RESUMEN
Interactions between dietary calcium (Ca) and lead (Pb) which influence serum levels of the vitamin D hormone, 1,25-dihydroxyvitamin D (1,25(OH)2D), intestinal Ca and Pb absorption, and body Pb retention were investigated in chicks. In a 5 x 5 (levels of Ca and Pb) design, response surface modeling was used to describe and compare the various interactions. Lead ingestion and Ca deficiency alone or in combination generally increased serum 1,25(OH)2D levels over most of the range of dietary Ca and Pb. However, in severe Ca deficiency, Pb ingestion resulted in marked decreases in hormone concentration. Overall similarities in response profiles for 1,25(OH)2D, intestinal Ca absorption and calbindin-D suggest that major interactions between Pb and Ca are mediated via changes in circulating 1,25(OH)2D concentration, rather than direct effects on the intestine. The response profiles for Ca and Pb absorption differed, in part, suggesting that intestinal transport of the two cations may not be identical. Kidney and bone Pb content also differed in response to varying Ca and Pb levels, providing evidence for additional tissue-specific interactions not related to 1,25(OH)2D. The present study provides a comprehensive basis on which to interpret the results of previous clinical and experimental results.
Asunto(s)
Calcio de la Dieta/metabolismo , Dihidroxicolecalciferoles/sangre , Plomo/metabolismo , Animales , Calbindinas , Calcio/deficiencia , Pollos , Relación Dosis-Respuesta a Droga , Absorción Intestinal/efectos de los fármacos , Marcaje Isotópico , Riñón/efectos de los fármacos , Riñón/metabolismo , Plomo/toxicidad , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
A combination of ion microscopic and conventional radionuclide techniques was employed to investigate the temporal-spatial dynamics of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated intestinal calcium (Ca) absorption. At varying times following the administration of a single intravenous dose of 1,25(OH)2D3 to vitamin D-deficient chicks, transepithelial transport and tissue retention of Ca were quantitated in vivo, using the ligated duodenal loop technique and 47Ca as the tracer. The localization of Ca in the intestinal tissue during absorption was monitored by ion microscopy, using the stable Ca isotope, 44Ca, as the absorbed species. There was little transepithelial absorption of Ca in the vitamin D-deficient animals despite a substantial tissue accumulation of luminally derived Ca, the latter localizing predominantly in the brush border region of the enterocyte, as shown by the 44Ca-ion microscopic images. The early (30 min-1 h) response to 1,25(OH)2D3 was an increased tissue uptake of luminal 47Ca, which also primarily associated with the brush border region, again as shown by ion microscopy. At 2-4 h after the 1,25(OH)2D3 dose, there was a progressive redistribution of Ca from the brush border region throughout the cytoplasm and into the lamina propria. At 8-16 h, 47Ca absorption was maximal and 44Ca was sparsely distributed in the intestinal tissue. 47Ca absorption gradually declined and reached pre-dose levels by 72 h. At this time, tissue 44Ca was again largely limited to the brush border region. These results provide support for the multiple actions of 1,25(OH)2D3 on the intestinal Ca absorption process. The ion microscopic images provided unique information on the specific time-dependent changes in the tissue localization of Ca during the process of its intestinal absorption as affected by 1,25(OH)2D3.
