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The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation
Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/130047-15$02.00/O
Experimental
THE
EFFECTS
Cell Research
OF INHIBITORS
PHOSPHODIESTERASE
OF ADENYLATE
ON D. DZSCOZDEUM CLAUDETTE
Edward
124 (1979) 205-213
CYCLASE
AND
AGGREGATION
KLEIN
A. Doisy Department of Biochemistry, Saint Louis School of Medicine, St Louis, MO 63104, USA
University,
SUMMARY The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ225364reated cells exhibited normal accumulation of their adenvlate cyclase activity as measured-in cell lysates but the amebae did not synthesize CAMP. The ability of this drug to compete for CAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of CAMP, suggest that SQ22536 functions as a CAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or CAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external CAMP.
Aggregation
of slime mold amebae, D. discoideum, is initiated when cells are nutritionally deprived. The chemotactic agent produced under such conditions, CAMP, serves not only to orient cell migration to central collection points, but also regulates the expression of various differentiated functions which characterize aggregationcompetent amebae [ 1, 21. The molecular sequence of events which leads to the production of the chemotactic signal are not known but apparently involve increases in adenylate cyclase activity [3] CAMP cell surface receptors, which are believed to mediate the cells responses to external CAMP [4], and phosphodiesterase activity [S, 61. Using a phosphodiesterase-minus mutant, Darmon et al. [7] have recently shown that cell aggregation requires the presence of only the extracellular form of this enzyme, probably to prevent the exter-
nal levels of CAMP from accumulating above that of the chemotactic signal. To investigate the events basic to the aggregation program, we have previously examined cell populations blocked at different stages in development, using in these studies various classes of aggregation-minus mutants [8], or cells inhibited from development by the addition of various drugs [9, lo] or nutrients [ 111. This approach has provided a great deal of information concerning both the events which lead to a functional adenylate cyclase, and the relationship between CAMP synthesis and the expression of CAMP-binding sites and phosphodiesterase activity. We have recently obtained two compounds, SQ 22536 and SQ20009, which, in higher eukaryotes, are inhibitors of adenylate cyclase and CAMP phosphodiesterase, respectively [12, 13, 141. In light of the critical roles of adenylate cyclase and phos-
206
C. Klein
phodiesterase in slime mold development, the effects of these two compounds on cell aggregation were of interest. The experiments reported here show that both drugs inhibit cell differentiation and aggregation. Biochemical and physiological analyses of cells treated with these compounds indicate that SQ22536 does inhibit adenylate cyclase activity and may do so because of its ability to function as a CAMP analogue. The effects of SQ20009, however, do not seem to be mediated by an inhibition of phosphodiesterase. Rather, cells are blocked at a very early stage in development, even prior to the increased production of this enzyme. The possible site(s) of action of each of these drugs is discussed. MATERIALS
AND
METHODS
Materials SQ20009 [I-Ethyl-4-(isoprophylidenehydrazino)-lHovrazolo-3.4-n) ovridine-5-carboxylic acid, ethyl ester], and SQ22.536[9-(tetrahydro-2ifuryl)adenine] were the generous gift of Dr Harris (Squibb Institute for Medical Research), [@P]ATP (12 Cilmmol) and [83H]cAMP (52 Ci/mmol) were purchased from Amersham. All other fine chemicals were obtained from Calbiochem. Silicone oil was the gift of Mr Connell (Walker Chemicals).
Cell culture and starvation conditions Ax-2 amoebae [15] were maintained as exponentially growing cultures in HL-5 medium [ 151. Starvation was initiated by washing cells twice with 17 mM phosphate buffer pH 6.4 and resuspending them at a density of 10’ cells/ml. Cells were starved as spinner suspensions in the presence of various concentrations of each inhibitor and/or phosphodiesterase as indicated in the experiment. Extracellular phosphodiesterase was prepared from Ax-2 cells starved for 6 h or A53 cells which had been starved for 2 h and then pulsed with CAMP for 6 h [l]. The media were concentrated lofold by Amicon filtration using a PM50 membrane. Enzyme preparations were filter sterilized and kept at 4°C. A detailed description of the procedure and the A53 strain are available [1, 71. Cell differentiation to aggregation competence was monitored by plating aliquots of cell suspensions on tissue culture plates. Amebae which appeared elongated and formed polar contacts were considered aggregation competent. When indicated, cells were pulsed with CAMP as described elsewhere [ 11. The concentration of the CAMP pulses refers to the final concentration obtained after the first drop of CAMP is released into the medium. Exp Cell Res 124 ( 1979)
All exueriments, except those involving- applied CAMP _. pulses, were also pe;formed using cells starved on tissue culture dishes as previously described [9]. The results were identical to those obtained with spinner suspensions. Mutant HPX235 was maintained on SM plates in association with Enterobacrer nerogenese [7]. Starvation was induced by washing cells free of bacteria and plating 3x 10’ cells on 100x20 mm tissue culture dishes in the presence or absence of 10m4M CAMP.
Assays At the indicated times, cells were washed twice with phosphate buffer and then either frozen as pellets or resuspended in phosphate buffer containing 10 mM dithiothreitol and immediately assayed fo; CAMPbinding sites. For this, cells were incubated at 4°C with 5% lOm8M [3H]cAMP for 30 set, pelleted through a layer of silicone oil, and the cell-associated activity determined. Details of the procedure are available L8J. For phosphodiesterase activity, frozen cell pellets were-thawed in 50 mM Tris buffer pH 8, 4 mMMgC1, and assayed as previously described [ 14. A unit of enzyme activity is defined-as the hydrolysis of 1 nmole of cAMP/min at 30°C When extracellular phosphodiesterase was measured, the medium as first dialysed against 50 mM Tris pH 8, 4 mM MgCl* and then assayed for enzymic activity. When cells were monitored for adenylate cyclase activity, they were washed once with 20 mM MES (2-(N-morpholino)ethanesulfonate) buffer pH 6.4 and immediately frozen in dry ice-ethanol. Enzyme activity was determined by following the conversion of [@P]ATP to [3ZP]cAMP by the method of Solomon rl71 usinn assav conditions described nreviously [3]. one unit of activity refers to the forma&on of 1 pmole cAMP/min at 27°C. CAMP determinations were-performed using the CAMP kit purchased from Amersham. Cells starved for 5 or 6 h in the presence or absence of added compounds were centrifuged and resuspended at a density of 108cells/ml. The cells were main&ned in suspensibn by gently bubbling oxygen through the medium. Every minute for 12 min, duplicate 1 ml aliquots were centrifuged in a Beckman minifuge for 10 set throuah a laver of silicone oil resting above a 50 ~1 drop of-5 % T-CA, 5 % glucose, containing 1000 cnm of f3HlcAMP. Cell pellets were immediately frozen in dry-ice and the supematant and silicone oil layers removed. The tubes were then thawed, and centrifuged to eliminate precipitated cell constituents. The supematants from the first 6 min points were combined, as were those of the last 6 points. Each were extracted with H,O-saturated ether and assayed for CAMP levels. Recovery was determined by following the dilution of [3H]cAMP during this orocedure. Chemotactic assays were performed as described by Koniin rl81 bv placing a 1 ul drop of cells which had been-&&d-for 4 h-in spinner-suspension on agar plates containing various amounts of the products to be tested. A positive response constitutes a stimulation of cell migration outward from the droplet in which they had been deposited. Alternatively, 1 ~1 droplets containing various amounts of each compound was placed in the center of an agar plate and 1 ~1 droplets of cells were placed 1, 2, 3 or 4 mm from the center
Inhibitors
Fig. 1. Abscissa: time (hours); ordinate: U/10’ cells. Effects of SQ2000!4 and SQ22536 on the developmental changes in adenylate cyclase and phosphodiesterase. Ax-2 amebae were starved in spinner suspension in the presence of 0, lo+ M SQZooaS; 0, 1O-4 M SQ22536, or +, in the absence of any added compounds. At the indicated times, cells were assayed for phosphodiesterase (-) and adenylate cyclase (---) activities. The results are representative of 3 expts. at 90” angles from each other. In this assay, no movement, movement toward, or away from the central drop could be observed. Control experiments using various concentrations of CAMP showed strong positive responses between lo-” to lo+ M. No response was seen in the absence of any added compounds. SQ22536 and SQ20009 were tested at concentrations ranging from lOmEto 10m2M.
