Nitric oxide‐sensitive guanylyl cyclase is the only nitric oxide receptor mediating platelet inhibition

See also Gordge MP. Nitric oxide: a one‐trick pony? This issue, pp 1340–2. Summary. Background: The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling cascade is involved in the precise regulation of platelet responses. NO released from the endothelium is known to activate NO‐sensitive guanylyl cyclase (NO‐GC) in platelets. By the generation of cGMP and subsequent activation of cGMP‐dependent protein kinase (PKG), NO‐GC mediates the reduction of the intracellular calcium and inhibits platelet adhesion and aggregation. However, NO has been postulated to influence these platelet functions also via cGMP‐independent mechanisms. Objective: We studied the effect of NO on platelets lacking NO‐sensitive guanylyl cyclase with regards to aggregation, adhesion, calcium mobilization and bleeding time. Methods and results: Here, we show that NO signaling leading to inhibition of agonist‐induced platelet aggregation is totally abrogated in platelets from mice deficient in NO‐GC (GCKO). Even at millimolar concentrations none of the several different NO donors inhibited collagen‐induced aggregation of GCKO platelets. In addition, NO neither affected adenosine 5′‐diphosphate (ADP)‐induced adhesion nor thrombin‐induced calcium release in GCKO platelets. Although the NO‐induced cGMP signal transduction was totally abrogated cyclic adenosine monophosphate (cAMP) signaling was still functional; however, cGMP/cAMP crosstalk was disturbed on the level of phosphodiesterase type 3 (PDE3). These in vitro data are completed by a reduced bleeding time indicating the lack of NO effect in vivo. Conclusions: We conclude that NO‐GC is the only NO receptor in murine platelets mediating the inhibition of calcium release, adhesion and aggregation: lack of the enzyme leads to disturbance of primary hemostasis.     

NO-synthase-/NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity

Summary.  Objectives: Platelets, specialized adhesive cells, play key roles in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions which are inhibited by nitric oxide (NO). Platelets have been reported to be regulated not only by exogenous endothelium-derived NO, but also by two isoforms of NO synthase, endothelial (eNOS) and inducible (iNOS), endogenously expressed in platelets. However, data concerning expression, regulation and function of eNOS and iNOS in platelets remain controversial. Methods and results: Using important positive (endothelial cells, stimulated macrophages) and negative (eNOS/iNOS knock-out mouse) controls, as well as human platelets highly purified by a newly developed protocol, we now demonstrate that human and mouse platelets do not contain eNOS/iNOS proteins or mRNA. NOS substrate ( l -arginine), NOS inhibitors (L-NAME, L-NMMA), and eNOS/iNOS deficiency did not produce detectable functional effects on human and mouse platelets. von Willebrand factor (VWF)/ristocetin treatment of platelets increased cGMP by NO-independent activation of soluble guanylyl cyclase (sGC) which correlated with Src kinase-dependent phosphorylation of sGC β₁-subunit-Tyr¹⁹². Conclusions: Human and mouse platelets do not express eNOS/iNOS. VWF/ristocetin-mediated activation of the sGC/cGMP signaling pathway may contribute to feedback platelet inhibition.     

Sodium azide, a bacteriostatic preservative contained in commercially available laboratory reagents, influences the responses of human platelets via the cGMP/PKG/VASP pathway

OBJECTIVE: The bacteriostatic preservative sodium azide (NaN(3)) activates soluble guanylate cyclase (sGC) in vascular tissues, thus elevating cellular 3',5'-cyclic guanosine monophosphate (cGMP). Because the sGC/cGMP pathway is involved in the control of platelet aggregation, we investigated whether in human platelets NaN(3) influences the responses to agonists, cGMP levels and cGMP-regulated pathways. DESIGN AND METHOD: Concentration- and time-dependent effects of NaN(3) (1-100 micromol/L; 5-60 min incubation) on ADP- and collagen-induced aggregation, NO synthase (NOS) activity, cGMP synthesis and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239 were investigated in platelets from 21 healthy individuals. RESULTS: NaN(3) exerted concentration- and time-dependent antiaggregatory effects starting from 1 micromol/L (IC(50) with 5-min incubation: 2.77+/-0.35 micromol/L with ADP and 4.64+/-0.48 micromol/L with collagen) and significantly increased intraplatelet cGMP levels and phosphorylation of VASP at Ser239 at 1-100 micromol/L; these effects were prevented by sGC inhibition, but not by NOS inhibition. CONCLUSIONS: NaN(3) exerts antiaggregatory effects in human platelets via activation of the sGC/cGMP/VASP pathway. This biological effect must be considered when azide-containing reagents are used for in vitro studies on platelet function.     

