cAMP controls cytosolic Ca2+ levels in Dictyostelium discoideum

Abstract

Background: Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i.

Results: We analyzed Ca2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced.

Conclusion: We conclude that there is a link between the second messengers cAMP and Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance.

Figure 1

Ca²⁺-influx after cAMP stimulation is reduced in regA^- cells. The amount of influx (pmol/10⁷ cells) after addition of 1 μM cAMP is plotted versus extracellular [Ca²⁺]. Average influx was higher in wild type than in regA^- amoebae (mean ± s.d. from at least 6 determinations in 3 independent experiments each).

Figure 2

Measurement of cAMP activated [Ca²⁺]_i-changes in wild type and mutant amoebae. Cells were stimulated with 1 μM cAMP in the presence or absence of 1 mM external CaCl₂. In wild type amoebae a [Ca²⁺]_i-transient was observed in the presence of external Ca²⁺. The graph shows the average increase (mean ± s.e.m.).

Figure 3

Measurement of cAMP activated [Ca²⁺]_i-transients in wild type and mutant amoebae in the cytosolic presence of IBMX. IBMX led to an elevation of basal [Ca²⁺]_i. Upon stimulation with 1 μM cAMP in the absence of external CaCl₂ a [Ca²⁺]_i-transient was observed in both, mutant and wild type amoebae (mean ± s.e.m.).

Figure 4

[Ca²⁺]_i-changes in regA^- cells in the presence of a Ca²⁺-chelator. Amoebae in nominally Ca²⁺-free medium were challenged with 1 mM BAPTA (final concentration) and subsequently with 1 μM cAMP. Arrows indicate the time point of addition of agents. The graph shows the average increase (mean ± s.e.m.).

Figure 5

Light scattering and [Ca²⁺]_e oscillations of regA^- cells. Light scattering and [Ca²⁺]_e were recorded as outlined in Methods. (a, b) Regular light scattering oscillations with a phase length of roughly 4–5 min but with strongly reduced amplitude as compared to wild type oscillations (see also [3]). (c) Irregular light scattering changes. (d) Oscillations of cAMP levels in the regA^- strain were less pronounced than in the wild type; the graph shows examples of one cAMP oscillation each, determined during one spike of light scattering oscillations.

Figure 6

Light scattering response upon addition of 1 μM cAMP. (a) Wild type cells displayed two peaks of light scattering which subsequently returned to the baseline. (b) In regA^- cells there was a strong decrease in light scattering after the second peak. One out of 7 independent experiments is shown.

Figure 7

[Ca²⁺]_e oscillations in wild type and regA^- cell suspensions. (a) Regular [Ca²⁺]_e oscillations were recorded in wild type cell suspensions (see also [2]). (b) Similar to light scattering oscillations the pattern of [Ca²⁺]_e oscillations in regA^- was irregular. One out of 5 independent experiments is shown.

Figure 8

Chemotactic speed of wild type and regA^- amoebae. The effect of preincubation with EGTA for 30 min was assayed. Chemotactic velocity of amoebae was affected dose dependently by EGTA treatment; when compared to the wild type the speed of the regA^- strain was significantly reduced at lower concentrations of EGTA. Velocity of wild type and regA^- cells is shown (median of at least 2 independent experiments).