RESUMEN
Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased approximately 10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4-6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Aceite de Maíz/administración & dosificación , Intestinos/enzimología , Lisofosfatidilcolinas/metabolismo , Fosfatasa Alcalina/genética , Animales , Antígenos de Neoplasias/metabolismo , Células CACO-2 , Duodeno/enzimología , Células Epiteliales/enzimología , Proteínas Ligadas a GPI , Humanos , Isoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Micelas , Microvellosidades/enzimología , Periodo Posprandial , Transporte de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Intestinal alkaline phosphatase (IAP) is involved in the process of fat absorption, a conclusion confirmed by an altered lipid transport and a faster body weight gain from 10 to 30 wk in both male and female mice with a homozygous null mutation of the IAP coding gene (Akp3(-/-) mice). This study was aimed to delineate morphologically and quantitatively the accelerated lipid absorption in male Akp3(-/-) mice. Feeding a corn oil bolus produced an earlier peak of triacylglycerol in serum (2 vs. 4 h for Akp3(-/-) and wild-type mice, respectively) and an approximately twofold increase in serum triacylglycerol concentration in Akp3(-/-) mice injected with a lipolysis inhibitor, Triton WR-1339. A corn oil load induced the threefold enlargement of the Golgi vacuoles in male wild-type mice but not in Akp3(-/-) mice, indicating that absorbed lipids rarely reached the Golgi complex and that the transcytosis of lipid droplets does not follow the normal pathway in male Akp3(-/-) mice. Force feeding an exaggerated fat intake by a 30% fat chow for 10 wk induced obesity in both male Akp3(-/-) and wild-type mice, and therefore no phenotypic difference was observed between the two. On the other hand, the forced high-fat chow induced an 18% greater body weight gain, hepatic steatosis, and visceral fat accumulation in female Akp3(-/-) mice but not in female wild-type controls. These results provide further evidence that IAP is involved in the regulation of the lipid absorption process and that its absence leads to progressive metabolic abnormalities in certain fat-forced conditions.
Asunto(s)
Fosfatasa Alcalina/genética , Hígado Graso/etiología , Absorción Intestinal/efectos de los fármacos , Intestino Delgado/enzimología , Grasa Intraabdominal/crecimiento & desarrollo , Metabolismo de los Lípidos/efectos de los fármacos , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Animales , Grasas de la Dieta/administración & dosificación , Femenino , Grasa Intraabdominal/efectos de los fármacos , Lipoproteína Lipasa/antagonistas & inhibidores , Masculino , Ratones , Polietilenglicoles/farmacología , Triglicéridos/sangreRESUMEN
Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous cell carcinoma tissue.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Neoplasias Pulmonares/enzimología , Surfactantes Pulmonares/metabolismo , Humanos , Isoenzimas/metabolismo , Neoplasias Pulmonares/patología , Fosfolípidos/metabolismo , Transporte de Proteínas , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Tensoactivos/aislamiento & purificación , Extractos de TejidosRESUMEN
Serum levels of intestinal alkaline phosphatase (IAP), a protein implicated in transcellular transport of chylomicrons, vary among ABO blood groups. In rat enterocytes, IAP is associated with chylomicron secretion, but the rat expresses only blood group A. It is not known whether chylomicron secretion may be affected in humans who express multiple blood group types. Serum samples from 40 healthy subjects were obtained after overnight fast and 3h after a high-fat meal, and assayed for IAP and apolipoprotein B-48 (apoB-48), both proteins exclusive to intestine, although only apoB-48 is found in chylomicrons. The two proteins were greater in subjects without blood antigen A (B and O) than in those with this antigen (A and AB); 2.4- and 4.7-fold for IAP and 1.5- and 2.0-fold for apoB-48 before and after the meal, respectively. Moreover, IAP and apoB-48 levels were strongly correlated in the subjects with the secretor phenotype (r > 0.81). These results indicate that IAP is strongly involved in chylomicron formation and fatty acid metabolism might change among ABO blood type. In addition, ABO blood type classification in apoB-48 measurement would improve the diagnostic value in the evaluation of metabolic syndrome.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/sangre , Fosfatasa Alcalina/sangre , Antígenos de Neoplasias/sangre , Apolipoproteínas B/sangre , Grasas de la Dieta/metabolismo , Mucosa Intestinal/metabolismo , Antígenos del Grupo Sanguíneo de Lewis/sangre , Adolescente , Adulto , Apolipoproteína B-48 , Femenino , Proteínas Ligadas a GPI , Humanos , Masculino , Estadística como AsuntoRESUMEN
