RESUMO
A recent genome-wide association meta-analysis for Alzheimer's disease (AD) identified 19 risk loci (in addition to APOE) in which the functional genes are unknown. Using Drosophila, we screened 296 constructs targeting orthologs of 54 candidate risk genes within these loci for their ability to modify Tau neurotoxicity by quantifying the size of >6000 eyes. Besides Drosophila Amph (ortholog of BIN1), which we previously implicated in Tau pathology, we identified p130CAS (CASS4), Eph (EPHA1), Fak (PTK2B) and Rab3-GEF (MADD) as Tau toxicity modulators. Of these, the focal adhesion kinase Fak behaved as a strong Tau toxicity suppressor in both the eye and an independent focal adhesion-related wing blister assay. Accordingly, the human Tau and PTK2B proteins biochemically interacted in vitro and PTK2B co-localized with hyperphosphorylated and oligomeric Tau in progressive pathological stages in the brains of AD patients and transgenic Tau mice. These data indicate that PTK2B acts as an early marker and in vivo modulator of Tau toxicity.
Assuntos
Quinase 2 de Adesão Focal/genética , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Animais , Biomarcadores , Modelos Animais de Doenças , Drosophila/genética , Quinase 2 de Adesão Focal/metabolismo , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Fatores de Risco , Proteínas tau/genéticaRESUMO
The peptidyl prolyl cis-trans isomerase Pin1 and the Inhibitor of Apoptosis Protein (IAP) Survivin are two major proteins involved in cancer. They both modulate apoptosis, mitosis, centrosome duplication and neuronal development but until now no functional relationship has been reported between these two proteins. We tested Pin1-induced regulation of Survivin in neuroblastoma cells. Pin1 overexpression in SY5Y neuroblastoma cells decreased Survivin levels. Immunocytochemical studies indicated that they partially co-localized in interphase and mitotic cells. Co-immunoprecipitation further demonstrates the existence of a Pin1/Survivin complex. Pin1-induced effect on Survivin was confirmed in COS cells. RT-PCR and mutagenesis experiments suggested that this Pin1-induced decrease of Survivin occurred at the protein level. Survivin downregulation depended on the binding ability of Pin1 but was not related to the single Thr-Pro site, suggesting an indirect relationship into a protein complex. Finally, this functional regulation of Survivin by Pin1 is reciprocal since Pin1 silencing led to an increase in Survivin levels. The characterization of this functional relationship between Pin1 and Survivin might help to better understand mitosis control and cancer mechanisms.
Assuntos
Regulação para Baixo , Proteínas Inibidoras de Apoptose/metabolismo , Peptidilprolil Isomerase/metabolismo , Animais , Sítios de Ligação , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA Complementar , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Peptidilprolil Isomerase/genética , Testes de Precipitina , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
In this work in vitro pharmacological profiles of two analogues of the C-terminal heptapeptide of cholecystokinin (CCK) were evaluated. The analogue Boc-[Nle28, Nle31]-CCK-7, a stable analogue of CCK-8, has the same activity profile as CCK-8, and was found to be very potent in stimulating amylase secretion, phospholipid breakdown and [Ca2+]i mobilization from rat pancreatic acini. It can be used as a probe for studying CCK-actions. The CCK-analogue Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester, (JMV180), which stimulates amylase secretion without inhibition at supramaximal concentrations, has different effects on phospholipid hydrolysis and [Ca2+]i mobilization, compared to CCK-8 and Boc-[Nle28, Nle31]-CCK-7. Compound JMV180 was unable to significantly promote phospholipid breakdown, and was only 50%-60% as efficacious as Boc-[Nle28, Nle31]-CCK-7 in promoting [Ca2+]i mobilization. These findings suggest that low affinity CCK-receptors might be responsible for the supra-maximal inhibition of amylase secretion, and are correlated with phospholipid breakdown and maximal [Ca2+]i mobilization.
