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Stimulation and inhibition of secretion by phorbol myristate acetate in different cell types
Vol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 950-955
March 29, 1985
STIMULATION AND INHIBITION OF SECRETION BY PI-DRBOL MYRISTATE ACETATE ~1 DIFFERENT CELL TYPES G.3. Bleckwell, R.W. Bonser, J. Dawson and L.G. Garland Departments of Biochemistry and Pro staglandin Research, The Wellcome Research Laboratories, Beckenham, Kent, BR3 3BS, England Received February 12, 1985
In washed human platelets and in HL60 granuloeytes phorbol myristate acetate (PMA, 1 - 2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL6O cells maintained a maximal response at PMA concentrations between 30 - 300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast ceils. However, in the mast ceil PMA concentrations above 10nM reduced maximum histamine release in a dose- dependant manner. © 1985AcademicPress, Inc.
There are numerous reports that the protein kinase- C activator phorbol myristate acetate (PMA) synergises with stimuli that provoke secretion in various cell types, including mast cells (1), neutrophils (2) and platelets (3). Recently, it has been suggested that PMA, possibly through protein kinase-C activation, also acts to inhibit calcium signals in such ceils. Thus, Drummond et al. (4) reported that PMA inhibited agonist-induced phosphoinositide metabolism and calcium flux in human platelets. In addition, PMA at the relatively high concentration of 75nM reduced the level of intracellular free calcium, raised by prior antigen challenge of sensitised rat basophil Ieukaemia cells (RBL 2H3).
This effect was associated with a reduction by PMA
(75nM) of antigen-induced release of histamine from RBL 2H3 cells (5). Furthermore, Naocache et al. (6) report that preincubation of rabbit neutrophils with PMA inhibits the increase in intracellular concentration of free calcium induced by chemotactic factor.
Consequently, we have investigated whether inhibition of secretion by high
concentrations of PMA occurs widely among different cell types. For this purpose we Abbreviations used
are:
DMSO, dimethylsulphoxide; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; fMet-Leu-Phe, f methionyl-leucyl-phenylalanine; U46619, l l a , 9 a epoxymethano PHG 2. 0006-291X/85 $1.50 Copyright © 1985 by Academic Press, Inc. All rights of reproduction m any form reserved.
950
ol. 127, No 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
have examined extended dose-response curves for P M A , using the secretory responses
of rat peritoneal mast cells, human platelets and a model of granulocyte function using DMSO - differentiated HL60 cells which develop a number of functions characteristic of mature neutrophils, including the generation of an oxidative metabolic burst (7, 8) and a secretory response (9, I0).
In addition to degranulation, PMA-induced
production of superoxide by HL60 cells was also studied. In each cell type experiments were designed to include the direct effects of PMA as well as synergism with the calcium ionophore A23187 and, in the case of platelets, the prostaglandin endoperoxide analogue U46619. Materials and Methods:.
Suspensions of rat peritoneal cells, containing approximately 5% mast cells, were used without further purification as described previously (ii). Cells were suspended in oxygenated, modified Tyrode's solution (pH 7.4) having the following composition (mmol/l): NaCI, 137; HEPES, 11; NaH?PO~., 0.3; KCI, 0.2; MgCI2, 1.0; CaCI 2, 0.2; dextrose, 5.6. The calcium concentra[ion-was lower than normal in order to l i m i t histamine release by the ionophore A23187. H~tamine release was measured fluorimetrically (12, 13) after 5 rain. incubation at 37 C with the appropriate stimulus. In the absence of PMA, release of histamine by A23187 (IBM), expressed as percentage of total histamine, was 5.7 + 0.64% when corrected for spontaneous release of 4.6 + 0.65% (both values being m e ~ + S.E.M. of 5 experiments, each in duplicate).
HL60 cell cultures were induced to differentiate along recognisable myeloid lines usingTDMSO as described previously (lZ;). Differentiated cells were suspended (2 x 10 /ml) in Hanks' balanced salt solution containing 30mM HEPES buffer (pH 7.2) and CaCI 2 1.25mM. Degranulation was measured as the release of N-acetyl-B-glucuronidase (15) in the presence of cytochalasin-B (5Bg/ml). In the absence of any stimulus, the spontaneous release of enzyme was < 1% of total. Superoxide production was measured continuously at 37 C as the (superoxide ~smutase-mhlbitable) increase m absorption (550nm) when l m l of HL60 ceils (2 x 10 cells/ml) in oxygenated Hanks' solution was incubated with an equal volume of 1501JM ferricytochrome-c (16). The ceils produced no absorbance change during 45 rain. incubation in the absence of PMA. •
O
.
