Abstract
Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.
Highlights
Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2؉ entry by buffering subplasmalemmal Ca2؉, their contribution to endoplasmic reticulum (ER) Ca2؉ refilling was not subjected to detailed analysis so far
ER Ca2؉ refilling took place even without an associated Ca2؉ elevation in the deeper cytosol, indicating that superficial ER domains mimic mitochondrial Ca2؉ buffering and efficiently sequester subplasmalemmal Ca2؉ and facilitate capacitative Ca2؉ entry. These data point to different contribution of mitochondria in the process of ER Ca2؉ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2؉ refilling from trans-mitochondrial Ca2؉ flux
ER Ca2ϩ Refilling Is Only Moderately Affected by IP3—As published recently [19], the removal of extracellular Ca2ϩ during stimulation with histamine accelerated the rate of ER Ca2ϩ depletion in endothelial cells, whereas Ca2ϩ re-addition yielded a rapid Ca2ϩ refilling of the ER (Fig. 1)
Summary
Inhibition of NCXmito Yields Ca2ϩ Accumulation in the Mitochondria and Spoils Their Capacity to Sequester Entering Ca2ϩ Independently of the Presence or Absence of an Agonist— The impact of Ca2ϩ influx on mitochondrial Ca2ϩ dynamics was investigated by the removal and subsequent re-addition of extracellular Ca2ϩ during and after cell stimulation with histamine. Inhibition of NCXmito with CGP 37157 reduced the cytosolic Ca2ϩ elevation upon re-addition of extracellular Ca2ϩ in the presence of histamine by ϳ40% (Fig. 2B, left panel).
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