Assuntos
Pâncreas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sincalida/análogos & derivados , Sincalida/farmacologia , Amilases/metabolismo , Animais , Cálcio/metabolismo , Técnicas In Vitro , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Masculino , Pâncreas/metabolismo , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos , Receptores da Colecistocinina/efeitos dos fármacos , Receptores da Colecistocinina/metabolismoRESUMO
Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.
Assuntos
Células Cromafins/fisiologia , Exocitose/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Actinas/fisiologia , Actinas/ultraestrutura , Animais , Grânulos Cromafim/fisiologia , Fusão de Membrana , Proteínas do Tecido Nervoso/metabolismo , Transdução de SinaisRESUMO
Syntheses of some partially modified retro-inverso analogues of the C-terminal octa- or heptapeptide of cholecystokinin are described. These analogues (in which the C-terminal carboxamide was deleted or not) were obtained by reverting one or several peptide bonds in the parent molecule. All these compounds were able to inhibit binding of labeled CCK-8 to rat pancreatic acini and guinea pig brain membranes and to stimulate amylase release from rat pancreatic acini with various potencies. Some of these derivatives reproduce only part of the biological response of CCK on amylase release.
Assuntos
Colecistocinina/análogos & derivados , Colecistocinina/síntese química , Receptores da Colecistocinina/efeitos dos fármacos , Sequência de Aminoácidos , Amilases/metabolismo , Animais , Encéfalo/metabolismo , Membrana Celular/metabolismo , Colecistocinina/farmacologia , Cobaias , Cinética , Masculino , Dados de Sequência Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/enzimologia , Pâncreas/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Ratos , Receptores da Colecistocinina/metabolismo , Sincalida/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of pseudopeptide analogues of the C-terminal heptapeptide of cholecystokinin in which each peptide bond, one at a time, has been replaced by a CH2NH bond were synthesized: Z-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp psi-(CH2NH)Phe-NH2 (1), Z-Tyr(SO3-)-Nle-Gly-Trp-Nle psi (CH2NH)Asp-Phe-NH2 (2), Z-Tyr(SO3-)-Nle-Gly-Trp psi-(CH2NH)Nle-Asp-Phe-NH2 (3), Z-Tyr(SO3-)-Nle-Gly psi(CH2NH)Trp-Nle-Asp-Phe-NH2 (4), Z-Tyr(SO3-)-Nle psi-(CH2NH)Gly-Trp-Nle-Asp-Phe-NH2 (5), Z-Tyr(SO3-)-Met-Gly-Trp-Nle-Asp psi (CH2NH)Phe-NH2 (6), Z-Tyr-(SO3-)-Met-Gly-Trp-Nle psi (CH2NH)Asp-Phe-NH2 (7), Z-Tyr(SO3-)-Met-Gly-Trp psi (CH2NH)Nle-Asp-Phe-NH2 (8). These derivatives were studied for their ability to stimulate amylase release from rat pancreatic acini and to inhibit the binding of labeled CCK-9 to rat pancreatic acini and to guinea pig brain membrane CCK receptors. They were compared to the potent CCK-8 analogue Boc-Asp-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2. All of these pseudopeptides were able to stimulate amylase secretion with the same efficacy as CCK-8 but with varying potencies. These compounds were also potent in inhibiting the binding of labeled CCK-9 to CCK receptors from rat pancreatic acini and from guinea pig brain membranes.
