cyclic-gmp and 3-(5--hydroxymethyl-2--furyl)-1-benzylindazole

Since the first article on YC-1 was published in 1994, it has been popularly used as a pharmacological tool to activate soluble guanylate cyclase and to increase cyclic GMP levels in cultured cells or isolated tissues. In terms of the pharmacological actions of YC-1, previous studies tend to be limited to it inhibition of platelet aggregation and vascular concentration. However, recent studies have demonstrated that YC-1 has versatile pharmacological effects other than the anti-platelet and vasodilatory effects. In particular, two recent reports suggest that YC-1 could be developed as a new class of anticancer agent for rapidly growing solid tumors, because it inhibits hypoxia-inducible factor 1 (HIF-1) activity, and has been reported to halt tumor growth in vivo. We here review the cyclic GMP-dependent and independent pharmacological actions of YC-1, and its anti-HIF-1, anticancer effect.

Topics: Animals; Antineoplastic Agents; Cyclic GMP; Guanylate Cyclase; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Neoplasms; Platelet Aggregation Inhibitors; Transcription Factors

The soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) second messenger system provides a complex and highly regulated mechanism for signal transduction events and ensuing functional responses through a cascade of serine/threonine protein kinase-dependent pathways. Nitric oxide (NO) and carbon monoxide (CO), two unique diatomic gases endogenously produced by the respective enzymes nitric oxide synthase (NOS) and heme oxygenase (HO), stimulate cellular sGC and synthesize cGMP within the vasculature. Emerging evidence suggests that the independent NOS and HO systems provide reciprocal and complimentary approaches that act to regulate cardiovascular and hematological homeostasis as well as provide protection to the vasculature in response to inimical stimuli or following the onset of vasoproliferative disease. Recent results from our laboratory and others suggest that the newly identified, chemically synthesized benzyl indazole derivative YC-1 is capable of exerting multifunctional and broad-ranging effects in the cardiovascular and hematological systems. YC-1 has been demonstrated to possess redundant biochemical mechanisms that confer significant stimulation upon NO- and CO-regulated, cyclase-dependent events. Ultimately, these acute molecular processes eventuate in YC-1-dependent modulation of platelet and vascular smooth muscle cell (SMC) and endothelial cell (EC) function under both eutrophic and deleterious conditions. Based on accumulating evidence, YC-1 has been suggested to serve as a potential therapeutic adjuvant to be used in interventional medicine, and these results may indicate the existence of an endogenous " YC-1-like" compound that would be the focus of much anticipated investigation. The purpose of this review, therefore, is to provide update information on the mechanisms and physiologic and pathophysiologic roles of the pivotal new multifunctional agent YC-1 in the cardiovascular and hematological systems, and to provide evidence for its potential use as a clinically relevant salutary agent.

Topics: Animals; Blood; Cardiovascular System; Cyclic GMP; Enzyme Activators; Humans; Indazoles; Nitric Oxide; Platelet Aggregation Inhibitors

Topics: Animals; Brain; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Humans; Indazoles; Nitric Oxide; Signal Transduction

3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) is a potential anticancer drug that may activate soluble guanylyl cyclase (sGC) and increase the level of cyclic guanosine monophosphate (cGMP). The aim of this study was to explore the effects of YC-1 on lipid droplet accumulation and foam cell formation in macrophages.. Human-oxidized low density lipoprotein (ox-LDL) was used to induce accumulation of lipid droplets in a murine macrophage cell line, RAW 264.7. Oil red O staining showed that treatment with 20 μM YC-1 for 24 h increased the area of intracellular lipid droplets in macrophages. The results of high content screening (HCS) with the AdipoRed™ assay further revealed that YC-1 enhanced ox-LDL-induced foam cell formation. This was evidenced by an increase in the total area of lipid droplets and the mean fluorescence intensity per cell. Inhibition of cGMP-dependent protein kinase (PKG) using KT5823 significantly reduced YC-1-enhanced lipid droplet formation in ox-LDL-induced macrophage foam cells.. YC-1 induces lipid droplet formation in macrophages, possibly through the sGC/cGMP/PKG signaling pathway. This chemical should be tested with caution in future clinical trials.

Topics: Animals; Cell Line; Cyclic GMP; Indazoles; Lipid Droplets; Lipoproteins, LDL; Macrophages; Mice; Second Messenger Systems

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

Topics: Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Cyclic GMP; Cysteine; Diltiazem; Female; Femoral Artery; Fetus; Indazoles; Male; Mesenteric Arteries; Nifedipine; Oxadiazoles; Quinoxalines; S-Nitrosoglutathione; S-Nitrosothiols; Sheep; Soluble Guanylyl Cyclase; Vasodilation; Vasodilator Agents

The present study was designed to investigate the effects of YC-1, a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, a NO donor, on smooth muscle responses in the preeclampsia model with suramin-treated rats and on the levels of cyclic guanosine monophosphate (cGMP) of thoracic aorta rings isolated from term-pregnant rats. Rats of 2 groups, control group and suramin group, were given intraperitoneal injection of saline or suramin, respectively. Suramin injection caused increased blood pressure, protein in urine, and fetal growth retardation. Thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction and papaverine relaxation responses were similar. Relaxation responses of YC-1 and DEA/NO decreased in suramin group. In both groups in the presence of ODQ, a sGC inhibitor, the relaxation responses of YC-1 and DEA/NO decreased. The cGMP content was determined by radioimmunoassay technique. The content of cGMP in the suramin group decreased. In the presence of YC-1 and DEA/NO in both groups, cGMP content increased, but in ODQ-added groups, there was a significant decrease. We conclude that in preeclampsia, the decrease of relaxation responses and the decrease of cGMP content could be due to the reduction in stimulation of sGC and the decrease in cGMP levels.

Topics: Animals; Aorta, Thoracic; Blood Pressure; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Female; Fetal Growth Retardation; Guanylate Cyclase; Indazoles; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Pre-Eclampsia; Pregnancy; Proteinuria; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Second Messenger Systems; Suramin; Vasodilation; Vasodilator Agents

The aim of the present study was to evaluate the effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) on multidrug resistance. Expression of human P-glycoprotein was assessed by realtime quantitative RT-PCR and western blot. The efflux function of P-glycoprotein was evaluated by rhodamine 123 accumulation and calcein-AM uptake models. The mechanisms of action of YC-1 on different signaling pathways were studied using series of antagonists and the kinetics was also assessed. Cytotoxicity was evaluated by MTT assay. The results demonstrated that increased intracellular accumulation of rhodamine 123 and increased fluorescence of calcein were observed after YC-1 treatment. Furthermore, increased YC-1 concentration resulted in significant decrease in Vmax while K(M) remained unchanged suggested that YC-1 acted as a noncompetitive inhibitor of P-glycoprotein. Moreover, the inhibition of Pgp efflux function by YC-1 was significantly reversed by NO synthase inhibitor, (L)-NAME, the sGC inhibitor, ODQ, the PKG inhibitor, Rp-8-Br-PET-cGMPS, and the PKG inhibitor KT5823. In addition, ERK kinase inhibitor PD98059 also significantly restored YC-1 inhibited Pgp efflux function. These results indicated that YC-1 inhibited Pgp efflux via the NO-cGMP-PKG-ERK signaling pathway through noncompetitive inhibition. The present study revealed that YC-1 could be a good candidate for development as a MDR modulator.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Western; Cell Line; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Extracellular Signal-Regulated MAP Kinases; Fluoresceins; Fluorescent Dyes; Gene Expression Regulation; Humans; Indazoles; Nitric Oxide; Reverse Transcriptase Polymerase Chain Reaction; Rhodamine 123; Signal Transduction

Glutamate, one of the major neurotransmitters in the central nervous system, is released into the synaptic spaces and bound to the glutamate receptors which facilitate normal synaptic transmission, synaptic plasticity, and brain development. Past studies have shown that glutamate with high concentration is a potent neurotoxin capable of destroying neurons through many signal pathways. In this research, our main purpose was to determine whether the specific soluble guanylyl cyclase activator YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole) had effect on glutamate-induced apoptosis in cultured PC12 cells. The differentiated PC12 cells impaired by glutamate were used as the cell model of excitability, and were exposed to YC-1 or/and ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) with gradient concentrations for 24 h. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) assay was used to detect the cellular viability. Radioimmunoassay (RIA) was used to detect the cGMP (cyclic guanosine monophosphate) concentrations in PC12 cells. Hoechst 33258 staining and flow cytometric analysis were used to detect the cell apoptosis. The cellular viability was decreased and the apoptotic rate was increased when PC12 cells were treated with glutamate. Cells treated with YC-1 or/and ODQ showed no significant differences in the cell viability and intracellular cGMP levels compared with those of control group. The specific soluble guanylyl cyclase (sGC) inhibitor ODQ showed an inhibitory effect on cGMP level and aggravated the apoptosis of PC12 cells induced by glutamate. YC-1 elevated cGMP level thus decreased PC12 cell apoptosis induced by glutamate, but this effect could be reversed by ODQ. These results revealed that YC-1 might attenuate glutamate-induced PC12 cell apoptosis via a sGC-cGMP involved pathway.

Topics: Animals; Apoptosis; Bisbenzimidazole; Cell Shape; Cell Survival; Cyclic GMP; Drug Synergism; Glutamates; Indazoles; Oxadiazoles; PC12 Cells; Protective Agents; Quinoxalines; Rats; Staining and Labeling

Although the phenomenon of opioid tolerance has been widely investigated, neither opioid nor nonopioid mechanisms are completely understood. The aim of the present study was to investigate the role of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in the development of morphine-induced analgesia tolerance. The study was carried out on male Wistar albino rats (weighing 180-210 g; n = 126). To develop morphine tolerance, animals were given morphine (50 mg/kg; s.c.) once daily for 3 days. After the last dose of morphine was injected on day 4, morphine tolerance was evaluated. The analgesic effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), BAY 41-2272, S-nitroso-N-acetylpenicillamine (SNAP), N(G)-nitro-L-arginine methyl ester (L-NAME), and morphine were considered at 15 or 30 min intervals (0, 15, 30, 60, 90, and 120 min) by tail-flick and hot-plate analgesia tests (n = 6 in each study group). The results showed that YC-1 and BAY 41-2272, a NO-independent activator of soluble guanylate cyclase (sGC), significantly increased the development and expression of morphine tolerance, and L-NAME, a NO synthase (NOS) inhibitor, significantly decreased the development of morphine tolerance. In conclusion, these data demonstrate that the nitric oxide-cGMP signal pathway plays a pivotal role in developing tolerance to the analgesic effect of morphine.

Topics: Analgesics, Opioid; Animals; Cyclic GMP; Drug Tolerance; Guanylate Cyclase; Indazoles; Male; Morphine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Soluble Guanylyl Cyclase

In addition to increasing cGMP, the soluble guanylyl cyclase (sGC) activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) can elevate intracellular cAMP levels. This response was assumed to be as a result of cGMP-dependent inhibition of cAMP phosphodiesterases; however, in this study, we show that YC-1-induced cAMP production in the rat pancreatic beta cell line INS-1E occurs independent of its function as a sGC activator and independent of its ability to inhibit phosphodiesterases. This YC-1-induced cAMP increase is dependent upon soluble adenylyl cyclase and not on transmembrane adenylyl cyclase activity. We previously showed that soluble adenylyl cyclase-generated cAMP can lead to extracellular signal-regulated kinase activation and that YC-1-stimulated cAMP production also stimulates extracellular signal-regulated kinase. Although YC-1 has been used as a tool for investigating sGC and cGMP-mediated pathways, this study reveals cGMP-independent pharmacological actions of this compound.

Topics: Animals; Blotting, Western; Cell Line; Cyclic AMP; Cyclic GMP; Enzyme Activators; Enzyme Inhibitors; Glucose; Guanylate Cyclase; Indazoles; Insulin-Secreting Cells; Mitogen-Activated Protein Kinases; Morpholines; Oxadiazoles; Oxazines; Phosphoric Diester Hydrolases; Pyrimidines; Quinoxalines; Rats; Transcription, Genetic

The subject of the study compare the influences of YC-1 guanylyl cyclase activator with ODQ guanylyl cyclase inhibitor on the tracheal smooth muscle contraction induced by carbachol. The study specified the influence of increasing concentrations of soluble guanylyl cyclase activators YC-1 and 8Br cGMP on the reaction of tracheal smooth muscle contraction released by carbachol. The author also examined the effect of increasing concentrations of soluble guanylyl cyclase inhibitor ODQ on the concentration-effect curves for carbachol.. Testing was conducted on an isolated trachea of both sexes of Wistar rats with weight ranging between 350 g and 450 g. Tracheas were prepared in accordance with the Akcasu (1959) method in Szadujkis-Szadurski (1996) modification. Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin (2006) modification.. According to conducted testing, activation of soluble guanylyl cyclase with the use of YC-1 and 8Br cGMP caused reduced reaction of the tracheal smooth muscle with carbachol on average to 80%. Comparing concentration-effect curves for carbachol before and after the use of 8Br cGMP, similar results were obtained for those released by YC-1. On the other hand, increasing concentrations of guanylyl cyclase inhibitor - ODQ cause shift of curves to the left, decrease of EC(50) value and an increase of maximum reaction to carbachol.. Carbachol, depending on concentration, causes tracheal smooth muscle contraction. According to testing, we can confirm that activation of guanylyl cyclase leads to reduction of the reaction of tracheal smooth muscle to carbachol on average up to 80%

Topics: Animals; Carbachol; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Enzyme Inhibitors; Female; Guanylate Cyclase; Indazoles; Male; Muscle, Smooth; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Trachea

NK cells play crucial roles in innate immunity and adaptive immunity. The detailed mechanisms, however, governing NK cell development remains unclear. In this study, we report that YC-1 significantly enhances NK cell populations differentiated from human umbilical cord blood hematopoietic stem cells (HSCs). NK cells increased by YC-1 display both phenotypic and functional features of fully mature NK (mNK) cells, but YC-1 does not affect the activation of mNK cells. YC-1 did not affect cGMP production and phosphorylation of STAT-5 which is essential for IL-15R signaling. On the other hand, YC-1 increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, p38 inhibitor SB203580 inhibited the differentiation of NK cells enhanced by YC-1. Taken together, these data suggest that YC-1 enhances NK cell differentiation through the activation of p38 MAPK which is involved in NK cell differentiation.

Topics: Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cyclic GMP; Enzyme Activators; Enzyme Inhibitors; Fetal Blood; Flow Cytometry; Hematopoietic Stem Cells; Humans; Imidazoles; Indazoles; Killer Cells, Natural; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Receptors, Interleukin-15; Signal Transduction; STAT5 Transcription Factor

We followed the development of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) system during locust embryogenesis in whole mount nervous systems and brain sections by using various cytochemical techniques. We visualized NO-sensitive neurons by cGMP immunofluorescence after incubation with an NO donor in the presence of the soluble guanylyl cyclase (sGC) activator YC-1 and the phosphodiesterase-inhibitor isobutyl-methyl-xanthine (IBMX). Central nervous system (CNS) cells respond to NO as early as 38% embryogenesis. By using the NADPH-diaphorase technique, we identified somata and neurites of possible NO-synthesizing cells in the CNS. The first NADPH-diaphorase-positive cell bodies appear around 40% embryogenesis in the brain and at 47% in the ventral nerve cord. The number of positive cells reaches the full complement of adult cells at 80%. In the brain, some structures, e.g., the mushroom bodies acquire NADPH-diaphorase staining only postembryonically. Immunolocalization of L-citrulline confirmed the presence of NOS in NADPH-diaphorase-stained neurons and, in addition, indicated enzymatic activity in vivo. In whole mount ventral nerve cords, citrulline immunolabeling was present in varying subsets of NADPH-diaphorase-positive cells, but staining was very variable and often weak. However, in a regeneration paradigm in which one of the two connectives between ganglia had been crushed, strong, reliable staining was observed as early as 60% embryogenesis. Thus, citrulline immunolabeling appears to reflect specific activity of NOS. However, in younger embryos, NOS may not always be constitutively active or may be so at a very low level, below the citrulline antibody detection threshold. For the CNS, histochemical markers for NOS do not provide conclusive evidence for a developmental role of this enzyme.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Brain; Citrulline; Cyclic GMP; Embryo, Nonmammalian; Enzyme Activators; Ganglia, Invertebrate; Indazoles; Locusta migratoria; NADPH Dehydrogenase; Nerve Regeneration; Nervous System; Neurites; Neurons; Neuropil; Nitric Oxide; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Signal Transduction

There is a correlation between cell volume changes and changes in the rate of aqueous humor outflow; agents that decrease trabecular meshwork (TM) cell volume increase the rate of aqueous humor outflow. This study investigated the effects of the nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC), YC-1, and BAY-58-2667 on TM cell volume and the signal transduction pathways and ion channel involved.. Cell volume was measured with the use of calcein AM fluorescent dye, detected by confocal microscopy. Inhibitors and activators of sGC, 3',5'-cyclic guanosine monophosphate (cGMP), protein kinase G (PKG), and the BK(Ca) channel were used to characterize their involvement in the YC-1- and BAY-58-2667-induced regulation of TM cell volume. cGMP was assayed by an enzyme immunoassay.. YC-1 (10 nM-200 microM) and BAY-58-2667 (10 nM-100 microM) each elicited a biphasic effect on TM cell volume. YC-1 (1 microM) increased TM cell volume, but higher concentrations decreased TM cell volume. Similarly, BAY-58-2667 (100 nM) increased TM cell volume, but higher concentrations decreased cell volume. The YC-1-induced cell volume decrease was mimicked by 8-Br-cGMP and abolished by the sGC inhibitor ODQ, the PKG inhibitor (RP)-8-Br-PET-cGMP-S, and the BK(Ca) channel inhibitor IBTX. The BAY-58-2667-induced cell volume decrease was mimicked by 8-Br-cGMP and was abolished by the PKG inhibitor and the BK(Ca) channel inhibitor. Unlike the YC-1 response, ODQ potentiated the BAY-58-2667-induced decreases in cell volume.. These data suggest that the NO-independent decrease in TM cell volume is mediated by the sGC/cGMP/PKG pathway and involves K(+) efflux.

