Vol. 175, No. 2, 1991 March 15, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 532-536
SYNERGISTIC EFFECT OF MAGNESIUM AND CALCIUM IONS IN THE ACTIVATION OF PHOSPHOLJPASE A, OF LIVER MACROPHAGES
Hellmut Krause, Peter Dieter, Agnes Schulze-Specking, Annette Ballhorn, Ernst Ferber’, and Karl Decker Biochemisches Institut der Albert-Ludwigs-Universitat and Max-Planck-Institut fur Immunbiologie’, Freiburg,Germany Received
January
21,
1991
Summary: In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A, was found to be strongly activated at free Ca2+ concentrations from 100 nM to 1 PM in the presence of 4 mM free Mg2+. This is within the range of intracellular free Cat’ reported for basal and various stimulated conditions, respectively. Ca2’ alone increased phospholipase A, activity at high Ca2+ concentrations (1mM) whereas Mg*+ alone had only little stimulatory effect. Calmodulin does not seem to participate in the regulation of phospholipase A, although it relieved the inhibition of phospholipase A, activity by calmodulin antagonists. 0 1991 RcademlcPress, 1°C.
Kupffer cells, the resident macrophages of the liver, contain an intracellular
form of
phospholipase A, (PLA,; EC 3.1.1.4) that cleaves arachidonic acid from the a-2 position of membrane phospholipids
(1). A role for Ca2+ as a second messenger involved in the
activation of PLA, in primary cultures of Kupffer cells was strongly suggested by the finding that raising the extracellular concentration of Ca*’ from 10m7to 10d M or stimulation with calcium ionophore A23187 resulted in an increased synthesis of prostaglandin
E,, an
arachidonic acid metabolite (2). In previous studies, PLA, was characterized and found to require the presence of Ca2’ concentrations in the mM range (1). The intracellular levels of free Cazf , however, are between 10m7to 10d M in unstimulated and stimulated cells, respectively (3). The aim of this study was to resolve this contradiction and to investigate the role of Ca2’, Mg2+ and calmodulin in PLA, regulation. MATERIALS
AND METHODS
EGTA, nitrilotriacetic acid (free acid), EDTA (free acid), phenylSepharose CL-4B, calmodulin, compound B24571 (calmidazolium), trifluoperazin, W7 and W5 were purchased from Sigma (Mtinchen, Germany). 1-Stearoyl-Z[ l-‘4C]arachidonoyl-L-3-phosphatidylcholine (55.9 mCi/mmol) was from Amersham (Frankfurt, Germany). Calcium standard solutions and 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
532
Vol.
175,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the F2112Ca-Selectrode were obtained from Radiometer (Copenhagen, Denmark). All other chemicals were of analytical grade. Preparation of cell cultures (4) and determination of PLA, activity in Kupffer cell homogenate (15) have been described already. Unless otherwise indicated, standard assay mixtures (100~1) contained 100 mM Tris-HCl, pH 9.0, 2.5 rg Kupffer cell protein, 1 PM free Ca2+, 10 mM Mg2+ (uncomplexed to chelators) and 2.2 PM [“Clphosphatidylcholine adsorbed to fatty-acid-free albumin (0.1% (w/v) final concentration). Protein was determined by the Spector modification (6) of the method of Bradford (standard: bovine serum albumin from Boehringer Mannheim, Germany). Determination (7) and purification of calmodulin by affinity chromatography (8) have been described. A 1 ml phenyl-Sepharose column was equilibrated with buffer I containing 100 mM Tris-HCl, pH 9.0,0.5 M NaCl and 1 mM CaCl,. The Kupffer cell homogenate was spun at 400,OOOg for 45 min in the presence of 1 mM of EGTA. One ml supematant was applied to the column (Ca2+ cont. 1 mM) and washed with 10 ml buffer I (2.5 ml/min) followed by elution of calmodulin with 10 ml buffer I containing 5 mM EGTA instead of CaCl,. The free Ca2+ concentrations were calculated from published data (9,lO) using the constants from (11) and subsequently confirmed by measurement of [Ca2’] with a calcium electrode (12) (Fig. 1). Unless otherwise indicated results are means + SD from duplicates of at least three independent experiments.
