Identification of bacterial guanylate cyclases

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRP_G, constructed here. While wild‐type CRP is activated exclusively by cAMP, CRP_G can be activated by either cAMP or cGMP. Using CRP‐ and CRP_G‐dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties. Proteins 2015; 83:799–804. © 2015 Wiley Periodicals, Inc.     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum

Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum. cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1?2}\text{s}$) and a class of slow-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15?150}\text{s}$). Exposure of D. discoideum cells to 1 mM cAMP for 30 min induces a reduction of cAMP binding (down-regulation; Klein, C. and Juliani, M.H. (1977) Cell 10, 329–335). The number of fast-dissociating sites was reduced by 80–90% in down-regulated cells. These sites are composed of two forms with high and low affinity which interconvert during the binding reaction. In down-regulated cells this transition still occurred in the residual sites. The accumulation of cellular cAMP levels induced by a saturating stimulus decreased by 80–90%. The number of slow-dissociating sites was not significantly reduced in down-regulated cells, but their affinity decreased about 10-fold. The accumulation of cellular cGMP levels induced by a saturating stimulus was not decreased; however, about 20-fold higher cAMP concentrations were required to induce the same response. These results demonstrate that the cAMP transduction pathways to adenylate cyclase and guanylate cyclase are down-regulated differently. Furthermore, the results suggest that the fast-dissociating sites are involved in the activation of adenylate cyclase, while the slow-dissociating sites are coupled to guanylate cyclase.     

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to adenylate cyclase.     

Cellular responsiveness to cyclic AMP in a phosphodiesterase-defective mutant of Dictyostelium discoideum

Responsiveness of Dictyostelium discoideum amoebae to cAMP, a chemotactic mediator, was investigated in a strain defective in cAMP-phosphodiesterase production. Cells were subjected to a high cAMP signal (10(-6) M) in the presence or absence of exogenous phosphodiesterase, and the changes of intracellular cAMP and cGMP concentrations and of adenylate cyclase activity were measured. In the presence of cAMP hydrolysis, both adenylate and guanylate cyclases are transiently activated. In the absence of hydrolysis, the high and constant extracellular cAMP concentration is sufficient to elicit a re-activation of adenylate cyclase a few minutes after the first transient response. In contrast, levels of cGMP remain basal for at least 20 min after termination of the initial response to the cAMP addition.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

Biochemical activity and multiple locations of particulate guanylate cyclase in Rhyacophila dorsalis acutidens (Insecta: Trichoptera) provide insights into the cGMP signalling pathway in Malpighian tubules

In insect renal physiology, cGMP and cAMP have important regulatory roles. In Drosophila melanogaster, considered a good model for molecular physiology studies, and in other insects, cGMP and cAMP act as signalling molecules in the Malpighian tubules (MTs).However, many questions related to cyclic nucleotide functions are unsolved in principal cells (PC) and stellate cells (SC), the two cell types that compose the MT. In PC, despite the large body of information available on soluble guanylate cyclase (sGC) in the cGMP pathway, the functional circuit of particulate guanylate cyclase (pGC) remains obscure. In SC, on the other side, the synthesis and physiological role of the cGMP are still unknown. Our biochemical data regarding the presence of cyclic nucleotides in the MTs of Rhyacophila dorsalis acutidens revealed a cGMP level above the 50%, in comparison with the cAMP. The specific activity values for the membrane-bound guanylate cyclase were also recorded, implying that, besides the sGC, pGC is a physiologically relevant source of cGMP in MTs. Cytochemical studies showed ultrastructurally that there was a great deal of pGC on the basolateral membranes of both the principal and stellate cells. In addition, pGC was also detected in the contact zone between the two cell types and in the apical microvillar region of the stellate cells bordering the tubule lumen. The pGC signal is so well represented in PC and, unexpectedly in SC of MTs, that it is possible to hypothesize the existence of still uncharacterized physiological processes regulated by the pGC-cGMP system.     

Activation of guanylate cyclase by E. coli heat-stable enterotoxin (STa). Modulation by NAD and pertussis toxin

The heat-stable enterotoxin (STa) of E. coli activates intestinal guanylate cyclase and leads to increased cGMP levels by an as yet undetermined mechanism. In comparing this cGMP system to other known toxin-mediated alterations in cAMP metabolism, we observed that pertussis toxin caused lower levels of intestinal cGMP synthesis in response to purified STa. Another participant in ADP-ribosylation reactions, NAD, enhanced the ability of STa to activate guanylate cyclase, yet had no effect on basal enzyme activity. Niacinamide and isoniacinamide also had no effect on basal activity, but attenuated the STa activation. These results are discussed in relation to current models of hormone/toxin-sensitive adenylate cyclase, and may suggest an involvement of guanine-nucleotide-binding proteins in intestinal cGMP metabolism.     

