Ca2+ refilling of the endoplasmic reticulum is largely preserved albeit reduced Ca2+ entry in endothelial cells

Abstract

In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca2+ refilling and the extent of Ca2+ entry was investigated in endothelial cells. ER and mitochondrial Ca2+ concentration were measured using genetically encoded Ca2+ sensors, while the amount of entering Ca2+ was controlled by varying either the extracellular Ca2+ or the electrical driving force for Ca2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca2+ replenishment was fully accomplished even if the Ca2+ concentration applied was reduced from 2 to 0.5mM. A similar strong efficiency of ER Ca2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca2+ refilling depended on mitochondrial Ca2+ transport and was more susceptible to membrane depolarization. Store-operated Ca2+ entry (SOCE), was strongly reduced under low Ca2+ and depolarizing conditions but increased if ER Ca2+ uptake was blocked or if ER Ca2+ was released continuously by IP(3). A correlation of the kinetics of ER Ca2+refilling with cytosolic Ca2+ signals revealed that termination of SOCE is a rapid event that is not delayed compared to ER refilling. Our data indicate that ER refilling occurs in priority to, and independently from the cytosolic Ca2+ elevation upon Ca2+ entry and that this important process is widely achieved even under conditions of diminished Ca2+entry.

Fig. 1

Modulation of store-operated Ca²⁺ entry (SOCE) by membrane depolarization or decreased extracellular Ca²⁺ concentration. Cytosolic Ca²⁺ signals were recorded using fura-2. In order to deplete the ER, cells were stimulated with 100 μM histamine in the absence of extracellular Ca²⁺. As a measure of SOCE, cytosolic Ca²⁺ elevation upon Ca²⁺ re-addition after agonist washout was obtained. (A) To induce plasma membrane depolarization, extracellular K⁺ concentration was increased from 5 to 130 mM 2 min prior Ca²⁺ re-addition (filled symbols, dotted line, n = 47). For comparison, experiments in physiological medium (i.e. 5 mM K⁺) are shown (open symbols, continuous line, n = 43). ^*P < 0.05 vs. control. (B) Left panel: as indicated, different Ca²⁺ concentrations ranging from nominal Ca²⁺-free to 2 mM Ca²⁺ were added (n = 32–91). Right panel: concentration–response curve of the cytosolic Ca²⁺ elevation achieved by the various extracellular Ca²⁺ buffers. (C) Left panel: a comparison of the effect of membrane depolarization with that of various extracellular Ca²⁺ concentration (values are expressed in percentage while the maximal effect under control conditions was set to 100%). Right panel: schematic illustration of the three experimental conditions: maximal [Ca²⁺]_cyto elevation was obtained in normal K⁺-containing buffer and addition of 2 mM Ca²⁺ (a, control), reduced extracellular Ca²⁺ in normal K⁺-containing buffer (b, low [Ca²⁺]_e) and depolarizing conditions in normal Ca²⁺-containing solution but high extracellular K⁺ (c, 130 mM K⁺).

Fig. 2

ER Ca²⁺ refilling is largely preserved under conditions of membrane depolarization. (A) Following the experimental protocol shown in Fig. 1A in which the ER of pre-stimulated cells was refilled by the addition of 2 mM Ca²⁺ in physiological extracellular K⁺ (i.e. 5 mM K⁺, open symbols, continuous line, n = 43) or under high extracellular K⁺ conditions (i.e. 130 mM K⁺, filled symbols, dotted line, n = 47) (grey in the present figure), ER Ca²⁺ content was estimated by a further stimulation with 100 μM histamine in the absence of extracellular Ca²⁺. ^*P < 0.05 vs. cytosolic Ca²⁺ elevation upon Ca²⁺ re-addition (grey line). (B) In EA.hy 926 cells that were transiently transfected with vYC4er, ER Ca²⁺ content was monitored upon stimulation with 100 μM histamine followed by Ca²⁺ refilling after agonist washout under control conditions (open symbols, continuous line, n = 12) and in high K⁺-containing buffer (filled symbols, dotted line, n = 15).

Fig. 3

ER Ca²⁺ refilling is largely assured even under conditions of reduced Ca²⁺ influx. (A) The impact of the concentration of extracellular Ca²⁺ on ER Ca²⁺ refilling was assessed in cells transiently transfected with YC4er (n = 6–15). (B) Calculation of the rate of ER Ca²⁺ refilling from the external Ca²⁺ concentration. The kinetics of ER Ca²⁺ loading under conditions with different external Ca²⁺ concentrations were fitted according to a Boltzmann sigmoid curve (upper panel) and used to calculate the derivation dRatio/dt. (C) Concentration–response curves of the rate (bold line, n = 6–15) and extent of ER Ca²⁺ refilling (bold dotted line, n = 6–15) and [Ca²⁺]_cyto elevation (grey line and symbols; Fig. 1C) upon Ca²⁺ re-addition. (D) After addition of different extracellular Ca²⁺ concentration (nominal Ca²⁺ free to 2 mM) to pre-stimulated cells (grey lines represent this protocol already described under Fig. 1B), cells were stimulated a second time with 100 μM histamine in the absence of extracellular Ca²⁺ (n = 32–91). (E) Concentration–response curves of the second histamine-induced cytosolic Ca²⁺ elevation (bold continuous line) compared with recurrences of the ER Ca²⁺ refilling (grey dotted line) and [Ca²⁺]_cyto elevation upon Ca²⁺ re-addition (grey continuous line). ^*P < 0.05 vs. cytosolic Ca²⁺ elevation upon Ca²⁺ re-addition (grey line).

