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The soluble form of guanylyl cyclase (sGC) is the main receptor for the signaling agent nitric oxide (NO), which regulates cardiomyocyte contractile function and attenuates cardiomyocyte hypertrophy. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), a regulator of vascular tone, and cardiac NO-sGC-cGMP signaling modulates cardiac stress responses, including ischemia and reperfusion (IR) injury. Here, we investigated the role of GUCY1B3 (the β subunit of sGC) in cardiomyocyte IR injury and myocardial infarction (MI) in vitro and in vivo. GUCY1B3 was upregulated in neonatal rat ventricular myocytes in response to IR injury, and GUCY1B3 overexpression restored IR-induced cell death and apoptosis. Treatment with specific inhibitors of PKCδ, PKCε, and Akt suggested that the protective effects of GUCY1B3 were mediated by PKCε/Akt signaling. In a mouse model of coronary artery ligation-induced MI, GUCY1B3 silencing aggravated MI-induced cardiac dysfunction and increased infarct size and exacerbated cardiomyocyte apoptosis in association with the inactivation of PKCε and Akt. Our results suggest that GUCY1B3 exerts cardioprotective effects through the modulation of the PKCε/Akt activity and identify a potential mechanism involved in NO-sGC-cGMP signaling in the heart.

Soluble guanylyl cyclase (sGC) plays a crucial role in cyclic nucleotide signaling that regulates numerous important physiological processes. To identify new sGC inhibitors that may prevent the formation of the active catalytic domain conformation, we carried out an in silico docking screen targeting a ‘backside pocket’ of the inactive sGC catalytic domain structure. Compounds 1 and 2 were discovered to inhibit sGC even at high/saturating nitric oxide concentrations. Both compounds also inhibit the BAY 58-2667-activated sGC as well as BAY 41-2272-stimulated sGC activity. Additional biochemical analyses showed that compound 2 also inhibits the isolated catalytic domain, thus demonstrating functional binding to this domain. Both compounds have micromolar affinity for sGC and are potential leads to develop more potent sGC inhibitors.

Hypertension is a dominating risk factor for cardiovascular disease. To characterize the genomic response to hypertension, we administered vehicle or angiotensin II to mice and performed gene expression analyses. AngII treatment resulted in a robust increase in blood pressure and altered expression of 235 genes in the aorta, including Gucy1a3 and Gucy1b3 which encode subunits of soluble guanylyl cyclase (sGC). Western blotting and immunohistochemistry confirmed repression of sGC associated with curtailed relaxation via sGC activation. Analysis of transcription factor binding motifs in promoters of differentially expressed genes identified enrichment of motifs for RBPJ, a component of the Notch signaling pathway, and the Notch coactivators FRYL and MAML2 were reduced. Gain and loss of function experiments demonstrated that JAG/NOTCH signaling controls sGC expression together with MAML2 and FRYL. Reduced expression of sGC, correlating with differential expression of MAML2, in stroke prone and spontaneously hypertensive rats was also seen, and RNA-Seq data demonstrated correlations between JAG1, NOTCH3, MAML2 and FRYL and the sGC subunits GUCY1A3 and GUCY1B3 in human coronary artery. Notch signaling thus provides a constitutive drive on expression of the major nitric oxide receptor (GUCY1A3/GUCY1B3) in arteries from mice, rats, and humans, and this control mechanism is disturbed in hypertension.

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine-rich repeat receptor-like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.

The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells including soluble guanylate cyclase (sGC). hsp90 associates with the heme-free (apo) sGC-β1 subunit and helps to drive heme insertion during maturation of sGC to its NO-responsive active form. Here, we found that NO caused apo-sGC-β1 to rapidly and transiently dissociate from hsp90 and associate with sGC-α1 in cells. This NO response (i) required that hsp90 be active and that cellular heme be available and be capable of inserting into apo-sGC-β1; (ii) was associated with an increase in sGC-β1 heme content; (iii) could be mimicked by the heme-independent sGC activator BAY 60-2770; and (iv) was followed by desensitization of sGC toward NO, sGC-α1 disassociation, and reassociation with hsp90. Thus, NO promoted a rapid, transient, and hsp90-dependent heme insertion into the apo-sGC-β1 subpopulation in cells, which enabled it to combine with the sGC-α1 subunit to form the mature enzyme. The driving mechanism likely involves conformational changes near the heme site in sGC-β1 that can be mimicked by the pharmacologic sGC activator. Such dynamic interplay between hsp90, apo-sGC-β1, and sGC-α1 in response to NO is unprecedented and represent new steps by which cells can modulate the heme content and activity of sGC for signaling cascades.

