RESUMEN

Few studies have addressed the ultrastructure and morphology of neurons in primary pure culture. We therefore use immunohistochemistry and electron microscopy to investigate the ultrastructure of cultured neurons during extended incubation in vitro. Rat cerebral cortex neurons were cultured in Neurobasal™ medium. Adherent cells developed as networks of single neurons or clusters depending on the plating density. Almost all surviving cells were neurons as demonstrated by neurofilament immunolabeling. The number of cultured neurons increased substantially to 14-21 days in vitro (DIV) and then plateaued and subsequently declined. From DIV 1-10 neurons extended large neurites, followed by the development of fine and dense neurites, and neurones survived until DIV 30-50. Notably, numerous mitochondria were observed along fibrous elements within neurites, suggestive of active intracellular trafficking. Electron microscopy also revealed that multiple types of synapses were formed between neurons. These ultrastructural results confirm previous reports of electrophysiological activity in cultured neurons. However many neurons contained distorted mitochondria and abnormal organelles including multilamellar vesicles and multivesicular myeloid bodies. The proportion of neurons containing abnormal organelles increased significantly in culture medium supplemented with antibiotics. On long-term culture neuronal death and apoptotic nuclei were observed. Despite the presence of abnormal organelles, the ultrastructure of cultured neurons was very similar to that of in vivo neurons; in vitro culture therefore provides a useful tool for studies on neuronal development, aging, and neurotransmission.

Asunto(s)

Corteza Cerebral/citología , Neuronas/ultraestructura , Animales , Antibacterianos/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Embrión de Mamíferos , Etiquetado Corte-Fin in Situ , L-Lactato Deshidrogenasa/metabolismo , Microscopía Electrónica de Transmisión , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Factores de Tiempo

RESUMEN

Abnormalities of carbohydrate metabolism and monoamine neurotransmitters have been widely implicated in the pathoetiology of human epilepsy, and glucose hypometabolism and/or tryptophan utilization can be used to localize epileptic foci in the human brain. To investigate the neurochemical changes that underlie seizure susceptibility we studied four strains of mice that respond differently to the convulsant methionine sulfoximine (MSO). Seizures in CBA/J strain were induced by MSO at a dosage half that necessary to provoke seizures in C57BL/6J, BALB/c, or Swiss mice. We report that brain glycogen content in response to MSO administration was markedly increased in all four strains of mice. Of the monoamine neurotransmitters studied, the most prominent change was in brain serotonin (5-hydroxytryptamine, 5-HT) levels that showed a significant reduction following MSO administration. MSO also lowered the concentration of the 5-HT precursor tryptophan. Notably, inhibition of the fall in 5-HT levels by coadministration of 5-hydroxytryptophan delayed the onset of MSO-induced seizures. These results indicate that increased glycogen content and decreased brain levels of 5-HT and tryptophan are hallmarks of MSO action in mice, and suggest that defective serotonergic neurotransmission could trigger glycogen increase and seizure genesis.

Asunto(s)

Convulsivantes/farmacología , Epilepsia/metabolismo , Glucógeno/metabolismo , Metionina Sulfoximina/farmacología , Serotonina/deficiencia , Serotonina/fisiología , Transmisión Sináptica/fisiología , Animales , Epilepsia/inducido químicamente , Epilepsia/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Transmisión Sináptica/efectos de los fármacos

