Biochimica et Biophysica Acta, 1092 (1991) 85-88 © 1991 ElsevierScience Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100124L

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Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostefium discoideum Masakazu Oyama and Kou Kubota Laboratory of Biology, Himefi Institute of Technology, Himeji Hyogo (Japan)

(Received 25 June 1990) (Revised manuscriptreceived 10 October 1990)

Key words: Adenylatecyclaseactivation; Dithiothreitol, Polyamine; Calciumion; EDTA; cyclicAMP binding; (D. discmdeum)

Binding of cAMP to cell surface receptors evokes the transient activation of adenylate ¢.yclase in Dictyosteli~m discoideum. Dithiothreitol is also known as an activator of this enzyme. We found that the dithiothreitol-i~duced activation was specifically enhanced by extracellular polyamines or divalent cations. Furthermore, EDTA, a chelating agent of divalent cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase while EDTA did not inhibit the cAMP-induced activation. The inhibition was nullified by addition of polyamines or divalent cations. These results suggest that extracellular polyamines and divalent cations play a specific role in the dithiothreitol-induced activation of adenylate cyclase.

Introduction Two types of substances have been known to activate adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. One is cyclic AMP (cAMP), an intrinsic agonist [1], which binds to the cell surface receptors [2-4] and induces a transient activation of this enzyme [5,6]. The other is a reducing reagent such as dithiothreitol [7,8] or 2,3-dimercapto-l-propanol [9]. It has been reported that Ca 2+ is involved in intracellular signal transduction from the receptor [10-13] and that extracellular divalent cations modify the states of the receptors [14] and the cAMP-induced activation of adenylate cyclase [15] in this organism. In spite of the importance of Ca 2+ in signal transduction, there is uncertainty about the role of extracellular Ca 2+ in this organism. That is, cAMP induces a transient uptake of extracellular Ca 2+ [18] while various cellular events are induced normally by cAMP stimulation even under the conditions where extracellular Ca 2+ is chelated [16,17].

Abbreviation: PBK, 20 mM phosphate buffer (pH 6.4) containing 10 mM KCI. Correspondence: M. Oyama, Laboratoryof Biology, Himeji Institute of Technology, Shosha 2167, Himeji Hyogo, 671-22, Japan.

We investigated effect of extraceUular Ca 2+ on the activation of adenylate cyclase by the two types of stimulants, cAMP and dithiothreitol. We also investigated effect of polyamines since polyarmnes are a potential mediator of Ca 2+ mobilization and are known to modify a large variety of physiological functions [19]. We report here that the dithiothreitoMnduced activation requires extracellular divalent cations or polyamines, but that the cAMP-induced activation does not.

Materials and Methods Dictyostelium discoideum NC4 cells were grown in a shake-culture with Escherichia coil B/r. Shortly after exhaustion of food bacteria, cells were harvested, washed and placed on a nitrocellulose filter. Cells on the filter were incubated at 21 ° C. Cells at the mid aggregation stage were harvested, washed and resuspended in 20 mM phosphate buffer (pH 6.4) containing 10 mM KCI (PBK) at 1.10 7 cells/ml. For stimulation, 1 ml of the cell suspension was shaken at 120 rpm at 21°C in a 20 ml glass vial for at least 30 min and then 10 pl of the stimulant were added to the suspension. 100/LI of the suspension were sampled into a nucrotest tube containing 100 /~1 of 1070 trichloroacetic acid or 3.570 HCIO4. The samples were centrifugated and total (cellular + extracellular) cAMP

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in the supernatant was assayed with an Amersham cAMP assay kit. We purchased ornithine • HCI from Seikagaku Kogyo, putrescine. 2HC! from Nakarai Tesque, spermidine3HCI and spermine. 4HCI from Wako Pure Chemical Industries and dcAMP and dithiothreitol from Sigma.

