Haastert, Peter J.M. van

Konijn, Theo M.

Krens, Frans A.

Mato, José M.

English

Suspensions of 3':5'-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3':5'-cyclic GMP (cGMP) levels. Under the assay conditions used (2 × 10^8 cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 × 10-8 M final concentration) increased cGMP levels from 1 pmol per 10^7 cells to 7 pmol per 10^7 cells in 10 sec and basal levels were recovered in 20-25 sec. cGMP accumulation did not occur when cells were in the cAMP-insensitive stage. cAMP-sensitive cells responded with increased cGMP levels when triggered by 5 × 10-8 M 5'-CH2-cAMP or 10-5 M adenosine-5'-methylmonophosphate (5'-AMPMe) but not after addition of 5 × 10-8 M 3':5'-cyclic IMP (cIMP) or 5 × 10-8 M 5'-AMP. As agonists of cAMP, 5'-CH2-cAMP and 5'-AMPMe have, respectively, more than 10% and 1% the chemotactic activity of cAMP, while cIMP has 0.01% the activity of cAMP and 5-AMP is inactive up to a concentration of 10-3 M. cAMP-mediated cGMP formation was dependent upon cAMP concentration, with a half-maximal cAMP concentration of about 10-8 M. This cAMP concentration agrees closely with that necessary for half-maximal receptor occupation. cAMP-mediated cGMP formation was independent of the presence of extracellular Ca2+; cell aggregation and chemotaxis were also independent of the presence of external Ca2+. Therefore, cAMP action does not depend on stimulation of the Ca2+ influx. cAMP was found to mediate desensitization of cAMP-dependent cGMP formation. Additon of 5 × 10-8 M cAMP to sensitive cells induced a desensitization period that lasted 1-5 min. Desensitization was dependent on the cAMP concentration. Finally, we propose that the translation of a chemotactic signal from the cell surface to pseudopod formation in Dictyostelium involves changes in the levels of cGMP