Asunto(s)
Calcitriol/farmacología , Calcio/metabolismo , Duodeno/metabolismo , Absorción Intestinal , Espectrometría de Masa de Ion Secundario , Animales , Radioisótopos de Calcio , Pollos , Citoplasma/metabolismo , Procesamiento de Imagen Asistido por Computador , Mucosa Intestinal/metabolismo , Masculino , Microvellosidades/metabolismo , Deficiencia de Vitamina D/metabolismoRESUMEN
The intestinal absorption of Ca2+ occurs by both a saturable, transcellular process and a nonsaturable, paracellular path. The transcellular path is a multistep process, comprised of the transfer of luminal Ca2+ into the enterocyte, the translocation of Ca2+ from point of entry (the microvillus border or membrane) to the basolateral membrane, and the active extrusion from the cell into the circulatory system. Each step in the transcellular movement of Ca2+ has a vitamin D-dependent component. The paracellular path also appears to be affected by vitamin D status. This review emphasizes some aspects of the Ca2+ absorptive process that require resolution and/or further experimental support. The following are discussed: evidence for participation in the active transport of Ca2+ by all segments of the small intestine; a hypothetical model for the feedback control of entry of luminal Ca2+; the current views on vitamin D-dependent movement of Ca2+ through the cytosolic compartment of the enterocyte; the stimulated synthesis of the plasma membrane Ca2+ pump and its gene expression by vitamin D; and the vitamin D-dependency of the paracellular transfer of Ca2+ with a comment on the physiological significance of the rapid response of the Ca2+ absorptive system in vitamin D-replete animals to 1,25-dihydroxyvitamin D.
Asunto(s)
Calcio/metabolismo , Absorción Intestinal/fisiología , Vitamina D/metabolismo , Animales , Transporte Biológico Activo/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Humanos , Modelos Biológicos , Sodio/metabolismoRESUMEN
The combined effects of dietary calcium level and lead level on several indices of vitamin D endocrine function were examined in young, growing chicks. Day-old animals fed a nutritionally adequate diet for 2 wk were fed diets either adequate (1.2%) or low (0.1%) in calcium, and containing 0, 0.2 or 0.8% lead for an additional 1 or 2 wk. In the calcium-adequate group, lead ingestion significantly elevated intestinal calbindin-D28k protein and mRNA levels as well as plasma 1,25-dihydroxyvitamin D concentration compared with the control animals fed a lead-free diet. The effect was apparent after 1 wk of treatment and continued through wk 2. In the calcium-deficient group, the early (1 wk) increases in plasma 1,25-dihydroxyvitamin D and calbindin-D28k protein and mRNA were significantly reversed by lead ingestion over the 2-wk trial period in a dose-dependent fashion. In these circumstances, vitamin D endocrine function is severely compromised. Therefore, lead ingestion may result in either enhanced or diminished circulating 1,25-dihydroxyvitamin D concentrations and ensuing intestinal responses, depending of dietary calcium level and the duration of lead intake. These results provide possible explanations for several apparently conflicting sets of observations regarding lead-calcium interactions.
Asunto(s)
Calcitriol/sangre , Calcio de la Dieta/farmacología , Calcio/deficiencia , Plomo/toxicidad , Animales , Calbindinas , Calcio de la Dieta/administración & dosificación , Pollos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Riñón/metabolismo , Plomo/farmacocinética , Masculino , ARN Mensajero/análisis , ARN Mensajero/genética , Proteína G de Unión al Calcio S100/sangre , Proteína G de Unión al Calcio S100/genética , Tibia/metabolismo , Factores de TiempoRESUMEN
A monoclonal antibody produced against the human erythrocyte plasma membrane calcium pump (PMCA) was shown to react immunohistochemically with an epitope of the PMCA in avian and mammalian cerebellum. Western blot analysis of purified synaptosomes and homogenates from avian cerebellum revealed major immunoreactive proteins with molecular masses (130 kDa and 138 kDa) similar to those of purified erythrocyte PMCA. Dual-imaging confocal immunofluorescence microscopy of avian cerebellum showed that the PMCA antibody stained the periphery of the soma whereas calbindin-D28k was located in the cytosol. PMCA heavily stained the more distal dendrites of the Purkinje cells and, within the resolution of the fluorescence procedure, colocalized with calbindin-D28k. By using alkaline phosphatase-conjugated second antibody, PMCA was again localized to the peripheral soma, to a segmental pattern in dendrites, and to presumed spiny elements. The soma periphery and dendrites of Purkinje cells of the rat cerebellum were also prominently stained with anti-PMCA antibody and compared to parvalbumin localization. Dendritic depolarization and dendritic spiking behavior are significant Ca(2+)-dependent events of Purkinje cells. The rapid decline of intracellular free Ca2+ after the rapid rise time of Ca2+ transients is considered to be due to sequestration by Ca2+ buffers, uptake by intracellular stores, and Ca2+ extrusion mechanisms, the latter a function of PMCA now shown immunohistochemically to be a prominent feature of Purkinje cell dendrites.
