RESUMEN
Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC). The NO-GC/cGMP pathway in platelets has been extensively studied. However, its role in regulating the biomechanical properties of platelets has not yet been addressed and remains unknown. We therefore investigated the stiffness of living platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat using scanning ion conductance microscopy (SICM). Stimulation of human and murine platelets with cGMP-modulating drugs decreased cellular stiffness and downregulated P-selectin, a marker for platelet activation. We also quantified changes in platelet shape using deep learning-based platelet morphometry, finding that platelets become more circular upon treatment with cGMP-modulating drugs. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulfate (ABC)-containing regimens. Our results corroborate a functional role of the NO-GC/cGMP pathway in platelet biomechanics, indicating that biomechanical properties such as stiffness or shape could be used as novel biomarkers in clinical research.
Increased platelet activation and development of thrombosis has been linked to a dysfunctional NO-GC/cGMP signaling pathway. How this pathway affects platelet stiffness, however, has not been studied yet. For the first time, we used novel microscopy techniques to investigate stiffness and shape of platelets in human and murine blood samples treated with cGMP modifying drugs. Stiffness contains information about biomechanical properties of the cytoskeleton, and shape quantifies the spreading behavior of platelets. We showed that the NO-GC/cGMP signaling pathway affects platelet stiffness, shape, and activation in human and murine blood. HIV-positive patients are often treated with medication that may disrupt the NO-GC/cGMP signaling pathway, leading to increased cardiovascular risk. We showed that treatment with cGMP-modifying drugs altered platelet shape and aggregation in blood from HIV-negative volunteers but not from HIV-positive patients treated with medication. Our study suggests that platelet stiffness and shape can be biomarkers for estimating cardiovascular risk.
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Plaquetas , Transducción de Señal , Humanos , Ratones , Animales , Fenómenos Biomecánicos , Plaquetas/metabolismo , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Activación Plaquetaria , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Óxido Nítrico/metabolismo , Agregación PlaquetariaRESUMEN
Natriuretic peptides exert not only blood-lowering but also kidney-protective effects through guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. Signaling through GC-A has been shown to protect podocytes from aldosterone-induced glomerular injury, and a p38 mitogen-activated protein kinase (MAPK) inhibitor reduced glomerular injury in aldosterone-infused podocyte-specific GC-A knockout mice. To explore the role of p38 MAPK in podocytes, we constructed podocyte-specific p38 MAPK and GC-A double knockout mice (pod-double knockout mice). Unexpectedly, aldosterone-infused and high salt-fed (B-ALDO)-treated pod-double knockout mice resulted in elevated serum creatinine, massive albuminuria, macrophage infiltration, foot process effacement, nephrin and podocin reduction, and additionally, intra-capillary fibrin thrombi, indicating endothelial injury. Microarray analysis showed increased plasminogen activator inhibitor-1 (PAI-1) in glomeruli of B-ALDO-treated pod-double knockout mice. In B-ALDO-treated pod-double knockout mice, PAI-1 increased in podocytes, and treatment with PAI-1 neutralizing antibody ameliorated intra-capillary thrombus formation. In vitro, deletion of p38 MAPK by the CRISPR/Cas9 system and knockdown of GC-A in human cultured podocytes upregulated PAI-1 and transforming growth factor- ß1 (TGF-ß1). When p38 MAPK knockout podocytes, transfected with a small interfering RNA to suppress GC-A, were co-cultured with glomerular endothelial cells in a transwell system, the expression of TGF-ß1 was increased in glomerular endothelial cells. PAI-1 inhibition ameliorated both podocyte and endothelial injury in the transwell system signifying elevated PAI-1 in podocytes is a factor disrupting normal podocyte-endothelial crosstalk. Thus, our results indicate that genetic dual deletion of p38 MAPK and GC-A in podocytes accelerates both podocyte and endothelial injuries, suggesting these two molecules play indispensable roles in podocyte function.
