Cyclic di-GMP as a second messenger

In many bacteria bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling determines the timing and amplitude of complex biological processes from biofilm formation and virulence to photosynthesis. Thereby, the tightly regulated temporal and spatial activity patterns of GGDEF and EAL domain proteins, which synthesize and degrade c-di-GMP, respectively, are currently being resolved. Although details of the mechanisms of c-di-GMP signaling are not yet determined, the recent presentation of PilZ as a candidate c-di-GMP binding-domain opens the field for experimental investigations. Besides its role as an intracellular signaling molecule in bacteria, c-di-GMP also acts as an intercellular signaling molecule between prokaryotes and also has effects in eukaryotes that could provide a perspective in cancer treatment.     

Cyclic GMP is a second messenger by which nitric oxide inhibits diaphragm contraction

We have shown that endogenous nitrogen oxides (NOx) modulate excitation-contraction coupling in diaphragm. Because cyclic GMP (cGMP) is a second messenger for nitric oxide (NO) inhibition of smooth muscle contraction, we rested the hypothesis that NO acts via cGMP in diaphragm. Fiber bundles from rat diaphragm were studied in vitro. Immunohistochemical analysis using a cGMP-specific monoclonal antibody confirmed the presence of cGMP in the subsarcolemmal region, near nitric oxide synthase (NOS). cGMP measured by ELISA in control muscle (0.27 pmol/mg +/- 0.01 SE) was significantly increased by the NO donor S-nitroso-N-acetylcysteine 1 mM (0.55+/-0.05; N = 6; P < 0.001). Contractile studies showed that the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) 10 mM increased submaximal (40 Hz) tetanic force (P < 0.0001). L-NNA effects were exaggerated by the guanylate cyclase inhibitor LY83583 5-10 microM; force at 40 Hz was increased (P < 0.001). L-NNA effects were partially reversed by 8-bromo-cGMP 1 mM (8-Br-GMP; a cell-permeable cGMP analogue; P < 0.0001) or dipyridamole 10 microM (DPM; a phosphodiesterase inhibitor; P < 0.0001). 8-Br-GMP and DPM produced more-complete L-NNA reversal in combination (P < 0.0001). We conclude that cGMP functions as a second messenger by which NO inhibits diaphragm contraction.     

Cyclic ADP-ribose as a potential second messenger for neuronal Ca2+ signaling

Cyclic ADP-ribose (cADPR), a known endogenous modulator of ryanodine receptor Ca2+ releasing channels, is found in the nervous system. Injection of cADPR into neuronal cells primarily induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i), and/or secondarily potentiates [Ca2+]i increases that are the result of depolarization-induced Ca2+ influx. Acetylcholine release from cholinergic neurons is facilitated by cADPR. cADPR modifies K+ currents or elicits Ca2+-dependent inward currents. cADPR is synthesized by both membrane-bound and cytosolic forms of ADP-ribosyl cyclase in neuronal cells. cADPR hydrolase activity is weak in the membrane fraction, but high in the cytoplasm. Cytosolic ADP-ribosyl cyclase activity is upregulated by nitric oxide/cyclic GMP-dependent phosphorylation. Stimulation of muscarinic and beta-adrenergic receptors activates membrane-bound ADP-ribosyl cyclase via G proteins within membranes of neuronal tumor cells and cortical astrocytes. These findings strongly suggest that cADPR is a second messenger in Ca2+ signaling in the nervous system, although many intriguing issues remain to be addressed before this identity is confirmed.     

The second messenger cyclic GMP mediates activation in Ancylostoma caninum infective larvae