Assuntos
Colecistocinina/análogos & derivados , Oligopeptídeos/farmacologia , Amilases/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Membrana Celular/metabolismo , Fenômenos Químicos , Química , Colecistocinina/metabolismo , Cobaias , Masculino , Oligopeptídeos/síntese química , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Endogâmicos , Receptores da Colecistocinina/metabolismo , Sincalida/análogos & derivados , Sincalida/farmacologia , Relação Estrutura-AtividadeRESUMO
Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester (JMV180), an analog of the C-terminal octapeptide of cholecystokinin (CCK-8), shows interesting biological activities behaving as an agonist at the high-affinity CCK binding sites and as an antagonist at the low-affinity CCK binding sites in rat pancreatic acini. Although we did not observe any major hydrolysis of the ester bond of Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester in our in vitro studies, we were aware of a possible and rapid cleavage of this ester bond during in vivo studies. To improve the stability of Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester, we decided to synthesize analogs in which the ester bond would be replaced by a carba (CH2-CH2) linkage. We synthesized the 3-amino-7-phenylheptanoic acid (beta-homo-Aph) with the R configuration in order to mimic the Asp-2-phenylethyl ester moiety and the 3-amino-6-(phenyloxy)hexanoic acid (H-beta-homo-App-OH), an analog of H-beta-homo-Aph-OH in which a methylene group has been replaced by an oxygen. (R)-beta-Homo-Aph and (R)-H-beta-homo-App-OH were introduced in the CCK-8 sequence to produce Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-(R)-beta-homo-Aph-OH and Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-(R)-beta-homo-App-OH. Both compounds were able to recognize the CCK receptor on rat pancreatic acini (IC50 = 12 +/- 8 nM and 13 +/- 5 nM, respectively), on brain membranes (IC50 = 32 +/- 2 nM and 57 +/- 5 nM, respectively), and on Jurkat T cells (IC50 = 75 +/- 15 nM and 65 +/- 21 nM, respectively). Like Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester, both compounds produced maximal stimulation of amylase secretion (EC50 = 6 +/- 2 nM and 4 +/- 2 nM, respectively) with no decrease of the secretion at high concentration indicating that these compounds probably act as agonists at the high-affinity peripheral CCK-receptor and as antagonists at the low-affinity CCK-receptor. Replacing the tryptophan by a D-tryptophan in such analogs produced full CCK-receptor antagonists. All these analogs might be more suitable for in vivo studies than Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester.
Assuntos
Caproatos/química , Colecistocinina/análogos & derivados , Ácidos Heptanoicos/química , Sequência de Aminoácidos , Amilases/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Membrana Celular/metabolismo , Cobaias , Espectroscopia de Ressonância Magnética , Masculino , Dados de Sequência Molecular , Estrutura Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Ratos , Receptores da Colecistocinina/metabolismo , Sincalida/análogos & derivados , Sincalida/química , Sincalida/metabolismo , Sincalida/farmacologiaRESUMO
Supramaximal doses of cholecystokinin (CCK) induce in vitro submaximal biological responses (i.e., smaller by 50% than the response to a maximal dose of CCK), desensitization and residual stimulation, and in vivo secretory inhibition and edematous pancreatitis. It has been reported previously that supramaximal doses of Boc-[Nle28-Nle31]CCK(27-32)/- phenylethylester (JMV 180) do not produce these effects. The aim of this study was to analyze the in vivo response of pancreatic secretion of the rat to a wide dose range of Boc-[Nle28-Nle31]CCK(26-33) (JMV118), an analog of CCK8 with the same activity spectrum as CCK8, to JMV180 and to Boc-[Nle28-Nle31]CCK(27-32)-phenylethylamide (JMV170). The three peptides were administered as intravenous infusions and as bolus intravenous injections. In the case of infusions, the same maximal effect was observed with all three peptides. It was obtained with 22.5 pmol/kg.min of JMV118; JMV180 and JMV170 were about 700 times less potent. In the case of bolus injections, the maximal response to JMV118 was observed with 450 pmol/kg, and the response peaked 10-15 min after the injection. Higher doses of JMV118 induced a secretory peak that was smaller and delayed relative to the moment of injection. JMV180 and JMV170 were about 500 times less potent: the maximal response was observed with 218700 pmol/kg and peaked 10-15 min after the injection. Larger doses of JMV180 and JMV170 produced neither supramaximal inhibition nor a delayed peak response, but induced a sustained stimulation of pancreatic secretion that could last more than 3 h after the injection.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Pâncreas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sincalida/análogos & derivados , Animais , Infusões Intravenosas , Injeções Intravenosas , Masculino , Pâncreas/metabolismo , Ratos , Ratos Wistar , Sincalida/farmacologiaRESUMO
