CELL REGULATION, Vol. 2, 915-925, November 1991
Regulation of Ca2l influx during mitosis: Ca2l influx and depletion of intracellular Ca2l stores are coupled in interphase but not mitosis
Susan F. Preston, Ramadan 1. Sha'afi, and Richard D. Berlin Department of Physiology University of Connecticut Health Center Farmington, Connecticut 06030 Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2l ([Ca2+J) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2"] results from two sources: an initial release of Ca2l from intracellular stores followed by an influx of extracellular Ca2". These two phases, release from intracellular stores and Ca2l influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2l from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2"], response in interphase HeLa cells: a rapid rise in [Ca2"] followed by a sustained elevation, the latter dependent entirely on extracellular Ca2". In mitotic cells only the initial elevation, derived by Ca2l release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2l influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2", being replenished by reuptake of Ca2l that is retained within the cell. To ensure the depletion of Ca2" stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2" from intracellular stores by inhibition of a specific Ca2+-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2" stores, stimulates Ca2l influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+1, that is dependent on extracellular Ca2` and is associated with a strong stimulation of "5Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+], elevation that is © 1991 by The American Society for Cell Biology
initially comparable in magnitude and largely independent of extracellular Ca2". However, unlike interphase cells, in mitotic cells the elevation of [Ca2"], is not sustained and "5Ca2" influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2" influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2" influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2" influx and sustained elevations of [CaJ2+]. Introduction
A wide range of membrane properties is altered during mitosis. This includes endocytosis (Berlin et aL, 1978), exocytosis (Hesketh et al., 1984), receptor recycling (Sager et al., 1984; Warren et al., 1985), glycosaminoglycan secretion (Preston et al., 1985), glycoprotein processing (Warren et al., 1983), and, we have shown recently, membrane signal transduction (Volpi and Berlin, 1988). In interphase HeLa cells, activation of the histamine Hi receptor leads to an elevation of intracellular free calcium ([Ca2+]D). The rise in [Ca2+]i is characterized by an initial peak dependent at least partly on the release of Ca2` from intracellular stores, followed by a plateau that is entirely dependent on the influx of extracellular Ca2+. This pattern is common to many nonexcitable cells stimulated by other hormones and has been confirmed recently by Tilly et al. (1990) for histamine in HeLa cells. In mitotic cells only the initial peak elevation of [Ca2+]i occurs. Measurements of 45Ca2` uptake suggested that Ca2" influx was stimulated by histamine in interphase but not mitotic cells. Activation of histamine Hi receptors stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (OP3) in all cell types that have been studied (e.g., Hollingworth, 1986), including HeLa cells (Tilly et al., 1990). Although there is general agreement that IP3 induces Ca2" release from intracellular stores, the mechanisms regulating Ca2" influx are poorly understood (Ber915
S.F. Preston et al.
ridge and Irvine, 1989). However, it is precisely this influx that appears to be blocked during mitosis. In this paper we have continued comparative studies of the responses of interphase and mitotic HeLa cells in an attempt to substantiate the specific inhibition of hormone-induced Ca2" influx in mitosis and to reveal the mechanisms controlling Ca2" influx. We first show that histamine stimulates a rapid efflux of [Ca2+]i in interphase but not mitotic cells. By controlling efflux we were able to measure unidirectional 45Ca2" influx and provide additional evidence that Ca2" influx is selectively blocked during mitosis. From this we infer that the [Ca2+]i transient in mitotic cells is essentially independent of Ca2" traffic across the plasma membrane, derived entirely from its release and sequestration by intracellular organelles. What then is the basis for the inhibition of