Thapsigargin induces cytoplasmic free Ca2+ oscillations in mouse oocytes

CM ca/cium (1995) 17, 154-164 Q Pearson Professional Ltd 1995

Thapsigargin oscillations Y. M. LAWRENCE Department

induces cytoplasmic in mouse oocytes

free Ca*’

and K. S. R. CUTHBERTSON

of Human Anatomy and Cell Biology, University of Liverpool, Liverpool, UK

Abstract - The mechanisms of calcium signalling in mammalian oocytes during maturation and fertilization are controversial. In this study we measured intracellular free Ca2+ concentrations ([Ca2’]i) with the photoprotein aequorin microinjected into immature mouse oocytes. Immature mouse oocytes typically produced [Ca2+]i responses to muscarinic acetylcholine (ACh) stimulation with two types of component. The first component consisted of a broad transient rise in [Ca2’]i lasting about 1 min. The second component consisted of pulsatile oscillations which could occur before, during or after the broad transient, but typically occurred on the rising phase of the broad transient, with a duration of about 5 8. Removal of external Ca2+ ([Ca2’]o) abolished the Ca2’ responses to ACh. Exposure of oocytes to the specific microsomal Ca2+ -ATPase inhibitors thapsigargin (TG) and cyclopiaronic acid unexpectedly produced sustained oscillations in [Ca2’]i which were sensitive to the concentration of Ca2+ in the external milieu. The frequency of these oscillations was slow, and ceased, sometimes after several cycles, when Ca2+0 was removed. Raised [Ca2’lo significantly increased the frequency in cells oscillating to TG and stimulated nonoscillating cells to begin oscillating. The majority of responsive oocytes which did not produce oscillations to ACh alone (70%), did so after TG treatment. Detailed data analysis indicated that these oscillations were identical to those generated by TG alone, with a similar sensitivity to changes in [Ca2’lo. Exposure of oocytes to ryanodine did not inhibit oscillatory behaviour. These results suggest that immature mouse oocytes possess a store which is insensitive to both TG and ryanodine and is capable of supporting [Ca2fli oscillations.

During the in vitro fertilization of mature ovulated mammalian eggs a series of repeated pulsatile intracellular Ca*’ ([Ca*+]i) transients occur which are sustained for several hours [1,49]. It is thought that these oscillations provide the trigger for the beginnin of the early stages of development [2,3], but 9, [Ca ]i oscillations have also been observed in oocytes earlier in their development [4,5]. These immature germinal vesicle intact oocytes are main-

tained in meiotic arrest (prophase I) until the appropriate hormonal signal is given to resume meiosis and progress to metaphase II. This constitutes the maturation process, which can occur spontaneously if the oocytes are released from antral follicles [6,7]. It is known that a fall in oocyte CAMP concentration is sufficient to trigger maturation, but it has also been suggested that [Ca*+]i signals are involved in releasing the oocytes from meiotic arrest [8]. We 154

