Search

The basic components of nitric oxide (NO) signaling involve nitric oxide synthase to synthesize NO and the soluble isoform of guanylate cyclase (sGC) to trap NO. Once activated by NO, sGC converts guanosine triphosphate to guanosine 3′5′-cyclic monophosphate (cGMP), leading to cGMP-dependent physiological responses such as vasodilation. Termination of the cGMP signal involves a cyclic nucleotide phosphodiesterase (PDE). There are multiple PDE isoforms, and selective inhibition of specific isoforms has proven to be clinically useful (for example, sidenifil (viagra) in the treatment of erectile dysfunction). sGC must also be turned off; however, the in vivo mechanism for this remains unknown. Catalytic activity of constitutive NOS isoforms involved in signaling is controlled by Ca2 + and calmodulin via receptor mediated events that trigger Ca2 + release from intracellular stores or uptake from external sources

Soluble guanylyl cyclase is recognized as the most sensitive physiologic receptor for nitric oxide. Binding of nitric oxide to the heme moiety of the cyclase induces its capacity to synthesize the second messenger cGMP. Although the changes in the state of the heme moiety upon exposure of enzyme to NO and its correlation to the stimulation of sGC catalytic activity are well documented, the exact mechanism of such coupling is not understood. Structure-functional studies are required to elucidate this process. In this chapter, we describe the method of expression and purification of recombinant human alpha1/beta1 isoform of sGC in insect cells, which can be a useful tool for such studies. Several approaches that enable characterization of the binding of NO to sGC heme moiety are also described.

This chapter summarizes the current evidence regarding mechanisms which account for activation of guanylate cyclase, adenylate cyclase, and potassium channels in cerebral vascular muscle. This chapter also focuses on endothelium-dependent relaxation and mechanism of vasomotor regulation. Two forms of guanylate cyclase are present in vascular muscle and contribute to regulation of vascular tone. The first form of guanylate cyclase is soluble, or cytosolic, and the second form is particulate (associated with the plasma membrane). Formation of nitric oxide occurs in cerebral blood vessels under basal conditions and activity of nitric oxide synthase can be further stimulated by increases in intracellular calcium that occur in response to receptor mediated agonists like acetylcholine or in response to increases in shear stress. After release by endothelium, nitric oxide diffuses to vascular muscle and stimulates soluble guanylate cyclase, resulting in a rise in intracellular concentrations of cyclic GMP and relaxation. Under normal conditions, nitric oxide is released both lumenally and ablumenally by endothelium. Release of nitric oxide into the vascular lumen contributes to the antithrombogenic properties of endothelium because nitric oxide inhibits aggregation of platelets and adherence of leukocytes to endothelium.

Publisher Summary This chapter illustrates that the signal transducing enzyme guanylate cyclase is activated in response to specific extracellular signals, leading to the formation of the intracellular second messenger—cyclic guanosine monophosphate (GMP). Two isoenzyme forms of guanylate cyclase exist in cells, which are physically and biochemically distinct and regulated by different agents. The soluble isoenzyme of guanylate cyclase, located in the cytosol, is a heterodimer composed of 70- and 82-kDa subunits and is activated by nitrate containing compounds, endothelial-derived relaxing factors, and oxytocin. A second isoenzyme called “particulate guanylate cyclase” is located in the cell membrane. Enterotoxigenic Escherichia coli elaborate heat-stable enterotoxin (ST), a plasmid-encoded low-molecular-weight peptide exotoxin that produces profuse watery diarrhea by increasing fluid and electrolyte secretion in the intestinal mucosa. A mammalian homologue of this peptide termed “guanylin” has been identified in small intestinal cells and presumably mediates local fluid and electrolyte balance in that organ. Atrial natriuretic peptide (ANP) has been shown to promote vascular smooth muscle relaxation, inhibit the secretion of renin, vasopressin and aldosterone, and increase the secretion of testosterone and progesterone. It is clear that many of the cellular effects of natriuretic peptides (NP) and ST are mediated by the activation of particulate guanylate cyclase and increase in intracellular concentrations of cyclic GMP. The chapter describes the membrane receptors for ANP and ST and particulate guanylate cyclase to which they are coupled.

Publisher Summary In vertebrate rod outer segments (ROSs), guanosine 3′,5′-cyclic monophosphate (cGMP) appears to be directly involved in the process of visual transduction. The illumination of rhodopsin leads to the activation of cGMP phosphodiesterase (PDE) through transducin. The resulting fall in cytoplasmic cGMP concentration contributes to closure of cGMP-sensitive cation channels, causing the hyperpolarization of plasma membranes. Moreover, recovery of the dark state requires the resynthesis of cGMP by guanylate cyclase. To explain the mechanisms for the recovery of the dark state, isolation and reconstitution of guanylate cyclase in detergent-free systems are essential. The chapter also describes the basic characterization of guanylate cyclase from amphibian and bovine ROS. Guanylate cyclase is analyzed by measuring the amount of [ 32 P]cGMP that is derived from [α- 32 P]GTP in the presence of [ 3 H]cGMP, 3-isobutyl-l-methylxanthine, and a GTP-generating system.

