Correspondence to: S. Brian Andrews, Building 36, Room 2A-21, 36 Convent Drive, National Institutes of Health Bethesda, MD 20892-4062. Fax:(301) 480-1485 E-mail:sba{at}helix.nih.gov.
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Abstract |
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CICR from an intracellular store, here directly characterized as the ER, usually refers to net Ca2+ release that amplifies evoked elevations in cytosolic free calcium ([Ca2+]i). However, the companion paper (Albrecht, M.A., S.L. Colegrove, J. Hongpaisan, N.B. Pivovarova, S.B. Andrews, and D.D. Friel. 2001. J. Gen. Physiol. 118:83100) shows that in sympathetic neurons, small [Ca2+]i elevations evoked by weak depolarization stimulate ER Ca accumulation, but at a rate attenuated by activation of a ryanodine-sensitive CICR pathway. Here, we have measured depolarization-evoked changes in total ER Ca concentration ([Ca]ER) as a function of [Ca2+]i, and found that progressively larger [Ca2+]i elevations cause a graded transition from ER Ca accumulation to net release, consistent with the expression of multiple modes of CICR. [Ca]ER is relatively high at rest (12.8 ± 0.9 mmol/kg dry weight, mean ± SEM) and is reduced by thapsigargin or ryanodine (5.5 ± 0.7 and 4.7 ± 1.1 mmol/kg, respectively). [Ca]ER rises during weak depolarization (to 17.0 ± 1.6 mmol/kg over 120s, [Ca2+]i less than 350 nM), changes little in response to stronger depolarization (12.1 ± 1.1 mmol/kg, [Ca2+]i
700 nM), and declines (to 6.5 ± 1.0 mmol/kg) with larger [Ca2+]i elevations (>1 µM) evoked by the same depolarization when mitochondrial Ca2+ uptake is inhibited (FCCP). Thus, net ER Ca2+ transport exhibits a biphasic dependence on [Ca2+]i. With mitochondrial Ca2+ uptake enabled, [Ca]ER rises after repolarization (to 16.6 ± 1.8 mmol/kg at 15 min) as [Ca2+]i falls within the permissive range for ER Ca accumulation over a period lengthened by mitochondrial Ca2+ release. Finally, although spatially averaged [Ca]ER is unchanged during strong depolarization, net ER Ca2+ release still occurs, but only in the outermost
5-µm cytoplasmic shell where [Ca2+]i should reach its highest levels. Since mitochondrial Ca accumulation occurs preferentially in peripheral cytoplasm, as demonstrated here by electron energy loss Ca maps, the Ca content of ER and mitochondria exhibit reciprocal dependencies on proximity to sites of Ca2+ entry, possibly reflecting indirect mitochondrial regulation of ER Ca2+ transport.
Key Words: calcium signaling, mitochondria, ryanodine, electron probe X-ray microanalysis, electron energy loss spectrum imaging
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INTRODUCTION |
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Neurons respond to depolarizing stimuli with a rise in the concentration of free cytosolic calcium ([Ca2+]i). This rise, initiated by Ca2+ entry through voltage-gated channels, is strongly influenced by the activity of intracellular calcium stores (
In that study, a model was presented that describes the interplay between [Ca2+]i-dependent uptake and release processes as the critical factor in determining the direction and rate of net ER Ca2+ transport. This model distinguishes three distinct "modes" of CICR; it accounts for Ca2+ buffering at low [Ca2+]i (Mode 1), but also predicts that in certain cases buffering strength will be further attenuated, and eventually eliminated, by graded activation of the Ca2+ release pathway. If activation of the CICR pathway is sufficiently strong at high [Ca2+]i that the rate of Ca2+ release can exceed the rate of uptake, stimulation should lead to net Ca2+ release. In one mode (moderate [Ca2+]i, Mode 2) the transition is gradual, whereas for the other (high [Ca2+]i, Mode 3) CICR may be regenerative, although both cases correspond to classical CICR and are expected to amplify depolarization-evoked [Ca2+]i elevations in concert with a reduction in the Ca load of the ER.