Ca2" influx in mitotic cells? We have addressed this question in terms of the widely accepted view that Ca2" entry is regulated, at least in part, by the Ca2" concentration within the IP3-sensitive intracellular pool (Irvine, 1990). According to this capacitative model proposed originally by Putney (1986), the intracellular pool is depleted by the action of IP3; IP3 itself does not directly affect entry because a pool that is held empty can stimulate entry long after IP3 levels have returned to control values (Takemura et al., 1989). Because histamine induces a transient elevation of [Ca2]i by the release of Ca2" from stores in mitotic cells, it is suggested that the coupling between the depleted IP3-sensitive store and Ca2l influx might be specifically inhibited in mitotic cells. However, in the absence of Ca2" efflux (shown here) the loss of Ca2" from stores within mitotic cells would be limited by complete reuptake into the storage sites. To clarify the contribution of reuptake to the suppression of influx in mitotic cells, we employed the sesquiterpenelactone, thapsigargin. Thapsigargin effects the release of Ca2" from the IP3-sensitive and perhaps other intracellular stores and prevents reuptake (Thastrup et al., 1990). Its action depends on inhibition of the Ca2+-ATPase of endoplasmic reticulum (but not plasma membrane). Consistent with the capacitative model, we show that in interphase cells thapsigargin induces a sustained Ca2" influx; HeLa cells are thus similar to the majority of cell types previously tested (Thastrup et al., 1989). By contrast, in mitotic cells, although thapsigargin induced an elevation of [Ca2"] and Ca2" efflux, leading to a depletion of intracellular stores, influx was not stimulated. Thus, in mi916
tosis there appears to be a specific uncoupling of the Ca2" pool (the "capacitance") from Ca2" entry. Results Histamine rapidly activates Ca2" efflux from interphase cells The importance of evaluating Ca2" efflux is twofold. First, efflux can diminish the apparent influx; efflux can potentially bring [Ca2"Ji toward baseline even in the face of continued Ca2" influx and second, in isotopic studies, the rate of influx even if constant will appear to decline if efflux is stimulated. Thus, our previous measurements of 45Ca2" influx, although showing a stimulation by histamine in interphase but not mitotic cells, were not linear; the rate of 45Ca2+ accumulation fell rapidly. Two explanations for the time course of declining uptake were considered: 1) that the influx was offset by a simultaneous efflux yielding a net decreasing rate of 45Ca2' accumulation or 2) that the period of influx was actually brief. The following experiments demonstrated that a stimulated, rapid efflux was the principle basis of the nonlinearity of 45Ca2+ uptake. Figure 1 A illustrates the effect of histamine on the Ca2+ content of 45Ca2+-preloaded interphase cells. A rapid efflux was observed that appeared to be -80% complete within 30 s. Ca2' efflux is generally thought to occur either by a Ca2+/Na+ exchange or by a plasma membrane Ca2+ pump; the former a low-affinity, highcapacity system and the latter a high-affinity, low-capacity system that is probably activated by calmodulin (Carifoli, 1987). 45Ca2+ can also be exchanged for extracellular Ca2+. Removing extracellular Ca2' by ethylene glycol-bis(fl-aminoethyl ether)-N,N,NV,N-tetraacetic acid (EGTA) addition inhibited 45Ca2' efflux but only partially (evidence for limited Ca2+-Ca2' exchange) (Figure 1 B). The cells were poorly tolerant of the removal of Na+ so that the Ca2+/Na+ exchange could not be tested directly. The calmodulin inhibitors W7 and calmidazolium blocked efflux by 50-70% at a concentration of 25 AM (data not shown). This is consistent with the activation of the membrane Ca2+ ATPase by a Ca2+-calmodulin complex reported in other systems (Carifoli, 1987), but we did not pursue further the analysis of mechanism. Histamine-induced influx of 45Ca2" into bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-loaded interphase cells Because the rapid efflux induced by histamine complicated the measurement of 45Ca21 influx, CELL REGULATION
Ca2" influx during mitosis A
1987) and results presumably from the rapid
-HA 90 c u 080
Ca2" influx that gradually overwhelms the Ca2" buffer capacity of the BAPTA. A , 1 00(
0 (U-
u'
70
+HA
0
+HA
60 L
. BAPTA
m30(
+
HA
Control
50 50
100
150
Time (sec)
B
60 seconds
1 00 4.,L Gc
0
,o u 4J0
U
_-
B
90
~L.