.
.
.
.
.
.
Washed human platelets were prepared a#cording to methods previously described (17). The pliaz~elets, suspended (3 x 10g/ml) in Tyrode's solution contain~g 1.0mM CaCI 9 and [ C] 5 hydroxytryptamine 0.5BM, were stored overnight at 4~C before use. f_Jnder these conditions > 95% of the radioactivity was incorporated into platelet granules. Platelet aggregation was monitored in a Payton dual channel Aggregometer using O.Sml samples of the piatelet suspension. The samples were incubated in the cuvette for 1.5 min. before the addition of PMA or vehicle (1BI ethanol), U46619 or vehicle (5BI 20mM Tris buffer pH 7.5) being added at 2 min. After a further 5 rain, the sample was transferred to a 2ml centrifuge tube containing l m l fixing buffer (3% glutaraldehyde in 0.1M cacodylate buffer pH 7.2). The samples were centrifuged at 1O,000 xg for 2 min., then a portion (0.75ml) of the supernatant was removed for determination of radioactivity. Results were expressed as a percentage of the total radioactivity in the sample. 5-Hydroxy [side chain 2 - 14C] tryptamine creatinine sulphate Batch 103, 56mCi/mmol, was obtained from Amersham International plc; 48-Phorbol 12-myristate 13-acetate was obtained from Sigma Chemical Company Limited; A23187 was obtained from Calbiochem Limited.
951
VoI 127, NO. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Rssulta and Dlscu~lon: The addition of PMA (l - 300nM) to rat peritoneal cells caused no significant release of histamine, the estimate a f t e r correction for spontaneous release being -0.09
+ 0.09% of total (mean + S.E.M. of 52 measurements).
However, when
combined with ionophore A2]187 (IIJM), PMA stimulated a marked increase in release above the threshold response to ionophore alone (Figure 1). response relationship for
PMA reached
an optimum at
3 - 10nM, higher concentrations being less effective.
The c o n c e n t r a t i o n -
concentrations between
The release stimulated by 3nM
PMA was three times more than that stimulated by 30gnM. These observations extend those of Katakami et al. (1) and Sagi-Eisenberg et al. (5). The former authors described synergistic histamine release by A23187 and PMA ( 1 6 - 49nM) but in the limited concentration range studied PMA had no inhibitory effect. The latter authors, using a related cell type (RBL 2H3), observed sub-optimal synergy between antigen and PMA (75nM) but did not describe the full concentrationresponse curve. It is interesting that in the present study PMA alone caused no release of histamine, whereas Katakami et al. (1) found that PMA (49nM) released 10% of total histamine.
Two differences between the studies may account for this: (i) in the
present experiments cells had not been subjected to density gradient centrifugation to purify mast cells; (ii) release was measured after 5 min. incubation with PMA compared with 10 min. used by Katakami et aI. (1). In DMSO-differentiated HL60 cells the calcium ionophore A23187 and the chemotactic peptide fMet-Leu-Phe both stimulate lysosomal enzyme release (9, 10). We have confirmed these observations (results not shown) and in addition report that PMA also stimulates enzyme release (Figure 2).
In contrast to rat peritoneal mast
ceils, ionophore-induced degranulation of HE60 cells was potentiated by PMA to a sustained maximum rather than to an optimum (Figure 2).
Similarly~ in these cells
superoxide generation reached a maximum at 10nM PMA and higher concentrations of the phorbol ester were equally effective (Figure 3). In washed human platelets PMA caused a dose-related secretion (Figure 4), release of 5HT increasing progressively rather than exhibiting an optimum. 952
Thus,
' o l 127, N o 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
I.C
0.5 E 5
E "E0,5 <
0 1
3
10 30 100 PMA (nM)
300
Q
3x10.7 M
1
3
10
100
30
300
PMA (nM)
A23187
Figure I :
Effect of various concentrations of PMA, added either alone or in combination with the calcium ionophore A23187 (lgM), on histamine release from rat peritoneal mast ceils. Results are the mean + SJ~.M. of 5 experiments each in duplication. O PMA, • PMA + A23187. Maximum response = lr2.5 + 8A% release of total histamine (mean + S.E.M. of 5 experiments). -
Figure 2:
Degranulation (release of N-ecetyl-B-glucosaminidase) from I-L60 granulocytes by PMA added either alone or in combination with the calcium ionophore A23187. Results are the mean + S.D. of 3 experiments each in duplicate. O PMA, • PMA + A23187. M e , m u m response = 36.9 + 7.3% release of t o t a l cellular enzyme (mean + S ~ . M . of 3 experiments).-
60
1.0
~ ( 2 , - - - - - - - - e (2) ' • (2)
~/(6)
50
c-
.= •
E _E
~
:.-:.',
•
~:-: .......... ":'~~'::~i!~!~
I- 3 0 ,.r
05
i,o
E
(5)