RESULTS Effects of SQ20009 and SQ22536 on cell aggregation Amebae starved either in spinner suspensions or on tissue culture plates in the presence of 10s4 M SQ20009 remained as individual cells and displayed none of the morphological characteristics of aggregation-competent amebae. Correspondingly, they did not show any increase in adenylate cyclase or phosphodiesterase activities (fig. 1). A similar inhibition of the developmental changes in CAMP-binding sites and extracellular phosphodiesterase was also observed (data not shown). Starvation of ame13%791814
of slime mold aggregation
207
bae in buffer containing lop4 M SQ22536 produced a slightly different result. Although cells showed no morphological signs of aggregation-competence and did not increase their levels of cellular or extracellular phosphodiesterase, or surface CAMPbinding sites, they did show increased levels of adenylate cyclase activity as measured in cell extracts. The time course and extent of this rise was similar to that seen in untreated control cells. It would appear that cells incubated with SQ22536 can respond to starvation, at least by increasing their levels of adenylate cyclase while SQ20009 cells are blocked at a step preceding this rise. In both cases, the inhibition of aggregation by these compounds was reversible. Cells incubated with either product for g-10 h, washed and resuspended in buffer void of any added compound, aggregated within the next 10-12 h. Total inhibition of cell aggregation was not observed with lofold lower concentrations of each compound. The ability of applied CAMP pulses to overcome the developmental blocks imposed by these two drugs was examined. Amebae starved in the presence of lop4 M SQ22536 were induced to aggregate when pulsed with CAMP (lo-’ M), indicating that they were able to perceive and respond normally to the chemotactic signal. Cells treated with SQ20009, however, were not induced to aggregate or express any of the characteristics of aggregation-competent cells when starved in the presence of applied CAMP pulses. SQ20009 does not appear to inhibit differentiation via its effect on phosphodiesterase SQ20009 is a known inhibitor of CAMP phosphodiesterase and in the case of slime mold, blocks this enzymic activity by apExp Cd
Ret
124 (1979)
208
C. Klein
Table
1. The ability
of inhibitor-treated cells to synthesize or respond to LAMP
Treatment Control
Length of treatment (hours)
pd” activity U/10’ cells
CAMP levels (pmol/ IO7 cells)
2
0.2 4.3
3.1
5 1O-3 M CAMP 1O-4 M SQ20009
lo-“M SQ20009 + 1O-3 M CAMP 1O-4 M SQ22536 Fig. 2. Abscissa: time (hours); ordinate:pmol CAMP formed/min. Effect of CAMP on the developmental rise in adenylate cyclase. HPX235 amebae were starved on tissue culture plates in O-O, the presence or +-+, absence of 10m3M CAMP. At the indicated times, cells were harvested and assayed for anylate cyclase activity. The levels depicted represent the enzymic activity of 2~ 10’ cells. The data are representative of 3 expts.
prox. 40% at 1O-4 M (Harris, personal communication). Since aggregation requires only an extracellular phosphodiesterase activity [7], and SQ20009-treated cells produced little or no such enzyme, this compound may act by decreasing the existing external phosphodiesterase activity below that necessary for development to proceed. This was examined by starving cells in the presence of lop4 M SQ20009 and adding various amounts of extracellular phosphodiesterase prepared from starved Ax-2 or A53 amebae. Even when the starvation medium contained 50-fold higher levels of phosphodiesterase than that normally produced by aggregating cells, SQ20009treated amoebae were still unable to undertake their developmental cycle. The same result was obtained with SQ22536-treated cells starved in the presence of added phosphodiesterase suggesting that in this case Exp Cell
Res 124 (1979)
2
5.0
5
ND
2 5
-c
2 5 2
ND
2 5
ND ND ND 0.4
ND ND ND 0.7
5
1O-J M SQ22536 + 1O-3 M CAMP
ND”
3.0
ND
ND ND
a pd, Cellular phosphodiesterase activity. b ND, Not determined. c -, Below the limit of detection.
their inability to aggregate is also not a direct consequence of their inability to synthesize the enzyme. It is also possible that SQ20009 acts like a CAMP analogue which is not hydrolyzable by phosphodiesterase. Under such conditions, the inhibition of cell aggregation would result from the continued presence of a high external concentration of “CAMP”. This possibility was examined by starving mutant HPX235 in the presence of lop4 M CAMP and comparing the developmental change in adenylate cyclase activity in these cells to that of untreated controls. Because this mutant is defective in phosphodiesterase production [7], the levels of CAMP added to the medium remain relatively constant. Unlike SQ20009, the presence of 10e4 M CAMP did not alter either the time, or the extent, of the rise in CAMP production (fig. 2). Similar results were obtained with wild-type cells, although in this case hydrolysis of the added CAMP by
Inhibitors
Fig. 3. Abscissa: molarity (lo-“); ordinate: % binding. Effects of SQ2000!9 and SQ22536 on CAMP bindina. Ax-2 cells were starved for 51 h. at which time th;y showed a significant rise in the levels of surface CAMP-binding sites. The levels of binding activity were measured by incubating cells for 30 set in the presence of 5x lO-s M [3H]~AMP (100% binding) or in the presence of 5x 1O-8 M [3H]~AMP and various concentrations of +-+, SQ2000!3 or O-O, SQ22536. The degrees to which 10e4 M and 10e3 M CAMP can prevent 3H-binding to cells were also determined (O-O). The data is representative of 3 expts.
