RESUMEN
NEW FINDINGS: What is the central question of this study? Gastric slow waves originating from the interstitial cells of Cajal-smooth muscle syncytium are usually studied in culture or in tissue segments, but nobody has described recordings of slow waves from awake, freely moving mice. Can radiotelemetry be used to record slow waves, and do they respond predictably to drug treatment? What is the main finding and its importance? Radiotelemetry can be used to record slow waves from awake, freely moving mice, permitting an examination of drug actions in vivo, which is crucial to drug discovery projects for characterizing the effects of drugs and metabolites on gastrointestinal function. ABSTRACT: The mouse is the most commonly used species in preclinical research, and isolated tissues are used to study slow waves from the interstitial cells of Cajal-smooth muscle syncytium of the gastrointestinal tract. The aim of this study was to establish a radiotelemetric technique in awake mice to record gastric myoelectric activity from the antrum to gain insight into the effects of endogenous modulatory systems on slow waves. Under general anaesthesia, two biopotential wires from a telemetry transmitter were sutured into the antrum of male ICR (imprinting control region) mice. The animals were allowed 1 week to recover from surgery before the i.p. administration of drugs to stimulate or inhibit slow waves. The basal dominant frequency of slow waves was 6.96 ± 0.43 c.p.m., and the percentages of power in the bradygastric, normogastric and tachygastric ranges were 6.89 ± 0.98, 37.32 ± 1.72 and 34.38 ± 0.77%, respectively (n = 74). Nicotine at 1 mg kg-1 increased normogastric power, but at 3 mg kg-1 it increased bradygastric power (P < 0.05). Metoclopramide at 10 mg kg-1 increased normogastric power; sodium nitroprusside at 10 mg kg-1 had latent effects on tachygastric power (P < 0.05); and l-NAME at 10 mg kg-1 had no effect (P > 0.05). Nicotine and bethanechol also caused varying degrees of hypothermia (>1°C reductions; P < 0.05). In conclusion, radiotelemetry can be used to record slow waves from awake, freely moving mice. In light of our findings, we recommend that studies assessing slow waves should also assess body temperature simultaneously.
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Músculo Liso/metabolismo , Músculo Liso/fisiología , Neurotransmisores/metabolismo , Estómago/fisiología , Animales , Temperatura Corporal/fisiología , Hipotermia/metabolismo , Hipotermia/fisiopatología , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos ICR , NG-Nitroarginina Metil Éster/metabolismo , Nitroprusiato/metabolismo , RegistrosRESUMEN
Glucagon-like peptide-1 (GLP-1) receptor agonists are indicated for the treatment of Type 2 diabetes and obesity, but can cause nausea and emesis in some patients. GLP-1 receptors are distributed widely in the brain, where they contribute to mechanisms of emesis, reduced appetite and aversion, but it is not known if these centrally located receptors also contribute to a modulation of gastric slow wave activity, which is linked causally to nausea. Our aim was to investigate the potential of the GLP-1 receptor agonist, exendin-4, administered into the 3rd ventricle to modulate emesis, feeding and gastric slow wave activity. Thermoregulation and cardiovascular parameters were also monitored, as they are disturbed during nausea. Ferrets were used as common laboratory rodents do not have an emetic reflex. A guide cannula was implanted into the 3rd ventricle for delivering a previously established dose of exendin-4 (10nmol), which had been shown to induce emesis and behaviours indicative of 'nausea'. Radiotelemetry recorded gastric myoelectric activity (GMA; slow waves), blood pressure and heart rate variability (HRV), and core temperature; food intake and behaviour were also assessed. Exendin-4 (10nmol, i.c.v.) decreased the dominant frequency of GMA, with an associated increase in the percentage of bradygastric power (lasting ~4h). Food intake was inhibited in all animals, with 63% exhibiting emesis. Exendin-4 also increased blood pressure (lasting ~24h) and heart rate (lasting ~7h), decreased HRV (lasting ~24h), and caused transient hyperthermia. None of the above parameters were emesis-dependent. The present study shows for the first time that gastric slow waves may be modulated by GLP-1 receptors in the brain through mechanisms that appear independent from emesis. Taken together with a reduction in HRV, the findings are consistent with changes associated with the occurrence of nausea in humans.