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have evidence that treatments which induce a [Ca2+]i signal increase both the rate of entry into maturation and the final proportion of oocytes undergoing maturation (E. Seraspe, Y.M. Lawrence, P.H. Cobbold and K.S.R. Cuthbertson, unpublished observations). Muscarinic acetylcholine receptors have been demonstrated to occur on the oolemma of several mammalian species such as mouse [9], monkey, rabbit [lo] and human [ 111. These receptors are thought to act through the phosphoinositide (PI) signalling system present in the oocyte [6]. As in a wide variety of other cells types, one of the ways [Ca2+]i is thought to increase is as a result of the activation of phospholipase C by receptor-activated GTP-binding proteins [ 12,131. The resulting hydrolysis of phosphatidylinositol 4,5-bisphosphate releases the soluble second messenger inositol (1,4,5)trisphosphate (IP3), which can mobilize Ca2’ from intracellular, nonmitochondrial stores by specific interactions with its receptor [ 141. Several different theoretical models have been proposed to explain how [Ca2+]i oscillations are generated in cells [ 15-21,481. Most models are based on IPs-induced Ca2’ release (IICR), and some involve a second Ca2+-sensitive store, in addition to the ubiquitous IPs-sensitive store, which generates the oscillations by Ca2+-induced Ca2’ release (CICR), with inactivation depending on store depletion [20,21]. The sesquiterpene lactone, thapsigargin (TG), has been used as a tool to interfere with Ca2’ store function and content [22]. Its mode of action is thought to be by the specific inhibition of nonmitochondrial microsomal Ca2+-ATPase activity [26], without affecting inositol phosphate levels [23-251. In some cells this inhibition leads to a rapid loss of Ca2’ from the TG-sensitive pool, which is often apparently the same as the IPs-sensitive pool. Recent studies have demonstrated that this depletion activates influx pathways at the plasma membrane [27,28], which in some cells appear to be the same as those activated by agonists [31-331. This mechanism would serve to replace Ca 2+ lost from the cell by surface Ca2+-ATPase pumping during [Ca2+]i transients, and so sustain the oscillations according to the needs of the finite Ca2+ store. It has also been proposed that during IP3-induced [Ca2+]i oscillations there may be direct activation of surface

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Ca2’ channels by IP3 [24,30] or IP4 [29]. The role of Ca2’ influx in the generation and maintenance of [Ca2+]i oscillations is one issue we address here. The aim of the present study was to examine agonist-induced responses before and after thapsigargin treatment in mouse oocytes. We show that thapsigargin induces sustained oscillations in [Ca2+]i which are frequency modulated by external Ca2’ levels. We also demonstrate that cells which cease oscillating to thapsigargin alone can resume oscillatory behaviour with the addition of acetylcholine and discuss the possible mechanisms that might be involved in the generation of these oscillations.

Materials and methods

The medium used for preparation of oocytes and for [Ca2+]i measurements was M16H, which is Whittingham’s bicarbonate-buffered medium M 16 modified with 25 mM additional HEPES buffer [l]. Oocytes were isolated from the ovaries of 6-8 week-old CBA mice by puncturing antral follicles with a pair of 25 gauge hypodermic needles, releasing them into medium M16H supplemented with 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX; Sigma) to maintain meiotic arrest. Oocytes were mechanically freed from surrounding granulosa cells by repeated trituration with a micropipette. Single healthy germinal vesicle intact oocytes were selected and prepared for microinjection by placing them individually into microslides (Camlab, 0.1 mm path length; cut to about 0.5 mm lengthwise) filled with 1.2% type VII agarose (Sigma) solution supplemented with 0.2 mM IBMX and overlaid with liquid paraffin. The oocytes were held in place in the microslides by gelling the agarose at 4°C for 3 min. They were then incubated at 37°C in a humidified incubator under 5% CO2 in air for at least 15 min after Single oocytes were microinjected refrigeration. with the calcium-sensitive photoprotein aequorin as described previously [l], and were loaded into a superperfusion chamber held at 37°C under a cooled photon-counting photomultiplier. The superperfusion system had a dead-time of 18 s, which was taken into account when reporting the results. Collection and computer analysis of the data were performed as described previously [ 1,491. Statistical analysis of

156 (4

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(b) 600

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300 200

100

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6

Fig. 1 (a) Intracelhdar free Ca*’ response obtained from aequorin-injected mouse oocytes perfused with 2 and 5 pM ACh, and 5 PM ACh in nominally Ca*‘-free medium. (b) Examples where the large intracellular free Ca” response is followed by oscillations of variable frequency in two different cells exposed to 2 pM ACh from the start of each trace. The time constants were 4 s for resting concentrations of free Ca*’ and 0.4 s for transients.

the data was performed with Students t-test. Stock solutions of thapsigargin and ryanodine (both Calbiochem; 10 mM) were dissolved in dimethylsulphoxide (DMSO; BDH; final concentration 0.1%) and stored in small aliquots at -20°C. Acetylcholine (Sigma) stock solutions were made fresh daily dissolved in distilled water. All agents were added to M16H for perfusion of oocytes.

