623

Journal of Physiology (1992), 455, pp. 623-640 With 9 figures Printed in Great Britain

LUMINAL Ca2+ PROMOTING SPONTANEOUS Ca2+ RELEASE FROM INOSITOL TRISPHOSPHATE-SENSITIVE STORES IN RAT HEPATOCYTES BY LUDWIG MISSIAEN*, COLIN W. TAYLORt AND MICHAEL J. BERRIDGE* From the * AFRC Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ and the tDepartment of Pharmacology, University of Cambridge, Cambridge CB2 1QJ

(Received 29 August 1991) SUMMARY

1. Spontaneous Ca2" release from the inositol 1,4,5-trisphosphate (InsP3)-sensitive stores in permeabilized hepatocytes was monitored using Fluo-3 to measure the free [Ca2+] of the medium bathing the cells. 2. Permeabilized cells rapidly sequestered Ca2+, reducing the [Ca2+] to 103 + 5 nM. Under conditions that depended critically upon cell density and the amount of Ca2+ in the medium, this was followed by a slow increase in [Ca2+] culminating in a substantial Ca2+ spike representing synchronous discharge from the InsP3-sensitive stores. 3. During the latency preceding the Ca2+ spike, the stores increased their sensitivity to InsP3. This sensitization seemed to be an all-or-none phenomenon. 4. Oxidized glutathione and thimerosal promoted the spontaneous release by sensitizing the InsP3 receptor. 5. An increase in the [Caa21] within the stores was required for both the increased sensitivity to InsP3 and the subsequent spike. 6. Caffeine (6 mM) antagonized the effect of very low InsP3 concentrations and abolished the Ca2+ spike, without itself releasing Ca2 . 7. Our results suggesting that luminal Ca2+ may sensitize InsP3-sensitive stores leading to spontaneous Ca2+ mobilization will be discussed in the light of a modified version of the two-pool model for explaining cytosolic Ca2+ oscillations. INTRODUCTION

Hepatocytes and many other non-excitable cells exhibit cytosolic Ca21 oscillations (Woods, Cuthbertson & Cobbold, 1986; Rooney, Sass & Thomas, 1989; Berridge, 1990). These oscillations are often evoked by agonists that stimulate inositol 1,4,5trisphosphate (InsP3) formation, although they can also be triggered, in the absence of an increase in [InsP3], by other agents that cause mobilization of intracellular Ca2+ (Rooney, Renard, Sass & Thomas, 1991). Variations of two basic models have been proposed to account for cytosolic Ca2+ oscillations: the first assumes that InsP3 levels MS 9676

624 624

L.

MISSLA4EN H.C ATAYLOR AND Ml. J. BERRIDGE

oscillate and the second assumes that Cal+ mobilization is episodic despite a sustained increase in cytosolic [InsP3] (Berridge & Galione. 1988). The two-pool model is one variation of the latter and proposes that a sustained increase in causes Ca`± mobilization leading to a sustained phase of Ca 2 entry which then causes stores to become overloaded and eventually to release their non-mitochondrial of a release. The ryanodine receptor (Lai, Erickson, process Ca2+-induced by Rousseau, Liu & Meissner, 1988) could mediate this response because it is activated by both cytosolic and luminal Ca2+ (Jaffe, 1991), but recent work suggests that the 1990; Finch, Turner & Goldin, InsP3 receptor is also activated by cytosolic and luminal Watras & Ca21 (Missiaen, Taylor & Ehrlich, 1991) 1991; Bezprozvanny, pools in the of Nunn intracellular & Taylor, 1992). Overloading Berridge, 1991; release from of a constant level of can a trigger spontaneous presence spontaneous release InsP3-sensitive Ca2+ pools (Missiaen et al. 1991). Since this resembles the Ca2+ spikes evoked by extracellular stimuli, this phenomenon has been oscillator in non-excitable included in a modified two-pool model to explain the

[Ins31'] Ca2+

Ca2+

(lino,

Ca2+ Ca2+

InsP3

Ca2+

cells.

Ca2+

release from The aim of this work was to further characterize the spontaneous questions following The pools when become overloaded. they InsP3-sensitive Ca2+ were addressed. (1) How did the sensitivity to change as a function of time during the latency preceding the spike? Evidence for an all-or-none sensitization to InsP3 will be presented. (2) How did sulphydryl agents promote this spontaneous release? We will show that they dramatically increased the sensitivity of the InsP3 rise, it was receptor. (3) Since the overloading of the pools caused a pacemaker rise in luminal the necessary to further investigate whether the spike depended on as a result of the overloading. We [Ca2+], or on the pacemaker rise inin cytosolic luminal was an absolute requirement to will demonstrate that the rise induce the spike. (4) Why did permeabilized hepatocytes never produce a second uptake and release followed spike? The possibility that asynchronous cycles of in permeabilized hepatocytes the spike will be considered. (5) Was the inhibited by caffeine in a similar manner to the spikes in intact cells (Osipehuk, Gallacher & Osipehuk & Petersen, 1990; Wakui, Wakui, Yule, Petersen, 1990; Parker & Ivorra, 1991; Harootunian, Kao, Paranjape & Tsien, Berridge, 1991; We will where caffeine to exert its effect without itself releasing seems 1991), this demonstrate that caffeine was able to inhibit the spike without releasing receptor. The action of caffeine was probably related to its ability to inhibit the oscillations relevance of these findings for our understanding of agonist-induced

InsP3

[Ca2+]

[Ca2+] [Ca2+]

Ca2+

Ca2+ spike Ca2+

Ca2.? Ca2+; InsP3 Ca2`

will be discussed.

