RESUMEN
Vulvodynia (vulvar pain syndrome) is a chronic multifactorial disease affecting almost 13 million women in the USA and can lead to morbidity and a reduced quality of life. We hypothesize that an initial microbiological insult in the vagina causes modifications in the biological vaginal milieu and/or an alteration on the lactobacilli flora. The vaginal milieu responds to the insult by developing an inflammatory reaction with abnormal cytokine production. These hypotheses were tested quantifying vaginal lactobacillus and cytokines, in patients with vulvodynia compared to matched healthy controls. Our preliminary data suggest a vaginal flora alteration and an immunological response involving Candida in patients with vulvodynia. Ongoing studies will assist us to clarify these findings.
Asunto(s)
Hongos/patogenicidad , Vulvodinia/microbiología , Femenino , HumanosRESUMEN
The K+-Cl- cotransport (COT) regulatory pathways recently uncovered in our laboratory and their implication in disease state are reviewed. Three mechanisms of K+-Cl- COT regulation can be identified in vascular cells: (1) the Li+-sensitive pathway, (2) the platelet-derived growth factor (PDGF)-sensitive pathway and (3) the nitric oxide (NO)-dependent pathway. Ion fluxes, Western blotting, semi-quantitative RT-PCR, immunofluorescence and confocal microscopy were used. Li+, used in the treatment of manic depression, stimulates volume-sensitive K+-Cl- COT of low K+ sheep red blood cells at cellular concentrations <1 mM and inhibits at >3 mM, causes cell swelling, and appears to regulate K+-Cl- COT through a protein kinase C-dependent pathway. PDGF, a potent serum mitogen for vascular smooth muscle cells (VSMCs), regulates membrane transport and is involved in atherosclerosis. PDGF stimulates VSM K+-Cl- COT in a time- and concentration-dependent manner, both acutely and chronically, through the PDGF receptor. The acute effect occurs at the post-translational level whereas the chronic effect may involve regulation through gene expression. Regulation by PDGF involves the signalling molecules phosphoinositides 3-kinase and protein phosphatase-1. Finally, the NO/cGMP/protein kinase G pathway, involved in vasodilation and hence cardiovascular disease, regulates K+-Cl- COT in VSMCs at the mRNA expression and transport levels. A complex and diverse array of mechanisms and effectors regulate K+-Cl- COT and thus cell volume homeostasis, setting the stage for abnormalities at the genetic and/or regulatory level thus effecting or being affected by various pathological conditions.
Asunto(s)
Aterosclerosis/metabolismo , Músculo Liso Vascular/metabolismo , Transducción de Señal/fisiología , Simportadores/metabolismo , Vasodilatación/fisiología , Enfermedades Cardiovasculares/metabolismo , Tamaño de la Célula , Regulación de la Expresión Génica , Humanos , Transporte Iónico , Músculo Liso Vascular/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Simportadores/genética , Cotransportadores de K ClRESUMEN
The binding of pro-opiomelanocortin,(POMC), pro-insulin, pro-enkephalin and chromogranin A (CGA) to the regulated secretory pathway sorting receptor, carboxypeptidase E (CPE), in bovine pituitary secretory granule (SG) membranes was investigated. N-POMC1-26, which contains the POMC sorting signal, bound to CPE in the SG membranes with low affinity and the binding was ion independent. Pro-insulin bound CPE with similar kinetics. Pro-enkephalin, but not CGA bound to CPE with similar IC50 as pro-insulin and N-POMC1-26. Crosslinking studies showed that pro-insulin and pro-enkephalin bound specifically to SG membrane CPE, similar to N-POMC1-26 reported previously. CPE was extracted from the SG membranes with NaHCO3 or KSCN, but not Triton X-100/1 M NaCl. The results show that CPE is tightly associated with SG membranes and binds several prohormones, but not CGA, with similar kinetics, providing further evidence that membrane CPE has the characteristics to function as a common sorting receptor for targeting prohormones to the regulated secretory pathway.
