Mitochondrial modulation of Ca2+ sparks and transient KCa currents in smooth muscle cells of rat cerebral arteries

Abstract

Mitochondria sequester and release calcium (Ca(2+)) and regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) in eukaryotic cells. However, the regulation of different Ca(2+) signalling modalities by mitochondria in smooth muscle cells is poorly understood. Here, we investigated the regulation of Ca(2+) sparks, Ca(2+) waves and global [Ca(2+)](i) by mitochondria in cerebral artery smooth muscle cells. CCCP (a protonophore; 1 microm) and rotenone (an electron transport chain complex I inhibitor; 10 microm) depolarized mitochondria, reduced Ca(2+) spark and wave frequency, and elevated global [Ca(2+)](i) in smooth muscle cells of intact arteries. In voltage-clamped (-40 mV) cells, mitochondrial depolarization elevated global [Ca(2+)](i), reduced Ca(2+) spark amplitude, spatial spread and the effective coupling of sparks to large-conductance Ca(2+)-activated potassium (K(Ca)) channels, and decreased transient K(Ca) current frequency and amplitude. Inhibition of Ca(2+) sparks and transient K(Ca) currents by mitochondrial depolarization could not be explained by a decrease in intracellular ATP or a reduction in sarcoplasmic reticulum Ca(2+) load, and occurred in the presence of diltiazem, a voltage-dependent Ca(2+) channel blocker. Ru360 (10 microm), a mitochondrial Ca(2+) uptake blocker, and lonidamine (100 microm), a permeability transition pore (PTP) opener, inhibited transient K(Ca) currents similarly to mitochondrial depolarization. In contrast, CGP37157 (10 microm), a mitochondrial Na(+)-Ca(2+) exchange blocker, activated these events. The PTP blockers bongkrekic acid and cyclosporin A both reduced inhibition of transient K(Ca) currents by mitochondrial depolarization. These results indicate that mitochondrial depolarization leads to a voltage-independent elevation in global [Ca(2+)](i) and Ca(2+) spark and transient K(Ca) current inhibition. Data also suggest that mitochondrial depolarization inhibits Ca(2+) sparks and transient K(Ca) currents via PTP opening and a decrease in intramitochondrial [Ca(2+)].

Figure 1. Rotenone and CCCP depolarize mitochondria in cerebral artery smooth muscle cells

Original recordings illustrating the mean percentage change in TMRM fluorescence in isolated arterial smooth muscle cells following application (indicated by arrow) of rotenone (10 μm), CCCP (1 μm) or oligomycin B (1 μm). Each trace represents the mean fluorescence change of 7, 10, and 11 cells for rotenone, CCCP and oligomycin B, respectively.

Figure 10. Mitochondrial PTP blockers reduce rotenone inhibition of transient K_Ca currents

A, original recording of transient K_Ca currents in a cerebral artery smooth muscle cell voltage-clamped at −40 mV. Bongkrekic acid (10 μm) reduced inhibition of transient K_Ca currents by rotenone (10 μm). B, frequency histogram illustrating the time course of transient K_Ca current inhibition by rotenone applied alone (from Fig. 3A, black bars) or in the presence of bongkrekic acid (from Fig. 10A, hatched bars). C, average relative effects on transient K_Ca current frequency (left panel) and amplitude (right panel) of bongkrekic acid (BA, 10 μm, n= 3), cyclosporin A (CsA, 1 μm, n= 4), rotenone (10 μm, n= 5) alone, or rotenone applied in the presence of bongkrekic acid (10 μm, n= 6) or cyclosporin A (1 μm, n= 7). *P < 0.05 using Student-Newman-Keuls test.

Figure 2. Regulation of Ca²⁺ sparks, Ca²⁺ waves and global [Ca²⁺]_i in smooth muscle cells of intact cerebral arteries by rotenone, CCCP and oligomycin B

A, average fluorescence (100 of 600 images) over 10 s of two different 56.3 μm × 52.8 μm areas of the same cerebral artery in control and 5 min after application of rotenone (10 μm). The locations of Ca²⁺ sparks that occurred during 10 s are indicated by white boxes (1.54 μm × 1.54 μm). Representative localized F/F₀ changes over time are illustrated below respective images and labelled accordingly. Average relative effects on Ca²⁺ spark (B) and wave frequency (C) and global [Ca²⁺]_i (D) of a 5 min application of rotenone, CCCP or oligomycin B. Rotenone decreased mean Ca²⁺ spark frequency from 1.18 ± 0.23 to 0.75 ± 0.12 Hz, Ca²⁺ spark amplitude (F/F₀) from 1.31 ± 0.01 to 1.24 ± 0.01, and wave frequency from 0.39 ± 0.07 to 0.09 ± 0.06 Hz (n= 6 arteries). CCCP decreased mean Ca²⁺ spark frequency from 1.05 ± 0.25 to 0.33 ± 0.01 Hz, spark amplitude (F/F₀) from 1.40 ± 0.01 to 1.25 ± 0.01, and wave frequency from 0.31 ± 0.04 to 0.04 ± 0.02 Hz (n= 6 arteries). Oligomycin B did not alter mean Ca²⁺ spark frequency (control, 1.03 ± 0.12; oligomycin B, 1.00 ± 0.38 Hz), spark amplitude (control, 1.34 ± 0.02; oligomycin B, 1.37 ± 0.02), or wave frequency (control, 0.31 ± 0.05; oligomycin B, 0.30 ± 0.04 Hz (n= 4 arteries). *P < 0.05 using Students t test.