Activation of the P2Y purinoceptor on turkey erythrocytes results in a G11-mediated activation of a phospholipase C-beta isoenzyme and hydrolysis of polyphosphoinositides. The role of the protein kinase C and Ca(2+)-mobilizing arms of the inositol lipid signalling cascade in P2Y purinoceptor-induced desensitization of phospholipase C has been examined using erythrocytes as a model system. Preincubation of intact erythrocytes with either P2Y purinoceptor agonist, ADP beta S, or the protein kinase C-activating phorbol ester, phorbol 12-myristate, 13 acetate (PMA), resulted in a time of preincubation-dependent decrease in guanine nucleotide-, P2Y purinoceptor-, and beta-adrenoceptor-stimulated phospholipase C activities in membranes isolated from these cells. The extent of heterologous desensitization induced by ADP beta S and PMA were additive suggesting that they did not share a common mechanism. A lack of involvement of activation of protein kinase C in P2Y purinoceptor-induced heterologous desensitization was further supported by the observation that although protein kinase C inhibitors or down-regulation of protein kinase C resulted in a loss of PMA-induced desensitization, neither treatment affected the extent of P2Y purinoceptor-induced desensitization. In addition, elevation of intracellular Ca2+ or prevention of its elevation did not induce heterologous desensitization and had no effect on the desensitization induced by ADP beta S. Thus, neither the protein kinase C nor Ca2+ mobilizing arms of the inositol lipid signalling pathway appear to be involved in P2Y purinoceptor promoted heterologous desensitization of phospholipase C. These results are consistent with the existence of a novel feedback pathway for agonist-induced heterologous desensitization of a second messenger generating enzyme. Preincubation of cells with ADP beta S or the beta-adrenoceptor agonist, isoproterenol, followed by rechallenge with each of the receptor agonists revealed that receptor-specific desensitization occurs in addition to heterologous desensitization. Thus, multiple mechanisms account for agonist-induced desensitization of the inositol lipid signalling system of turkey erythrocytes.
Assuntos
Eritrócitos/metabolismo , Receptores Purinérgicos/fisiologia , Fosfolipases Tipo C/metabolismo , Animais , Relação Dose-Resposta a Droga , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/farmacologia , Isoproterenol/farmacologia , Receptores Purinérgicos/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo , Perus , Fosfolipases Tipo C/fisiologiaRESUMO
The interaction of the cyclic AMP and inositol lipid signalling systems was studied in turkey erythrocytes. Elevation of intracellular cyclic AMP concentrations by pretreatment of the cells with forskolin or 8-Br-cAMP resulted in a marked decrease in responsiveness of phospholipase C to G-protein activators in membranes prepared from treated cells. Decreases in responsiveness occurred with a t1/2 of approximately 5 min and were reversible after transfer of desensitized cells to drug-free medium. Pretreatment of the cells with forskolin inhibited inositol phosphate formation in a concentration-dependent manner and addition of the phosphodiesterase inhibitor IBMX 93-isobutyl-1-methylxanthine) during pretreatment increased the capacity of forskolin to desensitize phospholipase C activity. IBMX also produced a similar potentiation of forskolin-stimulated accumulation of cyclic AMP in turkey erythrocytes. Isoproterenol pretreatment of the cells induced, like forskolin, partial inhibition of inositol phosphate generation in response to G-protein activators and to P2y purinoceptor and beta-adrenoceptor agonists. The capacity of isoproterenol to induce desensitization of phospholipase C activity also was increased by the presence of IBMX during pretreatment of the cells. H8 (N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide), an inhibitor of cyclic AMP-regulated protein kinase, completely prevented forskolin-induced desensitization but only partially blocked isoproterenol-induced desensitization. These results indicate that the cyclic AMP signalling cascade has a major inhibitory influence on receptor- and G-protein-activated inositol lipid signaling.
