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Possible role for a calcium-activated, phospholipid-dependent protein kinase in mode of action of DSCG
TIPS - May 1985
198 References 1 Harpey, J. P. (1973) Adverse Drug React. Bull. 43, 140-143 2 Sim, E., Gill, E. W. and Sim, R. B. (1984) Lancet ii, 422--424 3 Perry, H. M., Tan, E. M., Carmody, S. and Sakamoto, A. (1970) J. Lab. Clin, Med. 76, 114--125 4 Godeau, P., Aukert, M., Imbert, |-C.
5 6 7 8 9
and Herreman, G. (1974) Ann. Med. Interne 124, 181-186 Lachmann, P. J. (1984) Philos. Trans. R. Soc, London Ser. B. 306, 419--430 Sire, E. and Law, S. K. A. (1985) FEBS Lett. (in press) Perry, H. M. (1973) Am. J. Med. 54, 58--72 Christophidis, N. (1984) Lancet ii, 868 Hughes, G. R. V. in Connective Tissue
Possible role for a calci u m-activated, phospholipid-dependent protein kinase in mode of action of DSCG Ronit Sagi-Eisenberg Ronit Sagi-Eisenberg suggests t h a t the anti-allergic drug disodium cromoglycate (DSCG) exerts its inhibitory activity on mast cell degranulation by interacting with a Cat+- and phospholipid-dependent protein kinase C involved in the stimulus-secretion coupling of these cells. Her hypothesis is based on similarities between the conditions required to activate this kinase and those needed to evoke secretion. In addition, binding of DSCG to mast cells leads to protein phosphorylation; protein kinase C has been shown to play a dual role in the activation and termination of the secretory process in RBL2H3 cells. Hence, the Ca2+ phospholipid-dependent protein kinase C appears to be an attractive candidate for the protective action of DSCG. The anti-allergic drug disodium cromoglycate (DSCG), widely used as a prophylactic drug in the treatment of bronchial asthma, has been subjected to extensive studies in attempts to resolve its mode of action. Clearly, elucidation of the molecular mechanism by which this drug exerts its protective effect is of both pharmacological and biochemical importance as it might shed new light on the mechanism of allergy and asthma. Early studies established the inhibitory effect of DSCG on the allergic response in h u m a n lung in vivo, provided it is administered before the allergen. Subsequent in-vitro experiments demonstratRonit Sagi-Eisenberg is a post-doctoral fellow in the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel
ed that DSCG inhibits the release of the mediators of immediatetype hypersensitivity from sensitized lung fragments as well as from isolated rat peritoneal mast cells 1. O n the basis of these observations, it has been suggested that DSCG exhibits its pharmacological activity by stabilizing the mast cell membrane, thereby preventing degranulation. However, alt h o u g h DSCG markedly reduces degranulation of mast cells in certain tissues, in others no activity is demonstrated 2. Differences in m e m b r a n e structure or composition of these ceils may account for this reduced drug-cell interaction. In fact mast cells from different species or even from individual tissues within a single animal were shown to vary significantly in their response to given histamine releases 2. Inhibition of mast cells degran-
1985,ElsevierSciencePublishersB.V.,Amsterdam 0165- 6147/85/$02.00
Disease. (1977) p. 44, Blackwell 10 Ludden, T. M., McNay, J. L., Shepherd, A. M. M. and Lin, M. S. (1981) Arthritis Rheum. 24, 987-994 11 Reece, P. A., Cozamanis, l. and Zacest, R. (1980) Clin. Pharmacol. Ther. 28, 769--
778 12 Durant, P. J. and Harris, R. A. (1980) N. Engl. J, Med. 303, 584-585
ulation by DSCG is undoubtedly of major importance in its protective mode of action although its very particular effectiveness against asthma raises the possibility that cells other than mast cells are also involved in the mode of action of this drug. Characteristics of the DSCG-mast cell interaction Inhibition of mast cell degranulation by DSCG has been shown to be accompanied by blocking of antigen-induced Ca 2+ influx 3. Since Ca 2+ influx is essential for antigen-induced cell degranulation 4, the inhibitory action of DSCG may result from interference with the cellular Ca 2+ gating mechanism. Indeed, low concentrations of DSCG which completely inhibit the antigen-induced release (10-50 ~tM), fail to significantly affect release induced by the Ca 2+ ionophore A23187 (calimycin) which bypasses the Ca 2+ gating mechanisms 5. Furthermore, a specific, Ca2+-dependent, binding site for DSCG on the outer membrane of mast cells has been demonstrated 6. This has been subsequently shown to be a membrane protein, and has been isolated from rat basophilic leukemia cells (RBL-2H3). Possible involvement of this protein in the Ca 2+ gating mechanism of t h e s e cells is implied by its ability to restore Ca 2+ uptake activity of RBL variants impaired in both binding of DSCG and Ca 2+ uptake 7. However, recent reports have revealed that high concentrations of DSCG (> 100 ~u~4)do inhibit release induced by sub-optimal concentrations of A23187 (Ref. 8). It therefore appears that this drug has additional modes of action. The divergent dose-response suggests that either there are at least two different receptors for DSCG or that the cellular target for this drug's action is multifunctional. A clue to the molecular mechanism by which DSCG might exert its activity has been recently ob-