Topics: Adult; Aged; Aged, 80 and over; Benzoates; Cell Size; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activators; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Guanylate Cyclase; Humans; Indazoles; Large-Conductance Calcium-Activated Potassium Channels; Microscopy, Confocal; Nitric Oxide; Oxadiazoles; Quinoxalines; Trabecular Meshwork

Common bile duct ligation (CBDL) in the guinea pig is a well-defined model of acalculous cholecystitis. Nitric oxide (NO) mediates smooth muscle relaxation by stimulating the activity of soluble guanylate cyclase. The aim of this study was to determine whether the NO/cyclic guanosine monophosphate pathway plays a role in gallbladder relaxant response after CBDL.. Relaxant response of gallbladder muscle strips from CBDL and sham-operated guinea pigs was studied in vitro. Animals were treated with saline, aminoguanidine or an aminoguanidine + L-arginine combination in vivo. Concentration-response curves of papaverine, diethylamine/NO, YC-1, sildenafil and amrinone were obtained and relaxations in each group were calculated as the percent of the contractions induced by carbachol (10(-6) M).. There was a significant decrease in the gallbladder muscle relaxant responses to these substances in CBDL and aminoguanidine groups compared with sham surgical controls. The decreased relaxant response was reversed by aminoguanidine + L-arginine but not by aminoguanidine alone.. Decreased relaxant responses might be due to the reduced guanylate cyclase enzyme activity, but further studies are required.

Topics: Amrinone; Animals; Arginine; Bile Ducts; Cyclic GMP; Gallbladder; Guanidines; Guinea Pigs; Hydrazines; In Vitro Techniques; Indazoles; Ligation; Male; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Papaverine; Piperazines; Purines; Sildenafil Citrate; Sulfones

Guanosine-specific cyclic nucleotide signaling is suggested to serve protective actions in the vasculature; however, the influence of selective pharmacologic modulation of cyclic guanosine monophosphate- synthesizing soluble guanylate cyclase or cyclic guanosine monophosphate-degrading phosphodiesterase on vessel remodeling has not been thoroughly examined. In this study, rat carotid artery balloon injury was performed and the growth-modulating effects of the soluble guanylate cyclase stimulator YC-1 or the cyclic guanosine monophosphate-dependent phosphodiesterase-V inhibitor zaprinast were examined. YC-1 or zaprinast elevated vessel cyclic guanosine monophosphate content, reduced medial wall and neointimal cell proliferation, stimulated medial and neointimal cellular apoptosis, and markedly attenuated neointimal remodeling in comparable fashion. Interestingly, soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one failed to noticeably alter neointimal growth, and concomitant zaprinast with YC-1 did not modify any parameter compared to individual treatments. These results provide novel in vivo evidence that YC-1 and zaprinast inhibit injury-induced vascular remodeling through antimitogenic and proapoptotic actions and may offer promising therapeutic approaches against vasoproliferative disorders.

Topics: Animals; Apoptosis; Carotid Artery Injuries; Cell Proliferation; Cyclic GMP; Disease Models, Animal; Enzyme Activators; Guanylate Cyclase; Indazoles; Male; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley

Endothelial nitric oxide synthase (NOS3) regulates the functional response to beta-adrenergic (beta-AR) stimulation via modulation of the L-type Ca(2+) current (I(Ca)). However, the NOS3 signaling pathway modulating I(Ca) is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I(Ca). Myocytes were isolated from NOS3 knockout (NOS3(-/-)) and wildtype (WT) mice. We measured I(Ca) (whole-cell voltage-clamp), and simultaneously measured Ca(2+) transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased beta-AR stimulated (isoproterenol, ISO)-I(Ca), and Ca(2+) transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I(Ca), but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3(-/-) myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I(Ca), or Ca(2+) transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I(Ca), and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I(Ca); YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca(2+) transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I(Ca) and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating beta-AR stimulated I(Ca), while limiting effects on cardiac contraction.

Topics: Animals; Calcium Channels, L-Type; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Activation; Guanylate Cyclase; Indazoles; Ion Channel Gating; Isoproterenol; Mice; Models, Biological; Myocardial Contraction; Myocytes, Cardiac; Nitric Oxide Synthase Type III; Phosphodiesterase 5 Inhibitors; Purinones; Receptors, Adrenergic, beta; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase

Carbon monoxide (CO) has been shown to cause enteric smooth muscle relaxation by activating soluble guanylyl cyclase (sGC). In gastric fundus, the sGCalpha1beta1 heterodimer is believed to be the most important isoform. The aim of our study was to investigate the role of the sGCalpha1/alpha2 subunits in the relaxant effect of CO and CORM-2 in murine gastric fundus using wild-type (WT) and sGCalpha1 knock-out (KO) mice. In WT mice, CO (bolus)-induced relaxations were abolished by the sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), while CORM-2- and CO (infusion)-induced relaxations were only partially inhibited by ODQ. In sGCalpha1 KO mice, relaxant responses to CO and CORM-2 were significantly reduced when compared with WT mice, but ODQ still had an inhibitory effect. The sGC sensitizer 1-benzyl-3-(5'-hydroxymethyl-2'-furyl-)-indazol (YC-1) was able to potentiate CO- and CORM-2-induced relaxations in WT mice but lost this potentiating effect in sGCalpha1 KO mice. Both in WT and sGCalpha1 KO mice, CO-evoked relaxations were associated with a significant cGMP increase; however, basal and CO-elicited cGMP levels were markedly lower in sGCalpha1 KO mice. These data indicate that besides the predominant sGCalpha1beta1 isoform, also the less abundantly expressed sGCalpha2beta1 isoform plays an important role in the relaxant effect of CO in murine gastric fundus; however, the sGC stimulator YC-1 loses its potentiating effect towards CO in sGCalpha1 KO mice. Prolonged administration of CO-either by the addition of CORM-2 or by continuous infusion of CO-mediates gastric fundus relaxation in both a sGC-dependent and sGC-independent manner.

Topics: Animals; Carbon Monoxide; Cyclic GMP; Gastric Fundus; Guanylate Cyclase; Indazoles; Male; Mice; Mice, Knockout; Organometallic Compounds; Oxadiazoles; Protein Subunits; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase

Although much has been learned about the role of the amygdala in Pavlovian fear conditioning, relatively little is known about the signaling pathway involved in the acquisition of an active avoidance reaction. The aim of this study is to investigate the potentiating effects of the NO-guanylate cyclase activator YC-1 on learning and memory of shuttle avoidance test in rats. YC-1 enhanced the induction of long-term potentiation (LTP) in amygdala through NO-cGMP-PKG-ERK pathway and the increase of BDNF expression. The Western blot and PCR methods were used to examine the signaling pathways involved in fear memory. It was found that YC-1 increased the avoidance responses during learning period and the memory retention lasted longer than one week. The enhancement of learning behavior by YC-1 was antagonized by intracerebroventricular injection of NOS inhibitor l-NAME, PKG inhibitor Rp-8-Br-PET-cGMPS and MEK inhibitor PD98059, indicating that NO-cGMP-PKG and ERK pathways are involved in the learning potentiating action of YC-1. In addition, YC-1 increased the activation of ERK and Akt 30 min after Day-1 training in amygdala. YC-1 also potentiated the expression of BDNF and CREB in response to fear memory test. Taken together, these findings suggest that NO-cGMP-PKG-ERK signaling pathway is involved in the action of YC-1 in enhancing the fear memory.

Topics: Amygdala; Animals; Avoidance Learning; Brain-Derived Neurotrophic Factor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Extracellular Signal-Regulated MAP Kinases; Indazoles; Long-Term Potentiation; Male; MAP Kinase Signaling System; Nitric Oxide; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; RNA, Messenger

In rat hepatocytes, atrial natriuretic peptide (ANP) elevates cGMP through activation of particulate guanylyl cyclase and attenuates Ca(2+) signals by stimulating net plasma membrane Ca(2+) efflux. We show here that ANP-stimulated hepatocyte Ca(2+) efflux is mediated by protein kinase G (PKG) isotype I. Furthermore, we show that ANP recruits endogenous PKGIalpha, but not PKGIbeta, to the plasma membrane. These effects are mimicked by 8-bromo-cGMP, but not by the soluble guanylyl cyclase activators, sodium nitroprusside and YC-1. We propose that ANP, through localized cGMP elevation, promotes plasma membrane recruitment of PKGIalpha, which, in turn, stimulates Ca(2+) efflux.

Topics: Animals; Atrial Natriuretic Factor; Calcium; Cell Membrane; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Hepatocytes; Indazoles; Male; Nitroprusside; Rats; Rats, Wistar; Thionucleotides

Nitric oxide (NO) exerts physiological effects by activating specialized receptors that are coupled to guanylyl cyclase activity, resulting in cGMP synthesis from GTP. Despite its widespread importance as a signal transduction pathway, the way it operates is still understood only in descriptive terms. The present work aimed to elucidate a formal mechanism for NO receptor activation and its modulation by GTP, ATP, and allosteric agents, such as YC-1 and BAY 41-2272. The model comprised a module in which NO, the nucleotides, and allosteric agents bind and the protein undergoes a conformational change, dovetailing with a catalytic module where GTP is converted to cGMP and pyrophosphate. Experiments on NO-activated guanylyl cyclase purified from bovine lung allowed values for all of the binding and isomerization constants to be derived. The catalytic module was a modified version of one describing the kinetics of adenylyl cyclase. The resulting enzyme-linked receptor mechanism faithfully reproduces all of the main functional properties of NO-activated guanylyl cyclase reported to date and provides a thermodynamically sound interpretation of those properties. With appropriate modification, it also replicates activation by carbon monoxide and the remarkable enhancement of that activity brought about by the allosteric agents. In addition, the mechanism enhances understanding of the behavior of the receptor in a cellular setting.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Allosteric Regulation; Animals; Cattle; Cyclic GMP; Enzyme Activation; Guanosine Triphosphate; Guanylate Cyclase; Indazoles; Kinetics; Lung; Models, Chemical; Nitric Oxide; Protein Structure, Tertiary; Pyrazoles; Pyridines; Pyrophosphatases; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Thermodynamics

The activation of NO/cGMP pathways can induce pro-apoptotic pathways in cardiomyocytes although only a small number of cardiomyocytes fulfill the criteria of apoptosis. The same pathways reduce the contractile performance of cardiomyocytes. In the present study, we tested the hypothesis that exposure of cells to NO/cGMP for 24 h decrease their contractile performance due to an activation of pro-apoptotic pathways. Experiments were performed on freshly isolated and cultured adult ventricular rat cardiomyocytes. Cells were incubated with 8-bromo-cyclo-GMP (100 nmol/L-1 micromol/L), the NO donor SNAP (1 nmol/L-100 micromol/L), or the guanylyl cyclase activator YC-1 (3 micromol/L). Cell shortening, contraction and relaxation velocities, and diastolic cell lengths were determined at beating frequencies of 0.5, 1, and 2 Hz 24 h later. The activation of pro-apoptotic pathways was determined by staining of cardiomyocytes with an antibody directed against active caspase-3 and quantification of the number of apoptotic cells (annexin staining). Caspase-3 activation and an increase in the number of apoptotic cells was observed, but only at the highest concentrations tested (8-bromo-cyclo-GMP: 1-10 mmol/L; SNAP: 1-100 micromol/L). At these concentrations, none of the drugs decreased the mean cell shortening of cardiomyocytes. However, at concentrations lower than those required for induction of apoptotic cell death, the diastolic cell lengths and sarcomere lengths increased but cell shortening decreased. In conclusion, low concentrations of either NO or cGMP cause a desensitization of myofibrils, as indicated by elongated cell shapes, increased sarcomere lengths and reduced load-free cell shortening. High concentrations of NO/cGMP induce caspase-3 activation and increase the number of cells fulfilling the criteria of apoptotic cell death but did not impair cell function. Therefore, induction of apoptotic cell death per se seems not to contribute to the loss of contractile efficiency on the cellular level.

Topics: Animals; Apoptosis; Caspase 3; Cell Movement; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Indazoles; Male; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Donors; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Signal Transduction

1. Application of the nitric oxide (NO) donor, sodium nitrite and the NO synthase substrate l-arginine had no effect on nerve-evoked transmitter release in the rat isolated phrenic nerve/hemidiaphragm preparation; however, when adenosine A(1) receptors were blocked with the adenosine A(1) receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) prior to application of sodium nitrate or l-arginine, a significant increase in transmitter release was observed. In addition, the NO donor s-nitroso-N-acetylpenicillamine (SNAP) significantly increased transmitter release in the presence of DPCPX. In the present study, we have made the assumption that these NO donors elevate the level of NO in the tissue. Future studies should test other NO-donating compounds and also monitor the NO concentrations in the tissue to ensure that these effects are, in fact, NO induced. 2. Elevation of cGMP in this preparation with the guanylyl cyclase activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) significantly enhanced transmitter release. In the presence of DPCPX and the selective guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which blocks the production of cGMP, the excitatory effects of sodium nitrite and l-arginine were abolished. 3. These results suggest that NO serves to enhance transmitter release at the rat neuromuscular junction (NMJ) via a cGMP pathway and this facilitation of transmitter release can be blocked with adenosine. Previously, we demonstrated that adenosine inhibits N-type calcium channels. Because NO only affects transmitter release when adenosine A(1) receptors are blocked, we suggest that NO enhances transmitter release by enhancing calcium influx via N-type calcium channels. Further studies are needed to confirm that NO alters transmitter release via cGMP and that this action involves the N-type calcium channel. 4. The results of the present study are consistent with a model of NO neuromodulation that has been proposed for the mammalian vagal-atrial junction. This model suggests that NO acts on NO-sensitive guanylyl cyclase to increase the intracellular levels of cGMP. In turn, cGMP inhibits phosphodiesterase-3, increasing levels of cAMP, which then acts on the N-type calcium channels to enhance calcium influx, leading to an increase in transmitter release. Our only modification to this model for the NMJ is that adenosine serves to block the modulation of transmitter release by NO.

Topics: Animals; Arginine; Cyclic GMP; Diaphragm; Drug Synergism; Electric Stimulation; Enzyme Activators; Exocytosis; Guanylate Cyclase; Indazoles; Intracellular Fluid; Motor Endplate; Neuromuscular Junction; Neurotransmitter Agents; Nitric Oxide; Nitric Oxide Donors; Oxadiazoles; Phrenic Nerve; Purinergic P1 Receptor Antagonists; Quinoxalines; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Sodium Nitrite; Xanthines

An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC-1, a known activator of soluble guanylyl cyclase, against LPS-induced inflammatory responses in microglia.. To understand the inhibitory effects of YC-1 on LPS-induced neuroinflammation, primary cultures of rat microglia and the microglia cell line BV-2 were used. To examine the mechanism of action of YC-1, LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, iNOS, COX-2 and cytokine expression were analyzed by Griess reaction, ELISA, Western blotting and RT-PCR, respectively. The effect of YC-1 on LPS-induced activation of nuclear factor kappa B (NF-kappaB) was studied by NF-kappaB reporter assay and immunofluorocytochemistry.. YC-1 inhibited LPS-induced production of NO and PGE2 in a concentration-dependent manner. The protein and mRNA expression of iNOS and COX-2 in response to LPS application were also decreased by YC-1. In addition, YC-1 effectively reduced LPS-induced expression of the mRNA for the proinflammatory cytokines, TNF-alpha and IL-1beta. Furthermore, YC-1 inhibited LPS-induced NF-kappaB activation in microglia.. YC-1 was able to inhibit LPS-induced iNOS and COX-2 expression and NF-kappaB activation, indicating that YC-1 may be developed as an anti-inflammatory neuroprotective agent.

Topics: Animals; Blotting, Western; Cell Line; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Enzyme Activators; Gene Expression; Guanylate Cyclase; Indazoles; Lipopolysaccharides; Luciferases; Microglia; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Oxadiazoles; Proline; Quinoxalines; Rats; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiocarbamates; Thionucleotides

Cerebral microvascular endothelial cells form the anatomical basis of the blood-brain barrier (BBB), and the tight junctions of the BBB are critical for maintaining brain homeostasis and low permeability. Ischemia/reperfusion is known to damage the tight junctions of BBB and lead to permeability changes. Here we investigated the protective role of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), against chemical hypoxia and hypoxia/reoxygenation (H/R)-induced BBB hyperpermeability using adult rat brain endothelial cell culture (ARBEC). YC-1 significantly decreased CoCl2- and H/R-induced hyperpermeability of fluorescein isothiocyanate (FITC)-dextran in cell culture inserts. It was found that the decrease and disorganization of tight junction protein zonular occludens-1 (ZO-1) in response to CoCl2, and H/R was antagonized by YC-1. The protection of YC-1 may result from the inhibition of HIF-1alpha accumulation and production of its downstream target vascular endothelial growth factor (VEGF). VEGF alone significantly increased FITC-dextran permeability and down-regulated mRNA and protein levels of ZO-1 in ARBECs. We further used animal model to examine the effect of YC-1 on BBB permeability after cerebral ischemia/reperfusion. It was found that YC-1 significantly protected the BBB against ischemia/reperfusion-induced injury. Taken together, these results indicate that YC-1 may inhibit HIF-1alpha accumulation and VEGF production, which in turn protect BBB from injury caused by hypoxia.

