RESUMO
There is an increasing histological clinical evidence that both hepatic fibrosis and some degree of cirrhosis reversal can improve prognosis. Hepatic fibrosis involves a variety of cells and steps, and its reversal mechanism is also very complex, mainly including the reduction of hepatocyte necrosis and regeneration, the apoptosis and inactivation of activated hepatic stellate cells, and the reversal of hepatic sinusoidal endothelial cells and microvessels, restorative hepatic macrophages polarization and cell-to-cell interactions. Furthermore, the biochemical basis for reversal of hepatic fibrosis is decreased expression of matrix metalloproteinase inhibitors, up-regulation of matrix metalloproteinase activity, and increased degradation of extracellular matrix. However, at present, there are few studies on the clinicopathological mechanism of liver fibrosis reversal, and the key target groups of different etiologies with different degrees are still unclear, and the corresponding translational application research is lacking. Therefore, an in-depth and systematic understanding of the characteristics and mechanisms of hepatic fibrosis reversal can not only enrich the understanding of the natural history of hepatic fibrosis and cirrhosis, but also provide reference for the development and clinical application of anti-hepatic fibrotic drugs.
Assuntos
Células Endoteliais , Hepatopatias , Fibrose , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/patologia , Cirrose Hepática/patologia , Hepatopatias/patologiaRESUMO
Oxidative stress, especially mediated by peroxynitrite (ONOO-), plays a key role in diabetes. Mitochondria, as the generating source of ONOO-, may also be the major damaging target of ONOO-. Whether ONOO--induced protein nitration is responsible for renal mitochondrial damage in diabetes is not fully known. This study was aimed to clarify the relationship between nitration of entire mitochondrial proteins induced by ONOO- and the renal mitochondrial damage in diabetes. Sprague-Dawley male rats were injected ip with streptozotocin to induce diabetes. After 10 weeks, inducible nitric oxide synthase (iNOS) mRNA expression and protein content in renal cortex were detected. Distribution of nitrotyrosine (NT), a specific marker of ONOO-, in renal cortex and NT content in mitochondrial proteins were detected. The ultrastructure of glomerulus was observed. Aminoguanidine was used as a selective inhibitor of iNOS to reduce the derivation of ONOO-. In diabetic rat, increasing levels of iNOS mRNA and protein content, and NT content were observed, in accord with the pathological alterations of glomerulus. In aminoguanidine group, these alterations were attenuated significantly. In conclusion, ONOO- could induce entire mitochondrial proteins nitration, responsible for the damage of renal mitochondria in diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Glomérulos Renais/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Ácido Peroxinitroso/administração & dosagem , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Imuno-Histoquímica , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/enzimologia , Glomérulos Renais/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácido Peroxinitroso/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
High glucose (HG) state is closely related to diabetic complications, and among these one main pathogenesis involves apoptosis of important functional cells. However, the mechanism of osteoblast (OB) apoptosis induced by HG state is not clear. Peroxynitrite (ONOO-), the strongest oxidant, can mediate apoptosis in various kinds of cells, including OB. Therefore, this study was aimed at investigating whether HG state could induce OB apoptosis through ONOO-. Cultured OB from rat calvariae explanted from E21 fetuses were treated with HG solution. The ratio of OB apoptosis and ONOOcontent was assayed by TUNEL, flow cytometry, and immunohistochemistry. The results showed that HG state could induce ONOO- overformation (p<0.01), and excessive OB apoptosis (p<0.001). However, the HG-induced OB apoptosis could be attenuated by peonol, a potent scavenger of ONOO-. In conclusion, HG state might trigger excessive ONOO- formation, mediating OB apoptosis.
