CELL REGULATION, Vol. 2, 513-522, July 1991
Ca2l-induced Ca2l release amplifies the Ca 2+ response elicited by inositol trisphosphate in macrophages
Clotilde Randriamampita,*t Georges Bismuth,* and Alain Trautmann* *Laboratoire de Neurobiologie, URA 295 CNRS Ecole Normale Sup6rieure 75005 Paris, France t4Laboratoire d'lmmunologie Cellulaire et Tissulaire C. E.R.V.I. CH Piti6 Salpetri6re 75013 Paris, France We have studied the rise in intracellular calcium concentration ([Ca2+J) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]J increase begins with a massive and rapid release of Ca2" from intracellular stores. We have examined the mechanism of this Ca2" release, which has been generally assumed to be triggered by inositol trisphosphate (1P3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2"1, rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimicks the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca2+-induced Ca2+ release (CICR) in the development of the Ca2' response. lonomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2]1, CICR appears as a primary mechanism of positive feedback in the Ca2' response. Taken together, the results suggest that the response to PAF involves an IP3induced [Ca2J1, rise followed by CICR.
Introduction In living cells, intracellular calcium concentration ([Ca2+JD) is maintained at a low level by various t Present address: Howard Hughes Medical Institute, Cellular and Molecular Medicine M-047, University of California, San Diego, La Jolla, CA 92093-0647.
C 1991 by The American Society for Cell Biology
active processes that tend either to extrude Ca2" ions out of the cell or to concentrate them in intracellular organelles. These Ca2" stores constitute reserves of Ca2" that may be mobilized, for instance, during cell stimulation. Two major second messengers that induce the release of Ca2" from these intracellular stores are known: inositol trisphosphate (WP3) and calcium itself. As will be shown, each of these two Ca21 release pathways can lead to explosive [Ca2]i increases. After the discovery of the widespread capacity of IP3 to release Ca2+ from intracellular stores (see Berridge and Irvine, 1989 for a review), it is often assumed that when cell stimulation results both in a production of IP3 and in a rise in [Ca2+J, the Ca2' released comes directly from IP3-sensitive Ca21 stores. Under this hypothesis, the very fast rise of [Ca2+]i after stimulation can be explained in several ways. First, by a cooperative action of IP3, because it has been suggested that three or more IP3 molecules are necessary to open one Ca21 channel (Meyer et al., 1988; Parker and Miledi, 1989). Another possibility is that the IP3-sensitive Ca21 pools may be a collection of independent, localized compartments that release Ca2" in an all-ornone manner (Parker and Ivorra, 1990). In addition, it has been suggested that phospholipase C activation could be enhanced by Ca21 (see Harootunian et al., 1991), although in vivo studies suggest that this might not always be true (Rana and Hokin, 1990). On the other hand, other Ca21 stores have also been described that release Ca21 when [Ca2] reaches a threshold level. This phenomenon has been referred to as Ca2"-induced Ca21 release (CICR), has been thoroughly described in skeletal and cardiac muscles, and plays a fundamental role in cardiac contraction (Fabiato, 1983; Moutin and Dupont, 1988). CICR is in essence a positive feedback mechanism in which a small initial increase of [Ca21]i activates a massive release of Ca2 . More recently, CICR has also been observed in neurons (Kostyuk et al., 1989), exocrine cells (Marty and Tan, 1989; Osipchuk et al., 1990), reticulum vesicles from exocrine cells (Dehlinger-Kremer et al., 1991), and chromaffin cells (Malgaroli et al., 1990). It 513
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has been proposed that in some of these cells, CICR could be triggered by an initial IP3-dependent Ca2l rise (Berridge, 1988; Marty and Tan, 1989; Osipchuk et al., 1990). During a prolonged stimulation, similar interaction between two Ca2l pools (sensitive to IP3 or not) could be the basis of sustained oscillations (Goldbeter et al., 1990), although other interpretations have been proposed (see Harootunian et al., 1991). The physiological relevance of CICR in these cells is becoming more and more probable. However, an important piece of information is still lacking, i.e., the demonstration that inhibition of CICR affects the Ca2+ response elicited by the agonist. We have addressed this question in murine peritoneal macrophages. Stimulation of these cells by various agonists such as platelet-activating factor (PAF) leads to the activation of phospholipase C, the subsequent production of IP3 (Prpic et al., 1988), and a biphasic increase of [Ca2+]J (Prpic et al., 1988; Randriamampita and Trautmann, 1989). The first phase of the [Ca2+1J response results from the release of Ca2' from intracellular stores, whereas the second is the consequence of an influx of extracellular Ca2l into the cell. We have examined the origins of the Ca2' released during PAF stimulation. First, we have confirmed that the release of Ca2+ from IP3-sensitive Ca2+ stores is an essential step of the response to PAF. Then, we have demonstrated that CICR does take place in macrophages and that this phenomenon contributes a major part of the rise in [Ca2+], observed in PAFactivated macrophages. We propose that during PAF stimulation, Ca2+ released by IP3-sensitive Ca2+ stores activates CICR, which induces a large and rapid increase in [Ca2+],.
tion and the intracellular compartment (Figure 1). Similar responses were observed with 50 (Figure 1) and with 20 ,M IP3 (not shown). No variation of [Ca2+J1 was observed when IP3 was not included in the pipette solution (data not shown). The biphasic increase in [Ca2+]j evoked by IP3 was very similar to that observed during PAF stimulation (Figure 2). IP3 stimulation also activated an outward current at -60 mV (Figure 1, upper trace; see also. Figures 2 and 3). This current is most likely the result of the opening of Ca2 -activated K+ channels (Randriamampita and Trautmann, 1987; Gallin and McKinney, 1988). It should be noted that their activation did not parallel exactly the [Ca2+]i rise indicated by the fura-2 signal. Indeed, although [Ca21]i remained at a high level after the initial [Ca21], transient, Ca2+-activated K+ channels showed only a transient activation. This result could be explained if the channels were only activated by [Ca2+J1 in excess of a threshold of several hundred nanomolars (Randriamampita and Trautmann, 1987), if they underwent inactivation, or if the local [Ca2+], concentration directly below the plasma membrane varied differently from the bulk intracellular [Ca2+], concentration (see Foskett et aL, 1989). We observed a very similar activation of these Ca2+-activated K+ channels after the photolysis of "caged" IP3 (Walker et al., 1987) (results not
shown).