RESUMEN

A novel pyridine derivative, 8-{4-[(6-methoxy-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine-3-ylmethyl)-amino]-butyl}-8-aza-spiro[4.5]decane-7,9-dione hydrochloride, termed JB-788, was designed to selectively target 5-HT(1A) receptors. In the present study, the pharmacological profile of JB-788 was characterized in vitro using radioligands binding tests and in vivo using neurochemical and behavioural experiments. JB-788 bound tightly to human 5-HT(1A) receptor expressed in human embryonic kidney 293 (HEK-293) cells with a K(i) value of 0.8 nM. Its binding affinity is in the same range as that observed for the (+/-)8-OH-DPAT, a reference 5HT(1A) agonist compound. Notably, JB-788 only bound weakly to 5-HT(1B) or 5-HT(2A) receptors and moreover the drug displayed only weak or indetectable binding to muscarinic, alpha(2), beta(1) and beta(2) adrenergic receptors, or dopaminergic D(1) receptors. JB-788 was found to display substantial binding affinity for dopaminergic D(2) receptors and, to a lesser extend to alpha(1) adrenoreceptors. JB-788 dose-dependently decreased forskolin-induced cAMP accumulation in HEK cells expressing human 5-HT(1A), thus acting as a potent 5-HT(1A) receptor agonist (E(max.) 75%, EC(50) 3.5 nM). JB-788 did not exhibit any D(2) receptor agonism but progressively inhibited the effects of quinpirole, a D(2) receptor agonist, in the cAMP accumulation test with a K(i) value of 250 nM. JB-788 induced a weak change in cAMP levels in mouse brain but, like some antipsychotics, transiently increased glycogen contents in various brain regions. Behavioral effects were investigated in mice using the elevated plus-maze. JB-788 was found to increase the time duration spent by animals in anxiogenic situations. Locomotor hyperactivity induced by methamphetamine in mouse, a model of antipsychotic activity, was dose-dependently inhibited by JB-788. Altogether, these results suggest that JB-788 displays pharmacological properties, which could be of interest in the area of anxiolytic and antipsychotic drugs.

Asunto(s)

Aprendizaje por Laberinto/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Piridinas/farmacología , Receptor de Serotonina 5-HT1A/fisiología , Agonistas del Receptor de Serotonina 5-HT1/farmacología , Compuestos de Espiro/farmacología , Animales , Ansiolíticos/farmacología , Antipsicóticos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Antagonistas de los Receptores de Dopamina D2 , Glucógeno/metabolismo , Humanos , Masculino , Ratones , Ensayo de Unión Radioligante , Receptores de Dopamina D2/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores

RESUMEN

Growth factors such as insulin-like growth factor type I (IGF-I), epidermal growth factor (EGF), vascular-endothelial growth factor (VEGF) and transforming growth factor beta (TGF-beta) are present during the development of the CNS. When they reappear in the mature brain they are overexpressed in neoplastic glia, participating in the development of the most common human brain malignant tumor, glioblastoma multiforme, which is invariably fatal. Progress in treatment of this disease involves an increase in median survival from 8 to 11 months to an average of 15 months, rarely to 18 months. We do not know any therapy, which can make a complete stop of this neoplasm. To inhibit this process various anti-growth factor therapies have been proposed. We describe actual applications of growth factor inhibitors and antisense approaches. The review highlights results obtained with the promising treatment of glioblastoma multiforme: using inhibitors and antisense targeting growth factors, including IGF-I, their receptors, and their downstream signaling effectors including glycogenesis and oncogenes. The antisense strategies have been the subject of many clinical trials, especially the IGF-I antisense approach. Such antisense therapies, already introduced in clinical trial in the USA, Europe and Asia, will soon become the preferred alternative treatment for human glioblastoma multiforme. The inhibition of signal transduction pathways common to growth factors and glycogenesis appears as a parallel challenge to glioblastoma multiforme inhibition studies.

Asunto(s)

Neoplasias Encefálicas/fisiopatología , Glioma/fisiopatología , Factor I del Crecimiento Similar a la Insulina/fisiología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/terapia , Glioblastoma/mortalidad , Glioblastoma/fisiopatología , Glioma/mortalidad , Glioma/terapia , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Análisis de Supervivencia

RESUMEN

Glutamine synthetase (GS) plays a key role in the regulation of glutamate availability to neurons. In the present study glutamine synthetase was immobilized on a silica-based immobilized artificial membrane liquid chromatographic stationary phase (IAM-SP) to create the GS-IAM. The stability of GS was improved by immobilization, but the enzyme's affinity for the substrates L-glutamate and D-glutamate was significantly decreased. In contrast, immobilization significantly increased GS sensitivity to inhibition by methionine sulfoximine. The GS-IAM was packed into a chromatography column to create an immobilized enzyme reactor (GS-IMER). On-line experiments with the GS-IMER demonstrated that the immobilized enzyme was comparable to the non-immobilized enzyme with regards to retention of activity and selectivity toward substrates and inhibitors and was reusable for several weeks.