~
20
0
10 LI.
@(4)
®
-: f,:.*;.'l
I
I
I
0.3
1
3
I
i
10 30 PMA (nM)
I
0
I
100 300 Q
i
i
28.5~1 U46619
0.2 2 20 20O 2000 PMA (nM)
Figure 3:
Superoxide production by HL60 granulocytes stimulated with PMA. Values are the mean + 5.E.M• of several observations (numbers in parentheses).
Figure 4:
Release of 5-HT from washed human platelets by PMA added either alone or in combination with U46619• Hatched areas correspond to the effect of P M A alone. Results from a single, representative, experiment are shown, each value being the m e a n and range of duplicates• 953
1000
Vol. 127, No. 3. 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
concentrations as high as 2~M showed no inhibition of secretion.
Furthermore,
synergistic combination of PMA with a non-secretory concentration of U46619 exhibited a sustained maximum response.
In the three types of cell studied, occupancy of specific membrane receptors initiates a sequence of events which culminates in the secretion of stored intracel]ular constituents.
This phenomenon is associated with a rise in cytosolic free calcium
which can be achieved at least in part by the calcium-mobilising products of polyphosphoinositide metabolism (18).
Secretion can also be augmented by stimulators of
protein kinase C such as diacylglycerol and PMA (19). However, three recent reports ( 4 - 6) describe inhibition by PMA of polyphosphoinositide metabolism and calcium flux.
We have also illustrated that in rat peritoneal mast cells the secretion of
histamine was inhibited by concentrations of PMA in excess of 10nM. If this effect were important within secretory mechanisms generally, then the 'bell-shaped' doseresponse curve for PMA-induced synergy would be obtained in other cells as well as in rat mast cells. However, it seems that this is not the case. There was no evidence for inhibition of secretion in either human platelets or HL60 ceils, even when PMA was used at concentrations as high as 2UM.
This result was obtained when the primary
stimulus was PMA itself or when the PMA response was synergistic with either ionophore or the thromboxane - receptor agonist U46619.
Sagi-I£isenberg et al. (5) suggested that inhibition by PMA of antigen-stimulated histamine release from R B L - 2H3 ceils resulted from
a reduced calcium-influx
possibly mediated via altered polyphosphoinositide metabolism.
However, in our
experiments we observed the same effect when the ionophore A23187 was the primary agonist.
Thus, a more likely interpretation is that PMA facilitates either calcium
effiux or re-uptake by intraceliular organelles.
In conclusion, reduction by PMA at high concentrations of agonist-induced secretion is not a general phenomenon. The reason for this difference among different secretory ceils is not yet clear. However, the present results highlight the probability that the control of calcium homoeostasis varies among different cell types.
954
tol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgments: We wish to acknowledge the assistance of Mr Graham Spacey and Miss 3udith Montague.
Reference~ 1.
Katakami, Y., Kaibuchi, K., Sawamura, M., Takai, Y., and Nishizuka, Y. (1984) Biochem. Biophys. Res. C o m m u n . 121, 573-578.
2.
Kajikawa, N., Kaibuchi, K., Matsubara, T., Kikkawa, U., Takai, Y., and Nishizuka, Y. (1983) Biochem. Biophys. Res. Commun. 116, 743-750.
3.
Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikura, T., and Nishizuka, Y. (1983) 3. Biol. Chem. 25.8, 6701-6704.
4.
Drummond, A.H., Maclntyre, D.E., McNieol, A., and Rossi, A. (1984) Proceedings of the British Pharmacological Society, December C.48.
5.
Sagi-Eisenbarg, R., Lieman, H., and Pecht, I. (1985) Nature 315, 59-60.
