RESUMEN

The present research report describes Na+/H+ antiport by brush border membrane vesicles isolated from whole larvae of Aedes aegypti (AeBBMVw). Our hypothesis is that acid quenching of acridine orange by AeBBMVw is predominantly mediated by Na+/H+ antiport via the NHA1 component of the AeBBMVw in the absence of amino acids and ATP. AeNHA1 is a Na+/H+ antiporter that has been postulated to exchange Na+ and H+ across the apical plasma membrane in posterior midgut of A. aegypti larvae. Its principal function is to recycle the H+ and Na+ that are transported during amino acid uptake, e.g., phenylalanine. This uptake is mediated, in part, by a voltage-driven, Na+-coupled, nutrient amino acid transporter (AeNAT8). The voltage is generated by an H+ V-ATPase. All three components, V-ATPase, antiporter, and nutrient amino acid transporter (VAN), are present in brush border membrane vesicles isolated from whole larvae of A. aegypti. By omitting ATP and amino acids, Na+/H+ antiport was measured by fluorescence quenching of acridine orange (AO) caused by acidification of either the internal vesicle medium (Na+in > Na+out) or the external fluid-membrane interface (Na+in < Na+out). Vesicles with 100 micromolar Na+ inside and 10 micromolar Na+ outside or with 0.01 micromolar Na+ inside and 100 micromolar Na+ outside quenched fluorescence of AO by as much as 30%. Acidification did not occur in the absence of AeBBMVw. Preincubation of AeBBMVw with antibodies to NHA1 inhibit Na+/H+ antiport dependent fluorescence quenching, indicating that AeNHA1 has a significant role in Na+/H+ exchange.

Asunto(s)

Aedes/efectos de los fármacos , Aedes/metabolismo , Anticuerpos/farmacología , Vesículas Citoplasmáticas/metabolismo , Microvellosidades/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Animales , Transporte Biológico/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Concentración de Iones de Hidrógeno , Iones/metabolismo , Cinética , Larva , Microvellosidades/ultraestructura , Modelos Biológicos

RESUMEN

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-D-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl2/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-D-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-D-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J(max) and lowered K(m) in both salts. ³H-D-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis-Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-D-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (D-galactose, α-methyl-D-gluco-pyranoside, unlabeled D-glucose, D-fructose, and D-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-D-glucose influx. Only unlabeled D-glucose, D-fructose, and D-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of D-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-D-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-D-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, D-fructose, and D-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.

Asunto(s)

Proteínas de Artrópodos/metabolismo , Epitelio/metabolismo , Hepatopáncreas/metabolismo , Penaeidae/metabolismo , Potasio/metabolismo , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Animales , Proteínas de Artrópodos/antagonistas & inhibidores , Océano Atlántico , Transporte Biológico/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/ultraestructura , Florida , Fructosa/metabolismo , Glucosa/antagonistas & inhibidores , Glucosa/metabolismo , Hepatopáncreas/efectos de los fármacos , Hepatopáncreas/ultraestructura , Cinética , Manosa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Microvellosidades/metabolismo , Florizina/farmacología , Sodio/metabolismo , Proteínas de Transporte de Sodio-Glucosa/antagonistas & inhibidores , Tritio

RESUMEN

Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to study Na(+)-coupled, (3)H-labeled, phenylalanine (Phe) uptake. The affinity for all components of the uptake was very high with half maximal values in the sub-micromolar range. By contrast a K(0.5)(Phe) of 0.2mM and a K(0.5)(Na) of 26 mM were calculated from Phe-induced electrical currents in Xenopus oocytes that were heterologously expressing the Anopheles gambiae symporter (co-transporter), AgNAT8, in a buffer with 98 mM Na(+). What accounts for the >1000-fold discrepancy in affinity for substrates between the BBMV and oocyte experiments? Is it because Ae. aegypti were used to isolate BBMVw whereas An. gambiae were used to transfect oocytes? More likely, it is because BBMVw were exposed to [Na(+)] in the micromolar range with the transporter(s) being surrounded by native lipids. By contrast, the oocyte measurements were made at [Na(+)] 100,000 times higher with AgNAT8 surrounded by foreign frog lipids. The results show that AaBBMVw are osmotically sealed; the time-course has a Na(+)-induced overshoot, the pH optimum is ∼7 and the K(0.5) values for Phe and Na(+) are very low. The transport is virtually unchanged when Na(+) is replaced by K(+) or Li(+) but decreased by Rb(+). This approach to resolving discrepancies between electrical data on solute transporters such as AgNAT8 that are over-expressed in oocytes and flux data on corresponding transporters that are highly expressed in native membrane vesicles, may serve as a model for similar studies that add membrane biochemistry to molecular biology in efforts to identify targets for the development of new methods to control disease-vector mosquitoes.