199
TIPS - M a y 1985
tained from experiments revealing that DSCG affects phosphorylation of a single mast cell protein 9. Furthermore, this observation points to a possible linkage between Ca 2+ gating and protein phosphorylation. Indeed, as the Ca 2+ gates are regulated in a potential-independent manner :° , changes in their state of phosphorylation might be the actual mode by which their opening is regulated. Several lines of circumstantial evidence imply that a kinase C-type enzyme might be involved in the stimulus-secretion coupling of mast cells. Furthermore, this enzyme might be the cellular target for the inhibitory action of DSCG. Characteristics of protein kinase C activation Protein kinase C has been shown to be widely distributed in mammalian tissues, in both cytosolic and membrane fractions. The enzyme requires both Ca 2+ (in the mM range) and phosphatidylserine for attaining full activity::. Diacylglycerol, the hydrolysis product of phosphatidylinositol, dramatically increases its affinity for Ca 2+, thereby initiating its activation at less than micromolar concentrations of Ca 2+ (Ref. 12). The level of diacylglycerol is normally low and only rises during the receptor-dependent breakdown of phosphatidylinositol. This activity of protein kinase C appears to be modulated in a receptor-dependent manner. Furthermore, its activity is intimately related to a process (phosphatidylinositol turnover) previously suggested to be involved in the generation of a Ca 2+ signal by ligand-receptor interactions 13. The degranulation of mast cells is associated with an enhanced metabolism of phosphatidylinosito114 as well as with phosphorylation of several proteins 9. It is therefore reasonable to postulate that protein kinase C might be involved in the stimulus-secretion coupling of these cells. In this context, it is interesting to note that the degranulation of certain types of mast cells is increased in the presence of exogenously added phosphatidylserine TM. Moreover, the capacity of phosphatidylserine to enhance degranulation results from its potentiating effect on the Ca 2+ uptake activity,
indicating a role in the Ca 2+ gating mechanism :6. Thus, the possible involvement of the phosphatidylserine-dependent protein kinase C in the Ca 2+ gating mechanism might explain the potentiating effect phosphatidylserine exhibits on the Ca 2+ influx process, although a different mechanism cannot be excluded. Characteristics of the DSCG-Ca 2+ interaction In spite of the fact that binding of DSCG to mast cells shows an absolute dependency on the presence of Ca 2+, attempts to demonstrate Ca 2+ binding by DSCG in aqueous solutions have failed 3. An interaction between Ca 2+ and DSCG could, however, be shown in organic solvents such as propanol and methanol. Since DSCG is associated with the lipid components of cells, it appears likely
I
antigen
I
~I~
IgE receptor
~ aggregated receptor
diacylglycerol
~
that phospholipids are involved in DSCG binding in the presence of Ca 2+. Consistent with this proposal is the fact that high concentrations of exogenous phosphatidylserine overcome the inhibitory action of DSCG :7. As DSCG binding appears to involve Ca 2+, phosphatidylserine and a membrane protein, and since this binding eventually effects protein phosphorylation, it is possible that DSCG exerts its inhibitory effect by interacting with protein kinase C. This enzyme might be involved both in Ca 2+ gating regulation and in a step distal to Ca2+ influx, in the sequence of events leading to secretion. This would explain the dual action of DSCG, previously described. This hypothesis also raises a possible identity between protein kinase C and the DSCG-binding protein, isolated from the RBL cells 7, al-
~-~ kinase C:
C
a
2+, PS
kinase C2
X---~X-P
V
synergism
A ~
V'
synergism
secretion Fig. 1. Proposed mode/of stimulus-secret~n coupling in RBL cells. PS, phosphatidyl serine ; Ptlns., inos#ol phosphotipids ; TPA, 12-O-tetradecanoylphorbol-13-acetate ; X, endogenous substrate(s) for kinase C.
