RESUMEN
The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/ß-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.
Asunto(s)
Neuropéptidos/metabolismo , Fragmentos de Péptidos/farmacología , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Composición Corporal , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/metabolismo , Masculino , Ratones , Células 3T3 NIH , Factores de Crecimiento Nervioso , Obesidad/inducido químicamente , Obesidad/metabolismo , Unión Proteica , Transporte de Proteínas , Receptores de Superficie CelularRESUMEN
Daily injections of human parathyroid hormone (1-34), hPTH(1-34), provide a highly effective treatment option for severe osteoporosis. However, PTH analogs shorter than 28 amino acids do not retain any bone augmenting potential. Here, we present ZP2307 ([Ac5c¹, Aib³, Leu8, Gln¹°, Har¹¹, Ala¹², Trp¹4, Asp¹7]PTH(1-17)-NH2), a novel, chemically modified and cyclized hPTH(1-17) analog, that augments bone mass in ovariectomized, osteopenic rats. Subcutaneous administration of this structurally constrained, K¹³-D¹7 side-chain-to-side-chain cyclized peptide reversed bone loss and increased bone mineral density (BMD) up to or above baseline levels in rat long bones and vertebrae. Highly significant effects of ZP2307 were achieved at doses of 40-320 nmol/kg. Micro-CT and histomorphometric analyses showed that ZP2307 improved quantitative and qualitative parameters of bone structure. Biomechanical testing of rat femora confirmed that ZP2307 dramatically increased bone strength. Over a broad maximally effective dose range (40-160 nmol/kg) ZP2307 did not increase serum concentrations of ionized free calcium above normal levels. Only at the highest dose (320 nmol/kg) ZP2307 induced hypercalcemic calcium levels in the ovariectomized rats. To our knowledge ZP2307 is the smallest PTH peptide analog known to exert augmentation of bone. Our findings suggest that ZP2307 has the potential to effectively augment bone mass over a broad dose range without a concomitant increase in the serum concentration of ionized free calcium above the normal range.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Densidad Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Absorciometría de Fotón , Animales , Conservadores de la Densidad Ósea/farmacocinética , Calcio/sangre , AMP Cíclico/metabolismo , Femenino , Ratas , Ratas Endogámicas F344 , Tomografía Computarizada por Rayos XRESUMEN
Metabolic syndrome and obesity-related diseases are affecting more and more people in the Western world. The basis for an effective treatment of these patients is a better understanding of the underlying pathophysiology. Here, we characterize fructose- and fat-fed rats (FFFRs) as a new animal model of metabolic syndrome. Sprague-Dawley rats were fed a 60 kcal/100 kcal fat diet with 10% fructose in the drinking water. After 6, 12, 18, 24, 36, and 48 wk of feeding, blood pressure, glucose tolerance, plasma insulin, glucose, and lipid levels were measured. Cardiac function was examined by in vivo pressure volume measurements, and intramyocardial lipid accumulation was analyzed by confocal microscopy. Cardiac AMP-activated kinase (AMPK) and hepatic phosphoenolpyruvate carboxykinase (PEPCK) levels were measured by Western blotting. Finally, an ischemia-reperfusion study was performed after 56 wk of feeding. FFFRs developed severe obesity, decreased glucose tolerance, increased serum insulin and triglyceride levels, and an initial increased fasting glucose, which returned to control levels after 24 wk of feeding. The diet had no effect on blood pressure but decreased hepatic PEPCK levels. FFFRs showed significant intramyocardial lipid accumulation, and cardiac hypertrophy became pronounced between 24 and 36 wk of feeding. FFFRs showed no signs of cardiac dysfunction during unstressed conditions, but their hearts were much more vulnerable to ischemia-reperfusion and had a decreased level of phosphorylated AMPK at 6 wk of feeding. This study characterizes a new animal model of the metabolic syndrome that could be beneficial in future studies of metabolic syndrome and cardiac complications.
