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Chemical genetics and a global comparative analysis of phosphorylation and phospholipids in vivo shows that PKG is the upstream regulator that induces calcium signals that enables Plasmodium to progress through its complex life cycle., Many critical events in the Plasmodium life cycle rely on the controlled release of Ca2+ from intracellular stores to activate stage-specific Ca2+-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca2+ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca2+ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca2+ effectors, PKG emerges as a unifying factor to control multiple cellular Ca2+ signals essential for malaria parasite development and transmission., Author Summary Malaria, caused by Plasmodium spp. parasites, is a profound human health problem. Plasmodium parasites progress through a complex life cycle as they move between infected humans and blood-feeding mosquitoes. We know that tight regulation of calcium ion levels within the cytosol of the parasite is critical to control multiple signalling events in their life cycle. However, how these calcium levels are controlled remains a mystery. Here, we show that a single protein kinase, the cGMP-dependent protein kinase G (PKG), controls the calcium signals that are critical at three different points of the life cycle: (1) for the exit of the merozoite form of the parasite from human erythrocytes (red blood cells), (2) for the cellular activation that happens when Plasmodium sexual transmission stages are ingested by a blood-feeding mosquito, and (3) for the productive gliding of the ookinete, which is the parasite stage that invades the mosquito midgut. We provide initial evidence that the universal role of PKG relies on the production of lipid precursors which then give rise to inositol (1,4,5)-trisphosphate (IP3), a messenger molecule that serves as a signal for the release of calcium from stores within the parasite. This signalling pathway provides a potential target to block both malaria development in the human host and transmission to the mosquito vector.

Changes in metabolic state alter foraging behavior and food preference in animals. Here I show that normally attractive food becomes repulsive to C. elegans if animals are chronically under-nourished due to alimentary tract defects. This behavior plasticity is achieved in two ways: increased food leaving and induction of aversive behaviour towards food. A particularly strong food avoider is defective in the chitin synthase that makes the pharyngeal lining. Food avoidance induced by underfeeding is mediated by cGMP signaling in the olfactory neurons AWC and AWB, the gustatory neurons ASJ and ASK. Food avoidance is enhanced by increased population density and is reduced if the animals are unable to correctly interpret their nutritional state due to defects in the AMP kinase or TOR/S6kinase pathways. The TGF-β/DBL-1 suppresses food avoidance and the cellular basis for this is distinct from its role in aversive olfactory learning to harmful food. This study suggests that nutritional state feedback via nutrient sensors, population size, and olfactory neurons guide food preference in C. elegans.

Background Membrane bound guanylyl cyclase-G (mGC-G), a novel form of GC mediates ischemia and reperfusion (IR)-induced renal injury. We investigated the roles of mGC-G in intestinal IR-induced jejunal damage, inflammation, and apoptosis. Materials and methods Male C57BL/6 wild-type (WT) and mGC-G gene knockout (KO) mice were treated with a sham operation or 45 min of superior mesenteric arterial obstruction followed by 3, 6, 12, or 24 h of reperfusion. Results Sham-operated KO mice had significantly lower plasma nitrate and nitrite (NOx) levels and jejunal villus height, crypt depth, and protein expression of phosphorylated-nuclear factor-kappa-B (NF-κB), phosphorylated-c-Jun N-terminal kinases (JNK) 2/3, phosphorylated-p38, and B-cell lymphoma-2 (Bcl-2). They had significantly greater jejunal interleukin-6 mRNA, cytochrome c protein, and apoptotic index compared with sham-operated WT mice. Intestinal IR significantly decreased plasma NOx in WT mice and increased plasma NOx in KO mice. The jejunal apoptotic index and caspase 3 activities were significantly increased, and nuclear phosphorylated-NF-κB and phosphorylated-p38 protein were significantly decreased in WT, but not KO mice with intestinal IR. After reperfusion, KO mice had an earlier decrease in jejunal cyclic GMP, and WT mice had an earlier increase in jejunal proliferation and a later increase in cytosol inhibitor of kappa-B-alpha. Intestinal IR induced greater increases in plasma and jejunal interleukin-6 protein in WT mice and a greater increase in jejunal interleukin-6 mRNA in KO mice. Conclusions mGC-G is involved in the maintenance of jejunal integrity and intestinal IR-induced inflammation and apoptosis. These results suggest that targeting cGMP pathway might be a potential strategy to alleviate IR-induced jejunal damages.

