RESUMEN
Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARgamma dependent pathway. RARgamma repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARgamma repression is due to selective repression of RARgamma but not RARbeta transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARgamma nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARgamma selective agonist preferentially signals differentiation of NT2/D1 cells, while RARalpha/beta, RARbeta, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 microM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARgamma was shown to potently induce growth inhibition and apoptosis in both NT2/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT - PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARgamma induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARgamma transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARgamma independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Embrionario/metabolismo , Fenretinida/farmacología , Receptores de Ácido Retinoico/fisiología , Retinoides/metabolismo , Anticarcinógenos/farmacología , Apoptosis/genética , Carcinoma Embrionario/genética , Diferenciación Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/genética , Humanos , Neuronas/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Receptores de Ácido Retinoico/genética , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Factores de Tiempo , Tretinoina/farmacología , Células Tumorales Cultivadas , Receptor de Ácido Retinoico gammaRESUMEN
Cancer mortality usually results from the tumor invading the local environment and metastasizing to vital organs, e.g. liver, lung, and brain. Degradation of the extracellular matrix is, therefore, the sine qua non of tumor cell invasion. this degradation is mediated mainly by MMPs, and thus, inhibition of MMP synthesis is a target for anticancer agents. Tumor cells must traverse both the basement membrane (type IV collagen) and the interstitial stroma (type I collagen). Therefore, we used scanning electron microscopy to examine the invasive behavior of several aggressive tumor cell lines, A2058 melanoma cells, and SCC and FaDu squamous cell carcinomas through these matrices; and we monitored the ability of all-trans retinoic acid and several RAR-specific ligands to block invasion. We demonstrate that several retinoids, which are specific RAR alpha, beta, or gamma agonists/antagonists, selectively inhibited MMP synthesis in the three tumor cell lines. However, there was not a common pattern of MMP inhibition by a particular retinoid. For instance, a RAR alpha antagonist suppressed MMP-1 and MMP-2 synthesis in the melanoma cell line, but not in the FaDu or SCC-25 cells. On the other hand, synthesis of MMP-1 and MMP-9 by the FaDu cells was affected hardly at all, while a RAR gamma antagonist reduced the levels of MMP-2. Only all-trans retinoic acid reduced MMP-1 synthesis in these cells. We postulate that the differences may be related to a differential pattern of RAR expression in each of these cells, and that the RARs expressed by each cell line may not be targets of these RAR specific compounds. All-trans retinoic acid is a pan ligand, binding to all three RARs and, therefore, may modulate gene expression more generally. We conclude that the power of these new ligands lies in their specificity, which can be directed towards modulating expression of certain RARs and, thus, of certain MMPs. By blocking MMP synthesis, retinoids may be effective in cancer therapy by decreasing tumor invasiveness.
Asunto(s)
Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Invasividad Neoplásica/prevención & control , Inhibidores de Proteasas/farmacología , Retinoides/farmacología , Carcinoma de Células Escamosas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanoma , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Gene transcription is often regulated by small ligands, enabling cells to respond to external and metabolic stimuli. Of particular interest are the mechanisms by which hydrophobic hormones modulate the transcriptional activities of proteins of the nuclear receptor superfamily. It was previously shown that, in the absence of ligand, the retinoid X receptor (RXRalpha) forms tetramers with a high affinity and a pronounced positive co-operativity such that tetramers become the receptor's predominant species tat concentrations as low as 60-70 nM. It was shown further that while RXR tetramers are remarkably stable in the absence of ligand, ligand-binding induces their rapid dissociation into smaller species, dimers and monomers. Here, the functional consequences of the self-association properties of RXR were studied by examining two point mutants of RXR that displayed aberrant oligomerization behaviors. One mutant, mRXRalpha-R321A, was found to form tetramers with a wild-type affinity, but these tetramers failed to dissociate upon ligand-binding. This mutant was found to be impaired in its ability to associate with the nuclear receptor co-activator p/CIP and to activate transcription in response to the RXR ligand 9-cis-retinoic acid. The other mutant, mRXRalpha-F318A, self-associated into dimers with a wild-type affinity, but was unable to form tetramers. This mutant displayed substantial transcriptional activity even in the absence of ligand. We previously proposed, based on in vitro studies that RXR acts as an auto-silencer by sequestering itself into tetramers, and that an important role for the ligand in activating this receptor is to release active species, dimers and monomers, from the transcriptionally inactive tetrameric pool. The observations reported here provide in-cell evidence in support of this model and indicate that ligand induced dissociation of tetramers is the first step in signalling by RXR.
