RESUMO
We previously reported that atrial natriuretic factor (ANF) stimulates secretin-evoked cAMP efflux through multidrug resistance-associated protein 4 (MRP4) in the exocrine pancreas. Here we sought to establish in vivo whether this mechanism was involved in acute pancreatitis onset in the rat. Rats pretreated with or without probenecid (MRPs general inhibitor) were infused with secretin alone or with ANF. A set of these animals were given repetitive cerulein injections to induce acute pancreatitis. Plasma amylase and intrapancreatic trypsin activities were measured and histological examination of the pancreas performed. Secretin alone activated trypsinogen but induced no pancreatic histological changes. Blockade by probenecid in secretin-treated rats increased trypsin and also induced vacuolization, a hallmark of acute pancreatitis. ANF prevented the secretin response but in the absence of probenecid. In rats with acute pancreatitis, pretreatment with secretin aggravated the disease, but ANF prevented secretin-induced changes. Blockade of MRPs in rats with acute pancreatitis induced trypsinogen activation and larger cytoplasmic vacuoles as well as larger areas of necrosis and edema that were aggravated by secretin but not prevented by ANF. The temporal resolution of intracellular cAMP levels seems critical in the onset of acute pancreatitis, since secretin-evoked cAMP in a context of MRP inhibition makes the pancreas prone to injury in normal rats and aggravates the onset of acute pancreatitis. Present findings support a protective role for ANF mediated by cAMP extrusion through MRP4 and further suggest that the regulation of MRP4 by ANF would be relevant to maintain pancreatic acinar cell homeostasis.
Assuntos
Fator Natriurético Atrial/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Pancreatite/metabolismo , Células Acinares/metabolismo , Doença Aguda , Animais , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Espaço Intracelular/metabolismo , Modelos Biológicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transporte Proteico , Ratos , Tripsinogênio/metabolismoRESUMO
Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.
Assuntos
Proteínas Alimentares/metabolismo , Sistema Digestório/enzimologia , Invertebrados/enzimologia , Tripsina/metabolismo , Tripsinogênio/metabolismo , Adaptação Fisiológica , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Crustáceos/enzimologia , Sistema Endócrino/enzimologia , Ativação Enzimática , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Hidrólise , Hormônios de Inseto/metabolismo , Proteínas de Insetos/metabolismo , Insetos/enzimologia , Invertebrados/genética , Isoenzimas , Dados de Sequência Molecular , Conformação Proteica , Transcrição Gênica , Tripsina/química , Tripsina/genética , Tripsinogênio/química , Tripsinogênio/genéticaRESUMO
Mice fed genetically modified (GM) soybean were not affected in nutritional performance, but pancreatic microscopic features were disturbed. The mechanisms for these contradictory findings are unknown. This study analysed the histology of acinar pancreatic cells and the expression of pancreatitis-associated protein (PAP) and trypsinogen mRNA in rats fed GM soy protein. Two bioassays were run, each one with 34 Wistar rats distributed into two groups fed with non-GM or GM-soy protein (18% protein) for 0, 1, 3, 5, 15 and 30 days. Nutritional evaluation, plasma amylase levels, pancreatic histological analysis and quantification of PAP and trypsinogen mRNAs levels using quantitative real-time RT-PCR were done. No differences in nutritional performance among rats fed non-GM and GM diets were found. The GM, but not the non-GM, diet induced zymogen-granule depletion after 15 days feeding, returning to normal levels after 30 days (P < 0.05). Acinar disorganization started as early as 5 days after initiation of the GM diet and it recovered after 30 days. Levels of PAP mRNA significantly increased in the GM diet between day 1 and day 3 and decreased to the basal level by day 15. Trypsinogen mRNA peaked at two different times; at day 1 and at day 15, decreasing to basal levels after 30 days. Plasma amylase levels remained unchanged at all times. This indicates that GM soy protein intake affected pancreas function, evidenced by the early acute PAP mRNA increased levels and pancreas cellular changes followed by recuperation of acinar cells after 30 days.
