RESUMEN

Essentials A rapid test to detect thrombin inhibition by dabigatran would be valuable in acute situations. A thrombin-based trigger was applied in whole blood using rotation thromboelastometry. Effects of dabigatran were assessed in vitro and in samples from patients on dabigatran. The test produced data rapidly and was sensitive to dabigatran concentrations from 20 to 500 ng mL-1 . SUMMARY: Background Rapid determination of the anticoagulant effect of dabigatran is essential in emergency situations. Objective To study a viscoelastic test (rotational thromboelastometry [ROTEM]) for rapid determination of dabigatran effects in whole blood samples. Method ROTEM measurements were performed with comparison of two triggers (thrombin-based versus the commercial tissue factor-based trigger Ex-tem) in samples from 10 healthy donors spiked with dabigatran (20-500 ng mL-1 ) and in samples from 35 patients receiving dabigatran treatment; 10 healthy subjects served as controls. Clotting time (CT) and the difference in CT without versus with addition of the dabigatran antidote idarucizumab (CTdiff ) were measured. Addition of idarucizumab reveals the contribution of dabigatran to ROTEM measurements and its potential reversibility. Results In vitro studies showed that thrombin CT and thrombin CTdiff were more sensitive than Ex-tem CT and Ex-tem CTdiff in detecting dabigatran in whole blood samples. In patient samples, when thrombin CT and thrombin CTdiff were used, it was possible to detect dabigatran with a cut-off of dabigatran at 20 ng mL-1 , whereas, when Ex-tem CT and Ex-tem CTdiff were used, the method was less sensitive. Data from patient samples were obtained within 15 min of blood sampling. Conclusions ROTEM CT with a thrombin-based trigger is more sensitive to dabigatran effects than Ex-tem CT, and detects anticoagulant effects of drug concentrations in the low-very low therapeutic range. Analysis with idarucizumab (CTdiff ) reveals dabigatran-specific effects. As data are rapidly obtained, this method could, with further development and validation of its performance, be suitable for detecting clinically significant dabigatran effects in emergency situations.

Asunto(s)

Antitrombinas/sangre , Fibrilación Atrial/tratamiento farmacológico , Coagulación Sanguínea/efectos de los fármacos , Dabigatrán/sangre , Monitoreo de Drogas/métodos , Pruebas en el Punto de Atención , Tromboelastografía , Trombina/metabolismo , Adulto , Anciano , Antitrombinas/uso terapéutico , Fibrilación Atrial/sangre , Fibrilación Atrial/diagnóstico , Estudios de Casos y Controles , Dabigatrán/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Prueba de Estudio Conceptual , Flujo de Trabajo

RESUMEN

Essentials It is unclear if platelet micro-RNAs can regulate de novo protein synthesis of platelets. Platelet de novo protein synthesis of thrombospondin-1 (TSP-1) was induced by thrombin. Thrombin stimulation in vitro altered platelet microRNA profiles, including decreased miR-27b. Decreased miR-27b hampers platelet angiogenic activities via enhancing de novo TSP-1 synthesis. SUMMARY: Background Platelets can synthesize proteins upon activation. Platelets contain a number of microRNAs (miRNA) and a fully functional miRNA effector machinery. It is, however, unclear if platelet miRNAs can regulate protein synthesis of platelets, and whether the regulation may produce a physiological impact. Objectives To investigate if and how platelet miRNAs regulate de novo syntheses of angiogenic regulators and subsequently modulate platelet angiogenic activities. Methods and Results Microarray-based miRNA profiling showed that thrombin stimulation in vitro down- or up-regulated a number of platelet miRNAs, both in the total platelet miRNAs and in Ago2-associated miRNAs. Among those altered miRNAs, miR-27b was down-regulated in both the total and Ago2-immunoprecipitated miRNA profiles of platelets, which was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Using western blotting assays, we showed that thrombin induced platelet de novo synthesis of thrombospondin-1, and that the level of thrombospondin-1 synthesis could reach a level of 3-5-fold higher than that before thrombin stimulation. With either the platelet precursor megakaryocyte cell line MEG-01 cells or mature platelets, we demonstrated that transfection of miR-27b mimic, but not the negative control of miRNA mimic, markedly reduced thrombospondin-1 protein levels. The latter subsequently enhanced platelet-dependent endothelial tube formation on matrigel. Conclusions Thrombin stimulation in vitro reduces platelet miR-27b levels that may markedly enhance thrombin-evoked platelet de novo synthesis of thrombospondin-1. Elevation of platelet miR-27b by transfection inhibits thrombospondin-1 synthesis, and subsequently enhances platelet pro-angiogenic activities. Hence, platelet activation-dependent reduction of miR-27b levels may represent a novel negative regulatory mechanism of platelet angiogenic activities.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Plaquetas/efectos de los fármacos , MicroARNs/sangre , Neovascularización Fisiológica/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Trombina/farmacología , Trombospondina 1/biosíntesis , Adulto , Proteínas Argonautas/sangre , Plaquetas/metabolismo , Línea Celular , Células Progenitoras Endoteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Transducción de Señal , Trombospondina 1/sangre , Trombospondina 1/genética , Adulto Joven

RESUMEN

Although there is no doubt about the scientific value of randomized controlled clinical trials, they are usually conducted in selected populations different from those treated in clinical practice. Therefore, it is important to optimize real-time postmarketing evaluation of the effectiveness, safety, and cost of new drugs. Using electronic health records and administrative health databases from a well-defined region with universal access to healthcare, we have built a framework for real-time sequential monitoring of the effectiveness of newly marketed drugs in routine care. We chose the antiarrhythmic agent dronedarone as the study drug and flecainide as the comparator drug for illustration of the model. We demonstrate that this model produces consistent results with increasing precision over time as data accumulates in the clinical systems. We believe that use of this model at the introduction of new drugs can provide complementary evidence, especially in settings of adaptive licensing of new drugs.