The second phase of the IP3-induced [Ca2+], increase appeared to result from an influx of
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Results PAF-induced [Ca2+], increase can be mimicked by intracellular IP3 The stimulation of murine macrophages by PAF caused a marked and rapid increase in 1,4,5IP3 (Prpic et al., 1988) (see also below on Figure 3B) peaking at - 15 s after PAF addition. To relate the increases in IP3 and [Ca2+]j to PAF stimulation, we have initially examined to what
extent the PAF-induced [Ca2]i increase could be mimicked by an increase in intracellular IP3 and second whether this was blocked by heparin, an antagonist of the IP3 receptor (Ehrlich and Watras, 1988; Mollard et al., 1990). When IP3 was added to the patch pipette solution, a transient increase in [Ca2+J was observed immediately after break-in, establishing direct contact between the pipette filling solu514
O mV
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Figure 1. IP3 induces a biphasic increase in [Ca21]1. In this and in Figures 2 and 3, the bottom trace shows the value of [Ca2+J as a function of time, the middle trace shows the voltage imposed to the cell, and the top trace shows the current as a function of time. Zero voltage and current are indicated by dotted lines. Fifty micromolars of IP3 were included in the patch electrode filling solution. The establishment of the whole-cell recording configuration is indicated by the arrow. CELL REGULATION
Ca2"-induced Ca2l release in macrophages
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Figure 2. Heparin blocks the PAF-induced [Ca21, rise. Cells were dialyzed with a control internal solution (A) or with 2 mg/ ml heparin (B) for 4 min before stimulation with PAF (20 nM). The voltage was repeatedly stepped between -70 and 0 mV. The outward current observed at 0 mV is a K+ current that was activated at the peak of the PAF-induced Ca2` transient.
extracellular Ca2l because this phase was sensitive to changes in membrane potential. When the cell was depolarized from -60 to 0 mV (as shown in the middle trace of Figure 1), (Ca2+]i returned to its basal level. This result could be explained by the fact that the driving force for Ca2l ions was reduced when the cell was depolarized. It is possible, in addition, that depolarization reduced the number of open channels in these cells or accelerated Ca2l extrusion. A similar effect of membrane potential on [Ca2+]J was observed when Ca2" influx was evoked after activation of different membrane receptors in macrophages (Figure 2A), lymphocytes (Lewis and Cahalan, 1989), or mast cells (Penner et al., 1988). Figure 2 illustrates the effect of intracellular heparin on the response to PAF. Low-molecularweight heparin can be introduced into macrophages through a patch clamp electrode. To reach a sufficient intracellular antagonist concentration, cells were dialyzed for 4 min with a pipette filled with a heparin (2 mg/ml) solution before PAF stimulation. Under these conditions, PAF was no longer able to release Ca2l from intracellular stores or to activate Ca2+ influx. Indeed no change in [Ca2+], was observed after PAF stimulation or after altering membrane potential (Figure 2B). A similar result was obtained in nine different cells. Furthermore, as shown in the upper trace of Figure 2B, we did not obVol. 2, July 1991
activation of Ca2+-activated K+ channels that could have revealed localized increases in [Ca2+Ji just below the plasma membrane. We investigated whether this absence of a PAF response was due to a washout phenomenon, i.e., to the loss into the patch electrode of intracellular molecules required for the response to PAF. When a similar waiting period was allowed with a control internal solution (i.e., without heparin), PAF was still able to induce a biphasic [Ca2+], increase, and the second phase was sensitive to cell depolarization (Figure 2A). In addition, Ca2+-activated K+ currents (upper trace) were also activated, and as before this current progressively vanished before [Ca2+]1 reached its basal level. The results so far show that IP3 plays a key role in the triggering of the response to PAF. They further suggest the presence of a strong relation between the two phases of the PAF response (Ca2+ release from intracellular stores and Ca2+ influx), which are triggered together either by PAF or by IP3, and are inhibited together by heparin. serve any
lonomycin induces a biphasic increase of [Ca2+]i independently of the IP3 pathway In another series of experiments using the Ca2+ ionophore, ionomycin, which is generally assumed to make the plasma membrane perme515
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Figure 3. lonomycin induces a biphasic increase in [Ca2+J, that is not mediated by IP3. (A) [Ca2+], response elicited by 1 ,uM ionomycin in a cell dialyzed with a control internal solution. The second phase of the Ca2' response was reduced when the cell was depolarized. (B) No change in IP3 production occurred after stimulation with 500 nM ionomycin (A), whereas 20 nM PAF induced a clear increase in IP3 (0). Each point is the average (±SD) of three measurements. Similar results were obtained in two experiments.