Asunto(s)
Grasas/farmacología , Fructosa/farmacología , Obesidad/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Glucosa/metabolismo , Corazón/efectos de los fármacos , Insulina/sangre , Lípidos/sangre , Hígado/metabolismo , Masculino , Síndrome Metabólico/metabolismo , Miocardio/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
INTRODUCTION: High cholesterol-fructose (HCF) fed rats have previously been described as an animal model of impaired cardiac insulin signaling and decreased contractile performance. In this study, we evaluated the metabolic and cardiac effects of a HCF diet in rats. METHODS: Male Sprague-Dawley rats received a HCF diet for 16 to 17weeks. Body weight was measured weekly and mean arterial blood pressure, fasting blood glucose, fasting plasma insulin, glucose tolerance, and blood lipid levels were measured following 15weeks of feeding. One to 2weeks later, while still on the HCF diet, cardiac function was examined by in vivo pressure-volume measurements in the left ventricle. Finally, protein and glucose content in the urine was measured and all organs were weighed at the end of the study. RESULTS: Rats fed a HCF diet showed increased cholesterol and decreased high-density lipoprotein (HDL) levels in serum compared to control fed rats and they had more than a twofold increase in liver weight. However, in contrast to what has previously been reported, HCF diet had no effect on body weight, blood pressure, fasting blood glucose, fasting plasma insulin, glucose tolerance, or cardiac function during unstressed conditions. DISCUSSION: We were unable to reproduce previous findings that a HCF diet causes changes in glucose tolerance and cardiac contractile performance. Therefore, further studies are warranted to evaluate specific interactions between genetic, environmental, and dietary factors on metabolic and cardiovascular disease progression associated with intake of a westernized diet.
Asunto(s)
Fenómenos Fisiológicos Cardiovasculares , Colesterol/administración & dosificación , Fructosa/administración & dosificación , Hígado/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Colesterol/sangre , HDL-Colesterol/sangre , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/sangre , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/sangre , Fructosa/sangre , Insulina/sangre , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The antiarrhythmic dipeptide, GAP-134, ([2S,4R]-1[2-aminoacetyl]-4-benzamido-pyrrolidine-2-carboxylic acid) was evaluated in canine ischemia/reperfusion model. In dogs subjected to 60-minute ischemia and 4-hour reperfusion, GAP-134 was administered 10 minutes before reperfusion as a bolus + intravenous (IV) infusion. The doses administered were 0.25 microg/kg bolus + 0.19 microg/kg per hour infusion; 2.5 microg/kg + 1.9 microg/kg per hour; 25 mg/kg + 19 mg/kg per hour; 75 mg/kg + 57 mg/kg per hour. Ventricular ectopy was quantified during reperfusion, including premature ventricular contractions (PVC) and ventricular tachycardia (VT). Total incidence of VT was reduced significantly with the 2 highest doses of GAP-134 (1.7 + 0.8; 2.2 + 1.4 events; P < .05) compared to controls (23.0 + 6.1). Total PVCs were reduced significantly from 11.1 + 1.6% in control animals to 2.0% + 0.7% and 1.8% + 0.8% after the 2 highest doses of GAP-134. Infarct size, expressed as percentage of left ventricle, was reduced significantly from 19.0% + 3.5% in controls to 7.9% + 1.5% and 7.1% + 0.8% (P < .05) at the 2 highest doses of GAP-134. GAP-134 is an effective antiarrhythmic agent with potential to reduce ischemia/reperfusion injury.
