Assuntos
Proteínas de Choque Térmico HSP70/genética , Estresse Oxidativo/fisiologia , Carcinoma Hepatocelular , Citomegalovirus/genética , Vetores Genéticos , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Neoplasias Hepáticas , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The intrarenal localization and role of the V1a vasopressin receptor in body fluid homeostasis are unclear. We investigated the intranephron localization of V1a receptor mRNA and protein using reverse transcription (RT)-competitive polymerase chain reaction (PCR) and immunohistochemistry with a specific polyclonal antibody. To determine whether the V1a receptor is involved in the regulation of acid-base balance, we also examined the effects of acute and chronic metabolic acidosis and dehydration on V1a receptor expression. V1a mRNA was expressed most abundantly in the cortical collecting ducts (CCD) and decreased in the deeper CD. Expression in the glomeruli and thick ascending limbs was low. The immunohistochemical study revealed the presence of the V1a receptor in the glomeruli, the thick ascending limbs and the CD. Dehydration decreased V1a mRNA expression in the CD. Chronic metabolic acidosis increased V1a receptor mRNA expression in the CD but decreased V2 receptor mRNA expression. Western blot analysis revealed up-regulation of the V1a receptor protein in chronic metabolic acidosis. Incubation of microdissected CCD or outer medullary CD (OMCD) in a low-pH (or or low-HCO3-) medium increased the levels of V1a receptor mRNA but decreased V2 receptor mRNA expression. Incubating OMCD with arginine vasopressin (AVP) and the V1a receptor antagonist (OPC21268) increased V2 receptor mRNA expression compared with incubation with AVP alone. These data suggest that V1a receptors are present primarily in the principal and intercalated cells in the CD and that these receptors are involved in the regulation of water and acid-base balance.
Assuntos
Acidose/metabolismo , Desidratação/metabolismo , Néfrons/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Arginina Vasopressina/análogos & derivados , Arginina Vasopressina/farmacologia , Bicarbonatos/farmacologia , Regulação para Baixo , Homeostase , Imuno-Histoquímica , Masculino , Néfrons/anatomia & histologia , Piperidinas/farmacologia , Reação em Cadeia da Polimerase/métodos , Quinolonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/genética , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Heat shock proteins (HSPs) 60 and 10 are stress-inducible mitochondrial matrix proteins that form a chaperonin complex that is important for mitochondrial protein folding and function. The effect of cerebral ischemia on mitochondrial HSPs is unclear. The topographical and chronological patterns of HSP60 and HSP10 messenger ribonucleic acid (mRNA) expression and induction were investigated in the rat focal cerebral ischemia model. Focal cerebral ischemia was produced by transient middle cerebral artery occlusion for 30 or 90 min. Expression of mRNAs was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR analysis showed that both HSP60 and HSP10 mRNA levels increased significantly in the ischemic cortex from 4 to 24 h of reperfusion after 30 min of occlusion. In situ hybridization analysis demonstrated significant induction of both mRNAs in the whole ischemic cortex after 30 min of occlusion and in the dorsomedial border (penumbra) of the ischemic cortex and ipsilateral hippocampus after 90 min of occlusion. Expression patterns and the timing of the induction of both HSP60 and HSP10 mRNAs were identical throughout the experiments. Simultaneous induction of the mRNAs for the mitochondrial chaperonins, HSP60 and HSP10, in various regions in focal cerebral ischemia demonstrates that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in focal cerebral ischemia.
