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1.
Cancer Gene Ther ; 19(12): 828-38, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23079673

RESUMEN

The INSM1 gene encodes a transcriptional repressor that is exclusively expressed in neuronal and neuroendocrine tissue during embryonic development that is re-activated in neuroendocrine tumors. Using the 1.7 kbp INSM1 promoter, an adenoviral HSV thymidine kinase gene therapy was tested for the treatment of neuroendocrine tumors. An unforeseen interference on the INSM1 promoter specificity from the adenoviral genome was observed. Attempts were made to protect the INSM1 promoter from the influence of essential adenoviral sequences and to further enhance the tissue specificity of the INSM1 promoter region. Using the chicken ß-globin HS4 insulator sequence, we eliminated off-target tissue expression from the Ad-INSM1 promoter-luciferase2 constructs in vivo. In addition, inclusion of two copies of the mouse nicotinic acetylcholine receptor (n(AchR)) neuronal-restrictive silencer element (NRSE) reduced nonspecific activation of the INSM1 promoter both in vitro and in vivo. Further, inclusion of both the HS4 insulator with the n(AchR) 2 × NRSE modification showed a two log increase in luciferase activity measured from the NCI-H1155 xenograft tumors compared with the original adenovirus construct. The alterations increase the therapeutic potential of adenoviral INSM1 promoter-driven suicide gene therapy for the treatment of a variety of neuroendocrine tumors.


Asunto(s)
Adenoviridae/genética , Carcinoma Neuroendocrino/genética , Carcinoma Neuroendocrino/terapia , Terapia Genética/métodos , Proteínas Represoras/genética , Adenoviridae/metabolismo , Animales , Células COS , Carcinoma Neuroendocrino/virología , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Masculino , Ratones , Ratones Desnudos , Factores de Crecimiento Nervioso/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Receptores Nicotínicos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/biosíntesis , Proteínas Represoras/metabolismo , Elementos de Respuesta , Timidina Quinasa/biosíntesis , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Distribución Tisular , Transfección/métodos
2.
Int J Obes Relat Metab Disord ; 27(3): 313-8, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12629557

RESUMEN

OBJECTIVE: Peripherally administered exendin-4 is in clinical trials for the treatment of diabetes mellitus and obesity. Since its effects on food intake are mediated centrally, we determined the degree and type of its blood-to-brain penetration of the mouse blood-brain barrier (BBB). MEASUREMENTS AND RESULTS: High-performance liquid chromatography showed that exendin-4 was stable in blood, with most of the injected peptide reaching the brain intact. Capillary depletion studies with washout showed that the injected exendin-4 reached brain parenchyma rather than being trapped in the endothelial cells composing the BBB. Multiple-time regression analysis showed that exendin-4 crossed the BBB directly at a fast rate. The rapid brain entry of exendin-4, helped by its high lipophilicity as demonstrated by the octanol/buffer partition coefficient, was not dependent upon circumventricular organs and was not affected by food deprivation for 24 h. The simultaneous i.v. injection of high doses of unlabeled exendin-4 resulted in self-inhibition (saturation) that only became statistically significant (P<0.05) when the results of four experiments were combined; this suggests a possible limit to the amount of peripherally administered exendin-4 that can reach the brain after injection of high doses. CONCLUSION: The results indicate that exendin-4 is well conformed for exerting central effects involved in the control of obesity.


Asunto(s)
Encéfalo/metabolismo , Péptidos/farmacocinética , Ponzoñas , Animales , Barrera Hematoencefálica , Cromatografía Líquida de Alta Presión/métodos , Evaluación Preclínica de Medicamentos , Exenatida , Enlace de Hidrógeno , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos , Octanoles , Péptidos/sangre , Péptidos/química
3.
Neuroendocrinology ; 74(6): 423-32, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11752898