The present study tests several predictions of this model by directly measuring changes in total calcium concentrations within the ER ([Ca]ER) that occur in response to changes in [Ca2+]i during depolarization and/or in the presence of agents that affect intracellular Ca2+ transport. A basic analysis of elemental composition and Ca2+-handling characteristics confirmed that the ER is the structural correlate of the ryanodine-sensitive Ca2+ store. We then examined how [Ca]ER changes during and after steady depolarization as a function of [Ca2+]i, time, and intracellular location. The results show that as depolarization-induced increases in [Ca2+]i become larger, there is a switchover from Ca2+ buffering to net Ca2+ release, providing direct experimental support for Mode 2 or 3 CICR in these cells. They also reveal a dependence of ER Ca2+ handling on radial position within the cell that is reciprocal to the described previously radial gradient of mitochondrial Ca content (
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MATERIALS AND METHODS |
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Cell Preparation and Measurement of [Ca2+]i
Preparations of bullfrog sympathetic neurons, as isolated cells in primary culture or as dispersed ganglia, were obtained as described previously (
Measurement of [Ca]ER
The concentration of total calcium within individual cisternae of ER ([Ca]ER) was measured by energy-dispersive X-ray (EDX)* microanalysis of freeze-dried cryosections obtained from rapidly frozen dispersed ganglia, as described (63-nm probe was adequate to determine ER content without significant contamination from adjacent cytosol. To avoid overlying cytosol, only cisternae wholly contained within the section were analyzed.
Depolarization-induced changes in [Ca]ER were measured by transferring ganglia from normal Ringer's to depolarizing media (30 K+ or 50 K+ Ringer's, isotonic substitution of Na+) for times specified. For 10-s depolarizations, the solution change was accomplished by continuous superfusion on the stage of the freezing machine. Repolarization experiments were performed by transferring ganglia that had been depolarized for 2 min in 50 K+ back to normal Ringer's for specified times. The effect of ryanodine was assayed by preincubating ganglia in normal Ringer's containing 1 µM ryanodine plus 10 mM caffeine for 5 min; the latter facilitates the use-dependent effects of ryanodine on the CICR store. For [Ca]ER measurements in unstimulated cells, ganglia were then transferred to normal Ringers plus 1 µM ryanodine without caffeine 1 min before rapid freezing. For measurements of depolarization-evoked [Ca]ER changes, ganglia were preincubated with caffeine plus ryanodine followed by ryanodine only as described, then transferred to high K+ Ringer's plus ryanodine before rapid freezing.
Electron Energy Loss Spectrum Imaging
Spectrum images (EELSI's) are x-y arrays of pixels, i.e., images, containing a segment of an electron energy loss spectrum at every pixel. EELSI's were acquired using a Gatan model 766 DigiPEELS (parallel-detection electron energy loss spectrometer; Gatan, Inc.) mounted on the HB501 STEM, as described previously (1 nm with a current of
1 nA. The spectrometer was equipped with a 1024-channel photodiode array detector with a read-out noise of
2 counts (rms; equivalent to
20 fast electrons). Frozen-hydrated cryosections were transferred into the STEM at liquid nitrogen temperature and were freeze-dried at -100°C before recooling to -160°C for analysis. Annular dark-field images and EELS-images were recorded using a Gatan DigiScan acquisition system together with Digital Micrograph software (V3.3.1) running on a Power Macintosh 9600/233 computer that incorporated the Gatan spectrum-imaging package of
Calcium and carbon maps were computed by fitting the background in a pre-edge window according to an inverse power law. The extrapolated background was subtracted from the post-edge window to obtain the integrated elemental signals for the Ca L23 and C K core-edges at each pixel in the image. Post-edge windows of width = 10 eV (from 345 to 355 eV) for calcium and width
= 50 eV (from 285 to 335 eV) for carbon were selected. Pre-edge windows of similar width were selected to perform the background extrapolation. Atomic fractions of NCa/NC were computed from the ratios of signal intensities ICa/IC by dividing by the ratios of partial scattering cross-sections obtained from the Gatan EL/P program. The calcium concentrations in mmol/kg dry weight were obtained from the measured atomic fraction of NCa/NC using the formula: [Ca] = NCa/NC x 4.5 x 104 (
= 10 eV) and 4.99 x 103 barns for C (
= 50 eV).
Data Analysis, Reagents, and Supplies
Population results are expressed as mean ± SEM. Statistical significance was assessed using a sequentially rejective Bonferroni procedure for multiple comparisons against a single control, FWE = 0.05 (
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RESULTS |
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Characterization of the Endoplasmic Reticulum as the Ryanodine-sensitive Calcium Storage Organelle
Sympathetic neurons express an abundant network of ER (Fig 1). They also contain internal Ca stores that take up Ca2+ using sarco- and endoplasmic reticulum Ca ATPase (SERCA) pumps and release this ion mainly via a caffeine- and ryanodine-sensitive CICR pathway (
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To test these predictions, changes in the mean total Ca concentration within the ER ([Ca]ER) were measured by EDX microanalysis of rapidly frozen cells in isolated ganglia, in parallel with measurements of free cytosolic Ca2+ ([Ca2+]i) in fura-2loaded cultured neurons. The first observation is that resting [Ca]ER, spatially averaged over the cell soma, was notably high, 12.8 ± 0.9 mmol/kg dry weight (±SEM; equivalent to 3.6 ± 0.3 mmol/liter hydrated tissue; Table 1, Fig 2 A; see
300 nM (30 mM K+ for 2 min; Table 1; see Fig 3 in
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These results identify the ER as the principal, probably the only, Ca store responsible for CICR, but leave open the question of whether the ER Ca pool is a single, homogeneous functional entity. Arguments based on the effects of ryanodine on basal Ca2+ uptake rates and supporting a single pool model were presented in the companion paper (see
A [Ca2+]i-dependent Transition from ER Ca Accumulation to Net Ca Release
In the companion paper (see
Fig 3 A illustrates a typical [Ca2+]i response to strong depolarization (50 mM K+, 2 min) elicited in the absence of drugs or CICR modifiers. During depolarization, [Ca2+]i rapidly rises to a peak and declines toward a steady level of 600800 nM that is sustained for several minutes. Under these conditions, mitochondria avidly and continuously accumulate Ca (
300 nM under otherwise equivalent conditions (Fig 3 D, top left data point). One interpretation is that although both Ca2+-dependent Ca2+ uptake and release by the ER are accelerated when [Ca2+]i reaches
700 nM, the average rates of these processes are equal. In effect, the ER is cycling Ca2+, i.e., "spinning its wheels."
In contrast to untreated cells, cells depolarized in the presence of 1 µM FCCP attain [Ca2+]i levels in excess of 1 µM for 1 min before undergoing a rapid decay to a steady, low level of
200300 nM (Fig 3 B). After repolarization, [Ca2+]i recovers to its prestimulus level with a plateau phase that is greatly reduced compared with the plateau observed under control conditions. The larger [Ca2+]i response and the more rapid recovery are both consistent with suppression of mitochondrial Ca2+ uptake. Quite strikingly, depolarization in the presence of FCCP (45 s) reduces [Ca]ER to 6.5 ± 1.0 mmol/kg (Fig 3 D, bottom right data point), showing that when [Ca2+]i is very high, the ER loses net Ca. Net ER Ca2+ release would also explain the time course of [Ca2+]i, especially the >1-min-long "hump" (Fig 3, compare B with C) seen under these conditions. The hump consists of a prolonged [Ca2+]i elevation followed by a rapid decline (at arrow, Fig 3 B). In cells treated with Tg, in addition to FCCP, the hump is not observed, consistent with the idea that it reflects net Ca2+ release from the ER. The basis for the accelerated recovery after
2 min of FCCP exposure is not known, but, regardless of specific mechanisms, the prompt return to normal resting [Ca2+]i levels after repolarization indicates that exposure to FCCP had not depleted ATP sufficiently to affect normal Ca2+ clearance mechanisms; if it had, a return to a much higher basal [Ca2+]i level would be expected.
To summarize, there is a biphasic relationship between the [Ca2+]i levels reached during depolarization and parallel changes in [Ca]ER (Fig 3 D). Increasing [Ca2+]i from basal levels to 300 nM leads to ER Ca accumulation, whereas with larger [Ca2+]i elevations, [Ca]ER declines. To this point, the evidence for this relationship at high [Ca2+]i depends on measurements obtained while FCCP was present for the purpose of inhibiting mitochondrial Ca2+ uptake. FCCP is generally thought to be a highly specific protonophore without short-term effects on mitochondrial structure at the concentration and exposure times used here (
Time Course of Stimulus-evoked Changes in [Ca]ER at Intermediate [Ca2+]i Levels
Results just presented support the idea that with increasing [Ca2+]i, ER Ca accumulation slows, leading to lower and eventually to no net accumulation as Ca2+ uptake and release come into balance. The finding that such a steady-state condition occurs when [Ca2+]i is 600 nM (Fig 3 D) is further reinforced in Fig 4, a plot of [Ca]ER as a function of time during and after strong depolarization, which shows that [Ca]ER changes little if at all during maintained strong depolarization. (The apparent, statistically insignificant decline at 10 s is discussed below). This behavior contrasts with the very large increases in the concentration of total mitochondrial Ca ([Ca]mito) that occur under the same conditions (Fig 4;
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Upon repolarization, intracellular Ca2+ dynamics change dramatically as [Ca2+]i recovers. How does [Ca]ER respond during this recovery phase? After repolarization (from 50 mM K+, 2-min depolarization), [Ca2+]i recovers in several kinetically distinct phases, the first two of which are strongly influenced by mitochondrial Ca2+ transport. The earliest phase is speeded by mitochondrial Ca accumulation, whereas the long second phase is slowed by net mitochondrial Ca2+ release (Fig 3 A; 200300 nM (Fig 3 A) while [Ca]mito declines (Fig 4, Fig 2 Fig 3 Fig 4 Fig 5 min). Over this same period of the recovery, [Ca]ER rises significantly (Fig 4), which is consistent with the [Ca]ER rise observed when [Ca2+]i is within this same range during steady weak depolarization (see
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Fig 4 also shows that [Ca]ER is elevated for >10 min after repolarization-induced loading. Average [Ca]ER in many cells has begun to decline by 15 min, although that is not evident in Fig 4, owing to substantial cellcell variability (which also accounts for the large error bars at 7 and 17 min). In any case, [Ca]ER recovers quite slowly. Presumably, this reflects a relatively small imbalance between the rates of ER Ca2+ uptake and release, as well as potentially slow rates of release at low [Ca2+]i, both factors contributing to the slow tail phase of [Ca2+]i recovery that outlasts the period of mitochondrial Ca2+ release (Fig 3 A). These observations are reminiscent of similar findings for the ER in these and other neurons (
Spatial Dependence of Depolarization-evoked Changes in [Ca]ER and Its Relationship to Mitochondrial Ca2+ Uptake
The results presented so far indicate that the ER accumulates Ca at low [Ca2+]i, releases net Ca2+ at high [Ca2+]i, and transports little net Ca2+ at intermediate levels. These conclusions are based on spatially averaged measurements of [Ca]ER from multiple cells after steady depolarizations that were much longer than necessary for decay of the initially large, stimulus-evoked radial [Ca2+]i gradients, which dissipate in only a few seconds in these cells (600800 nM, such measurements do not detect significant changes in average [Ca]ER, but there is a tendency for (averaged) [Ca]ER to become lower as the length of depolarization shortens (Fig 4). Therefore, we considered factors that might quantitatively depress spatially averaged [Ca]ER at short depolarization times. One potential contribution is spatial nonuniformity of [Ca2+]i during initial periods of Ca2+ entry. We have provided evidence for the existence of radial gradients in the concentration of mitochondrial total Ca ([Ca]mito) after long depolarization times (e.g., 45 s), and proposed that mitochondria maintain a record of previous, spatially heterogeneous [Ca2+]i, signals when the stimulus raises [Ca2+]i, high enough that mitochondria are forced into their so-called "buffering mode," thereby accumulating Ca continuously (
An analysis of [Ca]ER as a function of distance from the plasma membrane in cells depolarized briefly (10 s) with 50 mM K+ reveals that net Ca2+ release does in fact occur, but only in the outermost 5-µm shell of cytoplasm (Fig 5 A). One likely explanation for this [Ca]ER gradient is that it reflects the transient radial gradients in [Ca2+]i that arise from Ca2+ entry through voltage-sensitive Ca2+ channels during the initial seconds of depolarization (600800 nM in this case, [Ca]ER in these shells would change little, as observed. Furthermore, the dominance of these shells in the spatially averaged [Ca]ER values would largely obscure any local decline of [Ca]ER within peripheral cisternae (Fig 4).
According to this general idea, radial [Ca2+]i gradients drive spatial heterogeneities of [Ca]ER, which, therefore, should be increasingly prominent as the strength of depolarization increases. In addition, for a stimulus of given strength, gradients would be most pronounced during the initial moments of stimulation. As [Ca2+]i gradients dissipate, [Ca]ER would relax, independently of location, toward a value that depends on (now spatially uniform) [Ca2+]i. In contrast, such a relaxation would not occur for [Ca]mito if [Ca2+]i remained above the level where mitochondria continuously accumulate Ca2+. Thus, it appears that different regimes of CICR can coexist in different parts of the cell at a given time, with consequent effects on the direction of net Ca2+ transport. Viewed another way, the radial gradient of [Ca]ER observed after short depolarization recapitulates in a single cell the [Ca2+]i dependence of CICR first deduced from spatially averaged [Ca2+]i measurements after long depolarizations in cell populations (Fig 3 D).
