Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed ICC from the rabbit urethra

The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co Louth, Ireland.
The Journal of Physiology (Impact Factor: 5.04). 09/2008; 586(Pt 19):4631-42. DOI: 10.1113/jphysiol.2008.159194
Source: PubMed


Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca(2+) waves. The purpose of this study was to investigate if this activity was influenced by Ca(2+) uptake into mitochondria. Spontaneous Ca(2+) waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 microm) and antimycin A (5 microm) abolished Ca(2+) waves and increased basal Ca(2+) levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 microm) and CCCP (1 microm). Spontaneous Ca(2+) waves were not inhibited by the ATP synthase inhibitor oligomycin (1 microm), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at -60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 microm) also inhibited STICS. Stimulation of Ca(2+) uptake into mitochondria using the plant flavonoid kaempferol (10 microm) induced a series of propagating Ca(2+) waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca(2+), but was not significantly affected by the IP(3) receptor blocker 2-APB (100 microm). These data suggest that spontaneous Ca(2+) waves in urethral ICC are regulated by buffering of cytoplasmic Ca(2+) by mitochondria.

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    • "Ca 2+ waves rely on Ca 2+ release from the intracellular ER Ca 2+ stores, with release via ryanodine receptors (RyRs) being responsible for Ca 2+ wave initiation and inositol 1,4,5-triphosphate (IP 3 ) sensitive stores contributing to Ca 2+ wave propagation [7]. Other factors that can modulate Ca 2+ waves include the external Ca 2+ concentration [7] as well as Ca 2+ handling by mitochondria [8]. The action of various protein kinases have been noted to affect intracellular Ca 2+ signalling by altering the sensitivity of ER Ca 2+ release channels in a variety of cells [9]. "
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    ABSTRACT: Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca(2+) waves that rely on Ca(2+) release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca(2+) waves in ICC. Intracellular [Ca(2+)] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10μM) significantly inhibited the generation of spontaneous Ca(2+) waves in ICC and this was associated with a significant decrease in the ER Ca(2+) load, measured with 10mM caffeine responses. Ca(2+) waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP3Rs) with 10μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca(2+) wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca(2+) load sufficient to inactivate IP3Rs but not RyRs.
    Cell Calcium 09/2014; 56(3). DOI:10.1016/j.ceca.2014.07.002 · 3.51 Impact Factor
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    • "In cardiac ventricular myocytes, the Ca2+ diffusion distance between the RyR clusters at the junctional SR and the ends of the mitochondria is approximately 50-100 nM [17], which may create a micro-domain for local Ca2+ and generate functional interaction between mitochondria and the SR [18]. While some studies have shown mitochondrial Ca handling may affect the dynamics and magnitudes of Cai2+ oscillations in other cell types [19–22], it is not well understood how the mitochondria and the SR are functionally coupled with relation to the regulation of Cai2+ homeostasis and generation of Cai2+ waves (CaWs) and triggered activities (TAs) in cardiac ventricular myocytes. "
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    ABSTRACT: Recent studies have suggested that mitochondria may play important roles in the Ca(2+) homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+) flux can regulate the generation of Ca(2+) waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+) (Cai (2+)) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and CaWs were induced in the presence of high (4 mM) external Ca(2+) (Cao (2+)). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai (2+) levels even after depletion of SR Ca(2+) in the absence of Cao (2+) , suggesting Ca(2+) release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m ) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m ) or Ru360 (a mitochondrial Ca(2+) uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+) uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+) release and uptake exquisitely control the local Ca(2+) level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.
    PLoS ONE 11/2013; 8(11):e80574. DOI:10.1371/journal.pone.0080574 · 3.23 Impact Factor
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    • "Because reducing the electrical driving force for ATP synthesis with CCCP did not critically reduce the Ca 2+ content of the ER (Figure 5), mitochondrial ATP production may not be fundamental for a functioning SERCA, at least over the short period of our experiments. This view is supported by the recent finding that oligomycin, a blocker of mitochondrial ATP synthetase, did not affect spontaneous Ca 2+ waves in urethral ICC-LCs (Sergeant et al., 2008). "
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    ABSTRACT: Although spontaneous Ca(2+) waves in interstitial cells of Cajal (ICC)-like cells (ICC-LCs) primarily arise from endoplasmic reticulum (ER) Ca(2+) release, the interactions among mitochondrial Ca(2+) buffering, cellular energetics and ER Ca(2+) release in determining the spatiotemporal dynamics of intracellular Ca(2+) remain to be elucidated. Spontaneous Ca(2+) transients in freshly isolated ICC-LCs of the rabbit urethra were visualized using fluo-4 Ca(2+) imaging, while the intracellular distribution of mitochondria was viewed with MitoTracker Red. Spontaneous Ca(2+) waves invariably originated from the perinuclear region where clusters of mitochondria surround the nucleus. Perinuclear Ca(2+) dynamics were characterized by a gradual rise in basal Ca(2+) that preceded each regenerative Ca(2+) transient. Caffeine evoked oscillatory Ca(2+) waves originating from anywhere within ICC-LCs. Ryanodine or cyclopiazonic acid prevented Ca(2+) wave generation with a rise in basal Ca(2+), and subsequent caffeine evoked a single rudimentary Ca(2+) transient. Inhibition of glycolysis with 2-deoxy-glucose or carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial protonophore, increased basal Ca(2+) and abolished Ca(2+) waves. However, caffeine still induced oscillatory Ca(2+) transients. Mitochondrial Ca(2+) uptake inhibition with RU360 attenuated Ca(2+) wave amplitudes, while mitochondrial Ca(2+) efflux inhibition with CGP37157 suppressed the initial Ca(2+) rise to reduce Ca(2+) wave frequency. Perinuclear mitochondria in ICC-LCs play a dominant role in the spatial regulation of Ca(2+) wave generation and may regulate ER Ca(2+) release frequency by buffering Ca(2+) within microdomains between both organelles. Glycolysis inhibition reduced mitochondrial Ca(2+) buffering without critically disrupting ER function. Perinuclear mitochondria may function as sensors of intracellular metabolites.
    British Journal of Pharmacology 10/2010; 161(3):680-94. DOI:10.1111/j.1476-5381.2010.00902.x · 4.84 Impact Factor
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