Asunto(s)

Antiarrítmicos/uso terapéutico , Dronedarona/uso terapéutico , Monitoreo de Drogas/métodos , Anciano , Anciano de 80 o más Años , Fibrilación Atrial/tratamiento farmacológico , Estudios de Cohortes , Sistemas de Computación , Bases de Datos Factuales , Aprobación de Drogas , Registros Electrónicos de Salud , Femenino , Flecainida/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Vigilancia de Productos Comercializados , Puntaje de Propensión , Suecia , Resultado del Tratamiento

RESUMEN

UNLABELLED: Essentials Simple and fast assaying of different anticoagulants (ACs) is useful in emergent situations. We used highly diluted prothrombin time (dPT) or highly diluted Fiix-PT (dFiix-PT) to assay ACs. Both tests could quantify target specific anticoagulants and warfarin anticoagulation. Improved results were consistently observed with the dFiix-PT compared with the dPT. SUMMARY: Background Assaying anticoagulants is useful in emergency situations or before surgery. Different specific assays are currently needed depending on the anticoagulant. Objectives We hypothesized that levels of warfarin, dabigatran, rivaroxaban, apixaban, and heparins could be measured with use of the diluted prothrombin time (dPT) and diluted Fiix-PT (dFiix-PT), using highly diluted thromboplastin (TP). The latter test is affected only by reduced levels of active factors II and X but corrects test plasma for other deficiencies Methods Increasing TP dilutions were used to identify suitable dilutions to measure dabigatran, rivaroxaban, apixaban, unfractionated heparin (UFH), and enoxaparin. Calibrators containing known amounts of direct oral anticoagulants (DOACs) were used to make standard curves. Citrated plasma samples were obtained from patients taking warfarin or DOACs with known drug concentrations as determined by specific assays. Results The dFiix-PT at a TP dilution of 1:1156 could be used to measure all of the drugs tested at therapeutic concentrations except for fondaparinux. The dPT achieved the same but required two TP dilutions (1:750 and 1:300). The warfarin effect could be assessed by using dFiix-PT at 1:1156 with a PT ratio identical to the international normalized ratio. Six different TPs yielded similar results, but two were less sensitive. Dabigatran, rivaroxaban, and apixaban could be accurately measured in patient samples using both dilute PT assays, but a better correlation was consistently observed between the dFiix-PT and specific assays than with the dPT. Conclusion The dFiix-PT using a single dilution of TP may be suitable to assess the anticoagulant effects of warfarin, dabigatran, rivaroxaban, apixaban, heparin, and enoxaparin.

Asunto(s)

Pruebas de Coagulación Sanguínea/métodos , Dabigatrán/sangre , Enoxaparina/sangre , Heparina/sangre , Pirazoles/sangre , Piridonas/sangre , Rivaroxabán/sangre , Warfarina/sangre , Anticoagulantes/química , Donantes de Sangre , Calibración , Factor X/química , Femenino , Fondaparinux , Humanos , Relación Normalizada Internacional , Masculino , Polisacáridos/sangre , Protrombina/química , Tiempo de Protrombina , Reproducibilidad de los Resultados , Tromboplastina/química

RESUMEN

Ways to monitor dabigatran etexilate (DE) therapy would be useful in certain situations. Functional assays such as aPTT or Hemoclot® Thrombin Inhibitor (HTI) have been proposed to evaluate dabigatran concentrations, but previous findings are based on in vitro studies and results must be confirmed in clinical samples. The aim of this study was to compare aPTT and HTI measurements with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements of dabigatran in plasma samples from DE treated patients. Seventy-one plasma samples were included. aPTT was performed using STA-CKPrest® and SynthASil®. HTI was performed according to instructions from the manufacturer. The LC-MS/MS method utilised dabigatran-d3 as internal standard. The plasma concentration range was 0 to 645 ng/ml as measured by LC-MS/MS. Overall, the HTI and LC-MS/MS analyses correlated well (r²=0.97). The Bland-Altman analysis showed a mean difference of 9 ng/ml (SD: 20 ng/ml). However, the HTI performed poorly at concentrations <50 ng/ml. LC-MS/MS was sensitive (limit of quantification 1.1 ng/ml) and specific for dabigatran. The aPTT methods did not correlate well with plasma concentrations measured by LC-MS/MS (r² = 0.59 with SynthASil® and 0.50 with STA-CKPrest®). In conclusion, the poor sensitivity, the important inter-individual variability, and the poor correlation with LC-MS/MS preclude the use of aPTT to estimate dabigatran concentrations. Due to its small inter-individual variability and good agreement with LC-MS/MS measurements, we recommend the use of HTI assays to rather accurately estimate concentrations of dabigatran >50 ng/ml. Quantification of lower dabigatran levels in DE-treated patients requires the "reference" LC-MS/MS method.

