RESUMEN
Type 1 diabetes generally results from autoimmune destruction of pancreatic islet beta-cells, with consequent absolute insulin deficiency and complete dependence on exogenous insulin treatment. The relative paucity of donations for pancreas or islet allograft transplantation has prompted the search for alternative sources for beta-cell replacement therapy. In the current study, we used pluripotent undifferentiated human embryonic stem (hES) cells as a model system for lineage-specific differentiation. Using hES cells in both adherent and suspension culture conditions, we observed spontaneous in vitro differentiation that included the generation of cells with characteristics of insulin-producing beta-cells. Immunohistochemical staining for insulin was observed in a surprisingly high percentage of cells. Secretion of insulin into the medium was observed in a differentiation-dependent manner and was associated with the appearance of other beta-cell markers. These findings validate the hES cell model system as a potential basis for enrichment of human beta-cells or their precursors, as a possible future source for cell replacement therapy in diabetes.
Asunto(s)
Insulina/genética , Islotes Pancreáticos/fisiología , Células Madre/fisiología , Animales , Adhesión Celular , Diferenciación Celular , División Celular , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/fisiología , Glucoquinasa/genética , Humanos , Insulina/análisis , Insulina/biosíntesis , Islotes Pancreáticos/citología , Ratones , Proteínas de Transporte de Monosacáridos/genética , Páncreas/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citologíaRESUMEN
The telomerase RNA-protein complex responsible for maintenance of telomeric DNA at chromosome ends, is usually inactive in most primary somatic human cells, but is specifically activated with in vitro immortalization and during tumorigenesis. Although expression of the RNA component of telomerase appears to be constitutive, the expression pattern of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is correlated with measured enzyme activity. In particular, a >80% concordance has been reported between telomerase activity and hTERT mRNA expression in ovarian tumors. Accordingly, to learn more about the mechanism regulating hTERT gene expression in ovarian carcinoma, we have performed a detailed analysis of the 5'-flanking promoter region of the hTERT gene. We have reported previously the isolation and analysis of a 5.8-kb genomic fragment containing the human hTERT gene promoter (M. Tzukerman et al., Mol. Biol. Cell, 11: 4381-4391, 2000). Deletion analysis of this promoter was carried out using transient transfection of promoter-reporter constructs in four different telomerase-expressing, ovarian carcinoma-derived cell lines, the tumorigenic properties of which have been characterized, and was compared with telomerase-negative primary human fibroblasts and nontransformed ovarian epithelial cells. These assays have shown that the hTERT promoter is inactive in telomerase-negative cells and is active in telomerase-positive cell lines. A core promoter of 283 bp upstream of the transcription initiation site (TI) was found to be sufficient for maximum promoter activity, suggesting the presence of inhibitory elements within the larger promoter sequence. Gel shift analysis of the core promoter using nuclear extracts from the ovarian and control cell lines revealed specific transcription factor binding using extracts from telomerase-positive cells. Among the binding elements, we identified two E-boxes (CACGTG) as well as a novel element (MT-box), which we identified recently in a number of differentiation systems. Site-directed mutagenesis was used to introduce mutations into this novel transcription factor binding element. These mutations significantly affect the transcriptional activity of hTERT promoter in a cell type-specific manner and suggest that the transcription factors that bind to the E-box and the novel element cooperatively function as major determinants of hTERT expression and telomerase activity in ovarian cancer. Further comparison of promoter activity, telomerase activity, and telomere length among the different ovarian cancer cells indicated that a threshold level of telomerase activity is apparently sufficient to protect telomere integrity and permit the immortal state of the different ovarian cancer cell lines.