RESULTS PLA, activity could be enhanced by Ca2+, Mg2+ and Ni2+ (Fig.2).
The biphasic
dependence on Cat’ (Fig.3) suggested the presence of a high- and a low-affinity binding site (10e7 to 10d M and 10e4to 10” M, respectively). In the presence of 10 mM Mg2+ the low affinity site disappeared yielding a monophasic Cat’ dependence of PLA, activity. 10 mM Mg’+ uncomplexed to chelators was calculated to correspond to approximately 4 mM free z E a q 1.0
-2 - -3 z ;t a -4 0 ii! 5 -5
z ,P x C .g 0.5 0 N
P -6
01
-7 10
0
6 PCs
4
2
02
0.0 Ca2+Mg2+
Ni2+
Mn2+
Cu2+Co2+
cd*+
Addition
Fig. 1 Dependence of nCa on the concentration of total Ca’+. Gpen symbols without, filled symbols with 10 mM uncomplexed Mg’+. Triangles, absence of chelator; circles, 1 mM nitrilotriacetic acid; square, 1 mM EGTA; inversed triangle, 0.99 mM EDTA+ 10pM EGTA; diamond, 1mM EDTA. Measurements were taken in 100 mM Tris-HCl buffer, pH 9.00 f 0.02, at 37°C (pH 9.00 was measured at 21°C; pH was then remeasured for all experimental conditions). pCa, negative lg of the concentration of free Cat’. Fip.2 PLA, activation bv addition of 1 mM divalent cations, The activity of PLA, in the absence of added ions is due to 400-800 nM contaminating free Ca” in the assay mixture (measured by atomic absorption). AA, arachidonic acid. 533
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BIOCHEMICAL
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4
3
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BIOPHYSICAL
Y,
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2
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COMMUNICATIONS
2 -763543210
8 PCs
PMg
Fie.3 Cat+-dependence of PLA, activity, Opencircleswithout (< 40 nM Mg2+, measured by atomic absorption),filled circles with 10 mM uncomplexedMg2+. For chelatorand divalent cation concentrationsseeFig. 1. AA, arachidonicacid; pCa, negativeIg of the concentrationof free Ca2+. Fig.4 Me’+ denendence of PLA, activity. Opencircleswithout (< lo-‘* M Ca2’), filled circleswith 1 FM free Ca2+.Calculationof the chelatoranddivalentcationconcentrations wascarriedout asdescribed in MaterialsandMethods.AA, arachidonic acid; pMg, negative lg of the concentrationof free Mg2+. Mg2+ according to Debye-Hiickel law which agreeswith reported intracellular levels of free Mg2+ (0.7 - 2 mM (13)). In the absenceof Ca2+ (free [Ca2’] < 1O“c M) Mg2+ had only little activating effect on PLA, (Fig.4). These data indicate a synergistic effect of Mg2+ on
Ca’+-induced PLA, activation in vivo. The dependence of PLA, involvement
of calmodulin
activity on panomolar Ca2+ concentrations suggested an in PLA, regulation (14). The calmodulin
inhibitors
R24571,
trifluoperazin, W7, and W5 were tested in the cell-free system. R24571 was found to inhibit PLA, activity in a dose-dependentmanner (Fig.5). The inhibition could be reversed by addition of calmodulin (Fig.6). In further studiesPLA, and calmodulin were separated on a phenyl-Sepharosecolumn (seeMethods). PLA, eluted within the first two ml of buffer I. This fraction contained s 1 pM calmodulin but its activity could not be enhancedby addition of
Fie.5 Inhibitionof PLA, bv calmodulin inhibitors. Results are means + SD of two to four independent experiments. 534
Vol.
