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Purpose: We comprehensively evaluated the mutational spectrum of Leber congenital amaurosis (LCA) and investigated the molecular diagnostic rate and genotype-phenotype correlation in a Korean cohort., Methods: This single-center retrospective case series included 50 Korean patients with LCA between June 2015 and March 2019. Molecular analysis was conducted using targeted panel-based next-generation sequencing, including deep intronic and regulatory variants or whole exome sequencing. The molecular diagnosis was made based on the inheritance pattern, zygosity, and pathogenicity., Results: Among the 50 patients, 27 patients (54%) were male, and 11 (22%) showed systemic features. Genetic variants highly likely to be causative were identified in 78% (39/50) of cases and segregated into families. We detected two pathogenic or likely pathogenic variants in a gene linked to a recessive trait without segregation analysis in three cases (6.0%). GUCY2D (20%), NMNAT1 (18%), and CEP290 (16%) were the most frequently mutated genes in Korean LCA. Copy number variations were found in three patients, which accounted for 6% of LCA cases. A possible dual molecular diagnosis (Senior-Løken syndrome along with Leigh syndrome, and Joubert syndrome with transposition of the great arteries) was made in two patients (4%). Three of 50 patients were medically or surgically actionable: one patient for RPE65 gene therapy and two patients with WDR19 Senior-Løken syndrome for early preparation for kidney and liver transplantations., Conclusions: This study demonstrated that approximately 4% of patients may have dual molecular diagnoses, and 6% were surgically or medically actionable in LCA. Therefore, accurate molecular diagnosis and careful interpretation of next-generation sequencing results can be of great help in patients with LCA., (Copyright © 2020 Molecular Vision.)

Insulin resistance (IR) is found in chronic hepatitis C (CHC) more frequently than in other chronic liver diseases.Prospective cross-sectional study to evaluate a wide multitest panel to identify factors related with IR in CHC and their possible interactions.In 76 patients with CHC we performed a series of routine laboratory analysis as well as specifically designed serum biochemical tests [retinol, retinol-binding protein 4 (RBP4), 25-OH vitamin D, Vitamin E, lipopolysaccharide-binding protein (LBP), interleukin-6 (IL-6), and cystatin C]. The single nucleotide polymorphisms rs7041 and rs4588 GC-DBP (group-specific component-Vitamin D-binding protein), rs738409 PNPLA3 (patatin-like phospholipase domain containing 3), and rs12979860 IL28B (interleukin-28 B) genes were determined. Insulin sensitivity was established with the HOMA-IR and IR was diagnosed when HOMA-IR > 3. Fibrosis staging was assessed with liver biopsy or transient elastography.After backward logistic regression analysis, independent variables associated with IR were Gc1s/Gc1s DBP phenotype, that results from the homozygous carriage of the rs7041G/rs4588C haplotype (P = 0.033); low retinol/RBP4 ratio, reflecting a greater rate of unbound RBP4 (P = 0.005); older age (P = 0.01); high serum tryglicerides (P = 0.026); and advanced (F3-F4) fibrosis stage. The AUROC provided by the multivariate model was 0.950 (95% CI = 0.906-0.993).In addition to previously known ones, the Gc1s/Gc1s phenotype variant of DBP and the unbound fraction of plasma RBP4 may be considered as factors related with the incidence, and possibly the risk, of IR in CHC patients., Competing Interests: The authors have no conflicts of interest to disclose.

The nitric oxide (NO)/soluble guanylate cyclase (sGC) system is fundamental to endothelial control of vascular tone, but also plays a major role in the negative modulation of platelet aggregation. The phenomenon of platelet NO resistance, or decreased antiaggregatory response to NO, occurs increasingly with advanced age, as well as in the context of cardiovascular disease states such as heart failure, ischemic heart disease, and aortic valve disease. The central causes of NO resistance are "scavenging" of NO and dysfunction of sGC. In the current review, we discuss the roles of several modulators of NO synthesis and of the NO/sGC cascade on changes in platelet physiology with aging, together with potential therapeutic options to reduce associated thrombotic risk., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Impaired tissue responsiveness to nitric oxide (NO) occurs in many cardiovascular diseases as well as with advanced age and is a correlate of poor outcomes. This phenomenon results from oxidative stress, with NO "scavenging" and dysfunction of soluble guanylate cyclase (sGC). Thioredoxin-interacting protein (TXNIP) is a major intracellular regulator of inflammatory activation and redox stress, but its interactions with NO/sGC are poorly understood. We have now evaluated the relationship between platelet TXNIP expression and function of the NO/sGC axis in subjects of varying age and during therapy with ramipril., Methods & Results: Young (n=42) and aging (n=49) subjects underwent evaluation of platelet TXNIP content. Aging subjects additionally had measurements of platelet NO responsiveness and routine biochemistry. Platelet TXNIP content was greater (376±33 units) in the aging compared to younger subjects (289±13 units; p<0.05). In the aging subjects there was a significant negative correlation (r=-0.50, p<0.001) between platelet TXNIP content and NO responsiveness. In a separate cohort of 15 subjects two week treatment with ramipril, which reversed platelet NO resistance and potentiated sGC activity, also decreased platelet TXNIP content by 40% (p=0.011)., Conclusions: Platelet TXNIP content increases with aging, varies inversely with responsiveness to NO, and diminishes rapidly following treatment with ramipril. These data suggest that TXNIP-induced oxidative stress may be a critical modulator of tissue resistance to NO, a fundamental basis for cardiovascular disease. Analogously suppression of TXNIP expression can potentially be utilized as an index of restoration of cardiovascular homeostasis., (© 2013.)