Assuntos
Chaperonina 10/genética , Chaperonina 60/genética , Ataque Isquêmico Transitório/fisiopatologia , Animais , Northern Blotting , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Hipocampo/irrigação sanguínea , Hipocampo/fisiopatologia , Hibridização In Situ , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Several investigations have postulated evidence of the involvement of apoptosis in delayed neuronal death following brief periods of global cerebral ischemia. Apoptosis may be closely linked to mitochondrial dysfunction. Heat shock protein (HSP) 60 and HSP10 are mitochondrial matrix proteins induced by stress and form the chaperonin complex that is implicated in protein folding and assembly within the mitochondria. This study investigated the induction of these mitochondrial stress protein genes in the hippocampal CA1 region and less vulnerable regions following transient forebrain ischemia. In situ hybridization analysis revealed that the induction pattern of HSP60 mRNA was identical to that of HSP10 mRNA throughout the entire ischemic course. No changes occurred in the expression of both mRNAs after 2 min ischemia. Strong induction of both mRNAs occurred in the CA1 region after 10 min ischemia and persisted until 1 d after reperfusion. In contrast, induction of both mRNAs in the less vulnerable regions was terminated by 1 d after reperfusion. These results demonstrate that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in regions vulnerable to transient forebrain ischemia.
Assuntos
Isquemia Encefálica/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Mitocôndrias/genética , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Animais , Apoptose , Northern Blotting , Isquemia Encefálica/patologia , Chaperonina 10/genética , Chaperonina 60/genética , Hipocampo/metabolismo , Hipocampo/patologia , Hibridização In Situ , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
In order to study the cytoprotective function of colonic heat shock proteins (HSPs) in vivo, the effect of specific preinduction of HSP60 by thyrotropin-releasing hormone (TRH) administration on the development of acetic acid-induced colonic mucosal lesion was investigated. Expression of 60-kDa, 72-kDa, and 90-kDa heat shock proteins (HSP60, HSP72, and HSP90, respectively) in rat colonic mucosa was investigated by western blot and immunohistochemical analyses before and after TRH administration. Following pretreatment with or without TRH administration, the rats received intrarectal infusion of 5% acetic acid. The colonic mucosal damage was macroscopically evaluated 24 hr after the intrarectal infusion of acetic acid. Expression of HSP60 was significantly increased by TRH administration in the colonic mucosa, whereas HSP72 and HSP90 did not increase. Immunohistochemical study also showed a significant increase in HSP60 in colonic mucosal cells, especially at the surface of the colonic mucosa after TRH administration. No histopathologic alteration was observed in the colonic mucosa after TRH administration. The colonic mucosal damage caused by intrarectal infusion of 5% acetic acid was not prevented by preinduction of HSP60. We demonstrated that specific preinduction of HSP60 by TRH administration did not show cytoprotective function in the colonic mucosa, although this protein plays a crucial role for cytoprotection in the pancreatic acinar cells. Our results indicate that the role of HSP60 may be different in each organ with respect to cytoprotection.
Assuntos
Ácido Acético/farmacologia , Chaperonina 60/metabolismo , Colo/efeitos dos fármacos , Doenças do Colo/induzido quimicamente , Doenças do Colo/prevenção & controle , Mucosa Intestinal/efeitos dos fármacos , Hormônio Liberador de Tireotropina/farmacologia , Animais , Colo/patologia , Doenças do Colo/patologia , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Mucosa Intestinal/patologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
We demonstrated previously that expression of rat pICln in Escherichia coli conferred a strong resistance to hypotonic stress. To define the intramolecular functional domain responsible for the resistance, molecular dissection of pICln was performed and the obtained peptides were expressed in E. coli. The cells expressing the peptides were exposed to a hypotonic solution, and their 'survival rates' were observed. The cells expressing only the peptides including the second acidic domain of pICln exhibited significantly higher 'survival rates' after hypotonic stress. The functional domain against hypotonicity was finally narrowed down to a peptide consisting of a 46-amino acid residue, P107-152. We conclude that the expression of P107-152 in E. coli cells could enhance their resistance to a hypotonic environment.
