RESUMEN
The blood-brain barrier (BBB), composed of the microvessels of cerebral capillary endothelial cells, regulates the passage of peptides into the brain in several ways, mainly by saturable transport or passive diffusion. Here we describe an additional mechanism by which this regulatory function can occur. Cerebral microvessels were isolated from different regions of the brain and incubated with the mu-opiate selective endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) or the opiate-modulating Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), both tetrapeptides selectively tritiated at the Pro. Degradation was determined by HPLC. For both peptides, the metabolism by microvessels from the cerebral cortex was much greater than that by microvessels from the hypothalamus or pons. For endomorphin-1, the least degradation was in the pons; for Tyr-MIF-1 there was no difference in metabolism by microvessels from the pons or hypothalamus. The results show a novel mechanism at the BBB by which the BBB can selectively regulate the activity of different peptides in different regions of the brain.
Asunto(s)
Barrera Hematoencefálica/fisiología , Corteza Cerebral/metabolismo , Hormona Inhibidora de la Liberación de MSH/metabolismo , Oligopéptidos/metabolismo , Animales , Capilares/metabolismo , Corteza Cerebral/irrigación sanguínea , Cromatografía Líquida de Alta Presión , Endotelio Vascular/metabolismo , Hipotálamo/irrigación sanguínea , Hipotálamo/metabolismo , Hormona Inhibidora de la Liberación de MSH/análogos & derivados , Masculino , Puente/irrigación sanguínea , Puente/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Two endogenous brain peptides (Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2)), a cyclized analog and two fragments of Tyr-W-MIF-1, and hemorphin (Tyr-Pro-Trp-Thr) were tested for binding to mu 1 and mu 2 opiate receptor. All these peptides bound to both mu 1 and mu 2 sites in assays optimized to discriminate these subtypes of the mu opiate receptor in membranes from bovine thalamus. The cyclized analog of Tyr-W-MIF-1, previously shown to have potency near that of Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) and morphine in producing analgesia after intracerebroventricular (i.c.v.) injection, bound to mu 1 and mu 2 sites with affinities similar to those of DAMGO. Tyr-W-MIF-1, previously shown to induce analgesia after i.c.v. injection but with much higher potency after intrathecal (i.t.) injection, also bound to both mu 1 and mu 2 sites with an affinity between that of morphiceptin and hemorphin. Although the highest ratios of Ki's for mu 2/mu 1 were shown by hemorphin, Tyr-W-MIF-1, and Tyr-W-MIF-1, none of the compounds were significantly different in selectivity. The results indicate that the relatively lower potency of Tyr-W-MIF-1 after i.c.v., compared with i.t. injection, is not due to a lack of binding to mu 1 sites. They suggest that it has relatively high efficacy at mu 2, but low efficacy at mu 1 sites, a possibility that might explain some of the novel properties of these peptides.
Asunto(s)
Unión Competitiva , Hormona Inhibidora de la Liberación de MSH/análogos & derivados , Antagonistas de Narcóticos/farmacología , Péptidos/farmacología , Receptores Opioides mu/efectos de los fármacos , Tálamo/efectos de los fármacos , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Hormona Inhibidora de la Liberación de MSH/farmacologíaRESUMEN
Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) was recently isolated from human brain cortex. We have now isolated it from bovine hypothalami by solid phase extraction and several consecutive rpHPLC steps monitored by an RIA originally developed for the endogenous brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2). Determination of the sequence of the purified material and comparison of its chromatographic behavior with synthetic Tyr-W-MIF-1 confirmed the structure. The synthetic peptide and the isolated material showed almost identical binding to mu opiate receptors.
Asunto(s)
Hipotálamo/química , Hormona Inhibidora de la Liberación de MSH/análogos & derivados , Neuropéptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Bovinos , Hormona Inhibidora de la Liberación de MSH/aislamiento & purificación , Hormona Inhibidora de la Liberación de MSH/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Receptores Opioides mu/metabolismoRESUMEN
Previous studies have shown that some of CNS actions of an endogenous peptide Tyr-MIF-1, are mediated by dopamine (DA) receptors. To study the effect of DA receptor blockade on the antiopiate properties of Tyr-MIF-1, the opiate form of footshock-induced analgesia was elicited in the rat. The nociceptive responses were determined using the hot-plate test (52.5 degrees C). Intraperitoneal pre-treatment with haloperidol (500 micrograms/kg), SCH 23390 (150 micrograms/kg), or spiroperidol (150 micrograms/kg) potentiated the antinociceptive effect of the footshock and blocked the antagonistic action of Tyr-MIF-1 (200 micrograms/kg and 2.0 mg/kg). A dose of haloperidol too small to potentiate the antinociceptive effect of the footshock (100 micrograms/kg) was still able to block the action of Tyr-MIF-1 (200 micrograms/kg). The results suggest that activation of DA receptors mediates the antagonizing effect of Tyr-MIF-1 on the opiate form of footshock-induced analgesia in the rat.