RESUMEN

Galanin-like peptide (GALP) was recently isolated from the hypothalamus, where its expression is influenced by leptin and food deprivation. Since leptin crosses the blood-brain barrier (BBB) by a saturable transport system that is downregulated by fasting, we examined the effect of leptin and fasting on the entry of GALP into mouse brain. Multiple-time regression analysis showed that the basal influx of 125I-GALP from blood was rapid (K(i) = 9.49 +/- 0.72 x 10(-4) ml/g x min). This influx was not affected by leptin but was significantly decreased by food deprivation for 24 or 48 h, accompanied by decreased immunoreactive plasma GALP at 48 h, but not at 24 h. By contrast, pretreatment of mice fasted for 24 h with glucose resulted in a significant increase in the blood-to-brain influx of GALP that was not accompanied by increased immunoreactive plasma GALP. HPLC showed that most of the GALP crossed the BBB in an intact form, and capillary depletion studies showed that more than 93% of the GALP crossing entered the parenchyma of the brain rather than being bound to the endothelial cells of the capillaries composing the BBB or being reversibly associated with the vasculature. Efflux of 125I-GALP occurred at the rate of the normal reabsorption of CSF, and the octanol-buffer partition coefficient showed insufficient lipophilicity to explain the fast rate of influx. When 125I-GALP was perfused in blood-free buffer, the self-inhibition characteristic of a saturable transport system was evident even though capillary gel electrophoresis showed GALP aggregating as a trimer. Capillary zone electrophoresis showed protein binding of GALP in serum, perhaps facilitating its interactions at the BBB. In particular, these studies show for the first time (1) that immunoreactive GALP is present in blood where (2) its concentrations are reduced by food deprivation, and (3) that there is a rapid blood-to-brain influx of intact GALP (4) which is decreased by fasting and (5) increased by pretreatment with glucose.


Asunto(s)
Encéfalo/metabolismo , Privación de Alimentos/fisiología , Proteínas del Tejido Nervioso/farmacocinética , Animales , Barrera Hematoencefálica , Tampones (Química) , Capilares/metabolismo , Cromatografía , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Electroforesis , Péptido Similar a Galanina , Glucosa/farmacología , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas del Tejido Nervioso/administración & dosificación , Proteínas del Tejido Nervioso/sangre , Perfusión , Radioinmunoensayo
4.
Neurosci Lett ; 310(1): 69-71, 2001 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-11524160

RESUMEN

Loss of ovarian function, such as occurs with menopause in human beings and ovariectomy in rodents, results in weight gain. Using multiple-time regression analysis, a sensitive technique for quantifying blood-to-brain transport of peptides and polypeptides, we found that mice ovariectomized for at least 5 weeks had markedly reduced entry of the satiety factor leptin into brain. The rate of entry of leptin into brain remained reduced half a year later. The results suggest that the weight gain resulting from loss of ovarian function could be explained by decreased transport of leptin into the brain.


Asunto(s)
Química Encefálica/fisiología , Leptina/metabolismo , Ovario/fisiología , Animales , Barrera Hematoencefálica , Femenino , Radioisótopos de Yodo , Ratones , Ovariectomía , Aumento de Peso/fisiología
5.
Peptides ; 22(5): 829-34, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-11337097

RESUMEN

Although urocortin is a potent inhibitor of food ingestion after peripheral administration, it was recently shown that under normal conditions this peptide crosses the blood-brain barrier (BBB) at a very slow rate. We examined whether hyperglycemia could stimulate the rate of entry (K(i)) of (125)I-urocortin into the mouse brain. In euglycemic mice, (125)I-urocortin injected iv entered the brain at a rate similar to that of the vascular marker (99m)Tc-albumin. However, injection of glucose (3 g/kg, ip) 0.5, 1, or 2 h before the (125)I-urocortin greatly increased the influx of urocortin. Without the glucose, the self-inhibition characteristic of a saturable transport system was not apparent. Self-inhibition could be demonstrated after the glucose injection, indicating activation of a transport system for urocortin that was saturable. Injection of insulin (10 U/kg, ip) 1 or 2 h before the (125)I-urocortin decreased the K(i). Thus, the entry of urocortin into brain can be activated by changes in the concentration of blood glucose, illustrating the responsiveness of the BBB to regulatory influences.


Asunto(s)
Encéfalo/metabolismo , Hormona Liberadora de Corticotropina/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacocinética , Glucosa/agonistas , Insulina/farmacología , Animales , Glucemia/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Hiperglucemia/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Estreptozocina , Urocortinas
6.
Neuroendocrinology ; 73(4): 237-42, 2001 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11340337

