Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes

Cell, Vol. 69, 263-294,

April 17, 1992, Copyright

0 1992 by Cell Press

Molecular Mechanisms of Intracellular Excitability in X. laevis Oocytes James D. Lechleiter’ and David E. Clapham Department of Pharmacology Mayo Foundation Rochester, Minnesota 55905

Summary Following receptor activation in Xenopus ooctyes, spiral waves of intracellular Ca2+ release were observed. We have identified key molecular elements in the pathway that give rise to Ca*+ excitability. The patterns of Ca*+ release produced by GTP-r-S and by inositol 1,4,5-trisphosphate (IPI) are indistinguishable from receptor-induced Ca*+ patterns. The regenerative Ca*+ activity is critically dependent on the presence of IPs and on the concentration of intracellular Ca2+, but is independent of extracellular Ca2+. Broad regions of the intracellular milieu can be synchronously excited to initiate Ca2+ waves and produce pulsating foci of Ca2+ release. By testing the temperature dependence of wavefront propagation, we provide evidence for an underlying process limited by diffusion, consistent with the elementary theory of excitable media. We propose a model for intracellular Ca2+ signaling in which wave propagation is controlled by IP3-mediated Ca2+ release from internal stores, but is modulated by the cytoplasmic concentration and diffusion of Ca2+. Introduction Intracellular Ca’+ release is a familiar convergence point for many receptor-induced cell signals, controlling processes ranging from secretion and heart rate to transcription and cell division (Berridge, 1987; Berridge and Irvine, 1989). However, the molecular mechanism(s) controlling Ca*+ release are incompletely understood, and still less is known about how specific cellular instructions are encoded. Previous work has suggested that cell signals are encoded in periodic Ca2+ changes (oscillations), which are highly organized both temporally and spatially (Berridge et al., 1988; DuPont and Goldbeter, 1989; Berridge, 1990; Petersen and Wakui, 1990; Tsien and Tsien, 1990; Berridge and Moreton, 1991; Cuthbertson and Chay, 1991; Meyer, 1991; Tsunoda, 1991). Using confocal imaging techniques, we recently described the process of receptor-induced Ca2+ release in Xenopus laevis oocytes (Lechleiteret al., 1991a, 1991 b). We found that these large cells exhibited complex spatial and temporal patterns of Ca2+ release in the form of regenerative circular and spiral waves of Ca2+ release. We proposed that these complex patterns were generated by an underlying excitable medium composed of Ca2+ release processes. *Present address: Department of Neurosciences, ginia School of Medicine, Charlottesville, Virginia

University 22908-0002.

of Vir-

Calcium

Excitability is a property common to other chemical and biological systems, including the Belousov-Zhabotinsky (BZ) reaction, aggregating slime mold Dictyostelium discoideum, as well as in the electrical activity in cardiac and neural cells (Zaikin and Zhabotinsky, 1970; Allesie et al., 1973; Devreotes et al., 1983; Goroleva and Bures, 1983; Winfree, 1987). Any process that Undt?rgOeE a large excursion away from and then back to steady-state, when perturbed by a suprathreshold stimulus, may be considered excitable. An excitable medium is defined as a population of excitatory processes, coupled byacommon stimulatory signal through diffusion (Winfree, 1990). In such a medium, suprathreshold stimuli are propagated from one excitatory process to the neighboring excitatory processes by the coupling signal, creating waves of excitation. Small subthreshold perturbationsawayfrom steady-state, on the other hand, are quickly damped out. Using excitability as a model for Ca2+ release, we previously obtained estimates for a refractory period of excitability, the minimal area necessary to initiate wave propagation (critical radius), and a diffusion constant of the excitatory signal suggesting that Ca2+ itself was the coupling stimulatory signal for receptor-induced Ca2+ release in Xenopus oocytes (Lechleiter et al., 1991b). The first objective of the work presented here was to identify, at the molecular level, the key elements involved in Ca2+ excitability. To accomplish this, we directly injected second messengers into the oocytes and released caged compounds by UV laser scanning (Bliton et al., 1992) thus bypassing receptor activation and directly manipulating the Ca2+ release machinery. The second objective of this work was to test some of the predictions of our model for intracellular Ca’+ release. To this end, we artificially induced synchronous excitation of Ca*+ stores in a band defined by the caged release of inositol 1,4,5-trisphosphate (IPs) with UV laser scanning. We also examined the effects of temperature dependence of Ca2+ wave propagation. The combined results of these experiments indicate the predominant roles of IP3 receptor release sites and Ca2+ as key regulators of the Ca*+ release process in oocytes. Results G Protein-Mediated Calcium Excitability Complex spatiotemporal patterns of Ca2+ release are clearly present in hormone receptor-mediated signaling in Xenopus oocytes. We have interpreted these data as evidence for an excitable medium composed of the collection of excitable processes of Ca2+ release. Theoretically, such a medium is composed of a homogenous distribution of excitable processes (Winfree, 1990). However, the evidence for this model of Ca*+ excitability could be affected by a complex spatial distribution of receptors. We wanted to examine the dependence of pattern formation on receptor distribution, by bypassing receptor-induced Caz+ release and directly activating G protein-mediated signal