Asunto(s)
ATPasas Transportadoras de Calcio/análisis , Cerebelo/citología , Parvalbúminas/análisis , Células de Purkinje/citología , Proteína G de Unión al Calcio S100/análisis , Fosfatasa Alcalina , Animales , Anticuerpos , Western Blotting , Calbindina 1 , Calbindinas , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Pollos , Inmunohistoquímica/métodos , Microscopía Fluorescente , Células de Purkinje/enzimología , Ratas , Sinaptosomas/química , Sinaptosomas/ultraestructuraRESUMEN
1. We have examined the ability of the Ca(2+)-binding proteins (CABP) calbindin D28k and paravalbumin to modulate increases in the intracellular free Ca2+ concentration ([Ca2+]i), produced by brief depolarizations, in rat dorsal root ganglion (DRG) neurones. 2. In order to obtain good voltage control, we replated DRG neurones prior to performing these experiments. Immunocytochemical staining of these cells revealed that approximately 10% stained for CABPs. 3. Using fluorescently labelled parvalbumin, we demonstrated that in the whole-cell voltage clamp mode the protein freely entered the cell soma with a mean half-life t0.5 of 6 min 22 s +/- 54 s. 4. Analysis of the effects of calbindin D28k (370 microM) and parvalbumin (1 mM) on Ca2+ currents in the whole-cell voltage clamp mode, revealed that neither protein changed the rate of inactivation of the Ca2+ current or its rate of run-down. 5. Introducing either calbindin D28k (370 microM) or parvalbumin (1 mM) into the cell soma did not significantly alter the basal [Ca2+]i when compared to control cells. 6. Compared to control cells, both CABPs significantly reduced the peak [Ca2+]i obtained for a Ca2+ influx of an equivalent charge density, whereas lysozyme (1 mM), a protein with low affinity for Ca2+, failed to do so. 7. Calbindin D28k caused an 8-fold decrease in the rate of rise in [Ca2+]i and altered the kinetics of decay of [Ca2+]i to a single slow component. Parvalbumin also slowed the rate of rise in [Ca2+]i. Parvalbumin selectively increased a fast component in the decay of the Ca2+ signal. 8. These data demonstrate that both calbindin D28k and paravalbumin effectively buffer Ca2+ in a cellular environment and may therefore regulate Ca(2+)-dependent aspects of neuronal function.
Asunto(s)
Calcio/metabolismo , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Parvalbúminas/farmacología , Proteína G de Unión al Calcio S100/farmacología , Animales , Tampones (Química) , Calbindina 1 , Calbindinas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Técnicas In Vitro , Líquido Intracelular/metabolismo , Cinética , Parvalbúminas/metabolismo , Ratas , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
Provision of Ca2+ for egg shell calcification in the avian uterus [egg shell gland (ESG)] derives mostly from vitamin D-dependent intestinal Ca2+ absorption from the diet. Ca2+ absorption is strongly linked to the intestinal vitamin D-dependent calbindin D28K (D28K) concentration. The laying hen ESG also contains D28K, and again, Ca2+ transport into the shell appeared to be linked to the ESG D28K concentration. However, evidence is now presented that ESG D28K synthesis may be estradiol (E2) dependent and vitamin D independent under certain conditions. One-day-old female chicks fed a vitamin D-free diet for as long as 6 weeks and then repeatedly injected im with E2 for up to 3 more weeks developed frank rickets, but possessed precociously matured reproductive tracts. While the tiny presumptive ESGs of nonestrogenized vitamin D-depleted chicks were devoid of D28K, the highly developed ESG, including the isthmus, of estrogenized chicks contained D28K. The ESGs of nonestrogenized, vitamin D-replete chicks also exhibited no development or detectable D28K. Regardless of whether vitamin D depleted or replete, estrogenized chick ESG contained similar D28K and D28K mRNA concentrations. Immunohistochemical techniques showed that the endometrial cellular localization of both D28K and Ca(2+)-ATPase (Ca2+ pump) in estrogenized chicks was similar to that in mature laying hens. There was no trace of D28K, nor was there any stimulation of Ca2+ absorption, in duodenum of vitamin D-free, immature chicks regardless of E2 treatment. As expected, both D28K and D28K mRNA were present in vitamin D-replete chick duodenum. We conclude that in E2-treated chicks, ESG D28K gene expression may be vitamin D independent and E2 dependent. This is the first clear demonstration of hormone-dependent tissue-specific D28K gene expression in the chick.