To understand the differences between the rat intestinal alkaline phosphatase isozymes rIAP-I and rIAP-II, we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP). Our models of rIAP-I and rIAP-II displayed a typical alpha/beta topology, but the crown domain of rIAP-I contained an additional beta-sheet, while the embracing arm region of rIAP-II lacked the alpha-helix, when each model was compared to hPLAP. The representations of surface potential in the rIAPs were predominantly positive at the base of the active site. The coordinated metal at the active site was predicted to be a zinc triad in rIAP-I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP-II. Using metal-depleted extracts from rat duodenum or jejunum and hPLAP, we performed enzyme assays under restricted metal conditions. With the duodenal and jejunal extract, but not with hPLAP, enzyme activity was restored by the addition of zinc, whereas in nonchelated extracts, the addition of zinc inhibited duodenal IAP and hPLAP, but not jejunal IAP. Western blotting revealed that nearly all of the rIAP in the jejunum extracts was rIAP-I, whereas in duodenum the percentage of rIAP-I (55%) correlated with the degree of AP activation (60% relative to that seen with jejunal extracts). These data are consistent with the presence of a triad of zinc atoms at the active site of rIAP-I, but not rIAP-II or hPLAP. Although no differences in amino acid alignment in the vicinity of metal-binding site 3 were predicted between the rIAPs and hPLAP, the His153 residue of both rIAPs was closer to the metal position than that in hPLAP. Between the rIAPs, a difference was observed at amino acid position 317 that is indirectly related to the coordination of the metal at metal-binding site 3 and water molecules. These findings suggest that the side-chain position of His153, and the alignment of Q317, might be the major determinants for activation of the zinc triad in rIAP-I.
Asunto(s)
Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Estructura Terciaria de Proteína , Fosfatasa Alcalina/genética , Animales , Humanos , Mucosa Intestinal/enzimología , Isoenzimas/genética , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Placenta/enzimología , Ratas , Ratas Endogámicas , Zinc/metabolismoRESUMEN
Alkaline phosphatases (APs) have been suggested to detoxify lipopolysaccharide (LPS) by dephosphorylation. Ambroxol, a bronchial expectorant, is known to accelerate the secretion of pulmonary surfactant particles including AP molecules as a pharmacological action. In the present study, some beneficial effects of ambroxol on LPS toxicity in the rat lung were investigated. In an experiment using the rat lung organ culture, AP activities were enhanced in a time-dependent manner by incubation with 25 microM of ambroxol in both the tissue and the medium. Western blot analysis indicated that AP activity was elevated by the treatment with ambroxol, due to the induction of surfactant proteins (SPs) and AP molecules. In the in vivo experiment, the serum LPS content was markedly increased after LPS administration to rats by intratracheal instillation of 20 mg/kg. However, when the rats were pretreated with oral ambroxol (1.0 mg/kg) at 1 h before LPS challenge, the area under the concentration--time curve (AUC) of serum LPS was significantly decreased. These results suggest that ambroxol inhibits the translocation of LPS from the lung into the circulation as well as its detoxification effect via the elevation of AP activity. Bromhexine, another expectorant, is less effective than ambroxol as an LPS detoxificant. Maintenance of high AP activity level in the lung suggests APs to have physiological significant effects against the inflammatory events induced by LPS.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Ambroxol/farmacología , Lipopolisacáridos/metabolismo , Pulmón/metabolismo , Administración Oral , Animales , Área Bajo la Curva , Western Blotting , Bronquios/patología , Relación Dosis-Respuesta a Droga , Expectorantes/farmacología , Lipopolisacáridos/sangre , Pulmón/patología , Masculino , Técnicas de Cultivo de Órganos , Fosforilación , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Gene expressions of alpha-amylase isozymes in rat tissues were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR), followed by EcoRI digestion. This procedure is based on evidence that an RT-PCR product from mouse pancreas RNA is sensitive to EcoRI, but not the product from the salivary gland or liver RNAs. The method was applied to the analysis of alpha-amylase expression in rat liver after partial hepatectomy, in which a potent expression of pancreas type isozyme was observed. However, no expression of the pancreatic isozyme in the regenerating liver was found. We also analyzed the expression of alpha-amylase gene in several additional rat tissues. In intestine, stomach, testis and skeletal muscle, the corresponding PCR products were amplified, but few were detected in heart or spleen. Intestine and stomach expressed a pancreatic isozyme of alpha-amylase. Analyses of the alpha-amylase activity and protein indicated the presence of the enzyme in those tissues. Immunohistochemical analysis also indicated that the amylase proteins were specifically present in epithelial cells of rat intestinal mucosa. This is a convenient method for identification of alpha-amylase isozyme mRNA in rodent tissues.
