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The Sarcoplasmic Reticulum, Ca²⁺ Trapping, and Wave Mechanisms in Smooth Muscle

John G. McCarron, Karen N. Bradley, Debbi MacMillan, Susan Chalmers and Thomas C. Muir

Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, University of Glasgow, Glasgow G12 8QQ, UK

	Abstract

 
The sarcoplasmic reticulum (SR) and apposed regions of the sarcolemma passively trap Ca²⁺ entering the cell to limit the rise in cytoplasmic Ca²⁺ concentration without SR pump involvement. When "leaky" the SR facilitates Ca²⁺ entry to the cytoplasm. SR Ca²⁺ release via inositol 1,4,5-trisphosphate receptors (IP₃Rs) propagates as calcium waves; IP₃Rs alone account for wave propagation.

	Introduction

Top Introduction The wave components The decline of the... References

 
The ability to change the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) is an inherent characteristic of eukaryotic cells. The amplitude, duration, frequency, and location of each of these [Ca²⁺]_c changes are important determinants of the Ca²⁺ signal; they combine to target specific cellular activities such as gene expression and metabolism and so control the nature of the overall cellular response. The cell’s ability to modulate each of these aspects of Ca²⁺ change provides the flexibility required to enable a single messenger to generate and manage many different and often opposing cellular functions.

Two main sources of [Ca²⁺]_c are recognized: the extracellular fluid and the intracellular stores of the sarcoplasmic reticulum (SR). One of the main Ca²⁺ entry pathways from the extracellular fluid is the voltage-dependent Ca²⁺ channel in the sarcolemma. Membrane depolarization increases the activity of these channels to enhance Ca²⁺ entry and raise [Ca²⁺]_c. The other main [Ca²⁺]_c source is the internal SR store from which release proceeds via two main receptor-controlled channels: the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) and the ryanodine receptor (RyR). The SR may liberate Ca²⁺ in response to activation of IP₃R as a result of agonist activity at the sarcolemma that increases IP₃ production. Ca²⁺ influx from outside may activate RyR in the process of Ca²⁺-induced Ca²⁺ release (CICR). The SR may also release Ca²⁺, via RyR, when the store’s Ca²⁺ content exceeds normal physiological values, i.e., in "store overload." Indeed, store overload may facilitate the occurrence of CICR in cells that do not normally display this feature.