Asunto(s)
Cáscara de Huevo , Estradiol/farmacología , Glándulas Exocrinas/metabolismo , Homeostasis , Proteína G de Unión al Calcio S100/biosíntesis , Deficiencia de Vitamina D/metabolismo , Animales , Calbindinas , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Pollos , Dieta , Duodeno/efectos de los fármacos , Duodeno/metabolismo , Glándulas Exocrinas/efectos de los fármacos , Femenino , Inmunohistoquímica , Absorción Intestinal/efectos de los fármacos , Oviductos/efectos de los fármacos , Oviductos/crecimiento & desarrollo , Oviductos/metabolismoRESUMEN
We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.
Asunto(s)
Calcio de la Dieta/administración & dosificación , Fósforo Dietético/administración & dosificación , ARN Mensajero/metabolismo , Vitamina D/administración & dosificación , Animales , Northern Blotting , Calbindinas , Calcitriol , Pollos , Duodeno/química , ARN/aislamiento & purificación , Receptores de Calcitriol , Receptores de Esteroides/genética , Proteína G de Unión al Calcio S100/genéticaRESUMEN
Any consideration of calcium entry into the cell must recognize that it is the initial event in a complex sequentially integrated process. Any step in this process, when viewed individually and in isolation, may appear to be of overwhelming importance, but this need not be an accurate reflection of its relative role in the overall process. Calcium entry may be of substantial importance in terms of calcium transport rate or capacity under certain circumstances, but it is most likely not the sole limiting step in calcium absorption. The Symposium papers that follow stress the importance of additional factors and events that have been implicated in intestinal calcium absorption.
Asunto(s)
Calcio/metabolismo , Absorción Intestinal , Animales , Intestinos/ultraestructura , Microvellosidades/metabolismo , Vitamina D/fisiología , Deficiencia de Vitamina D/metabolismoRESUMEN
The basolateral membrane of the enterocyte was previously shown to contain an adenosine triphosphate-dependent calcium pump. Using immunological procedures, the localization of the Ca2+ pump in chick intestine, and the effect of dietary variables on the concentration of the pump, were studied. A monoclonal antibody produced against the human erythrocyte calcium pump was shown to cross-react with a chick intestinal Ca2+ pump epitope. The most intense staining of intestinal tissue, as determined immunohistochemically, occurred at the basolateral membrane of the duodenum, jejunum, ileum, and colon, with minor staining elsewhere. By the Western blotting procedure, vitamin D repletion of vitamin D-deficient chicks was shown to significantly increase the concentration of the Ca2+ pump epitope of duodenal, jejunal, and ileal mucosa by a factor of 2-3. Chicks were also fed diets deficient in calcium or phosphorus, a situation known to result in the stimulation of the synthesis of calbindin-D28k and an enhancement of the efficiency of Ca2+ absorption. Adaptation of the chicks to these deficient diets was verified by an increase in intestinal levels of calbindin-D28k, and is now shown to increase the Ca2+ pump epitope. From these immunological studies, it seems apparent that dietary variables that enhance intestinal Ca2+ absorption also increase the amount of the intestinal basolateral Ca2+ pump.