Asunto(s)

Encéfalo/enzimología , Cromatografía Liquida/instrumentación , Glutamato-Amoníaco Ligasa/análisis , Animales , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Glutamato-Amoníaco Ligasa/metabolismo , Ácido Glutámico/metabolismo , Ovinos , Especificidad por Sustrato

RESUMEN

Methionine sulfoximine is a xenobiotic amino acid derived from methionine. One of its major properties is to display a glycogenic activity in the brain. After studying this property, we investigate here a possible action of this xenobiotic on the expression of genes related to carbohydrate anabolism in the brain. Glycogen was studied by the means of electron microscopy. Astrocytes were cultured and the influence of methionine sulfoximine on carbohydrate anabolism in these cells was investigated. In vivo, methionine sulfoximine induced a large increase in glycogen accumulation. It also enhanced the glycogen accumulation in cultured astrocytes principally, when the medium was enriched in glucose. The gluconeogenic enzyme fructose-1,6-bisphosphatase may account for glycogen accumulation. Plasmids were built using antisens cDNA to permanently block the expression of fructose-1,6-bisphosphatase. An eukaryotic vector was used and the expression of fructose-1,6-bisphosphatase gene was under the control of the promoter of the glial fibrillary acidic protein. In this case, the glycogen content in cultured astrocytes largely decreased. This work shows that methionine sulfoximine enhances energy carbohydrate synthesis in the brain. Since this xenobiotic also enhances the expression of some genes related to one of the key step of glucose synthesis, it is possible that genes may be one target of methionine sulfoximine. Next investigations will study the actual effect of methionine sulfoximine in the cells.

Asunto(s)

Química Encefálica/efectos de los fármacos , Química Encefálica/genética , Metabolismo de los Hidratos de Carbono , Expresión Génica/efectos de los fármacos , Metionina Sulfoximina/toxicidad , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Clonación Molecular , Fructosa-Bifosfatasa/biosíntesis , Glucógeno/metabolismo , Masculino , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Transfección

RESUMEN

It is now well established that in epileptic patients, hypometabolic foci appear during interictal periods. The meaning and the mechanism of such an hypometabolism are as yet unclear. The aim of the present investigation was to look for a putative relationship between glucose metabolism in the brain and the genesis of seizures in mice using administration of the convulsant, methionine sulfoximine. Besides its epileptic action, methionine sulfoximine is a powerful glycogenic agent. We analyzed the epileptogenic and glycogenic effects of methionine sulfoximine in two inbred mouse strains with different susceptibility towards the convulsant. CBA/J mice displayed high response to methionine sulfoximine. The tonic convulsions appeared 5-6 h after MSO administration, without brain glycogen content variations during the preconvulsive period. These mice died of status epilepticus during the first seizure(s). Conversely, C57BL/6J mice displayed low response to MSO. The tonic and clonic seizures appeared 8 to 14 h after MSO administration with only 2% mortality. The seizures were preceded by an increase in brain glycogen content during the preconvulsive period. Moreover, during seizures, C57BL/6J mice were able to mobilize this accumulated brain glycogen, that returned to high value after seizures. The epileptic and glycogenic responses of the parental strains were also observed in mice of the F2 generation. The F2 mice that convulsed early (16%) did not utilize their small increase in brain glycogen content, and resembled CBA/J mice; while the F2 mice that seized tardily (24%) increased their brain glycogen content before convulsion, utilized it during convulsions, and resembled C57BL/6J mice. Sixty percent of the F2 mice presented an intermediate pattern in epileptogenic responses to the convulsant. These data suggest a possible genetic link between the two MSO effects, epileptiform seizures and increase in brain glycogen content. The increase in brain glycogen content and the capability of its mobilization during seizures could delay the seizure's onset and could be considered a "resistance factor" against the seizures.