The complete structure of the assembled domains of nitric oxide-sensitive guanylate cyclase (NOsGC) remains to be determined. It is also unknown how binding of NO to heme in guanylate cyclase is communicated to the catalytic domain. In the current study the conformational change of guanylate cyclase on activation by NO was studied using FRET. Endogenous tryptophan residues were used as donors, the substrate analog 2'-Mant-3'-dGTP as acceptor. The enzyme contains five tryptophan residues distributed evenly over all four functional domains. This provides a unique opportunity to detect the movement of the functional domains relative to the substrate-binding catalytic region. FRET measurements indicate that NO brings tryptophan 22 in the αB helix of the β1 heme NO binding domain and tryptophan 466 in the second short helix of the α1 coiled-coil domain closer to the catalytic domain. We propose that the respective domains act as a pair of tongs forcing the catalytic domain into the nitric oxide-activated conformation.

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.

Nitric oxide (NO), a signaling molecule in the cardiovascular system, has been receiving increasing attention since Furchgott, Ignarro, and Murad were awarded the Nobel Prize in Physiology and Medicine for the discovery in 1998. Soluble guanylate cyclase (sGC), as an NO receptor, is a key metalloprotein in mediating NO signaling transduction. sGC is activated by NO to catalyze the conversion of guanosine 5′-triphosphate (GTP) to cyclic guanylate monophosphate (cGMP). The dysfunction of NO signaling results in many pathological disorders, including several cardiovascular diseases, such as arterial hypertension, pulmonary hypertension, heart failure and so on. Significant advances in its structure, function, mechanism, and physiological and pathological roles have been made throughout the past 15 years. We herein review the progress of sGC on structural, functional investigations, as well as the proposed activation/deactivation mechanism. The heme-dependent sGC stimulators and heme-independent sGC activators have also been summarized briefly.

Membrane-bound guanylate cyclases harbor a region called the dimerization or linker domain, which aids the enzymes in adopting an optimal monomer-monomer arrangement for catalysis. One subgroup of these guanylate cyclases is expressed in rod and cone cells of vertebrate retina, and mutations in the dimerization domain of rod outer segment guanylate cyclase 1 (ROS-GC1, encoded by the GUCY2D gene) correlate with retinal cone-rod dystrophies. We investigate how a Q847L/K848Q double mutation, which was found in patients suffering from cone-rod dystrophy, and the Q847L and K848Q single-point mutations affect the regulatory mechanism of ROS-GC1. Both the wild type and mutants of heterologously expressed ROS-GC1 were present in membranes. However, the mutations affected the catalytic properties of ROS-GC1 in different manners. All mutants had higher basal guanylate cyclase activities but lower levels of activation by Ca²⁺-sensing guanylate cyclase-activating proteins (GCAPs). Further, incubation with wild-type GCAP1 and GCAP2 revealed for all ROS-GC1 mutants a shift in Ca²⁺ sensitivity, but activation of the K848Q mutant by GCAPs was severely impaired. Apparent affinities for GCAP1 and GCAP2 were different for the double mutant and the wild type. Circular dichroism spectra of the dimerization domain showed that the wild type and mutants adopt a prevalently α-helical structure, but mutants exhibited lower thermal stability. Our results indicate that the dimerization domain serves as a Ca²⁺-sensitive control module. Although it is per se not a Ca²⁺-sensing unit, it seems to integrate and process information regarding Ca²⁺ sensing by sensor proteins and regulator effector affinity.

Zebrafish express in the retina a large variety of three different membrane-bound guanylate cyclases and six different guanylate cyclase-activating proteins (zGCAPs) belonging to the family of neuronal calcium sensor proteins. Although these proteins are predominantly localized in rod and cone photoreceptor cells of the retina, they differ in their spatial-temporal expression profiles. Further, each zGCAP has a different affinity for Ca(2+) and displays different Ca(2+)-sensitivities of guanylate cyclase activation. Thus, zGCAPs operate as cytoplasmic Ca(2+)-sensors that sense incremental changes of cytoplasmic Ca(2+)-concentration in rod and cone cells and control the activity of their target guanylate cyclases in a Ca(2+)-relay mode fashion.

Background Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets. Methods To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASPS239) by flow cytometry and Western blot. ANOVA followed by Bonferroni’s test and Student’s t-test were used as appropriate. Results We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite. Conclusions All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.