able to [Ca2+],, we made the unexpected observation that the [Ca21]i rise induced by ionomycin (1 MM) followed a time course very similar to that evoked by PAF: it started with a large transient followed by a sustained increase in [Ca2+J1, which could persist for several minutes after washing off ionomycin. Ca2+-activated K+ channels were also simultaneously activated. Similar results were obtained with A23187, another Ca2` ionophore (data not shown). The second phase of the ionomycin response was strongly depressed during the application of a Ca2+-free solution (not shown) or when the cell was depolarized from -70 to 0 mV (Figure 3A, middle trace), similar to the PAF response shown above. The initial peak in the ionomycin-induced [Ca2+], increase was due, to a large extent, to 516
the emptying of intracellular Ca2" stores. As a matter of fact, this [Ca2+]i transient could still be recorded in a Ca2"-free solution (not shown). A similar observation has been reported in parotid acinar cells (Merritt and Rink, 1987). With low concentrations of ionomycin (50-200 nM), the amplitude of the [Ca2+]J transient was small (100-200 nM), and the second phase of the response much reduced in amplitude and eventually absent. A similar finding was previously described for low PAF doses (Randriamampita and Trautmann, 1989), suggesting that the Ca2+ influx is activated only after a sufficient rise in [Ca2+J,. The striking similarity between the time course of the [Ca2+]i responses evoked by ionomycin and PAF led us to examine to what extent the initial [Ca2+]i rise elicited by the Ca2+ ionophore was triggering secondary cellular mechanisms that could contribute to shape the Ca2' response of the cell. It has been reported that under certain conditions phospholipase C activity can be enhanced by a rise in [Ca2+], (see Rana and Hokin, 1990 for a review). One could then wonder whether ionomycin could activate the production of IP3, in which case part of the response to ionomycin could be explained by an IP3-induced Ca2+ release. The answer was negative: no increase in IP3 was observed in macrophages activated by ionomycin (500 nM), even after 1 min of stimulation, whereas in the same batch of cells, 20 nM PAF induced a marked increase in IP3 (Figure 3B). To strengthen this point, we next examined whether the ionomycin-induced [Ca2+]j increase was sensitive to the presence of heparin (2 mg/ml) in the intracellular solution in experiments similar to those illustrated in Figure 2. This was not the case: the amplitude of the ionomycin-induced [Ca2+]i rise measured in the presence of heparin was 101 ± 110/ (n = 6 cells) of the control value. Both of these results argue against any significant role of IP3 during the ionomycininduced [Ca2+], increase.
CICR contributes to the ionomycin-induced [Ca2+j, increase Another cellular mechanism that could amplify an initial [Ca2+]i rise (evoked by ionomycin) is CICR. In skeletal and cardiac muscle, CICR results from the opening of Ca2+-activated Ca21 channels located on the sarcoplasmic reticulum (Fabiato, 1983; Moutin and Dupont, 1988), which have been identified as ryanodine receptors (Lai et al., 1988). These channels are activated above a [Ca2+]i threshold and are known to be blocked by ruthenium red (Fabiato, 1983; Lai et al., 1988; Moutin and Dupont, 1988) or CELL REGULATION
Ca2l-induced Ca2l release in macrophages
octanol (Ma et al., 1988). Furthermore, their Ca2+ sensitivity is increased by caffeine (Moutin and Dupont, 1988). However, it is not known until now whether macrophages are also endowed with such a mechanism or not. Figure 4 shows an experiment in which cells from the same batch were stimulated with 500 nM ionomycin first in control conditions (Figure 4A), then after adding 1 mM octanol to the bath (Figure 4B), and finally after washing octanol from the bath (Figure 4C). The average responses obtained with three or four cells in each condition are shown in Figure 4. One can see that both phases of the response to ionomycin were markedly and reversibly inhibited by octanol. In Figure 4D, two typical traces obtained before and after adding octanol to the bath are shown superimposed, on an expanded time scale. One can observe a large reduction of the rate of rise of the Ca2' response, from 256 nM/ s in control conditions to 33 nM/s in the presence of octanol. It also appears in Figure 4D that, in the presence of octanol, the [Ca2+]i response rose as long as ionomycin was present, at least for several seconds. The time course of the response induced by ionomycin in the presence of octanol might represent the true time course of the Ca2` permeability changes directly due to of the insertion of the Ca2+ ionophore in.the plasma membrane and also possibly in
the membrane of intracellular stores. Although octanol might have other actions, its effect was consistent with the hypothesis that ionomycin activates a phenomenon of CICR in macrophages. To confirm this hypothesis, we have tested other pharmacological agents that affect CICR. lonomycin-induced [Ca2+]i increase was strongly reduced in the presence of ruthenium red (50 M) in the external medium (Figure 5, A and B). The average amplitude of the [Ca2+]j response to ionomycin (500 nM) recorded on different cells from the same dish was 647 ± 255 nM (n = 3) in control conditions (Figure 5A), whereas it was significantly reduced (p < 0.05) to 294 ± 101 nM (n = 5) by ruthenium red (Figure 5B). On the other hand, caffeine (5 mM) potentiated increases of [Ca2+]J induced by a low dose of ionomycin (50 nM) (Figure 5, C and D). The average amplitude of [Ca21]i responses was significantly increased (p < 0.1) from 227 ± 96 nM (n = 3) in control conditions (Figure 5C) to 444 ± 163 (n = 5) when cells were kept in a caffeine-containing solution for 10-30 min (Figure 5D). PAF stimulation activates CICR Next, we examined whether CICR was involved in the PAF-induced [Ca21], transient. Figure 6,
Mc
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Cai (nM) Figure 4. The response to ionomycin is reversibly inhibited by octanol. Responses to 500 nM ionomycin in control conditions (A), after adding 1 mM octanol to the bath (B), and after washing octanol (C). Each trace is the average of four (A) or three (B and C) individual responses from different cells of the same dish. Two typical responses obtained in control con) or in the ditions ( presence of octanol (... ) are shown on an expanded time scale in D. The applications of ionomycin are indicated by horizontal bars. In D the longer application corresponds to the dotted trace. Vol. 2, July 1991
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Figure 5. The response to lonomycin is inhibited by ruthenium red and potentiated by caffeine. Responses to ionomycin (500 nM in A and B and 50 nM in C and D) in control conditions (A and C), in the presence of 50 ltM ruthenium red (B), or of 5 mM caffeine (D). Each trace is the average of three (A and C) and five (B and D) individual responses from different cells of the same dish. In the experiment shown in C and D, caffeine induced an increase in the basal level of [Ca2]j. However, such an effect of caffeine was not systematically observed (see, for example, Figure 6, C and D).
