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.     

A novel guanylyl cyclase receptor, BdmGC-1, is highly expressed during the development of the oriental fruit fly Bactrocera dorsalis (Hendel)

A novel receptor guanylyl cyclase (GC) has been identified from the oriental fruit fly Bactrocera dorsalis (Hendel) and has been designated BdmGC-1. Protein domain analysis revealed that BdmGC-1 possesses a characteristic domain organization similar to all known receptor GCs but with a unique carboxyl-terminal extension. When overexpressed in 293T cells, BdmGC-1 manifests as a cell-surface glycoprotein with a marked cGMP-generating activity but is unresponsive to all ligands known to activate mammalian receptor GCs. BdmGC-1 mRNAs were highly expressed during development but had low or no expression in adult tissues. On the basis of its unique sequence and distinct developmental expression pattern, BdmGC-1 represents a novel receptor GC that may play a critical role during the development of B. dorsalis.     

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⁺.     

Wound healing action of nitric oxide‐releasing self‐expandable collagen sponge

Mounting evidence showing that local nitric oxide (NO) delivery may significantly improve the wound healing process has stimulated the development of wound dressings capable of releasing NO topically. Herein, we describe the preparation of a self‐expandable NO‐releasing hydrolyzed collagen sponge (CS), charged with the endogenously found NO donor, S‐nitrosoglutathione (GSNO). We show that cold pressed and GSNO‐charged CS (CS/GSNO) undergo self‐expansion to its original 3D shape upon water absorption to a swelling degree of 2,300 wt%, triggering the release of free NO. Topical application of compressed CS/GSNO on wounds in an animal model showed that exudate absorption by CS/GSNO leads to the release of higher NO doses during the inflammatory phase and progressively lower NO doses at later stages of the healing process. Moreover, treated animals showed significant increase in the mRNA expression levels of monocyte chemoattractant protein‐1 (MCP‐1), murine macrophage marker (F4/80), transforming growth factor beta (TGF‐β), stromal cell‐derived factor 1 (SDF‐1), insulin‐like growth factor‐1 (IGF‐1), nitric oxide synthase(iNOS), and matrix metalloproteinase(MMP‐9). Cluster differentiation 31 (CD31), vascular endothelial growth factor (VEGF), and F4/80 were measured on Days 7 and 12 by immunohistochemistry in the cicatricial tissue. These results indicate that the topical delivery of NO enhances the migration and infiltration of leucocytes, macrophages, and keratinocytes to the wounded tissue, as well as the neovascularization and collagen deposition, which are correlated with an accelerated wound closure. Thus, self‐expandable CS/GSNO may represent a novel biocompatible and active wound dress for the topical delivery of NO on wounds.     

Nitric oxide specifically inhibits integrin‐mediated platelet adhesion and spreading on collagen

Summary. Background: Nitric oxide (NO) inhibits platelet adhesion to collagen, although the precise molecular mechanisms underlying this process are unclear. Objectives: Collagen‐mediated adhesion is a multifaceted event requiring multiple receptors and platelet‐derived soluble agonists. We investigated the influence of NO on these processes. Results: S‐nitrosoglutathione (GSNO) induced a concentration‐dependent inhibition of platelet adhesion to immobilized collagen. Maximal adhesion to collagen required platelet‐derived ADP and TxA₂. GSNO‐mediated inhibition was lost in the presence of apyrase and indomethacin, suggesting that NO reduced the availability of, or signaling by, ADP and TxA₂. Exogenous ADP, but not the TxA₂ analogue U46619, reversed the inhibitory actions of GSNO on adhesion. Under adhesive conditions NO inhibited dense granule secretion but did not influence TxA₂ generation. These data indicated that NO may block signaling by TxA₂ required for dense granule secretion, thereby reducing the availability of ADP. Indeed, we found TxA₂‐mediated activation of PKC was required to drive dense granule secretion, a pathway that was inhibited by NO. Because our data demonstrated that NO only inhibited the activation‐dependent component of adhesion, we investigated the effects of NO on individual collagen receptors. GSNO inhibited platelet adhesion and spreading on α₂β₁ specific peptide ligand GFOGER. In contrast, GSNO did not inhibit GPVI‐mediated adhesion to collagen, or adhesion to the GPVI specific ligand, collagen related peptide (CRP). Conclusions: NO targets activation‐dependent adhesion mediated by α₂β₁, possibly by reducing bioavailability of platelet‐derived ADP, but has no effect on activation‐independent adhesion mediated by GPVI. Thus, NO regulates platelet spreading and stable adhesion to collagen.     