Asunto(s)
Hormona Inhibidora de la Liberación de MSH/análogos & derivados , Dolor/fisiopatología , Receptores Dopaminérgicos/fisiología , Secuencia de Aminoácidos , Animales , Benzazepinas/farmacología , Antagonistas de Dopamina , Sinergismo Farmacológico , Pie , Haloperidol/farmacología , Calor , Hormona Inhibidora de la Liberación de MSH/farmacología , Masculino , Datos de Secuencia Molecular , Naloxona/farmacología , Ratas , Ratas Endogámicas , Espiperona/farmacología , Estimulación Eléctrica Transcutánea del NervioRESUMEN
Evidence is presented that the small peptides MIF-1/Tyr-MIF-1 are part of an endogenous antiopiate system that may function to balance the opiate system. We review the biological activity, behavioral activity, and functional effects of this proposed opiate antagonist system. In addition, we suggest, based on antinociceptive mechanisms, that the individual components of the antiopiate system might function differently from naloxone.
Asunto(s)
Hormona Inhibidora de la Liberación de MSH/análogos & derivados , Hormona Inhibidora de la Liberación de MSH/fisiología , Antagonistas de Narcóticos , Analgesia , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hipotálamo/metabolismo , Cinética , Naloxona/farmacología , Receptores Opioides/metabolismoRESUMEN
The effects of melanotrophin release inhibiting factor (Pro-Leu-Gly-NH2, MIF), its possible metabolites, Pro-Leu-OH, Leu-Gly-NH2, Leu-Gly-OH and an analogue, cyclo(Leu-Gly), on [3H]spiroperidol binding sites in the striatum and on [3H]apomorphine binding sites in the striatum and hypothalamus of male Sprague-Dawley rats were determined. [3H]Spiroperidol binding to dopamine receptors in striatal membranes was unaffected by any of the above peptides in concentration up to 0.1 mM. The binding of [3H]apomorphine was enhanced by MIF, Pro-Leu-OH and cyclo(Leu-Gly) in both striatal and hypothalamic membranes in submicromolar concentrations. Leu-Gly-NH2 and Leu-Gly-OH did not affect [3H]apomorphine binding to dopamine receptors in striatum of hypothalamus. The enhancement in binding of [3H]apomorphine by MIF and cyclo(Leu-Gly) was not related to the changes in the number of binding sites but to an increase in the affinity to the receptors. The results indicate that MIF and some of its related peptides do not affect dopamine receptor binding sites labeled by the neuroleptic [3H]spiroperidol but facilitate the transmission in those sites labeled by [3H]apomorphine. Since [3H]apomorphine and [3H]spiroperidol predominantly label pre- and post-synaptic dopamine receptors, it is concluded that MIF and its active analogs interact with presynaptic dopamine receptors.
Asunto(s)
Apomorfina/metabolismo , Butirofenonas/metabolismo , Hormona Inhibidora de la Liberación de MSH/análogos & derivados , Hormona Inhibidora de la Liberación de MSH/farmacología , Receptores Dopaminérgicos/efectos de los fármacos , Espiperona/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Técnicas In Vitro , Masculino , Membranas/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Binding sites for N-Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1), a novel peptide structurally related but immunoreactively different from Pro-Leu-Gly-NH2 (MIF-1), were investigated. The presence of Tyr-MIF-1-like material in brain tissue has previously been demonstrated by RIA and its levels were shown to vary with the diurnal cycle and after pinealectomy. We now demonstrate a high affinity binding site in rat brain that is saturable and specific for Tyr-MIF-1. Crude P2 synaptosomal fractions from rat brains were incubated with 125I-Tyr-MIF-1 in the presence or absence of 10 microM unlabeled Tyr-MIF-1 (nonspecific binding). Binding reached equilibrium at 30-40 min at 23 degrees C and at about 4 hr on ice, after which it was relatively stable for at least 18 hr. None of the other peptides (including MIF-1) or amino acid residues tested were found to effectively compete for 125I-Tyr-MIF-1 binding. Binding was linear with protein from 280 micrograms to at least 1.1 mg protein per tube. Scatchard analysis of the striatum-thalamus revealed the presence of binding sites with an apparent Kp of 91 nM and maximum number of sites in the range of 45 fmol/mg tissue. Analysis of several brain areas revealed a differential distribution of the binding sites with relatively high concentrations in cortex, striatum, and amygdala and low concentrations in pons-medulla. Together with the previously published RIA results, the demonstration of a receptor for Tyr-Pro-Leu-Gly-NH2 supports the concept of the presence of this novel peptide and its receptor in the brain.