Assuntos
Apoptose/fisiologia , Glucose/administração & dosagem , Osteoblastos/citologia , Osteoblastos/metabolismo , Ácido Peroxinitroso/fisiologia , Acetofenonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Sequestradores de Radicais Livres/farmacologia , Glucose/antagonistas & inibidores , Osteoblastos/efeitos dos fármacos , Ácido Peroxinitroso/antagonistas & inibidores , RatosRESUMO
BACKGROUND AND PURPOSE: While investigating the effects of systemic urotensin II (U-II), a potent vasoactive peptide acting at the UT receptor, we observed ear pinna flushing after systemic administration to conscious rats. In the present study, U-II-induced ear flushing was quantified in terms of ear pinna temperature change and potential mechanisms were explored. EXPERIMENTAL APPROACH: U-II-induced ear flushing was quantified by measuring lateral ear pinna temperature changes and compared to that of calcitonin gene-related peptide (CGRP), a known cutaneous vasodilator. Further, the effects of a variety of pharmacological agents on U-II-induced ear flushing were explored. KEY RESULTS: Subcutaneous injection of U-II (9 microg kg(-1))produced localized ear pinna flushing with an onset of approximately 15 min, a duration of approximately 30 min and a maximal temperature change of 9 degrees C. In contrast, CGRP caused cutaneous flushing within multiple cutaneous beds including the ear pinna with a shorter onset and greater duration than U-II. A potent UT receptor antagonist, urantide, blocked U-II-induced ear flushing but did not affect CGRP-induced ear flushing. Pretreatment with indomethacin or L-Nomega-nitroarginine methylester (L-NAME) abolished U-II-induced ear flushing. Mecamylamine or propranolol did not affect this response to U-II. Direct intracerebroventricular injection studies suggested that the ear flushing response to U-II was not mediated directly by the CNS. CONCLUSION AND IMPLICATIONS: Our results suggest that U-II-induced ear flushing and temperature increase is mediated by peripheral activation of the UT receptor and involves prostaglandin- and nitric oxide-mediated vasodilation of small capillary beds in the rat ear pinna.
Assuntos
Orelha Externa/irrigação sanguínea , Rubor/induzido quimicamente , Urotensinas/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Temperatura Corporal/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Inibidores Enzimáticos/farmacologia , Indometacina/farmacologia , Injeções Subcutâneas , Masculino , Mecamilamina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Antagonistas Nicotínicos/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Propranolol/farmacologia , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Urotensinas/administração & dosagem , Urotensinas/antagonistas & inibidores , Vasodilatadores/farmacologiaRESUMO
Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with the in vivo studies indicating that p53 blocks constitutive activation but not ligand-dependent stimulation. These studies provide insight into mechanisms by which unliganded nuclear hormone receptors can modulate gene expression and may provide an explanation for the mechanism of action of the v-erbA oncoprotein, a retroviral homolog of chicken T3R alpha.
Assuntos
Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Galinhas , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Técnicas In Vitro , Ligantes , Modelos Biológicos , Mutação , Regiões Promotoras Genéticas , Ratos , Transfecção , Tri-Iodotironina/metabolismoRESUMO
The mdm2 gene is positively regulated by p53 through a p53-responsive DNA element in the first intron of the mdm2 gene. mdm2 binds p53, thereby abrogating the ability of p53 to activate the mdm2 gene, and thus forming an autoregulatory loop of mdm2 gene regulation. Although the mdm2 gene is thought to act as an oncogene by blocking the activity of p53, recent studies indicate that mdm2 can act independently of p53 and block the G1 cell cycle arrest mediated by members of the retinoblastoma gene family and can activate E2F1/DP1 and the cyclin A gene promoter. In addition, factors other than p53 have recently been shown to regulate the mdm2 gene. In this article, we report that thyroid hormone (T3) receptors (T3Rs), but not the closely related members of the nuclear thyroid hormone/retinoid receptor gene family (retinoic acid receptor, vitamin D receptor, peroxisome proliferation activation receptor, or retinoid X receptor), regulate mdm2 through the same intron sequences that are modulated by p53. Chicken ovalbumin upstream promoter transcription factor I, an orphan nuclear receptor which normally acts as a transcriptional repressor, also activates mdm2 through the same intron region of the mdm2 gene. Two T3R-responsive DNA elements were identified and further mapped to sequences within each of the p53 binding sites of the mdm2 intron. A 10-amino-acid sequence in the N-terminal region of T3Ralpha that is important for transactivation and interaction with TFIIB was also found to be important for activation of the mdm2 gene response element. T3 was found to stimulate the endogenous mdm2 gene in GH4C1 cells. These cells are known to express T3Rs, and T3 is known to stimulate replication of these cells via an effect in the G1 phase of the cell cycle. Our findings, which indicate that T3Rs can regulate the mdm2 gene independently of p53, provide an explanation for certain known effects of T3 and T3Rs on cell proliferation. In addition, these findings provide further evidence for p53-independent regulation of mdm2 which could lead to the development of tumors from cells that express low levels of p53 or that express p53 mutants defective in binding to and activating the mdm2 gene.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Receptores dos Hormônios Tireóideos/metabolismo , Animais , Fator I de Transcrição COUP , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Íntrons , Ligantes , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-mdm2 , Proto-Oncogenes , Ratos , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta , Receptores X de Retinoides , Relação Estrutura-Atividade , Fator de Transcrição TFIIB , Fatores de Transcrição/metabolismo , Tri-Iodotironina/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LBD of RXRbeta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad or T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity. This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements. These studies indicate that the LBD has diverse functional roles in gene regulation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Família Multigênica , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas de Saccharomyces cerevisiae , Ativação Transcricional , Animais , Sítios de Ligação , Linhagem Celular , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Hipófise/metabolismo , Multimerização Proteica , RNA Mensageiro/metabolismo , Ratos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Tri-Iodotironina/farmacologiaRESUMO
The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.