The developmentally arrested infective larva (L(3)) of hookworms encounters a host-specific signal during infection that initiates previously suspended developmental pathways. Activated L(3) express a parasitic gene set that encodes proteins involved in moulting, growth and development to the adult stage. Early events in this activation to parasitism can be investigated using an in vitro larval feeding assay. When Ancylostoma caninum L(3) are exposed to a host-like stimulus, they resume feeding and release molecules involved in infection. The dauer larva of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm L(3). Recovery from the dauer stage has been proposed as a model for the transition to parasitism in hookworm. Dauer formation and recovery involve several tightly regulated pathways, including a cyclic GMP mediated signalling pathway. To determine if hookworm L(3) activation uses a similar pathway, 8-bromo-cyclic GMP, a membrane permeant analogue of cyclic GMP, was tested for its ability to stimulate feeding. Populations of L(3) incubated with 0.5 mM 8-bromo-cyclic GMP began feeding, and reached maximum feeding at 3.5-5.0 mM. Unlike the serum stimulus, which triggers feeding after a short exposure, 8-bromo-cyclic GMP must be present throughout the entire incubation. Both serum stimulated and 8-bromo-cyclic GMP stimulated L(3) secreted Ancylostoma secreted protein 1, indicating that the stimuli activate the same pathway. Serum and 8-bromo-cyclic GMP stimulated feeding was inhibited by atropine, a muscarinic receptor antagonist. However, only serum stimulated feeding was inhibited by 4,7-phenanthroline, a non-chelating isomer of the metalloprotease inhibitor 1,10-phenantholine. The results indicate that cyclic GMP mediates activation in hookworm larvae, and that a muscarinic receptor is involved in activation. This also suggests that hookworm activation and dauer recovery share similar signalling pathways, and that C. elegans dauer recovery can be used as a model for the transition to parasitism in hookworms.     

Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated activation of murine N9 microglial cell line

Lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll-like receptor complex (TLR-4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca2+]i) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide-stimulated murine N9 microglial cells, cyclic ADP-ribose, a universal and potent Ca2+ mobiliser generated from NAD+ by ADP-ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7-fold increase in the concentration of intracellular cyclic ADP-ribose. This event was paralleled by doubling of the basal [Ca2+]i levels, which was largely prevented by the cyclic ADP-ribose antagonists 8-Br-cyclic ADP-ribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide-induced microglia-activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor alpha. The identification of cyclic ADP-ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro-inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.     

Cyclic AMP as the second hormonal messenger in the in vitro folliculogenesis of the foetal thyroid gland of the rat

Thyroid-stimulating hormone, the catecholamine isoproterenol, and prostaglandins E1 and E2, all substances known to increase cAMP concentration in thyroid tissue, accelerate the formation of follicular cavities in explanted thyroid of 15-day-old rat foetuses. Dibutyryl-cAMP added to the medium, but not sodium fluoride, also stimulates the folliculogenesis. Since fluoride stimulates membrane adenylate cyclase but does not increase the intracellular cAMP level, these results show that cAMP is involved as a second messenger in the activation of foetal thyroid morphogenesis induced by hormones. They indicate also that the thyroid gland of the foetal rat is capable of responding to hormonal stimulation as early as the 15th day of pregnancy; this implies that on day 15, the foetal thyroid possesses receptors not only for the thyroid-stimulating hormone, but also for catecholamines and prostaglandins.     

Cyclic GMP regulates free cytosolic calcium in the pancreatic acinar cell

The present studies were performed in order to measure the effects of cyclic GMP (cGMP) on the regulation of free cytosolic calcium [( Ca2+]i) in the pancreatic acinar cell. In guinea pig dispersed pancreatic acini the findings demonstrated that the Ca2+ ionophore, Br A23187, caused a sustained increase in [Ca2+]i in the presence of 3 mM CaCl2 in the media and a transient 20 fold rise in cellular cGMP followed by a sustained 3-4 fold rise in cellular cGMP. Increasing cellular cGMP with nitroprusside, hydroxylamine or dibutyryl cGMP had no effect on resting [Ca2+]i. However, these agents attenuated the increase in [Ca2+]i resulting from Br A23187-induced Ca2+ influx. Nitroprusside also attenuated the carbachol-induced sustained rise in [Ca2+]i that resulted from Ca2+ influx. The nitroprusside effect on carbachol-stimulated acini occurred without decreasing Ca2+ influx across the plasma membrane or alteration in the mobilization of Ca2+ from the intracellular agonist-sensitive pool. Inhibition of the increase in cellular cGMP caused by Br A23187 by the guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), resulted in augmentation of the increase in [Ca2+]i. This augmentation was reversed with dibutyryl cGMP. These results indicated that cGMP regulated [Ca2+]i in the pancreatic acinar cell. The mechanism involves the removal of Ca2+ from the cytoplasm.     