METHODS

Male Wistar rats (180-250 g) were stunned by a blow on the head and subsequently killed by cervical dislocation. Hepatocytes were prepared by collagenase digestion and kept in cold (w/v) bovine serum albumin until required Eagle's medium containing NaHCO3-buffered (Nunn & Taylor, 1990). Cells were normally washed twice and resuspended at a density of 30 mm-HEPES (pH 7-3), 1 5x 106 cells/ml in the following medium: 120 they 25 mM-phosphocreatine, 25 units/ml creatine phosphokinase and 5 mM-ATP, 1were at 37 in used within 1 h. Cells (300 were transferred to a stirred thermostatted a Perkin-Elmer LS5 luminescence spectrometer (excitation, 503 nm; emission, 530 nm) and

2%

mm-KCl,

,ul)

mM-MgCl2, /tM-Fluo-3; cuvette °CCa2+

SPONTANEOUS Ca2+ RELEASE IN HEPATOCYTES

625

uptake started by adding 50 #uM-digitonin to permeabilize the plasma membrane. The tracings were calibrated using the equation:

[Ca2"] = 864 nM F-in

Fmax

F

where F is fluorescence. Fmax was obtained by adding 1 mM-CaCl2 to the cuvette and Fmin by subsequently adding 10 mM-K-EGTA (pH 7 3). Each trace is typical of at least three experiments performed on different batches of cells. ATP, creatine phosphokinase and phosphocreatine were from Boehringer. Caffeine, digitonin, NaN3, oxidized glutathione, methylene diphosphonic acid, thimerosal, calmodulin and 2,2'dithiodipyridine were from Sigma. Fluo-3 was from Molecular Probes. InsP3 was a gift from Dr Robin Irvine. All other reagents were of the highest purity commercially available.

RESULTS

Spontaneous Ca2O release from InsP3-sensitive Ca2+ pools Figure 1 A illustrates how permeabilized hepatocytes rapidly sequestered Ca2+ from the medium and lowered the free [Ca2+] to 103 + 5 nM (n = 20). Much of the initial Ca2+ uptake was into the mitochondria from which it later leaked to further load the non-mitochondrial stores, inducing a pacemaker [Ca2+] rise culminating in a spontaneous spike (Missiaen et al. 1991). We previously demonstrated that the spike represented a spontaneous Ca2+ release from the InsP3-sensitive Ca2+ pools once they became overloaded. When cells were resuspended at 5 x 106/ml, this Ca2+ release was dependent on the presence of oxidized glutathione (GSSG) or thimerosal. It is important to emphasize that spontaneous Ca2+ spikes occurred only under precisely defined conditions. The following were critical. (1) When first isolated, the cells must look healthy and then be stored in Eagle's medium for no more than 8 h. (2) A rather low ATP concentration (1 mM) in the assay medium, because increasing the [ATP] to 5 mm prevented the occurrence of the spike, possibly by antagonizing the binding of InsP3 to its receptor (Nunn & Taylor, 1990; Maeda, Kawasaki, Nakade, Yokota, Taguchi, Kasai & Mikoshiba, 1991). (3) A pH of 7 3, because the spike did not occur at pH 6-9, possibly because InsP3 then binds with lower affinity to its receptor (Joseph, Rice & Williamson, 1989). (4) Cell density was critical and had to be separately determined for each batch of cells. Once the optimal cell density (typically 5 x 106/ml) was established, a change of as little as 20 % usually abolished the spike. (5) The balance between cell density and the Ca2+ content of the medium was also critical; to achieve this, it was sometimes necessary to either add extra Ca2+ or to wash the intact cells up to 3 times to remove contaminating Ca2+. Given these strict requirements, it is not surprising that only one in five preparations yielded a clear Ca2+ spike. But once the optimal conditions were established for a certain cell preparation, the traces were very reproducible between different experiments on the same batch of cells.

All-or-none sensitization to InsP3 during the latency In an earlier report (Missiaen et al. 1991), we showed that the latency preceding the Ca2+ spike was associated with an increase in the amount of Ca2+ released by a low InsP3 concentration as the intracellular stores progressively loaded with Ca2+ leaking

L. MISSIAEN, C. W. TAYLOR AND M. J. BERRIDGE

626

from the mitochondria. In Fig. lB and C, we further investigated the time course of the release induced by 25 nM-InsP3. When added immediately before the Ca2l spike, 25 nM-InsP3 produced a large Ca2" release (Fig. LB), but, when added earlier, it was almost ineffective (Fig. 1 C). The onset of the spike was, therefore, accompanied by an abrupt increase in the responsiveness to InsP3. A

GSSG

B

GSSG

GSSG

.

730 C

543

12

521

1C2

124]tZ 5 n-

5m

Fig. 1. Response to InsP3 during the latency preceding spontaneous Ca2+ release. A represents the spontaneous spike after a latency. B illustrates the pronounced Ca2+ release after adding 25 nM-InsP3 immediately preceding the spike. C illustrates the ineffectiveness of the earlier additions of 25 nM-InsP3. GSSG (4 mM) was present throughout each incubation.

