Reactive oxygen species up-regulate CD11b in microglia via nitric oxide: Implications for neurodegenerative diseases

Microglial activation is considered as a hallmark of several neurodegenerative disorders. During microglial activation, the expression of CD11b, the beta-integrin marker of microglia, is increased. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of reactive oxygen species (ROS) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) stimulated the expression of CD11b in mouse BV-2 microglial cells and primary microglia, the effect that was blocked by antioxidants such as N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Furthermore, comicroinjection of either NAC or PDTC with LPS was also able to suppress LPS-stimulated expression of CD11b in striatum in vivo. Similarly, other neurotoxic molecules, such as interleukin-1beta (IL-1beta), IL-12 p40(2), fibrillar amyloid-beta (Abeta) peptides, HIV-1 gp120, and double-stranded RNA (poly(IC)), also stimulated the expression of CD11b in microglia through the involvement of ROS. Complete inhibition of LPS-stimulated expression of CD11b by catalase, induction of CD11b expression by H2O2 alone, and inhibition of superoxide-stimulated CD11b expression by catalase suggest that H2O2, but not superoxide, is in fact involved in the expression of CD11b. Interestingly, we also demonstrate that ROS stimulated the expression of CD11b after the induction of nitric oxide (NO) production and failed to stimulate CD11b when NO production was inhibited by either 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) or L-N6-(1-iminoethyl)-L-lysine (L-NIL). Taken together, these studies suggest that the up-regulation of CD11b in microglia is redox sensitive and that ROS up-regulates CD11b via NO.     

Nitric oxide induces phosphodiesterase 4B expression in rat pulmonary artery smooth muscle cells

Phosphodiesterases (PDE) metabolize cyclic nucleotides limiting the effects of vasodilators such as prostacyclin and nitric oxide (NO). In this study, DNA microarray techniques were used to assess the impact of NO on expression of PDE genes in rat pulmonary arterial smooth muscle cells (rPASMC). Incubation of rPASMC with S-nitroso-l-glutathione (GSNO) increased expression of a PDE isoform that specifically metabolizes cAMP (PDE4B) in a dose- and time-dependent manner. GSNO increased PDE4B protein levels, and rolipram-inhibitable PDE activity was 2.3 +/- 1.0-fold greater in GSNO-treated rPASMC than in untreated cells. The soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one, and the cAMP-dependent protein kinase inhibitor, H89, prevented induction of PDE4B gene expression by GSNO, but the protein kinase G (PKG) inhibitors, Rp-8-pCPT-cGMPs and KT-5823, did not. Incubation of rPASMC with IL-1beta and tumor necrosis factor-alpha induced PDE4B gene expression, an effect that was inhibited by l-N(6)-(1-iminoethyl)lysine, an antagonist of NO synthase 2 (NOS2). The GSNO-induced increase in PDE4B mRNA levels was blocked by actinomycin D but augmented by cycloheximide. Infection of rPASMC with an adenovirus specifying a dominant negative cAMP response element binding protein (CREB) mutant inhibited the GSNO-induced increase of PDE4B gene expression. These results suggest that exposure of rPASMC to NO induces expression of PDE4B via a mechanism that requires cGMP synthesis by sGC but not PKG. The GSNO-induced increase of PDE4B gene expression is CREB dependent. These findings demonstrate that NO increases expression of a cAMP-specific PDE and provide evidence for a novel "cross talk" mechanism between cGMP and cAMP signaling pathways.     

Fibrillar amyloid-beta peptides activate microglia via TLR2: implications for Alzheimer's disease

Microglial activation is an important pathological component in brains of patients with Alzheimer's disease (AD), and fibrillar amyloid-beta (Abeta) peptides play an important role in microglial activation in AD. However, mechanisms by which Abeta peptides induce the activation of microglia are poorly understood. The present study underlines the importance of TLR2 in mediating Abeta peptide-induced activation of microglia. Fibrillar Abeta1-42 peptides induced the expression of inducible NO synthase, proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-6), and integrin markers (CD11b, CD11c, and CD68) in mouse primary microglia and BV-2 microglial cells. However, either antisense knockdown of TLR2 or functional blocking Abs against TLR2 suppressed Abeta1-42-induced expression of proinflammatory molecules and integrin markers in microglia. Abeta1-42 peptides were also unable to induce the expression of proinflammatory molecules and increase the expression of CD11b in microglia isolated from TLR2(-/-) mice. Finally, the inability of Abeta1-42 peptides to induce the expression of inducible NO synthase and to stimulate the expression of CD11b in vivo in the cortex of TLR2(-/-) mice highlights the importance of TLR2 in Abeta-induced microglial activation. In addition, ligation of TLR2 alone was also sufficient to induce microglial activation. Consistent to the importance of MyD88 in mediating the function of various TLRs, antisense knockdown of MyD88 also inhibited Abeta1-42 peptide-induced expression of proinflammatory molecules. Taken together, these studies delineate a novel role of TLR2 signaling pathway in mediating fibrillar Abeta peptide-induced activation of microglia.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.     

