RESUMEN
Naturally occurring benzodiazepines have been identified in regular food such as wheat and potato, but there is still no evidence that potato extracts can affect CNS responses in vivo. Here we found that undiluted potato juice and potato juice diluted with saline 1 : 2 administered 10 min intracisternally ( I. C.) and 30 min per os before bicuculline exerted significant anticonvulsant activity in the bicuculline-induced seizure threshold test in mice. In vitro, potato juice from different harvests at dilution series from 10 % to 0.000001 %, diluted 100,000-fold, displaced 50 % of gamma-aminobutyric acid (GABA) receptor ligand [ (3)H]GABA and diluted 40-fold displaced 50 % of [(3)H]flunitrazepam from binding sites in mice forebrain membranes. The low content of diazepam (0.04 +/- 0.01 mg/kg) determined by HPLC and mass spectrometry in the potato extracts could not sustain the anticonvulsant activity of potato juice in vivo; therefore we hypothesized that potato juice might contain GABA (A) receptor GABA-site active compounds. The findings of this study suggest that potato juice as well as potato taken as food may have the capacity of influencing brain GABA-ergic activity.
Asunto(s)
Anticonvulsivantes/análisis , Conducta Animal/efectos de los fármacos , Diazepam/análisis , Extractos Vegetales/farmacología , Receptores de GABA/metabolismo , Solanum tuberosum/química , Animales , Anticonvulsivantes/farmacología , Bicuculina , Cromatografía Líquida de Alta Presión , Diazepam/farmacología , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos ICR , Receptores de GABA/efectos de los fármacosRESUMEN
The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.
Asunto(s)
Anticonvulsivantes/farmacología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Receptores de GABA/metabolismo , Triterpenos/metabolismo , Triterpenos/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Bicuculina/farmacología , Flunitrazepam/metabolismo , Moduladores del GABA/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Tono Muscular/efectos de los fármacos , Triterpenos Pentacíclicos , Equilibrio Postural/efectos de los fármacos , Convulsiones/inducido químicamente , Convulsiones/psicología , Ácido gamma-Aminobutírico/metabolismo , Ácido BetulínicoRESUMEN
Betulin is a principal component of birch bark and is known to possess a broad range of biological activities, including antiinflammatory, antiviral and anticancer actions. The present study was carried out in vitro to clarify the influence of betulin on melanocortin (MC) receptor-ergic signalling by using COS-7 cells transfected with corresponding human MC receptor DNA. The results showed that betulin binds to the human melanocortin MC1, three to five receptors with selectivity to the MC1 subtype (K(i) value 1.022 +/- 0.115 microM). Betulin binds to the MC receptors with the following potency order-MC > MC3 > MC5 > MC4. Betulin itself does not stimulate cAMP generation, however, it slightly antagonizes alpha-melanocyte-stimulating hormone (alpha-MSH)-induced cAMP accumulation in the mouse melanoma cell line B16-F1. As a water-insoluble substance, betulin was dissolved in DMSO therefore DMSO competition with the labelled ligand NDP-MSH for the binding to the MC receptors was tested in the identical experimental set-up. We found that DMSO competes for binding to all the MC receptor subtypes, at 20% concentration and above. Selectivity for one or another receptor subtype was not observed. We have demonstrated for the first time, the ability of the plant compound betulin to bind to the MC receptors. One may suggest MC receptor MC1 subtype as the essential target for the antimelanoma action of betulin and its structurally close molecules such as betulinic acid. Moreover, we have found a new non-peptide small molecule MC mimetic, that is betulin. Thus, we report a new chemical motif for the binding to the MC receptors that could be used as a template for the search of more selective MC mimetics.
Asunto(s)
AMP Cíclico/metabolismo , Melanoma/patología , Receptores de Melanocortina/metabolismo , Triterpenos/metabolismo , alfa-MSH/análogos & derivados , Animales , Unión Competitiva/efectos de los fármacos , Células COS , Chlorocebus aethiops , Humanos , Cinética , Ratones , alfa-MSH/farmacologíaRESUMEN
Melanocortins exert multiple physiological effects that include the modulation of immune responses, inflammation processes, and pain transmission. In the present study we investigated the peripheral activity of natural melanocortins - alpha-, beta-, gamma1- and gamma2-melanocyte stimulating hormone (MSH) - and melanocortin receptor subtypes 3 and 4 (MC3/4 receptor) antagonist HS014 in pain (formalin and tail flick) tests after peptide subcutaneous administration in mice. In the formalin test, among all substances tested only alpha-MSH (1 micromol/kg) statistically significantly inhibited the formalin-induced first phase pain response, however, all tested peptides (except gamma1-MSH) at the dose of 1 micromol/kg produced a pronounced inhibitory effect on nociceptive behavior in the second phase and this activity was comparable with that of indomethacin (reference drug, 5 mg/kg intraperitoneally); beta-MSH was also active at a dose 0.1 micromol/kg. In the tail flick test, alpha-MSH (1 micromol/kg) showed algesic, whereas HS014 (0.5 micromol/kg) and indomethacin (10 mg/kg) exerted analgesic activity. Other peptides did not exert any activity in the tail flick test. These data indicate that peripherally administered melanocortin receptor agonists alpha-MSH, beta-MSH and gamma2-MSH, as well as MC3/4 receptor antagonist HS014 induced antinociception on pain/inflammatory events caused by formalin suggesting a predominant anti-inflammatory role of these peptides.