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01 cAMP [5,6] and dithiothreitol [7,8] are known as an activator of adenylate cyclase in D. discoideum. We investigated the effects of polyamines (putrescine, spermidine and spermine which have di, tri and tetra positive charges, respectively) on activation of this enzyme. dcAMP, an analogue of cAMP, binds to the cAMP receptors and induces receptor-mediated events without interfering with the isotope dilution assay for cAMP [20]. Addition of spermine (1 mM) did not show any effect when adenylate cyclase was stimulated with dcAMP (Fig. 1, left). Total (cellular + extracellular) cAMP gradually increased and peaked at 2 min after the stimulation and then decreased due to degradation by cAMP.phosphodiesterase produced by Dictyostelium cells (Fig. 1). In contrast to dcAMP stimulation, addition of spermine enhanced the cAMP production by more than 50~ when adenylate cyclase was activated by dithiothreitol (Fig. 1, right). Total cAMP increased gradually after the stimulation by dithiothreitol and reached a plateau at 6 min after the stimulation (Fig. 1). Total cAMP did not decrease since dithiothreitol is an inhibitor of cAMP-phosphodiesterase [2]. Other polyamines

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Fig. 1. The effect of spermine on accumulation of total cAMP induced by dcAMP (left) or dithiothreitol (right). Aggregating cells were harvested and shaken in PBK. 10 /=M dcAMP (square) or 5 mM dithiothreitol (circles and triangles),with (closed symbols) or without (open symbols) 1 mM spermine was added to the medium at 0 time. Cell suspension was sampled at indicated times for cAMP assay. In the right graph, circles and triangles show the data from two separate experiments.

(putrescine and spermidine) also enhanced the cAMP production induced by dithiothreitol while they were a weaker enhancer than spermine (Table I). Stimulation with polyamines alone did not induce any activation (data not shown). To clarify the role of polyamines, we tested the effects of CaCI2 and MgSO4. We fouad that CaCl2 also enhanced the cAMP accumulation induced by dithiothreitol but not by dcAMP (Table I). MgSO4 showed weaker effect on the activation (Table I). Importance of extraceUular divalent cations in the activation by dithiothreitol was clearly demonstrated by the result that EDTA, a chelating agent of divalent

TABLE !

Effect of polyamines and divalent cations on accumulation of total cAMP induced by dithiothreitol or dcAMP Aggregating cells were harvested and shaken in PBK with or without EDTA (1 mM for Exp. 1 or 2 mM for Exps. 2, 3, 4) and then stimulated by 5 mM dithiothreitol (DTT) or 10 taM dcAMP with polyamines (1 raM), CaCI 2 (1 mM for EXp. 1, 5 mM for Exps. 2, 3 or 10 mM for Exp. 4) or MgSO4 (5 mM for Exp. 3 or 10 mM for Exp. 4) as indicated. Cell suspension was sampled at 2 rain (dcAMP) or l0 rain (dithiothreitol) after the stimulation for cAMP assay, n.t., not tested. Culture

Stimulation

medium PBK PBK PBK PBK PBK + EDTA

none dcAMP dcAMP + spermine dcAMP+ CaCI 2 dcAMP

PBK PBK PBK PBK PBK PBK PBK PBK+ EDTA

DTT D'IT + ornithine D T I ' + putrescine DTT + spermidine D ' I T + spermine D T r ÷CaC! 2 D T F + MgSO4 DTI'

Total cAMP (pmol/1 • 107 cells)

normalized

Exp. 1

Exp. 2

Exp. 3

4A 48 48 33 63

4.5 46 52 33 59 67 53 n.t. n.t. 107 95 n.t. 7.2

79 101 104 135 129 89 n.t. 20

Exp. 4

mean + S.D.

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14 n.t. n.t. n.t. n.t.

100 94 + 25 70+ 2 117 + 21

83 70 104 96 140 119 82 8.7

82 n.t. 91 102 103 84 97 16

100 97 + 27 123 + 11 137 + 30 155 + 19 127:1:18 109 16 + 7

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amine synthesis, showed no effect either on enhancement or on nullification of the inhibition by EDTA (Table I, Fig. 3).

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Fig. 2. Inhibition of activation of adenylate cyclase by EDTA. Aggregating cells were harvested and shaken in PBK with (closed symbols) or without (open symbols) 2 mM EDTA. Cells were stimulated by 5 mM dithiothreitol with 1 mM spermine (triangles) or 5 mM Cael 2 (squares) or without either of them (circles). Cell suspension was sampled at indicated times for cAMP assay.

cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase (Table I, Fig. 2). EGTA also inhibited the dithiothreitol-induced activation (data not shown). This inhibition was not due to the general toxicity of EDTA since EDTA did not inhibit but rather slightly enhanced the activation of adenylate cyclase induced by dcAMP (Table I). The inhibition by EDTA could be nullified by addition of CaCI 2, MgSO4 or polyamines (Figs. 2, 3). While CaCI 2 or polyamines consistently nullified the inhibition by EDTA as seen in Figs. 2 and 3, the effect of MgSO4 was partial in the separate experiment even at 10 mM (data not shown). Spermine was the most effective. Only 10/~M spermine resulted in a half-maximum recovery while 100 #M spermidine or 1 mM putrescine was required (Fig. 3). Ornithine, a precursor of poly-

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Fig. 3. Nullification of the EDTAinduced inhibition by various concentration of polyamines and CaCI2. Aggregating cells were harvested and shaken in PBK containing 2 mM EDTA. Cells were stimulated by 5 mM dithiothreitol with various concentrations of orn/thirie (cross), putrescine (open circle), spermidine (closed circle), spermine (open triangle), CaCI2 (closed triangle) or MgSO4 (open square). Cell suspension was sampled at 10 rain after the stimulation for cAMP assay.

This report shows that the dithiothreitol-induced activation of adenylate cyclase depends on extracellular divalent cations a n d / o r polyamines while the cAMP-induced activation does not. That is, (1) the activation by dithiothreitol but not by dcAMP is enhanced by CaCI 2 or polyamines (Fig. 1, Table I). (2) The dithiothreitolinduced activation is inhibited by EDTA while EDTA rather slightly enhances the production of cAMP induced by dcAMP (Table I, Fig. 2). (3) The inhibition by EDTA is nullified by addition of CaCl 2, MgSO 4 or polyamines (Figs. 2, 3). The reducing reagent is known to inhibit cAMPphosphodiesterase [2]. Thus, it has been generally believed that the dithiothreitol-induced activation of adcnylate cyclas¢ is due to interaction of the receptor and gradually accumulated cAMP [7,8]. The clear differences between the reducing reagent-induced and the dcAMP-induced activation of adenylate cyclase reported here have driven us to investigate differences between them. Oyama (unpublished data) and Oyama et al. [9] found that the reducing reagent can induce the activation even under the conditions where the receptors have been already occupied with supersaturated dcAMP or where the cAMP-induced activation is inhibited by a protein kinase inhibitor, K252a. We proposed that the reducing reagent has an independent effect from the inhibition of phosphodiesterase and the receptor-cAMP interaction in the activation of adenylate eyclase. It is possible that the specific requirement of extracellular polyeation for the dithiothreitol-ieduced activation refleets the independent action of the reducing reagent from the receptor-cAMP interaction. Abe et al. [10] have shown that cAMP stimP.lation induces an elevation of cytoplasmic free Ca 2+. We found that divalent cations (for example, 1 to 10 mM CaCI2) or polyamines alone can activate adenylate cyelase with the identical time-course to dcAMP stimulation in permeabilized cells with saponin, while the maximum amount of cAMP induced by divalent cations or polyamines is about 70% of that by dcAMP (unpublished data). Snaar-Jagalska et al. [21] reported that myo-inositol 1,4,5-triphosphate is required for the activation of adenylate eyclase in a mutant which is defective in the transmembrane signal transduction [22]. Myo-inositol 1,4,5-triphosphate is known to mobilize intracellular store of Ca 2+ [23]. These reports suggest that cytoplasmic Ca 2+ plays a role in the activation of adenylate cyclase. Thus, dithiothreitol may utilize extracellular divalent cations or polyamines to modify

88 cytoplasmic free C a 2+ while the receptor-cAMP interaction induces mobilization of intracellular Ca 2+. Brenner and Thoms [24] and Theibert and Devreotes [25] reported that caffeine which is known to mobilize intracellular Ca 2+ and A23187, a calcium ionophore, inhibit the cAMP-induced activation of adenylate cyclase. Bumann et al. [26] proposed that Ca 2+ is a mediator of adaptation which inhibits adenylate cyclase. De Young et al. [27] hypothesized that intracellular Ca 2+ works as an inhibitor of adenylate cyclase to construct a mathematical model for regulation of adenylate cyclase. While they proposed that Ca 2+ inhibits the activation of adenylate cyclase, these reports may only superficially conflict with our proposal since it becomes clear that Ca 2+ concentration is regulated differently among cellular compartments and that Ca '+ concentration of a specific compartment plays a specific role [28]. Therefore, roles of Ca 2+ in activation and inhibition of adenylate cyclase are not established until it is clarified from where, in which compartment and at what timing Ca 2+ mobilization is induced by caffeine, A23187, dithiothreitol or cAMP and how much Ca 2+ is mobilized by them. The effect of polyamines found in this report is interesting since Koenig et al. [29] reported that polyamines mediate hormone-induced Ca 2+ mobilization in mouse kidney, while it is not clear whether polyamines play an intrinsic role in the activation of adenylate cyclase in this organism.