Asunto(s)
Páncreas/enzimología , alfa-Amilasas/metabolismo , Animales , Desoxirribonucleasa EcoRI , Duodeno/enzimología , Expresión Génica , Inmunohistoquímica , Isoenzimas/análisis , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/enzimología , Masculino , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa-Amilasas/análisis , alfa-Amilasas/genéticaRESUMEN
We first determined whether oxidized high-density lipoprotein (ox-HDL) activates transcription factor nuclear factor-kappa B (NF-kappa B) in cultured human umbilical vein endothelial cells (HUVECs). Treatment for 7h with 100 microg/ml ox-HDL elicited a marked downregulation of I kappa B alpha and upregulation of the phosphorylated form of I kappa B alpha in HUVECs in a manner dependent on the dose of ox-HDL. Electrophoretic mobility shift assay in nuclear fraction from HUVECs showed translocation of NF-kappa B to the nucleus and binding of NF-kappa B to NF-kappa B consensus oligonucleotides during ox-HDL exposure for 7h, suggesting that ox-HDL brings about NF-kappa B activation in endothelial cells. To clarify the mechanism of NF-kappa B activation in HUVECs treated with ox-HDL, we investigated the effect of ox-HDL treatment on intracellular production of reactive oxygen species (ROS) in HUVECs. Ox-HDL induced a significant dose-dependent increase in ROS production during 4h incubation and this enhanced production of ROS was inhibited in the presence of probucol or diphenylene iodonium (DPI), an inhibitor of NADPH oxidase. In addition, pretreatment with probucol or DPI suppressed the phosphorylation and degradation of I kappa B alpha protein induced by ox-HDL, demonstrating that increased generation of ROS by ox-HDL may be associated with NF-kappa B activation. Pretreatment with antibody against oxidized low-density lipoprotein receptor-1 (LOX-1) significantly suppressed the ox-HDL-induced downregulation of I kappa B alpha, suggesting that LOX-1 mediates NF-kappa B activation in endothelial cells stimulated with ox-HDL. Taking all of the above findings together, ox-HDL activates NF-kappa B via binding to LOX-1 on the cell surface, followed by enhancement of intracellular ROS production in endothelial cells.
Asunto(s)
Endotelio/metabolismo , Lipoproteínas LDL/metabolismo , FN-kappa B/metabolismo , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Lipoproteínas HDL/metabolismo , Oxígeno/metabolismo , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
A nucleotide phosphomonoesterase activity that preferably hydrolyzed dCMP was detected in rabbit liver and purified approximately 20-fold. The enzyme was similar in the catalytic and molecular properties to pyrimidine 5'-nucleotidase subclass I (P5N-I), which distributed specifically in vertebrate erythrocytes. In addition to liver, the activity was found in rabbit kidney, spleen, heart, intestine, but was not detected in any rat or chicken tissues tested. The rabbit enzyme protein reacted with antibodies against chicken P5N-I. Its pI was estimated to be approximately 5.3, and the enzyme was concluded to consist of single polypeptide of an approximately 38 kDa based on gel filtration and Western blot analysis. The partially purified enzyme preferentially hydrolyzes dCMP, UMP and CMP, K(m) values for these substrates are approximately 0.3 mM, the optimal pH is approximately 7, and the enzyme requires Mg(2+). This nucleotidase may contribute to the regulation of intracellular pyrimidine nucleotides in the rabbit.