Metabolic syndrome (MetS) is a combination of clinical disorders that together increase the risk for cardiovascular disease and diabetes. SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP.ZF) rats with MetS show impaired nitric oxide-mediated relaxation in coronary and mesenteric arteries, and angiotensin II receptor type 1 blockers protect against dysfunction and oxidative-nitrosative stress independently of metabolic effects. We hypothesize that superoxide contributes to functional deterioration in SHRSP.ZF rats. To test our hypothesis, we studied effects of treatment with tempol, a membrane-permeable radical scavenger, on impaired vasodilation in SHRSP.ZF rats. Tempol did not alter body weight, high blood pressure, or metabolic abnormalities, but prevented impairment of acetylcholine-induced and nitroprusside-induced vasodilation in the coronary and mesenteric arteries. Furthermore, tempol reduced the levels of serum thiobarbituric acid reactive substance (TBARS) and 3-nitrotyrosine content in mesenteric arteries. Systemic administration of tempol elevated the expression of soluble guanylate cyclase (sGC) above basal levels in mesenteric arteries of SHRSP.ZF rats. However, acute treatment with tempol or ebselen, a peroxynitrite scavenger, did not ameliorate impaired relaxation of isolated mesenteric arteries. No nitration of tyrosine residues in sGC was observed; however, sGC mRNA expression levels in the arteries of SHRSP.ZF rats were lower than those in the arteries of Wistar-Kyoto rats. Levels of Thr(496)- and Ser(1177)-phosphorylated endothelial nitric oxide synthase (eNOS) were lower in arteries of SHRSP.ZF rats, and acetylcholine decreased Thr(496)-phosphorylated eNOS levels. These results indicated that prolonged superoxide production, leading to oxidative-nitrosative stress, was associated with impaired vasodilation in SHRSP.ZF rats with MetS. Down-regulated sGC expression may be linked to dysfunction, while reduced NO bioavailability/eNOS activity and modified sGC activity due to superoxide production were excluded as pivotal mechanisms., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We assessed the safety, tolerability and preliminary efficacy of riociguat, a soluble guanylate cyclase stimulator, in patients with pulmonary hypertension associated with interstitial lung disease (PH-ILD). In this open-label, uncontrolled pilot trial, patients received oral riociguat (1.0-2.5 mg three times daily) for 12 weeks (n=22), followed by an ongoing long-term extension (interim analysis at 12 months) in those eligible (n=15). Primary end-points were safety and tolerability. Secondary end-points included haemodynamic changes and 6-min walk distance (6MWD). Overall, 104 adverse events were reported, of which 25 were serious; eight of the latter were considered drug-related. After 12 weeks of therapy, mean cardiac output increased (4.4 ± 1.5 L · min(-1) to 5.5 ± 1.8 L · min(-1)), pulmonary vascular resistance (PVR) decreased (648 ± 207 dyn · s(-1) · cm(-5) to 528 ± 181 dyn · s(-1) · cm(-5)) and mean pulmonary artery pressure (mPAP) remained unchanged compared with baseline. Arterial oxygen saturation decreased but mixed-venous oxygen saturation slightly increased. The 6MWD increased from 325 ± 96 m at baseline to 351 ± 111 m after 12 weeks. Riociguat was well tolerated by most patients and improved cardiac output and PVR, but not mPAP. Further studies are necessary to evaluate the safety and efficacy of riociguat in patients with PH-ILD.

Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, β/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 μM) dose-dependently enhanced the activity and intracellular level of PPAR-β/γ in platelets. In the presence of selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-β/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-β/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-β/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-β/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-β/γ-dependent pathways., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) is recognized as one of the most important cardiovascular signaling molecules, with multiple regulatory effects on myocardial and vascular tissue as well as on other tissues and organ systems. With the growth in understanding of the range and mechanisms of NO effects on the cardiovascular system, it is now possible to consider pharmaceutical interventions that directly target NO or key steps in NO effector pathways. This article reviews aspects of the cardiovascular effects of NO, abnormalities in NO regulation in heart failure, and clinical trials of drugs that target specific aspects of NO signaling pathways., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Adiposity greatly increases the risk of atherothrombotic events, a pathological condition where a chronic state of oxidative stress is reported to play a major role. This study aimed to investigate the involvement of (NO)-soluble guanylyl cyclase (sGC) signaling pathway in the platelet dysfunction from high fat-fed (HFF) rats., Methods: Male Wistar rats were fed for 10 weeks with standard chow (SCD) or high-fat diet (HFD). ADP (10 μM)- and thrombin (100 mU/ml)-induced washed platelet aggregation were evaluated. Measurement of intracellular levels of ROS levels was carried out using flow cytometry. Cyclic GMP levels were evaluated using ELISA kits., Results: High-fat fed rats exhibited significant increases in body weight, epididymal fat, fasting glucose levels and glucose intolerance compared with SCD group. Platelet aggregation induced by ADP (n = 8) and thrombin from HFD rats (n = 8) were significantly greater (P < 0.05) compared with SCD group. Platelet activation with ADP increased by 54% the intraplatelet ROS production in HFD group, as measured by flow cytometry (n = 6). N-acetylcysteine (NAC; 1 mM) and PEG-catalase (1000 U/ml) fully prevented the increased ROS production and platelet hyperaggregability in HFD group. The NO donors sodium nitroprusside (SNP; 10 μM) and SNAP (10 μM), as well as the NO-independent soluble guanylyl cyclase stimulator BAY 41-2272 (10 μM) inhibited the platelet aggregation in HFD group with lower efficacy (P < 0.05) compared with SCD group. The cGMP levels in response to these agents were also markedly lower in HFD group (P < 0.05). The prostacyclin analogue iloprost (1 μM) reduced platelet aggregation in HFD and SCD rats in a similar fashion (n = 4)., Conclusions: Metabolic abnormalities as consequence of HFD cause platelet hyperaggregability involving enhanced intraplatelet ROS production and decreased NO bioavailability that appear to be accompanied by potential defects in the prosthetic haem group of soluble guanylyl cyclase.

Objective: The soluble guanylate cyclase (sGC) activator Cinaciguat (BAY 58-2667) represents a novel class of drugs that selectively activate oxidised sGC. The extent of oxidised sGC depends on the patient's oxidative burden. We here describe two platelet-based assays that allow determining the extent of oxidised sGC and thus provide a basis for an individualised pharmacotherapy., Methods/results: Platelets obtained from patients with (n=12) and without (n=12) coronary artery disease (CAD) were examined by flow cytometry (P-selectin expression), and Western blots (vasodilator associated phosphoprotein, VASP-phosphorylation). Results were compared to maximal oxidation of sGC achieved by the oxidising agent ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). Treatment of platelets with Cinaciguat resulted in differential activation of oxidised sGC. Platelet P-selectin expression and VASP-phosphorylation revealed significant differences (p=0.012, p=0.039, respectively) between CAD and non-CAD patients., Conclusion: We describe platelet-based assays that allow the determination of patients' oxidative status and thus allow the prediction of pharmacological response to direct sGC activators., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca(2+)](i)) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca(2+)](i) which suggests that a small portion of the thrombin mediated increase in [Ca(2+)](i) was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca(2+)](i) by stimulating store-operated Ca(2+) entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca(2+)](i) however it inhibited the ability of thrombin to increase [Ca(2+)](i) and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca(2+)](i) whereas higher concentrations (>200 nM) of SNP inhibited thrombin induced increases in [Ca(2+)](i). Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba(2+) influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (>200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba(2+) influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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 beta(1)-subunit-Tyr(192)., 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.