Results Most single aequorin-injected oocytes exhibited an intracellular free Ca2+ response to acetylcholine (ACh) with a threshold concentration of l-2 uM (50/80). Some sensitive oocytes (7/80) were responsive to lower doses (0.245 PM), whilst others (23/80) were completely unresponsive even to 100

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PM ACh. Usually a single application of ACh produced a single free Ca*’ response (duration typically GO 500

l/d4

TG I

400

[Ca*+li nM 300

(b)

1200. 1pM TG I

1000

000 Tree Ca (nM) 600

400

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s

h

Fig. 2 Two types of thapsigargin-induced Ca2’ responses: (a) monophasic and (b) oscillations. Time constant for resting signals 20 s, and 1 s for transients.

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about 1 min, but ranging from 0.5-2 min), which could be repeated with a second application after a recovery period of about 15 min. Too long an exposure, or too short a wash period, resulted in a reduced response or in some cases the complete loss of the response on the next application of ACh. Oocytes which were responsive to repeated applications of ACh displayed a dose-dependent relationship. The intracellular free Ca*’ transients featured in Figure la were typical of most responsive oocytes and were composed of two distinct components. One component consisted of rapid oscillations whilst the other component (which we refer to as the ‘monophasic response’ in this paper) was a more prolonged release phase which terminated abruptly to resting levels. This two-component transient predominated, occurring in 70% of oocytes (Fig. la), whilst a smaller proportion of responsive oocytes (30%) developed oscillations to ACh after this transient. As the examples in Figure lb illustrate, both the frequency and the transient durations of these ACh-induced oscillations varied widely between individual oocytes. These transients could be as short as 5 s or as long as 30 s. The effect of changes in extracellular Ca2’ ([Ca*+],) on ACh transients was examined. For these experiments, [Ca2+lo was reduced to about 100 pM or below and Figure la shows the result of such an experiment. In all cells tested, the response to ACh was blocked on removal of Ca2+o. In 4/9 of these cells, the control response to ACh could be regenerated with the readmission of normal [Ca2+lo (1.7 mM). In 4/9 cells, there was no further response to ACh on readmission of Ca2’o, even after a long period (about 30-40 min) in normal [Ca2+lo. In 119 cells, the response on readmission of Ca2+o was restricted to a short spike. We examined the effect of thapsi argin, an inhibitor of Ca2’ store function, on [Ca 9+]i responses in oocytes. Doses of l-2 pM TG were p+und to be maximally effective in generating a Ca response in parotid acinar cells [31], lacrimal acinar cells [25], platelets [23], hepatocytes [22], and Xenopus oocytes [47] and were also used here in the following experiments. When oocytes were exposed to 1 uM TG there was a long delay of 6-9 min followed by two quite different [Ca2+]i responses. The first type of effect with TG was a slow monophasic re-

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a00 (a)

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sponse (Fig. 2a) that lasted 2.0-2.5 min and rose from basal levels of about 0.1-0.2 PM to a peak [Ca2+]i of about 0.4-0.5 PM. This occurred in 22% of responding oocytes. The second and most surprising type of respnse involved the generation of oscillations in [Ca +]i as shown in Figure 2b. This usually consisted of an initial group of small high frequency oscillations followed by much larger transients peaking at 0.8-1.2 PM [Ca2+]i. The frequencies of the larger transients were slow with mean intertransient periods of 5.06 f 2.17 min (range 4.5 10 min). This occurred in 78% of responding oocytes. The monophasic type effect with TG was comparable to the slow component of the ACh response with similar peak heights and duration, but unlike the response to ACh, it could not be reproduced in the same oocyte and did not show a sharp fall to resting levels in the falling phase. The majority of oocytes (78%) produced oscillations for over an hour which were quite unlike the single transients produced by most responsive oocytes to ACh (duration about 1 min), as the sharp rise and fall in free Ca2’ resulted in transients with mean durations of 17.87 f 1.80 s (n = 112 transients). Oocytes which were unresponsive, even to high doses of ACh were also unresponsive to TG. When higher doses of TG (20-50 p.M) were used, Ca2+ oscillations were induced which were similar to those initiated by l-2 PM (3 oocytes). However, no oscillations were observed with concentrations of TG between 50-200 nM. The experiments with TG were repeated with