Asunto(s)
Carboxipeptidasas/química , Gránulos Citoplasmáticos/enzimología , Receptores de Superficie Celular/química , Animales , Calcio , Carboxipeptidasa H , Carboxipeptidasas/metabolismo , Bovinos , Cromogranina A , Cromograninas/química , Cromograninas/metabolismo , Reactivos de Enlaces Cruzados , Membranas Intracelulares/enzimología , Cinética , Octoxinol , Hipófisis/enzimología , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Peptide hormones, neuropeptides, and other molecules such as the granins are specifically packaged into granules of the regulated secretory pathway and released in a calcium-dependent manner upon stimulation. Many of these molecules are synthesized as larger precursors (prohormones) that are processed to biologically active products within the granules. It has now become apparent that prohormones, proneuropeptides, and the granins contain conformation-dependent sorting signal motifs that facilitate their specific sorting and packaging into regulated secretory granules. Recently, a receptor to which these sorting signals bind has been identified as the membrane form of carboxypeptidase E (CPE) and localized to the Golgi apparatus, where sorting occurs, specifically at the trans-Golgi network. In this article, we review the evidence for a sorting signal-receptor-mediated mechanism for routing peptide hormones and prohormones to the regulated secretory granules. We also describe a mouse model, Cpe(fat), which has the CPE gene naturally mutated. Pituitary hormones were misrouted and secreted in an unregulated manner via the constitutive pathway in these Cpe(fat) mice, leading to endocrine disorders.
RESUMEN
A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated secretory pathway proteins, including prohormones, but not constitutively secreted proteins. We show that in the Cpe(fat) mutant mouse lacking CPE, the pituitary prohormone, pro-opiomelanocortin, was missorted to the constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting receptor, leads to multiple endocrine disorders in these genetically defective mice, including hyperproinsulinemia and infertility.
Asunto(s)
Carboxipeptidasas/fisiología , Gránulos Citoplasmáticos/enzimología , Enfermedades del Sistema Endocrino/enzimología , Membranas Intracelulares/enzimología , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Carboxipeptidasa H , Carboxipeptidasas/metabolismo , Bovinos , Línea Celular , Cromogranina A , Cromograninas/metabolismo , Dopamina/farmacología , Femenino , Aparato de Golgi/enzimología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Sistemas Neurosecretores/citología , Hipófisis/metabolismo , Proopiomelanocortina/metabolismo , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína , ARN sin SentidoRESUMEN
The subcellular localization and functionality of transfected yeast aspartic protease 3 (YAP3p) in a mammalian cell line were investigated. The complementary DNAs encoding the prohormone-processing enzyme (YAP3p) and a prohormone, bovine POMC, were cotransfected into PC12 (rat pheochromocytoma) cells. Immunocytochemical analysis of the cells using a YAP3p antibody showed a perinuclear punctate distribution of YAP3p in the cell body as well as immunostaining in the tips of the neurites. This pattern of immunostaining indicates localization of YAP3p in secretory granules. Analysis of the processing of POMC showed that in cells transfected with the POMC complementary DNA alone, only POMC was found, indicating a lack of processing of the prohormone. However, in cells coexpressing YAP3p, the POMC was completely processed to yield ACTH-(1-39) and ACTH-(1-14), consistent with the specificity of YAP3p found in vitro. Pulse-chase studies showed that POMC was processed after 20 min of chase, suggesting that processing occurred in the late Golgi network and continued in the secretory granules. Western blot analysis determined that YAP3p was secreted from the cells in a regulated manner. This study provides the first demonstration that a yeast prohormone-processing enzyme (YAP3p) of the aspartic protease class can be sorted correctly to secretory granules and activated to process a prohormone (POMC) in a highly efficient manner in mammalian cells.