Background and Purpose: Oncocalyxone A (OncoA) has a concentration-dependent anti-platelet activity. The present study aimed to further understand the mechanisms related to this effect., Experimental Approach: Human platelet aggregation was measured by means of a turbidimetric method. OncoA (32-256 microM) was tested against several platelet-aggregating agents, such as adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin and thrombin., Key Results: OncoA completely inhibited platelet aggregation with a calculated mean inhibitory concentration (IC50-microM) of 122 for ADP, 161 for collagen, 159 for AA, 169 for ristocetin and 85 for thrombin. The anti-aggregatory activity of OncoA was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). OncoA, at a concentration that caused no significant anti-aggregatory activity, potentiated sodium nitroprusside (SNP) anti-aggregatory activity (18.8+/-2.9%-SNP vs 85.0+/-8.2%-SNP+OncoA). The levels of nitric oxide (NO) or cAMP were not altered by OncoA while cGMP levels were increased more than 10-fold by OncoA in resting or ADP-activated platelets. Flow cytometry revealed that OncoA does not interact with receptors for fibrinogen, collagen or P-selectin. Nevertheless, OncoA decreased the binding of antibodies to GP Ibalpha, a glycoprotein that is related both to von Willebrand factor and to thrombin-induced platelet aggregation., Conclusion and Implications: OncoA showed anti-aggregatory activity in platelets that was associated with increased cGMP levels, not dependent on NO and with blocking GP Ibalpha glycoprotein. This new mechanism has the prospect of leading to new anti-thrombotic drugs.

Circulating tumor cell (CTC) detection in peripheral blood of colon and other epithelial cancer patients is becoming a scientifically recognised indicator for the presence of primary tumors and/or metastasis. The resulting need to further develop CTC detection-based systems for improved diagnosis, prognosis and assessment of therapy efficacy in tumour patients has prompted the application of different approaches, including expression analysis of tissue-specific and epithelial genes. In this context, lack of specificity of the analysed genes remains a fundamental problem for reliable CTC detection. In this study, we have selected a panel of highly specific epithelial genes: cytokeratin 20 (CK20), cytokeratin 19 (CK19), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC), and performed RT-PCR analysis to assess their expression in total blood and in different cell fractions of peripheral blood (PBMC and CD45-negative population) of cancer patients and healthy controls. Our results demonstrate that analysis of a single gene in a CTC-enriched population (CD45(-) peripheral blood cells) of cancer patients allows detection of a CTC molecular signature in at most 63.3% of cases, while analysis of all four genes performed in all three sample types increases the detection of positive patient samples to 87.7%. Healthy controls did not show positivity for any combination of these genes, although positivity was observed for the CEA marker alone, which was detected in 3 (6.6%) out of 45 donors, and only in the CD45(-) fraction. Here, we demonstrate that combined analysis of the genes above, in multiple blood fractions, results in a highly specific and sensitive CTC detection system in patients with metastatic solid tumors. Therefore, we believe that validation on a large scale of this approach, which demonstrates higher specificity in patients compared to controls, could become a relevant CTC screening test in patients with established metastatic disease, and furthermore, may also be useful for evaluating the possible presence of CTCs before the onset of clinically manifested metastatic spreading.

It is unknown whether long-term pharmacological stimulation of soluble guanylate cyclase (sGC), elevating intracellular cGMP levels, has a beneficial effect on hypertension. The purpose of this study is to investigate the effects of BAY41-2272, an orally available sGC stimulator, on cardiovascular remodeling in hypertensive rats. Eight-week-old male Wistar rats with hypertension induced by angiotensin II infused subcutaneously at 250 ng/kg per minute were treated orally with a low ([L] 2 mg/kg per day) or high ([H] 10 mg/kg per day) dose of BAY41-2272 for 14 days. BAY41-2272-H partially suppressed the rise in blood pressure and reduced the heart weight (4.20+/-0.34 versus 3.68+/-0.20 mg/g; P<0.01), whereas BAY41-2272-L had no effect. However, both doses decreased the angiotensin II-induced left ventricular accumulation of collagen in the perivascular area (L, -20%, P<0.05; H, -30%, P<0.01) and myocardial interstitium (L, -21%, P<0.05; H, -38%, P<0.01), reducing the number of activated fibroblasts surrounding coronary arteries (L, -74%; H, -79%; P<0.05). BAY41-2272 downregulated the angiotensin II-induced left ventricular gene expression of type 1 collagen (L, -41%, P<0.05; H, -49%, P<0.01) and transforming growth factor-beta1 (L, -49%, P<0.05; H, -65%, P<0.01). cGMP levels were elevated by BAY41-2272 not only in the left ventricle, but also in cultured cardiac fibroblasts, resulting in reduced thymidine incorporation into the cells. Thus, stimulation of sGC by BAY41-2272 attenuates fibrosis of the left ventricle in rats with angiotensin II-induced hypertension partly in a pressure-independent manner, suggesting an important role for sGC generating cGMP in inhibiting cardiovascular remodeling.

Changes in cerebral cytochrome oxidase (COX) activity, nitric oxide (NO)-cyclic GMP (cGMP) pathway and cholinergic muscarinic receptors (MRs) have been reported in rodents acutely exposed to carbon monoxide (CO). These endpoints measurable in lymphocytes may serve as peripheral markers of CO neurotoxicity. The early and delayed effects of repeated and acute in vivo CO inhalation were investigated on COX activity, cGMP formation and MR binding in rat brain and lymphocytes to assess whether each endpoint was similarly affected both centrally and peripherally. Male Wistar rats either inhaled 500 ppm CO, 6 h/day, 5 days/week, 4 weeks (repeated exposure) or 2,400 ppm, 1 h (single exposure). Neither treatment altered brain or lymphocyte COX activity 1 and 7 days post-treatment. Also ineffective were repeated and acute CO treatments towards (3)H-quinuclidinyl benzilate (QNB) binding to MRs in cerebral cortex, hippocampus, striatum, cerebellum (respective controls, mean+/-S.D.: 171 +/- 45, 245 +/- 53, 263 +/- 14 and 77 +/- 7 fmol/mg protein) and lymphocytes (24 +/- 10 fmol/million cells) at the same time points. In lymphocytes control cGMP levels averaged 1.98 +/- 0.99 pmol/mg protein under basal conditions, and 3.94 +/- 0.55 pmol/mg protein after NO-stimulation. One day after chronic treatment cessation, the CO-treated group displayed about a 50% decrease in both basal and NO-stimulated cGMP values, which persisted up to 7 days after, compared to air-exposed rats. Acutely, CO caused a delayed enhancement (+140%) of NO-induced activation of soluble guanylate cyclase. The finding that the NO-cGMP pathway is a target for the delayed effects of CO in peripheral blood cells is in accordance with our data in brain [Hernández-Viadel, M., Castoldi, A.F., Coccini, T., Manzo, L., Erceg, S., Felipo, V., 2004. In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum. Journal of Neurochemistry 89, 1,157-1,165], and supports the use of this peripheral endpoint as a biomarker of CO central effects.