Dexamethasone-mediated inhibition of human T cell growth factor and gamma-interferon messenger RNA

Glucocorticoids suppress the proliferation of human T lymphocytes. Activated T lymphocytes require T cell growth factor (TCGF) for proliferation. TCGF is produced by a subset of T lymphocytes, and this production is regulated at the TCGF mRNA level. Dexamethasone, a synthetic glucocorticoid, strongly inhibits the synthesis of TCGF mRNA in human normal peripheral blood lymphocytes stimulated in culture with phytohemagglutinin. It also inhibits the accumulation of gamma-interferon mRNA in these cells. This dual effect may in part explain some of the immunosuppressive and anti-inflammatory effects of glucocorticoids.     

Cyclic ADP-ribose: a novel Ca2+-mobilising second messenger.

Cyclic ADP-ribose (cADPR) was discovered as a potent Ca2+-mobilising natural compound in sea urchin eggs. Recently, cADPR was reported to stimulate Ca2+ signalling in several higher eukaryotic cell systems (e.g., smooth and cardiac muscle cells, neuronal cells, adrenal chromaffin cells, macrophages, pancreatic acinar cells and T-lymphocytes). The following aspects of the role of cADPR as a Ca2+-mobilising second messenger are reviewed: coupling of metabolism of cADPR to stimulation of receptors in the plasma membrane, properties and pharmacology of Ca2+ release by cADPR and the involvement of cADPR in Ca2+ entry.     

Enhanced production of human gamma-interferon in nylon column-fractionated cell cultures

The production of human gamma-interferon (HuIFN-gamma) in unfractionated and nylon wool column-fractionated leukocyte cell cultures stimulated with PMA and PHA was investigated. Production was studied with normal and reduced autologous serum protein levels in 96-hr spinner cultures. A 10- to 15-fold enhancement of production and a 50-fold increase in specific activity of crude HuIFN-gamma was demonstrated in nylon column-fractionated/reduced serum cell cultures. Kinetic analysis revealed a production rate maximum within 6 hr of induction in unprocessed cell cultures, whereas production occurred at an essentially constant rate for 48 hr in fractionated cell cultures.     

Neural cell adhesion molecules influence second messenger systems

We have investigated the influence of the neural cell adhesion molecules L1 and N-CAM on second messenger systems using a PC12 rat pheochromocytoma cell line as a model and triggering cell surface receptors by specific antibody binding. Antibodies directed against L1 and N-CAM, but not against other cell surface components, reduce intracellular levels of the inositol phosphates IP2 and IP3, while intracellular levels of cAMP are unaffected. Antibodies against L1 and N-CAM also reduce intracellular pH and increase intracellular Ca2+ by opening Ca2+ channels in a pertussis toxin-inhibitable manner, suggesting the involvement of a G protein in the signal transduction process. Cross-linking of the adhesion molecules on the surface membrane is not required for the effects to occur. Furthermore, adhesion of single PC12 cells to each other elicits effects on intracellular pH and Ca2+ similar to those seen after application, underscoring the physiological significance of the observed changes.     

Pertussis toxin triggers rapid second messenger production in human T lymphocytes

Pertussis toxin (PT) is a known mitogen for T lymphocytes. The mechanism by which the toxin stimulates proliferation has remained obscure and paradoxical because, in some types of cells, the toxin also inhibits growth factor-mediated signal transduction. It has previously been shown that the adenosine-diphosphate ribosyltransferase activity of the toxin is not required to produce the mitogenic effect. A biochemical explanation for the mitogenic activity has therefore remained obscure. We investigated the biochemical basis for the mitogenic activity of PT by using the transformed human T cell line, Jurkat. PT stimulated a rapid rise in cytosolic-free [Ca2+] from both intra- and extracellular sources. This was associated with an increase in the cellular diacylglycerol and inositol triphosphate levels with a concomitant decrease in the levels of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. The half-maximal effective dose of PT was 1.7 nM. PT also stimulated the production of interleukin 2. Only the holotoxin or B-oligomer (the presumptive membrane-binding subunit) was capable of stimulating an increase in [Ca2+] in these cells. This activity of PT mimicked that of some anti-T3-T cell antigen receptor complex monoclonal antibodies that also stimulate increases in the second messengers, diacylglycerol and Ca2+. The effects of PT and anti-T3 complex antibody were identical and not additive in Jurkat cells, suggesting that both agents were activating the same signal transduction pathway. These data provide a mechanistic explanation for the mitogenic effects of PT and suggest that the toxin may be interacting with a specific receptor in the T lymphocyte plasma membrane.     