Topics: Animals; Blood-Brain Barrier; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Ischemia; Male; Membrane Proteins; Permeability; Phosphoproteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Vascular Endothelial Growth Factor A; Zonula Occludens-1 Protein

Thrombin potently regulates human platelets by the G protein-coupled receptors protease-activated receptor (PAR) 1 and PAR4. Platelet activation by thrombin and other agonists is broadly inhibited by prostacyclin and nitric oxide acting through adenylyl and guanylyl cyclases to elevate cAMP and cGMP levels, respectively. Using forskolin and YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] to selectively activate the adenylyl and guanylyl cyclases, respectively, and the membrane-permeable analogs N(6),2'-O-dibutyryladenosine-3'-5'-cAMP (dibutyryl-cAMP) and 8-(4-parachlorophenylthoi)-cGMP (8-pCPT-cGMP), we sought to identify key antiplatelet steps for cyclic nucleotide actions in blocking platelet activation by PAR1 versus PAR4. Platelet aggregation by PAR1 or PAR4 was inhibited with similar EC(50) of 1.2 to 2.1 microM forskolin, 31 to 33 microM YC-1, 57 to 150 microM dibutyryl-cAMP, and 220 to 410 microM 8-pCPT-cGMP. There was a marked left shift in the inhibitory potencies of forskolin and YC-1 for alpha-granule release and glycoprotein IIbIIIa/integrin alphaIIbbeta3 activation (i.e., EC(50) of 1-60 and 40-1300 nM, respectively) that was not observed for dibutyryl-cAMP and 8-pCPT-cGMP (i.e., EC(50) of 200-600 and 40-140 microM, respectively). This inhibition was essentially instantaneous, and measurements of cyclic nucleotide levels and kinase activities support a model of compartmentation involving the cyclic nucleotide effectors and regulators and the key molecular targets for this platelet inhibition. The different sensitivities of PAR1 and PAR4 to inhibition of calcium mobilization and dense granule release identify key antiplatelet steps for cyclic nucleotide actions and are consistent with the signaling models for these receptors. Specifically, PAR4 inhibition depends on the regulation of both calcium mobilization and dense granule release, and PAR1 inhibition depends predominantly on the regulation of dense granule release.

Topics: 1-Methyl-3-isobutylxanthine; Blood Platelets; Bucladesine; Calcium; Cell Adhesion Molecules; Colforsin; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Humans; Indazoles; Kinetics; Microfilament Proteins; Nucleotides, Cyclic; Oligopeptides; Peptide Fragments; Phosphoproteins; Phosphorylation; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptor, PAR-1; Receptors, Thrombin; Signal Transduction; Thionucleotides; Thrombin

Soluble guanylyl cyclase (sGC), the predominant receptor for nitric oxide (NO), exists in 2 active isoforms (alpha(2)beta(1) and alpha(1)beta(1)). In vascular tissue sGCalpha(1)beta(1) is believed to be the most important. The aim of our study was to investigate the functional importance of the sGCalpha(1)-subunit in vasorelaxation.. Aortic and femoral artery segments from male and/or female sGCalpha(1)(-/-) mice and wild-type littermates were mounted in a small-vessel myograph for isometric tension recording. This was supplemented with biochemical measurements of the cGMP concentration and sGC enzyme activity.. The functional importance of sGCalpha(1)beta(1) was demonstrated by the significantly decreased relaxing effects of acetylcholine (ACh), sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), NO gas, YC-1, BAY 41-2272 and T-1032 in the sGCalpha(1)(-/-) mice of both genders. Moreover, the basal and SNP-stimulated cGMP levels and basal sGC activity were significantly lower in the sGCalpha(1)(-/-) mice. However, the relaxing effects of NO, BAY 41-2272 and YC-1 seen in blood vessels from sGCalpha(1)(-/-) mice indicate a role for an sGCalpha(1)beta(1)-independent mechanism. The increase in sGC activity after addition of BAY 41-2272 and the inhibition of the ACh-, SNP-, SNAP- and NO gas-induced response by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the sGCalpha(1)(-/-) mice are observations suggesting that the sGCalpha(2)beta(1) isoform is also functionally active. However, the insignificant increase in cGMP in response to SNP and the non-upregulated sGCalpha(2) expression level in the sGCalpha(1)(-/-) mice suggest rather the involvement of (an) sGC-independent mechanism(s).. We conclude that sGCalpha(1)beta(1) is involved in the vasorelaxation induced by NO-dependent and NO-independent sGC activators in both genders. However, the remaining relaxation seen in the sGCalpha(1)(-/-) mice suggests that besides sGCalpha(1)beta(1) also the minor isoform sGCalpha(2)beta(1) and/or (an) sGC-independent mechanism(s) play(s) a substantial role.

Topics: Acetylcholine; Animals; Aorta; Cyclic GMP; Female; Guanylate Cyclase; In Vitro Techniques; Indazoles; Isoenzymes; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxadiazoles; Pyrazoles; Pyridines; Quinoxalines; Receptors, Cytoplasmic and Nuclear; S-Nitroso-N-Acetylpenicillamine; Soluble Guanylyl Cyclase; Vasodilator Agents

The radial artery (RA) is used as a spastic coronary bypass graft. This study was designed to investigate the mechanism of vasorelaxant effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO (diethylamine/nitric oxide), a NO-nucleophile adduct, on the human RA. RA segments (n = 25) were obtained from coronary artery bypass grafting patients and were divided into 3-4 mm vascular rings. Using the isolated tissue bath technique, the endothelium-independent vasodilatation function was tested in vitro by the addition of cumulative concentrations of YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) following vasocontraction by phenylephrine in the presence or absence of 10-5 mol/L ODQ (1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one), the selective sGC inhibitor, 10-7 mol/L iberiotoxin, a blocker of Ca2+-activated K+ channels, or 10-5 mol/L ODQ plus 10-7 mol/L iberiotoxin. We also evaluated the effect of YC-1 and DEA/NO on the cGMP levels in vascular rings obtained from human radial artery (n = 6 for each drug). YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) caused the concentration-dependent vasorelaxation in RA rings precontracted with phenylephrine (10-5 mol/L) (n = 20 for each drug). Pre-incubation of RA rings with ODQ, iberiotoxin, or ODQ plus iberiotoxin significantly inhibited the vasorelaxant effect of YC-1, but the inhibitor effect of ODQ plus iberiotoxin was significantly more than that of ODQ and iberiotoxin alone (p < 0.05). The vasorelaxant effect of DEA/NO almost completely abolished in the presence of ODQ and iberiotoxin plus ODQ, but did not significantly change in the presence of iberiotoxin alone (p > 0.05). The pEC50 value of DEA/NO was significantly lower than those for YC-1 (p < 0.01), with no change Emax values in RA rings. In addition, YC-1-stimulated RA rings showed more elevation in cGMP than that of DEA/NO (p < 0.05). These findings indicate that YC-1 is a more potent relaxant than DEA/NO in the human RA. The relaxant effects of YC-1 could be due to the stimulation of the sGC and Ca2+-sensitive K+channels, whereas the relaxant effects of DEA/NO could be completely due to the stimulation of the sGC. YC-1 and DEA/NO may be effective as vasodilator for the short-term treatment of perioperative spasm of coronary bypass grafts.

Topics: Aged; Analysis of Variance; Coronary Artery Bypass; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Enzyme Inhibitors; Female; Humans; Hydrazines; In Vitro Techniques; Indazoles; Male; Middle Aged; Nitric Oxide Donors; Oxadiazoles; Peptides; Phenylephrine; Potassium Channels, Calcium-Activated; Quinoxalines; Radial Artery; Vasoconstrictor Agents; Vasodilation

This study evaluated how YC-1, a guanylate cyclase activator, affects the maturation of human monocyte-derived dendritic cells. Maturation markers and intracellular signaling pathways were evaluated. YC-1 inhibited the lipopolysaccharide up-regulation of mature markers, including CD40, CD80 or CD86 in a concentration-dependent manner with IC(50) values of 4.6+/-0.4, 4.9+/-0.6 or 4.5+/-0.5 microM, respectively. YC-1, at a higher concentration, inhibited lipopolysaccharide-induced HLADR expression. These effects of YC-1 were not reversed by ODQ (10 microM), which is a soluble guanylate cyclase inhibitor, nor by KT5823 (1 microM), which is a PKG inhibitor. Additionally, YC-1 did not increase levels of cyclic nucleotides in dendritic cells, supporting the claim that YC-1 affects dendritic cells maturation in a cGMP-independent manner. YC-1, in a cGMP-independent manner, inhibited lipopolysaccharide-induced Akt activation, IkappaBalpha degradation and NF-kappaB translocation, all of which are associated with co-stimulatory molecules expression. YC-1 inhibited the capacity of dendritic cell to activate allogenic T cells with an IC(50) value of 1.2+/-0.3 microM. YC-1-treated dendritic cells have mature phenotypes that exhibit up-regulated CCR7, enhanced IL-10 release and low phagocytosis activity in the presence of lipopolysaccharide. In conclusion, YC-1 inhibited the lipopolysaccharide-induced co-stimulatory molecular expression of dendritic cells by inhibiting Akt activation, IkappaBalpha degradation and NF-kappaB translocation. These inhibitory effects on co-stimulatory molecules suppressed the capacity of dendritic cells to activate allogenic T cells. Additionally, YC-1 treated dendritic cells exhibit the up-regulation of CCR7, enhanced IL-10 release and the down-regulation of phagocytosis in the presence of lipopolysaccharide. Accordingly, YC-1 might be a useful tool for evaluation of dendritic cells on autoimmune or allergic disease.

Topics: Cell Differentiation; Cell Proliferation; Cyclic AMP; Cyclic GMP; Dendritic Cells; Guanylate Cyclase; Humans; Immunologic Factors; Indazoles; Interleukin-10; Mitogen-Activated Protein Kinases; Monocytes; NF-kappa B; Phosphorylation; Receptors, CCR7; Receptors, Chemokine; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; T-Lymphocytes

Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, and postrevascularization production of vascular smooth muscle cells may play key roles in development of arterial restenosis. We investigated the inhibitory effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a benzyl indazole compound, on MMP-2 and MMP-9 activity in a balloon-injury rat carotid artery model. Injury was induced by inserting a balloon catheter through the common carotid artery; after 14 days, histopathological analysis using immunostaining and Western blotting revealed significant restenosis with neointimal formation that was associated with enhanced protein expression of MMP-2 and MMP-9. However, these effects were dose-dependently reduced by orally administered YC-1 (1-10 mg/kg). In addition, gelatin zymography demonstrated that increased MMP-2 and MMP-9 activity was diminished by YC-1 treatment. On the other hand, YC-1 inhibited hydrolysis of the fluorogenic quenching substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH(2) by recombinant MMP-2 and MMP-9 with IC(50) values = 2.07 and 8.20 muM, respectively. Reverse transcription-polymerase chain reaction analysis of MMP-2 and MMP-9 mRNA revealed that YC-1 significantly inhibited mRNA levels of MMPs. Finally, for the YC-1 treatment group, we did not observe elevation of cGMP levels using enzyme-linked immunosorbent assay, suggesting that YC-1 inhibition of neointimal formation is not through a cGMP-elevating pathway. These data show YC-1 suppression of neointimal formation is dependent on its influence on MMP-2 and MMP-9 protein, mRNA expression, and activity, but not through a cGMP-elevating effect. YC-1 shows therapeutic potential for treatment of restenosis after angioplasty.

Topics: Angiogenesis Inhibitors; Animals; Carotid Arteries; Catheterization; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Immunohistochemistry; Indazoles; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neovascularization, Pathologic; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Nitric oxide (NO) is a major inhibitor in various parts of the gastrointestinal tract. This study was designed to compare the effects of YC-1, NO-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, NO-nucleophile adduct, on sheep sphincters of Oddi (SO).. SO rings were mounted in a tissue bath and tested for changes in isometric tension in response to 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1, 10(-10)-10(-5)M), diethylamine/NO complex (DEA/NO, 10(-8)-10(-4)M). We also evaluated the effect of YC-1 (10(-6) and 10(-5)M) and DEA/NO (10(-5) and 10(-4)M) on the levels cyclic GMP (cGMP) in isolated SO.. YC-1 (10(-10)-10(-5) M) and DEA/NO (10(-8)-10(-4)M) induced concentration-dependent relaxation of isolated SO rings precontracted with carbachol (10(-6)M). The pEC(50) value of DEA/NO was significantly lower than those for YC-1 (p < 0.05), with no change of E(max) values. YC-1 increased cGMP levels more than control, carbachol and DEA/NO groups (p < 0.05).. These results show that YC-1 is a more potent relaxant than DEA/NO and causes more elevation of cGMP levels in isolated SO rings.

Topics: Animals; Cyclic GMP; Hydrazines; In Vitro Techniques; Indazoles; Male; Muscle Relaxation; Neuromuscular Blocking Agents; Nitrogen Oxides; Sheep; Sphincter of Oddi

The nitric oxide (NO)/cGMP pathway plays a key role in the regulation of pulmonary vascular tone during the transition from the fetal to the neonatal circulation, and it is impaired in pathophysiological conditions such as pulmonary hypertension. In the present study, we have analyzed the changes in the function and expression of soluble guanylyl cyclase (sGC) in pulmonary arteries during early postnatal maturation in isolated third-branch pulmonary arteries from newborn (3-18 h of age) and 2-wk-old piglets. The expression of sGC beta(1)-subunit in pulmonary arteries increased with postnatal age both at the level of mRNA and protein. The catalytic region of porcine sGC beta(1) was sequenced, showing a 92% homology with the human sequence. This age-dependent increase in sGC expression correlated with increased vasorelaxant responses to the physiological sGC activator NO and to the exogenous sGC activator YC-1, but not to the membrane-permeable cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate. In conclusion, an increased expression of sGC in pulmonary conduit arteries from 2-wk-old compared with newborn piglets explains, at least partly, the age-dependent increase in the vasorelaxant response of NO and other activators of sGC.

Topics: Aging; Animals; Animals, Newborn; Base Sequence; Cyclic GMP; Enzyme Activators; Guanylate Cyclase; Indazoles; Molecular Sequence Data; Nitric Oxide; Pulmonary Artery; RNA, Messenger; Sequence Homology, Nucleic Acid; Solubility; Swine; Vasodilation

This study delineates the antiproliferative activities and in vivo efficacy of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] in human hepatocellular carcinoma cells. YC-1 inhibited the growth of HA22T and Hep3B cells in a concentration-dependent manner without significant cytotoxicity. YC-1 induced G(1) phase arrest in the cell cycle, as detected by an increase in the proportion of cells in the G(1) phase using FAC-Scan flow cytometric analysis. It was further shown that cGMP, p42/p44 mitogen-activated protein kinase, or AKT kinase-mediated signaling pathways did not contribute to the YC-1-induced effect. Of note, YC-1 induced a dramatic increase in the expression of cyclin-dependent kinase (CDK)-inhibitory protein, p21(CIP1/WAP1), and a modest increase in p27(KIP1). The association of p21(CIP1/WAP1) with CDK2 was markedly increased in cells responsive to YC-1. YC-1 did not modify the expression of cyclin D1, cyclin E, CDK2, or CDK4. In a corollary in vivo study, YC-1 induced dose-dependent inhibition of tumor growth in mice inoculated with HA22T cells. Immunohistochemical analysis revealed an inverse relationship between the staining of p21(CIP1/WAF) and the staining of Ki-67, a cell proliferation marker. Based on the results reported herein, we suggest that YC-1 induces cell cycle arrest and inhibits tumor growth both in vitro and in vivo via the up-regulation of p21(CIP1/WAP1) expression in HA22T cells. Because of this, YC-1 is a potential antitumor agent worthy of further investigation.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Proliferation; Cyclic GMP; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; G1 Phase; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; Transcription Factors; Tumor Suppressor Proteins

Classically, nitric oxide (NO) formed by endothelial NO synthase (eNOS) freely diffuses from its generation site to smooth muscle cells where it activates soluble guanylyl cyclase (sGC), producing cGMP. Subsequently, cGMP activates both cGMP- and cAMP-dependent protein kinases [cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively], leading to smooth muscle relaxation. In endothelial cells, eNOS has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine (ACh)-induced relaxation due to alteration in caveolar structure. Given the nature of NO to be more soluble in a hydrophobic environment than in water, and assuming that colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation, we hypothesize that sGC, PKA, and PKG activation may occur at the plasma membrane caveolae. In endothelium-intact rat aortic rings, the relaxation induced by ACh, by the sGC activator 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), and by 8-bromo-cGMP was impaired in the presence of methyl-beta-cyclodextrin, a drug that disassembles caveolae by sequestering cholesterol from the membrane. sGC, PKG, and PKA were colocalized with caveolin-1 in aortic endothelium, and this colocalization was abolished by methyl-beta-cyclodextrin. Methyl-beta-cyclodextrin efficiently disassembled caveolae in endothelium. In summary, our results provide evidence of compartmentalization of sGC, PKG, and PKA in endothelial caveolae contributing to NO signaling cascade, giving new insights by which the endothelium mediates vascular smooth muscle relaxation.