RESUMEN

Since fasting is one of the few factors found to change the rate of entry of leptin into brain, we used multiple-time regression analysis to study the effects of pretreatment with glucose or insulin on leptin transport across the blood-brain barrier (BBB). Two hours after intraperitoneal injection of glucose (3 g/kg), there was a statistically significant increase in the entry rate (K(i)) of leptin in fasted (from 4.91 +/- 0.70 x 10(-4) ml/g x min to 9.03 +/- 1.00 x 10(-4) ml/g x min) but not (p = 0.15) in nonfasted normal (from 4.90 +/- 1.21 x 10(-4) ml/g x min to 6.42 +/- 1.79 x 10(-4) ml/g x min) or fasted streptozotocin (STZ)-treated diabetic mice (from 4.043 +/- 0.959 x 10(-4) ml/g min to 5.395 +/- 1.355 x 10(-4) ml/g min). Insulin (10 U/kg) increased leptin influx in fasted (from 4.77 +/- 0.26 x 10(-4) ml/g x min to 10.6 +/- 0.15 x 10(-4) ml/g x min at 0.5 h) and nonfasted (from 4.64 +/- 0.75 x 10(-4) ml/g x min to 7.46 +/- 1.48 x 10(-4) ml/g x min at 0.5 h) normal mice, but not in STZ-diabetic mice deficient in insulin (and leptin), even though basal concentrations of glucose were similarly increased in the nonfasted normal and STZ-treated mice. Moreover, the basal rate of leptin influx was the same in overnight fasted normal mice, nonfasted normal mice and STZ-diabetic mice. The results indicate that glucose and insulin can increase leptin transport, but they probably are not the principal factors responsible for the regulatory effect of the BBB on leptin entry into the brain.


Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Glucosa/farmacología , Hipoglucemiantes/farmacología , Insulina/farmacología , Leptina/metabolismo , Animales , Glucemia/metabolismo , Glucosa/administración & dosificación , Hipoglucemiantes/administración & dosificación , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Insulina/administración & dosificación , Insulina/sangre , Leptina/farmacocinética , Ratones , Ratones Endogámicos ICR , Agregado de Albúmina Marcado con Tecnecio Tc 99m
7.
Horm Metab Res ; 33(1): 19-25, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11280710

RESUMEN

Adrenomedullin (ADM) is present both in the periphery and brain. In addition to its peripheral effects, this peptide can exert central effects such as decreasing food ingestion. We used multiple-time regression analysis to determine that labeled ADM can cross from blood to brain with an apparent influx constant (K(I)) of 5.83 +/- 1.44 x 10(-4) ml/g-min, much faster than that of albumin, the vascular control. HPLC showed that almost all of the injected 125I-ADM in the brain was intact, and capillary depletion showed that it could reach the parenchyma of the brain. However, more 125I-ADM was reversibly associated with the brain vasculature than we have seen with any other peptide tested by these methods. After intracerebroventricular injection, 125I-ADM exited the brain with the bulk reabsorption of cerebrospinal fluid at an efflux rate comparable to that of albumin. Although there was no blood-to-brain saturation, in situ brain perfusion of 125I-ADM in blood-free physiological buffer showed self-inhibition by excess unlabeled ADM. This, along with evidence of the lack of protein binding shown by capillary zone electrophoresis, indicated competition for the binding site of ADM at the BBB. The low lipophilicity of ADM determined by the octanol/buffer partition coefficient was also consistent with the prominent reversible association of ADM with the vasculature of the BBB. This suggests a function for ADM at the cerebral blood vessels, such as altering cerebral blood flow and perfusion, without disruption of the BBB.


Asunto(s)
Barrera Hematoencefálica/fisiología , Péptidos/farmacocinética , Adrenomedulina , Animales , Tampones (Química) , Capilares/fisiología , Circulación Cerebrovascular/fisiología , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Inyecciones Intraventriculares , Radioisótopos de Yodo , Leptina/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Nitroglicerina/farmacología , Péptidos/sangre , Análisis de Regresión , Vasodilatadores/farmacología
8.
Peptides ; 22(12): 2127-36, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11786200

RESUMEN

Multiple-time regression analysis has been used to study the influx of radiolabeled peptides and polypeptides across the blood-brain barrier (BBB). This study used both tritiated and iodinated leptin to clarify several issues associated with these measurements. Recombinant murine leptin was radiolabeled with 3H by derivatization or with 125I by the iodobead method and each studied separately in mice. Intact 3H-leptin had a higher apparent influx rate from blood to brain than did intact 125I-leptin, correlating with its higher proportion of reversible association with the capillary lumen that would misleadingly appear to reflect entry. Yet the majority of 3H-leptin and 125I-leptin reached brain parenchyma. There was no significant difference in the influx rate between cerebral cortex and the subcortical regions, thus ruling out a predominant contribution of simple diffusion through the circumventricular organs or choroid plexuses outside the BBB. The influx of radiolabeled leptin, especially 125I-leptin, was decreased by excess unlabeled leptin, supporting the presence of a saturable transport system for leptin at the BBB. To identify the specificity of the transport system and determine whether it is shared by 3H-leptin and 125I-leptin, these radioactively labeled leptins were heat-denatured. Denaturation had no effect on the fast influx of 3H-leptin, but abolished the entry of 125I-leptin into brain; excess denatured leptin failed to inhibit the influx of either 3H-leptin or 125I-leptin. This indicates that the conformation of 125I-leptin is similar to that of native unlabeled leptin, so that iodination would be the better choice for investigating the interaction of leptin with the BBB. However, 3H-leptin can use the same transport system, as shown by inhibition of its influx by unlabeled leptin, whereas the derivatization procedure altered its biophysical properties such that its non-saturated influx was greatly enhanced. Finally, the rapid influx of radioactively labeled leptin contrasted greatly with that of the reference compounds 99mTc-albumin and 3H-inulin which had no significant penetration of the BBB. Thus, with additional considerations such as stability and interactions with the vasculature, multiple-time regression analysis is sensitive and selective for study of the penetration of peptides across the BBB.