Asunto(s)
Mutación , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Sitios de Unión , Electroforesis/métodos , Fluorescencia , Histona Acetiltransferasas , Humanos , Coactivador 3 de Receptor Nuclear , Receptores de Ácido Retinoico/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores X Retinoide , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/química , Tretinoina/metabolismoRESUMEN
The transcriptional response mediated by retinoic acid involves a complex series of events beginning with ligand recognition by a nuclear receptor. To dissect the amino acid contacts important for receptor-specific ligand recognition, a series of retinoic acid receptor (RAR) mutants were constructed. Transcriptional studies revealed that serine 232 (Ser232) in RARalpha and methionine 272 (Met272) in RARgamma are critical residues for the recognition of their respective receptor-selective analogs. The identification of these key amino acids in the ligand binding pocket is confirmed by the reported crystal structure of RARgamma. Interestingly, the serine at position 232 in RARalpha gives an explanation for the observed differences in the affinity of the naturally occurring ligand, all-trans-retinoic acid (t-RA), in this receptor compared with that for the other receptors, since hydrogen bonding would not be permitted between the hydroxyl of serine and the hydrophobic linker of t-RA. Using this model, a molecular mechanism for the transcriptional antagonism of a synthetic analog is suggested that involves an alteration in the structure of the receptor protein in the region around the AF2 domain in helix 12.
Asunto(s)
Metionina/metabolismo , Receptores de Ácido Retinoico/metabolismo , Serina/metabolismo , Células HeLa , Humanos , Ligandos , Unión Proteica , Receptores de Ácido Retinoico/clasificación , Receptores de Ácido Retinoico/genética , Activación TranscripcionalRESUMEN
The retinoid X receptor (RXR), a member of the superfamily of hormone nuclear receptors, is a ligand-inducible transcription factor that is activated by the vitamin A derivative 9-cis-retinoic acid. We previously showed that RXR self-associates into tetramers with a high affinity and that ligand binding induces rapid dissociation of receptor tetramers to smaller species. Here, the RXR region that is responsible for mediating tetramer formation is identified. It is shown that this interface, which we term the "tetramerization domain," critically contains two consecutive phenylalanine residues located at the C-terminal region of the receptor. Mutation of these residues is sufficient to disrupt RXR tetramers without affecting the overall fold of the protein or interfering with ligand binding, dimer formation, or DNA binding by the receptor. Nevertheless, the tetramer-impaired mutant was found to be transcriptionally defective. The newly characterized tetramerization domain and the previously identified main dimerization interface of RXR act autonomously to affect separate intersubunit interactions that, overall, lead to formation of tetramers. Protein-protein interactions mediated by the tetramerization domain, but not those that involve the dimerization interface, are disrupted following ligand binding by RXR. Overall, these data attest to the specificity of the interaction and implicate the tetramerization interface in playing a direct role in regulating transcriptional activation by RXR.
Asunto(s)
Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , ADN/metabolismo , Células HeLa , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Pliegue de Proteína , Receptores de Ácido Retinoico/genética , Receptores X Retinoide , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Factores de Transcripción/genética , UreaRESUMEN
The mechanism by which the naturally occurring ligand for a nuclear hormone receptor regulates transcription remains largely unknown. One approach combines the specificity of monoclonal antibodies, which recognize a three-dimensional epitope, with ligand binding. Using purified retinoic acid receptor gamma D and E domains, a panel of six unique monoclonal antibodies were isolated and characterized using solid-state receptor binding and retinoic acid receptor (RAR)-RXR heterodimer supershift formation. Three antibodies are specific for RARgamma (mAbI, mAbII, and mAbV) and four recognize a three-dimensional epitope (mAbI, mAbIV, mAbV, and mAbVI). Three antibodies (mAbIII, mAbV, and mAbVI) dissociate from the receptor in electrophoretic mobility shift assays upon the addition of retinoic acid. In particular, the binding characteristics of mAbIII, whose epitope was mapped to a region identified as an omega-loop (amino acids 207-222), suggest a model for ligand binding to the receptor. In this model, ligand binding causes a positioning of helix 12 into a favorable conformation for interaction with the transcriptional machinery. The Omega-loop then closes in order to stabilize this "active" position. The results reported here also suggest that a region of the hinge or D domain of the receptor (amino acids 156-188), an area that can play a role in protein-protein interactions, may also be important in ligand-induced functional changes.