Novel roles of cAMP/cGMP‐dependent signaling in platelets

Summary. Endothelial prostacyclin and nitric oxide potently inhibit platelet functions. Prostacyclin and nitric oxide actions are mediated by platelet adenylyl and guanylyl cyclases, which synthesize cyclic AMP (cAMP) and cyclic GMP (cGMP), respectively. Cyclic nucleotides stimulate cAMP‐dependent protein kinase (protein kinase A [PKA]I and PKAII) and cGMP‐dependent protein kinase (protein kinase G [PKG]I) to phosphorylate a broad panel of substrate proteins. Substrate phosphorylation results in the inactivation of small G‐proteins of the Ras and Rho families, inhibition of the release of Ca²⁺ from intracellular stores, and modulation of actin cytoskeleton dynamics. Thus, PKA/PKG substrates translate prostacyclin and nitric oxide signals into a block of platelet adhesion, granule release, and aggregation. cAMP and cGMP are degraded by phosphodiesterases, which might restrict signaling to specific subcellular compartments. An emerging principle of cyclic nucleotide signaling in platelets is the high degree of interconnection between activating and cAMP/cGMP‐dependent inhibitory signaling pathways at all levels, including cAMP/cGMP synthesis and breakdown, and PKA/PKG‐mediated substrate phosphorylation. Furthermore, defects in cAMP/cGMP pathways might contribute to platelet hyperreactivity in cardiovascular disease. This article focuses on recent insights into the regulation of the cAMP/cGMP signaling network and on new targets of PKA and PKG in platelets.     

Nitric oxide modifies the sarcoplasmic reticular calcium release channel in endotoxemia by both guanosine-3',5' (cyclic) phosphate-dependent and independent pathways

OBJECTIVES: a) To determine whether decreased sarcoplasmic calcium release channel (CRC) activity is a mechanism by which myocardial contractility is reduced in endotoxemia; b) to determine whether nitric oxide modulates CRC activity in endotoxemia; and c) to examine two nitric oxide signaling pathways in relation to CRC function in endotoxemia. DESIGN: Randomized, prospective using a rat model of endotoxemia. SETTING:: Research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Endotoxemia was induced by lipopolysaccharide administration. The effects of nitric oxide were studied using the highly selective inducible nitric oxide synthase inhibitor N-(3-(aminomethyl)benzyl)acetamidine dihydrochloride (1400W) and the specific guanylyl cyclase inhibitor 1-H (1, 2, 4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). MEASUREMENTS AND MAIN RESULTS: We assessed myocardial contractility, myocardial nitric oxide content, and guanosine-3',5' (cyclic) phosphate (cGMP) content. We determined CRC activity by calcium release and ryanodine binding assays. We followed these variables at four time points through the course of endotoxemia. We found that myocardial contractility and CRC activity were decreased in late but not in early endotoxemia. Furthermore, inducible nitric oxide synthase inhibition with 1400W restored contractility and CRC activity in late endotoxemia but paradoxically worsened these variables in early endotoxemia. Through the use of the guanylyl cyclase inhibitor ODQ, we demonstrate that nitric oxide acts through cGMP-mediated mechanisms in early and late endotoxemia. We investigated cGMP-independent pathways by assessing the oxidative status of the CRC. We found that in late endotoxemia, nitric oxide decreased the number of free thiols, demonstrating that nitric oxide also acts through cGMP-independent pathways. CONCLUSIONS: Nitric oxide has a dual effect on the CRC in endotoxemia. At low concentrations, as measured in early endotoxemia, nitric oxide stabilizes the CRC through cGMP-mediated mechanisms. In late endotoxemia, high nitric oxide concentrations decrease channel activity through both cGMP-dependent and cGMP-independent mechanisms.     