6.
Naccache, P.H., Molski, T.F.P., Borgeat, P., White, J.R., and Sha'afi, R.I. (1984) J. Biol. Chem. Submitted for publication.
7.
Collins, S.3., Ruscetti, F.W., Gallagher, R.E., and Gallo, R.C. (1979) Med. 149, 969-974.
8.
Newburger, P.E.; Chovaniec, M.E., Greenberger, 33. and Cohen, H.3. (1979) J. Cell. Biol. 8___2315-322. 2,
9.
Naccache, P.H., Molski, T.F.P., Spinnelli, B., Borgeat, P. and Abboud, C.N. (1984) J. Cell Physiol. 119,241-246.
10.
Chaplinski, T.3. and Niedel, J.E. (1982) J. Clin. Invest. 70, 95]-964.
11.
Garland, L.G., and Mongar, 3.L. (1976) Int. Arch. Allergy. appl. Immunol. 50, 27-42.
12.
Leoffler, L.3., Lovenberg, W., and Sjoerdsma, A. (1971) Biochem. Pharmacol. 213, 2287-2297. Hakanson, R., Ronnberg, A.L., and Sjolund, K. (1972) Ana|yticaL Biochem. 4../7, 556-570.
13. 14. 15.
J. Exp.
Bonser, R.W., Siegel, M.I., McConnell, R.T., and Cuatrecasas, P. (1981) Biochem. Biophys. Res. Commun. 102, 1269-1275. Ringrose, P.S., Parr, M.A., and McLaren, M. (1975) Biochem. Pharmacol. 24, 607-614.
16.
Cohen, H.J. and Chovaniec, M.E. (1978) 3. Clin. Invest. 6_.1, 1081-1087.
17.
BIackwell, G.3., Radomski, M., Vargas, R., and Moncada, S. (1982) Biochim. Biophys. Acts., 718, 60-65.
18.
Berridge, M.3., and Irvine, R.F. (1984) Nature, 512, 515-521.
19.
Nishizuka, Y., Takai, Y., Kishimoto, A., Kikkawa, U. and Kaibuchi, K. Progress in Hormone Research, 40, 301-345.
955
(1984)
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 950-955
March 29, 1985
STIMULATION AND INHIBITION OF SECRETION BY PI-DRBOL MYRISTATE ACETATE ~1 DIFFERENT CELL TYPES G.3. Bleckwell, R.W. Bonser, J. Dawson and L.G. Garland Departments of Biochemistry and Pro staglandin Research, The Wellcome Research Laboratories, Beckenham, Kent, BR3 3BS, England Received February 12, 1985
In washed human platelets and in HL60 granuloeytes phorbol myristate acetate (PMA, 1 - 2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL6O cells maintained a maximal response at PMA concentrations between 30 - 300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast ceils. However, in the mast ceil PMA concentrations above 10nM reduced maximum histamine release in a dose- dependant manner. © 1985AcademicPress, Inc.
There are numerous reports that the protein kinase- C activator phorbol myristate acetate (PMA) synergises with stimuli that provoke secretion in various cell types, including mast cells (1), neutrophils (2) and platelets (3). Recently, it has been suggested that PMA, possibly through protein kinase-C activation, also acts to inhibit calcium signals in such ceils. Thus, Drummond et al. (4) reported that PMA inhibited agonist-induced phosphoinositide metabolism and calcium flux in human platelets. In addition, PMA at the relatively high concentration of 75nM reduced the level of intracellular free calcium, raised by prior antigen challenge of sensitised rat basophil Ieukaemia cells (RBL 2H3).
This effect was associated with a reduction by PMA
(75nM) of antigen-induced release of histamine from RBL 2H3 cells (5). Furthermore, Naocache et al. (6) report that preincubation of rabbit neutrophils with PMA inhibits the increase in intracellular concentration of free calcium induced by chemotactic factor.
Consequently, we have investigated whether inhibition of secretion by high
concentrations of PMA occurs widely among different cell types. For this purpose we Abbreviations used
are:
DMSO, dimethylsulphoxide; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; fMet-Leu-Phe, f methionyl-leucyl-phenylalanine; U46619, l l a , 9 a epoxymethano PHG 2. 0006-291X/85 $1.50 Copyright © 1985 by Academic Press, Inc. All rights of reproduction m any form reserved.