In plants, nitric oxide-mediated 3′, 5′- cyclic guanosine monophosphate (cGMP) synthesis plays an important role during pathogenic stress response, stomata closure upon osmotic stress, the development of adventitious roots and transcript regulation. The nitric oxide-cGMP dependent pathway is well characterized in mammals. The binding of nitric oxide to soluble guanylate cyclase enzymes (GCs) initiates the synthesis of cGMP from guanosine triphosphate. The produced cGMP alters various cellular responses, such as the function of protein kinase activity, cyclic nucleotide gated ion channels and cGMP-regulated phosphodiesterases. The signal generated by the second messenger is terminated by 3′, 5′- cyclic nucleotide phosphodiesterase (PDEs) enzymes that hydrolyze cGMP to a non cyclic 5′- guanosine monophosphate. To date, no homologues of mammalian cGMP-synthesizing and degrading enzymes have been found in higher plants. In the last decade, six receptor proteins from Arabidopsis thaliana have been reported to have guanylate cyclase activity in vitro. Of the six receptors, one was shown to be a nitric oxide dependent guanylate cyclase enzyme (NOGC1). However, the role of these proteins in planta remains to be elucidated. Enzymes involved in the degradation of cGMP remain elusive, albeit, PDE activity has been detected in crude protein extracts from various plants. Additionally, several research groups have partially purified and characterized PDE enzymatic activity from crude protein extracts. In this review, we focus on presenting advances toward the identification of enzymes involved in the cGMP metabolism pathway in higher plants.

Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with N(ω)-nitro-l-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway.

Soluble guanylate cyclase (sGC), a heterodimeric heme protein, catalyses the conversion of GTP in to cyclic GMP, which acts as a second messenger in cellular signaling. Nitric oxide activates this enzyme several hundred folds over its basal level. Carbon monoxide, along with some activator molecules like YC-1 and BAY, also synergistically activate sGC. Mechanism of this synergistic activation is a matter of debate. Here we review the existing literature to identify the possible binding site for YC-1 and BAY on bovine lung sGC and its mechanism of activation. These two exogenous compounds bind sGC on alpha subunit inside a pocket and thus exert allosteric effect via subunit interface, which is relayed to the catalytic site. We used docking studies to further validate this hypothesis. We propose that the binding of YC-1/BAY inside the sensory domain of the alpha subunit modulates the interactions on the subunit interface resulting in rearrangements in the catalytic site into active conformation and this partly induces the cleavage of Fe-His bond.

Vertebrate phototransduction depends on the reciprocal relationship between two-second messengers, cyclic GMP and Ca(2+). The concentration of both is reciprocally regulated including the dynamic synthesis of cyclic GMP by a membrane bound guanylate cyclase. Different from hormone receptor guanylate cyclases, the cyclases operating in phototransduction are regulated by the intracellular Ca(2+)-concentration via small Ca(2+)-binding proteins. Based on the site of their expression and their Ca(2+) modulation, this sub-branch of the cyclase family was named sensory guanylate cyclases, of which the retina specific forms are named ROS-GCs (rod outer segment guanylate cyclases). This review focuses on the structure and function of the ROS-GC subfamily present in the mammalian retinal neurons: photoreceptors and inner layers of the retinal neurons. Portions and excerpts of the review are from a previous chapter (Curr Top Biochem Res 6:111-144, 2004).

Production of cGMP in bacteria has been studied since the early 1970s. From the beginning on it proved to be a challenging topic. In Escherichia coli the cGMP levels were two orders of magnitude lower than the corresponding cAMP levels. Furthermore, no specific cGMP receptor protein was identified in the bacterium and a physiological role of cGMP in the bacterium was not substantiated. Consequently in 1977, compelling evidence was given that cGMP is a by-product of E. coli adenylate cyclase in vivo. This may be the reason why also work on cGMP in other bacteria like Bacillus licheniformis and Caulobacter crescentus was not pursued any further. However, recent study on cGMP and guanylate cyclase in the cyanobacterium Synechocysis PCC 6803 brought cGMP signaling in bacteria back to attention. In Synechocystis cGMP levels are of similar magnitude as those of cAMP and deletion of the cya2 gene markedly reduced the amount of cGMP without affecting cAMP. A few months ago the Cya2 gene product has been biochemically and structurally characterized. It behaves as a specific guanylate cyclase in vitro and a single amino acid substitution transforms the enzyme into a specific adenylate cyclase. These data point toward the existence of a true bacterial cGMP-signaling pathway, which needs to be explored and established by future experiments.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC), like the other members of the membrane guanylate cyclase family, is a single transmembrane-spanning protein. The transmembrane domain separates the protein into two regions, extracellular and intracellular. The extracellular region contains the ANF-binding domain and the intracellular region the catalytic domain located at the C-terminus of the protein. Preceding the catalytic domain, the intracellular region is comprised of the following functional domains: juxtaposed 40 amino acids to the transmembrane domain is the ATP-regulated module (ARM) domain [also termed the kinase homology domain (KHD)], and the putative dimerization domain. The ANF-RGC signaling is initiated by hormone, ANF, binding to its extracellular binding site. The binding signal is transduced through the transmembrane domain to the intracellular portion where ATP binding to the ARM domain partially activates the cyclase and prepares it for subsequent steps involving phosphorylation and attaining the fully activated state. This chapter reviews the signaling modules of ANF-RGC.