The increasing response to low concentrations of InsP3 was not merely the consequence of there being more Ca2+ in the stores as the pools progressively loaded during the latency, but in addition represented an increase in the sensitivity of the stores to InsP3. Figure 2 illustrates how the relative magnitudes of the responses to low (50 nM) and maximal (10 glM) JnsP3 concentrations changed during the latency. Ca2r released At the beginning of the latency, 50 nM-InsP3 released 21+3 % of the by a maximal InsP3 concentration (Fig. 2A), whereas it released 51+3% any riserepresented a immediately before the spike (Fig. 2B) (n = 3). Since[Ca2t] Ca2d release and reuptake, we had to exclude the balance between the rate of possibility that the increasing [Ca2m] rise upon addition of 50 ng-InsP3 during the of resequestration. The finding that the latency was due to a decreased rateCa2n [Ca2+] rise induced by 0i two 5 nmol Ca2+ pulses did not increase during the latency (Fig. 2 C), argues against a decreasing rate of Ca2+ sequestration being responsible for the increasing [Ca2+] rise in response to 50 nM-InsP3. It can therefore be concluded that the pools became more sensitive to InsP3 as they loaded with Ca2+ during the

SPONTANEOUS Ca2+ RELEASE IN HEPATOCYTES

627

latency preceding the spike. The increased amount of Ca2+ released in response to 10 gM-InsP3 (Fig. 2A and B) in addition proves that the InsP3-sensitive Ca2+ pools further loaded up during the latency. The endogenous [InsP3], measured with an InsP3 binding protein kit from Amersham, was 47+4 nm at the beginning of the latency and rose slightly to GSSG

A

B

GSSG

C

GSSG

1305 1175

~~~~785

C

1005 M-IfnsP3

05 nmol Ca`

E2

416

402

l10#M-InsP3 183

188

t 50

17 nM-ImlP3 137

~~t

180 145

50 nM-InsP3

5 min

Fig. 2. The pools become more sensitive to InsP3 during the latency preceding the spike. Fifty nanomolar InsP3, immediately followed by an addition of 10 /ZM-InsP3, was added at the beginning of the latency (A) or immediately preceding the spike (B). C illustrates the [Ca2+] rises upon adding two pulses of 0-5 nmol Ca2 . If all the Ca2+ were to be taken up, the addition of 0-5 nmol Ca2+ would represent a loading of cell Ca2+ of about 20 JiM, which represents a tiny percentage of the total cell-associated Ca2+. GSSG (4 mM) was present throughout each incubation.

59 + 4 nM at the time of the spike (n = 3). These levels were unaffected by the presence of 4 mM-GSSG. This small [InsP3] rise during the latency could not have been the trigger for the spike, because elevating the [InsP3] from 47 to 72 nm at the beginning of the latency by adding 25 nm-InsP3 did not trigger the spike (Fig. 1 C). These basal levels of InsP3 are very similar to those measured in permeabilized osteosarcoma cells (50 nM; Zhao, Khademazad & Muallem, 1990 a).

Effect of sulphydryl agents At low cell densities (5 x 106/ml), the Ca2+ spike occurred only in the presence of GSSG or thimerosal; at higher cell densities (107/ml), they were not needed (Missiaen et al. 1991). The sensitizing effect of sulphydryl agents on the InsP3 receptor, which underlies their promoting effect on the spike, was further investigated in Fig. 3. We

L. MISSIAEN, C. W. TAYLOR AND M. J. BERRIDGE

628

previously observed that 4 mM-GSSG dramatically increased the [Ca2+] rise in response to 50 nM-InsP3. Since sulphydryl agents are known to inhibit Ca21 pumps, we had to exclude the possibility that the increased [Ca2+] rise was simply the manifestation of impaired Ca2+ sequestration. We therefore compared the ratio A

B

C

4 mMGSSG

25 ,Mthimerosal

D

501M-

E

thimerosal

100 sM2,2'- DTDP

1288

C

~~~~763 598-

K

~~~~~372

C;+

396

163 106 88

Fig. 3. Sulphydryl agents increase the Ca2l release in response to 50 nM-InsP3. Ca2 , 0-35 nmol, followed by 50 nM-InsP3, was added in the presence of 4 mM-GSSG (B), 25 (C) and 50 (D) /tM-thimerosal, 100 /zm-2,2'-DTDP (E), or in the absence of sulphydryl agents (A).

between the [Ca2+] rise after a 50 nM-InsP3 addition with that after a 0-35 nmol Ca2+ pulse (a measure for the rate of Ca2+ sequestration). This ratio was 0-53 in the absence of sulphydryl agents (Fig. 3A) but in the presence of 4 mm-GSSG, it increased to 4-8 (Fig. 3B), indicating that relatively more Ca2+ had been released by InsP3 in the presence of the sulphydryl reagent. Note that at 4 mm, GSSG did not significantly affect the initial rate of Ca2+ sequestration following permeabilization, nor did it affect the amplitude of the Ca2+ spike induced by the 0-35 nmol Ca2+ pulse (131 nM in the presence of GSSG, while 108 nm in its absence), implying that GSSG increased the InsP3-induced Ca2+ release without significantly inhibiting the Ca2+ pumps. The same enhancing effect on the InsP3-induced Ca2+ release was observed with thimerosal: the ratio of the [Ca2+] rise induced by 50 nM-InsP3 over the [Ca2+] rise after addition of 0-35 nmol Ca2+ was 2-6 in the presence of 25 fM- (Fig. 3 C) and 5-5 in the presence of 50 ftM-thimerosal (Fig. 3D). However, 50 4uM-thimerosal significantly

SPONTANEOUS Ca2+ RELEASE IN HEPATOCYTES

629

inhibited the Ca2l pumps, judged from the slower rate of initial Ca2+ sequestration, the higher steady-state [Ca2+], and the much higher [Ca2+] rise after the 0 35 nmol Ca2+ pulse (186 nm versus 108 nm in the control). Figure 3E illustrates that 100 JLM2,2'-dithiodipyridine (2,2'-DTDP) also increased the Ca2+ release by 50 nM-InsP3 A

B

GSSG

C

Thimerosal

136i:1425 ~~1305

2,2'- DTDP

1315

_

1360

D

2~~~~