Asunto(s)
Barrera Hematoencefálica , Leptina/farmacocinética , Animales , Capilares/metabolismo , Cromatografía Líquida de Alta Presión , Hidrólisis , Yodo/química , Leptina/química , Masculino , Ratones , Ratones Endogámicos ICR , Análisis de Regresión , Tritio/química
9.
Metabolism ; 49(11): 1444-8, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11092509

RESUMEN

Agouti-related protein (AgRP), expressed in both the periphery and the brain, can result in obesity. Its active C-terminal fragment, AgRP(83-132), was recently reported to increase feeding and antagonize alpha-melanocyte-stimulating hormone (alpha-MSH) and leptin. We used multiple-time regression analysis to show that the rate at which AgRP(83-132) crossed the blood-brain barrier (BBB) from the blood to the brain was very slow (Ki = 0.6 x 10(-4) mL/g x min). Entry was not self-inhibited by excess AgRP(83-132) after either intravenous (i.v.) injection or perfusion in blood-free medium, indicating the absence of a saturable transport system, and was not cross-inhibited by alpha-MSH or leptin. Not only did AgRP(83-132) cross much slower than the saturably entering leptin, but the entry was slower than almost all other non-saturably entering endogenous peptides or neurotrophins. Nevertheless, high-performance liquid chromatography (HPLC) showed that the small amount of AgRP(83-132) crossing the BBB did so in intact form, and capillary depletion showed that it entered the brain parenchyma rather than binding to capillary endothelial cells or adhering to vascular components. There was no rapid efflux system out of the brain that might have misleadingly appeared as slow entry for AgRP(83-132). Poor lipophilicity was shown by a low octanol/buffer partition coefficient. By size-exclusion chromatography, AgRP(83-132) appeared as a 17-kd substance in both blood and buffer. Since protein was absent from the buffer, the 17-kd peak probably represented a trimer of the 5.7-kd AgRP(83-132). Capillary electrophoresis confirmed that most of the AgRP(83-132) existed as a trimer, with much smaller amounts as a dimer and monomer. Thus, although intact AgRP(83-132) can cross the BBB from the blood to the brain, its nonsaturable rate of entry is very slow, probably influenced by aggregation.


Asunto(s)
Barrera Hematoencefálica , Fragmentos de Péptidos/farmacocinética , Proteína Relacionada con Agouti , Animales , Encéfalo/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Semivida , Masculino , Ratones , Ratones Endogámicos ICR
10.
J Pharmacol Exp Ther ; 294(2): 633-6, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10900242

RESUMEN

The mouse mahogany gene encodes a protein that is involved in the suppression of diet-induced obesity. We studied the ability of its widely conserved C-terminal fragment to cross the blood-brain barrier (BBB) in mice. Multiple-time regression analysis showed that the entry rate (K(i)) of (125)I-mahogany (1377-1428) from blood-to-brain was 5.5 x 10(-4) ml/g. min. After coinjection of unlabeled mahogany (1377-1428), the K(i) was significantly decreased, showing the self-inhibition characteristic of a saturable transport mechanism. The excess mahogany (1377-1428) did not change the influx rate of (99m)Tcalbumin, the vascular control, indicating a lack of disruption of the BBB. Statistically significant cross-inhibition was not seen with agouti-related protein (83-132), melanin-concentrating hormone, epidermal growth factor, leptin, a melanocortin-4 receptor antagonist, or alpha-melanocyte-stimulating hormone. HPLC showed that most of the injected (125)I-mahogany (1377-1428) reached the brain intact, and capillary depletion with washout showed that most of it reached the parenchyma. There was no brain-to-blood efflux system for mahogany (1377-1428) but rather retention after i.c.v. administration, and the octanol/buffer partition coefficient showed low lipophilicity. Thus, the results show that the C-terminal peptide product encoded by the mahogany gene crosses the BBB by a transport mechanism that is saturable. The ability of this system to be regulated indicates the therapeutic potential of mahogany (1377-1428) in the treatment of obesity.