Asunto(s)
Antiarrítmicos/farmacología , Benzamidas/farmacología , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocardio/patología , Prolina/análogos & derivados , Taquicardia Ventricular/prevención & control , Complejos Prematuros Ventriculares/prevención & control , Animales , Antiarrítmicos/administración & dosificación , Benzamidas/administración & dosificación , Circulación Coronaria/efectos de los fármacos , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Infusiones Intravenosas , Infarto del Miocardio/etiología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Prolina/administración & dosificación , Prolina/farmacología , Taquicardia Ventricular/etiología , Taquicardia Ventricular/patología , Taquicardia Ventricular/fisiopatología , Factores de Tiempo , Complejos Prematuros Ventriculares/etiología , Complejos Prematuros Ventriculares/patología , Complejos Prematuros Ventriculares/fisiopatologíaRESUMEN
The anti-arrhythmic peptide AAP10 has previously been shown to acutely upregulate electrical cell-to-cell coupling mediated via connexin 43 gap junctions. In the present work, we have further examined the connexin (Cx) specificity and mechanism of action of this peptide in HeLa cells expressing Cx43, Cx40 or Cx26. The ability of cells to transfer the small fluorescent dyes Alexa 488 (MW 570) or Alexa 594 (MW 759), as markers for metabolic coupling mediated via gap junctions, before and after exposure to AAP10 and/or the protein kinase C inhibitor chelerythrine for 5 h was determined by microinjection analysis. Immunofluorescence analysis assessed the effect of AAP10 on the spatial localisation of each Cx sub-type. Cell extracts were isolated for Western blot and reverse transcription polymerase chain reaction analysis at 0, 5, 10, 18 and 24 h following exposure to AAP10 and the relative Cx expression profiles determined. AAP10 enhanced the ability of Cx43 and, to a lesser extent, Cx40 to transfer Alexa 488. It also enhanced the ability of Cx43 to transfer Alexa 594 but not Cx40. Inhibition of protein kinase C blocked this enhanced response in both Cx sub-types. Western blot analysis determined that AAP10 induced Cx40 protein expression over periods of up to 24 h with an associated increase in the localisation of Cx40 at points of cell-to-cell contact following 24-h exposure. Cx43 expression was transiently induced following exposure to the peptide for 5-10 h, with an associated increase in Cx43 at points of cell-to-cell contact, returning to control levels by 18-24 h, via a post-translational mechanism independent of chelerythrine. A transient increase in Cx40 mRNA expression but not Cx43 mRNA expression was also observed. By contrast, AAP10 had no effect on the ability of Cx26 gap junctions to transfer the dyes or on the level of Cx26 expression. We propose that AAP10 is a versatile peptide that remodels metabolic coupling via Cx43 and to a lesser extent Cx40 gap junction channels via an initial protein-kinase-C-dependent pathway modifying local responses at the plasma membrane. This is followed by enhanced Cx43 or Cx40 protein expression.
Asunto(s)
Antiarrítmicos/farmacología , Conexina 43/efectos de los fármacos , Conexinas/efectos de los fármacos , Oligopéptidos/farmacología , Benzofenantridinas/farmacología , Western Blotting , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Conexina 26 , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Colorantes Fluorescentes/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Proteína Quinasa C/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Proteína alfa-5 de Unión ComunicanteRESUMEN
Gap junction uncoupling can alter conduction pathways and promote cardiac re-entry mechanisms that potentiate many supraventricular arrhythmias, such as atrial fibrillation (AF) and atrial flutter (AFL). Our objective was to determine whether GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], a small dipeptide gap junction modifier, can improve conduction and ultimately prevent AF/AFL. In rat atrial strips subjected to metabolic stress, GAP-134 prevented significantly conduction velocity slowing at 10 nM compared with vehicle (p < 0.01). In the canine sterile pericarditis model, conduction time (CT; n = 5), atrial effective refractory period (AERP; n = 3), and AF/AFL duration/inducibility (n = 16) were measured 2 to 3 days postoperatively in conscious dogs. CT was significantly faster after GAP-134 infusion (average plasma concentration, 250 nM) at cycle lengths of 300 ms (66.2 +/- 1.0 versus 62.0 +/- 1.0 ms; p < 0.001) and 200 ms (64.4 +/- 0.9 versus 61.0 +/- 1.3 ms; p < 0.001). No significant changes in AERP were noted after GAP-134 infusion. The mean number of AF/AFL inductions per animal was significantly decreased after GAP-134 infusion (2.7 +/- 0.6 versus 1.6 +/- 0.8; p < 0.01), with total AF/AFL burden being decreased from 12,280 to 6063 s. Western blot experiments showed no change in connexin 43 expression. At concentrations exceeding those described in the AF/AFL experiments, GAP-134 had no effect on heart rate, blood pressure, or any electrocardiogram parameters. In conclusion, GAP-134 shows consistent efficacy on measures of conduction and AF/AFL inducibility in the canine sterile pericarditis model. These findings, along with its oral bioavailability, underscore its potential antiarrhythmic efficacy.