Nitric oxide augments single Ca2+ channel currents via cGMP-dependent protein kinase in Kenyon cells isolated from the mushroom body of the cricket brain

Behavioral and pharmacological studies in insects have suggested that the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is involved in the formation of long-term memory (LTM) associated with olfactory learning. However, the target molecules of NO and the downstream signaling pathway are still not known. In this study, we investigated the action of NO on single voltage-dependent Ca²⁺ channels in the intrinsic neurons known as Kenyon cells within the mushroom body of the cricket brain, using the cell-attached configuration of the patch-clamp technique. Application of the NO donor S-nitrosoglutathione (GSNO) increased the open probability (NP_O) of single Ca²⁺ channel currents. This GSNO-induced increase was blocked by ODQ, a soluble guanylate cyclase (sGC) inhibitor, suggesting that the NO generated by GSNO acts via sGC to raise cGMP levels. The membrane-permeable cGMP analog 8-Bro-cGMP also increased the NP_O of single Ca²⁺ channel currents. Pretreatment of cells with KT5823, a protein kinase G blocker, abolished the excitatory effect of GSNO. These results suggest that NO augments the activity of single Ca²⁺ channels via the cGMP/PKG signaling pathway. To gain insight into the physiological role of NO, we examined the effect of GSNO on action potentials of Kenyon cells under current-clamp conditions. Application of GSNO increased the frequency of action potentials elicited by depolarizing current injections, indicating that NO acts as a modulator resulting in a stimulatory signal in Kenyon cells. We discuss the increased Ca²⁺ influx through these Ca²⁺ channels via the NO/cGMP signaling cascade in relation to the formation of olfactory LTM.     

Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMP-dependent modulation of NF-κB activity

Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (≤10μM) and decreased at higher (>100μM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-κB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-κB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway.     

Involvement of cyclic GMP and protein kinase G in the regulation of apoptosis and survival in neural cells

Our current understanding of nitric oxide (NO), cyclic GMP (cGMP) and protein kinase G (PKG) signaling pathways in the nervous systems has its origins in the early studies conducted on vascular tissues during the late 1970s and early to mid-1980s. The pioneering research into the NO/cGMP/PKG pathway in blood vessels conducted by the laboratories of Drs. Ferid Murad, Louis Ignarro and Robert Furchgott ultimately led to the awarding of the 1998 Nobel Prize in Physiology or Medicine to these three scientists. On the basis of further pioneering studies by Drs. John Garthwaite, Solomon Snyder, Steven Vincent and many other neuroscientists during the late 1980s and throughout the 1990s, it became recognized that NO serves as a neurotransmitter/neuromodulator in the central and peripheral nervous systems and that certain neural cells possess a cGMP signaling pathway similar to that in vascular smooth muscle cells. Although NO (at high concentrations) is toxic and thought to participate in neuronal cell death during stroke and neurodegenerative diseases (e.g. amyotrophic lateral sclerosis, Alzheimer's disease, HIV dementia and Parkinson's disease), recent evidence suggests that NO at low physiological concentrations can act as an antiapoptotic/prosurvival factor in certain neural cells (e.g. PC12 cells, motor neurons and neurons of dorsal root ganglia, hippocampus and sympathetic nerves). The antiapoptotic effects of NO are mediated, in part, by cGMP and a downstream target protein, PKG. Other cGMP-elevating factors (e.g. atrial and brain natriuretic peptides) and direct PKG activator (e.g. 8-bromo-cGMP) also have antiapoptotic effects which have been quantified by the new capillary electrophoresis with laser-induced fluorescence detector technology. Inhibition of soluble guanylyl cyclase and lowering of basal cGMP levels cause apoptosis in unstressed neural cells (NG108-15 and N1E-115 cells). The cGMP/PKG pathway appears to play an essential role in preventing activation of a proapoptotic pathway, thus promoting neural cell survival.     