Asunto(s)
Neoplasias Ováricas/genética , Regiones Promotoras Genéticas/genética , ARN , Telomerasa/genética , Secuencia de Bases , Sitios de Unión/genética , Unión Competitiva , Línea Celular , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Neoplasias Ováricas/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Células Tumorales CultivadasRESUMEN
Three different cell differentiation experimental model systems (human embryonic stem cells, mouse F9 cells, and human HL-60 promyelocytic cells) were used to determine the relationship between the reduction in telomerase activity after differentiation and the regulation of the promoter for the hTERT gene. Promoter constructs of three different lengths were subcloned into the PGL3-basic luciferase reporter vector. In all three experimental systems, all three promoter constructs drove high levels of reporter activity in the nondifferentiated state, with a marked and time-dependent reduction after the induction of differentiation. In all cases, the smallest core promoter construct (283 nt upstream of the ATG) gave the highest activity. Electrophoretic mobility shift assays revealed transcription factor binding to two E-box domains within the core promoter. There was also a marked time-dependent reduction in this binding with differentiation. In addition, a distinct and novel element was identified within the core promoter, which also underwent time-dependent reduction in transcription factor binding with differentiation. Site-directed mutagenesis of this novel element revealed a correlation between transcription factor binding and promoter activity. Taken together, the results indicate that regulation of overall telomerase activity with differentiation is mediated at least in part at the level of the TERT promoter and provides new information regarding details of the regulatory interactions that are involved in this process.
Asunto(s)
Diferenciación Celular , Regiones Promotoras Genéticas , ARN , Telomerasa/genética , Factores de Transcripción/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Proteínas de Unión al ADN , Humanos , Ratones , Mutación , Células Madre/citología , Células Madre/enzimología , Telomerasa/metabolismoRESUMEN
Expression of the Npt2 gene, encoding the type II sodium-dependent phosphate cotransporter, is restricted to renal proximal tubule epithelium. We have isolated a 4,740-bp fragment of the 5'-flanking sequence of the rat Npt2 gene, identified the transcription initiation site, and demonstrated that this 5'-flanking sequence drives luciferase-reporter gene expression, following transfection in the proximal tubule cell-derived opossum kidney (OK) cell line but not in unrelated cell lines. Analysis of the promoter sequence revealed the presence of 10 consensus binding motifs for the AP2 transcription factor. Transient transfection assays revealed an important effect of the number of tandemly repeated AP2 sites in enhancing promoter activity. The promoter sequence also revealed a pair of inverted repeats enclosing 1,324 bp of intervening sequence and containing 8 of the total 10 AP2 consensus sites in the promoter sequence. Deletion or reversal of orientation of the distal inverted repeat resulted in marked enhancement of promoter activity. Electrophoretic mobility shift analysis revealed a distinct pattern of transcription factor binding to oligonucleotides containing AP2 sites, using nuclear extracts from OK cells, compared with unrelated cell lines. Taken together, these results suggest an important role for AP2 consensus binding sites in regulating Npt2 gene expression and suggest a mechanism of regulation mediated by the interaction of inverted repeats enclosing these sites.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regiones Promotoras Genéticas/fisiología , Simportadores , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Células 3T3 , Animales , Secuencia de Bases/genética , Sitios de Unión/fisiología , Proteínas Portadoras/farmacología , Línea Celular , Secuencia de Consenso/genética , Secuencia de Consenso/fisiología , Humanos , Ratones , Datos de Secuencia Molecular , Zarigüeyas , Ratas , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III , Especificidad de la Especie , Factor de Transcripción AP-2 , Transcripción Genética/fisiologíaRESUMEN
Telomeres are distinct structures, composed of short, repeated sequences, at the ends of all eukaryotic chromosomes. Telomeres have been shown in yeast to induce late replication in S phase and to silence transcription of neighboring genes. To examine the possibility of similar effects in human chromosomes, we studied cells from a subject with a microdeletion of 130 kb at the end of one copy of chromosome arm 22q, repaired by the addition of telomere repeats. Using fluorescence in situ hybridization of S phase nuclei, a distinct difference was found in the replication timing of the breakpoint region between the intact and truncated copies of chromosome 22. This difference was evident as a shift from middle to late replication time of the breakpoint region adjacent to the repaired telomere. This finding suggests that the human telomere sequence influences activation of adjacent replication origin(s). The difference in replication timing between the two chromosomes was not associated with differences in sensitivity to digestion by DNase I or with methylation of regions immediately adjacent to the breakpoint. Furthermore, both alleles of arylsulfatase A, a gene located at a distance of approximately 54 kb from the breakpoint, were expressed. We conclude that as in yeast, the proximity of telomeric DNA may induce a positional effect that delays the replication of adjacent chromosomal regions in humans.
Asunto(s)
Replicación del ADN , Telómero , Línea Celular , Metilación de ADN , Desoxirribonucleasa I/farmacología , Humanos , Factores de TiempoRESUMEN
The kinases and regulatory proteins that convey signals initiated by transforming growth factor-beta (TGF-beta) to the nucleus are poorly characterized. To study the role of the extracellular signal-regulated kinase (ERK) pathway in this process, we transiently transfected NIH 3T3 fibroblasts with TGF-beta-responsive luciferase reporter genes and expression vectors designed to interrupt this kinase cascade. Mitogen-activated protein (MAP) kinase phosphatase-1 and a dominant negative MAP/ERK kinase 1 mutant reduced stimulation of plasminogen activator inhibitor-1 (PAI-1) promoter activity by TGF-beta1 from 11.5- to 4-fold and 4.9-fold, respectively. Similar results were observed with the type I collagen promoters. TGF-beta1 increased ERK1 activity 4.5-fold at 5 min and 3. 1-fold at 3 h, while Jun kinase and p38 activity were not affected. Cotransfection of a dominant negative mutant of the small G protein, Rac, but not dominant negative Ras, Cdc42, or Rho mutants, reduced the effects of TGF-beta1 on the PAI-1 promoter by approximately half. In support of a role for Rac in signaling by TGF-beta, GTP binding to Rac was increased 3.7-fold following exposure of NIH 3T3 cells to TGF-beta1 for 3 min. These findings indicate that TGF-beta1 modulates gene expression partly through ERK and Rac in NIH 3T3 cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas de Unión al GTP/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas Quinasas Activadas por Mitógenos , Factor de Crecimiento Transformador beta/fisiología , Células 3T3 , Animales , Colágeno/genética , Ratones , Proteína Quinasa 3 Activada por Mitógenos , Inhibidor 1 de Activador Plasminogénico/genética , Regiones Promotoras Genéticas , Proteínas de Unión al GTP racRESUMEN
1. In insulin-dependent diabetes mellitus, hyperglycaemia has a profound effect on renal and systemic haemodynamic function. The mechanism for this is unknown. 2. We conducted a study in 11 males with insulin-dependent diabetes mellitus, within 6 years of diagnosis. We examined the neurohumoral, haemodynamic and renal variables during euglycaemia (4.0-6.0 mmol/l) and after a 12 h period of hyperglycaemia (8.5-10.5 mmol/l). Subjects were examined in a sodium-replete state during supine rest and during simulated orthostatic stress induced by lower body negative pressure at -15 mmHg. 3. Variations in glycaemia markedly influenced plasma renin activity, which was increased at baseline during hyperglycaemia (3.82 +/- 0.66 pmol of angiotensin I h-1 ml-1 compared with 2.13 +/- 0.33 pmol of angiotensin I h-1 ml-1 during euglycaemia, P = 0.009), and rose further during simulated orthostatic stress. Mean arterial pressure was also elevated during hyperglycaemia (89 +/- 2 mmHg compared with 81 +/- 3 mmHg during euglycaemia, P = 0.03), both at rest and during orthostatic stress. 4. The renal haemodynamic effects of hyperglycaemia included maintenance of glomerular filtration rate in the face of significant declines in renal blood flow, and a probable increase in filtration fraction (0.21 +/- 0.01 compared with 0.18 +/- 0.01 during euglycaemia, P = 0.06). The responses of mean arterial pressure and renal blood flow to simulated orthostatic stress were not affected by hyperglycaemia, but the forearm vascular response was significantly augmented. 5. These data suggest that sustained hyperglycaemia activates the renin-angiotensin system, thereby increasing systemic and renal vasomotor tone. Over time such changes may have deleterious microvascular consequences.
Asunto(s)
Diabetes Mellitus Tipo 1/fisiopatología , Hiperglucemia/fisiopatología , Riñón/fisiopatología , Renina/sangre , Adulto , Presión Sanguínea/fisiología , Diabetes Mellitus Tipo 1/sangre , Humanos , Hiperglucemia/sangre , Masculino , Norepinefrina/sangre , Presión , Sistema Renina-Angiotensina/fisiologíaRESUMEN
1. The precursor of eicosanoids is arachidonic acid, which emanates from the cleavage of the sn-2 position of phospholipids by phospholipase A2 (PLA2). Eicosanoids have diverse physiological and pathophysiological effects in the kidney. The regulation of phospholipase A2 has important implications for kidney function. 2. In the current communication we focus our attention on mesangial cell cytosolic PLA2 (cPLA2) and its regulation at the post-translational and post-transcriptional level. 3. At the post-translational level, using site directed mutagenesis of cPLA2 and a dominant negative ras, we have demonstrated that cPLA2 can be phosphorylated by mitogen activated protein (MAP-2) kinase leading to increased cPLA2 enzymatic activity. 4. At the post-transcriptional level we show that the half-life of cPLA2 mRNA in mesangial cells is significantly increased when mesangial cells are stimulated by mitogens. We further demonstrate the presence of three ATTTA motifs in the 3' untranslated region (3' UTR) of the cPLA2 cDNA. 5. Using chimeric constructs bearing the 3' UTR from rat cPLA2 fused downstream of the luciferase reporter, we demonstrate that this region exerts a destabilizing effect on cPLA2. 6. We have isolated and mapped genomic DNA and polymorphic markers for cPLA2 in the human and rat.
Asunto(s)
Mesangio Glomerular/fisiología , Fosfolipasas A/fisiología , Animales , Riñón/metabolismo , Fosfolipasas A2 , Fosfotransferasas/metabolismo , RatasRESUMEN
Levels of mRNA expressed by the multidrug resistance gene MDR1 were examined in 23 renal cell carcinoma samples and adjacent normal kidney cortex using reverse-transcription PCR. Comparison of MDR1 levels between histological types revealed that there was on average significantly more MDR1 in clear cell tumors than in oncocytomas (0. 89 +/- 0.10 versus 0.28 +/- 0.20, ratio of MDR1 in tumor cells to the drug-resistant cell line KB-8, P < 0.05). The mean MDR1 level of all of the non-oncocytoma tumors was not significantly different from the mean MDR1 level of normal adjacent kidney (0.89 +/- 0.10 versus 1.11 +/- 0.12, P = 0.07). However, the mean MDR1 level of the more undifferentiated clear cell tumors was significantly lower than the mean MDR1 level of adjacent normal kidney (0.74 +/- 0.10 versus 1.11 +/- 0.12, P < 0.05). MDR1 levels in early stage, clear cell tumors (n = 14) were lower than in tumors that had spread into perinephric tissue or had metastasized (n = 6) (0.77 +/- 0.08 versus 1.24 +/- 0.30, P < 0.05). In conclusion, MDR1 expression decreases in the more undifferentiated tumors, but still remains at levels high enough to be drug resistant. Higher MDR1 expression in the invasive tumors compared with noninvasive tumors suggests that MDR1 expression and invasiveness may be linked.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Carcinoma de Células Renales/genética , Genes MDR , Neoplasias Renales/genética , Proteínas de Neoplasias/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patología , Adenoma Oxifílico/genética , Adenoma Oxifílico/metabolismo , Adenoma Oxifílico/patología , Adulto , Anciano , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Femenino , Expresión Génica , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Estadificación de NeoplasiasRESUMEN
Transmembrane signal transduction is the process whereby a ligand binds to the external surface of the cell membrane and elicits a physiological response specific for that ligand and cell type. It is now appreciated that numerous disease states represent disturbances in normal transmembrane signaling mechanisms. In the current paper, we focus our attention on the mesangial cell of the glomerular microcirculation as a prototypical model system for understanding normal and abnormal transmembrane signaling processes. Among the major receptor and effector mechanisms for transmembrane signal transduction in the mesangial cell, this paper emphasizes the phospholipase effector response to growth factors and vasoactive hormones. The post-translational and transcriptional pathways for regulation of phospholipase C and phospholipase A2 are described, including consideration of perturbations in these systems that characterize two disease models, namely: acute cyclosporine nephrotoxicity and early diabetic glomerulopathy.
Asunto(s)
Enfermedades Renales/fisiopatología , Riñón/fisiología , Transducción de Señal/fisiología , Animales , Membrana Celular/fisiología , Humanos , Riñón/fisiopatologíaRESUMEN
A phase I-II clinical trial was conducted to determine the maximum-tolerated dose (MTD) of oral cyclosporine (CsA) and vinblastine in patients with metastatic renal cell cancer (RCC) as well as to estimate the response rate. Sixteen patients received a 5 mg/kg oral loading dose of CsA followed by 3 days of CsA in 4 divided daily doses escalating from 10 mg/kg per day up to 17 mg/kg per day. Vinblastine (Vbl) was administered as an intravenous bolus on the morning of the 3rd day with dose escalation from 6 to 10 mg/m2. Cycles were repeated every 4 weeks until tumor progression. Forty-nine cycles of CsA with vinblastine were administered. The maximum tolerated dose of Vbl was 10 mg/m2, with neutropenia as the dose-limiting toxicity resulting in one death. CsA could not be escalated above 17 mg/kg per day because of nausea and vomiting. Other toxicities included constipation (100%), malaise (100%), temporary increase in pain (36%), and one seizure that may have been drug-related. There were no clinically significant changes in renal function or serum bilirubin. Mean peak whole-blood CsA level at the highest CsA dose level was 919 ng/ml (range: 414-1,827) with a trough prior to Vbl injection of 451 ng/ml (range: 128-1,229). There were no tumor responses. The combination of oral CsA and Vbl is not nephrotoxic but is poorly tolerated. In most patients optimal blood levels of CsA for reversal of MDR cannot be reliably achieved, and vinblastine dose intensity must be compromised because of the significant toxicity of this regimen.
Asunto(s)
Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/secundario , Ciclosporina/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Vinblastina/uso terapéutico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Administración Oral , Adulto , Anciano , Carcinoma de Células Renales/metabolismo , Ciclosporina/administración & dosificación , Ciclosporina/efectos adversos , Ciclosporina/farmacocinética , Esquema de Medicación , Quimioterapia Combinada , Femenino , Expresión Génica , Humanos , Neoplasias Renales/metabolismo , Masculino , Persona de Mediana Edad , Vinblastina/administración & dosificación , Vinblastina/efectos adversosRESUMEN
The Rb family of proteins includes pRb, p107, and p130. These nuclear polypeptides associate with cyclins and transcription factors involved in the control of cell proliferation. This has suggested that members of the pRb family may modulate cell growth, at least in part, by regulating gene transcription. We have investigated the ability of p107 to modulate transcription and compared it with that of pRb. Whereas pRb inhibition of the c-myc promoter required the presence of E2F sites, p107 inhibition did not. Moreover, p107, but not pRb, repressed transcription from other promoters including fibronectin, herpes virus thymidine kinase, and a synthetic promoter containing a SV40 repeat activator motif upstream from the adenovirus major late-promoter TATA box. In contrast, the activity of the TATA-lacking promoters from the epidermal growth factor receptor and the cytoplasmic phospholipase A2 genes was unaffected by either p107 or pRb. Likewise, overexpression of p107 or pRb had no effect on the activity of a synthetic promoter lacking a TATA box and containing the SV40 repeat motif upstream from the terminal transferase gene initiator element. The domains in p107 required for transcriptional repression included the A segment of the pocket region and parts of the B segment, but not the spacer domain. In spite of their structural similarities, p107 and pRb may contribute to the control of cell proliferation by modulating the transcription of different genes.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Genes de Retinoblastoma , Familia de Multigenes , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Factores de Transcripción E2F , Genes myc , Células HeLa , Humanos , Regiones Promotoras Genéticas , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , TATA Box , Factor de Transcripción DP1 , Células Tumorales Cultivadas , Proteínas Virales/fisiologíaRESUMEN
Agonists which stimulate the inositol 1,4,5 trisphosphate ([1,4,5]-IP3)-dependent mobilization of Ca2+ from intracellular stores also stimulate entry of divalent cations across the cell membrane. Under appropriate experimental conditions, divalent cation entry across the cell membrane can be monitored as the rate at which the intracellular fluorescence of divalent cation indicators is quenched by the addition of Mn2+ to the extracellular medium. We report that addition of vasopressin to fura-2-loaded glomerular mesangial cells in culture markedly accelerated the rate at which Mn2+ quenched fura-2 fluorescence at its Ca(2+)-insensitive wavelength in the presence of extracellular NaCl, but that this quench response was attenuated when Cl- was removed from the extracellular medium by equimolar substitution with impermeant anions (gluconate, methanesulfonate, acetate, lactate). Similarly, loss of agonist-induced quench also occurred when Cl- was substituted with gluconate in K(+)-containing media. Addition of the Cl- channel inhibitor, 5-nitro-2-(3-phenylpropylaminobenzoic acid) (NPPB), also inhibited Mn(2+)-induced quench of fura-2 fluorescence following vasopressin addition. In contrast, in the presence of gramicidin to provide an alternate conductance pathway to accompany divalent cation entry, agonist-dependent Mn2+ quench occurred even in the absence of extracellular Cl-, indicating that the requirement for Cl- was not the result of cotransport on a common transporter nor the result of Cl- serving as a necessary cofactor for divalent cation entry. A similar dependence on extracellular Cl- was observed for other Ca(2+)-mobilizing agonists such as endothelin, as well as the intracellular Ca2+ ATPase inhibitor, thapsigargin. Extracellular Cl- dependence for agonist-induced divalent cation entry was also reflected in a corresponding extracellular Cl- dependence for agonist-induced mesangial cell contraction. It has been previously shown by ourselves (Kremer et al., 1992a, Am. J. Physiol., 262:F668-F678) and others that agonist-stimulated calcium mobilization in mesangial cells is accompanied by inhibition of K+ conductance and increased Cl- conductance. Accordingly, we conclude that the current findings suggest that activation of Cl- conductance provides regulated charge compensation for receptor-mediated divalent cation entry in response to Ca(2+)-mobilizing vasoconstrictor agonists in mesangial cells.
Asunto(s)
Cationes Bivalentes/farmacocinética , Permeabilidad de la Membrana Celular/fisiología , Cloruros/farmacología , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Receptores de Superficie Celular/fisiología , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Canales de Cloruro/antagonistas & inhibidores , Endotelinas/farmacología , Fluorescencia , Mesangio Glomerular/ultraestructura , Gluconatos/farmacología , Gramicidina/farmacología , Concentración de Iones de Hidrógeno , Manganeso/farmacología , Nitrobenzoatos/farmacología , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Cloruro de Sodio/farmacología , Terpenos/farmacología , Tapsigargina , Vasopresinas/farmacologíaRESUMEN
Vinblastine and erythromycin are among the most commonly used chemotherapeutic and antimicrobial agents, respectively. No interaction between the two has ever been reported. Towards the end of a phase I study of vinblastine plus oral cyclosporin (to reverse multidrug resistance), three patients also received erythromycin to raise their cyclosporin levels. All developed severe toxicity consistent with a much higher vinblastine dose than was actually given. This apparent potentiation of vinblastine toxicity has not been previously described.
Asunto(s)
Carcinoma de Células Renales/tratamiento farmacológico , Eritromicina/efectos adversos , Neoplasias Renales/tratamiento farmacológico , Vinblastina/efectos adversos , Anciano , Carcinoma de Células Renales/secundario , Estreñimiento/inducido químicamente , Ciclosporina/uso terapéutico , Sinergismo Farmacológico , Quimioterapia Combinada , Eritromicina/uso terapéutico , Humanos , Neoplasias Renales/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Metástasis Linfática , Masculino , Persona de Mediana Edad , Espasticidad Muscular/inducido químicamente , Neutropenia/inducido químicamente , Dolor/inducido químicamente , Vinblastina/uso terapéuticoRESUMEN
Hyperinsulinemia and insulin resistance are implicated in the etiology of hypertension, but the mechanisms involved have not been established. The objectives of this study were to determine whether untreated essential hypertensive patients are more sensitive to the antinatriuretic action of insulin and more resistant to the counteracting natriuretic effect of atrial natriuretic peptide in contrast to age- and sex-matched normotensive control subjects. Urinary sodium excretion was measured at baseline, during hyperinsulinemic euglycemic clamp, and during coadministration of insulin and atrial natriuretic peptide. Baseline urinary sodium excretion was not significantly different in the normotensive subjects (415 +/- 47 mumol/min, n = 12) and hypertensive patients (381 +/- 18 mumol/min, n = 10); with the institution of insulin infusion, there was a similar and significant decline from baseline (P < .001) to 289 +/- 35 mumol/min in normotensive subjects and 235 +/- 17 mumol/min in hypertensive patients. Atrial natriuretic peptide was able to oppose the antinatriuretic action of insulin in normotensive subjects, increasing urinary sodium excretion significantly to a mean level of 352 +/- 31 mumol/min (P < .05), which did not differ significantly from baseline. In the hypertensive group, atrial natriuretic peptide infusion had no effect on urinary sodium excretion (238 +/- 18 mumol/min), and the difference from baseline remained highly significant (P < .001). The hypertensive patients were significantly less insulin sensitive than their normotensive counterparts, as reflected by a lower glucose utilization rate and higher mean baseline plasma insulin level (P < .05 for each).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factor Natriurético Atrial/fisiología , Hipertensión/fisiopatología , Insulina/fisiología , Natriuresis/efectos de los fármacos , Adulto , Técnica de Clampeo de la Glucosa , Hemodinámica , Humanos , Insulina/sangre , Resistencia a la Insulina , Túbulos Renales/metabolismo , Masculino , Persona de Mediana Edad , Circulación Renal , Sodio/metabolismoRESUMEN
Plasma atrial natriuretic factor (ANF) concentrations are increased in subjects with insulin-dependent diabetes mellitus (IDDM). A potential contribution of ANF to the maintenance of abnormalities in renal hemodynamic function has been considered but not proven in human diabetic subjects. The aim of these experiments was to determine the response of renal blood flow (RBF), glomerular filtration rate (GFR), filtration fraction (FF), and urinary sodium excretion (UNaV) to a reduction of plasma ANF concentrations induced by application of nonhypotensive lower body negative pressure (LBNP) in a group of subjects with early, uncomplicated, well-controlled IDDM compared with control subjects. Baseline supine measurements before LBNP revealed the diabetic subjects to have a significantly higher plasma ANF (31 +/- 2 vs. 24 +/- 2 pg/ml, P = 0.05). GFR tended to be higher (118 +/- 11 vs. 104 +/- 9 ml/min) and UNaV tended to be depressed (188 +/- 25 vs. 240 +/- 25 mumol/min) despite equal sodium intake, but not significantly so. In addition IDDM subjects exhibited significantly lower baseline plasma norepinephrine (PNE) concentrations (0.91 +/- 0.20 vs. 1.60 +/- 0.2 nmol/l, P = 0.03). Forearm vascular resistance (FVR) was not significantly different between the two groups (29 +/- 5 vs. 33 +/- 5 units). LBNP induced comparable decreases in ANF and central venous