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   University of Groningen3'Mato, José M.; Krens, Frans A.; Haastert, Peter J.M. van; Konijn, Theo M.Published in:FEBS LettersIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1977Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mato, J. M., Krens, F. A., Haastert, P. J. M. V., & Konijn, T. M. (1977). 3': 5'-Cyclic AMP-dependent 3'.FEBS Letters, 79(2).CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022Proc. Natl. Acad. Sci. USAVol. 74, No. 6, pp. 2348-2351, June 1977Biochemistry3':5'-Cyclic AMP-dependent 3':5'-cyclic GMP accumulation inDictyostelium discoideum(chemotaxis/desensitization/Ca2+/cellular slime molds/cell aggregation)JOSE M. MATO, FRANS A. KRENS, PETER J. M. VAN HAASTERT, AND THEO M. KONIJNCell Biology and Morphogenesis Unit, Zoological Laboratory, University of Leiden, Kaiserstraat 63, Leiden, The NetherlandsCommunicated by J. T. Bonner, March 28, 1977ABSTRACT Suspensions of 3':5'-cyclic AMP (cAMP)-sen-sitive cells of Dictyostelium discoideum responded to a cAMPpulse with increased 3':5'-cyclic GMP (cGMP) levels. Under theassay conditions used (2 X 108 cells per ml in 10 mM phosphatebuffer, H 6.0) cAMP (5 X 10-8M final concentration) increasedcGMP levels from 1 pmol per 107 cells to 7 pmol per 107 cells in10 sec and basal levels were recovered in 20-25 sec. cGMP ac-cumulation did not occur when cells were in the cAMP-insen-sitive stage. cAMP-sensitive cells responded with increasedcGMP levels when triggered by 5 X 10-8 M 5'-CH2-cAMP or10-5 M adenosine-5'-methylmonophosphate (5'-AMPMe) butnot after addition of-5 X 10-8 M 3':5'-cyclic IMP (cIMP) or 5 X10-8 M 5'-AMP. As agonists of cAMP, 5'-CH2-cAMP and 5'-AMPMe have, respectively, more than 10% and 1% the che-motactic activity of -cAMP, while cIMP has 0.01% the activityof cAMP and 5-AMP is inactive up to a concentration of 10-M. cAMP-mediated cGMP formation was dependent uponcAMP concentration, with a half-maximal cAMP concentrationof about 10-8 M. This cAMP concentration agrees closely withthat necessary for half-maximal receptor occupation. cAMP-mediated cGMP formation was independent of the presenceof extracellular Ca2+; cell aggregation and chemotaxis were alsoindependent of the presence of external Ca2+. Therefore, cAMPaction does not depend on stimulation of the Ca2+ influx. cAMPwas found to mediate desensitization of cAMP-dependentcGMP formation. Additon of 5 X 10-8M cAMP to sensitive cellsinduced a desensitization period that lasted 1-5 min. Desensi-tization was dependent on the cAMP concentration. Finally,we propose that the translation of a chemotactic signal from thecell surface to pseudopod formation in Dictyostelium involveschanges in the levels of cGMP.Cell aggregation in the cellular slime molds is mediated bychemotaxis (1). cAMP is the chemotactic molecule (acrasin) inthe larger species of Dictyostelium (2, 3). In D. discoideum theinput signal for chemotaxis is a spatial gradient of 3':5'-cyclicAMP (cAMP) concentration (4) measured by specific cell-sur-face-bound receptors (5-8). The cAMP-receptor interactiontriggers the following two processes necessary for cell aggre-gation: (i) pseudopod formation towards the attractant source(9) and (ii) signal amplification or relay (10, 11). Pseudopodformation is achieved within 10 sec after cAMP application (9),while intracellular peaks of cAMP pccur at 60 sec and are fol-lowed by extracellular peaks 30 sec later (10, 12). The chemo-tactic response lasts for about 100 sec (13); therefore, the in-tracellular rise in cAMP does not coincide with the beginningof the chemotactic response but with its end. In an attempt tounderstand the role of cyclic nucleotides during chemotaxis inDictyostelium, we measured early changes in cyclic nucleotidesassociated with acrasin addition to pre-aggregative amebae ofAbbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; cGMP,guanosine 3':5'-cyclic monophosphate; cIMP, inosine 3':5'-cyclicmonophosphate; 5'-CH2-cAMP, 5'-deoxy-5'-methylene adenosine3':5'-cyclic monophosphate; 5'-AMPMe, adenosine 5'-methylmono-phosphate; EGTA, ethylene glycol-bis(&-aminoethyl ether)-N',N'-tetraacetic acid.D. lacteum. * In this species cAMP is not used as attractant andthere is no evidence for signal relay. Addition of the purifiedattractant (14) to pre-aggregative amebae of D. lacteum in-duces a 2-fold increase in the intracellular concentration of3':5'-cyclic GMP (cGMP) within 10 sec without modifying thecAMP levels.