Asunto(s)
5'-Nucleotidasa/metabolismo , Hígado/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , 5'-Nucleotidasa/análisis , 5'-Nucleotidasa/aislamiento & purificación , Animales , Western Blotting , Catálisis , Citidina/metabolismo , Eritrocitos/enzimología , Perfilación de la Expresión Génica , Hidrólisis , Extractos Hepáticos , Monoéster Fosfórico Hidrolasas/análisis , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Conejos , Ratas , Ratas Wistar , Especificidad por SustratoRESUMEN
Nonenzymatic glycosylation of plasma proteins may contribute to the excess risk of developing atherosclerosis in patients with diabetes mellitus. Although it is believed that high-density lipoprotein (HDL) is glycosylated at an increased level in diabetic individuals, little is known about a possible linkage between glycated HDL and endothelial dysfunction in diabetes. To clarify whether glucose-modified HDL affects the function of endothelial cells, we first examined herein the level of H(2)O(2) generation from cultured human aortic endothelial cells (HAECs) exposed to a glycated oxidized HDL (gly-ox-HDL) prepared in vitro. Incubation for 48 hours with 100 microg/mL of gly-ox-HDL induced significant release of H(2)O(2) from cells and gly-ox-HDL-induced H(2)O(2) formation was inhibited in the presence of diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, stimulation of HAECs with gly-ox-HDL for 48 hours elicited a marked downregulation of catalase and Cu(2+), Zn(2+)-superoxide dismutase (CuZn-SOD), suggesting H(2)O(2) formation by gly-ox-HDL to be due to a disturbance involving oxidant and antioxidant enzymes in the cells. Treatment of HAECs with gly-ox-HDL attenuated the expression of endothelial nitric oxide synthase (eNOS), but not inducible nitric oxide synthase (iNOS), and this was followed by decreased production of nitric oxide (NO) by the cells. Furthermore, in vitro experiments with glycated HDL (gly-HDL) in the presence of 2 mmol/L EDTA and Cu(2+)-oxidized HDL suggested the effect of gly-HDL on endothelial function to be possibly potentiated by additional oxidative modification. Taking all of the above findings together, gly-ox-HDL may lead to the deterioration of vascular function through altered production of reactive oxygen species and reactive nitrogen species in endothelial cells.
Asunto(s)
Endotelio Vascular/metabolismo , Glicoproteínas/farmacología , Lipoproteínas HDL/farmacología , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Western Blotting , Catalasa/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Glicoproteínas/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Lipoproteínas HDL/metabolismo , Oxidación-Reducción , Desnaturalización Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismoRESUMEN
We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Proteínas de Choque Térmico/metabolismo , Calor , Intestinos/enzimología , Fosfatasa Alcalina/química , Animales , Arsenitos/farmacología , Secuencia de Bases , Western Blotting , Línea Celular , Núcleo Celular/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dactinomicina/farmacología , Glutatión/farmacología , Proteínas de Choque Térmico/química , Isoenzimas/química , Isoenzimas/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Intestinal alkaline phosphatase (AP), as a host defense factor, was first investigated in vivo using rats orally exposed to lipopolysaccharide (LPS). After the oral administration of LPS to rats, serum LPS content was increased within 2 hr and then decreased to 6 hr. In contrast, when L-phenylalanine (L-Phe), an inhibitor of intestinal-type AP isozymes, was simultaneously administered