200 though more than one receptor might be involved in the action of DSCG.
The role of protein kinase C in the stimulus--secretion coupling of the histamine secreting rat basophilic leukaemia cells (RBL-2H3) RBL-2H3 cells, which degranulate on aggregation of IgE receptors, contain kinase C activity; partially-purified enzyme can be directly activated by the phorbal ester, TPA, increasing its affinity for Ca 2+ by three orders of magnitude TM. TPA is structurally related to diacylglycerol. However, when added to intact RBL cells, TPA failed to trigger release although simultaneous addition of a suboptimal concentration of A23187 significantly enhanced secretion TM. These observations indicate that, while activation of kinase C by itself is insufficient to cause release, kinase C-mediated phosphorylation and ionophoreinduced rise in cytosolic Ca 2+ concentration act synergistically. At concentrations lower than 15 riM, TPA also synergized with the antigen-induced release TM. However, at higher concentrations, this potentiating effect was reversed leading to inhibition of secretion rather than to activation. Thus, protein kinase C appears to participate in both activation and termination of the secretory process. Since TPA selectively inhibits release induced by antigen without affecting that evoked by the Ca 2+ ionophore, it may act by limiting receptor-dependent Ca 2+ influx. The effect of TPA on Ca 2+ fluxes has been studied directly employing quin-2 as a monitor TM. Addition of TPA to quin-2-1oaded RBL cells failed to cause any change in the internal Ca 2+ concentration TM. However, when added prior to antigen, TPA completely blocked the antigeninduced Ca 2+ signal. In contrast, TPA had no effect on the Ca 2+ signal triggered by the Ca2+ ionophore TM. Furthermore, when added subsequently to antigen, TPA rapidly reversed the signal. These results indicate that kinase C,
T I P S - M a y 1985
which is considered the cellular target through which TPA exerts its actions, does indeed interfere with the Ca 2+ gating mechanism. Interestingly, when added subsequently to antigen, although TPA dramatically accelerated the decay of the Ca2+ signal, release was potentiated rather than inhibited TM. This observation further supports the notion that kinase C is involved in both activation and termination of release. An unexpected but important finding was that low concentrations of TPA (< 15 riM), which synergize with antigen-induced release, also block Ca 2+ influx. Thus, provided that kinase C is activated and the receptors crosslinked, RBL cells degranulate in the absence of any detectable rise in Ca2+i In the absence of antigen, TPA failed to trigger release. We therefore conclude that receptor aggregation elicits an additional, as yet unknown, signal which, together with kinase C activation, yields a cellular response in the absence of any rise in Ca 2+ (Fig. 1). This 'signal' may be an endogenous substrate, which becomes available for kinase C phosphorylation only upon aggregation of the receptors. It remains to be clarified why antigen-induced release is inhibited in the presence of higher concentrations of TPA. The existence of two forms of kinase C in equilibrium, one responsible for regulation of Ca 2+ and the other involved in activation could exlain this finding (Fig. 1). This rather speculative, yet attractive possibility could also explain why: (1) exogenously-added phosphatidylserine enhances Ca 2+ uptake; (2) exogenously-added phosphatidylserine also delays desensitization, attributed to closure of the Ca 2 gate; (3) exogenously-added phosphatidylserine overcomes the inhibitory action of DSCG; and (4) DSCG effects protein phosphorylotion and inhibits Ca 2+ uptake. Circumstantial evidence strongly points to the possibility that DSCG exerts its inhibitory effect on mast cell degranulation by interacting with a Ca2+-activated,
phospholipid-dependent, protein kinase C involved in the secretory process. Future studies may verify this proposal. Meanwhile, studies aimed at testing this hypothesis will help resolve the mode of action of this drug, which provides a useful tool for determining the sequence of events leading to mast cell degranulation.
Acknowledgements I would like to thank Professor Israel Pecht from the Department of Chemical Immunology, The Weizmann Institute of Science and Dr John C. Foreman, from the Department of Pharmacology, University College London, for many fruitful discussions.
