RESUMEN
Hypertensive disorders of pregnancy globally affect 6-8% of gestation and remain a major cause of both foetal and maternal morbidity and mortality. However, the antihypertensive medications for the patients of this disease are strictly limited due to the teratogenic potentials. Here, we found that tele-methylhistamine (tMH) increased in response to the administration of hydralazine (Hdz), a vasodilative agent, in the pregnancy-associated hypertensive (PAH) mice. Hdz abrogated the degradation of tMH catalyzed by monoamine oxidase B (MAO-B) in vitro. These results suggested that Hdz inhibited the MAO-B activity and consequently tMH increased in the maternal circulation of PAH mice.
Asunto(s)
Hidralazina/farmacología , Hipertensión Inducida en el Embarazo/tratamiento farmacológico , Metilhistaminas/metabolismo , Monoaminooxidasa/metabolismo , Aminas/sangre , Animales , Antihipertensivos/farmacología , Biocatálisis/efectos de los fármacos , Cromatografía Líquida de Alta Presión/métodos , Femenino , Humanos , Hipertensión Inducida en el Embarazo/enzimología , Hipertensión Inducida en el Embarazo/metabolismo , Metilhistaminas/sangre , Ratones , Embarazo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
Since serotonin, homocysteine and oxytocin are known to fluctuate during mammalian gestation, we screened amines altered in pregnant-associated hypertensive (PAH) mice by tagging their amino groups with 6-aminoquinoline carbamoyl (AQC) group in concert with ultra high-performance liquid chromatography (UPLC). Interestingly, a candidate amine significantly increased in PAH mice was recovered to the basal level, when treated with antihypertensive drugs. Mass spectrometric analyses indicated that the molecular mass of this amine was 61.2, which was identified as ethanolamine.
Asunto(s)
Antihipertensivos/uso terapéutico , Cromatografía Líquida de Alta Presión/métodos , Etanolamina/análisis , Feto , Hipertensión Inducida en el Embarazo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Antihipertensivos/farmacología , Etanolamina/metabolismo , Femenino , Feto/efectos de los fármacos , Feto/metabolismo , Hipertensión Inducida en el Embarazo/tratamiento farmacológico , Hipertensión Inducida en el Embarazo/metabolismo , Masculino , Ratones , EmbarazoRESUMEN
Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.
Asunto(s)
Aminacrina/metabolismo , Biocatálisis , Colina/metabolismo , Pruebas de Enzimas/métodos , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Células HEK293 , Humanos , Hidrólisis , Límite de DetecciónRESUMEN
Histamine (HA), a mediator of inflammation, type I allergic responses and neurotransmission, is synthesized from L-histidine, the reaction of which is catalyzed by histidine decarboxylase (HDC). HDC has been reported to be induced by various stimuli, not only in mast cells and basophils, but also in T lymphocytes and macrophages. Although its mRNA has been shown to be increased in Jurkat cells when treated with phorbol 12-myristate 13-acetate (TPA), little is known concerning the induced production of HA by HDC. The present study quantified the trace amounts of intracellular HA using ultra-high liquid chromatography in combination with the 6-aminoquinoline carbamate-derivatization technique. To test whether the cellular level of HA is elevated by the induction of HDC in Jurkat cells treated with TPA, the peak corresponding to authentic HA in the cell lysate was fractioned and its molecular weight determined by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry. The results of this study show that the HA level is increased by the induction of HDC expression by TPA in Jurkat cells. Therefore, this method is useful in elucidating the physiological significance of HA production.
Asunto(s)
Histamina/metabolismo , Histidina Descarboxilasa/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Cromatografía Líquida de Alta Presión , Histidina Descarboxilasa/genética , Humanos , Células Jurkat , Quinolinas/química , ARN Mensajero/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). It is well established that PRMTs are implicated in various cellular processes, but their physiological roles remain unclear. Using nematodes with a loss-of-function mutation, we show that prmt-1, the major asymmetric arginine methyltransferase, is a positive regulator of longevity in C. elegans. This regulation is dependent on both its enzymatic activity and DAF-16/FoxO transcription factor, which is negatively regulated by AKT-mediated phosphorylation downstream of the DAF-2/insulin signaling. prmt-1 is also required for stress tolerance and fat storage but not dauer formation in daf-2 mutants. Biochemical analyses indicate that PRMT-1 methylates DAF-16, thereby blocking its phosphorylation by AKT. Disruption of PRMT-1 induces phosphorylation of DAF-16 with a concomitant reduction in the expression of longevity-related genes. Thus, we provide a mechanism by which asymmetric arginine dimethylation acts as an antiaging modification in C. elegans.
Asunto(s)
Arginina/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Longevidad/genética , Metilación , Proteína-Arginina N-Metiltransferasas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Inmunoprecipitación , Insulina/genética , Insulina/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Reacción en Cadena de la Polimerasa , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
ω-NG-monomethylarginine (MMA) and asymmetric ω-NG, ω-NG-dimethylarginine (ADMA), are endogenous competitive inhibitors for three isoforms of nitric oxide synthase (NOS). Although free methylarginines are thought to be liberated through the intracellular proteolysis of proteins methylated by protein arginine methyltransferases (PRMTs), the degradation pathways of the arginine-methylated proteins involved in the biosynthesis of free methylarginines have yet to be determined. In this study, the biosynthesis of free methylarginines with cultured cells was analyzed as follows: first, we established a method for quantifying trace amounts of free intracellular methylarginines by means of ultra highperformance liquid chromatography (UPLC). Second, we determined the type of methylation produced in the cultured cell lines using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry (MALDI-QIT-TOF/MS). Finally, we investigated whether methylarginines are generated via the proteasome and autophagy pathways, the primary intracellular protein degradation systems. By using specific inhibitors for each pathway, we found that the blockade of proteasome activity reduced the amount of free ADMA and symmetric ω-NG, ω-N'G-dimethylarginine (SDMA), while the inhibition of autophagy significantly reduced cellular ADMA only. These results suggest that both the proteasome and autophagy pathways play an essential role in the production of free methylarginines.