Globular adiponectin increases cGMP formation in blood platelets independently of nitric oxide

Summary. Background: Platelet‐derived nitric oxide (NO) has been shown to play conflicting roles in platelet function, although it is accepted that NO mediates its actions through soluble guanylyl cyclase (sGC). This confusion concerning the roles of platelet NO may have arisen because of an uncharacterized mechanism for activation of sGC. Objectives: To examine the ability of the novel platelet agonist globular adiponectin (gAd) to stimulate the NO‐independent cGMP–protein kinase G (PKG) signaling cascade. Methods: We used three independent markers of NO signaling, [³H]l ‐citrulline production, cGMP accrual, and immunoblotting of vasodilator‐stimulated phosphoprotein (VASP), to examine the NO signaling cascade in response to gAd. Results: gAd increased platelet cGMP formation, resulting in a dose‐ and time‐dependent increase in phospho‐VASP^157/239. Phosphorylation of VASP in response to gAd was mediated by both protein kinase A and PKG. Importantly, cGMP formation occurred in the absence of NO synthase (NOS) activation and in the presence of NOS inhibitors. Indeed, inhibition of the NOS signaling cascade had no influence on gAd‐mediated platelet aggregation. Exploration of the mechanism demonstrated that NO‐independent cGMP formation, phosphorylation of VASP and association of sGCα₁ with heat shock protein‐90 induced by gAd were blocked under conditions that inhibited Src kinases, implying a tyrosine kinase‐dependent mechanism. Indeed, sGCα1 was reversibly tyrosine phosphorylated in response to gAd, collagen, and collagen‐related peptide, an effect that required Src kinases and downstream Ca²⁺ mobilization. Conclusions: These data demonstrate activation of the platelet cGMP signaling cascade by a novel tyrosine kinase‐dependent mechanism in the absence of NO.     

Endothelial cells play an important role in the antiaggregatory effect of nitric oxide

OBJECTIVE: To investigate the role of endothelial cyclooxygenase in the antiaggregatory effect of nitric oxide, and to investigate the significance of the time span between contact of nitric oxide and platelets and laboratory evaluation by platelet aggregation. DESIGN: Prospective, controlled, in vitro study. SETTING: Research laboratory of a university hospital. PARTICIPANTS: Three healthy volunteers. INTERVENTIONS: Incubation of platelets with different concentrations (30 microM, 100 microM, 500 microM, 1000 microM) of the nitric oxide-donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) for varying incubation times (0 hrs, 1 hr, 2 hrs, 4 hrs) with and without endothelial cells. Induction of platelet aggregation with adenosine diphosphate. Inhibition of the effect of SNAP by 100 microM of the guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). Inhibition of prostacyclin production by endothelial cells with COX inhibitors acetyl salicylic acid (1 mM) and indomethacin (10 microM). MEASUREMENTS AND MAIN RESULTS: Incubation with endothelial cells (= controls) had no effect on platelet aggregation. Platelet aggregation was significantly inhibited by all concentrations of SNAP. Time course studies with 30 microM of SNAP showed an inhibitory effect only after 0, 1, and 2 hrs of incubation, whereas after 4 hrs of incubation the inhibition of platelet aggregation could not be detected any more. Endothelial cells significantly increased the inhibitory effect of SNAP after 1 and 2 hrs of incubation. Incubation with ODQ with and without endothelial cells reversed the SNAP-mediated inhibition of maximum platelet aggregation regardless of the incubation time. Pretreatment of the endothelial cells with the COX inhibitors acetyl salicylic acid and indomethacin blocked the increased inhibitory effect of the endothelial cells after 1 and 2 hrs of incubation. CONCLUSIONS: The time span between nitric oxide contact with platelets and induction of platelet aggregation by adenosine 5'-diphosphate is important for correct estimation of the antiaggregatory effect of nitric oxide. Endothelial cyclooxygenase plays an important role in the nitric oxide-mediated inhibition of platelet aggregation.     