To date, the calcium-regulated membrane guanylate cyclase Rod Outer Segment Guanylate Cyclase type 1 (ROS-GC1) transduction system in addition to photoreceptors is known to be expressed in three other types of neuronal cells: in the pinealocytes, mitral cells of the olfactory bulb and the gustatory epithelium of tongue. Very recent studies from our laboratory show that expression of ROS-GC1 is not restricted to the neuronal cells; the male gonads and the spermatozoa also express ROS-GC1. In this presentation, the authors review the existing information on the localization and function of guanylate cyclase with special emphasis on Ca(2+)-modulated membrane guanylate cyclase, ROS-GC1, in the testes. The role of ROS-GC1 and its Ca(2+)-sensing modulators in the processes of spermatogenesis and fertilization are discussed.

Three membrane bound sensory guanylate cyclases are expressed in photoreceptor cells of the developing and adult zebrafish retina. First appearance of mRNA transcripts was detected by in situ hybridization techniques for all guanylate cyclases between 3 and 4 days post fertilization (dpf), but only one isoform (guanylate cyclase 3) appeared to be specifically expressed in cones of the adult retina. Transcripts of three cone specific guanylate cyclase-activating proteins (zGCAP3, zGCAP4 and zGCAP7) were also detected at 3–4 dpf. The expression onset of the guanylate cyclases and these neuronal calcium sensor proteins mainly overlapped. High guanylate cyclase activities in larval eye preparations and the precisely controlled coexpression of guanylate cyclases and zGCAPs coincide with the onset of visual function at 3–4 dpf.

The Ca(2+)-modulated ONE-GC membrane guanylate cyclase is a central component of the cyclic GMP signaling in odorant transduction. It is a single transmembrane spanning modular protein. Its intracellular region contains Ca(2+) sensor recognition domains linked to GCAP1 and to neurocalcin delta, and a catalytic module. These domains sense increments in free Ca(2+) and stimulate the catalytic module. The present study makes three significant mechanistic advancements. First, to date no ligand for the extracellular (ext) domain is known, for this reason ONE-GC has been deemed as an orphan receptor. The present study identifies its ligand. Uroguanylin stimulates ONE-GC through its ext domain. Second, so far no ligand is known that directly stimulates the catalytic module of any membrane guanylate cyclase. The presented evidence shows that in the presence of the semimicromolar range of free Ca(2+), neurocalcin binds to the catalytic module and stimulates ONE-GC. Thus, ONE-GC has trimodal regulation, two occurring intracellularly and one extracellularly. Third, guanylin, a urine odorant, does not directly stimulate ONE-GC. This challenges the proposed hypothesis that the guanylin odorant signal occurs via ONE-GC [T. Leinders-Zufall, R.E. Cockerham, S. Michalakis, M. Biel, D.L. Garbers, R.R. Reed, F. Zufall, S.D. Munger, Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium, Proc. Natl. Acad. Sci. USA. 104 (2007) 14507-14512].

Nitric oxide (NO) is a physiologically relevant activator of the hemoprotein soluble guanylate cyclase (sGC). In the presence of NO, sGC is activated several hundredfold above the basal level by a mechanism that remains to be elucidated. The heme ligand n-butyl isocyanide (BIC) was used to probe the mechanism of NO activation of sGC. Electronic absorption spectroscopy was used to show that BIC binds to the sGC heme, forming a 6-coordinate complex with an absorbance maximum at 429 nm. BIC activates sGC 2-5-fold, and synergizes with the allosteric activator YC-1, to activate the enzyme 15-25-fold. YC-1 activates the sGC-BIC complex, and leads to an increase in both the Vmax and Km. BIC was also used to probe the mechanism of NO activation. The activity of the sGC-BIC complex increases 15-fold in the presence of NO, without displacing BIC at the heme, which is consistent with previous reports that proposed the involvement of a non-heme NO binding site in the activation process.

Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.

The lymphatic vascular system regulates tissue fluid homeostasis and the afferent phase of the immune response, and it is also involved in tumor metastasis. There is increasing evidence that lymphatic vessels also mediate acute and chronic inflammation. However, the mechanisms and functional consequences of lymphangiogenesis under inflammatory conditions are largely unknown. Here, we show that lymphatic endothelial cells (LECs) specifically express the alpha1beta1 isoform of soluble guanylate cyclase (sGC), that vascular endothelial growth factor-A potently induces sGCalpha1beta1, and that nitric oxide (NO) -induced LEC proliferation, migration, and cGMP production in LECs are specifically dependent on sGCalpha1beta1. Moreover, the specific sGC inhibitor NS-2028 completely prevents ultraviolet B-irradiation-induced lymphatic vessel enlargement, edema formation, and skin inflammation in vivo. These findings identify a crucial role of the NO/sGCalpha1beta1/cGMP pathway in modulating lymphatic vessel function. The blockade of sGCalpha1beta1 signaling might serve as a novel therapeutic strategy for inhibiting lymphangiogenesis and inflammation, in addition to its effects on the blood vasculature.