Topics: Acetylcholine; Animals; beta-Cyclodextrins; Caveolae; Caveolin 1; Caveolins; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium, Vascular; Enzyme Activation; Guanylate Cyclase; Immunohistochemistry; In Vitro Techniques; Indazoles; Isometric Contraction; Male; Microscopy, Electron; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Signal Transduction; Vasodilator Agents

To evaluate the anti-sepsis potential of YC-1, we have examined the effect of YC-1 on the regulation of cytokine production in human leukocytes and endotoxemic mice. The data demonstrated that YC-1 showed a preferential inhibition on proinflammatory cytokine production without inhibition of cell growth or induction of cytotoxicity in human leukocytes. On the other hand, in the septic mouse model, treatment with an intraperitoneal application of LPS caused a cumulative death within 27 hours. The post-treatment administration of YC-1 significantly increased the survival rate in endotoxemic mice. Furthermore, several mediators were detected and the data showed that YC-1 profoundly blocked LPS-induced NO as well as TNF-alpha production, and prevented lung damage by histological examination. Samples from the animal model showed that LPS-induced NF-kappaB/DNA binding activity and consequent up-regulation of iNOS expression in tissues were abolished by post-administration of YC-1. Furthermore, YC-1, by itself, did not modify cGMP content while significantly inhibit LPS-induced cGMP formation, suggesting that YC-1-mediated effect was not through a cGMP-elevating pathway. Taken together, it is evident that the post-treatment administration of YC-1 after LPS application significantly inhibits NF-kappaB activation, iNOS expression, NO over-production, and cytokine release reaction resulting in an improved survival rate in endotoxemic mice. It is suggested that YC-1 may be a potential agent for the therapeutic treatment of sepsis.

Topics: Animals; Blotting, Western; Cyclic GMP; Cytokines; Disease Models, Animal; DNA; Endotoxemia; Humans; Immunohistochemistry; Indazoles; Leukocytes; Leukocytes, Mononuclear; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred ICR; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Shock, Septic; Time Factors; Up-Regulation

Grasshopper sound production, in the context of mate finding, courtship, and rivalry, is controlled by the central body complex in the protocerebrum. Stimulation of muscarinic acetylcholine receptors in the central complex has been demonstrated to stimulate specific singing in various grasshoppers including the species Chorthippus biguttulus. Sound production elicited by stimulation of muscarinic acetylcholine receptors in the central complex is inhibited by co-applications of various drugs activating the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway. The nitric oxide-donor sodium nitroprusside caused a reversible suppression of muscarine-stimulated sound production that could be blocked by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one (ODQ), which prevents the formation of cGMP by specifically inhibiting soluble guanylyl cyclase. Furthermore, injections of both the membrane-permeable cGMP analog 8-Br-cGMP and the specific inhibitor of the cGMP-degrading phosphodiesterase Zaprinast reversibly inhibited singing. To identify putative sources of nitric oxide, brains of Ch. biguttulus were subjected to both nitric oxide synthase immunocytochemistry and NADPH-diaphorase staining. Among other areas known to express nitric oxide synthase, both procedures consistently labeled peripheral layers in the upper division of the central body complex, suggesting that neurons supplying this neuropil contain nitric oxide synthase and may generate nitric oxide upon activation. Exposure of dissected brains to nitric oxide and 3-(5'hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) induced cGMP-associated immunoreactivity in both the upper and lower division. Therefore, both the morphological and pharmacological data presented in this study strongly suggest a contribution of the nitric oxide/cGMP signaling pathway to the central control of grasshopper sound production.

Topics: Animals; Behavior, Animal; Brain; Cyclic GMP; Drug Interactions; Enzyme Activators; Enzyme Inhibitors; Functional Laterality; Grasshoppers; Immunohistochemistry; Indazoles; Male; Muscarine; Muscarinic Agonists; Muscle, Smooth, Vascular; Nitric Oxide; Nitrophenols; Organophosphorus Compounds; Signal Transduction; Time Factors; Vocalization, Animal

This study was designed to investigate the effect of 1-benzyl-3-(5'-hydroxymethyl-2'-furyl) indazole (YC-1), a guanylate cyclase activator, upon the proliferation of rat mesangial cells and its underlying mechanism. YC-1 inhibited cell proliferation and DNA synthesis in a dose- and time-dependent manner. Flow cytometry cell-cycle studies revealed that YC-1 prevented the entry of cells from G1 into S phase. The expression of cyclin D1 and the kinase activity of cyclin D1/cyclin-dependent kinase (CDK)4 were lower within YC-1-treated cells, revealed by Western blotting, Northern blotting and kinase assays. YC-1 did not increase the intracellular cGMP concentration in mesangial cells. Inhibitors of soluble guanylate cyclase, protein kinase G, or protein kinase A also did not reverse the inhibitory effect elicited by YC-1, while co-treatment with p38 mitogen-activated protein kinase (MAPK) inhibitor could partially reverse the suppressive effect. YC-1 inhibited proliferation of mesangial cells and induced cell-cycle arrest by the reduction of cyclin D1 synthesis and cyclin D1/CDK4 kinase activity. This effect acts partially through p38 MAPK signal transduction activation and is independent of cGMP-signaling pathways.

Topics: Animals; Blotting, Northern; Blotting, Western; Bromodeoxyuridine; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Enzyme Inhibitors; G1 Phase; Gene Expression; Glomerular Mesangium; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Guanylate Cyclase; Imidazoles; Indazoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Protein Kinase C; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyridines; Rats; RNA, Messenger; Signal Transduction; Solubility; Tumor Suppressor Proteins

This study was designed to compare the effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase activator, and diethylenetriamine-NONOate (DETA/NO), a NO donor, on spontaneous contractions and the levels of cyclic GMP (cGMP) of myometrial strips isolated from timed-pregnant rats. Myometrial strips were obtained from timed-pregnant Wistar albino rats (n=10) and were mounted in organ baths and tested for changes in isometric tension in response to YC-1 and DETA/NO. We also evaluated the effect of YC-1 and DETA/NO on the levels of cGMP in myometrial strips obtained from timed-pregnant rat uterine horns (n=20). YC-1 (10(-9)-3x10(-5) M) and DETA/NO (10(-7)-10(-4) M) concentration-dependently decreased the amplitude and frequency of spontaneous contractions of myometrial strips isolated from term-pregnant rats. The inhibitions of the amplitude and frequency of spontaneous contractions by YC-1 and DETA/NO were antagonized with methylene-blue (10(-5) M). Antagonistic effect of methylene-blue (10(-5) M) was more on DETA/NO responses than that of YC-1 (P<0.05). In addition, YC-1-stimulated myometrial strips showed more elevation in myometrial cGMP than that of DETA/NO (P<0.05). We demonstrated that YC-1 and DETA/NO induce relaxations in the amplitude and frequency of spontaneous contractions of myometrial strips with different potencies. We also found that YC-1 and DETA/NO-induced relaxations are associated with significant increases in cGMP. These results might suggest that the relaxant effects of YC-1 and DETA/NO on the rat myometrium could be due to the stimulation of the soluble guanylate cyclase and cGMP may play a role for the maintenance of uterine quiescence during pregnancy.

Topics: Animals; Cyclic GMP; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Indazoles; Male; Methylene Blue; Muscle Relaxation; Myometrium; Nitric Oxide Donors; Pregnancy; Rats; Rats, Wistar; Time Factors; Triazenes; Uterine Contraction

N-Formyl-Met-Leu-Phe (fMLP) activated neutrophils and then induced neutrophil-platelet complex formation in co-incubation condition. In addition, fMLP induce intracellular calcium mobilization in platelets, only when it is incubated along with neutrophils. This data established that fMLP-stimulated neutrophils activate platelets. 9E1, a monoclonal antibody of P-selectin, significantly blocks the formation of neutrophil-platelet complex induced by fMLP, indicating the involvement of P-selectin in the neutrophil-platelet complex formation. 3-(5'-hydroxymethyl-2'-furyl-1-benzylindazole (YC-1), an unique nitric oxide-independent activator of soluble guanylate cyclase, was evaluated for its effect on neutrophil-platelet complex. YC-1 inhibits fMLP-induced neutrophil-platelet complex formation in a concentration-dependent manner with an IC50 value of 15.3+/-3.5 microM. However, this effect of YC-1 is partially reversed by pre-treatment of 1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ; 10 microM), which is a soluble guanylate cyclase inhibitor. Pre-treatment of either neutrophils or platelets with YC-1 (50 microM) prevent the fMLP-induced neutrophil-platelet complex formation, indicating that YC-1 could potentially exert its effects individually on either neutrophils or platelets alone. Cathepsin G released from fMLP-stimulated neutrophil activates the nearby platelets. YC-1 was also shown to inhibit this release of cathepsin G in a concentration-dependent manner. The IC50 value was 6.2+/-0.2 microM. This inhibitory effect of YC-1 on cathepsin G release is reversed by ODQ (10 microM) and a protein kinase G inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carbooxylic acid methyl ester (KT5835); 1 microM]. YC-1 inhibits cathepsin G-induced P-selectin expression on human platelet at the IC50 value of 32.5+/-2.6 microM. A further study showed that YC-1 inhibits fMLP-induced neutrophil-platelet complex formation in whole blood at the IC50 value of 35.8+/-8.1 microM in a concentration-dependent manner. According to these data, it was hypothesized that fMLP stimulates neutrophils to release cathepsin G, which subsequently activates the nearby platelets, creating neutrophil-platelet complexes. YC-1 inhibits fMLP-induced neutrophil from releasing cathepsin G via a cGMP-dependent pathway. This inhibitory effect of YC-1 on cathepsin G release is a major mechanism for affecting fMLP-induced neutrophil-platelet complex. YC-

Topics: Adult; Antibodies; Blood Platelets; Calcium; Cathepsin G; Cathepsins; Cell Adhesion; Cell Communication; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Flow Cytometry; Guanylate Cyclase; Humans; Indazoles; Intracellular Space; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxadiazoles; P-Selectin; Quinoxalines; Serine Endopeptidases; Time Factors

We established previously that nitric oxide (NO) in the hippocampal formation (HF) participates actively in negative feedback regulation of penile erection. This study further evaluated whether this process engaged soluble guanylyl cyclase (sGC)/cGMP cascade or peroxynitrite in the HF. Intracavernous pressure (ICP) recorded from the penis in adult, male Sprague-Dawley rats anesthetized with chloral hydrate was employed as our experimental index for penile erection. Microinjection bilaterally of a NO-independent sGC activator, YC-1 (0.1 or 1 nmol) or a cGMP analog, 8-Bromo-cGMP (0.1 or 1 nmol), into the HF elicited a significant reduction in baseline ICP. Bilateral application into the HF of equimolar doses (0.5 or 1 nmol) of a sGC inhibitor, LY83583 or a NO-sensitive sGC inhibitor, ODQ significantly antagonized the decrease in baseline ICP induced by co-administration of the NO precursor, L-arginine (5 nmol), along with significant enhancement of the magnitude of papaverine-induced elevation in ICP. In contrast, a peroxynitrite scavenger, L-cysteine (50 or 100 pmol), or an active peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis-(N-methyl-4'-pyridyl)-porphyrinato iron (III) (10 or 50 pmol), was ineffective in both events. These results suggest that NO may participate in negative feedback regulation of penile erection by activating the sGC/cGMP cascade in the HF selectively.

Topics: Aminoquinolines; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activators; Enzyme Inhibitors; Feedback; Hippocampus; Indazoles; Male; Microinjections; Nitric Oxide; Penile Erection; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Venous Pressure

We have evaluated the hypothesis that cGMP may serve as an intracellular messenger regulating the viability of pancreatic beta-cells. A direct activator of soluble guanylyl cyclase, YC-1, caused a time- and dose-dependent loss of viability in clonal BRIN-BD11 beta-cells. This was accompanied by a rise in cGMP and was antagonised by Rp-8-pCPT-cGMPS, a selective inhibitor of protein kinase G (PKG). Reverse transcription polymerase chain reaction analysis confirmed that BRIN-BD11 cells (and human islets) express all three known isoforms of PKG (PKG-Ialpha, -Ibeta and II). Cell death induced by YC-1 was not sensitive to cell-permeable caspase inhibitors and was not accompanied by oligonucleosomal DNA fragmentation. The response was, however, inhibited by actinomycin D, suggesting that a transcription-dependent pathway of programmed cell death is involved in the actions of cGMP.

Topics: Animals; Apoptosis; Cell Line; Cell Survival; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinase Type II; Cyclic GMP-Dependent Protein Kinases; Dactinomycin; Guanylate Cyclase; Indazoles; Insulin; Insulin Secretion; Islets of Langerhans; Protein Isoforms; Rats; Signal Transduction; Thionucleotides; Transcription, Genetic

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.

Topics: Amino Acid Sequence; Animals; Asparagine; Binding Sites; COS Cells; Cyclic GMP; Cysteine; Cytosol; DNA Mutational Analysis; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Guanylate Cyclase; Indazoles; Kinetics; Methionine; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitric Oxide; Phenotype; Protein Subunits; Pyrazoles; Pyridines; Rats; Solubility; Tyrosine

By the formation of cGMP, nitric oxide (NO)-sensitive guanylyl cyclase (GC) acts as the effector for the signaling molecule NO and mediates the relaxation of vascular smooth muscle and the inhibition of platelet aggregation. The compounds YC-1 and BAY 41-2272 are regarded as NO-independent activators and sensitizers of NO-sensitive GC. In vivo effects, for example, lowering blood pressure and prolonging tail-bleeding times, turn the compounds into promising candidates for the therapy of cardiovascular diseases. However, YC-1 has also been shown to inhibit the major cGMP-degrading enzyme phosphodiesterase type 5 (PDE5). The synergistic properties of YC-1 on cGMP formation and degradation lead to an excessive NO-induced cGMP accumulation in cells, explaining the observed physiological effects. We assessed a potential inhibition of PDE5 by the new GC activator BAY 41-2272.. The effects of BAY 41-2272 on NO-sensitive GC and PDE5 activities were tested in vitro. BAY 41-2272 not only sensitized NO-sensitive GC toward activation by NO but also, with comparable potency, inhibited cGMP degradation by PDE5. In intact platelets, BAY 41-2272 greatly potentiated the NO-induced cGMP response that was caused by a synergistic effect of BAY 41-2272 on cGMP formation and degradation.. The physiological effects of BAY 41-2272, which are commonly ascribed to the NO-independent activation of NO-sensitive GC, are rather due to the synergism of sensitization of NO-sensitive GC and inhibition of PDE5.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cell Line; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Activation; Guanylate Cyclase; Humans; Indazoles; Kidney; Nitric Oxide; Nitric Oxide Donors; Phosphodiesterase Inhibitors; Pyrazoles; Pyridines; Quaternary Ammonium Compounds; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; S-Nitrosoglutathione; Soluble Guanylyl Cyclase; Transfection; Vasodilation

No studies have specifically addressed whether cAMP can influence nitric oxide (NO)/cGMP-induced cerebral vasodilation. In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase 5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs). To that end, we evaluated, in male rats, the effects of knockdown [via antisense oligodeoxynucleotide (ODN) applications] of the cGMP efflux protein multidrug resistance protein 5 (MRP5) and PDE5 inhibition on pial arteriolar NO donor [S-nitroso-N-acetyl penicillamine (SNAP)]-induced dilations in the absence and presence of cAMP elevations via forskolin. Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats. In control (missense ODN treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (approximately 50% reduction in SNAP EC50). However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response). In the presence of the PDE5 inhibitor MY-5445, the repression of the NO donor response accompanying forskolin was prevented. These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases. On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux. On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown. However, because increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP stimulation of cGMP hydrolysis.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Arterioles; Cerebrovascular Circulation; Colforsin; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Activators; Hydrolysis; Indazoles; Male; Multidrug Resistance-Associated Proteins; Nitric Oxide; Nitric Oxide Donors; Phosphoric Diester Hydrolases; Rats; Rats, Sprague-Dawley; Vasodilation

It is well known that the muscarinic cholinergic agonists, carbachol and methacholine, enhance nitric oxide synthase (NOS) activity, and also stimulate salivary secretion. In the present study, we investigated whether salivary secretion by muscarinic cholinergic stimulation is mediated through the NO/cGMP signaling pathway in rat salivary glands. Since NO activates soluble guanylyl cyclase (sGC) and cGMP may function as a mediator, the localisation of sGC was investigated in the salivary glands. sGC was localized in both the acinar and duct cells of the rat parotid and sublingual glands, and localized only in the acinar cells of the submandibular glands. S-Nitroso-glutathione (NO generator; GSNO) and YC-1 (NO-independent sGC activator) stimulated sGC in the cytosol to synthesise cGMP. The combination of GSNO and YC-1 stimulated sGC synergistically. Carbachol, GSNO and YC-1 enhanced amylase release from the rat parotid glands. Amylase release stimulated by carbachol and GSNO was inhibited by addition of the sGC inhibitor, ODQ, and cGMP-dependent protein kinase inhibitor, KT-5823. These results indicate that amylase release may be mediated through the NO/cGMP signaling pathway.