Assuntos
Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/antagonistas & inibidores , Animais , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Proteína Vmw65 do Vírus do Herpes Simples/fisiologia , Humanos , Técnicas In Vitro , Ligantes , Substâncias Macromoleculares , Ratos , Receptores dos Hormônios Tireóideos/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
Oxidative stress has been implicated in protein phosphorylation and dephosphorylation in cells. In our current studies, H2O2 was shown to reversibly inhibit protein tyrosine phosphatase (PTPase) activity in HER14 cells. H2O2 (150 mM) resulted in 40% inhibition of PTPase activity by 15 min and recovery from inhibition was nearly complete by 60 min. H2O2-induced inhibition or recovery of PTPase activity was not affected by cycloheximide, a protein synthesis inhibitor. L-Buthionine-[S,R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, had no effect on H2O2-induced inhibition of PTPase activity but retarded the recovery of activity. Epidermal growth factor (EGF) and EGTA, a Ca2+ chelator, did not influence H2O2-induced inhibition or recovery of PTPase activity. These results suggest that at least 40% of fibroblast PTPase activity can be regulated by cellular redox activity.
Assuntos
Peróxido de Hidrogênio/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Animais , Linhagem Celular , Cicloeximida/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Radicais Livres , Humanos , Camundongos , OxirreduçãoRESUMO
We have identified a splice variant of human neutrophil collagenase (MMP-8) transcript (MMP-8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP-8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP-8, the translation product of MMP-8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP-8alt protein may lead to uncover novel roles it plays in cellular physiology.
Assuntos
Processamento Alternativo , Colagenases/genética , Sequência de Aminoácidos , Sequência de Bases , Condrócitos , Colagenases/metabolismo , Ativação Enzimática , Humanos , Metaloproteinase 8 da Matriz , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNA , Células U937RESUMO
Inside-out membrane patches excised from rat hippocampal neurons were used to test if ion channels could be formed by fragment 31-35 of amyloid beta-protein. The results showed: (1) after application of fragment 31-35 of amyloid beta-protein (5 microM) to either the inner or outer side of the patches, spontaneous currents could be recorded from those patches that had previously been 'silent'; (2) the fragment 31-35-induced conductance was cation-selective with a permeability ratio of P(Cs)/P(Cl)=23; (3) different levels of conductance, ranging from 25 to 500 pS, could be recorded in different patches, and in some cases, different conductances and spontaneous transitions among them could be recorded in a single patch; and (4) application of ZnCl(2) (1 mM) to the inner side of the patches reversibly blocked the newly formed channel activity; a similar effect was observed after application of CdCl(2) (1 mM). These results show that fragment 31-35 of amyloid beta-protein can insert into membrane patches from both sides and form cation-selective, Zn(2+)- and Cd(2+)-sensitive ion channels. It is proposed that fragment 31-35 in amyloid beta-protein might be the shortest active sequence known to date to form ion channels across neuronal membranes.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Canais Iônicos/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Cádmio/farmacologia , Cátions/metabolismo , Membrana Celular/metabolismo , Eletrofisiologia , Hipocampo/citologia , Técnicas In Vitro , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/fisiologia , Neurônios/ultraestrutura , Ratos , Ratos Wistar , Zinco/farmacologiaRESUMO
By using extracellular recordings of field potential, the exact pathway by which the endogenous ACh influencing the induction of long-term potentiation (LTP) in CA1 area was analysed in slices of rat hippocampus. The results showed that: (1) the application of (-) huperzine A, an AChE inhibitor extracted from Chinese herb Qian Ceng Ta (Huperzia Serrata), could enhance the induction of LTP, while this drug showed little effect on the second components of multiple population spikes that were recorded in Mg(2+)-free medium and had proven to be N-methyl-D-aspartate (NMDA) receptor-mediated response; and (2) scopolamine, a muscarinic receptor antagonist, could significantly suppressed the induction of LTP, while most of the suppressive effect of scopolamine was blocked when slices were pretreated by bicuculline, a gamma-aminobutyric acid (GABA(A)) receptor antagonist. These results suggest that endogenous ACh potentiates the induction of LTP through the inhibition of GABAergic interneurons that modulate pyramidal neurons, but not through the activation of NMDA receptors located on pyramidal neurons.
