Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process (original) (raw)
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The Journal of Physiology, 2000
In a wide variety of non-excitable cell types, release of Ca¥ from intracellular Ca¥ stores initiates a store depletiondependent Ca¥ influx mechanism, termed capacitative Ca¥ entry (for review see Berridge, 1995; Putney, 1997; Parekh & Penner, 1997; Holda et al. 1998). This store-operated or Ca¥ release-dependent capacitative Ca¥ entry (CCE) plays a key role in maintaining the Ca¥ load of intracellular stores (endoplasmic reticulum, ER), but has also been implicated in important cellular functions such as secretion, regulation of adenylate cyclase, gene transcription, cell cycle and proliferation, apoptosis and modulation of [Ca¥]é oscillations and [Ca¥]é waves (for review see e.g. Parekh & Penner, 1997). Furthermore, in endothelial cells CCE has been shown to be a preferential Ca¥ source for the regulation of the Ca¥-calmodulin-dependent NO synthase activity (Xu et al. 1994; Wang et al. 1996). Hoth & Penner (1992) were the first to demonstrate a membrane conductance directly related to Ca¥ store depletion, which subsequently was termed Ca¥ release-activated Ca¥ current (ICRAC). A similar membrane current has been identified in vascular endothelial cells (Fasolato & Nilius, 1998). ICRAC is the best characterized store-operated Ca¥ current, although in recent years an increasing number of store-dependent membrane conductances have been characterized in different cell types that differ from the original ICRAC in their biophysical properties and modes of regulation and modulation (see Clapham, 1995; Parekh & Penner, 1997). Several key questions regarding regulation of CCE have remained unanswered. For example, the signalling mechanism that communicates the degree of store filling to the surface membrane Ca¥-permeable channel remains elusive. Arguments in favour of or against a mechanism based on diffusible signalling messengers or protein-protein interactions between release channel and surface membrane channel are abundant (e.g. Berridge, 1996; Csutora et al.
Two different storemoperated Ca2+ entry pathways in MDCK cells
Cell Calcium, 1996
Whole cell patch clamp experiments in conjunction with Fura-fluorescence microscopy were performed to study the mechanisms of 'store-operated' (capacitative) Ca2+ entry. In MDCK cells, depletion of inositol 1,4,5trisphosphate (IPJ-sensitive Ca2+ stores activates a store-operated cation current (SOCC) predominantly selective for Ca*+ than for Na+ or Mn2+ [Delles C., Haller T., Diet1 P. A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells. J fhysioll995; 486: 557-5691. In the presence of extracellular Ca*+, thapsigargin (TG) stimulated both SOCC and a Ca2+-dependent K+ current (I k&, reflecting stimulation of store-operated Ca*+ entry. The Caz+ entry blocker 1-[3-(4-methoxyphenyl)
The Journal of Physiology, 2000
In rat basophilic leukaemia (RBL_1) cells, an experimental model for mucosal mast cells, calcium (Ca¥) influx is a central event in the secretion of inflammatory mediators (Ali et al. 1990; Kim et al. 1997). Activation of cell-surface receptors that couple to inositol 1,4,5-trisphosphate (IP×) production evokes a biphasic increase in intracellular Ca¥: an initial Ca¥ release phase is followed by a smaller but sustained Ca¥ influx component (Berridge, 1993). In RBL_1 cells, like other non-excitable cells, emptying of the intracellular Ca¥ stores activates a Ca¥ current called ICRAC (Ca¥ releaseactivated Ca¥ current; Hoth & Penner, 1992; Parekh & Penner, 1997). The relationship between IP×-evoked Ca¥ release and subsequent activation of ICRAC is complex. A partial dissociation between Ca¥ release and store-operated Ca¥ influx has been found in several cells including RBL_1 cells (Parekh et al. 1997; Hartmann & Verkhratsky, 1998; Liu et al. 1998). Despite its importance, the mechanisms underlying this widespread phenomenon are not known. When ICRAC is studied under conditons of physiological cytoplasmic Ca¥ buffering and IP× is used to deplete the intracellular stores, the current is not detectable, although intracellular fluorescent dyes reveal modest activation of Ca¥ influx following IP× elevation (Parekh et al. 1997; Huang et al. 1998). It is widely accepted that the stores are fully depleted under these conditions, and the inability to record any macroscopic ICRAC reflects Ca¥-dependent
Cell Calcium, 2007
In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca 2+ refilling and the extent of Ca 2+ entry was investigated in endothelial cells. ER and mitochondrial Ca 2+ concentration were measured using genetically encoded Ca 2+ sensors, while the amount of entering Ca 2+ was controlled by varying either the extracellular Ca 2+ or the electrical driving force for Ca 2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca 2+ replenishment was fully accomplished even if the Ca 2+ concentration applied was reduced from 2 to 0.5 mM. A similar strong efficiency of ER Ca 2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca 2+ refilling depended on mitochondrial Ca 2+ transport and was more susceptible to membrane depolarization. Store-operated Ca 2+ entry (SOCE), was strongly reduced under low Ca 2+ and depolarizing conditions but increased if ER Ca 2+ uptake was blocked or if ER Ca 2+ was released continuously by IP 3. A correlation of the kinetics of ER Ca 2+ refilling with cytosolic Ca 2+ 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 Ca 2+ elevation upon Ca 2+ entry and that this important process is widely achieved even under conditions of diminished Ca 2+ entry.