In our studies, depolarization activates voltage-dependent Ca²⁺ channels to increase [Ca²⁺]_c uniformly throughout the bulk of the cytoplasm (Fig. 1A). The increase can be explained solely by Ca²⁺ influx and does not involve the SR or CICR (3). Thus depolarization does not increase [Ca²⁺]_c in the presence of voltage-dependent Ca²⁺ channel blockers, in the absence of extracellular Ca²⁺, or when the membrane potential is held at the equilibrium potential for Ca²⁺. Each of these findings demonstrates that, in response to sarcolemma depolarization, Ca²⁺ influx is essential for a rise in [Ca²⁺]_c and that channel activity or membrane potential changes themselves failed to alter [Ca²⁺]_c in the absence of influx. Moreover, Ca²⁺ influx produced by depolarization of the sarcolemma is quantitatively adequate to account for the entire [Ca²⁺]_c increase, without SR involvement (3). At least 100 times more Ca²⁺ enters the cell than appears as free Ca²⁺ in the cytosol (3). Not surprisingly, therefore, depletion of the SR Ca²⁺ stores using the Ca²⁺ pump inhibitors thapsigargin or cyclopiazonic acid or by ryanodine and caffeine to open RyR on the SR (Fig. 1B) does not reduce depolarization-evoked [Ca²⁺]_c increases (3). These results suggest that the SR does not contribute to the rise in [Ca²⁺]_c evoked by Ca²⁺ influx via CICR, although controversy persists as to the precise contribution of CICR in smooth muscle. Significantly, in equine tracheal myocytes, Ca²⁺ influx may activate, albeit after a considerable delay, Ca²⁺ release from the internal SR store in a process referred to as "loosely coupled CICR" (10). The delayed Ca²⁺ release from the SR was attributed to the time required for subsarcolemmal [Ca²⁺] to build up in the gap between the voltage-dependent Ca²⁺ channels in the sarcolemma and the RyR in the SR to the value necessary to activate CICR. However, the delay between Ca²⁺ influx and Ca²⁺ release from the SR may also reflect the time required for the SR store to accumulate and subsequently discharge Ca²⁺ via RyR when store overload conditions had developed. In those cells in which "loosely coupled CICR" has been described, experiments are required to distinguish between store overload-evoked Ca²⁺ release and the proposed loosely coupled CICR.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Depolarization-evoked cytoplasmic Ca2+ concentration ([Ca2+]c) increases and the passive Ca2+ trap in myocytes. A: depolarization (–70 mV to +10 mV; iv) activated membrane currents (iii) and increased [Ca2+]c as represented by the color changes in i, frames a–f (blue represents low and red represents high [Ca2+]c) and by the fluorescence transients (F/F0; ii). The images in the frames in i were taken before (a), during (b, c), and after (d–f) depolarization and show the resulting [Ca2+]c changes. The time points at which images were obtained are indicated by their respective letters (a–f) above the Ca2+ transients in ii; letters correspond to those in i. Changes in the fluorescence ratio with time (ii) come from 1-pixel-wide lines (536 nm) across the cell at 10-µm intervals. The positions from which the transients (ii) were obtained are indicated in i, frame a (drawn at 2-pixel widths to facilitate visualization). A simultaneous and uniform increase in [Ca2+]c, rather than a propagated wave, occurred throughout the cell during depolarization. [Ca2+]c was imaged at 10-ms intervals. Reproduced from Journal of Biological Chemistry (13) with permission. B: ryanodine depleted the sarcoplasmic reticulum (SR) Ca2+content but increased the depolarization-evoked rise in [Ca2+]c. Depolarization (–70 mV to –10 mV; c) triggered an inward Ca2+ current (d) and increased [Ca2+]c (a). Caffeine (CAF; 10 mM by pressure ejection; b) and inositol 1,4,5-trisphosphate (IP3; photolyzed from caged IP3; a, arrow) each increased [Ca2+]c (a). Ryanodine (50 µM), after repeated application of caffeine to open RyR, abolished both caffeine- and IP3-evoked Ca2+ transients (a), indicating that the SR Ca2+ content had been significantly reduced. In contrast, the depolarization-evoked rise in [Ca2+]c (c) was increased by ryanodine (a) despite a substantially smaller Ca2+ influx (d). The amount of Ca2+ entering the cell by depolarization, i.e., the "calculated" [Ca2+]c (red dotted line; see Ref. 3 for method of calculation) in control (e, i) and in ryanodine (e, ii) was compared with the "measured" [Ca2+]c (blue dashed line) as computed from the Ca2+ transient in the same cell as assessed by fluorimetry. The time course of the measured [Ca2+]c followed that of the calculated increase over the period of depolarization, although more Ca2+ entered than appeared as free Ca2+ in the cell. Ryanodine increased the measured [Ca2+]c value for a given calculated Ca2+ value compared with that in its absence (control; n = 15); i.e., ryanodine decreased the apparent Ca2+ buffer capacity of the cell. Reproduced from Cell Calcium (2) with permission. C: thapsigargin depleted the SR Ca2+content but did not increase the depolarization-evoked rise in [Ca2+]c. Depolarization (–70 mV to 0 mV, c) triggered an inward Ca2+ current (d) and a Ca2+ transient (a). Caffeine (10 mM, b) and IP3 (arrow) each produced Ca2+ transients (a). Thapsigargin (500 nM) abolished both (a; note the residual flash artifact indicated by the arrow), indicating that the SR Ca2+ content had been significantly reduced. Thapsigargin (500 nM) reduced peak amplitude of the Ca2+ current (d and e), and as a consequence the magnitude of the depolarization-evoked Ca2+ transient (a). However, in contrast to the effects of ryanodine, the relationship between the amount of Ca2+ entering the cell by depolarization i.e., the calculated [Ca2+]c (see Ref. 3 for method of calculation) and the measured [Ca2+]c was not significantly altered by thapsigargin (n = 8; P > 0.05); i.e., the apparent Ca2+ buffer capacity of the cell was not decreased by thapsigargin. Reproduced from Cell Calcium (2) with permission. D: passive Ca2+ trap in smooth muscle. Ca2+ entering the cell is retained in a buffer region from which the ion only slowly escapes by negotiating a restricted space between the SR and sarcolemma (a). The SR may also restrict access of bulk [Ca2+]c to the sarcolemma (b). When RyR are locked open, the SR is rendered "leaky." Ca2+ is not retained in the trap but may move more freely through the SR structure to exit into the bulk of the cytoplasm; i.e., more of the Ca2+ entering the cell during transient depolarizations appears as free Ca2+ in the bulk cytosol. With a leaky SR (c), when Ca2+ influx is terminated and removal mechanisms dominate to control [Ca2+]c, the Ca2+ gradients within the cell are inverted and then the process is reversed; Ca2+ must reach the sarcolemma to be expelled from the cell. SR Ca2+ pump (SERCA) activity and a leaky SR may increase the rate at which the ion reaches the sarcolemma. SLCA, sarcolemma Ca2+ pump. Reproduced from Cell Calcium (2) with permission.