with LPS, serum LPS content significantly increased from 1 hr and the area under the concentration-time curve of serum LPS was augmented approximately 2-fold, suggesting that APs in the gastrointestinal tract reduced serum LPS content. In addition, LPS toxicity diminished by a treatment in vitro with intestinal APs, were recovered by the treatment in the co-presence of L-Phe. In the experiment using human aortic endothelial cells (HAECs), we observed that the cell viability decreased in a dose-dependent manner of LPS-exposure, and the LPS dose, exhibiting 50% viability of the cells, was 0.05 microg/ml. When the cells were exposed to LPS pretreated with 50 nIU/ml of intestinal AP at pH 10.0 and 8.0, the 50% viability was at 2.0 microg/ml of LPS. These results strongly suggest that the APs reduced the toxicity of LPS, as a host defense factor against LPS.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/toxicidad , Administración Oral , Animales , Antígenos de Neoplasias/metabolismo , Western Blotting , Duodeno/efectos de los fármacos , Duodeno/enzimología , Proteínas Ligadas a GPI , Técnicas In Vitro , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/sangre , Masculino , Óxido Nítrico/análisis , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Fosforilación , Ratas , Ratas WistarRESUMEN
This paper reviews working procedures for the separation and detection of oxidized high-density lipoproteins (ox-HDL) and their constituents. It begins with an introductory overview of structural alterations of the HDL particle and its constituents generated during oxidation. The main body of the review delineates various procedures for the isolation and detection of ox-HDL as well as the purification and separation of phosphatidylcholine metabolites and denatured apolipoproteins in the particle. The useful methods published more recently are picked up and the utility of the separation techniques is described. The last section covers a clinical evaluation of changes in these factors in ox-HDL as well as future directions of ox-HDL research.
Asunto(s)
Lipoproteínas HDL/química , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Peróxidos Lipídicos/química , Lipoproteínas HDL/sangre , Oxidación-ReducciónRESUMEN
Liver amylase activity in rodents was assayed with Blue Starch as substrate, and found to be higher than in humans or pigs. Based on the result of concanavalin A affinity chromatography, we found that the sugar moieties of amylase molecules increased in parallel with amylase activity in the tested mammals. However, the amounts of amylase proteins determined by Western bloting with anti-human salivary-type antibody as the probe, were similar to the levels in mammalian livers. Moreover, a similar expression of amylase mRNA was also detected in the mammalian livers by a reverse transcriptional-polymerase chain reaction using primers specific for the human salivary and/or pancreatic amylase complementary DNA (cDNA) sequences. The amylase was detected at the catalytic activity, protein molecule and mRNA levels in rat liver at all ages from fetus to adult. Salivary-type liver amylase activity increased up to one week after birth, and was maintained at the adult level thereafter. However, based on the results of the electrophoretic mobility test, livers with accelerated amylase activity, e.g., at 2-4 weeks after birth or during liver regeneration after partial hepatectomy, were also found to express an amylase electrophoretical identical to pancreatic-type amylase in addition to salivary-type activity. These results suggest that the liver may express an etopic amylase in a certain condition.
