Inositol 1,4,5-trisphosphate induces cyclic GMP formation in Dictyostelium discoideum

Chemotactic signalling in the cellular slime mould Dictyostelium discoideum employs signalling molecules such as folate and cyclic AMP. These bind to specific cell surface receptors and rapidly trigger internal responses that induce chemotactic movement of the amoebae. Previous studies have shown that actin is polymerised within 3-5 sec of cyclic AMP or folate binding and that a peak of cyclic GMP is formed within 9-12 sec. Release of Ca2+ from intracellular stores has been implicated as a secondary messenger. Here we present evidence that D-myo-inositol 1,4,5-trisphosphate, when added to permeabilized amoebae of Dictyostelium, can mimic the action of chemoattractants on normal intact amoebae in inducing cyclic GMP formation. Our data suggest that IP3, which is known to act as an intermediary messenger between cell surface hormone receptors and release of Ca2+ from internal stores in mammalian cells, functions in a similar capacity during chemotaxis of this primitive eukaryote.     

Specific binding proteins for cyclic AMP and cyclic GMP in Dictyostelium discoideum.

In Dictyostelium discoideum both cyclic AMP and cyclic GMP are regulated by chemotactic stimuli. Binding proteins specific for cAMP and cGMP have been found in aggregation competent cells as well as in cells harvested during growth. The activity of binding proteins was, on the average, lower in the growth phase cells. cAMP binding proteins were separated into 3 fractions, whereas the cGMP binding activity appeared in 1 major peak both on DEAE-cellulose and Sephadex G-200. Protein kinase activity was present in most but not all cyclic necleotide binding fractions; evidence for a relationship is however missing.     

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.     

Cyclic AMP relay and cyclic AMP-induced cyclic GMP accumulation during development of Dictyostelium discoideum

Abstract Cyclic AMP-induced cAMP and cGMP responses during development of Dictyostelium discoideum were investigated. The cAMP-induced cGMP response is maximal when aggregation is in full progress, and then decreases to about 10% of the maximal level during further multicellular development. The cAMP response increases upon starvation, reaches its maximum at the onset of aggregation, and then decreases to about 8% of the maximum level. The dynamics of the post-aggregative cAMP response are in qualitative agreement with the dynamics of the cAMP relay response in aggregation-competent cells.     

Triiodothyronine induces an increase in cyclic GMP in bullfrog tadpole tissues

Cyclic GMP (cGMP) and cyclic AMP (cAMP) were determined in bullfrog tadpole liver and tail fin using 125I-RIA. cGMP increased approx. 100% 1-6 h after the injection of T3 (3 X 10(-10) mol/g body wt.). Reducing the dose of T3 to 1, 3, and 10 X 10(-11) mol/g body wt. provided increases in cGMP of 50-100% above the control value after 2 h. In contrast, only small increases (less than 20%) in cAMP were observed 2-24 h after T3 injection. We conclude that T3 produces a rapid and significant increase in cGMP in the liver and tail fins of premetamorphic tadpoles. These results suggest that thyroid hormones in amphibia may not be an exclusively nucleus-mediated hormone.     

Streamers: chemotactic mutants of Dictyostelium discoideum with altered cyclic GMP metabolism

Mutants of Dictyostelium discoideum that developed huge aggregation streams in expanding clones were investigated using optical and biochemical techniques. Representatives of the six complementation groups previously identified (stmA-stmF) were found to be similar to the parental wild-type strain XP55 in both the extent and timing of their ability to initiate and relay chemotactic signals and in the formation of cyclic AMP receptors and phosphodiesterases. The mutants differed from the wild-type in producing an abnormal chemotactic (movement) response visible using both dark-field optics with synchronously aggregating amoebae on solid substrata and light scattering techniques with oxygenated cell suspensions. Mutants of complementation group stmF showed chemotactic movement responses lasting up to 520 s, rather than 100 s as seen in the parental and other strains. Measurements of cyclic GMP formed intracellularly in response to chemotactic pulses of cyclic AMP in stmF mutants showed that abnormally high concentrations of this nucleotide were formed within 10 s and were not rapidly degraded. A causal correlation between defective cyclic GMP metabolism and the altered chemotactic response is suggested, and a model is proposed that accounts for the formation of huge aggregation streams in clones of these mutants.U     

Evidence for a messenger function of cyclic GMP during phosphodiesterase induction in Dictyostelium discoideum

