Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation

12/15-Lipoxygenase (LOX) activity is elevated in vascular diseases associated with impaired nitric oxide (⋅NO) bioactivity, such as hypertension and atherosclerosis. In this study, primary porcine monocytes expressing 12/15-LOX, rat A10 smooth muscle cells transfected with murine 12/15-LOX, and purified porcine 12/15-LOX all consumed ⋅NO in the presence of lipid substrate. Suppression of LOX diene conjugation by ⋅NO was also found, although the lipid product profile was unchanged. ⋅NO consumption by porcine monocytes was inhibited by the LOX inhibitor, eicosatetraynoic acid. Rates of arachidonate (AA)- or linoleate (LA)-dependent ⋅NO depletion by porcine monocytes (2.68 ± 0.03 nmol ⋅ min−1 ⋅ 106 cells−1 and 1.5 ± 0.25 nmol ⋅ min−1 ⋅ 106 cells−1, respectively) were several-fold greater than rates of ⋅NO generation by cytokine-activated macrophages (0.1–0.2 nmol ⋅ min−1 ⋅ 106 cells−1) and LA-dependent ⋅NO consumption by primary porcine monocytes inhibited ⋅NO activation of soluble guanylate cyclase. These data indicate that catalytic ⋅NO consumption by 12/15-LOX modulates monocyte ⋅NO signaling and suggest that LOXs may contribute to vascular dysfunction not only by the bioactivity of their lipid products, but also by serving as catalytic sinks for ⋅NO in the vasculature.

Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.

Interaction of the two cytosolic domains of mammalian adenylyl cyclase.

Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli. We have synthesized and purified the C1a domain of type I adenylyl cyclase and the C2 domain of the type II enzyme to assess their interactions with each other and with the activators Gsalpha and forskolin. In the absence of an activator, the fragments associate with low affinity and display low catalytic activity. This basal activity can be stimulated more than 100-fold by either forskolin or activated Gsalpha. Further, the addition of these activators increases the apparent affinity of the fragments for each other. Stimulation of catalysis by Gsalpha and forskolin is synergistic. These data suggest a model wherein either Gsalpha or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the C1 and C2 domains of the enzyme.

The heart communicates with the kidney exclusively through the guanylyl cyclase-A receptor: acute handling of sodium and water in response to volume expansion.

Disruption of guanylyl cyclase-A (GC-A) results in mice displaying an elevated blood pressure, which is not altered by high or low dietary salt. However, atrial natriuretic peptide (ANP), a proposed ligand for GC-A, has been suggested as critical for the maintenance of normal blood pressure during high salt intake. In this report, we show that infusion of ANP results in substantial natriuresis and diuresis in wild-type mice but fails to cause significant changes in sodium excretion or urine output in GC-A-deficient mice. ANP, therefore, appears to signal through GC-A in the kidney. Other natriuretic/diuretic factors could be released from the heart. Therefore, acute volume expansion was used as a means to cause release of granules from the atrium of the heart. That granule release occurred was confirmed by measurements of plasma ANP concentrations, which were markedly elevated in both wild-type and GC-A-null mice. After volume expansion, urine output as well as urinary sodium and cyclic GMP excretion increased rapidly and markedly in wild-type mice, but the rapid increases were abolished in GC-A-deficient animals. These results strongly suggest that natriuretic/diuretic factors released from the heart function exclusively through GC-A.

Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 alpha-/-mice.

The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.

Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase.

The phosphoprotein phosducin (Pd) regulates many guanine nucleotide binding protein (G protein)-linked signaling pathways. In visual signal transduction, unphosphorylated Pd blocks the interaction of light-activated rhodopsin with its G protein (Gt) by binding to the beta gamma subunits of Gt and preventing their association with the Gt alpha subunit. When Pd is phosphorylated by cAMP-dependent protein kinase, it no longer inhibits Gt subunit interactions. Thus, factors that determine the phosphorylation state of Pd in rod outer segments are important in controlling the number of Gts available for activation by rhodopsin. The cyclic nucleotide dependencies of the rate of Pd phosphorylation by endogenous cAMP-dependent protein kinase suggest that cAMP, and not cGMP, controls Pd phosphorylation. The synthesis of cAMP by adenylyl cyclase in rod outer segment preparations was found to be dependent on Ca2+ and calmodulin. The Ca2+ dependence was within the physiological range of Ca2+ concentrations in rods (K1/2 = 230 +/- 9 nM) and was highly cooperative (n app = 3.6 +/- 0.5). Through its effect on adenylyl cyclase and cAMP-dependent protein kinase, physiologically high Ca2+ (1100 nM) was found to increase the rate of Pd phosphorylation 3-fold compared to the rate of phosphorylation at physiologically low Ca2+ (8 nM). No evidence for Pd phosphorylation by other (Ca2+)-dependent kinases was found. These results suggest that Ca2+ can regulate the light response at the level of Gt activation through its effect on the phosphorylation state of Pd.

cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase.