Pharmacological evidence of involvement of nitric oxide pathway in anti‐pruritic effects of sumatriptan in chloroquine‐induced scratching in mice

Chloroquine (CQ) induces histamine‐independent itch in human and mice. We recently reported the role of intradermal nitric oxide (NO)/cyclic guanosine monophosphate pathway in CQ‐evoked scratching in mice. Chloroquine stimulates neuronal nitric oxide synthase (nNOS) activity to over‐producing NO in the skin. Sumatriptan, a 5‐hydroxytryptamine 1b/1d receptors (5‐HTR1b/1d) agonist, is involved in pain and used to treat migraine and cluster headaches. According to previous studies, sumatriptan inhibits NOS activity. Thus, we aimed to investigate the effect of sumatriptan on CQ‐induced scratching. We used the rostral back model of itch. Chloroquine was injected intradermally into the rostral back of NMRI mice, and the scratching behavior was evaluated by measuring the number of bouts over 30 min. We evaluated the effect of sumatriptan and combination of sumatriptan and a non‐selective NO synthase inhibitor, L‐N‐nitro arginine methyl ester (L‐NAME), on the scratching behavior. Additionally, the changes of skin, hippocampus, and cortical nitrite level after different treatments were studied. Intraperitoneal and intradermal sumatriptan attenuates CQ‐induced itch which reversed by GR‐127935, the selective 5‐HTR1b and 5‐HTR1d antagonist. Co‐administration of subeffective doses of sumatriptan and L‐NAME significantly decreases the scratching behavior. Intradermal injection of CQ significantly increases the intradermal nitrite levels while it does not have any significant effects on hippocampal or cortical nitrite concentrations. Likewise, the effective doses of intraperitoneal and intradermal sumatriptan significantly reduce intradermal nitrite levels. We concluded that sumatriptan suppresses CQ‐induced itch most likely by activating 5‐HT1b/1d receptors. This effect probably mediates through NO pathway.     

Effects of nitric oxide synthase inhibitors 1‐(2‐trifluoromethylphenyl) – imidazole (TRIM) and 7‐nitroindazole (7‐NI) on learning and memory in mice

Nitric oxide (NO) plays an important role in hippocampal long‐term potentiation (LTP), which is involved in memory processes. This led to the hypothesis that nitric oxide synthase (NOS) inhibitors will have disturbing effects on learning and memory. The aim of our study was to investigate the effects of the new selective neuronal and inducible NOS inhibitor 1‐ (2‐trifluoromethylphenyl) imidazole (TRIM) (10–50 mg/kg) on learning and memory and compare it to the nonselective NOS inhibitor 7‐NI (15–45 mg/kg) using different behavioral tests in Swiss mice, thus clarifying the role of neuronal NOS (nNOS) and endothelial NOS (eNOS) in cognitive processes. TRIM had no specific effect on either learning or memory parameters, while 7‐NI (30 mg/kg) disturbed spatial memory in the probe trial of the Morris water maze test, which was performed on the last day of the test. No differences between TRIM and the control groups were observed, while 7‐NI (30 and 45 mg/kg) significantly disturbed memory in the novel object recognition test. In the social transmission of food preference test, both TRIM (50 mg/kg) and 7‐NI (45 mg/kg) impaired hippocampal olfactory memory, but the total food consumption was also significantly decreased at these doses. In the passive avoidance test, TRIM did not disturb the performance, while memory impairment was observed, even with lower doses of 7‐NI. All of these results suggest that TRIM has no clear effect on cognitive impairment compared to 7‐NI and that inhibition of both nNOS and eNOS are necessary for the deterioration of memory processes.     