Although SR Ca²⁺ pump activity does not limit the rise in [Ca²⁺]_c, the SR itself nonetheless restricts the increase in bulk [Ca²⁺]_c that follows influx by forming a passive Ca²⁺ trap. Inducing leakiness of the SR store, using ryanodine together with caffeine (and fully depleting the SR of Ca²⁺), significantly increases the [Ca²⁺]_c rise that occurs after depolarization and Ca²⁺ influx (Fig. 1B). Yet fully depleting the SR by using either thapsigargin (Fig. 1C) or cyclopiazonic acid (each of which inhibits the SR Ca²⁺ pump) does not increase the amplitude of the depolarization-evoked Ca²⁺ transient (Fig. 1C). These results suggest that, under physiological conditions, the SR store limits the rise in [Ca²⁺]_c that occurs following influx. However, pump activity is not required for this limiting action of the SR store; neither thapsigargin nor cyclopiazonic acid increased the amplitude of the depolarization-evoked [Ca²⁺]_c rise. Rather, the store forms a physical barrier with the sarcolemma to passively limit the access of Ca²⁺ that has entered by influx to the bulk of the cytoplasm, i.e., a passive "Ca²⁺ trap" (2) (Fig. 1D). As much as 40% of the Ca²⁺ entering the cell is held in the trap, generating high subsarcolemma [Ca²⁺] in the tens of micromolar range (2).

One possible role for the Ca²⁺ trap may be to activate low-affinity Ca²⁺-sensitive ion channels in the sarcolemma to modulate Ca²⁺ entry via membrane potential changes. Another role may be to maintain the SR Ca²⁺ content itself. Ca²⁺ influx is essential to maintain the IP₃-sensitive store content of Ca²⁺. Two influx pathways, one voltage insensitive (i.e., not voltage gated) and one voltage sensitive (i.e., voltage gated), allow store refilling to be maintained over a wide membrane voltage range (12). Refilling appears to take place substantially from a subsarcolemma Ca²⁺ pool in which Ca²⁺ concentration exceeds bulk average cytoplasmic values. The Ca²⁺ trap may contribute to the generation and maintenance of the high subsarcolemma Ca²⁺ concentration to permit store refilling (12).