c- c 0
80
Cu
u
0)
c Li U
0j
=
4-i
70 4
U, un
6
60
50
Time (sec)
Time (sec)
Figure 1. Histamine-stimulated "Ca2+ effiux from interphase HeLa cells. Cells were loaded with 'Ca2+ as described in Materials and methods. When present, histamine (100 ,M) and EGTA (2 mM) were added to cell suspensions, and measurement of efflux was begun immediately. Rep*) resentative curves show: (A) efflux in CMH with (or without (O 0) histamine (HA) and (B) histamine-induced efflux with (0) EGTA (3 0) or without (O experiments).
sought experimental conditions that would reduce it. To this end and to establish the role of [Ca2+], in the activation of influx, we preincubated cell suspensions in the membranepermeant Ca2+ chelator, BAPTA-AM (Tsien, 1980). After entry and hydrolysis of the acetoxymethyl esters, the free BAPTA accumulates and buffers [Ca2]J,. This was shown by measurement of [Ca2]I with the fluorescent [Ca2+] indicator Indo 1 (Figure 2A). As described previously (Volpi and Berlin, 1988; Tilly et al., 1990), histamine induced a rise in [Ca2]J, characterized by a peak and elevated plateau. After BAPTA loading, the [Ca2]Ji elevation was delayed, rising gradually after a lag of 1-2 min. This is similar to the pattern observed in other nonexcitable cells such as leukocytes (Nasmith and Grinstein, we
Vol. 2, November 1991
10,000'
C
1-
4a 4,
O+HA
E
cL
-VI
ur
O 10- _CX
Regulation of Ca2l influx during mitosis: Ca2l influx and depletion of intracellular Ca2l stores are coupled in interphase but not mitosis
Susan F. Preston, Ramadan 1. Sha'afi, and Richard D. Berlin Department of Physiology University of Connecticut Health Center Farmington, Connecticut 06030 Activation of a wide variety of membrane receptors leads to a sustained elevation of intracellular Ca2l ([Ca2+J) that is pivotal to subsequent cell responses. In general, in nonexcitable cells this elevation of [Ca2"] results from two sources: an initial release of Ca2l from intracellular stores followed by an influx of extracellular Ca2". These two phases, release from intracellular stores and Ca2l influx, are generally coupled: stimulation of influx is coordinated with depletion of Ca2l from stores, although the mechanism of coupling is unclear. We have previously shown that histamine effects a typical [Ca2"], response in interphase HeLa cells: a rapid rise in [Ca2"] followed by a sustained elevation, the latter dependent entirely on extracellular Ca2". In mitotic cells only the initial elevation, derived by Ca2l release from intracellular stores, occurs. Thus, in mitotic cells the coupling of stores to influx may be specifically broken. In this report we first provide additional evidence that histamine-stimulated Ca2l influx is strongly inhibited in mitotic cells. We show that efflux is also strongly stimulated by histamine in interphase cells but not in mitotics. It is possible, thus, that in mitotics intracellular stores are only very briefly depleted of Ca2", being replenished by reuptake of Ca2l that is retained within the cell. To ensure the depletion of Ca2" stores in mitotic cells, we employed the sesquiterpenelactone, thapsigargin, that is known to affect the selective release of Ca2" from intracellular stores by inhibition of a specific Ca2+-ATPase; reuptake is inhibited. In most cells, and in accord with Putney's capacitative model (1990), thapsigargin, presumably by depleting intracellular Ca2" stores, stimulates Ca2l influx. This is the case for interphase HeLa cells. Thapsigargin induces an increase in [Ca2+1, that is dependent on extracellular Ca2` and is associated with a strong stimulation of "5Ca2+ influx. In mitotic cells thapsigargin also induces a [Ca2+], elevation that is © 1991 by The American Society for Cell Biology
initially comparable in magnitude and largely independent of extracellular Ca2". However, unlike interphase cells, in mitotic cells the elevation of [Ca2"], is not sustained and "5Ca2" influx is not stimulated by thapsigargin. Thus, the coupling between depletion of intracellular stores and Ca2" influx is specifically broken in mitotic cells. Uncoupling could account for the failure of histamine to stimulate Ca2" influx during mitosis and would effectively block all stimuli whose effects are mediated by Ca2" influx and sustained elevations of [CaJ2+]. Introduction