Asunto(s)
Fibrilación Atrial/tratamiento farmacológico , Aleteo Atrial/tratamiento farmacológico , Benzamidas/uso terapéutico , Dipéptidos/uso terapéutico , Uniones Comunicantes/efectos de los fármacos , Sistema de Conducción Cardíaco/efectos de los fármacos , Pericarditis/tratamiento farmacológico , Prolina/análogos & derivados , Animales , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Fibrilación Atrial/fisiopatología , Aleteo Atrial/fisiopatología , Benzamidas/farmacología , Conexina 43/metabolismo , Dipéptidos/efectos adversos , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Perros , Conductividad Eléctrica , Femenino , Uniones Comunicantes/fisiología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Sistema de Conducción Cardíaco/fisiología , Masculino , Estructura Molecular , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Pericarditis/fisiopatología , Complicaciones Posoperatorias/tratamiento farmacológico , Complicaciones Posoperatorias/fisiopatología , Prolina/farmacología , Prolina/uso terapéutico , Ratas , Ratas Sprague-Dawley , Periodo Refractario Electrofisiológico/efectos de los fármacosRESUMEN
Rotigaptide (3) is an antiarrhythmic peptide that improves cardiac conduction by modifying gap-junction communication. Small molecule gap-junction modifiers with improved physical properties were identified from a Zealand Pharma peptide library using pharmaceutical profiling, established SAR around 3, and a putative pharmacophore model for rotigaptide. Activity of the compounds was confirmed in a mouse cardiac conduction block model of arrhythmia. Dipeptide 9f (GAP-134) was identified as a potent, orally active gap-junction modifier for clinical development.
Asunto(s)
Antiarrítmicos/química , Fibrilación Atrial/tratamiento farmacológico , Benzamidas/farmacología , Uniones Comunicantes/efectos de los fármacos , Prolina/análogos & derivados , Administración Oral , Animales , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Benzamidas/química , Benzamidas/uso terapéutico , Dipéptidos/química , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Ratones , Biblioteca de Péptidos , Prolina/química , Prolina/farmacología , Prolina/uso terapéutico , Relación Estructura-ActividadRESUMEN
ZP120 is a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further investigate the ZP120 pharmacological profile using mouse and rat preparations, J-113397 and UFP-101, as well as NOP receptor knockout (NOP(-/-)) mice. Electrically stimulated mouse and rat vas deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies the tail-withdrawal assay was performed in wild type (NOP(+/+)) and NOP knockout (NOP(-/-)) mice. In the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ and ZP120 effects showing similar pK(B) values ( approximately 7.8). UFP-101 antagonized the actions of N/OFQ (pK(B) values approximately 7.3) but did not modify the effects of ZP120. The inhibitory effects of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP(-/-) mice. In NOP(+/+) mice subjected to the tail-withdrawal assay, ZP120 (1 nmol) mimicked the pronociceptive action of N/OFQ (10 nmol), producing longer lasting effects. The effects of both peptides were absent in NOP(-/-) animals. The NOP receptor ligand ZP120 is a high potency NOP selective partial agonist able to evoke long-lasting effects; its diverse antagonist sensitivity in comparison with N/OFQ may derive from different modality of binding to the NOP receptor.
