RESUMEN
The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Nucleares , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Complejo CD3/metabolismo , Calcio/metabolismo , Línea Celular , Reactivos de Enlaces Cruzados/farmacología , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Genes Reporteros , Humanos , Isoenzimas/metabolismo , Células Jurkat , Cinética , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Factores de Transcripción NFATC , Fosfolipasa C gamma , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Fosfolipasas de Tipo C/metabolismo , Regulación hacia Arriba , Proteína Tirosina Quinasa ZAP-70RESUMEN
The regulation of Col2a1, which encodes type II collagen, likely results from a balance of both positive and negative proteins. Here we present evidence that the transcription factor deltaEF1 participates in the negative regulation of Col2a1 transcription. A deletion analysis suggested that a region between -100 and -307 of the rat Col2a1 gene was required for activity in differentiating chick limb bud mesenchymal cells; however, mutation of a conserved E2 box site in this region actually increased promoter activity. Supershift analysis demonstrated that deltaEF1, a known transcriptional repressor, bound to the E2 box in a sequence-dependent manner. Chick limb bud mesenchymal cells, which do not express type II collagen, expressed abundant deltaEF1 mRNA, but, following differentiation in micromass culture, deltaEF1 mRNA expression was lost. Primary embryonic chick sternal chondrocytes, which express abundant type II collagen, displayed minimal levels of deltaEF1 mRNA. The inhibition of Col2a1 transcription following treatment of chick sternal chondrocytes with growth factors was accompanied by increased deltaEF1 expression. Overexpression of deltaEF1 in differentiated chondrocytes resulted in decreased expression of a reporter construct containing a collagen II promoter/enhancer insert; however, this negative regulation was not dependent on the proximal E2 box. This is the first report of a specific transcription factor involved in the negative regulation of Col2a1.
Asunto(s)
Condrocitos/metabolismo , Colágeno/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Animales , Colágeno/genética , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Bcl-2 is widely expressed in a variety of cell types and is known to block apoptosis through a conserved pathway. However, recent reports have demonstrated that Bcl-2 regulates cell behavior independent of its control of apoptosis. Chondrocytes express a unique set of matrix proteins, including the proteoglycan aggrecan, and have been widely used to study the relationship between trophic factors and apoptosis. In this article, we report that Bcl-2 affects the morphology and regulates the expression of aggrecan in a rat chondrocyte cell line (IRC). Endogenous Bcl-2 and aggrecan mRNA were both down-regulated in response to serum withdrawal in parental IRC cells, while constitutive expression of Bcl-2 maintained aggrecan levels under conditions of serum withdrawal. In addition, expression of anti-sense Bcl-2 resulted in decreased aggrecan mRNA and produced a fibroblastic morphology compared with parental cells. The caspase inhibitor ZVAD-fmk effectively blocked full apoptosis of IRC cells in response to serum withdrawal or anti-sense Bcl-2 but did not prevent the down-regulation of aggrecan expression from either signal. These results suggest a novel role for Bcl-2 in regulating the differentiated phenotype of chondrocytes and the expression of a differentiation-specific gene independent of its control of apoptosis.
Asunto(s)
Apoptosis/genética , Caspasas/metabolismo , Condrocitos/citología , Condrocitos/metabolismo , Proteínas de la Matriz Extracelular , Genes bcl-2 , Proteoglicanos/genética , Agrecanos , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Inhibidores de Caspasas , Línea Celular , Condrocitos/efectos de los fármacos , Medio de Cultivo Libre de Suero , Inhibidores de Cisteína Proteinasa/farmacología , Regulación hacia Abajo , Activación Enzimática/genética , Regulación de la Expresión Génica/efectos de los fármacos , Lectinas Tipo C , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
The regulation of chondrocyte apoptosis in articular cartilage may underlay age-associated changes in cartilage and the development of osteoarthritis. Here we demonstrate the importance of Bcl-2 in regulating articular chondrocyte apoptosis in response to both serum withdrawal and retinoic acid treatment. Both stimuli induced apoptosis of primary human articular chondrocytes and a rat chondrocyte cell line as evidenced by the formation of DNA ladders. Apoptosis was accompanied by decreased expression of aggrecan, a chondrocyte specific matrix protein. The expression of Bcl-2 was downregulated by both agents based on Northern and Western analysis, while the level of Bax expression remained unchanged compared to control cells. The importance of Bcl-2 in regulating chondrocyte apoptosis was confirmed by creating cell lines overexpressing sense and antisense Bcl-2 mRNA. Multiple cell lines expressing antisense Bcl-2 displayed increased apoptosis even in the presence of 10% serum as compared to wild-type cells. In contrast, chondrocytes overexpressing Bcl-2 were resistant to apoptosis induced by both serum withdrawal and retinoic acid treatment. Finally, the expression of Bcl-2 did not block the decreased aggrecan expression in IRC cells treated with retinoic acid. We conclude that Bcl-2 plays an important role in the maintenance of articular chondrocyte survival and that retinoic acid inhibits aggrecan expression independent of the apoptotic process.