Staging patients with colorectal cancer defines their prognosis and therapeutic management. Unfortunately, histopathology, the current standard for staging, is relatively insensitive for detecting occult micrometastases and a significant fraction of patients are understaged and, consequently, undertreated. Similarly, current approaches to postoperative surveillance of patients with colorectal cancer detect disease recurrence at a point when interventions have little impact on survival. The detection of rare cells in tissue, for accurately staging patients, and in blood, for detecting disease recurrence, could be facilitated by employing sensitive and specific markers of disease. Guanylyl cyclase C (GCC), the receptor for the diarrheagenic bacterial heat-stable enterotoxin, is expressed selectively by cells derived from intestinal mucosa, including normal intestinal cells and colorectal tumor cells, but not by extragastrointestinal tissues and tumors. The nearly uniform expression of relatively high levels by metastatic colorectal tumors suggests that GCC may be a sensitive and specific molecular marker for metastatic colorectal cancer cells. Employing GCC reverse transcriptase PCR, occult colorectal cancer micrometastases were detected in lymph nodes that escaped detection by histopathology. Moreover, marker expression correlated with the risk of disease recurrence. Similarly, GCC reverse transcriptase PCR revealed the presence of tumor cells in blood of all patients examined with metastatic colorectal cancer and, in some studies, was associated with an increased risk of disease recurrence and mortality. These observations suggest that GCC reverse transcriptase PCR is a sensitive and specific technique for identifying tumor cells in extraintestinal sites and may be useful for staging and postoperative surveillance of patients with colorectal cancer.

The molecular regulation of nitric oxide synthase (NOS) in blood platelets is an uncharacterised area of platelet biology. We investigated the mechanism of collagen-stimulated NO synthesis in platelets. Our aim was to identify the key collagen receptor and downstream signalling mechanisms linking collagen to NOS activation. Collagen and the GpVI-specific platelet activator collagen-related peptide (CRP-XL) stimulated NO synthesis, as evidenced by increased [(3)H]L-citrulline production, and cyclic GMP (cGMP) formation. After platelet activation by collagen and CRP-XL was normalised, we found no differences in NOS activation or cGMP formation in response to these agonists. Blocking the interaction of collagen with integrin alpha(2)beta(1), a second collagen receptor, failed to affect NOS activation by collagen. These data indicate that collagen-induced NO synthesis is linked to GpVI activation. cGMP formation in response to collagen and CRP-XL required increased intracellular Ca(2+), Src family kinases, phosphatidylinositol 3-kinase (PI3-K) and protein kinase C. By comparison, Gp VI-independent cGMP formation induced by thrombin was Src kinase-dependent, but was independent of PI3-K and PKC. Thus the mechanisms of collagen- and CRP-XL-induced NOS activation were identical, but distinct from that of thrombin. Platelet activation in response to collagen leads to secretion of adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Our results demonstrate that collagen-stimulated cGMP synthesis was enhanced significantly by platelet-derived ADP and TxA(2). These results reveal that collagen stimulates platelet NOS activation through a specific Ca(2+)-dependent GpVI receptor signalling cascade, and demonstrate that collagen-induced cGMP accrual requires the release of secondary platelet agonists.

Activation of soluble guanylate cyclase (sGC) has been reported to up-regulate gamma-globin gene transcription in erythroid cell lines and primary erythroblasts. sGC is activated by nitric oxide (NO), subsequently catalysing the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP), which mediates various physiological responses. To study the importance of this mechanism in the erythroid cells of sickle cell patients, cGMP levels were measured in the red blood cells (RBC) of normal individuals, steady-state sickle cell patients (SS) and SS patients on hydroxyurea (HU) therapy (SS + HU). cGMP levels were found to be significantly higher in RBC of SS patients (SS RBC) than in RBC of normal individuals, and were further increased in RBC of SS + HU patients. cGMP levels correlated with fetal haemoglobin (HbF) levels in SS/SS + HU patients, but not with reticulocyte count. Furthermore, NO-stimulated sGC activity, following incubation of cells with a NO donor, was significantly greater in SS RBC than in normal RBC. These results demonstrate, for the first time, an increased metabolism of NO mediated by sGC in the SS RBC, which is further increased by hydroxyurea. Augmentation of cGMP levels by NO in erythroid cells may constitute a mechanism for induction of HbF and other erythrocyte functions and represent a possible therapeutic target for treatment of sickle cell disease.

Mice with a genetic deletion of the atrial natriuretic peptide (ANP) receptor, guanylyl cyclase A (GC-A -/-), have chronic arterial hypertension and cardiac hypertrophy from the first day of life. To characterize the role of the angiotensin II and endothelin systems in the development of this cardiovascular phenotype, the effects of chronic treatment with either the angiotensin type I (AT1) receptor antagonist losartan or the endothelin A receptor antagonist BSF208075 were tested. Losartan almost completely reversed systemic arterial hypertension and left ventricular hypertrophy of GC-A -/- mice. This was accompanied by a marked regression of the left ventricular mRNA expression of cardiac hypertrophy markers such as ANP and brain natriuretic peptide and a significant reduction of left ventricular and pulmonary interstitial collagen accumulation. BSF208075 had no effect on any of these cardiovascular parameters. Intriguingly, GC-A -/- mice also showed a very marked right ventricular hypertrophy, which was not reversed by losartan or BSF208075 treatment. Analyses of components of the renin-angiotensin system (RAS) revealed an inhibition of renal and systemic RAS contrasting with increased local left ventricular angiotensin II levels in GC-A -/- mice. Collectively, the results suggest that RAS plays a more important role than the endothelin system in the pathogenesis of arterial hypertension as well as left ventricular hypertrophy and fibrosis in GC-A gene-disrupted mice.

Micrometastasis are defined by the existence of cells or groups of cells in target organs. In the particular cas of colon cancers, although lymph node involvement is frequent, metastatic medullary involvement (while rarely at the origin of identified metastasis) can also be observed. Furthermore, micrometastatics cells can be identified in the circulating blood. This research relies on recent technics of immunocytochemistry with image analysis or molecular biology technics (generally PCR or RT-PCR). It is essential to have a specific reliable marker of metastatic cells. The prognostic value of identifying micrometastasis in organs also remains to be defined.

1. The aim of the present study was to determine whether the regulation of vascular natriuretic peptide receptors (NPR) is related to the local renin-angiotensin system (RAS). 2. Male Sprague-Dawley rats were made two-kidney, one-clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt hypertensive to activate and inhibit the RAS, respectively. Another model of hypertension was induced by treatment with an inhibitor of nitric oxide synthesis, namely NG-nitro-L-arginine methyl ester (L-NAME). 3. The mRNA expression of NPR-A, NPR-C, angiotensin- converting enzyme (ACE) and angiotensin AT1 receptors was determined in the thoracic aorta by semiquantitative reverse transcription-polymerase chain reaction. The particulate guanylyl cyclase activity stimulated by atrial natriuretic peptide (ANP) was also determined in the membrane fraction of the thoracic aorta. 4. The plasma concentrations of ANP were increased significantly in the three models of hypertension. Plasma renin activity was increased in 2K1C hypertension, decreased in DOCA-salt hypertension and not significantly altered in L-NAME hypertension. 5. The mRNA expression of NPR-A and NPR-C was decreased, whereas that of ACE and AT1 receptors was increased in 2K1C and L-NAME hypertension. The mRNA expression of NPR-A and NPR-C was increased, whereas that of ACE and AT1 receptors was decreased in DOCA-salt hypertension. 6. The particulate guanylyl cyclase activity was decreased in 2K1C and L-NAME hypertension and increased in DOCA-salt hypertension. 7. The vascular expression of NPR may be reciprocally regulated by local RAS activity.