Asunto(s)
Oligopéptidos/farmacología , Péptidos Opioides/farmacología , Animales , Estimulación Eléctrica , Ligandos , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Antagonistas de Narcóticos , Ratas , Ratas Sprague-Dawley , Receptores Opioides/genética , Receptores Opioides/metabolismo , Conducto Deferente/efectos de los fármacos , Vasodilatadores/farmacología , NociceptinaRESUMEN
AIMS: Rotigaptide is proposed to exert its anti-arrhythmic effects by improving myocardial gap-junction communication. To directly investigate the mechanisms of rotigaptide action, we treated cultured neonatal murine ventricular cardiomyocytes with clinical pharmacological doses of rotigaptide and directly determined its effects on gap-junctional currents. METHODS AND RESULTS: Neonatal murine ventricular cardiomyocytes were enzymatically isolated and cultured for 1-4 days. Primary culture cell pairs were subjected to dual whole cell patch-clamp procedures to directly measure gap-junctional currents (I(j)) and voltage (V(j)). Rotigaptide (0-350 nM) was applied overnight or acutely perfused into 35 mm culture dishes. Rotigaptide (35-100 nM) acutely and chronically increased the resting gap-junction conductance (g(j)), and normalized steady-state minimum g(j) (G(min)) by 5-20%. Higher concentrations produced a diminishing response, which mimics the observed therapeutic efficacy of the drug. The inactivation kinetics was similarly slowed in a therapeutic concentration-dependent manner without affecting the V(j) dependence of inactivation or recovery. The effects of 0-100 nM rotigaptide on ventricular g(j) during cardiac action potential propagation were accurately modelled by computer simulations which demonstrate that clinically effective concentrations of rotigaptide can partially reverse conduction slowing due to decreases in g(j) and inactivation. CONCLUSION: These results demonstrate that therapeutic concentrations of rotigaptide increase the resting gap-junction conductance and reduce the magnitude and kinetics of steady-state inactivation in a concentration-dependent manner. Rotigaptide may be effective in treating re-entrant forms of cardiac arrhythmias by improving conduction and preventing the formation of re-entrant circuits in partially uncoupled myocardium.
Asunto(s)
Antiarrítmicos/farmacología , Comunicación Celular/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Oligopéptidos/farmacología , Potenciales de Acción , Animales , Animales Recién Nacidos , Células Cultivadas , Simulación por Computador , Relación Dosis-Respuesta a Droga , Uniones Comunicantes/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Cinética , Ratones , Ratones Endogámicos C57BL , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismoRESUMEN
Existing anti-arrhythmic therapy is hampered by lack of efficacy and unacceptable side effects. Thus, ventricular tachycardia and fibrillation remains the strongest predictor of in-hospital mortality in patients with myocardial infarction. In atrial fibrillation, rhythm control with conventional ion channel blockers provide no therapeutic benefit relative to rate control. Several lines of research indicate that impaired gap junctional cell-to-cell coupling between neighbouring cardiomyocytes is critical for the development of cardiac re-entry arrhythmias. Rotigaptide is the first drug that has been developed to prevent arrhythmias by re-establishing gap junctional intercellular communication. During conditions with acute cardiac ischaemia, rotigaptide effectively prevents induction of both ventricular and atrial tachyarrhythmia. Moreover, rotigaptide effectively prevents ischaemia reperfusion arrhythmias. At the cellular level, rotigaptide inhibits ischaemia-induced dephosphorylation of Ser297 and Ser368, which is considered important for the gating of connexin43 gap junction channels. No drug-related toxicity has been demonstrated at plasma concentrations 77,000 times above therapeutic concentrations. In rats and dogs, rotigaptide reduces infarct size following myocardial infarction. A series of phase I trials has been completed in which rotigaptide has been administered intravenously to ~200 healthy persons. No drug-related side effects have been demonstrated in healthy human beings. Clinical safety, tolerability and efficacy in patients with heart disease are being evaluated in ongoing clinical trials. Rotigaptide represents a pioneering pharmacological principle with a highly favourable preclinical and clinical safety profile, which makes this molecule a promising drug candidate for the prevention of cardiac arrhythmias.