Asunto(s)

Bencimidazoles/administración & dosificación , Monitoreo de Drogas/métodos , Tiempo de Tromboplastina Parcial/métodos , Piridinas/administración & dosificación , Tiempo de Trombina/métodos , Anticoagulantes/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Calibración , Cromatografía Liquida/métodos , Dabigatrán , Voluntarios Sanos , Humanos , Valores de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , Trombina/antagonistas & inhibidores

RESUMEN

BACKGROUND: Encouraging the prescribing of ACEIs first line vs. angiotensin receptor blockers (ARBs) has been a health authority focus with generic ACEIs as ACEIs and ARBs have similar effectiveness and there is limited coughing with ACEIs. This includes Sweden with its multiple initiatives keeping expenditure on renin-angiotensin inhibitor drugs similar between 2001 and 2007 despite appreciably increased volumes. Generic losartan became available and was reimbursed in March 2010 providing further opportunities for the authorities in Sweden to save costs with all ARBs seen as similar in managing hypertension and CHF at appropriate doses. AIMS: The main aim of this study was to assess changes in the utilisation of losartan vs. other single ARBs after generic losartan alongside accompanying demand-side measures. Additional aims were to (i) assess changes in the price of generic losartan and single ARB expenditure over time; (ii) suggest additional programmes, if needed; and (iii) analyse utilisation of ARB FDCs and compare with ACEI FDCs. METHODS: Retrospective observational study using an interrupted time series design. RESULTS: Multiple demand-side measures introduced among the 21 Counties in Sweden significantly enhanced the utilisation of generic losartan, growing from 26% to 27% of total ARBs (DDD basis) before generic losartan to 40% by August 2011. Losartan was principally generics (97% by August 2011). Expenditure/DDD for generic losartan was 10% of the pre-patent loss price in August 2011. This reduced total single ARB expenditure by 26% by the study end despite a 16% increase in utilisation. Greater utilisation of ARB FDCs than seen with ACEI FDCs. This may be due to similarities in prices between single and FDC ARBs. DISCUSSION: Multiple demand-side measures appreciably enhanced ARB prescribing efficiency, mirroring other studies. No significant increase in losartan utilisation following generics was seen in European countries where no specific measures were instigated. Losartan price reduction was in line with expectations. CONCLUSION: Multiple and intensive demand-side measures are needed to change physician prescribing habits. Authorities cannot rely on physicians transferring their activities from one class to another without interventions.

Asunto(s)

Antagonistas de Receptores de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Losartán/uso terapéutico , Antagonistas de Receptores de Angiotensina/economía , Antihipertensivos/economía , Ahorro de Costo , Revisión de la Utilización de Medicamentos , Medicamentos Genéricos/economía , Medicamentos Genéricos/uso terapéutico , Gastos en Salud , Humanos , Hipertensión/economía , Losartán/economía , Pautas de la Práctica en Medicina/economía , Análisis de Regresión , Estudios Retrospectivos , Suecia

RESUMEN

One-lung ventilation (OLV) is applied during esophagectomy to improve exposure during the thoracic part of the operation. Collapse of lung tissue, shunting of pulmonary blood flow, and changes in alveolar oxygenation during and after OLV may possibly induce an ischemia-reperfusion response in the lung, which may affect the pulmonary endothelium. Such a reaction might thereby contribute to the frequently occurring respiratory complications among these patients. In this small trial, 30 patients were randomized to either OLV (n= 16) or two-lung ventilation (TLV, n= 14) during esophagectomy. Central venous and arterial plasma samples were taken before and after OLV/TLV for analysis of nitrite and a metabolite of nitric oxide (NO), and also during the 1st, 2nd, 3rd, and 10th postoperative day for analysis of endothelin, another endothelium-derived vasoactive mediator. Lung biopsies were taken before and after OLV or TLV, and analyzed regarding immunofluorescence for isoform of NO synthase, a protein upregulated during inflammatory response and also vascular congestion. No changes in lung isoform of NO synthase immunofluorescence or vascular congestion were registered after neither OLV nor TLV. Plasma nitrite and endothelin levels were similar in the two study groups. We conclude that OLV does not seem to have any influence on key regulators of pulmonary vascular tone and inflammation, i.e. NO and endothelin. From this perspective, OLV seems to be a safe method, which defends its clinical position to facilitate surgical exposure during thoracoabdominal esophagectomy.

Asunto(s)

Endotelina-1/metabolismo , Esofagectomía/métodos , Lesión Pulmonar/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , Ventilación Unipulmonar/métodos , Daño por Reperfusión/metabolismo , Adenocarcinoma/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/metabolismo , Carcinoma de Células Escamosas/cirugía , Estudios de Cohortes , Neoplasias Esofágicas/cirugía , Carcinoma de Células Escamosas de Esófago , Femenino , Humanos , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Lesión Pulmonar/etiología , Masculino , Persona de Mediana Edad , Ventilación Unipulmonar/efectos adversos , Daño por Reperfusión/etiología , Respiración Artificial/métodos

Asunto(s)

Inhibidores de Agregación Plaquetaria/administración & dosificación , Recuento de Plaquetas/métodos , Recuento de Plaquetas/estadística & datos numéricos , Sistemas de Atención de Punto/estadística & datos numéricos , Trombosis/sangre , Trombosis/epidemiología , Trombosis/prevención & control , Ticlopidina/análogos & derivados , Clopidogrel , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Suecia/epidemiología , Ticlopidina/administración & dosificación