Assuntos
Canais de Cloreto/fisiologia , Escherichia coli/fisiologia , Peptídeos/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Clonagem Molecular , Contagem de Colônia Microbiana , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Soluções Hipotônicas/farmacologia , Canais Iônicos/antagonistas & inibidores , Pressão Osmótica , Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , RatosRESUMO
It has been reported that immunosuppressant cyclosporin A or FK506 binds to immunophilins in the cell and that these immunophilins make a complex with molecular chaperones HSP70 or HSP90. Although mizoribine has been used clinically as an immunosuppressant, immunophilins of the agent have not yet been fully understood. We investigated their specific binding proteins using mizoribine affinity column chromatography and porcine kidney cytosols. By increasing mizoribine in the eluant from the column, two major proteins (with molecular masses of 60 and 43 kDa) were detected by SDS-polyacrylamide gel electrophoresis. Based on the amino acid sequence analysis of these proteins, 60- and 43-kDa mizoribine-binding proteins were identified with HSP60 and cytosolic actin, respectively. A considerable amount of actin was also eluted from the affinity column by nucleotides, but a very low quantity of HSP60 was eluted under the same conditions. On the other hand, HSP60 was eluted as a major protein in the eluant that was eluted preferentially, with nucleotide followed by mizoribine. Actin was also detected in the eluant, but the quantity of the protein was very low. These results indicated that HSP60 has high affinity to mizoribine, and the interaction was also observed on surface plasmon resonance analysis. Although HSP60 or GroE facilitated refolding of citrate synthase in vitro, mizoribine interfered with the chaperone activity of HSP60. On different types of mizoribine affinity columns, HSP60 or actin recognized the NH(2) group of mizoribine, and this group may be a functional group of the agent.
Assuntos
Chaperonina 60/efeitos dos fármacos , Chaperonina 60/metabolismo , Imunossupressores/farmacologia , Ribonucleosídeos/farmacologia , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Bovinos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Proteínas de Choque Térmico HSP70/metabolismo , Fígado/metabolismo , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Nucleotídeos/farmacologia , Ligação Proteica , Ribonucleosídeos/química , Ribonucleosídeos/metabolismo , Serina Endopeptidases/metabolismo , Ressonância de Plasmônio de Superfície , Suínos , Fatores de TempoRESUMO
The 90-kDa heat shock protein (HSP90) acts as a specific molecular chaperone in the folding and regulates a wide range of associated proteins such as steroid hormone receptors. It is known that HSP90 possesses two different chaperone sites, both in the N- and C-domains, and that the chaperone activity of HSP90 is blocked by binding of geldanamycin (GA) to the N-domain, the same as the ATP-binding site. Here we show that Cisplatin [cis-diamminedichloroplatinum (II), CDDP], an antineoplastic agent, associates with HSP90 and reduces its chaperone activity. In order to analyse the binding proteins, bovine brain cytosols were applied to a CDDP-affinity column and binding proteins were eluted by CDDP. In the elutants, only 90-kDa protein bands were detected on SDS/PAGE, and the protein was cross-reacted with the anti-HSP90 antibody on immunoblotting. No protein bands were detected in the elutants from the control column on SDS/PAGE. These results indicated that CDDP has a high affinity for HSP90. On CD spectrum analysis, the binding of CDDP to HSP90 resulted in a conformational change in the protein. Although HSP90 inhibited the aggregation of citrate synthase as a molecular chaperone in vitro, the activity was suppressed almost completely in the presence of CDDP. Mg/ATP has an influence on the chaperone activity to some extent. The CDDP binding region of HSP90 is near the C-terminal which is quite different from the GA-binding site. Our results suggest that the chaperone activity of HSP90 may be inhibited by the binding of CDDP or GA by different mechanisms.