Asunto(s)
Antiarrítmicos , Arritmias Cardíacas/prevención & control , Uniones Comunicantes/efectos de los fármacos , Oligopéptidos , Animales , Antiarrítmicos/efectos adversos , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/fisiopatología , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/prevención & control , Uniones Comunicantes/fisiología , Humanos , Oligopéptidos/efectos adversos , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Taquicardia Ventricular/fisiopatología , Taquicardia Ventricular/prevención & controlRESUMEN
We previously showed that aquaresis induced by the peripherally acting nociceptin/orphanin FQ receptor agonist ZP120 is associated with a decreased protein level of aquaporin-2 (AQP2) in whole-kidney homogenates. We now examined the effects of Ac-RYYRWKKKKKKK-NH(2) (ZP120) (1 nmol/kg/min i.v. for 4 h) on renal regional expression (cortex/outer stripe of outer medulla, inner stripe of outer medulla, and inner medulla) and subcellular localization of aquaporin-2. Responses to ZP120 were compared to the effects of an equi-aquaretic dose ( approximately 40% inhibition of distal water reabsorption) of the vasopressin type-2 receptor antagonist 5-dimethylamine-1-[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzapine (OPC31260) (32 nmol/kg/min). ZP120 decreased the aquaporin-2 protein level in the rat cortex/outer stripe of outer medulla and decreased apical plasma membrane localization of aquaporin-2 in the cortex (P = 0.002) and in the inner medulla (P = 0.06). These effects were not accompanied by a decrease in the aquaporin-2 mRNA level. OPC31260-induced aquaresis was associated with a decreased aquaporin-2 protein level in both the cortex/outer stripe of outer medulla and in the inner stripe of outer medulla. Apical localization of aquaporin-2 was decreased throughout all kidney zones, and OPC31260 decreased the AQP2 mRNA level in the inner medulla. We conclude that equi-aquaretic doses of ZP120 and OPC31260 produce different patterns of aquaporin-2 down-regulation, suggesting different signaling pathways.
Asunto(s)
Acuaporina 2/antagonistas & inhibidores , Riñón/efectos de los fármacos , Receptores Opioides/fisiología , Receptores de Vasopresinas/fisiología , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Acuaporina 2/genética , Acuaporina 2/fisiología , Benzazepinas/farmacología , Diuresis/efectos de los fármacos , Regulación hacia Abajo , Riñón/metabolismo , Masculino , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores Opioides/agonistas , Sodio/metabolismo , Receptor de NociceptinaRESUMEN
Rotigaptide (formerly ZP123) is a novel antiarrhythmic peptide that prevents uncoupling of connexin 43 (Cx43)-mediated, gap junction communication during acute metabolic stress. Since rotigaptide's long-term effects on Cx43 are unknown, we studied its effect on Cx43 protein levels at 24 h in neonatal ventricular myocytes. As determined by Western blot analysis, rotigaptide produced a dose-dependent increase in Cx43 protein expression that reached a maximum level at 100 nM. Furthermore, 100 nM rotigaptide markedly increased Cx43 immunoreactivity and Cx43-positive gap junctions as observed in immunocytochemical studies. Cycloheximide, an inhibitor of protein synthesis, was used to investigate rotigaptide's mechanism of action. Cycloheximide (10 microg/ml) reduced Cx43 protein levels to 39% of vehicle (17 mM ethanol) whereas cotreatment of 10 microg/ml cycloheximide with 100 nM rotigaptide reduced Cx43 protein levels to 56% of vehicle. Our findings suggest that rotigaptide's effect on Cx43 expression is partly due to increased biosynthesis.