Assuntos
Acetilcolina/fisiologia , Inibidores da Colinesterase/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Sesquiterpenos/farmacologia , Alcaloides , Animais , Bicuculina/farmacologia , Estimulação Elétrica , Antagonistas GABAérgicos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Antagonistas Muscarínicos/farmacologia , Células Piramidais/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Escopolamina/farmacologiaRESUMO
To clarify the shortest essential active sequence in amyloid beta-protein (AbetaP) responsible for affecting neuronal electrophysiological properties, the effects of fragments 31-35 and 25-35 of AbetaP on the large conductance Ca(2+)-activated potassium (BK) channels were investigated in the "inside-out" membrane patches excised from hippocampal neurons of rats. After application of AbetaP 3l-35 (5 micromol/L, n=10), the mean P(o) and open frequency of BK channels decreased by 85.8+/-l3.5 percent;percent; (P<0.01) and 72.1+/-22.8 percent; (P<0.01), respectively, and the mean open time decreased by 41.l+/-l8.5 percent; (P<0.0l), while the mean current amplitude was not significantly affected (P>0.05). Application of AbetaP 25-35 (5 micromol/L) also induced a decrease of 85.5+/-22.l percent; (P<0.0l) in mean P(o) and of 5l.4+/-18.3 percent; (P<0.05) in mean open time within l~3 min after application. These results suggest that the functional alteration in BK channels elicited by AbetaP fragments may play an important role in the mechanisms underlying AbetaP neurotoxicity, and AbetaP 31-35 may be the shortest active sequence in AbetaP responsible for affecting the electrophysiological properties of neurons.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Hipocampo/citologia , Fragmentos de Peptídeos/farmacologia , Canais de Potássio Cálcio-Ativados/fisiologia , Animais , Cálcio/farmacologia , Separação Celular , Eletrofisiologia , Hipocampo/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Neurônios/fisiologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
We have applied microelectrode technique to record 118 spontaneously firing units from the hypothalamus in rats. Detection of the recording sites showed that 84 were in the paraventricular nucleus (PVN) and 34 were near the PVN (near-PVN). After intracerebroventricular (i.c.v.) administration of atrial natriuretic polypeptide (ANP), 91% (P less than 0.005) of the PVN neurones and 71% (P greater than 0.05) of near-PVN neurones sensitive to ANP showed a significant decrease in spontaneously firing rate. After i.c.v. administration of hypertonic NaCl solution, 64.7% (P less than 0.005) of the PVN neurones and 61.1% (P greater than 0.05) of near-PVN neurones showed a significant increase in firing rate. The results indicate that i.c.v. administration of ANP profoundly inhibits the electrical activity of the PVN neurones, but hypertonic NaCl solution markedly stimulates the PVN neurones.