Assuntos
Encéfalo/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Benzoquinonas , Sítios de Ligação , Bovinos , Cromatografia de Afinidade , Dicroísmo Circular , Cisplatino/farmacologia , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/isolamento & purificação , Lactamas Macrocíclicas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Quinonas/farmacologiaRESUMO
It is well documented that gastric mucosa can increase its resistance to mucosal damage caused by aspirin during repeated long-term administration of aspirin. However, the underlying mechanism of this adaptation is not well established. In the present study, we investigated the effect of long-term (chronic) administration of aspirin on expression of heat shock proteins (HSPs), which are known as endogenous cytoprotectants, in rat gastric mucosa. Rats were administered aspirin (100 mg/kg) daily for up to 20 days. After various periods of aspirin administration, a high dose of aspirin (250 mg/kg) was administered, and the mucosal damage was assessed. Expression of heat shock proteins (HSPs) in gastric mucosa was evaluated by Western blot. Intracellular localization of each HSP was studied immunohistochemically. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels were also investigated. Long-term aspirin administration resulted in development of resistance to aspirin-induced mucosal damage, and the increase of HSP72 expression correlated with mucosal resistance to aspirin. No significant increase was observed in HSP60 and HSP90 levels. Immunohistochemical study showed an increase of HSP72 in the cytoplasm of mucosal surface cells. The PGE2 level was suppressed and no change in the level of LTB4 was observed. It is possible that HSP72 could play important roles in gastric mucosal adaptation when the PGE2 level is suppressed by NSAIDs.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Aspirina/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Adaptação Fisiológica/genética , Animais , Dinoprostona/metabolismo , Mucosa Gástrica/patologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP72 , Técnicas Imunoenzimáticas , Leucotrieno B4/metabolismo , Assistência de Longa Duração , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
To elucidate the function of pI(Cln), its localization in subcellular organellae was investigated. A specific polyclonal anti-pI(Cln) antibody detected the soluble 38-kDa pI(Cln) exclusively in the cytosols of rat heart, lung, liver, spleen, skeletal muscle, testis, and brain, but not rat kidney. pI(Cln)-associated proteins in skeletal muscle were also analyzed. Native-gradient PAGE showed a single 340-kDa protein band reactive to anti-pI(Cln) antibody. This band also stained with anti-actin antibody. Two-dimensional PAGE and immunoprecipitation analysis indicated that all of the pI(Cln) was present in association with actin of a constant length: the molecular ratio of pI(Cln) to actin was roughly 1:7. In addition, all actin in the cytosol fractions was found in association with pI(Cln). These results suggest the possibility that skeletal muscle pI(Cln) controls the length of cytosolic F-actin.
Assuntos
Actinas/química , Canais de Cloreto/química , Canais Iônicos , Músculo Esquelético/química , Sequência de Aminoácidos , Animais , Canais de Cloreto/genética , Citosol/química , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Peso Molecular , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/química , Distribuição TecidualRESUMO
BACKGROUND: Recent studies have shown that the 72-kDa heat shock protein (HSP72) can be induced in renal tubular cells by a variety of stress conditions, and suggested its cytoprotective function. We have tested this hypothesis directly by transfection studies. METHODS: LLC-PK1 cells (porcine renal tubular epithelial cells) were stably transfected with pBK-CMV or pBK-CMV containing the human HSP72 gene (pBK-CMV-HSP72). These cells were then treated with various concentrations of hydrogen peroxide or cisplatin. The cell viability and lytic cell damage were determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release assay. RESULTS: Immunoblot and immunocytochemical analyses showed the high level expression of HSP72 in LLC-PK1 cells transfected with pBK-CMV-HSP72. In addition, the expression of other major HSPs (HSP90, HSP73, HSP60 and HSP27) was not affected by transfection. LLC-PK1 cells overexpressing HSP72 were significantly more resistant to hydrogen peroxide and cisplatin treatments than control cells. CONCLUSION: These results indicate that overexpressed HSP72 plays a direct role in protecting renal tubular cells against oxidative injury and cisplatin toxicity.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Proteínas de Choque Térmico/fisiologia , Túbulos Renais/efeitos dos fármacos , Estresse Oxidativo , Animais , Células Cultivadas , Proteínas de Choque Térmico HSP72 , Humanos , Peróxido de Hidrogênio/toxicidade , Túbulos Renais/metabolismo , SuínosRESUMO
Endothelin-1 (ET-1) is thought to play a significant role in acute renal failure induced by cyclosporin A (CsA). The cDNA sequence encoding endothelin-converting enzyme-1 (ECE-1), which produces the active form of ET-1 from big ET-1, was recently reported. To elicit the role of ECE-1 in the glomerular and tubular dysfunction induced by CsA, the effects of CsA on mRNA and protein expression of ECE-1 in rat kidney and on mRNA expression of prepro-ET-1 and ET A- and B-type receptors in glomeruli were studied. ECE-1 mRNA was detected in glomeruli and in whole nephron segments. ECE-1 mRNA expression was downregulated in all nephron segments at 24 h after CsA injection. Protein levels were also downregulated in glomeruli and in the outer and inner medulla. CsA rapidly increased prepro-ET-1 mRNA expression in glomeruli at 30 to 60 min after injection; this rapid increase was followed by an increase in plasma ET-1 levels. These increases were followed by decreased expression of ECE-1, ET A-type receptor, and ET B-type receptor mRNA at 6 h after injection, and serum creatinine levels were increased at 24 h after CsA injection. It is suggested that downregulation of glomerular and tubular ECE-1 expression may be caused by increased ET-1 synthesis in CsA-induced acute renal failure.