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Fenómenos Fisiológicos Cardiovasculares , Motilidad Gastrointestinal , Receptor del Péptido 1 Similar al Glucagón/fisiología , Náusea/inducido químicamente , Péptidos/administración & dosificación , Ponzoñas/administración & dosificación , Vómitos/inducido químicamente , Animales , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Exenatida , Hurones , Motilidad Gastrointestinal/efectos de los fármacos , Receptor del Péptido 1 Similar al Glucagón/agonistas , Frecuencia Cardíaca/efectos de los fármacos , MasculinoRESUMEN
BACKGROUND: GLP-1 receptor agonists are utilised for the treatment of Type-2 diabetes but can be associated with undesirable effects of nausea and vomiting. OBJECTIVES: To investigate the role of GLP-1 receptors in mechanisms of emesis, behaviours indicative of nausea (BIN) and food intake in the ferret. RESULTS: Exendin-4 (10 and 30nmol, i.c.v.) induced emesis, inhibited food intake, and increased the frequency of BIN. Increases in c-Fos in the brainstem, midbrain and forebrain occurred in animals exhibiting emesis; no activation of the brainstem occurred in animals not vomiting. Exendin-4 (10nmol, i.c.v.) when preceded by i.c.v. saline (15µl), was not emetic but induced BIN and inhibited food intake; exendin (9-39) (100nmol) reduced BIN only. c-Fos showed that consistent with the absence of emesis in saline/exendin-4 treated animals there was no increase in c-Fos in the brainstem, but it increased in midbrain and forebrain nuclei. Excepting the amygdala, exendin (9-39) was without efffect on the increases in c-Fos. Analysis of c-Fos data showed a positive linear relationship between midbrain and forebrain areas irrespective of the occurrence of emesis induced by exendin-4. In contrast, brainstem and midbrain c-Fos levels were positively correlated, but only in animals with emesis. CONCLUSIONS: The brainstem is critical for exendin-4-induced emesis but suppression of food intake and BIN involves more rostral brain sites. Exendin-4-induced BIN and c-Fos activation of the amygdala are sensitive to exendin (9-39), whereas the suppression of food intake is not implicating separate control mechanisms for emesis and BIN.
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Encéfalo/efectos de los fármacos , Eméticos/farmacología , Náusea/inducido químicamente , Péptidos/farmacología , Ponzoñas/farmacología , Vómitos/inducido químicamente , Animales , Encéfalo/metabolismo , Encéfalo/patología , Catéteres de Permanencia , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Exenatida , Hurones , Péptido 1 Similar al Glucagón/agonistas , Péptido 1 Similar al Glucagón/metabolismo , Inmunohistoquímica , Inyecciones Intraventriculares , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Náusea/metabolismo , Náusea/patología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Vómitos/metabolismo , Vómitos/patologíaRESUMEN
BACKGROUND: Rodents are incapable of emesis and consequently the emetic potential of glucagon-like peptide-1 receptor (GLP-1R) agonists in studies designed to assess a potential blood glucose lowering action of the compound was missed. Therefore, we investigated if the ferret, a carnivore with demonstrated translation capability in emesis research, would identify the emetic potential of the GLP-1R agonist, exendin-4, and any associated effects on gastric motor function, appetite and cardiovascular homeostasis. METHODS: The biological activity of the GLP-1R ligands was investigated in vivo using a glucose tolerance test in pentobarbitone-anesthetised ferrets and in vitro using organ bath studies. Radiotelemetry was used to investigate the effect of exendin-4 on gastric myoelectric activity (GMA) and cardiovascular function in conscious ferrets; behaviour was also simultaneously assessed. Western blot was used to characterize GLP-1R distribution in the gastrointestinal and brain tissues. RESULTS: In anesthetised ferrets, exendin-4 (30 nmol/kg, s.c.) reduced experimentally elevated blood glucose levels by 36.3%, whereas the GLP-1R antagonist, exendin (9-39) (300 nmol/kg, s.c.) antagonised the effect and increased AUC0-120 by 31.0% when injected alone (P < 0.05). In animals with radiotelemetry devices, exendin-4 (100 nmol/kg, s.c.) induced emesis in 1/9 ferrets, but inhibited food intake and decreased heart rate variability (HRV) in all animals (P < 0.05). In the animals not exhibiting emesis, there was no effect on GMA, mean arterial blood pressure, heart rate, or core body temperature. In the ferret exhibiting emesis, there was a shift in the GMA towards bradygastria with a decrease in power, and a concomitant decrease in HRV. Western blot revealed GLP-1R throughout the gastrointestinal tract but exendin-4 (up to 300 nM) and exendin (9-39), failed to contract or relax isolated ferret gut tissues. GLP-1R were found in all major brain regions and the levels were comparable those in the vagus nerve. CONCLUSIONS: Peripherally administered exendin-4 reduced blood glucose and inhibited feeding with a low emetic potential similar to that in humans (11% vs 12.8%). A disrupted GMA only occurred in the animal exhibiting emesis raising the possibility that disruption of the GMA may influence the probability of emesis occurring in response to treatment with GLP-1R agonists.