175,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
COMMUNICATIONS
B
‘a .[ 0.1 i 5 5E > .T ‘S 0 .Y C0.0 Control x,
z
RESEARCH
CaM
1324571
CaM
noCaM
CaM
+R24571
Fig.6 PLA, and calmodulin. A) Measurement of PLA, activity in cell-free extract of Kupffer cells in the presence of 10e4M Ca‘+ . Control, PLA, activity without addition of calmodulin and calmodulin inhibitors (since 1 mol calmodulin binds 4 mol CaZf a second control was done with 20 PM Ca2+; it gave the same result). CaM, 20 FM calmodulin; R24571, 10 PM compound R2457 1. B) Measurement of PLA, activity in a calmodulin-free PLA, preparation. No CaM, [CaM] 5 1 pM calmodulin; CaM, 20 PM calmodulin added. Results in A) and B) are means f SD of three independentexperiments.
calmodulin
(Fig.6)& Calmodulin
was detectable. in the second elution in a concentration of
about 1 nM and was thus clearly separated from PLA,.
These data argue against a
participation of calmodulin in the regulation of PLA, of rat Kupffer cells. DISCUSSION Besides Ca’+ and Mg*+ , other divalent cations are known to activate PLA2, e.g. Nit+ (Fig.2), S?‘,
Bazf, and Mn*+ (15), suggesting that they can mimick Ca*+-and/or Mg*+-
dependent effects, The synergistic effect between Ca2+ and Mg”
on PLA, activation has not
been observed before. The existence of two Ca*‘-dependent
activity plateaus of intracellular
reported (15). According to Lenting et z&(16) the low-affinity
PLA, has been
binding site of intracellular
PLA, is due to a Ca*+ -induced transition of the phospholipid structure. An improved enzymephospholipid interaction could also be the mechanism of the synergistic effect of Mg*+ as it is in line with the disappearence of the low affinity Ca*+-binding site (Fig.3). Ca*+ uptake is known to be an early event during stimulation of cultured Kupffer cells, e.g. with zymosan (17). The prostaglandin E, release from these cells by various stimuli, e.g. zymosan, is increased in the presence of 2 10d M extracellular Ca*+ (2). It is interesting to note that the same Ca*’ concentration is required for the activation of PLA, in the presence of Mg*+ (Fig.3). This suggests that PLA, activity can respond to physiological fluctuations of the intracellular level of free Ca*+ in vivo. It is still a matter of controversy whether cellular PLA, is regulated by calmodulin (15,18). Half-maximal
activation of most calmodulin-dependent 535
enzymes occurs at about 4 FM
Vol.
BIOCHEMICAL
175, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNKATIONS
calmodulin (19). The intracellular fluid space of Kupffer cells contains approximately 25 f 10 PM calmodulin
(7) evidently in excess to the content of free Ca’+ that is found at 5 I PM
in most cell types (3). Although calmodulin reversed the inhibitory effect of R24571, it did not increase the activity of PLA, in a calmodulin-depleted
preparation (Fig.6). Thus, it is
more likely that R24571 acts in a different way. An analogy of the Ca2+-binding sites of both PLA, and calmodulin could be a possible explanation. This assumption would be in line with data obtained with cell cultures: calmodulin inhibitors diminished the effects of those stimuli that are supposed to act via Ca’+ -induced PLA, activation, e.g. the calcium ionophore A23187 and zymosan (2). Acknowledements:
This
Forschungsgemeinschaft
work
was supported by a grant
from
the Deutsche
(Bonn) through SFB 154 and from the Fonds der Chemischen
Industrie (Frankfurt). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15.