endogenous phosphodiesterase continuously alters the concentration of the cyclic nucleotide in the medium. Other results also suggest that SQ20009 does not function as an analogue of CAMP in inhibiting cell aggregation. As seen previously, treated cells showed no increase in phosphodiesterase activity. In contrast, amebae incubated with 10e4 M CAMP exhibit a precocious induction of this enzyme [ 161. In fact, we observed that cells treated with lop4 M SQ20009 could no longer respond to added concentrations of CAMP as high as 10e3 M by increasing their levels of phosphodiesterase (table 1). SQ20009 does not affect CAMP binding
CAMP-binding proteins on the cell surface are believed to mediate the cells’ responses to external CAMP. These responses involve both chemotaxis and the differentiation of the population to aggregation competence.
of slime mold aggregation
209
Fig. 4. Abscissa: time (min); ordinate: % binding. Ability of SQ20009 and SQ22536 to compete for pre-bound [3H]~AMP. 54 h starved Ax-2 cells were incubated for 30 set with 5X lo-* M [3H]cAMP after which time O-O, SQ22536; O-O, SQ20009; or X-X, CAMP at lo+ M was added. Control cells (+-+) received no additions. Levels of CAMP binding are expressed as a percentage of that seen on control cells. The results are representative of 2 expts.
The above experiments indicate that SQ20009-treated cells do not show normal responses to CAMP, either at high concentrations or when added in the form of imposed pulses of lo-’ or lo-* M. To examine the relationship between the effects of SQ20009 on aggregation and the CAMP surface receptor, we assessed the ability of SQ20009 to inhibit [3H]cAMP binding to the cell surface (fig. 3). At the concentration which inhibited development, SQ20009 did not interfere with CAMP binding to cells. At 3.0~ 10s3 M, SQ20009, only a slight competition for CAMP binding was observed. Similar results were obtained when lops M [3H]cAMP was used to measure binding activity predominantly due to CAMP receptor sites of high affinity [19]. SQ20009 was also found to be a poor competitor of [3H]cAMP which had been bound to cells prior to the addition of the drug (fig. 4). Pre-incubation of amebae for 30 set with SQ20009 did not alter the time course or degree of binding of [3H]~AMP added subsequently. Exp Cd
Res 124 (19791
210
C. Klein
SQ22536 competes for CAMP-binding sites In contrast to SQ20009, approx. 50% less [3H]cAMP was bound by cells incubated in the presence of 1O-4 M SQ22536 (fig. 3). The same loss of CAMP binding could be seen with cells which had first bound [3H]CAMP and were then incubated with lop4 M SQ22536 (fig. 4). In this case, loss of [3H]CAMP binding occurred within 30 set after the addition of SQ22536. A similar rapid loss of cell-bound radioactivity was seen when lop4 M CAMP was added to cells which had been preincubated with [3H]CAMP. However, this treatment resulted in an 85% decrease in bound radioactivity. The ability of SQ22536 to compete for CAMP binding to cells was also examined by incubating cells with this compound for 30 set and then adding [3H]~AMP to measure the level of CAMP-binding activity. Under these conditions, amebae also showed only 50% of the binding of untreated cells. This level of binding was observed within 30 set after [3H]~AMP addition and did not change during the 2-3 min of the experiment. In light of the observation that the cellsurface CAMP receptor may recognize SQ22536 as a CAMP analogue, we tested the possibility that this compound could function, to some degree, like CAMP in affecting cell differentiation to aggregation competence or chemotaxis. Since cells normally produce CAMP in the form of pulses, we first attempted to stimulate cell differentiation by starving amebae in the presence of applied pulses of SQ22536. The concentration of the pulses ranged from 1OF to lo-” M, similar to that of applied CAMP pulses which have been shown to stimulate cell differentiation [ 1, 23. SQ22536 pulses did not affect cell differentiation as judged both by morphological criteria and the time course of the rise in phosphodiExp Celi
Res 124 ((979)
esterase activity. The latter is a biochemical marker of aggregation competence [20]. When tested for its ability to induce a chemotactic response in starved cells, SQ22536 did not cause amebae to migrate outward from the droplet in which they had been deposited. The same result was obtained using SQ20009. It would appear that, although SQ22536 can bind to the CAMP receptor, it is unable to initiate events leading to cell differentiation or chemotaxis. SQ22536-treated amebae do not synthesize CAMP Competition by SQ22536 for CAMP surfacebinding sites, apparently, does not render cells unresponsive to CAMP. As mentioned above, amebae stimulated with an exogenous source of CAMP pulses overcame the inhibition of aggregation effected by the drug. The cells’ ability to respond to CAMP was also evidenced in experiments analyzing the induction of phosphodiesterase by 10e3 M CAMP in amebae treated with SQ22536 (table 1). The time-course of phosphodiesterase induction was similar to that seen in cells treated with lop3 M CAMP alone but the degree of enzyme induction was approx. 30-40% less. We should perhaps mention that it is unknown if the induction of the enzyme by high external concentrations of CAMP is mediated by the cell surface receptor and we do not wish to correlate the two. This experiment is provided only as further evidence that the SQ22536-treated amebae can respond to extracellular CAMP similarly to amebae which have not been treated with the drug. Given that drug-treated cells remain responsive to added CAMP, we examined if their failure to aggregate was related to their inability to synthesize CAMP. Drug-treated and control cells were harvested and analyzed for CAMP levels. Amoebae treated
Inhibitors
of slime mold aggregation
211
with SQ22536 showed extremely low levels of CAMP as compared to untreated, aggregation competent cells (table 1). The levels observed in SQ22536-treated cells were similar to those measured in amebae starved in the presence of SQ20009 and which do not show any developmental regulation of their adenylate cyclase activity. ’ Although SQ22536 almost totally blocked adenylate cyclase activity in intact amebae, the enzyme in broken cell preparations was relatively insensitive to the compound. A final concentration of lop3 M SQ22536 inhibited adenylate cyclase in cell-free lysates by approx. 50% while lo-* M SQ22536, which was sufficient to block development, could inhibit only approx. 20% of the activity (data not shown).