Insulin activates vascular endothelial growth factor in vascular smooth muscle cells: influence of nitric oxide and of insulin resistance

BACKGROUND: We aimed to evaluate whether insulin influences vascular endothelial growth factor (VEGF) synthesis and secretion in cultured vascular smooth muscle cells (VSMCs) via nitric oxide (NO) and whether these putative effects are lost in insulin-resistant states. MATERIALS AND METHODS: In VSMC derived from human arterioles and from aortas of insulin-sensitive Zucker fa/+rats and insulin-resistant Zucker fa/fa rats incubated with different concentrations of human regular insulin with or without inhibitors of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3-K), mitogen-activated protein kinase (MAPK), nitric oxide synthase (NOS) and guanosine 3',5'cyclic monophosphate(cGMP)-dependent protein kinase (PKG), we measured protein expression (Western blot) and secretion (ELISA) of VEGF. RESULTS: We found that in VSMCs from humans and from insulin-sensitive Zucker fa/+rats, insulin increases VEGF protein expression and secretion, with mechanisms blunted by wortmannin and LY294002 (PI3-K inhibitors), PD98059 (MAPK inhibitor), L-NMMA (NOS inhibitor) and Rp-8pCT-cGMPs (PKG inhibitor). Also the NO donor sodium nitroprusside (SNP) and the cGMP analogue 8-Bromo-cGMP increase VEGF protein expression and secretion, with mechanisms inhibited by wortmannin and PD98059. The insulin effects on VEGF are impaired in VSMCs from Zucker fa/fa rats, which also present a reduced insulin ability to increase NO. CONCLUSIONS: In VSMCs from humans and insulin-sensitive Zucker fa/+rats: (i) insulin increases VEGF protein expression and secretion via both PI3-K and MAPK; (ii) the insulin effects on VEGF are mediated by nitric oxide. The insulin action on both nitric oxide and VEGF is impaired in VSMCs from Zucker fa/fa rats, an animal model of metabolic and vascular insulin-resistance.     

The possible role of the NO-cGMP pathway in nociception: different spinal and supraspinal action of enzyme blockers on rat dorsal horn neurones

In the literature, the pro- or antinociceptive effects of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) are discussed controversially. Our laboratory and others have reported that in the spinal cord a local lack of NO has an excitatory action on the ongoing (background) activity of dorsal horn neurones. Here, we tested the hypothesis that this effect of NO is mediated by cGMP and that part of the controversy is due to differences in the spinal and supraspinal actions of both compounds. In anaesthetised rats, impulse activity of lumbar dorsal horn neurones was recorded, and blockers of NO- and cGMP-synthesis, as well as the phosphodiesterase 5 (PDE5) inhibitor sildenafil (which increases the cGMP level), or 8-Bromo-cGMP (a membrane permeable cGMP analogue) were administered spinally or supraspinally. Topical superfusion of the spinal cord with a blocker of the guanylyl cyclase (ODQ) to reduce the cGMP level led to an increase in background activity of nociceptive lumbar dorsal horn neurones similar to that caused by l-NAME, a blocker of the NO synthase. Spinal superfusion with sildenafil or 8-Bromo-cGMP had no excitatory effect. In contrast, injections of sildenafil or 8-Bromo-cGMP into the third cerebral ventricle caused an increased background activity in lumbar dorsal horn neurones, while l-NAME and ODQ were ineffective. The results show that at the spinal level, a lack of cGMP and NO has an excitatory action on dorsal horn neurones, whereas supraspinally an elevated level of cGMP is excitatory.     