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proopiomelanocortina/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Inmunohistoquímica , Células PC12/metabolismo , Radioinmunoensayo , Ratas , Proteínas de Saccharomyces cerevisiae , Factores de Tiempo , TransfecciónRESUMEN
The processing of pro-opiomelanocortin (POMC) was examined in GH3 cells, a rat sommatomammotrope cell line, by transiently-transfecting the cells with mouse POMC cDNA. The peptide products were extracted, chromatographed on HPLC and identified by specific radioimmunoassay. POMC was processed to generate ACTH-related peptides, beta-endorphin and Lys-gamma 3- MSH, with complete disappearance of the POMC precursor. The ACTH-related molecules were identified as ACTH1-14, ACTH1-15, ACTH1-17, as well as ACTH1-39. GH3 cells which were not transfected with POMC cDNA did not contain endogenous POMC-related peptides. RT-PCR demonstrated that GH3 cells contain prohormone convertase 2 (PC2) mRNA but no PC1 mRNA. To determine if PC2 was the enzyme responsible for POMC processing in this cell line, GH3 cells were stably-transfected with PC2 antisense cDNA. A cell line was obtained which showed an absence of PC2 protein compared to control untransfected GH3 cells, indicating successful hybridization of PC2 antisense mRNA to the endogenous PC2 mRNA. When this cell line was then transiently-transfected with POMC cDNA, POMC was not processed. The results from these experiments suggest that PC2 alone can correctly process POMC to biologically active smaller peptides in vivo. Additionally, the GH3 cell line with and without incorporation of PC2 antisense cDNA can be used as a model system to study the role of PC2 in the post-translational processing of other prohormones and proproteins in vivo.
Asunto(s)
Proopiomelanocortina/metabolismo , ARN sin Sentido/genética , ARN Mensajero/genética , Subtilisinas/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , ADN Complementario/genética , Ratones , Datos de Secuencia Molecular , Proopiomelanocortina/química , Proopiomelanocortina/genética , Proproteína Convertasa 2 , Procesamiento Proteico-Postraduccional , Ratas , TransfecciónRESUMEN
Mouse neuroblastoma Neuro-2a cells were examined for the expression of pro-enkephalin mRNA, protein, and Met-enkephalin ([Met]-Enk) peptide. Reverse transcriptase/polymerase chain reaction (RT/PCR) and in situ hybridization demonstrated the presence of pro-enkephalin mRNA in these cells. Immunocytochemistry using an antibody which recognizes pro-enkephalin and high pressure liquid chromatography (HPLC) followed by radioimmunoassay indicated that pro-enkephalin was synthesized in these cells and processed to yield the bioactive pentapeptide, [Met]-Enk. Furthermore, release studies showed that the [Met]-Enk was secreted from these cells with high K+ stimulation. Using double labeling, in situ hybridization combined with immunocytochemistry, we demonstrated that prohormone convertase 2 (PC2) mRNA is colocalized with pro-enkephalin in the same Neuro-2a cells, suggesting that this enzyme may be responsible for processing this precursor. we also showed the presence of vasopressin mRNA and arginine-vasopressin peptide in these cells using in situ hybridization and immunocytochemistry, respectively. Thus, the Neuro-2a cells are a multiple neuropeptide-producing cell line and an excellent model for studying the mechanisms involved in the synthesis, intracellular targeting and processing of endogenous pro-enkephalin and pro-vasopressin, as well as other transfected neuropeptide precursors.
