1887

Abstract

A simple assay has been developed to quantify the cAMP-relay in . The assay is based on the stimulation of cells, in the presence of a phosphodiesterase inhibitor, with 2-deoxyadenosine 3′,5′-monophosphate (dcAMP) at a concentration which saturates cell surface cAMP receptors. The accumulation of cAMP is measured by an isotope dilution assay using a cAMP-binding protein with such a low affinity for dcAMP that purification of cAMP can be omitted.

The accumulation of extracellular and total cAMP was measured at 20 °C and at 0 °C. At 20 °C the rateof cAMP synthesis increased immediately after stimulation, reaching a maximum after 1 min, with a return to undetectable levels after about 4 min. The secretion of cAMP at this temperature was proportional to the intracellular cAMP concentration and reached a maximum at about 2 min. At 20 °C the cAMP-relay was essentially completed within 3·4 min, yielding about 35 pmol cAMP per 10 cells. At 0 °C the same amount of cAMP was relayed, but the rate of cAMP synthesis was reduced about 2·5-fold. The secretion rate depended on the intracellular cAMP concentration with a delay period of 1·25 ± 0·5 min, and was reduced about 5·5-fold compared to secretion at 20 °C. Due to the relatively slow secretion and fast synthesis of cAMP, an approximately twofold higher intracellular cAMP concentration was reached at 0 °C compared to 20 °C, and this persisted for about 15 min. Taking into account that the secretion rate is proportional to intracellular cAMP concentration, these results indicate that the mechanism underlying cAMP secretion is slowed 13-fold when the temperature is reduced from 20°C to 0°C.

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1984-10-01
2021-10-20
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References

  1. Devreotes P.N., Steck T.L. 1979; Cyclic 3′.5′-AMP relay in Dictyostelium discoideum II. Requirements for initiation and termination of the response. Journal of Cell Biology 80:300–309
    [Google Scholar]
  2. Devreotes P.N., Derstine P.L., Steck T.L. 1979; Cyclic 3₹,5₹-AMP relay in Dictyosielium discoideum I. A technique to monitor responses to controlled stimuli. Journal of Cell Biology 80:291–299
    [Google Scholar]
  3. De Wit R.J.W., Hoppe J., Stec W.J., Baraniak J., Jastorff B. 1982; Interaction of cAMP derivatives with the ‘stable’ cAMP-binding site in the cAMP-dependent protein kinase type I. European Journal of Biochemistry 122:95–99
    [Google Scholar]
  4. Dinauer M.C., MacKay S.A., Devreotes P.N. 1980a; Cyclic 3₹,5₹-AMP relay in Dictyostelium discoideum III. The relationship of cAMP synthesis and secretion during the cAMP signalling response. Journal of Cell Biology 86:537–544
    [Google Scholar]
  5. Dinauer M.C., Steck T.L., Devreotes P.N. 1980b; Cyclic 3₹,5₹-AMP relay in Dictyosielium discoideum IV. Recovery of the cAMP signalling response after adaptation to cAMP. Journal of Cell Biology 86:545–553
    [Google Scholar]
  6. Dinauer M.C., Steck T.L., Devreotes P.N. 1980c; Cyclic 3₹.5₹-AMP relay in Dictyostelium discoideum V. Adaptation of the cAMP signalling response during cAMP stimulation. Journal of Cell Biology 86:554–561
    [Google Scholar]
  7. Gerisch G., Wick U. 1975; Intracellular oscillations and release of cyclic AMP from Dictyosteliumcells. Biochemical and Biophysical Research Communications 65:364–370
    [Google Scholar]
  8. Konijn T.M. 1974; The chemotaclic effect of cyclic AMP and its analogues in the Acrasieae. Antibiotics and Chemotherapy 19:96–110
    [Google Scholar]
  9. Konijn T.M., Vande Meene J.G.C., Bonner J.T., Barkley D.S. 1967; The acrasin activity of adenosine 3₹,5₹-cyclic phosphate. Proceedings of the National Academy of Sciences of the United States of America 58:1152–1154
    [Google Scholar]
  10. Maeda Y., Gerisch G. 1977; Vesicle formation in Dictyostelium discoideum cells during oscillations of cAMP synthesis and release. Experimental Cell Research 110:119–126
    [Google Scholar]
  11. Malchow D., Gerisch G. 1974; Short term binding and hydrolysis of cyclic 3₹,5₹-adenosine monophosphate by aggregating Dictyostelium cells. Proceedings of the National Academy of Sciences of the United States of America 71:2423–2427
    [Google Scholar]
  12. Mato J.M., Jastorff B., Morr M., Konijn T.M. 1978; A model for cyclic AMP-chemoreceptor interaction in Dictyostelium discoideum. Biochimica et biophysica acta 544:309–314
    [Google Scholar]
  13. Nanjundiah V., Hara K., Konijn T.M. 1976; Effect of temperature on morphogenetic oscillations in Dictyostelium discoideum. Nature; London: 260705
    [Google Scholar]
  14. Shaffer B.M. 1975; Secretion of cyclic AMP induced by cyclic AMP in the cellular slime mouldDictyostelium discoideum. Nature; London: 255549–552
    [Google Scholar]
  15. Tomchik K.J., Devreotes P.N. 1981; Adenosine 3₹,5₹-monophosphate waves in Dictyostelium discoideum: a demonstration by isotope dilulion-fluoro-graphy. Science 212:443–446
    [Google Scholar]
  16. VanHaastert P.J.M. 1983a; Sensory adaptation of Dictyostelium discoideum cells to chemolactic signals. Journal of Cell Biology 96:1559–1565
    [Google Scholar]
  17. VanHaastert P.J.M. 1983b; Binding of cAMP and adenosine derivatives to Dictyostelium discoideum cells. Relationships of binding, chemotactic and antagonistic activities. Journal of Biological Chemistry 258:9643–9648
    [Google Scholar]
  18. VanHaastert P.J.M., Kien E. 1983; Binding of cAMP derivatives to Dictyostelium discoideum cells. Activation mechanism of the cell surface cAMP receptor. Journal of Biological Chemistry 258:9636–9642
    [Google Scholar]
  19. VanHaastert P.J.M., Van Der Heijden P.R. 1983; Excitation, adaptation and deadaptation of the cAMP-mediated cGMP response in Dictyostelium discoideum. Journal of Cell Biology 96:347–353
    [Google Scholar]
  20. Wurster B. 1976; Temperature dependence of biochemical oscillations in cell suspensions of Dictyostelium discoideum. Nature; London: 260703–704
    [Google Scholar]
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