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Depresores del Apetito/farmacología , Eméticos/farmacología , Hipoglucemiantes/farmacología , Péptidos/farmacología , Ponzoñas/farmacología , Animales , Glucemia/metabolismo , Exenatida , Hurones , MasculinoRESUMEN
The use of glucagon-like peptide-1 (7-36) amide (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus is commonly associated with nausea and vomiting. Previous studies using Suncus murinus revealed that the GLP-1 receptor agonist, exendin-4, induces emesis via the brainstem and/or hypothalamus. The present study investigated the mechanism of exendin-4-induced emesis in more detail. Ondansetron (1 mg/kg, s.c.) and CP-99,994 (10 mg/kg, s.c) failed to reduce emesis induced by exendin-4 (3 nmol, i.c.v.), suggesting that 5-HT3 and NK1 receptors are not involved in the mechanism. In other studies, the GLP-1 receptor antagonist, exendin (9-39), antagonised emesis and c-Fos expression in the brainstem and the paraventricular hypothalamus induced by the chemotherapeutic drug cisplatin (30 mg/kg, i.p.; p < 0.05), but not the emesis induced by nicotine (5 mg/kg, s.c.; p > 0.05), or copper sulphate pentahydrate (120 mg/kg, p.o.; p > 0.05). GLP-1 receptors may therefore represent a potential target for drugs to prevent chemotherapy-induced emesis in situations where 5-HT3 and NK1 receptor antagonists fail.
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Antieméticos/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Glucagón/antagonistas & inhibidores , Animales , Encéfalo/metabolismo , Exenatida , Receptor del Péptido 1 Similar al Glucagón , Masculino , Ondansetrón/farmacología , Péptidos , Piperidinas/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Musarañas , Ponzoñas , Vómitos/inducido químicamente , Vómitos/metabolismoRESUMEN
The use of glucagon-like peptide-1 (7-36) amide (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus is commonly associated with nausea and vomiting. Therefore, the present studies investigated the potential of GLP-1 receptor ligands to modulate emesis and feeding in Suncus murinus. Exendin-4, a selective GLP-1 receptor agonist, was administered subcutaneously (1-30 nmol/kg) or intracerebroventricularly (0.03-3 nmol) after 12-h of fasting. In other studies, animals were pretreated with the GLP-1 receptor antagonist, exendin (9-39), or saline (5 µl) 15 min prior to exendin-4 (3 nmol, i.c.v.). Behaviour of animals and food and water intake were then recorded for 1-2 h; c-Fos expression was also assessed in the brains of animals in the i.c.v. studies. The subcutaneous administration of exendin-4 reduced food and water intake (p < 0.001) and induced emesis in 40% of animals (p > 0.05). The intracerebroventricular administration of exendin-4 also prevented feeding, and induced emesis (p < 0.01). In these studies, exendin (9-39) (30 nmol, i.c.v.) antagonised emesis induced by exendin-4 and the increased c-Fos expressions in the brainstem and hypothalamus (p < 0.05), but it was ineffective in reversing the exendin-4-induced inhibition of food and water intake (p > 0.05). These data suggest that exendin-4 exerts its emetic effects in the brainstem and/or hypothalamus via GLP-1 receptors. The action of exendin-4 to suppress feeding may involve non-classical GLP-1 receptors or other mechanisms.
Asunto(s)
Depresores del Apetito/farmacología , Eméticos/farmacología , Péptidos/farmacología , Receptores de Glucagón/agonistas , Musarañas , Ponzoñas/farmacología , Animales , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/metabolismo , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos/efectos de los fármacos , Interacciones Farmacológicas , Ingestión de Alimentos/efectos de los fármacos , Exenatida , Femenino , Receptor del Péptido 1 Similar al Glucagón , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Inyecciones Intraventriculares , Inyecciones Subcutáneas , Actividad Motora/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Péptidos/administración & dosificación , Péptidos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Glucagón/antagonistas & inhibidores , Ponzoñas/administración & dosificaciónRESUMEN
The microelectrode array (MEA) can be used to study extracellular field potentials (exFPs) of electrogenic cells. Microcontact printing, which must be repeated after each experiment, is often used to promote accurate positioning of cells onto electrodes. The present study used MEAs with evenly spaced detection electrodes aligning along permanent SU-8 topographical guidance channels to measure propagation direction and speed. Chronotropic agents, isoproterenol (ISO, 1 nM-1 mM), and verapamil (VP, 1 nM-10 µM); and potassium channel openers (KCOs), pinacidil (PIN), and SDZ PCO400 (SDZ), were used to characterize these MEA chips. ISO (1 mM) enhanced the propagation speed from 247.25 ± 50.58 µm/ms 381.29 ± 92.01 µm/ms (n = 9, p < 0.05), whereas VP (10 µM) reduced the propagation speed completely (n = 12, p < 0.001). PIN (1 mM) significantly reduced the propagation speed from 278.6 ± 43.7 µm/ms to 49.7 ± 27.7 µm/ms (n = 10, p < 0.001), whereas SDZ (1 mM) completely stopped the propagation (n = 9, p < 0.001). Both KCOs induced conduction pattern changes similar to those observed in cardiac arrhythmia. The MEA chips with SU-8 guidance channels may be used to study cardiovascular diseases that are related to conduction disruption.