Asunto(s)
Encefalina Metionina/genética , Expresión Génica , Neuroblastoma/metabolismo , ARN Mensajero/metabolismo , Vasopresinas/genética , Animales , Arginina Vasopresina/análisis , Cromatografía Líquida de Alta Presión , Encefalina Metionina/metabolismo , Encefalinas/análisis , Encefalinas/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Reacción en Cadena de la Polimerasa , Proproteína Convertasa 2 , Precursores de Proteínas/análisis , Precursores de Proteínas/genética , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARN , Subtilisinas/genética , Células Tumorales Cultivadas , Vasopresinas/análisisRESUMEN
The NH2-terminal region of pro-opiomelanocortin (POMC) is highly conserved across species, having two disulfide bridges that cause the formation of an amphipathic hairpin loop structure between the 2nd and 3rd cysteine residues (Cys8 to Cys20). The role that the NH2-terminal region of pro-opiomelanocortin plays in acting as a molecular sorting signal for the regulated secretory pathway was investigated by using site-directed mutagenesis either to disrupt one or more of the disulfide bridges or to delete the amphipathic loop entirely. When POMC was expressed in Neuro-2a cells, ACTH immunoreactive material was localized in punctate secretory granules in the cell body and along the neurites, with heavy labeling at the tips. ACTH was secreted from these POMC-transfected cells in a regulated manner. Disruption of both disulfide bridges or the second disulfide bridge or removal of the amphipathic hairpin loop resulted in constitutive secretion of the mutant POMC from the cells and a lack of punctate secretory granule immunostaining within the cells. We have modeled the NH2-terminal POMC Cys8 to Cys20 domain and have identified it as an amphipathic loop containing four highly conserved hydrophobic and acidic amino acid residues (Asp10-Leu11-Glu14-Leu1). Thus the sorting signal for POMC to the regulated secretory pathway appears to be encoded by a specific conformational motif comprised of a 13-amino acid amphipathic loop structure stabilized by a disulfide bridge, located at the NH2 terminus of the molecule.
Asunto(s)
Proopiomelanocortina/metabolismo , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Bovinos , Línea Celular , Disulfuros/metabolismo , Humanos , Lisina/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proopiomelanocortina/química , Proopiomelanocortina/genética , Conformación Proteica , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Alineación de SecuenciaRESUMEN
The N-terminal 26 amino acids of the prohormone pro-opiomelanocortin (POMC) were investigated to determine whether this region has the capacity to act as a sorting signal for the regulated secretory pathway. Constructs were made using the N-terminal 101, 50, 26 or 10 amino acids of POMC fused to the chloramphenicol acetyltransferase (CAT) reporter protein and expressed in AtT20 cells to show that at least the first 26 amino acids were required to sort CAT to the regulated secretory pathway. Full length POMC was mutated by deleting amino acids 2-26 from the N-terminal region. Analysis of Neuro-2a cells expressing this mutation compared to wild type POMC indicated that these 26 amino acids contain information essential for sorting POMC to the regulated secretory pathway. The results presented here suggest the presence of a conformation-dependent signal in the N-terminal 26 amino acids of POMC responsible for sorting POMC to the regulated secretory pathway.
Asunto(s)
Proopiomelanocortina/metabolismo , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Proopiomelanocortina/análisis , Proopiomelanocortina/química , Señales de Clasificación de Proteína/química , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Treatment of confluent cultures of JAR human placental choriocarcinoma cells with cholera toxin or forskolin for 16 h markedly stimulated (2.4-fold) serotonin transport activity in these cells. Cycloheximide, an inhibitor of protein synthesis or actinomycin D, an inhibitor of mRNA synthesis effectively blocked this stimulation. Northern blot analysis revealed that treatment with cholera toxin resulted in severalfold increase in the concentrations of the three mRNA species (6.8, 4.9 and 3.0 kilobases in size) which hybridized to the human placental serotonin transporter cDNA. Under similar conditions, the concentrations of the mRNA species which hybridized to the human placental taurine transporter cDNA or to the human beta-actin cDNA were not affected. Analysis of paroxetine-sensitive binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane to the membranes prepared from control and cholera toxin-treated cells indicated that the maximal binding capacity was increased 2.5-fold by cholera toxin, with no significant change in the binding affinity. Thus, stimulation of serotonin transporter activity in the placental choriocarcinoma cells following cholera toxin treatment is likely a result of an increase in cell surface density of the serotonin transporter protein as a consequence of increased steady state serotonin transporter mRNA levels.