References 1 Cox, l- S. C. (1967) Nature (London) 216, 1328-1329 2 Pearce, F. L. (1982) Klin. Wochenschr. 60, 954-957 3 Spataro, A.C. and Bosmann, H.B. (1976) Biochem. Pharmacol. 25, 505-510 4 Foreman, J.C., Garland, L.G. and Mongar, J. L. (1976) Symp. Soc. Exp. Biol. 30, 193-218 5 Foreman, J.C., HaUett, M.B. and Mongar, J.L. (1977) Br. J. Pharmacol. 473--474 6 Mazurek, N., Berger, G. and Pecht, I. (1980) Nature (London) 286, 722-723 7 Mazurek, N., Bashkin, P., Loyter, A. and Pecht, I. (1983) Proc. Natl Acad. Sci. USA 80, 6014-6018 8 Pearce, F.L. and Truneh, A. (1981) Agents and Actions, 11, 44-50 9 Theoharides, T.C., Sieghart, W., Greengard, P. a n d Douglas, W.W. (1980) Science 207, 80-82 10 Sagi-Eisenberg, R. and Pecht, I. (1984) EMBO J. 3, 497-500 11 Kikkawa, U., Takai, Y., Minakuchi, R., lnohara, S. and Nishizuka, Y. (1982) J. Biol. Chem., 257, 13341-13348 12 Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276 13 Michell, R.H. (1979) Trends Biochem. Sci. 4, 128-131 14 Kennerly, D.A., Sullivan, T.J., Sylvester, P. and Parker, C.W. (1979) J. Exp. Med. 150, 1034-1039 15 Goth, A., Adams, H.R. and Knoohuizen, M. (1971) Science 173, 1034-1035 16 Sagi-Eisenberg, R., Geller-Bemstein, C., Ben-Neriah, Z. and Pecht, I. (1983) FEBS Lett. 161, 37-40 17 Garland, L. G. and Mongar, J. L. (1974) Br. J. Pharmacol. 50, 137-143 18 Sagi-Eisenberg, R. and Pecht, I. (1984) Immunol. Lett. 8, 237-241 19 Sagi-Eisenberg, R., Lieman, H. and Pecht, I. (1985) Nature (London) 8, 59-60
198 References 1 Harpey, J. P. (1973) Adverse Drug React. Bull. 43, 140-143 2 Sim, E., Gill, E. W. and Sim, R. B. (1984) Lancet ii, 422--424 3 Perry, H. M., Tan, E. M., Carmody, S. and Sakamoto, A. (1970) J. Lab. Clin, Med. 76, 114--125 4 Godeau, P., Aukert, M., Imbert, |-C.
5 6 7 8 9
and Herreman, G. (1974) Ann. Med. Interne 124, 181-186 Lachmann, P. J. (1984) Philos. Trans. R. Soc, London Ser. B. 306, 419--430 Sire, E. and Law, S. K. A. (1985) FEBS Lett. (in press) Perry, H. M. (1973) Am. J. Med. 54, 58--72 Christophidis, N. (1984) Lancet ii, 868 Hughes, G. R. V. in Connective Tissue
Possible role for a calci u m-activated, phospholipid-dependent protein kinase in mode of action of DSCG Ronit Sagi-Eisenberg Ronit Sagi-Eisenberg suggests t h a t the anti-allergic drug disodium cromoglycate (DSCG) exerts its inhibitory activity on mast cell degranulation by interacting with a Cat+- and phospholipid-dependent protein kinase C involved in the stimulus-secretion coupling of these cells. Her hypothesis is based on similarities between the conditions required to activate this kinase and those needed to evoke secretion. In addition, binding of DSCG to mast cells leads to protein phosphorylation; protein kinase C has been shown to play a dual role in the activation and termination of the secretory process in RBL2H3 cells. Hence, the Ca2+ phospholipid-dependent protein kinase C appears to be an attractive candidate for the protective action of DSCG. The anti-allergic drug disodium cromoglycate (DSCG), widely used as a prophylactic drug in the treatment of bronchial asthma, has been subjected to extensive studies in attempts to resolve its mode of action. Clearly, elucidation of the molecular mechanism by which this drug exerts its protective effect is of both pharmacological and biochemical importance as it might shed new light on the mechanism of allergy and asthma. Early studies established the inhibitory effect of DSCG on the allergic response in h u m a n lung in vivo, provided it is administered before the allergen. Subsequent in-vitro experiments demonstratRonit Sagi-Eisenberg is a post-doctoral fellow in the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel
ed that DSCG inhibits the release of the mediators of immediatetype hypersensitivity from sensitized lung fragments as well as from isolated rat peritoneal mast cells 1. O n the basis of these observations, it has been suggested that DSCG exhibits its pharmacological activity by stabilizing the mast cell membrane, thereby preventing degranulation. However, alt h o u g h DSCG markedly reduces degranulation of mast cells in certain tissues, in others no activity is demonstrated 2. Differences in m e m b r a n e structure or composition of these ceils may account for this reduced drug-cell interaction. In fact mast cells from different species or even from individual tissues within a single animal were shown to vary significantly in their response to given histamine releases 2. Inhibition of mast cells degran-