Mitochondria as Sensors of Subplasma Membrane [Ca2+]i Gradients
We have previously reported that mitochondria within a similarly sized 5-µm peripheral shell of cytoplasm accumulate disproportionately large amounts of Ca (
Here, we have applied a novel analytical approachelectron energy loss spectrum imaging (EELSI;
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DISCUSSION |
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The goal of this study was to characterize the functional contributions of the ryanodine-sensitive Ca store to depolarization-evoked changes in [Ca2+]i, with a particular eye toward determining whether in sympathetic neurons this store acts as a Ca2+ source or as a sink. The study was prompted by our seemingly paradoxical observation that, although this store exhibits many of the hallmarks of classical CICR (
The ER Is the Structural Correlate of the Ryanodine-sensitive Ca Store
Since an important aspect of our approach depended on directly measuring changes in the Ca content of the ryanodine-sensitive store, it was necessary to know the corresponding subcellular structure. The Ca content of the ER ([Ca]ER), as well as its pharmacological profile, directly identifies the ER as the principal, if not the only, structural correlate of this Ca store. Although this result is not surprising, it was essential to demonstrate, considering that other organelles may function as Ca stores (
The ER Is a Tunable Ca2+ Buffer that Retains a Record of Signaling Events
The companion article (see 600800 nM, there is little net change in [Ca]ER, suggesting that at these [Ca2+]i levels, enhanced CICR via RyRs largely balances Ca2+ uptake, even though both inward and outward fluxes are larger than basal levels. Therefore, we conclude that sympathetic neurons express either Mode 2 or Mode 3 CICR, since these are the types characterized by a transition to net Ca2+ release.
After repolarization (from 50 K+), [Ca]ER increased during the plateau phase of recovery, a period during which [Ca2+]i is maintained at 300 nM by net mitochondrial Ca2+ release (
The ER of sympathetic neurons appears to differ from certain other neuronal cell types in at least two important respects. First, the distribution of [Ca]ER measurements, at rest and in response to depolarization and a variety of drugs, is generally normal. Therefore, the results are consistent with the idea that the somatic ER of sympathetic neurons is a single functional entity, and provide no evidence for the existence of discrete ER domains that differ in their capacity to transport Ca2+. Although this agrees with results from some nonmuscle cell types (
[Ca2+]i Dependence of ER Ca2+ Transport Confers Location Dependence on the Transition from Ca2+ Sink to Source
Given the acceleration of ER Ca2+ release that occurs with stronger stimuli, we tested whether even larger [Ca2+]i elevations lead to net ER Ca2+ release. After disabling mitochondrial Ca2+ uptake (with FCCP), the same strong depolarizing stimuli (50 mM K+) raised [Ca2+]i to >1 µM, with a concomitant decline in [Ca]ER. This decline reflects Ca2+ release as expected during classical CICR, which, in turn, appears to have synergistically amplified the [Ca2+]i response (Fig 3 B) beyond that attributable to just Ca2+ entry.
It is increasingly clear that intracellular Ca2+ signaling depends on both time and space (
Spatiotemporal Interactions between the ER, Mitochondria, and Cytosolic Calcium
An important component of this study is the confirmation and quantitative refinement of the depolarization-induced radial gradient in [Ca]mito. We previously reported such a gradient at 45 s after strong depolarization, but the EDX data underlying this analysis was scattered owing to the punctate distribution of mitochondrially sequestered Ca (
The cytoplasmic shell where maximal mitochondrial Ca2+ uptake occurs coincides with the shell of maximal ER Ca release. This and other observationsfor example, the rapid [Ca2+]i decline followed by a plateau seen after caffeine/ryanodine-evoked dumping of the ER Ca store (Fig 2 C)suggest functional coupling between these two organelles and are consistent with the idea of direct Ca transfer within organized, functional complexes (
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Footnotes |
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* Abbreviations used in this paper: EDX, energy-dispersive X-ray; EELS, electron energy loss spectroscopy; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; InsP3, D-myo-inositol-1,4,5-trisphosphate; RyR, ryanodine receptor; SERCA, sarco- and endoplasmic reticulum Ca ATPase; Tg, thapsigargin.
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Acknowledgements |
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We thank Drs. T.S. Reese and E.S. Ralston for helpful discussions and critical evaluation and Dr. C.A. Brantner for excellent technical assistance.
This work was supported by a grant (No. NS-33514) from the NIH to D.D. Friel and by the NIH Intramural Research Program.
Submitted: 6 October 2000
Revised: 15 May 2001
Accepted: 17 May 2001
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