Asunto(s)

Encéfalo/metabolismo , Convulsivantes/farmacología , Glucógeno/metabolismo , Metionina Sulfoximina/farmacología , Convulsiones/metabolismo , Animales , Encéfalo/efectos de los fármacos , Cruzamientos Genéticos , Femenino , Predisposición Genética a la Enfermedad , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Convulsiones/inducido químicamente , Convulsiones/genética

RESUMEN

On the basis of paradigms in development wherein discrete transcriptional events are pivotal regulatory steps, we tested the hypothesis that transcriptional sodium (Na+)-response mechanisms are involved in in vivo Na+-induced responses relevant to normal (homeostatic) and pathophysiological (salt-sensitive hypertension) conditions. We used Na,K-ATPase alpha-subunit genes as molecular probes and the Na+ ionophore monensin to induce a dose-specific incremental increase in [Na+]i in rat A10 embryonic aortic smooth muscle cells. RNA blot analysis of rat A10 cells revealed a dose-specific (0.022 to 30 micromol/L monensin) upregulation of alpha1-, alpha2-, and beta1-subunit Na,K-ATPase RNA levels. Control beta-actin and alpha-tropomyosin RNA levels did not change. With the use of chloramphenicol acetyltransferase (CAT) as reporter gene, CAT assays of rat alpha1[-1288]CAT and human alpha2[-798]CAT promoter constructs exhibited induction of CAT activity in monensin (10 micromol/L)-treated A10 cells compared with untreated A10 cells. Promoter deletion constructs for rat alpha1[-1288]CAT defined a positive Na+-response regulatory region within -358 to -169 that is distinct from the basal transcriptional activation region of -155 to -49 previously defined. Similarly, a positive Na+-response regulatory region is delimited to within -301 in the human alpha2 Na,K-ATPase 5' flanking region. Analysis of transgenic TgH alpha2[-798]CAT rats demonstrated sodium activation of human alpha2[-798]CAT transgene expression in aorta parallel to observations made in rat A10 aortic tissue culture cells. Southwestern blot analysis of nuclear extracts from monensin (10 micromol/L)-treated and control untreated A10 cells revealed a nuclear DNA binding protein (approximately 95 kD) that is upregulated by increased [Na+]i. These data provide initial characterization of a transcriptional Na+-response mechanism delimiting a positive Na+-response regulatory region in two target genes (alpha1 and alpha2 Na,K-ATPase) as well as detection of a Na+-response nuclear DNA binding protein. The in vitro data are corroborated by in vivo experimental and transgenic promoter expression studies, thus validating the biological relevance of the observations.

Asunto(s)

Sodio/metabolismo , Transcripción Genética , Animales , Animales Modificados Genéticamente/genética , Línea Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica/fisiología , Regulación de la Expresión Génica , Genes , Humanos , Ionóforos/farmacología , Isoenzimas/genética , Monensina/farmacología , Ratas , Sodio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genética , Transgenes

RESUMEN

The mouse fructose-1,6-bisphosphatase (FBPase) cDNA was previously cloned from testicular teratocarcinoma cultured cells (F9 cells). Using this published nucleotide sequence four primer sets were defined and used to amplify FBPase transcript from cerebral cortex, heart, kidney, liver and testis of male C57B1/6 mice. Only one primer set was efficient in all total RNA prepared from the various tissues. The restriction maps of these RNA amplification products suggested the existence of three different FBPase transcripts; this was confirmed by the nucleotide sequences of the FBPase transcripts and by the deduced amino acid sequences. These data are consistent with the existence of three different FBPase genes. This may be relevant in neurological disease in which abnormalities of brain glucose metabolism are involved.