A wide range of membrane properties is altered during mitosis. This includes endocytosis (Berlin et aL, 1978), exocytosis (Hesketh et al., 1984), receptor recycling (Sager et al., 1984; Warren et al., 1985), glycosaminoglycan secretion (Preston et al., 1985), glycoprotein processing (Warren et al., 1983), and, we have shown recently, membrane signal transduction (Volpi and Berlin, 1988). In interphase HeLa cells, activation of the histamine Hi receptor leads to an elevation of intracellular free calcium ([Ca2+]D). The rise in [Ca2+]i is characterized by an initial peak dependent at least partly on the release of Ca2` from intracellular stores, followed by a plateau that is entirely dependent on the influx of extracellular Ca2+. This pattern is common to many nonexcitable cells stimulated by other hormones and has been confirmed recently by Tilly et al. (1990) for histamine in HeLa cells. In mitotic cells only the initial peak elevation of [Ca2+]i occurs. Measurements of 45Ca2` uptake suggested that Ca2" influx was stimulated by histamine in interphase but not mitotic cells. Activation of histamine Hi receptors stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (OP3) in all cell types that have been studied (e.g., Hollingworth, 1986), including HeLa cells (Tilly et al., 1990). Although there is general agreement that IP3 induces Ca2" release from intracellular stores, the mechanisms regulating Ca2" influx are poorly understood (Ber915
S.F. Preston et al.
ridge and Irvine, 1989). However, it is precisely this influx that appears to be blocked during mitosis. In this paper we have continued comparative studies of the responses of interphase and mitotic HeLa cells in an attempt to substantiate the specific inhibition of hormone-induced Ca2" influx in mitosis and to reveal the mechanisms controlling Ca2" influx. We first show that histamine stimulates a rapid efflux of [Ca2+]i in interphase but not mitotic cells. By controlling efflux we were able to measure unidirectional 45Ca2" influx and provide additional evidence that Ca2" influx is selectively blocked during mitosis. From this we infer that the [Ca2+]i transient in mitotic cells is essentially independent of Ca2" traffic across the plasma membrane, derived entirely from its release and sequestration by intracellular organelles. What then is the basis for the inhibition of Ca2" influx in mitotic cells? We have addressed this question in terms of the widely accepted view that Ca2" entry is regulated, at least in part, by the Ca2" concentration within the IP3-sensitive intracellular pool (Irvine, 1990). According to this capacitative model proposed originally by Putney (1986), the intracellular pool is depleted by the action of IP3; IP3 itself does not directly affect entry because a pool that is held empty can stimulate entry long after IP3 levels have returned to control values (Takemura et al., 1989). Because histamine induces a transient elevation of [Ca2]i by the release of Ca2" from stores in mitotic cells, it is suggested that the coupling between the depleted IP3-sensitive store and Ca2l influx might be specifically inhibited in mitotic cells. However, in the absence of Ca2" efflux (shown here) the loss of Ca2" from stores within mitotic cells would be limited by complete reuptake into the storage sites. To clarify the contribution of reuptake to the suppression of influx in mitotic cells, we employed the sesquiterpenelactone, thapsigargin. Thapsigargin effects the release of Ca2" from the IP3-sensitive and perhaps other intracellular stores and prevents reuptake (Thastrup et al., 1990). Its action depends on inhibition of the Ca2+-ATPase of endoplasmic reticulum (but not plasma membrane). Consistent with the capacitative model, we show that in interphase cells thapsigargin induces a sustained Ca2" influx; HeLa cells are thus similar to the majority of cell types previously tested (Thastrup et al., 1989). By contrast, in mitotic cells, although thapsigargin induced an elevation of [Ca2"] and Ca2" efflux, leading to a depletion of intracellular stores, influx was not stimulated. Thus, in mi916