did not occur in drug-treated cells. Using a phosphodiesterase-minus mutant, HPX235, we observed that the constant presence of high external CAMP levels during cell starvation does not alter the developmental changes in adenylate cyclase. This again would suggest that SQ20009 does not function either by inhibiting phosphodiesterase activity (and allowing exter,tal CAMP levels to accumulate) or otherwise as a CAMP analogue. Amebae starved in the presence of SQ20009 remained relatively insensitive to CAMP: their differentiation to aggregation competence could not be elicited by applied CAMP pulses, and no induction of phosphodiesterase was observed when lop3 M CAMP was added to cells starved with this compound. The inability of these cells to respond to external CAMP does not appear to be related to an action of this comDISCUSSION pound on the plasma membrane CAMP reThe experiments reported here describe the ceptor since no competition by this product inhibition of Dictyostelium discoideum ag- for CAMP bound to the cell surface could gregation by two adenine analogues, be demonstrated. If SQ20009 does function SQ20009 and SQ22536. These compounds extracellularly to inhibit development, then are reported inhibitors of CAMP phosphowe would conjecture that it functions at a diesterase and adenylate cyclase, respec- site other than the CAMP receptor or the tively, in a variety of systems [12, 13, 141. CAMP phosphodiesterase. To date, it is unBoth were shown here to inhibit aggregation known if all the effects of CAMP on cell and differentiation of D. discoideum ame- differentiation and aggregation are medibae. Although SQ20009 can inhibit the ated by the surface receptors measured in phosphodiesterase activity in enzyme prep- the CAMP-binding assays. For example, exarations from slime mold (Harris, personal ternal CAMP may cause changes in ion percommunication), several experiments sug- meability of the plasma membrane which gest that this is not the primary site of ac- initiate a sequence(s) of events leading to tion of the drug. The addition of excessive differential gene expression. Alternatively, amounts of phosphodiesterase to drug- some CAMP may have to enter the cell in treated cells could not overcome the inhibiorder to influence differentiation and it is tion. Also, amebae are blocked in developthis intracellular site(s) which is blocked by SQ20009. If SQ20009 must be internalized ment at a stage prior to the time that their phosphodiesterase activity increases. Even to inhibit development, the high concentrathe rise in adenylate cyclase activity, one of tions of the drug required to do so may to the earliest known events involved in the reflect the cells relative impermeability production of the chemotactic signal [21], this compound. 14-791814
Exp Cell
Res 124 (1979)
212
C. Klein
The inhibition of cell differentiation by SQ22536 occurs at a step subsequent to the developmental rise in adenylate cyclase and prior to the increase in phosphodiesterase and CAMP surface-binding sites. However, the cyclase activity measured in cell extracts is not functional in intact amebae as evidenced by the very low levels of CAMP found in drug-treated cells. This finding is consistent with the known role of SQ22536 as an inhibitor of adenylate cyclase activity. The developmental block produced by this compound resembles that found in glucosetreated amebae [l l] and in several of our developmental mutants [8]. In each case, increasing adenylate cyclase activity can be measured in cell extracts even though little or no CAMP is actually produced by the cell. Correspondingly, no increases in phosphodiesterase or CAMP-binding sites occur. As in these cases, the adenylate cyclase of SQ22536-treated cells can also be activated by applied CAMP pulses. Such treatment results in cell differentiation to aggregationcompetence and if the pulses are arrested after an initial time period, amebae are able to continue their aggregation program. The mechanism by which SQ22536 inhibits development of amebae may be related to its ability to bind to the plasma membrane CAMP receptor. It was shown that this compound can prevent CAMP binding to this site by approx. 50%. It is possible that the compound binds to the CAMP receptor but is unable to initiate the subsequent events which lead to an activation of adenylate cyclase. The observations that pulses of low levels of SQ22536 could not affect cell differentiation to aggregation competence and that the drug itself is not a chemoattractant would be consistent with such a hypothesis. Since adenylate cyclase remains inactive, little or no CAMP is synthesized and therefore available to Exp Cell
RPS 124 (1979)
compete for the sites occupied by the drug. If cells are stimulated by an exogenous source of CAMP, as in the case of applied CAMP pulses, the external CAMP can compete for binding sites occupied by SQ22536 and activate the adenylate cyclase. Once this occurs, and enzyme activity is sufficient to produce enough CAMP to continue to compete for the existing drug, then aggregation can proceed in the absence of exogeneously generated CAMP pulses. Alternatively, the ability of SQ22536 to inhibit CAMP binding to its cell surface may only be an indication that the compound bears some structural relationship to CAMP rather than the mechanism by which it affects development. For example, SQ22536 may enter the cell and inhibit adenylate cyclase activity by a direct interaction with the enzyme. Experiments designed to assess the ability of the drug to inhibit adenylate cyclase activity in cell lysates have shown only slight decreases in activity. These results are difficult to interpret, however, since lop3 M CAMP is also present in the assay mixture to inhibit phosphodiesterase activity [3]. As this CAMP may compete for any sites which bind the drug, the relative insensitivity of adenylate cyclase activity in cell extracts to SQ22536 may not be a true indication that the drug does not function intracellularly. Continued research into the mechanisms by which SQ22536 and SQ20009 inhibit cell aggregation should provide important insight into the regulatory events vital to this process. The author wishes to thank dr Harris for initially SURgesting the use of these compounds and for r&i& available his preliminary data and MS Cynthia Nettrour for her excellent iechnical assistance. This research was supported by a Grant GM 25080-01 from the NIH.
REFERENCES 1. Darmon, M, Brachet, P & da Silva, P, Proc natl acad sci US 72 (1975) 3136.
Inhibitors of slime mold aggregation 2. Gerisch, G, Fromm, H, Heresgen, A & Wick, U, Nature 255 (1975) 547. 3. Klein, C, FEBS lett 68 (1976) 125. 4. Malchow, D & Gerisch, G, Proc natl acad sci US 71 (1974) 2423. 5. Chang, Y Y, Science 160 (1%8) 57. 6. Pannbacker. K G & Bravard. L G. Science 175 (1972) 1014.’ 7. Darmon. M. Barra. J L Brachet. P. J cell sci 31 (1978)233. 8. Juliani, M H & Klein, C, Dev biol62 (1978) 162. 9. Brachet, P & Klein, C, Exp cell res 93 (1975) 159. 10. Klein, C & Brachet, P, Nature 254 (1975) 432. 11. Dan-non, M & Klein, C, Dev bio163 (1978) 377. 12. Fain, J E, Pointer, R H &Ward, W F, J biol them 247 (1972) 6866. 13. Haslam, R J, Davidson, M M, Davies, T, Lynham, J A & McClenaghan, M D, Adv cyclic nucleotide res 9 (1978) 533.
213
14. Salzman, E W, Biochim biophys acta 499 (1977) 48. 15. Watts, D J & Ashworth, J M, Biochem j 119 (1970) 171. 16. Klein, C, J biol them 250 (1975) 7134. 17. Solomon, Y, Landos, C & Rodbell, M, Anal biochem 58 (1974) 541. 18. Konijn, T, Advances in cyclic nucleotide research (ed P Greengard & G A Robinson) vol. 1. Raven Press, New York (1974). 19. Green, A & Newell, P C, Cell 6 (1975) 129. 20. Klein, C & Darmon, M, Biochem biophys res commun 67 (1975) 440. 21. Klein, C, FEBS lett 1(1977) 17.