~~~~~~785

C

~~~~~~~10aum-

co

InsP3 4.-

651 490 ~~~~~~~~~~~~~~101tM-

InsP3

E

2

~~~~~~~~~~~~~~~~~~~~~~~~~1 0 t

5 min

Fig. 4. Sulphydryl agents sensitize the InsP3-induced Ca2+-release mechanism. Fifty nanomolar InsP3, immediately followed by 10 ,uM-InsP3, was added as indicated in the absence (A) and in the presence of 4 mM-GSSG (B), 37 /tM-thimerosal (C) and 100 fM-2,2'DTDP (D).

(amounting to 1-8 times the [Ca2+] rise induced by the 0 35 nmol Ca2` pulse). However, the slower rate of Ca21 sequestration, the higher steady-state [Ca2+] and the 181 nm [Ca2+] rise after the 0 35 nmol Ca21 pulse again indicate that the pumps were significantly inhibited. The increased Ca2' release in response to 50 nM-InsP3 in the presence of these sulphydryl agents seemed to depend upon a sensitization of the InsP3-induced Ca2+release mechanism (Fig. 4). In the absence of sulphydryl agents, 50 nM-InsP3 released only 3 % of the Ca2' released by a subsequent addition of 10 /LM-InsP3 (Fig. 4A). In the presence of 4 mM-GSSG (Fig. 4B), 37 ftM-thimerosal (Fig. 4C) or 100 1M-2,2'DTDP (Fig. 4D), 50 nM-InsP3 released 53, 38 and 24°% respectively of the Ca2+ released by 10 /,M-InsP3. These sulphydryl agents therefore sensitized the JnsP3induced Ca2' release mechanism and this may account for their ability to promote Ca21 spikes.

630

L. MISSIAEN, C. W. TA YLOR AND M. J. BERRIDGE

The role of luminal [Ca2+] in spike initiation We previously ascribed the sensitization to InsP3, and the subsequent Ca2+ release, to the gradual overloading of non-mitochondrial pools with Ca2+ leaking from the mitochondria (Missiaen et al. 1991). Additional evidence in favour of this proposal A

I

B

I

GSSG

1210

ia

-

0

eq

E 0

236 181

10/1tM-lnsP3 50 nM-lnsP3

-65 min

,

Fig. 5. Phosphate prevents the progressive sensitization to InsP3. Fifty nanomolar InsP3, immediately followed by an addition of 10 /M-InsP3, was added as indicated, in the presence of 10 mM-phosphate. GSSG (4 mM) was present.

comes from an experiment in which the assay medium was supplemented with 10 mm-phosphate. This precipitating anion will prevent overloading since it enlarges the capacity of the non-mitochondrial Ca2+ pools in hepatocytes (Fulceri, Bellomo, Gamberucci & Benedetti, 1990). Adding 10 mM-phosphate to the bathing medium abolished the usual increase in InsP3 sensitivity (compare Fig. 5 with parts A and B of Fig. 2). Essentially similar results were obtained with two other precipitating anions, oxalate (5 mM) and methylene diphosphonic acid (0 5 mM) (not shown). Our original evidence that the Ca2+ leak from the mitochondria was related to the onset of the spike was the finding that the mitochondria progressively lost their Ca2+ during the course of the experiment (Missiaen et al. 1991). Additional evidence in favour of this proposal that the spike was related to overloading of non-mitochondrial pools is provided by experiments in which we specifically increased the mitochondrial Ca21 leak with NaN3. Figure 6A shows that NaN3 (10 mm) increased the mitochondrial Ca2+ leak because the uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP, 10 tM) caused substantial Ca2+ release in the absence of NaN3, but not after its addition. The addition of a low concentration of

SPONTANEOUS Ca2+ RELEASE IN HEPATOCYTES 631 NaN3 (3 mM) substantially reduced the latency preceding the spike (Fig. 6B). The earlier spike did not occur in the absence of GSSG (Fig. 6C) suggesting that it resulted from premature sensitization of the InsP3-sensitive release mechanism rather than reflecting direct release of Ca2+ from the mitochondria. The increased GSSG + phosphate A \10 mM-NaN3