Fig. 3 Frequency regulation of TG-induced oscillations by the level of [Ca*+],. (a) Nominally Cazf-free medium prevents oscillatory behaviour. (b) Exchanging normal 1.7 mM Ca*+-containing medium for high [Ca*+J, medium (6.8 mM) increases the frequency of osciilations and reduces the amplitude. (c) Oscillations persist in oocytes exposed to Ca*+-free medium after pretreatment with high (6.8 mM) [Ca*‘], Time constants for resting signals 30 s and for transients 1 s.

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Fig. 4 (a) Resumptionof oscillatorybehaviourwith additionof ACh after spontaneous cessationof TG-inducedoscillations(break= 50 min) and (b) frequencychangesof theseACh oscillationswhen [Ca’+k is raisedfrom 1.7 mM to 3.4 mM and 6.8 r&l. Time constants for resting signals 30 s and 1 s for transients.

25 pM cyclopiazonic acid (CPA), which has a similar action to TG, except that it is easily reversible. As expected, CPA produced the same range of both monophasic and oscillatory responses, with a slightly shorter lag phase (typically about 4 min, perhaps reflecting quicker access across the cytoplasm to the ER), and with both reversibility and repeatability. Oscillations ceased with the removal of CPA and resumed with its return, and a second monophasic response to CPA could be induced after a recovery period without CPA. Experiments were performed to test the effect of omitting Ca2+o on the TG-induced oscillations. The result of this type of experiment is shown in Figure 3a. TG oscillations consistently ceased on removal of Ca2+o (7/7 oocytes), but readily resumed with similar peak heights and frequencies on re-addition of normal [Ca2+lo. The sensitivity of the oscillations to the presence of Ca2+o was further tested by raising [Ca2+], 4 fold from 1.7 mM to 6.8 mM. This elevation of [Ca2+lo resulted in a dramatic increase in frequency of the oscillations and a concomitant reduction in peak height shown in Figure 3b. When oocytes previously exposed to high [Ca*+], (6.8 mM) were exposed to 0 mM [Ca2+], plus 0.5 mM

EGTA, several cycles of Ca2+ release occurred before the transients stopped (Fig. 3c; 2/2 oocytes). This ability of the TG-induced oscillations to continue in the absence of extracellular Ca2’, and the reduction in amplitude with increased [Ca2’lo, confirm the intracellular origin of the Ca2’ release. Cells which did not oscillate with TG in normal [Ca2+lo developed oscillatory behaviour when [Ca2+lo was raised to 6.8 mM. We allowed oscillating cells to continue to do so until the oscillations spontaneously ceased, and then added ACh. ACh then initiated ongoing oscillations, similar to those induced by TG alone (Fig. 4a), which continued for as long as the agonist was present. Many oocytes could oscillate for 3-4 h without any signs of the desensitization which usually occurs in the continued presence of ACh prior to TG treatment. These oscillations showed a similar sensitivity to the presence of Ca2’ in the external medium as those induced by TG alone, in that they ceased on Ca2+ removal, returned on Ca 2’ re-addition and increased in frequency with increasing external Ca2’ levels. Figure 4b shows the frequency and amplitude changes of series of ACh-stimulated Ca2’ spikes after TG treatment when [Ca2+lo was

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w

‘u

Fig. 5 An example where 100 FM ryanodine had a minor effect on TG-induced oscillations. Time constants for resting signals 30 s and 1 s for transients.