The importance of the second messengers, Ca 2+ and cyclic GMP, for the process of fertilization is well established; themechanisms for their intracellular regulations in the testes are,however, poorly understood. This study documents the biochemical,molecular, and functional identity of a Ca 2+ -modulated membraneguanylate cyclase transduction machinery in bovine testes. Themachinery is both inhibited and stimulated by free Ca 2+ levels. TheCa 2+ - sensor component of the inhibitory mode of the machinery isGCAP1 (guanylate cyclase activating protein type 1) and for thestimulatory mode is S100B. The transduction component is a Ca 2+ -driven rod outer segment membrane guanylate cyclase type 1, ROS-GC1. The cyclase is predominantly expressed in spermatogeniccells. GCAP1 expression is restricted to a small population ofspermatogonia, whereas S100B is present in the majority ofspermatocytes and spermatids. The expression of GCAP1 andS100B in spermatocytes and spermatids is mutually exclusive.Key words: ROS-GC1, GCAP1, S100B, signal transduction.J Androl 2007;28:50–58

The heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the beta1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate Fe(II)-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate Fe(II)-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal Fe(II)-His bond and suggests that in both the 5- and 6-coordinate Fe(II)-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes.

Vascular endothelial growth factor-A (VEGF), which binds to both VEGF receptor-1 (Flt1) and VEGFR-2 (KDR/Flk-1), requires nitric oxide (NO) to induce angiogenesis in a cGMP-dependent manner. Here we show that VEGF-E, a VEGFR-2-selective ligand stimulates NO release and tube formation in human umbilical vein endothelial cells (HUVEC). Inhibition of phospholipase Cgamma (PLCgamma) with U73122 abrogated VEGF-E induced endothelial cell migration, tube formation and NO release. Inhibition of endothelial nitric oxide synthase (eNOS) using l-NNA blocked VEGF-E-induced NO release and angiogenesis. Pre-incubation of HUVEC with the soluble guanylate cyclase inhibitor, ODQ, or the protein kinase G (PKG) inhibitor, KT-5823, had no effect on angiogenesis suggesting that the action of VEGF-E is cGMP-independent. Our data provide the first demonstration that VEGFR-2-mediated NO signaling and subsequent angiogenesis is through a mechanism that is dependent on PLCgamma but independent of cGMP and PKG.

Atrial natriuretic factor (ANF) receptor guanylate cyclase ANF-RGC is a single transmembrane spanning modular protein. Juxtaposed to each side of the transmembrane module is a Cys423-Cys432 disulfide ANF signaling module motif and the ATP-regulated transduction module (ARM) motif. The signaling module motif is conserved in nearly all membrane guanylate cyclases and is believed to be critical in the signaling activities of all membrane guanylate cyclases. The present study with the model system of the olfactory membrane guanylate cyclase shows that this concept is not valid. Furthermore, the study shows that in ANF-GC the signaling motif works through the ARM domain. A new signaling model is proposed where in its natural state the disulfide structural motif represses the ARM domain activity, which, in turn, represses the catalytic module activity of ANF-RGC. ANF signaling relieves the disulfide structural motif restraint on the ARM inhibition and stimulates the catalytic module of the cyclase.

The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.

Adaptation to hypoxia and maintenance of O(2) homeostasis involve a wide range of responses that occur at different organizational levels in the body. One of the most important transcription factors that activate the expression of O(2)-regulated genes is hypoxia-inducible factor 1 (HIF-1). Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels and cytotoxicity of activated macrophages. We investigated the effect of the clinically used nitrates nitroglycerin (NTG), isosorbide dinitrate (ISDN), and sodium nitroprusside (SNP) on HIF-1-mediated transcriptional responses to hypoxia. We demonstrate that among the three nitrates, only SNP inhibits HIF-1 activation in response to hypoxia. In contrast, NTG or ISDN does not affect HIF-1 activity. SNP inhibits the accumulation of HIF-1alpha, the regulatory subunit of HIF-1, and the transcriptional activation of HIF-1alpha via a mechanism that is not dependent on either NO or soluble guanylate cyclase.

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.

GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 °C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N-acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.