Autotaxin protects microglial cells against oxidative stress

Oxidative stress occurs when antioxidant defenses are overwhelmed by oxygen-reactive species and can lead to cellular damage, as seen in several neurodegenerative disorders. Microglia are specialized cells in the central nervous system that act as the first and main form of active immune defense in the response to pathological events. Autotaxin (ATX) plays an important role in the modulation of critical cellular functions, through its enzymatic production of lysophosphatidic acid (LPA). In this study, we investigated the potential role of ATX in the response of microglial cells to oxidative stress. We show that treatment of a microglial BV2 cell line with hydrogen peroxide (H(2)O(2)) stimulates ATX expression and LPA production. Stable overexpression of ATX inhibits microglial activation (CD11b expression) and protects against H(2)O(2)-treatment-induced cellular damage. This protective effect of ATX was partially reduced in the presence of the LPA-receptor antagonist Ki16425. ATX overexpression was also associated with a reduction in intracellular ROS formation, carbonylated protein accumulation, proteasomal activity, and catalase expression. Our results suggest that up-regulation of ATX expression in microglia could be a mechanism for protection against oxidative stress, thereby reducing inflammation in the nervous system.     

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.     

Protective effects of S-nitrosoglutathione against amyloid beta-peptide neurotoxicity

Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. We have previously demonstrated ceramide production secondary to Abeta-induced activation of neutral sphingomyelinase (nSMase) in cerebral endothelial cells and oligodendrocytes, which may contribute to cellular injury during progression of AD. In this study, we first established the "Abeta --> nSMase --> ceramide --> free radical --> cell death" pathway in primary cultures of fetal rat cortical neurons. We also provided experimental evidence showing that S-nitrosoglutathione (GSNO), a potent endogenous antioxidant derived from the interaction between nitric oxide (NO) and glutathione, caused dose-dependent protective effects against Abeta/ceramide neurotoxicity via inhibition of caspase activation and production of reactive oxygen species (ROS). This GSNO-mediated neuroprotection appeared to involve activation of cGMP-dependent protein kinase (PKG), phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinase (ERK). Activation of the cGMP/PKG pathway induced expression of thioredoxin and Bcl-2 that were beneficial to cortical neurons in antagonizing Abeta/ceramide toxicity. Consistently, exogenous application of thioredoxin exerted remarkable neuroprotective efficacy in our experimental paradigm. Results derived from the present study establish a neuroprotective role of GSNO, an endogenous NO carrier, against Abeta toxicity via multiple signaling pathways.     

Gangliosides activate cultured rat brain microglia

Microglia, brain resident macrophages, are activated in brain injuries and several neurodegenerative diseases. However, microglial activators that are produced in the brain are not yet defined. In this study, we showed that gangliosides, sialic acid-containing glycosphingolipids, could be a microglial activator. Gangliosides induced production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) and expression of cyclooxygenase-2 (COX-2). The effect of gangliosides on NO release increased dose-dependently in the range of 10-100 microgram/ml; however, the effect decreased at concentrations higher than 200 microgram/ml. Specific types of gangliosides showed differential effects on microglial activation. Similar to gangliosides, GT1b induced production of NO and TNF-alpha and expression of COX-2. However, GM1 and GD1a induced expression of COX-2 but had little effect on NO and TNF-alpha release. The effect of gangliosides and GT1b on NO release was reduced in the presence of neuraminidase, which removes sialic acid residues from gangliosides and GT1b. Gangliosides activated extracellular signal-regulated kinase significantly but activated c-jun N-terminal kinase/stress-activated protein kinase and p38 relatively weakly. The inhibition of extracellular signal-regulated kinase by PD98059 reduced NO release from both gangliosides- and GT1b-treated microglia whereas inhibition of p38 by SB203580 increased it rather slightly. Gangliosides activated NF-kappaB, and N-acetyl cystein, an inhibitor of NF-kappaB, reduced NO release. These results suggest that gangliosides could be a microglial activator that functions via activation of mitogen-activated protein kinase and NF-kappaB.     

Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B-cell-directed cytotoxicity of cytokines in insulin-dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-cell line HIT-T15 in a dose- and time-dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 μM) and dibutyryl cGMP (300 μM) were able to fully relieve this inhibition. In addition, incubation of HIT-T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose-dependent over the concentration range of 10–250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT-T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.     

Role of protein kinase G in nitric oxide- and cGMP-induced relaxation of newborn ovine pulmonary veins.

