RESUMEN
The adult human pituitary lacks a well defined intermediate lobe, and it is uncertain whether the POMC cells that remain in the zona intermedia represent melanotropes or corticotropes. In the present study, we investigated whether the N-acetylated beta-endorphin- and alpha-MSH-related peptides that are characteristically produced by melanotropes in the rat and other species are localized in the human pituitary. Sequential gel filtration and ion exchange HPLC analysis revealed that small amounts of alpha-N-acetyl-beta-endorphin-(1-31), as well as beta-endorphin-(1-27) and beta-endorphin-(1-26), were detectable in human pituitary extracts, although beta-endorphin-(1-31) was clearly the major form. Consistent with this analysis, low levels of alpha-MSH, but not N,O-diacetyl-alpha-MSH, were identified by reverse-phase HPLC, although again, the desacetyl form of alpha-MSH predominated. Immunohistochemistry revealed that N-acetyl-beta-endorphin immunoreactivity was colocalized with ACTH and beta-endorphin in a subpopulation of zona intermedia cells. Unexpectedly, immunoreactive N-acetyl-beta-endorphin was also observed in a comparable proportion of corticotropes dispersed throughout the anterior lobe. alpha-MSH immunoreactivity was similarly distributed. These results indicate that N-acetylation is not restricted to the zona intermedia, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in the rat and other species does not extend to the human pituitary.
Asunto(s)
Hipófisis/metabolismo , alfa-MSH/metabolismo , betaendorfina/análogos & derivados , Hormona Adrenocorticotrópica/metabolismo , Anciano , Anciano de 80 o más Años , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Adenohipófisis/metabolismo , Radioinmunoensayo , Distribución Tisular , betaendorfina/metabolismo , beta-Lipotropina/metabolismoRESUMEN
POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that beta-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to beta-endorphin- and alpha-MSH-related peptides. Ion exchange HPLC analysis revealed that beta-endorphin(1-31) was further processed to alpha-N-acetyl-beta-endorphin(1-31), which comprised 35.9 +/- 0.1% of total immunoreactivity, and smaller amounts of beta-endorphin(1-27), beta-endorphin(1-26), and their alpha-N-acetylated derivates. The predominant alpha-MSH immunoreactive peptides coeluted with alpha-MSH and N,O-diacetyl-alpha-MSH by reverse-phase HPLC, although small amounts of ACTH(1-13)-NH2 were also present. Thus, multiple forms of beta-endorphin and alpha-MSH are localized in rat heart. beta-Endorphin(1-31) is a minor constituent, however, indicating that nonopioid beta-endorphin peptides predominate.
Asunto(s)
Miocardio/química , alfa-MSH/análisis , betaendorfina/análisis , Hormona Adrenocorticotrópica/análisis , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Masculino , Ratas , Ratas Sprague-Dawley , beta-Lipotropina/análisisAsunto(s)
Hormona Adrenocorticotrópica/biosíntesis , Expresión Génica , Miocardio/metabolismo , Proopiomelanocortina/metabolismo , ARN Mensajero/metabolismo , alfa-MSH/análogos & derivados , alfa-MSH/biosíntesis , betaendorfina/biosíntesis , Hormona Adrenocorticotrópica/aislamiento & purificación , Animales , Cromatografía Líquida de Alta Presión , Ventrículos Cardíacos , Hibridación in Situ , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , alfa-MSH/aislamiento & purificación , betaendorfina/análisisRESUMEN
To assess the effects of long-term pressure overload on sympathetic presynaptic components in the left ventricle, young adult male rats were subjected to surgical constriction of the suprarenal abdominal aorta. At 4 and 8 wk postsurgery, but not at 1 wk, left ventricular sympathetic activity, measured by the net fractional norepinephrine (NE) decrease after alpha-methyl-p-tyrosine methyl ester administration, was elevated in the aortic-banded rats. However, left ventricular NE was reduced only at 8 wk. Scatchard analysis of saturation binding of [3H]nisoxetine, a specific ligand for NE uptake sites, determined that left ventricular NE transporter sites were also reduced at 8 wk, suggesting a relationship between a reduced number of uptake sites and loss of NE stores. In contrast, aortic constriction did not reduce neuropeptide Y (NPY), tyrosine hydroxylase, dopamine beta-hydroxylase, nervegrowth factor, and low-affinity nerve growth factor receptors at any time point. Thus long-term pressure overload can cause a selective reduction in ventricular NE stores without a reduction in NPY, a colocalized sympathetic neurotransmitter.
Asunto(s)
Hipertensión/fisiopatología , Terminales Presinápticos/fisiología , Sistema Nervioso Simpático/fisiopatología , Simportadores , Función Ventricular Izquierda , Animales , Aorta Abdominal , Sitios de Unión , Proteínas Portadoras/metabolismo , Fluoxetina/análogos & derivados , Fluoxetina/metabolismo , Ligadura , Masculino , Metiltirosinas/farmacología , Miocardio/metabolismo , Neuropéptido Y/metabolismo , Norepinefrina/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Previous attempts to design inhibitors of mammalian folylpolyglutamate synthetase (FPGS) have resulted in three classes of active compounds, all of which have charged moieties in the side chain, but structural alteration of the rest of the folate molecule has not seemed to be an avenue for drug discovery. However, groups in the side chain of folate analogs that bear charge distributions different from that of glutamic acid appear to prevent efficient transport into mammalian cells on the reduced folate carrier system. We now report that substituents at the 7-, 2'-, or 3'-position of 2-desamino-2-methyl-4-hydroxyquinazoline antifolates decrease or prevent the catalysis of diglutamate formation by FPGS but are compatible with efficient binding to the reduced folate carrier system. Thus, 5,8-dideazafolates with a 3'-alkyl group had a lower Vmax for FPGS than did the corresponding unsubstituted quinazolines, by a factor of 4-12, but these compounds inhibited the reaction of control FPGS substrates, indicating that the 3'-groups had much larger effects on catalytic activity than on binding to enzyme. A 7-methyl substituent affected the Vmax of a series of 5,8-dideazafolate compounds by a factor of 2-8, but this decrease in the catalytic rate was also accompanied by an increase in the Km of the substituted compounds by a factor of 10-100. The extent of the effect of a 7-methyl substituent on Vmax appeared to be dependent on the size of the substituent at N10. Different substituents at the 2'-position affected the kinetics of the FPGS reaction with one of three patterns, i.e., 1) a 2'-fluoro substituent both increased Vmax and decreased Km slightly, 2) either -OH or -NH2 decreased the Vmax without affecting the Km, and 3) 2'-Cl-, -CH3, -CF3, or -OCH3 substituents were found to both decrease Vmax and increase Km. Substitutions at the 7-, 2'-, or 3'-position had only minor effects on the ability of 2-desamino-2-methyl-4-oxoquinazolines to interfere with the transport of [3H]methotrexate into L1210 cells. Hence, these classes of compounds are likely to be efficiently transported by the reduced folate carrier system. We conclude that the region of the folate molecule bounded by the 7-, 6-, 9-, 10-, 3'-, and 2'-positions, the "bay region," is of major importance both for the binding of folates and folate analogs to FPGS and for the assumption of a conformation of the enzyme-substrate complex compatible with catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)