Decreased aortic smooth muscle contraction in a rat model of multibacterial sepsis

We investigated whether blockade of the smooth muscle cell (SMC) inducible nitric oxide synthase (iNOS)-soluble guanylyl cyclase (sGC) vasodilator pathway would restore the fall in vasoreactivity produced by sepsis following cecal ligation and perforation (CLP) in rats. Contraction of adjacent aortic rings paired for the presence or absence of endothelial cells (EC) was recorded following high [K(+)](e) (40 mm) or norepinephrine (NE, 10(-8) to 10(-5) m) in the presence of the nitric oxide synthase inhibitor (NOS), N(G)-nitro-l-arginine methyl ester (l-NAME, 0.3 mm) or the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 5 mum). In EC-denuded rings, sepsis halved SMC contraction induced by high [K(+)](e) or NE; neither l-NAME nor ODQ produced an increase in NE E(max) or high [K(+)](e)-evoked contraction. In conclusion, SMC contractility is globally reduced in CLP; this reduction does not appear to be explained by induction of SMC NOS in this CLP model.     

Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway

Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process.Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.     

Signaling mechanisms coupled to presynaptic A(1)- and H(3)-receptors in the inhibition of cholinergic contractile responses of the guinea pig ileum

The mechanisms coupled to adenosine A(1)- and histamine H(3)-receptors have been examined in the presynaptic inhibition of acetylcholine (ACh) release from the guinea pig ileum. Electrically evoked twitch contractions were used as a measure of neuronal ACh release. A(1)- and H(3)-receptors were activated by adenosine and R-(alpha)-methylhistamine (RAMH), respectively. The neuroinhibitory effect of adenosine and RAMH was augmented in the presence of the N-type Ca(2+) channel blocker, omega-conotoxin GVIA but unaffected by the L-type Ca(2+) channel blocker, nifedipine. The irreversible adenylyl cyclase inhibitor, MDL-12330A, potentiated the action of both adenosine and RAMH. Conversely, neither agonist was affected by the cAMP phosphodiesterase III and IV inhibitors, SKF-95654 and Ro-20-1724, respectively, or the cAMP antagonist, (R(p))-adenosine 3',5'-cyclic monophosphorothioate triethylamine. The neuromodulatory effect of adenosine, only, was potentiated by the cGMP phosphodiesterase V inhibitors, SKF-96231 and 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)- pyrazolo[3, 4-d]pyrimidin-4-(5H)-one but was unmodified by the cGMP analog, 8-bromo-cGMP or the guanylyl cyclase inhibitors, N-methylhydroxylamine and 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxaline-1-one (ODQ). N-Methylhydroxylamine reduced, and ODQ potentiated, the inhibitory action of H(3)-receptor activation, but 8-bromo-cGMP was without effect. The study suggests that presynaptic A(1)- and H(3)-receptors inhibit cholinergic neurotransmission in the guinea pig ileum by limiting the availability of intraneuronal Ca(2+) via inhibition of N-type Ca(2+) channels. The balance of evidence does not support the involvement of the adenylyl cyclase/cAMP or guanylyl cyclase/cGMP systems.     

Effects of oral combined hormone replacement therapy on platelet aggregation in postmenopausal women

The effects of combined oestrogen/progestin hormone replacement therapy (HRT) on platelet aggregation were studied using women on HRT or placebo. The study involved 32 postmenopausal women (aged 50-75 years) who were enrolled in a double-blind randomized controlled trial, and who received either oral continuous combined HRT (Kliogest(R); 2 mg of oestradiol+1 mg of norethisterone) or placebo for a minimum of 6 months. Platelet aggregation was measured by whole-blood impedance aggregometry in response to the agonists collagen, arachidonic acid and ADP. To determine whether the effects of oestrogen on platelets were influenced by platelet-derived nitric oxide, exposure to collagen was repeated in the presence of the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Mean platelet volume was similar in the two groups. Compared with the placebo group, the women on HRT had similar rates and maximum values of platelet aggregation in response to collagen, arachidonic acid and ADP. Addition of L-NMMA did not alter the aggregation response to collagen in either the HRT or the placebo group. In conclusion, postmenopausal women on oral combined continuous HRT comprising oestradiol and norethisterone had similar whole-blood platelet aggregation rates and maximum platelet aggregation responses to higher doses of platelet agonists when compared with those on placebo. The endogenous platelet nitric oxide system did not appear to affect aggregation in either group.     

Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells.

Both nitric oxide (NO) and carbon monoxide (CO) are vessel wall-derived messenger molecules that cause platelet inhibition and vasodilation by activating guanylyl cyclase in target cells. Since vascular smooth muscle cells (SMCs) are exposed to shear and tensile stresses, this study examined the effects of these hemodynamic forces on the enzymes that generate NO and CO in SMCs. Monolayers of cultured rat aortic SMCs were subjected to shear stress using a modified cone and plate viscometer, or cyclic elongational stretch using a compliant silastic culture membrane. Shear stress stimulated time-dependent increases in mRNA and protein for inducible heme oxygenase-1 (HO-1), the enzyme which forms CO as a byproduct of heme degradation. The threshold level of shear necessary to induce HO-1 expression was between 5 and 10 dynes/cm2. In contrast, shear stress did not stimulate inducible NO synthase (iNOS) expression. Cyclic stretch also induced the expression of HO-1 but not of iNOS mRNA. Exposure of vascular SMCs to shear stress stimulated the production and release of CO as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. In addition, ADP-stimulated aggregation was inhibited in platelets exposed to sheared SMCs but not in platelets exposed to untreated control SMCs. Treatment of sheared SMCs with the HO-1 inhibitor, tin protoporphyrin-IX, blocked the antiaggregatory effect of the cells, whereas the iNOS inhibitor, methyl--arginine, had no effect. These results indicate that hemodynamic forces induce HO-1 gene expression and CO production in vascular SMCs, and that SMC-derived CO inhibits platelet aggregation. Thus, CO is a novel endogenous vessel wall-derived messenger molecule that may be selectively induced by hemodynamic forces to inhibit platelet reactivity and preserve blood fluidity at sites of vascular injury.     

A 4-trifluoromethyl derivative of salicylate, triflusal, stimulates nitric oxide production by human neutrophils: role in platelet function

BACKGROUND: The thrombotic process is a multicellular phenomenon in which not only platelets but also neutrophils are involved. Recent in vitro studies performed in our laboratory have demonstrated that triflusal, a 4-trifluoromethyl derivative of salicylate, reduced platelet aggregation not only by inhibiting thromboxane A2 production but also by stimulating nitric oxide (NO) generation by neutrophils. The aim of the present study was to evaluate whether oral treatment of healthy volunteers with triflusal could modify the ability of their neutrophils to produce NO and to test the role of the NO released by neutrophils in the modulation of ADP-induced platelet aggregation and alpha-granule secretion. METHODS: The study was performed in 12 healthy volunteers who were orally treated with triflusal (600 mg day-1) for 5 days. Flow cytometric detection of platelet surface expression of P-selectin was used as a measure of the ability of platelets to release the contents of their alpha-granules. RESULTS: After treatment with triflusal, there was an increase in NO production by neutrophils and an increase in endothelial nitric oxide synthase (eNOS) protein expression in neutrophils. A potentiation of the inhibition of platelet aggregation by neutrophils was reversed by incubating neutrophils with both an L-arginine antagonist, NG-nitro-L-arginine methyl ester (L-NAME) and an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline 1-oxyl 3-oxide (C-PTIO). A slight decrease in P-selectin surface expression on platelets was found which was not modified by the presence of neutrophils and therefore by the neutrophil-derived NO. Exogenous NO released by sodium nitroprusside dose-dependently inhibited both ADP-stimulated alpha-granule secretion and platelet aggregation. Therefore, platelet aggregation showed a greater sensitivity to be inhibited by exogenous NO than P-selectin expression. CONCLUSION: Oral treatment of healthy volunteers with triflusal stimulated NO production and eNOS protein expression in their neutrophils. After triflusal treatment, the neutrophils demonstrated a higher ability to prevent ADP-induced platelet aggregation. However, the neutrophils and the endogenous NO generated by them failed to modify P-selectin expression in ADP-activated platelets.     