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Microelectrodos , Miocitos Cardíacos/fisiología , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/fisiopatología , Ingeniería Biomédica , Fármacos Cardiovasculares/farmacología , Células Cultivadas , Fenómenos Electrofisiológicos , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica de Rastreo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Canales de Potasio/agonistas , RatasRESUMEN
The microelectrode array was used to study the pharmacologic preconditioning effect of adenosine triphosphate-sensitive channel activation using potassium channel openers (KCOs) on rat cardiomyocytes over 90 minutes of ischemia. Cell viability and electrophysiological changes between KCOs pretreated and untreated cardiomyocytes were compared. Ischemia caused significant increases in beat frequency, extracellular field potential amplitude, and propagation velocity of spontaneously beating untreated cardiomyocytes. However, these electrophysiological parameters reduced as the duration of ischemia increased. The electrophysiological changes on ischemic cardiomyocytes were abolished by pretreating the cells with KCOs. Pinacidil pretreated cardiomyocytes retained a significantly higher viability than the untreated cardiomyocytes after 90 minutes of ischemia. Because Connexin 43 has a direct correlation with the propagation velocity, the Connexin 43 protein expression was also investigated. Connexin 43 expression levels were lower in KCOs pretreated cardiomyocytes than that of the untreated controls, and this correlated with the propagation velocity results obtained from the microelectrode array. The effect of pinacidil (sarcolemmal adenosine triphosphate-sensitive channel opener) was more prominent than that of diazoxide (mitochondrial K adenosine triphosphate-sensitive channel opener) on ischemic cardiomyocytes as indicated in the present acute ischemia study.
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Precondicionamiento Isquémico Miocárdico/métodos , Canales KATP/metabolismo , Miocitos Cardíacos/metabolismo , Vasodilatadores/farmacología , Animales , Supervivencia Celular , Células Cultivadas , Conexina 43/genética , Diazóxido/farmacología , Electrofisiología , Regulación de la Expresión Génica/efectos de los fármacos , Canales KATP/efectos de los fármacos , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Pinacidilo/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Anti-emetic drugs such as the tachykinin NK(1) receptor antagonists are useful to control emesis induced by diverse challenges. Evidence suggests pungent capsaicin-like TRPV1 activators also have broad inhibitory anti-emetic activity. However, pungent compounds are associated with undesirable effects including adverse actions on the cardiovascular system and on temperature homeostasis. In the present investigations using the ferret, we examine if the non-pungent vanilloid, olvanil, has useful anti-emetic properties without adversely affecting behaviour, blood pressure or temperature control. Olvanil (0.05-5 mg/kg, s.c.) was compared to the pungent vanilloid, resiniferatoxin (RTX; 0.1 mg/kg, s.c.), and to the anandamide reuptake inhibitor, AM404 (10 mg/kg, s.c.), for a potential to inhibit emesis induced by apomorphine (0.25 mg/kg, s.c.), copper sulphate (50 mg/kg, intragastric), and cisplatin (10 mg/kg, i.p.). Changes in blood pressure and temperature were also recorded using radiotelemetry implants. In peripheral administration studies, RTX caused transient hypertension, hypothermia and reduced food and water intake, but also significantly inhibited emesis induced by apomorphine, copper sulphate, or cisplatin. Olvanil did not have a similar adverse profile, and antagonised apomorphine- and cisplatin-induced emesis but not that induced by copper sulphate. AM404 reduced only emesis induced by cisplatin without affecting other parameters measured. Following intracerebral administration only olvanil antagonised cisplatin-induced emesis, but this was associated with transient hypothermia. In conclusion, olvanil demonstrated clear anti-emetic activity in the absence of overt cardiovascular, homeostatic, or behavioural effects associated with the pungent vanilloid, RTX. Our studies indicate that non-pungent vanilloids may have a useful spectrum of anti-emetic properties via central and/or peripheral mechanisms after peripheral administration.