D c ~~~BGSSGG ,_CDGSSG

752

54061 ~~~~~458

0

~~~~~~~~~~~~~~~~~~~~~~~~9

M

E3

63I3MNN m-aN

NaN,

361

FCCP 167 107-

FC

7

169

149

162

113

105

103 mi

Fig. 6. Increasing the mitochondrial Ca2+ leak triggers an earlier spike. A illustrates that 10 mM-NaN3 increased the mitochondrial Ca2+ leak, judged from the much reduced Ca2+ release upon addition of 10 /LM-carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP). The assay medium also contained 15 mM-phosphate, which suppressed the spontaneous spike. B illustrates how the addition of 3 mM-NaN3 reduced the latency. This earlier spike was prevented in C by omission of GSSG. D shows how adding the indicated amount (in nmol) of Ca2+ immediately before permeabilization affected the spike.

peak [Ca2+] and the increased [Ca2+] during the latency in the presence of NaN3 (Fig. 6B) resembled the result of adding 9 nmol extra Ca2+ to the assay medium (Fig. 6D). As reported previously (Missiaen et al. 1991), the addition of less extra Ca2+ (6 nmol) triggered an earlier spike of the same amplitude (Fig. 6D). Since overloading of the pools was inevitably associated with a rise in medium free [Ca2+], we had to further investigate the relative contributions of luminal and cytosolic [Ca2+] to triggering the spike. Figure 7A illustrates that supplementing the assay medium with 10 or 20 mM-phosphate, prevented the spike. Phosphate has two effects: it reduces the rise in luminal free [Ca2+] in the endoplasmic reticulum (Fulceri et al. 1990), and it increases the cytosolic free [Ca2+] (judged from the slower rate of Ca2+ uptake in Fig. 7A, which is probably related to a decreased rate of Ca2+ pumping (Smith, Steele & Crichton, 1991)). The finding that phosphate, which decreased the luminal free [Ca2+] and at the same time increased the cytosolic free [Ca2+], prevented the spike, suggests that a rise in luminal [Ca2+] was an absolute requirement for initiating the spike. This is further supported by Fig. 7B, where elevating the cytosolic [Ca2+] at a time when the pools were not yet overloaded (i.e. 21

PHY 455

632

L. MISSIA EN, C. W. TA YLOR AND M. J. BERRIDGE

during the latency) to a higher level than at the end of the pacemaker [Ca2+] rise, was unable to trigger the spike. The converse experiment (decreasing the medium free [Ca2+] while increasing the luminal free [Ca2+]) cannot be performed, and it was therefore impossible to assess the contribution of the pacemaker [Ca2+] rise in inducing the spike once the pools are overloaded. GSSG

A

GSSG

B

634 2

618-

nmol ~~~~~~~0-4 2+

5)

Ca

E

.3

~~10mM

5)~ ~~posht

235-

IbI 1144

phosphate 5 min

Fig. 7. A rise in luminal [Ca2"] was required to trigger the spike. A illustrates how supplementing the assay medium with the indicated amount of phosphate decreased the initial rate of Ca2" sequestration and abolished the spike. B shows that addition of 0 4 nmol Ca2" during the latency did not trigger the spike. GSSG (4 mM) was present.

Asynchronous cycles of Ca2" uptake and release may follow the spike The spontaneous spike was never repeated, probably because the individual release elements enter asynchronous cycles of Ca2" uptake and release. Such cycles would explain why the rate of reuptake of Ca2" after the spike was consistently slower than the initial rate of uptake following permeabilization (see Fig. 7B). The existence of such cycles implies that a proportion of the Ca21 stores will be empty and this was supported by the observation that a maximal concentration of InsP3 (10 /tM) caused much less Ca2' release when given after the spontaneous Ca21 spike than when given before the spike (Fig. 8A) or to cells where the spike was prevented by omission of GSSG (Fig. 8B). This indicates that there was no appreciable reloading of the InsP3-sensitive pools after the spike. If the proposed cycle of reuptake and InsP3induced release was interrupted by adding heparin (50 ,ug/ml), there was a rapid reuptake of Ca2" which was particularly marked in some experiments where the reuptake phase was very slow (Fig. 8C).

SPONTANEOUS Ca2+ RELEASE IN HEPATOCYTES

633

A

1280C

co

' 632 481 CD

159 101

Fig. 8. Permeabilized hepatocytes do not produce a second spike. The state of filling of the InsP3-sensitive Ca2+ pools in the presence (A) and absence (B) of 4 mM-GSSG was assessed from the [Ca2+] rise induced by 10 /zM-InsP3. C illustrates how heparin (50 itg/ml) immediately lowered the [Ca2+] in cells where the re-uptake phase of the spike was extremely slow.

C

GSSG I-

1-

a

u

co

a U)

0

0

6 mM-

E

caffeine

5 min

Fig. 9. Caffeine inhibits the spontaneous spike. A illustrates how supplementing the assay medium with 6 mM-caffeine abolished the spike, but not the pacemaker [Ca2+] rise. B illustrates how 6 mM-caffeine inhibited the Ca2+ release by 25 nM-InsP3. C shows the absence of any Ca2+ release upon addition of 6 mM-caffeine. Because 6 mM-caffeine quenched the Fluo-3 fluorescence by 7 %, trace C was not calibrated. GSSG (4 mM) was present in all experiments. 21-2

634

L. MISSIAEN, C W. TAYLOR AND M. J. BERRIDGE