A and B shows that the PAF response was strongly depressed by ruthenium red (50 ,uM). The average amplitude of the responses to 20 nM PAF was 795 nM before (Figure 6A) and 367 nM after adding ruthenium red to the bath (Figure 6B). Similar results were obtained in three experiments. Altogether, the responses measured in the presence of ruthenium red were 60 ± 24% (n = 12) of the corresponding controls. The SD of the 14 controls was 170/o. The reduction of the PAF response by ruthenium red is statistically significant (p < 0.001). Similar results were obtained in three other batches of cells with 1 mM octanol, which reduced the PAF responses to 38 ± 210% (n = 12) of the control responses (Student's t test, p < 0.001). On the other hand, Figure 6, C and D shows that the response to a low dose of PAF (0.4 nM) was clearly potentiated by caffeine (5 mM); the average amplitude of the Ca2l transients was 103 nM in control conditions (Figure 6C) and 243 nM in the presence of caffeine (Figure 6D). In two experiments, the response to PAF measured in the presence of caffeine was 199 ± 54% (n = 8) of the control response (100 518
± 49%, n = 8). This difference is statistically significant (p < 0.001). Note that in macrophages, as in pancreatic cells in which [Ca2+J is lightly buffered (Osipchuk et al., 1990), caffeine does not discharge Ca2l but simply potentiates the Ca2' release evoked by an initial rise in [Ca21],, in line with the original observations made in muscle cells (Endo, 1977). Finally, we investigated the possibility that the reduction of the [Ca21], transients observed in the presence of ruthenium red could be explained by an inhibition of the PAF-induced IP3 production. When cells were kept for 15 min in 50,uM ruthenium red, PAF (20 nM) induced an IP3 increase that had amplitude and kinetics similar to those observed in control conditions on the same batch of cells (Figure 7).
Discussion The aim of this work was to determine the respective roles of two intracellular messengers, IP3 and Ca2", in the induction of the explosive release of Ca2+ from intracellular stores that follows the stimulation of macrophages by PAF. CELL REGULATION
Ca2"-induced Ca2l release in macrophages
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Figure 6. The response to PAF is inhibited by ruthenium red and potentiated by caffeine. Responses to PAF (20 nM in A and B and 0.4 nM in C and D) in control conditions (A and C), in the presence of 50 MM ruthenium red (B), or 5 mM caffeine (D). Each trace is the average of four (A, C, and D) and five (B) individual responses from different cells of the same dish.
Before discussing this central point, let us make some comments on the influx of Ca2+ that allows sustained but relatively slow changes in [Ca2+]J after the initial Ca2+ transient. Our results give some indications on the activation mechanism of this influx, which is still a matter of debate. For instance, it has been proposed that IP3 (Gardner, 1990) and/or IP4 (Irvine and Moor, 1986; Morris et al., 1987) could lead to the activation of Ca2+ influx in some nonexcitable cells, but there is not a general agreement on this point (Downes, 1988; Matthews et aL, 1989). We have observed that Ca2+ influx was activated after ionomycin-induced Ca2+ transients of large amplitude, even though ionomycin does not induce any change in IP3 concentration in these cells. This shows, in agreement with previous results obtained with thapsigargin (Gouy et aL, 1990; Bismuth, Randriamampita, Trautmann, unpublished experiments), that Ca2+ influx can be stimulated in the absence of IP3 (and probably IP4) production. In addition, the experiment illustrated in Figure 2B shows that in the presence of heparin, the increase in IP3 and thus in IP4, which had presumably been elicited by PAF, was unable to induce a Ca2+ influx in the cell. Vol. 2, July 1991
On the other hand, as already mentioned, the amplitude of the initial [Ca2"J rise usually seems to be of fundamental importance for the triggering of the Ca2l influx. One possibility is that Ca2"-dependent enzymes are activated by this 3000
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Figure 7. The production of IPN by 20 nM PAF was not affected by the presence of 50 MM ruthenium red in the bath. The dotted line shows the control IP3 production after stimulation with 20 nM PAF, as shown in Figure 3. Each point is the average (±SD) of three measurements. Similar results were obtained in two experiments.
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initial [Ca2"Ji rise. Alternatively, the emptying of the intracellular stores could be the trigger for the subsequent Ca2" influx, as suggested by Takemura and Putney (1989). More recently, Takemura et al. (1989) showed that their "capacitative" model was probably oversimplified and that a second messenger is probably required between the emptying of intracellular stores and the subsequent Ca2" influx. One of the most prominent features of Ca2" release from intracellular stores in macrophages and other cell types is that it is a very rapid phenomenon; in macrophages, the peak of the PAF-induced [Ca2+]J response is reached in
Ca2l-induced Ca2l release amplifies the Ca 2+ response elicited by inositol trisphosphate in macrophages
Clotilde Randriamampita,*t Georges Bismuth,* and Alain Trautmann* *Laboratoire de Neurobiologie, URA 295 CNRS Ecole Normale Sup6rieure 75005 Paris, France t4Laboratoire d'lmmunologie Cellulaire et Tissulaire C. E.R.V.I. CH Piti6 Salpetri6re 75013 Paris, France We have studied the rise in intracellular calcium concentration ([Ca2+J) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]J increase begins with a massive and rapid release of Ca2" from intracellular stores. We have examined the mechanism of this Ca2" release, which has been generally assumed to be triggered by inositol trisphosphate (1P3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2"1, rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimicks the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca2+-induced Ca2+ release (CICR) in the development of the Ca2' response. lonomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2]1, CICR appears as a primary mechanism of positive feedback in the Ca2' response. Taken together, the results suggest that the response to PAF involves an IP3induced [Ca2J1, rise followed by CICR.
Introduction In living cells, intracellular calcium concentration ([Ca2+JD) is maintained at a low level by various t Present address: Howard Hughes Medical Institute, Cellular and Molecular Medicine M-047, University of California, San Diego, La Jolla, CA 92093-0647.
C 1991 by The American Society for Cell Biology
active processes that tend either to extrude Ca2" ions out of the cell or to concentrate them in intracellular organelles. These Ca2" stores constitute reserves of Ca2" that may be mobilized, for instance, during cell stimulation. Two major second messengers that induce the release of Ca2" from these intracellular stores are known: inositol trisphosphate (WP3) and calcium itself. As will be shown, each of these two Ca21 release pathways can lead to explosive [Ca2]i increases. After the discovery of the widespread capacity of IP3 to release Ca2+ from intracellular stores (see Berridge and Irvine, 1989 for a review), it is often assumed that when cell stimulation results both in a production of IP3 and in a rise in [Ca2+J, the Ca2' released comes directly from IP3-sensitive Ca21 stores. Under this hypothesis, the very fast rise of [Ca2+]i after stimulation can be explained in several ways. First, by a cooperative action of IP3, because it has been suggested that three or more IP3 molecules are necessary to open one Ca21 channel (Meyer et al., 1988; Parker and Miledi, 1989). Another possibility is that the IP3-sensitive Ca21 pools may be a collection of independent, localized compartments that release Ca2" in an all-ornone manner (Parker and Ivorra, 1990). In addition, it has been suggested that phospholipase C activation could be enhanced by Ca21 (see Harootunian et al., 1991), although in vivo studies suggest that this might not always be true (Rana and Hokin, 1990). On the other hand, other Ca21 stores have also been described that release Ca21 when [Ca2] reaches a threshold level. This phenomenon has been referred to as Ca2"-induced Ca21 release (CICR), has been thoroughly described in skeletal and cardiac muscles, and plays a fundamental role in cardiac contraction (Fabiato, 1983; Moutin and Dupont, 1988). CICR is in essence a positive feedback mechanism in which a small initial increase of [Ca21]i activates a massive release of Ca2 . More recently, CICR has also been observed in neurons (Kostyuk et al., 1989), exocrine cells (Marty and Tan, 1989; Osipchuk et al., 1990), reticulum vesicles from exocrine cells (Dehlinger-Kremer et al., 1991), and chromaffin cells (Malgaroli et al., 1990). It 513
C. Randriamampita et al.
has been proposed that in some of these cells, CICR could be triggered by an initial IP3-dependent Ca2l rise (Berridge, 1988; Marty and Tan, 1989; Osipchuk et al., 1990). During a prolonged stimulation, similar interaction between two Ca2l pools (sensitive to IP3 or not) could be the basis of sustained oscillations (Goldbeter et al., 1990), although other interpretations have been proposed (see Harootunian et al., 1991). The physiological relevance of CICR in these cells is becoming more and more probable. However, an important piece of information is still lacking, i.e., the demonstration that inhibition of CICR affects the Ca2+ response elicited by the agonist. We have addressed this question in murine peritoneal macrophages. Stimulation of these cells by various agonists such as platelet-activating factor (PAF) leads to the activation of phospholipase C, the subsequent production of IP3 (Prpic et al., 1988), and a biphasic increase of [Ca2+]J (Prpic et al., 1988; Randriamampita and Trautmann, 1989). The first phase of the [Ca2+1J response results from the release of Ca2' from intracellular stores, whereas the second is the consequence of an influx of extracellular Ca2l into the cell. We have examined the origins of the Ca2' released during PAF stimulation. First, we have confirmed that the release of Ca2+ from IP3-sensitive Ca2+ stores is an essential step of the response to PAF. Then, we have demonstrated that CICR does take place in macrophages and that this phenomenon contributes a major part of the rise in [Ca2+], observed in PAFactivated macrophages. We propose that during PAF stimulation, Ca2+ released by IP3-sensitive Ca2+ stores activates CICR, which induces a large and rapid increase in [Ca2+],.
tion and the intracellular compartment (Figure 1). Similar responses were observed with 50 (Figure 1) and with 20 ,M IP3 (not shown). No variation of [Ca2+J1 was observed when IP3 was not included in the pipette solution (data not shown). The biphasic increase in [Ca2+]j evoked by IP3 was very similar to that observed during PAF stimulation (Figure 2). IP3 stimulation also activated an outward current at -60 mV (Figure 1, upper trace; see also. Figures 2 and 3). This current is most likely the result of the opening of Ca2 -activated K+ channels (Randriamampita and Trautmann, 1987; Gallin and McKinney, 1988). It should be noted that their activation did not parallel exactly the [Ca2+]i rise indicated by the fura-2 signal. Indeed, although [Ca21]i remained at a high level after the initial [Ca21], transient, Ca2+-activated K+ channels showed only a transient activation. This result could be explained if the channels were only activated by [Ca2+J1 in excess of a threshold of several hundred nanomolars (Randriamampita and Trautmann, 1987), if they underwent inactivation, or if the local [Ca2+], concentration directly below the plasma membrane varied differently from the bulk intracellular [Ca2+], concentration (see Foskett et aL, 1989). We observed a very similar activation of these Ca2+-activated K+ channels after the photolysis of "caged" IP3 (Walker et al., 1987) (results not
shown).
The second phase of the IP3-induced [Ca2+], increase appeared to result from an influx of
200 0 pA 400
Results PAF-induced [Ca2+], increase can be mimicked by intracellular IP3 The stimulation of murine macrophages by PAF caused a marked and rapid increase in 1,4,5IP3 (Prpic et al., 1988) (see also below on Figure 3B) peaking at - 15 s after PAF addition. To relate the increases in IP3 and [Ca2+]j to PAF stimulation, we have initially examined to what
extent the PAF-induced [Ca2]i increase could be mimicked by an increase in intracellular IP3 and second whether this was blocked by heparin, an antagonist of the IP3 receptor (Ehrlich and Watras, 1988; Mollard et al., 1990). When IP3 was added to the patch pipette solution, a transient increase in [Ca2+J was observed immediately after break-in, establishing direct contact between the pipette filling solu514
O mV
-10(0 Cai (nM)
0 0
2
4 min
Figure 1. IP3 induces a biphasic increase in [Ca21]1. In this and in Figures 2 and 3, the bottom trace shows the value of [Ca2+J as a function of time, the middle trace shows the voltage imposed to the cell, and the top trace shows the current as a function of time. Zero voltage and current are indicated by dotted lines. Fifty micromolars of IP3 were included in the patch electrode filling solution. The establishment of the whole-cell recording configuration is indicated by the arrow. CELL REGULATION
Ca2"-induced Ca2l release in macrophages
B
A
500
500
oliOpA
-sik~bM~m-I O"I APA PAF rnn .
&AM,AAAAA.AMMUSS
-100
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1000 r
PAF
-immimm.,
'*
1
-100
Cai (nM)
Heparin o
0
1
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3
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5 min
1
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Figure 2. Heparin blocks the PAF-induced [Ca21, rise. Cells were dialyzed with a control internal solution (A) or with 2 mg/ ml heparin (B) for 4 min before stimulation with PAF (20 nM). The voltage was repeatedly stepped between -70 and 0 mV. The outward current observed at 0 mV is a K+ current that was activated at the peak of the PAF-induced Ca2` transient.
extracellular Ca2l because this phase was sensitive to changes in membrane potential. When the cell was depolarized from -60 to 0 mV (as shown in the middle trace of Figure 1), (Ca2+]i returned to its basal level. This result could be explained by the fact that the driving force for Ca2l ions was reduced when the cell was depolarized. It is possible, in addition, that depolarization reduced the number of open channels in these cells or accelerated Ca2l extrusion. A similar effect of membrane potential on [Ca2+]J was observed when Ca2" influx was evoked after activation of different membrane receptors in macrophages (Figure 2A), lymphocytes (Lewis and Cahalan, 1989), or mast cells (Penner et al., 1988). Figure 2 illustrates the effect of intracellular heparin on the response to PAF. Low-molecularweight heparin can be introduced into macrophages through a patch clamp electrode. To reach a sufficient intracellular antagonist concentration, cells were dialyzed for 4 min with a pipette filled with a heparin (2 mg/ml) solution before PAF stimulation. Under these conditions, PAF was no longer able to release Ca2l from intracellular stores or to activate Ca2+ influx. Indeed no change in [Ca2+], was observed after PAF stimulation or after altering membrane potential (Figure 2B). A similar result was obtained in nine different cells. Furthermore, as shown in the upper trace of Figure 2B, we did not obVol. 2, July 1991
activation of Ca2+-activated K+ channels that could have revealed localized increases in [Ca2+Ji just below the plasma membrane. We investigated whether this absence of a PAF response was due to a washout phenomenon, i.e., to the loss into the patch electrode of intracellular molecules required for the response to PAF. When a similar waiting period was allowed with a control internal solution (i.e., without heparin), PAF was still able to induce a biphasic [Ca2+], increase, and the second phase was sensitive to cell depolarization (Figure 2A). In addition, Ca2+-activated K+ currents (upper trace) were also activated, and as before this current progressively vanished before [Ca2+]1 reached its basal level. The results so far show that IP3 plays a key role in the triggering of the response to PAF. They further suggest the presence of a strong relation between the two phases of the PAF response (Ca2+ release from intracellular stores and Ca2+ influx), which are triggered together either by PAF or by IP3, and are inhibited together by heparin. serve any
lonomycin induces a biphasic increase of [Ca2+]i independently of the IP3 pathway In another series of experiments using the Ca2+ ionophore, ionomycin, which is generally assumed to make the plasma membrane perme515
C. Randriamampita et al.
U'..... 400 r
A
200
I0 pA
Iono
0 mV
RI -100
Cai (nM'
B
3000 v) --
u
2000-
E 1000' 0
20 40 time (sec)
60
Figure 3. lonomycin induces a biphasic increase in [Ca2+J, that is not mediated by IP3. (A) [Ca2+], response elicited by 1 ,uM ionomycin in a cell dialyzed with a control internal solution. The second phase of the Ca2' response was reduced when the cell was depolarized. (B) No change in IP3 production occurred after stimulation with 500 nM ionomycin (A), whereas 20 nM PAF induced a clear increase in IP3 (0). Each point is the average (±SD) of three measurements. Similar results were obtained in two experiments.