Asunto(s)
ATPasas Transportadoras de Calcio/efectos de los fármacos , Mucosa Intestinal/metabolismo , Minerales/farmacología , Animales , Western Blotting , Calbindina 1 , Calbindinas , Calcio/sangre , Calcio/deficiencia , Pollos , Colecalciferol/farmacología , Densitometría , Dieta/efectos adversos , Duodeno/metabolismo , Técnica del Anticuerpo Fluorescente , Íleon/metabolismo , Yeyuno/metabolismo , Fósforo/sangre , Fósforo/deficiencia , Proteína G de Unión al Calcio S100/biosíntesisRESUMEN
The intestinal absorption of calcium has been proposed to occur by the transcellular transfer of Ca2+ through the enterocyte proper and between the cells of the intestinal epithelium, i.e., the paracellular path. Attention in this report is given to the transcellular models of Ca2+ absorption and, more specifically, the Ca2+ extrusion events occurring at the basolateral membrane. These extrusion processes include the operation of an ATP-dependent Ca2+ pump and a Na+/Ca2+ exchanger, as well as exocytosis as the terminal event in a proposed vesicular transport mechanism. Evidence for the presence of an ATP-dependent Ca2+ pump at the basolateral membrane is documented and illustrated with biochemical and immunological data from studies on the avian intestinal basolateral membrane. As shown immunohistochemically, the Ca2+ pump was primarily localized on the enterocyte basolateral membrane. The ATP-dependency and vitamin D enhancement of Ca2+ uptake by isolated basolateral membrane vesicles are shown. Western blot analysis of intestinal mucosa, by using a monoclonal antibody produced against the erythrocyte Ca2+ pump, indicated that the number of pump units is increased by 1,25-dihydroxycholecalciferol. The possible involvement of calbindin-D28K as a direct stimulator of the Ca2+ pump is discussed, and the quantitative relationship between Ca2+ transport rates and Ca2+ pumping activity has been estimated. Information related to the basolateral membrane Na+/Ca2+ exchanger and the vesicular transport model of Ca2+ absorption is also briefly reviewed.
Asunto(s)
Calcio/metabolismo , Absorción Intestinal , Mucosa Intestinal/metabolismo , Animales , Transporte Biológico Activo , ATPasas Transportadoras de Calcio/fisiología , Membrana Celular/metabolismo , Humanos , Intestinos/ultraestructuraRESUMEN
Computer simulations of transcellular Ca2+ transport in enterocytes were carried out using the simulation program SPICE. The program incorporated a negative-feedback entry of Ca2+ at the brush-border membrane that was characterized by an inhibitor constant of 0.5 microM cytosolic Ca2+ concentration ([Ca2+]). The basolateral Ca(2+)-ATPase was simulated by a four-step mechanism that resulted in Michaelis-Menten kinetics with a Michaelis constant of 0.24 microM [Ca2+]. The cytosolic diffusion of Ca2+ was simulated by dividing the cytosol into 10 slabs of equal width. Ca2+ binding to calbindin-D9K was simulated in each slab, and diffusion of free Ca2+, free calbindin, and Ca(2+)-laden calbindin was simulated between each slab. The cytosolic [Ca2+] of the simulated cells was regulated within the physiological range. Calbindin-D9K reduced the cytosolic [Ca2+] gradient, increased Ca2+ entry into the cell by removing the negative-feedback inhibition of Ca2+ entry, increased cytosolic Ca2+ flow, and increased the efflux of Ca2+ across the basolateral membrane by increasing the free [Ca2+] immediately adjacent to the pump. The enhancement of transcellular Ca2+ transport was nearly linearly dependent on calbindin-D9K concentration. The values of the dissociation constant (Kd) for calbindin-D9K were previously obtained experimentally in the presence and absence of KCl. Calbindin with the Kd obtained in the presence of KCl enhanced the simulated Ca2+ transport more than with the Kd obtained in the absence of KCl. This result suggests that the physiological Kd of calbindin is optimal for the enhancement of transcellular Ca2+ transport. The simulated Ca2+ flow was less than that predicted from the "near-equilibrium" analytic solution of the reaction-diffusion problem.