* These results suggest a possible function of cGMPduring the chemotactic process. In this study, changes in cGMPassociated with cAMP addition have been examined in D.discoideum. Our results provide evidence that cGMP is in-volved in the translation of the chemotactic signal responsiblefor movement during cell aggregation in cAMP-sensitive andcAMP-insensitive species of Dictyostelium.METHODS AND MATERIALSOrganism. D. discoideum NC-4(H) was used for all exper-iments. Cells were grown on a solid medium and harvested asdescribed (15).Cell Aggregation. After harvesting, cells were suspended incold 10 mM phosphate buffer (pH 6.0) in the presence orabsence of 1 mM ethylene glycol-bis(O-aminoethyl ether)-N',N'-tetraacetic acid (EGTA) and washed twice in the samebuffer. The cell suspension was adjusted to 107 cells per ml. Cellaggregation was studied by placing small drops of an amebalsuspension on hydrophobic agar (15) buffered with 10 mMphosphate buffer (pH 6.0) to which 1 mM EGTA or 1 mMCaCl2 was added. The amebal drops were incubated at 22° indarkness and the appearance of aggregation centers wasmonitored at 1-hr intervals.Assay for Chemotaxis. The chemotactic response of amebaeto cAMP was determined as described (16).cGMP Measurements. Cells were harvested as describedabove, suspended in phosphate buffer, and starved by shaking(17). After shaking (4 hr if not stated otherwise), cells werecentrifuged, washed three times in cold phosphate buffer, andadjusted to 2 X 108 cells per ml. Air was bubbled through for10 min. Samples of 100 Al were equilibrated for 10 min at 220under vigorous shaking, after which 20 pl of cAMP (final con-centration 5 X 10-8 M if not stated differently) was added. Atthe tithe indicated 200 Al of ice-cold ethanol/HCI (60 volumesof ethanol + 1 volume of 11 M HCI) was pipetted into the tubes.After standing 15 min at 00, the ethanol/HC1 extracts (to which1500 cpm of [8-3H]cGMP had been added) were centrifuged(8000 X g, 2 min), the pellet was washed with 200 pA of etha-nol/HCl, and the pooled supernatants were dried at 650. Afterdrying, samples were dissolved in 500 Ml of water and extractedtwice with water-saturated ether. The aqueous phase was driedat 650 and then dissolved in 300,gl of 50 mM Tris-HC1 buffer(pH 7.5) containing 4 mM EDTA. cGMP recovery was then* J. M. Mato, F. A. Krens, P. J. M. van Haastert, and T. M. Konijn,unpublished data.2348Proc. Nati. Acad. Sci. USA 74 (1977) 23498C.DC-0cV-a.)5aE6420 10 20 30SecondsFIG. 1. Time course ofcGMP formation after D. discoideum cellswere triggered with 5 X 10-8M cAMP. cAMP was added at time zeroand the formation ofcGMP was followed. (0) Cells starved for 4 hr;(0) cells starved for 0.5 hr.calculated (60-70%) and cGMP levels were measured. cGMPwas measured in duplicate by the method of Steiner et al. (18)using antibody and [8-3H]cGMP supplied by Amersham (spe-cific activity 20 Ci/mmol). The high specificity of the antibodyfor cGMP allows cGMP measurements without further samplepurification. The amount of cAMP added to the amebae didnot show crossreactivity with the antibody. Preincubation ofthe samples with phosphodiesterase (Boehringer) abolishedreactivity with the antibody.Materials. 5'-CH2-cAMP was kindly provided by Dr. J. G.Moffatt; adenosine 5'-methylmonophosphate (5'-AMPMe) wasa gift of Dr. F. Eckstein; cAMP and 3':5'-cyclic IMP (cIMP)were purchased from Boehringer; and 5'-AMP and EGTA werefrom Sigma.RESULTS AND DISCUSSIONFig. 1 shows the time course of cGMP formation after additionof cAMP using cells starved for 0.5 hr or 4 hr. In cells starvedfor 0.5 hr, cAMP addition did not change cGMP levels, whilein cells starved for 4 hr cAMP induced, within 2 sec, an increasein the cGMP concentration. cGMP level reached its peak 10 secafter cAMP addition and then fell to the basal level at ap-proximately 20 sec. cGMP levels increased by a factor of 7 whencells were triggered with 5 X 10-8 M cAMP. Basal cGMP levelsin the cells starved for 0.5 hr were two to three times higher thanin the cells starved for 4 hr. Under our conditions cells reachedthe aggregation-competence stage after 5-6 hr of starvation.To avoid autonomous cAMP oscillations (12), we used cellsstarved for 4 hr. At this stage cells respond chemotactically andbind cAMP while cells starved only for 0.5 hr do not bind cAMPand are about 1% as sensitive to cAMP as attractant (5, 6,19).To study the specificity of cGMP formation induced bycAMP, we added various nucleotides. In Fig. 2 is shown thatwhile the addition of 5'-AMP or cIMP did not influence cGMPlevels, the addition of 5'-CH2-cAMP or 5'-AMPMe induced anincrease in the cGMP levels. 