High on‐aspirin platelet reactivity as measured with aggregation‐based,cyclooxygenase‐1 inhibition sensitive platelet function tests is associated with the occurrence of atherothrombotic events

Summary. Background: High on‐aspirin platelet reactivity (HAPR) is associated with atherothrombotic events following percutaneous coronary intervention (PCI). The aim of the present study was to identify the platelet function test sensitive for platelet cyclooxygenase‐1 inhibition that best predicts atherothrombotic events. Methods and results: Nine hundred and fifty‐one consecutive patients on dual antiplatelet therapy undergoing elective PCI were enrolled. On‐aspirin platelet reactivity was measured in parallel by arachidonic acid (AA)‐induced light transmittance aggregometry (AA‐induced LTA), the VerifyNow® Aspirin Assay (VerifyNow® Aspirin Assay), the arachidonic acid prestimulated IMPACT‐R (IMPACT‐R AA) and the PFA‐100 collagen/epinephrine cartridge (PFA COL/EPI). Cut‐offs for HAPR were established by receiver‐operator characteristic curve analysis. At 1‐year follow‐up, the composite of all‐cause death, non‐fatal acute myocardial infarction, stent thrombosis and ischemic stroke occurred more frequently in patients with HAPR when assessed by LTA [10.1% vs. 6.0%, P = 0.020 (n = 925)] and VerifyNow^® [13.3% vs. 5.9%, P = 0.015 (n = 422)]. The VerifyNow^® ASA assay (AUC = 0.78) and, to a lesser extent, AA‐induced LTA (AUC = 0.73) added significantly to a model consisting of clinical and procedural risk factors in predicting atherothrombotic events. In contrast, the IMPACT‐R (n = 791) and the PFA Collagen/Epinephrine (n = 719) were unable to discriminate between patients with and without primary endpoint at 1‐year follow‐up. None of the platelet function tests was able to identify patients at risk for bleeding. Conclusions: AA‐induced LTA and the VerifyNow^® ASA test were able to identify aspirin‐treated patients undergoing PCI with stenting at risk for atherothrombotic events. The VerifyNow^® Aspirin Assay had the highest predictive accuracy. None of the tests was able to identify patients at higher risk of bleeding.     

Anticonvulsive effect of atorvastatin on pentylenetetrazole‐induced seizures in mice: the role of nitric oxide pathway

Atorvastatin has shown to possess neuroprotective, antiexcitotoxic, and antiepileptic effects besides its cholesterol‐lowering properties. Nitric oxide (NO) may be responsible for a group of these effects. In the present study, a model of clonic seizure induced by pentylenetetrazole (PTZ) in male NMRI mice was used to investigate the anticonvulsive effects of atorvastatin through NO‐dependent pathways. Atorvastatin (5 mg/kg) significantly increased the seizure threshold (P < 0.001). Moreover, L‐arginine (a precursor of NO) significantly (P < 0.01) potentiated the anticonvulsive effects of subeffective doses of atorvastatin (1 mg/kg). Finally, L‐NAME [L‐arginine methyl ester dihydrochloride], a nonspecific NO synthase inhibitor, completely abolished the anticonvulsive properties of atorvastatin. Our findings demonstrated the role of atorvastatin as an anticonvulsive agent and showed the effects to be mediated through NO‐related pathways.     

Nitric oxide inhibits von Willebrand factor‐mediated platelet adhesion and spreading through regulation of integrin αIIbβ3 and myosin light chain

Summary. Background: von Willebrand factor (VWF)‐mediated platelet adhesion and spreading at sites of vascular injury is a critical step in hemostasis. This process requires two individual receptors: glycoprotein Ib (GPIb)‐V‐IX and integrin α_IIbβ₃. However, little is known about the negative regulation of these events. Objectives: To examine if the endogenous platelet inhibitor nitric oxide (NO) has differential effects on adhesion, spreading and aggregation induced by immobilized VWF. Results: S‐nitrosoglutathione (GSNO) inhibited platelet aggregation on immobilized VWF under static and flow conditions, but had no effect on platelet adhesion. Primary signaling events underpinning the actions of NO required cyclic GMP but not protein kinase A. Dissecting the roles of GPIb and integrin α_IIbβ₃ demonstrated that NO targeted α_IIbβ₃‐mediated aggregation and spreading, but did not significantly influence GPIb‐mediated adhesion. To understand the relationship between the effects of NO on adhesion and subsequent aggregation, we evaluated the activation of α_IIbβ₃ on adherent platelets. NO reduced the phosphorylation of extracellular stimuli‐responsive kinase (ERK) and p38, required for integrin activation resulting in reduced binding of the activated α_IIbβ₃‐specific antibody PAC‐1 on adherent platelets. Detailed analysis of platelet spreading initiated by VWF demonstrated key roles for integrin α_IIbβ₃ and myosin light chain (MLC) phosphorylation. NO targeted both of these pathways by directly modulating integrin affinity and activating MLC phosphatase. Conclusion: These data demonstrate that initial activation‐independent platelet adhesion to VWF via GPIb is resistant to NO, however, NO inhibits GPIb‐mediated activation of α_IIbβ₃ and MLC leading to reduced platelet spreading and aggregation.     