Asunto(s)
Amilasas/análisis , Amilasas/clasificación , Hígado/enzimología , Filogenia , Amilasas/genética , Amilasas/metabolismo , Animales , Western Blotting , Electroforesis en Acetato de Celulosa , Humanos , Hígado/crecimiento & desarrollo , Regeneración Hepática , Páncreas/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/enzimología , PorcinosRESUMEN
BACKGROUND: The alpha-amylase isozymes can be detected separately by electrophoresis; however, sometimes the identification is difficult because of their microheterogeneity. In the present study, we tried to establish a convenient method for the detection of alpha-amylase isozyme expression. METHODS: The procedure is based on three different restriction sites presented in those genes; a PstI site in both AMY 2A and 2B genes, a HaeII site in both AMY 1 and 2A genes, and a BamHI site in AMY 2B gene. After amplification from total tissue RNAs by RT-PCR with primers that were able to cover each exon, the products were cleaved with corresponding restriction endonucleases. RESULTS: This method was applied to human samples from the parotid gland, liver (non-hepatoma), hepatoma and white blood cells (WBCs). The results indicated that the parotid gland and hepatoma (also liver) clearly expressed AMY 1 and AMY 2B genes, respectively. However, AMY 2B gene was also expressed apparently in WBCs, which produced salivary-type isozyme of the alpha-amylase, although the amylase protein was not able to identify for the hepatic isozyme. CONCLUSIONS: The method presented here might be convenient and useful for the determination of alpha-amylase isozyme expression in humans.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , alfa-Amilasas/análisis , alfa-Amilasas/genética , Carcinoma Hepatocelular/enzimología , Humanos , Isoenzimas/análisis , Isoenzimas/genética , Leucocitos/enzimología , Hígado/enzimología , Glándula Parótida/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The characteristics of lipopolysaccharide (LPS)-induced alkaline phosphatase (AP) isozymes on the various pulmonary surfactant subtypes were investigated. We used continuous sucrose-gradient centrifugation to separate surfactant into subtypes. The density of each surfactant subtype isolated from LPS-instilled rats was greater than that of the subtypes from the control rats; and the proportion of light surfactant was lower, thereby decreasing the ratio of light to heavy surfactant. The results of an inhibition study revealed the main AP isozyme in bronchoalveolar fluid (BAF) to be tissue-nonspecific AP (TNAP), but some of the activity was characteristic of intestinal-type AP (IAP). IAP, in addition to TNAP and surfactant-associated protein A (SP-A), was detected on heavy surfactant, and LPS induced both APs. To examine the expression of IAP in the lungs, we prepared primers to detect the cDNAs of two types of rat IAP mRNA, IAP-I and -II, and amplified their cDNAs. LPS instillation induced IAP-I mRNA, but not IAP-II mRNA or TNAP mRNA. Immunohistochemical localization of IAP and TNAP revealed reaction products for both in type II cells. The present study thus demonstrated that, in rats, type II cells produce both IAP and TNAP and that these surfactants bearing AP isozymes are secreted into the alveolar space following induction by intratracheal instillation of LPS.
Asunto(s)
Intestinos/química , Isoenzimas/análisis , Isoenzimas/biosíntesis , Lipopolisacáridos/efectos adversos , Surfactantes Pulmonares/análisis , Fosfatasa Alcalina , Animales , Líquido del Lavado Bronquioalveolar , Endotoxinas/efectos adversos , Endotoxinas/toxicidad , Neumonía/diagnóstico , Proteolípidos/análisis , Proteolípidos/biosíntesis , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/biosíntesis , RatasRESUMEN
We reported previously that Ca and Pi levels are elevated and alkaline phosphatase (ALP) activity and 1alpha,25(OH)2D3 levels are reduced in chlorpromazine (CPZ)-challenged rats. In the present study, we determined the serum levels of interleukin (IL)-6 and acid phosphatase (ACP) in CPZ-challenged rats, in addition to levels of ALP protein. ALP mRNA and coccyx morphology were examined in CPZ-challenged rats as well as the effect of CPZ on 1alpha(OH)D3 production in vivo. Although Ca, Pi, IL-6, and ACP activity levels in CPZ-challenged rats were markedly increased on day 30, the elevated serum levels were restored to within normal ranges by the in vivo addition of 1alpha(OH)D3 to CPZ administration. The gain in body weight in CPZ-treated rats was significantly improved by the addition of 1alpha(OH)D3. Reduced levels of 1alpha,25(OH)2D3 in CPZ-treated rats were restored to normal levels by the administration of 1alpha(OH)D3. Moreover, the decreased ALP activity and ALP mRNA levels in the rat coccyx marrow in CPZ-treated rats were also restored by the administration of 1alpha(OH)D3 with CPZ. However, the molecular sizes of rat ALP molecules and ALP mRNA were the same for each group. Furthermore, bone morphometry showed that trabecular bone in the rat coccyx was decreased in CPZ-treated rats. However, the reduced volume of trabecular bone in CPZ-treated rats was restored by the addition of 1alpha(OH)D3 to CPZ administration. Taken together, altered bone metabolism in CPZ-treated rats can be improved by the addition of 1alpha(OH)D3.