Asunto(s)
Calcio/farmacocinética , Mucosa Intestinal/metabolismo , Proteína G de Unión al Calcio S100/fisiología , Absorción , Animales , Transporte Biológico , Calbindinas , Simulación por Computador , Difusión , Humanos , Microvellosidades/metabolismo , Modelos BiológicosRESUMEN
The biological interactions between lead and calcium are complex and not well-understood, but can be demonstrated in virtually every tissue. High affinity lead-binding to intracellular calcium receptor and transport proteins as well as the involvement of lead in calcium-activated and calcium-regulating processes may provide a molecular basis for the broad spectrum cellular and systemic effects. The intestinal absorptive cells are responsible for transporting the entire body complement of calcium and most of the body lead burden. They represent, therefore, the first critical step in maintaining systemic and cellular calcium homeostasis, as well as the first line of defense against lead poisoning. Any interactions which occur at this level, either to enhance the body burden of lead or to diminish the transport of calcium, may have serious health-related repercussions. This article reviews research concerning those interactions involving calcium, lead and the vitamin D endocrine system which ultimately influence intestinal function, calcium homeostasis and body lead retention.
Asunto(s)
Calcio/farmacocinética , Absorción Intestinal/fisiología , Plomo/farmacocinética , Animales , Proteínas de Unión al Calcio/fisiología , Interacciones Farmacológicas , Glándulas Endocrinas/fisiología , Humanos , Plomo/toxicidad , Vitamina D/fisiologíaRESUMEN
The combined effects of dietary calcium (Ca) and lead (Pb) status on intestinal Ca and Pb absorption and related parameters were investigated in young growing chicks. Dietary Pb intake resulted in two remarkable, apparently independent and essentially opposite effects on intestinal Ca and Pb absorption, depending on dietary Ca and Pb levels and duration of treatment. The initial response (1 week) to Ca deficiency was stimulated Ca absorption and calbindin-D level, regardless of dietary Pb intake. The later response (2 weeks) was a reversal, by Pb, of the early phase stimulation. Intestinal Pb absorption was similarly enhanced by Ca deficiency initially, and this response was also inhibited by prolonged dietary Pb intake. Ingestion of Pb by chicks fed adequate Ca resulted in generally elevated intestinal Ca absorption and calbindin-D levels after both 1 and 2 weeks. Intestinal Pb absorption was also increased in the adequate Ca situation, but only after 2 weeks at the lower levels of dietary Pb. The results underscore the complicated nature of Pb-Ca interactions and demonstrate the importance of thorough characterization of the animal model system.
Asunto(s)
Calcio de la Dieta/farmacocinética , Absorción Intestinal/efectos de los fármacos , Plomo/farmacocinética , Administración Oral , Animales , Calbindinas , Calcio de la Dieta/sangre , Calcio de la Dieta/farmacología , Pollos , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Intestinos/fisiología , Plomo/sangre , Plomo/farmacología , Fósforo/sangre , Proteína G de Unión al Calcio S100/sangre , Factores de TiempoRESUMEN
The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces expression of the gene encoding calbindin-D28K, a protein involved in intestinal Ca2+ transport. Glucocorticoids stimulate intestinal development and function, and presumed interaction with 1,25-(OH)2D3 has been intensively studied. Most studies involved administration of high doses of glucocorticoids in vivo, which inhibits intestinal Ca2+ transport by an unknown mechanism. However, it is now known from studies of the duodenal organ culture model that low concentrations of glucocorticoids enhance 1,25-(OH)2D3-dependent calbindin-D28K biosynthesis and Ca2+ transport. High concentrations mimic the action of administered glucocorticoids in vivo, suggesting that a distinct pharmacological or toxic mechanism causes inhibition of Ca2+ absorption. This report further shows that dexamethasone (DEX) rapidly enhanced calbindin-D28K gene expression, that is de novo calbindin-D28K mRNA biosynthesis. DEX also markedly reduced the actions of RNA and protein synthesis inhibitors on calbindin-D28K gene expression, although no evidence for an action of DEX or 1,25-(OH)2D3 at the translational level was obtained. Ca2+ transport activity was highly correlated with calbindin-D28K concentration regardless of treatment. Washout permitted complete reversal of inhibition, verifying the specificity of inhibitor activity. These results appear to show positive contranscriptional regulation of calbindin-D28K gene expression by 1,25-(OH)2D3 and glucocorticoids. The use of this model should continue to clarify the interactive roles of nuclear-acting hormones on the Ca2+ absorptive mechanism and on complex physiological and pathological processes in general.
