Ovariectomy upregulates expression of estrogen receptors,NOS, and HSPs in porcine platelets

Platelets participate in normal and pathological thrombotic processes. Hormone replacement in postmenopausal women is associated with increase risk for thrombosis. However, little is known regarding how platelets are affected by hormonal status. Nitric oxide (NO) modulates platelet functions and is modulated by hormones. Therefore, the present study was designed to determine how loss of ovarian hormones changes expression of estrogen receptors and regulatory proteins for NO synthase (NOS) in platelets. Estrogen receptors (ER alpha and ER beta), NOS, heat shock proteins 70 and 90 (HSP70 and HSP90), caveolin-1, -2, and -3, calmodulin, NOS activity, and cGMP were analyzed in a lysate of platelets from gonadally intact and ovariectomized female pigs. Expression of ER beta and ER alpha receptors, endothelial NOS (eNOS), HSP70, and HSP90 increased with ovariectomy. NOS activity and cGMP also increased; calmodulin was unchanged. Caveolins were not detected. These results suggest that ovarian hormones influence expression of estrogen receptors and eNOS in platelets. Changes in estrogen receptors and NOS could affect platelet aggregation in response to hormone replacement.     

Dehydroepiandrosterone stimulates nitric oxide release in vascular endothelial cells: evidence for a cell surface receptor

Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.     

Adiponectin inhibits hyperlipidemia-induced platelet aggregation via attenuating oxidative/nitrative stress

Adiponectin acts as an endogenous antithrombotic factor. However, the mechanisms underlying the inhibition of platelet aggregation by adiponectin still remain elusive. The present study was designed to test whether adiponectin inhibits platelet aggregation by attenuation of oxidative/nitrative stress. Adult rats were fed a regular or high-fat diet for 14 weeks. The platelet was immediately separated and stimulated with recombinant full-length adiponectin (rAPN) or not. The platelet aggregation, nitric oxide (NO) and superoxide production, endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) expression, and antioxidant capacity were determined. Treatment with rAPN inhibited hyperlipidemia-induced platelet aggregation (P<0.05). Interestingly, total NO, a crucial molecule depressing platelet aggregation and thrombus formation?was significantly reduced, rather than increased in rAPN-treated platelets. Treatment with rAPN markedly decreased superoxide production (-62 %, P<0.05) and enhanced antioxidant capacity (+38 %, P<0.05) in hyperlipidemic platelets. Hyperlipidemia-induced reduced eNOS phosphorylation and increased iNOS expression were significantly reversed following rAPN treatment (P<0.05, P<0.01, respectively). Taken together, these data suggest that adiponectin is an adipokine that suppresses platelet aggregation by enhancing eNOS activation and attenuating oxidative/nitrative stress including blocking iNOS expression and superoxide production.     

PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act upstream upregulating this pathway.     

The anandamide effect on NO/cGMP pathway in human platelets

In this study the effect of the endocannabinoid anandamide on platelet nitric oxide (NO)/cGMP pathway was investigated. Data report that anandamide in a dose-and time-dependent manner increased NO and cGMP levels and stimulated endothelial nitric oxide synthase (eNOS) activity. These parameters were significantly reduced by LY294002, selective inhibitor of PI3K and by MK2206, specific inhibitor of AKT. Moreover anandamide stimulated both eNOSser1177 and AKTser473 phosphorylation. Finally the anandamide effect on NO and cGMP levels, eNOS and AKT phosphorylation/activation were inhibited by SR141716, specific cannabinoid receptor 1 antagonist, supporting the involvement of anandamide binding to this receptor. Overall data of this report indicate that low concentrations of anandamide, through PI3K/AKT pathway activation, stimulates eNOS activity and increases NO levels in human platelets. In such way anandamide contributes to extend platelet survival.     

(-)-Epicatechin induces calcium and translocation independent eNOS activation in arterial endothelial cells

The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (-)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca(2+) depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca(2+)-dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca(2+)-free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca(2+) depletion. Thus, under Ca(2+)-free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca(2+)-deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-L-arginine methyl ester, suggesting a functional relevance for this phenomenon.     

The arachidonic acid effect on platelet nitric oxide level

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A₂ mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A₂ receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.     

Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were "uncoupling" NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.     