Purpose: This study was designed to show, in certain patients, that colonic epithelial cells can be present in peripheral blood while absent in portal venous blood., Methods: The circulating colorectal epithelial cells were detected by a reverse transcriptase-polymerase chain reaction assay, which involved amplifying guanylyl cyclase C transcripts. Portal venous and peripheral blood samples were obtained at intervals from 58 patients undergoing colorectal cancer surgery., Results: Circulating colonic epithelial cells were more frequently detected in portal venous blood than in peripheral blood only before mobilization of the tumor-bearing colon segment in patients with tumors of Stage B. In five other patients, before mobilization of their tumor-bearing colon segments, and in another three patients, during the mobilization, colorectal epithelial cells were detected in peripheral blood but not in portal venous blood. These eight patients had Stage C or D tumors., Conclusion: In 8 of 58 patients, colorectal epithelial cells were detected in peripheral but not in portal venous blood. Metastatic deposits in lymphatic vessels or liver might be the source of these cells.

Natriuretic peptides ANP, BNP and CNP with their receptors A, B, and C play an important role in maintaining the homeostasis. They have vasodilating, diuretic and natriuretic actions and regulate the systemic resistance. These peptides have been proved to contribute in pathogenesis of many diseases, for example heart failure, hypertension, acute coronary events or hepatic and renal insufficiency. The aim of our study was the estimation whether ANP or BNP could be markers of postinfarct heart failure in patients after thrombolytic therapy. The survey was made in 96 patients with acute myocardial infarction (AMI), who were treated in Department of Cardiology of University School of Medicine in Wrocław. Patients were divided into 2 groups. The first group consisted of 56 patients with reperfusion after thrombolytic therapy and the second group created 40 patients without reperfusion. All patients were administered acethylsalicylic acid, 100 mg r-tPA (recombined tissue plasminogen activator) i.v. within 90 min and heparin in typical doses for the first 3-5 days. Venous blood samples for ANP and BNP estimation were taken before thrombolysis and then in 1st, 3rd, 5th and 30th day after treatment. We also measured the ejection fraction and activity of CPK and CK-MB in all the patients. Patients stayed under clinical observation for 12 months. Our study showed that the levels of ANP and BNP increase in 1st day after treatment in all patients with AMI. The normalization of ANP and BNP occurs in 3rd day after treatment in patients with reperfusion and in patients without reperfusion there are increased levels of these peptides even in 30th day. The increased level of BNP (> 160 pg/ml) is a significant risk factor of left ventricle systolic dysfunction, renewed AMI and sudden death in all patients with AMI after thrombolytic therapy whereas the increased level of ANP (> 100 pg/ml)--only in patients without reperfusion. Taking BNP as a marker of postinfarct heart failure it is possible to asses the risk of this complication in first day after thrombolytic therapy and choose the most proper treatment.

Purpose: To examine the utility of guanylyl cyclase C (GC-C)-specific nested reverse transcriptase polymerase chain reaction (RT-PCR) to detect circulating tumor cells in patients with colorectal cancer., Patients and Methods: Peripheral-blood mononuclear cells from 24 patients with Dukes' stage D colorectal cancer were analyzed by GC-C-specific nested RT-PCR using 1 microg of total RNA. Peripheral-blood mononuclear cells from 20 healthy volunteers served as controls. Additionally, peripheral-blood CD34+ progenitor cells were assayed for the expression of both GC-C and other epithelial cell-specific markers., Results: GC-C mRNA was detected in blood mononuclear cells from all 24 patients with colorectal cancer and all healthy volunteers. These unexpected positive results reflected low-level ectopic transcription of GC-C in CD34+ progenitor cells. Moreover, CD34+ progenitor cells expressed other epithelial cell-specific markers, including prostate-specific antigen, prostate-specific membrane antigen, carcinoembryonic antigen, CK-19, CK-20, mucin 1, and GA733.2. Limiting the quantity of mononuclear cell total RNA analyzed to < or = 0.8 microg eliminated detection of GC-C and other tissue-specific transcripts in blood of healthy volunteers. However, under the same conditions, GC-C mRNA was detected in mononuclear cells from all 24 patients with metastatic colorectal cancer. Using 0.5 microg of total RNA and GC-C-specific primers, nested RT-PCR detected a single human colon carcinoma cell (approximately 20 to 200 GC-C transcripts/cell) in 10(6) to 10(7) mononuclear blood cells., Conclusion: These data suggest that GC-C may be useful for detecting circulating colorectal cancer cells. They also demonstrate that CD34+ cells are a source of ectopically expressed epithelial cell-specific markers and that CD34+ cells may contribute to the high false-positive rate generally observed when those markers are used to detect rare circulating metastatic cancer cells by RT-PCR.

In previous studies, a strong synergism between low concentrations of hydrogen peroxide and nitric oxide in the inhibition of agonist-induced platelet aggregation has been established and may be due to enhanced formation of cyclic GMP. In this investigation, hydrogen peroxide and NO had no effect on the activity of pure soluble guanylyl cyclase or its activity in platelet lysates and cytosol. H(2)O(2) was found to increase the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), increasing the amount of the 50-kDa form that results from phosphorylation at serine(157). This occurs both in the presence and in the absence of low concentrations of NO, even at submicromolar concentrations of the peroxide, which alone was not inhibitory to platelets. These actions of H(2)O(2) were inhibited to a large extent by an inhibitor of cyclic AMP-dependent protein kinase, even though H(2)O(2) did not increase cyclic AMP. This inhibitor reversed the inhibition of platelets induced by combinations of NO and H(2)O(2) at low concentrations. The results suggest that the action on VASP may be one site of action of H(2)O(2) but that this event alone does not lead to inhibition of platelets; another unspecified action of NO is required to complete the events required for inhibition., (Copyright 2001 Academic Press.)

Background: N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability., Materials and Methods: 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin)., Results: N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets., Conclusion: N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.

Natriuretic peptide binding sites on platelets have been hypothesized to act as clearance receptors; however, there is no clear definition of the function of this receptor. The aim of the study was: 1) to characterize natriuretic peptide receptors in human platelets by original competition study; 2) to evaluate a possible age modulation of these binding sites, since a delayed clearance of ANP in the elderly has been observed. The binding of 125I-ANP to intact platelets was completely inhibited by h-ANP, h-BNP, h-CNP and c-ANP, the selective ligand of the clearance receptor. IC50 values were 0.089+/-0.029, 0.703+/-0.104 and 1.19+/-0.13, 3.84+/-0.04 nmol/l, mean+/-SE, respectively (p<0.001 for IC50 value of h-ANP compared to the other natriuretic peptides). This observation on the receptor selectivity of natriuretic peptides in human platelets provides new evidence for the presence of the clearance receptor on platelets. In control subjects the Kd was 34.6+/-4.0 pmol/l and Bmax 13.6+/-0.92 fmol/10(9) platelets (mean+/-SE), (no.=46, mean age 41.7+/-2.1 years). Bmax was significantly reduced in older subjects (no.=25, mean age 53.2+/-1.5 years) with respect to the younger group (no.=21, mean age 28.0+/-0.87 years): 11.4+/-1.1 vs 16.1+/-1.4 fmol/10(9) cells, p=0.0096, respectively; moreover, a significant inverse relationship between Bmax and the subject's age was observed. This observation suggests a possible reduction of the natriuretic peptide clearance with aging, associated to a significant increase of plasma levels of natriuretic peptides.