RESUMEN

AIM: The vascular endothelium produces several substances, including nitric oxide (NO) and endothelin-1 (ET-1), which participate in the regulation of vascular tone in humans. Both these substances may exert other actions of importance for cardiovascular disease, e.g. effects on vascular smooth muscle cell proliferation and inflammation, and NO inhibits platelet function. Experiments were designed to investigate the effect of ET-1 on endothelium-dependent vasodilatation and attenuation of platelet activation. METHODS: In 25 healthy male subjects (25 +/- 1 years), forearm blood flow was measured by venous occlusion plethysmography, and platelet activity was assessed by whole blood flow cytometry (platelet fibrinogen binding and P-selectin expression) in unstimulated and adenosine diphosphate (ADP)-stimulated samples during administration of ET-1, the endothelium-dependent vasodilator acetylcholine and the NO synthase inhibitor l-NMMA. RESULTS: Acetylcholine increased forearm blood flow and significantly inhibited platelet activation in both unstimulated and ADP-stimulated samples. In samples stimulated with 0.3 microm ADP, fibrinogen binding decreased from 41 +/- 4% to 31 +/- 3% (P < 0.01, n = 11) after acetylcholine administration. The vasodilator response to acetylcholine was significantly impaired during infusions of ET-1 and l-NMMA. ET-1 did not affect platelet activity per se, whereas l-NMMA increased platelet P-selectin expression. Both ET-1 and l-NMMA attenuated the acetylcholine-induced inhibition of platelet activity. CONCLUSIONS: Our study indicates that, further to inhibiting endothelium-dependent vasodilatation, ET-1 may also attenuate endothelium-dependent inhibition of platelet activation induced by acetylcholine. An enhanced ET-1 activity, as suggested in endothelial dysfunction, may affect endothelium-dependent platelet modulation and thereby have pathophysiological implications.

Asunto(s)

Plaquetas/patología , Endotelinas/farmacología , Endotelio/fisiología , Flujo Sanguíneo Regional/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Adulto , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Vasos Sanguíneos , Arteria Braquial/fisiología , Endotelina-1/farmacología , Endotelio Vascular , Antebrazo/fisiología , Humanos , Masculino , Óxido Nítrico/farmacología , Flujo Sanguíneo Regional/fisiología , Vasodilatación/fisiología , Vasodilatadores , omega-N-Metilarginina/farmacología

RESUMEN

Diminished levels of L-arginine and endothelial nitric oxide synthase (eNOS) uncoupling through deficiency of tetrahydrobiopterin (BH(4)) may contribute to endothelial dysfunction. We investigated the effect of L-arginine and BH(4) administration on ischemia-reperfusion (I/R)-induced endothelial dysfunction in patients with type 2 diabetes and coronary artery disease (CAD). Forearm blood flow was measured by venous occlusion plethysmography in 12 patients with type 2 diabetes or impaired glucose tolerance and CAD. Forearm ischemia was induced for 20 min, followed by 60 min of reperfusion. The patients received a 15 min intra-brachial infusion of L-arginine (20 mg/min) and BH(4) (500 microg/min) or 0.9% saline starting at 15 min of ischemia on two separate study occasions. Compared with pre-ischemia the endothelium-dependent vasodilatation (EDV) induced by acetylcholine was significantly reduced at 15 and 30 min of reperfusion when saline was infused (P<0.001), but not following L-arginine and BH(4) infusion. EDV was also significantly less reduced at 15 and 30 min of reperfusion following L-arginine and BH(4) infusion, compared to saline infusion (P<0.02). Endothelium-independent vasodilatation (EIDV) induced by nitroprusside was unaffected by I/R. Venous total biopterin levels in the infused arm increased from 37+/-7 at baseline to 6644+/-1240 nmol/l during infusion of L-arginine and BH(4) (P<0.0001), whereas there was no difference in biopterin levels during saline infusion. In conclusion L-arginine and BH(4) supplementation reduces I/R-induced endothelial dysfunction, a finding which may represent a novel treatment strategy to limit I/R injury in patients with type 2 diabetes and CAD.

Asunto(s)

Arginina/administración & dosificación , Biopterinas/análogos & derivados , Fármacos Cardiovasculares/administración & dosificación , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Endotelio Vascular/efectos de los fármacos , Antebrazo/irrigación sanguínea , Daño por Reperfusión/prevención & control , Vasodilatación/efectos de los fármacos , Anciano , Biopterinas/administración & dosificación , Velocidad del Flujo Sanguíneo , Enfermedad de la Arteria Coronaria/fisiopatología , Estudios Cruzados , Diabetes Mellitus Tipo 2/fisiopatología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Endotelio Vascular/fisiopatología , Femenino , Humanos , Infusiones Intraarteriales , Masculino , Flujo Sanguíneo Regional , Daño por Reperfusión/fisiopatología , Resultado del Tratamiento , Vasodilatadores/administración & dosificación

RESUMEN

The effects of the first selective, non-peptide, NPY Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamid (BIIE0246) were studied on splenic vascular responses evoked in the pig in vivo. BIIE0246 abolished the splenic vasoconstrictor response to the NPY Y2 receptor agonist N-acetyl[Leu25Leu31]NPY(24-36), but did not affect the response to the NPY Y1 receptor agonist [Leu31Pro34]NPY, which in turn was abolished by the selective NPY Y1 receptor antagonist (2R)-5-([amino(imino)methyl]amino)-2-[(2,2-diphenylacetyl)amino]-N-[(IR)-1-(4-hydroxyphenyl)ethyl]-pentanamide (H 409/22). Furthermore, the PYY-evoked splenic vasoconstrictor response was partially antagonized by BIIE0246 and subsequently almost abolished by the addition of H 409/22. It is concluded that BIIE0246 exerts selective (vs the NPY Y1 receptor) NPY Y2 receptor antagonism, and thus represents an interesting tool for classification of NPY receptors, in vivo. In addition, evidence for NPY Y2 receptor mediated vasoconstriction was presented. Furthermore, both NPY Y1 and Y2 receptors are involved in the splenic vasoconstrictor response to PYY.