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Antieméticos/farmacología , Capsaicina/análogos & derivados , Canales Catiónicos TRPV/metabolismo , Vómitos/tratamiento farmacológico , Animales , Antieméticos/administración & dosificación , Antieméticos/efectos adversos , Apomorfina , Ácidos Araquidónicos/farmacología , Conducta Animal/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Capsaicina/administración & dosificación , Capsaicina/efectos adversos , Capsaicina/farmacología , Cisplatino , Sulfato de Cobre , Diterpenos/efectos adversos , Diterpenos/farmacología , Relación Dosis-Respuesta a Droga , Hurones , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Canales Catiónicos TRPV/agonistas , Vómitos/inducido químicamenteRESUMEN
Pungent transient receptor potential vanilloid (TRPV1) channel activators have been shown to have broad inhibitory anti-emetic activity against centrally- and peripherally acting challenges but only at doses that have adverse effects on the cardiovascular system and on temperature homeostasis. In the present studies, we investigated the anti-emetic potential of the non-pungent TRPV1 activator, olvanil (0.05-5 mg/kg, s.c., 3 times per day, for 3 days) to antagonise the acute and delayed emesis induced by cisplatin (5 mg/kg, i.p.) in ferrets that had been implanted with radiotelemetry devices to enable an analysis of heart rate and temperature. Cisplatin induced an acute (day 1: 48.0+/-18.3 retches+vomits) and delayed (day 2: 111.7+/-35.5; day 3: 147.5+/-20.2 retches+vomits) emetic response that was associated with reduced food (-98.7% at day 3, P<0.001) and water consumption (-70.2% at day 3, P<0.001) and progressive weight loss (-12.0% at day 3, P<0.001). Olvanil did not prevent either emesis or the weight loss and negative effects on food and water consumption (P>0.05); the effect on food consumption appeared potentiated by a further 21.2% at 0.05 mg/kg (P<0.05) and 19.9% at 0.5 mg/kg (P<0.05). Cisplatin did not alter body temperature (basal: 37.7+/-0.1 degrees C) or heart rate (basal: 233.7+/-5.5 beats per min (BPM); P>0.05), but hypothermia (-1.6 degrees C) and increases in locomotor activity (50-90%) were recorded in animals concomitantly treated with olvanil (P<0.05). These data indicate that non-pungent activators as exemplified by olvanil are unlikely to be useful clinically for the control of the gastrointestinal side effects induced by cisplatin.
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Antineoplásicos/efectos adversos , Capsaicina/análogos & derivados , Cisplatino/efectos adversos , Canales Catiónicos TRPV/agonistas , Vómitos/tratamiento farmacológico , Animales , Temperatura Corporal/efectos de los fármacos , Capsaicina/farmacología , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Hurones , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Actividad Motora/efectos de los fármacos , Vómitos/inducido químicamenteRESUMEN
To determine whether changes in membrane potential affect the extent of mast cell degranulation, compound 48/80 was added to rat peritoneal mast cell suspensions in the absence or presence of potassium channel openers (KCOs). Changes were compared between the field potential (FP) and the amount of histamine released. The results demonstrated that (i) the onset and duration of FP, which reflects the hyperpolarizing nature of the response, increased as the concentration of compound 48/80 increased; (ii) both FP and the amount of histamine released increased as the concentration of compound 48/80 increased; (iii) although both KCOs (SDZ PCO400, a benzopyran derivative, and P1060, a cyanoguanidine derivative) potentiated compound 48/80-induced increases in FP and histamine release, without compound 48/80, they had no effect on either parameter; (iv) both glibenclamide and charybdotoxin significantly attenuated the compound 48/80-induced increase in FP; and (v) glibenclamide was able to attenuate the KCO-induced potentiation of FP. The results show that drugs presumably causing hyperpolarization can affect histamine release from rat peritoneal mast cells. The effect of KCOs on compound 48/80-induced response appears to be potentiation in nature rather than synergism. It is possible that KCO hyperpolarizes the cell membrane, enhances Ca2+ influx, and thus increases histamine release. As such, selective blockers of K+ channels may be useful for the treatment of immunological disorders.