Heart failure with preserved ejection fraction (HFpEF) is a growing public health problem that accounts for approximately half of all prevalent heart failure (HF).[1][1]–[2][2] Once believed to carry a favorable prognosis compared with HF and reduced ejection fraction (HFrEF), contemporary data

Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In this study we examined established human breast cancer cell lines and patients’ primary breast cancer tissues for expression of genetic components of phosphodiesterase 6 (PDE6), a cGMP-specific PDE involved in transduction of the light signal, and previously thought to be selectively expressed in photoreceptors. By microarray analysis we find highly significant expression of mRNA for the PDE6B, PDE6C, and PDE6D genes in both the cell lines and patients’ tissues, minimal expression of PDE6A and PDE6G and no expression of PDE6H. Using antibody specific for PDE6β, we find expression of PDE6B protein in a wide range of patients’ tissues by immunohistochemistry, and in MCF-7 breast cancer cells by immunofluorescence and Western blot analysis. Considerable expression of key circadian genes, PERIOD 2, CLOCK, TIMELESS, CRYPTOCHROME 1, and CRYPTOCHROME 2 was also seen in all breast cancer cell lines and all patients’ breast cancer tissues. These studies indicate that genes for PDE6 and control of circadian rhythm are expressed in human breast cancer cells and tissues and may play a role in transducing the effects of light on breast cancer.

In Caenorhabditis elegans, satiety quiescence mimics behavioral aspects of satiety and postprandial sleep in mammals. On the basis of calcium-imaging, genetics, and behavioral studies, here we report that a pair of amphid neurons, ASI, is activated by nutrition and regulates worms' behavioral states specifically promoting satiety quiescence; ASI inhibits the switch from quiescence to dwelling (a browsing state) and accelerates the switch from dwelling to quiescence. The canonical TGFβ pathway, whose ligand is released from ASI, regulates satiety quiescence. The mutants of a ligand, a receptor and SMADs in the TGFβ pathway all eat more and show less quiescence than wild-type. The TGFβ receptor in downstream neurons RIM and RIC is sufficient for worms to exhibit satiety quiescence, suggesting neuronal connection from ASI to RIM and RIC is essential for feeding regulation through the TGFβ pathway. ASI also regulates satiety quiescence partly through cGMP signaling; restoring cGMP signaling in ASI rescues the satiety quiescence defect of cGMP signaling mutants. From these results, we propose that TGFβ and cGMP pathways in ASI connect nutritional status to promotion of satiety quiescence, a sleep-like behavioral state.

As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) also act as second messengers. Hydrolysis by phosphodiesterases (PDEs) is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-)labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs) are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.

In the hippocampus, as in many other CNS areas, nitric oxide (NO) participates in synaptic plasticity, manifested as changes in pre- and/or postsynaptic function. While it is known that these changes are brought about by cGMP following activation of guanylyl cyclase-coupled NO receptors attempts to locate cGMP by immunocytochemistry in hippocampal slices in response to NO have failed to detect the cGMP elevation where expected, i.e. in the pyramidal neurones. Instead, astrocytes, unidentified varicose fibres and GABA-ergic nerve terminals are reported to be the prominent NO targets, raising the possibility that NO acts indirectly via other cells. We have re-investigated the distribution of cGMP generated in response to endogenous and exogenous NO in hippocampal slices using immunohistochemistry and new conditions designed to optimise cGMP accumulation and, hence, its detectability. The conditions included use of tissue from the developing rat hippocampus, a potent inhibitor of phosphodiesterase-2, and an allosteric enhancer of the NO-receptive guanylyl cyclase. Under these conditions, cGMP was formed in response to endogenous NO and was found in a population of pyramidal cell somata in area CA3 and subiculum as well as in structures described previously. The additional presence of exogenous NO resulted in hippocampal cGMP reaching the highest level recorded for brain tissue (1700 pmol/mg protein) and in cGMP immunolabelling throughout the pyramidal cell layer. Populations of axons and interneurones were also stained. According with these results, immunohistochemistry for the common NO receptor β1-subunit indicated widespread expression. A similar staining pattern for the α1-subunit with an antibody used previously in the hippocampus and elsewhere, however, proved to be artefactual. The results indicate that the targets of NO in the hippocampus are more varied and extensive than previous evidence had suggested and, in particular, that the pyramidal neurones participating in NO-dependent synaptic plasticity are direct NO targets.