In a variety of systemic blood vessels, protein kinase G (PKG) plays a critical role in mediating relaxation induced by agents that elevate cGMP, such as nitric oxide. The role of PKG in nitric oxide- and cGMP-induced relaxation is less certain in the pulmonary circulation. In the present study, we examined the effects of inhibitors of PKG on the responses of isolated fourth-generation pulmonary veins of newborn lambs (10 +/- 1 days of age) to nitric oxide and cGMP. In vessels preconstricted with endothelin-1, nitric oxide and 8-bromo-cGMP (a cell-membrane-permeable cGMP analog) induced concentration-dependent relaxation. The relaxation was significantly attenuated by beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Rp-8-Br-PET-cGMPS; a PKG inhibitor) and N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide [H-8; an inhibitor of PKG and protein kinase A (PKA)] but was not affected by KT-5720 (a PKA inhibitor). Biochemical study showed that PKG activity in newborn ovine pulmonary veins was inhibited by 8-Br-PET-cGMPS and H-8 but not by KT-5720. PKA activity was not affected by 8-Br-PET-cGMPS but was inhibited by H-8 and KT-5720. These results suggest that PKG is involved in relaxation of pulmonary veins of newborn lambs induced by nitric oxide and cGMP.     

Immunosuppression,peripheral inflammation and invasive infection from endogenous gut microbiota activate retinal microglia in mouse models

Although its actual role in the progression of degenerative processes is not fully known, the persistent activated state of retinal microglia and the concurrent secretion of inflammatory mediators may contribute to neuronal death and permanent vision loss. Our objective was to determine whether non‐ocular conditions (immunosuppression and peripheral inflammation) could lead to activation of retinal microglia. Mouse models of immunosuppression induced by cyclophosphamide and/or peripheral inflammation by chemically induced sublethal colitis in C57BL/6J mice were used. Retinal microglia morphology, spatial distribution and complexity, as well as MHCII and CD11b expression levels were determined by flow cytometry and confocal immunofluorescence analysis with anti‐CD11b, anti‐IBA1 and anti‐MHCIIRT1B antibodies. Retinas of mice with double treatment showed changes in microglial morphology, spatial distribution and expression levels of CD11b and MHCII. These effects were higher than those observed with any treatment separately. In addition, we also observed in these mice: (i) translocation of endogenous bacteria from gut to liver, and (ii) upregulation of TLR2 expression in retinal microglia. Using a mouse model of immunosuppression and gut colonization by Candida albicans, translocation of fungal cells was confirmed to occur in wild type and, to a higher extent, in TLR2 KO mice, which are more susceptible to fungal invasion; interestingly microglial changes were also higher in TLR2 KO mice. Hence, non‐ocular injuries (immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota) can activate retinal microglia and therefore could affect the progression of neurodegenerative disorders and should be taken into account to improve therapeutic options.     

TGF-beta1 represses activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity

Overactivation of microglial cells may cause severe brain tissue damage in various neurodegenerative diseases. Therefore, the overactivation of microglia should be repressed by any means. The present study investigated the potential mechanism and signaling pathway for the repressive effect of TGF-beta1, a major anti-inflammatory cytokine, on overactivation and resultant death of microglial cells. A bacterial endotoxin LPS stimulated expression of inducible NO synthase (iNOS) and caused death in cultured microglial cells. TGF-beta1 markedly blocked these LPS effects. However, the LPS-evoked death of microglial cells was not solely attributed to excess production of NO. Because phosphatidylinositol 3-kinase (PI3K) was previously shown to play a crucial role in iNOS expression and cell survival signals, we further studied whether PI3K signaling was associated with the suppressive effect of TGF-beta1. Like TGF-beta1, the PI3K inhibitor LY294002 blocked iNOS expression and death in cultured microglial cells. Both TGF-beta1 and LY294002 decreased the activation of caspases 3 and 11 and the mRNA expression of various kinds of inflammatory molecules caused by LPS. TGF-beta1 was further found to decrease LPS-induced activation of PI3K and Akt. TGF-beta1 and LY294002 suppressed LPS-induced p38 mitogen-activated kinase and c-Jun N-terminal kinase activity. In contrast, TGF-beta1 and LY294002 enhanced LPS-induced NF-kappaB activity. Our data indicate that TGF-beta1 protect normal or damaged brain tissue by repressing overactivation of microglial cells via inhibition of PI3K and its downstream signaling molecules.     