Asunto(s)

Arginina/análogos & derivados , Neuropéptido Y/metabolismo , Receptores de Neuropéptido Y/metabolismo , Bazo/metabolismo , Amidas/farmacología , Animales , Arginina/farmacología , Benzazepinas/farmacología , Masculino , Receptores de Neuropéptido Y/antagonistas & inhibidores , Porcinos

RESUMEN

BIIE0246, a recently introduced non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was pharmacologically characterized in vivo, on vascular responses evoked in the anaesthetized pig. The NPY Y(2) receptor agonist N-acetyl[Leu(28)Leu(31)]NPY(24-36) evoked dose-dependent vasoconstriction in spleen. These vascular responses were potently and dose-dependently antagonized by BIIE0246. Significant inhibition was seen already at 1 nmol kg(-1), whereas at 100 nmol kg(-1) of BIIE0246 these responses were completely abolished. The ID(50) value for this antagonism was 2.1 nmol kg(-1). Peptide YY (PYY) evoked dose-dependent vasoconstriction in both kidney and spleen, vascular responses mediated by the NPY Y(1) receptor and both NPY Y(1) and Y(2) receptors, respectively. Only the splenic response was inhibited by BIIE0246, the effect of which reached significance at 1 nmol kg(-1). Already 30 min after the last dose of BIIE0246 there was a significant recovery of the PYY-evoked splenic vasoconstriction, and a further 60 min later, this response was no longer significantly inhibited compared to control. BIIE0246 (100 nmol kg(-1)) did not affect renal and splenic vasoconstrictor responses either to the NPY Y(1) receptor agonist [Leu(31)Pro(34)]NPY, the alpha(1)-adrenoceptor agonist phenylephrine, the P2X(1)-purinoceptor agonist alpha,beta-methylene ATP or angiotensin II, demonstrating both selectivity and specificity for the NPY Y(2) receptor in vivo. It is concluded that BIIE0246 is a highly potent and selective NPY Y(2) receptor antagonist, albeit with rather short duration of action, in vivo. BIIE0246 thus represents the first interesting tool for studies on NPY Y(2) receptor-mediated transmission in vivo.

Asunto(s)

Arginina/análogos & derivados , Arginina/farmacología , Benzazepinas/farmacología , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Receptores de Neuropéptido Y/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Riñón/irrigación sanguínea , Masculino , Neuropéptido Y/análogos & derivados , Neuropéptido Y/farmacología , Péptido YY/farmacología , Receptores de Neuropéptido Y/agonistas , Bazo/irrigación sanguínea , Porcinos , Vasoconstricción/efectos de los fármacos

RESUMEN

The object of the present paper was to investigate the in vivo pharmacological profile of the dihydropyridine neuropeptide Y Y(1) receptor antagonist 1,4-Dihydro-4-[3-[[[[3-[spiro(indene-4,1'-piperidin-1-yl)]propyl]amino]carbonyl]amino]phenyl]-2,6-dimethyl-3,5-pyridine dicarboxylic acid, dimethylester (H 394/84). The renal vasoconstrictor response to neuropeptide Y in anaesthetized rats was dose-dependently antagonized by H 394/84 (ID(50) value=41+/-4 nmol/kg/min), whereas the renal vascular responses to noradrenaline and angiotensin II were only slightly affected by H 394/84 (500 nmol/kg/min). In pigs pretreated with reserpine and transection of sympathetic nerves (depleted of noradrenaline), H 394/84 dose-dependently antagonized renal and femoral vasoconstrictor responses evoked by sympathetic nerve activation (neuronally released neuropeptide Y) and exogenous neuropeptide Y. Significant inhibition was seen already at 1.0 nmol/kg/min, when plasma levels of the antagonist reached 29+/-4 nM. Around 70% of the antagonism remained 90 min after H 394/84 was given. The disposition of H 394/84 fits a biexponential model with initial and terminal half-lives of 2.6 and 48 min, respectively. H 394/84 (100 nmol/kg/min) did not inhibit vascular responses to neuropeptide Y Y(2) receptor-, alpha-adrenoceptor- or purinoceptor-activation in the pig in vivo. It is concluded that H 394/84 is a potent neuropeptide Y Y(1) receptor antagonist with rather long duration of action in vivo. The selectivity and specificity in vivo is more than 100-fold, and H 394/84 antagonizes vascular responses to exogenous and endogenous, neuronally released, neuropeptide Y with similar potency.