Calreticulin Transacetylase mediated activation of human platelet nitric oxide synthase by acetyl group donor compounds

Polyphenols have attracted immense interest because of their diverse biological and pharmacological activities. Surprisingly, not much is documented about the biological activities of acetoxy derivatives of polyphenol called polyphenolic acetates (PA). In our previous reports, we have conclusively established the Calreticulin Transacetylase (CRTAase) catalyzed activation of neuronal nitric oxide synthase (nNOS) and tumor necrosis factor-α (TNF-α) induced nitric oxide synthase (iNOS) by PA. In the present work, specificity of CRTAase to various classes of PA was characterized in human platelet. The effect of PA, on platelet NOS and intracellular cyclic guanosine monophosphate (cGMP), and adenosine diphosphate (ADP)-induced platelet aggregation were studied in an elaborated manner. Platelet CRTAase exhibited differential specificities to polyphenolic acetates upon incubation with l-arginine leading to activation of NOS. The intraplatelet generation of NO was studied by flowcytometry using DCFH-DA. The differential specificities of CRTAase to PA were found to positively correlate with increased production of NO upon incubation of PRP with PA and l-arginine. Further, the inhibitory effect of l-NAME on PA induced NO formation in platelets substantiated the CRTAase catalyzed activation of NOS. The real-time RT-PCR profile of NOS isoforms confirmed the preponderance of eNOS over iNOS in human platelets on treatment with PA. Western blot analysis also reiterated the differential pattern of acetylation of eNOS by PA. PA were also found effective in increasing the intraplatelet cGMP levels and inhibiting ADP-induced platelet aggregation. It is worth mentioning that the effects of PA were found to be in tune with the specificities of platelet CRTAase to PA as the substrates.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5?5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca²⁺]_i mobilization, thromboxane A₂ (TxA₂) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH^●) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA₂ cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.     

Usefulness of experimental models to understand the vascular effects of estrogens

Hormonal replacement therapy does not prevent cardiovascular events in postmenopausal women. In contrast, the incidence of cardiovascular diseases is higher in men than in premenopausal women but increases in postmenopausal women, and all animal studies demonstrate a prevention of fatty streak deposit by estradiol. Although estradiol improves the lipoprotein profile, this effect can account for only a minor part of the protective effect. Endothelium appears to be an important target for estradiol, because this hormone potentiates endothelial nitric oxide (NO) production, thus promoting the beneficial effects of NO, such as vasorelaxation and inhibition of platelet aggregation. Estradiol accelerates endothelial regrowth, thus favoring vascular healing, and prevents apoptosis of endothelial cells. Estradiol prevents fatty streak deposit through a mechanism which is clearly independent of NO. The immuno-inflammatory system appears to play a key role in the development of fatty streak deposit as well as in atherosclerotic plaque rupture. Mice deficient either in monocyte-macrophages or in lymphocytes are partially protected against fatty streak deposit. Interestingly, the atheroprotective effect of estradiol is absent in mice deficient in T and B lymphocytes. Most of these effects of estradiol are mediated by estrogen receptor alpha, and are independent of estrogen receptor beta. Thus, the inflammatory-immune system appears to be also a major target of estrogens. However, the effects of estrogens on the immuno-inflammatory system appear ambiguous, as in some models, estradiol rather promotes inflammation (by increasing interferon gamma which could elicit plaque destabilization). A better understanding of the mechanisms of estrogens on the normal and atheromatous arteries is required and should help to optimize the prevention of cardiovascular disease after menopause.     

A cell-based nitric oxide reporter assay useful for the identification and characterization of modulators of the nitric oxide/guanosine 3',5'-cyclic monophosphate pathway

Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca(2+)-dependent and Ca(2+)-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.     

Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.     

N(omega)-nitro-L-arginine inhibits inducible HSP-70 via Ca(2+), PKC, and PKA in human intestinal epithelial T84 cells

The nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45 degrees C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC(50) of 0.0471 +/- 0.0007 microM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca(2+) dependent, we measured [Ca(2+)](i) after treating cells with L-NNA. L-NNA at 100 microM significantly decreased resting [Ca(2+)](i). Likewise, treatment with 1 microM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca(2+)](i) and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca(2+)](i) and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca(2+)](i) and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.     