Adenylate cyclase in cilia from Paramecium: Localization and partial characterization

A particulate adenylate cyclase was identified in the excitable ciliary membrane from Paramecium tetraurelia. MnATP was preferentially used as substrate, the K_m was 67 μM, V_max was 1 nmol cAMP.min^?1.mg^?1, a marked temperature optimum of 37°C was observed. Adenylate cyclase was not inhibited by 100 μM EGTA or 100 μM La³⁺, whereas under these conditions guanylate cyclase activity was abolished. Fractionation of ciliary membrane vesicles by a Percoll density gradient yielded two vesicle populations with adenylate cyclase activity. In contrast, calmodulin/Ca-dependent guanylate cyclase was associated with vesicles of high buoyant density only.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.     

Multiple regulation of the activity of adenylate cyclase in Escherichia coli

Summary We have studied the correlation between the activities of adenylate cyclase (ATP pyrophosphatelyase-(cyclizing); EC 4.6.1.1) and in vivo rates of synthesis and intracellular concentrations of adenosine 3,5 cyclic monophosphate (cAMP) under various growth conditions in wild-type Escherichia coli and in mutants lacking or overproducing the cAMP receptor protein (CAP). We showed that when wild-type bacteria are grown in the presence of a variety of carbon sources the intracellular concentrations of cAMP are inversely related to the adenylate cyclase activities determined in permeabilized cells, suggesting that the carbon source-dependent modulation of cAMP levels is not directly related to the regulation of adenylate cyclase activity. In mutants lacking functional CAP (crp) the in vivo rates of cAMP synthesis are several hundred-fold higher than in the wild-type parent without a parallel increase of adenylate cyclase activities. In a strain carrying multiple copies of the crp gene and overproducing CAP the activity of adenylate cyclase is severely inhibited, although the in vivo rate of cAMP synthesis is similar to the parental strain. We interpret these results as indicating that CAP controls mainly the activity rather than the synthesis of adenylate cyclase.     

Activation of kidney guanylate cyclase by cobalt.

Guanylate cyclase activity and cyclic nucleotide content were studied in individual slices from guinea pig kidneys. Basal guanylate cyclase activity, assayed in homogenates or in particulate fractions (100,000g × 1 h), and the tissue content of cGMP and cAMP were greater in the inner than in the outer (entirely cortical) slices. The fraction of guanylate cyclase activity recovered in the supernatant was greater in the cortex. Taurodeoxycholate increased activity of the particulate cyclase but decreased that of the supernatant enzyme. Activity of the particulate was increased ca. 200% and that of the supernatant >500% by 1 mm NaN₃. Supernatant activity was markedly increased by 0.1 mm Co²⁺, which had no effect on the particulate enzyme. (Incubation of kidney slices with 2 mm Co²⁺ did not alter their cGMP content, but caused a small increase in the cAMP content of slices containing medullary tissue.) Basal guanylate cyclase activity in fresh supernatants increased linearly with pH from 5.9 to 9, whereas in the presence of Co²⁺ there was a clear maximum at pH 7.3 to 7.5. Incubation of fresh supernatant fractions at 37 °C for 3 h increased guanylate cyclase activity and abolished Co²⁺ activation. The relationship between Co²⁺ activation and that resulting from incubation remains to be defined. It seems probable, however, that these phenomena reflect regulatory properties of the supernatant guanylate cyclases of kidney and other tissues.     

Improved two-step method for the assay of adenylate and guanylate cyclase.

A two-step assay for adenylate and guanylate cyclase is described utilizing α-³²P-labeled ATP or GTP as substrate and involving purification of the resulting ³²P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 mm HCl for cGMP and 10 mm HClO₄ for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the ³²P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.     

The role of Ca2+ and cyclic nucleotides in progesterone initiation of the meiotic divisions in amphibian oocytes

Progesterone appears to be the physiological inducer of the resumption of the meiotic divisions in amphibian oocytes. Within minutes following exposure to progesterone there is a release of Ca²⁺ and a transient rise in [cAMP]_i followed by a fall in [cAMP]_i and rise in [cGMP]_i over the next 1–3 h. Agents that induce a fall in [cAMP]_i induce meiosis whereas those that prevent the fall and/or elevate [cAMP]_i block meiosis. A comparison of the conversion of injected [³H]-ATP to [³H]-cAMP and rate of hydrolysis of injected [³H]-cAMP following exposure to meiotic agonists and antagonists indicates that adenylate cyclase and not phosphodiesterase is the rate-limiting step in regulating [cAMP]_i in the oocyte. These findings are consistent with a model in which progesterone initiates the resumption of the meiotic divisions by down-regulation of membrane adenylate cyclase via Ca²⁺ release from specific membrane sites and that a translocation of Ca²⁺ produces a coordinate activation of guanylate cyclase.