Asunto(s)

Riñón/efectos de los fármacos , Neuropéptido Y/antagonistas & inhibidores , Niacina/farmacología , Compuestos de Fenilurea/farmacología , Receptores de Neuropéptido Y/antagonistas & inhibidores , Vasoconstricción/efectos de los fármacos , Anestesia , Angiotensina II/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Semivida , Frecuencia Cardíaca/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Riñón/irrigación sanguínea , Masculino , Neuropéptido Y/farmacología , Niacina/análogos & derivados , Niacina/sangre , Niacina/química , Norepinefrina/farmacología , Farmacocinética , Compuestos de Fenilurea/sangre , Compuestos de Fenilurea/química , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/metabolismo , Circulación Renal/efectos de los fármacos , Reserpina/farmacología , Especificidad por Sustrato , Porcinos , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiología

RESUMEN

The object of the present study was to investigate the involvement of nitric oxide (NO) in the regulation of renal vasoconstrictor responses to sympathetic nerve activation, and each of the known sympathetic cotransmitters separately, in the pig in vivo. Renal vasoconstrictor responses were elicited by sympathetic nerve stimulation, the alpha(1)-adrenoceptor agonist phenylephrine (10 nmol kg(-1), injected iv), neuropeptide Y (NPY, 120 pmol kg(-1), iv) acting on the NPY Y(1) receptor, and the stable ATP-analogue alpha,beta-methylene ATP (mATP, 10 nmol kg(-1)) presumably acting on the P2X(1) purinoceptor. Infusion of the NO-donor sodium nitroprusside, at a dose (0.1 mg kg(-1) h(-1), iv) that elevated renal blood flow (by 14 +/- 7%) and lowered mean arterial pressure (by 30 +/- 5%), inhibited renal vasoconstrictor responses to sympathetic nerve stimulation, phenylephrine, and NPY, but not to mATP. In contrast, injection of the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester, at a dose (10 mg kg(-1), iv) that lowered renal blood flow (by 47 +/- 4%) and elevated mean arterial pressure (by 28 +/- 8%), potentiated the renal vasoconstriction evoked by sympathetic nerve stimulation, phenylephrine, and NPY, but not mATP. It is concluded that endogenous NO may function as an inhibitory modulator of vasoconstrictor responses to the sympathetic cotransmitters norepinephrine and NPY. In contrast, NO seems not to modify vasoconstrictor responses to the sympathetic cotransmitter ATP, a discrepancy that may be due to differences in the types of receptors and intracellular effector mechanisms.

Asunto(s)

Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Nitroprusiato/farmacología , Porcinos/fisiología , Vasoconstricción/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Estimulación Eléctrica , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Riñón/inervación , Riñón/fisiología , Masculino , Neuropéptido Y/farmacología , Fenilefrina/farmacología , Circulación Renal/efectos de los fármacos , Sistema Nervioso Simpático/fisiología

RESUMEN

The object of the present study was to investigate the effects of the sympathetic cotransmitter neuropeptide Y (NPY), and the closely related gut hormone peptide YY (PYY), on splanchnic blood flow regulation in the anaesthetized pig in vivo. Systemic injections of NPY, PYY and the NPY Y(1) receptor agonist [Leu(31)Pro(34)]NPY (470 pmol kg(-1) each) evoked pressor and mesenteric vasoconstrictor responses that were largely abolished by the selective NPY Y(1) receptor antagonist H 409/22 (60 nmol kg(-1) min(-1)). In contrast, the NPY Y(2) receptor agonist N-acetyl[Leu(28)Leu(31)]NPY(24-36) (1.1 nmol kg(-1)), a dose of which potently evoked splenic NPY Y(2) receptor mediated (not affected by H 409/22) vasoconstriction, did not evoke any mesenteric vascular response. Mesenteric vascular responses to angiotensin II (10 pmol kg(-1)), alpha,beta-methylene ATP (10 nmol kg(-1)) and the alpha(1)-adrenoceptor agonist phenylephrine (15 nmol kg(-1)), were not inhibited by H 409/22. It is concluded that NPY and PYY evokes porcine mesenteric vasoconstriction mediated by the NPY Y(1) receptor subtype, as demonstrated by selective and specific inhibition exerted by the NPY Y(1) receptor antagonist H 409/22, in vivo.

Asunto(s)

Arteria Mesentérica Superior/fisiología , Neuropéptido Y/farmacología , Péptido YY/farmacología , Receptores de Neuropéptido Y/fisiología , Circulación Esplácnica/fisiología , Vasoconstricción/fisiología , Angiotensina II/farmacología , Animales , Femenino , Masculino , Arteria Mesentérica Superior/efectos de los fármacos , Neuropéptido Y/análogos & derivados , Fragmentos de Péptidos/farmacología , Fenilefrina/farmacología , Receptores de Neuropéptido Y/agonistas , Receptores de Neuropéptido Y/antagonistas & inhibidores , Circulación Esplácnica/efectos de los fármacos , Porcinos , Vasoconstricción/efectos de los fármacos

RESUMEN

We studied the effects of the novel neuropeptide Y (NPY) Y1 receptor antagonist H 409/22, and its inactive enantiomer H 510/45, on vascular responses evoked by endogenous and exogenous NPY in the pig in vivo. H 409/22 and H 510/45 were given as 30-min infusions, and the antagonistic effects and circulating plasma concentrations were measured. The initial and terminal half-lives of H 409/22 in plasma were approximately 3 and 30 min, respectively. In pigs pretreated with reserpine and transection of sympathetic nerves (depletion of noradrenaline), sympathetic nerve stimulation evoked nonadrenergic vasoconstrictor responses in kidney and hindlimb, mediated by neuronally released NPY. Significant inhibition of these vasoconstrictor responses, as well as of vascular responses to injections of exogenous NPY, were seen during a low-dose infusion of H 409/22 (1.8 nmol/kg/min), when plasma levels of the antagonist reached 77 +/- 8 nM. Greatest inhibitory effects were seen at the highest dose of H 409/22 (180 nmol/kg/min, giving plasma levels of 7.4 +/- 0.6 microM) when all vascular responses evoked by NPY were strongly attenuated or largely abolished. H 510/45 did not affect any of the vascular responses studied. It is concluded that H 409/22 potently and dose-dependently antagonizes vascular responses to exogenous and endogenous NPY in the pig, and thus represents an interesting tool for studies on NPY Y1 receptor-mediated effects in vivo.