Assuntos
AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Fosfolipases Tipo C/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Colforsina/farmacologia , Interações Medicamentosas , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Isoproterenol/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Simpatomiméticos/farmacologia , PerusRESUMO
Preliminary studies on CCK receptors in the central nervous system were carried out on guinea pig cerebral cortical synaptoneurosome preparations. In binding assays, the range of affinity of CCK-8, Boc-[Nle28,Nle31]CCK-7, a potent CCK analog, Boc-[Leu31]CCK-4 and of the two benzodiazepine CCK receptor antagonists L-365,260 and MK-329, is in agreement with the presence of CCKB receptors on this model. The effects of Boc-[Nle28,Nle31]CCK-7 on inositol phosphates, cAMP accumulation and 45Ca2+ efflux were investigated. Neither inositol phosphate nor cAMP accumulations could be observed. On the other hand, evidence of Boc-[Nle28,Nle31]CCK-7-, CCK-8- and Boc-[Leu31]CCK-4-induced 45Ca2+ efflux was found in a dose-dependent manner. The CCKB-selective receptor antagonist L-365,260 and, with a weaker efficiency, the CCKA-selective receptor antagonist MK-329, are able to block a maximal effect of Boc-[Nle28,Nle31]CCK-7-induced 45Ca2+ efflux, suggesting that CCKB receptors may regulate calcium ion mobilization.
Assuntos
Cálcio/metabolismo , Córtex Cerebral/efeitos dos fármacos , Compostos de Fenilureia , Receptores da Colecistocinina/metabolismo , Sinaptossomos/efeitos dos fármacos , Animais , Benzodiazepinonas/metabolismo , Benzodiazepinonas/farmacologia , Sítios de Ligação , Córtex Cerebral/metabolismo , Colecistocinina/análogos & derivados , Colecistocinina/metabolismo , Colecistocinina/farmacologia , AMP Cíclico/metabolismo , Devazepida , Cobaias , Fosfatos de Inositol/metabolismo , Masculino , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Sincalida/análogos & derivados , Sincalida/metabolismo , Sincalida/farmacologia , Sinaptossomos/metabolismo , Tetragastrina/análogos & derivados , Tetragastrina/metabolismo , Tetragastrina/farmacologiaRESUMO
The cholecystokinin octapeptide (CCK8)-derived synthetic peptides Boc-Tyr(SO3H)-Nle-Gly-DTrp-Nle-Asp-O-CH2-CH2-C6H5 (JMV179) and Boc-Tyr(SO3H)-Nle-Gly-DTrp-Nle-Asp-NH-CH2-CH2-C6H5 (JMV167) are antagonists of peripheral cholecystokinin (CCK) receptors in vitro. In the present study, antagonist activity of these peptides was studied on rat pancreatic secretion in vivo, and compared to those of other peptidic molecules and to the non-peptidic antagonists L364718, D-, L-, DL-lorglumide, and proglumide. The decreasing order of antagonist potencies on amylase release in vitro was L364718 greater than JMV179 greater than lorglumide greater than JMV167 greater than proglumide; JMV179 was 25 times less potent than L364718 and 300 times more potent than JMV167. The decreasing order of antagonist potencies on protein output in pancreatic juice in vivo was L364718 greater than JMV167 greater than JMV179 greater than lorglumide greater than proglumide; JMV167 was two times more potent than JMV179 and only 8 times less potent than L364718. Increased potency of JMV167, relative to JMV179 under in vivo conditions, is probably due to the slower rate of catabolism of the phenylethylamide group, relative to the phenylethylester group, since the metabolite issued from hydrolysis of the ester bond was totally inactive. This study shows that it is possible to obtain peptidic CCK antagonists, which are active and potent in vivo, and provides a quantitative measurement of potency changes occurring in vivo for several peptidic and non-peptidic antagonists.