Asunto(s)
Fragmentos de Péptidos/química , Receptores de Ácido Retinoico/química , Tretinoina/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Mapeo Epitopo , Epítopos , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/inmunología , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Receptor de Ácido Retinoico gammaRESUMEN
Signalling by all-trans retinoic acid is mediated through RXR-RAR retinoid receptor heterodimers, in which RXR has been considered to act as a transcriptionally silent partner. However, we show here that in cultured NB4 (ref. 6) human acute promyelocytic leukaemia cells treated with either an RAR-alpha-selective agonist alone, or certain RAR-alpha antagonists in combination with an RXR agonist, receptor-DNA binding is induced in vivo, resulting in expression of the target genes of retinoic acid as well as acute promyelocytic leukaemia protein (PML) relocation to nuclear bodies and differentiation before apoptosis. These results indicate that RAR-alpha ligands can induce two separate events: one enables RXR-RAR-alpha heterodimers to bind to DNA in vivo and allows RXR agonists to act; the other induces transcriptional activity of RAR-alpha. The availability of receptor-specific synthetic retinoids that can induce distinct receptor functions has potential in extending the therapeutic repertoire of retinoids.
Asunto(s)
Proteínas de Neoplasias , Proteínas Nucleares , Receptores de Ácido Retinoico/metabolismo , Tretinoina/farmacología , Animales , Apoptosis , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Sinergismo Farmacológico , Humanos , Ligandos , Ratones , Proteína de la Leucemia Promielocítica , Unión Proteica , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Retinoides/farmacología , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.
Asunto(s)
Regulación de la Expresión Génica , Genes Homeobox , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Madre de Carcinoma Embrionario , Células Madre Neoplásicas , ARN Mensajero/genética , Receptores de Ácido Retinoico/efectos de los fármacos , Retinoides/farmacologíaRESUMEN
In search for retinoic acid receptor (RAR) selective ligands, a series of 6-substituted 2-naphthoic acid retinoids were synthesized and evaluated in vitro in a transactivation assay and a competition binding assay for all RARs. These derivatives, in general, showed RAR beta,gamma selectivity. Among these naphthoic acids, oxime derivative 12 was identified as a potent RAR gamma-selective retinoid, while olefinic derivative 11 was found to be comparable to retinoic acid and slightly RAR beta,gamma selective. For the bioassays, a general correlation was observed between the binding affinity of the ligand to the receptors and the potency of the compounds in the transactivation assay. The structure-activity relationship of these naphthoic acids will be discussed.