Asunto(s)

Amidas/farmacología , Receptores de Neuropéptido Y/antagonistas & inhibidores , Amidas/sangre , Animales , Presión Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Miembro Posterior/irrigación sanguínea , Técnicas In Vitro , Masculino , Neuropéptido Y/antagonistas & inhibidores , Neuropéptido Y/farmacología , Flujo Sanguíneo Regional/efectos de los fármacos , Circulación Renal/efectos de los fármacos , Reserpina/farmacología , Estereoisomerismo , Porcinos , Sistema Nervioso Simpático/efectos de los fármacos , Simpaticolíticos/farmacología , Vasoconstricción/efectos de los fármacos

RESUMEN

Neuropeptide Y (NPY) -receptor subtypes were studied in the rat kidney in vivo by systemic administration of NPY, the two agonists [Leu(31), Pro(34)]NPY (Y1-receptor agonist) and NPY (13-36) (Y2-receptor agonist), or the Y1-receptor antagonist BIBP 3226. Effects on mean arterial blood pressure (MAP) and renal arterial blood flow were recorded. The Y1-receptor agonist evoked a dose-dependent increase in MAP concomitantly with a reduction in renal blood flow. At the largest dose administered (1.42 pmol/g), the Y1-agonist [Leu(31), Pro(34)] NPY increased MAP by 20 +/- 6 mmHg and reduced the renal vascular conductance by more than 50%. The same dose of the Y2-agonist NPY (13-36) did not evoke any clear-cut effects on the renal blood flow or MAP. Furthermore, administration of the Y1-receptor antagonist BIBP 3226 reduced the NPY-induced renal vasoconstriction, but did not affect the response to angiotensin II or phenylephrine. The effects evoked by 0.71 pmol/g NPY were almost abolished by 3 mg/kg BIBP 3226. In situ hybridization histochemistry was used to study the expression of Y1-receptor mRNA in the developing rat kidney. The levels of Y1-receptor mRNA expression in the vascular smooth muscle of the rat kidney varied at different ages, with low levels at postnatal day 10 and high levels at 20 days and again low levels at 40 days. In summary, the present study show a maturation-specific expression pattern of NPY Y1-receptor mRNA as well as functional effects of vascular NPY receptors of the Y1-subtype in the rat kidney.

Asunto(s)

Riñón/irrigación sanguínea , Riñón/química , Receptores de Neuropéptido Y/genética , Circulación Renal/fisiología , Vasoconstricción/fisiología , Angiotensina II/farmacología , Animales , Autorradiografía , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Hibridación in Situ , Masculino , Neuropéptido Y/análogos & derivados , Neuropéptido Y/farmacología , Fragmentos de Péptidos/farmacología , Fenilefrina/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Receptores de Neuropéptido Y/metabolismo , Circulación Renal/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología

RESUMEN

Cloning with subsequent in vitro and in vivo characterization of vascular neuropeptide Y (NPY) receptor subtypes in porcine and canine peripheral tissues was performed. RT-PCR with Y1 and Y2 receptor-specific primers, indicated expression of Y1 receptors in both kidney and spleen of dog and pig, and expression of Y2 receptors in pig spleen. In pig kidney, expression of Y1 receptor mRNA was located to intrarenal arteries, as demonstrated with in situ hybridization using human probes. The cloned and sequenced canine Y1, porcine Y1 and Y2 receptors revealed high homologies to previously characterized mammalian NPY receptors. Membrane and autoradiographic receptor binding studies showed specific high-affinity binding sites for the purported Y1-selective radioligands 125I-[Leu31Pro34]peptide YY (PYY) and 3H-BIBP 3226 in dog spleen, and for the putative Y2-selective 125I-PYY(3-36) in dog and pig spleen. In the pig in vivo, [Leu31Pro34]PYY, administered i.v., evoked vasoconstriction in spleen and kidney, actions that were potently inhibited by the non-peptide Y receptor antagonist SR 120107A. In contrast, PYY(3-36) evoked vasoconstriction only in spleen and this effect was not influenced by SR 120107A. NPY evoked renal and splenic vasoconstriction in the dog in vivo, vascular responses that were inhibited by both BIBP 3226 and SR 120107A. Furthermore, the Y1 receptor agonist [Leu31Pro34]NPY also caused vasoconstriction in dog kidney and spleen, whereas the putative Y2 agonist N-acetyl[Leu28Leu31]NPY(24-36) evoked no such vascular responses. It is concluded that the pig spleen is likely to contain Y1 and Y2 receptors, both involved in splenic vasoconstriction. In contrast, the Y1 receptor seems to be the sole vascular NPY receptor subtype in pig kidney. Moreover, Y1 receptors predominate in dog spleen and kidney. Furthermore, the cloned canine Y1 receptor and the porcine Y1 and Y2 receptors show great homologies to, and possess ligand requirement profiles in accordance with, the human forms.