Asunto(s)

Aedes/metabolismo , Microvellosidades/metabolismo , Fenilalanina/metabolismo , Vesículas Transportadoras/metabolismo , Aedes/ultraestructura , Animales , Concentración de Iones de Hidrógeno , Cinética , Larva/metabolismo , Larva/ultraestructura , Ósmosis , Sodio/metabolismo , Cloruro de Sodio/metabolismo , Tritio , Xenopus

RESUMEN

Transepithelial transport of dietary D-glucose and d-fructose was examined in the lobster Homarus americanus intestine using D-[(3)H]glucose and D-[(3)H]fructose. Lobster intestines were mounted in a perfusion chamber to determine transepithelial mucosal to serosal (MS) and serosal to mucosal (SM) transport mechanisms of glucose and fructose. Both MS glucose and fructose transport, as functions of luminal sugar concentration, increased in a hyperbolic manner, suggesting the presence of mucosal transport proteins. Phloridizin inhibited the MS flux of glucose, but not that of fructose, suggesting the presence of a sodium-dependent (SGLT1)-like glucose co-transporter. Immunohistochemical analysis, using a goat anti-rabbit GLUT5 polyclonal antibody, revealed the localization of a brush border GLUT5-like fructose transport protein. MS fructose transport was decreased in the presence of mucosal phloretin in warm spring/summer animals, but the same effect was not observed in cold autumn/winter animals, suggesting a seasonal regulation of sugar transporters. Mucosal phloretin had no effect on MS glucose transport. Both SM glucose and SM fructose transport were decreased in the presence of increasing concentrations of serosal phloretin, providing evidence for the presence of a shared serosal GLUT2 transport protein for the two sugars. The transport of d-glucose and d-fructose across lobster intestine is similar to sugar uptake in mammalian intestine, suggesting evolutionarily conserved absorption processes for these solutes.

Asunto(s)

Fructosa/metabolismo , Glucosa/metabolismo , Mucosa Intestinal/metabolismo , Nephropidae/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Fructosa/farmacología , Glucosa/farmacología , Transportador de Glucosa de Tipo 5/metabolismo , Inmunohistoquímica , Mucosa Intestinal/efectos de los fármacos , Intestinos/citología , Intestinos/efectos de los fármacos , Cinética , Masculino , Nephropidae/efectos de los fármacos , Floretina/farmacología , Florizina/farmacología , Estaciones del Año , Membrana Serosa/citología , Membrana Serosa/efectos de los fármacos , Membrana Serosa/metabolismo , Tritio/metabolismo

RESUMEN

Rat CYP1A1 promoter-luciferase, transiently transfected wild-type and 4S PAH receptor (glycine N-methyl transferase, GNMT)-transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild-type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT-transformed CHO cells and wild-type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno-precipitated with anti-GNMT antibodies, separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti-aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1ß) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero-oligomer (dimer?) with ARNT/HIF-1ß which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor-1 function with respect to the HIF-1ß subunit (ARNT).