Pflügers Archiv : European journal of physiology, 2010
In endothelial cells, agonist-induced Ca(2+) entry takes place via the store-operated Ca(2+) entry pathway and/or via channel(s) gated by second messengers. As cell stimulation leads to both a partial Ca(2+) store depletion as well as the production of second messengers, these two pathways are problematic to distinguish. We showed that passive endoplasmic reticulum (ER) depletion by thapsigargin or cell stimulation by histamine activated a similar Ca(2+)-release-activated Ca(2+) current (CRAC)-like current when 10 mM Ba(2+)/2 mM Ca(2+) was present in the extracellular solution. Importantly, during voltage clamp recordings, histamine stimulation largely depleted the ER Ca(2+) store, explaining the activation of a CRAC-like current (due to store depletion) upon histamine in Ba(2+) medium. On the contrary, in the presence of 10 mM Ca(2+), the ER Ca(2+) content remained elevated, and histamine induced an outward rectifying current that was inhibited by Ni(2+) and KB-R7943, two blockers ...
Calcium release and influx colocalize to the endoplasmic reticulum
Current Biology, 1997
Intracellular Ca 2+ is released from intracellular stores in the endoplasmic reticulum (ER) in response to the second messenger inositol (1,4,5) trisphosphate (InsP 3) [1,2]. Then, a poorly understood cellular mechanism, termed capacitative Ca 2+ entry, is activated [3,4]; this permits Ca 2+ to enter cells through Ca 2+-selective Ca 2+-release-activated ion channels [5,6] as well as through less selective store-operated channels [7]. The level of stored Ca 2+ is sensed by Ca 2+-permeant channels in the plasma membrane, but the identity of these channels, and the link between them and Ca 2+ stores, remain unknown. It has been argued that either a diffusible second messenger (Ca 2+ influx factor; CIF) [8] or a physical link [9,10] connects the ER Ca 2+-release channel and storeoperated channels; strong evidence for either mechanism is lacking, however [7,10]. Petersen and Berridge [11] showed that activation of the lysophosphatidic acid receptor in a restricted region of the oocyte membrane results in stimulation of Ca 2+ influx only in that region, and concluded that a diffusible messenger was unlikely. To investigate the relationship between ER stores and Ca 2+ influx, we used centrifugation to redistribute into specific layers the organelles inside intact Xenopus laevis oocytes, and used laser scanning confocal microscopy with the two-photon technique to 'uncage' InsP 3 while recording intracellular Ca 2+ concentration. Ca 2+ release was localized to the stratified ER layer and Ca 2+ entry to regions of the membrane directly adjacent to this layer. We conclude that Ca 2+ depletion and entry colocalize to the ER and that the mechanism linking Ca 2+ stores to Ca 2+ entry is similarly locally constrained.
Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells
Pfl�gers Archiv European Journal of Physiology, 1993
There is increasing evidence that some agonists not only induce intracellular Ca 2+ increases, due to store release and transmembranous influx, but also that they stimulate Ca 2+ effiux. We have investigated the agonist-stimulated response on the intracellular Ca 2+ activity ([Ca2+]i) in the presence of thapsigargin (10 -8 mol/1, TG) in HT29 and CFPAC-1 cells. For CFPAC-I the agonists ATP (10-7-10 -3 tool/l, n = 9), carbachol (10 6_ 10 -3 mol/1, n = 5) and neurotensin (10-1~ -7 mol/1, n = 6) all induced a concentration-dependent decrease in [Ca2+]i in the presence of TG. Similar results were obtained with HT29 ceils. This decrease of [Ca2+]i could be caused by a reduced Ca 2+ inflUX, either due to a reduced driving force for Ca 2+ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca 2+ permeability. Using the fura-2 Mn 2+ quenching technique we demonstrated that ATP did not slow the TG-induced Mn 2+ quench. This indicates that the agonist-induced [Ca2+]i decrease in the presence of TG was not due to a reduced influx of Ca 2+ into the cell, but rather due to stimulation of Ca 2+ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of -49 + 3.6 mV (n = 9). Within the first 3 rain ATP was still able to induce a depolarization which could be attributed to an increase in C1-conductance. This was expected, since at this time after TG stimulation all Ca 2+ agonists still liberated some [Ca2+]~. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca2+]~ decrease cannot be explained by the reduction of the driving force for Ca > into the cell. In the same cells hypotonic swelling (160 mosmol/1, n = 15) still induced a further [Ca2+]~ increase in the presence of TG and concomitantly induced C1-and K + conductances. We conclude that the agonist-induced decrease of [Ca2+]~ in the presence of TG probably unmasks a stimulation of [Ca2+]~ export.
Pflügers Archiv, 1998
Ca 2+ -dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 µM free Ca 2+ and 300 µM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) resulted in prominent capacitance increases. However, dialysis with either Ca 2+ (225 nM to 10 µM) or GTP[γ-S] alone failed to induce a capacitance change. Under conditions of weak Ca 2+ buffering (0.1 mM EGTA), activation of Ca 2+ -release-activated Ca 2+ (CRAC) channels by dialysis with inositol 1,4,5trisphosphate (InsP 3 ) failed to induce a capacitance increase even in the presence of GTP[γ-S]. However, when Ca 2+ ATPases were inhibited by thapsigargin, InsP 3 and GTP[γ-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca 2+ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca 2+ influx because it was effectively suppressed when external Ca 2+ was removed. Our results demonstrate that I CRAC represents an important source of Ca 2+ for triggering a secretory response.