Fura-2 antagonises calcium-induced calcium release

Calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER) takes place through ryanodine receptors (RyRs) and it is often revealed by an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by caffeine. Using fura-2-loaded cells, we find such an effect in bovine adrenal chromaffin cells, but not in cerebellar granule neurones or in HEK-293 cells. In contrast, a caffeine-induced [Ca(2+)](c) increase was clearly visible with either fluo-3 or cytosolic aequorin. Simultaneous loading with fura-2 prevented the [Ca(2+)](c) increase reported by the other Ca(2+) probes. Caffeine-induced Ca(2+) release was also measured by following changes of [Ca(2+)] inside the ER ([Ca(2+)](ER)) with ER-targeted aequorin in HEK-293 cells. Fura-2 loading did not modify Ca(2+) release from the ER. Thus, fura-2, but not fluo-3, antagonises the generation of the cytosolic Ca(2+) signal induced by activation of RyRs. Cytosolic Ca(2+) buffering and/or acceleration of Ca(2+) diffusion through the cytosol may contribute to these actions. Both effects may interfere with the generation of microdomains of high [Ca(2+)](c) near the ER release channels, which are essential for the propagation of the Ca(2+) wave through the cytosol. In any case, our results caution the use of fura-2 to study CICR.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.     

Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum

We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca(2+) release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca(2+) uptake and (ii) in maintaining a sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)). For this purpose, we expressed in HeLa cells aequorin-based Ca(2+)-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H(1) receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca(2+)](c) rise and drop in ER luminal [Ca(2+)] ([Ca(2+)](er)), and then the ER refilled with [Ca(2+)](c) at resting values. With histamine, [Ca(2+)](c) after the initial peak stabilized at a sustained plateau, and [Ca(2+)](er) decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca(2+) response than glutamate ( approximately 15 versus approximately 65 microm). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca(2+)](c) plateau and the sustained ER Ca(2+) release and markedly increased the mitochondrial Ca(2+) response. Conversely, mitochondrial Ca(2+) uptake evoked by histamine was drastically reduced by very transient ( approximately 2-s) agonist applications. These data indicate that efficient mitochondrial Ca(2+) uptake depends on the preservation of high Ca(2+) microdomains at the mouth of ER Ca(2+) release sites close to mitochondria. This in turn depends on continuous Ca(2+) release balanced by Ca(2+) reuptake into the ER and maintained by Ca(2+) influx from the extracellular space.     

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca(2+)] control that operates between IP(3)receptor Ca(2+)release channels (IP(3)R) and mitochondrial Ca(2+)uptake sites, and displays functional similarities to synaptic transmission. Activation of IP(3)R by IP(3)is known to evoke quantal Ca(2+)mobilization that is associated with incremental elevations of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Here we report that activation of IP(3)R by adenophostin-A (AP) yields non-quantal Ca(2+)mobilization in mast cells. We also show that the AP-induced continuous Ca(2+)release causes relatively small [Ca(2+)](m)responses, in particular, the sustained phase of Ca(2+)release is not sensed by the mitochondria. Inhibition of ER Ca(2+)pumps by thapsigargin slightly increases IP(3)-induced [Ca(2+)](m)responses, but augments AP-induced [Ca(2+)](m)responses in a large extent. In adherent permeabilized cells exposed to elevated [Ca(2+)], ER Ca(2+)uptake fails to affect global cytosolic [Ca(2+)], but attenuates [Ca(2+)](m)responses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca(2+)pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca(2+)uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca(2+)release through the IP(3)R or during gradual Ca(2+)influx to the junction between ER and mitochondria.     

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.     

Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.     

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value. Using electrophysiological depolarization, we directly demonstrate the process of Ca(2+)-induced Ca(2+) release triggered by Ca(2+) entry through voltage-gated Ca(2+) channels. The amplitude of Ca(2+) release from the ER lumen was linearly dependent on I(Ca).     

Ca(2+)-induced Ca(2+) release via inositol 1,4,5-trisphosphate receptors is amplified by protein kinase A and triggers exocytosis in pancreatic beta-cells

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca(2+)-induced Ca(2+) release (CICR) in pancreatic beta-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca(2+) spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca(2+) spiking at low or even in the absence of depolarization-dependent elevation of [Ca(2+)](i). The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca(2+), failed to mobilize intracellular Ca(2+). On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Therefore, the cell-permeable IP(3)R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic beta-cells were followed by [Ca(2+)](i) spikes in neighboring human erythroleukemia cells, used to report secretory events in the beta-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP(3)Rs is part of the mechanism by which cAMP amplifies insulin release.     

High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling

The Ca(2+) coupling between endoplasmic reticulum (ER) and mitochondria is central to multiple cell survival and cell death mechanisms. Cytoplasmic [Ca(2+)] ([Ca(2+)](c)) spikes and oscillations produced by ER Ca(2+) release are effectively delivered to the mitochondria. Propagation of [Ca(2+)](c) signals to the mitochondria requires the passage of Ca(2+) across three membranes, namely the ER membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Strategic positioning of the mitochondria by cytoskeletal transport and interorganellar tethers provides a means to promote the local transfer of Ca(2+) between the ER membrane and OMM. In this setting, even >100 microM [Ca(2+)] may be attained to activate the low affinity mitochondrial Ca(2+) uptake. However, a mitochondrial [Ca(2+)] rise has also been documented during submicromolar [Ca(2+)](c) elevations. Evidence has been emerging that Ca(2+) exerts allosteric control on the Ca(2+) transport sites at each membrane, providing mechanisms that may facilitate the Ca(2+) delivery to the mitochondria. Here we discuss the fundamental mechanisms of ER and mitochondrial Ca(2+) transport, particularly the control of their activity by Ca(2+) and evaluate both high- and low-[Ca(2+)]-activated mitochondrial calcium signals in the context of cell physiology.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.     

Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.     

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.     

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.     

Multiple modes of calcium-induced calcium release in sympathetic neurons I: attenuation of endoplasmic reticulum Ca2+ accumulation at low [Ca2+](i) during weak depolarization.

Many cells express ryanodine receptors (RyRs) whose activation is thought to amplify depolarization-evoked elevations in cytoplasmic Ca2+ concentration [Ca2+](i) through a process of Ca2+ -induced Ca2+ release (CICR). In neurons, it is usually assumed that CICR triggers net Ca2+ release from an ER Ca2+ store. However, since net ER Ca 2+ transport depends on the relative rates of Ca2+ uptake and release via distinct pathways, weak activation of a CICR pathway during periods of ER Ca accumulation would have a totally different effect: attenuation of Ca2+ accumulation. Stronger CICR activation at higher [Ca2+](i) could further attenuate Ca2+ accumulation or trigger net Ca2+ release, depending on the quantitative properties of the underlying Ca2+ transporters. This and the companion study (Hongpaisan, J., N.B. Pivovarova, S.L. Colgrove, R.D. Leapman, and D.D. Friel, and S.B. Andrews. 2001. J. Gen. Physiol. 118:101-112) investigate which of these CICR "modes" operate during depolarization-induced Ca2+ entry in sympathetic neurons. The present study focuses on small [Ca2+](i) elevations (less than approximately 350 nM) evoked by weak depolarization. The following two approaches were used: (1) Ca2+ fluxes were estimated from simultaneous measurements of [Ca2+](i) and I(Ca) in fura-2-loaded cells (perforated patch conditions), and (2) total ER Ca concentrations ([Ca](ER)) were measured using X-ray microanalysis. Flux analysis revealed triggered net Ca2+ release during depolarization in the presence but not the absence of caffeine, and [Ca2+](i) responses were accelerated by SERCA inhibitors, implicating ER Ca2+ accumulation, which was confirmed by direct [Ca](ER) measurements. Ryanodine abolished caffeine-induced CICR and enhanced depolarization-induced ER Ca2+ accumulation, indicating that activation of the CICR pathway normally attenuates ER Ca2+ accumulation, which is a novel mechanism for accelerating evoked [Ca2+](i) responses. Theory shows how such a low gain mode of CICR can operate during weak stimulation and switch to net Ca2+ release at high [Ca2+](i), a transition demonstrated in the companion study. These results emphasize the importance of the relative rates of Ca2+ uptake and release in defining ER contributions to depolarization-induced Ca2+ signals.     

The endoplasmic reticulum and neuronal calcium signalling

The endoplasmic reticulum (ER) is a multifunctional signalling organelle regulating a wide range of neuronal functional responses. The ER is intimately involved in intracellular Ca(2+) signalling, producing local or global cytosolic calcium fluctuations via Ca(2+)-induced Ca(2+) release (CICR) or inositol-1,4,5-trisphosphate-induced Ca(2+) release (IICR). The CICR and IICR are controlled by two subsets of Ca(2+) release channels residing in the ER membrane, the Ca(2+)-gated Ca(2+) release channels, generally known as ryanodine receptors (RyRs) and InsP(3)-gated Ca(2+) release channels, referred to as InsP(3)-receptors (InsP(3)Rs). Both types of Ca(2+) release channels are expressed abundantly in nerve cells and their activation triggers cytoplasmic Ca(2+) signals important for synaptic transmission and plasticity. The RyRs and InsP(3)Rs show heterogeneous localisation in distinct cellular sub-compartments, conferring thus specificity in local Ca(2+) signals. At the same time, the ER Ca(2+) store emerges as a single interconnected pool fenced by the endomembrane. The continuity of the ER Ca(2+) store could play an important role in various aspects of neuronal signalling. For example, Ca(2+) ions may diffuse within the ER lumen with comparative ease, endowing this organelle with the capacity for "Ca(2+) tunnelling". Thus, continuous intra-ER Ca(2+) highways may be very important for the rapid replenishment of parts of the pool subjected to excessive stimulation (e.g. in small compartments within dendritic spines), the facilitated removal of localised Ca(2+) loads, and finally in conveying Ca(2+) signals from the site of entry towards the cell interior and nucleus.     

Calcium-induced calcium release in neurosecretory insect neurons: fast and slow responses

The dynamics of intracellular free Ca(2+)([Ca(2+)](i)) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca(2+) channels caused a steep increase in [Ca(2+)](i). Depolarizations lasting for < 100ms led to Ca(2+) release from intracellular stores as is indicated by the finding that the rise of [Ca(2+)](i) was greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca(2+)current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca(2+) influx enhanced in this way again caused a rise of [Ca(2+)](i) sensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca(2+)responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca(2+) responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca(2+) rises after having been treated with NHD. Thus, the variability of Ca(2+) signals might be caused by different filling states of Ca(2+) stores, and the resting Ca(2+) current seems to represent a source to fill empty Ca(2+) stores. In line with this notion, block of the endoplasmic Ca(2+) pump by thapsigargin (1 microM) produced either no or largely varying Ca(2+) responses. The Ca(2+) signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca(2+) release channels. Block of mitochondrial Ca(2+) uptake with carbonyl cyanide m -chlorophenylhydrazone (CCCP, 1 microM) completely abolished cADPR-induced Ca(2+) signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca(2+) uptake pathways and that some of these pathways involve mitochondria.