Assuntos
Pâncreas/metabolismo , Receptores da Colecistocinina/antagonistas & inibidores , Sincalida/análogos & derivados , Sequência de Aminoácidos , Amilases/metabolismo , Animais , Benzodiazepinonas/farmacologia , Devazepida , Masculino , Dados de Sequência Molecular , Pâncreas/efeitos dos fármacos , Suco Pancreático/metabolismo , Proglumida/análogos & derivados , Proglumida/farmacologia , Ratos , Ratos Endogâmicos , Sincalida/química , Sincalida/farmacologiaRESUMO
Analogues of the C-terminal octapeptide of cholecystokinin (CCK) modified in the Met28-Gly29 region, were tested for their ability to interact with peripheral cholecystokinin receptors on rat pancreatic acini and to stimulate amylase secretion. These analogues were further evaluated for their ability to recognize central CCK receptors on guinea pig brain membranes. The behavioral effect of these analogues was also tested after intrastriatal injection into mice. It appeared that these analogues were full CCK agonists in the peripheral system. Although some induced dopaminomimetic effects after intrastriatal injection into mice, being as potent as the C-terminal octapeptide of cholecystokinin (CCK-8), others did not have any effect and were able to antagonize CCK-8 actions in the striatum. The results of this study confirm that one can obtain very potent CCK analogues by modifying the peptide bond between Met28 and Gly29, and that this modification can produce either CCK agonists or antagonists of CCK-induced dopamine transmission in the striatum.
Assuntos
Colecistocinina/análogos & derivados , Colecistocinina/farmacologia , Sequência de Aminoácidos , Amilases/metabolismo , Animais , Encéfalo/metabolismo , Colecistocinina/química , Corpo Estriado , Dopamina/fisiologia , Cobaias , Injeções , Masculino , Camundongos , Dados de Sequência Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/enzimologia , Ratos , Ratos Endogâmicos , Receptores da Colecistocinina/efeitos dos fármacos , Receptores da Colecistocinina/metabolismoRESUMO
New analogues of cholecystokinin-7 (CCK-7) modified at amino acid residues 5 and 7 were assayed for their effect on gall bladder, pancreatic secretion, food intake (anorectic activity), amount of rearing (sedative activity) and analgesia, as well as their ability to inhibit 125I-CCK-8 binding to pancreatic cell membrane receptors and brain membrane receptors. The results were compared to the activities of standard compounds, CCK-8, cerulein, BOC-CCK-7 (BOC = tertbutyloxycarbonyl) and BOC-[Nle2,Nle5]CCK-7. All analogues exhibited agonistic effects. Their anorectic activity was significantly prolonged.
Assuntos
Amilases/metabolismo , Anorexia/induzido quimicamente , Colecistocinina/análogos & derivados , Pâncreas/efeitos dos fármacos , Analgesia , Animais , Colecistocinina/metabolismo , Relação Dose-Resposta a Droga , Vesícula Biliar/efeitos dos fármacos , Cobaias , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores da Colecistocinina/efeitos dos fármacosRESUMO
Structure-activity relationship on cholecystokinin is presented. C-terminal heptapeptide analogues of cholecystokinin exhibiting selective agonist or antagonist activities were synthesized and their biological and pharmacological properties studied. We showed that: 1) Suppression of the C-terminal phenylalanine residue leads to peripheral as well as central cholecystokinin receptor antagonists; 2) Suppression of the C-terminal amide function produces "partial agonists" exhibiting interesting biological and pharmacological activities; 3) Replacement of L-tryptophan residue by D-tryptophan in such "partial agonist analogues" resulted in potent CCK receptor antagonists; 4) The peptide bond between methionine28 and glycine29, as well as the glycine residue are quite significant for the central biological activity; 5) It is possible to obtain highly potent and selective CCK analogues for the central receptor (CCK-B) by cyclization including the C-terminal tetrapeptide. Synthesis and pharmacological studies with these analogues have allowed to precise the significance of some amino acid residues as well as of some peptide bonds. They are significant pharmacological tools for the study of CCK-A (peripheral) and CCK-B (central) receptors, their biological actions and their associated intracellular messengers.