In addition to the extracellular fluid source, the SR also plays an integral role in the generation of those Ca²⁺ signals that give rise to the biological response. These signals emanate from the SR and appear as localized events: Ca²⁺ "puffs" and "sparks" that are restricted to small areas of the cell. In turn, these localized events may activate sarcolemma ion channels such as Ca²⁺-activated K⁺ channels and generate spontaneous transient outward currents (STOCs). Under certain, as yet unspecified conditions the cytoplasm becomes excitable, and Ca²⁺ puffs and/or sparks may coalesce to give rise to repetitive Ca²⁺ transients called "oscillations," which may in turn propagate as Ca²⁺ waves (Fig. 2A). Significantly, removal of external Ca²⁺ inhibits both waves and oscillations. Yet external Ca²⁺ may serve primarily to fill the SR store or to maintain [Ca²⁺]_c at a level that can support waves rather than provide the underlying mechanisms for wave initiation (8). Nonetheless, it is the ordered release of Ca²⁺ from the SR and the characteristics of this release that influence the location, frequency, and amplitude of the Ca²⁺ signals that in turn generate the biological response.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Carbachol (CCh)-evoked Ca2+ waves and the effects of ryanodine on IP3R. A: CCh-increased [Ca2+]c as represented by color changes in the frames (ii; in a–h, blue represents low and red represents high [Ca2+]c) and fluorescence transients (F/F0; iii, 1–7). i: Brightfield image of the cell (left); the calibration applies to all frames in i and ii. [Ca2+]c increases to CCh began in one part of the cell and progressed throughout the cell as a wave (right to left; ii, b–h). The precise time points from which the cell [Ca2+]c images were obtained (ii) are indicated by the corresponding letters above the Ca2+ transients in iii. The F/F0 changes plotted against time (iii) come from 1-pixel-wide lines (536 nm) across the cell at 20-µm intervals. (The positions of the lines are indicated in i, right, drawn at 2-pixel widths to facilitate visualization). Peak [Ca2+]c occurring throughout the cell was well maintained (iv). B: effect of ryanodine on the smooth muscle RyR reconstituted into planar bilayers. Single-channel currents from an isolated RyR are shown as upward deflections from the closed state (c). Ryanodine (10 µM; bottom trace) persistently opened the channel, albeit to a subconductance state (modified from Ref. 19; reproduced with permission, copyright (1994) National Academy of Sciences USA). C: Ca2+ released from IP3R did not activate RyR in myocytes. In single voltage-clamped cells, IP3 (arrow) reproducibly increased [Ca2+]c (a, b), the amplitude of which was not significantly altered by ryanodine (50 µM). a, Summary data from 5 cells (means ± SE) from 8 experiments. These results show that IP3-mediated Ca2+ release does not activate Ca2+-induced Ca2+ release (CICR) at RyR. Reproduced from Journal of Biological Chemistry (5) with permission. D: depletion of the caffeine-sensitive store inhibited the response to IP3. In single voltage-clamped myocytes, IP3 (arrow) and caffeine (caff; c) each reproducibly raised [Ca2+]c (a, b). Ryanodine (50 µM) reduced the [Ca2+]c increase produced by caffeine (c), presumably as a result of depleting the SR of Ca2+, and as a result the subsequent responses to IP3 were also inhibited (arrow; a, b). Summary data from 5 cells are shown in a. *Significant inhibition of the IP3-evoked Ca2+ transient (P < 0.05). Reproduced from Journal of Biological Chemistry (5) with permission. E: ryanodine reduced IP3-mediated Ca2+ transients when RyR were active. Depolarization (to –20 mV; d) from a membrane potential (VM) of –69 mV activated spontaneous transient outward currents (STOCs; a and b) that increased in frequency and amplitude even as [Ca2+]c (c) declined. The current amplitude of STOCs varied (a and b). F: at a holding potential of –20 mV to activate RyR (d) IP3 (arrow) increased [Ca2+]c (b); ryanodine (50 µM) markedly reduced these transients (a, b). Activation of RyR by caffeine (10 mM, c) increased [Ca2+]c (b). A second application of caffeine some 60 s later almost abolished both the [Ca2+]c transient, presumably by depleting the SR store, and the IP3 response (arrow), leaving only the artifact (b). Because the IP3-evoked Ca2+ transient was blocked after caffeine in the presence of ryanodine, leaving only the flash artifact, IP3R and RyR may share a common Ca2+ store that can be partially depleted of Ca2+ by ryanodine, when RyR are active, to reduce IP3-evoked Ca2+ transients. Reproduced from Journal of Cell Science (11) with permission.

	The wave components

Top Introduction The wave components The decline of the... References

 
Waves are induced from the IP₃R on the SR by IP₃-generating agonists and proceed, at nearly constant amplitude, by sequential Ca²⁺ release from one receptor to the next throughout the store (Fig. 2A). Each wave comprises both temporally and spatially related elements, and both a rising and a declining phase have been identified. The rising phase consists of a localized "initiation" component derived from the release of Ca²⁺ from the IP₃R followed by an "amplification" component during which this release is augmented by CICR by positive feedback at either the IP₃R or RyR. The positive feedback process thus amplifies the initiation (local) response to produce the rising phase of the wave.