The paper deals with the molecular mechanisms of antihypertensive and antiaggregatory actions of nitric oxide (NO). These effects of NO are shown to be directly associated with the activation of soluble guanylate cyclase and with the accumulation of 3',5'-guanosine monophosphate. The paper discusses the mechanism of activation of guanylate cyclase with new donors: the role and value of a nitrosyl-hemic complex formed in the interaction of guanylate cyclase with NO. New approaches to studying the mechanism of antihypertensive and antiaggregatory actions of the NO donors in question provided fundamental and priority data that lead to the development of the molecular bases of goal-oriented search and the synthesis of new effective vasodilators and antiaggregants. Examining the molecular mechanisms of targeted activation of soluble guanylate cyclase with new NO donors has revealed new enzyme activators that generate NO in the body and participate in the regulation of homeostasis and vascular tone.

In the present study, we showed that 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a nitric oxide (NO)-independent activator of soluble guanylate cyclase, could potentiate H2O2-induced inhibition of platelet aggregation and increase of platelet cGMP levels. The synergistic effect of YC-1 and H2O2 on platelet aggregation and increases of cGMP were almost completely prevented by catalase and a selective soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or partially attenuated by the hydroxyl radical scavenger mannitol. In contrast, superoxide dismutase failed to influence H2O2/YC-1-induced inhibition of aggregation. Furthermore, YC-1 could enhance the activation of soluble guanylate cyclase caused by FeSO4/H2O2 and, this effect was prevented markedly by mannitol. These results suggest that YC-1 may enhance the antiaggregatory effect of H2O2 via the sensitization of platelet soluble guanylate cyclase. In addition, this phenomenon is, at least in part, dependent on H2O2-derived hydroxyl radical.

The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5'-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies.

Aim: To study effects of long-term administration of isosorbide-5-mononitrate (monochinquare retard-MR) on functional activity of soluble guanylate cyclase (GC), cardiohemodynamics and exercise tolerance in patients with coronary heart disease (CHD) complicated by chronic cardiac failure (CCF)., Materials and Methods: The trial included 20 males aged 58 to 77 years with CHD, effort angina (NYHA class II-III), CCF (NYHA class I-II). Endothelial function was assessed before the treatment and 12 weeks after it by indirect measurement of production of endothelial relaxing factor (NO)--by activation of platelet soluble GC in the presence of its activators: L-arginine, disulphide, sodium nitroprusside and protoporfirine IX. Hemodynamic parameters, morphofunctional condition of the heart, exercise tolerance test results were compared with changes in GC functional activity after 12 weeks of MR monotherapy., Results: Two types of the changes were revealed. Type I (n = 14): high GC basal activity and low activity before the treatment with normalization of all the parameters after the treatment. Type II (n = 6): initially normal basal activity and GC activation with their lowering after the treatment. MR in a single daily dose 50 mg given for 12 weeks has improved morphofunctional cardiac parameters with a reduction of left ventricular volume, a 8.3% increase in the ejection fraction and a 117% increase in exercise tolerance. GC activity inhibition was recorded in some cases, in angina class III, in particular. In patients with normalization of GC activity the response was higher than in those who demonstrated inhibition of GC activity., Conclusion: MR therapeutic action may be due to enhancement of soluble GC activity which was observed in 70% of the cases.

Guanylate cyclase activity in human erythrocytes is investigated by evaluating the intracellular guanosine 3',5'-cyclic monophosphate (cGMP) content in the presence of various agents that exert specific effects on soluble or particulate guanylate cyclase. The increase in the intraerythrocyte cGMP content by the soluble guanylate cyclase activators nitroprusside and NaNO2 suggests the presence of this enzyme in human erythrocytes. The effects of four different atrial natriuretic peptide (ANP) fragments on the intraerythrocyte cGMP content is also studied. ANP II and ANP III increase the intraerythrocyte cGMP content, whereas ANP I and des-Ser5,des-Ser6-ANP III are ineffective. Thus our data show that human erythrocytes possess particulate guanylate cyclase together with the soluble enzyme. The ANP fragments ANP II and ANP III also activate the erythrocyte Na+/H+ exchange. Nitroprusside, M & B 22948 (an inhibitor of cGMP phosphodiesterase), and the cGMP analogues dibutyryl cGMP and 8-bromoguanosine 3',5'-cyclic monophosphate also increase the erythrocyte Na+/H+ exchange rate. The latter data also suggest that the erythrocyte Na+/H+ exchange is regulated by cGMP.

Platelet hyperactivity plays an important role in the pathogenesis of cardio-vascular diseases. In patients with stable angina pectoris, we have recently demonstrated that nitroglycerin suppressed the increased platelet aggregability. The anti-aggregating effect of NTG and other nitrovasodilators is mediated by platelet guanylate cyclase, which generates cyclic GMP (cGMP) in response to nitric oxide (NO) liberated from the nitrovasodilator molecule. In the current study we utilised a more "direct" NO donor, sodium nitroprusside (SNP), to examine reversal of ADP-induced platelet aggregation in comparison with intraplatelet cGMP elevation in platelets from normal subjects (n = 22) and patients with stable angina pectoris (n = 23). Concentrations of SNP associated with 50% reversal of aggregation were 2.7 +/- 0.4 x 10(-7) mol/L with normal subjects and 4.5 +/- 0.5 x 10(-6) mol/L with patients (P < 0.01). SNP produced a concentration-dependent elevation of intraplatelet cGMP content: with 10(-4) mol/L SNP this was 17-fold for normals and 5-fold for patients (P < 0.01). An increase in cAMP content was seen only with 10(-4) mol/L SNP, and was 157 +/- 11% of baseline in platelets from normal subjects and 138 +/- 14% in patients. There was a strong interrelationship between cGMP-stimulating and anti-aggregating effects of SNP. The decrease in cGMP responsiveness to SNP was not related to a dysfunction of platelet guanylate cyclase; neither basal nor SNP-stimulated activity of the enzyme varied significantly between normal subjects and patients. Lipophilic derivatives of cGMP (db-cGMP) and cAMP (db-cAMP) caused reversal of aggregation; there was a nonsignificant trend towards decreased responsiveness of platelets from patients to both db-cGMP and db-cAMP. The observed decrease in responsiveness of platelets from angina patients to anti-aggregating effects of the exogenous NO donor, SNP, can therefore be attributed to suppressed cGMP accumulation. These results imply reduced platelet sensitivity to endogenous NO (endothelium-derived relaxing factor): this might contribute to platelet hyperaggregability observed in angina pectoris.