Asunto(s)
Proteínas Portadoras/metabolismo , Toxina del Cólera/farmacología , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , ARN Mensajero/metabolismo , Serotonina/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Coriocarcinoma , AMP Cíclico/farmacología , Cicloheximida/farmacología , ADN Complementario , Dactinomicina/farmacología , Femenino , Humanos , Ligandos , Glicoproteínas de Membrana/genética , Placenta/citología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Células Tumorales CultivadasRESUMEN
The characteristics of taurine transport were studied in a human retinal pigment epithelial cell line (HRPE). Uptake of taurine into monolayer cultures of the HRPE cells was markedly stimulated by the presence of NaCl in the uptake medium whereas the uptake was negligible in its absence. This NaCl-dependent uptake was an active process as the cells were able to accumulate taurine against a concentration gradient. The uptake rate of taurine was found to be many-fold greater than that of gamma-aminobutyric acid (GABA). Unlabeled taurine and GABA competed with radiolabeled taurine for the uptake process, the former being more effective than the latter. However, uptake of radiolabeled GABA was not affected by unlabeled taurine and GABA. Substrate specificity studies revealed strong interaction of beta-amino acids with the transport system responsible for taurine uptake. alpha-Amino acids failed to inhibit taurine uptake. A specific anion requirement was observed for optimal activity of the taurine transport system and Cl- was the most supportive among several anions tested. Kinetic analyses showed that multiple Na+ and one Cl- were involved in transfer of one taurine molecule. The transport process consisted of a single saturable system with a Michaelis-Menten constant of 2.0 +/- 0.1 microM. These results show that the HRPE cell line expresses a high-affinity taurine transport system. This is the first demonstration of the presence of the taurine transporter in the human retinal pigment epithelium and the HRPE cell line may provide a useful model system for future studies involving taurine transport in the retinal pigment epithelium.
Asunto(s)
Proteínas de Transporte de Membrana , Epitelio Pigmentado Ocular/metabolismo , Taurina/metabolismo , Adulto , Aminoácidos/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Línea Celular , Células Cultivadas , Humanos , Cinética , Masculino , Glicoproteínas de Membrana/metabolismo , Cloruro de Sodio/metabolismo , Especificidad por Sustrato , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The 5-hydroxytryptamine (5-HT; serotonin) transporter was solubilized from purified human placental brush border membranes by cholate in the presence of urea, and the solubilized transporter was reconstituted into proteoliposomes in a functionally active form. Solubilization of the membranes with cholate in the absence of urea inactivated the transporter. The reconstitution procedure involved precipitation of the solubilized proteins and simultaneous removal of cholate and urea by poly(ethylene glycol), and incorporation of the precipitated proteins into proteoliposomes in the presence of asolectin by a freeze-thaw/sonication technique. Optimal conditions included the use of 6% poly(ethylene glycol) during the precipitation step and an asolectin/protein ratio of 10:1 during the reconstitution step. K+ was present in the reconstitution medium. The reconstituted proteoliposomes showed the ability to transport 5-HT against a concentration gradient when an inwardly directed NaCl gradient was imposed. The 5-HT transport system in the proteoliposomes had an absolute requirement for Na+ and Cl-. The system was specific for 5-HT and was inhibited by imipramine, paroxetine and fluoxetine. The Na+/Cl-/5-HT stoichiometry was found to be 1:1:1. The transport process was electrically silent, indicating that one K+ ion was countertransported for each 5-HT molecule. The reconstituted 5-HT transporter showed high affinity for 5-HT, with an apparent Michaelis-Menten constant of 0.34 +/- 0.01 microM. It is concluded that the human placental 5-HT transporter can be solubilized and reconstituted into proteoliposomes in a transport-competent form and that the characteristics of the reconstituted transporter are similar to those of the native transporter.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Microvellosidades/metabolismo , Proteínas del Tejido Nervioso , Placenta/metabolismo , Serotonina/metabolismo , Aniones , Proteínas Portadoras/aislamiento & purificación , Cationes , Fraccionamiento Celular , Ácido Cólico , Ácidos Cólicos , Femenino , Humanos , Cinética , Liposomas , Glicoproteínas de Membrana/aislamiento & purificación , Embarazo , Proteolípidos/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Solubilidad , UreaRESUMEN
The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.
