Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.
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PMID:Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. 169 82

We investigated the molecular mechanisms whereby Ca2+ enters the endothelial cytosol and regulates endothelial nitric oxide synthesis L-arginine-dependent nitric oxide synthesis by isolated endothelial cytosol as quantified by activation of a purified soluble guanylate cyclase was concentration-dependently enhanced by free Ca2+ (EC50 0.3 microM). The Ca(2+)-dependent activation was inhibited by the calmodulin antagonists mastoparan, melittin, and calcineurin (IC50 450, 350, and 60 nM, respectively) in a calmodulin-reversible manner. After removal of endogenous calmodulin the Ca(2+)-dependency of endothelial NO synthase was lost, but could be reconstituted with exogenous calmodulin. The results indicate that Ca(2+)-calmodulin directly activates the endothelial nitric oxide synthase, thereby transducing agonist-induced increases in intracellular free Ca2+ concentration to nitric oxide formation from L-arginine, K(+)-induced depolarization of the endothelial cells markedly inhibited the sustained, but not initial phase of the intracellular Ca2+ response to bradykinin, indicating that K(+)-induced depolarization depresses the transmembrane Ca2+ influx. On the contrary, the K+ channel activator Hoe 234 which elicits hyperpolarization of the endothelial cell membrane, augmented the sustained phase of the agonist-induced intracellular Ca2+ signal, but not the resting intracellular Ca2+ level. The effects of K+ and Hoe 234 on the agonist-induced Ca(2+)-response were reflected by corresponding changes in agonist-induced EDRF/NO release. From these data, we suggest that the endothelial membrane potential may play an important role for the extent of agonist-induced Ca2+ influx and, thereby, the endothelial EDRF/NO synthesis.
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PMID:Cellular mechanisms controlling EDRF/NO formation in endothelial cells. 171 54

Protein phosphorylation has been recognized as a major mechanism by which cellular functions are controlled by neurotransmitters and hormones. In this review, applications of molecular biological techniques to the analyses of regulatory mechanisms of protein phosphorylation by four major second messengers, cAMP, cGMP, diacylglycerol, and Ca2+, are described. 1) Complementary DNA of the regulatory subunit of the cAMP-dependent protein kinase was cloned and expressed in E. coli. Point mutations were introduced in order to analyze functional domains of the subunit. 2) The soluble isoform of guanylate cyclase was purified, and a cDNA of its 70-KD subunit was cloned. Cyclic GMP binding to purified cGMP-dependent protein kinase was characterized using a rapid filtration assay. 3) Primary structure of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A) was determined and the presence of the second isoform of the enzyme was shown by the cDNA cloning technique. 4) The regulatory domain of the protein kinase C was expressed in E. coli. Analysis using site-directed mutagenesis revealed that a "zinc finger"-like structure is responsible for the binding of phorbol esters. In these studies, the molecular biological approach has proven to be useful for clarifying the molecular mechanisms of cellular signal transduction related to second messengers and protein phosphorylation.
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PMID:[Second messengers and protein phosphorylation in cellular signal transduction]. 222 19

The localization of cGMP, cGMP-dependent protein kinase, calmodulin and the calmodulin-binding protein calcineurin in Paramecium tetrauelia cells has been examined with immunocytochemical methods. These molecules appeared to be localized to a large extent in the cilia of this protozoan. To ascertain that antibodies had access to all cellular compartments we have used three different preparations for immunocytochemistry: (i) with 'whole cell' preparations immunofluorescent staining for the four molecules was mainly visible in the cilia; (ii) in 'deciliated' Paramecium, staining for cGMP and calmodulin was found in regular patterns on the cell surface most likely representing kinetosomes; (iii) using 'sectioned cells', additional cytoplasmic calmodulin appeared to be associated with glycogen particles as evidenced by the disappearance of the granular staining pattern after preincubation with alpha-amylase. In contrast, cGMP, cGMP-dependent protein kinase and calcineurin fluorescence was only very weak and diffuse in cell bodies. No nuclear fluorescence was detectable after staining with any of the antibodies. Because of the colocalization of cGMP, cGMP-dependent protein kinase, a guanylate cyclase-calmodulin-complex, and calcineurin in cilia from Paramecium, an involvement of these components in the regulation of ciliary activity is discussed.
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PMID:Immunocytochemical localization of cyclic GMP, cGMP-dependent protein kinase, calmodulin and calcineurin in Paramecium tetraurelia. 632 Nov 86

Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.
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PMID:Ca(2+)-binding proteins in the retina: from discovery to etiology of human disease(1). 1110 66