Assuntos
Colecistocinina/análogos & derivados , Sequência de Aminoácidos , Animais , Colecistocinina/síntese química , Dados de Sequência Molecular , Ratos , Relação Estrutura-AtividadeAssuntos
Amilases/metabolismo , Pâncreas/metabolismo , Receptores da Colecistocinina/metabolismo , Sincalida/análogos & derivados , Sequência de Aminoácidos , Animais , Cinética , Dados de Sequência Molecular , Pâncreas/efeitos dos fármacos , Ratos , Receptores da Colecistocinina/efeitos dos fármacos , Sincalida/síntese química , Sincalida/metabolismo , Sincalida/farmacologia , Relação Estrutura-AtividadeRESUMO
So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.
Assuntos
Receptores da Colecistocinina/efeitos dos fármacos , Sincalida/análogos & derivados , Amilases/metabolismo , Animais , Membrana Celular/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Colecistocinina/análogos & derivados , Colecistocinina/antagonistas & inibidores , Cobaias , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Receptores da Colecistocinina/metabolismo , Sincalida/farmacologiaRESUMO
Cholecystokinin (CCK) analogues (JMV310, JMV320, JMV328, and JMV332), obtained by side chain to side chain cyclization of a lysine residue in position 28 with a lysine residue in position 31, were found to be highly selective for the brain CCK receptor (CCK-B receptor), both in guinea pig and rat. In these analogues, the C-terminal tetrapeptide region of the molecule, which is the crucial determinant for binding to CCK-B receptors, has been constrained by cyclization. These analogues were highly potent in inhibiting binding of labeled CCK-8 to rat and guinea pig brain membranes (apparent affinity in the nanomolar range) but were poorly efficacious in inhibiting binding of labeled CCK-8 to rat or guinea pig pancreatic acini. In agreement with their low affinity for the pancreatic receptor, these CCK analogues were not very potent in stimulating amylase secretion. These cyclic CCK analogues were demonstrated to be highly selective for the brain CCK receptors.
Assuntos
Encéfalo/metabolismo , Colecistocinina/análogos & derivados , Receptores da Colecistocinina/metabolismo , Amilases/metabolismo , Animais , Sítios de Ligação , Colecistocinina/metabolismo , Colecistocinina/farmacologia , Relação Dose-Resposta a Droga , Cobaias , Masculino , Ratos , Ratos Endogâmicos , Sincalida/metabolismoRESUMO
Cyclic CCK analogues in which positions 28 and 31 have been replaced by lysine residues and whose side chains are bridged by a succinic moiety, were synthesized. They were tested for their ability to inhibit the binding of 125I-BH-CCK-8 to isolated rat pancreatic acini and to guinea pig brain membranes. These cyclic CCK-analogues were compared to the potent CCK analogue Boc-[Nle28,31]-CCK-7 and to Boc-Trp-Leu-Asp-Phe-NH2, analogue of CCK-4. These cyclic compounds appeared to be highly selective for central CCK receptors.
Assuntos
Colecistocinina/análogos & derivados , Receptores da Colecistocinina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Encéfalo/metabolismo , Fenômenos Químicos , Química , Colecistocinina/síntese química , Colecistocinina/metabolismo , Ciclização , Cobaias , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Estrutura Molecular , Pâncreas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Dynamin proteins have been implicated in many aspects of endocytosis, including clathrin-mediated endocytosis, internalization of caveolae, synaptic vesicle recycling, and, more recently, vesicular trafficking to and from the Golgi complex. To provide further insight into the function(s) of dynamin in neuroendocrine cells, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation, immunoreplica analysis, and confocal immunofluorescence. We found that dynamin, presumably the dynamin-2 isoform, is associated specifically with the membrane of purified secretory chromaffin granules. Oligomerization state analysis by sucrose density velocity gradients indicated that the granule-associated dynamin is in a monomeric form. Immunoprecipitation experiments coupled to double-labeling immunofluorescence cytochemistry revealed that the granular dynamin is associated with a syntaxin component that is not involved in the granule-bound SNARE complex. The possibility that dynamin participates in the coupling of the exocytotic and endocytotic reaction through the building of a granular membrane subset of proteins is discussed.