Soluble guanylate cyclases (sGCs) are heterodimeric enzymes consisting of alpha and beta subunits and catalyze the formation of cGMP from GTP. The beta1 subunit has been characterized in detail, whereas the function and physiological role of the beta2 subunit are poorly understood. In this study, I isolated two distinct cDNA fragments for the beta2 subunit of sGC (beta2a and beta2b) from a rat brain cDNA library by 3' rapid amplification of cDNA ends using degenerate sense primers based on amino acid sequences conserved among membrane-bound guanylate cyclases. The deduced amino acid sequence of beta2a is identical with the corresponding sequence of the previously described beta2 subunit, whereas that of beta2b is C-terminally shorter by 46 amino acids and thus does not contain a consensus sequence for isoprenylation/carboxymethylation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that both variants are expressed in various tissues, including kidney, liver, and brain. Although the functional significance of the C-terminal region containing the consensus sequence for isoprenylation/carboxymethylation of beta2a remains unclear yet, it is likely that these beta2 subunits play some physiological or pathophysiological role in various tissues.

Natriuretic peptides (NP) activate particulate guanylate cyclase (pGC) and nitric oxide (NO) activates soluble guanylate cyclase (sGC). Both guanylate cyclases catalyse the formation of the same second messenger, cyclic guanosine 3',5'-monophosphate (cGMP), which activates the cGMP-dependent protein kinases (PKG). PKG then starts a signalling cascade that mediates many cardiovascular and renal effects, such as smooth muscle relaxation and diuresis. Many cell types possess both sGC and pGC. Because both GC-cGMP systems play complementary roles, an interaction between the two pathways might represent an important physiological control mechanism. In this report we demonstrate an interaction between the two pathways. C-type natriuretic peptide (CNP) decreased the beta-subunit of sGC (sGC-beta) steady-state protein levels and enzymatic activity in cultured human mesangial cells (HMC) in a time- and dose-dependent manner. This down-regulation was not dependent on changes in sGC-beta mRNA levels. Treatment of the cells with the stable cGMP analogue 8-Br-cGMP or the phosphodiesterase type-5 inhibitor Zaprinast produced the same down-regulatory effect. Inhibition of PKG or proteasome activity prevented the CNP-induced reduction of sGC-beta protein levels and activity. Taken together, these results demonstrate that pGC activation induces a post-transductional down-regulation of sGC by a mechanism involving PKG and the proteasome pathway.

Hydrophobic bile acids such as deoxycholate are known tumor promoters in the gastrointestinal tract. We have previously shown that deoxycholate induces apoptosis in colon epithelial cells and that these cells can be made resistant to deoxycholate-induced apoptosis. We now show that the nitric oxide synthase/nitric oxide/guanylate cyclase/cyclic guanosine monophosphate/cGMP-activated protein kinase (NOS/NO/GC/cGMP/PKG) signaling module contributes, in part, to the observed resistance of the cultured DOC-resistant colon epithelial cells (HCT-116R) using pharmacological inhibitors/antagonists (NS2028, Rp-8pCPT-cGMP, KT5823) of members of this signaling module. A novel finding from this study is the caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis of deoxycholate-sensitive HCT-116SA cells and the absence of guanylate cyclase alpha 1 cleavage in deoxycholate-treated HCT-116R resistant cells using Western blot analyses. This cleavage was specific to caspases as lysosomal, proteasomal, serine protease, cathepsin and calpain inhibitors failed to prevent the cleavage, whereas a general caspase inhibitor and a specific caspase-6 inhibitor did prevent guanylate cyclase alpha 1 cleavage.

Although nitric oxide (NO) was shown not only to exert biological activities through activation of soluble guanylate cyclase (sGC), but also to cause oxidative stress, mechanisms for switching these pathways are unknown. This study aimed to examine aberrant utilization of NO under disease conditions such as diabetes mellitus. Diabetes was induced in male Wistar rats by injecting streptozotocin (STZ; 50 mg/kg body weight, i.p.). Retina was perfusion-fixed for immunohistochemistry to detect the gas-mediated activation of sGC by anti-sGC antibodies that are function-sensitive [monoclonal antibody (MoAb) 3221] and -insensitive (MoAb28131). Regional lipid peroxidation was also examined by an anti-acrolein MoAb. At 6 weeks after STZ injection, inducible NO synthase induction became evident, coinciding with the overproduction of nitrotyrosine, followed by that of acrolein. Despite such NO overproduction, sGC did not exhibit any notable activation. When STZ-treated animals were posttreated with a derivative of superoxide dismutase that stays in circulation without undergoing renal ultrafiltration, immunoreactivities to MoAb3221 but not to MoAb28131 increased markedly in diabetic retina, suggesting that superoxide cancels free NO for local sGC activation. These results provide evidence of aberrant utilization of NO and suggest that superoxide plays a role in interfering with NO-mediated sGC activation for phototransducing events in this neural tissue.