1985,ElsevierSciencePublishersB.V.,Amsterdam 0165- 6147/85/$02.00
Disease. (1977) p. 44, Blackwell 10 Ludden, T. M., McNay, J. L., Shepherd, A. M. M. and Lin, M. S. (1981) Arthritis Rheum. 24, 987-994 11 Reece, P. A., Cozamanis, l. and Zacest, R. (1980) Clin. Pharmacol. Ther. 28, 769--
778 12 Durant, P. J. and Harris, R. A. (1980) N. Engl. J, Med. 303, 584-585
ulation by DSCG is undoubtedly of major importance in its protective mode of action although its very particular effectiveness against asthma raises the possibility that cells other than mast cells are also involved in the mode of action of this drug. Characteristics of the DSCG-mast cell interaction Inhibition of mast cell degranulation by DSCG has been shown to be accompanied by blocking of antigen-induced Ca 2+ influx 3. Since Ca 2+ influx is essential for antigen-induced cell degranulation 4, the inhibitory action of DSCG may result from interference with the cellular Ca 2+ gating mechanism. Indeed, low concentrations of DSCG which completely inhibit the antigen-induced release (10-50 ~tM), fail to significantly affect release induced by the Ca 2+ ionophore A23187 (calimycin) which bypasses the Ca 2+ gating mechanisms 5. Furthermore, a specific, Ca2+-dependent, binding site for DSCG on the outer membrane of mast cells has been demonstrated 6. This has been subsequently shown to be a membrane protein, and has been isolated from rat basophilic leukemia cells (RBL-2H3). Possible involvement of this protein in the Ca 2+ gating mechanism of t h e s e cells is implied by its ability to restore Ca 2+ uptake activity of RBL variants impaired in both binding of DSCG and Ca 2+ uptake 7. However, recent reports have revealed that high concentrations of DSCG (> 100 ~u~4)do inhibit release induced by sub-optimal concentrations of A23187 (Ref. 8). It therefore appears that this drug has additional modes of action. The divergent dose-response suggests that either there are at least two different receptors for DSCG or that the cellular target for this drug's action is multifunctional. A clue to the molecular mechanism by which DSCG might exert its activity has been recently ob-
199
TIPS - M a y 1985
tained from experiments revealing that DSCG affects phosphorylation of a single mast cell protein 9. Furthermore, this observation points to a possible linkage between Ca 2+ gating and protein phosphorylation. Indeed, as the Ca 2+ gates are regulated in a potential-independent manner :° , changes in their state of phosphorylation might be the actual mode by which their opening is regulated. Several lines of circumstantial evidence imply that a kinase C-type enzyme might be involved in the stimulus-secretion coupling of mast cells. Furthermore, this enzyme might be the cellular target for the inhibitory action of DSCG. Characteristics of protein kinase C activation Protein kinase C has been shown to be widely distributed in mammalian tissues, in both cytosolic and membrane fractions. The enzyme requires both Ca 2+ (in the mM range) and phosphatidylserine for attaining full activity::. Diacylglycerol, the hydrolysis product of phosphatidylinositol, dramatically increases its affinity for Ca 2+, thereby initiating its activation at less than micromolar concentrations of Ca 2+ (Ref. 12). The level of diacylglycerol is normally low and only rises during the receptor-dependent breakdown of phosphatidylinositol. This activity of protein kinase C appears to be modulated in a receptor-dependent manner. Furthermore, its activity is intimately related to a process (phosphatidylinositol turnover) previously suggested to be involved in the generation of a Ca 2+ signal by ligand-receptor interactions 13. The degranulation of mast cells is associated with an enhanced metabolism of phosphatidylinosito114 as well as with phosphorylation of several proteins 9. It is therefore reasonable to postulate that protein kinase C might be involved in the stimulus-secretion coupling of these cells. In this context, it is interesting to note that the degranulation of certain types of mast cells is increased in the presence of exogenously added phosphatidylserine TM. Moreover, the capacity of phosphatidylserine to enhance degranulation results from its potentiating effect on the Ca 2+ uptake activity,
