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It is clear that multiple signalling pathways regulate the critical balance between cell death and survival in myocardial ischaemia–reperfusion. Recent attention has focused on the activation of survival or salvage kinases, particularly during reperfusion, as a common mechanism of many cardioprotective interventions. The phosphatidyl inositol 3′‐hydroxy kinase/Akt complex (PI3K/Akt) and p42/p44 mitogen‐activated protein kinase cascades have been widely promoted in this respect but the cyclic guanosine 3′,5′‐monophosphate/cGMP‐dependent protein kinase (cGMP/PKG) signal transduction cassette has been less systematically investigated as a survival cascade. We propose that activation of the cGMP/PKG signalling pathway, following activation of soluble or particulate guanylate cyclases, may play a pivotal role in survival signalling in ischaemia–reperfusion, especially in the classical preconditioning, delayed preconditioning and postconditioning paradigms. The resurgence of interest in reperfusion injury, largely as a result of postconditioning‐related research, has confirmed that the cGMP/PKG pathway is a pivotal salvage mechanism in reperfusion. Numerous studies suggest that the infarct‐limiting effects of preconditioning and postconditioning, exogenously donated nitric oxide (NO), natriuretic peptides, phosphodiesterase inhibitors, and other diverse drugs and mediators such as HMG co‐A reductase inhibitors (statins), Rho‐kinase inhibitors and adrenomedullin, whether given before and during ischaemia, or specifically at the onset of reperfusion, may be mediated by activation or enhancement of the cGMP pathway, either directly or indirectly via endogenous NO generation downstream of PI3K/Akt. Putative mechanisms of protection include PKG regulation of Ca2+ homeostasis through the modification of sarcoplasmic reticulum Ca2+ uptake mechanisms, and PKG‐induced opening of ATP‐sensitive K+ channels during ischaemia and/or reperfusion. At present, significant technical obstacles in defining the precise roles played by cGMP/PKG signalling include the heavy reliance on pharmacological PKG inhibitors of uncertain selectivity, difficulties in determining PKG activity in intact tissue, and the growing recognition that intracellular compartmentalisation of the cGMP pool may contribute markedly to the nucleotide's biological actions and biochemical determination. Overall, the body of experimental evidence suggests that cGMP/PKG survival signalling ameliorates irreversible injury associated with ischaemia–reperfusion and may be a tractable therapeutic target. British Journal of Pharmacology (2007) 152, 855–869; doi:10.1038/sj.bjp.0707409; published online 13 August 2007

Agents that enhance production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) ameliorate the progression of renal fibrosis. However, the molecular mechanism of this process is not fully understood. We hypothesize that the antifibrotic effects of cGMP and cGMP‐dependent kinase I (cGKI) are mediated via regulation of the TGFβ signalling pathway, both via ERK and the Smad‐dependent route. Kidney fibrosis was induced by unilateral ureter obstruction (UUO) in wild‐type and cGKI‐deficient (cGKI‐KO) mice. The cGMP/cGKI signalling pathway was activated by application of the soluble guanylate cyclase (sGC) stimulator BAY 41‐8543 (BAY), beginning 1 day after UUO. After 7 days, the antifibrotic effects of BAY were analysed by measuring mRNA and protein expression of characteristic fibrotic biomarkers. The effects of cGMP/TGFβ on cultured fibroblasts were also analysed in vitro. BAY application influenced the activity of the extracellular matrix (ECM)‐degrading matrix metalloproteases (MMP2 and MMP9) and their inhibitor tissue inhibitors of metalloproteinase‐1, the secretion of cytokines (e.g. IL‐6) and the expression pattern of ECM proteins (e.g. collagen, fibronectin) and profibrotic mediators (e.g. connective tissue growth factors and plasminogen‐activator inhibitor‐1). Activation of the cGMP/cGKI signalling pathway showed protective effects against fibrosis which were mediated by inhibition of P‐Erk1/2 and translocation of P‐smad3. The elucidation of these signalling mechanisms might support the development of new therapeutic options regarding cGMP/cGKI‐mediated antifibrotic actions. In renal fibrosis, activation of cGMP/cGMP‐dependent kinase I signalling by application of soluble guanylate cyclase stimulator BAY 41‐8543 showed antifibrotic effects by inhibition of P‐Erk1/2 and P‐smad3 translocation. Thereby, BAY influenced the activity of matrix metalloproteases (MMP2 and MMP9), expression pattern of extracellular matrix proteins (collagen, fibronectin) and profibrotic mediators (connective tissue growth factors, plasminogen‐activator inhibitor‐1).