Krause,H., Dieter,P., Schulz+Specklng,A. ,and Decker,K.( 1989) ExpCell Biol. 57,102. Dieter,P., Schulze-Specking,A.,and Decker,K.(1988) Eur.J.Biochem. 177,61-67. Pozzan,T., Arslan,P., Tsien,R.Y. and Rink,T.J.(1982) J,Cell Biol. 94,335-340. Dieter,P., Krause,H. ,and Schulze-Specking,A.( 1990) Eicosanoids 3,45-5 1. Flesch,I. ,and F?rber,E.(1986) Biochim.Biophys.Acta 889,6-14. Spector,T.(1978) Analyt.Biochem. 86,142-146. Birmelin,M., MarmC,D., Ferber,E. ,and Decker,K.( 1984) Eur. J.Biochem. 140,55-61. Gopalakrishna,R.,and Anderson,W.B.(1982) Biochem.Biophys.Res.Comm. 104,830-836. Fabiato,A.,and Fabiato,F.(1979) J.Physiol.(Paris) 75, 463-505. Harrison,S.M.,and Bers,D.M.(1989) Am.J.Physiol. 256, C1250-C1256. Martell,A.E.,and Smith,R.M.(1974) Critical stability constants, Plenum Press, New York. Bers,D.M.(1982) Am.J.Physiol. 242,C404-C408. Rink,T.J., Tsien,R.Y. and Pozzan,T.(1982) J.Cell Biol. 95,189-196. Dieter,P.(1987) in: Models in plant physiology and biochemistry (Newman,D.W.,and Wilson,K.G.,eds) ~01.3, pp.ll-14, CRC Press, New York. de Winter,J.M., Korpancova,J.,and van den Bosch,H.(1984) Arch.Biochem.Biophys. 234,243-252.
16. 17.
18. 19.
Lenting,H.B.M., Neys,F.W. and van den Bosch,H.(1988) Biochim.Biophys.Acta 961,129-138. Birmelin,M.,and Decker,K.(1983) Eur.J.Biochem. 131,539-543. Moskowitz,N., Andres, A., Silva,W., Shapiro,L., Schook,W. and,Puszkin,S. (1985) Arch.Biochem.Biophys. 241,413-417. Hidaka,H., Yamaki,T., Naka,M., Tam&T., Hayashi,H. ,and Kobayashi,R.( 1979) Mol.Pharmacol. 17,66-72.
536
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 532-536
SYNERGISTIC EFFECT OF MAGNESIUM AND CALCIUM IONS IN THE ACTIVATION OF PHOSPHOLJPASE A, OF LIVER MACROPHAGES
Hellmut Krause, Peter Dieter, Agnes Schulze-Specking, Annette Ballhorn, Ernst Ferber’, and Karl Decker Biochemisches Institut der Albert-Ludwigs-Universitat and Max-Planck-Institut fur Immunbiologie’, Freiburg,Germany Received
January
21,
1991
Summary: In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A, was found to be strongly activated at free Ca2+ concentrations from 100 nM to 1 PM in the presence of 4 mM free Mg2+. This is within the range of intracellular free Cat’ reported for basal and various stimulated conditions, respectively. Ca2’ alone increased phospholipase A, activity at high Ca2+ concentrations (1mM) whereas Mg*+ alone had only little stimulatory effect. Calmodulin does not seem to participate in the regulation of phospholipase A, although it relieved the inhibition of phospholipase A, activity by calmodulin antagonists. 0 1991 RcademlcPress, 1°C.
Kupffer cells, the resident macrophages of the liver, contain an intracellular
form of
phospholipase A, (PLA,; EC 3.1.1.4) that cleaves arachidonic acid from the a-2 position of membrane phospholipids
(1). A role for Ca2+ as a second messenger involved in the
activation of PLA, in primary cultures of Kupffer cells was strongly suggested by the finding that raising the extracellular concentration of Ca*’ from 10m7to 10d M or stimulation with calcium ionophore A23187 resulted in an increased synthesis of prostaglandin
E,, an
arachidonic acid metabolite (2). In previous studies, PLA, was characterized and found to require the presence of Ca2’ concentrations in the mM range (1). The intracellular levels of free Cazf , however, are between 10m7to 10d M in unstimulated and stimulated cells, respectively (3). The aim of this study was to resolve this contradiction and to investigate the role of Ca2’, Mg2+ and calmodulin in PLA, regulation. MATERIALS
AND METHODS
EGTA, nitrilotriacetic acid (free acid), EDTA (free acid), phenylSepharose CL-4B, calmodulin, compound B24571 (calmidazolium), trifluoperazin, W7 and W5 were purchased from Sigma (Mtinchen, Germany). 1-Stearoyl-Z[ l-‘4C]arachidonoyl-L-3-phosphatidylcholine (55.9 mCi/mmol) was from Amersham (Frankfurt, Germany). Calcium standard solutions and 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
532
Vol.