Topics: Amylases; Animals; Carbachol; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Guanylate Cyclase; Indazoles; Male; Muscarinic Agonists; Nitric Oxide; Nitric Oxide Donors; Parotid Gland; Rats; Rats, Wistar; S-Nitrosoglutathione; Signal Transduction

We demonstrated previously that 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), an activator of soluble guanylate cyclase (sGC), induces cyclooxygenase-2 (COX-2) expression via cGMP- and p44/42 mitogen-activated protein kinase-dependent pathways in human pulmonary epithelial A549 cells. In this study, we explore the role of Ras, phosphoinositide-3-OH-kinase (PI3K), Akt, and transcription factor nuclear factor-kappaB (NF-kappaB) in YC-1-induced COX-2 expression in A549 cells. A Ras inhibitor (manumycin A), a PI3K inhibitor (wortmannin), an Akt inhibitor (1l-6-Hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate]), and an NF-kappaB inhibitor [pyrrolidine dithiocarbamate (PDTC)] all reduced YC-1-induced COX-2 expression. The YC-1-induced increase in COX activity was also blocked by manumycin A, wortmannin, PDTC, and the dominant-negative mutants for Ras (RasN17), Akt (Akt DN), and IkappaBalpha (IkappaBalphaM). The YC-1-induced increase in Ras activity was inhibited by an sGC inhibitor [1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ)], a protein kinase G (PKG) inhibitor [1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT-5823)], and manumycin A. YC-1-induced Akt activation was also inhibited by ODQ, KT-5823, manumycin A, and wortmannin. YC-1 caused the formation of an NF-kappaB-specific DNA-protein complex and an increase in kappaB-luciferase activity. YC-1-induced kappaB-luciferase activity was inhibited by ODQ, KT-5823, manumycin A, wortmannin, an Akt inhibitor, PDTC, RasN17, Akt DN, and IkappaBalphaM. Likewise, YC-1-induced IKKalpha/beta activation was inhibited by ODQ, KT-5823, manumycin A, wortmannin, and an Akt inhibitor. Furthermore, YC-1-induced COX-2 promoter activity was inhibited by manumycin A, RasN17, Akt DN, PDTC, and IkappaBalphaM. Taken together, these results indicate that YC-1 might activate the sGC/cGMP/PKG pathway to induce Ras and PI3K/Akt activation, which in turn initiates IKKalpha/beta and NF-kappaB activation and finally induces COX-2 expression in A549 cells.

Topics: Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Humans; I-kappa B Kinase; I-kappa B Proteins; Indazoles; Interleukin-1; Isoenzymes; Lung; Membrane Proteins; Mutation; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphatidylinositol 3-Kinases; Polyenes; Polyunsaturated Alkamides; Proline; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; ras Proteins; Thiocarbamates; Transcription Factors

Soluble guanylyl cyclase (SGC) is the main receptor for the gaseous signalling molecule nitric oxide (NO) in vertebrates and invertebrates. Recently, a novel class of drugs that regulate mammalian SGC by NO-independent allosteric mechanisms has been identified [e.g. 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, YC-1]. To assess the evolutionary conservation and hence the potential physiological relevance of these mechanisms, we have tested YC-1 on the brains of two model insects, the cockroach Periplaneta americana and the locust Schistocerca gregaria. YC-1 strongly potentiated the NO-induced elevation of total cyclic 3',5'-guanosine monophosphate (cGMP) and amplified the intensity and consistency of NO-induced cGMP-immunoreactivity in the brain. Our data indicate that the effect of YC-1 was independent of phosphodiesterase inhibition and thus mediated by direct sensitization of SGC. Immunohistopharmacology and co-labelling with antibodies against the SGC alpha-subunit confirmed that cGMP induced by co-application of NO and YC-1 is predominantly attributable to SGC. The staggering number of NO-responsive neurons revealed by YC-1 suggests that previous studies may have considerably underestimated the number of cellular targets for NO in the insect brain. Moreover, a subset of these targets exhibited cGMP-immunoreactivity without application of exogenous NO, demonstrating that YC-1 can be exploited for visualization of physiological cGMP signals in response to endogenous NO production. In conclusion, our discovery that YC-1 is a potent sensitizer of insect SGC indicates that a NO-independent regulatory site is an evolutionarily conserved feature of SGC. Our findings add considerable momentum to the concept of an as yet unidentified endogenous ligand that regulates the gain of the NO-cGMP signalling pathway.

Topics: Amino Acid Sequence; Animals; Brain; Conserved Sequence; Cyclic GMP; Evolution, Molecular; Female; Grasshoppers; Guanylate Cyclase; Indazoles; Insect Proteins; Male; Molecular Sequence Data; Nitric Oxide; Periplaneta; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Up-Regulation

To demonstrate the expression of soluble guanylyl cyclase (sGC) alpha and beta subunits in rat granulosa cells and determine the effects sGC activation on levels of cyclic GMP (cGMP), E2, and cAMP.. Basic research study.. University research laboratory.. Estrogen-treated immature Sprague-Dawley female rats from which primary cell culture of granulosa cells was obtained.. Functionally immature rat granulosa cells were incubated for 48 hours with media alone, FSH, or FSH plus YC-1, a specific activator of sGC.. Expression of sGC alpha and beta subunits was determined by immunoblot analysis. Media concentrations of E2, cAMP, and cGMP were measured by radioimmunoassays.. Immunoblot analysis of granulosa cells revealed the expression of sGC alpha and beta subunits. While cGMP accumulation was low in cells incubated with media alone or with FSH, cotreatment with FSH plus YC-1 increased cGMP levels approximately five-fold. Incubation of cells with FSH stimulated E2 production in a dose-dependent manner. However, cotreatment of cells with FSH plus YC-1 significantly decreased E2 concentrations at all doses of FSH tested. Similarly, while FSH increased cAMP accumulation from granulosa cells, cotreatment with YC-1 markedly inhibited FSH-stimulated cAMP levels.. These findings demonstrate the expression of sGC subunits in rat granulosa cells and indicate that activation of sGC increases cGMP levels, which are associated with inhibition of FSH-stimulated E2 production and cAMP accumulation.

Topics: Animals; Cells, Cultured; Cyclic AMP; Cyclic GMP; Drug Synergism; Enzyme Activation; Enzyme Activators; Estradiol; Female; Follicle Stimulating Hormone; Granulosa Cells; Guanylate Cyclase; Hormones; Immunoblotting; Indazoles; Isoenzymes; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Solubility

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.

Topics: Acrylamides; Animals; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Enzyme Inhibitors; Guanylate Cyclase; Humans; In Vitro Techniques; Indazoles; Male; Muscle Relaxation; Muscle, Smooth; Muscle, Smooth, Vascular; Penile Erection; Rabbits; Rats; Rats, Wistar; Spodoptera

The aim of present study was to characterize the in vitro and in vivo pharmacological effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a soluble guanylate cyclase activator, on corpus cavernosal smooth muscle and penile erectile activity. YC-1 relaxed phenylephrine precontracted cavernosal smooth muscle (EC(50)=4.4 microM) and this effect was partially antagonized by 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ). ODQ is a selective soluble guanylate cyclase inhibitor that completely blocked the relaxation induced by sodium nitroprusside, suggesting that YC-1 binds to soluble guanylate cyclase at a different site from nitric oxide (NO). Both YC-1 and sodium nitroprusside, but not sildenafil (1-100 microM) caused concentration-dependent increases in cyclic GMP levels in cultured rabbit cavernosal smooth muscle cells and produced synergistic effects. Intraperitoneal administration of YC-1 (10 micromol/kg) evoked penile erection in rats with 70% incidence. More importantly, YC-1 was able to significantly augment the pro-erectile effects of a suboptimal dose of apomorphine. These results suggest that the soluble guanylate cyclase activator YC-1 increases cyclic GMP levels, leading to relaxation of cavernosal smooth muscle. These biochemical events may be related to the pro-erectile properties of YC-1 in vivo.

Topics: Animals; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Indazoles; Male; Muscle Contraction; Muscle, Smooth; Nitric Oxide; Nitroprusside; Oxadiazoles; Penile Erection; Penis; Phenylephrine; Quinoxalines; Rabbits; Rats; Rats, Wistar; Signal Transduction; Vasoconstrictor Agents; Vasodilator Agents

The effect of 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) on nitric oxide (NO) mediated responses and NO generation from NO donors were studied in vitro. In precontracted rat isolated anococcygeus muscles, relaxations induced by NO donors, electrical field stimulation and 5-[1-(phenylmethyl)-1H-indazole-3-yl]-2-furanmethanol (YC-1) were significantly inhibited by ebselen (100 microM), whereas responses elicited by papaverine and theophylline were not affected; those by 8-bromo-cyclic-guanosine-monophosphate (8-Br-cGMP) were slightly enhanced. NO generation from NO gas aqueous solution or acidified nitrite was not affected, but that from S-nitroso-N-acetyl-penicillamine (SNAP) was attenuated by ebselen, and the attenuation was reserved by glutathione. Both glutathione and cupric sulphate altered the ultraviolet spectrum of ebselen. These findings suggest that ebselen at high concentrations nonselectively inhibited NO-mediated responses, possibly through inhibiting soluble guanylate cyclase. Ebselen does not appear to directly interact with NO, but it may inhibit NO release from nitrosothiols by a thiol- and/or copper-dependent mechanism.

Topics: Animals; Aorta; Azoles; Copper; Cyclic GMP; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Electric Stimulation; Endothelium, Vascular; Enzyme Activators; Enzyme Inhibitors; Glutathione; In Vitro Techniques; Indazoles; Isoindoles; Male; Muscle Relaxation; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Organoselenium Compounds; Papaverine; Penicillamine; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Spectrophotometry, Ultraviolet; Vasodilation

Using cultured rat alveolar NR 8383 macrophages, this study investigated the effect of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole], a soluble guanylyl cyclase (sGC) activator, on the production of tumor necrosis factor-alpha (TNF alpha). YC-1 enhanced lipopolysaccharide and interferon-gamma (LPS/IFN gamma)-induced TNF alpha formation in a concentration- and time-dependent fashion. YC-1 also caused an increasing effect on the TNF alpha mRNA level, suggesting that the transcriptional process was involved. However, further studies suggested that cyclic GMP did not mediate the potentiation of YC-1 on TNF alpha release, because (a) the sGC inhibitor and the protein kinase G inhibitor failed to block the effect; and (b) the cyclic GMP analogues, on the contrary, concentration-dependently diminished LPS/IFN gamma-induced TNF alpha synthesis. In agreement with this finding, YC-1 produced changes in cell function but no changes in cyclic GMP and cyclic AMP levels or sGC activity. Pretreatment of the cells with cyclooxygenase inhibitors, a p38 mitogen-activated protein kinase inhibitor, a mitogen-activated protein kinase kinase (MEK) inhibitor, and a tyrosine kinase inhibitor did not attenuate the potentiation of TNF alpha release by YC-1. Cycloheximide prevented the YC-1-enhanced TNF alpha formation, implying that new protein synthesis was required. Interestingly, protein kinase C inhibitors enhanced the potentiation of YC-1 to a greater extent. Nevertheless, a protein kinase C activator, phorbol 12-myristate 13-acetate, failed to suppress the potentiation of TNFalpha production by YC-1. In summary, potentiation of TNF alpha release by YC-1 in LPS/IFN gamma-activated alveolar macrophages is an additional mode of action of this compound that is independent of the elevation of cyclic GMP. Thus, caution needs to be used in attributing the YC-1-mediated response to the activation of sGC.

Topics: Animals; Cells, Cultured; Cyclic AMP; Cyclic GMP; Drug Interactions; Enzyme Inhibitors; Guanylate Cyclase; Indazoles; Macrophages, Alveolar; Rats; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

This study was designed to investigate the effect of YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, in human umbilical vein endothelial cells (HUVECs) proliferation and its underlying mechanism. YC-1 at a range of concentrations (5-50 microM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a dose- and time-dependent manner. YC-1 was not cytotoxic at these concentrations. [3H]thymidine incorporation and flow cytometry analyses revealed that YC-1 treatment decreased DNA synthesis and arrested the cells at the G0/G1 phase of the cell cycle. Western blot analysis demonstrated that YC-1 (5-50 microM) increased the levels of cyclin-dependent kinase (CDK)-inhibitory proteins (CKIs), p21 and p27, but did not induce any significant changes of cyclins and CDKs. In the YC-1-treated HUVEC, the formation of CDK2-p21 complex, but not CDK2-p27 complex, was increased and the assayable CDK2 kinase activity was decreased. These changes were in a dose-dependent manner. In contrast, the formations of CDK4-p21 and CDK4-p27 complex were slightly increased and the assayable CDK4 kinase activity was slightly decreased (if there were any changes). Pretreatment with guanylyl cyclase inhibitors, 1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one (ODQ) and methylene blue, inhibited the YC-1-induced increase of cyclic GMP level, but did not change significantly the magnitude of the YC-1-induced inhibition of thymidine incorporation and cell number in HUVEC. These results indicate that YC-1-induced cell cycle arrest in HUVEC occurred when the cyclin-CDK system was inhibited just as p21 and p27 protein levels were augmented. This YC-1-induced antiproliferation effect in HUVEC is via a cyclic GMP-independent pathway.

Topics: Cell Division; Cyclic GMP; DNA; Endothelium, Vascular; G1 Phase; Humans; Indazoles; Platelet Aggregation Inhibitors; Resting Phase, Cell Cycle; Thymidine; Tritium

Soluble guanylate cyclase (sGC), a heterodimer consisting of alpha- and beta-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the beta-subunit via His(105) is crucial for the activation of the enzyme by NO. In addition to this NO binding capability, the heme status of the enzyme influences the activity of non-NO sGC activators and sGC inhibitors. Different sGC activity profiles were observed in the presence, absence, or the oxidized form of heme. Modulating the heme status is therefore crucial for the investigation of the mechanism of sGC activation. Here, we present a simple and reliable procedure for the removal of the heme moiety of sGC that is capable of eliminating any traces of unbound heme and detergent from the sample mixture in one single step. Samples containing 15 microg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 degrees C for 10 min and loaded onto centrifugal ion exchange columns. After centrifugation, heme was bound entirely to the ion exchanger and could not be eluted, even after incubation with 1M NaCl. Tween 20 was found completely within the flowthrough. Heme-free sGC was eluted from the ion exchanger after application of 300 mM NaCl. The absence of the heme moiety was confirmed by UV/Vis spectra and determination of the enzymatic activity. In summary, the described procedure is suitable for the preparation of very small amounts of highly purified heme-free sGC for the investigation of the mechanism of action of different types of sGC activators.

Topics: Benzoates; Chromatography, Ion Exchange; Cyclic GMP; Enzyme Activation; Enzyme Activators; Guanosine Triphosphate; Guanylate Cyclase; Heme; Hemeproteins; Histidine; Indazoles; Nitroprusside; Polysorbates; Proteins; Protoporphyrins; Spectrophotometry; Zinc

3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a novel type of soluble guanylyl cyclase (sGC) activator, is useful in investigating the signaling of cGMP and may provide a new approach for treating cardiovascular diseases. Herein, YC-1 was demonstrated to inhibit the generation of superoxide anion (O2-) and the release of beta-glucuronidase release, to diminish the membrane-associated p47phox and to accelerate resequestration of cytosolic calcium in formyl-l-methionyl-l-leucyl-l-phenylalanine-activated human neutrophils. YC-1 not only directly promoted sGC activity and cGMP formation but also dramatically potentiated sodium nitroprusside-induced sGC activity and cGMP formation in human neutrophils. However, the synergistic increase in the amount of cGMP was inconsistent with its cellular response. Moreover, neither an sGC inhibitor nor protein kinase G inhibitors reversed the inhibitory effect of YC-1. Interestingly, YC-1 also increased the cAMP concentration and protein kinase (PK)A activity. The inhibitory effect of YC-1 was significantly enhanced by prostaglandin (PG)E1 and isoproterenol, and almost abolished by PKA inhibitors. These results show that cAMP, but not cGMP, mediates the YC-1-induced inhibition of human neutrophils. YC-1 increased the PGE1- and forskolin-induced but not 3-isobutyl-1-methylxanthine-produced cAMP formation, suggesting inhibition of phosphodiesterase. These findings thus reveal novel mechanism-mediated anti-inflammatory properties of YC-1 in human neutrophils, which can influence the progression of cardiovascular disease. cAMP, but not cGMP, plays an important role in the regulation of respiratory burst and degranulation in human neutrophils.

