Dataset Information

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 ?M 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⁺.

SUBMITTER: Di Giuro CML

PROVIDER: S-EPMC5590033 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Di Giuro Cristiana M L CML Shrestha Niroj N Malli Roland R Groschner Klaus K van Breemen Cornelis C Fameli Nicola N

Pflugers Archiv : European journal of physiology 20170511 10

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 ...[more]

PMID: 28497275

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Project description:BackgroundWhile calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated.ResultsPartial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation.ConclusionsInhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

Dataset Information

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

Publications

Na <sup>+</sup>/Ca<sup>2+</sup> exchangers and Orai channels jointly refill endoplasmic reticulum (ER) Ca<sup>2+</sup> via ER nanojunctions in vascular endothelial cells.

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