pressure (CVP) in both groups. The anticipated renal response to ANF reduction (declines in GFR, FF, and UNaV) occurred only in the normal control group. The percent decline in GFR (11% vs. 34.5%, P = 0.01) was markedly attenuated in IDDM subjects. The expected reflexive increase in PNE and FVR also did not occur in IDDM subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factor Natriurético Atrial/sangre , Presión Sanguínea , Diabetes Mellitus Tipo 1/fisiopatología , Tasa de Filtración Glomerular , Hipotensión Ortostática/fisiopatología , Músculos/irrigación sanguínea , Circulación Renal , Resistencia Vascular , Adulto , Aldosterona/sangre , GMP Cíclico/sangre , Diabetes Mellitus Tipo 1/sangre , Diástole , Antebrazo/irrigación sanguínea , Frecuencia Cardíaca , Humanos , Masculino , Norepinefrina/sangre , Valores de Referencia , Renina/sangre , Sodio/orina , Posición Supina , Sístole , Factores de TiempoRESUMEN
Recent findings have suggested that certain ligand-dependent responses to EGF may be propagated in a manner that is not dependent on the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGF-R, Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538) or, alternatively, that these responses may occur through the interaction of the human tyrosine kinase-deficient EGF-R with an as yet unidentified kinase (Selva, E., Raden, D. L., and Davis, R. J. (1993) J. Biol. Chem. 268, 2250-2254). These conclusions represent a significant departure from our current understanding of signal transduction by receptor tyrosine kinases. Therefore we examined the effect of expression of tyrosine kinase-negative human EGF receptor in murine NIH-3T3-2.2 cells on the EGF-dependent phosphorylation of mitogen-activated protein (MAP-2) kinase. In parental cells (NIH-3T3-2.2) that express low levels of endogenous murine EGF-R, there was no demonstrable EGF-dependent coupling to MAP-2 kinase. In NIH-3T3-2.2 cells transfected with tyrosine kinase-negative human EGF-R, there was unexpected EGF-dependent phosphorylation of MAP-2 kinase. Analysis of the tyrosine kinase-negative human EGF-R in these cells revealed significant tyrosine phosphorylation of the EGF-R. A low level of endogenous murine EGF-R present in these cells were also phosphorylated on tyrosine residues and displayed autokinase activity. Similar results were obtained using an unrelated cell line (B82L cells), in which EGF-dependent phosphorylation of MAP-2 kinase was previously attributed to signal propagation through a tyrosine kinase-negative human EGF-R (Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538). Taken together, these results suggest that the tyrosine kinase-negative human EGF-R are able to amplify the response to activation of low levels of endogenous murine EGF-R, thus leading to EGF-dependent phosphorylation of MAP-2 kinase in cells expressing tyrosine kinase-negative human EGF-R.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Células 3T3 , Animales , Receptores ErbB/genética , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Fosforilación , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal growth factor (EGF)-stimulated arachidonic acid release in rat-1 fibroblasts transfected with the N-17 dominant negative mutation of ras. Cells transfected with the N-17 ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-17-mutant expressing cells. No differences were detected between sham-transfected and N-17 mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-17 mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal growth factor-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal growth factor and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Genes ras/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Línea Celular , Receptores ErbB/análisis , Fibroblastos/química , Regulación de la Expresión Génica/genética , Genes ras/genética , Mutación/genética , Fosfolipasas A/análisis , Fosfolipasas A2 , Fosforilación , Pruebas de Precipitina , Ratas , Acetato de Tetradecanoilforbol/farmacología , Transfección , Tritio , Tirosina/metabolismoRESUMEN
We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.