able to [Ca2+],, we made the unexpected observation that the [Ca21]i rise induced by ionomycin (1 MM) followed a time course very similar to that evoked by PAF: it started with a large transient followed by a sustained increase in [Ca2+J1, which could persist for several minutes after washing off ionomycin. Ca2+-activated K+ channels were also simultaneously activated. Similar results were obtained with A23187, another Ca2` ionophore (data not shown). The second phase of the ionomycin response was strongly depressed during the application of a Ca2+-free solution (not shown) or when the cell was depolarized from -70 to 0 mV (Figure 3A, middle trace), similar to the PAF response shown above. The initial peak in the ionomycin-induced [Ca2+], increase was due, to a large extent, to 516
the emptying of intracellular Ca2" stores. As a matter of fact, this [Ca2+]i transient could still be recorded in a Ca2"-free solution (not shown). A similar observation has been reported in parotid acinar cells (Merritt and Rink, 1987). With low concentrations of ionomycin (50-200 nM), the amplitude of the [Ca2+]J transient was small (100-200 nM), and the second phase of the response much reduced in amplitude and eventually absent. A similar finding was previously described for low PAF doses (Randriamampita and Trautmann, 1989), suggesting that the Ca2+ influx is activated only after a sufficient rise in [Ca2+J,. The striking similarity between the time course of the [Ca2+]i responses evoked by ionomycin and PAF led us to examine to what extent the initial [Ca2+]i rise elicited by the Ca2+ ionophore was triggering secondary cellular mechanisms that could contribute to shape the Ca2' response of the cell. It has been reported that under certain conditions phospholipase C activity can be enhanced by a rise in [Ca2+], (see Rana and Hokin, 1990 for a review). One could then wonder whether ionomycin could activate the production of IP3, in which case part of the response to ionomycin could be explained by an IP3-induced Ca2+ release. The answer was negative: no increase in IP3 was observed in macrophages activated by ionomycin (500 nM), even after 1 min of stimulation, whereas in the same batch of cells, 20 nM PAF induced a marked increase in IP3 (Figure 3B). To strengthen this point, we next examined whether the ionomycin-induced [Ca2+]j increase was sensitive to the presence of heparin (2 mg/ml) in the intracellular solution in experiments similar to those illustrated in Figure 2. This was not the case: the amplitude of the ionomycin-induced [Ca2+]i rise measured in the presence of heparin was 101 ± 110/ (n = 6 cells) of the control value. Both of these results argue against any significant role of IP3 during the ionomycininduced [Ca2+], increase.
CICR contributes to the ionomycin-induced [Ca2+j, increase Another cellular mechanism that could amplify an initial [Ca2+]i rise (evoked by ionomycin) is CICR. In skeletal and cardiac muscle, CICR results from the opening of Ca2+-activated Ca21 channels located on the sarcoplasmic reticulum (Fabiato, 1983; Moutin and Dupont, 1988), which have been identified as ryanodine receptors (Lai et al., 1988). These channels are activated above a [Ca2+]i threshold and are known to be blocked by ruthenium red (Fabiato, 1983; Lai et al., 1988; Moutin and Dupont, 1988) or CELL REGULATION
Ca2l-induced Ca2l release in macrophages
octanol (Ma et al., 1988). Furthermore, their Ca2+ sensitivity is increased by caffeine (Moutin and Dupont, 1988). However, it is not known until now whether macrophages are also endowed with such a mechanism or not. Figure 4 shows an experiment in which cells from the same batch were stimulated with 500 nM ionomycin first in control conditions (Figure 4A), then after adding 1 mM octanol to the bath (Figure 4B), and finally after washing octanol from the bath (Figure 4C). The average responses obtained with three or four cells in each condition are shown in Figure 4. One can see that both phases of the response to ionomycin were markedly and reversibly inhibited by octanol. In Figure 4D, two typical traces obtained before and after adding octanol to the bath are shown superimposed, on an expanded time scale. One can observe a large reduction of the rate of rise of the Ca2' response, from 256 nM/ s in control conditions to 33 nM/s in the presence of octanol. It also appears in Figure 4D that, in the presence of octanol, the [Ca2+]i response rose as long as ionomycin was present, at least for several seconds. The time course of the response induced by ionomycin in the presence of octanol might represent the true time course of the Ca2` permeability changes directly due to of the insertion of the Ca2+ ionophore in.the plasma membrane and also possibly in
the membrane of intracellular stores. Although octanol might have other actions, its effect was consistent with the hypothesis that ionomycin activates a phenomenon of CICR in macrophages. To confirm this hypothesis, we have tested other pharmacological agents that affect CICR. lonomycin-induced [Ca2+]i increase was strongly reduced in the presence of ruthenium red (50 M) in the external medium (Figure 5, A and B). The average amplitude of the [Ca2+]j response to ionomycin (500 nM) recorded on different cells from the same dish was 647 ± 255 nM (n = 3) in control conditions (Figure 5A), whereas it was significantly reduced (p < 0.05) to 294 ± 101 nM (n = 5) by ruthenium red (Figure 5B). On the other hand, caffeine (5 mM) potentiated increases of [Ca2+]J induced by a low dose of ionomycin (50 nM) (Figure 5, C and D). The average amplitude of [Ca21]i responses was significantly increased (p < 0.1) from 227 ± 96 nM (n = 3) in control conditions (Figure 5C) to 444 ± 163 (n = 5) when cells were kept in a caffeine-containing solution for 10-30 min (Figure 5D). PAF stimulation activates CICR Next, we examined whether CICR was involved in the PAF-induced [Ca21], transient. Figure 6,
Mc
A
1200
Cai (nM) Figure 4. The response to ionomycin is reversibly inhibited by octanol. Responses to 500 nM ionomycin in control conditions (A), after adding 1 mM octanol to the bath (B), and after washing octanol (C). Each trace is the average of four (A) or three (B and C) individual responses from different cells of the same dish. Two typical responses obtained in control con) or in the ditions ( presence of octanol (... ) are shown on an expanded time scale in D. The applications of ionomycin are indicated by horizontal bars. In D the longer application corresponds to the dotted trace. Vol. 2, July 1991
0
.