changed from 1.7 mM to 3.4 mM and 6.8 mM. The Table shows statistical comparisons made between the detailed data analysis of spikes produced by TG alone and those produced by ACh after TG treatment. The mean intertransient period for ACh control spikes post TG treatment (1.7 mM external Table

Comparison

of oscillations

induced by thapsigargin Peak Cazf hW

Ca*“) was 5.10 f 1.77 min. This period was reduced by 36% and 76% in 3.4 and 6.8 mM [Ca2+lo respectively, and the average peak [Ca*+]i was also reduced, from 1006 r&l to 883 and 815 nM. There was no significant difference between the profile of individual Ca2’ spikes induced by TG alone and those induced by ACh post TG treatment when [Ca2+Jo was normal (1.7 mM). The possibility that the Ca*+-influx pathway may sustain oscillations by replenishing I%-insensitive stores which might be ryanodine-sensitive was examined. Ryanodine binds specifically to Ca2+ release channels in sarcoplasmic reticulum of skeletal muscle which locks the channel open and inhibits CICR. Oscillations induced by TG alone (Fig. 5) or those induced by ACh post TG treatment, were not inhibited by ryanodine (20-100 PM) in 6/7 cells after exposure to the alkaloid for up to 60 min. In two of these cells, the first spike following the addition of ryanodine occurred after a prolonged period and was longer in duration than control spikes (see Fig. 5). In 4 oocytes this did not occur. In the seventh cell where oscillations ceased to 50 yM ryanodine, the oocyte appeared unhealthy and died after 20 min.

Discussion We have previously described intracellular free Ca*’ oscillations with aequorin during fertilization of mature mouse oocytes [l], but the generation of Ca2’ oscillations in immature GV intact mouse oocytes alone and acetylcholine Duration fs)

post thapsigargin

treatment.

Period ($1

1 pM TG only 1.7 mM [Cal, (112 tram) 6.8 mM [Cal, (67 trans)

1015.5 + 63.58 707.25 f 31.60

17.87 f 1.80 20.75 k. 0.70

303.62 f 130.23 128.25 + 8.43*

2 i.tM ACh (post 1.7 mM 3.4 mM 6.8 mM

1006.5 zk22.16 883.37 f 25.75 815 rt: 18.31

17.12 3~2.30 15.37 zk0.52*,+ 15.75 f 0.46*,+

303.42 + 106.27 196.12 f 9.46*,+ 74.00 * 3.07*,+

TG treatment) [Cal, (130 tram) [Cal, (50 tram) [Cal, (45 trans)

Means of samples different at P = 0.05 level of significance: *for comparisons with TG only in normal Ca*+-containing and ‘comparisons with Ach stimulation post TG treatment in normal Ca2+-containing medium (1.7 mM). Abbreviation:

medium (1.7mM), trans = transients.