Asunto(s)
Barrera Hematoencefálica/fisiología , Encéfalo/metabolismo , Proteínas de la Membrana/farmacocinética , Fragmentos de Péptidos/farmacocinética , Proteína Relacionada con Agouti , Animales , Transporte Biológico Activo/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Tampones (Química) , Capilares/metabolismo , Cromatografía Líquida de Alta Presión , Factor de Crecimiento Epidérmico/farmacología , Semivida , Péptidos y Proteínas de Señalización Intercelular , Radioisótopos de Yodo , Cinética , Leptina/farmacología , Masculino , Hormonas Estimuladoras de los Melanocitos/farmacología , Proteínas de la Membrana/sangre , Ratones , Ratones Endogámicos ICR , Octanoles/química , Fragmentos de Péptidos/sangre , Proteínas/farmacología , Ratas , Solubilidad , alfa-MSH/farmacología
11.
Peptides ; 21(5): 679-82, 2000 May.
Artículo en Inglés | MEDLINE | ID: mdl-10876050

RESUMEN

Food deprivation and adrenalectomy are associated with low concentrations of leptin in blood and the absence of obesity. Because leptin is known to cross the blood-brain barrier (BBB) by a saturable transport system, we examined whether fasting and adrenalectomy (ADX) also act at the BBB. Multiple-time regression analysis showed that fasting, but not ADX, significantly decreased the entry of leptin into mouse brain. After 3 days of food deprivation, the influx of leptin became indistinguishable from that of the vascular control (albumin); 5 h of refeeding significantly reversed this reduced rate of influx. Thus, the results indicate that the BBB provides a dynamic site for the regulation of physiological processes involving leptin.


Asunto(s)
Adrenalectomía , Ayuno , Leptina/metabolismo , Animales , Transporte Biológico , Barrera Hematoencefálica , Masculino , Ratones , Ratones Endogámicos ICR
12.
J Neurochem ; 74(1): 385-91, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10617143

RESUMEN

Melanin-concentrating hormone (MCH), found both peripherally and centrally, is involved in food ingestion. Although its expression in brain is increased by fasting, it is not known whether it crosses the blood-brain barrier (BBB). Use of the sensitive method of multiple-time regression analysis has shown that almost all of the peptides and polypeptides tested cross the BBB at a rate faster than the vascular marker albumin. With this same method, however, we found that the 19-amino acid 125I-Phe13,Tyr19-MCH did not cross faster than 99mTc-albumin. Several mechanisms were excluded as possible explanations for the slow rate of influx. These included degradation, association with capillary endothelial cells, and transport from brain to blood. When Phe13,Tyr19-MCH was perfused in blood-free buffer, however, it entered the brain significantly faster than albumin. This suggested protein binding as an explanation for the slow rate of influx when the MCH was administered in blood. Protein binding was confirmed by capillary zone electrophoresis, which showed that almost all of the Phe13,Tyr19-MCH added to blood migrated with a large-molecular-weight substance. Sodium dodecyl sulfate-capillary gel electrophoresis of Phe13,Tyr19-MCH in buffer additionally showed that the MCH aggregated as a trimer, a factor not preventing its influx by blood-free perfusion. Thus, the results show that blood-borne Phe13,Tyr19-MCH does not significantly cross the BBB, probably because of its binding to serum proteins.


Asunto(s)
Barrera Hematoencefálica , Hormonas Hipotalámicas/farmacocinética , Melaninas/farmacocinética , Hormonas Hipofisarias/farmacocinética , Animales , Proteínas Sanguíneas/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Capilares/metabolismo , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Inyecciones Intravenosas , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos ICR , Perfusión , Albúmina Sérica/farmacocinética
13.
Peptides ; 21(12): 1811-7, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11150641