Role of platelet endothelial form of nitric oxide synthase in collagen-platelet interaction: regulation by phosphorylation

Different pathways have been reported to be involved in platelet-collagen interaction. We have reported that the platelet endothelial form of nitric oxide synthase (eNOS) and the platelet receptor for type I collagen, p65, are closely associated. But the controlling mechanism underlying the generation of nitric oxide (NO) by the eNOS has not been fully explored. In this investigation, Western blot analyses of time course samples with anti-phosphorylated tyrosine, and anti-serine/threonine showed a marked increase in serine/threonine phosphorylation of eNOS during type I collagen-induced platelet aggregation. Meanwhile, the eNOS activity measured by the conversion of [3H]-arginine to [3H]-citrulline is significantly decreased. Correlation of type I collagen-induced platelet aggregation and the activity of eNOS in the presence of the serine/threonine phosphatase inhibitor, okadiac acid and the tyrosine phosphatase inhibitor, vanadate were performed with PRP. Results show the decrease in eNOS activity by adding okadiac acid correlated with the inhibitory effect on platelet aggregation in a dose-dependent manner. On the other hand, vanadate significantly inhibits platelet aggregation and also inhibits eNOS activity when the concentration of vanadate is greater than 2 mM. These results suggest that phosphorylation of serine/threonine and tyrosine residues control the activity of eNOS through different mechanisms to affect collagen-induced platelet aggregation.     

Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.     

Increased nitric oxide production in platelets from severe chronic renal failure patients

Nitric oxide (NO) production occurs through oxidation of the amino acid L-arginine by NO synthase (NOS). NO inhibits platelet activation by increasing the levels of cyclic guanosine monophosphate (cGMP), thus maintaining vascular homeostasis. Our group previously demonstrated (da Silva et al. 2005) an enhancement of the L-arginine-NO-cGMP pathway in platelets taken from chronic renal failure (CRF) patients on haemodialysis associated with reduced platelet aggregation. We investigate the platelet L-arginine-NO-cGMP pathway, platelet function, and inflammation from patients in CRF on conservative treatment. A total of 42 CRF patients and 42 controls (creatinine clearance = 27 ± 3 vs. 93 ± 1 mL per min per 1.73 m2, respectively) participated in this study. NOS activity and expression and cGMP concentration were measured in platelets. Platelet aggregation induced by collagen or ADP was evaluated and plasma levels of fibrinogen were determined by the Clauss method. A marked increase in basal NOS activity was seen in undialysed CRF patients compared with controls, accompanied by an elevation of fibrinogen plasma levels. There were no differences in expression of NOS and in cGMP levels. In this context, platelet aggregation was not affected. We provide the first evidence of increased intraplatelet NO biosynthesis in undialysed CRF patients, which can be an early marker of future haemostatic abnormalities during dialysis treatment.     

Nitric Oxide Participates in Cold-Inhibited Camellia sinensis Pollen Germination and Tube Growth Partly via cGMP In Vitro

Nitric oxide (NO) plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS)-like activity, cGMP content and proline (Pro) accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC) inhibitor ODQ or phosphodiesterase (PDE) inhibitor Viagra at 25°C (control) or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis.     

豚鼠耳蜗中ATP对一氧化氮/环磷酸鸟苷途径的激活作用

实验研究了豚鼠耳蜗中ATP和一氧化氮／环磷酸鸟苷途径(nitric oxide／cyclic guanosine monophosphate,NO／cGMP pathway)的关系。将40只耳廓反射灵敏的健康白色豚鼠随机分为5组,分别对其离体的耳蜗即刻灌流人工外淋巴基础液(artificial perilymph basic solution,APBS)以及溶于人工外淋巴基础液的ATP、一氧化氮合酶抑制剂左旋-N^G-硝基精氨酸(L-N^G-nitroarginine,L-NNA) ATP、可溶性鸟苷酸环化酶抑制剂1H-[1,2,4]草酸重氮[4,3-a]喹恶啉(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one,ODQ) ATP和A-23187(Ca^2 载体),收集耳蜗组织标本,利用放射免疫方法测定耳蜗组织中的cGMP的平均含量,比较各组之间耳蜗组织cGMF平均含量的差异。试验结果显示,向刚离体的耳蜗中灌流ATP和A-23187可以引起耳蜗组织中的cGMP含量升高,而灌流L-NNA和ODQ则可以抑制ATP所引起的耳蜗组织中cGMP含量的升高,提示在耳蜗组织中ATP可以通过升高细胞内Ca^2 浓度的作用而激活NO／cGMF途径。从本实验结果可以提出假说：耳蜗中ATP从神经末梢释放,通过提高细胞内Ca^2 的浓度,有激活NO／cGMP途径的作用,而NO／cGMP又能对ATP进行负反馈调节,两者共同调节耳蜗的生理功能,在耳蜗中存在ATP／Ca^2 -NO／cGMP通路。