Integration of multiple signals into the canonical BMP/Smad pathway poses a big challenge during the course of embryogenesis and tissue homeostasis. Here, we show that cyclic guanosine 3′,5′‐monophosphate (cGMP)‐dependent kinase I (cGKI) modulates BMP receptors and Smads, providing a novel mechanism enhancing BMP signalling. cGKI, a key mediator of vasodilation and hypertension diseases, interacts with and phosphorylates the BMP type II receptor (BMPRII). In response to BMP‐2, cGKI then dissociates from the receptors, associates with activated Smads, and undergoes nuclear translocation. In the nucleus, cGKI binds with Smad1 and the general transcription factor TFII‐I to promoters of BMP target genes such as Id1 to enhance transcriptional activation. Accordingly, cGKI has a dual function in BMP signalling: (1) it modulates BMP receptor/Smad activity at the plasma membrane and (2) after redistribution to the nucleus, it further regulates transcription as a nuclear co‐factor for Smads. Consequently, cellular defects caused by mutations in BMPRII, found in pulmonary arterial hypertension patients, were compensated through cGKI, supporting the positive action of cGKI on BMP‐induced Smad signalling downstream of the receptors.

Nitric oxide (NO) is a ubiquitous signalling molecule with widespread distribution in prokaryotes and eukaryotes where it is involved in countless physiological processes. While the mechanisms governing NO synthesis and signalling are well established in animals, the situation is less clear in the green lineage. Recent investigations have shown that NO synthase (NOS), the major enzymatic source for NO in animals, is absent in land plants but present in a limited number of algae. First detailed analysis highlighted that these new NOSs are functional but display specific structural features and probably original catalytic activities. Completing this picture, analyses were undertaken in order to investigate whether major components of the prototypic NO/cyclic GMP signalling cascades mediating many physiological effects of NO in animals were also present in plants. Only few homologues of soluble guanylate cyclases, cGMP-dependent protein kinases, cyclic nucleotide-gated channels and cGMP-regulated phosphodiesterases, were identified in some algal species and their presence did not correlate with that of NOSs. In contrast, GSNO reductase, a critical regulator of S-nitrosothiols, was recurrently found. Overall, these findings highlight that plants do not mediate NO signalling through the classical NO/cGMP-signalling module and support the concept that S-nitrosation is a ubiquitous NO-dependent signalling mechanism.

Our previous data demonstrated that type II cGMP-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced MAPK/ERK/JNK-mediated signal transduction through inhibiting the phosphorylation/activation of the epidermal growth factor receptor (EGFR). Since the EGFR also binds with several other ligands as well as EGF, the present study was designed to investigate whether PKG II inhibited transforming growth factor-α (TGF-α), betacellulin (BTC) and epiregulin (EPR) induced phosphorylation/activation of the EGFR and consequent MAPK/ERK-mediated signaling. The human gastric cancer cell line AGS, was infected with adenoviral constructs encoding cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II and was treated with 8-pCPT-cGMP to activate the kinase. Western blotting was applied to detect the phosphorylation of EGFR and MAPK/ERK. The results demonstrated that treatment with EGF (100 ng/ml, 5 min), TGF-α (100 ng/ml, 5 min), BTC (100 ng/ml, 5 min) and EPR (100 ng/ml, 5 min) increased the tyrosine (tyr) 1068 phosphorylation of the EGFR and the threonine (thr) 202/tyr 204 phosphorylation of MAPK/ERK. Infecting the cells with Ad-PKG II and stimulating the kinase with 8-pCPT-cGMP efficiently inhibited the phosphorylation of the EGFR and MAPK/ERK induced by EGF, TGF-α, BTC and EPR. The results indicated that PKG II also inhibits the activation of the EGFR caused by diverse ligands of the receptor.

Microalgae are competitive and commercial sources for health-benefit carotenoids. In this study, a Chromochloris zofingiensis mutant (Cz-pkg), which does not shut off its photosystem and stays green upon glucose treatment, was generated and characterized. Cz-pkg was developed by treating the algal cells with a chemical mutagen as N-methyl-N’-nitro-N-nitrosoguanidine and followed by a color-based colony screening approach. Cz-pkg was found to contain a dysfunctional cGMP-dependent protein kinase (PKG). By cultivated with CO2 aeration under mixotrophy, the mutant accumulated lutein up to 31.93 ± 1.91 mg L−1 with a productivity of 10.57 ± 0.73 mg L−1 day−1, which were about 2.5- and 8.5-fold of its mother strain. Besides, the lutein content of Cz-pkg could reach 7.73 ± 0.52 mg g−1 of dry weight, which is much higher than that of marigold flower, the most common commercial source of lutein. Transcriptomic analysis revealed that in the mutant Cz-pkg, most of the genes involved in the biosynthesis of lutein and chlorophylls were not down-regulated upon glucose addition, suggesting that PKG may regulate the metabolisms of photosynthetic pigments. This study demonstrated that Cz-pkg could serve as a promising strain for both lutein production and glucose sensing study.

Bitter taste receptors (T2Rs) are GPCRs involved in detection of bitter compounds by type 2 taste cells of the tongue, but are also expressed in other tissues throughout the body, including the airways, gastrointestinal tract, and brain. These T2Rs can be activated by several bacterial products and regulate innate immune responses in several cell types. Expression of T2Rs has been demonstrated in immune cells like neutrophils; however, the molecular details of their signaling are unknown. We examined mechanisms of T2R signaling in primary human monocyte-derived unprimed (M0) macrophages (MΦs) using live cell imaging techniques. Known bitter compounds and bacterial T2R agonists activated low-level calcium signals through a pertussis toxin (PTX)-sensitive, phospholipase C-dependent, and inositol trisphosphate receptor-dependent calcium release pathway. These calcium signals activated low-level nitric oxide (NO) production via endothelial and neuronal NO synthase (NOS) isoforms. NO production increased cellular cGMP and enhanced acute phagocytosis ~ threefold over 30–60 min via protein kinase G. In parallel with calcium elevation, T2R activation lowered cAMP, also through a PTX-sensitive pathway. The cAMP decrease also contributed to enhanced phagocytosis. Moreover, a co-culture model with airway epithelial cells demonstrated that NO produced by epithelial cells can also acutely enhance MΦ phagocytosis. Together, these data define MΦ T2R signal transduction and support an immune recognition role for T2Rs in MΦ cell physiology.