RESUMEN

There are several transport systems for peptides and polypeptides at the blood-brain barrier (BBB) which facilitate the passage of bioactive substances from blood to brain or from brain to blood. Nonetheless, it would be a novel concept for one peptide or polypeptide to activate the transport of another peptide with a similar function but unrelated structure. In this study, we report the first observation of such a phenomenon: activation of a urocortin transport system at the BBB by leptin. Urocortin, a corticotropin-releasing factor (CRF)-related neuropeptide, is a more potent suppressor of food intake than leptin or CRF when injected peripherally. Radiolabeled urocortin ((125)I-urocortin) was used for these in vivo studies in mice; it remained stable and intact during the experimental period. Unlike CRF, urocortin was not saturably transported out of the brain. There was no substantial entry of (125)I-urocortin into brain as determined by sensitive multiple-time regression analysis after iv bolus injection. Addition of leptin, however, caused a dose-related increase in the influx of (125)I-urocortin and greatly facilitated its entry into brain parenchyma; this effect disappeared at higher doses of leptin. Moreover, in the presence of an activating dose of leptin, the entry of (125)I-urocortin into brain was saturable. The results indicate that the presence of leptin contributes to the potent satiety effects of urocortin after peripheral administration. Thus, the action of leptin in the periphery extends beyond its direct passage across the BBB and involves acute modulation of an inert transport system. We believe that these findings have broad physiological implications and indicate a unique function of the BBB as a regulatory interface.


Asunto(s)
Encéfalo/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Leptina/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Capilares/efectos de los fármacos , Capilares/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos ICR , Octanoles/farmacología , Transporte de Proteínas , Análisis de Regresión , Factores de Tiempo , Urocortinas
14.
Am J Physiol ; 277(5): E901-4, 1999 11.
Artículo en Inglés | MEDLINE | ID: mdl-10567018

RESUMEN

Cocaine- and amphetamine-regulated transcript (CART) is a new anorectic peptide found in the brain and periphery. It is closely associated with leptin, an anorectic agent saturably transported across the blood-brain barrier (BBB). Using multiple time-regression analysis, we found that CART has a rapid rate of entry into brain from blood. However, there was no self-inhibition with CART, even when perfused in blood-free buffer or in fasted mice, showing a lack of saturation. HPLC showed that at least 58% of the injected CART reached brain tissue in intact form, and capillary depletion with and without washout showed that the CART was not bound to endothelial cells or adherent to vascular components. There was no evidence for an efflux system out of the brain for CART. Thus CART can cross the BBB from blood to brain, but its rapid rate of entry is not inhibited by excess CART or leptin.


Asunto(s)
Barrera Hematoencefálica/fisiología , Leptina/farmacología , Proteínas del Tejido Nervioso/farmacocinética , Albúminas/farmacocinética , Animales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Tampones (Química) , Cromatografía Líquida de Alta Presión , Retroalimentación/efectos de los fármacos , Retroalimentación/fisiología , Conducta Alimentaria/fisiología , Inyecciones Intravenosas , Radioisótopos de Yodo/farmacocinética , Masculino , Ratones , Ratones Endogámicos ICR , Octanoles , Análisis de Regresión , Tecnecio/farmacocinética
15.
Am J Physiol ; 276(3): E479-82, 1999 03.
Artículo en Inglés | MEDLINE | ID: mdl-10070013

RESUMEN

Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 x 10(-4) ml. g. -1. min-1. However, this rate of entry was not significantly changed by injection of 10 microgram/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB.


Asunto(s)
Encéfalo/metabolismo , Neuropéptido Y/farmacocinética , Animales , Barrera Hematoencefálica/fisiología , Tampones (Química) , Capilares/fisiología , Circulación Cerebrovascular/fisiología , Cromatografía Líquida de Alta Presión , Masculino , Ratones , Ratones Endogámicos ICR , Perfusión
16.
J Pharmacol Exp Ther ; 289(1): 219-23, 1999 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10087007

RESUMEN

We determined the ability of orexin A and orexin B, recently discovered endogenous appetite enhancers, to cross the blood-brain barrier (BBB) of mice. Multiple time-regression analysis showed that an i.v. bolus of 125I-orexin A rapidly entered the brain from the blood, with an influx rate (Ki = 2.5 +/- 0.3 x 10(-4) ml/g.min) many times faster than that of the 99mTc-albumin control. This relatively rapid rate of entry was not reduced by administration of excess orexin A (or leptin) or by fasting for 22 h, even when penetration into only the hypothalamus was measured. Lack of saturability also was shown by perfusion in blood-free buffer. HPLC revealed that most of the injected 125I-orexin A reached the brain as intact peptide. Capillary depletion studies showed that the administered peptide did not remain bound to the endothelial cells comprising the BBB but reached the brain parenchyma. Efflux of 125I-orexin A from the brain occurred at the same rate as 99mTc-albumin. The octanol/buffer partition coefficient of 0.232 showed that orexin A was highly lipophilic, whereas the value for orexin B was only 0.030. Orexin B, moreover, was rapidly degraded in blood, so no 125I-orexin B could be detected in intact form in brain when injected peripherally. Thus, although orexin B is rapidly metabolized in blood and has low lipophilicity, orexin A rapidly crosses the BBB from blood to reach brain tissue by the process of simple diffusion.


