Na +/Ca2+ exchangers and Orai channels jointly refill endoplasmic reticulum (ER) Ca2+ via ER nanojunctions in vascular endothelial cells

Abstract

We investigated the role of Na+/ Ca2+ exchange (NCX) in the refilling of endoplasmic reticulum (ER) Ca2+ in vascular endothelial cells under various conditions of cell stimulation and plasma membrane (PM) polarization. Better understanding of the mechanisms behind basic ER Ca2+ content regulation is important, since current hypotheses on the possible ultimate causes of ER stress point to deterioration of the Ca2+ transport mechanism to/from ER itself. We measured [Ca2+]i temporal changes by Fura-2 fluorescence under experimental protocols that inhibit a host of transporters (NCX, Orai, non-selective transient receptor potential canonical (TRPC) channels, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), Na+/ K+ ATPase (NKA)) involved in the Ca2+ communication between the extracellular space and the ER. Following histamine-stimulated ER Ca2+ release, blockade of NCX Ca2+-influx mode (by 10 μM KB-R7943) diminished the ER refilling capacity by about 40%, while in Orai1 dominant negative-transfected cells NCX blockade attenuated ER refilling by about 60%. Conversely, inhibiting the ouabain sensitive NKA (10 nM ouabain), which may be localized in PM-ER junctions, increased the ER Ca2+ releasable fraction by about 20%, thereby supporting the hypothesis that this process of privileged ER refilling is junction-mediated. Junctions were observed in the cell ultrastructure and their main parameters of membrane separation and linear extension were (9.6 ± 3.8) nm and (128 ± 63) nm, respectively. Our findings point to a process of privileged refilling of the ER, in which NCX and store-operated Ca2+ entry via the stromal interaction molecule (STIM)-Orai system are the sole protagonists. These results shed light on the molecular machinery involved in the function of a previously hypothesized subplasmalemmal Ca2+ control unit during ER refilling with extracellular Ca2+.Electronic supplementary material The online version of this article (doi:10.1007/s00424-017-1989-8) contains supplementary material, which is available to authorized users.

Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells

“…The amplitude of the Ca 2+ elevation was, however, significantly lower whereas its duration was dramatically curtailed. Third, the selective blockade of the reverse mode NCX with KB-R mimicked the effect of Na + withdrawal on NaHS- and capsaicin-induced intracellular Ca 2+ signals [ 62 , 64 ], thereby confirming that the reverse mode NCX is recruited by TRPV1-mediated extracellular Na + entry to sustain the ensuing increase in [Ca 2+ ] i . These data are consistent with those reported in native endothelium of rat aorta [ 62 ] and confirm a recent report on in the CRC cell line, DLD1 [ 20 ].…”

“…The amplitude of the Ca 2+ signal in the responding cells (66 out of 245; 27%) was significantly ( p < 0.05) smaller as compared to the control ( Figure 11 C). In order to confirm NCX role in NaHS-dependent Ca 2+ signaling, we probed the effect of KB-R 7943 (20 μM, 20 min), a selective inhibitor of the reverse-mode NCX at this concentration [ 62 , 64 ]. In agreement with the results presented above, NaHS failed to elicit an [Ca 2+ ] i elevation in 146 out of 182 (80.2%) cells pre-treated with the blocker ( Figure 11 D,E).…”

Type 2 Diabetes Alters Intracellular Ca2+ Handling in Native Endothelium of Excised Rat Aorta

“…Therefore, the dramatic increase in plateau amplitude observed in the presence of CPA is not due to further SOCE activation, but to the impairment of Ca 2+ sequestration into ER lumen, which results in Ca 2+ accumulation within the cytosol. It has been shown that ER Ca 2+ refilling occurs at plasma membrane/ER nanojunctions [55,56], which recruit SERCA upon massive ER Ca 2+ depletion [57,58], in vascular endothelial cells. Accordingly, InsP 3 -induced drop in ER Ca 2+ concentration causes SERCA migration and co-localization with Stim1 and Orai1, thereby resulting in more efficient Ca 2+ pumping into the ER [57,58].…”

Reactive Oxygen Species and Endothelial Ca2+ Signaling: Brothers in Arms or Partners in Crime?

“…Herein, STIM1 physically interacts with and gates Orai1, which provides the pore forming subunit of a store-operated channel termed the Ca 2+ release-activated Ca 2+ (CRAC) channel. STIM1 and Orai1 were also shown to mediate SOCE in HAECs [ 105 , 173 ], in human pulmonary artery endothelial cells (HPAECs) [ 170 ], and in the HUVEC-derived endothelial cell line, EA.hy926 [ 174 , 175 ]. Vascular endothelial cells also express the STIM1 and Orai1 paralogues, i.e., STIM2, Orai2, and Orai3 [ 109 , 110 , 173 ].…”