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lono
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riv
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Figure 5. The response to lonomycin is inhibited by ruthenium red and potentiated by caffeine. Responses to ionomycin (500 nM in A and B and 50 nM in C and D) in control conditions (A and C), in the presence of 50 ltM ruthenium red (B), or of 5 mM caffeine (D). Each trace is the average of three (A and C) and five (B and D) individual responses from different cells of the same dish. In the experiment shown in C and D, caffeine induced an increase in the basal level of [Ca2]j. However, such an effect of caffeine was not systematically observed (see, for example, Figure 6, C and D).
A and B shows that the PAF response was strongly depressed by ruthenium red (50 ,uM). The average amplitude of the responses to 20 nM PAF was 795 nM before (Figure 6A) and 367 nM after adding ruthenium red to the bath (Figure 6B). Similar results were obtained in three experiments. Altogether, the responses measured in the presence of ruthenium red were 60 ± 24% (n = 12) of the corresponding controls. The SD of the 14 controls was 170/o. The reduction of the PAF response by ruthenium red is statistically significant (p < 0.001). Similar results were obtained in three other batches of cells with 1 mM octanol, which reduced the PAF responses to 38 ± 210% (n = 12) of the control responses (Student's t test, p < 0.001). On the other hand, Figure 6, C and D shows that the response to a low dose of PAF (0.4 nM) was clearly potentiated by caffeine (5 mM); the average amplitude of the Ca2l transients was 103 nM in control conditions (Figure 6C) and 243 nM in the presence of caffeine (Figure 6D). In two experiments, the response to PAF measured in the presence of caffeine was 199 ± 54% (n = 8) of the control response (100 518
± 49%, n = 8). This difference is statistically significant (p < 0.001). Note that in macrophages, as in pancreatic cells in which [Ca2+J is lightly buffered (Osipchuk et al., 1990), caffeine does not discharge Ca2l but simply potentiates the Ca2' release evoked by an initial rise in [Ca21],, in line with the original observations made in muscle cells (Endo, 1977). Finally, we investigated the possibility that the reduction of the [Ca21], transients observed in the presence of ruthenium red could be explained by an inhibition of the PAF-induced IP3 production. When cells were kept for 15 min in 50,uM ruthenium red, PAF (20 nM) induced an IP3 increase that had amplitude and kinetics similar to those observed in control conditions on the same batch of cells (Figure 7).
Discussion The aim of this work was to determine the respective roles of two intracellular messengers, IP3 and Ca2", in the induction of the explosive release of Ca2+ from intracellular stores that follows the stimulation of macrophages by PAF. CELL REGULATION
Ca2"-induced Ca2l release in macrophages
800
800
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aF
PA
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Ruth. Red
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4Q0r
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Caffeine
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n v
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2 mnu
1
Figure 6. The response to PAF is inhibited by ruthenium red and potentiated by caffeine. Responses to PAF (20 nM in A and B and 0.4 nM in C and D) in control conditions (A and C), in the presence of 50 MM ruthenium red (B), or 5 mM caffeine (D). Each trace is the average of four (A, C, and D) and five (B) individual responses from different cells of the same dish.
Before discussing this central point, let us make some comments on the influx of Ca2+ that allows sustained but relatively slow changes in [Ca2+]J after the initial Ca2+ transient. Our results give some indications on the activation mechanism of this influx, which is still a matter of debate. For instance, it has been proposed that IP3 (Gardner, 1990) and/or IP4 (Irvine and Moor, 1986; Morris et al., 1987) could lead to the activation of Ca2+ influx in some nonexcitable cells, but there is not a general agreement on this point (Downes, 1988; Matthews et aL, 1989). We have observed that Ca2+ influx was activated after ionomycin-induced Ca2+ transients of large amplitude, even though ionomycin does not induce any change in IP3 concentration in these cells. This shows, in agreement with previous results obtained with thapsigargin (Gouy et aL, 1990; Bismuth, Randriamampita, Trautmann, unpublished experiments), that Ca2+ influx can be stimulated in the absence of IP3 (and probably IP4) production. In addition, the experiment illustrated in Figure 2B shows that in the presence of heparin, the increase in IP3 and thus in IP4, which had presumably been elicited by PAF, was unable to induce a Ca2+ influx in the cell. Vol. 2, July 1991
On the other hand, as already mentioned, the amplitude of the initial [Ca2"J rise usually seems to be of fundamental importance for the triggering of the Ca2l influx. One possibility is that Ca2"-dependent enzymes are activated by this 3000
:20) u
t ECL,
2000
r-4
"O
1000
c
0
20
40 time (sec)
60
Figure 7. The production of IPN by 20 nM PAF was not affected by the presence of 50 MM ruthenium red in the bath. The dotted line shows the control IP3 production after stimulation with 20 nM PAF, as shown in Figure 3. Each point is the average (±SD) of three measurements. Similar results were obtained in two experiments.
519
C. Randriamampita et aL.
initial [Ca2"Ji rise. Alternatively, the emptying of the intracellular stores could be the trigger for the subsequent Ca2" influx, as suggested by Takemura and Putney (1989). More recently, Takemura et al. (1989) showed that their "capacitative" model was probably oversimplified and that a second messenger is probably required between the emptying of intracellular stores and the subsequent Ca2" influx. One of the most prominent features of Ca2" release from intracellular stores in macrophages and other cell types is that it is a very rapid phenomenon; in macrophages, the peak of the PAF-induced [Ca2+]J response is reached in