Earlier studies have shown that inhibition of aggregation of washed platelets (WP) by NO was enhanced almost 100-fold by H2O2. In the present study, the interactions of H2O2 with nitrosothiols, the influence of the presence of plasma and the mechanism of the synergism were investigated. H2O2 strongly enhanced the inhibitory effects of S-nitrosoglutathione (GSNO) on thrombin-induced aggregation of WP. S-Nitrosoalbumin also inhibited platelets, and this was similarly enhanced by H2O2. The synergism with H2O2 was demonstrable for both exogenous GSNO and NO in the presence of plasma when platelets were stimulated with collagen. The inhibition of platelets by GSNO and H2O2 was completely inhibited by guanylate cyclase inhibitors. Synergism was also observed whether the H2O2 was added simultaneously or 1 min before or after the GSNO (or NO). This suggests that the action of H2O2 follows the occupation by NO of haem sites in guanylate cyclase and that a prior reaction between NO and H2O2 was not required. In the absence of exogenous GSNO or NO, H2O2 inhibited activation of platelets in plasma, an effect abolished by guanylate cyclase inhibitors. This suggested that endogenous NO donors in plasma or NO synthesized in platelets may interact with H2O2. Addition of NG-nitro-L-arginine methyl ester (hydrochloride) (L-NAME) decreased the effects of the H2O2 by 25%, indicating that the major endogenous source of NO in platelet-rich plasma was not derived from platelet synthesis of NO but from NO donors in plasma, such as nitrosothiols. Inhibition by H2O2 was also enhanced by beta-mercaptosuccinate, a glutathione peroxidase inhibitor that protects the H2O2. These results suggest a potent synergism of H2O2 with endogenous plasma nitrosothiols that inhibit platelet function through an intracellular mechanism involving guanylate cyclase.

Soluble guanylate cyclase is a key enzyme of nitric oxide (NO)-related intracellular signal transduction in platelets. In the present study, we investigated the effects of sex and age on the enzyme activity in human platelets. Soluble guanylate cyclase activity was determined by generation of cyclic GMP in platelet cytosol. No significant differences in the basal activity of soluble guanylate cyclase were observed between in men and women, and between in young and old subjects. However, soluble guanylate cyclase activity in response to sodium nitroprusside, an exogenous NO donor, was higher in young men than in young and old women. Furthermore, the enzyme activity was lower in old than in young men, but there were no differences in female platelets between from young and old subjects. The present data suggest that NO-related signal transduction system in the platelet is affected by sex and age, which, to certain extent, contributes to different sensitivity of human platelets.

Background: One of the possible mechanisms responsible for pre-eclampsia is a loss of efficiency of the L-arginine-nitric oxide pathway with subsequent inactivation of the guanylyl cyclases of the vascular smooth muscle cells. As a result there should be a decrease in plasma cyclic 3'-5' guanosine monophosphate (cGMP) concentrations in pre-eclampsia. We assessed the behavior of this nucleotid in the plasma of pre-eclamptic women., Subjects and Methods: Sixteen pre-eclamptic women, 16 normotensive pregnant women matched for gestational age and six nonpregnant controls were investigated. Arterial blood pressure was recorded at inclusion time and then once-a-day until the fourth day after delivery concomitantly with the collection of blood samples for determining plasma cGMP, atrial natriuretic peptides (ANP), creatinine, uric acid and platelet counts. Also 24 h urines were simultaneously collected to calculate renal clearance of cGMP., Results: Before the initiation of antihypertensive treatment, plasma cGMP levels were significantly higher (p < 0.01) in pre-eclampsia women as compared both to pregnant normotensive controls and nonpregnant women (7.02 +/- 0.9 versus 4.8 +/- 0.76 versus 1.93 +/- 0.15 pmol.ml-1, p < 0.01). Under antihypertensive treatment, cGMP levels decreased significantly (p < 0.05) to 5.48 +/- 0.9 pmol.ml-1. The increase of plasma cGMP was associated with high ANP levels; the likelihood that a renal impairment could account for an increase in plasma cGMP was ruled out because the clearance of creatinine was not impaired. Similarly the possibility of a significant linear correlation between cGMP levels and blood pressure values or biological data was excluded in these women., Conclusion: Plasma cGMP concentrations are increased in pre-eclampsia. They decrease to control values when blood pressure returns to normal values; they indicate enhanced guanylyl cyclase activation by ANP and additional factors, but cannot be considered as a direct index of the severity of pre-eclampsia.

Effects of aminoethylisothiuronium bromide (AET), known as radioprotector, on human platelet soluble guanylate cyclase and on ADP-induced human platelets aggregation were studied. It was shown that AET - in Tris buffer and at certain pH values - is converted, via transguanidine rearrangement, to mercaptoethylguanidine. The latter contains in its molecule both the guanidine and SH groups which act as donor and acceptor of nitric oxide (NO), respectively. It was demonstrated that AET, after its rearrangement to mercaptoethylguanidine, is able to activate human platelet soluble guanylate cyclase, as well as to inhibit ADP-induced human stimulatory effect of AET is dependent on the effectiveness of its transguanidine rearrangement to mercaptoethylguanidine. The molecular mechanism of the hypotensive by - effect of AET is proposed.

The molecular mechanism of the synergistic platelet inhibition by activators of adenylate cyclase and guanylate cyclase in human platelets was investigated. The adenylate cyclase activators iloprost and prostaglandin E1 and the guanylate cyclase activator 3-morpholino-syndnonimine (SIN-1) dose-dependently inhibited thrombin-induced aggregation of washed human platelets. Furthermore, SIN-1 at a concentration inhibiting platelet aggregation by only 10% shifted the IC50 values of iloprost and prostaglandin E1 by one order of magnitude to the left, indicating a synergistic action of adenylate cyclase and guanylate cyclase activators. Iloprost and prostaglandin E1 dose-dependently elevated platelet cAMP without a significant influence on cGMP. In contrast, the platelet cGMP level was dose-dependently elevated by SIN-1. In addiiton, SIN-1 markedly increased cAMP level induced by low concentrations of adenylate cyclase activators (0.1-0.3 nM iloprost or 10-150 nM prostaglandin E1). In contrast, the rise in cAMP induced by higher adenylate cyclase activator concentrations (3 nM iloprost or 30 microM prostaglandin E1) was significantly reduced in the presence of SIN-1. The same biphasic mode of action of SIN-1 was observed with forskolin, an adenylate cyclase stimulator acting receptor independently, indicating a prostacyclin-receptor independent mechanism. The cAMP elevating effect of SIN-1 in the presence of low prostanoid concentrations was completely abolished by piroximone, a selective inhibitor of phosphodiesterase type III. Therefore, the inhibition of phosphodiesterase III by cGMP seems to be the mechanism for the elevation of cAMP levels by SIN-1 in the presence of low concentration of adenylate cyclase activators in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] inhibited the aggregation of and ATP release from washed rabbit platelets induced by arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), and thrombin in a concentration-dependent manner. YC-1 also disaggregated the clumped platelets caused by these inducers. The thromboxane B2 formation caused by collagen, PAF, and thrombin was inhibited by concentrations of YC-1 that did not affect formation of thromboxane B2 and prostaglandin D2 caused by AA. YC-1 suppressed the increase of intracellular Ca2+ concentration and generation of inositol 1,4,5-trisphosphate caused by these five aggregation inducers. Both the cAMP and cGMP contents of platelets were increased by YC-1 in a concentration- and time-dependent manner. Like sodium nitroprusside, YC-1 potentiated formation of cAMP caused by prostaglandin E1 but not that by 3-isobutyl-1-methylxanthine. Adenylate cyclase and cAMP phosphodiesterase activities were not altered by YC-1. Activity of cGMP phosphodiesterase was unaffected by YC-1. Activities of guanylate cyclase in platelet homogenate and cytosolic fraction were activated by YC-1, whereas particulate guanylate cyclase activity was unaffected. The antiplatelet effect of sodium nitroprusside but not that of YC-1 was blocked by hemoglobin and potentiated by superoxide dismutase. After intraperitoneal administration for 30 minutes, YC-1 prolonged the tail bleeding time of conscious mice. These data indicate that YC-1 is a direct soluble guanylate cyclase activator in rabbit platelets. It may also possess antithrombotic potential in vivo.