Asunto(s)
Naftalenos/química , Receptores de Ácido Retinoico/efectos de los fármacos , Retinoides/farmacología , Unión Competitiva , Diseño de Fármacos , Genes Reporteros , Células HeLa/efectos de los fármacos , Humanos , Ligandos , Estructura Molecular , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Retinoides/síntesis química , Retinoides/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Activación Transcripcional/efectos de los fármacos , Tretinoina/metabolismo , Receptor de Ácido Retinoico gammaRESUMEN
Retinoic acid exerts its many biological effects by interaction with a nuclear protein, the retinoic acid receptor (RAR). The details of this interaction are unknown due mainly to the lack of sufficient quantities of pure functional receptor protein for biochemical and structural studies. We have recently subcloned the D and E domains of human RAR gamma for expression in Escherichia coli. Using nickel-chelation affinity chromatography with a polyhistidine amino-terminal tail, purification of the DE peptide with a pI of 5.18 was accomplished to greater than 98% purity. Scatchard analysis and fluorescence quenching techniques using the purified protein indicate a very high percentage of functional molecules ( > 95%) with a Kd for retinoic acid (t-RA) of 0.6 +/- 0.1 nM. Circular dichroism spectra of the purified domains predict a predominantly alpha-helical structure (approximately 56%) with little beta sheet present. No significant changes in these structural characteristics were observed upon binding of t-RA. Inspection of the amino acid sequence within these domains identified a single tryptophan residue at position 227. Modeling the amino acid sequence in this region as an alpha-helical structure indicates that this tryptophan is adjacent to alanine 234, which corresponds to alanine 225 in RAR beta that has previously been linked to the ligand binding site. Fluorescence of this tryptophan was quenched in a dose-dependent manner on the addition of t-RA, confirming that Trp-227 is within the ligand binding site. Tryptophan fluorescence quenching analysis also demonstrates that a single retinoic acid molecule is bound per receptor and suggests that receptor-ligand interactions occur within the amino-terminal portion of the predominantly alpha-helical ligand binding domain.
Asunto(s)
Estructura Secundaria de Proteína , Receptores de Ácido Retinoico/química , Secuencia de Bases , Sitios de Unión , Fluorescencia , Humanos , Ligandos , Datos de Secuencia Molecular , Receptores de Ácido Retinoico/metabolismo , TriptófanoRESUMEN
Utilizing a strategy involving domain exchange between retinoic acid receptors alpha and beta (RAR alpha and RAR beta) and monitoring the transcriptional activity of the resulting chimeric receptors with receptor-selective retinoids, we identified a 70-aa region within the N-terminal portion of the RAR alpha and -beta domain E which is important for an RAR alpha- or RAR beta-specific response. Two amino acid residues within this region, serine-232 (S232) and threonine-239 (T239) in RAR alpha and the corresponding alanine-225 (A225) and isoleucine-232 (I232) in RAR beta, were found to be essential for this effect. In addition, binding studies using the chimeric receptors expressed in Escherichia coli showed that the N-terminal portion of domain E was also important for the characteristic binding profile of t-RA and various retinoids with RAR alpha or RAR beta. Structural predictions of the primary amino acid sequence in this region indicate the presence of an amphipathic helix-turn-helix structure with five hydrophobic amino acids that resemble a leucine zipper motif. The amino acid residues identified by domain swapping, S232 and T239 in RAR alpha and A225 and I232 in RAR beta, were found within the hydrophobic face of an alpha-helix in close proximity to this zipper motif, suggesting that the ligand may interact with the receptor in the region adjacent to a surface involved in protein-protein interactions. This finding may link ligand binding to other processes important for transcriptional activation.
Asunto(s)
Estructura Secundaria de Proteína , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Tretinoina/análogos & derivados , Tretinoina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , Escherichia coli , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Ácido Retinoico/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Receptor alfa de Ácido Retinoico , Homología de Secuencia de Aminoácido , Activación Transcripcional , TransfecciónRESUMEN
The three retinoic acid receptors (RAR alpha, RAR beta and RAR gamma) are known to modulate the transcription of target genes through interaction of the individual receptors with their naturally occurring ligand, retinoic acid (RA). Since RA has multiple effects in vivo, considerable effort has recently been devoted to finding selective compounds to elucidate the functions of individual receptors and to relate these functions to specific in vivo effects. The racemic synthetic retinoid 6-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)hydroxy-methyl]-2- naphthalene carboxylic acid has recently been identified as an RAR gamma-selective agonist. A synthetic method involving lipase-mediated transformation has been developed to prepare the individual enantiomers. Discrimination between the two enantiomers is seen in both transcriptional activity and binding to recombinant receptors with the (S)-enantiomer being the more active. Differences between the two compounds are also seen in the Rhino mouse utriculi reduction assay and the rabbit irritation model. In both animal models, the (S)-enantiomer consistently gave a greater response. Taken together, these results suggest that the activity and irritation seen with RA and related compounds is receptor mediated. Further, the strong selectivity of the compounds reported here for RAR gamma suggests that this receptor plays an important role in these in vivo biological activities. The discrimination between these enantiomers may be useful in the design of novel retinoids with uniquely defined biological properties.