Gender differences in protein kinase G-mediated vasorelaxation of rat aorta

Although gender and oestrogen treatment influence production of the vasorelaxant, NO, their influence on factors downstream in the NO signal-transduction pathway, specifically protein kinase G (PKG), remains unknown. We aimed to study the influence of sex hormones on PKG, along with the endothelial modulation of these effects, in rat thoracic aortic rings in two separate groups, control male and female rats and ovariectomized female rats after treatment with oestrogen or vehicle. Vessel preparations were preconstricted with phenylephrine (0.1 microM). Constrictions were greater in male than female aortas. This differential effect was attenuated by endothelium removal, addition of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM) and the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA, 100 microM), supporting the role of NO in maintenance of basal relaxation and vascular tone in females. We have examined the relative activity of the specific PKG subtypes 1 alpha and 1 beta in vascular smooth muscle, based on relaxation of rat aortas by two cGMP analogues with different selectivity, beta-phenyl-l-N(2)-ethano-8-bromo-cGMP (8-Br-PET-cGMP) and 8-(2-aminophenylthio)cGMP (8-APT-cGMP). 8-Br-PET-cGMP was more potent than 8-APT-cGMP in both sexes, suggesting that PKG 1 alpha is the primary subtype involved in vasorelaxation. The gender differences in PKG activity were examined based on relaxation responses in male and female rat aortas. Both 8-Br-PET-cGMP and 8-APT-cGMP were more potent in aortas from male than female rats. In further studies on the endothelial modulation of relaxation with 8-APT-cGMP, the differential gender-vasorelaxation response was negated by endothelium removal and addition of the guanylate cyclase inhibitor ODQ (1 microM), but not by the NOS inhibitor L-NMMA (100 microM), suggesting that an endothelial-dependent factor other than NO may be responsible for the observed differential PKG-mediated vasorelaxation between the sexes. To further investigate oestrogen influence on PKG, treated female rats were studied. Contrary to our hypothesis, in the presence of 1 microM ODQ, there were no differences in either the phenylephrine constriction, or the relaxation with 8-APT-cGMP from either sham-operated, vehicle-treated or oestrogen-treated ovariectomized rats. In conclusion, female rat aortas have greater basal NO production compared with males. Relaxant responses to PKG activation are greater in aortas from male compared with female rats. These findings suggest hormonal regulation of PKG; however, oestrogen treatment of ovariectomized rats did not affect PKG activity, suggesting factors other than oestrogen may be responsible for the gender differences noted in this study.     

Signaling through NO and cGMP‐dependent protein kinases

The gaseous molecule nitric oxide (NO) modulates a large variety of physiological functions including vascular tone, intestinal motility, platelet aggregation, proliferation, apoptosis, and neurotransmission. NO initiates diverse cellular signaling cascades which comprise nitrosylation of proteins, adenosine 5′‐diphosphate (ADP)‐ribosylation, or stimulation of soluble guanylyl cyclases which catalyze intracellular guanosine 3′,5′‐cyclic monophosphate (cGMP) synthesis. cGMP activates cGMP‐dependent protein kinases (cGK) which mediate localized and global signaling. Furthermore, cGMP regulates the activity of phosphodiesterases (PDE) which modulate the duration and amplitude of cyclic nucleotide signaling. Two different types of cGK are expressed in mammals, cGKI and cGKII. Activation of the NO/cGMP/cGKI pathway induces relaxation of smooth muscle by lowering the cytosolic calcium level and/or by calcium desensitization of the contractile elements.     