Asunto(s)
Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Placenta/metabolismo , Serotonina/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Antidepresivos/farmacología , Transporte Biológico Activo/efectos de los fármacos , Bucladesina/farmacología , Toxina del Cólera/farmacología , Coriocarcinoma , Colforsina/farmacología , Femenino , Gramicidina/farmacología , Humanos , Cinética , Nigericina/farmacología , Ouabaína/farmacología , Reserpina/farmacología , Sodio/metabolismo , Células Tumorales CultivadasRESUMEN
The JAR human placental choriocarcinoma cell line transports taurine, concentrating it over 1000-fold inside the cell. The transport system is energized by a Na+ gradient and exhibits an absolute requirement for Cl-. Neutral beta-amino acids such as beta-alanine and hypotaurine effectively compete with the system, whereas neutral alpha-amino acids such as alanine, leucine and alpha-aminoisobutyric acid do not. The transport system interacts with gamma-aminobutyric acid to an appreciable extent. Kinetic analysis reveals that the taurine transport system in this cell line is of a high-affinity and low-capacity type (apparent dissociation constant 2.3 +/- 0.3 microM; maximal velocity 88.5 +/- 5.0 pmol/3 min per mg of protein). Pretreatment of the JAR choriocarcinoma cells with phorbol 12-myristate 13-acetate results in the inhibition of the taurine transport system in a dose-dependent manner. The inhibition is blocked by co-treatment of the cells with staurosporine, an inhibitor of protein kinase C. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, has no effect on the transport system. These data show that the choriocarcinoma cells express a taurine transporter with characteristics similar to those of the taurine transporter described in the normal human placenta, and that the activity of the transporter in these cells is under the regulatory control of protein kinase C.
Asunto(s)
Proteína Quinasa C/metabolismo , Taurina/metabolismo , Alcaloides/farmacología , Aminoácidos/farmacología , Aniones , Transporte Biológico/efectos de los fármacos , Línea Celular , Coriocarcinoma , Femenino , Humanos , Cinética , Ésteres del Forbol/farmacología , Placenta , Embarazo , Proteína Quinasa C/antagonistas & inhibidores , Sodio/farmacología , Estaurosporina , Especificidad por Sustrato , Neoplasias UterinasRESUMEN
The interaction of fluoxetine, a non-tricyclic antidepressant, with the human placental serotonin transporter was investigated by studying its influence on [3H]paroxetine binding to the transporter and on [3H]serotonin uptake via the transporter. These studies were done using brush-border membrane vesicles purified from normal term human placentas. Fluoxetine inhibited binding of paroxetine to the membrane vesicles in a concentration-dependent manner, with a Ki value of 3 nM. Kinetic analysis revealed that the inhibition was competitive because the presence of 10 nM fluoxetine increased the Kd for paroxetine from 72 to 461 pM, but had no effect on the Bmax. Fluoxetine also caused a time-dependent dissociation of paroxetine already bound to the transporter. The dissociation followed first-order kinetics. Uptake of serotonin in these membrane vesicles was also inhibited by fluoxetine. The inhibition was concentration dependent with a Ki value of 66 nM at pH 7.5 and 80 nM at pH 6.5. The effect of fluoxetine on the uptake kinetics was to increase the apparent dissociation constant (Kt) for serotonin without influencing the maximal transport capacity (Vmax). The results demonstrate that fluoxetine is a high-affinity ligand and a potent inhibitor of the serotonin transporter found in the human placental brush-border membrane.