Assuntos
Fator Natriurético Atrial/farmacologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Animais , Eletrofisiologia , Injeções Intraventriculares , Masculino , Microeletrodos , Núcleo Hipotalâmico Paraventricular/fisiologia , Ratos , Ratos EndogâmicosRESUMO
Alzheimer's disease (AD) is a progressive and degenerative disorder accompanied by cognitive impairment, but effective strategies against AD are currently not available. Interestingly, glucagon-like peptide-1 (GLP-1) used in type 2 diabetes mellitus (T2DM) has shown neuroprotective effects in preclinical studies of AD. Lixisenatide, an effective GLP-1 receptor (GLP-1R) agonist with much longer half life than GLP-1, has been licensed in the EU as a treatment for T2DM. However, the neuroprotective effects of lixisenatide in the brain remain to be clarified. In the present study, we report for the first time the effects of lixisenatide on the amyloid ß (Aß) protein-induced impairments in spatial learning and memory of rats, and investigated its electrophysiological and molecular mechanisms. We found that: (1) bilateral intrahippocampal injection of Aß25-35 resulted in a significant decline in spatial learning and memory of rats, as well as a suppression of in vivo hippocampal long-term potentiation (LTP); (2) lixisenatide treatment effectively prevented the Aß25-35-induced impairments; (3) lixisenatide inhibited the Aß25-35 injection-induced activation of glycogen synthase kinase 3ß (GSK3ß), with a significant increase in the phosphorylation of ser9 and a significant decrease in the phosphorylation of Y216. These results indicate that lixisenatide, by affecting the PI3K-Akt-GSK3ß pathway, can prevent Aß-related impairments in synaptic plasticity and spatial memory of rats, suggesting that lixisenatide may be a novel and effective treatment for AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Memória Espacial/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Animais , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Potenciação de Longa Duração/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/fisiopatologia , Fosforilação/efeitos dos fármacos , Distribuição Aleatória , Ratos Sprague-Dawley , Receptores de Glucagon/agonistas , Receptores de Glucagon/metabolismo , Memória Espacial/fisiologiaRESUMO
Amyloid ß protein (Aß) is considered to be partly responsible for the impairment of learning and memory in Alzheimer disease (AD). In addition, it has been found recently that type 2 diabetes mellitus (T2DM) is a risk factor for developing AD. One promising treatment for AD is using analogues for the insulin-release facilitating gut hormone glucagon-like peptide-1 (GLP-1) that has been developed as a T2DM therapy. GLP-1 has been shown to have neuroprotective properties. However, if GLP-1 can protect the late phase-long term potentiation (L-LTP) and related cognitive function against Aß-induced impairment it is still an open question. To further characterize the neuroprotective function of GLP-1 in the brain, we investigated the effects of i.c.v. injected Val(8)-GLP-1(7-36) on the Aß fragment-induced impairment of in vivo hippocampal L-LTP and spatial learning and memory in rats. The results showed that (1) Aß1-40 (5 nmol) injection did not affect the baseline field excitatory postsynaptic potentials (fEPSPs), but significantly suppressed multiple high frequency stimulation (HFS)-induced L-LTP in hippocampal CA1 region; (2) Val(8)-GLP-1(7-36) (0.05 pmol) administration alone did not affect the baseline synaptic transmission and the maintenance of L-LTP; (3) pretreatment with Val(8)-GLP-1(7-36) (0.05 pmol) effectively prevented Aß1-40-induced deficit of L-LTP; (4) i.c.v. injection of 5 nmol Aß1-40 resulted in a significant decline learning a spatial Morris water maze (MWM) test; (5) Val(8)-GLP-1(7-36) (0.05 pmol) administration alone did not affect spatial learning in this task, while pretreatment with Val(8)-GLP-1(7-36) effectively reversed the impairment of spatial learning and memory induced by Aß1-40. At the same time, the swim speeds and escape latencies of rats among all groups in the visible platform tests did not show any difference. These results suggest that increase of GLP-1 signalling in the brain may be a promising strategy to ameliorate the degenerative processes observed in AD.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Região CA1 Hipocampal/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Região CA1 Hipocampal/fisiologia , Potenciais Pós-Sinápticos Excitadores , Masculino , Memória/efeitos dos fármacos , Ratos , Ratos Wistar , Natação , Transmissão Sináptica/efeitos dos fármacos , Visão Ocular/efeitos dos fármacosRESUMO