Identification of second messenger mediating signal transduction in the olfactory receptor cell

One of the biggest controversial issues in the research of olfaction has been the mechanism underlying response generation to odorants that have been shown to fail to produce cAMP when tested by biochemical assays with olfactory ciliary preparations. Such observations are actually the original source proposing a possibility for the presence of multiple and parallel transduction pathways. In this study the activity of transduction channels in the olfactory cilia was recorded in cells that retained their abilities of responding to odorants that have been reported to produce InsP3 (instead of producing cAMP, and therefore tentatively termed "InsP3 odorants"). At the same time, the cytoplasmic cNMP concentration ([cNMP]i) was manipulated through the photolysis of caged compounds to examine their real-time interactions with odorant responses. Properties of responses induced by both InsP3 odorants and cytoplasmic cNMP resembled each other in their unique characteristics. Reversal potentials of currents were 2 mV for InsP3 odorant responses and 3 mV for responses induced by cNMP. Current and voltage (I-V) relations showed slight outward rectification. Both responses showed voltage-dependent adaptation when examined with double pulse protocols. When brief pulses of the InsP3 odorant and cytoplasmic cNMP were applied alternatively, responses expressed cross-adaptation with each other. Furthermore, both responses were additive in a manner as predicted quantitatively by the theory that signal transduction is mediated by the increase in cytoplasmic cAMP. With InsP3 odorants, actually, remarkable responses could be detected in a small fraction of cells ( approximately 2%), explaining the observation for a small production of cAMP in ciliary preparations obtained from the entire epithelium. The data will provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

A second look at the second messenger hypothesis

Several hundred hormones, neurotransmitters, growth factors and other "first messengers" bind to specific cell membrane receptors and induce a myriad of effects: short term, transport, metabolic, mitotic and regulation of thousands of specific genes. Yet, less than a dozen "second messengers" have been clearly established to date. Even allowing for the discovery of a large number of additional second messengers, there remains a paradox in terms of information-transfer within the cell: how can so many specific signals produce so many effects through so few relatively nonspecific intermediates? We consider several possible solutions to this paradox, including the hypothesis that signal specificity is encoded in part in the primary structure of the receptor.     

Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell

The present studies were performed to determine the role of cyclic GMP in regulating agonist mediated calcium entry in the pancreatic acinar cell. In guinea pig-dispersed pancreatic acini the findings demonstrated that carbachol stimulated a transient 20-40-fold rise in cellular cyclic GMP followed by a sustained 3-4-fold rise in cellular cyclic GMP. The guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), caused a dose-dependent inhibition of carbachol-stimulated increases in cellular cyclic GMP both during the initial transient large increase in cyclic GMP and the sustained increase in cyclic GMP. LY83583 also inhibited cellular Ca2+ influx during carbachol stimulation and reloading of the agonist-sensitive pool of Ca2+ at the termination of carbachol stimulation with atropine. The effect of the inhibition on reloading of the agonist-sensitive pool was secondary to its effects on the plasma membrane C2+ entry. The addition of dibutyryl cyclic GMP to LY83583-treated acini restored Ca2+ influx across the plasma membrane. Nitroprusside increased both cellular cyclic GMP and the rate of Ca2+ influx. During periods when plasma membrane Ca2+ entry was activated, cellular cyclic GMP levels were increased. These results suggest that agonist-induced increases in cellular cyclic GMP are necessary and sufficient to mediate the effects of the agonist on the plasma membrane Ca2+ entry mechanism.