One interesting but disputed aspect of wave initiation is whether or not IP₃ concentration oscillations are required, i.e., are IP₃ concentration changes coupled to wave production? In the "cross-coupling hypothesis," IP₃R activity, from an as yet ill-defined initiating stimulus, releases Ca²⁺, which results in more IP₃ production in a positive feedback loop until the store presumably is depleted of Ca²⁺, at which time IP₃ production falls to basal levels. However, in other cases changes in IP₃ levels, which occur for example following agonist activity, are not mirrored by corresponding changes in Ca²⁺ levels. Indeed, Ca²⁺ oscillations and waves can be induced by application of constant concentrations of IP₃ (17). Implicit in these findings is the view that IP₃R open and close in a coordinated fashion in the presence of constant concentrations of IP₃, i.e., oscillating IP₃ levels are not necessary for rhythmic activity. It may be that the IP₃R responds to released Ca²⁺ by increasing its open probability, i.e., synergy exists between Ca²⁺ and IP₃ in gating the receptor channel (7) to account for the rising phase of the wave.

Another area of particular interest and controversy concerns the precise nature of the receptors involved in the amplification phase of wave production, especially as to whether IP₃R and RyR each contribute to the process. In one proposal waves initiated by agonists propagate exclusively as a result of IP₃R activity. Here waves originate from properties inherent in the IP₃R themselves that allow them to open and close (deactivate) in the continued presence of a constant concentration of IP₃. This proposal requires that 1) synergism exists between Ca²⁺ and IP₃ in which the IP₃R may act as CICR site, 2) a high [Ca²⁺]_c inhibits IP₃R opening, and 3) a refractory period for the IP₃-gated channel follows channel opening; this period may be induced by IP₃ itself or by cytoplasmic and/or luminal Ca²⁺ concentration.

In another proposal, the IP₃-mediated Ca²⁺ release serves only to initiate the wave and thereafter the role of the IP₃R ends (6). The initial IP₃-mediated Ca²⁺ release then triggers a more substantial Ca²⁺ release from the RyR via CICR. This in turn activates more RyR so that a positive feedback cycle of Ca²⁺ release ensues exclusively at RyR. In support of RyR involvement, drugs that alter RyR activity (ryanodine, ruthenium red, or tetracaine) sometimes abolish [Ca²⁺]_c oscillations (e.g., Ref. 9). Yet different interpretations of these findings are possible. For example, the experimental protocol may have created abnormal store overload conditions in which the sensitivity of RyR to Ca²⁺ may have increased, thus predisposing the cell to wave production at RyR (e.g., Ref. 4), an event that may not occur under physiological conditions.

Alternatively, the pharmacological agents used to assess the contribution of RyR may lack specificity and also block IP₃R. Ryanodine, tetracaine, and ruthenium red may each inhibit IP₃-mediated Ca²⁺ signals independently of RyR involvement (e.g., Refs. 5, 8, 11, 15, 16, and 20). The actions of ryanodine are particularly complex. The drug binds to the open state of the RyR and may prolong receptor open time, albeit at a lower conductance (e.g., Ref. 19; Fig. 2B). Persistently open RyR would presumably increase Ca²⁺ leak from the SR and lower its Ca²⁺ content. Under such conditions, depletion of these stores to which IP₃R and RyR have common access would indirectly inhibit IP₃-mediated responses. This seems a more likely explanation for the inhibition than the proposed amplification of Ca²⁺ release by CICR at RyR. Several of our own observations support this view. First, ryanodine by itself does not inhibit IP₃-mediated Ca²⁺ transients (at a membrane potential of –70 mV), suggesting that Ca²⁺ released via the IP₃R does not activate RyR (Fig. 2C). On the other hand, after transient activation of RyR by caffeine, IP₃-mediated Ca²⁺ release is inhibited (Fig. 2D). Caffeine opens RyR, allowing ryanodine to persistently activate the channel and deplete the store of Ca²⁺. Thus the IP₃-mediated Ca²⁺ transient arises from IP₃R activity alone without RyR involvement. Block of IP₃-mediated Ca²⁺ transients by ryanodine occurs indirectly as a result of a reduction in the Ca²⁺ content of the SR store to which IP₃R and RyR have common access rather than from RyR involvement in the IP₃ response. Ryanodine may also inhibit IP₃ responses without prior activation of RyR by caffeine. For example, under experimental conditions that increase the SR Ca²⁺ content (e.g., depolarization to –20 mV) and activate RyR (as evidenced by the occurrence of STOCs; Fig. 2E), ryanodine reduces the IP₃-evoked Ca²⁺ transient (Fig. 2F). These results can again be explained by ryanodine’s maintaining the RyR in an open configuration to reduce the store content and attenuate the IP₃-evoked Ca²⁺ transients.