Assuntos
Injúria Renal Aguda/enzimologia , Ácido Aspártico Endopeptidases/metabolismo , Néfrons/enzimologia , Animais , Ácido Aspártico Endopeptidases/genética , Sequência de Bases , Western Blotting , Creatinina/sangue , Técnicas de Cultura , Ciclosporina/sangue , Modelos Animais de Doenças , Enzimas Conversoras de Endotelina , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/enzimologia , Masculino , Metaloendopeptidases , Dados de Sequência Molecular , Néfrons/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Valores de ReferênciaRESUMO
Bowel dysfunction such as irritable bowel syndrome caused by stress is well described. Previous reports suggest that stress is known to cause the release of endogenous substances such as catecholamine, beta-endorphine, 5-hydroxytryptamine, corticotropin-releasing factor, and thyrotropin-releasing hormone (TRH). However, the role played by these neurohormonal mediators in bowel dysfunction under stress conditions is not well known. We investigated the influence of water-immersion stress or TRH administration on the expression of 60-kDa, 72-kDa, and 90-kDa heat-shock proteins (HSP60, HSP72, and HSP90, respectively) in rat small intestinal mucosa by Western blot and immunohistochemical analyses. The cytoprotective function of preinduced HSPs on experimentally induced mucosal damage also was studied. In order to investigate the influence of preinduction of HSP60 on small intestinal damage, the small intestinal lumen was perfused with 1.5% acetic acid 1 ml/min for 15 min with or without pretreatment with water-immersion stress or TRH administration. Expression of HSP60 was significantly increased by water-immersion stress or TRH administration in the small intestinal mucosa, whereas HSP72 and HSP90 did not increase. Interestingly, expression of this protein showed the biphasic peak pattern after water-immersion stress or TRH administration. Each peak was observed 3-6 hr and 21-24 hr after the initiation of water-immersion stress or TRH administration. Immunohistochemical study also showed a significant increment of HSP60 in both the cytoplasm and nuclei of the small intestinal mucosal cells. No histopathologic alteration was observed in rat small intestinal mucosa after each treatment. Small intestinal damage caused by 1.5% acetic acid perfusion was not influenced by preinduction of HSP60. We demonstrated that water-immersion stress or TRH administration specifically induced HSP60, although preinduction of this protein did not show a cytoprotective function in the small intestinal mucosa.
Assuntos
Proteínas de Choque Térmico/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Ácido Acético , Animais , Chaperonina 60/metabolismo , Proteínas de Choque Térmico HSP72 , Imersão/efeitos adversos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Estresse Fisiológico/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Fatores de Tempo , Regulação para Cima , ÁguaRESUMO
We amplified the coding region DNA sequence from a rat renal pI(Cln) cDNA by PCR and expressed the protein in Escherichia coli cells. The cells were exposed to hypotonic conditions followed by spreading them onto LB plates for subsequent colony survival assay. The present study demonstrated that the cells expressing pI(Cln) exhibit a strong resistance to hypotonic stress. Moreover, the resistance was specifically inhibited by extracellular ATP and some anion channel inhibitors. These findings indicate that the expression of pI(Cln) directly confers tolerance to hypotonic stress, and pI(Cln) is concluded to be an important molecule for cell-volume regulation.