Asunto(s)

Encéfalo/enzimología , Fructosa-Bifosfatasa/biosíntesis , Isoenzimas/biosíntesis , Riñón/enzimología , Hígado/enzimología , Miocardio/enzimología , Transcripción Genética , Secuencia de Aminoácidos , Animales , Cartilla de ADN , Fructosa-Bifosfatasa/química , Humanos , Isoenzimas/química , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Teratoma , Células Tumorales Cultivadas

RESUMEN

alpha 2-adrenoceptors are involved in various renal functions regulating blood pressure. They were classified in subtypes whom genes were identified in both humans and rats. In rat renal cortex it was evidenced that the alpha 2B isoform is predominant. This result was confirmed in Sabra rats. However, the renal cortex alpha 2B density is higher in salt-sensitive (SBH) than in salt-resistant (SBN) Sabra rats. alpha 2B-adrenoceptors were recently subclassified in two pharmacologically distinct subtypes exhibiting high and low affinity for guanoxabenz and respectively called alpha 2B1 and alpha 2B2. We studied sodium loading effect on alpha 2B1 and alpha 2B2 distribution in Sabra rat renal cortex using competition experiments between [3H]-yohimbine and guanoxabenz. The rats were submitted to normal (0.2%) or high sodium diet (8%) for six weeks. Under normal diet, proportion alpha 2B1 and alpha 2B2 was similar in SBH and SBN. Nevertheless, their respective densities were significantly higher in SBH as compared to SBN (alpha 2B1: 90.6 +/- 4.1 vs 57.4 +/- 2.5 fmoles/mg prot, p < 0.0001; n = 5; alpha 2B2: 102.7 +/- 4.0 vs 66.4 +/- 4.6 fmoles/mg prot; p < 0.0001; n = 5). Under high sodium diet the distribution of these two isoforms was altered. The densities of alpha 2B1 were decreased by 27.0 +/- 5.9% in SBH (68.0 +/- 4.0 fmoles/mg prot; p < 0.0001, n = 5) and by 47.3 +/- 7.4% for SBN (29.2 +/- 3.1 fmoles/mg prot; p < 0.0001; n = 5). Conversely, the densities of alpha 2B2 were increased by 28.3 +/- 5.4% in SBH (131.1 +/- 9.5 fmoles/mg prot; p < 0.001; n = 5) and by 75.0 +/- 17% in SBN (123.2 +/- 9.1 fmoles/mg prot; p < 0.0001; n = 5). In conclusion, alpha 2B1- and alpha 2B2-adrenoceptor subtypes are found in renal cortex of both SBH and SBN. Our data demonstrated an equal distribution of these two isoforms between SBH and SBN under normal salt diet. This distribution is largely altered, especially in SBN, by the high sodium diet. From these modifications might result differential renal responses to activation of alpha 2B-adrenoceptors between SBH and SBN, and consequently responsible for normal or high blood pressure after high sodium diet.

Asunto(s)

Corteza Renal/metabolismo , Receptores Adrenérgicos alfa 2/análisis , Sodio en la Dieta/efectos adversos , Antagonistas Adrenérgicos alfa/metabolismo , Animales , Antihipertensivos , Unión Competitiva , Guanabenzo/análogos & derivados , Guanabenzo/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Oximetazolina/metabolismo , Prazosina/metabolismo , Ratas , Receptores Adrenérgicos alfa 2/metabolismo , Yohimbina/metabolismo

RESUMEN

OBJECTIVE: To assess whether alterations of alpha 2-adrenoceptor subtypes in distribution and gene expression in the renal cortex could explain the predisposition to salt-sensitivity or salt-resistance in Sabra rats. DESIGN: Studies were performed using plasma membranes and RNA preparations from renal cortex of 8- to 10-week-old Sabra salt-sensitive (SBH) and salt-resistant (SBN) rats on a normal-sodium diet. METHODS: The alpha 2-adrenoceptor subtypes were determined by competition experiments with [3H]-yohimbine or [3H]-RX821002. Their gene expression was studied by RNA-directed complementary DNA synthesis followed by Taq DNA polymerase amplification. RESULTS: Binding studies showed that alpha 2B- and alpha 2A-adrenoceptor subtypes represented in SBN 72 and 28% of the maximal binding capacities of the two radioligands, respectively. In contrast, only the alpha 2B subtype was detected in the SBH rat. However, the use of guanoxabenz disclosed alpha 2B-adrenoceptors in alpha 2B1 and alpha 2B2 subtypes. The densities of those alpha 2B subtypes appeared to be higher in the SBH rat than in the SBN rat. Messenger RNA corresponding to alpha 2A and alpha 2B subtypes were found both in SBH rats and in SBN rats. The expression of the alpha 2B subtype was permanently higher in the SBH rats than in the SBN rats. The expression of the alpha 2A gene in the SBH rats suggests a specific SBH post-transcriptional regulation resulting in the absence of alpha 2A-adrenoceptor. CONCLUSIONS: Differences exist in the renal cortex concerning expression and distribution of alpha 2-adrenoceptor subtypes between SBH and SBN rats. From these differences there might result different alpha 2-adrenoceptor-mediated renal functions in SBH and in SBN rats, which could lead to a predisposition to sensitivity or resistance to a high sodium intake.