Asunto(s)

Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Glicina N-Metiltransferasa/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Multimerización de Proteína , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Benzo(a)pireno/farmacología , Células CHO , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Cricetinae , Cricetulus , Citocromo P-450 CYP1A1/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Glicina N-Metiltransferasa/genética , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Estructura Cuaternaria de Proteína , Ratas

RESUMEN

The mouse COL3A1 first intron is 9684 bp. RNA's of approximately 1.6 and 3.0 kb were detected by Northern hybridization analysis of poly-A RNA from fetal mice and total RNA from suckling and adult mouse intestine using (32)P-labeled, anti-sense RNA synthesized from a mouse COL3A1 first intron, 5 prime region, 5.4 kb Xba I fragment (1655-7030 bp), recombinant plasmid (pPI5.4x). Expression of the 1.6 and 3.0 kb RNA's was significantly reduced in adult mouse intestine, indicating that these RNAs are developmentally regulated. "BLAST" analysis indicated that the mouse first intron 5 prime sequence has 94-100% identity to 13 mouse ESTs. These mouse first intron EST's lie within the 5.4 Xba I fragment of the mouse COL3A1 first intron. Two of the mouse first intron EST's have significant identity to known miRNA, mature sequences, mmu-miR-466f-3P, mmu-miR-1187, and mmu-miR-574-5P as well as others. Predicted targets for mmu-miR-466f-3P include COL1A1, COL19A1, COL11A2, COL4A1, and COL4A5 indicating that COL3A1 intronic miRNAs may regulate the expression of other collagen genes in development.

Asunto(s)

Colágeno Tipo III/genética , Etiquetas de Secuencia Expresada , MicroARNs/genética , Poli A/genética , Animales , Northern Blotting , Biología Computacional , Electroforesis en Gel de Agar , Humanos , Intrones , Ratones , Ratas

RESUMEN

[(3)H]-fructose and [(3)H]-glucose transport activities were determined in brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from Limulus polyphemus (horseshoe crab) hepatopancreas. Glucose transport was equilibrative in the absence of sodium and sodium dependent in the presence of sodium in BBMV, suggesting GLUT-like and SGLT-like transport activity. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and sodium dependent in the presence of sodium. Western blot analysis using a rabbit anti-mouse SGLT-1 polyclonal antibody indicated the presence of a cross-reacting horseshoe crab BBMV protein of similar molecular weight to the mammalian SGLT1. Sequence alignment of the mouse SGLT-4 and SGLT1 with a translated, horseshoe crab-expressed sequence tag also indicated significant identity between species. Fructose and glucose uptake in the absence and presence of sodium by hepatopancreas BBMV and BLMV indicated the presence of sodium-dependent transport activity for each sugar that may result from the presence of transporters similar to those described for other species.

Asunto(s)

Fructosa/metabolismo , Glucosa/metabolismo , Hepatopáncreas/metabolismo , Cangrejos Herradura/metabolismo , Microvellosidades/metabolismo , Sodio/fisiología , Secuencia de Aminoácidos , Animales , Membranas/metabolismo , Ratones , ARN Mensajero/química , Alineación de Secuencia , Transportador 1 de Sodio-Glucosa/química

RESUMEN

Brush border membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs) contain an H(+) V-ATPase (V), a Na(+)/H(+) antiporter, NHA1 (A) and a Na(+)-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles. AgNAT8 was cloned from Anopheles gambiae, localized in BBMs and characterized in Xenopus laevis oocytes. AgNHA1 was cloned and localized in BBMs but characterization in oocytes was compromised by an endogenous cation conductance. AeBBMVWs complement Xenopus oocytes for characterizing membrane proteins, can be energized by voltage from the V-ATPase and are in their natural lipid environment. BBMVs from caterpillars were used in radio-labeled solute uptake experiments but approximately 10,000 mosquito larvae are needed to equal 10 caterpillars. By contrast, functional AeBBMVWs can be prepared from 10,000 whole larvae in 4h. Na(+)-coupled (3H)phenylalanine uptake mediated by AeNAT8 in AeBBMVs can be compared to the Phe-induced inward Na(+) currents mediated by AgNAT8 in oocytes. Western blots and light micrographs of samples taken during AeBBMVW isolation are labeled with antibodies against all of the VAN components. The use of AeBBMVWs to study coupling between electrogenic V-ATPases and the electrophoretic transporters is discussed.