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Podocitos , Trombosis , Animales , Humanos , Ratones , Aldosterona/farmacología , Aldosterona/metabolismo , Células Endoteliales/metabolismo , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Ratones Noqueados , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/farmacología , Podocitos/metabolismo , Trombosis/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteína Quinasa 14 Activada por MitógenosRESUMEN
Diabetic kidney disease (DKD) is the most common cause of renal failure. Therapeutics development is hampered by our incomplete understanding of animal models on a cellular level. We show that ZSF1 rats recapitulate human DKD on a phenotypic and transcriptomic level. Tensor decomposition prioritizes proximal tubule (PT) and stroma as phenotype-relevant cell types exhibiting a continuous lineage relationship. As DKD features endothelial dysfunction, oxidative stress, and nitric oxide depletion, soluble guanylate cyclase (sGC) is a promising DKD drug target. sGC expression is specifically enriched in PT and stroma. In ZSF1 rats, pharmacological sGC activation confers considerable benefits over stimulation and is mechanistically related to improved oxidative stress regulation, resulting in enhanced downstream cGMP effects. Finally, we define sGC gene co-expression modules, which allow stratification of human kidney samples by DKD prevalence and disease-relevant measures such as kidney function, proteinuria, and fibrosis, underscoring the relevance of the sGC pathway to patients.
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Diabetes Mellitus , Nefropatías Diabéticas , Humanos , Ratas , Animales , Guanilil Ciclasa Soluble/metabolismo , Guanilil Ciclasa Soluble/farmacología , Guanilil Ciclasa Soluble/uso terapéutico , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/patología , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Riñón/metabolismo , FibrosisRESUMEN
RATIONALE: Re-exposing an animal to an environment previously paired with an aversive stimulus evokes large alterations in behavioral and cardiovascular parameters. Dorsal hippocampus (dHC) receives important cholinergic inputs from the basal forebrain, and respective acetylcholine (ACh) levels are described to influence defensive behavior. Activation of muscarinic M1 and M3 receptors facilitates autonomic and behavioral responses along threats. Evidence show activation of cholinergic receptors promoting formation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) in dHC. Altogether, the action of ACh and NO on conditioned responses appears to converge within dHC. OBJECTIVES: As answer about how ACh and NO interact to modulate defensive responses has so far been barely addressed, we aimed to shed additional light on this topic. METHODS: Male Wistar rats had guide cannula implanted into the dHC before being submitted to the contextual fear conditioning (3footshocks/085 mA/2 s). A catheter was implanted in the femoral artery the next day for cardiovascular recordings. Drugs were delivered into dHC 10 min before contextual re-exposure, which occurred 48 h after the conditioning procedure. RESULTS: Neostigmine (Neo) amplified the retrieval of conditioned responses. Neo effects (1 nmol) were prevented by the prior infusion of a M1-M3 antagonist (fumarate), a neuronal nitric oxide synthase inhibitor (NPLA), a NO scavenger (cPTIO), a guanylyl cyclase inhibitor (ODQ), and a NMDA antagonist (AP-7). Pretreatment with a selective M1 antagonist (pirenzepine) only prevented the increase in autonomic responses induced by Neo. CONCLUSION: The results show that modulation in the retrieval of contextual fear responses involves coordination of the dHC M1-M3/NO/cGMP/NMDA pathway.
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N-Metilaspartato , Óxido Nítrico , Acetilcolina , Animales , Colinérgicos/farmacología , Miedo/fisiología , Fumaratos/farmacología , Guanosina Monofosfato/farmacología , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Hipocampo , Masculino , N-Metilaspartato/farmacología , Neostigmina/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Pirenzepina/farmacología , Ratas , Ratas Wistar , Receptores Colinérgicos/metabolismo , Receptores de N-Metil-D-Aspartato , Transmisión SinápticaRESUMEN
Platelets are the "guardians" of the blood circulatory system. At sites of vessel injury, they ensure hemostasis and promote immunity and vessel repair. However, their uncontrolled activation is one of the main drivers of thrombosis. To keep circulating platelets in a quiescent state, the endothelium releases platelet antagonists including nitric oxide (NO) that acts by stimulating the intracellular receptor guanylyl cyclase (GC). The latter produces the second messenger cyclic guanosine-3',5'-monophosphate (cGMP) that inhibits platelet activation by stimulating protein kinase G, which phosphorylates hundreds of intracellular targets. Intracellular cGMP pools are tightly regulated by a fine balance between GC and phosphodiesterases (PDEs) that are responsible for the hydrolysis of cyclic nucleotides. Phosphodiesterase type 5 (PDE5) is a cGMP-specific PDE, broadly expressed in most tissues in humans and rodents. In clinical practice, PDE5 inhibitors (PDE5i) are used as first-line therapy for erectile dysfunction, pulmonary artery hypertension, and lower urinary tract symptoms. However, several studies have shown that PDE5i may ameliorate the outcome of various other conditions, like heart failure and stroke. Interestingly, NO donors and cGMP analogs increase the capacity of anti-platelet drugs targeting the purinergic receptor type Y, subtype 12 (P2Y12) receptor to block platelet aggregation, and preclinical studies have shown that PDE5i inhibits platelet functions. This review summarizes the molecular mechanisms underlying the effect of PDE5i on platelet activation and aggregation focusing on the therapeutic potential of PDE5i in platelet disorders, and the outcomes of a combined therapy with PDE5i and NO donors to inhibit platelet activation.