C‐type natriuretic peptide (CNP) and its receptor guanylyl cyclase (GC‐B) are expressed in the heart and modulate cardiac contractility in a cGMP‐dependent manner. Since the distal cellular signalling pathways remain unclear, we evaluated the peptide effects on cardiac function and calcium regulation in wild‐type (WT) and transgenic mice with cardiac overexpression of cGMP‐dependent protein kinase I (PKG ITG). In isolated, perfused working WT hearts, CNP (10 nM) provoked an immediate increase in the maximal rates of contraction and relaxation, a small increase in the left ventricular systolic pressure and a decrease in the time of relaxation. These changes in cardiac function were accompanied by a marked increase in the levels of Ser16‐phosphorylated phospholamban (PLB). In PKG ITG hearts, the effects of CNP on cardiac contractility and relaxation as well as on PLB phosphorylation were markedly enhanced. CNP increased cell shortening and systolic Cai2+ levels, and accelerated Cai2+ decay in isolated, Indo‐1/AM‐loaded WT cardiomyocytes, and these effects were enhanced in PKG I‐overexpressing cardiomyocytes. 8‐pCPT‐cGMP, a membrane‐permeable PKG activator, mimicked the contractile and molecular actions of CNP, the effects again being more pronounced in PKG ITG hearts. In contrast, the cardiac reponses to β‐adrenergic stimulation were not different between genotypes. Taken together, our data indicate that PKG I is a downstream target activated by the CNP/GC‐B/cGMP‐signalling pathway in cardiac myocytes. cGMP/PKG I‐stimulated phosphorylation of PLB and subsequent activation of the sarcoplasmic reticulum Ca2+ pump appear to mediate the positive inotropic and lusitropic responses to CNP. British Journal of Pharmacology (2003) 140, 1227–1236. doi:10.1038/sj.bjp.0705567

Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4 ⁻/⁻ mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430–460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor. Significance Non–image-forming opsins such as Opn4 regulate important physiological functions such as circadian photo-entrainment and affect. The recent discovery that melanopsin (Opn4) functions outside the central nervous system prompted us to explore a potential role for this receptor in blood vessel regulation. We hypothesized that Opn4-mediated signaling might explain the phenomenon of photorelaxation, for which a mechanism has remained elusive. We report the presence in blood vessels of Opn4 and demonstrate that it mediates wavelength-specific, light-dependent vascular relaxation. This photorelaxation signal transduction involves cGMP and phosphodiesterase 6, but not protein kinase G. Furthermore it is regulated by G protein-coupled receptor kinase 2 and involves vascular hyperpolarization. This receptor pathway can be harnessed for wavelength-specific light-based therapy in the treatment of diseases that involve altered vasoreactivity.

Nitric oxide (NO) and the C‐type natriuretic peptide (CNP) exert their action via stimulation of the cyclic GMP (cGMP)‐signaling pathway, which includes the activation of cGMP‐dependent protein kinases (PKG). The present report shows that the activation of PKG by local application of 8‐bromo‐cGMP in the caudate–putamen reduced the expression of the epigenetic markers, methyl‐CpG‐binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2), in dopaminergic projection areas of cocaine‐treated rats. An effect of lesser amplitude was observed when rats were not injected with cocaine. We also studied the effect of PKG overexpression by injecting a plasmid vector containing the human PKG‐Iα cDNA in either the caudate–putamen or the ventral tegmental area. Injection in the caudate–putamen reduced the epigenetic parameters with higher amplitude than the cGMP analog. The effect was abolished by the injection of a selective PKG inhibitor, confirming that it was due to PKG‐dependent phosphorylation. As MeCP2 and HDAC2 modulate dynamic functions in the adult brain such as memory formation and synaptic plasticity, the downregulation of expression by PKG suggests that the cGMP pathway affects cognitive processes through a mechanism that comprises the MeCP2/HDAC2 complex and the subsequent control of gene silencing. The present report shows that activation of cGMP‐dependent protein kinase by local application of 8‐bromo‐cGMP in the caudate–putamen reduced the expression of the epigenetic markers, methyl‐CpG‐binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) , in dopaminergic projection areas. We also studied the effect of PKG overexpression by injecting a plasmid vector containing the human PKG‐Iα cDNA in either the ventral tegmental area or the caudate–putamen. Data suggest that the cGMP pathway affects cognitive processes through a mechanism that comprises the MeCP2/HDAC2 complex and the subsequent control of gene silencing.