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with the specific microsomal Ca2+-ATPase inhibitor, thapsigargin, was unexpected and novel. These oscillations were frequency modulated by the level of Ca2’ contained in the external milieu. Ca2’ oscillations during fertilization of hamster eggs were similarly dependent on extracellular Ca2+ in their frequency and abolished in Ca’+-free medium [46], as were spontaneous oscillations in mouse oocytes [6]. The acetylcholine receptor controlled [Ca2+]i transients (Fig. la), which displayed desensitization characteristics, were also converted to oscillations after Ca2+-pump inhibition with TG. The acetylcholine response is probably linked to IP3 production as in other cell types [34,35]. The direct release of IP3 from its photo-releasable caged form in mouse oocytes [4,5] gives similar responses to acetylcholine. The lag phase most probably reflects the early events following receptor occupation, such as the accumulation of sufficient activated G-protein to promote PI turnover, since raising ACh levels, which would raise the rate of active G-protein accumulation, reduces this lag. Our finding of complex phases with ACh stimulation was similar to the complex phases found with the photo-release of caged IP3 [5]. However, the [Ca2+]i response of the latter was not strongly dependent on extracellular Ca2’ and was considered to represent mainly an intracellular redistribution of Ca2+. In contrast, our finding indicated that the [Ca2+]i response to ACh required an element of ,Ca2’ influx. This may reflect differences between receptor controlled Ca2+ release and Ca2’ release by injected IP3, such as G-protein and PLC activation [36] which the latter would bypass. PLC activation in particular may be sensitive to local [Ca2+]l and therefore to calcium influx. The refractory period following the monophasic response to ACh can be explained either by desensitization of the muscarinic receptor, or by Ca2+-store depletion, or, quite plausibly, by both. Desensitization and downregulation of muscarinic receptors have been reported in mouse oocytes [9] and other cell types which express this receptor [37]. Such a desensitization mechanism would account for the requirement for a recovery period without ACh before an additional response can be induced. The single monophasic response typical for ACh never occurred after treatment with TG, which is consistent with the idea that TG depletes, at least functionally,

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the IP3-sensitive store responsible for this part of the ACh response. The similarity in amplitude and duration (though not shape) of the ACh- and TG-induced monophasic responses suggests that store depletion could at least contribute to the inactivation of Ca2+ release during the former. Following TG treatment, instead of a monophasic response, ACh induced ongoing oscillations. These oscillations were produced in the continued presence of ACh which would normally have resulted in apparent receptor desensitization, implying that at least some muscarinic receptor function remained intact. If Ca2+-pumps are extremely active in re-sequestering Ca2+ [38], it would be expected that their sudden inhibition by TG or CPA would result in the immediate loss of the store’s Ca 2+ load. The monophasic response appears to fit such an off-loading of the Ca2’ stores content, and is similar to responses recorded in a neuronal cell line [22], but only after a long delay. This long lag phase typically lasted 6-9 min and is difficult to explain by a simple inhibition of Ca2+-ATPases. In contrast, many cell types, such as parotid acinar cells, react to TG almost immediately [31]. Studies on Ca2’ transport activity of sarcoplasmic reticulum purified from skeletal muscle found that the enzyme containing bound Ca2’ was found to reside in two states [39,40]. As only one state was reactive to TG, only the enzyme population residing in this state reacted immediately with TG, forming a catalytically inactive dead-end complex. The population residing in the unreactive state had to undergo slow conversion to the reactive state before being affected by TG. A conversion of Ca2’pumps to a more TG reactive state has also been reported with vesicles of cardiac SR when phosphorylated with protein kinase A [41]. The long lag phase demonstrated here might similarly be due to ER Ca2+-ATPases that reside in a state not immediately susceptible to TG inhibition. Any alternative explanation based on delayed access for TG to the ER would require that delay to be nearly constant for the majority of the TG, so delivering a pulse increase in [TG] at the ER, since modelling shows that a delay represented by a first order process cannot deliver the kind of pulse response observed. Our finding that oocytes which had spontaneously stopped oscillating with TG alone resumed oscillatory behaviour on subsequent addition of PI