Asunto(s)
Apoptosis , Sangre , Cartílago Articular/citología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Tretinoina/farmacología , Anciano , Animales , Apoptosis/efectos de los fármacos , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Células Cultivadas , Expresión Génica/efectos de los fármacos , Humanos , Conejos , RatasRESUMEN
The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase (MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant to cartilage degradation. Both interleukin-1beta and tumor necrosis factor alpha induced a 4- to 9-fold increase in the level of MMP-9 expression in conditioned media, and a 17- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic fibroblast growth factor and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines. Transforming growth factor beta had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this process.
Asunto(s)
Condrocitos/citología , Condrocitos/efectos de los fármacos , Citocinas/farmacología , Metaloendopeptidasas/biosíntesis , Óxido Nítrico/farmacología , Animales , Línea Celular Transformada , Condrocitos/enzimología , Inducción Enzimática/efectos de los fármacos , Interleucina-1/farmacología , Ratas , Estaurosporina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
OBJECTIVE: The aggrecan proteoglycan is a major component of articular cartilage and supports the biomechanical function of this tissue. A variable number tandem repeat (VNTR) polymorphism has been discovered recently in a region of the human aggrecan gene that codes for the chondroitin sulfate attachment sites. We examined whether alleles of this polymorphism displayed a non-random association with bilateral hand or knee osteoarthritis (OA) in men from the Baltimore Longitudinal Study of Aging (BLSA). DESIGN: DNA was obtained from 93 Caucasian men, aged 60 and above, who had bilateral hand and standing knee radiographs read for changes of OA. The DNA was analyzed by polymerase chain reaction (PCR) and/or Southern blotting for the presence of the VNTR alleles. RESULTS: Bilateral hand OA and knee OA were present in 46 and 30% of the men respectively. The following distribution of alleles was observed: allele 33 (0.5%), 29 (2.2%), 28 (31.7%), 27 (43.0%), 26 (16.7%), 25 (3.2%), 22 (2.2%) and 19 (0.5%). This distribution was similar to that detected in a random population of individuals from a separate study. In multiple logistic regression analysis, adjusting for age and body mass index, the presence of allele 27 was associated with bilateral hand OA with an odds ratio (OR) = 3.23 (95% confidence intervals (CI): 1.24-8.41). No other alleles showed an association with bilateral hand OA and the association between allele 27 and bilateral knee OA was not statistically significant (OR = 1.14; 95% CI: 0.45-2.88). CONCLUSIONS: These data demonstrate the first association between a human aggrecan gene polymorphic allele and hand OA. This finding supports the concept that genetic factors may play a role in the development and/or progression of some forms of age-onset OA.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Proteínas de la Matriz Extracelular , Osteoartritis/genética , Polimorfismo Genético , Proteoglicanos/genética , Anciano , Agrecanos , Alelos , Índice de Masa Corporal , Mano , Humanos , Lectinas Tipo C , Estudios Longitudinales , Masculino , Repeticiones de Minisatélite/genética , Osteoartritis de la Rodilla/genética , Reacción en Cadena de la PolimerasaRESUMEN
Transforming growth factor beta and basic fibroblast growth factor are multipotential factors found in bone and cartilage that may be involved in both the proliferation and differentiation of chondrocytes. It was previously reported that TGF-beta plus FGF caused a modulation of chondrocyte phenotype that included the down-regulation of steady-state level of the collagen II transcript. In this report, the results of nuclear run-off data indicate that repression of transcript initiation from the collagen II gene is the primary mechanism involved in the growth factor induced inhibition. Transient transfection assays with CAT expression vectors containing portions of the collagen II gene show that the TGF-beta/FGF induced transrepression requires a region in the first intron previously reported to have transcriptional enhancer activity and to bind chondrocyte nuclear proteins. In addition, silencer elements in the promoter also appear to play a role. Protein data as well as transient transfection experiments indicate that the activation of protein kinase C is necessary for the growth factor-induced down-regulation of collagen II expression. These studies suggest that a cascade initiating with PKC activation is responsible for modifying transcription factors that interact with regulatory sequences in the collagen II gene. A detailed understanding of the factors involved in cartilage-specific gene regulation in chondrocytes would facilitate development of therapeutic protocols for the repair of degenerated cartilage in diseases such as osteoarthritis.