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Óxido Nítrico , Inhibidores de Fosfodiesterasa 5 , Humanos , Masculino , Plaquetas/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/farmacología , Guanosina/metabolismo , Guanosina/farmacología , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/metabolismo , Nucleótidos Cíclicos/metabolismo , Nucleótidos Cíclicos/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Inhibidores de Fosfodiesterasa 5/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Proteínas Quinasas/metabolismoRESUMEN
Hinokitiol is a natural bioactive compound with numerous pharmacological properties. Here, we aimed to examine hinokitiol's effects on vascular relaxation. Cumulative relaxation responses to hinokitiol were assessed in isolated aortae from normotensive and angiotensin II-induced hypertensive rats in the presence and absence of selective inhibitors. Hinokitiol produced vasodilation of phenylephrine preconstricted aortae using both normotensive and hypertensive rats. In normotensive rats, hinokitiol's vasodilation was reduced by endothelial denudation and nitric oxide synthase (NOS), guanylate cyclase, and cyclooxygenase inhibition. Also, hinokitiol vasodilation was attenuated by ß-receptors, adenylate cyclase, Ca2+-activated K+ channels and hyperpolarization inhibition. Moreover, hinokitiol exhibited a blocking activity on Ca2+ mobilization through voltage dependent Ca2+ channels (VDCC). However, its effect was not changed by muscarinic receptor and Sarc-K+ ATP channels blocking but was enhanced by blocking voltage-dependent K+ channels. However, in angiotensin II-induced hypertension, hinokitiol vasodilating activity was attenuated by NOS inhibition and it blocked Ca2+ mobilization through VDCC, while its vasodilation was partially attenuated by Sarc-K+ ATP channels blocking. However, the vasodilating effect of hinokitiol was not attenuated by either cyclooxygenase, ß-receptor, Ca2+-activated K+ channels, or voltage-dependent potassium channels inhibition, but was enhanced by blocking hyperpolarization. Hinokitiol's vasodilating effect in normotensive and hypertensive vessels is mediated through both endothelium-dependent and endothelium-independent mechanisms.
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Hipertensión , Vasodilatación , Adenosina Trifosfato/metabolismo , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/farmacología , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Canales de Calcio Tipo L/metabolismo , Endotelio Vascular/metabolismo , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Óxido Nítrico Sintasa , Fenilefrina/farmacología , Canales de Potasio/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/farmacología , RatasRESUMEN
AIMS: Intimal hyperplasia is a common feature of vascular remodelling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo. METHODS AND RESULTS: RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodelling of cultured human saphenous vein segments. CONCLUSIONS: Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signalling and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease.
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Músculo Liso Vascular , Lesiones del Sistema Vascular , Animales , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacología , Proliferación Celular , Células Cultivadas , GMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Ciclina D1/metabolismo , Células Endoteliales/metabolismo , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , ARN Mensajero/metabolismo , Remodelación Vascular , Lesiones del Sistema Vascular/tratamiento farmacológico , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/metabolismoRESUMEN
INTRODUCTION: Human dental pulp cells (HDPCs) are recalcitrant to hypoxic stress. We investigated whether hypoxia-induced autophagy of HDPCs offered these cells a survival advantage and the underlying mechanism of this resistance. METHODS: The viability and apoptosis of HDPCs were examined after exposure to hypoxia by Vi-CELL cell viability analyzer and flow cytometry. Autophagy was assessed by using immunofluorescence, acridine orange staining, real-time polymerase chain reaction, and Western blotting. Either 3-methyladenine or expression vectors encoding dominant negative ATG5 were used to inhibit autophagy. Rapamycin was used as an autophagic inducer. To explore the mechanisms of autophagy, adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway and hypoxia-inducible transcription factor-1 were suppressed by chemical inhibitors Compound C and YC-1, respectively. RESULTS: The exposure of HDPCs to hypoxia had no effect on viability and resulted in increasing acidic vesicular organelle-positive cells, autophagosome formation, and up-regulation of autophagy genes. Inhibition of autophagy with 3- methyladenine or expression vectors encoding dominant negative ATG5 abrogated the protective effects of HDPCs. The phosphorylation of AMPK was up-regulated, whereas the phosphorylation of mTOR was down-regulated in hypoxia-treated HDPCs, which were both attenuated by Compound C. Furthermore, treatment with Compound C rather than YC-1 reduced the autophagy. CONCLUSIONS: Our results suggested that autophagy of HDPCs might be cytoprotective against hypoxic stress via the AMPK/mTOR signaling pathway.