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linked ACh suggests that TG treatment leaves an IP3-sensitive Ca + store functioning sufficiently well to allow ACh to promote oscillatory behaviour. Some reports confirm that in addition to some SR and plasma membrane Ca2+-pumps, which are unaffected by TG, various endoplasmic reticulum Ca2’pumps also have different sensitivities towards TG inhibition [39-41], and there is evidence for considerable heterogeneity for the molecular constituents of Ca2+ stores in some cell types [14]. Thus mouse oocytes might possess both IP3-sensitive and IPs-insensitive ER membranes which contain Ca2+-pumps whose sensitivity to TG is low. The existence of at least one such store is required to explain the rapid falling phase of each transient in the oscillations occurring in the presence of TG, given that these oscillations can continue for over 30 min in the absence of Ca2’, (Fig. 3~). Such TG-insensitive stores could also be the basis for the generation of oscillations in exocrine acinar cells, as suggested by Foskett [31,32], which are the only other cells that have been shown to produce oscillations with TG. In those cells, the Ca2+ oscillations can be enhanced by caffeine and inhibited by ryanodine, and are thought to be driven by periodic Ca2’ release from a TGand IP’s-insensitive Ca2+ store with properties similar to SR of excitable cells [31,32]. In contrast, the TG oscillations shown here in mouse oocytes were not inhibited by concentrations of ryanodine 5 times greater than that required to inhibit TG oscillations in parotid acinar cells. It has been argued that high exogenous concentrations of ryanodine in the mM range are required to inhibit CICR in mouse oocytes, with the suggestion that ryanodine does not accumulate readily in egg cytoplasm [43]. However, since we observed a clear, though minor, effect with ryanodine at 100 l.rM, the problem is not one of access. The effect of ryanodine was limited to the spike occurring immediately after its addition (see Fig. 5). This suggests that ryanodine-sensitive stores were recruited during the longer spike but did not participate in subsequent spikes, perhaps because the store is relatively small and becomes depleted. The oscillations induced by TG are probably due to positive feedback by Ca2’. The most likely targets for Ca2’ positive feedback are PLC [ 17,481 and Ca2+-release channels on intracellular stores [21]. While models involving Ca2+ positive feedback on

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PLC may be appropriate for parts of the ACh responses (e.g. the oscillations shown in the first part of Fig. lb), for the TG-induced oscillations PLC is unlikely to be the target for positive feedback since there is no activation of PLC by G proteins or tyrosine kinases above basal levels, and TG does not directly induce phosphoinositide turnover [26]. Therefore, we propose that the mechanism underlying TG-oscillations involves the sensitization of IP3 receptors to endogenous levels of IP3 by Ca2’ [45], though we cannot formally rule out the involvement of some other, as yet unidentified, Ca2+-release channel in the same family wl$h is insensitive to ryanodine. In this scheme, Ca exerts a positive feedback effect by promoting the Ca2+ mobilizing action of IP3. Any increase in the net flux of Ca2+ into the cytoplasm, whether from enhanced Ca2’ influx due to a capacitative refilling mechanism [27], or due to increased [Ca2+]e, or resulting from Ca2’pump inhibition, would have the same effect on the promotion of oscillations, and increased levels of IP3, for example in response to ACh, would also promote similar oscillations. For the switch off of Ca2’ release we prefer a process that inactivates the Ca2+-release channel to the emptying of the calcium store [21], since the potential of the oscillations to continue in the absence of extracellular Ca2’ indicates a substantially larger calcium store than a depletion mechanism would require. Also the constancy of the pulse duration fits better with an oscillator mechanism depending on Ca2’ channel kinetics, which would be independent of the cell, than with a mechanism depending on store size, which could be expected to vary from cell to cell. The burst of faster and briefer Ca2+ transients at the start of some TG responses (e.g. Figs 2b, 3b & 5) are presumably due to another Ca2+-release channel with faster inactivation and reactivation kinetics, which may be the ryanodine receptor. In summary, we have demonstrated that the microsomal Ca2+-ATPase inhibitor, thapsigargin, stimulates sustained [Ca2+]i oscillations which are frequency modulated by external Ca2+ levels and appear to involve stores which are TG- and ryanodine-insensitive. These stores either are insensitive to TG but sensitive to IP3, or possess a novel calcium channel insensitive to both IP3 and ryanodine but capable of CICR. At least three cal-

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cium oscillator mechanisms are required to explain the range of oscillatory phenomena reported here, with characteristic pulse durations of 5 s, 17.5 s and about 30 s, respectively.

Acknowledgements KSRC would like to thank the Royal Society for support, and the University of Liverpool for funding for YL. We thank Prof. Peter Cobbold for laboratory facilities and helpful discussions.

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