950
ol. 127, No 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
have examined extended dose-response curves for P M A , using the secretory responses
of rat peritoneal mast cells, human platelets and a model of granulocyte function using DMSO - differentiated HL60 cells which develop a number of functions characteristic of mature neutrophils, including the generation of an oxidative metabolic burst (7, 8) and a secretory response (9, I0).
In addition to degranulation, PMA-induced
production of superoxide by HL60 cells was also studied. In each cell type experiments were designed to include the direct effects of PMA as well as synergism with the calcium ionophore A23187 and, in the case of platelets, the prostaglandin endoperoxide analogue U46619. Materials and Methods:.
Suspensions of rat peritoneal cells, containing approximately 5% mast cells, were used without further purification as described previously (ii). Cells were suspended in oxygenated, modified Tyrode's solution (pH 7.4) having the following composition (mmol/l): NaCI, 137; HEPES, 11; NaH?PO~., 0.3; KCI, 0.2; MgCI2, 1.0; CaCI 2, 0.2; dextrose, 5.6. The calcium concentra[ion-was lower than normal in order to l i m i t histamine release by the ionophore A23187. H~tamine release was measured fluorimetrically (12, 13) after 5 rain. incubation at 37 C with the appropriate stimulus. In the absence of PMA, release of histamine by A23187 (IBM), expressed as percentage of total histamine, was 5.7 + 0.64% when corrected for spontaneous release of 4.6 + 0.65% (both values being m e ~ + S.E.M. of 5 experiments, each in duplicate).
HL60 cell cultures were induced to differentiate along recognisable myeloid lines usingTDMSO as described previously (lZ;). Differentiated cells were suspended (2 x 10 /ml) in Hanks' balanced salt solution containing 30mM HEPES buffer (pH 7.2) and CaCI 2 1.25mM. Degranulation was measured as the release of N-acetyl-B-glucuronidase (15) in the presence of cytochalasin-B (5Bg/ml). In the absence of any stimulus, the spontaneous release of enzyme was < 1% of total. Superoxide production was measured continuously at 37 C as the (superoxide ~smutase-mhlbitable) increase m absorption (550nm) when l m l of HL60 ceils (2 x 10 cells/ml) in oxygenated Hanks' solution was incubated with an equal volume of 1501JM ferricytochrome-c (16). The ceils produced no absorbance change during 45 rain. incubation in the absence of PMA. •
O
.
.
.
.
.
.
.
Washed human platelets were prepared a#cording to methods previously described (17). The pliaz~elets, suspended (3 x 10g/ml) in Tyrode's solution contain~g 1.0mM CaCI 9 and [ C] 5 hydroxytryptamine 0.5BM, were stored overnight at 4~C before use. f_Jnder these conditions > 95% of the radioactivity was incorporated into platelet granules. Platelet aggregation was monitored in a Payton dual channel Aggregometer using O.Sml samples of the piatelet suspension. The samples were incubated in the cuvette for 1.5 min. before the addition of PMA or vehicle (1BI ethanol), U46619 or vehicle (5BI 20mM Tris buffer pH 7.5) being added at 2 min. After a further 5 rain, the sample was transferred to a 2ml centrifuge tube containing l m l fixing buffer (3% glutaraldehyde in 0.1M cacodylate buffer pH 7.2). The samples were centrifuged at 1O,000 xg for 2 min., then a portion (0.75ml) of the supernatant was removed for determination of radioactivity. Results were expressed as a percentage of the total radioactivity in the sample. 5-Hydroxy [side chain 2 - 14C] tryptamine creatinine sulphate Batch 103, 56mCi/mmol, was obtained from Amersham International plc; 48-Phorbol 12-myristate 13-acetate was obtained from Sigma Chemical Company Limited; A23187 was obtained from Calbiochem Limited.
951
VoI 127, NO. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Rssulta and Dlscu~lon: The addition of PMA (l - 300nM) to rat peritoneal cells caused no significant release of histamine, the estimate a f t e r correction for spontaneous release being -0.09
+ 0.09% of total (mean + S.E.M. of 52 measurements).
However, when
combined with ionophore A2]187 (IIJM), PMA stimulated a marked increase in release above the threshold response to ionophore alone (Figure 1). response relationship for
PMA reached
an optimum at
3 - 10nM, higher concentrations being less effective.