Asunto(s)

Receptores de Neuropéptido Y/genética , Receptores de Neuropéptido Y/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Perros , Humanos , Hibridación in Situ , Riñón/metabolismo , Datos de Secuencia Molecular , Péptido YY/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Neuropéptido Y/clasificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Bazo/metabolismo , Porcinos , Vasoconstricción/efectos de los fármacos

RESUMEN

We have investigated the effects of ischaemia on neuropeptide Y (NPY) mechanisms involved in sympathetic vascular control of the pig kidney in vivo. Reperfusion after 2 h of renal ischaemia was associated with local overflow of noradrenaline (NA) but not of NPY-like immunoreactivity (-LI). Renal sympathetic nerve stimulation 10 min into reperfusion evoked markedly reduced vasoconstrictor effects and significantly less overflow of NA (reduced by 70% from the pre-ischaemic conditions), whereas NPY-LI overflow was unaltered. Renal vasoconstrictor responses to exogenous peptide YY (PYY), phenylephrine and angiotensin II were strongly attenuated after this ischaemic period, while vasoconstriction to alpha, beta-methylene ATP was maintained to a larger extent. The renal vascular responses and NA overflow had become partially normalized within a 2 h recovery period. In contrast, the renal vasoconstrictor response and the overflow of NPY-LI upon sympathetic nerve stimulation were enhanced after 15 min of renal ischaemia. In parallel, the PYY-evoked renal vasoconstriction was selectively and markedly prolonged after the 15 min of ischaemia. In the presence of the NPYY1 receptor antagonist BIBP 3226, the augmented vascular response to nerve stimulation was significantly attenuated. We conclude that reperfusion after 2 h of renal ischaemia is associated with local overflow of NA, whereas the sympathetic nerve-evoked release of NA and the reactivity of the renal vasculature to vasoconstrictor stimuli are reversibly reduced. Furthermore, possibly due to an impaired local degradation, the role of neurogenically released NPY in renal sympathetic vasoconstriction is enhanced after short-term (15 min) ischaemia compared with control conditions.

Asunto(s)

Neuropéptido Y/fisiología , Circulación Renal/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Estimulación Eléctrica , Femenino , Isquemia/fisiopatología , Masculino , Neuropéptido Y/sangre , Norepinefrina/sangre , Norepinefrina/metabolismo , Péptido YY/metabolismo , Fenilefrina/farmacología , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/metabolismo , Circulación Renal/efectos de los fármacos , Daño por Reperfusión/fisiopatología , Porcinos , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiología , Factores de Tiempo , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología

RESUMEN

1. The antagonistic effects of the neuropeptide Y (NPY) Y1 receptor antagonist BIBP 3226 on equally prominent vascular responses evoked by sympathetic nerve stimulation and exogenous NPY were compared during different intravenous (i.v.) infusions of the antagonist (0.19-190 nmol kg-1 min-1 for 30 min). 2. High frequency sympathetic nerve stimulation in reserpine-treated pigs in vivo evoked non-adrenergic vasoconstrictor responses in kidney and hind limb, in the latter followed by a long-lasting phase of decreased blood flow. The vascular response in the kidney and the long-lasting phase in hind limb resembled the effects of exogenous NPY administered i.v. (kidney) and intraarterially (i.a.) (in the hind limb, which only responded to higher NPY doses). 3. Plasma levels of BIBP 3226 reached a plateau within 20 min and the inhibitory effects on vascular responses were studied during the last ten minutes of infusion. The elimination of BIBP 3226 from plasma was found to fit a two-compartment model with an alpha-phase of 2.0 +/- 0.2 min and a beta-phase of 20.1 +/- 0.9 min. 4. Significant inhibition of presumably Y1 receptor-mediated vascular responses evoked by both endogenous and exogenous NPY in kidney and hind limb was seen even during low-dose infusion of BIBP 3226 (1.9 nmol kg-1 min-1), when plasma levels of the antagonist reached 59 +/- 8 nM. The maximum inhibitory effects of BIBP 3226 were seen during the highest-dose infusion (190 nmol kg-1 min-1), when the long-lasting vasoconstrictor responses in hind limb to sympathetic nerve stimulation and exogenous NPY administration (i.a.) were abolished. Simultaneously, the vascular responses in kidney to exogenous NPY were inhibited by 89% and to sympathetic nerve stimulation by 60%. 5. It is concluded that BIBP 3226 has a short half-life in plasma and should preferably be given by i.v. infusions to maintain blockade and avoid non-specific effects. Furthermore, BIBP 3226 dose-dependently and with similar potency antagonizes vascular responses to exogenous and endogenous NPY both in the kidney and hind limb of the reserpine-treated pig in vivo. Thus, inhibition of vascular responses to exogenous NPY may be a good indicator of the dose of this antagonist needed to inhibit sympathetic Y1 receptor-transmission.

Asunto(s)

Arginina/análogos & derivados , Vasos Sanguíneos/efectos de los fármacos , Neuropéptido Y/farmacología , Receptores de Neuropéptido Y/antagonistas & inhibidores , Animales , Arginina/sangre , Arginina/farmacología , Vasos Sanguíneos/fisiología , Estimulación Eléctrica , Femenino , Masculino , Porcinos , Sistema Nervioso Simpático/fisiología