Assuntos
Canais de Cloreto/fisiologia , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Adaptação Biológica/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Soluções Hipotônicas , Dados de Sequência Molecular , Plasmídeos , RatosRESUMO
We produced a highly specific antibody to the C-terminal peptide sequence of pICln. It recognized pICln with a 38-kDa molecular mass on SDS-polyacrylamide gel electrophoresis, coinciding with that previously reported. During native polyacrylamide gel electrophoresis, three immunoreactive bands (38, 70, and 130 kDa) were detected. The isoelectric point of pICln was calculated to be 4.0. Subcellular localization study showed the presence of pICln in the soluble and microsomal fraction. pICln can be easily solubilized from the membrane fraction with Triton X-100. From immunohistochemical observations, we found pICln to be obviously located on the luminal surface membranes of the distal tubules and Henle's loop ascending limbs, and it can also be found inside proximal tubular cells. The present results suggested that pICln functions as a "cytosolic anchor = membrane insertion" model, and it plays important roles in the "urine dilution segment" cells of nephrons.
Assuntos
Canais de Cloreto/análise , Canais Iônicos , Túbulos Renais/química , Alça do Néfron/química , Animais , Fracionamento Celular , Tamanho Celular/fisiologia , Humanos , Imuno-Histoquímica , Ponto Isoelétrico , Rim/citologia , Rim/fisiologia , Proteínas de Membrana/química , Octoxinol/farmacologia , Pressão Osmótica , Solubilidade , SuínosRESUMO
The expression of the fructose 1,6-bisphosphatase gene in HL-60 cells was induced by retinoic acid. The levels of mRNA, enzyme activity and enzyme protein in the cell line began to rapidly increase after culturing with retinoic acid for 72 h. Retinoic acid dose-dependently increased the enzyme activity with maximal stimulation at 1 microM. The responses of the fructose 1,6-bisphosphatase gene expression by retinoic acid were markedly slower than those of the enzyme expression by 1alpha,25-dihydroxyvitamin D3. When HL-60 cells were cultured in the presence of both retinoic acid and 1alpha,25-dihydroxyvitamin D3, the effects of the two agents on enzyme activity, protein and mRNA were additive.
Assuntos
Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Células HL-60/enzimologia , Tretinoína/farmacologia , Western Blotting , Calcitriol/farmacologia , Relação Dose-Resposta a Droga , Frutose-Bifosfatase/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Humanos , RNA MensageiroAssuntos
Imersão , Isquemia/patologia , Pâncreas/irrigação sanguínea , Traumatismo por Reperfusão/patologia , Estresse Fisiológico/fisiopatologia , Água , Animais , Chaperonina 60/metabolismo , Temperatura Alta , Masculino , Pâncreas/metabolismo , Pâncreas/patologia , Ratos , Ratos Wistar , Estresse Fisiológico/etiologiaRESUMO
Heat shock proteins are ubiquitous intracellular proteins induced by various physiological stress-related events. A 72-kDa heat shock protein (HSP72) has been reported to be an endogenous cytoprotectant in variety of cells in vitro. In order to study the cytoprotective function of HSP72 in the liver, the effect of preinduction of HSP72 in rat liver by systemic hyperthermia on thioacetamide-induced hepatic injury was investigated in this study. Expression of HSP72 in the liver was investigated by immunoblot and densitometric analysis. Rats were injected with thioacetamide (100 mg/kg, subcutaneously) with or without preinduction of HSP72 by hyperthermia. Serum AST and ALT concentrations were measured before and after thioacetamide injection in both group. Histologic alteration of the liver was evaluated also. Systemic hyperthermia (42.5 degrees C, 20 min) significantly induced HSP72 in the liver. Thioacetamide-induced hepatic injury was clearly prevented by preinduction of HSP72 by hyperthermia. Prevention of hepatocyte damage was more clear in the area around central veins where HSP72 induction was apparent. Our findings might suggest that HSP72 has an important function in the liver with respect to cytoprotection. These results might be important for understanding the mechanism of "adaptive cytoprotection" in the liver mediated by the function of heat shock proteins as "molecular chaperons" as reported in vitro.