Tetracaine also lacks specificity and blocks IP₃-mediated Ca²⁺ release (e.g., Ref. 15). In our experiments, tetracaine blocked submaximal (unpublished results) but not maximal (11) responses to IP₃. Ruthenium red also lacks specificity and exerts complex actions on IP₃-mediated Ca²⁺ responses. For example, in avian atria ruthenium red inhibits the response to IP₃ by an action unrelated to RyR since the response to caffeine was potentiated (16). Clearly there is a problem of lack of specificity of those drugs used to distinguish between the contributions of RyR and IP₃R. Control experiments demonstrating that IP₃R are not inhibited by "RyR blockers" are required to permit the correct interpretation of drug effects on Ca²⁺ signals.

In our studies of Ca²⁺ waves (13), the potential difficulties introduced by poor drug specificity were avoided as far as possible and the conditions proposed to generate waves were employed, i.e., a localized increase in IP₃ and Ca²⁺ that subsequently, it is proposed, activates RyR. Even though IP₃-generating agonists evoked waves (Fig. 2A), the localized release of IP₃ (by localized photolysis of the caged phosphoinositide) did not (Fig. 3A). If activation of RyR had been involved in propagation, it would be expected that the resulting localized increase in Ca²⁺ following photolysis would propagate throughout the cell as a wave. Clearly, our results showed that a localized increase in Ca²⁺ originating from IP₃R activity was, by itself, insufficient to trigger a Ca²⁺ wave, i.e., RyR were not recruited by localized increases in Ca²⁺ from the IP₃R even under conditions where RyR were active and generating STOCs (i.e., at –20 mV) (13) (Fig. 3A). On the other hand, when IP₃ concentrations throughout the cell were elevated, e.g., by subthreshold concentrations of IP₃-generating agonists (Fig. 3B) or following dialysis of the cell with IP₃ to sensitize and prime the IP₃R to release Ca²⁺ (13), a local increase in Ca²⁺ from the activated IP₃R following a localized photolysis of the caged compound successfully evoked a Ca²⁺ wave. Thus a global elevation in IP₃, i.e., a general increase throughout the cell, is required for wave production. One proposal to accommodate these findings is that a global elevation in IP₃ sensitizes the IP₃R to Ca²⁺. When Ca²⁺ release occurs, propagation throughout the cell takes place by the activity of the Ca²⁺-sensitized IP₃R. This accounts for the rising phase (the leading edge) of the propagated Ca²⁺ wave. Wave propagation as implied by the present findings extends only to those cellular sites already sensitized to Ca²⁺ by an increased IP₃ concentration. This permits a spatial control of the Ca²⁺ signal by the cell.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Localized IP3 increased [Ca2+]c in myocytes but produced no waves. A: at a VM of –20 mV (at which RyR are active), local photolyzed IP3 (arrow, 10-µm-diameter region; release position indicated by the bright spot in i, left, which also shows the position of patch electrode) increased [Ca2+]c (ii and iii). [Ca2+]c changes in the cell are represented by color changes in frames a–f (blue represents low and red represents high [Ca2+]c). These increases were maximal at and decreased with each 10-µm increment away from the release site (ii and iii). Numbers indicating the positions of each measurement (regions 1–6; iii) correspond to those in i, right. [Ca2+]c decreased with distance from the release site in the same cell i.e., there was no evidence of wave propagation under these conditions where RyR were active (iv). The photolysis spot appears larger than 10 µm in diameter because the image has been defocused to facilitate visualization of the cell. B: IP3 evoked [Ca2+]c increases and together with CCh produced propagated waves. At –70 mV, local photolyzed increases in IP3 (arrow, 10-µm-diameter region at bright spot a, left; position of patch electrode also shown) increased [Ca2+]c (b, i). Increases were maximal at and decreased with each 10 µm from the release site (b, i; c, i; summary, d; n = 7). The region of each [Ca2+]c measurement (b, i–iii) corresponds to those shown in a, right; the numbers beside the line traces (b, i–iii) also correspond to the regions in a, right. In low subthreshold concentrations of CCh, localized increases in IP3 increased [Ca2+]c (b, ii). These increases were maintained throughout the cell (b, ii; c, ii; summary, d); i.e., they produced a propagated Ca2+ wave. After washout of CCh, photolysis of IP3 increased [Ca2+]c (b, iii). These were maximal at the release site and decreased with each 10 µm from the release site (b, iii; c, iii; summary, d; n = 7), i.e., they produced no waves. Reproduced from Journal of Biological Chemistry (13) with permission. C: proposed structural organization of the SR Ca2+ store. While the wave front progresses, Ca2+ declines at the back of the wave. The decline occurs despite the availability of Ca2+ within the store, as evidenced by its maintained release at the front of the wave. The question therefore arises as to why Ca2+ declines. One possibility is that the store structure is not in luminal continuity with adequate Ca2+ reserves but exists as a series of discontinuous elements, each containing a limited measure of Ca2+. The sequential release and depletion of each of the discontinuous elements could account for the progression of the wave front and the decline in Ca2+ at the back as each of the compartments is depleted of Ca2+. In the event (see Fig. 4), the SR behaves as a functionally compartmentalized entity and is structurally luminally continuous.