tosis there appears to be a specific uncoupling of the Ca2" pool (the "capacitance") from Ca2" entry. Results Histamine rapidly activates Ca2" efflux from interphase cells The importance of evaluating Ca2" efflux is twofold. First, efflux can diminish the apparent influx; efflux can potentially bring [Ca2"Ji toward baseline even in the face of continued Ca2" influx and second, in isotopic studies, the rate of influx even if constant will appear to decline if efflux is stimulated. Thus, our previous measurements of 45Ca2" influx, although showing a stimulation by histamine in interphase but not mitotic cells, were not linear; the rate of 45Ca2+ accumulation fell rapidly. Two explanations for the time course of declining uptake were considered: 1) that the influx was offset by a simultaneous efflux yielding a net decreasing rate of 45Ca2' accumulation or 2) that the period of influx was actually brief. The following experiments demonstrated that a stimulated, rapid efflux was the principle basis of the nonlinearity of 45Ca2+ uptake. Figure 1 A illustrates the effect of histamine on the Ca2+ content of 45Ca2+-preloaded interphase cells. A rapid efflux was observed that appeared to be -80% complete within 30 s. Ca2' efflux is generally thought to occur either by a Ca2+/Na+ exchange or by a plasma membrane Ca2+ pump; the former a low-affinity, highcapacity system and the latter a high-affinity, low-capacity system that is probably activated by calmodulin (Carifoli, 1987). 45Ca2+ can also be exchanged for extracellular Ca2+. Removing extracellular Ca2' by ethylene glycol-bis(fl-aminoethyl ether)-N,N,NV,N-tetraacetic acid (EGTA) addition inhibited 45Ca2' efflux but only partially (evidence for limited Ca2+-Ca2' exchange) (Figure 1 B). The cells were poorly tolerant of the removal of Na+ so that the Ca2+/Na+ exchange could not be tested directly. The calmodulin inhibitors W7 and calmidazolium blocked efflux by 50-70% at a concentration of 25 AM (data not shown). This is consistent with the activation of the membrane Ca2+ ATPase by a Ca2+-calmodulin complex reported in other systems (Carifoli, 1987), but we did not pursue further the analysis of mechanism. Histamine-induced influx of 45Ca2" into bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-loaded interphase cells Because the rapid efflux induced by histamine complicated the measurement of 45Ca21 influx, CELL REGULATION
Ca2" influx during mitosis A
1987) and results presumably from the rapid
-HA 90 c u 080
Ca2" influx that gradually overwhelms the Ca2" buffer capacity of the BAPTA. A , 1 00(
0 (U-
u'
70
+HA
0
+HA
60 L
. BAPTA
m30(
+
HA
Control
50 50
100
150
Time (sec)
B
60 seconds
1 00 4.,L Gc
0
,o u 4J0
U
_-
B
90
~L.
c- c 0
80
Cu
u
0)
c Li U
0j
=
4-i
70 4
U, un
6
60
50
Time (sec)
Time (sec)
Figure 1. Histamine-stimulated "Ca2+ effiux from interphase HeLa cells. Cells were loaded with 'Ca2+ as described in Materials and methods. When present, histamine (100 ,M) and EGTA (2 mM) were added to cell suspensions, and measurement of efflux was begun immediately. Rep*) resentative curves show: (A) efflux in CMH with (or without (O 0) histamine (HA) and (B) histamine-induced efflux with (0) EGTA (3 0) or without (O experiments).
sought experimental conditions that would reduce it. To this end and to establish the role of [Ca2+], in the activation of influx, we preincubated cell suspensions in the membranepermeant Ca2+ chelator, BAPTA-AM (Tsien, 1980). After entry and hydrolysis of the acetoxymethyl esters, the free BAPTA accumulates and buffers [Ca2]J,. This was shown by measurement of [Ca2]I with the fluorescent [Ca2+] indicator Indo 1 (Figure 2A). As described previously (Volpi and Berlin, 1988; Tilly et al., 1990), histamine induced a rise in [Ca2]J, characterized by a peak and elevated plateau. After BAPTA loading, the [Ca2]Ji elevation was delayed, rising gradually after a lag of 1-2 min. This is similar to the pattern observed in other nonexcitable cells such as leukocytes (Nasmith and Grinstein, we
Vol. 2, November 1991
10,000'
C
1-
4a 4,
O+HA
E
cL
-VI
ur
O 10- _CX