175,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the F2112Ca-Selectrode were obtained from Radiometer (Copenhagen, Denmark). All other chemicals were of analytical grade. Preparation of cell cultures (4) and determination of PLA, activity in Kupffer cell homogenate (15) have been described already. Unless otherwise indicated, standard assay mixtures (100~1) contained 100 mM Tris-HCl, pH 9.0, 2.5 rg Kupffer cell protein, 1 PM free Ca2+, 10 mM Mg2+ (uncomplexed to chelators) and 2.2 PM [“Clphosphatidylcholine adsorbed to fatty-acid-free albumin (0.1% (w/v) final concentration). Protein was determined by the Spector modification (6) of the method of Bradford (standard: bovine serum albumin from Boehringer Mannheim, Germany). Determination (7) and purification of calmodulin by affinity chromatography (8) have been described. A 1 ml phenyl-Sepharose column was equilibrated with buffer I containing 100 mM Tris-HCl, pH 9.0,0.5 M NaCl and 1 mM CaCl,. The Kupffer cell homogenate was spun at 400,OOOg for 45 min in the presence of 1 mM of EGTA. One ml supematant was applied to the column (Ca2+ cont. 1 mM) and washed with 10 ml buffer I (2.5 ml/min) followed by elution of calmodulin with 10 ml buffer I containing 5 mM EGTA instead of CaCl,. The free Ca2+ concentrations were calculated from published data (9,lO) using the constants from (11) and subsequently confirmed by measurement of [Ca2’] with a calcium electrode (12) (Fig. 1). Unless otherwise indicated results are means + SD from duplicates of at least three independent experiments.
RESULTS PLA, activity could be enhanced by Ca2+, Mg2+ and Ni2+ (Fig.2).
The biphasic
dependence on Cat’ (Fig.3) suggested the presence of a high- and a low-affinity binding site (10e7 to 10d M and 10e4to 10” M, respectively). In the presence of 10 mM Mg2+ the low affinity site disappeared yielding a monophasic Cat’ dependence of PLA, activity. 10 mM Mg’+ uncomplexed to chelators was calculated to correspond to approximately 4 mM free z E a q 1.0
-2 - -3 z ;t a -4 0 ii! 5 -5
z ,P x C .g 0.5 0 N
P -6
01
-7 10
0
6 PCs
4
2
02
0.0 Ca2+Mg2+
Ni2+
Mn2+
Cu2+Co2+
cd*+
Addition
Fig. 1 Dependence of nCa on the concentration of total Ca’+. Gpen symbols without, filled symbols with 10 mM uncomplexed Mg’+. Triangles, absence of chelator; circles, 1 mM nitrilotriacetic acid; square, 1 mM EGTA; inversed triangle, 0.99 mM EDTA+ 10pM EGTA; diamond, 1mM EDTA. Measurements were taken in 100 mM Tris-HCl buffer, pH 9.00 f 0.02, at 37°C (pH 9.00 was measured at 21°C; pH was then remeasured for all experimental conditions). pCa, negative lg of the concentration of free Cat’. Fip.2 PLA, activation bv addition of 1 mM divalent cations, The activity of PLA, in the absence of added ions is due to 400-800 nM contaminating free Ca” in the assay mixture (measured by atomic absorption). AA, arachidonic acid. 533
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BIOCHEMICAL
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4
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BIOPHYSICAL
Y,
4
2
RESEARCH
COMMUNICATIONS
2 -763543210
8 PCs
PMg
Fie.3 Cat+-dependence of PLA, activity, Opencircleswithout (< 40 nM Mg2+, measured by atomic absorption),filled circles with 10 mM uncomplexedMg2+. For chelatorand divalent cation concentrationsseeFig. 1. AA, arachidonicacid; pCa, negativeIg of the concentrationof free Ca2+. Fig.4 Me’+ denendence of PLA, activity. Opencircleswithout (< lo-‘* M Ca2’), filled circleswith 1 FM free Ca2+.Calculationof the chelatoranddivalentcationconcentrations wascarriedout asdescribed in MaterialsandMethods.AA, arachidonic acid; pMg, negative lg of the concentrationof free Mg2+. Mg2+ according to Debye-Hiickel law which agreeswith reported intracellular levels of free Mg2+ (0.7 - 2 mM (13)). In the absenceof Ca2+ (free [Ca2’] < 1O“c M) Mg2+ had only little activating effect on PLA, (Fig.4). These data indicate a synergistic effect of Mg2+ on