Chemotactic stimulation of vegetative or aggregative Dictyostelium discoideum cells induced a transient elevation of cyclic GMP levels. The addition of chemoattractants to postvegetative cells by pulsing induced phosphodiesterase activity. The following lines of evidence suggest a messenger function for cyclic GMP in the induction of phosphodiesterase: (i) Folic acid and cyclic AMP increased cyclic GMP levels and induced phosphodiesterase activity. (ii) Cyclic AMP induced both cyclic GMP accumulation and phosphodiesterase activity by binding to a rate receptor. (iii) The effects of chemical modification of cyclic AMP or folic acid on cyclic GMP accumulation and phosphodiesterase induction were closely correlated. (iv) A close correlation existed between the increase of cyclic GMP levels and the amount of phosphodiesterase induced, independent of the type of chemoattractant by which this cyclic GMP accumulation was produced. (v) Computer simulation of cyclic GMP binding to intracellular cyclic GMP-binding proteins indicates that half-maximal occupation by cyclic GMP required the same chemoattractant concentration as did half-maximal phosphodiesterase induction.     

2-Deaminofolic acid elicits desensitization without excitation of the cyclic GMP response in Dictyostelium discoideum

Folic acid induces a rapid transient increase in cellular cGMP levels in D. discoideum. A half-maximal cGMP response is effected by about 0.2 μM folate. In addition, extracellular folate is rapidly inactivated by deaminases. The main product 2-deaminofolic acid generates no measurable cGMP response at concentrations of up to 0.1 mM. At 1 mM a 10% response is observed. In contrast, 1 mM deaminofolic acid inhibits the cGMP accumulation as induced by 0.3 μM folic acid by 90%. This antagonism by deaminofolate is competitive with a K_I value of 20 μM. When the cells are preincubated with deaminofolic acid before stimulation with folic acid, the inhibition changes to a non-competitive type (K_I = 20 μM). It is concluded that although deaminofolate does not elicit cGMP accumulation, some cellular process is activated resulting in a diminished cGMP response to saturating folate stimuli. This process of desensitization is characterized by a first-order rate constant of 0.04 s⁻¹, while folic acid-induced desensitization proceeds with a higher rate of 0.6 s⁻¹. We discuss that the latter rate constant is more likely to reflect the rate of detection of a desensitizing signal, rather than the rate of the desensitization process itself.     

Calcium and cyclic GMP regulation of light-sensitive protein phosphorylation in frog photoreceptor membranes

In frog photoreceptor membranes, light induces a dephosphorylation of two small proteins and a phosphorylation of rhodopsin. The level of phosphorylation of the two small proteins is influenced by cyclic GMP. Measurement of their phosphorylation as a function of cyclic GMP concentration shows fivefold stimulation as cyclic GMP is increased from 10(-5) to 10(-3) M. This includes the concentration range over which light activation of a cyclic GMP phosphodiesterase causes cyclic GMP levels to fall in vivo. Cyclic AMP does not affect the phosphorylations. Calcium ions inhibit the phosphorylation reactions. Calcium inhibits the cyclic GMP-stimulated phosphorylation of the small proteins as its concentration is increased from 10(-6) to 10(-3) M, with maximal inhibition of 70% being observed. Rhodopsin phosphorylation is not stimulated by cyclic nucleotides, but is inhibited by calcium, with 50% inhibition being observed as the Ca++ concentration is increased from 10(-9) to 10(-3) M. A nucleotide binding site appears to regulate rhodopsin phosphorylation. Several properties of the rhodopsin phosphorylation suggest that it does not play a role in a rapid ATP-dependent regulation of the cyclic GMP pathway. Calcium inhibition of protein phosphorylation is a distinctive feature of this system, and it is suggested that Ca++ regulation of protein phosphorylation plays a role in the visual adaptation process. Furthermore, the data provide support for the idea that calcium and cyclic GMP pathways interact in regulating the light-sensitive conductance.     

Calcium channel activation and inactivation inParamecium biochemically measured by cyclic GMP production

Summary The Ca-inward current ofParamecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba²⁺ increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec^–1, or about 1.2 ·10⁸ molecules/cell·sec^–1. Because no other factors than the Ca-inward current were found to affect cGMP formation inParamecium, we used it as a quantitative measure of Ca²⁺ channel activity. After a transient stimulation of cGMP formation by 1mm Ba²⁺, an additional increase of Ba²⁺ to 5mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1mm Ba²⁺ revealed a time-dependent inactivation of the Ca²⁺ channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization ofParamecium reduced the maximal response, but did not shift the dose-response curve for Ba²⁺. Veratridine, which activates the Ca²⁺ channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response ofParamecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol.The extremely high rate of cGMP turnover inParamecium is reminiscent of findings in visual cells. A model for regulation of the voltage-dependent Ca channel ofParamecium is proposed.     