indicating a role in the Ca 2+ gating mechanism :6. Thus, the possible involvement of the phosphatidylserine-dependent protein kinase C in the Ca 2+ gating mechanism might explain the potentiating effect phosphatidylserine exhibits on the Ca 2+ influx process, although a different mechanism cannot be excluded. Characteristics of the DSCG-Ca 2+ interaction In spite of the fact that binding of DSCG to mast cells shows an absolute dependency on the presence of Ca 2+, attempts to demonstrate Ca 2+ binding by DSCG in aqueous solutions have failed 3. An interaction between Ca 2+ and DSCG could, however, be shown in organic solvents such as propanol and methanol. Since DSCG is associated with the lipid components of cells, it appears likely
I
antigen
I
~I~
IgE receptor
~ aggregated receptor
diacylglycerol
~
that phospholipids are involved in DSCG binding in the presence of Ca 2+. Consistent with this proposal is the fact that high concentrations of exogenous phosphatidylserine overcome the inhibitory action of DSCG :7. As DSCG binding appears to involve Ca 2+, phosphatidylserine and a membrane protein, and since this binding eventually effects protein phosphorylation, it is possible that DSCG exerts its inhibitory effect by interacting with protein kinase C. This enzyme might be involved both in Ca 2+ gating regulation and in a step distal to Ca2+ influx, in the sequence of events leading to secretion. This would explain the dual action of DSCG, previously described. This hypothesis also raises a possible identity between protein kinase C and the DSCG-binding protein, isolated from the RBL cells 7, al-
~-~ kinase C:
C
a
2+, PS
kinase C2
X---~X-P
V
synergism
A ~
V'
synergism
secretion Fig. 1. Proposed mode/of stimulus-secret~n coupling in RBL cells. PS, phosphatidyl serine ; Ptlns., inos#ol phosphotipids ; TPA, 12-O-tetradecanoylphorbol-13-acetate ; X, endogenous substrate(s) for kinase C.
200 though more than one receptor might be involved in the action of DSCG.
The role of protein kinase C in the stimulus--secretion coupling of the histamine secreting rat basophilic leukaemia cells (RBL-2H3) RBL-2H3 cells, which degranulate on aggregation of IgE receptors, contain kinase C activity; partially-purified enzyme can be directly activated by the phorbal ester, TPA, increasing its affinity for Ca 2+ by three orders of magnitude TM. TPA is structurally related to diacylglycerol. However, when added to intact RBL cells, TPA failed to trigger release although simultaneous addition of a suboptimal concentration of A23187 significantly enhanced secretion TM. These observations indicate that, while activation of kinase C by itself is insufficient to cause release, kinase C-mediated phosphorylation and ionophoreinduced rise in cytosolic Ca 2+ concentration act synergistically. At concentrations lower than 15 riM, TPA also synergized with the antigen-induced release TM. However, at higher concentrations, this potentiating effect was reversed leading to inhibition of secretion rather than to activation. Thus, protein kinase C appears to participate in both activation and termination of the secretory process. Since TPA selectively inhibits release induced by antigen without affecting that evoked by the Ca 2+ ionophore, it may act by limiting receptor-dependent Ca 2+ influx. The effect of TPA on Ca 2+ fluxes has been studied directly employing quin-2 as a monitor TM. Addition of TPA to quin-2-1oaded RBL cells failed to cause any change in the internal Ca 2+ concentration TM. However, when added prior to antigen, TPA completely blocked the antigeninduced Ca 2+ signal. In contrast, TPA had no effect on the Ca 2+ signal triggered by the Ca2+ ionophore TM. Furthermore, when added subsequently to antigen, TPA rapidly reversed the signal. These results indicate that kinase C,