Asunto(s)

Hipertensión/metabolismo , Corteza Renal/química , ARN Mensajero/análisis , Receptores Adrenérgicos alfa 2/análisis , Cloruro de Sodio/farmacología , Animales , Secuencia de Bases , Northern Blotting , Amplificación de Genes , Masculino , Datos de Secuencia Molecular , Ratas , Receptores Adrenérgicos alfa 2/genética , Yohimbina/metabolismo

RESUMEN

A defect in sodium modulation of density and agonist affinity of renal alpha 2-adrenoceptor exists in normotensive salt-resistant Sabra (SBN) rats when compared to hypertensive salt-sensitive (SBH). A highly conserved aspartic acid residue in the second helix has been implicated in sodium regulation of alpha 2-adrenoceptor-ligand interactions. As the alpha 2B-adrenoceptor subtype is preponderantly present in kidney of SBH and SBN rats, a mutation might distinguish this subtype between SBH and SBN rats. From this study, no difference between SBH and SBN alpha 2B-adrenoceptor gene could be demonstrated in terms of nucleotide sequence. These data suggest that in Sabra rats, the differential sodium regulation in density and agonist affinity between renal SBH and SBN alpha 2-adrenoceptor may have another origin than the alpha 2B-adrenoceptor encoding gene.

Asunto(s)

ADN/análisis , Hipertensión/metabolismo , Receptores Adrenérgicos alfa 2/genética , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Electroforesis , Amplificación de Genes , Genoma , Masculino , Datos de Secuencia Molecular , Mutación , Ratas

RESUMEN

In vivo, testosterone-treatment of female hamsters for 4 days promotes a doubling of alpha 2-adrenoreceptor protein in parametrial adipocytes, with a concomitant accumulation of the alpha 2A-adrenoreceptor subtype mRNA. During in vitro incubation of minced parametrial fat pads for 6 to 48h with testosterone or dihydrotestosterone (100 nM), alpha 2A-adrenoreceptor protein and mRNA levels were also increased and remained to control levels when an antiandrogen or actinomycin D were added in the medium. It is concluded that in hamster adipocytes, androgens upregulate alpha 2A-adrenoreceptor subtype expression at the mRNA level by an androgen receptor-dependent transcriptional activation.

Asunto(s)

Adipocitos/metabolismo , Expresión Génica/efectos de los fármacos , Receptores Adrenérgicos alfa 2/biosíntesis , Testosterona/farmacología , Adipocitos/efectos de los fármacos , Antagonistas Adrenérgicos alfa/metabolismo , Animales , Secuencia de Bases , Southern Blotting , Cricetinae , ADN/análisis , ADN/metabolismo , Cartilla de ADN , Dioxanos/metabolismo , Femenino , Idazoxan/análogos & derivados , Técnicas In Vitro , Cinética , Masculino , Mesocricetus , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Orquiectomía , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Receptores Adrenérgicos alfa 2/metabolismo , Mapeo Restrictivo , Factores Sexuales , Factores de Tiempo