Asunto(s)

Aedes/enzimología , Proteínas de Insectos/metabolismo , Vesículas Secretoras/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Aedes/clasificación , Aedes/genética , Aedes/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Proteínas de Insectos/genética , Larva/clasificación , Larva/enzimología , Larva/genética , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Filogenia , Vesículas Secretoras/genética , ATPasas de Translocación de Protón Vacuolares/genética

RESUMEN

[(3)H]Fructose and [(3)H]glucose transport were determined in brush-border membrane vesicles (BBMV), basolateral membrane vesicles (BLMV) and isolated cells (E, R, F, B) of H. americanus (Atlantic lobster) hepatopancreas. Glucose transport in BBMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. Sodium-dependent glucose transport by BBMV was not inhibited by a tenfold molar excess of fructose. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. This enhancement was not affected by a tenfold molar excess of glucose in the presence of sodium. E-, F- and B-cells showed sodium-dependent uptake of fructose, while R-cells did not. Sodium-dependent fructose uptake by E-cells was not inhibited by a tenfold molar excess of glucose or mannose. Western blot analysis of BBMV, BLMV and E-, R-, F- and B-cells using rabbit polyclonal antibodies directed against epitopes of mammalian GLUT2, GLUT5, SGLT1 and SGLT4 indicated the presence of cross-reacting lobster proteins. Sequence alignment of the mammalian proteins with translated, lobster expressed sequence tags also indicated significant identity between species. Comparison of fructose and glucose uptake in the absence and presence of sodium by BBMV, BLMV and isolated cells indicated the presence of a distinct sodium-dependent transport activity for each sugar in the Atlantic lobster.

Asunto(s)

Fructosa/metabolismo , Hepatopáncreas/metabolismo , Nephropidae/metabolismo , Sodio/metabolismo , Simportadores/fisiología , Secuencia de Aminoácidos , Animales , Transporte Biológico , Etiquetas de Secuencia Expresada , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/análisis , Proteínas Facilitadoras del Transporte de la Glucosa/química , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Hexosas/metabolismo , Microvellosidades/metabolismo , Datos de Secuencia Molecular , Nephropidae/genética , Alineación de Secuencia , Proteínas de Transporte de Sodio-Glucosa/análisis , Proteínas de Transporte de Sodio-Glucosa/química , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Simportadores/química

RESUMEN

Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and 65Zn2+, 36Cl-, 35SO(4)2- and 14C-oxalate2- were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO4(2-) or PO(4)3- led to a threefold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl- or oxalate2-. The stimulation of 65Zn2+ uptake by intravesicular sulfate was SO(4)2- concentration dependent with a maximal enhancement at 500 micromol l(-1). Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn+/3H+. 35SO4(2-) and 14C-oxalate2- uptakes were both enhanced in vesicles loaded with intravesicular Cl- compared to vesicles containing mannitol, suggesting the presence of anion countertransport. 35SO4(2-) influx was a sigmoidal function of external [SO(4)2-] with 25 mmol l(-1) internal [Cl-], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl- or OH- ions. 36Cl- influx was a sigmoidal function of external [Cl-] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl- for 1 OH-. 14C-oxalate influx was a hyperbolic function of external [oxalate2-] with 25 mmol l(-1) internal [Cl-], suggesting a 1:1 exchange of oxalate2- for Cl-. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO4(2-) and PO4(3-)) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.

Asunto(s)

Aniones/metabolismo , Células Epiteliales/metabolismo , Lisosomas/metabolismo , Metales Pesados/metabolismo , Nephropidae/metabolismo , Animales , Transporte Biológico Activo , Cloruros , Células Epiteliales/citología , Hepatopáncreas/citología , Hepatopáncreas/metabolismo , Hidróxidos , Sulfatos