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Benzopiranos/farmacología , Ciclopentanos/farmacología , Guanidinas/farmacología , Liberación de Histamina/fisiología , Canales KATP/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Mastocitos/fisiología , Peritoneo/fisiología , p-Metoxi-N-metilfenetilamina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Liberación de Histamina/efectos de los fármacos , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Peritoneo/citología , Peritoneo/efectos de los fármacos , Peritoneo/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
INTRODUCTION: Simultaneous recording of electrical potentials from multiple cells may be useful for physiological and pharmacological research. The present study aimed to establish an in vitro cardiac hypoxia experimental platform on the microelectrode array (MEA). METHODS: Embryonic rat cardiac myocytes were cultured on the MEAs. Following >or=90 min of hypoxia, changes in lactate production (mM), pH, beat frequency (beats per min, bpm), extracellular action potential (exAP) amplitude, and propagation velocity between the normoxic and hypoxic cells were compared. RESULTS: Under hypoxia, the beat frequency of cells increased and peaked at around 42.5 min (08.1+/-3.2 bpm). The exAP amplitude reduced as soon as the cells were exposed to the hypoxic medium, and this reduction increased significantly after approximately 20 min of hypoxia. The propagation velocity of the hypoxic cells was significantly lower than that of the control throughout the entire 90+ min of hypoxia. The rate of depolarisation and Na(+) signal gradually reduced over time, and these changes had a direct effect on the exAP duration. DISCUSSION: The extracellular electrophysiological measurements allow a partial reconstruction of the signal shape and time course of the underlying hypoxia-associated physiological changes. The present study showed that the cardiac myocyte-integrated MEA may be used as an experimental platform for the pharmacological studies of cardiovascular diseases in the future.
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Miocitos Cardíacos/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Hipoxia de la Célula , Células Cultivadas , Electrofisiología , Corazón/efectos de los fármacos , Corazón/fisiología , Concentración de Iones de Hidrógeno , Lactatos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Concentración Osmolar , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-DawleyRESUMEN
We performed this study to demonstrate the applicability of the microelectrode array (MEA) to study electrophysiological changes of rat peritoneal mast cells in the presence of compound 48/80 under normal, Ca(2+)-free, Ca(2+)-free with EDTA, and Cl(-)-free conditions. The use of high extracellular K(+) (KCl, 150 mM), charybdotoxin (ChTX, 100 nM), and Cl(-)-free containing ChTX buffers verified that the hyperpolarizing signal was due to the activation of mainly K(+) and, to a lesser extent, Cl(-) channels. Compound 48/80 concentration-dependently shortened the latent periods (the onset of response) and increased both the spatial (the K(+) and Cl(-) hyperpolarizing field potentials, HFP) and temporal measurements (the duration of response). Ca(2+)-free buffer had no effect on the latent period of compound 48/80 but increased the HFP at high concentrations. The latent period increased while the HFP diminished when cells were equilibrated in Ca(2+)-free buffer containing EDTA. Durations of the HFP were generally longer when cells were in either Ca(2+)-free or Ca(2+)-free containing EDTA buffers than when cells were in normal buffer. The EC(50) values confirmed that effects were only affected in Ca(2+)-free buffer containing EDTA but not in Ca(2+)-free or Cl(-)-free buffers, further reinforcing the hypothesis that the presence of Ca(2+) is not essential to the action of compound 48/80. The present study is the first application of MEA to study rat peritoneal mast cells, and our results indicate that it could be of value in future pharmacological research on other non-excitable cells.
Asunto(s)
Mastocitos/fisiología , Análisis por Micromatrices/métodos , Microelectrodos , Cavidad Peritoneal/citología , Animales , Calcio/metabolismo , Células Cultivadas , Canales de Cloruro/fisiología , Ácido Edético/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Polyphenols in foods may chelate dietary Fe and lower its bioavailability. Concentrations of phenols are higher in red beans than in white beans. The aim of this study was to compare iron bioavailabilities from red and white beans in a piglet hemoglobin repletion model. Fe deficient cross bred piglets (Hampshire x Landrace x Yorkshire) were used. Nutritionally balanced diets (except for Fe) were formulated to contain 50% precooked, dehydrated beans (either small red or Great Northern white). At age 5 weeks, the piglets were assigned to two groups and fed diets containing either red or white beans for 4 weeks. Weight and hemoglobin (Hb) concentrations were monitored weekly. Feed intakes were measured daily. Hemoglobin repletion efficiency (HRE) was calculated as the gain in total body hemoglobin Fe (Hb-Fe) divided by Fe intake. Hb concentrations, Hb-Fe gains, and HRE were not different between the groups at any time point ( p > 0.05). HRE values in the red bean group were 50% in the first week and 30% over the entire 4 week period. In the white bean group, they were 56 and 26%, respectively. Proline-rich protein mRNA concentrations in parotid glands were higher in the red bean group compared to the white bean group. These results show that iron bioavailabilities from red and white beans are similar and suggest that pigs adapt to the inhibitory effects of polyphenols on iron absorption by increasing the secretion of protective proline-rich proteins in the saliva.