Asunto(s)
Calcitriol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Intestinos/fisiología , Proteína G de Unión al Calcio S100/genética , Transcripción Genética , Animales , Calbindinas , Calcio/farmacocinética , Embrión de Pollo , Cicloheximida/farmacología , Dactinomicina/farmacología , Dexametasona/farmacología , Interacciones Farmacológicas , ARN Mensajero/metabolismo , Proteína G de Unión al Calcio S100/farmacocinéticaRESUMEN
The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca2+ pump was not detectable in the infundibulum or the magnum. Calbindin-D28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca2+ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D28k differed, co-localizing with the Ca2+ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca2+ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.
Asunto(s)
ATPasas Transportadoras de Calcio , Oviductos/química , Proteína G de Unión al Calcio S100/análisis , Animales , Anticuerpos Monoclonales , Western Blotting , Calbindina 1 , Calbindinas , Calcio/metabolismo , Pollos , Femenino , InmunohistoquímicaRESUMEN
A solution hybridization assay employing specific synthetic oligodeoxynucleotide probes was developed to study the regulation of intestinal calbindin-D28K mRNA. The technique is rapid and quantitative and eliminates the need for sample transfer, blotting, autoradiography, and densitometry. Following validation of the assay, chick intestinal calbindin-D and calbindin-D mRNA levels were compared under conditions known to stimulate intestinal calcium (Ca) transport. Both the protein and its mRNA were undetectable in 25-day-old vitamin D-deficient chicks. Following acute administration of vitamin D3, calbindin-D mRNA levels increased somewhat more rapidly than calbindin-D protein, but overall, the correlation was excellent. Chicks fed a nutritionally adequate diet for 14 days and then changed to a low Ca (0.1%) diet responded with increased calbindin-D and calbindin-D mRNA levels. Again the correlation was excellent over the ensuing 14-day experimental period. The combined effects of vitamin D repletion and dietary Ca status were also investigated with respect to calbindin D and its mRNA. Fourteen-day-old vitamin D-deficient chicks were changed to diets containing vitamin D and either adequate (1.2%) or low (0.3%) in Ca. The intestinal responses were measured at intervals up to 14 days. In the normal Ca situation, there were initial increases in both calbindin mRNA levels, which peaked at between 4 and 7 days, and calbindin protein levels, which peaked at 7 days. Both values subsequently declined during the remaining 7 days of the experimental period. In the low Ca situation, there were similar increases in calbindin mRNA and protein levels through 4 and 7 days respectively, but these levels remained high for the remainder of the 14-day experimental period. The present results demonstrate that intestinal tissue levels of calbindin D and its mRNA respond similarly to vitamin D repletion and dietary Ca restriction as well as the combination of these stimuli. There is no evidence to support significant post-transcriptional regulation of calbindin-D by Ca.
Asunto(s)
Duodeno/metabolismo , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Proteína G de Unión al Calcio S100/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calbindinas , Pollos , Sondas de ADN , Cinética , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genéticaRESUMEN
The intestinal absorption of calcium includes at least three definable steps; transfer across the microvillar membrane, movement through the cytosolic compartment, and energy-dependent extrusion into the lamina propria, Tracing the movement of calcium through the epithelium has been hampered by lack of suitable techniques and, in this study, advantage was taken of ion microscopy in conjunction with cryosectioning and use of the stable isotope 44Ca to visualize calcium in transit during the absorptive process. The effect of vitamin D, required for optimal calcium absorption, was investigated. Twenty millimolar 44Ca was injected into the duodenal lumen in situ of vitamin D-deficient and vitamin D-replete chickens. At 2.5, 5.0, and 20.0 min after injection, duodenal tissue was obtained and processed for ion microscopic imaging. At 2.5 min. 44Ca was seen to be concentrated in the region subjacent to the microvillar membrane in tissue from both groups. At 5.0 and 20.0 min, a similar pattern of localization was evident in D-deficient tissues. In D-replete tissues, the distribution of 44Ca became more homogenous, indicating that vitamin D increased the rate of transfer of Ca2+ from the apical to the basolateral membrane, a function previously ascribed to the vitamin D-induced calcium-binding protein (28-kDa calbindin-D). Quantitative aspects of the calcium absorptive process were determined in parallel experiments with the radionuclide 47Ca. Complementary information on the localization of the naturally occurring isotopes of calcium (40Ca) and potassium (39K) is also described.