RESUMEN

The organic cation, tetraethylammonium (TEA(+)), is actively secreted by mammalian nephrons and crustacean urinary bladders by similar processes in both animal groups. These mechanisms consist of a basolateral Organic Cation Transporter (OCT family) that employs the transmembrane electrical potential as a driving force for organic cation uptake from the blood and a brush border secondary active transport process that exchanges luminal protons for TEA(+). The present study examined the nature of (14)C-TEA(+) transport across the perfused intestinal epithelium of the American lobster, Homarus americanus, to ascertain whether the gut complemented the kidneys in the clearance of these organic metabolites from the blood. Unidirectional mucosa to serosa (M to S) (14)C-TEA(+) fluxes in anterior and posterior intestine were hyperbolic functions of luminal [TEA(+)] and significantly (P<0.01) exceeded the respective serosa to mucosa (S to M) fluxes. Luminal quinine (1 mM) significantly (P<0.05) inhibited M to S flux of the organic cation, while serosal addition of the drug had no effect on S to M transfer of TEA(+). Reducing serosal pH from 7.20 to 6.02 significantly (P<0.01) stimulated M to S transfer of 0.1 mM (14)C-TEA(+), but significantly (P<0.05) lowered S to M transfer of the metabolite. Addition of 2.0 mM unlabelled serosal TEA(+) trans-stimulated the M to S flux of 0.1 mM (14)C-TEA and doubled the transfer rate of the organic cation from lumen to blood compared to its transport in the absence of TEA(+) in the bath. Results suggest that this organic cation is absorbed across lobster intestine by the combination of a brush border OCT-1-like transporter coupled with a basolateral H(+)/TEA(+) exchanger. A working model is presented for intestinal organic cation absorption in crustaceans and compared to the secretory transport model for this class of metabolites previously reported for crustacean and mammalian kidneys.

Asunto(s)

Nephropidae/metabolismo , Tetraetilamonio/metabolismo , Absorción , Animales , Transporte Biológico/efectos de los fármacos , Cationes/metabolismo , Concentración de Iones de Hidrógeno , Mucosa Intestinal/metabolismo , Membrana Mucosa/metabolismo , Nephropidae/anatomía & histología , Proteínas de Transporte de Catión Orgánico/fisiología , Quinina/farmacología , Membrana Serosa/metabolismo

RESUMEN

This article demonstrates how glucocorticoids decrease collagen synthesis. The parameters used to assess procollagen synthesis in our laboratory will be compared to those used by others. This article will note all the pertinent literature on the molecular mechanisms of this down regulation of procollagen synthesis. For example, what are the effects of glucocorticoids at the levels of transcription and translation of collagen mRNAs? Finally, we will define a molecular mechanism to inhibit Type I collagen synthesis by decreasing the binding of the TGF-beta activator protein complex to the TGF-beta element in the distal promoter of the proalpha1 Type I collagen gene, preventing the 2:1 ratio of alpha1 to alpha2 chains in the processed Type I collagen molecule. We will next ask "How do sense oligo decoys decrease Type I collagen synthesis at the in vivo and at the cell levels?" In primary fibrotic cell culture, the double-stranded phosphorothioate oligodeoxynucleotide decoys were more effective than their sense single-stranded counterparts. The molecular mechanism for the decrease in Type I collagen synthesis is the same as glucocorticoids, that is by decreasing the binding of the TGF-beta activator protein complex to the TGF-beta element in the distal promoter of the proalpha1 Type I collagen gene for the transcription of the proalpha1 mRNAs. The reason for using sense oligo decoys as anti-fibrotic agents as compared to the anti-fibrotic glucocorticoids, is that presently marketed and FDA approved glucocorticoids have many untoward side effects which the sense oligo decoys do not have.

Asunto(s)