Topics: Adolescent; Adult; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Guanylate Cyclase; Humans; Indazoles; Neutrophils; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Superoxides

Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K-Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0+/-0.4 to 0.9+/-0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

Topics: Animals; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression Regulation; Guanylate Cyclase; Indazoles; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Protein Isoforms; Rats; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Signal Transduction; Soluble Guanylyl Cyclase; Symporters; Thionucleotides

Heme oxygenase (HO) and carbon monoxide (CO) participate in the homeostatic control of cardiovascular functions, including the regulation of blood pressure (BP). Upregulation of the HO/CO system has been shown to lower BP in young (8 weeks) but not in adult (20 weeks) spontaneously hypertensive rats (SHR). The underlying mechanism for this selective effect, however, has been unknown and was investigated in the present study. The administration of hemin resulted in a marked decrease in BP (from 148.6+/-3.2 to 125.8+/-2.6 mm Hg, P<0.01) in young but not in prehypertensive (4 weeks) or adult SHR or Wistar-Kyoto rats at all ages. The inhibition of HO with chromium mesoporphyrin abrogated the BP-lowering effect of hemin. Significantly lower expression levels of HO-1 and soluble gyanylyl cyclase (sGC) as well as reduced cGMP content were detected in 8-week SHR but not in adult SHR or Wistar-Kyoto rats of all ages. These deficiencies were all corrected by hemin treatment. The expression of HO-2 protein was not different among all animal groups tested and not affected by hemin treatment. Desensitization of the sGC/cGMP pathway in adult SHR was demonstrated by the reduced vasorelaxant potency of the sGC activator 3-(5' -hydroxymethyl-2-'furyl)-1-benzylindazole. Thus, in young and prehypertensive SHR, a defective HO/CO-sGC/cGMP system might constitute a pathogenic mechanism for the development of hypertension. The HO/CO-sGC/cGMP system appears normal in adult SHR, but desensitization of the downstream targets of the system to sGC/cGMP may endow SHR at this stage a persistent hypertension status.

Topics: Animals; Blood Pressure; Culture Techniques; Cyclic GMP; Guanylate Cyclase; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemin; Hypertension; Indazoles; Male; Mesenteric Arteries; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Vasodilator Agents

1. Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-N-methyl-N-[6-(N-methylammoniohexyl)amino]diazen-1-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and to investigate the possible role of activation of sarco-endoplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2-10 micro g ml(-1)) and adenosine diphosphate (ADP; 2 micro M) in platelet rich plasma. It was (i). more effective at inhibiting aggregation induced by collagen than by ADP, and (ii). less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3. ODQ (10 micro M) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4. The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole; 1-100 micro M), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1-1 mM), caused minimal inhibition. 5. On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 micro M present) were abolished by thapsigargin (200 nM). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6. Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7. Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

Topics: Adenosine Diphosphate; Animals; Catalase; Collagen; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activators; Enzyme Inhibitors; Guanylate Cyclase; Hydrazines; In Vitro Techniques; Indazoles; Male; Nitric Oxide Donors; Oxadiazoles; Platelet Aggregation; Platelet Aggregation Inhibitors; Pulmonary Artery; Quinoxalines; Rats; Rats, Wistar; S-Nitrosoglutathione; Superoxide Dismutase; Thapsigargin; Thionucleotides; Vasoconstriction

In the rat isolated optic nerve, nitric oxide (NO) activates soluble guanylyl cyclase (sGC), resulting in a selective accumulation of cGMP in the axons. The axons are also selectively vulnerable to NO toxicity. The experiments initially aimed to determine any causative link between these two effects. It was shown, using a NONOate donor, that NO-induced axonal damage occurred independently of cGMP. Unexpectedly, however, the compound YC-1, which is an allosteric activator of sGC, potently inhibited NO-induced axonopathy (IC(50) = 3 microM). This effect was not attributable to increased cGMP accumulation. YC-1 (30 microM) also protected the axons against damage by simulated ischemia, which (like NO toxicity) is sensitive to Na(+) channel inhibition. Although chemically unrelated to any known Na(+) channel inhibitor, YC-1 was effective in two biochemical assays for activity on Na(+) channels in synaptosomes. Electrophysiological recording from hippocampal neurons showed that YC-1 inhibited Na(+) currents in a voltage-dependent manner. At a concentration giving maximal protection of optic nerve axons from NO toxicity (30 microM), YC-1 did not affect normal axon conduction. It is concluded that the powerful axonoprotective action of YC-1 is unrelated to its activity on sGC but is explained by a novel action on voltage-dependent Na(+) channels. The unusual ability of YC-1 to protect axons so effectively without interfering with their normal function suggests that the molecule could serve as a prototype for the development of more selective Na(+) channel inhibitors with potential utility in neurological and neurodegenerative disorders.

Topics: Animals; Axons; Cyclic GMP; Drug Interactions; Enzyme Activators; Glucose; Guanylate Cyclase; Indazoles; Male; Nitric Oxide; Oxygen; Rats; Rats, Wistar; Sodium Channel Blockers; Sodium Channels

L-arginine-nitric oxide (NO) pathway participates in the physiology and in many pathological processes in the eye, such as glaucoma. The aim of the present study was to compare the ocular hypotensive effect of different NO-donors, and to get more information on the role of cyclic guanosine 3',5'-monophosphate (cGMP) in this process. The test compounds were administered topically or intravitreally in the eye of a normotensive rabbit. Intraocular pressure (IOP) was measured with a pneumatonometer after topical anesthesia. The metabolites of NO (nitrite, nitrate, NOx) and cGMP were assayed from the aqueous humor and plasma. NO-synthase (NOS) protein expression was assayed in the ciliary body by Western blotting. The maximal lowering of IOP was achieved as follows: atriopeptin III (concentration 78 (microM, decrease in IOP 50%), atriopeptin II (84 (microM 37%). 8-Br-cGMP (90 mM, 37%), zaprinast + 8-Br-cGMP (1 mM + 90 mM, 34%), L-arginine (1 mM, 29%), SNP (40 mM, 28%), nitrosocaptopril (100 mM, 28%), S-nitrosothiol (SNOG) (10 mM, 27%), YC-1 (10 (microM, 25%), zaprinast + SNP (1 mM + 40 mM, 22%), spermine NONOate (100 mM, 20%) [corrected]. The decrease in IOP lasted for 2-5 hr, except with atriopeptin II and III, when IOP values were first normalized in 6 hr and 2 days, respectively. In conclusion, the results of the present study indicate that by increasing the activity of L-arginine/NO/cGMP-pathway it is possible to lower IOP in rabbits equally to the currently used antiglaucomatous drugs.

Topics: Animals; Arginine; Cyclic GMP; Female; Indazoles; Intraocular Pressure; Male; Nitric Oxide Donors; Nitric Oxide Synthase; Purinones; Rabbits

YC-1, a synthetic benzyl indazole derivative, is capable of stimulating endogenous vessel wall cyclic guanosine monophosphate (cGMP) production and attenuating the remodeling response to experimental arterial angioplasty. In an effort to investigate the mechanisms of this YC-1-mediated vasoprotection, we examined the influence of soluble YC-1 or YC-1 incorporated in a polyethylene glycol (PEG) hydrogel on cultured rat vascular smooth muscle cell (SMC) cGMP synthesis, SMC proliferation, and platelet function. Results demonstrate that soluble YC-1 stimulated SMC cGMP production in a dose-dependent fashion, while both soluble and hydrogel-released YC-1 inhibited vascular SMC proliferation in a dose-dependent fashion without effects on cell viability. Platelet aggregation and adherence to collagen were both significantly inhibited in a dose-dependent fashion by soluble and hydrogel-released YC-1. Arterial neointima formation following experimental balloon injury was significantly attenuated by perivascular hydrogel-released YC-1. These results suggest that YC-1 is a potent, physiologically active agent with major anti-proliferative and anti-platelet properties that may provide protection against vascular injury through cGMP-dependent mechanisms.

Topics: Angioplasty, Balloon; Animals; Arterial Occlusive Diseases; Blood Platelets; Carotid Stenosis; Cell Adhesion; Cell Division; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Hydrogels; Indazoles; Muscle, Smooth, Vascular; Platelet Aggregation Inhibitors; Polyethylene Glycols; Rats; Rats, Sprague-Dawley

Insulin inhibits contraction and migration of primary confluent, cultured canine vascular smooth muscle cells (VSMCs) with inducible nitric oxide synthase (iNOS) by stimulating cyclic GMP (cGMP) production. The present study was performed to determine how insulin stimulates guanylate cyclase activity in these cells.. Primary cultured VSMC were obtained from canine femoral arteries. Lactate and pyruvate levels were measured by enzymatic assays, cGMP production by radioimmunoassay, iNOS activity by conversion of arginine to citrulline, and cell contraction by photomicroscopy.. Insulin (1 nmol/L) increased cGMP production fivefold in VSMC with iNOS while raising the lactate-to-pyruvate ratio (LPR) from 3.1 +/- 0.5 to 10.0 +/- 1.6 (P < .05), indicating a rise in the ratio of reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD+) redox state of the cell. Insulin's stimulation of cGMP production was blocked by 0.1 mmol/L NG-monomethyl-L-arginine (L-NMMA) indicating dependence on iNOS activity, but insulin did not affect iNOS activity. Blocking insulin's increase in LPR by pyruvate (0.5 mmol/L) or oxaloacetate (0.5 mmol/L) completely inhibited the insulin-stimulated component of cGMP production. Pyruvate also blocked insulin's inhibition of serotonin-induced contraction in nonproliferated cells. In the absence of insulin, 5 mmol/L lactate or isocitrate increased the LPR by 420% +/- 47% and 167% +/- 20%, respectively (both P < .05), and stimulated cGMP production by 1,045% +/- 272% and 278% +/- 33%, respectively (both P < .05) by an L-NMMA-inhibitable mechanism. Although cGMP production in cells with iNOS was increased by insulin, the stimulation of cGMP production in cells without iNOS by 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) was not affected by insulin, suggesting that insulin does not stimulate guanylate cyclase activity directly.. We conclude that insulin increases cGMP production in VSMC with iNOS by raising the cell NADH/NAD+ redox state, which may increase the availability of iNOS-derived NO.

Topics: Animals; Cells, Cultured; Cyclic GMP; Dogs; Guanylate Cyclase; Indazoles; Insulin; Isocitrates; Lactic Acid; Muscle, Smooth, Vascular; NAD; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxaloacetic Acid; Oxidation-Reduction; Pyruvic Acid; Stimulation, Chemical

Nitric oxide (NO) activates corpus cavernosum smooth muscle soluble guanylate cyclase (sGC) and increases the synthesis of cGMP that results in smooth muscle relaxation and ultimately, penile erection. To characterize sGC and define the potential synergy between NO and the allosteric activator YC-1 in corpus cavernosum, rat sGC was activated by either sodium nitroprusside (SNP) or YC-1, and YC-1 potentiated the effects of SNP with a 200-fold activation of sGC. Both SNP and YC-1 decreased the Km and increased the Vmax. ODQ significantly inhibited sGC activated by SNP with IC50 of 0.5 nM, but did not affect the sGC activated by YC-1 as well as basal sGC activity. SNP and YC-1 synergistically increased intracellular cGMP levels in rabbit corpus cavernosum smooth muscle cell cultures. YC-1 significantly relaxed rabbit cavernosum tissue strips in organ baths with an EC50 of 8.4 microM. In the presence of L-nitroarginine methyl ester to block endogenous NO production, co-administration of SNP shifted the dose response of YC-1 to the left, showing the synergism of SNP and YC-1 in tissue strips. In view of the clinical efficacy of phosphodiesterase-5 inhibitors, activation of sGC may provide an alternative means for enhancing the activity of neurally derived NO during sexual stimulation in the corpus cavernosum, representing a novel approach for the treatment of erectile dysfunction.

Topics: Animals; Cell Line; Cloning, Molecular; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; Humans; In Vitro Techniques; Indazoles; Isoenzymes; Male; Muscle, Smooth; Nitric Oxide; Penis; Rabbits; Rats; Recombinant Proteins

3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a soluble guanylyl cyclase (sGC) activator, inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O(2)*(-)) generation and O(2) consumption in rat neutrophils (IC(50) values of 12.7+/-3.1 and 17.7+/-6.9 microM, respectively). Inhibition of O(2)*(-) generation by YC-1 was partially reversed by the cyclic GMP-lowering agent 6-anilinoquinoline-5,8-quinone (LY83583) and by the Rp isomer of 8-(4-chlorophenylthio)guanosine-3',5'-monophosphorothioate (Rp-8-pCPT-cGMPS), a cyclic GMP-dependent protein kinase inhibitor. In cell-free systems, YC-1 failed to alter O(2)*(-) generation during dihydroxyfumaric acid autoxidation, phorbol 12-myristate 13-acetate (PMA)-activated neutrophil particulate NADPH oxidase preparation, and arachidonic acid-induced NADPH oxidase activation. YC-1 increased cellular cyclic GMP levels through the activation of sGC and the inhibition of cyclic GMP-hydrolyzing phosphodiesterase activity. The plateau phase, but not the initial spike, of fMLP-induced [Ca(2+)](i) changes was inhibited by YC-1 (IC(50) about 15 microM). fMLP- but not PMA-induced phospholipase D activation was inhibited by YC-1 (IC(50) about 28 microM). Membrane-associated ADP-ribosylation factor and Rho A in cell activation was also reduced by YC-1 at a similar concentration range. Neither cytosolic protein kinase C (PKC) activity nor PKC membrane translocation was altered by YC-1. YC-1 did not affect either fMLP-induced phosphatidylinositol 3-kinase activation or p38 mitogen-activated protein kinase phosphorylation, but slightly attenuated the phosphorylation of extracellular signal-regulated kinase. Collectively, these results indicate that the inhibition of the fMLP-induced respiratory burst by YC-1 is mediated by cyclic GMP-dependent and -independent signaling mechanisms.

Topics: Animals; Calcium; Cell-Free System; Cyclic GMP; Enzyme Activators; In Vitro Techniques; Indazoles; Mitogen-Activated Protein Kinases; Neutrophils; Phosphatidylinositol 3-Kinases; Phospholipase D; Phosphorylation; Protein Kinase C; Rats; Respiratory Burst; Superoxides

YC-1 is a direct activator of soluble guanylyl cyclase (sGC) and sensitizes the enzyme for activation by nitric oxide (NO) and CO. Because the potentiating effect of YC-1 on NO-induced cGMP formation in platelets and smooth muscle cells has been shown to be substantially higher than observed with the purified enzyme, the synergism between heme ligands and YC-1 is apparently more pronounced in intact cells than in cell-free systems. Here, we investigated the mechanisms underlying the synergistic activation of sGC by YC-1 and NO in endothelial cells. Stimulation of the cells with YC-1 enhanced cGMP accumulation up to approximately 100-fold. The maximal effect of YC-1 was more pronounced than that of the NO donor DEA/NO (approximately 20-fold increase in cGMP accumulation) and markedly diminished in the presence of L-N(G)-nitroarginine, EGTA, or oxyhemoglobin. Because YC-1 did not activate endothelial NO synthase, the pronounced effect of YC-1 on cGMP accumulation was apparently caused by a synergistic activation of sGC by YC-1 and basal NO. The effect of YC-1 was further enhanced by addition of DEA/NO, resulting in a approximately 160-fold stimulation of cGMP accumulation. Thus, YC-1 increased the NO-induced accumulation of cGMP in intact cells by approximately 8-fold. Addition of endothelial cell homogenate increased the stimulatory effect of YC-1 on NO-activated purified sGC from 1.2- to 3.7-fold. This effect was not observed with heat-denatured homogenates, suggesting that a heat-labile factor present in endothelial cells potentiates the effect of YC-1 on NO-activated sGC.

Topics: Animals; Cells, Cultured; Cyclic GMP; Drug Synergism; Endothelium, Vascular; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Guanylate Cyclase; Indazoles; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Swine

A major limitation of the use of organic nitrates in cardiovascular medicine is the development of tolerance, which has been attributed, in part, to a decrease in their metabolic activation in the vascular smooth muscle cell. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was shown to potentiate vascular smooth muscle responsiveness to glyceryl trinitrate (GTN), sodium nitroprusside, and the nitric oxide donor NOC 18, in organic nitrate-naive vascular smooth muscle. We used GTN-tolerant rabbit aortic rings (RARs) to test the hypothesis that a non-vasorelaxant concentration of YC-1 enhances the ability of the prototypical organic nitrate GTN to relax vascular smooth muscle and elevate intravascular cGMP under conditions of GTN tolerance. Treatment with YC-1 (3 microM) produced a left shift of the GTN concentration-response curve and decreased the EC50 value for GTN-induced relaxation in both GTN-tolerant and non-tolerant RARs (P < 0.05). Intravascular cGMP elevation induced by GTN was enhanced in the presence of YC-1 in GTN-tolerant and non-tolerant RARs (P < 0.05). These observations indicate that YC-1, or similarly acting drugs, may be useful in overcoming the tolerance that develops during sustained GTN therapy, and that its mechanism may involve enhanced cGMP formation.