Ca’+-induced PLA, activation in vivo. The dependence of PLA, involvement
of calmodulin
activity on panomolar Ca2+ concentrations suggested an in PLA, regulation (14). The calmodulin
inhibitors
R24571,
trifluoperazin, W7, and W5 were tested in the cell-free system. R24571 was found to inhibit PLA, activity in a dose-dependentmanner (Fig.5). The inhibition could be reversed by addition of calmodulin (Fig.6). In further studiesPLA, and calmodulin were separated on a phenyl-Sepharosecolumn (seeMethods). PLA, eluted within the first two ml of buffer I. This fraction contained s 1 pM calmodulin but its activity could not be enhancedby addition of
Fie.5 Inhibitionof PLA, bv calmodulin inhibitors. Results are means + SD of two to four independent experiments. 534
Vol.
175,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
COMMUNICATIONS
B
‘a .[ 0.1 i 5 5E > .T ‘S 0 .Y C0.0 Control x,
z
RESEARCH
CaM
1324571
CaM
noCaM
CaM
+R24571
Fig.6 PLA, and calmodulin. A) Measurement of PLA, activity in cell-free extract of Kupffer cells in the presence of 10e4M Ca‘+ . Control, PLA, activity without addition of calmodulin and calmodulin inhibitors (since 1 mol calmodulin binds 4 mol CaZf a second control was done with 20 PM Ca2+; it gave the same result). CaM, 20 FM calmodulin; R24571, 10 PM compound R2457 1. B) Measurement of PLA, activity in a calmodulin-free PLA, preparation. No CaM, [CaM] 5 1 pM calmodulin; CaM, 20 PM calmodulin added. Results in A) and B) are means f SD of three independentexperiments.
calmodulin
(Fig.6)& Calmodulin
was detectable. in the second elution in a concentration of
about 1 nM and was thus clearly separated from PLA,.
These data argue against a
participation of calmodulin in the regulation of PLA, of rat Kupffer cells. DISCUSSION Besides Ca’+ and Mg*+ , other divalent cations are known to activate PLA2, e.g. Nit+ (Fig.2), S?‘,
Bazf, and Mn*+ (15), suggesting that they can mimick Ca*+-and/or Mg*+-
dependent effects, The synergistic effect between Ca2+ and Mg”
on PLA, activation has not
been observed before. The existence of two Ca*‘-dependent
activity plateaus of intracellular
reported (15). According to Lenting et z&(16) the low-affinity
PLA, has been
binding site of intracellular
PLA, is due to a Ca*+ -induced transition of the phospholipid structure. An improved enzymephospholipid interaction could also be the mechanism of the synergistic effect of Mg*+ as it is in line with the disappearence of the low affinity Ca*+-binding site (Fig.3). Ca*+ uptake is known to be an early event during stimulation of cultured Kupffer cells, e.g. with zymosan (17). The prostaglandin E, release from these cells by various stimuli, e.g. zymosan, is increased in the presence of 2 10d M extracellular Ca*+ (2). It is interesting to note that the same Ca*’ concentration is required for the activation of PLA, in the presence of Mg*+ (Fig.3). This suggests that PLA, activity can respond to physiological fluctuations of the intracellular level of free Ca*+ in vivo. It is still a matter of controversy whether cellular PLA, is regulated by calmodulin (15,18). Half-maximal
activation of most calmodulin-dependent 535
enzymes occurs at about 4 FM
Vol.