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (IC50 = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 approximately 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.

Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements.

Agonist-induced desensitization of dopamine D1 receptor-stimulated adenylyl cyclase activity is temporally and biochemically separated from D1 receptor internalization.

The regulation of the dopamine D1 receptor was investigated by using c-myc epitope-tagged D1 receptors expressed in Sf9 (fall armyworm ovary) cells. Treatment of D1 receptors with 10 microM dopamine for 15 min led to a loss of the dopamine-detected high-affinity state of the receptor accompanying a 40% reduction in the ability of the receptor to mediate maximal dopamine stimulation of adenylyl cyclase activity. After 60 min of agonist exposure, 45 min after the occurrence of desensitization, 28% of the cell surface receptors were internalized into an intracellular light vesicular membrane fraction as determined by radioligand binding and supported by photoaffinity labeling, immunocytochemical staining, and immunoblot analysis. Pretreatment of cells with concanavalin A or sucrose completely blocked agonist-induced D1 receptor internalization without preventing agonist-induced desensitization, indicating a biochemical separation of these processes. Collectively, these findings indicate that the desensitization of D1 receptor-coupled adenylyl cyclase activity and D1 receptor internalization are temporarily and biochemically distinct mechanisms regulating D1 receptor function following agonist activation.

Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.

A cDNA encoding rat oxidosqualene lanosterol-cyclase [lanosterol synthase; (S)-2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization. An open reading frame of 2199 bp encodes a M(r) 83,321 protein with 733 amino acids. The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-). Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif. The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs. The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide. Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain. A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae. The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor [3H]29-MOS.

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.

Cloning and expression of a second photoreceptor-specific membrane retina guanylyl cyclase (RetGC), RetGC-2.

One of the membrane guanylyl cyclases (GCs), RetGC, is expressed predominantly in photoreceptors. No extracellular ligand has been described for RetGC, but it is sensitive to activation by a soluble 24-kDa protein (p24) and is inhibited by Ca2+. This enzyme is, therefore, thought to play a role in resynthesizing cGMP for photoreceptor recovery or adaptation. By screening a human retinal cDNA library at low stringency with the cytoplasmic domains from four cyclases, we cloned cDNAs encoding a membrane CG that is most closely related to RetGC. We have named this GC RetGC-2, and now term the initially described RetGC RetGC-1. By in situ hybridization, mRNA encoding RetGC-2 is found only in the outer nuclear layer and inner segments of photoreceptor cells. By using synthetic peptide antiserum specific for each RetGC subtype, RetGC-2 can be distinguished from RetGC-1 as a slightly smaller protein in immunoblots of bovine rod outer segments. Membrane GC activity of recombinant RetGC-2 expressed in human embryonic kidney 293 cells is stimulated by the activator p24 and is inhibited by Ca2+ with an EC50 value of 50-100 nM. Our data reveal a previously unappreciated diversity of photoreceptor GCs.

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

We have cloned an additional member (GC-D) of the membrane receptor guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] family that is specifically expressed in a subpopulation of olfactory sensory neurons. The extracellular, putative ligand-binding domain of the olfactory cyclase is similar in primary structure to two guanylyl cyclases expressed in the retina but diverges considerably from other known guanylyl cyclases. The expression of GC-D RNA is restricted to a small, randomly dispersed population of neurons that is within a single topographic zone in the olfactory neuroepithelium and resembles the pattern of the more diverse seven-transmembrane-domain odorant receptors. These observations suggest that GC-D may function directly in odor recognition or in modulating the sensitivity of a subpopulation of sensory neurons to specific odors.

Basis of guanylate cyclase activation by carbon monoxide.

Kinetics of CO association with guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] and dissociation from carboxy guanylate cyclase have been studied at pH 7.5 by flash photolysis, yielding rate constants at 23 degrees C of 1.2 +/- 0.1 x 10(5) M-1.sec-1 and 28 +/- 2 sec-1, respectively. While the CO combination rate constant is the same as for the T state of hemoglobin, the CO dissociation rate constant is much higher than expected for a six-coordinate carboxyheme protein; yet the absorption spectrum is indicative of a six-coordinate heme. The two observations are reconciled by a reaction mechanism in which CO dissociation proceeds via a five-coordinate intermediate. This intermediate is structurally very similar to the five-coordinate nitrosyl heme derivative of guanylate cyclase and is presumably responsible for the observed 4-fold activation of guanylate cyclase by CO. Thus, we provide a model that explains enzyme activities of the nitrosyl and carboxy forms of the enzyme on the basis of a common mechanism.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.