The plasma membrane forms of guanylate cyclase contain a highly conserved catalytic domain, which is also conserved in the soluble form of the enzyme and in mammalian adenylate cyclase. A protein kinase-like domain lies to the amino-terminal side of the catalytic domain and appears to be required for signaling via cGMP; it might also signal, itself, through phosphotransferase activity. This domain is present in the growth factor receptors, but appears not to be a component of other guanylate cyclases or adenylate cyclases. A single transmembrane domain then separates the cyclase catalytic and protein kinase-like domains from the putative ligand-binding domain. At least two plasma membrane forms of gunaylate cyclase (i.e., GC-A and GC-B) have now been identified, and their ligand specificities appear to be distinctly different. The tissue/cellular distribution of this family of receptors is now of potential importance, since specific agonists might differentially regulate physiological processes via the secondary messenger, cGMP, dependent on cellular distribution of the receptors.

Publisher Summary This chapter discusses the coordinate interactions of cyclic nucleotide- and phospholipid-metabolizing pathways in Ca2+ -dependent processes, which may help to explain how cells consolidate multiple signals into an integrated response. Complexities are implicit in the interactions among Ca2+, cyclic nucleotides, and phospholipid-metabolizing pathways in cell signal transduction. The interactions are varied and intricate, often involving several levels of cell amplification mechanisms. Upsetting the balance of fatty acids in membrane phospholipids can have detrimental effects on adenylate cyclase. Thus, n - 3 fatty acid enrichment of phospholipids suppresses adenylate cyclase activity. The ingestion of large quantities of these n - 3 fatty acids can alter the profile of cyclooxygenase and lipoxygenase products produced in cells. Also there appears to be a clear distinction between the regulatory properties of eicosanoids regarding adenylate and guanylate cyclase activities. Whereas prostaglandins often stimulate adenylate cyclase activity, they have little effect on guanylate cyclase activity. In the intact cell, there is a strong implication that the dual stimulation of guanylate cyclase by Ca2+ and fatty acid evokes optimal enzyme activity.

Publisher Summary This chapter describes the purification procedure of enzyme guanylate kinase from Escherichia coli. Guanylate kinase from E. coli actively phosphorylates guanine and 2-aminopurine nucleofides but shows only slight activity with other purine nucleotides and no activity with pyrimidine nucleofides. The activity of the enzyme is markedly stimulated by K+ and NH4+, but the mechanism of activation by the two ions appears to be quite different. The spectrophotometric assay is the more convenient of the two assays presented but is subject to high blank values with crude extracts. Ninety percent of the interfering activities can be removed from crude extracts by treatment with acetone, permitting the use of the spectrophotometric assay at all stages of the purification. The activity of guanylate kinase is specific for purine nucleotides. Guanylate kinase requires Mg2+ for activity. These results indicate that the guanylate kinase activity is associated with a single protein, native molecular weight 88,000, that does not contain tryptophan.

The mechanism by which acetylcholine and other transmitters stimulate the synthesis of cyclic GMP was studied at the cellular and molecular levels using neuroblastoma N1E 115 cells and rat brain. (i) Phosphatidic acid added to the medium stimulated the influx of calcium and elevated intracellular cyclic GMP content in the neuroblastoma cells. Treatment of the cells with phospholipase C increased the 32 Pi incorporation into phosphatidic acid and, in parallel, stimulated calcium influx and cyclic GMP synthesis in the cells. (ii) Guanylate cyclase was readily solubilized from neuroblastoma cells by homogenization with hypotonic buffer. The supernatant also contained an endogenous activator of small molecule for guanylate cyclase. The activator was also detected in the supernatant of rat brain and was purified to a high purity. Calcium was required for the activation of guanylate cyclase by the endogenous activator. (iii) Monoclonal antibody to the soluble guanylate cyclase from rat brain cross-reacted with the soluble enzyme from various tissues of rat, but not with the soluble enzyme of mouse brain and rabbit brain indicating a speciecs difference of soluble guanylate cyclase. Immunochemical titration study revealed that guanylate cyclase in the synaptosomal soluble fraction of rat brain was in a highly activated state.

Publisher Summary This chapter discusses the use of incubated retinas in investigating the effects of calcium and other ions on cyclic-nucleotide levels in photoreceptors. Photoreceptors contain both cyclic adenosine 3´,5´-monophosphate (cAMP) and cyclic guanosine 3´,5´-monophosphate (cGMP). The chapter discusses the hypothesis that explains the effect of calcium chelation on cGMP levels, which is the disinhibition of the guanylate cyclase of the outer segment. The calcium chelation that increases cGMP is, thus, far unique to the retina among nervous tissues. In addition to the particulate Ca 2+ -inhibitable guanylate cyclase of the outer segment, the ground squirrel retina may have a soluble Ca 2+ -activated guanylate cyclase in its cone inner segments. At present, there is no evidence that photoreceptors are postsynaptic to any cell using a chemical transmitter that activates adenylate cyclase in postsynaptic membranes. However, cAMP might be associated with glycogen metabolism, microtubule assembly, the modulation of protein synthesis, or photovoltage-controlled activities.