BIOCHEMICAL
175, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNKATIONS
calmodulin (19). The intracellular fluid space of Kupffer cells contains approximately 25 f 10 PM calmodulin
(7) evidently in excess to the content of free Ca’+ that is found at 5 I PM
in most cell types (3). Although calmodulin reversed the inhibitory effect of R24571, it did not increase the activity of PLA, in a calmodulin-depleted
preparation (Fig.6). Thus, it is
more likely that R24571 acts in a different way. An analogy of the Ca2+-binding sites of both PLA, and calmodulin could be a possible explanation. This assumption would be in line with data obtained with cell cultures: calmodulin inhibitors diminished the effects of those stimuli that are supposed to act via Ca’+ -induced PLA, activation, e.g. the calcium ionophore A23187 and zymosan (2). Acknowledements:
This
Forschungsgemeinschaft
work
was supported by a grant
from
the Deutsche
(Bonn) through SFB 154 and from the Fonds der Chemischen
Industrie (Frankfurt). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15.
Krause,H., Dieter,P., Schulz+Specklng,A. ,and Decker,K.( 1989) ExpCell Biol. 57,102. Dieter,P., Schulze-Specking,A.,and Decker,K.(1988) Eur.J.Biochem. 177,61-67. Pozzan,T., Arslan,P., Tsien,R.Y. and Rink,T.J.(1982) J,Cell Biol. 94,335-340. Dieter,P., Krause,H. ,and Schulze-Specking,A.( 1990) Eicosanoids 3,45-5 1. Flesch,I. ,and F?rber,E.(1986) Biochim.Biophys.Acta 889,6-14. Spector,T.(1978) Analyt.Biochem. 86,142-146. Birmelin,M., MarmC,D., Ferber,E. ,and Decker,K.( 1984) Eur. J.Biochem. 140,55-61. Gopalakrishna,R.,and Anderson,W.B.(1982) Biochem.Biophys.Res.Comm. 104,830-836. Fabiato,A.,and Fabiato,F.(1979) J.Physiol.(Paris) 75, 463-505. Harrison,S.M.,and Bers,D.M.(1989) Am.J.Physiol. 256, C1250-C1256. Martell,A.E.,and Smith,R.M.(1974) Critical stability constants, Plenum Press, New York. Bers,D.M.(1982) Am.J.Physiol. 242,C404-C408. Rink,T.J., Tsien,R.Y. and Pozzan,T.(1982) J.Cell Biol. 95,189-196. Dieter,P.(1987) in: Models in plant physiology and biochemistry (Newman,D.W.,and Wilson,K.G.,eds) ~01.3, pp.ll-14, CRC Press, New York. de Winter,J.M., Korpancova,J.,and van den Bosch,H.(1984) Arch.Biochem.Biophys. 234,243-252.
16. 17.
18. 19.
Lenting,H.B.M., Neys,F.W. and van den Bosch,H.(1988) Biochim.Biophys.Acta 961,129-138. Birmelin,M.,and Decker,K.(1983) Eur.J.Biochem. 131,539-543. Moskowitz,N., Andres, A., Silva,W., Shapiro,L., Schook,W. and,Puszkin,S. (1985) Arch.Biochem.Biophys. 241,413-417. Hidaka,H., Yamaki,T., Naka,M., Tam&T., Hayashi,H. ,and Kobayashi,R.( 1979) Mol.Pharmacol. 17,66-72.
536