Asunto(s)
Analgésicos/farmacología , Melanocortinas/farmacología , Dimensión del Dolor , Animales , Masculino , Ratones , Ratones Endogámicos ICR , Óxido Nítrico/fisiología , Péptidos Cíclicos/farmacología , Receptores de Melanocortina/fisiología , alfa-MSH/farmacología , beta-MSH , gamma-MSH/farmacologíaRESUMEN
The anti-inflammatory effects of melanocortin peptides have been demonstrated in different inflammation models. This is the first report describing the molecular mechanisms for the beta-MSH-induced suppression of bacterial lipopolisaccharide (LPS)-caused brain inflammation. We found that beta-MSH suppresses LPS-induced nuclear translocation of the transcription factor NF-kappaB, and inhibits the expression of inducible nitric oxide synthase, and the following nitric oxide overproduction in the brain, in vivo. Moreover, administering the preferentially MC(4) receptor selective antagonist HS014 blocked completely these effects, suggesting a tentative MC(4) receptor mediated mechanism of action for the beta-MSH. However, as HS014 shows quite low selectivity vis-à-vis the MC(3) receptor, a role for the MC(3) receptor cannot be excluded. In conclusion, our results show that beta-MSH is capable of inhibiting brain inflammation via activation of melanocortin receptors, of the subtypes 4 and/or 3.
Asunto(s)
Encefalitis/tratamiento farmacológico , Hormonas/uso terapéutico , FN-kappa B/metabolismo , Receptor de Melanocortina Tipo 3/fisiología , Receptor de Melanocortina Tipo 4/fisiología , Transducción de Señal/fisiología , beta-MSH/uso terapéutico , Animales , Química Encefálica/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Espectroscopía de Resonancia por Spin del Electrón/métodos , Ensayo de Cambio de Movilidad Electroforética/métodos , Encefalitis/inducido químicamente , Inmunoquímica/métodos , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos ICR , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Péptidos Cíclicos/farmacologíaRESUMEN
The pro-opiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) mediates many diverse physiological actions, including anti-inflammatory and immunomodulatory effects. However, little is known about the physiological roles of the other melanocortins, beta- and gamma-MSH. Here, we investigated the effects of melanocortin peptides in an in vivo neuroinflammation model. Six hours following intracisternal (i.c.) administration of 10 microg lipopolysaccharide (LPS) to mice a five-fold increase in the nitric oxide (NO) level was seen in the animals' brains, when detected by electron paramagnetic resonance (EPR). All tested melanocortins, alpha-, beta-, gamma1- and gamma2-MSH (0.001-10 nmol/mouse i.c.), dose dependently reduced the LPS induced increases in brain NO, with an order of effectiveness: beta-MSH > or = gamma1-MSH=gamma2-MSH>alpha-MSH. Our results suggest specialized functions of beta- and gamma-MSH melanocortins in inflammatory signal modulation in the brain.
Asunto(s)
Encéfalo/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Óxido Nítrico/metabolismo , beta-MSH/metabolismo , gamma-MSH/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/fisiología , Masculino , Ratones , Ratones Endogámicos ICR , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , alfa-MSH/metabolismo , alfa-MSH/farmacología , beta-MSH/farmacología , gamma-MSH/farmacologíaRESUMEN
The C-terminal tripeptide of melanocyte-stimulating hormone, MSH (11-13) (Lys-Pro-Val), possesses strong anti-inflammatory actions, which are mediated via mechanisms that are not fully understood. To shed more light into these mechanisms we have here synthesised and evaluated the activities of L- and D-Val substituted cyclic modifications of MSH (11-13) on nitric oxide (NO) in macrophage RAW 264.7 cells, as well as on binding to melanocortin receptors (MCRs) in B16-F1 and MCR expressing insect cells, and for effects on cAMP. MSH (11-13) and its analogues did neither bind to MCRs nor stimulate cAMP in RAW 264.7 and B16-F1 cells, except H-, which showed a tendency to increase cAMP at high (10-100 microM) concentrations. However, all investigated peptides dose dependently inhibited NO in LPS/IFN-gamma-stimulated RAW 264.7, cells with a structure activity relationship suggesting the existence of a distinct receptive site. This site appears to be distinct from the MCRs and not linked with cAMP.