Asunto(s)
Absorción Intestinal , Hierro/metabolismo , Hierro/farmacocinética , Phaseolus/metabolismo , Animales , Disponibilidad Biológica , Células CACO-2 , Color , Hemoglobinas/metabolismo , Humanos , Phaseolus/química , Distribución Aleatoria , PorcinosRESUMEN
Sodium iron(III) ethylenediaminetetraacetate (NaFeEDTA) has considerable promise as an iron fortificant because of its high bioavailability in foods containing iron absorption inhibitors. In this study, uptakes of iron from NaFeEDTA, FeSO4, and FeCl3 by Caco-2 cells were compared in the absence or presence of ascorbic acid (AA), an iron absorption enhancer; at selected pH levels; and in the absence or presence of an iron absorption inhibitor, bathophenanthroline disulfonic acid (BPDS). Ferritin formation in the cells was used as the indicator of iron uptake. Uptake from all three Fe sources was similar in the absence of AA. Adding AA at a 5:1 molar excess as compared to Fe increased uptake by 5.4-, 5.1-, and 2.8-fold for FeSO4, FeCl3, and NaFeEDTA, respectively. The smaller effect of AA on uptake from NaFeEDTA may be related to the higher solubility of NaFeEDTA and/or the strong binding affinity of EDTA for Fe3+, which may prevent AA and duodenal cytochrome b from effectively reducing EDTA-bound Fe. Uptake was inversely related to the pH of the media over a range of 5.8-7.2. Because uptake by DMT-1 is proton-coupled, the inverse relationship between pH and Fe uptake in all three iron sources suggests that they all follow the DMT-1 pathway into the cell. Adding BPDS to the media inhibited uptake from all three iron compounds equally. Because BPDS binds Fe2+ but not Fe3+ and because only Fe2+ is transported by DMT-1, the finding that BPDS inhibited uptake from NaFeEDTA suggests that at least some iron dissociates from EDTA and is reduced just as simple inorganic iron at the brush border membrane of the enterocyte. Taken together, these results suggest that uptake of iron from NaFeEDTA by intestinal enterocytes is regulated similarly to uptake from iron salts.
Asunto(s)
Ácido Ascórbico/farmacología , Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Quelantes del Hierro/farmacología , Hierro/metabolismo , Disponibilidad Biológica , Células CACO-2 , Ácido Edético/metabolismo , Ferritinas/metabolismo , Humanos , Concentración de Iones de HidrógenoRESUMEN
Sodium iron ethylenediaminetetraacetate (NaFeEDTA) has superior iron bioavailability especially in foods containing iron absorption inhibitors. However, mechanisms involved in the absorption and subsequent partitioning of iron complexed with EDTA are poorly understood. Our objectives were to compare retention and tissue distribution of iron administered to rats either as FeSO4 or NaFeEDTA, either orally (OR) or subcutaneously (SC). Weanling rats were fed semipurified diets supplemented with either FeSO4 or NaFeEDTA for 7 days. They were then given a meal containing 59Fe-labeled FeSO4 or NaFeEDTA, or they were injected SC with these two forms of radiolabeled Fe. 59Fe retention was measured by whole body counting. Urine was collected and counted at 24 h intervals throughout the counting period. Tissue samples were analyzed for nonheme iron and 59Fe activity. Absorption of iron from FeSO4 or NaFeEDTA was similar (57.7 and 53.4%, respectively). Seventy-seven percent of the injected Na59FeEDTA was excreted in the urine within 24 h, whereas only 0.5, 0.8, and 1.4% of the injected 59FeSO4, oral 59FeSO4, and oral Na59FeEDTA, respectively, was excreted in the urine. The nonheme iron content was lower in the liver and spleen, by 56.8 and 28.4%, respectively, among rats consuming the NaFeEDTA diet as compared to rats fed FeSO4. We conclude that iron is dissociated from EDTA prior to or during intestinal absorption and that some fraction of the dissociated EDTA is absorbed separately from the iron.