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Proteínas Quinasas Activadas por AMP/fisiología , Autofagia/fisiología , Hipoxia de la Célula/fisiología , Pulpa Dental/enzimología , Serina-Treonina Quinasas TOR/fisiología , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Adenina/análogos & derivados , Adenina/farmacología , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/análisis , Autofagia/efectos de los fármacos , Proteína 12 Relacionada con la Autofagia , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Técnicas de Cultivo de Célula , Supervivencia Celular/fisiología , Células Cultivadas , Pulpa Dental/citología , Vectores Genéticos/genética , Guanilato Ciclasa/farmacología , Humanos , Factor 1 Inducible por Hipoxia/análisis , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Indazoles/farmacología , Proteínas de la Membrana/análisis , Proteínas Asociadas a Microtúbulos/análisis , Plásmidos/genética , Proteínas Proto-Oncogénicas/análisis , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/análisis , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Supresoras de Tumor/análisisRESUMEN
PURPOSE: Because neuronal released endogenous H2S has a key role in relaxation of the bladder outflow region, we investigated the mechanisms involved in H2S dependent inhibitory neurotransmission to the pig bladder neck. MATERIALS AND METHODS: Bladder neck strips were mounted in myographs for isometric force recording and simultaneous measurement of intracellular Ca(2+) and tension. RESULTS: On phenylephrine contracted preparations electrical field stimulation and the H2S donor GYY4137 evoked frequency and concentration dependent relaxation, which was reduced by desensitizing capsaicin sensitive primary afferents with capsaicin, and the blockade of adenosine 5'-triphosphate dependent K(+) channels, cyclooxygenase and cyclooxygenase-1 with glibenclamide, indomethacin and SC560, respectively. Inhibition of vanilloid, transient receptor potential A1, transient receptor potential vanilloid 1, vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide and calcitonin gene-related peptide receptors with capsazepine, HC030031, AMG9810, PACAP6-38 and CGRP8-37, respectively, also decreased electrical field stimulation and GYY4137 responses. H2S relaxation was not changed by guanylyl cyclase, protein kinase A, or Ca(2+) activated or voltage gated K(+) channel inhibitors. GYY4137 inhibited the contractions induced by phenylephrine and by K(+) enriched (80 mM) physiological saline solution. To a lesser extent it decreased the phenylephrine and K(+) induced increases in intracellular Ca(2+). CONCLUSIONS: H2S produces pig bladder neck relaxation via activation of adenosine 5'-triphosphate dependent K(+) channel and by smooth muscle intracellular Ca(2+) desensitization dependent mechanisms. H2S also promotes the release of sensory neuropeptides and cyclooxygenase-1 pathway derived prostanoids from capsaicin sensitive primary afferents via transient receptor potential A1, transient receptor potential vanilloid 1 and/or related ion channel activation.
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Señalización del Calcio/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Canales KATP/metabolismo , Músculo Liso/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Transmisión Sináptica/efectos de los fármacos , Vejiga Urinaria/inervación , Vejiga Urinaria/metabolismo , Acetanilidas/farmacología , Acrilamidas/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Estimulación Eléctrica , Gliburida/farmacología , Guanilato Ciclasa/farmacología , Indometacina/farmacología , Morfolinas/farmacología , Compuestos Organotiofosforados/farmacología , Fenilefrina/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Purinas/farmacología , Pirazoles/farmacología , PorcinosRESUMEN
BACKGROUND: Portal pressure (PP) results from the interplay of vasoconstrictors and vasodilators. Recently, we have shown that Kupffer cell (KC) activation increases PP. AIMS: The role of the vasodilating compounds nitric oxide (NO) and carbon monoxide (CO) was studied. The hypothesis of the present study was that these vasodilators counteract the PP increase following KC activation. METHODS: Livers of rats weighing 180-200 g were isolated and perfused. KCs were activated by zymosan A (cell wall particles from yeast; 150 µg/ml). The effects of NO and guanylate cyclase (GC) were evaluated by the NO synthase inhibitor N(G)-nitro-L-arginine methylester (L-NAME; 0.3 mM, and the GC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028, 1.0 µM); the effects of the heme oxygenase (HO) derived compound CO were evaluated by direct administration of CO or inhibition of HO by zinc protoporphyrin IX (ZnPP IX, 1.0 µM). RESULTS: In isolated perfused rat livers, administration of L-NAME or NS-2028 further raised PP increase following KC activation. This effect could be reduced by the cGMP analogue 8-Br-cGMP. Inhibition of HO caused marked amplification of PP increase in zymosan-treated organs. CO prevented this PP increase cGMP independently. Interestingly, KC activation and simultaneous inhibition of HO augmented the production of prostaglandins D2 and F2α and of thromboxane A2. Accordingly, indomethacin blunted the increase of PP in zymosan/ZnPP-treated livers. CONCLUSIONS: NO restricts the initial PP increase after KC activation by GC-mediated cGMP. CO from heme degradation limits the increase of PP after KC activation eicosanoid dependently, but cGMP independently.