Asunto(s)
Queratolíticos/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Animales , Erupciones por Medicamentos/etiología , Femenino , Técnicas In Vitro , Queratolíticos/efectos adversos , Ratones , Conejos , Estereoisomerismo , Tretinoina/efectos adversosRESUMEN
Postsynthetic modifications of core histones by treatment of HeLa S3 cells with 5mM sodium butyrate lead to alterations in the structure of high molecular weight chromatin. Whole chromatin from butyrate-treated cells, which results in highly acetylated core histones, has an ellipticity [theta]282.5 of 3700 deg cm2 dmol-1 (0.2 mM EDTA, pH 7.4) that is 1200 deg c m2 dmol-1 less than chromatin from untreated HeLa cells, suggesting a more condensed structure. No difference in the circular dichroism spectra was observed in H1-stripped, high molecular weight chromatin obtained from control and butyrate-treated cells at low (0.2 mM EDTA, pH 7.4) ionic strength. Thermal denaturation profiles of high molecular weight chromatin were resolved into three transitions and exhibited a shifting of hyperchromicity from transition I to transitions III, at a higher Tm, with butyrate treatment of HeLa cells, further indicating a more compact structure. Thermal denaturation profiles of H1-stripped chromatin were not affected by butyrate treatment. Ionic strength studies in the range of 0-5 mM NaH2PO4, 0.2 mM EDTA, pH 7.4, of high molecular weight chromatin exhibited a decrease in [theta]282.5 and a shifting of hyperchromicity from transition I to transition III with increasing ionic strength. Control high molecular weight chromatin was more sensitive to changes in ionic strength than its highly acetylated counterpart. These results suggest that acetylation of histones alone does not result in a change in histone-DNA interactions but other changes associated with butyrate treatment most probably cause a more condensed structure, of the fraction studied herein, which is mediated by H1 or other materials removed during stripping in 0.35 M NaCl.
Asunto(s)
Butiratos/farmacología , Cromatina/efectos de los fármacos , Histonas/metabolismo , Nucleosomas/ultraestructura , Acetilación , Cromatina/ultraestructura , Dicroismo Circular , Desoxirribonucleasas , Femenino , Células HeLa , Calor , Humanos , Peso MolecularRESUMEN
Complexes formed by the interaction of histone H1 and the immunomodulator Pyran co-polymer, were evaluated using nephelometric titration. At a constant concentration of histone (10 microgram/ml), maximum turbidity was obtained at a Pyran concentration of 2 microgram/ml. Titration of H1 by Pyran was found to be sensitive to ionic strength. Concentration of Na+ greater than 200 mM or Mg+2 greater than 3 mM apparently blocked Pyran . H1 complex formation.
Asunto(s)
Histonas , Polímeros , Copolímero del Pirano , Fenómenos Químicos , Química , Nefelometría y TurbidimetríaRESUMEN
The permeability of Necturus proximal tubule to hydrophilic nonelectrolytes of varying molecular size was studied under control conditions and during isotonic expansion of the animal's extracellular volume. Transepithelial permeability was measured in perfused tubular segments under conditions of zero net water flux. During volume expansion, tubular permeability to urea increased slightly, whereas mannitol decreased slightly and permeability to sucrose was significantly decreased. Volume expansion had a greater effect on osmotic flow parameters; the NaCl reflection coefficient decreased from 0.64 to 0.47 (summer animals) and from 0.41 to 0.27 (winter animals). Osmotic water flux and hydraulic conductivity increased but only in the lumen-to-capillary direction. Reflection coefficients of nonelectrolytes measured at the apical surface were reduced during volume expansion for probing molecules greater than 3 A in radius and were unchanged for smaller molecules, less than 3 A, suggesting two pore populations. We propose that an increase in tight-junction permeability can account for modification of osmotic flow parameters, whereas the whole thickness of the epithelium, particularly the intercellular space, plays the dominant role in regulation of diffusional permeability.