Cyclic GMP specifically suppresses Type-Iα cGMP-dependent protein kinase expression by ubiquitination

Type I cGMP-dependent protein kinase (PKG-I) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. Expression of PKG-I in cultured smooth muscle cells depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-α, which are known to stimulate Type II NO synthase (iNOS) expression. We report here that the suppression of PKG-I protein levels in smooth muscle cells is triggered by the ubiquitin/26S proteasome pathway. Incubation of vascular smooth muscle cells with phosphodiesterase-resistant cyclic GMP analogs (e.g., 8-bromo-cGMP) decreases PKG-I protein level in a time- and concentration-dependent manner. To study this process, we tested the effects of 8-Br-cGMP on PKG-I protein level in Cos7 cells, which do not express endogenous type I PKG mRNA. 8-Br-cGMP induced the ubiquitination and down-regulation of PKG-Iα, but not PKG-Iβ. Treatment of cells with the 26S proteasome inhibitor, MG-132, increased ubiquitination of PKG. Blocking PKG-I catalytic activity using the cell-permeant specific PKG-I inhibitor, DT-2, inhibited cGMP-induced PKG-I ubiquitination and down-regulation, suggesting that PKG catalytic activity and autophosphorylation were required for suppression of PKG-I level. Mutation of the known autophosphorylation sites of PKG-Iα to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG-I level. The results suggest that chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and degradation of PKG-Iα in smooth muscle.     

Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa

Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+ -free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.     

Oxygen-mediated regulation of tumor cell invasiveness. Involvement of a nitric oxide signaling pathway

Tumor hypoxia is associated with a poor prognosis for patients with various cancers, often resulting in an increase in metastasis. Moreover, exposure to hypoxia increases the ability of breast carcinoma cells to invade the extracellular matrix, an important aspect of metastasis. Here, we demonstrate that the hypoxic up-regulation of invasiveness is linked to reduced nitric oxide signaling. Incubation of human breast carcinoma cells in 0.5% versus 20% oxygen increased their in vitro invasiveness and their expression of the urokinase receptor, an invasion-associated molecule. These effects of hypoxia were inhibited by nitric oxide-mimetic drugs; and in a manner similar to hypoxia, pharmacological inhibition of nitric oxide synthesis increased urokinase receptor expression. The nitric oxide signaling pathway involves activation of soluble guanylyl cyclase (sGC) and the subsequent activation of protein kinase G (PKG). Culture of tumor cells under hypoxic conditions (0.5% versus 20% oxygen) resulted in lower cGMP levels, an effect that could be prevented by incubation with glyceryl trinitrate. Inhibition of sGC activity with a selective blocker or with the heme biosynthesis inhibitor desferrioxamine increased urokinase receptor expression. These compounds also prevented the glyceryl trinitrate-mediated suppression of urokinase receptor expression in cells incubated under hypoxic conditions. In contrast, direct activation of PKG using 8-bromo-cGMP prevented the hypoxia- and desferrioxamine-induced increases in urokinase receptor expression as well as the hypoxia-mediated enhanced invasiveness. Further involvement of PKG in the regulation of invasion-associated phenotypes was established using a selective PKG inhibitor, which alone increased urokinase receptor expression. These findings reveal that an important mechanism by which hypoxia increases tumor cell invasiveness (and possibly metastasis) requires inhibition of the nitric oxide signaling pathway involving sGC and PKG activation.     

Chemosensory behaviour and ciliary cyclic GMP-dependent protein kinase in Tetrahymena thermophila

The role of protein kinase, in particular cyclic GMP-dependent protein kinase (PKG), in the control of chemotaxis was studied in Tetrahymena thermophila using the membrane-permeable cGMP analogue 8-bromo-cGMP and the NO-generator sodium nitroprusside (SNP) that stimulates cGMP production by activating guanylate cyclase. Stimulation of chemoattraction was observed in the presence of 8-bromo-cGMP and nitroprusside when used in 10–100 μM concentrations in vivo. In vitro stimulation of ciliary membrane PKG activity was observed when using similar concentrations of cGMP or 8-bromo-cGMP to those in the in vivo experiments. In contrast, the protein kinase flavonol inhibitors quercitin and kaempherol block chemoattraction and reduce ciliary membrane PGK activity in vitro. For the inhibition of PKG, the IC-50 s for quercitin and kaempherol are 22 and 19 μM, respectively. The results suggest a modulating function of PKG on adaptory processes in cilia-mediated chemotaxis.

The ciliary membrane-associated PKG was partially characterized. Without added external protein kinase substrate in vitro, an endogenous ciliary membrane kinase activity showed phosphorylation of 55 and 97 kDa Triton-X-100 soluble proteins when analyzed by SDS-PAGE under reducing conditions and with ³²P-γ-ATP as phosphorylation donor. Phosphoamino acid analysis of PKG-phosphorylated proteins showed ³²P-phosphate labeling of serine and threonine residues. Ciliary membrane-associated PKG was further purified by carboxy-methyl-sephadex-column chromatography. The membrane enzyme was Mg²⁺⁺-dependent and had a pH optimum at 6.4. The carboxy-methyl-sephadex-eluted PKG was analyzed by electrophoresis on sodium dodecyl sulphate polyacrylamide gels showing a molecular weight of 70–75 kDa.