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Monóxido de Carbono/farmacología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Macrófagos del Hígado/metabolismo , Óxido Nítrico/farmacología , Presión Portal/efectos de los fármacos , Prostaglandinas/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/farmacología , Hemo Oxigenasa (Desciclizante)/metabolismo , Hígado/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Oxadiazoles/farmacología , Oxazinas/farmacología , Protoporfirinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Intracerebroventricular (icv) administration of leptin facilitates lordosis behavior in ad libitum-fed, estrogen-primed rats. The cellular mechanism involved in this response is unknown. The present study tested the hypothesis that the nitric oxide-guanylyl cyclase, cGMP-dependent protein kinase (PKG) pathway is involved in the facilitation of lordosis behavior induced by the central administration of leptin. We tested the importance of the nitric oxide/cGMP pathway for lordosis stimulation by either icv infusion of a nitric oxide synthase inhibitor (L-NAME) or a nitric oxide-dependent, soluble guanylyl cyclase inhibitor (ODQ) 30 min before leptin administration (1 µg). This dose of leptin reliably induced lordosis behavior in ovariectomized estradiol benzoate treated rats. The lordosis induced by leptin at 1 and 2h after infusion was significantly reduced by the previous injection of either L-NAME or by ODQ. Intracerebroventricular infusion of the PKG inhibitor (KT5823) 30 min before leptin infusion, also significantly inhibited the lordosis behavior induced by leptin at 1 and 2h after hormone administration. These data support the hypothesis that the nitric oxide/cGMP/PKG pathway is involved in the facilitation of lordosis by leptin in estrogen-primed female rats.
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Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Leptina/administración & dosificación , Óxido Nítrico/metabolismo , Conducta Sexual Animal , Animales , Carbazoles/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Femenino , Guanilato Ciclasa/farmacología , Leptina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/fisiología , Ovariectomía , Postura , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/farmacología , Guanilil Ciclasa SolubleRESUMEN
BACKGROUND: We previously reported that chronic periodontal inflammation causes the accumulation of the transcriptional activator hypoxia-inducible factor-1α (HIF-1α) in human gingival fibroblasts (HGFs) in vivo. Here, evidence is provided that bacterial lipopolysaccharides (LPS) and cellular hypoxia, both associated with periodontitis, can individually, or in combination, lead to the accumulation and activation of HIF-1 in HGF in vitro. METHODS: Primary gingival fibroblasts were cultured from human gingival biopsies. HIF-1α peptide from HGFs treated with Escherichia coli LPS under normoxia or hypoxia was detected by nuclear protein extraction, immunoprecipitation, immunoblotting, and immunocytofluorescence. HIF-1α transcripts were detected using reverse transcription polymerase chain reaction (PCR). The transcript expression levels of vascular endothelial growth factor-A (VEGF-A), a downstream gene of HIF-1α, were assessed by quantitative real-time PCR. RESULTS: Two HIF-1α splicing transcription variants were found to be constitutively expressed in HGFs. E. coli LPS induced a dose- and time-dependent nuclear accumulation of HIF-1α peptide in HGFs. This accumulation could be attenuated by treatment with a Toll-like receptor 4 (TLR4)-neutralizing antibody. Under hypoxia, LPS further increased HIF-1α accumulation in HGFs. VEGF-A transcript expression was upregulated by LPS under both normoxia and hypoxia but was downregulated by pretreatment with TLR4-neutralizing antibody or the specific HIF-1α inhibitor 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole. CONCLUSION: LPS induces the nuclear accumulation of HIF-1α in HGFs and induces HIF-1 biologic activity under normoxia or hypoxia possibly through TLR4.
