25 nM, ii). (iii) Overlay of red TMRE on green MitoTracker images produces yellow regions in which the two indicators are co-localized. (iv) A bright-field image of the cell is also shown. (D) Separation of [Ca2+]c and m measurements in a voltage-clamped myocyte co-loaded with Fluo-4 (green) and TMRE (red). Plasmalemma depolarization (500 ms, –70 to +10 mV, ii) induced a large increase in Fluo-4 fluorescence with no change in TMRE fluorescence (i); subsequent CCCP (1 µM) treatment caused a large decrease in TMRE fluorescence with no decrease in Fluo-4 fluorescence. Scale bars: 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 3. m depolarization with CCCP and m hyperpolarization with oligomycin were each detected by changes in TMRE fluorescence of individual mitochondria. (A) CCCP (1 µM, white squares, n=4) caused a large, rapid decrease in the average TMRE fluorescence compared with controls (black triangles, n=9). Oligomycin (6 µM, white circles, n=3) caused a small but significant increase in TMRE fluorescence (**P<0.01 compared with controls). (B) Images of TMRE fluorescence from approximately 50% of a single smooth muscle cell before and after CCCP (1 µM) treatment. Left-most panel: the punctuate fluorescence comes from the TMRE signals from individual mitochondria. The loss of punctuate TMRE fluorescence in successive panels occurs as a result of the loss of TMRE from mitochondria as m is depolarized with CCCP. Images were obtained at the time-points indicated by connecting lines to panel A. Scale bars: 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Ca2+accumulated by mitochondria during Ins(1,4,5)P3- or ICa-evoked [Ca2+]c transients can be released by mitochondrial depolarization in the 15 seconds after [Ca2+]c elevation. Local photolysis of caged Ins(1,4,5)P3 (UV-flash release at , A) or plasmalemmal depolarization (–70 to +10 mV, 500 ms, C) each transiently increased [Ca2+]c, as indicated by an increase in Fluo-4 fluorescence (controls, black line). In a subsequent activation by Ins(1,4,5)P3 (A) or plasmalemmal depolarization (C) in the same cells, rapid m depolarization (A,C, as shown by a decrease in TMRE fluorescence, red line) by CCCP (20 µM, by pressure-ejection puffer pipette for 2 seconds as shown), caused a transient increase in [Ca2+]c (blue line). The magnitude of mitochondrial Ca2+ release was measured from the time-integrated Fluo-4 signal for a 10-second period immediately after CCCP application minus the time-integrated signal for the control (hashed area, A,C, shown in panels B,D, n=3 individual cells for each data point).

View larger version (26K): [in this window] [in a new window]   Fig. 5. Neither Ins(1,4,5)P3- nor ICa-evoked [Ca2+]c increases significantly altered m of individual mitochondria. ICa activation (A, by depolarizing from –70 to +10 mV, 500 ms, n=9, white squares) or local photolysis of caged Ins(1,4,5)P3 (B, , n=10, white circles) each increased [Ca2+]c (Ci and Di). Neither caused significant alteration in m (as shown by TMRE fluorescence, normalized to starting values, F/F0) compared with control (n=9, black triangles, P>0.05 for all data points). Occasionally, the m of individual mitochondria decreased during the period of observation of either ICa-evoked [Ca2+]c elevation (Ci, [Ca2+]c changes; Cii, holding potential, Vm; Ciii, m of six individual mitochondria) or Ins(1,4,5)P3-evoked [Ca2+]c elevation (Di, [Ca2+]c changes; Dii, activated by photolytic release of Ins(1,4,5)P3 at ; Diii, m of eight individual mitochondria); however, this was equally as likely to occur before, during or after [Ca2+]c elevation.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Neither Ins(1,4,5)P3 (IP3)- nor ICa-induced [Ca2+]c increases altered the m of the entire-cell mitochondrial complement when measured with TMRE in `quench' mode. m depolarization was not seen during increases of [Ca2+]c induced by either Ins(1,4,5)P3 (released by localized photolysis of caged Ins(1,4,5)P3, at , A), following a single plasmalemmal depolarization (+10 mV for 500 ms, B) or following a train of plasmalemmal depolarizations (+10 mV for 250 ms, 2 Hz, 5 seconds, C) in myocytes co-loaded with Fluo-4 (10 µM) and TMRE (150 nM, see text). By contrast, the mitochondrial inhibitors CCCP plus oligomycin (1 and 6 µM, respectively, D) significantly increased TMRE fluorescence when compared with untreated controls (E); **P<0.01; n.s. (not significant) P>0.05.

View larger version (44K): [in this window] [in a new window]   Fig. 7. m changes in cells undergoing spontaneous, repetitive [Ca2+]c oscillations. (A) During spontaneous, repetitive [Ca2+]c oscillations (Fluo-4 fluorescence normalized to baseline values, F/F0, i and iii), individual mitochondria (visualized as punctate TMRE fluorescence, shown in close-up still frames at the time points indicated during [Ca2+]c oscillations, ii and iv) depolarized. Localized fluorescence decreases (depolarizations) are indicated by blue arrows and localized fluorescence increases (repolarizations) are shown by white arrows (images pseudocolored to indicate fluorescence intensity, v). (B) TMRE-fluorescence changes in three individual mitochondria (lower panels) from three separate cells were of varying magnitude and duration, and did not correspond directly to changes in [Ca2+]c (upper panels, Fluo-4 fluorescence, F/F0). Scale bars, 5 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 8. m depolarization frequency increased with [TMRE] or intensity of excitation light and was inhibited by antioxidants or cyclosporin A. (A) The frequency of m depolarizations plotted against [TMRE] (10-150 nM) with (shaded bars) and without (white bars) the attenuation of excitation light by a neutral density filter (*P<0.05, **P<0.01). (B) The mPTP inhibitor cyclosporin A (CsA, 2 µM) or a combination of antioxidants [ascorbic acid (1 mM), catalase (250 units/ml), Trolox (1 mM) and TEMPO (500 µM)] each decreased the number of m depolarizations observed at 150 nM TMRE (**P<0.01). Cells were co-loaded with TMRE and Fluo-4 (10 µM) and fluorescent images acquired at 1 Hz.