5'-AMP has no chemotactic activityand cIMP has 0.01% the activity of cAMP (3). As a chemotacticattractant, 5'-CH2-cAMP is slightly less active than cAMP and5'-AMPMe has about 1% the activity of cAMP (3, 20). Thespecificity of cAMP-mediated cGMP formation is similar to thatof chemotaxis.Cells washed in phosphate buffer containing 1 mM EGTAand then plated on EGTA/hydrophobic agar started to ag-(4E..20 10 20 30SecondsFIG. 2. Effect of different nucleotides as agonists of cAMP-mediated cGMP formation. Cells were starved for 4 hr and stimulatedat time zero with: (&) 5 X 10-8M 5'-AMP; (0) 5 X 10-8M cIMP; (A)5 x 10-8 M 5'-CH2-cAMP; and (0) 10-5 M 5'-AMPMe.gregate within the same time interval (9-10 hr) as they did inthe absence of EGTA. The chemotactic sensitivity of cells onEGTA/agar plates was also in the same range (10-9-10-8 M)as in control plates. Preincubation of cells with 1 mM EGTAprior to cAMP stimulation does not affect cGMP formation(Fig. 3). These results indicate that extracellular calcium isnecessary neither for cell aggregation nor for cAMP-mediatedcGMP formation.In various systems ligand-induced cGMP accumulation de-pends on stimulation of the Ca2+ influx (21). In D. discoideumcGMP elevation is independent of external Ca2+, which rulesout the above model. Mason et al. (22) have reported that cellaggregation in D. discoideum depends on extracellular Ca2+;however, we have not been able to confirm their results. Theprimary action of the cAMP-receptor interaction in D. dis-coideum may be to change the intracellular Ca2+ distributionor to increase guanylate cyclase activity without mediation ofCa2+. Other possibilities, such as a direct action on phospho-diesterase, although less likely, cannot be excluded.As shown in Fig. 4, cGMP formation was dependent on theconcentration of cAMP added. Half-maximal cGMP formationoccurred at about 10-8 M cAMP. This concentration corre-sponds closely to that which causes half-maximal occupancyof the cAMP receptors and to that present in the background-n6.)(4E2.20 10 20 30SecondsFIG. 3. Time course of cAMP-dependent cGMP formation in thepresence of 1 mM EGTA. Cells were starved for 4 hr, washed threetimes in 1 mM EGTA/phosphate buffer, suspended in the samebuffer, and, after 10 min of equilibration, stimulated with 5 X i0-8M cAMP. Under these conditions the concentration of free Ca2+ isbelow 107 M (26).I:IBiochemistry: Mato et al.Proc. Nati. Acad. Sci. USA 74 (1977)80[O 60-C._ 40-40"201C.T-0~(-a.0.-11 -10 -9 -85x10 5x10 5x10 5X10 5x10cAMP, MFIG. 4. cAMP concentration dependence of cAMP-mediatedcGMP formation. Cells were starved for 4 hr and stimulated withvarious cAMP concentrations. Samples were taken at 0, 4, 6, 8, 10, 15,20, and 30 sec after cAMP stimulation. cGMP peaks were obtainedat 10 sec. The maximal cGMP increase with respect to basal concen-trations was then calculated and plotted, taking the increase obtainedwith 5 X 10-7 M cAMP as 100.when cells are triggered by a threshold chemotactic signal (4,6).Fig. 5 shows the effect of cAMP when cells are triggeredrepeatedly. cGMP formation was not induced by a secondcAMP pulse (5 X 10-8 M) when given 15, 30, or 60 sec after afirst cAMP pulse of the same magnitude. The time necessaryfor resensitizing the cAMP-mediated cGMP formation wasvariable, ranging from 1 to 5 min (not shown). When cells werefirst triggered with a cAMP pulse of 5 X 10-9 M and a secondtime with a cAMP pulse of 5 X -8 M, cGMP formation was in-duced by the second pulse (Fig. 6). Apparently, desensitizationof cAMP-mediated cGMP formation is dependent on the cAMPconcentration. The existence of a desensitized (refractory)period has been hypothesized to explain unidirectional prop-agation of the chemotactic signal (23-25). The present resultsindicate that the period and magnitude of refractoriness de-pends on the magnitude of the signal. Thus, a given cell mayrespond within a few seconds to a new signal if the first signal5E 2t/ 2 'o'° J10 20 30 40 50 60SecondsFIG. 