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Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Lipopolisacáridos/farmacología , Empalme Alternativo/genética , Anticuerpos Neutralizantes/farmacología , Técnicas de Cultivo de Célula , Hipoxia de la Célula/fisiología , Núcleo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Activadores de Enzimas/farmacología , Escherichia coli , Exones/genética , Técnica del Anticuerpo Fluorescente , Variación Genética/genética , Encía/citología , Guanilato Ciclasa/farmacología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Immunoblotting , Inmunoprecipitación , Indazoles/farmacología , Consumo de Oxígeno/fisiología , Factores de Tiempo , Receptor Toll-Like 4/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genéticaAsunto(s)
Terapia Genética/métodos , Guanilato Ciclasa/deficiencia , Guanilato Ciclasa/farmacología , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/terapia , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Receptores de Superficie Celular/deficiencia , Visión Ocular/efectos de los fármacos , AnimalesRESUMEN
Leber congenital amaurosis (LCA) is a severe retinal dystrophy manifesting from early infancy as poor vision or blindness. Loss-of-function mutations in GUCY2D cause LCA1 and are one of the most common causes of LCA, accounting for 20% of all cases. Human GUCY2D and mouse Gucy2e genes encode guanylate cyclase-1 (GC1), which is responsible for restoring the dark state in photoreceptors after light exposure. The Gucy2e(-/-) mouse shows partially diminished rod function, but an absence of cone function before degeneration. Although the cones appear morphologically normal, they exhibit mislocalization of proteins involved in phototransduction. In this study we tested the efficacy of an rAAV2/8 vector containing the human rhodopsin kinase promoter and the human GUCY2D gene. Following subretinal delivery of the vector in Gucy2e(-/-) mice, GC1 protein was detected in the rod and cone outer segments, and in transduced areas of retina cone transducin was appropriately localized to cone outer segments. Moreover, we observed a dose-dependent restoration of rod and cone function and an improvement in visual behavior of the treated mice. Most importantly, cone preservation was observed in transduced areas up to 6 months post injection. To date, this is the most effective rescue of the Gucy2e(-/-) mouse model of LCA and we propose that a vector, similar to the one used in this study, could be suitable for use in a clinical trial of gene therapy for LCA1.
Asunto(s)
Terapia Genética/métodos , Guanilato Ciclasa/deficiencia , Guanilato Ciclasa/farmacología , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/terapia , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Receptores de Superficie Celular/deficiencia , Visión Ocular/efectos de los fármacos , Análisis de Varianza , Animales , Western Blotting , Cartilla de ADN/genética , Dependovirus , Relación Dosis-Respuesta a Droga , Electrorretinografía , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Guanilato Ciclasa/administración & dosificación , Guanilato Ciclasa/genética , Inmunohistoquímica , Amaurosis Congénita de Leber/enzimología , Ratones , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Superficie Celular/administración & dosificación , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Septic shock is a major cause of morbidity and mortality in the intensive care unit, and effective therapies are limited. Methylene blue is a selective inhibitor of guanylate cyclase, a second messenger involved in nitric oxide-mediated vasodilation. The use of methylene blue in the treatment of septic shock has been repeatedly evaluated over the past 20 years, but data remain scarce. To evaluate the safety and efficacy of methylene blue for the treatment of septic shock, we conducted a literature search of the EMBASE (1974-June 2009), MEDLINE (1966-June 2009), and International Pharmaceutical Abstracts (1970-June 2009) databases. All available studies published in English were reviewed. Observational studies with methylene blue have demonstrated beneficial effects on hemodynamic parameters and oxygen delivery, but use of methylene blue may be limited by adverse pulmonary effects. Methylene blue administration is associated with increases in mean arterial pressure while reducing catecholamine requirements in patients experiencing septic shock; however, its effects on morbidity and mortality remain unknown. Well-designed, prospective evaluations are needed to define the role of methylene blue as treatment of septic shock.
Asunto(s)
Azul de Metileno/farmacología , Choque Séptico/tratamiento farmacológico , Ensayos Clínicos como Asunto , Guanilato Ciclasa/farmacología , Hemodinámica/efectos de los fármacos , Humanos , Pulmón/efectos de los fármacos , Óxido Nítrico/farmacología , Ensayos Clínicos Controlados Aleatorios como Asunto , Estudios Retrospectivos , Choque Séptico/fisiopatología , Vasodilatación/efectos de los fármacosRESUMEN
Adipose tissue releases an "adipocyte-derived relaxing factor" (ADRF) lowering tone of isolated arteries. The potential influence of hypoxia on the vasorelaxing properties of adipose tissue was investigated. Aortas from male Swiss mice with or without adherent adipose tissue were mounted in a wire myograph for isometric tension recording. Hypoxia (bubbling with 95% N(2), 5% CO(2)) relaxed precontracted (norephinephrine, 5 microM) aorta with adipose tissue while only a minimal vasorelaxing effect was observed in arteries without adipose tissue. This effect was also seen after precontraction with prostaglandin F(2alpha) (30 microM) or U-46619 (10 nM). Precontraction with 60 or 120 mM K(+), incubation with tetraethylammoniumchloride (3 mM) or glibenclamide (30 microM) significantly impaired the hypoxic response. Glibenclamide (30 microM) enhanced the vasorelaxing effect of NaHS (except at high concentrations of NaHS). Lactate (10 nM to 1 mM) had no effect on preparations with or without adipose tissue. 8-(p-sulfophenyl)theophylline (0.1 mM), zinc protoporphyrin IX (10 microM), 1 H-[1, 2, 4]oxadiazolo[4,3-A]quinoxalin-1-one (10 microM) and removal of the endothelium did not influence the hypoxic relaxation. Our findings indicate that hypoxia has a relaxing influence on mice aorta that is dependent on the presence of adherent adipose tissue. This relaxation is partly mediated by opening K(ATP) channels and independent of the endothelium and soluble guanylyl cyclase. Neither lactate, adenosine, CO nor H(2)S seems to be involved in this hypoxic response. However, the involvement of the as yet unidentified "adipocyte-derived relaxing factor" (ADRF) cannot be excluded.
Asunto(s)
Tejido Adiposo/metabolismo , Aorta Torácica/fisiología , Hipoxia de la Célula/fisiología , Vasodilatación/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Adenosina/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Arterias/efectos de los fármacos , Dinoprost/farmacología , Gliburida/farmacología , Guanilato Ciclasa/farmacología , Masculino , Ratones , Norepinefrina/farmacología , Potasio/farmacología , Prostaglandinas/farmacología , Receptores Citoplasmáticos y Nucleares/farmacología , Guanilil Ciclasa Soluble , Sulfuros/farmacología , Tetraetilamonio/farmacología , Teofilina/análogos & derivados , Teofilina/farmacologíaRESUMEN
We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (K(Ca)), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [-90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of K(Ca) channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.
Asunto(s)
Endotelio Vascular/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/biosíntesis , Arterias Mesentéricas/metabolismo , Canales de Potasio Calcio-Activados/biosíntesis , Acetilcolina/farmacología , Animales , Factores Biológicos/fisiología , Inhibidores de la Ciclooxigenasa/farmacología , Endotelio Vascular/efectos de los fármacos , Guanilato Ciclasa/farmacología , Indoles/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/agonistas , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Masculino , Arterias Mesentéricas/efectos de los fármacos , Contracción Muscular , Relajación Muscular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oximas/farmacología , Canales de Potasio Calcio-Activados/agonistas , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Ratas , Ratas Endogámicas WKY , Receptores Citoplasmáticos y Nucleares/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Guanilil Ciclasa Soluble , Circulación Esplácnica , Estrés MecánicoRESUMEN
Pulmonary arterial hypertension (PAH) is a severe, progressive and often fatal disease for which only a limited number of drugs have proven to be of clinical benefit. One of the therapeutic approaches for this disease is the induction of pulmonary vasodilation via stimulation of the nitric oxide (NO)-mediated pathway. Abnormalities in the NO/soluble guanylate cyclase (sGC) axis and enhanced PDE5 activity render currently available drugs ineffective in many patients with PAH. Bayer AG is developing riociguat, an oral sGC stimulator, for the potential treatment of patients with PAH. Treatment with riociguat abrogated the severity of pulmonary hypertension in rodent models of the disease. Published data from phase I and II clinical trials demonstrated that riociguat was well tolerated, with single doses significantly decreasing pulmonary arterial pressure and increasing cardiac output and physical-exercise tolerance in patients with PAH. A decrease in systemic arterial diastolic pressure was the only significant side effect reported. Ongoing phase II and III trials for riociguat have been designed to address the long-term safety and clinical effectiveness of the drug in different types of pulmonary hypertension. Should the results of these trials demonstrate that riociguat is superior to current therapies, such as cyclic AMP-dependent drugs and endothelin receptor antagonists, the drug could become the preferred pharmacological treatment for patients with PAH.