Nitric oxide (NO) participates in the regulation of the daily activities of cells as well as in cytotoxic events. Elucidating the mechanism(s) by which NO carries out its diverse functions has been the goal of numerous laboratories. In the cardiovascular system, evidence indicates that NO mediates its effects via an activation of soluble guanylyl cyclase (sGC). In other tissues, it is not clear if sGC is an exclusive target for NO or what the functions of cGMP might be. It is also unlikely that the diversity of NO actions is explained solely by changes in cGMP. This review focuses on the evidence that NO modulates cAMP signalling, with specific attention to the effects of NO on adenylyl cyclase (AC) as the target of NO regulation.

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Two membrane bound guanylate cyclases are expressed in vertebrate photoreceptor cells. They serve a key function in photoreceptor physiology as they synthesize the intracellular transmitter of photoexcitation guanosine 3′,5′-cyclic monophosphate (cGMP). Both cyclases named ROS-GC1 and ROS-GC2 form a subclass of membrane bound cyclases and differ in many aspects from hormone peptide receptor guanylate cyclases. One unique feature is their regulation by three small Ca2+-binding proteins called GCAPs. These regulatory proteins sense changes in the cytoplasmic Ca2+-concentration [Ca2+] during illumination and activate ROS-GCs when the [Ca2+] decreases below the value in a dark adapted cell of 500–600 nM. Recent work has identified the target regions of GCAP-1 in ROS-GC1. In addition to GCAPs several other proteins including aktin, tubulin, a glutamic-acid-rich protein and a GTPase accelerating protein (RGS9) were found to interact with ROS-GC1 and probably form a multiprotein complex.

Almost four decades of research in the field of membrane guanylate cyclases is discussed in this review. Primarily, it focuses on the chronological development of the field, recognizes major contributions of the original investigators, corrects certain misplaced facts, and projects its future trend.

The broad role of nitric oxide (NO) and cyclic GMP in biochemistry and biology as important messenger molecules is evident from the numerous publications in this research field. NO and cGMP have been known as components of the key signaling pathway in regulating numerous processes such as vascular dilation, blood pressure, neurotransmission, cardiovascular function, and renal function. In spite of almost 150,000 publications with nitric oxide and cyclic GMP, there are few publications regarding the effects of these messenger molecules on gene regulation, cell differentiation and cell proliferation. Our research data with embryonic stem cells and several cancer cell lines suggest that nitric oxide, its receptor soluble guanylyl cyclase (sGC) and sGC's product cyclic GMP can regulate the processes of proliferation and differentiation. Furthermore, we have found that undifferentiated stem cells and some malignant tumors such as human glioma have decreased levels of sGC and translocation of the sGCβ1 subunit to the nucleus. We propose that sGC and cyclic GMP function as tumor suppressors. An understanding of the mechanisms of the translocation of the sGCβ1 subunit into the nucleus and the possible regulation of gene expression of NO and/or cyclic CMP could lead to novel and innovative approaches to cancer therapy and stem cell proliferation and differentiation.

We tested whether adenosine, a cytoprotective mediator and trigger of preconditioning, could protect endothelial cells from inflammation-induced deficits in mitochondrial biogenesis and function. We examined this question using human microvascular endothelial cells exposed to TNFα. TNFα produced time and dose-dependent decreases in mitochondrial membrane potential, cellular ATP levels, and mitochondrial mass, preceding an increase in apoptosis. These effects were prevented by co-incubation with adenosine, a nitric oxide (NO) donor, a guanylate cyclase (GC) activator, or a cell-permeant cyclic GMP (cGMP) analog. The effects of adenosine were blocked by a nitric oxide synthase inhibitor, a soluble guanylate cyclase inhibitor, a morpholino antisense oligonucleotide to endothelial nitric oxide synthase (eNOS), or siRNA knockdown of the transcriptional coactivator, PGC-1α. Incubation with exogenous NO, a GC activator, or a cGMP analog reversed the effect of eNOS knockdown, while the effect of NO was blocked by inhibition of GC. The protective effects of NO and cGMP analog were prevented by siRNA to PGC-1α. TNFα also decreased expression of eNOS, cellular NO levels, and PGC-1α expression, which were reversed by adenosine. Exogenous NO, but not adenosine, rescued expression of PGC-1α in cells in which eNOS expression was knocked down by eNOS antisense treatment. Thus, TNFα elicits decreases in endothelial mitochondrial function and mass, and an increase in apoptosis. These effects were reversed by adenosine, an effect mediated by eNOS-synthesized NO, acting via soluble guanylate cyclase/cGMP to activate a mitochondrial biogenesis regulatory program under the control of PGC-1α. These results support the existence of an adenosine-triggered, mito-and cytoprotective mechanism dependent upon an eNOS-PGC-1α regulatory pathway, which acts to preserve endothelial mitochondrial function and mass during inflammatory challenge.

Guanylate cyclase-activating protein 1 (GCAP-1) is a Ca(2+)-sensing protein in vertebrate photoreceptor cells. It activates a membrane-bound guanylate cyclase. Three of four cysteines present in wild-type GCAP-1 were accessible to the thiol-modifying reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) in the presence of Ca(2+). Only Cys106 became exposed to the solvent after Ca(2+)-chelation. Since Cys106 is located in EF-hand 3, we could determine an apparent K(D) of 2.9 microM for Ca(2+) binding to this site with a fast off-rate (t approximately 2 ms). We conclude that the rapid dissociation of Ca(2+) from EF-hand 3 in GCAP-1 triggers activation of guanylate cyclase in rod cells.

This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.

In this study we analyzed the role of vascular NAD(P)H oxidase in the generation of O−2 and the endothelial impairment of NO signal transduction pathway in hypertension. In aortic rings of 15-month-old stroke-prone spontaneously hypertensive rats (SHR15) we found a 10-fold increased expression of NAD(P)H oxidase subunit gp91phox mRNA associated with a 3-fold increased production of O−2 comparedto age-matched Wistar rats (WIS15). Vasorelaxationstudies in aortas of SHR15 showed a strongly diminished response to acetylcholine, NO-donor S-nitroso-N-acetyl- d,l -penicillamine, and organic nitrate glyceryl trinitrate compared to WIS15. Soluble guanylate cyclase (sGC) activity and sGC β1-subunit protein expression was downregulated in aortas and lungs of SHR15. These data suggest an upregulation of vascular NAD(P)H oxidase and an impairment of the NO signal transduction pathway in hypertension.

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.

The second messenger systems involved in the final stages of the phototransduction cascade in Limulus photoreceptors remain unclear. Excised patches of transducing membrane contain cGMP-gated channels, suggesting the involvement of cGMP in the excitation process. To further explore this possibility, we tested the effects of inhibitors and agonists of guanylate cyclase. The active site cyclase inhibitors guanosine 5′-tetraphosphate and adenosine 5′-tetraphosphate produced a reversible reduction of the response to light without affecting resting membrane properties. The cyclase inhibitor Rp-GTPαS produced a similar reduction, but the effect was only slightly reversible. The reduction in the response produced by these inhibitors was robust, often producing over a 95% decrease in the amplitude of the light response. Previous work had shown that an end-product cyclase inhibitor, imidodiphosphate, also inhibited the response. The consistent results with four different guanylate cyclase inhibitors strongly support the involvement of this enzyme in the phototransduction cascade. To determine whether the guanylate cyclase involved is the NO-dependent soluble form, we applied inhibitors and activators of the nitric oxide synthase/guanylate cyclase pathway such as L-N5-(1-iminoethyl) ornithine, sodium nitroprusside, and carboxy-PTIO. None of these agents had any substantial effect on phototransduction. Taken together, these results support a role for a particulate guanylate cyclase in Limulus photoreceptor excitation.

SYNOPSIS. Photoreceptors undergoing target selection in the optic lobe of Drosophila express a nitric oxide sensitive soluble guanylate cyclase (sGC). At the same time, cells in the target region of the optic lobe express nitric oxide synthase (NOS). Pharmacological inhibition of NOS, NO or sGC leads to disruption of the retinal projection pattern in vitro, and the extension of individual retinal axons beyond their appropriate targets. The disruptive effects of NOS inhibition in vitro are prevented by adding a cGMP analog. Mutations in the sGC alpha subunit gene, Gc a1, reduce sGC expression and attenuate NO-sensitive retinal cGMP production in the visual system. Although the retinal projection pattern is undisturbed in Gca1 mutants, they lack positive phototaxis as adults, suggesting inappropriate connections exist between the photoreceptors and optic lobe interneurons in these flies. Preliminary results show that heat-shock expression of wild-type Gc a1 during metamorphosis can restore positive phototaxis in severe Gca1 mutants. These in vivo results support the in vitro findings that NOS and sGC activity are required to promote the appropriate retinal innervation of the optic lobe.

The distribution of nitric oxide synthase in both neuronal and non-neuronal pancreatic tissues and the role of nitric oxide in the control of exocrine pancreatic secretion are reviewed in this article. Earlier reports based on in vivo studies suggested that nitric oxide can affect the secretory activity of the exocrine pancreas through changes in pancreatic blood flow. More recently, the employment of either nitric oxide synthase inhibitors or nitric oxide donors in in vitro preparations has provided evidence that nitric oxide can exert a direct action on this gland independently on its vascular effects. Most research in this area seems to indicate that modulation of exocrine pancreatic function by nitric oxide is exerted via activation of guanylate cyclase and generation of cGMP, although other pathways cannot be excluded. Experiments performed over the last year in our laboratory reveal a novel and interesting mechanism based on the ability of nitric oxide to control the release of endogenous neurotransmitter in the pancreas and, subsequently, the nerve-mediated enzyme secretion.