一氧化氮合酶抑制剂对严重烧伤大鼠的作用研究

目的 :研究一氧化氮合酶 (NOS)抑制剂对严重烧伤大鼠体内一氧化氮 (NO)含量及 NOS表达的影响及其与预后的关系。方法 :复制大鼠重症烧伤模型 ,检测应用非选择性 NOS抑制剂 N 硝基 L 精氨酸甲酯(L NAME)和选择性诱生型 NOS(i NOS)抑制剂氨基胍 (AG)后大鼠血液中 NO代谢产物 (NO- 2 /NO- 3 )以及肺和十二指肠组织中神经型 NOS(n NOS) m RNA的表达水平 ,同时统计各组大鼠的存活率。结果 :烧伤后大鼠血液中 NO- 2 /NO- 3 含量明显增高 ,L NAME和 AG都能抑制 NO2 /NO- 3 的升高 ,L NAME作用更为明显 ;烧伤后 n NOS的 m RNA表达在肺和十二指肠中均有不同程度升高 ,AG和 L NAME使 n NOS表达增加 ,AG作用更为明显 ;L NAME组动物存活时间较对照组显著缩短 ,AG组动物存活时间明显延长。结论 :结构型NOS(c NOS)与 i NOS在烧伤休克病理生理过程中的作用明显不同 ,i NOS活性过度增高与烧伤休克发病关系密切     

Effects induced by inhibitors of the phosphatidylinositol 3‐kinase/Akt and nitric oxide synthase/guanylyl cyclase pathways on the isometric contraction in rat aorta: a comparative study

This work was aimed to determine whether isometric contraction in Wistar rat aorta is related to the phosphatidylinositol 3‐kinase (PI3K)/Akt‐dependent activation of endothelial nitric oxide synthase (eNOS). Basically, we hypothesized that additional increases in active tone occur after the pharmacological inhibition of a transduction pathway involved in NO synthesis or action. In intact aortic rings contracted with phenylephrine or high K⁺, the cumulative administration of the PI3K inhibitor, LY294002, elicited significant decreases – but not supplementary increases – in tone. In endothelium‐intact tissues, on the other hand, the Akt1/2 kinase inhibitor did not alter phenylephrine‐ and K⁺‐induced isometric contractions. The PI3K inhibitor wortmannin (1 × 10^?7 m ) produced a significant supplementary contraction only in endothelium‐intact aortic rings precontracted with phenylephrine. Higher concentrations of this inhibitor produced relaxations of phenylephrine and high K⁺‐constricted endothelium‐intact and endothelium‐denuded aortic rings. LY294002 and wortmannin did not cause any potentiating effect on phenylephrine‐ and angiotensin II‐induced concentration‐dependent contractile responses in endothelium‐intact tissues. In intact aortic rings contracted with phenylephrine or high K⁺, the addition of the NOS inhibitor, L‐NAME, or the guanylyl cyclase inhibitor, ODQ, further augmented tone in a concentration‐dependent manner, and these supplementary contractions were significantly reduced by endothelium removal. Taken together, our data suggest that the PI3K/Akt pathway is not counteracting aortic isometric contractions by activation of the eNOS. It appears, on the other hand, that the smooth muscle PI3K can stimulate contraction without activation of the protein kinase Akt in response to GPCR agonists and high K⁺.     

Signaling through NO and cGMP-dependent protein kinases

The gaseous molecule nitric oxide (NO) modulates a large variety of physiological functions including vascular tone, intestinal motility, platelet aggregation, proliferation, apoptosis, and neurotransmission. NO initiates diverse cellular signaling cascades which comprise nitrosylation of proteins, adenosine 5'-diphosphate (ADP)-ribosylation, or stimulation of soluble guanylyl cyclases which catalyze intracellular guanosine 3',5'-cyclic monophosphate (cGMP) synthesis. cGMP activates cGMP-dependent protein kinases (cGK) which mediate localized and global signaling. Furthermore, cGMP regulates the activity of phosphodiesterases (PDE) which modulate the duration and amplitude of cyclic nucleotide signaling. Two different types of cGK are expressed in mammals, cGKI and cGKII. Activation of the NO/cGMP/cGKI pathway induces relaxation of smooth muscle by lowering the cytosolic calcium level and/or by calcium desensitization of the contractile elements.