Publisher Summary This chapter discusses the localization and assay of guanylate cyclase. Vertebrate retinal rod outer segments contain high levels of guanylate cyclase activity. Its localization in the outer segment has been demonstrated by two sorts of experiment. After retinal homogenates have been subjected to isopycnic centrifugation in continuous sucrose density gradients, the distribution of guanylate cyclase activity approximately parallels the distribution of rhodopsin. The guanylate cyclase found in almost all other tissues either is soluble or can be solubilized by nonionic detergents. However, attempts to solubilize the guanylate cyclase of retinal rod outer segments have met with very limited success. While developing a procedure for isolating axonemes from bovine retinal rods, it is observed that guanylate cyclase copurifies with structures that include rod axonemes, basal bodies, and centrioles.

Publisher Summary This chapter illustrates the principles of adenylate and guanyIate cyclase assays, the procedures utilizing labeled substrate are also emphasized. Factors relating to assay sensitivity and precision will be discussed. However, the possible interaction of hormone receptors or regulatory factors, e.g., trace metals, are not considered. Adenylate cyclase and guanylate cyclase catalyze apparently analogous reactions. The substrates appear to be complexes of the respective nucleoside triphosphate and a divalent cation, i.e., Me.ATP or Me.GTP. Additional free Me2+ appears to be necessary for the maximal activity of both enzymes. Inorganic pyrophosphate is a product of the adenylate cyclase reaction, but this has not yet been established for the guanylate cyclase reaction.

Publisher Summary Guanylate cyclase activity is measured by estimating radioactive cGMP formation from [3H] GTP. The nature of the phosphate product formed in the reaction has not been reported, although it is known that the α-phosphate of GTP is incorporated into cGMP. Guanylate cyclase is found in much higher activity in sea urchin sperm than in mammalian tissues. The activity in homogenates of sea urchin (Strongylocentrotus purpuratus) sperm ranges from 15 to 60 nmoles of cGMP formed main-1 mg protein-1 at 37°c at pH 7.8 (0.3 mM GTP, 5 mM Mn2+). When measured under similar conditions, the activity in rat lung homogenates ranges from about 0.1 to 0.4 nmole of cGMP formed MaiN-1 mg protein-1. Although guanylate cyclase is largely soluble in most mammalian tissues, the sea urchin sperm enzyme is found in particulate fractions. The enzyme is primarily of flagellar origin and appears to be membrane associated.

Publisher Summary Adenylate cyclase and guanylate cyclase catalyze the formation of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), respectively, from adenosine triphosphate (ATP) and guanosine triphosphate (GTP) in the presence of a metal. With the exception of testes, mammalian adenylate cyclase is exclusively membrane bound and guanylate cyclase is distributed in particulate and cytosolic compartments. This chapter focuses on two highly sensitive assay methods applicable to both enzyme systems. The radiometric assay using [ α - 32 P]ATP or [ α - 32 P]GTP is extremely rapid, whereas the second method employing nonlabeled substrate and radioimmunoassay (RIA) of the product is relatively slow. However, it offers the most sensitive, convenient, and least expensive method to measure cyclic nucleotides in adenylate and guanylate cyclase incubations. After incubation with the cyclase, the excess substrate [ α - 32 P]ATP or [ α - 32 P]GTP is separated from the product cyclic[ 32 P]AMP or cyclic[ 32 P]GMP. This procedure combines chromatography of nucleotides on Dowex-50 columns and neutral alumina (aluminum oxide). The amount of labeled cyclic nucleotide formed is then determined.

The relationship between Ca2+-dependent actions of lymphocyte mitogens to increase cyclic GMP levels and phospholipid turnover is investigated. Guanylate cyclase activity of human peripheral blood lymphocytes, measured in cytoplasmic or membrane fractions, is increased up to 4-fold by incubation of intact cells for 10 min with PHA.1 These increases require extracellular Ca2+ and are prevented by DTE, indicating the requirement for an oxidative step. Guanylate cyclase activation is also prevented by 4 inhibitors of phospholipase A2 (DB-cyclic AMP, DDPH, PBB, quinacrine) and 2 inhibitors of lipoxygenase (ETYA, NDGA) but not by an inhibitor of cyclooxygenase (indomethacin). Addition of phospholipase A2 or of arachidonate to lymphocytes or homogenates results in guanylate cyclase stimulation. 5-, 11- and 12-HETE, products of arachidonate metabolized via lipoxygenase, but not PGE1 or PGF2α, products via cyclooxygenase, stimulate guanylate cyclase. We propose that the mitogen:guanylate cyclase activation mechanism involves the following steps: 1) activation of membrane-bound, Ca2+-dependent phospholipase A2, releasing arachidonate from membrane phospholipids; 2) oxidation of arachidonate by way of the Ca2+-dependent lipoxygenase pathway to hydroperoxy and hydroxy derivatives of arachidonate (HPETE and HETE); and 3) oxidative activation by one or both of these derivatives of guanylate cyclase.