Caffeine inhibits the spontaneous spike Caffeine (6 mM) prevented the spontaneous Ca2+ spike without affecting either the initial Ca21 uptake or the subsequent pacemaker [Ca2+] rise (Fig. 9A). Since the Ca21 spike was the result of Ca2' release from the stores that were most sensitive to InsP3 (Missiaen et al. 1991), we have investigated the effect of caffeine on responses to InsP3. Caffeine (6 mM) substantially inhibited the response to a very low InsP3 concentration (25 nm, Fig. 9B), inhibited the response to 50 nm by about 50 %, and scarcely affected the response to 100 nm-InsP3 (data not shown). Addition of 6 mmcaffeine during the latency did not trigger any Ca2+ release on its own (Fig. 9C). Caffeine may therefore be a low-affinity antagonist for the InsP3 receptor. DISCUSSION

The role of luminal [Ca2+] in spike initiation The following arguments indicate that the overloading of the InsP3-sensitive pools can initiate Ca2+ release by sensitizing them to the ambient low level of InsP3. (1) The pools spontaneously released Ca21 under conditions where they were allowed to load with Ca2+ (Missiaen et al. 1991). (2) Speeding up the Ca2+ loading, either by adding extra Ca2+ (Fig. 6D), or by specifically increasing the Ca2+ leak from the mitochondria with NaN3 (Fig. 6B) triggered an earlier spike. The increased [Ca2+] during the latency, as well as the increased peak [Ca2+] when the Ca2+ supply to the pools was increased by adding much extra Ca2+ or NaN3, corresponds with oscillatory behaviour in AR42J cells (Zhao, Loessberg, Sachs & Muallem, 1990b), where an elevation of the external [Ca2+] also increased the baseline and peak [Ca2+]. (3) Increasing the capacity of the pools with precipitating anions prevented the progressive sensitization to InsP3 (Fig. 5) and the subsequent spike (Fig. 7A). The rise in luminal [Ca2+] was an absolute requirement for setting the stage for Ca2+ release, but the role of the pacemaker-like rise in cytosolic [Ca2+] could not be directly ascertained. The pacemaker LCa2+] rise is a consequence of overloading the non-mitochondrial Ca2+ pools such that they can no longer buffer the Ca2+ leaking from the mitochondria. The rise in luminal [Ca2+] sensitized the pools to low levels of InsP3 (Fig. 2) and once release begins, the opening of the channel appears to be an all-or-none phenomenon (Fig. 1). A similar all-or-none behaviour of the InsP3 receptor has already been reported for the Ca2+ release triggered by a rise in cytosolie [InsP3] (Miyazaki, 1988; Parker & Ivorra, 1990). That the InsP3 receptor could be regulated by luminal [Ca2 ] has already been proposed by Irvine (1990) and modelled by Tregear, Dawson & Irvine (1991). The observation that partially emptying the pools of permeabilized hepatocytes with ionomycin reduces their InsP3 sensitivity also points to the regulatory role of luminal Ca2+ (Nunn & Taylor, 1992). Luminal Ca2+ might affect the InsP3 receptor, either by directly interacting with the channel, or, alternatively, by interacting with some associated protein in the lumen of the endoplasmic reticulum. The latter mechanism has been proposed for the ryanodine receptor, where [Ca2+]-dependent conformational changes of calsequestrin affect the Ca2+ channel function (Ikemoto, Ronjat, Meszaros & Koshita, 1989).

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Effects of sulphydryl reagents Rooney et al. (1991) reported that hepatocytes pretreated with tert-butyl hydroperoxide, which oxidizes glutathione (GSH) to GSSG, were more sensitive to InsP3.'We can confirm their findings in that the sulphydryl reagents GSSG and thimerosal sensitized the Ca2`-release mechanism from the InsP3-sensitive pool (Fig. 4). It is unlikely that this sensitization was caused by an increased Ca2+ supply to the InsP3 -sensitive Caa2+ pools. Firstly, they do not affect the mitochondrial Ca2+ leak (Missiaen et al. 1991). Secondly, they had no effect on the response to a maximal dose of InsP3 whereas this was doubled by adding 100 1aM-GTP (data not shown) which increases the Ca2+ supply to the InsP3-sensitive pools by connecting them to InsP3insensitive Ca2+ pools (Ghosh, Mullaney, Tarazi & Gill, 1989). A more likely possibility, therefore, is that GSSG and thimerosal exerted a direct effect on the InsP3 receptor. The latter consists of four large molecules which have membranespanning regions located in their C-terminal domains which come together to form the Ca2+ channel. The interaction of these subunits is affected by the redox state (Maeda et al. 1991) which may influence two cysteine-residues located in this Cterminal region which are highly conserved in both mouse and Drosophila InsP3 receptors, and even the ryanodine receptor (Miyawaki, Furuichi, Ryou, Yoshikawa, Nakagawa, Saitoh & Mikoshiba, 1991). It remains to be determined whether these Cterminal cysteines represent the site of action of GSSG and thimerosal and therefore whether the action of these sulphydryl agents it due to the monomer to tetramer transition. Experiments in which we tried to mimic the tetramer association with cross-linkers were unsuccessful, since they resulted in a complete loss of InsP3mediated Ca2+ mobilization. Alternatively, these sulphydryl agents could interact with the InsP3-binding site of the receptor, which is located in the N-terminal domain. Indeed, preliminary [3H]InsP3-binding studies on liver membranes suggest that GSSG may stimulate InsP3 binding to its receptor (A. Richardson & C. W. Taylor, unpublished). Whatever the exact mechanism of action, thiol oxidation and formation of disulphide bridges seem to activate the InsP3 receptor, as is also the case for the Na+-Ca2+ exchanger (Longoni & Carafoli, 1987), and this therefore contrasts with the loss of activity normally occurring with most other proteins. The finding that 2,2'-DTDP stimulated the InsP3 receptor is intriguing, because this compound also activated the release channel in muscle sarcoplasmic reticulum (Zaidi, Lagenaur, Abramson, Pessah & Salama, 1989). 