Cyclic GMP, produced in response to nitric oxide and natriuretic peptides, is a key regulator of vascular smooth muscle cell contractility, growth, and differentiation, and is implicated in opposing the pathophysiology of hypertension, cardiac hypertrophy, atherosclerosis, and vascular injury/restenosis. cGMP regulates gene expression both positively and negatively at transcriptional as well as at posttranscriptional levels. cGMP-regulated transcription factors include the cAMP-response element binding protein CREB, the serum response factor SRF, and the nuclear factor of activated T cells NF/AT. cGMP can regulate CREB directly, through phosphorylation by cGMP-dependent protein kinase, or indirectly, through activation of mitogen-activated protein kinase pathways; regulation of SRF and NF/AT by cGMP is indirect, through modulation of RhoA and calcineurin signaling, respectively. Downregulation of the RNA-binding protein HuR by cGMP leads to destabilization of guanylate cyclase mRNA, but this posttranscriptional mechanism may affect many more cGMP-regulated genes. In this review, we discuss the role of cGMP-regulated gene expression in (patho)physiological processes most relevant to the cardiovascular system, such as regulation of vascular tone, cardiac hypertrophy, phenotypic modulation of vascular smooth muscle cells, and regulation of cell proliferation and apoptosis.
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PMID:Regulation of gene expression by cyclic GMP. 1464 34

The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.
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PMID:Local atrial natriuretic peptide signaling prevents hypertensive cardiac hypertrophy in endothelial nitric-oxide synthase-deficient mice. 1579 9

Ca2+ signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the < or =10(3) dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Ca2+-dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-localised, and characterised as phosphoglucomutase, the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo, followed by MALDI analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial Ser/Thr residues. Although present also in exo(-) mutants, pp63/pf is selectively de-phosphorylated only in exo(+) strains during synchronous exocytosis (80 ms) and re-phosphorylated within approximately 20 s, i.e., the time required to re-establish [Ca2+] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider Ca2+/calmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Ca2+-activated guanylate cyclase. Another kinase, casein kinase 2, is inhibited by Ca2+ and, hence, activated with some delay in parallel to decreasing [Ca2+] after exocytosis. In total, several Ca2+-sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo(-) mutants) and NADH formation, with effects on Ca2+ signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca2+ dynamics during synchronous exo- and endocytosis.
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PMID:Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells. 1610 20

The natriuretic peptide receptor-A (NPR-A) mediates natriuretic, hypotensive, and antihypertrophic effects of natriuretic peptides through the production of cGMP. In pathological conditions such as heart failure, these effects are attenuated by homologous and heterologous desensitization mechanisms resulting in the dephosphorylation of the cytosolic portion of the receptor. In contrast with natriuretic peptide-induced desensitization, pressor hormone-induced desensitization is dependent on protein kinase C (PKC) stimulation and (or) cytosolic calcium elevation. Mechanisms by which PKC and Ca(2+) promote NPR-A desensitization are not known. The role of cGMP and of the cytosolic Ca(2+) pathways in NPR-A desensitization were therefore studied. In contrast with the activation of NPR-A by its agonist, activation of soluble guanylyl cyclases of LLC-PK1 cells by sodium nitroprusside also leads to a production of cGMP but without altering NPR-A activation. Consequently, cGMP elevation per se does not appear to mediate homologous desensitization of NPR-A. In addition, cytosolic calcium increase is required only for the heterologous desensitization pathway since the calcium chelator BAPTA-AM blocks only PMA or ionomycin-induced desensitization. Calcineurin inhibitors block the NPR-A guanylyl cyclase heterologous desensitization induced by ionomycin, suggesting an essential role for this Ca(2+)-stimulated phosphatase in NPR-A desensitization. In summary, the present report demonstrates that neither cGMP nor Ca(2+) cytosolic elevation cause NPR-A homologous desensitization. Our results also indicate for the first time a role for calcineurin in NPR-A heterologous desensitization.
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PMID:Role of cyclic GMP and calcineurin in homologous and heterologous desensitization of natriuretic peptide receptor-A. 1690 99

The cardiac hormones atrial and brain natriuretic peptides (NPs) counteract the systemic, hypertensive, and hypervolemic actions of angiotensin II (Ang II) via their guanylyl cyclase-A (GC-A) receptor. In the present study, we took advantage of genetically modified mice with conditional, cardiomyocyte (CM)-restricted disruption of GC-A (CM GC-A knockout mice) to study whether NPs can moderate not only the endocrine but also the cardiac actions of Ang II in vivo. Fluorometric measurements of [Ca(2+)](i) transients in isolated, electrically paced adult CMs showed that atrial NP inhibits the stimulatory effects of Ang II on free cytosolic Ca(2+) transients via GC-A. Remarkably, GC-A-deficient CMs exhibited greatly enhanced [Ca(2+)](i) responses to Ang II, which was partly related to increased activation of the Na(+)/H(+)-exchanger NHE-1. Chronic administration of Ang II to control and CM GC-A knockout mice (300 ng/kg body weight per minute via osmotic minipumps during 2 wk) provoked significant cardiac hypertrophy, which was markedly exacerbated in the later genotype. This was concomitant to increased cardiac expression of NHE-1 and enhanced activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducers Ca(2+)/calmodulin-dependent kinase II and calcineurin. On the basis of these results, we conclude that NPs exert direct local, GC-A-mediated myocardial effects to antagonize the [Ca(2+)](i)-dependent hypertrophic growth response to Ang II.
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PMID:Local actions of atrial natriuretic peptide counteract angiotensin II stimulated cardiac remodeling. 1751 Feb 45


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