Asunto(s)
Colágeno/antagonistas & inhibidores , Colágeno/genética , Factores de Crecimiento de Fibroblastos/farmacología , Genes Reguladores , Proteína Quinasa C/fisiología , Factor de Crecimiento Transformador beta/farmacología , Animales , Cartílago/citología , Cartílago/metabolismo , Colágeno/metabolismo , Intrones , Mitosis , Regiones Promotoras Genéticas , Proteína Quinasa C/antagonistas & inhibidores , Estereoisomerismo , Transcripción GenéticaRESUMEN
The effect of dietary phosphorus on intestinal calcium uptake was examined in duodenal cells isolated from vitamin D-deficient chicks. Cells from chicks on a high phosphorus diet accumulated calcium at a rate 38% higher than cells from animals on a normal phosphorus diet. Diet high in calcium did not affect calcium absorption in duodenal cells. The dietary phosphorus effect on calcium absorption was specific. Uptake of alpha-methyl glucose was not altered. Increase in calcium absorption by a high phosphorus diet was not due to a change in cellular energy metabolism nor to the content of phosphorus in cells. Kinetically, a high phosphorus diet increased the Vmax of calcium uptake; the affinity for calcium was unaffected. The effectiveness of dietary phosphorus to enhance the intestinal calcium uptake could also be demonstrated in brush border membrane vesicles. The increase in calcium uptake was not due to an alteration in membrane binding capacity nor to calcium efflux from vesicles. To test the hypothesis that a high phosphorus diet may affect membrane transport by altering phospholipid metabolism in duodenal cells, we examined the phospholipid content in isolated brush border membranes. The content of phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine was not altered by the high phosphorus diet. These findings suggest that the vitamin D-independent and dietary phosphorus-dependent effect on intestinal calcium absorption was primarily due to a change in the calcium flux at the luminal side of the cells. However, the precise mechanism is still not clear.
Asunto(s)
Calcio/farmacocinética , Pollos/fisiología , Duodeno/metabolismo , Fósforo Dietético/farmacología , Deficiencia de Vitamina D/fisiopatología , Administración Oral , Animales , Transporte Biológico , Calcio/metabolismo , Calcio de la Dieta/farmacología , Membrana Celular/química , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Células Cultivadas , Relación Dosis-Respuesta a Droga , Duodeno/efectos de los fármacos , Duodeno/ultraestructura , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Metilglucósidos/farmacocinética , Microvellosidades/metabolismo , Microvellosidades/fisiología , Microvellosidades/ultraestructura , Modelos Biológicos , Concentración Osmolar , Fosfolípidos/análisis , Fosfolípidos/metabolismo , Deficiencia de Vitamina D/metabolismoAsunto(s)
Proteínas del Grupo de Alta Movilidad/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , ADN/genética , Humanos , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Ratas , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Cartilage is a unique tissue containing only one cell type, the chondrocyte, surrounded by an extensive extracellular matrix. One of the principal components of the cartilage matrix is type II collagen. The gene coding for type II collagen is relatively large and contains several distinct sequences that function to both up-regulate and down-regulate expression by interacting with chondrocyte transcription factors. Also, there appears to be regulation of collagen II expression by differential splicing of the collagen II mRNA to form different forms of the protein. Finally, the gene is a target for mutations that result in diseases of cartilage such as chondrodysplasias and some forms of osteoarthritis.
Asunto(s)
Enfermedades de los Cartílagos/genética , Colágeno/genética , Regulación de la Expresión Génica , Animales , Secuencia de Bases , Colágeno/metabolismo , ADN , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Collagen II is synthesized at high levels by differentiated chondrocytes. A 620-base pair DNA sequence in the first intron of the rat collagen II gene was previously determined to have chondrocyte-specific enhancer activity. Using mobility shift assays, we have defined a decamer sequence, 5'-CACAATGCAT-3', in the middle of the enhancer that binds a protein or protein complex expressed by chondrocytes but not by NIH3T3 cells or L2 rat fibroblasts. This protein was also induced during the differentiation of limb bud mesenchymal cells into chondrocytes. Mutational analyses, coupled with both in vitro binding studies and direct assays of enhancer activity following transfection into cells, demonstrated that this sequence is involved in the binding of the chondrocyte nuclear protein(s) and is necessary for the enhancer activity. This sequence has homology to the consensus binding sequence for helix-loop-helix transcription factors.
Asunto(s)
Cartílago/metabolismo , Núcleo Celular/metabolismo , Colágeno/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Células 3T3 , Animales , Secuencia de Bases , Cartílago/citología , Cartílago/enzimología , Cloranfenicol O-Acetiltransferasa/metabolismo , Ratones , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Ratas , TransfecciónRESUMEN
We have examined the effects of various analogs of vitamin D on the expression of the aggrecan proteoglycan by an immortalized rat chondrocyte cell line. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), produced a concentration-dependent reduction in the synthesis of aggrecan as monitored by histochemical staining of the matrix, incorporation of [35S]sulfate, and the level of aggrecan core protein. Other analogs of vitamin D were much less potent or had no activity whatsoever. The reduced expression of aggrecan was caused by a dramatic decrease in the steady-state level of the mRNA coding for the aggrecan core protein. A nuclear run-off analysis revealed that the rate of transcription of the aggrecan gene was not significantly altered by 1,25(OH)2D3 treatment, suggesting that the metabolite was acting through a post-transcriptional mechanism. Experiments using the transcriptional inhibitor actinomycin D also supported a nondirect effect of 1,25(OH)2D3 on the expression of the aggrecan gene. These results suggest that the vitamin D metabolite activates a new pattern of gene expression which results in a more rapid turnover of the aggrecan mRNA. This system should be useful for characterizing the regulation of chondrocyte gene expression by vitamin D.
Asunto(s)
Calcitriol/farmacología , Proteínas de la Matriz Extracelular , Proteoglicanos/metabolismo , Agrecanos , Animales , Northern Blotting , Cartílago/citología , Cartílago/metabolismo , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Dactinomicina/farmacología , Regulación hacia Abajo , Lectinas Tipo C , Proteoglicanos/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Transcripción GenéticaRESUMEN
Administration of ovine-prolactin (O-PRL) stimulated Ca2+ uptake in isolated duodenal cells prepared from vitamin D-deficient rats. The time course of this effect was biphasic: uptake activity reached a peak in 2.5 hrs followed by a decrease at 5 hrs to original levels. This stimulatory effect of O-PRL was observed in vitamin D-deficient male, but not in female rats. This stimulatory effect was observed in 16- and 26-week old, but not 9 week old, animals. Increase in Ca2+ uptake in duodenal cells was not due to a decrease in intracellular Ca2+ efflux. We measured serum Ca concentration in vitamin D-deficient female rats and found that serum Ca increased in D-deficient female rats between 16 and 52 weeks whereas a minimal increase was observed in D-deficient male rats. Although prolactin was shown to stimulate duodenal Ca2+ uptake, it appears that the source of the increase in levels of serum Ca in D-deficient female rats was not derived from an increase in Ca2+ uptake by prolactin in duodenum. The increase in serum calcium with time may explain why female D-deficient rats survive longer then male.
Asunto(s)
Calcio/metabolismo , Duodeno/efectos de los fármacos , Prolactina/farmacología , Deficiencia de Vitamina D/metabolismo , Envejecimiento/metabolismo , Animales , Calcio/sangre , Duodeno/metabolismo , Femenino , Masculino , Ratas , Factores SexualesRESUMEN
The in vivo and in vitro effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on calcium uptake by isolated chick duodenal cells were studied. In vivo, 1,25-(OH)2D3 given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion. In vitro, pre-incubation of 1,25-(OH)2D3 with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10(-15) M, was maximal at 10(-13) M, and was diminished at higher (10(-11) M) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D3 analogs was 1,25-(OH)2D3 = 1-(OH)D3 greater than 25-(OH)D3 greater than 1,24,25-(OH)3D3 greater than 24,25-(OH)2D3 greater than D3. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)2D3 was five orders of magnitude greater than D3. Kinetically, 1,25-(OH)2D3 increased the Vmax of calcium uptake; the affinity for calcium (Km = 0.54 mM) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)2D3, in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect on calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)2D3. These findings suggest that the in vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)2D3 induction of calcium transport and specific biochemical correlates.
Asunto(s)
Calcitriol/farmacología , Calcio/metabolismo , Duodeno/metabolismo , Deficiencia de Vitamina D/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Pollos , Duodeno/efectos de los fármacos , Técnicas In Vitro , Cinética , Masculino , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismoRESUMEN
The in vitro incubation of chick renal cells with parathyroid hormone (PTH) resulted in the inhibition of Na+-dependent phosphate uptake when the cells were isolated from 1,25-dihydroxycholecalciferol 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]-repleted chicks but not when the cells came from vitamin D-deficient animals. Na+-independent phosphate and Na+-dependent alpha-methylglucoside uptakes were not affected by PTH and the vitamin D status of the bird. The activation of chick renal cell adenylate cyclase by PTH was significantly blunted when the enzyme was from vitamin D-deficient animals relative to the activation of the enzyme from repleted cockerels. This alteration was due to a change in maximum velocity of the system rather than an effect on the affinity for hormone. The response of adenylate cyclase to other hormones, e.g., prostaglandin E2, and activators, e.g., 5' -guanylyl-imidodiphosphate and forskolin, was not affected by the vitamin D status of the animal. PTH had little effect in activating protein kinase in cells from vitamin D-deficient chicks. In cells from vitamin D-sufficient birds, PTH caused a fourfold increase in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Dibutyryl cAMP inhibited Na+-dependent phosphate uptake by cells from 1,25-(OH)2D3-repleted animals, but the cyclic nucleotide had no effect on phosphate uptake in cells from vitamin D-depleted chicks. This finding suggests that the loss of PTH receptor sites known to be concomitant with the secondary hyperparathyroidism associated with vitamin D deficiency is only a partial explanation for the failure of PTH to inhibit phosphate uptake in cells from vitamin D-deficient animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcitriol/farmacología , Riñón/fisiología , Hormona Paratiroidea/farmacología , Adenilil Ciclasas/metabolismo , Animales , Pollos , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/fisiopatología , Cinética , Masculino , Fosfatos/metabolismo , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
Renal cells isolated from vitamin D-deficient chicks had an increased Na+-dependent phosphate uptake when preincubated with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Phosphate uptake in the absence of Na+ and methyl alpha-glucoside uptake dependent on Na+ were not affected. Phosphate uptake was stimulated 15% by 0.010 pM 1,25-(OH)2D3. Maximal enhancement of 30% was obtained with 100 pM. The uptake when fully stimulated by preincubation in vitro approximated the uptake of cells isolated from chicks that were previously repleted with 1,25-(OH)2D3 in vivo. Cells from repleted chicks were not stimulated additionally when preincubated with 1,25-(OH)2D3 in vitro. The increase in phosphate uptake could be measured after a 1-hr preincubation period; full response required at least 2 hr. Phosphate uptake induced by 1,25-(OH)2D3 was blocked by cycloheximide and actinomycin D. Enhancement of phosphate uptake was relatively specific for the 1,25-(OH)2D3 analog of vitamin D3. The potency order was 1,25-(OH)2D3 greater than 25-(OH)D3 = 1-(OH)D3 greater than 24,25-(OH)2D3 greater than D3. Kinetically, 1,25-(OH)2D3 increased the Vmax of the phosphate uptake system; the affinity for phosphate was unaffected. 3H-Labeled 1,25-(OH)2D3 was taken up by the isolated renal cells. It was estimated that the stimulation of phosphate uptake might be initiated by very few molecules of 1,25-(OH)2D3 per cell. It is proposed that 1,25-(OH)2D3 contributes importantly to the mechanisms by which phosphate transport is regulated in the kidney.