Topics: Animals; Aorta, Thoracic; Cyclic GMP; Drug Tolerance; Enzyme Activators; In Vitro Techniques; Indazoles; Isometric Contraction; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Rabbits; Vasodilator Agents

The effects of vinpocetine, an inhibitor of cyclic GMP phosphodiesterase, on ionic currents were examined in rat pituitary GH3 lactotrophs with the aid of the patch-clamp technique. In GH3 cells bathed in normal Tyrode's solution, vinpocetine (10 microM) reversibly increased the amplitude of Ca2+-activated K+ current (I(K)Ca) with an EC50 value of 4 microM. When the recording pipettes were filled with 10 mM EGTA, vinpocetine also stimulated I(K)Ca. In the cell-attached configuration, application of vinpocetine to the bath increased the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels. In excised membrane patches, application of vinpocetine (10 microM) to the bath did not change the single-channel conductance of BK(Ca) channels; however, it did increase channel activity. In the inside-out configuration, neither 8-bromo cyclic GMP nor YC-1 applied intracellularly affected BK(Ca) channel activity. The vinpocetine-induced change in the kinetic behavior of BK(Ca) channels was due to an increase in mean open time and a decrease in mean closed time. Vinpocetine (10 microM) caused a leftward shift in the midpoint for the voltage-dependent opening. Under the current-clamp mode, vinpocetine (10 microM) decreased the firing rate of spontaneous action potentials induced by thyrotropin-releasing hormone (10 microM) in GH3 cells. In pheochromocytoma PC12 cells, vinpocetine (10 microM) applied intracellularly also enhanced the activity of BK(Ca) channels without altering single-channel conductance. Thus, the present study suggests that vinpocetine-mediated stimulation of I(K)Ca may result from the direct activation of BK(Ca) channels and indirectly from elevated cytosolic Ca2+.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Cyclic GMP; Drug Interactions; Electrophysiology; Enzyme Activators; Indazoles; Kinetics; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; PC12 Cells; Pheochromocytoma; Pituitary Gland; Potassium Channels; Potassium Channels, Calcium-Activated; Rats; Tumor Cells, Cultured; Vinca Alkaloids

YC-1 is a newly developed agent that inhibits platelet aggregation and vascular contraction. Although its effects are independent of nitric oxide (NO), it mimics some of the biological actions of NO. For example, it stimulates soluble guanylate cyclase (sGC) and increases intracellular cGMP concentration. Here, we tested the possibility that YC-1 inhibits hypoxia-inducible factor (HIF)-1-mediated hypoxic responses, as does NO. Hep3B cells were used during the course of this work to observe hypoxic induction of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the effects of YC-1 were compared with those of a NO donor, sodium nitropurruside (SNP). In hypoxic cells, YC-1 blocked the induction of EPO and VEGF mRNAs, and inhibited the DNA-binding activity of HIF-1. It suppressed the hypoxic accumulation of HIF-1alpha, but not its mRNA level. It also reduced HIF-1alpha accumulation induced by cobalt and desferrioxamine. Treatment with antioxidants did not recover the HIF-1alpha suppressed by YC-1. We examined whether these effects of YC-1 are related to the sGC/cGMP signal transduction system. Two sGC inhibitors examined failed to block the effects of YC-1, and 8-bromo-cGMP did not mimic actions of YC-1. The effects of YC-1 on the hypoxic responses were comparable with those of SNP. These results suggest that YC-1 and SNP suppressed the hypoxic responses by post-translationally inhibiting HIF-1alpha accumulation. The YC-1 effect may be linked with the metal-related oxygen sensing pathway, and is not due to the stimulation of sGC. This observation implies that the inhibitory effects of YC-1 on hypoxic responses can be developed to suppress EPO-overproduction by tumor cells and tumor angiogenesis.

Topics: Cell Hypoxia; Cyclic GMP; DNA-Binding Proteins; Drug Interactions; Endothelial Growth Factors; Enzyme Activators; Erythropoietin; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Lymphokines; Nuclear Proteins; Oxygen; Transcription Factors; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

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.

Topics: 1-Methyl-3-isobutylxanthine; Alkaloids; Blood Platelets; Blood Proteins; Carbazoles; Cell Adhesion Molecules; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytosol; Drug Synergism; Enzyme Inhibitors; Guanylate Cyclase; Humans; Hydrazines; Hydrogen Peroxide; In Vitro Techniques; Indazoles; Indoles; Kinetics; Microfilament Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Phosphoproteins; Phosphoserine; Platelet Aggregation; Platelet Aggregation Inhibitors; Subcellular Fractions; Thrombin

It is shown that the glutamate-NO-cGMP pathway is impaired in cerebellum of rats with portacaval anastomosis in vivo as assessed by in vivo brain microdialysis in freely moving rats. NMDA-induced increase in extracellular cGMP in the cerebellum was significantly reduced (by 27%) in rats with portacaval anastomosis. Activation of soluble guanylate cyclase by the NO-generating agent S-nitroso-N-acetyl-penicillamine and by the NO-independent activator YC-1 was also significantly reduced (by 35-40%), indicating that portacaval anastomosis leads to remarkable alterations in the modulation of guanylate cyclase in cerebellum. Moreover, the content of soluble guanylate cyclase was increased ca. two-fold in the cerebellum of rats with portacaval anastomosis. Activation of soluble guanylate cyclase by NO was higher in lymphocytes isolated from rats with portacaval anastomosis (3.3-fold) than in lymphocytes from control rats (2.1-fold). The results reported show that the content and modulation of soluble guanylate cyclase are altered in brain of rats with hepatic failure, resulting in altered function of the glutamate-NO-cGMP pathway in the rat in vivo. This may lead to alterations in cerebral processes such as intercellular communication, circadian rhythms, including the sleep-waking cycle, long-term potentiation, and some forms of learning and memory.

Topics: Animals; Cerebellum; Cyclic GMP; Disease Models, Animal; Enzyme Activators; Excitatory Amino Acid Agonists; Glutamic Acid; Guanylate Cyclase; Hepatic Encephalopathy; Hyperammonemia; Immunoblotting; Indazoles; Lymphocytes; Male; Microdialysis; N-Methylaspartate; Nitric Oxide; Nitric Oxide Donors; Penicillamine; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; S-Nitroso-N-Acetylpenicillamine; Synaptic Transmission

Recent evidence points to a potential role of cyclic GMP (cGMP) in the control of cardiac glucose utilization. The present work examines whether the glucose transport system of cardiac myocyte is a site of this cGMP-dependent regulation. Treatment of isolated rat cardiomyocytes (for 10 min) with the membrane-permeant cGMP analogue 8-(4-chlorophenylthio)-cGMP (8-p-CPT-cGMP, 200 microM) caused a decrease in glucose transport in non-stimulated (basal) myocytes, as well as in cells stimulated with insulin or with the mitochondrial inhibitor oligomycin B by up to 40%. An inhibitory effect was also observed with another cGMP analogue (8-bromo-cGMP), and in cells stimulated by hydrogen peroxide or anoxia. In contrast, 8-p-CPT-cAMP (200 microM), or the beta-adrenergic agonist isoprenaline (which increases cAMP levels) did not depress glucose transport, and even potentiated the effect of insulin. Blockade of endogenous cGMP formation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) significantly increased basal and insulin-dependent glucose transport (by 25%), whereas addition of the guanylate cyclase activator 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazol (YC-1, 30 microM) produced a depression of glucose transport (by 20%). Confocal laser scanning microscopic studies revealed that cGMP partially prevents the insulin-induced redistribution of the glucose transporter GLUT4 from intracellular stores to the cell surface. These observations suggest that the glucose transport system of cardiomyocytes represents a metabolic target of inhibition by cGMP, and that this regulation occurs at the level of the trafficking of glucose transporters.

Topics: Animals; Biological Transport; Cell Hypoxia; Cell Membrane Permeability; Cells, Cultured; Cyclic GMP; Deoxyglucose; Drug Combinations; Female; Glucose Transporter Type 4; Glycolysis; Hydrogen Peroxide; Indazoles; Insulin; Microscopy, Confocal; Monosaccharide Transport Proteins; Muscle Proteins; Myocardium; Oligomycins; Oxadiazoles; Rats; Rats, Sprague-Dawley; Rotenone

The effects of a soluble guanylyl cyclase (sGC) activator, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), on formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated [Ca(2+)](i) elevation in rat neutrophils were examined. YC-1 produced a concentration-dependent inhibition of [Ca(2+)](i) elevation. Pretreatment of neutrophils with YC-1 did not enhance its inhibitory effect. YC-1 also inhibited the [Ca(2+)](i) changes caused by ionomycin. In a biphasic model, measuring the [Ca(2+)](i) stimulation by fMLP in a Ca(2+)-free medium followed by reintroduction of Ca(2+), YC-1 mainly affected Ca(2+) influx. YC-1 also inhibited active and passive Mn(2+) influx, and this inhibitory effect was not attenuated by the sGC inhibitor 6-anilino-5,8-quinolinequinone (LY83583). Sodium nitroprusside did not affect the fMLP-stimulated [Ca(2+)](i) changes. Pretreatment of neutrophils with the cyclic GMP-dependent protein kinase inhibitor 8-(4-chlorophenylthio) guanosine-3',5'-monophosphorothioate, Rp-isomer (Rp-8-pCPT-cGMPS), LY83583, the protein phosphatase 2B inhibitor cyclosporin A, or the protein kinase inhibitor staurosporine did not attenuate the inhibition of [Ca(2+)](i) by YC-1. YC-1 inhibited the fMLP-stimulated protein tyrosine phosphorylation. These results indicate that cyclic GMP does not play an important role in the regulation of [Ca(2+)](i) in rat neutrophils. Inhibition of fMLP-stimulated [Ca(2+)](i) changes by YC-1 is mainly via the blockade of Ca(2+) entry through the inhibition of tyrosine kinase activity, but not the stimulation of protein kinase C and protein phosphatase 2B.

Topics: Animals; Calcium; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; In Vitro Techniques; Indazoles; Ion Transport; Manganese; Neutrophils; Phosphorylation; Platelet Aggregation Inhibitors; Rats; Solubility; Tyrosine

YC-1, a benzyl indazole derivative, is an NO-independent direct activator of soluble guanylyl cyclase (sGC), which presents a synergistic action with NO in stimulating cGMP synthesis. These properties have served to suggest YC-1 as an attractive therapeutic agent by permitting the reduction of nitrovasodilator dosage and regulating endogenous cGMP metabolism. Here we studied the effect of prolonged exposure of adrenomedullary endothelial and chromaffin cells to YC-1. We found that YC-1 increased cGMP in the two types of cells and this action was blocked by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, cytoskeletal disorganization and changes in membrane permeability after prolonged incubation with YC-1. Caspase-3-like protease activity and DNA fragments in the cytoplasm were increased in a dose-dependent manner by 16 h YC-1 treatment. The specific and cell permeable caspase-3-like protease inhibitor DEVD-CHO effectively inhibited YC-1-mediated caspase-3-like activation and DNA fragmentation. Moreover, YC-1 also induced cell shape changes accompanied by actin filament disorganization and alterations in membrane permeability. Cells incubated for 24h with YC-1 showed damaged membranes by binding to nucleic acid of a dye excluded by the intact plasma membrane of live cells. YC-1 also induced a decrease in the intracellular non-specific esterase activity, another indication of cell toxicity. Apoptotic phenomena were not prevented by the presence of ODQ although it effectively inhibited the YC-1-elicited cGMP increases. These findings indicate that YC-1 induces apoptosis by activating caspase-3-like protease through a mechanism independent of sGC activation.

Topics: Adrenal Medulla; Animals; Apoptosis; Cattle; Cell Adhesion; Cell Size; Cells, Cultured; Chromaffin Cells; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium; Enzyme Activators; Indazoles; Intracellular Fluid; Time Factors

Pathologic microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially neurotoxic agents, such as pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha). Using cultured N9 microglial cells, we have examined the regulation of TNF-alpha following endotoxic insult with lipopolysacharide (LPS), focusing on the role of the pro-inflammatory phospholipase A2/mitogen activated protein kinase/arachidonic acid/cyclo-oxygenase-2 cascade and the nitric oxide/cGMP pathway. Data show that various inhibitors of the PLA2 cascade markedly inhibit LPS-induced TNF-alpha release, supporting a key role of this pathway in the regulation of microglial activation. We also investigated the putative effects of cGMP-elevating agents on blocking microglial activation induced by LPS. Data show that each member of this class of cGMP-elevating compounds that we employed opposed microglial TNF-alpha release, suggesting that strengthening intracellular cGMP signaling mitigates against microglial activation. Taken together, our results suggest novel strategies for reducing microglial activation.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Cells, Cultured; Cyclic GMP; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dipyridamole; Enzyme Inhibitors; Flavonoids; Guanylate Cyclase; Imidazoles; Indazoles; Isoenzymes; Lipopolysaccharides; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mice; Microglia; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; p38 Mitogen-Activated Protein Kinases; Phospholipases A; Phospholipases A2; Phosphorylcholine; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyridines; Signal Transduction; Tumor Necrosis Factor-alpha

The pathobiologic process of arterial stenosis following balloon angioplasty continues to be an enigmatic problem in clinical settings. This research project investigates the ability of YC-1, a benzyl indazole derivative that sensitizes sGC/cGMP, to stimulate endogenous cGMP and attenuate balloon injury-induced neointima (NI) formation in the rat carotid artery. Northern and Western blot analyses revealed enhanced acute expression of iNOS and inducible heme oxygenase (HO-1) mRNA and protein in the injured artery. The contralateral uninjured artery also demonstrated acute HO-1 mRNA and protein induction without detectable iNOS expression. Perivascular application of YC-1 immediately following injury significantly stimulated acute vessel wall cGMP compared to untreated controls. YC-1 treated sections demonstrated significant reduction in NI area (-74%), NI area/medial wall area (-72%), and NI thickness (-76%) 2 weeks post-injury. These results directly implicate YC-1 as a potent new therapeutic agent capable of reducing post-angioplasty stenosis through endogenous CO- and/or NO-mediated, cGMP-dependent processes.

Topics: Angioplasty, Balloon; Animals; Carotid Arteries; Carotid Artery Injuries; Carotid Stenosis; Cyclic GMP; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Indazoles; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Recurrence; RNA, Messenger; Transcription, Genetic; Tunica Intima

Chronic hyperammonemia impairs the glutamate-nitric oxide-cGMP pathway in rat brain in vivo. The aims of this work were to assess whether hyperammonemia impairs modulation of soluble guanylate cyclase, and to look for a peripheral marker for impairment of this pathway in brain. We activated the pathway at different steps using glutamate, SNAP, or YC-1. In control neurons these compounds increased cGMP by 7.4-, 9.7- and 7.2-fold, respectively. In ammonia-treated neurons formation of cGMP induced by glutamate, SNAP, and YC-1 was reduced by 50%, 56%, and 52%, respectively, indicating that hyperammonemia impairs activation of guanylate cyclase. This enzyme is also present in lymphocytes. Activation of guanylate cyclase by SNAP or YC-1 was impaired in lymphocytes from hyperammonemic rats. These results suggest that determination of the activation of soluble guanylate cyclase in lymphocytes could serve as a peripheral marker for impairment of the neuronal glutamate-nitric oxide-cGMP pathway in brain.

Topics: Ammonia; Animals; Biomarkers; Cells, Cultured; Chronic Disease; Cyclic GMP; Enzyme Activation; Excitatory Amino Acid Antagonists; Glutamic Acid; Guanylate Cyclase; Hepatic Encephalopathy; Indazoles; Lymphocytes; Neurons; Penicillamine; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Time Factors

The vasomotor and cyclic GMP-elevating activity of YC-1, a novel NO-independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18. Similarly to SNP and NOC 18, YC-1 (0.3-300 microM) caused a concentration-dependent, endothelium-independent relaxation that was greatly reduced by the sGC inhibitor 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ 10 microM; 59% inhibition of dilation induced by 100 microM YC-1) suggesting the activation of sGC as one mechanism of action. Preincubation with YC-1 (3 and 30 microM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO-donors, SNP and NOC 18, by almost two orders of magnitude. Vasoactivity induced by SNP and YC-1 displayed different kinetics as evidenced by a longlasting inhibition by YC-1 (300 microM) on the phenylephrine (PE)-induced contractile response, which was not fully reversible even after extensive washout (150 min) of YC-1, and was accompanied by a long-lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30 microM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound. Surprisingly, YC-1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1-5 in vitro. In conclusion, YC-1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC-1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator.

Topics: Animals; Aorta, Thoracic; Cyclic GMP; Enzyme Activation; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Indazoles; Isoenzymes; Kinetics; Muscle Contraction; Muscle Relaxation; Muscle Tonus; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitroprusside; Nitroso Compounds; Oxadiazoles; Phosphoric Diester Hydrolases; Quinoxalines; Rabbits; Vasodilator Agents

1. The effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole) on tension, levels of cyclic GMP and cyclic AMP were investigated in guinea-pig trachea. We especially studied the combined effect of YC-1 with exogenous or endogenous nitric oxide on these parameters. 2. YC-1 at the concentration 3 or 10 microM, which caused only minor effect by itself, elicited concentration-dependent potentiation of sodium nitroprusside (SNP)-induced tracheal relaxation. This relaxation of YC-1 with SNP was reversed by ODQ. 3. Relaxant responses to electric field stimulation (EFS) in the presence of indomethacin, atropine, guanethidine, alpha-chymotrypsin and histamine were also markedly increased by YC-1 (10 microM). In the presence of L-NAME or ODQ, the relaxant effects to EFS were attenuated and the following addition of YC-1 did not further enhance relaxation. 4. YC-1 (10 microM) or SNP (0.3 microM) alone did not induce significant elevation of cyclic GMP levels in the presence of IBMX, whereas simultaneous application of both compounds markedly elevated the cyclic GMP accumulation. In contrast, the cyclic AMP levels were not altered even at the combination of YC-1 and SNP. Additionally, YC-1 also affected cyclic GMP metabolism, since it inhibited the activity of phosphodiesterase type V in human platelets. 5. YC-1 (30 microM) did not scavenge superoxide anion and had no effect on the removal of superoxide anion by superoxide dismutase in a xanthine/xanthine oxidase system. 6. In conclusion, these results indicate that although YC-1 elicits negligible relaxation of guinea-pig trachea by itself, it can potentiate the relaxant responses of exogenous or endogenous NO. This synergistic response of YC-1 is via the elevation of cyclic GMP contents.

Topics: Animals; Cyclic AMP; Cyclic GMP; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Indazoles; Male; Muscle Relaxation; Nitric Oxide; Superoxides; Trachea

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.