	The decline of the wave

Top Introduction The wave components The decline of the... References

View larger version (24K): [in this window] [in a new window]   FIGURE 4. [Ca2+]c-dependent deactivation of IP3R leads to refractoriness of the receptor and accounts for the back of the wave. A: IP3-evoked [Ca2+]c increases at 2 sites in the same cell. At –70 mV, local photolyzed IP3 in a 10 µm diameter region, released at the "test site" (bright spot in a, left; see also patch electrode) increased [Ca2+]c (arrow, b, i). Release was maximal at the release site and decreased with each 10 µm increment away from it (b, i, left). A second photolysis of IP3 ~12 s later at the same test site (b, i, right) generated a much smaller increase in [Ca2+]c than the first (b, i, left). The position of each region of measurement is shown in a, right, the figures of which correspond to those regions in b, i and b, ii. On the other hand, [Ca2+]c release at the test site was not reduced when preceded (by ~12 s) by [Ca2+]c release at the second site. The apparent decline in [Ca2+]c between release at the second and test sites (b, ii) arose from cell movement that accompanied repositioning of the photolysis spot to the test site from the second site. The reduction in release, with 2 photolysis (b, i), at the same site is likely to have been due to a failure of the store to respond to IP3 rather than to an absence of IP3 in the region because of exhaustion of the caged compound by previous photolysis. There was a constant supply of IP3 from the patch pipette, and assuming that the diffusion coefficient (D) of caged IP3 is similar to that of IP3 (estimated as 2 x 10–6 µm2/s), IP3 would have diffused the distance (S) of 5 µm in a time (t) of 625 ms (from t = S2/2D). In support, the time course of fluorescence recovery in the region of photolysis after photobleaching tetramethylrhodamine ethyl ester perchlorate (TMRE; a fluorophore with similar molecular weight and so presumably with a comparable diffusion coefficient to that of IP3) occurred within 1 s (770 ± 72 ms; n = 8) in keeping with our calculated value. Therefore, the time between photolysis (~12 s) would appear adequate to permit the reestablishment of basal conditions. Reproduced from Journal of Biological Chemistry (13) with permission. B: effect of prior photolysis of IP3 on caffeine-evoked increases in [Ca2+]c at the same site in the same cell. At –70 mV, caffeine increased [Ca2+]c (b, i) throughout the cell (the regions of measurement in b, i and b, ii correspond to those shown in a, right). The amplitude of the caffeine-evoked [Ca2+]c increase at region 1 (a; where photolysis occurred) was not significantly altered by prior local photolysis of IP3 (arrow, 10-µm-diameter) at region 1 (b, ii; position of photolysis indicated by the bright spot in a; this panel also shows the patch electrode), suggesting that the store retained Ca2+ after release by IP3. Reproduced from Journal of Biological Chemistry (13) with permission. C: effects of depolarization-evoked [Ca2+]c increases on IP3-mediated Ca2+ release. Depolarization (–70 mV to +10 mV) increased [Ca2+]c and subsequently inhibited IP3-mediated [Ca2+]c release (arrow) even when [Ca2+]c had returned to resting values. Depolarization, in the absence of a [Ca2+]c increase (by pulsing to the equilibrium potential for Ca2+, +132 mV) did not inhibit IP3-mediated Ca2+ release (not shown). D: significant [Ca2+]c-dependent inhibitions of IP3-mediated Ca2+ release occurred with pulse durations >1 s. Here the test photolysis of IP3 was applied 12 s after the end of the depolarization (–70 mV to +10 mV). E: [Ca2+]c-dependent inhibition persisted even after [Ca2+]c had been restored to resting values and remained so 30 s after depolarization ceased. Controls in D and E were taken as 100%, and each time point was compared with its own control (P < 0.05). Reproduced from Journal of Biological Chemistry (13) with permission. F: Ca2+ wave progression in smooth muscle. A global elevation in IP3 sensitizes IP3R to Ca2+. The ensuing Ca2+ release activates CICR at neighboring IP3R; CICR amplifies the initial IP3-mediated Ca2+ release and contributes to progression. The large Ca2+ increase so produced then initiates a slower and persistent negative feedback response, which results in deactivation of the IP3R and the demise of the wave.