Received April 18, 1979 Accepted May 18, 1979
Exp Cell Res 124 (1979)
Experimental
THE
EFFECTS
Cell Research
OF INHIBITORS
PHOSPHODIESTERASE
OF ADENYLATE
ON D. DZSCOZDEUM CLAUDETTE
Edward
124 (1979) 205-213
CYCLASE
AND
AGGREGATION
KLEIN
A. Doisy Department of Biochemistry, Saint Louis School of Medicine, St Louis, MO 63104, USA
University,
SUMMARY The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ225364reated cells exhibited normal accumulation of their adenvlate cyclase activity as measured-in cell lysates but the amebae did not synthesize CAMP. The ability of this drug to compete for CAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of CAMP, suggest that SQ22536 functions as a CAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or CAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external CAMP.
Aggregation
of slime mold amebae, D. discoideum, is initiated when cells are nutritionally deprived. The chemotactic agent produced under such conditions, CAMP, serves not only to orient cell migration to central collection points, but also regulates the expression of various differentiated functions which characterize aggregationcompetent amebae [ 1, 21. The molecular sequence of events which leads to the production of the chemotactic signal are not known but apparently involve increases in adenylate cyclase activity [3] CAMP cell surface receptors, which are believed to mediate the cells responses to external CAMP [4], and phosphodiesterase activity [S, 61. Using a phosphodiesterase-minus mutant, Darmon et al. [7] have recently shown that cell aggregation requires the presence of only the extracellular form of this enzyme, probably to prevent the exter-
nal levels of CAMP from accumulating above that of the chemotactic signal. To investigate the events basic to the aggregation program, we have previously examined cell populations blocked at different stages in development, using in these studies various classes of aggregation-minus mutants [8], or cells inhibited from development by the addition of various drugs [9, lo] or nutrients [ 111. This approach has provided a great deal of information concerning both the events which lead to a functional adenylate cyclase, and the relationship between CAMP synthesis and the expression of CAMP-binding sites and phosphodiesterase activity. We have recently obtained two compounds, SQ 22536 and SQ20009, which, in higher eukaryotes, are inhibitors of adenylate cyclase and CAMP phosphodiesterase, respectively [12, 13, 141. In light of the critical roles of adenylate cyclase and phos-
206
C. Klein
phodiesterase in slime mold development, the effects of these two compounds on cell aggregation were of interest. The experiments reported here show that both drugs inhibit cell differentiation and aggregation. Biochemical and physiological analyses of cells treated with these compounds indicate that SQ22536 does inhibit adenylate cyclase activity and may do so because of its ability to function as a CAMP analogue. The effects of SQ20009, however, do not seem to be mediated by an inhibition of phosphodiesterase. Rather, cells are blocked at a very early stage in development, even prior to the increased production of this enzyme. The possible site(s) of action of each of these drugs is discussed. MATERIALS
AND
METHODS
Materials SQ20009 [I-Ethyl-4-(isoprophylidenehydrazino)-lHovrazolo-3.4-n) ovridine-5-carboxylic acid, ethyl ester], and SQ22.536[9-(tetrahydro-2ifuryl)adenine] were the generous gift of Dr Harris (Squibb Institute for Medical Research), [@P]ATP (12 Cilmmol) and [83H]cAMP (52 Ci/mmol) were purchased from Amersham. All other fine chemicals were obtained from Calbiochem. Silicone oil was the gift of Mr Connell (Walker Chemicals).
Cell culture and starvation conditions Ax-2 amoebae [15] were maintained as exponentially growing cultures in HL-5 medium [ 151. Starvation was initiated by washing cells twice with 17 mM phosphate buffer pH 6.4 and resuspending them at a density of 10’ cells/ml. Cells were starved as spinner suspensions in the presence of various concentrations of each inhibitor and/or phosphodiesterase as indicated in the experiment. Extracellular phosphodiesterase was prepared from Ax-2 cells starved for 6 h or A53 cells which had been starved for 2 h and then pulsed with CAMP for 6 h [l]. The media were concentrated lofold by Amicon filtration using a PM50 membrane. Enzyme preparations were filter sterilized and kept at 4°C. A detailed description of the procedure and the A53 strain are available [1, 71. Cell differentiation to aggregation competence was monitored by plating aliquots of cell suspensions on tissue culture plates. Amebae which appeared elongated and formed polar contacts were considered aggregation competent. When indicated, cells were pulsed with CAMP as described elsewhere [ 11. The concentration of the CAMP pulses refers to the final concentration obtained after the first drop of CAMP is released into the medium. Exp Cell Res 124 ( 1979)
All exueriments, except those involving- applied CAMP _. pulses, were also pe;formed using cells starved on tissue culture dishes as previously described [9]. The results were identical to those obtained with spinner suspensions. Mutant HPX235 was maintained on SM plates in association with Enterobacrer nerogenese [7]. Starvation was induced by washing cells free of bacteria and plating 3x 10’ cells on 100x20 mm tissue culture dishes in the presence or absence of 10m4M CAMP.
Assays At the indicated times, cells were washed twice with phosphate buffer and then either frozen as pellets or resuspended in phosphate buffer containing 10 mM dithiothreitol and immediately assayed fo; CAMPbinding sites. For this, cells were incubated at 4°C with 5% lOm8M [3H]cAMP for 30 set, pelleted through a layer of silicone oil, and the cell-associated activity determined. Details of the procedure are available L8J. For phosphodiesterase activity, frozen cell pellets were-thawed in 50 mM Tris buffer pH 8, 4 mMMgC1, and assayed as previously described [ 14. A unit of enzyme activity is defined-as the hydrolysis of 1 nmole of cAMP/min at 30°C When extracellular phosphodiesterase was measured, the medium as first dialysed against 50 mM Tris pH 8, 4 mM MgCl* and then assayed for enzymic activity. When cells were monitored for adenylate cyclase activity, they were washed once with 20 mM MES (2-(N-morpholino)ethanesulfonate) buffer pH 6.4 and immediately frozen in dry ice-ethanol. Enzyme activity was determined by following the conversion of [@P]ATP to [3ZP]cAMP by the method of Solomon rl71 usinn assav conditions described nreviously [3]. one unit of activity refers to the forma&on of 1 pmole cAMP/min at 27°C. CAMP determinations were-performed using the CAMP kit purchased from Amersham. Cells starved for 5 or 6 h in the presence or absence of added compounds were centrifuged and resuspended at a density of 108cells/ml. The cells were main&ned in suspensibn by gently bubbling oxygen through the medium. Every minute for 12 min, duplicate 1 ml aliquots were centrifuged in a Beckman minifuge for 10 set throuah a laver of silicone oil resting above a 50 ~1 drop of-5 % T-CA, 5 % glucose, containing 1000 cnm of f3HlcAMP. Cell pellets were immediately frozen in dry-ice and the supematant and silicone oil layers removed. The tubes were then thawed, and centrifuged to eliminate precipitated cell constituents. The supematants from the first 6 min points were combined, as were those of the last 6 points. Each were extracted with H,O-saturated ether and assayed for CAMP levels. Recovery was determined by following the dilution of [3H]cAMP during this orocedure. Chemotactic assays were performed as described by Koniin rl81 bv placing a 1 ul drop of cells which had been-&&d-for 4 h-in spinner-suspension on agar plates containing various amounts of the products to be tested. A positive response constitutes a stimulation of cell migration outward from the droplet in which they had been deposited. Alternatively, 1 ~1 droplets containing various amounts of each compound was placed in the center of an agar plate and 1 ~1 droplets of cells were placed 1, 2, 3 or 4 mm from the center
Inhibitors
Fig. 1. Abscissa: time (hours); ordinate: U/10’ cells. Effects of SQ2000!4 and SQ22536 on the developmental changes in adenylate cyclase and phosphodiesterase. Ax-2 amebae were starved in spinner suspension in the presence of 0, lo+ M SQZooaS; 0, 1O-4 M SQ22536, or +, in the absence of any added compounds. At the indicated times, cells were assayed for phosphodiesterase (-) and adenylate cyclase (---) activities. The results are representative of 3 expts. at 90” angles from each other. In this assay, no movement, movement toward, or away from the central drop could be observed. Control experiments using various concentrations of CAMP showed strong positive responses between lo-” to lo+ M. No response was seen in the absence of any added compounds. SQ22536 and SQ20009 were tested at concentrations ranging from lOmEto 10m2M.