T I P S - M a y 1985
which is considered the cellular target through which TPA exerts its actions, does indeed interfere with the Ca 2+ gating mechanism. Interestingly, when added subsequently to antigen, although TPA dramatically accelerated the decay of the Ca2+ signal, release was potentiated rather than inhibited TM. This observation further supports the notion that kinase C is involved in both activation and termination of release. An unexpected but important finding was that low concentrations of TPA (< 15 riM), which synergize with antigen-induced release, also block Ca 2+ influx. Thus, provided that kinase C is activated and the receptors crosslinked, RBL cells degranulate in the absence of any detectable rise in Ca2+i In the absence of antigen, TPA failed to trigger release. We therefore conclude that receptor aggregation elicits an additional, as yet unknown, signal which, together with kinase C activation, yields a cellular response in the absence of any rise in Ca 2+ (Fig. 1). This 'signal' may be an endogenous substrate, which becomes available for kinase C phosphorylation only upon aggregation of the receptors. It remains to be clarified why antigen-induced release is inhibited in the presence of higher concentrations of TPA. The existence of two forms of kinase C in equilibrium, one responsible for regulation of Ca 2+ and the other involved in activation could exlain this finding (Fig. 1). This rather speculative, yet attractive possibility could also explain why: (1) exogenously-added phosphatidylserine enhances Ca 2+ uptake; (2) exogenously-added phosphatidylserine also delays desensitization, attributed to closure of the Ca 2 gate; (3) exogenously-added phosphatidylserine overcomes the inhibitory action of DSCG; and (4) DSCG effects protein phosphorylotion and inhibits Ca 2+ uptake. Circumstantial evidence strongly points to the possibility that DSCG exerts its inhibitory effect on mast cell degranulation by interacting with a Ca2+-activated,
phospholipid-dependent, protein kinase C involved in the secretory process. Future studies may verify this proposal. Meanwhile, studies aimed at testing this hypothesis will help resolve the mode of action of this drug, which provides a useful tool for determining the sequence of events leading to mast cell degranulation.
Acknowledgements I would like to thank Professor Israel Pecht from the Department of Chemical Immunology, The Weizmann Institute of Science and Dr John C. Foreman, from the Department of Pharmacology, University College London, for many fruitful discussions.
References 1 Cox, l- S. C. (1967) Nature (London) 216, 1328-1329 2 Pearce, F. L. (1982) Klin. Wochenschr. 60, 954-957 3 Spataro, A.C. and Bosmann, H.B. (1976) Biochem. Pharmacol. 25, 505-510 4 Foreman, J.C., Garland, L.G. and Mongar, J. L. (1976) Symp. Soc. Exp. Biol. 30, 193-218 5 Foreman, J.C., HaUett, M.B. and Mongar, J.L. (1977) Br. J. Pharmacol. 473--474 6 Mazurek, N., Berger, G. and Pecht, I. (1980) Nature (London) 286, 722-723 7 Mazurek, N., Bashkin, P., Loyter, A. and Pecht, I. (1983) Proc. Natl Acad. Sci. USA 80, 6014-6018 8 Pearce, F.L. and Truneh, A. (1981) Agents and Actions, 11, 44-50 9 Theoharides, T.C., Sieghart, W., Greengard, P. a n d Douglas, W.W. (1980) Science 207, 80-82 10 Sagi-Eisenberg, R. and Pecht, I. (1984) EMBO J. 3, 497-500 11 Kikkawa, U., Takai, Y., Minakuchi, R., lnohara, S. and Nishizuka, Y. (1982) J. Biol. Chem., 257, 13341-13348 12 Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276 13 Michell, R.H. (1979) Trends Biochem. Sci. 4, 128-131 14 Kennerly, D.A., Sullivan, T.J., Sylvester, P. and Parker, C.W. (1979) J. Exp. Med. 150, 1034-1039 15 Goth, A., Adams, H.R. and Knoohuizen, M. (1971) Science 173, 1034-1035 16 Sagi-Eisenberg, R., Geller-Bemstein, C., Ben-Neriah, Z. and Pecht, I. (1983) FEBS Lett. 161, 37-40 17 Garland, L. G. and Mongar, J. L. (1974) Br. J. Pharmacol. 50, 137-143 18 Sagi-Eisenberg, R. and Pecht, I. (1984) Immunol. Lett. 8, 237-241 19 Sagi-Eisenberg, R., Lieman, H. and Pecht, I. (1985) Nature (London) 8, 59-60