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4-fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2-fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.

Human atrial natriuretic peptide (ANP) fragments ANP-(127-150) or ANP-III and ANP-(127-149) or ANP-II activate Na+/H+ exchange in human erythrocytes at concentrations as low as 1 pM. Both ANP-(127-147) or ANP-I and ANP-(129-150) or des-Ser5, Ser6-ANP-III have no effect on erythrocyte Na+/H+ exchange. ANP-III also produces a time-dependent increase of intraerythrocyte guanosine 3',5'-phosphate (cGMP) concentration. M&B 22,948, a specific inhibitor of cGMP phosphodiesterase, increases Na+/H+ exchange and the intracellular concentration of cGMP. Both 8-bromoguanosine 3',5'-phosphate (8-Br-cGMP) and dibutyryl-cGMP mimic the effect of ANP-III on erythrocyte Na+/H+ exchange. Our data suggest that human erythrocytes possess guanylate-cyclase activity stimulated by ANP-III and that activation of Na+/H+ exchange by this peptide is mediated by cGMP. Human erythrocytes display a high degree of sensitivity to ANP-III or ANP-II and a specificity for ANP-fragment structures just as cells with established ANP-specific receptors.

Nicorandil (nicotinamidoethyl nitrate) is a novel vasodilator. Its vasodilator properties are related both to the nicotinamide and nitrate moieties. Classic nitrates such as nitroglycerin (NTG) and isosorbide dinitrate demonstrate in vitro inhibition of ADP-induced platelet aggregation. Such effects have been shown to occur in a dose-related manner, are potentiated by reduced thiols and by increasing preincubation time, and are associated with increases in intracellular cyclic GMP. We explored the effect of nicorandil on ADP-induced human platelet aggregation and the role of reduced thiol N-acetylcysteine (NAC) in modulating this response. Nicorandil significantly inhibited aggregation to ADP dose dependently (IC50 3.0 mM). These effects were associated with inhibition of fibrinogen binding to the platelet surface (IC50 2 mM). Addition of nicorandil after maximal ADP-induced aggregation was achieved resulted in disaggregation. Addition of a source of reduced thiol (NAC) potentiated the antiaggregatory effects of nicorandil threefold (p < 0.05). Platelet inhibition by nicorandil was also augmented by increase in duration of preincubation, with maximal effects observed at 180 min. Preincubation of platelets with 10 mM nicorandil resulted in attenuated inhibition of platelet aggregation on gel filtration and subsequent exposure to additional nicorandil, indicative of tolerance induction. Methylene blue (MB), an inhibitor of guanylate cyclase, significantly reversed nicorandil-induced inhibition of platelet aggregation. Moreover, in accordance with this mechanism, nicorandil increased intracellular platelet cyclic GMP levels. Although the antiplatelet effect of nicotinamide was partially reversed by the K+ channel inhibitor iberotoxin, preincubation with iberotoxin had no impact on inhibition of platelet aggregation by nicorandil.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequestration of nitric oxide (NO) by subcellular fractions isolated from bovine pulmonary arterial medial layer (BPA) and rabbit platelets (RP) was studied utilizing a novel chemiluminescence--headspace gas technique. Sequestration in all fractions was similarly rapid (5 min) and remained constant for at least 30 min. When incubated with 108 pmol of NO, the BPA mitochondrial, microsomal, and nuclear fractions sequestered 22.8 +/- 1.9, 20.5 +/- 2.2 and 15.2 +/- 3.6% of the NO, respectively (n = 14). However, significantly more of the 108 pmol of NO, 36.8 +/- 2.8 and 32.9 +/- 3.6%, respectively, was sequestered by the BPA homogenate (about 2 mg protein/mL) and BPA cytosolic fraction (about 1 mg protein/mL) (n = 19). Also, RP cytosolic fraction (about 3 mg protein/mL) sequestered a greater amount of NO than any BPA fraction when incubated with 108 pmol of NO (83.0 +/- 1.0%; n = 3). Analysis of the binding data obtained for the BPA homogenate and cytosolic fraction was consistent with the existence of two binding sites, one site with a Kd of approximately 100 nM and another with a Kd of approximately 1 microM. Both the BPA homogenate fraction and the cytosolic fraction as well as the RP cytosolic fraction were shown to have soluble guanylyl cyclase activity. The nitrovasodilator sodium nitroprusside (SNP) caused a concentration-dependent increase in the activity of this enzyme in all these fractions. Maximum stimulations caused by 1 mM SNP in BPA homogenate fraction, BPA cytosolic fraction, and RP cytosolic fraction were equivalent to 2-, 4- and 3-fold increases in catalytic activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Derivatives of diazetidine-di-N-oxides have been found capable of the nonenzymatic generation of nitric oxide by the principally new mechanism of the nitric oxide splitting at physiological pH values. The effect of the synthesized compounds on human platelet soluble guanylate cyclase activity as well as their spasmolytic and hypotensive action were studied. Four of 7 derivatives studied exhibited a distinct correlation between the ability of being decomposed with the nitric oxide formation, activation of soluble guanylate cyclase, and spasmolytic and antihypertensive activities. Among them, 3-brom, 4-methyl-3,4-tetramethylene-diazetidine-di-N-oxide has proved to be most effective, its spasmolytic effect being commensurable with glyceryl trinittrate activity. The revealed correlation allows us to regard 1,2-diazetidine-1,2-di-N-oxide derivatives as vasodilatory agents of a new class.