Asunto(s)
Barrera Hematoencefálica , Encéfalo/metabolismo , Proteínas Portadoras/farmacocinética , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/farmacocinética , Animales , Tampones (Química) , Capilares/metabolismo , Proteínas Portadoras/sangre , Proteínas Portadoras/química , Cromatografía Líquida de Alta Presión , Difusión , Inyecciones Intravenosas , Radioisótopos de Yodo , Masculino , Ratones , Ratones Endogámicos ICR , Neuropéptidos/sangre , Neuropéptidos/química , Octanoles , Orexinas , Solubilidad , Agregado de Albúmina Marcado con Tecnecio Tc 99m
17.
J Cell Sci ; 111 ( Pt 4): 533-40, 1998 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9443901

RESUMEN

HIV-1 induces the AIDS dementia complex and infects brain endothelial and glial cells. Because the endothelial cells comprising the blood-brain barrier (BBB) do not possess CD4 receptors or galactosylceramide binding sites, it is unclear how HIV-1 negotiates the BBB. Previous work has suggested that gp120, the glycoprotein viral coat of HIV-1, is capable of inducing adsorptive endocytosis. Glycoprotein lectins like wheatgerm agglutinin induce adsorptive endocytosis and greatly potentiate the uptake by and passage across mouse endothelial cells in vivo and in vitro. We show here that the wheatgerm agglutinin-induced binding of gp120 is dose-dependent and involves components of the cytoskeleton. The uptake is partially dependent on temperature and energy and is modestly enhanced by potassium depletion. Glycosylation of gp120 is critical for its uptake by adsorptive endocytosis since the non-glycosylated form of gp120 is unaffected by wheatgerm agglutinin. Evidence is presented for the existence of a coreceptor sensitive to protamine sulfate that is primarily involved in membrane fusion after 125I-gp120 has bound to the cell membrane and is probably activated after internalization. This coreceptor probably contains a negatively charged heparin sulfate group and could be a member of the chemokine receptor family.


Asunto(s)
Barrera Hematoencefálica/fisiología , Endocitosis/fisiología , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Receptores del VIH/metabolismo , Adsorción , Animales , Membrana Celular/virología , Citoesqueleto/metabolismo , Endotelio Vascular/citología , Glucosa , Glicosilación , Humanos , Ratones , Polilisina/farmacología , Potasio/fisiología , Protaminas/farmacología , Temperatura , Aglutininas del Germen de Trigo/farmacología
18.
Life Sci ; 61(9): PL119-25, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9284088

RESUMEN

HIV-1 infects the brain and leads to AIDS dementia complex. The viral coat glycoprotein, gp120, may facilitate the passage of HIV-1 and HIV-infected immune cells across the blood-brain barrier (BBB). Since the endothelial cells of the BBB do not possess the CD4 or galactosylceramide binding sites used by gp120 to induce HIV-1 uptake into other cell types, how gp120 mediates entry into brain is unknown. We postulate that gp120 crosses the BBB and does so by acting as a weak lectin to induce adsorptive endocytosis (AE) in a fashion similar to other glycoproteins like wheatgerm agglutinin (WGA). We found in vivo that gp120 crosses the BBB and its passage is enhanced 18.7-fold by WGA. In vitro studies confirm that WGA enhances uptake of gp120 by brain endothelia; most of the uptake is membrane-associated, as expected in AE. Uptake is not dependent on clatharin, caveolae, calcium channels, or endosomal acidification. Our results suggest that gp120 crosses the BBB and does so by acting as a lectin to induce AE.