2,2'-DTDP (100 /aM) was unable to induce a Ca2+ spike in diluted cells (not shown), probably because of its low potency in activating the InsP3 receptor at doses that already significantly inhibit the Ca2+ pumps. The two-pool oscillatory model Thimerosal can induce Ca2+ oscillations in hamster eggs that are indistinguishable from those triggered by fertilization (Swann, 1991). Similarly, tert-butyl hydroperoxide can duplicate the noradrenaline-induced Ca2+ spikes in intact liver cells through a mechanism which may depend on the conversion of reduced glutathione to GSSG (Rooney et al. 1991). In the latter case, the GSSG-induced oscillations were not accompanied by any change in the resting level of InsP3 thus supporting the two-

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pool oscillatory model (Berridge & Galione, 1988; Berridge, 1990). As originally proposed, the two-pool model considered that oscillations resulted from an interaction between two pools - an InsP3-sensitive pool providing a constant flux of primer Ca2" which then charged up and triggered periodic spiking in an InsP3insensitive pool. Based on the finding that the kinetics as well as the sulphydrylsensitivity of the spontaneous release from overloaded InsP3-sensitive stores in permeabilized hepatocytes closely resemble the spikes in intact cells, we have recently revised the two-pool model to incorporate the possibility that Ca21 spikes originate from an InsP3-sensitive store (Missiaen et al. 1991). The mitochondria, which were used as the source of priming Ca21 to overload the InsP3-sensitive store in our permeabilized system, are probably in a steady-state balance in the intact cell, and will not provide the priming Ca21 to overload the InsP3-sensitive store. Since oscillations in the intact cell are ultimately dependent on a continuous Ca21 influx across the plasma membrane (Berridge, 1990), we suggest that this Ca21 influx provides the primer Ca21 to overload the InsP3-sensitive store. Since oscillations often occur after moderate stimulation of phosphoinositide turnover, we now envisage that such low concentrations of agonists only induce a significant [InsP3] rise in a microdomain of the cell near the plasma membrane. There is some evidence supporting a localized action of InsP3 (Marty, Horn, Tan & Zimmerberg, 1989; Zhao et al. 1990a; Yule & Williams, 1991). Emptying of InsP3-sensitive pools near the plasma membrane will then promote continuous Ca2+ entry into the cell (Putney, 1986). This incoming Ca21 would then charge InsP3-sensitive Ca2+ pools further from the membrane, where the [InsP3] rise might be insufficient to open the Ca2+ channel directly. The loading of these pools then sensitizes them to the ambient low level of InsP3 until a critical Ca2+ content is reached and a Ca21 spike is triggered (Missiaen et al. 1991). Ca21 will then be extruded from the cell, and the continuing Ca21 influx will then set up the next cycle. More intense stimulation of the phosphoinositidesignalling pathway (often obtained after maximal agonist stimulation) is assumed to induce a more general [InsP3] rise which then directly empties all InsP3-sensitive pools to induce a sustained [Ca2+] rise. Ca2+ spikes in intact cells have a very rapid rising phase, and some form of positive feedback must therefore be included in the model. It has recently become evident that the InsP3 -sensitive Ca2+-release channel is not only activated by InsP3 (Berridge & Irvine, 1989), but also by cytosolic Ca21 (lino, 1990; Finch et al. 1991 ; Bezprozvanny et al. 1991). Once release begins, therefore, Ca2+ may exert a positive feedback effect by promoting the Ca2+-mobilizing action of InsP3. Based on experiments with calmodulin inhibitors, Somogyi & Stucki (1991) recently proposed that calmodulin might be involved in this Ca2+ effect. However, using an experimental protocol similar to that of Fig. 4, we were unable to find any effect of 0-6 ,/M-calmodulin on the InsP3 receptor, which confirms the findings of Maeda et al. (1991). Another possible positive feedback pathway is Ca2+-stimulated InsP3 production, but it is unclear, at present, whether such a mechanism is important for generating oscillations (Meyer & Stryer, 1988; Harootunian et al. 1991) or is a secondary consequence of Ca2+ spiking (Dupont, Berridge & Goldbeter, 1991). How does the model explain that hepatocytes can generate a couple of spikes in the absence of extracellular Ca2+ (Rooney et al. 1989; Woods, Dixon, Cuthbertson &