Assuntos
Glycine max/genética , Pâncreas/patologia , Plantas Geneticamente Modificadas/metabolismo , Amilases/sangue , Fenômenos Fisiológicos da Nutrição Animal , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Pâncreas/enzimologia , Pâncreas/metabolismo , Proteínas Associadas a Pancreatite , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Tripsinogênio/genética , Tripsinogênio/metabolismoRESUMO
BACKGROUND: Newborn screening for cystic fibrosis (CF) with immunoreactive trypsinogen (IRT) and DeltaF508 analysis followed by sweat testing misses some infants with CF and detects more DeltaF508 carriers than expected. Some of the apparent DeltaF508 carriers may be DeltaF508 compound heterozygotes with normal sweat electrolyte levels. METHODS: Infants identified by newborn screening with an elevated IRT level, one DeltaF508 allele, and a sweat chloride level <60 mmol/L underwent CF mutation analysis, pancreatic stimulation testing, and repeat IRT analysis followed by clinical review and repeat sweat test at 12 months. RESULTS: Over a 24-month period we identified 122 DeltaF508 heterozygotes and recruited 57; 4 had borderline sweat chloride levels (40 to 60 mmol/L), 5 (8.8%, 95% CI 1.4, 16.2) had a second CF mutation (R117H), and 11 (20%, 95% CI 10, 30) had the intron 8 5T allele. Three had clinical CF at 12 months (initial sweat chloride levels: 53, 51, and 32 mmol/L). Pancreatic electrolyte secretion in the subjects with a borderline sweat chloride level was similar to that in patients with known CF. CONCLUSION: The excess of DeltaF508 heterozygotes detected by IRT/DNA screening is associated with the presence of a second mutation or the 5T allele in some infants. Screened infants with borderline sweat chloride levels almost certainly have CF, but long-term follow-up of the infants with the genotype DeltaF508/R117H and DeltaF508/5T is required to determine their outcome. In the meantime, newborn screening should be confined to severe mutations associated with classic CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Análise Mutacional de DNA , Triagem de Portadores Genéticos , Testes de Função Pancreática , Fibrose Cística/metabolismo , Feminino , Genótipo , Humanos , Recém-Nascido , Masculino , Triagem Neonatal , Tripsinogênio/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).
Assuntos
Besouros/enzimologia , Tripsina/genética , Tripsinogênio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Besouros/genética , DNA Complementar , Sistema Digestório , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Inibidores de Proteases , Análise de Sequência , Homologia de Sequência de Aminoácidos , Tripsina/metabolismo , Tripsinogênio/metabolismoRESUMO
The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was delta GH2O = 6.99 +/- 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 6.37 +/- 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 +/- 0.4 A to 26.0 +/- 0.3 A for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 +/- 0.3 A to 25.7 +/- 0.6 A for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.
Assuntos
Dobramento de Proteína , Tripsinogênio/metabolismo , Animais , Bovinos , Diuréticos Osmóticos/farmacologia , Guanidina/farmacologia , Parassimpatomiméticos/farmacologia , Desnaturação Proteica , Ureia/farmacologiaRESUMO
The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was delta GH2O = 6.99 + ou - 1.40 kcal/mol for guanidine hydrochloride and delta GH2O = 6.37 + ou - 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 + ou - 0.4 angstron to 26.0 + ou - 0.3 angstron 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 + ou - 0.3 angstron to 25.7 + ou - 0.6 angstron for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.