RESUMEN

The alpha tropomyosin (TM)/N5 enhancer is an SV40-like mammalian enhancer comprised of a 99 bp repeat with modular cis-acting regulatory elements exhibiting apparent hierarchical organization. The enhancer differentially regulates the alpha TM and N5 transcription units which exhibit distinct tissue-specific expression patterns and interacts with multiple myotube-associated nuclear DNA binding proteins that varied in size and amount. To further characterize the interaction with multiple myotube nuclear factors, comparative southwestern blot analyses were done with a panel of strategic DNA probes representative of modular enhancer sequences in the alpha TM/N5 enhancer and respective alpha TM and N5 promoter regions. Results demonstrate that multiple DNA binding proteins, which vary in size and amount, can interact with a particular enhancer modular sequence (delimited to 18 bp- to 38 bp-long); and that likewise, a DNA binding protein can bind specifically to different DNA enhancer modular sequences with apparent different affinities. Results also demonstrate DNA binding proteins that differentially bind to both enhancer modular sequences and respective promoter regions supporting a putative parsimonious mechanism for the approximation of enhancer and promoter elements as an alternative to the multi-protein stereospecific enhancer complex. Cogent to this interesting "head to head"/shared enhancer gene arrangement, we investigated the primary structure of the "other" transcription unit, N5. Nucleotide sequence analysis of the N5 cDNA reveals that it is a putative DNA binding protein representing a new structural class of transcription factors exhibiting a novel combinatorial motif: single zinc finger (DNA-binding)-leucine zipper (dimerization)--making it a z-ZIP instead of a b-ZIP (basic region/leucine zipper) protein.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Tropomiosina/genética , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Bovinos , Línea Celular , Núcleo Celular , ADN/metabolismo , Leucina Zippers , Ratones , Datos de Secuencia Molecular , Unión Proteica , Conejos , Factores de Transcripción , Transcripción Genética

RESUMEN

Existing evidences indicate that a crossed regulation between alpha 2-adrenergic receptors and Na+/H+ exchanger(s) exists, that Na decreases the affinity of alpha 2-adrenergic receptors for agonists and antagonists, that intracellular Na+ and H+ ion concentrations regulate Na+/H+ exchanger activity, that intracellular pH controls the affinity of the alpha 2-adrenergic receptors for their agonists and antagonists. Alterations of alpha 2-adrenergic receptor densities and allosteric regulation by sodium have been demonstrated in sodium-dependent hypertension in rats. Increased Na+/H+ exchanger activity has been reported in genetic hypertension. Nevertheless, cosegregation experiments and human genetic polymorphism suggest that the exchanger could not be related to hypertension. We propose the following hypothesis: the increased Na+/H+ exchanger characteristic of hypertension could be secondary to the abnormalities of the alpha 2-adrenergic receptors found in hypertension, probably through the alteration of the sodium allosteric effect on these receptors.

Asunto(s)

Hidrógeno/metabolismo , Hipertensión/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Sodio en la Dieta/farmacología , Sodio/metabolismo , Animales , Resistencia a Medicamentos , Hipertensión/etiología , Transporte Iónico , Ratas , Ratas Endogámicas , Sodio en la Dieta/efectos adversos

RESUMEN

A decrease in platelet 5-HT content linked to partial inhibition of 5-HT uptake has been described in essential hypertension. Transport of 5-HT through platelet membrane is dependent upon transmembranal Na+ and K+ gradients. It is inhibited by Na+, K+-ATPase inhibitors such as ouabain and endogenous digitalis-like compounds isolated from hemodiafiltrate. The activity of such compounds in plasma extracts, measured by inhibition of Na+,K+-ATPase or ouabain binding to human erythrocytes, and platelet 5-HT content were determined in parallel in essential hypertensive patients. Significant negative correlations were observed between these parameters in men, suggesting that high levels of digitalis-like compounds can affect platelet 5-HT content. In addition, in essential hypertensive patients, total plasma cholesterol was inversely related to both platelet 5-HT content (n = 15, r = -0.594, P less than 0.02) and maximal velocity of 5-HT uptake (n = 15, r = -0.717, P less than 0.003). In normotensive control subjects, no variation of platelet 5-HT content with cholesterol was observed. This suggests that the platelet membranes of essential hypertensive patients are more sensitive to increases in plasma cholesterol than those of normotensive subjects.