RESULTS Effects of SQ20009 and SQ22536 on cell aggregation Amebae starved either in spinner suspensions or on tissue culture plates in the presence of 10s4 M SQ20009 remained as individual cells and displayed none of the morphological characteristics of aggregation-competent amebae. Correspondingly, they did not show any increase in adenylate cyclase or phosphodiesterase activities (fig. 1). A similar inhibition of the developmental changes in CAMP-binding sites and extracellular phosphodiesterase was also observed (data not shown). Starvation of ame13%791814
of slime mold aggregation
207
bae in buffer containing lop4 M SQ22536 produced a slightly different result. Although cells showed no morphological signs of aggregation-competence and did not increase their levels of cellular or extracellular phosphodiesterase, or surface CAMPbinding sites, they did show increased levels of adenylate cyclase activity as measured in cell extracts. The time course and extent of this rise was similar to that seen in untreated control cells. It would appear that cells incubated with SQ22536 can respond to starvation, at least by increasing their levels of adenylate cyclase while SQ20009 cells are blocked at a step preceding this rise. In both cases, the inhibition of aggregation by these compounds was reversible. Cells incubated with either product for g-10 h, washed and resuspended in buffer void of any added compound, aggregated within the next 10-12 h. Total inhibition of cell aggregation was not observed with lofold lower concentrations of each compound. The ability of applied CAMP pulses to overcome the developmental blocks imposed by these two drugs was examined. Amebae starved in the presence of lop4 M SQ22536 were induced to aggregate when pulsed with CAMP (lo-’ M), indicating that they were able to perceive and respond normally to the chemotactic signal. Cells treated with SQ20009, however, were not induced to aggregate or express any of the characteristics of aggregation-competent cells when starved in the presence of applied CAMP pulses. SQ20009 does not appear to inhibit differentiation via its effect on phosphodiesterase SQ20009 is a known inhibitor of CAMP phosphodiesterase and in the case of slime mold, blocks this enzymic activity by apExp Cd
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Table
1. The ability
of inhibitor-treated cells to synthesize or respond to LAMP
Treatment Control
Length of treatment (hours)
pd” activity U/10’ cells
CAMP levels (pmol/ IO7 cells)
2
0.2 4.3
3.1
5 1O-3 M CAMP 1O-4 M SQ20009
lo-“M SQ20009 + 1O-3 M CAMP 1O-4 M SQ22536 Fig. 2. Abscissa: time (hours); ordinate:pmol CAMP formed/min. Effect of CAMP on the developmental rise in adenylate cyclase. HPX235 amebae were starved on tissue culture plates in O-O, the presence or +-+, absence of 10m3M CAMP. At the indicated times, cells were harvested and assayed for anylate cyclase activity. The levels depicted represent the enzymic activity of 2~ 10’ cells. The data are representative of 3 expts.
prox. 40% at 1O-4 M (Harris, personal communication). Since aggregation requires only an extracellular phosphodiesterase activity [7], and SQ20009-treated cells produced little or no such enzyme, this compound may act by decreasing the existing external phosphodiesterase activity below that necessary for development to proceed. This was examined by starving cells in the presence of lop4 M SQ20009 and adding various amounts of extracellular phosphodiesterase prepared from starved Ax-2 or A53 amebae. Even when the starvation medium contained 50-fold higher levels of phosphodiesterase than that normally produced by aggregating cells, SQ20009treated amoebae were still unable to undertake their developmental cycle. The same result was obtained with SQ22536-treated cells starved in the presence of added phosphodiesterase suggesting that in this case Exp Cell
Res 124 (1979)
2
5.0
5
ND
2 5
-c
2 5 2
ND
2 5
ND ND ND 0.4
ND ND ND 0.7
5
1O-J M SQ22536 + 1O-3 M CAMP
ND”
3.0
ND
ND ND
a pd, Cellular phosphodiesterase activity. b ND, Not determined. c -, Below the limit of detection.
their inability to aggregate is also not a direct consequence of their inability to synthesize the enzyme. It is also possible that SQ20009 acts like a CAMP analogue which is not hydrolyzable by phosphodiesterase. Under such conditions, the inhibition of cell aggregation would result from the continued presence of a high external concentration of “CAMP”. This possibility was examined by starving mutant HPX235 in the presence of lop4 M CAMP and comparing the developmental change in adenylate cyclase activity in these cells to that of untreated controls. Because this mutant is defective in phosphodiesterase production [7], the levels of CAMP added to the medium remain relatively constant. Unlike SQ20009, the presence of 10e4 M CAMP did not alter either the time, or the extent, of the rise in CAMP production (fig. 2). Similar results were obtained with wild-type cells, although in this case hydrolysis of the added CAMP by
Inhibitors
Fig. 3. Abscissa: molarity (lo-“); ordinate: % binding. Effects of SQ2000!9 and SQ22536 on CAMP bindina. Ax-2 cells were starved for 51 h. at which time th;y showed a significant rise in the levels of surface CAMP-binding sites. The levels of binding activity were measured by incubating cells for 30 set in the presence of 5x lO-s M [3H]~AMP (100% binding) or in the presence of 5x 1O-8 M [3H]~AMP and various concentrations of +-+, SQ2000!3 or O-O, SQ22536. The degrees to which 10e4 M and 10e3 M CAMP can prevent 3H-binding to cells were also determined (O-O). The data is representative of 3 expts.
