RESUMEN
ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swelling-activated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl(-) > I(-) selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.
Asunto(s)
Canales de Cloruro/metabolismo , Animales , Células CHO , Tamaño de la Célula , Canales de Cloruro/genética , Cricetinae , Conductividad Eléctrica , Hepatocitos/citología , Hepatocitos/metabolismo , Soluciones Hipotónicas , Activación del Canal Iónico , Soluciones Isotónicas , Cinética , Técnicas de Placa-Clamp , Ratas , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The molecular identities of functional chloride channels in hepatocytes are largely unknown. We examined the ClC-3 chloride channel in rat hepatocytes and found that mRNA for two different isoforms is present. A short form is identical to the previously reported sequence for rat ClC-3, and a long form contains a 176-bp insertion immediately upstream of the translation initiation site. This predicts a 58-amino acid NH(2) terminal insertion. Both long and short form mRNA was expressed in diverse tissues of the rat. Transient transfection of the long form in CHO-K1 cells resulted in currents with an I(-) > B(-) > Cl(-) selectivity sequence, outward rectification, and inactivation at positive voltages. Short form currents had identical ionic selectivity but displayed a more extreme outward rectification and showed no voltage-dependent inactivation. Immunofluorescence and immunoblots localized native ClC-3 preferentially but not exclusively to the canalicular membrane. We have therefore identified a new isoform of rat ClC-3 and shown that expression of both isoforms produces functional channels. In hepatocytes, ClC-3 is located in association with the canalicular membrane.
Asunto(s)
Canales de Cloruro , Hígado/química , Animales , Secuencia de Bases , Células CHO , Canales de Cloruro/análisis , Canales de Cloruro/química , Canales de Cloruro/genética , Cricetinae , Electrofisiología , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Activación del Canal Iónico/fisiología , Isomerismo , Hígado/citología , Masculino , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Peso Molecular , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120, 000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3'-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.
Asunto(s)
Encéfalo/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Médula Espinal/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Exones , Expresión Génica , Biblioteca Genómica , Humanos , Inmunohistoquímica , Intrones , Riñón , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/metabolismo , Proteoglicanos/química , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Sindecano-3 , TransfecciónRESUMEN
Syndecans are a family of transmembrane proteoglycans that have been implicated in cell-extracellular matrix adhesion and growth factor binding. We reported previously that syndecan-1 expression by cultured rate vascular smooth muscle cells (VSMCs) is induced by serum- or platelet-derived growth factor (PDGF). We now report that syndecan-4 mRNA is rapidly induced in cultured VSMCs in response to basic fibroblast growth factor (bFGF) or serum stimulation. In the presence of cycloheximide, induction of syndecan-4 mRNA was enhanced. These characteristics identified syndecan-4 as a primary-response gene product in VSMCs. In contrast, syndecan-1 mRNA expression in response to serum was completely blocked in the presence of cycloheximide. We also examined the expression of syndecan mRNAs in VSMCs in response to balloon catheter injury in vivo. A reverse transcriptase-polymerase chain reaction technique was developed that enabled us to amplify all four syndecan mRNAs in a single reaction tube and determine relative changes in their expression. All four syndecan mRNAs were detected in uninjured rat carotid arteries. In endothelium-denuded arteries, the medial layer (presumably VSMCs) accounted for 70% to 90% of the syndecan mRNAs in the vessel wall. The levels of syndecan-2 and syndecan-3 mRNAs were not altered significantly after balloon injury. In contrast, syndecan-4 mRNA was increased at early times after injury but then decreased to control level by 7 days. Syndecan-1 mRNA levels showed a slower but prolonged increase that reached a maximum at 7 days after injury. Immunostaining with anti-syndecan-4 antibodies demonstrated a rapid increase in syndecan-4 proteoglycan expression in the injured carotid artery.
Asunto(s)
Arterias Carótidas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas de Membrana/biosíntesis , Músculo Liso Vascular/metabolismo , Proteoglicanos/biosíntesis , Animales , Arterias Carótidas/patología , Cateterismo , ADN Complementario/genética , Masculino , Glicoproteínas de Membrana/genética , Músculo Liso Vascular/patología , Proteoglicanos/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Sindecano-4RESUMEN
The cDNA coding for a 67 kDa protein (p67) was isolated from a rat Schwann cell library. A recombinant form of p67 expressed in bacteria was used to produce polyclonal anti-p67 antibodies. By immunoblot analysis p67 was found to be expressed in most tissues and cell lines examined. Inspection of the deduced amino acid sequence revealed a COOH-terminal consensus sequence for isoprenylation. Consistent with this finding, p67 was a substrate for isoprenylation in vitro by geranylgeranylpyrophosphate. p67 was associated predominantly with the particulate fraction of rat smooth muscle cells. The rat p67 sequence was highly homologous to a family of recently described human and mouse gamma-interferon inducible, guanine nucleotide binding proteins.
Asunto(s)
Proteínas Portadoras/genética , ADN Complementario/aislamiento & purificación , Proteínas de Unión al GTP/genética , Células de Schwann/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Prenilación de Proteína , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
Rat aortic vascular smooth muscle (VSM) cells synthesize the transmembrane proteoglycan syndecan (Cizmeci-Smith, G., Asundi, V., Stahl, R. C., Teichman, L. J., Chernousov, M., Cowan, K., and Carey, D. J. (1992) J. Biol. Chem. 267, 15729-15736). The present work demonstrated that VSM cells synthesize the related transmembrane proteoglycan fibroglycan and that increased expression of these two proteoglycans is stimulated under different conditions. Fibroglycan synthesis by cultured rat aortic VSM cells was demonstrated by Northern blot analysis with a rat fibroglycan cDNA probe and immunoblot analysis with anti-rat fibroglycan antibodies. Effects of growth factors and vasoactive substances on syndecan and fibroglycan expression were examined by Northern blot analysis. Syndecan mRNA levels increased in response to stimulation of VSM cells with serum, platelet-derived growth factor, or angiotensin II. VSM cells stimulated with platelet-derived growth factor contained more syndecan core protein and processed syndecan than control cells. Fibroglycan mRNA levels either decreased or remained unchanged in response to these agents. Fibroglycan mRNA levels increased following transforming growth factor-beta stimulation, while syndecan mRNA levels decreased. Other agents, including basic fibroblast growth factor, endothelin, and carbacyclin did not alter the expression of either proteoglycan. Syndecan and fibroglycan mRNA levels also varied as a function of cell density. These data demonstrate that syndecan and fibroglycan expression are regulated differently in VSM cells and lend support to the hypothesis that these proteoglycans carry out distinct physiological functions.