Axonal branching is a key mechanism to form complex circuits in the mature nervous system. Depending on their functions, axons can branch extensively to send information to their distinct targets and establish a unique pattern of connectivity. However, signaling mechanisms regulating axonal branching during the development of the nervous system are still poorly understood. The cGMP signaling pathway including the secreted C-type natriuretic peptide (CNP), its receptor natriuretic peptide receptor 2 (Npr2) and the cGMP-dependent protein kinase Iα (cGKIα) has been shown to control the bifurcation of DRG (dorsal root ganglion) sensory neurons within the spinal cord. Using mutant mouse models it has been demonstrated that in the absence of one of these components, the sensory axons are unable to bifurcate at the entry zone of the spinal cord. Based on these findings, it is intriguing to ask whether other neuronal subpopulations besides the DRG neurons exist and if they use the above mentioned cGMP signaling pathway to form branches emerging from the bifurcating growth cone. To investigate the role of Npr2-dependent axonal branching in other neural systems than DRG neurons, a genetic strategy was used and two mouse models (Npr2LacZ and Npr2CreERT2) were generated, which allow studies on the expression pattern of Npr2 and its influence on axonal branching of single Npr2-expressing axons in the mouse nervous system. While the Npr2LacZ reporter mouse enabled detailed analysis of the localization of the signaling pathway components, the Npr2CreERT2 mouse is a valuable tool for visualizing single axonal projections after crossing with reporter strains. A prominent Npr2 expression was found not only in the DRG, but also in the cranial sensory ganglia of the developing mouse. Using immunohistochemistry, a co-localization of the receptor Npr2 and the kinase cGKIα was detected in neurons of cranial sensory ganglia. To follow the single projections of sensory axons from cranial sensory ganglia and study their branching behavior, the Npr2CreERT2 mouse was crossed with two different reporter strains (Z/AP and mGFP). After induction of Cre recombinase with tamoxifen or 4-hydroxytamoxifen at embryonic day 9.5 or 10.5, single axonal projections could be visualized at embryonic day 12.5 or 13.5, respectively. This genetic approach uncovered two new aspects about central projections of cranial sensory ganglia: (i) almost all central projections of cranial sensory ganglia bifurcate at the entry zone of the hindbrain and (ii) in the absence of Npr2 the sensory axons lose their bifurcational behavior when entering the hindbrain. Hence, the neurons of all cranial sensory ganglia - similar to DRG neurons - use cGMP signaling to bifurcate as soon as they enter the hindbrain., Die axonale Verzweigung ist ein wichtiger Mechanismus, um die komplexen Verschaltungen des Nervensystems zu bilden. Je nach ihrer Funktion können sich Axone weitläufig verzweigen, um die Informationen an ihre verschiedenen Ziele zu senden und ein einzigartiges Muster der Verschaltung zu etablieren. Allerdings sind die Signalmechanismen, die die axonale Verzweigung während der Entwicklung des Nervensystems regulieren, noch weitgehend unverstanden. Ein cGMP-Signalweg kontrolliert die Bifurkation der sensorischen DRG-Neurone (Dorsal root ganglia; Spinalganglien) im Rückenmark und besteht aus folgenden Komponenten: dem C-Typ natriuretischen Peptid (CNP), dem natriuretischen Peptid-Rezeptor-2 (Npr2) und der cGMP-abhängigen Proteinkinase I (cGKIα). In Mausmodellen konnte gezeigt werden, dass in Abwesenheit einer dieser Komponenten die sensorischen DRG-Neurone an der Eintrittszone des Rückenmarks nicht bifurkieren. Offen ist, ob es neben den DRG-Neuronen andere neuronale Subpopulationen gibt, die den cGMP-Signalweg im Rahmen der axonalen Verzweigung nutzen. Um die Rolle der Npr2-abhängigen axonalen Verzweigung in anderen Systemen als DRG-Neuronen zu untersuchen, wurden zwei transgene knock- in-Mausmodelle (Npr2LacZ und Npr2CreERT2) generiert, die es erlauben, das Expressionsmuster von Npr2 und die axonale Verzweigung einzelner Npr2-exprimierender Axone in der Maus zu studieren. Während die Npr2LacZ- Reportermaus es ermöglichte, eine detaillierte Analyse der Lokalisation der cGMP-Komponenten durchzuführen, stellte die Npr2CreERT2-Maus nach Kreuzung mit Reporter-Stämmen (Z/AP und mGFP) ein wertvolles Werkzeug für die Visualisierung einzelner axonaler Verzweigungen dar. Eine starke Npr2-Expression wurde nicht nur in den DRG-Neuronen gefunden, sondern auch in den kranialen sensorischen Ganglien. In Neuronen der kranialen sensorischen Ganglien wurde immunhistochemisch eine Ko-Expression des Rezeptors Npr2 und der Kinase cGKIα detektiert. Um das Bifurkationsverhalten einzelner Axone der kranialen sensorischen Neurone zu verfolgen, wurde die Npr2CreERT2-Maus mit den zwei Reporter-Stämmen gekreuzt. Nach Induktion der Cre-Rekombinase mit Tamoxifen am embryonalen Tag 9.5 bzw. 10.5 konnten einzelne axonalen Verweigungen am embryonalen Tag 12.5 bzw. 13.5 visualisiert werden. Dieser genetische Ansatz deckte zwei neue Aspekte der axonalen Bifurkation auf: (i) fast alle zentralen Projektionen der kranialen sensorischen Axone verzweigen an der Eingangszone des Hinterhirns und (ii) in Abwesenheit von Npr2 verlieren die sensorischen Axone ihre Fähigkeit beim Eintritt ins Hinterhirn zu bifurkieren. Zusammenfassend zeigen diese Untersuchungen, dass die kranialen sensorischen Neurone den cGMP-Signalweg nutzen, um beim Erreichen des Hinterhirns zu bifurkieren.

Calcium-signaling in cells requires a fine-tuned system of calcium-transport proteins involving ion channels, exchangers, and ion-pumps but also calcium-sensor proteins and their targets. Thus, control of physiological responses very often depends on incremental changes of the cytoplasmic calcium concentration, which are sensed by calcium-binding proteins and are further transmitted to specific target proteins. This Review will focus on calcium-signaling in vertebrate photoreceptor cells, where recent physiological and biochemical data indicate that a subset of neuronal calcium sensor proteins named guanylate cyclase-activating proteins (GCAPs) operate in a calcium-relay system, namely, to make gradual responses to small changes in calcium. We will further integrate this mechanism in an existing computational model of phototransduction showing that it is consistent and compatible with the dynamics that are characteristic for the precise operation of the phototransduction pathways.

The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the Gs protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both Gi- and Gs-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation.

Since the discovery of thrombospondin-1 (TSP1) in the 1970s, and the gradual unveiling of four more members of the mammalian TSP family, the field has been in pursuit of two fundamental questions: what are the physiological roles of these large multidomain glycoproteins and which receptors and signaling pathways mediate these functions? This has been a complex task because TSPs interact with multiple receptors and extracellular binding partners via their several domains. The multidomain structure of the TSP subunits, their trimeric or pentameric quaternary structure, and the promiscuity of their receptors combine to present us with a veritable “Rubic’s Cube” of possible functions and mechanisms of action. In this chapter we provide a current overview of progress in solving this puzzle. We present a brief overview of the structure and evolution of the TSP family members and the receptors that recognize various domains of TSPs. These advances have led to the identification of important pathophysiological roles for certain TSP family members and revealed opportunities to develop novel therapeutics.

Natriuretic peptides (NPs) are cardioprotective through the activation of guanylyl cyclase (GC) receptors A and B. CD-NP, also known as cenderitide, is a novel engineered NP that was designed to uniquely serve as a first-in-class dual GC receptor agonist. Recognizing the aldosterone suppressing actions of GC-A activation and the potent inhibitory actions on collagen synthesis and fibroblast proliferation through GC-B activation, the current study was designed to establish the anti-fibrotic actions of CD-NP, administered subcutaneously, in an experimental rat model of early cardiac fibrosis induced by unilateral nephrectomy (UNX). Our results demonstrate that a two week subcutaneous infusion of CD-NP significantly suppresses left ventricular fibrosis and circulating aldosterone, while preserving both systolic and diastolic function, in UNX rats compared to vehicle treated UNX rats. Additionally we also confirmed, in vitro, that CD-NP significantly generates the second messenger, cGMP, through both the GC-A and GC-B receptors. Taken together, this novel dual GC receptor activator may represent an innovative anti-fibrotic therapeutic agent.

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters. Engineering and Physical Sciences Research Council (EPSRC) Basic Technology Research Grant [grant number EP/E032745/1]. WK was also supported by Science Foundation Ireland [grant number 06/CE/B1129]. Deposited by bulk import

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity.

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ~20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation--processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200-1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor (32)PO(4) co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.

A growing body of evidence suggests that hydrogen sulfide (H₂S) is a signaling molecule in mammalian cells. In the cardiovascular system, H₂S enhances vasodilation and angiogenesis. H₂S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K(ATP)); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H₂S-induced vasorelaxation. The effect of H₂S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H₂S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H₂S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H₂S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H₂S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H₂S production) were reduced in vessels of PKG-I knockout mice (PKG-I⁻/⁻). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I⁻/⁻, suggesting that there is a cross-talk between K(ATP) and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.

Receptor desensitization is a ubiquitous regulatory mechanism that defines the activatable pool of receptors, and thus, the ability of cells to respond to environmental stimuli. In recent years, the molecular mechanisms controlling the desensitization of a variety of receptors have been established. However, little is known about the molecular mechanisms that underlie desensitization of natriuretic peptide receptors, including natriuretic peptide receptor-A (NPR-A). Here we report that calcineurin (protein phosphatase 2B, PP2B, PPP3C) regulates homologous desensitization of NPR-A in murine Leydig tumor (MA-10) cells. We demonstrate that both pharmacological inhibition of calcineurin activity and siRNA-mediated suppression of calcineurin expression potentiate atrial natriuretic peptide (ANP)-induced cGMP synthesis. Treatment of MA-10 cells with inhibitors of other phosphoprotein phosphatases had little or no effect on ANP-induced cGMP accumulation. In addition, overexpression of calcineurin blunts ANP-induced cGMP synthesis. We also present data indicating that the inhibition of calcineurin potentiates ANP-induced testosterone production. To better understand the contribution of calcineurin in the regulation of NPR-A activity, we examined the kinetics of ANP-induced cGMP signals. We observed transient ANP-induced cGMP signals, even in the presence of phosphodiesterase inhibitors. Inhibition of both calcineurin and phosphodiesterase dramatically slowed the decay in the response. These observations are consistent with a model in which calcineurin mediated dephosphorylation and desensitization of NPR-A is associated with significant inhibition of cGMP synthesis. PDE activity hydrolyzes cGMP, thus lowering intracellular cGMP toward the basal level. Taken together, these data suggest that calcineurin plays a previously unrecognized role in the desensitization of NPR-A and, thereby, inhibits ANP-mediated increases in testosterone production.

Objective There is clearly a necessity to identify novel non-dopaminergic mechanisms as new therapeutic targets for Parkinson's disease (PD). Among these, the soluble guanylyl cyclase (sGC)-cGMP signaling cascade is emerging as a promising candidate for second messenger-based therapies for the amelioration of PD symptoms. In the present study, we examined the utility of the selective sGC inhibitor 1H-[1], [2], [4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) for reversing basal ganglia dysfunction and akinesia in animal models of PD. Methods The utility of the selective sGC inhibitor ODQ for reversing biochemical, electrophysiological, histochemical, and behavioral correlates of experimental PD was performed in 6-OHDA-lesioned rats and mice chronically treated with MPTP. Results We found that one systemic administration of ODQ is sufficient to reverse the characteristic elevations in striatal cGMP levels, striatal output neuron activity, and metabolic activity in the subthalamic nucleus observed in 6-OHDA-lesioned rats. The latter outcome was reproduced after intrastriatal infusion of ODQ. Systemic administration of ODQ was also effective in improving deficits in forelimb akinesia induced by 6-OHDA and MPTP. Interpretation Pharmacological inhibition of the sGC-cGMP signaling pathway is a promising non-dopaminergic treatment strategy for restoring basal ganglia dysfunction and attenuating motor symptoms associated with PD.

Ciliopathies are pleiotropic and genetically heterogeneous disorders caused by defective development and function of the primary cilium. Bardet-Biedl syndrome (BBS) proteins localize to the base of cilia and undergo intraflagellar transport, and the loss of their functions leads to a multisystemic ciliopathy. Here we report the identification of mutations in guanylate cyclases (GCYs) as modifiers of Caenorhabditis elegans bbs endophenotypes. The loss of GCY-35 or GCY-36 results in suppression of the small body size, developmental delay, and exploration defects exhibited by multiple bbs mutants. Moreover, an effector of cGMP signalling, a cGMP-dependent protein kinase, EGL-4, also modifies bbs mutant defects. We propose that a misregulation of cGMP signalling, which underlies developmental and some behavioural defects of C. elegans bbs mutants, may also contribute to some BBS features in other organisms., Author Summary Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, multisystemic disorder. Defects to the cilium, an evolutionarily conserved organelle, cause ciliopathies, a growing class of diseases that includes BBS. BBS proteins are involved in the vesicular transport of proteins to the cilium and in the process of intraflagellar transport. Here we show that, in addition to sensory defects, Caenorhabditis elegans bbs mutants exhibit reduced body size and delayed developmental timing. The reduced body size phenotype is not fully recapitulated by IFT mutants, suggesting that BBS proteins may have additional functions beyond bridging IFT motors. We further identified that the loss of function mutations in the soluble guanylate cyclase complex, GCY-35/GCY-36, results in a suppression of these defects. Interestingly, GCY-35/GCY-36 influences the body size through a cGMP-dependent protein kinase EGL-4 in a group of body cavity neurons. BBS proteins, on the other hand, function through a non-overlapping set of ciliated sensory neurons to influence cGMP signalling in the body cavity neurons. In conclusion, this study reveals a non-cell autonomous role for sensory cilia in regulating cGMP signalling during development. We propose that aberrant cGMP signalling, essential for a number of cellular processes, may also contribute to some ciliopathy features in other systems.

Background Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. Methodology/Principal Findings We have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A) of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. Conclusions/Significance Our findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.

Nitric oxide signals through activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain) to the effector domain (catalytic domain), in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105) of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca(2+)-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca(2+)-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca(2+) around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca(2+)/Mg(2+)-dependent conformational changes and in the Ca(2+)-dependent monomer-dimer equilibrium. Direct Ca(2+)-binding revealed that all zGCAPs bound at least three Ca(2+). The corresponding apparent affinity constants reflect binding of Ca(2+) with high (≤ 100 nM), medium (0.1-5 µM) and/or low (≥ 5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca(2+)-sensor system in zebrafish rod and cone cells supporting a Ca(2+)-relay model of differential zGCAP operation in these cells.

Author(s): Chai, Yongping; Zhang, Dai-Min; Lin, Yu-Fung | Abstract: BackgroundCyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K(ATP)) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.Methods and findingsSingle-channel recordings of cardiac K(ATP) channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K(ATP)) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H(2)O(2) scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H(2)O(2) also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K(ATP) channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.ConclusionThe present study provides novel evidence that PKG exerts dual regulation of cardiac K(ATP) channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H(2)O(2) in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K(ATP) channels and contribute to cardiac protection against ischemia-reperfusion injury.

The present study examined the effect of the nitric oxide (NO) donor NOC18 on lipopolysaccharide (LPS)-induced NO production to investigate a regulation mechanism of NO production by microglial cells. LPS increased the levels of NO and inducible NO synthase (iNOS) protein in BV-2 murine microglial cells in a concentration-dependent manner. Pretreatment with NOC18 for 24 h concentration-dependently attenuated the LPS-induced iNOS protein expression and NO production. The inhibitory effect of NOC18 on LPS-induced NO production was partially blocked by LY83583, a soluble guanylate cyclase inhibitor. Pretreatment with dibutyryl guanosine-3′,5′-cyclic monophosphate (DBcGMP), a cell-permeable cGMP analogue, for 24 h attenuated partially LPS-induced iNOS protein expression and NO production. Furthermore, the effects of LPS on iNOS and NO production were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125, and LPS-induced phosphorylation of JNK and c-Jun was inhibited by NOC18 and DBcGMP. These results suggest that NO production by microglial cells is controlled by a negative feedback mechanism via the NO/cGMP signaling pathway. Keywords:: nitric oxide (NO), guanosine-3′,5′-cyclic monophosphate (cGMP), microglia, inducible nitric oxide synthase (iNOS), negative-feedback regulation

Background Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, tubular apoptosis and tubulointerstitial fibrosis. Pharmacological stimulation of soluble guanylate cyclase (sGC) and subsequent cGMP production have recently been found to be renoprotective. In this study, the sGC stimulator BAY 41-8543 was given to rats after relief of UUO in order to analyze the effects of enhanced NO/sGC/cGMP signaling on the subsequent renal disease recovery, concerning the renal atrophy, tubular apoptosis and tubulointerstitial fibrosis.

This article briefly reviews the background of endothelium-dependent vasorelaxation, describes the nitric oxide/cGMP/protein kinase pathway and its role in modulating pulmonary vascular tone and remodeling, and describes three approaches that target the nitric oxide/cGMP pathway in the treatment of patients with pulmonary arterial hypertension.

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the α isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A–induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.

Rationale The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated. Objective We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate. Methods and Results Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry. Conclusion These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.