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Proteínas de Choque Térmico/biossíntese , Fígado/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Doença Hepática Induzida por Substâncias e Drogas/patologia , Proteínas de Choque Térmico HSP72 , Hipertermia Induzida , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Pré-Medicação , Ratos , Ratos Sprague-Dawley , TioacetamidaRESUMO
A 30 kDa protein was purified from pig liver cytosol by using ATP-Sepharose and Green A column chromatography. The partial amino acid sequences of the protein (95 amino acid residues) had no similarity with any proteins recorded in data banks. The protein was able to form a stable complex with unfolded dihydrofolate reductase (DHFR). The spontaneous refolding of chemically denatured DHFR was arrested by the 30 kDa protein. This inhibition presumably results from the formation of a stable complex between the 30 kDa protein and DHFR. Bound DHFR could be released from the protein with ATP. The protein also showed protease resistance in an ATP-dependent manner. Incubation of the 30 kDa protein with 5 mM ATP resulted in its resistance to V8 protease or to trypsin treated with 1-chloro-4-phenyl-3-L-toluene-p-sulphonamidobutan-2-one. Divalent cations enhanced the ATP-protection effect. CD analysis of the 30 kDa protein showed that ATP induced an increase in the beta-pleated sheet content and a decrease in the alpha-helix content of the 30 kDa protein. These results suggest that the 30 kDa protein, a novel cytosolic protein, might have an affinity for ATP, a chaperonin activity, and and an ATP-protection effect against some proteases in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/isolamento & purificação , Chaperonas Moleculares/isolamento & purificação , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/efeitos dos fármacos , Dicroísmo Circular , Endopeptidases/metabolismo , Fígado/química , Chaperonas Moleculares/química , Chaperonas Moleculares/efeitos dos fármacos , Dados de Sequência Molecular , Peso Molecular , Suínos , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
We previously reported that water-immersion stress specifically induced the synthesis of a 60-kDa heat-shock protein (HSP60, chaperonin homolog) in pancreatic cells and preinduction of HSP60 completely prevented development of cerulein-induced pancreatitis in the rat in an HSP60 quantitatively dependent manner. In order to study the cytoprotective function of a 72-kDa heat-shock protein (HSP72, stress-inducible hsp70), the effect of specific preinduction of HSP72 by hyperthermia on cerulein-induced pancreatitis was investigated and compared with the effect of preinduction of HSP60 in this study. Expression of HSP60 and HSP72 in the pancreas was investigated by immunoblot before and after water immersion or hyperthermia. Following pretreatment with water-immersion stress or hyperthermia, the rats were injected with cerulein (40 micrograms/kg, intraperitoneally). The pancreas wet weight and serum amylase concentration were measured before and after cerulein injection. Hyperthermia (42.5 degrees C, 20 min) specifically induced HSP72 in the pancreas. The synthesis of HSP60 was specifically induced by water-immersion stress in the pancreas. Cerulein-induced pancreatitis was clearly prevented by specific preinduction of HSP60 by water-immersion stress. However, preinduction of HSP72 by hyperthermia had no preventive effect on cerulein-induced pancreatitis. Our findings suggest that HSP60 and HSP72 have distinct functions in the pancreas, and their induction mechanisms are also different in vivo. These results could be important for understanding the mechanism of "adaptive cytoprotection" in the pancreas mediated by heat-shock proteins.