Topics: Antioxidants; Blood Platelets; Catalase; Cyclic GMP; Glucose Oxidase; Glutathione Peroxidase; Guanylate Cyclase; Hemostatics; Humans; Hydrogen Peroxide; In Vitro Techniques; Indazoles; Mannitol; Platelet Aggregation Inhibitors; Superoxide Dismutase; Thrombin

1. The sensitivity of the soluble guanylate cyclase (sGC)-cyclic guanosine-3',5'-monophosphate (cyclic GMP) system to nitric oxide (NO) was investigated in mouse aorta from wild type (WT) and NO synthase (NOS) knockout (KO) animals. 2. The NO donor, spermine-NONOate (SPER-NO) was more potent in aortas from eNOS KO mice compared to WT (pEC50 7.30+/-0.06 and 6.56+/-0.04, respectively; n=6; P<0.05). In contrast, the non-NO based sGC activator, YC-1 was equipotent in vessels from eNOS WT and KO mice. The sensitivity of aortas from nNOS and iNOS KO animals to SPER-NO was unchanged. Forskolin (an adenylate cyclase activator), was equipotent in vessels from eNOS WT and KO animals. 3. The cyclic GMP analogue, 8-Br-cGMP was equipotent in eNOS WT and KO mice (pEC50 4. 38+/-0.04 and 4.40+/-0.05, respectively; n=5; P>0.05). Zaprinast (10-5 M) a phosphodiesterase type V (PDE V) inhibitor, had no effect on the response to SPER-NO in vessels from eNOS WT or KO mice. 4. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 M) increased the potency of SPER-NO in aortas from WT mice (pEC50 6. 64+/-0.02 and 7.37+/-0.02 in the absence and presence of L-NAME, respectively; n=4; P<0.05). 5. In summary, there is increased sensitivity of vessels from eNOS KO animals to NO. Cyclic AMP-mediated dilatation is unchanged, consistent with a specific up-regulation of sGC - cyclic GMP signalling. The functional activity of cyclic GMP-dependent protein kinase (G-kinase) and PDE V was also unchanged, suggesting that sGC is the site of up-regulation. These alterations in the sensitivity of the sGC - cyclic GMP pathway might represent a mechanism for the dynamic regulation of NO bioactivity.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Aorta, Thoracic; Colforsin; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Guanylate Cyclase; Homeostasis; Indazoles; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrogen Oxides; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Platelet Aggregation Inhibitors; Purinones; Spermine

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) play key roles in various signaling cascades and are structurally closely related. The crystal structure of a soluble AC revealed one binding site each for the substrate ATP and the activator forskolin. Recently, YC-1, a novel activator of the heterodimeric soluble GC (sGC), has been identified which acts like forskolin on AC. Here, we investigated the respective substrate and potential activator domains of sGC using point-mutated subunits. Whereas substitution of the conserved Cys-541 of the beta(1) subunit with serine led to an almost complete loss of activity, mutation of the respective homologue (Cys-596) in the alpha(1) subunit yielded an enzyme with an increased catalytic rate and higher sensitivity toward NO. This phenotype exhibits characteristics similar to those of the YC-1-treated wild-type enzyme. Conceivably, this domain which corresponds to the forskolin site of the ACs may comprise the binding site for YC-1.

Topics: Animals; Binding Sites; Catalytic Domain; Cattle; Conserved Sequence; Cyclic GMP; Dimerization; Enzyme Activators; Guanosine Triphosphate; Guanylate Cyclase; Indazoles; Magnesium; Manganese; Mutagenesis, Site-Directed; Nitric Oxide; Point Mutation; Solubility

1 In this study we examined the endothelium-dependent effect of YC-1 - a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC) - on vascular relaxation and nitric oxide (NO) and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. 2 In preconstricted rat aortic rings with intact endothelium, YC-1 produced a concentration-dependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC-1, when (i) the aortas were pre-treated with L-NG-nitroarginine methylester (L-NAME) or (ii) the endothelium was removed. 3 Incubation of bovine aortic endothelial cells (BAEC) with YC-1 produced a concentration-dependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre-incubating cells with L-NAME or with 8-bromo-cyclic GMP decreased this effect indicating that the YC-1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct effect of YC-1 on recombinant endothelial constitutive NO synthase activity was observed. 4 The YC-1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. 5 In human umbilical vein endothelial cells (HUVEC), YC-1 stimulated intracellular cyclic GMP production in a concentration- and time-dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC-1 compared to calcium ionophore A23187. Similar effects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). 6 When HUVEC and RMCEC were pre-treated with L-NG-nitroarginine (L-NOARG), the maximum YC-1 stimulated cyclic GMP increase was reduced by >/=50%. 7 These results indicate, that beside being a direct activator of sGC, YC-1 stimulates a NO-synthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.

Topics: Animals; Aorta, Thoracic; Bradykinin; Calcimycin; Cattle; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Activation; Guanylate Cyclase; Humans; In Vitro Techniques; Indazoles; Ionophores; Male; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroarginine; Rats; Rats, Wistar; Solubility; Time Factors

Soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to the second messenger cGMP, functions as the receptor for nitric oxide (NO) and nitrovasodilator drugs. Three distinct cDNA species of each subunit (alpha1-alpha3, beta1-beta3) have been reported from various species. From human sources, none of these have been expressed as functionally active enzyme. Here we describe the expression of human alpha/beta heterodimeric sGC in Sf9 cells yielding active recombinant enzyme that was stimulated by the nitrovasodilator sodium nitroprusside or the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1). At the protein level, both alpha and beta subunits were detected in human tissues, suggesting co-expression also in vivo. Moreover, resequencing of the human cDNA clones [originally termed alpha3 and beta3; Giuili, Scholl, Bulle and Guellaen (1992) FEBS Lett. 304, 83-88] revealed several sequencing errors in human alpha3; correction of these eliminated major regions of divergence from rat and bovine alpha1. As human beta3 also displays more than 98% similarity to rat and bovine beta1 at the amino acid level, alpha3 and beta3 represent the human homologues of rat and bovine alpha1 and beta1, and the isoenzyme family is decreased to two isoforms for each subunit (alpha1, alpha2; beta1, beta2). Having access to the human key enzyme of NO signalling will now permit the study of novel sGC-modulating compounds with therapeutic potential.

Topics: Amino Acid Sequence; Animals; Cattle; Cyclic GMP; Guanosine Triphosphate; Guanylate Cyclase; Humans; Indazoles; Isoenzymes; Molecular Sequence Data; Nitric Oxide; Nitroprusside; Platelet Aggregation Inhibitors; Protein Conformation; Rats; Recombinant Proteins; Solubility; Spodoptera; Vasodilator Agents

Nitric oxide (NO), the physiological activator of soluble guanylyl cyclase (sGC), induces inhibitory effects on platelet activation via elevation of cGMP levels and stimulation of the cGMP-dependent protein kinase. YC-1, a benzylindazole derivative, was shown to activate sGC in intact platelets, resulting in inhibition of platelet aggregation. In a previous study, we demonstrated that YC-1 not only stimulates purified sGC but also potentiates the stimulatory action of submaximally effective NO and carbon monoxide (CO) concentrations. Here, we investigated the potentiating effect of YC-1 in intact platelets. YC-1 together with NO or CO led to complete inhibition of platelet aggregation at concentrations that were ineffective by themselves. Maximally effective 2, 2-diethyl-1-nitroso-oxyhydrazine (3 microM) and YC-1 (100 microM) concentrations each elevated the cGMP levels in intact platelets approximately 13-fold, and administration of the two drugs together resulted in enormous potentiation of cGMP formation, which greatly exceeded the effect on the purified enzyme and yielded a >1300-fold increase in cGMP levels. Similar results were obtained using CO instead of NO. Furthermore, YC-1 not only stimulated sGC but also inhibited cGMP-hydrolyzing phosphodiesterases in platelets. The enormous elevation of cGMP levels led to enhanced phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein. Thus, by the combination of two effects (i.e., potentiation of NO-induced sGC stimulation and phosphodiesterase inhibition), YC-1-like substances are potent activators of the sGC/cGMP pathways and are therefore interesting candidates to act as modulators of cGMP-mediated effects, especially within the cardiovascular system.

Topics: 1-Methyl-3-isobutylxanthine; Blood Platelets; Carbon Monoxide; Cyclic AMP; Cyclic GMP; Drug Synergism; Humans; Indazoles; Nitric Oxide; Phosphodiesterase Inhibitors; Phosphorylation; Platelet Aggregation Inhibitors; Vasodilator Agents

1. We studied the effects of 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine-3':5' cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or superoxide anions on these parameters. 2. YC-1 elicited a direct concentration-dependent activation of sGC (EC50 18.6 +/- 2.0 microM), which was rapid in onset and quickly reversible upon dilution. YC-1 altered the enzyme kinetics with respect to GTP by decreasing KM and increasing Vmax. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC-1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H-(1,2,4)-oxdiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (NS 2028), abolished activation of the enzyme by either compound. 3. YC-1 induced a concentration-dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increases in response to SNP and YC-1 were additive. 4. YC-1 relaxed preconstricted endothelium-denuded rabbit aortic rings in a concentration-dependent manner (50% at 20 microM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (3 microM), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP- and NTG-induced cyclic GMP formation. 5. Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O-2) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (SOD). YC-1-stimulated sGC was also sensitive to inhibition by endogenously generated (O-2 in rat preconstricted endothelium-denuded aortic rings. Relaxation to YC-1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O-2 at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC-1. 6. In conclusion, these results indicate that YC-1 is an NO-independent

Topics: Animals; Cells, Cultured; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; In Vitro Techniques; Indazoles; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Rabbits; Rats; Rats, Inbred WKY; Superoxides; Vasodilator Agents

In the present study, we examined the effects and action mechanisms of cGMP-elevating agents on platelet adhesion to collagen fiber. YC-1, a nitric oxide (NO)-independent activator of soluble guanylate cyclase, inhibited both initial and long-term platelet adhesion to collagen, and the inhibitory effect was potentiated by dipyridamole, a selective inhibitor of cGMP-specific phosphodiesterase. Sodium nitroprusside (SNP), a NO-donor, and 8-bromo-cGMP also inhibited the initial platelet adhesion, but inhibited long-term adhesion only in the presence of dipyridamole. Collagen-induced intracellular Ca2+ mobilization and actin polymerization were prevented by YC-1, SNP and 8-bromo-cGMP. Since blockade of Ca2+ mobilization and actin polymerization caused by collagen led to decrease of platelet adhesion, we suggest that the inhibitory activity of cGMP-elevating agents on the adhesion of platelets to collagen is resulting from interference of these signaling pathways.

Topics: Actins; Adult; Blood Platelets; Calcium; Collagen; Cyclic GMP; Humans; Indazoles; Nitroprusside; Platelet Aggregation; Platelet Aggregation Inhibitors

1. The effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), an activator of soluble guanylyl cyclase, on tension, levels of cyclic GMP and cyclic AMP, and cardiac L-type Ca2+-current (I[Ca(L)]) were investigated in aortic smooth muscle and ventricular heart muscle from rat. 2. YC-1 (0.1-30 microM) induced a concentration-dependent relaxation in aortic rings precontracted with phenylephrine (3 microM). The relaxant effects of YC-1 were reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (30 microM; ODQ), potentiated by zaprinast (10 microM) and antagonized by Rp-8-Br-cGMPS (100 microM). 3. In ventricular heart muscle strips, YC-1 (30 microM) exhibited no effects on force of contraction (Fc) in the absence or presence of either zaprinast (10 microM) or 3-isobutyl-1-methylxanthine (30 microM). Fc was slightly increased by YC-1 (30 microM) in the presence of isoprenaline (100 nM), but this effect was not influenced by ODQ (30 microM). 4. Cardiac I[Ca(L)] was not significantly affected by YC-1 (30 microM), either in the absence or presence of isoprenaline (30 nM). 5. In aortic rings, cyclic GMP levels were increased almost 3 fold by YC-1 (30 microM); this effect was abolished by ODQ (30 microM). In isolated ventricular cardiomyocytes, cyclic GMP levels were not affected by YC-1 (30 microM) but almost doubled by activation of particular guanylyl cyclase with atriopeptin II (100 nM). 6. YC-1 (30 microM) did not increase cyclic AMP levels either in aortic rings or in ventricular cardiomyocytes. In contrast, isoprenaline (3 microM) increased cyclic AMP levels about two fold in both tissues. In cardiomyocytes, the effect of isoprenaline (3 microM) was slightly enhanced by YC-1 (30 microM). 7. It is concluded that relaxation of smooth muscle preparations by YC-1 is mediated mainly by activation of soluble guanylyl cyclase and subsequent increase in cyclic GMP levels. The failure of YC-1 to affect cardiac Fc, levels of cyclic GMP, and I[Ca(L)] suggests that soluble guanylyl cyclase is not influenced by YC-1 in rat heart muscle or only barely present in this tissue.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Cyclic AMP; Cyclic GMP; Enzyme Activation; Female; Guanylate Cyclase; Indazoles; Isoproterenol; Male; Muscle, Smooth, Vascular; Myocardial Contraction; Myocardium; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Vasoconstriction

An indazole derivative, YC-1, was identified in this study to be capable of reversibly and effectively inhibiting proliferation of rat A10 vascular smooth-muscle cells (VSMCs) in vitro. YC-1 (1-100 microM) dose-dependently inhibited [3H]thymidine incorporation into DNA in rat A10 VSMCs that were synchronized by serum depletion and then restimulated by addition of 10% foetal calf serum (FCS), whereas FCS-induced [3H]thymidine incorporation into rat synchronized endothelial cells was unaffected by this agent. The dose of YC-1 required to cause inhibition of FCS-induced proliferation was similar to that necessary for the formation of cellular cyclic GMP (cGMP). Guanylate cyclase activity in soluble fractions of VSMCs was activated by YC-1 (1-100 microM), whereas cGMP-specific phosphodiesterase activity was unaffected by this compound. The anti-proliferative effect of YC-1 was mimicked by 8-bromo-cGMP, a membrane-permeable cGMP analogue, and was antagonized by KT 5823 (0.2 microM), a selective inhibitor of protein kinase G. The anti-proliferative effect of YC-1 was also antagonized by Methylene Blue (50 microM), a guanylate cyclase inhibitor, and was potentiated by 3-isobutyl-1-methylxanthine (500 microM), a phosphodiesterase inhibitor. These results verified that YC-1 is a direct soluble guanylate cyclase activator in A10 VSMCs, and the anti-proliferative effect of YC-1 is mediated by cGMP. YC-1 still inhibited FCS-induced DNA synthesis even when added 10-18 h after restimulation of the serum-deprived A10 VSMCs with 10% FCS. Flow cytometry in synchronized populations revealed an acute blockage of FCS-inducible cell-cycle progression at a point in the G1/S-phase in YC-1 (100 microM)-treated cells. The inhibition of proliferation by YC-1 was demonstrated to be independent of cell damage, as documented by several criteria of cell viability. In conclusion, YC-1 reversibly and effectively inhibited the proliferation of VSMCs, suggesting that it has potential as a therapeutic agent in the prevention of vascular diseases.

Topics: Animals; Cell Cycle; Cell Division; Cells, Cultured; Cyclic GMP; Flow Cytometry; Indazoles; Muscle, Smooth, Vascular; Platelet Aggregation Inhibitors; Rats

1. Our previous study demonstrated that YC-1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC-1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. 2. In human washed platelets, YC-1 inhibited platelet aggregation and ATP released induced by U46619 (2 microM), collagen (10 micro ml(-1)) and thrombin (0.1 u ml(-1)) in a concentration-dependent manner with IC50 values of (microM) 2.1 +/- 0.03, 11.7 +/- 2.1 and 59.3 +/- 7.1, respectively. 3. In a 30,000 g supernatant fraction from human platelet homogenate, YC-1 (5-100 microM) increased sGC activity in a concentration-dependent manner. At the same concentration-range, YC-1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. 4. MY-5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC-1, and shifted the concentration-anti-aggregation curve of YC-1 to the left. In contrast, HL-725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti-aggregatory effect caused by YC-1. 5. Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC-1, and attenuated markedly the anti-aggregatory effect of YC-1. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA) did not affect YC-1-induced inhibition of platelet aggregation. 6. Haemoglobin, which binds NO, prevented the activation of sGC and anti-aggregatory effect caused by sodium nitroprusside, but did not affect YC-1 response. 7. These results would suggest that YC-1 activates sGC of human platelets by a NO-dependent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Blood Platelets; Collagen; Cyclic AMP; Cyclic GMP; Dideoxyadenosine; Dose-Response Relationship, Drug; Enzyme Activation; Furans; Guanylate Cyclase; Hemoglobins; Humans; Indazoles; Nitric Oxide; Phosphodiesterase Inhibitors; Phthalazines; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Solubility; Thrombin; Thromboxane A2; Vasoconstrictor Agents

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.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine Triphosphate; Alprostadil; Animals; Bleeding Time; Blood Platelets; Calcium; Colforsin; Collagen; Cyclic AMP; Cyclic GMP; Drug Interactions; Enzyme Activation; Guanylate Cyclase; Imidazoles; Indazoles; Indomethacin; Methemoglobin; Mice; Molecular Structure; Nitroprusside; Phosphoric Diester Hydrolases; Platelet Aggregation Inhibitors; Prostaglandin D2; Rabbits; Superoxide Dismutase; Thromboxane A2