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Cobbold, 1990)? Detailed kinetic studies of the InsP3-mediated Ca2" release suggest that low [InsP3] can trigger a slow release phase (Champeil, Combettes, Berthon, Doucet, Orlowski & Claret, 1989), which can last more than 90 s (Meyer & Stryer, 1990). It is therefore possible that the gradual emptying of InsP3-sensitive stores near the plasma membrane can overload the deeper pools more than once to generate a couple of spikes. The spike in our permeabilized cell system, which we assume to be representative of the Ca2" spikes in intact cells, could only be observed at rather low [ATP] (1 mM) and high levels of GSSG (4 mM). To what extent, therefore, is the mechanism involved in healthy cells (5 mm-ATP, 10 ,uM-GSSG and 5 mM-GSH)? It is possible that the competitive antagonism by 5 mM-ATP on the binding of endogenous InsP3 (47-59 nM) to the InsP3 receptor in our permeabilized hepatocytes, can be overcome in the intact cell by the much higher basal [InsP3]. We, furthermore, had to sensitize the InsP3 receptor with GSSG to make the system responsive to the 47-59 nm-InsP3. Such sensitization may not be necessary to produce the spike in intact cells because of the much higher basal InsP3 levels. It is not our intention to imply that modification of sulphydryl groups has any role to play in the normal function of the InsP3 receptor. Under the reducing conditions in intact cells, the levels of GSSG will be far too low to influence the InsP3 receptor. However, an increase in InsP3-receptor sensitivity following thiol oxidation may explain the Ca2+ spiking observed in hepatocytes (Rooney et al. 1991) or hamster eggs (Swann, 1991) during treatment with either tert-butyl hydroperoxide or thimerosal respectively. In the case of the former, Ca2+ spiking was observed in the absence of any elevation in the level of InsP3 and this would be consistent with a mechanism dependent on an increase in the sensitivity of the InsP3 receptor. A similar increase in receptor sensitivity may explain the ability of cyclic AMP to stimulate Ca2+ spiking in hepatocytes (Burgess, Bird, Obie & Putney, 1991). Under normal conditions, however, the increase in receptor sensitivity responsible for Ca2+ spiking is probably achieved by the build-up of luminal Ca21 within the endoplasmic reticulum. Effects of caffeine Caffeine can reduce the frequency or inhibit Ca2+ oscillations in Xenopus oocytes (Berridge, 1991; Parker & Ivorra, 1991), fibroblasts (Harootunian et al. 1991) and pancreatic acinar cells (Osipchuk et al. 1990; Wakui et al. 1990). In none of these cells does caffeine alone release Ca2+. The inability of caffeine to release Ca2+ in oocytes (Parker & Ivorra, 1991) and fibroblasts (Harootunian et al. 1991) was considered as sufficient evidence against the involvement of a ryanodine receptor in the generation of oscillations. The inhibitory effect of caffeine on the spontaneous Ca2+ spike in our permeabilized preparation (Fig. 9A) is unlikely to be due to an action of caffeine on a putative ryanodine receptor for the following reasons. (1) Caffeine has never been reported to inhibit the ryanodine receptor. (2) Various Ca2+ pulsing protocols were unable to functionally demonstrate Ca2+-induced Ca2' release in permeabilized hepatocytes (Missiaen et al. 1991). (3) Caffeine appears not to release Ca2+ in hepatocytes (Fig. 9 C; Burgess, McKinney, Fabiato, Leslie & Putney, 1983; Somogyi & Stucki, 1991). (4) mRNA for the ryanodine receptor could not be detected in rabbit (Otsu, Willard,

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Khanna, Zorzato, Green & MacLennan, 1990) or rat hepatocytes (Shoshan-Barmatz, Pressley, Higham & Kraus-Friedmann, 1991). (5) Ryanodine has no effect on agonist-induced Ca2" oscillations in intact rat hepatocytes (Cobbold, SanchezBueno & Dixon, 1991; Somogyi & Stucki, 1991). Despite the lack of any functional evidence for the presence of a ryanodine receptor in hepatocytes, ryanodine binds to liver membranes (Shoshan-Barmatz, 1990; Shoshan-Barmatz et al. 1991). ShoshanBarmatz (1990) reported that these binding sites cross-reacted with the skeletal muscle ryanodine receptor, while Shoshan-Barmatz et al. (1991) failed to observe any cross-reactivity. Since the spontaneous release in our permeabilized preparation represented the response of the most sensitive pools to the endogenous level of InsP3, and since caffeine blocked the Ca2" release by very low levels of InsP3 (Fig. 9B), we conclude that caffeine prevented the spike by inhibiting the InsP3 receptor. Parker & Ivorra (1991) came to the same conclusion for the inhibitory effect of caffeine on Ca2" oscillations in XenopUs oocytes. Low concentrations of caffeine enhance cytosolic Ca2" oscillations in cells that do express the ryanodine receptor: e.g. muscle (Benham & Bolton, 1986), neurones (Lipscombe, Madison, Poenie, Reuter, Tsien & Tsien, 1988) and chromaffin cells (Malgaroli, Fesce & Meldolesi, 1990). It remains to be determined, however, whether Ca2" is periodically released from caffeine-sensitive stores because it is possible that the oscillator in these cells may also be located in the InsP3-sensitive Ca2+ pool. The enhancing effect of caffeine would then be explained by this oscillator receiving an increased supply of Ca2+, due to the inability of the caffeine-sensitive pool to accumulate Ca2+. We thank Dr Robin Irvine for stimulating discussions and for his gift of the InsP3. L. M. is Senior Research Assistant of the NFXVO (Belgium) and was in receipt of an EMBO long-term fellowship. The research was supported by the Wellcome Trust (C. W. T.) and Otsuka Pharmaceutical Company (M. J. B.). REFERENCES

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