Amyloid beta protein (Abeta) is thought to be responsible for the deficit of learning and memory in Alzheimer's disease (AD), possibly through interfering with synaptic plasticity such as hippocampal long-term potentiation (LTP). Nicotinic acetylcholine receptors (nAChRs) participate in various cognitive brain functions. However, it is unclear whether nAChRs, especially alpha4beta2 subtype nAChRs, are involved in Abeta-induced impairment of hippocampal LTP. The present study investigates a possible role of nAChRs during the impairment of LTP by Abeta. Our results showed that: (1) intracerebroventricular injection of Abeta(1-40), Abeta(25-35) or Abeta(31-35) significantly suppressed high-frequency stimulation-induced LTP, while Abeta(35-31), a reversed sequence of Abeta(31-35), have no effect on the LTP; (2) epibatidine, a specific agonist of alpha4beta2 subtype of nAChRs, dose-dependently suppressed the induction of LTP; (3) co-injection of epibatidine together with Abeta(31-35) did not further enhance the suppression of LTP induced by Abeta(31-35) or epibatidine alone; (4) dihydro-beta-erythroidine, a selective antagonist against alpha4beta2 subtype of nAChRs, showed no effect on the induction of LTP, but significantly reversed Abeta(31-35)-induced LTP impairment. These results indicate that: (1) sequence 31-35 in Abeta molecule might be a shorter active center responsible for the neurotoxicity of full length of Abeta; (2) alpha4beta2 subtype of nAChRs is required for the suppressive action of Abeta on the hippocampal LTP in vivo. Thus, the present study provides further insight into the mechanisms by which Abeta impairs synaptic plasticity and cognitive function in the AD brain.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Hipocampo , Potenciação de Longa Duração/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores Nicotínicos/metabolismo , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/genética , Animais , Compostos Bicíclicos Heterocíclicos com Pontes , Di-Hidro-beta-Eritroidina/farmacologia , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/anatomia & histologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Potenciação de Longa Duração/fisiologia , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Agonistas Nicotínicos/farmacologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Piridinas , Ratos , Ratos Wistar , Receptores Nicotínicos/genéticaRESUMO
AIMS/HYPOTHESIS: The aim of this study was to determine the effects of preconditioning on injury and expression of heat shock proteins 70 in diabetic rat hearts. METHODS: Diabetes was induced by an intraperitoneal injection of 65 mg kg(-1) streptozotocin. Daily subcutaneous injection of 4 IU insulin started 2 weeks after streptozotocin treatment for 4 weeks. Rats were preconditioned by intravenous injection of 10 mg kg(-1) U50,488H, a selective kappa-opioid receptor agonist (U50,488H preconditioning). The effects of U50,488H preconditioning had previously been shown to be blocked by a selective kappa-opioid receptor antagonist, nor-binaltorphimine. Twenty-four hours later, rats were subjected to 30 min of regional ischaemia by occlusion of the left coronary artery followed by 4 h of reperfusion. Infarct size was determined at the end of reperfusion. Stress-inducible and constitutive heat shock proteins 70 were analysed at the end of ischaemia and reperfusion by Western blotting. RESULTS: Myocardial infarcts induced by ischaemia and reperfusion were greater in diabetic rats. U50,488H preconditioning significantly reduced the infarct size and increased the expression of stress-inducible heat-shock protein 70 in normal rats. The effects of U50,488H preconditioning were abolished in streptozotocin-induced diabetic rats, but restored by insulin replacement. CONCLUSION/INTERPRETATION: In addition to a greater susceptibility to ischaemic insults, the delayed cardioprotection of U50,488H preconditioning was lost, which could at least partly be due to impaired synthesis of stress-inducible heat-shock protein 70 in diabetic rats.
Assuntos
Cardiotônicos/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Proteínas de Choque Térmico HSP70/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Receptores Opioides kappa/antagonistas & inibidores , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Western Blotting , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSC70 , Coração/efeitos dos fármacos , Coração/fisiopatologia , Insulina/farmacologia , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
It has been demonstrated that atrial natriuretic polypeptide (ANP), or atriopeptin, is synthesized and stored not only in the cardiac atrial myocyte but also in the central nervous system, especially in the hypothalamus. ANP may play an important role in the regulation of fluid and electrolyte balance and blood pressure in the peripheral and central nervous systems. Some of the biological actions of ANP are opposite to those of angiotensin II and arginine vasopressin in the central nervous system. However, the mechanism of action of ANP remains, in a large part, to be defined.