Fig. 4

Entering Ca²⁺ is directed towards the cytosol upon SERCA inhibition. (A) The impact of SERCA inhibition with 15 μM BHQ on intracellular Ca²⁺ mobilization to 100 μM histamine followed by an addition of 2 mM extracellular Ca²⁺ in high K⁺-containing buffer. [Ca²⁺]_cyto signals were assessed in fura-2 loaded cells. Traces represent average curves of 15 measurement in the absence of BHQ (filled symbols, continuous line) and 63 recordings in the presence of BHQ (open symbols, dotted line). ^*P < 0.05 vs. in the absence of BHQ. (B) Left panel: in low K⁺ medium (i.e. 5 mM), different extracellular Ca²⁺ concentration ranging from nominal free Ca²⁺ to 2 mM were added to pre-stimulated cells in the continuous presence of 15 μM BHQ (n = 29–45). Right panel: the peak magnitudes reached upon the addition of given extracellular Ca²⁺ concentration in the presence of BHQ were normalized to the maximal amplitude and used for calculation of a concentration–response curve. For comparison replications from Fig. 1D and C are presented as grey curves. ^*P < 0.05 vs. cytosolic Ca²⁺ elevation upon Ca²⁺ re-addition in the absence of BHQ (grey line).

Fig. 5

A trans-ER Ca²⁺ flux maintains increased [Ca²⁺]_cyto levels under depolarizing conditions. (A) 2 mM extracellular Ca²⁺ were added to pre-stimulated cells (100 μM histamine) in the continuous presence of the agonist under conditions of physiological extracellular K⁺ (i.e. 5 mM K⁺, open circles, continuous line, n = 45) or in the presence of high extracellular K⁺ (i.e. 130 mM K⁺, closed circles, dotted line, n = 59), while [Ca²⁺]_cyto signals were recorded using fura-2. Alternatively histamine was removed before 2 mM Ca²⁺ was readded in the presence of 130 mM extracellular K⁺ (filled squares, dashed line, n = 15). *P < 0.05 vs. control. ^#P < 0.05 vs. in the absence of histamine. (B) Left panel: as indicated, cells were stimulated with 100 μM histamine in the presence of 2 mM extracellular Ca²⁺ under conditions of low extracellular K⁺ (i.e. 5 mM K⁺, open symbols, continuous line, n = 43) or in high extracellular K⁺ buffer (i.e. 130 mM K⁺, filled symbols, dotted line, n = 41). ^*P < 0.05 vs. control. Right panel: cells transiently transfected with D1_ER were used to investigate changes of the ER Ca²⁺ content upon stimulation with 100 μM histamine in low extracellular K⁺ buffer (i.e. 5 mM K⁺, open symbols, continuous line, n = 13) and high extracellular K⁺ medium (i.e. 130 mM K⁺, filled symbols, dotted line, n = 17). ^*P < 0.05 vs. control.

Fig. 6

Two routes for ER Ca²⁺ refilling. (A) Cells expressing RP-mt were used to measure mitochondrial Ca²⁺ sequestration upon Ca²⁺ re-addition in the presence of 100 μM histamine under conditions of physiological (i.e. 5 mM; n = 8) and high (i.e. 130 mM; n = 22) external K⁺. (B) Cells were transiently transfected with D1_ER and ER replenishment was monitored upon Ca²⁺ re-addition in the presence of 100 μM histamine under conditions of physiological (n = 6) and high (n = 5) external K⁺. (C) Schematic illustration of the two different Ca²⁺ paths that achieve ER Ca²⁺ refilling upon Ca²⁺ re-addition after agonist washout in physiological K⁺ (I) or high K⁺ buffer (II), and in the presence of histamine in physiological K⁺ (III) or high K⁺ buffer (IV). Accordingly, the respective conditions are indicated in (A) and (B) and Fig. 2B.

Fig. 7

Termination of SOCE. (A) ER Ca²⁺ stores were emptied by application of 15 μM BHQ, and 2 mM Ca²⁺ was then readded in high K⁺ buffer (130 mM K⁺). After 4 min, the medium was switched to physiological K⁺ concentration (5 mM K⁺) (n = 14). (B) ER Ca²⁺ stores were emptied by the application of 100 μM histamine and after the agonist washout, 2 mM Ca²⁺ were readded in high K⁺ buffer (130 mM K⁺). After 3 min, the medium was switched to physiological K⁺ concentration (5 mM K⁺) (n = 10). (C) The inset represents the protocol used to evaluate the termination of SOCE. After ER depletion, 2 mM Ca²⁺ were readded in high K⁺ buffer (130 mM K⁺), and after different time points (15 and 45 s are represented), the medium was switched to normal K⁺ concentration (5 mM K⁺). Columns represents the statistic evaluation of the impact of reducing the K⁺ concentration to normal level (5 mM K⁺) at different time points (15, 20, 30 and 45 s) after Ca²⁺ re-addition (n = 16–48).