Stimulation of cyclic GMP formation in smooth muscle cells by atriopeptin II

Addition of synthesized atriopeptin II (AP-2), a 23 amino acid peptide of rat atria, to rat thoracic aorta smooth muscle cells results in the stimulation of cyclic GMP production by the cells. The EC50 for the effect is 81 nM and a 7 fold increase occurs at 10 microM AP-2. Cyclic GMP levels increased within 15 seconds after the addition of AP-2 and were maximal at 5 minutes. Cyclic GMP levels in primary rabbit kidney cells were increased 15 fold by 10 microM AP-2. However, no increase in cyclic GMP was detected in WI-38 fibroblast cells after the addition of 10 microM AP-2. Cyclic AMP levels were not affected by AP-2 in any of these cell systems. The effect upon cyclic GMP accumulation was specific for AP-2; none of the other compounds or peptides tested affected cyclic GMP levels.     

Conditions that elevate intracellular cyclic AMP levels promote spore formation in Dictyostelium

We have been using sporogenous mutants of Dictyostelium discoideum strain V12M2 to study regulation of cell fate during terminal differentiation of spores and stalk cells. Analyses of intracellular cAMP accumulation, cAMP secretion, cAMP binding to cell surface receptors, and chemotactic sensitivity to exogenous cAMP during aggregation showed that all of these functions were identical in V12M2 and HB200, a sporogenous mutant. We used several methods of altering intracellular cAMP levels in HB200 cells to test the hypothesis that intracellular cAMP levels affect cell fate. First, HB200 amoebae were treated with 5 mM caffeine for 4 h during growth, washed, and allowed to develop in the absence of caffeine. Treated cells had normal levels of intracellular cAMP and adenylate cyclase activities at the beginning of differentiation; by 6 h development, they contained two to three times more intracellular cAMP and two times more GTP-dependent adenylate cyclase activity than untreated cells. However, their level of basal Mn++-dependent adenylate cyclase activity was the same as untreated controls. Thus, treatment of growing HB200 amoebae with caffeine for only 4 h leads to hyperinduction of a GTP-dependent regulator (or inhibition of a negative regulator) of adenylate cyclase during subsequent differentiation, without induction of basal activity. The fraction of amoebae forming spores increased twofold when HB200 amoebae were treated with caffeine during growth. Spore (but not stalk cell) differentiation by such treated cells was blocked by inhibitors of cAMP accumulation. Second, cells grown on nutrient agar accumulated higher levels of intracellular cAMP and formed more spores in vitro than cells grown in shaken suspension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin-3 reduces C-type natriuretic peptide-induced cyclic GMP formation in C6 glioma cells

The effect of endothelin-3 (ET-3) on C-type natriuretic peptide (CNP)-induced guanosine 3′,5′-cyclic monophosphate (cGMP) was examined in C6 glioma cells, CNP-induced cGMP formation was both time- and dose-dependent, with an EC₅₀ value of about 10 nM. While ET-3 and phorbol 12-myristate 13-acetate (PMA) had no effect on basal cGMP production, both compounds were potent inhibitors of CNP-induced cGMP formation, with IC₅₀ values of approximately 10 and 2 nM, respectively. Although protein kinase C (PKC) inhibitors had no effect on basal cGMP formation, Ro 31-8220, a PKC inhibitor, reversed the ET-3 inhibition on CNP-induced cGMP formation by 63% and that of PMA almost completely. Our findings suggest that stimulation of cGMP formation by CNP in C6 glioma cells is negatively modulated by PKC activation, and that the inhibitory action of ET-3 on CNP-stimulated cGMP formation is mediated partly by PKC.     

Cyclic GMP and cyclic AMP changes in response to folic acid pulses during cell development of Dictyostelium discoideum.

Folic acid pulses induced developmental processes in agip 71, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. Cells that had received folic acid pulses were able to form EDTA-stable cell aggregates and to complete full differentiation to fruiting bodies. In these cells no autonomous periodic activities were observed by light scattering. Folic acid pulses elicited increases in the concentrations of cyclic GMP and cyclic AMP. In undifferentiated cells, folic acid caused a rapid increase in the level of cyclic GMP without a significant change in the level of cyclic AMP. In an advanced developmental state folic acid caused an increase in cyclic AMP in addition to two successsive peaks of cyclic GMP. Experiments performed with the parent strain, Ax-2, also showed that during the development towards aggregation competence, cells acquired the ability to produce a cyclic AMP peak in response to folic acid.