Asunto(s)
Calcitriol/farmacología , Riñón/metabolismo , Fosfatos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Pollos , Cicloheximida/farmacología , Dactinomicina/farmacología , Cinética , Masculino , Potenciales de la Membrana , Sodio/fisiología , Deficiencia de Vitamina D/metabolismoRESUMEN
Renal cells from Vitamin D-deficient and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-repleted chicks were isolated by a collagenase-hyaluronidase procedure. Exclusion of trypan blue and respiratory measurements indicate that the cells were functionally intact and metabolically active. The uptakes of phosphate and alpha-methylglucoside were stimulated markedly by Na+ in the extracellular medium. Phosphate uptake in the presence of Na+ was saturable with respect to phosphate concentration; half-maximal activity was obtained with approximately 0.2 mM. Three hours after 1,25-(OH)2D3 was injected into vitamin D-deficient chicks the Na+-dependent phosphate uptake by the isolated cells had increased about 40%, i.e., 2.00 compared with 1.44 nmol.min-1.mg protein-1. Phosphate uptake in the presence of K+ in the extracellular medium and alpha-methylglucoside uptake in the presence or absence of Na+ were unchanged. In a secondary response found 17 h after 1,25-(OH)2D3 injection, Na+-dependent phosphate uptake decreased. Serum concentrations of phosphorus and calcium were not measurably changed in the 3-h repleted bird, but both levels were increased 17 h after treatment. Administration of phosphate into vitamin D-deficient chicks, so that the serum concentration of phosphorus was raised to that of the 17-h 1,25-(OH)2D3 repleted animal, effected a comparable decrease in phosphate uptake. Serum calcium levels were not altered by this treatment. The actions of parathyroid hormone in stimulating adenylate cyclase and in inhibiting phosphate uptake were notably blunted in the vitamin D-deficient chick. Sensitivity to parathyroid hormone was not restored until several days after 1,25-(OH)2D3 repletion. These findings suggest that the initial response to 1,25-(OH)2D3, to increase renal phosphate uptake, and the secondary response, to decrease phosphate uptake, were by parathyroid hormone-independent processes. The results also indicate that the isolated renal cell represents an excellent model for studying the mechanism by which 1,25-(OH)2D3 regulates phosphate transport in the kidney.
Asunto(s)
Animales Recién Nacidos/fisiología , Calcitriol/farmacología , Riñón/citología , Fosfatos/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Calcio/sangre , Pollos , Riñón/metabolismo , Metilglucósidos/metabolismo , Hormona Paratiroidea/fisiología , Fósforo/sangre , Vitamina D/fisiología , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/metabolismoAsunto(s)
Membrana Celular/enzimología , AMP Cíclico/farmacología , Isoenzimas/metabolismo , Corteza Renal/enzimología , Microvellosidades/enzimología , Proteínas Quinasas/metabolismo , Animales , Activación Enzimática , Isoenzimas/aislamiento & purificación , Cinética , Protamina Quinasa/aislamiento & purificación , Protamina Quinasa/metabolismo , Proteínas Quinasas/aislamiento & purificación , Conejos , Especificidad por SustratoAsunto(s)
AMP Cíclico/farmacología , Túbulos Renales Proximales/enzimología , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Membrana Celular/enzimología , Histonas/metabolismo , Cinética , Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/farmacología , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Protaminas/metabolismo , Inhibidores de Proteínas Quinasas , Conejos , Relación Estructura-ActividadRESUMEN
The binding of cyclic AMP to the proximal tubule luminal (brush border) membrane isolated from the rabbit renal cortex was studied. The rate of binding was dependent on temperature; at 37 degrees equilibrium was attained in 45 min, whereas at 0 degrees 120 min was required. The final levels of binding were identical. The binding of 3H-cyclic AMP was reversed by dilution or addition of unlabeled cyclic nucleotide. Debinding was markedly temperature sensitive. Binding was only partially saturable with respect to cyclic AMP concentration, apparently with more than one binding site. The cyclic AMP bound to the membrane was recovered unchanged. When bound to the membrane cyclic AMP was resistant to hydrolysis by endogenous membrane or exogenously added phosphodiesterase. The binding to the membranes was relatively specific for cyclic AMP, although other cyclic purine nucleotides inhibited, cyclic IMP greater than dibutyryl cyclic AMP greater than cyclic GMP. The renal membranes did bind cyclic GMP, but this binding was relatively non-specific. Hormones and drugs, that mediate cyclic AMP generation or renal function, as well as other compounds common to the proximal tubule were without significant effect on cyclic AMP binding. Binding was inhibited by sulfhydryl reacting agents and this inhibition could be blocked and partially reversed by mercaptoethanol.