Article

Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra.

The Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Co Louth, Ireland.
The Journal of Physiology (Impact Factor: 4.54). 09/2008; 586(Pt 19):4631-42. DOI: 10.1113/jphysiol.2008.159194
Source: PubMed

ABSTRACT 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.
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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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    • "In the mouse renal pelvis preparations where L-type Ca 2+ channels have been blocked, cyclopiazonic acid (CPA) readily prevents the generation of STDs and Ca 2+ transients in ATSMCs in a manner associated with a large increase in basal Ca 2+ (Lang et al., 2007b). In the present study, CCCP, at concentrations that evokes large increases in the basal Ca 2+ in ICC (Sergeant et al., 2008) or smooth muscle (Fukuta et al., 2002; Kubota et al., 2003), mimicked the effects of CPA in ATSMCs, suggesting that both organelles may work concurrently to regulate spontaneous Ca 2+ transient discharge. A recent study employing the simultaneous monitoring of cytoplasmic Ca 2+ concentration and Ca 2+ concentration in either ER or mitochondria clearly demonstrates that Ca 2+ shuttles between ER and mitochondria in phase with the Ca 2+ oscillations, thus revealing an essential requirement for mitochondria in generating Ca 2+ transients (Ishii et al., 2006). "
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