Assuntos
Animais , Bovinos , Dobramento de Proteína , Tripsinogênio/metabolismo , Cromatografia Líquida de Alta Pressão , Diuréticos Osmóticos/farmacologia , Guanidina/farmacologia , Parassimpatomiméticos/farmacologia , Desnaturação Proteica , Ureia/farmacologiaRESUMO
Acute pancreatitis was induced with sodium taurocholate 1% in two lots of rats fed during 21 days with diets that differed in lipid composition. Serum amylase, pancreatic tissue enzymes (trypsinogen, chymotrypsinogen and amylase), pancreatic tissue nucleotides (RNA and DNA) and biopsies for histological study were collected in normal pair fed animals, and in the experimental lots 1, 4, 7 and 15 days after AP was induced. ANOVA and Student t-test were used for the comparison of biochemical data (p less than 0.05). They showed that acute pancreatitis aggravated progressively until the fourth day independently of the regimen. On the 15th day, the histological and biochemical parameters reached normal values. The authors concluded that high lipidic diet was not the main factor responsible for progressive injury of the pancreas.
Assuntos
Gorduras na Dieta/farmacologia , Pâncreas/metabolismo , Pancreatite/metabolismo , Doença Aguda , Amilases/sangue , Análise de Variância , Animais , Quimotripsina/metabolismo , Quimotripsinogênio/metabolismo , Masculino , Pancreatite/induzido quimicamente , Pancreatite/patologia , Ratos , Ratos Endogâmicos , Ácido Taurocólico , Tripsina/metabolismo , Tripsinogênio/metabolismoRESUMO
1. The determination of the binding of 4,4'-diazoamino-bis-benzamidine (DABB) to alpha-trypsin by equilibrium measurements in columns indicated a stoichiometry of 2 mol ligand/mol enzyme. One molecule of ligand is bound to the active center, as shown by competitive experiments and Ki. The second molecule binds to the secondary binding site, with Ki2 = 0.63 mM at pH 8.0, 25 degrees C. 2. Bovine pancreatic trypsin inhibitor (BPTI) prevented binding of DABB to both sites, indicating that they are topographically close and within the interface of the trypsin-BPTI complex. 3. On the basis of data from the literature regarding the tertiary structure of the trypsin-BPTI complex, we concluded that the secondary binding site of trypsin is plausibly identified as the same site in trypsin that binds the Arg-17 residue of BPTI, i.e., Tyr-39 and Tyr-151 in bovine trypsin. This site would then correspond to subsite S'2 on the enzyme surface.
Assuntos
Benzamidinas/metabolismo , Tripsina/metabolismo , Benzamidinas/química , Sítios de Ligação , Cromatografia de Afinidade , Tripsinogênio/metabolismoRESUMO
Com a finalidade de estudar o efeito de regimes dietéticos diversos sobre a recuperaçäo das funçöes do pâncreas exócrino pós pancreatite aguda (PA), dois lotes de ratos submetidos, durante três semanas, a dietas isoprotéicas, diferindo apenas no teor de gordura (normo e hiperlipídica), foram distribuídos em grupos controles e com pancreatite aguda moderada, induzida com taurocolato de sódio a 1%. Amostras de sangue para dosagem de amilase sérica e fragmentos de tecido pancreático, para determinaçäo de enzimas triptolíticos, amilase, proenzimas e nucleotídeos foram colhidos nos grupos controles e nos com pancreatite após um, quatro, sete e 15 dias da induçäo da doença. Os resultados foram comparados estatisticamente por ANOVA, entre grupoos sob o mesmo regime alimentar e pelo teste "t" de Student, entre grupos correspondentes, sob regimes alimentares diversos (p <0.05). Os autores verificaram que, nas condiçöes experimentais utilizadas, a pancreatite agravou-se progressivamente até o quarto dia pós-PA, independentemente do regime alimentar prévio. A recuperaçäo funcional no órgäo manifestou-se a partir do sétimo dia pelo aumento de RNA/DNA, mas foi incompleta durante o período deste estudo. No décimo quinto dia pós-PA, ocorreu a normalizaçäo de parâmetros histopatológicos e bioquímicos, para os grupos com ambas as dietas. Os autores concluíram que, nas condiçöes experimentais deste estudo, o agravamento da doença, traduzido pela capacidade de síntese do pâncreas exócrino, foi progressivo até o quarto dia e independeu do teor de gordura na dieta