5. cAMP-stimulated desensitization of cAMP-dependentcGMP formation. Cells starved for 4 hr were triggered at time zerowith 5 X 10-8 M cAMP and the time course of cGMP formation wasfollowed during 60 sec (-). At the time indicated by the arrows (15,30, and 60 sec) a second cAMP pulse of the same magnitude was givento the cell suspension and the cGMP levels were measured (0). Underthese conditions 90% of the added cAMP was hydrolized to 5'-AMPin about 30 sec (data not shown).86420 10 20 30SecondsFIG. 6. At time zero cells starved for 4 hr were stimulated with5 X 10-9M cAMP (0) or 5 X 10-8M cAMP (0) and the time courseofcGMP formation was followed. At the time indicated by the arrow,cells that had been previously stimulated with a cAMP pulse ofmagnitude 5 x 10-9 M were triggered again with a cAMP pulse ofmagnitude 5 X 10-8 M and the time course of cGMP formation wasfollowed (a).was very weak and, after a stronger signal, may be refractoryfor as long as 1-5 min.Based on the following observations, we propose that cGMPmediates cAMP-induced chemotactic responses in Dictyo-stelium: (i) the increase of the cGMP level precedes pseudopodformation; (ii) cGMP levels increased only after the cells werechemotactically sensitive to cAMP; (Mi) chemotaxis and increasein cGMP show similar nucleotide specificity; (iv) a close cor-relation exists between cAMP receptor occupancy and cGMPincrease; and (v) the cGMP increase is not limited to cAMP-sensitive* species.The costs of publication of this article were defrayed in part by thepayment of page charges from funds made available to support theresearch which is the subject of the article. This article must thereforebe hereby marked "advertisement" in accordance with 18 U. S. C.§1734 solely to indicate this fact.1. Bonner, J. T. (1947) J. Exp. Zool. 106, 1-26.2. Konijn, T. M., Barkley, D. S., Chang, Y. Y. & Bonner, J. T. (1968)Am. Nat. 102,225-234.3. Konijn, T. M. (1972) Adv. Cyclic Nucleotide Res. 1, 17-31.4. Mato, J. M., Losada, A., Nanjundiah, V. & Konijn, T. M. (1975)Proc. NatI. Acad. Sci. USA 72,4991-4993.5. Malchow, D. & Gerisch, G. (1974) Proc. Natl. Acad. Sci. USA 71,2423-2427.6. Mato, J. M. & Konijn, T. M. (1975) Blochim. Blophys. Acta 385,173-179.7. Henderson, E. J. (1975) J. Biol. Chem. 250,4730-4736.8. Green, A. A. & Newell, P. C. (1975) Cell 6, 129-136.9. Gerisch, G., Hulser, D., Malchow, D. & Wick, U. (1975) Phil.Trans. R. Soc. London Ser. B. 272, 187-192.10. Roos, W., Nanjundiah, V., Malchow, D. & Gerisch, G. (1975)FEBS Lett. 53, 139-142.11. Shaffer, B. M. (1975) Nature 255,549-552.12. Gerisch, G. & Wick, U. (1975) Biochem. Biophys. Res. Commun.65,369-370.13. Cohen, M. H. & Robertson, A. (1971) J. Theor. Biol. 31, 119-130.14. Mato, J. M., Haastert, P. J. M. van, Krens, F. A. & Konijn, T. M.(1977) Dev. Biol. 57, 170-173.15. Konijn, T. M. & Raper, K. B. (1961) Dev. Biol. 3,725-756.16. Konijn, T. M. (1970) Experientia 26,367-369.17. Gerisch, G. (1962) Wilhelm Roux Arch. Entwicklungsmech. Org.153,603-620.T AI /AI' '| O2350 Biochemistry: Mato et al.IIBiochemistry: Mato et al.18. Steiner, A. L., Pagliara, A. S., Chase, L. R. & Kipnis, D. N. (1972)J. Biol. Chem. 247, 1114-1120.19. Bonner, J. T., Barkley, D. S., Hall, E. M., Konijn, T. M., Mason,J. W., O'Keefe, G. & Wolfe, P. B. (1969) Dev. Biol. 20,72-87.20. Mato, J. M. & Konijn, T. M. (1977) FEBS Lett., 75, 173-176.21. Schultz, G. & Hardman, J. G. (1975) Adv. Cyclic Nucleotide Res.5,339-351.22. Mason, J. W., Rasmussen, H. & Dibella, F. (1971) Exp. Cell Res.67, 156-160.Proc. Natl. Acad. Sci. USA 74 (1977) 235123. Shaffer, B. M. (1957) Am. Nat. 91, 19-35.24. Alcantara, F. & Monk, M. (1974) J. Gen. Microbiol. 85, 321-324.25. Gerisch, G., Malchow, D. & Hess, B. (1974) in Biochemistry ofSensory Functions, ed. Jaenicke, L. (Springer Verlag, Berlin-NewYork), pp. 279-298.26. Portzehl, H., Caldwell, P. C. & Ruegg, J. C. (1963) Biochim.Biophys. Acta 79,581-591.

3':5'-Cyclic AMP-dependent 3'

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3':5'-Cyclic AMP-dependent 3'

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