Asunto(s)
Antihipertensivos/uso terapéutico , Guanilato Ciclasa/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Receptores Citoplasmáticos y Nucleares/uso terapéutico , Vasodilatadores/uso terapéutico , Animales , Antihipertensivos/efectos adversos , Antihipertensivos/química , Antihipertensivos/farmacología , Química Farmacéutica , Ensayos Clínicos como Asunto , Diástole/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Guanilato Ciclasa/efectos adversos , Guanilato Ciclasa/química , Guanilato Ciclasa/farmacología , Humanos , Hipertensión Pulmonar/enzimología , Masculino , Estructura Molecular , Pirazoles/efectos adversos , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/efectos adversos , Pirimidinas/química , Pirimidinas/farmacología , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores Citoplasmáticos y Nucleares/efectos adversos , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/farmacología , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Guanilil Ciclasa Soluble , Relación Estructura-Actividad , Comprimidos , Resultado del Tratamiento , Vasodilatadores/administración & dosificaciónRESUMEN
We have shown that the peripheral and spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase-cGMP pathways play an important role in antinociception in the rat experimental formalin model. Our objective was to determine if there is synergism between peripheral (paw) and spinal HO-CO pathways in nociception. Rats were handled and adapted to the experimental environment for a few days before the formalin test, in which 50 microL of a 1% formalin was injected subcutaneously into the dorsal surface of the right hind paw. The animals were then observed for 1 h and the frequency of flinching behavior was taken to represent the nociceptive response. Thirty minutes before the test, rats were pretreated with intrathecal injections of the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is a substrate of the HO pathway. The paw treatments took place 20 min before the test. Low doses of ZnDPBG did not increase nociception, while a low heme-lysinate dose did not change flinching behavior after paw or spinal injections. Combined subactive spinal (50 nmol) and peripheral (40 nmol) low doses of ZnDPBG induced hypernociception (increase of 80% in the first and 25% in the second phase flinching), whereas combined spinal-peripheral heme-lysinate (50 and 30 nmol) led to second phase antinociception (40% reduction in flinching). These findings suggest a synergy between the peripheral and spinal HO-CO pathways. Local activation of the HO system probably regulates the nociception initiation in peripheral tissue and participates in buffering the emerging nociceptive signals at the peripheral and spinal sites of action. In short, an antinociceptive synergy exists between peripheral and spinal HO pathways, which may reduce the doses required and side effects.
Asunto(s)
Monóxido de Carbono/metabolismo , Guanilato Ciclasa/administración & dosificación , Hemo Oxigenasa (Desciclizante)/metabolismo , Nociceptores/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/administración & dosificación , Médula Espinal/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Guanilato Ciclasa/farmacología , Hemo Oxigenasa (Desciclizante)/efectos de los fármacos , Inyecciones Espinales , Masculino , Nociceptores/fisiología , Ratas , Ratas Wistar , Transducción de Señal , Guanilil Ciclasa Soluble , Médula Espinal/fisiologíaRESUMEN
We have shown that the peripheral and spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase-cGMP pathways play an important role in antinociception in the rat experimental formalin model. Our objective was to determine if there is synergism between peripheral (paw) and spinal HO-CO pathways in nociception. Rats were handled and adapted to the experimental environment for a few days before the formalin test, in which 50 µL of a 1 percent formalin was injected subcutaneously into the dorsal surface of the right hind paw. The animals were then observed for 1 h and the frequency of flinching behavior was taken to represent the nociceptive response. Thirty minutes before the test, rats were pretreated with intrathecal injections of the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is a substrate of the HO pathway. The paw treatments took place 20 min before the test. Low doses of ZnDPBG did not increase nociception, while a low heme-lysinate dose did not change flinching behavior after paw or spinal injections. Combined subactive spinal (50 nmol) and peripheral (40 nmol) low doses of ZnDPBG induced hypernociception (increase of 80 percent in the first and 25 percent in the second phase flinching), whereas combined spinal-peripheral heme-lysinate (50 and 30 nmol) led to second phase antinociception (40 percent reduction in flinching). These findings suggest a synergy between the peripheral and spinal HO-CO pathways. Local activation of the HO system probably regulates the nociception initiation in peripheral tissue and participates in buffering the emerging nociceptive signals at the peripheral and spinal sites of action. In short, an antinociceptive synergy exists between peripheral and spinal HO pathways, which may reduce the doses required and side effects.