The c o n c e n t r a t i o n -
concentrations between
The release stimulated by 3nM
PMA was three times more than that stimulated by 30gnM. These observations extend those of Katakami et al. (1) and Sagi-Eisenberg et al. (5). The former authors described synergistic histamine release by A23187 and PMA ( 1 6 - 49nM) but in the limited concentration range studied PMA had no inhibitory effect. The latter authors, using a related cell type (RBL 2H3), observed sub-optimal synergy between antigen and PMA (75nM) but did not describe the full concentrationresponse curve. It is interesting that in the present study PMA alone caused no release of histamine, whereas Katakami et al. (1) found that PMA (49nM) released 10% of total histamine.
Two differences between the studies may account for this: (i) in the
present experiments cells had not been subjected to density gradient centrifugation to purify mast cells; (ii) release was measured after 5 min. incubation with PMA compared with 10 min. used by Katakami et aI. (1). In DMSO-differentiated HL60 cells the calcium ionophore A23187 and the chemotactic peptide fMet-Leu-Phe both stimulate lysosomal enzyme release (9, 10). We have confirmed these observations (results not shown) and in addition report that PMA also stimulates enzyme release (Figure 2).
In contrast to rat peritoneal mast
ceils, ionophore-induced degranulation of HE60 cells was potentiated by PMA to a sustained maximum rather than to an optimum (Figure 2).
Similarly~ in these cells
superoxide generation reached a maximum at 10nM PMA and higher concentrations of the phorbol ester were equally effective (Figure 3). In washed human platelets PMA caused a dose-related secretion (Figure 4), release of 5HT increasing progressively rather than exhibiting an optimum. 952
Thus,
' o l 127, N o 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
I.C
0.5 E 5
E "E0,5 <
0 1
3
10 30 100 PMA (nM)
300
Q
3x10.7 M
1
3
10
100
30
300
PMA (nM)
A23187
Figure I :
Effect of various concentrations of PMA, added either alone or in combination with the calcium ionophore A23187 (lgM), on histamine release from rat peritoneal mast ceils. Results are the mean + SJ~.M. of 5 experiments each in duplication. O PMA, • PMA + A23187. Maximum response = lr2.5 + 8A% release of total histamine (mean + S.E.M. of 5 experiments). -
Figure 2:
Degranulation (release of N-ecetyl-B-glucosaminidase) from I-L60 granulocytes by PMA added either alone or in combination with the calcium ionophore A23187. Results are the mean + S.D. of 3 experiments each in duplicate. O PMA, • PMA + A23187. M e , m u m response = 36.9 + 7.3% release of t o t a l cellular enzyme (mean + S ~ . M . of 3 experiments).-
60
1.0
~ ( 2 , - - - - - - - - e (2) ' • (2)
~/(6)
50
c-
.= •
E _E
~
:.-:.',
•
~:-: .......... ":'~~'::~i!~!~
I- 3 0 ,.r
05
i,o
E
(5)
~
20
0
10 LI.
@(4)
®
-: f,:.*;.'l
I
I
I
0.3
1
3
I
i
10 30 PMA (nM)
I
0
I
100 300 Q
i
i
28.5~1 U46619
0.2 2 20 20O 2000 PMA (nM)
Figure 3:
Superoxide production by HL60 granulocytes stimulated with PMA. Values are the mean + 5.E.M• of several observations (numbers in parentheses).
Figure 4:
Release of 5-HT from washed human platelets by PMA added either alone or in combination with U46619• Hatched areas correspond to the effect of P M A alone. Results from a single, representative, experiment are shown, each value being the m e a n and range of duplicates• 953
1000
Vol. 127, No. 3. 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
concentrations as high as 2~M showed no inhibition of secretion.
Furthermore,
synergistic combination of PMA with a non-secretory concentration of U46619 exhibited a sustained maximum response.
In the three types of cell studied, occupancy of specific membrane receptors initiates a sequence of events which culminates in the secretion of stored intracel]ular constituents.
This phenomenon is associated with a rise in cytosolic free calcium
which can be achieved at least in part by the calcium-mobilising products of polyphosphoinositide metabolism (18).
Secretion can also be augmented by stimulators of
protein kinase C such as diacylglycerol and PMA (19). However, three recent reports ( 4 - 6) describe inhibition by PMA of polyphosphoinositide metabolism and calcium flux.