The role of nitric oxide‐ and prostacyclin‐independent vasodilatation in the human cutaneous microcirculation: effect of cytochrome P450 2C9 inhibition

The component of the flow‐ or agonist‐dependent vasodilatation, insensitive to inhibitors of nitric oxide (NO) synthases (NOS) or cyclooxygenases (COX), is suggested to reflect the production of an endothelium‐dependent hyperpolarizing factor (EDHF). The identity of EDHF in humans remains controversial; in coronary arterioles, it appears to be a cytochrome P450 (CYP) 2C9‐derived metabolite, whereas there are no data for human skin microcirculation. Therefore, the aim of our study was to investigate the role of the NO‐ and prostacyclin (PGI₂)‐independent mechanism, particularly the potential involvement of CYP 2C9, in skin microcirculation. We measured skin blood flow on the volar aspect of the forearm in 12 healthy subjects by laser‐Doppler fluxmetry (LDF). The inhibitors of NOS, N^ω‐monomethyl‐L‐arginine (L‐NMMA), and cyclooxygenase (COX), diclofenac, as well as sulfaphenazole, the specific CYP 2C9 inhibitor, and saline as control, were administered to the measurement sites by an intradermal microinjection in different combinations. Afterwards, baseline LDF was assessed and iontophoresis of acetycholine (ACh) applied. Combined NOS and COX inhibition had no effect on baseline LDF, whereas it significantly reduced the ACh‐induced increase in LDF (t‐test, P<0·05). Sulfaphenazole did not affect baseline LDF either in the control site or in the L‐NMMA‐ and diclofenac‐pretreated site. In addition, sulfaphenazole did not attenuate the ACh‐induced vasodilatation in either site. We conclude that a NO‐ and PGI₂‐independent vasodilator mechanism, potentially attributable to EDHF, contributes substantialy to the ACh‐induced vasodilatation in human skin microcirculation and that it is probably not a CYP 2C9‐derived metabolite.     

Insulin/IGF‐1 hybrid receptor expression on human platelets: consequences for the effect of insulin on platelet function

Summary. Objectives: As platelets express both insulin and insulin‐like growth factor‐1 (IGF‐1) receptors, their subunits may randomly heterodimerize to form insulin/IGF‐1 receptor hybrids, which avidly bind IGF‐1, but not insulin. This study investigated the possibility that platelets express hybrid receptors, which may affect insulin action on platelet function. Methods: Platelets were incubated with insulin and IGF‐1. Expression and phosphorylation of insulin/IGF‐1 receptors was determined by western blotting of immunoprecipitates, and compared with platelet functional responses. Relative expression of insulin and IGF‐1 receptors was estimated by competitive ligand binding and quantitative polymerase chain reaction. Results: We demonstrated the presence of insulin/IGF‐1 hybrid receptors on human platelets by detecting both insulin and IGF‐1 receptor β subunits in coimmunoprecipitation studies. Stimulation of platelets with insulin (1–100 nm ) resulted in tyrosine phosphorylation of insulin receptors, but not of hybrid receptors. High insulin concentrations (50–100 nm ) stimulated weak phosphorylation of IGF‐1 receptors and protein kinase B (Akt), and correlated with moderately increased aggregation and fibrinogen binding, whereas low insulin concentrations (1–10 nm ) had no effect. In contrast, IGF‐1 (1–100 nm ) induced strong phosphorylation of both hybrid and IGF‐1 receptors, and potentiated platelet aggregation and fibrinogen binding. Specific binding of [¹²⁵I]IGF‐1 (1.08% ± 0.16%) was significantly higher than that of [¹²⁵I]insulin (0.15% ± 0.03%). Accordingly, IGF‐1 receptor mRNA was more abundant than insulin receptor mRNA (IGF‐1 receptor/insulin receptor ratio 69 ± 3.8). Conclusions: Insulin has minimal effects on platelet function, which can be explained by the relatively low insulin receptor expression levels resulting in the majority of insulin receptor subunits being expressed as insulin/IGF‐1 hybrids.