Figure 3. Regulation of transient K_Ca currents in isolated voltage-clamped arterial smooth muscle cells by rotenone, CCCP and oligomycin B

A and B, original recordings of transient K_Ca currents in cerebral artery smooth muscle cells voltage-clamped at −40 mV using the perforated-patch configuration. Rotenone (10 μm, A) reduced transient K_Ca current frequency and amplitude, whereas oligomycin B (1 μm, B) did not alter transient K_Ca currents. C, average relative changes in transient K_Ca current frequency (left panel) and amplitude (right panel) when compared with control of a 5 min application of: rotenone (10 μm, p-p, n= 7), rotenone (10 μm, w-c, n= 10), CCCP (1 μm, p-p, n= 6), CCCP (1 μm, w-c, n= 7), CCCP (1 μm) + oligomycin B (1 μm, p-p, n= 7), and oligomycin B (1 μm) at 5 and 15 min (p-p, n= 6 for each). p-p, perforated-patch; w-c, conventional whole-cell configuration. *P < 0.05 using Students t test.

Figure 4. CCCP attenuates the coupling relationship between Ca²⁺ sparks and transient K_Ca currents

A, original simultaneous recordings of Ca²⁺ sparks and transient K_Ca currents in the same voltage-clamped (−40 mV) cerebral artery smooth muscle cell. The black trace illustrates whole cell K⁺ current. Red and green traces illustrate fluorescence changes (F/F₀) measured in two different areas of the cell where Ca²⁺ sparks occurred. Traces show activity in control and 5 min after CCCP (1 μm) and oligomycin B (1 μm). B, scatter plot of Ca²⁺ spark and evoked transient K_Ca current amplitude at −40 mV obtained in the same cells in control (black), and after CCCP (red, n= 5 cells). Ca²⁺ spark amplitude is calculated as the local elevation in [Ca²⁺]_i (i.e. Δ[Ca²⁺]_i) reported by fluo-4. First-order polynomial linear fits for control and CCCP data are illustrated with confidence bands. CCCP reduced the slope of the amplitude correlation between a spark and the evoked transient K_Ca current from 0.018 ± 0.001 to 0.011 ± 0.001 (P < 0.05). Ca²⁺ spark and associated transient K_Ca current amplitudes were significantly correlated for control and CCCP (P < 0.05 for each), but CCCP reduced the correlation coefficient (r) from 0.61 to 0.32.

Figure 5. Rotenone and CCCP activate K_Ca channels

A, original recordings of single K_Ca channels measured at 0 mV using the perforated-patch configuration. Ca²⁺ sparks, and thus transient K_Ca currents, were abolished with thapsigargin (100 nm). Rotenone increased K_Ca channel activity. ‘C’ indicates closed level. B, average relative effect of rotenone (10 μm) or CCCP (1 μm) on K_Ca channel activity (NP_o). Rotenone increased mean NP_o from 0.017 ± 0.003 to 0.044 ± 0.024 (n= 7 cells). CCCP increased mean NP_o from 0.021 ± 0.005 to 0.042 ± 0.016 (n= 5 cells). *P < 0.05 using Students t test.

Figure 6. Regulation of SR Ca²⁺ load by CCCP, rotenone and oligomycin B

A and B, original traces illustrating the regulation of intracellular Ca²⁺ concentration and caffeine (10 mm)-induced [Ca²⁺]_i transients in single cerebral artery smooth muscle cells by CCCP (1 μm, A) or oligomycin B (1 μm, B). C, average relative caffeine-induced [Ca²⁺]_i transients in control (two applications 5 min apart), and five, 10 and 15 min after CCCP (1 μm, n= 9 cells), rotenone (10 μm, n= 5 cells) or oligomycin B (1 μm, n= 11 cells). *P < 0.05, when compared with control using Student-Newman-Keuls test.

Figure 7. Rotenone blocks transient K_Ca currents when applied in the continued presence of diltiazem, and H₂O₂ activates transient K_Ca currents

Average relative effects of diltiazem (50 μm, n= 6), rotenone (10 μm) applied in the continued presence of diltiazem (50 μm, n= 6), or H₂O₂ (100 μm, n= 7) on transient K_Ca current frequency (A) and amplitude (B). *P < 0.05 using using Student-Newman-Keuls test.

Figure 8. Differential regulation of transient K_Ca currents by Ru360 and CGP37157

A, original recording obtained in a voltage-clamped (−40 mV) cerebral artery smooth muscle cell illustrating activation of transient K_Ca currents by CGP37157 (10 μm) and inhibition by Ru360 (10 μm). B, average relative effects on transient K_Ca current frequency (left panel) and amplitude (right panel) of Ru360 (n= 6), CGP37157 (n= 9), and Ru360 applied in the continued presence of CGP37157 (n= 5). *P < 0.05 using Students t test.

Figure 9. Lonidamine inhibits transient K_Ca currents

A, original recording illustrating inhibition of transient K_Ca currents by lonidamine (100 μm) in a cerebral artery smooth muscle cell voltage-clamped at −40 mV. B, average effects of lonidamine on transient K_Ca current frequency and amplitude (n= 10). *P < 0.05 using Students t test.