Asunto(s)
Proteínas Portadoras/metabolismo , Fluoxetina/farmacología , Piperidinas/metabolismo , Antagonistas de la Serotonina/metabolismo , Serotonina/metabolismo , Femenino , Humanos , Técnicas In Vitro , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Paroxetina , Placenta/efectos de los fármacos , Placenta/metabolismo , EmbarazoRESUMEN
We investigated the interaction of paroxetine, a nontricyclic antidepressant, with the serotonin transporter of the human placental brush-border membrane. Paroxetine bound to the purified placental brush-border membranes with a high affinity [dissociation constant (Kd) = 72 pM]. The maximal binding capacity (Bmax) was 3.9 pmol/mg protein. Imipramine, desipramine, and serotonin inhibited the binding in a dose-dependent manner with inhibition constant (Ki) values of 4.4 nM, 48.7 nM, and 1.77 microM, respectively, whereas reserpine, ketanserin, and 5-hydroxytryptophan did not have any effect. Imipramine and serotonin inhibited paroxetine binding by increasing the Kd with essentially no effect on Bmax. Binding of paroxetine to the membranes increased hyperbolically with increasing concentrations of Na+ in the assay medium. Cl- had little effect on the binding. The effect of Na+ was primarily to increase the affinity of the transporter for paroxetine with no effect on Bmax. The association constant (Ka) increased hyperbolically as the concentration of Na+ increased, indicating a 1Na+:1paroxetine stoichiometry. The maximal value for Ka was 12.1 +/- 2.5 x 10(12) M-1, and Kd for Na+ was 10.0 +/- 3.5 mM. Treatment of the membranes with tyrosyl group-specific reagents reduced the Na(+)-dependent binding, suggesting the involvement of tyrosyl residues in the binding process. This inhibition was, however, significantly reduced when treatment with the reagent was performed in the presence of Na+, suggesting that the reactive tyrosyl residues were located at or near the Na(+)-binding site. Paroxetine inhibited NaCl gradient-dependent serotonin uptake in placental brush-border membrane vesicles both at pH 6.5 and 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Portadoras/metabolismo , Microvellosidades/metabolismo , Piperidinas/metabolismo , Placenta/metabolismo , Antagonistas de la Serotonina/metabolismo , Serotonina/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacología , Unión Competitiva , Transporte Biológico , Femenino , Humanos , Imidazoles/farmacología , Cinética , Paroxetina , Embarazo , Sodio/farmacología , Tetranitrometano/farmacología , TirosinaRESUMEN
The modulation of serotonin uptake kinetics by Na+, Cl-, H+, and K+ was investigated in brush-border membrane vesicles prepared from normal human term placentas. The presence of Na+ and Cl- in the external medium was mandatory for the function of the serotonin transporter. In both cases, the initial uptake rate of serotonin was a hyperbolic function of the ion concentration, indicating involvement of one Na+ and one Cl- per transport of one serotonin molecule. The apparent dissociation constant for Na+ and Cl- was 145 and 79 mM, respectively. The external Na+ increased the Vmax of the transporter and also increased the affinity of the transporter for serotonin. The external Cl- also showed similar effects on the Vmax and the Kt, but its effect on the Kt was small compared to that of Na+. The presence of an inside-acidic pH, with or without a transmembrane pH gradient, stimulated the NaCl-dependent serotonin uptake. The effect of internal [H+] on the transport function was to increase the Vmax and decrease the affinity of the transporter for serotonin. The presence of K+ inside the vesicles also greatly stimulated the initial rates of serotonin uptake, and the stimulation was greater at pH 7.5 than at pH 6.5. This stimulation was a hyperbolic function of the internal K+ concentration at both pH values, indicating involvement of one K+ per transport of one serotonin molecule. The apparent dissociation constant for K+ was 5.6 mM at pH 6.5 and 4.0 mM at pH 7.5. The effects of internal [K+] on the uptake kinetics were similar to those of internal [H+].(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Microvellosidades/metabolismo , Placenta/metabolismo , Serotonina/metabolismo , Sodio/farmacología , Cloruros/farmacología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Cinética , Microvellosidades/efectos de los fármacos , Concentración Osmolar , Potasio/farmacología , EmbarazoRESUMEN
A copper slug heated with a soldering iron was used to produce full- and partial-thickness burns on the backs of mice. The untreated partial-thickness burns healed by outgrowth of epidermal cells from the hair follicles and adjoining skin, and the full-thickness burns formed linear scars. Skin equivalents containing isogeneic fibroblasts and epidermal cells were used to replace full-thickness burns; these grafts were fully vascularized and covered with a cornified epidermis within 2 weeks. The grafts maintained 34% of their original area at 180 days, but the full-thickness burns retained only 4.5% of the initial area. For the first 2 weeks, the splenic index in animals that received burns followed by surgical excision and grafting was significantly greater than in the animals that had burns not followed by excision, but the difference was no longer significant by 21 days.