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Figure 1. G Ca% Activity

Protein-Induced

Regenerative

(A and B) Spatial patterns of Ca2+ release for single optical slices recorded at 340 and 190 s, respectively. (C and D) Stereo view of the spatiotemporal pattern of Ca% release. Caz+ activity was rendered as a volume by sequentially stacking 550 images, captured at 1 s intervals, of a single optical slice in the x-y plane of the oocyte. By presenting a volume of Caz+ activity, it is possible to display the temporal changes of Ca2+ release in the space of a single figure, compared with the space required for the sequential display of hundreds of frames as in (A) and (B) (see text). A brightest-pixel algorithm was also used for presentation. This routine displaysonly the brightest pixel along theviewers line of sight and adds depth to the twodimensional image (Lechleiter et al., 1991a). Resting Cap+ isshown in blue, Ca2;’ increases in green, and purple indicates time when oocyte was scanned with UV light. Color scale intensity is shown in (E), where the absolute intensity of the central pixel is plotted; only 100 of a total of 255 intensity levels are shown. The z-axis is labeled in seconds.

transduction. Xenopus oocytes were simultaneously injected (50 nl) with the Ca2+ dye indicator flue-3 (25 nl of 1 mM; ~25 uM final concentration) and caged GTPr-S (25 nl of 66 mM; ~1.65 mM final concentration). After a 2030 min equilibration period, a single optical slice (760 x 760 x 40 urn) near the plasma membrane surface of the oocyte was confocally imaged at 1 s intervals (Figure 1). The bath solution contained less than 10 nM free Ca2+ to exclude extracellular Ca2+ as a significant source for intracellular Ca2+ release. From 26 to 30 s, the optical slice was laser scanned with ultraviolet (UV) light (shown in purple) to release GTP-r-S uniformly throughout the imaging plane. The integrated dwell time of the UV laser was less than 10 tus at any one location. A focal increase in Ca*+ was immediately apparent at 31 s. From this focus, a wave of Ca’+ irregularly propagated, in bursts, across the imaging plane. The Ca2+ concentration remained high and fairly constant throughout the image until 130-150 s, where it began to decline. At this time, several distinct regions distributed throughout the oocyte began to exhibit regenerative focal activity, initiating waves of Ca2+ release, propagation, and annihilation. This period of time is the regenerative phase of Ca’+ activity. The particular region that showed Ca* release immediately after UV scanning also developed into a prominent pulsating focus of Ca2+ release, producing circular patterns of wave propagation (Figure 1B). The ends of some incomplete arcs of Ca2+ developed into spiral waves (Figure 1A; see below). The complete temporal change in Ca2+ was rendered as a stereo volume by stacking 550 sequential images of the same optical slice, captured at 1 s intervals (Figures IC and 1 D). In these volumes of Ca2+ activity, time is represented by the z-axis. This form of presentation compresses over time Caw activity that is normally shown sequentially in hundreds of frames, as in Figures 1A and 16, into the space of a single figure. Interestingly, the peak

amplitudes for individual Ca2+ waves occurred during the regenerative phase of C$+ activity. This is demonstrated in Figure 1 E, where the intensity of a single pixel, located in the center of the imaging field, is plotted for the entire sequence of images. As the cytoplasmic concentrations continued to drop toward resting levels, the number of pulsating foci decreased. Prior focal regions still supported wave propagation, but the reduction in activity caused fewer annihilations and permitted longer, unbroken arcs of C3+ waves. The waves continued to decrease in frequency over time but were still present 20 min after the initial release of GTP-Y-S (data not shown). The spatiotemporal patterns of this and 7 of 6 other oocytes induced by uncaged GTP+S were qualitatively similar to receptor-induced patterns. In the one exception, an initial Cap+ wave was observed, but no regenerative activity occurred in its wake. We quantitated this similarity by examining the effects of curvature on wavefront velocities, since increasing curvature is predicted to result in faster propagation speeds in an excitable medium (Zykov, 1980; Keener, 1986). We restricted our analysis to circular patterns of pulsating Ca2+ release that were less than 4 s old. At greater times, wavefront edges were often less distinct, due to the slowing of wavefront velocity by partially recovered excitable regions. This dependence of propagation speed on the frequency of activity is referred to as the dispersion effect (Miller and Rinzel, 1981; Dockery et al., 1988). Ca*+ patterns were then processed by sequentially subtracting consecutive images, producing active Ca2+ wavefronts of 1 s duration. In Figure 2A, an expanding circular wavefront is presented. For clarity, only the wavefront edge plus one-fourth of the previous edge is shown in thelefthandcolumn. Fromthisfigure,it isalreadyapparent that larger velocities (note the difference in radii between wavefront edges) are associated with the larger curvatures at 3 and 4 s, compared with 1 and 2 s. A plot of curvature

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hormone receptor-mediated parameters for Ca2+ waves (Lechleiter et al., 1991 b). In terms of Ca2+ release, it will be interesting to determine whether these parameters are conserved in other cell types or if Ca2+ activity is scaled by cell size. We concluded from these experiments that Ca2+ excitability induced by receptors and that induced by uncaged GTP-r-S were indistinguishable, and that the excitable processes of Ca*+ release were distal to receptor activation. We focused the next series of experiments on the role of IPs in Ca2+ excitability, since receptor G protein coupling likely stimulated CaZ+ release through phosphatidyl inositol turnover (Berridge and Irvine, 1989).

- 40

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curvature Figure 2. Curvature Induced Ca2+ Waves (A) Expanding only wavefront Images were above resting (B) Curvature each indicated from (A).

Effect

(LO

0

(pm-l)

on Wavefront

Propagation

for GTP--r-S-

circular wave pattern shown at O-4 s. Left column shows edges plus one-fourth of the previous wavefront edge. sequentially subtracted to define the wavefront edge Ca% levels. versus velocity plot of four expanding circular waves, by different symbols. Closed circles are data plotted

versus velocity from four expanding Ca*+ wavefronts is shown in Figure 28. Here the difference in radii was used as an estimate of velocity, and the inverse of the radii midpoints was used as an estimate of the curvature. In agreement with theory, we observed an approximately linear relationship between increasing curvature and increasing velocity. A linear regression fit of these data gave a mean planar velocity (zero curvature) of 28 urn/s, a mean critical radius of 14.2 urn (zero velocity), and a mean diffusion constant of the excitatory signal (line slope) of 3.97 x lO-6 cm2/s. These estimates were quantitatively similar to

lPsDependent Ca*+ Excitability To examine the role of inositol triphosphates in Ca2+ excitability, preinjected oocytes (~50 uM flue3, -final concentration) were again injected 20-30 min later with either IPs (50 nl of 20 uM, ~1 uM final) or the nonhydrolyzable analog, inositol 1,4,5trisphosphorothioate(lP&) (also ml uM final), and then imaged confocally as described above. In short, the resultant spatiotemporal patterns of Ca*+ release induced by both IPs (9 of 12 oocytes; cf. Figure 3) and IP& (15 of 16 oocytes; cf. Figure 4) were similar to receptor- and G protein-induced Ca*+ activity. The first observation was generally a Ca*+ wave that propagated throughout the imaging plane, producing an elevated cytoplasmic Ca2+ concentration in its wake (cf. Figure 38). Second, the cytosolic Ca2+ decreased and regenerative Cd+ activity developed (Figures 3A, 3C, 3D, and 4B-4D; note the differences in time scale). The magnitude of individual wavefronts and the number of pulsating foci initially increased, but gradually, over a period of minutes, the trend reversed, with the number and frequency of pulsating foci decreasing. The magnitudes of the remaining Ca*+ wavefronts were not significantly smaller than peak amplitude (< 10%) for either IPa- or IP&-induced regenerative activity. This suggests that the metabolism of IPs is not responsible for the cessation of Ca*+ activity. To investigate further whether IP3-induced regenerative activity was similar to receptor- and G protein-mediated activity, the dependence of wavefront velocity on curvature was determined. Analysis of four expanding circular wavefronts from an oocyte injected with IP& yielded a mean planar velocity of 29.5 urn/s, a mean critical radius of 9.8 urn, and a mean diffusion constant for the excitatory signal of 2.94 x 1O+ cm2/s. The similarity between these estimates and those reported above suggested that Ca2+ activity was dominated by IP3-induced Ca2+ release and distal steps in regenerative release rather than prior steps in signal transduction. Stimulation of Ca2+ release, whether by an irreversibly activated G protein or by a nonhydrolyzable analog of IPa, did not result in gross alteration of the CaZ+ release pathway, since the measured activity was indistinguishable from both receptor-mediated and (directly mediated) IPs injections. Generation of Spiral Ca*+ Spiral waves are examples tions that can be generated ure 5A). The formation of

Waves of the complex pattern formaby an excitable medium (Figthis complex pattern can be

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Figure tivity

L. IR-induced

Regenerative

Ca2+ Ac-

(A and B) Spatial patterns of Ca2+ release recorded just prior (270 s) to the addition of caffeine (280 s) and during the initial wave of Ca*+ (95 s). (C and D) Stereo view of the spatiotemporal pattern of Ca2+ release. Ca% activity was rendered as in Figure 1. Gray scale intensity is shown in (D), where the intensity of the central pixel is plotted.

attributed to two properties of excitability when applied to intracellular Ca*+ release. The first is a finite refractory period subsequent to excitation. This period is equated to the time when the underlying Ca*+ processes are unable to release Ca2+. Refractory periods result in the annihilation of Ca*+ wavefronts when they impinge on recently excited areas. Refractory regions recover and support ensuing excitation and Ca*+ wave propagation. The second property is a regenerative stimulus occurring at multiple foci, equated here to the pulsatile release of Ca*+ at multiple foci. Individually, a suprathreshold stimulus would create circular patterns of Ca*+ release. However, when multiple foci are active, the multitude of collisions create incomplete arcs of Ca*+ release and, more importantly, create multiple regions with different degrees of recovering excitability. Since the speed of propagation is dependent on the extent of recovery (dispersion effect), differentially recovered regions can dictate the direction of wavefront propagation and may therefore regulate the direction of the Ca*+ signal within the cell. The sequence of Ca*+ images in Figure 5B show the key features in the development of a spiral wave. A single primary focus, centrally located at frames 3, 15, 27, and 39 s, initiated Cd+ waves at set intervals (&every 6 s). In total, eight circular waves were initiated prior to frame 40 s, but for presentation every other 6 s period has been omitted. Critical to the development of the spiral wave was the nearly concurrent activity of multiple foci along a line to the lower right of the primary focal site (~5 o’clock, frame 4 s). These sites interrupted the symmetrical circular wavefront and created an incomplete arc of Ca*+ release (frame 13 s). More importantly, these sites created a lane of recovering excitability, which directed the advancing end of the wavefront arc. Frequently, propagating Ca*+ arcs are blocked at refractory regions, but on occasion (frame 14 s), the propagating end is directed back around itself. At frames 15 and 16 s, the second pulse of the primary pulsating focus annihilated the turning Ca*+ arc. By frame 27 s, however, the end of the Ca2+ arc had turned faster than the primary

focus, initiating a spiral wavefront. Once initiated, the curvature effect of an excitable medium can maintain spiral wavefront propagation. This effect states that a convex (negative curvature) wavefront propagates more slowly than a planar wave, which in turn propagates more slowly than a concave wavefront (positive curvature; Zykov, 1980; Keener, 1966). Synchronous Excitation of Ca*+ Release Fundamental to our model of intracellular Ca*+ release are the individual excitable processes collectively referred to as the excitable medium. By definition, every process within this medium is excitable, and any small collection of excitable processes (>the critical radius; cf. Zykov, 1960; Keener, 1966; Foerster et al., 1989) is capable of initiating a Ca*+ wave, provided the region is not refractory. These characteristics provided a critical test for our model and predicted that a preselected region, when synchronously excited, would initiate a propagating Ca*+ wave. In agreement with theory, we found that extensive regions of the oocyte were capable of initiating Ca*+ waves, but that some regions were more easily excitable. We also found that under conditions of basal Ca*+ activity, the “hot spots” initiated repetitive pulses of Ca*+ release. Oocytes were initially injected with flue-3 (~25 FM final) and caged IPs (~170 uM final) and imaged confocally as described above. A restricted band of caged IPs (~50 x 760 urn) was then released using a laser scanned UV band (pass lasting 1130 second; see Experimental Procedures). Under these conditions, Ca*+ release was observed and propagated from the band into the adjacent regions of the oocyte. However, propagation was slow (
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Figure 4. IP&lnduced tivity

Regenerative

Ca*

Ac-

(A and B) Spatial patterns of Ca* release recorded after (240 s) and just before (160 s)addition of 20 mM caffeine at 175 s. (C and D) Stereo view of the spatiotemporal pattern of Caz+ release. Volume rendered as in Figure 1. Gray scale intensity is shown in (D), where the

intensity of the central pixel is plotted. Note the expanded time scale compared with Figure 3.

However, nonrefractory regions could still be selected, synchronously excited to release Ca*+, and tested as initiation sites for wave propagation. The oocyte shown in Figure 6 was synchronously excited with a restricted band of uncaged IPs, released by UV scanning at 11 s (purple band). The entire rectangular region defined by the sweeping laser beam evoked a planar Ca*+ wave, which maintained a propagation velocity (~25 pm/s) that was indistinguishable from normal Ca*+ activity (Figure 6A, panel Evk). Remarkably, the initial region became a focus of Ca* release, initiating spontaneous pulses of planar waves (Figure 6A, panels Spt). The magnitude of the Ca*+ wavefront became weaker on each subsequent pulse. By the third and fourth pulses, propagation of the wavefront was noticeably damped (Figure 7). Specifically, the leading edge of the wavefront was difficult to discern; in its place were small focal increases in Ca*+ (cf. insets of frames 43 and 45 s). Assuming the concentration of IPa decreased uniformly, presumably by diffusion and/or metabolic breakdown, the emergence of islands of Ca*+ release suggests regions with higher sensitivity to IPa (hot spots). These regions displayed pulsatile release, but apparently lacked sufficient stimulus to initiate a propagated wave. Although caged release of IPs could initiate pulsating foci, Ca% activity could also be inhibited under certain conditions. The oocyte shown in Figure 8 was injected, as above, with fluo-3, with caged IPa, and, subsequently, with IP& ~10 s prior to imaging. In the first seconds of imaging, IP3S3 had not diffused across the oocyte, as indicated by the advance of Ca*+ activity (Figure 8B, lower panel at 4 s). The initial sites of injection were not visible. At 5 s, UV laser light released a restricted band of IPa across the imaging plane. This produced a diff erential Ca*+ wave, which was larger and broader in the presence of IP& (6 s). As above, pulsatile Ca*+ release was induced

Figure

5. Generation

of a Spiral Ca2+ Wave

(A) Spiral Ca*+ wave pattern of wavelength s from frame 66 s in (B), where the previous

-150 vrn and period -6 65 s of history is shown.

Images (530 x 530 Km) are sequentially subtracted and show only the active Ca% wavefront. Every other 6 s period is omitted. Ca*+ activity generated with IP& (-1 PM final).

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Figure 6. Synchronous Excitation of Ca* Release InitiatesCaH Waves and Pulsating Focus (A) Spatial patterns of Ca- release recorded at 5 s (basal activity, panel Bsl), 12 s (evoked wave, panel Evk), 18 s, and 26 s (spontaneous waves, panels Spt). A band of caged IPJ was released (purple vertical strip) at 11 s with UV scanning. tmages (750 x 750 pm) are slanted 20° for display. (6) Stereo view of 30 consecutive images stacked as a volume. Initial Ca2+ activity generated with lP& (ml pM final). Note that the expanded time scale for this volume of Ca2+ activity permitted the display of individual images but produced intervening gaps, This contrasts with previous volumes (Figures 1,3, and 4) where sequential images werestacked in contact with oneanother. Color scale as in Figure 1.

Figure lease

7. Low Threshold

Regions

of CaH Re-

Ca*+ release patterns recorded from 25 to 47 s (every other frame) for the experiment in Figure 6. Images (750 x 375 urn) are rotated 90° from Figure 6 for presentation. Insets at 43 and 45 s are 375 x 107.5 urn.

Figure IPS

8. Inhibition

of Ca*+ Activity

by Caged

Spatial patterns of Ca2+ release patterns at 195 s (A) and at 4, 5, and 6 s (B). Caged IPs was released at 5 s (purple vertical strip) and 180 s (purple plane). Images (750 x 750 urn) are slanted 20°. Note the two time scales for the stereo view in (C). Initial Ca= activity generated with IP& (ml uM final). Color scale as in Fig ure 1.

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only in the region exposed to IP3S3. By 140 s, lP& had induced regenerative Ca2+ activity throughout the OOCyte. At 180 s, the entire confocal slice was scanned with UV light. The resultant increase in IPs caused a step increase in cytoplasmic Ca*+ and abruptly halted regenerative Ca2+ activity (Figures 8A and 8C). Subsequent release of IPs with restricted UV scans failed to initiate regenerative activity (data not shown). Ca*+ inhibition of l4-induced Ca2+ release has been previously demonstrated in Xenopus OOcytes by direct Ca*+ injections (Parker and Ivorra, 1990a) and is likely to account for our observed inhibition. This interpretation is further supported by the inhibition of IP&-induced Ca2+ activity by bath application of caffeine (20 mM; 12 of 12 oocytes tested; cf. Figures 3 and 4) a compound that releases Ca*+ from IPs-insensitive Ca”+ stores (Endo, 1985) although recent data suggest that caffeine itself may also directly inhibit the IPs receptor (IP$t) (Parker and Ivorra, 1991). Bath application of caffeine (l-20 mM) alone did not induce regenerative Ca*+ activity. Similarly, ryanodine a compound that binds to the same IP3-insensitive Ca*+ channel that is opened by caffeine and augments channel activity (Smith et al., 1988), was ineffective at inducing regenerative Ca2+ activity (0 of 5 oocytes; ~1 uM final). Bath application of thapsigargin, a compound that depletes IPs-sensitive Ca*+ stores(Thastrupet al., 1990), was also ineffective in inducing regenerative Ca*+ activity. Furthermore, oocytes pretreated with thapsigargin (1 uM for 4 hr in zero extracellular Ca’+) exhibited no basal Ca2’ activity when injected with IP& (0 of 11 oocytes), even though subsequent treatment with caffeine still elicited a small Ca*+ increase. These data indicate that Ca2+ excitability is not dependent on the ryanodine-sensitive channel, the channel that is commonly associated with Ca*+induced Ca*+ release (CICR) (Tsien and Tsien, 1990) nor is it dependent on a nonspecific increase in cytoplasmic Ca’+. Rather, our data suggest that Ca*+ excitability is dependent on an IP3R channel, which in turn is dependent on the concentration of cytoplasmic Ca*+. Our estimates of D (the diffusion parameter) implicated Ca2+ as the excitatory coupling signal, yet excitability was inhibited by high concentrations of cytoplasmic Ca*+. This apparent paradox can be resolved by recent reports, which demonstrate a dual regulatory role for Caz+ when regulating IP3R activity. At high concentrations Ca*+ inhibits IP3R channel activity, but at low concentrations Ca*+ is stimulatory (lino, 1990; Bezprozvanny et al., 1991; Finch et al., 1991). Thus, Ca*+ is initially excitatory. This form of CaZ+ feedback is considered here as another form of Ca2+induced CaZ+ release (CICR), albeit at the level of the IP3R channel. Ca*+ Dependence of Regenerative Activity We next examined the role of Ca*+ alone in Ca*+ excitability. We injected a 50 nl bolus of 200 uM Ca*+ into fluo3injected oocyte ~10 s prior to imaging. However, we recorded regenerative Ca2+ activity in only 1 of 13 oocytes. This contrasted with control oocytes injected with IPs (ml PM final), where 9 of 10 oocytes injected developed regenerative activity. This inability to induce CaZ+ activity by di-

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Top: Two expanding Caz+ waves shown at 2!YC and 38% in the same oocyte. Circles mark the edge of wavefronts at 1 s intervals from 0 to 4 s. Only the 1 s active Ca*+ wavefronts are presented. Bottom: Radii are plotted against time for five expanding foci at 25% (A) and for four expanding foci at 36% (B). Curvature versus velocity plots at 25OC (C) and 36% (D) are shown. Closed circles represent data from images.

rect Ca2+ injections is in agreement with previous reports where CaZ+ injections induced oscillations in the Ca2+sensitive chloride current in Xenopus oocytes, but at large Ca*+ concentrations and at a reduced success rate when compared with IPs injections (Miledi and Parker, 1984; Swann and Whitaker, 1988; DeLisle et al., 1990). We concluded that Ca*+ itself was unlikely to generate the complex spatiotemporal patterns of Cap+ release. We also examined the effects of caged Ca”+ release on excitability. At the onset, technical drawbacks limited the usefulness of the available caged compounds. Nitr-5 specifically bound Ca2+, but with a relatively low affinity (16 -150 nM for Ca*+; Gurney et al., 1987). Under our experimental conditions (UV laser scanning), we could not induce release of caged Ca*+ with initial resting Ca*+ concentrations less than 250 nM (Fabiato and Fabiato, 1979).

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Figure 10. Temperature Dependence Radius, and Diffusion Constant

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Velocity,

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Planar velocities (A) were estimated from the zero curvative intercepts, the critical radii (6) from the zero velocity intercepts, and the diffusion constants (C)from the slope of linear regression fits of the curvaturevelocity plots as represented in Figures 9C and 9D. Different symbols represent data from four oocytes (circles are data estimated from Figure 9). lndividualvalues(mean f SEM)calculatedfrom 34expanding circular foci. Temperature dependence of each parameter was then fit by linear regression. Co values are from 22% to 32%.

Temperature Dependence of Ce2+ Excitability An excitable medium is acollection of excitable processes linked by diffusion (Winfree, 1990). We also examined the effects of temperature on Ca*+ excitability to discriminate between enzymatic and diffusional processes. Regenerative Ca*+ activity was first established in fluo9-injected oocytes with a subsequent injection of IP& (ml PM final). The bath temperature was then stepped from 19OC to 40°C, dwelling long enough at each setting for several bursts of activity. Ca*+ patterns were processed by sequentially subtracting consecutive images, producing active Ca2+ wavefronts of one second duration. Two circular patterns of Ca*+ wavefronts are shown for 25OC and 36OC (Figure 9). Circles mark the edge of wavefronts at 1 s intervals. The most apparent affect of temperature on Ca2+ excitablility was the velocity of wavefront propagation. The 3-4s circles markvelocities (difference in radii per second) of 29 and 56 pm/s for 25OC and 36OC, respectively. The radii of several targets, at 1 s intervals, are plotted in Figures 9A and 9B. Their respective velocities are plotted in Figures 9C and 9D against curvature (inverse of the radii midpoint). From these graphs, the mean planar velocities, critical radii, and diffusion constants were determined and plotted as a function of temperature (Figure 10). Each of the estimated parameters increased with increasing temperature. For comparison, the change for a 1O°C increase, from 22OC to 32OC (Q,,), was calculated. The velocity and critical radius had QIo values less than 2, consistent with a biological process limited by diffusion (Segel, 1976) and therefore supportive of the treatment of intracellular Ca*+ release as a collection of excitable processes linked by diffusion. Discussion

DM-nitrophen, on the other hand, had a higher affinity for Ca2+ (‘~5 nM, decreasing to -3 mM with UV illumination) but also possessed micromolar affinity for Mg*+ (~5 PM; Kaplan and Ellis-Davies, 1988). Given the millimolar concentrations of intracellular Mg2+, it was necessary to use high concentrations of partially charged DM-nitrophen (~3.3 mM final oocyte concentration). At this concentration, we could induce caged release of Ca*+ inside oocytes with DM-nitrophen that was charged to 62.4%, 50.00/o, 37.6%,and22.7%(133mMDM-nitrophenand83.3,66.7, 50, and 30.3 mM CaCI,, respectively). However, in none of these experiments (0 of 9,11,7, and 4 oocytes at each charging, respectively) could we induce Ca2+-regenerative activity with UV scanning. We were also unable to induce Caa activity with subsequent injections of IPs (cf. above). Significantly, the resting Ca2+ at 62.40/b, 50%, and 37.6% charging was initially increased, which could have inhibited activity as discussed above. Alternatively, the necessarily high concentrations of caged Ca2+ compounds at all charging levels may have resulted in excessive Ca2+ buffering, thereby inhibiting Cam activity. In summary, Ca* itself is critical for Ca*+ activity, but must act in the presence of IPa. The data are consistent with the hypothesis that Ca*+ is the propagating signal between the excitatory processes of Ca2+ release and that IP3 is important at the excitatory processes.

By analyzing the spatiotemporal patterns of Ca*+ release in Xenopus oocytes, we have identified key elements involved in Ca*+ excitability and have tested several theoretical predictions of excitability. We discuss the Ca2+ signal in two phases as it develops within the cell and propose a molecular model for Ca*+ excitability. Finally, we suggest mechanisms by which an excitable medium is capable of encoding signal information. The Oocyte Ca*+ Wave Intracellular Ca*+ release is initiated by the well-established pathway of receptor G protein coupling. This phase of cell signaling produces a wave of Ca2-’ that envelops the entire oocyte. This signal is reminiscent of the Ca2+ wave that occurs during fertilization in Xenopus eggs (Busa et al., 1985; Busa and Nuccitelli, 1985; Kubota et al., 1987). In an analogous manner, a Caz+ wave is initiated at the site of sperm-egg contact and propagates to envelop the entire egg. Here, we discovered focal regions where Ca*+ release first occurs in response to uniform increases in GTPr-S (see Figure 1). At present we do not know the structural basis for these foci, but the initial Ca*+ foci could be accounted for by a higher density of Ca= stores and/ or by an increased sensitivity to IPa, possibly due to an increased number of IPaRs. In support of this hypothesis,

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Calcium

Excitability

we also can detect the existence of foci with caged release of IPs (see Figure 6; cf. Parker and Ivorra, 1990b). Biologically, the foci for Ca2+ release may provide the cell with a mechanism for controlling the direction of wave propagation. Furthermore, the strength of the initial release of Ca2+, in response to external inputs, could presumably be controlled by variations in focal sensitivity. Excitable Medium Model of Intracellular Ca2+ Release To create an excitable medium, the excitatory processes must be coupled. Based on the estimates of the diffusion constant, the temperature dependence of wave propagation, and the sensitivity of regenerative activity to Ca2+ buffering, we believe that Ca*+ itself is the coupling signal. In this context, wave propagation is due to a suprathreshold increase in the Ca2+ concentration at the neighboring excitatory processes (the IP3-bound lPIR channel; see below), which in turn release Cd’and excite their neighbors. This form of propagation is analogous to action potential propagation in neuronal and cardiac cells in terms of the all-or-none propagation of the signal. However, in contrast to voltage dependence for electrical propagation, our mechanism of feedback is Ca2+ release and is considered here as another form of CICR. The idea that CICR is responsible for Ca2+ wave propagation is a well-established hypothesis (Berridge and Irvine, 1969; Berridge, 1990; Meyer, 1991). What has previously been in doubt is the identity of the underlying Ca2+-excitable pool (Berridge, 1990) as well as the precise details of propagation. The possibility that the IP$l might be capable of such a phenomenom has been suggested by both Finch et al. (1991) and Missiaen et al. (1991). In this paper, we have presented data that support the idea that the IP’a-sensitive pool is the target of Ca2+ feedback (see discussion below) and provide a mechanism for wave propagation of undiminished amplitude. As argued above, the activity and location of multiple Ca2+ foci is responsible for the complex spatiotemporal patterns of Ca2+ release. Changes in the location and sensitivity of these foci ultimately affect the response of the cell to a particular input. Although we cannot attribute significance to a particular pattern of Ca2+ waves, there is another feature worth noting which may be important for encoding information. This is the observation that colliding wavefronts annihilate. In our model, this is due to the refractory property of the elementary excitatory process, either IPzR channel inhibition or depletion of Ca2+ stores (see below). The refractory behavior of Ca2+ release prevents propagation of a signal past the point of collision. This observation suggests, then, that one of the encoding mechanisms in the signaling process is directionally dependent, since a spatial location can receive input from only one source at a time. Thus, in addition to frequency, amplitude, and space, information can also be encoded by the phase of the signal input. Development of Regenerative Ca- Activity The next phase of signaling is marked by the reduction in cytoplasmic Cd+ concentration, most likely due to the

activity of the Ca2+ ATPase enzymes pumping.Ca’+ back into internal stores. It is during this time of signal reduction that regenerative activity begins. Regenerative C3+ release is a mechanism for using the available Ca2-’ to repetitively peak intracellular Cd’. Effectively, information is encoded in frequency rather than in amplitude, sustaining the initial Ca2+ signal over time. This is based on the observation that the peak amplitudes of regenerative Ca2+ waves remain undiminished for tens of minutes after the initial envelopment of the oocyte in Ca2+ (see Figures 3 and 4). The regenerative phase of Ca2+ signaling producescomplex spatiotemporal patterns of Ca2+ waves. Most frequently, there are several focal regions of Ca2+ release, where the multitude of propagating Ca2+ waves results in multiple collisions and creates incomplete arcs of Ca2+ release. The direction of propagation of the Ca2+ arcs is likely directed by partially refractory regions, since the speed of propagation within a region is dependent on the degree of recovering excitability. Thus, the regenerative foci ultimately control the direction of the Ca2+ signal within the cell. When only one focus is active, the pulsating focus produces circular waves of Ca2+ release. In other cases, spiral waves patterns are created. The structural identity of the regenerative foci is unknown, but they apparently share similarities with the foci involved in the initial Ca2+ waves that envelope the oocyte, since the same cellular loci frequently become sites of pulsating Ca2+ release during the regenerative phase of signaling (see Figure 1 B). Mechanism(s) of Regenerative Ca*+ Activity Models for Ca*+ oscillations have been proposed in many cell types and have involved various forms of both positive and negative feedback on the chain of events leading to Ca2+ release. Most models can be reduced to mechanisms where IPs remains constant and only Cap+ levels oscillate, or to mechanisms where the concentration of both Ca*+ and IPs levels oscillate (Berridge, 1990; Tsien and Tsien, 1990; Berridge and Moreton, 1991; Harootunian et al., 1991; Meyer, 1991). Recent work has shown that changes in IPs levels are not necessary, since nonhydrolyzable analogs of IPa are effective in producing Ca2+ oscillations (Wakui et al., 1969; DeLisle et al., 1990). However, the arguments become circular, since even at a constant concentration of basal IPJ, increases in Ca2+ could produce transient increases in PLC activity (Harootunian et al., 1991) and thereby induce oscillations. Similarily, we are unable to demonstate conclusively the likelihood of one model of regenerative Ca*+ activity over all others. However, based on recently published reports and the data presented within this paper, the possibilities have been narrowed considerably. Since we were unable to effectively induce regenerative Ca2+ activity with Ca*+ alone, and since activation of the caffeine-sensitive Ca2+ channel inhibited rather than induced regenerative Ca*+ activity, we have developed a model for the regenerative phase of Ca2+ signaling based on the role of IPa and its receptor. The similarity between the patternsof Ca*+ activity produced by IPs and the nonhydrolyzable analog IP& suggest that the IPs concentration remains relatively constant during this

Cell 292

period to the depletion of individual CaZ+ stores and refilling time, rather than to Ca*+ inhibition of the IPaR. The activity of the Ca2+ pump and store capacity would also determine the duration of this period. We have theoretically simulated the complex spatiotemporal patterns of Ca2+ release by equating Ca2+ depletion with the refractory period (Girard et al., 1992). At present, we cannot distinguish between the two alternative explanations for the refractory period. With the IP3R channel as the molecular analog for excitability, Ca2+ release becomes analogous to electrical excitability in neuronal and cardiac cells, where voltagesensitive channels play similar roles. We have summarized this model of Ca2+ release in Figure 11. Experimental

Figure

11. Molecular

Model for Regenerative

Ca2+ Signaling

Spiral and circular Ca2+ waves are generated at constant levels of IPJ. through the cyclical stimulation and inhibition of the lPIR channel by Ca2+. Ca2+ release within the propagating active zone (gray bands or gray receptors of inset) is due to Ca*+-induced Ca*+ release (CICR) from IPs (black spheres of inset)-bound IP&s (shown as a tetramer). The refractory zone is due to subsequent inhibition of the IP3Rs by high Ca2+ levels (see text for details). Ca*+ inhibition of the lPBR results in annihilation of colliding wavefronts.

time. At constant levels of IPS, the total number of bound IP3Rs would remain relatively constant, assuming that the affinity of the IP3R remains unchanged. Based on these observations, we suggest that regenerative activity is due to the cyclical nature of stimulation and inhibition of the IP3R channel as governed by cytoplasmic Ca’+ concentrations. Recent work has demonstrated that at low Ca*+ concentrations, Ca2+ acts as a coagonist with IPS to release Ca2+ from stores (Bezprozvanny et al., 1991; Finch et al., 1991). At high concentrations, CaZ+ is inhibitory (lino, 1990). Thus, the high Ca2+ in the wake of the initial Ca2+ wave would inhibit the IP3R channel activity until the pumps lower the Ca2+ concentration past a critical level. Once inhibition is removed, and the store is replenished to a required set level (Missiaen et al., 1991) the channel conducts Ca2+ into the cytoplasm. Critically, channel activity is now potentiated by Ca”, which in turn stimulates more Ca2+ release. When the cytoplasmic concentration climbs back past the critical threshold level, channel activity is again inhibited. Thus, there are periods of positive (Ca*+ release) and negative (refractory period) feedback. We interpret the positive feedback of Ca2+ on IP3R channel activity (considered here as CICR) as the likely molecular analog of the elementary excitatory process. The observed refractory period then corresponds to the time when Ca2+ concentrations are high and inhibitory. One variant to this model is to equate the observed refractory

Procedures

Oocyte Methods Experimental conditions and oocyte procedures were essentially as described previously (Lechleiter et al., 1991a, 1991 b). Albino female X. laevis frogs were obtained from Xenopus I (Ann Arbor, Ml) and Nasco (Fort Atkinson, WI). Stage V and VI oocytes (diameters 21000 urn) were mechanically defolliculated. Defolliculated oocytes were kept in L-l 5 supplemented medium (Gibco) at 19OC for no longer than 72 hr. Oocytes were placed in a zero Ca2+ solution (96 mM NaCI, 2 mM KCI, 2 mM MgCI,, 5 mM HEPES [Gibco], 1 mM EGTA [Sigma] [pH 7.51) for all injections. They were given in a 50 nl bolus using a 10 ul Drummond micropipette. An oocyte volume of 1 ul was used to estimate the final concentration of a compound. IPJ was obtained from Calbiochem (San Diego). IP& was obtained from New England Nuclear (Boston). Caged GTP-1-S and flue-3 were purchased from Molecular Probes (Eugene, OR).

Image Acquisition and Analysis Images (126 x 126 pixels) were acquired on a Lasersharp MRC-600 Bio-Rad confocal box adapted to an IM35 Zeiss microscope (10 x UV planapo Olympus objective, 0.4 NA) and analyzed using ANALYZE software (Mayo Foundation, Rochester, MN) on a Silicon Graphics Personal Iris computer. The confocal detector aperture was set at the largest opening (resulting in a40 x 750 x 750 urn optical slice) and the photomultiplier gain at the largest setting to maximize the fluorescence signal. All images were recorded at 1 s intervals and analyzed after lowpass filtering (7 x 7 sigma filter, o =20). The images in Figure 1 were processed as previously described (Lechleiter et al., 199Ia). Briefly, one optical image prior to UV exposure was arbitrarily chosen to represent resting [Ca%],. Ca2+ increases were obtained by subtracting the [Ca*+], image from each of the consecutive optical slices. The final image displays Ca*+ increases superimposed on the resting Ca=. All other figures represent actual CaZ+ gradients. CaZ+ concentrations were calibrated in vitro and placed O-20 intensity units between lo-40 nM with the maximum of 255 units corresponding to >300 nM, as previously reported (Lechleiter et al., 1991a). Image acquisitions were also recorded in the zero Cap+ solution given above. UV Laser Scanning Simultaneous UV and visible wavelength confocal scanning was possible due to a recent modification in our confocal microscope (Bliton et al., 1992). The duration of a scan was determined by a shutter placed only in the path of the UV laserbeam (Coherent lnova 90 argon laser with UV optics supplying 100 mW total power distributed at lines 334, 351, and 363 nm). Thus, 1130 s duration refers to the period of time when the oocyte is simultaneously scanned with UV and visible wavelengths of light. Acknowledgments We thank Steven Girard for helpful discussions on the theoretical treatment of excitability, Chris Bliton for her work in confocal UV scanning, and Dr.% Patricia Camacho and Ernest Peralta for their careful critiques of this manuscript. This work was supported by the American

Intracellular 293

Calcium

Excitability

Heart Association (J. D. L. and D. E. C.), by the Whitaker Foundation (D. E. C.), and by NIH (D. E. C). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenr” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

December

5, 1991; revised

January

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