Asunto(s)

Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Colesterol/sangre , Digoxina , Hipertensión/sangre , Saponinas , Serotonina/sangre , Adulto , Cardenólidos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ouabaína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/sangre

RESUMEN

Endogenous digitalis-like compounds are present in biological fluids and in some tissues. Little is known, so far, about their chemical nature and the tissues where they originate. Plasma and urinary levels of these compounds are elevated in essential and experimental arterial hypertension, and this rise may contribute to the genesis and maintenance of an abnormally high blood pressure. This is explained by an increase in intracellular calcium concentration resulting from inhibition of the sodium/potassium pump and increase of intracellular sodium. Concerning the chemical nature of these endogenous digitalis-like compounds two hypotheses have been put forward: peptides or steroids. Precise identification of these compounds and a knowledge of their mechanism of action on the sodium/potassium pump will probably lead to the development of new drugs in the field of cardiovascular and renal diseases.

Asunto(s)

Glicósidos Digitálicos , Hipertensión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Humanos , Hipertensión/sangre , Hipertensión/orina

RESUMEN

Endogenous digitalislike compound (or compounds) has been described as involved in some diseases. Questions remain concerning its chemical nature, origin, and biological properties. The methods of measuring the compound are based on biological properties of digitalis, mainly Na+, K+-adenosine triphosphatase (ATPase) inhibition and related properties. Chemically, digitalislike compound has been described as a peptide, as fatty acids, and as a steroid. Its origin could be the brain, particularly the hypothalamus and pituitary gland. The adrenal glands were also proposed as its origin. The reported biological properties of digitalislike compound are mainly dependent on Na+, K+-ATPase inhibition. No definitive conclusions can be drawn from the available data.

Asunto(s)

Proteínas Sanguíneas/aislamiento & purificación , Digoxina , Saponinas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Proteínas Sanguíneas/farmacología , Cardenólidos , Diuresis/efectos de los fármacos , Perros , Interacciones Farmacológicas , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Ácidos Grasos/aislamiento & purificación , Humanos , Mamíferos , Natriuresis/efectos de los fármacos , Ouabaína/farmacología , Péptidos/aislamiento & purificación , Potasio/farmacología , Ranidae , Ratas , Serotonina/metabolismo , Esteroides/aislamiento & purificación , Orina/análisis

RESUMEN

Plasma and urine levels of an endogenous digitalis-like compound (EDLC) are increased in low renin Na+-dependent experimental hypertension, in some normotensive offspring of hypertensive patients and in some essential hypertensive patients. Urine-drived EDLC was purified from 550 L of urine from essential hypertensive patients (n = 8) and from normotensive subjects with a family history of hypertension (n = 27), using flash chromatography on C18 reversed-phase, anion exchange chromatography and various reversed-phase high performance liquid chromatographies. The mechanism of Na+-K+ ATPase inhibition and the related effects of semipurified urine-derived EDLC were studied and compared with those of ouabain. Its action was similar to that of ouabain in 8 out of 10 of the tests applied. The main effects of such a compound were the depression of Na+-K+ pump activity of human erythrocytes, the inhibition of 5-hydroxytryptamine reuptake by human platelets, and the induction of natriuresis in urethanized rats. Therefore, EDLC may be considered as one of the natriuretic hormones whose mechanism of action closely resembles that of ouabain.

Asunto(s)

Proteínas Sanguíneas/orina , Digoxina , Saponinas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Adulto , Animales , Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/farmacología , Cardenólidos , Cromatografía Líquida de Alta Presión , Eritrocitos/metabolismo , Femenino , Humanos , Hipertensión/genética , Hipertensión/metabolismo , Riñón/enzimología , Cinética , Masculino , Natriuresis/efectos de los fármacos , Ouabaína/farmacología , Ratas , Ratas Endogámicas , Valores de Referencia , Serotonina/sangre , ATPasa Intercambiadora de Sodio-Potasio/sangre

RESUMEN

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.

Asunto(s)

Adenosina Trifosfatasas/antagonistas & inhibidores , Digoxina , Saponinas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Sitios de Unión , Plaquetas/metabolismo , Encéfalo/enzimología , Cardenólidos , Catálisis , Perros , Humanos , Riñón/enzimología , Ouabaína/análisis , Ouabaína/farmacología , Conejos , Serotonina/sangre , ATPasa Intercambiadora de Sodio-Potasio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/orina