Asunto(s)
Calcio/metabolismo , Duodeno/metabolismo , Absorción Intestinal , Deficiencia de Vitamina D/metabolismo , Animales , Transporte Biológico , Isótopos de Calcio , Radioisótopos de Calcio , Pollos , Duodeno/citología , Cinética , Masculino , Músculo Liso/citología , Músculo Liso/metabolismo , Valores de ReferenciaRESUMEN
This study was designed to investigate, in some detail, the relative effects of the hormonal form of vitamin D (1,25-dihydroxycholecalciferol) on duodenal Pb and Ca absorption as a function of dietary Pb level. When cholecalciferol-deficient chicks were chronically repleted with physiologic levels of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), as the sole source of the vitamin, 203Pb and 47Ca absorption were enhanced over 4- and 8-fold, respectively. Ingestion of Pb during the repletion period had no significant effect on the intestinal Ca absorption response to 1,25-(OH)2D3 even at a very high dietary Pb level. The efficiency of intestinal 203Pb absorption was, however, significantly diminished by dietary Pb, in an apparent dose-dependent fashion. The results indicate that the extent to which systemic Ca homeostatic mechanisms influence intestinal Pb absorption is dependent, in large part, on Pb status.
Asunto(s)
Calcitriol/administración & dosificación , Calcio/metabolismo , Duodeno/metabolismo , Absorción Intestinal , Plomo/metabolismo , Fosfatasa Alcalina/biosíntesis , Animales , Peso Corporal , Calbindinas , Calcio/sangre , Pollos , Colecalciferol/deficiencia , Dieta , Relación Dosis-Respuesta a Droga , Plomo/administración & dosificación , Masculino , Fósforo/sangre , Proteína G de Unión al Calcio S100/biosíntesis , Deficiencia de Vitamina D/metabolismoRESUMEN
Synthetic oligonucleotide probes complementary to chick calbindin-28 kDa-mRNA were used to study the latter's regulation and relationship to calbindin in the chick. The effects of vitamin D3 sources and dietary alteration on the genomic expression were characterized by Northern blot and solution hybridization. Intestinal calbindin and its mRNA were almost absent in vitamin D-deficient chicks and were not affected by dietary alteration. Renal calbindin and its mRNA were lower in the vitamin D-deficient than in vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-fed chicks. In the same animal, renal calbindin mRNA and calbindin were higher than intestinal. In vitamin D3-fed chicks, dietary calcium (Ca) or phosphorus (P) restriction induced, and high dietary Ca inhibited, intestinal calbindin and its mRNA synthesis. In the same chicks, dietary P restriction induced renal calbindin mRNA and calbindin synthesis. In 1,25-(OH)2D3-fed chicks, dietary P restriction induced and high dietary Ca inhibited the synthesis of intestinal and renal calbindin. The results suggest that: (a) most of the changes in renal and intestinal calbindin could be attributed to the changes in the mRNA; (b) the adaptation to dietary Ca and P alterations requires vitamin D metabolites; (c) high dietary Ca affects intestinal and renal calbindin-mRNA and calbindin via mechanisms independent of kidney 1-hydroxylase; and (d) plasma Ca and renal calbindin or its mRNA tend to change together in vitamin D-deficient or vitamin D3-fed, but not in 1,25(OH)2D3-fed chicks.