Asunto(s)
Compuestos Férricos/farmacocinética , Absorción Intestinal , Hierro/farmacocinética , Animales , Disponibilidad Biológica , Ácido Edético/metabolismo , Ácido Edético/farmacocinética , Compuestos Férricos/metabolismo , Hierro/análisis , Hierro/metabolismo , Radioisótopos de Hierro/análisis , Radioisótopos de Hierro/orina , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Although it has been shown that iron absorption from NaFeEDTA, a promising iron fortificant, is effectively down-regulated in iron-loaded rats, effects of prolonged exposure to high dietary levels of NaFeEDTA are not well understood. The objectives of this study were to determine whether rats can adapt to a high dietary level of NaFeEDTA by down-regulating iron absorption, and to determine effects on tissue iron distribution, with or without an iron absorption inhibitor. Male Sprague-Dawley rats were exposed to diets supplemented with FeSO4 or NaFeEDTA at 1200 mg of Fe/kg of diet, with or without tea, for 27 days. Iron absorption measured by whole-body counting before and after exposure showed that rats adapted to the high dietary level of FeSO4 or NaFeEDTA by down-regulating iron absorption to a similar extent. However, nonheme iron concentrations in liver and spleen were about 35-50% lower, whereas the concentration in kidney was about 300% higher in rats fed NaFeEDTA, compared to rats fed FeSO4. Tea had no major impact on iron absorption or iron status, regardless of iron source. Our results showed that although iron absorption was down-regulated similarly, body iron distribution was markedly different between rats exposed to FeSO4 and those exposed to NaFeEDTA. Further studies are warranted to determine the effects of prolonged exposure to dietary NaFeEDTA on kidney iron accumulation and kidney function.
Asunto(s)
Compuestos Férricos/administración & dosificación , Hierro de la Dieta/administración & dosificación , Hierro/farmacocinética , Absorción , Animales , Peso Corporal , Hemoglobinas/análisis , Hierro/análisis , Masculino , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Iron chelates, namely, ferrous bisglycinate and ferric EDTA, are promising alternatives to iron salts for food fortification. The objectives of this study were to compare iron uptake from radiolabeled ferrous sulfate, ferrous ascorbate, ferrous bisglycinate, ferric chloride, ferric citrate, and ferric EDTA by Caco-2 cells with different iron status and in the presence of divalent metal cations. Iron-loaded Caco-2 cells, with reduced DMT-1 and elevated HFE mRNA levels, down-regulated uptake from ferrous ascorbate and bisglycinate but not from ferric compounds. Nevertheless, iron uptake from all compounds was markedly inhibited in the presence of 100-fold molar excess of Co2+ and Mn2+ cations, with ferrous compounds showing a greater percent reduction. Our results suggest that ferrous iron is the predominant form of iron taken up by intestinal epithelial cells and the DMT-1 pathway is the major pathway for uptake. Iron uptake from chelates appears to follow the same pathway as uptake from salts.
Asunto(s)
Cationes Bivalentes/farmacología , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Hierro/metabolismo , Células CACO-2 , Cobalto/farmacología , Enterocitos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Humanos , Intestinos/química , Quelantes del Hierro/metabolismo , Manganeso/farmacologíaRESUMEN
The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.
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Potenciales de Acción/fisiología , Encéfalo/fisiología , Técnicas de Cultivo de Célula/instrumentación , Microelectrodos , Modelos Neurológicos , Neuronas/fisiología , Transistores Electrónicos , Animales , Encéfalo/embriología , Células Cultivadas , Simulación por Computador , Diseño de Equipo , Análisis de Falla de Equipo , Ratas , Ratas Sprague-DawleyRESUMEN
The present study was designed to evaluate the use of collagen gel loaded with human retinal pigment epithelium (ARPE19) in cellular transfer and to assess its viability within the gel. Collagen solution was prepared by dissolving calfskin in hydrochloric acid to make a final concentration of 2.0 mg/ml and this was mixed with 10,000 ARPE19 cells/ml. The cell viability in gel was determined using MTT assay. van Gieson stain and proliferating cell nuclear antigen (PCNA) were used to identify the location of collagen and to localize the site of cell proliferation, respectively. The ARPE19 cells in gel appeared to be healthy with a rounded morphology. The optimal collagen concentration was 1.9 mg/ml. When this concentration was used to hold cells for over 12 days, it could be seen that the growth rate was the same between day 2 and day 8 in gel and on plastic. When the cell-loaded gels were transferred onto standard tissue culture plastics, progressive cell migrations over time resembling cell migrations in organotypic explant cultures were observed. Upon intravitreal injection of cell-containing collagen suspension into a rabbit's eye, the gel became suspended within the vitreous a few hours after injection (day 0). However, it became obvious that the gel dispersed and spread around the vitreous even after just 24 h. These cells inside the vitreous were PCNA positive, indicating that the human ARPE19 cells have the capacity to proliferate even after 11 days. The present study demonstrated the potential use of collagen gel as a tool in the transfer of cellular matrix onto other substrates. The results show that the cell seeding number must be critically balanced with the concentration of gel for it to be used as transplant material.