We have also illustrated that in rat peritoneal mast cells the secretion of
histamine was inhibited by concentrations of PMA in excess of 10nM. If this effect were important within secretory mechanisms generally, then the 'bell-shaped' doseresponse curve for PMA-induced synergy would be obtained in other cells as well as in rat mast cells. However, it seems that this is not the case. There was no evidence for inhibition of secretion in either human platelets or HL60 ceils, even when PMA was used at concentrations as high as 2UM.
This result was obtained when the primary
stimulus was PMA itself or when the PMA response was synergistic with either ionophore or the thromboxane - receptor agonist U46619.
Sagi-I£isenberg et al. (5) suggested that inhibition by PMA of antigen-stimulated histamine release from R B L - 2H3 ceils resulted from
a reduced calcium-influx
possibly mediated via altered polyphosphoinositide metabolism.
However, in our
experiments we observed the same effect when the ionophore A23187 was the primary agonist.
Thus, a more likely interpretation is that PMA facilitates either calcium
effiux or re-uptake by intraceliular organelles.
In conclusion, reduction by PMA at high concentrations of agonist-induced secretion is not a general phenomenon. The reason for this difference among different secretory ceils is not yet clear. However, the present results highlight the probability that the control of calcium homoeostasis varies among different cell types.
954
tol. 127, No. 3, 1985
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgments: We wish to acknowledge the assistance of Mr Graham Spacey and Miss 3udith Montague.
Reference~ 1.
Katakami, Y., Kaibuchi, K., Sawamura, M., Takai, Y., and Nishizuka, Y. (1984) Biochem. Biophys. Res. C o m m u n . 121, 573-578.
2.
Kajikawa, N., Kaibuchi, K., Matsubara, T., Kikkawa, U., Takai, Y., and Nishizuka, Y. (1983) Biochem. Biophys. Res. Commun. 116, 743-750.
3.
Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikura, T., and Nishizuka, Y. (1983) 3. Biol. Chem. 25.8, 6701-6704.
4.
Drummond, A.H., Maclntyre, D.E., McNieol, A., and Rossi, A. (1984) Proceedings of the British Pharmacological Society, December C.48.
5.
Sagi-Eisenbarg, R., Lieman, H., and Pecht, I. (1985) Nature 315, 59-60.
6.
Naccache, P.H., Molski, T.F.P., Borgeat, P., White, J.R., and Sha'afi, R.I. (1984) J. Biol. Chem. Submitted for publication.
7.
Collins, S.3., Ruscetti, F.W., Gallagher, R.E., and Gallo, R.C. (1979) Med. 149, 969-974.
8.
Newburger, P.E.; Chovaniec, M.E., Greenberger, 33. and Cohen, H.3. (1979) J. Cell. Biol. 8___2315-322. 2,
9.
Naccache, P.H., Molski, T.F.P., Spinnelli, B., Borgeat, P. and Abboud, C.N. (1984) J. Cell Physiol. 119,241-246.
10.
Chaplinski, T.3. and Niedel, J.E. (1982) J. Clin. Invest. 70, 95]-964.
11.
Garland, L.G., and Mongar, 3.L. (1976) Int. Arch. Allergy. appl. Immunol. 50, 27-42.
12.
Leoffler, L.3., Lovenberg, W., and Sjoerdsma, A. (1971) Biochem. Pharmacol. 213, 2287-2297. Hakanson, R., Ronnberg, A.L., and Sjolund, K. (1972) Ana|yticaL Biochem. 4../7, 556-570.
13. 14. 15.
J. Exp.
Bonser, R.W., Siegel, M.I., McConnell, R.T., and Cuatrecasas, P. (1981) Biochem. Biophys. Res. Commun. 102, 1269-1275. Ringrose, P.S., Parr, M.A., and McLaren, M. (1975) Biochem. Pharmacol. 24, 607-614.
16.
Cohen, H.J. and Chovaniec, M.E. (1978) 3. Clin. Invest. 6_.1, 1081-1087.
17.
BIackwell, G.3., Radomski, M., Vargas, R., and Moncada, S. (1982) Biochim. Biophys. Acts., 718, 60-65.
18.
Berridge, M.3., and Irvine, R.F. (1984) Nature, 512, 515-521.
19.
Nishizuka, Y., Takai, Y., Kishimoto, A., Kikkawa, U. and Kaibuchi, K. Progress in Hormone Research, 40, 301-345.
955
(1984)