Asunto(s)
Antiarrítmicos/farmacología , Conexina 43/metabolismo , Miocardio/metabolismo , Oligopéptidos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Animales , Animales Recién Nacidos , Células Cultivadas , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Uniones Comunicantes/metabolismo , Miocardio/ultraestructura , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed.We conclude that the effects of rotigaptide were confined to cells expressing Cx43.
Asunto(s)
Antiarrítmicos/farmacología , Comunicación Celular/efectos de los fármacos , Conexina 43/fisiología , Uniones Comunicantes/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Oligopéptidos/farmacología , Animales , Conexina 26 , Conexina 43/análisis , Conexinas/análisis , Uniones Comunicantes/fisiología , Células HeLa , Humanos , Miocitos Cardíacos/química , Ratas , Ratas WistarRESUMEN
The role of gap junction intercellular communication (GJIC) in ischemia-induced focal ventricular tachycardia (VT) is unknown. We have developed a new, stable antiarrhythmic peptide analog named ZP123 that selectively increases GJIC and prevents reentrant VT. Our aim in this study was to use ZP123 as a tool to assess the role of GJIC on occurrence of ischemia-induced focal VT and triggered activity (TA) due to delayed afterdepolarizations (DADs). Focal VT was induced by programmed stimulation in alpha-chloralose-anesthetized, open-chest dogs 1-4 h after coronary artery occlusion. Three-dimensional activation mapping was done using 6 bipolar electrograms on each of 23 multipolar needles in the risk zone. Dogs were randomly assigned to receive either saline or ZP123 cumulatively at three dose levels (an intravenous bolus followed by a 30-min infusion per dose). Attempts to induce VT were repeated in each dose. Mass spectrometry was used to measure plasma ZP123 concentrations. Standard microelectrode techniques were used for in vitro study of DADs and TA. Twenty-six dogs with focal VT were included. ZP123 did not affect the inducibility of focal VT at any plasma concentrations vs. saline (0.8 +/- 0.1 nM, 77 vs. 75%; 7.8 +/- 0.4 nM, 86 vs. 77%; and 78.8 +/- 5.0 nM, 77 vs. 91%). In vitro, ZP123 did not affect the induction of DADs (12/12) and TAs (10/10) in ischemic tissues or tissue removed from the origin of focal VT (DADs, 8/8; TAs, 4/4). Therefore, although indirect, the data with the doses and concentrations used suggest that GJIC may not play a major role in the genesis of focal activity in the ischemic models studied.
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Uniones Comunicantes/efectos de los fármacos , Corazón/fisiopatología , Isquemia Miocárdica/fisiopatología , Oligopéptidos/farmacología , Taquicardia Ventricular/fisiopatología , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Perros , Femenino , Uniones Comunicantes/fisiología , Corazón/efectos de los fármacos , Masculino , Oligopéptidos/sangre , Taquicardia Ventricular/etiologíaRESUMEN
Nociceptin, the endogenous ligand of the inhibitory G protein-coupled opioid receptor-like 1 receptor, produces aquaresis (i.e., increases the excretion of solute-free urine) in rats. However, the mechanism underlying this effect has not yet been explained. Using immunohistochemistry, we found the opioid receptor-like 1 receptor in the rat kidney colocalized with the vasopressin-regulated water channel aquaporin-2 in inner medullary collecting ducts. We investigated the aquaretic effect of opioid receptor-like 1 receptor stimulation by infusing the selective nociceptin analog ZP120C; volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 50 mM glucose. ZP120C induced a marked and sustained aquaresis in normal and congestive heart failure rats in the absence of changes in vasopressin plasma concentrations. The ZP120C-induced aquaresis was associated with downregulation of the aquaporin-2 protein level in both rat groups, suggesting that opioid receptor-like 1 receptor stimulation produces aquaresis by inhibiting the vasopressin type-2 receptor-mediated stimulation on collecting duct water reabsorption. However, substantial amounts of PKA-mediated serine 256 phosphorylated aquaporin-2 were still present after 4 h of ZP120C treatment. Furthermore, neither preincubation with nociceptin nor ZP120C inhibited vasopressin-mediated cAMP accumulation in isolated collecting ducts. We conclude that renal opioid receptor-like 1 receptor stimulation in normal and congestive heart failure rats produces aquaresis by a direct renal effect, via aquaporin-2 downregulation, through a mechanism not involving inhibition of vasopressin type-2 receptor-mediated cAMP production.