endogenous phosphodiesterase continuously alters the concentration of the cyclic nucleotide in the medium. Other results also suggest that SQ20009 does not function as an analogue of CAMP in inhibiting cell aggregation. As seen previously, treated cells showed no increase in phosphodiesterase activity. In contrast, amebae incubated with 10e4 M CAMP exhibit a precocious induction of this enzyme [ 161. In fact, we observed that cells treated with lop4 M SQ20009 could no longer respond to added concentrations of CAMP as high as 10e3 M by increasing their levels of phosphodiesterase (table 1). SQ20009 does not affect CAMP binding
CAMP-binding proteins on the cell surface are believed to mediate the cells’ responses to external CAMP. These responses involve both chemotaxis and the differentiation of the population to aggregation competence.
of slime mold aggregation
209
Fig. 4. Abscissa: time (min); ordinate: % binding. Ability of SQ20009 and SQ22536 to compete for pre-bound [3H]~AMP. 54 h starved Ax-2 cells were incubated for 30 set with 5X lo-* M [3H]cAMP after which time O-O, SQ22536; O-O, SQ20009; or X-X, CAMP at lo+ M was added. Control cells (+-+) received no additions. Levels of CAMP binding are expressed as a percentage of that seen on control cells. The results are representative of 2 expts.
The above experiments indicate that SQ20009-treated cells do not show normal responses to CAMP, either at high concentrations or when added in the form of imposed pulses of lo-’ or lo-* M. To examine the relationship between the effects of SQ20009 on aggregation and the CAMP surface receptor, we assessed the ability of SQ20009 to inhibit [3H]cAMP binding to the cell surface (fig. 3). At the concentration which inhibited development, SQ20009 did not interfere with CAMP binding to cells. At 3.0~ 10s3 M, SQ20009, only a slight competition for CAMP binding was observed. Similar results were obtained when lops M [3H]cAMP was used to measure binding activity predominantly due to CAMP receptor sites of high affinity [19]. SQ20009 was also found to be a poor competitor of [3H]cAMP which had been bound to cells prior to the addition of the drug (fig. 4). Pre-incubation of amebae for 30 set with SQ20009 did not alter the time course or degree of binding of [3H]~AMP added subsequently. Exp Cd
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C. Klein
SQ22536 competes for CAMP-binding sites In contrast to SQ20009, approx. 50% less [3H]cAMP was bound by cells incubated in the presence of 1O-4 M SQ22536 (fig. 3). The same loss of CAMP binding could be seen with cells which had first bound [3H]CAMP and were then incubated with lop4 M SQ22536 (fig. 4). In this case, loss of [3H]CAMP binding occurred within 30 set after the addition of SQ22536. A similar rapid loss of cell-bound radioactivity was seen when lop4 M CAMP was added to cells which had been preincubated with [3H]CAMP. However, this treatment resulted in an 85% decrease in bound radioactivity. The ability of SQ22536 to compete for CAMP binding to cells was also examined by incubating cells with this compound for 30 set and then adding [3H]~AMP to measure the level of CAMP-binding activity. Under these conditions, amebae also showed only 50% of the binding of untreated cells. This level of binding was observed within 30 set after [3H]~AMP addition and did not change during the 2-3 min of the experiment. In light of the observation that the cellsurface CAMP receptor may recognize SQ22536 as a CAMP analogue, we tested the possibility that this compound could function, to some degree, like CAMP in affecting cell differentiation to aggregation competence or chemotaxis. Since cells normally produce CAMP in the form of pulses, we first attempted to stimulate cell differentiation by starving amebae in the presence of applied pulses of SQ22536. The concentration of the pulses ranged from 1OF to lo-” M, similar to that of applied CAMP pulses which have been shown to stimulate cell differentiation [ 1, 23. SQ22536 pulses did not affect cell differentiation as judged both by morphological criteria and the time course of the rise in phosphodiExp Celi
Res 124 ((979)
esterase activity. The latter is a biochemical marker of aggregation competence [20]. When tested for its ability to induce a chemotactic response in starved cells, SQ22536 did not cause amebae to migrate outward from the droplet in which they had been deposited. The same result was obtained using SQ20009. It would appear that, although SQ22536 can bind to the CAMP receptor, it is unable to initiate events leading to cell differentiation or chemotaxis. SQ22536-treated amebae do not synthesize CAMP Competition by SQ22536 for CAMP surfacebinding sites, apparently, does not render cells unresponsive to CAMP. As mentioned above, amebae stimulated with an exogenous source of CAMP pulses overcame the inhibition of aggregation effected by the drug. The cells’ ability to respond to CAMP was also evidenced in experiments analyzing the induction of phosphodiesterase by 10e3 M CAMP in amebae treated with SQ22536 (table 1). The time-course of phosphodiesterase induction was similar to that seen in cells treated with lop3 M CAMP alone but the degree of enzyme induction was approx. 30-40% less. We should perhaps mention that it is unknown if the induction of the enzyme by high external concentrations of CAMP is mediated by the cell surface receptor and we do not wish to correlate the two. This experiment is provided only as further evidence that the SQ22536-treated amebae can respond to extracellular CAMP similarly to amebae which have not been treated with the drug. Given that drug-treated cells remain responsive to added CAMP, we examined if their failure to aggregate was related to their inability to synthesize CAMP. Drug-treated and control cells were harvested and analyzed for CAMP levels. Amoebae treated
Inhibitors
of slime mold aggregation
211
with SQ22536 showed extremely low levels of CAMP as compared to untreated, aggregation competent cells (table 1). The levels observed in SQ22536-treated cells were similar to those measured in amebae starved in the presence of SQ20009 and which do not show any developmental regulation of their adenylate cyclase activity. ’ Although SQ22536 almost totally blocked adenylate cyclase activity in intact amebae, the enzyme in broken cell preparations was relatively insensitive to the compound. A final concentration of lop3 M SQ22536 inhibited adenylate cyclase in cell-free lysates by approx. 50% while lo-* M SQ22536, which was sufficient to block development, could inhibit only approx. 20% of the activity (data not shown).