Asunto(s)
Barrera Hematoencefálica/fisiología , Endocitosis/fisiología , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Adsorción , Animales , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Endocitosis/efectos de los fármacos , Endotelio/metabolismo , Ratones , Aglutininas del Germen de Trigo/farmacología
19.
Am J Hypertens ; 7(7 Pt 1): 583-90, 1994 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-7946158

RESUMEN

Endothelin modulates human mesangial cell (HMC) proliferation in response to angiotensin II (Ang II). Angiotensin converting enzyme inhibitors (ACEIs) have variable effects on HMC growth depending on culture conditions. No studies, however, have investigated the effects of ACEIs on HMC production of endothelin-1 in either actively proliferating or quiescent HMCs. The present study was designed to evaluate the effects of ACEIs on HMC-associated mitogenesis, cell counts, and endothelin-1 production in the presence and absence of insulin in both quiescent and proliferating HMCs. It tests the hypothesis that ACEIs attenuate HMC growth through a reduction in HMC-associated endothelin-1 generation. The effects of four different ACEIs, an Ang II receptor antagonist, losartan, and a monoclonal antibody to endothelin-1 were evaluated. ACEIs inhibited HMC mitogenesis and cell counts in proliferative but not quiescent cells. This was due to the absence of ACE activity in HMCs and its presence in 10% fetal calf serum. Both ACEIs and losartan reduced endothelin-1 production per cell. Compared to vehicle, losartan reduced the amount of endothelin-1 in conditioned media to a greater extent than any ACEI (2.2 +/- 0.3, captopril v 1.9 +/- 0.5, quinaprilat v 3.8 +/- 0.3 delta pg/cell x 10(-3) endothelin-1, losartan; P < .05). Moreover, insulin potentiated the antimitogenic effects of both ACEIs and losartan on HMCs. Lastly, the attenuated increase of endothelin-1 in conditioned media and associated antimitogenic effect on HMCs with losartan alone was not potentiated by the addition of any ACEI to losartan. These data provide indirect evidence that Ang II production may occur in culture media when both its precursors and a sufficient amount of converting enzyme activity are present. This is predicated on the observation that HMCs lack ACE activity and that ACEIs blunt mitogenesis of proliferating HMCs. The kinetics of this reaction, as well as the mechanism of how insulin potentiates the antimitogenic effects of ACEIs, were not studied.


Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Endotelinas/fisiología , Mesangio Glomerular/citología , Anticuerpos Monoclonales , Compuestos de Bifenilo/farmacología , División Celular/efectos de los fármacos , Endotelinas/antagonistas & inhibidores , Mesangio Glomerular/metabolismo , Humanos , Imidazoles/farmacología , Losartán , Mitosis/efectos de los fármacos , Peptidil-Dipeptidasa A/metabolismo , Tetrazoles/farmacología
20.
Am J Physiol ; 264(5 Pt 1): E723-9, 1993 May.
Artículo en Inglés | MEDLINE | ID: mdl-8498494

RESUMEN

Cyclo(His-Pro) (cHP) is a peptide widely distributed in the central nervous system (CNS) and peripheral tissues that can affect brain function after either peripheral or CNS administration. This suggests that cHP may be a neuromodulator capable of crossing the blood-brain barrier (BBB). We, therefore, studied the ability of radioactively labeled cHP (I-cHP) to cross the BBB. We found that I-cHP can cross the BBB in either the direction of blood to brain or brain to blood by nonsaturable mechanisms. The rate of entry of I-cHP into the CNS was low in comparison with other peptides, especially considering its relatively low molecular weight and high lipid solubility. However, this slow entry was offset by a long half-life in blood and extreme enzymatic resistance, allowing cHP to accumulate in the CNS. This accumulation was sufficient to allow intravenous cHP to reverse ethanol-induced narcosis, an effect mediated through the CNS. The rate of entry of I-cHP was resistant to conditions that alter the passage of some other substances across the BBB or that have been shown to affect cHP metabolism such as aging, diabetes, and pretreatment with aluminum. Entry of cHP into the brain was not retarded by binding to serum proteins. Significant amounts of I-cHP entered the serum, brain, and other tissues after intraperitoneal administration, the route used in many studies of cHP. Taken together, these results show that cHP is a highly stable peptide that, after intravenous injection, slowly enters the brain by a nonsaturable mechanism in amounts large enough to affect such aspects of the CNS as ethanol-induced narcosis.


Asunto(s)
Barrera Hematoencefálica , Ventrículos Cerebrales/fisiología , Etanol/farmacología , Inhibidores de la Captación de Neurotransmisores/farmacología , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Piperazinas/metabolismo , Piperazinas/farmacología , Sueño/efectos de los fármacos , Animales , Ventrículos Cerebrales/efectos de los fármacos , Inyecciones Intraventriculares , Cinética , Masculino , Tasa de Depuración Metabólica , Ratones , Inhibidores de la Captación de Neurotransmisores/metabolismo , Péptidos Cíclicos/farmacocinética , Piperazinas/farmacocinética , Sueño/fisiología , Factores de Tiempo , Distribución Tisular
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