Acknowledgments We thank Drs. Koji Okamoto, Shuji Ishida and Yoko Yamada (Kyoto University) for valuable discussions and readings.

References 1 Konijn, T,M,, Van De Meene, J.G.C., Bonnet, J.T. and Barkley, D,S. (1967) Proc. Natl. Acad. Sci. USA 58, 1152-1154. 2 Green, A.A. and Newell, P.C. (1975) Cell 6, 129-136.

3 Henderson, E.J. (1975) J. Biol. Chem. 250, 4730-4736. 4 Malchow, D. and Gerisch, G. (1973) Biochem. Biophys. Res. Commun. 55, 200-204. 5 Klein, C., Brachet, P. and Darmon, M. (1977) FEBS Lett. 76, 145-147. 6 Roos, W. and Gerisch, G. (1976) FEBS Left. 68, 170-172. 7 Devreotes, P.N., Derstine, P.L. and Steck, T.L. (1979) J. Cell Biol. 80, 291-299. 8 Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7705-7710. 9 0 y a m a , M., Kubota, K. and Okamoto, K. (1990) Biochem. Biophys. Res. Commun. 167, 767-771. 10 Abe, T., Maeda, Y. and lijima, T. (1988) Differentiation 39, 90-96. 11 Blumberg, D.D., Comer, J.F. and Walton, E.M. (1989) Differentiation 41, 14-21. 12 Bumann, J., Malchow, D. and Wurster, B. (1986) Differentiation 31, 85-91. 13 Newell, P.C., Europe-Finner, G.N. and Small, N.V. (1987) Microbiol. Sci. 4, 5-11. 14 Van Haastert, P.J.M. (1985) Biochim. Biophys. Acta 846, 324-333. 15 Gerisch, G., Malchow, D., Roos, W. and Wick, U. (1979) J. Exp. Biol. 81, 33-47. 16 Mato, J.M. and Konijn, T.M. (1977) in Development and Differentiation in the Cellular Slime Moulds (Cappuccinelli, P. and Ashworth, J.M., eds.), pp. 93-103, Elsevier, Amsterdam. 17 Saito, M. (1979) Exp. Cell Res. 123, 79-86. 18 Wick, U., Malchow, D. and Gerisch, G. (1978) Cell Biol. Int. Rep. 2, 71-79. 19 Tabor, C.W. and Tabor, H. (1984) Annu. Rev. Biochem. 53, 749-790. 20 Van Haastert, P.J.M. (1984) J. Gen. Microbiol. 130, 2559-2564. 21 Snaar-Jagalska, B.E., Kesbeke, F. and Van Haastert, P.J.M. (1988) in Molecular Biology of Dictyostelium Development (Kimmel, A.R., ed.), pp. 215-226, Alan R. Liss, New York. 22 Coukell, M.B., Lappano, S. and Cameron, A.M. (1983) Dev. Genet. 3, 283-297. 23 Europe-Finner, G.N. and Newell, P.C. (1986) Biochim. Biophys. Acta 887, 335-340. 24 Brenner, M. and Thoms, S.D. (1984) Dev. Biol. 101,136-146. 25 Theibert, A. and Devreotes, P.N. (1983) J. Cell Biol. 97, 173-177. 26 Bumann, J., Wurster, B. and Malt;how, D. (1984) J. Cell Biol. 98, 173-178. 27 De Young, G., Monk, P.B. and Othmrr, H.G. 0988) J. Math. Biol. 26, 487-517. 28 Cheek, T.R. (1989) J. Cell Sci. 93, 211-21o. 29 Koenig, H., Goldstone, A. and Lu, C.Y. (1983) Nature 305, 530-534.

Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of cAMP to cell surface receptors evokes the transient activation of of adenylate cyclase in Dictyostelium discoideum. Dithiothreitol is also ...
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