Colágeno/genética , Glucocorticoides/farmacología , Oligodesoxirribonucleótidos/farmacología , Unión Competitiva , Colágeno/biosíntesis , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Dexametasona/farmacología , Regulación hacia Abajo/genética , Fibroblastos/efectos de los fármacos , Fibrosis/tratamiento farmacológico , Expresión Génica/efectos de los fármacos , Glucocorticoides/uso terapéutico , Glucocorticoides/toxicidad , Humanos , Oligodesoxirribonucleótidos/uso terapéutico , Oligodesoxirribonucleótidos/toxicidad , Regiones Promotoras Genéticas , Elementos de Respuesta , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Elevated blood ATP and increased red blood cell (RBC) ATP transport is associated with cystic fibrosis (CF). In this report, we demonstrate the presence of the wild-type and the DeltaF508 mutant form of the CF transmembrane conductance regulator protein in RBC membranes and its putative interaction with ecto-apyrase, an ATP hydrolyzing enzyme also present in the RBC membrane. RBC membranes of control and DeltaF508 individuals and of wild-type and CF transmembrane conductance regulator-knockout mice were examined by immunoblot using several antibodies directed against different epitopes of this protein. These experiments indicated that human RBC membranes contain comparable amounts of the wild-type CF transmembrane conductance regulator protein and the DeltaF508 mutant form of the protein, respectively. CF transmembrane conductance regulator protein was also detected in wild-type mouse RBC membranes but not in the gene knockout mouse RBC membranes. Antibodies directed against ecto-apyrase co-immunoprecipitated CF transmembrane conductance regulator protein of human RBC membranes indicating a physical interaction between these two membrane proteins consistent with ATP transport and extracellular hydrolysis. We conclude that RBCs are a significant repository of CF transmembrane conductance regulator protein and should provide a novel system for evaluating its expression and function.

Asunto(s)

Adenosina Trifosfato/metabolismo , Apirasa/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Membrana Eritrocítica/metabolismo , Animales , Antígenos CD , Transporte Biológico/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Ratones , Ratones Noqueados , Mutación/genética , Unión Proteica/fisiología

RESUMEN

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.

Asunto(s)

Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Benzopirenos/farmacología , Línea Celular Tumoral , Humanos , Masculino

RESUMEN

Rat small intestinal epithelial cells and human colon adenocarcinoma cells cultured on Matrigel expressed the differentiation specific enzyme, sucrase-isomaltase, as determined by indirect immunofluorescence. Rat small intestinal epithelial cells, rat colonocytes, and human colon adenocarcinoma cells developed an altered morphology when cultured on Matrigel and became apoptotic within 24-48 h. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin caused a 2- and 5-fold induction, respectively, of ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on plastic was not detected. 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment caused a 14-fold induction of transfected, rat CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induced ethoxyresorufin-o-deethylase activity by 6- and 1.6-fold, respectively in rat colonocytes cultured on Matrigel. Induction of ethoxyresorufin-o-deethylase activity was not observed in rat colonocytes cultured on plastic. CYP1A1-promoter-luciferase activity was induced 3-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat colonocytes cultured on Matrigel. Induction of CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells or rat colonocytes cultured on plastic was not observed. Ethoxyresorufin-o-deethylase activity in human colon adenocarcinoma cells, cultured on either plastic or Matrigel, was induced 7-fold by benzo[a]pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity was 2-fold greater in human colon adenocarcinoma cells cultured on Matrigel compared to cells cultured on plastic. Extracellular matrix-mediated differentiation and apoptosis of intestinal cells provide in vitro systems for study of the regulation of CYP1A1 expression, carcinogen activation in the gut and mechanism(s) of apoptosis of colon cancer cells.

Asunto(s)

Apoptosis , Membrana Basal/enzimología , Colon/enzimología , Neoplasias del Colon/enzimología , Citocromo P-450 CYP1A1/metabolismo , Mucosa Intestinal/enzimología , Intestino Delgado/enzimología , Animales , Membrana Basal/citología , Benzo(a)pireno/farmacología , Células CACO-2 , Técnicas de Cultivo de Célula , Diferenciación Celular , Tamaño de la Célula , Colágeno , Colon/citología , Colon/efectos de los fármacos , Colon/patología , Neoplasias del Colon/patología , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Combinación de Medicamentos , Inducción Enzimática/efectos de los fármacos , Humanos , Mucosa Intestinal/citología , Intestino Delgado/citología , Laminina , Peso Molecular , Oligo-1,6-Glucosidasa/metabolismo , Plásticos , Regiones Promotoras Genéticas/genética , Proteoglicanos , Ratas , Sacarasa/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas