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.     

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.     

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.     

Calcium,cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca²⁺ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca²⁺ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.     

A staphylococcal GGDEF domain protein regulates biofilm formation independently of cyclic dimeric GMP

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.     

Genetic locus (stmF) associated with cyclic GMP phosphodiesterase activity in Dictyostelium discoideum maps in linkage group II.

Previous attempts to map the stmF locus in Dictyostelium discoideum, by using only clone morphology as a marker, have led to equivocal results. Since strains carrying mutations at the stmF locus possess very low cyclic GMP phosphodiesterase activity, we have remapped this locus using both morphological and biochemical markers. Our results indicate that mutations producing a stable "streamer" phenotype and reduced cyclic GMP phosphodiesterase activity are located in linkage group II, probably centromere distal to acrA.     

Ethylene induces zygote formation through an enhanced expression of zyg1 in Dictyostelium mucoroides

We have previously demonstrated that a potent plant hormone, ethylene induces sexual development including zygote formation in Dictyostelium cells, and that a novel gene (zyg1) is also involved in zygote formation. Based on these findings, the present work was mainly designed to reveal (1) the precise relationship between the ethylene amount and zygote formation, and (2) the relation of in situ ethylene synthesis to zyg1 expression, using transformants that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene biosynthesis. ACO(OE) cells overexpressing Dd-aco gene overproduced ethylene and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells, in which the expression of Dd-aco was suppressed by the RNAi method, showed a reduced level of ethylene production, thus resulting in inhibition of zygote formation. Importantly, the expression of zyg1 was affected by the amount of ethylene produced: Zyg1 expression was augmented in ACO(OE) cells, but was significantly suppressed in ACO-RNAi cells. In another experiment, we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the function of ethylene by binding specifically to ethylene receptors, greatly suppresses zygote formation. These results indicate that ethylene is capable of inducing zygote formation through the expression of zyg1.     

Cyclic AMP and cyclic GMP in hyperresponsiveness

Cyclic-AMP has been shown to cause a hyperresponse in blood pressure change in conjunction with norepinephrine in the anesthetized rat system. Recent experiments show that the antagonist to angiotensin II, Sar1-Thr8 angiotensin II, abolishes the hyperresponse produced by c-AMP. This is interpreted to mean that the added response caused by c-AMP is mediated through angiotensin II. When the antagonist is removed, the hyperresponse is observed to return. The experiments with cyclic-GMP indicate that the hyperresponse seen with c-AMP is not only absent, but a constant decrease in response to norepinephrine is observed as long as c-GMP is present. The decrease in blood pressure change in the presence of c-GMP suggests that the 10-5M c-GMP causes a relaxation of vascular smooth muscle. These two cyclic nucleotides seem to produce their effects by two completely different mechanisms.