did not occur in drug-treated cells. Using a phosphodiesterase-minus mutant, HPX235, we observed that the constant presence of high external CAMP levels during cell starvation does not alter the developmental changes in adenylate cyclase. This again would suggest that SQ20009 does not function either by inhibiting phosphodiesterase activity (and allowing exter,tal CAMP levels to accumulate) or otherwise as a CAMP analogue. Amebae starved in the presence of SQ20009 remained relatively insensitive to CAMP: their differentiation to aggregation competence could not be elicited by applied CAMP pulses, and no induction of phosphodiesterase was observed when lop3 M CAMP was added to cells starved with this compound. The inability of these cells to respond to external CAMP does not appear to be related to an action of this comDISCUSSION pound on the plasma membrane CAMP reThe experiments reported here describe the ceptor since no competition by this product inhibition of Dictyostelium discoideum ag- for CAMP bound to the cell surface could gregation by two adenine analogues, be demonstrated. If SQ20009 does function SQ20009 and SQ22536. These compounds extracellularly to inhibit development, then are reported inhibitors of CAMP phosphowe would conjecture that it functions at a diesterase and adenylate cyclase, respec- site other than the CAMP receptor or the tively, in a variety of systems [12, 13, 141. CAMP phosphodiesterase. To date, it is unBoth were shown here to inhibit aggregation known if all the effects of CAMP on cell and differentiation of D. discoideum ame- differentiation and aggregation are medibae. Although SQ20009 can inhibit the ated by the surface receptors measured in phosphodiesterase activity in enzyme prep- the CAMP-binding assays. For example, exarations from slime mold (Harris, personal ternal CAMP may cause changes in ion percommunication), several experiments sug- meability of the plasma membrane which gest that this is not the primary site of ac- initiate a sequence(s) of events leading to tion of the drug. The addition of excessive differential gene expression. Alternatively, amounts of phosphodiesterase to drug- some CAMP may have to enter the cell in treated cells could not overcome the inhibiorder to influence differentiation and it is tion. Also, amebae are blocked in developthis intracellular site(s) which is blocked by SQ20009. If SQ20009 must be internalized ment at a stage prior to the time that their phosphodiesterase activity increases. Even to inhibit development, the high concentrathe rise in adenylate cyclase activity, one of tions of the drug required to do so may to the earliest known events involved in the reflect the cells relative impermeability production of the chemotactic signal [21], this compound. 14-791814
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C. Klein
The inhibition of cell differentiation by SQ22536 occurs at a step subsequent to the developmental rise in adenylate cyclase and prior to the increase in phosphodiesterase and CAMP surface-binding sites. However, the cyclase activity measured in cell extracts is not functional in intact amebae as evidenced by the very low levels of CAMP found in drug-treated cells. This finding is consistent with the known role of SQ22536 as an inhibitor of adenylate cyclase activity. The developmental block produced by this compound resembles that found in glucosetreated amebae [l l] and in several of our developmental mutants [8]. In each case, increasing adenylate cyclase activity can be measured in cell extracts even though little or no CAMP is actually produced by the cell. Correspondingly, no increases in phosphodiesterase or CAMP-binding sites occur. As in these cases, the adenylate cyclase of SQ22536-treated cells can also be activated by applied CAMP pulses. Such treatment results in cell differentiation to aggregationcompetence and if the pulses are arrested after an initial time period, amebae are able to continue their aggregation program. The mechanism by which SQ22536 inhibits development of amebae may be related to its ability to bind to the plasma membrane CAMP receptor. It was shown that this compound can prevent CAMP binding to this site by approx. 50%. It is possible that the compound binds to the CAMP receptor but is unable to initiate the subsequent events which lead to an activation of adenylate cyclase. The observations that pulses of low levels of SQ22536 could not affect cell differentiation to aggregation competence and that the drug itself is not a chemoattractant would be consistent with such a hypothesis. Since adenylate cyclase remains inactive, little or no CAMP is synthesized and therefore available to Exp Cell
RPS 124 (1979)
compete for the sites occupied by the drug. If cells are stimulated by an exogenous source of CAMP, as in the case of applied CAMP pulses, the external CAMP can compete for binding sites occupied by SQ22536 and activate the adenylate cyclase. Once this occurs, and enzyme activity is sufficient to produce enough CAMP to continue to compete for the existing drug, then aggregation can proceed in the absence of exogeneously generated CAMP pulses. Alternatively, the ability of SQ22536 to inhibit CAMP binding to its cell surface may only be an indication that the compound bears some structural relationship to CAMP rather than the mechanism by which it affects development. For example, SQ22536 may enter the cell and inhibit adenylate cyclase activity by a direct interaction with the enzyme. Experiments designed to assess the ability of the drug to inhibit adenylate cyclase activity in cell lysates have shown only slight decreases in activity. These results are difficult to interpret, however, since lop3 M CAMP is also present in the assay mixture to inhibit phosphodiesterase activity [3]. As this CAMP may compete for any sites which bind the drug, the relative insensitivity of adenylate cyclase activity in cell extracts to SQ22536 may not be a true indication that the drug does not function intracellularly. Continued research into the mechanisms by which SQ22536 and SQ20009 inhibit cell aggregation should provide important insight into the regulatory events vital to this process. The author wishes to thank dr Harris for initially SURgesting the use of these compounds and for r&i& available his preliminary data and MS Cynthia Nettrour for her excellent iechnical assistance. This research was supported by a Grant GM 25080-01 from the NIH.
REFERENCES 1. Darmon, M, Brachet, P & da Silva, P, Proc natl acad sci US 72 (1975) 3136.
Inhibitors of slime mold aggregation 2. Gerisch, G, Fromm, H, Heresgen, A & Wick, U, Nature 255 (1975) 547. 3. Klein, C, FEBS lett 68 (1976) 125. 4. Malchow, D & Gerisch, G, Proc natl acad sci US 71 (1974) 2423. 5. Chang, Y Y, Science 160 (1%8) 57. 6. Pannbacker. K G & Bravard. L G. Science 175 (1972) 1014.’ 7. Darmon. M. Barra. J L Brachet. P. J cell sci 31 (1978)233. 8. Juliani, M H & Klein, C, Dev biol62 (1978) 162. 9. Brachet, P & Klein, C, Exp cell res 93 (1975) 159. 10. Klein, C & Brachet, P, Nature 254 (1975) 432. 11. Dan-non, M & Klein, C, Dev bio163 (1978) 377. 12. Fain, J E, Pointer, R H &Ward, W F, J biol them 247 (1972) 6866. 13. Haslam, R J, Davidson, M M, Davies, T, Lynham, J A & McClenaghan, M D, Adv cyclic nucleotide res 9 (1978) 533.
213
14. Salzman, E W, Biochim biophys acta 499 (1977) 48. 15. Watts, D J & Ashworth, J M, Biochem j 119 (1970) 171. 16. Klein, C, J biol them 250 (1975) 7134. 17. Solomon, Y, Landos, C & Rodbell, M, Anal biochem 58 (1974) 541. 18. Konijn, T, Advances in cyclic nucleotide research (ed P Greengard & G A Robinson) vol. 1. Raven Press, New York (1974). 19. Green, A & Newell, P C, Cell 6 (1975) 129. 20. Klein, C & Darmon, M, Biochem biophys res commun 67 (1975) 440. 21. Klein, C, FEBS lett 1(1977) 17.
Received April 18, 1979 Accepted May 18, 1979
Exp Cell Res 124 (1979)