Since the IP₃ response persisted after sustained global elevations in IP₃ (after dialysis with the phosphoinositide or activation with sarcolemma agonists), this suggests (by elimination) that an increased [Ca²⁺]_c may be sufficient to account for the refractoriness under the present experimental conditions. Indeed, the refractoriness of IP₃-mediated Ca²⁺ release was mimicked by an increased [Ca²⁺]_c in the absence of either an increased cytoplasmic IP₃ concentration or a reduced SR luminal [Ca²⁺] (Fig. 4C). The IP₃R type 1 (as exists in the present preparation; unpublished result) is activated only over a narrow range of Ca²⁺ concentrations (up to ~300 nM); higher concentrations inhibit the receptor. Previous studies in a variety of tissues and in isolated IP₃R in bilayers under steady-state conditions found that Ca²⁺ deactivation of IP₃R occurred at concentrations >300 nM (e.g., Ref. 7). The deactivation was rapid in onset with, for example, a t_1/2 of 50 ms in rat permeabilized hepatocytes (1). The Ca²⁺-dependent deactivation reported in our studies (11) was much slower in onset and required pulse generations >1 s to be significant (Fig. 4D). After onset, the deactivation persisted in our study for tens of seconds, whereas the recovery (Fig. 4E) from other steady-state studies took considerably less (cf. t_1/2 = 400 ms; Ref. 1). Others have, however, also reported a slow onset and recovery from deactivation. For example, in rat basophilic leukemia cells, a Ca²⁺-dependent suppression of the IP₃-mediated Ca²⁺ transient took 30–60 s for recovery (14). It seems possible that separate, different, Ca²⁺-dependent inhibitory mechanisms of IP₃-mediated Ca²⁺ release may exist, one rapid in onset and recovering quickly on Ca²⁺ removal, a second slow in onset and persistent. Indeed, deactivation of IP₃R, in the present study, once initiated by an increased [Ca²⁺]_c, persisted long after restoration of [Ca²⁺]_c even to resting values, i.e., the deactivation became, at least partially, Ca²⁺ independent (see also Ref. 14). Since some signaling pathways that are activated by Ca²⁺ subsequently become Ca²⁺ independent, e.g., Ca²⁺-dependent protein kinase II, a study of the ability of kinase and phosphatase inhibitors to modulate the deactivation of IP₃-mediated Ca²⁺ release seemed justified. In the event, no support for the participation of either of these enzymes emerged. Perhaps in response to substantial (~1 µM) transient elevations in Ca²⁺, a persistent inhibitory effect of Ca²⁺ on the receptor ensues.

In summary, depolarization evokes uniform increases in [Ca²⁺]_c throughout the bulk of the cytoplasm. However, a significant fraction of the Ca²⁺ entering the cell is retained in a Ca²⁺ trap created by the close apposition of the sarcolemma and SR structure to limit the [Ca²⁺]_c increase in the bulk of the cytoplasm and to create a high subsarcolemma Ca²⁺ concentration. IP₃-generating agonists evoke [Ca²⁺]_c waves, which progress through the cytoplasm by CICR acting on IP₃R without RyR involvement. A delayed onset and thereafter a persistent negative feedback accounts for the decline of the wave. Properties of the IP₃R are themselves sufficient to account for wave progression (Fig. 4F).

	Acknowledgments

 
The Wellcome Trust (060094/Z/00/Z) and British Heart Foundation (PG/2001079; PG/02/161) funded this work.

	References

Top Introduction The wave components The decline of the... References

 

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