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Acuaporinas/biosíntesis , Insuficiencia Cardíaca/fisiopatología , Túbulos Renales Colectores/fisiología , Péptidos Opioides/farmacología , Receptores Opioides/biosíntesis , Vasodilatadores/farmacología , Vasopresinas/farmacología , Animales , Acuaporina 2 , Acuaporinas/metabolismo , Regulación hacia Abajo , Insuficiencia Cardíaca/complicaciones , Inmunohistoquímica , Infusiones Intravenosas , Masculino , Ratas , Ratas Sprague-Dawley , Orina/química , Receptor de Nociceptina , NociceptinaRESUMEN
Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH(2)) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo. ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH(2)). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H(2)O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 +/- 6% and 146 +/- 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCalpha inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro, but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivo studies of antiarrhythmic peptides.
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Sistema de Conducción Cardíaco/efectos de los fármacos , Corazón/fisiopatología , Contracción Miocárdica/efectos de los fármacos , Oligopéptidos/farmacología , Proteína Quinasa C/metabolismo , Animales , Antiarrítmicos/farmacología , Conexina 43/metabolismo , Electrocardiografía , Inhibidores Enzimáticos/farmacología , Uniones Comunicantes/fisiología , Células HeLa , Corazón/efectos de los fármacos , Humanos , Masculino , Desnaturalización Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-alfa , Conejos , RatasRESUMEN
INTRODUCTION: The aim of this study was to determine if the stable antiarrhythmic peptide (AAP) analogue ZP123 increases gap junctional intercellular conductance and prevents reentrant ventricular tachycardia (VT) during coronary artery occlusion. METHODS AND RESULTS: Voltage clamp experiments demonstrated that 10 nM ZP123 improved gap junctional intercellular conductance by 69% +/- 20% in pairs of guinea pig ventricular myocytes. VT was induced by programmed stimulation in alpha-chloralose anaesthetized open chest dogs 1 to 4 hours after coronary artery occlusion. Three-dimensional activation mapping was done using six bipolar electrograms on each of 23 multipolar needles in the risk zone. When VT was reproducibly induced, dogs were randomly assigned to receive either saline or ZP123 cumulatively at three dose levels (intravenous bolus followed by 30-min infusion per dose). Attempts to induce VT were repeated in each infusion period. Mass spectrometry was used to measure ZP123 plasma concentrations. Twenty-six dogs with reentrant VT were included. ZP123 significantly prevented reentrant VT at all plasma concentrations vs saline: 1.0 +/- 0.2 nM: 6/12 vs 0/12; 7.7 +/- 0.6 nM: 7/13 vs 1/12; and 69.2 +/- 5.4 nM: 9/13 vs 1/13. The preventive effect of ZP123 on reentrant VT was closely correlated to reversal of functional, unidirectional conduction block. ZP123 did not affect effective refractory period, surface ECG parameters, mean arterial pressure, or infarct size. CONCLUSION: The stable AAP analogue ZP123 increased gap junctional intercellular conductance and specifically prevented the induction of reentrant VT during ischemia in a broad dose range without proarrhythmic or hemodynamic side effects. ZP123 is a promising candidate for use in preventing ischemia-induced VT.