RESUMO
Analogues of bombesin which incorporate dipeptide or turn mimetics have been synthesized. One of them (compound 11) containing a seven-membered lactam ring revealed a good affinity for GRP/BN receptors on rat pancreatic acini (K(i) value of 1.7 +/- 0.4 nM) and on Swiss 3T3 cells (K(i) value of 1.0 +/- 0.2 nM). On the basis of this observation, antagonists containing the same dipeptide mimic were obtained by modification of the C-terminal part of the bombesin analogues. The most potent constrained compounds (15 and 17) were able to antagonize 1 nM bombesin-stimulated amylase secretion from rat pancreatic acini with high potency (K(i) = 21 +/- 3 and 3.3 +/- 1.0 nM, respectively) and 10(-7) M bombesin-stimulated ¿(3)Hthymidine incorporation into Swiss 3T3 cells (K(i) = 7.8 +/- 2. 0 and 0.5 +/- 0.1 nM, respectively).
Assuntos
Bombesina/análogos & derivados , Bombesina/síntese química , Oligopeptídeos/síntese química , Receptores da Bombesina/agonistas , Receptores da Bombesina/antagonistas & inibidores , Células 3T3 , Amilases/metabolismo , Animais , Bombesina/química , Bombesina/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Mimetismo Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Ratos , Ratos Wistar , Receptores da Bombesina/metabolismo , Relação Estrutura-Atividade , Timidina/metabolismoRESUMO
The aim of this study was to analyse the antisecretory mechanism of L-365,260 in vitro in isolated rabbit gastric glands. We showed that compound L-365,260, described as a non-peptide specific competitive CCK-B receptor antagonist, was able to dose-dependently inhibit [14C]-aminopyrine accumulation induced by histamine (10(-4) M), carbachol (5x10(-5) M), 3-isobutyl-1-methyl-xanthine (IBMX) (5x10(-6) M) and forskolin (5x10(-7) M) with similar IC50 values respectively of 1.1+/-0.6x10(-7) M, 1.9+/-1.2x10(-7) M, 4.2+/-2.0x10(-7) M and 4.0+/-2.8x10(-7) M. We showed that L-365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+ -ATPase. We found that L-365,260 inhibited cyclic AMP-induced [14C]-aminopyrine accumulation in digitonin-permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP-dependent protein kinase (PKA) pathway.
Assuntos
Benzodiazepinonas/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ácido Gástrico/metabolismo , Compostos de Fenilureia/farmacologia , Receptores da Colecistocinina/antagonistas & inibidores , Aminopirina/metabolismo , Animais , AMP Cíclico/biossíntese , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/enzimologia , Mucosa Gástrica/metabolismo , Antagonistas dos Receptores H2 da Histamina/farmacologia , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Omeprazol/farmacologia , Inibidores da Bomba de Prótons , Coelhos , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
We have investigated the potential role of gastrin in the regulation of cell growth in human astrocytic tumors. To this end we have used synthetic analogs of gastrin and cholecystokinin (CCK) which behave as gastrin and/or CCK antagonists, e.g. compounds JMV-97, JMV-209 and JMV-179. Their effects on astrocytic tumor cell proliferation was investigated by the use of the colorimetric MTT assay. The in vitro biological models used in the present study included the SW1088, U87 and U373 astrocytic tumor cell lines. The results demonstrated marked influence of gastrin and CCK antagonists in the regulation of astrocytic tumor growth. This suggests that gastrin and/or CCK antagonists might be tested in experimental glioblastoma.
Assuntos
Astrócitos/patologia , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Colecistocinina/antagonistas & inibidores , Gastrinas/antagonistas & inibidores , Gastrinas/farmacologia , Glioblastoma/patologia , Fragmentos de Peptídeos/farmacologia , Sincalida/análogos & derivados , Colecistocinina/fisiologia , Gastrinas/fisiologia , Humanos , Sincalida/farmacologia , Células Tumorais CultivadasRESUMO
In the present study we compared various CCK(B) receptor antagonists and tried to detect a difference in biological activity between the C-terminal octapeptides of cholecystokinin (CCK-8) and [Leu11]gastrin-(5-17) in isolated rabbit gastric glands. Binding experiments showed that different CCK(B)/gastrin receptor agonists bound with high affinity and that antagonists inhibited this binding in accordance with a CCK(B)/gastrin pharmacological profile. [Leu11]gastrin-(5-17), CCK-8 and cionin were found to induce [14C]aminopyrine accumulation to 25% above the basal level. Under the same experimental conditions, histamine induced a response twice as great as the response obtained with [Leu11]gastrin-(5-17) or CCK-8. [Leu11]gastrin-(5-17) (10(-7) M), CCK-8 (10(-8) M) and cionin (10(-8) M) appeared to be full agonists. CCK(B)/gastrin receptor antagonists including L-365,260 (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-N-(3-methylphenyl) urea), L-364,718 (3S-(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-1H-indole-2-carboximide) (a selective CCK(A) receptor antagonist), PD-135,158 (4([2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2. 2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl] amino-4-oxo-[1S-1alpha.2beta[S*(S*)]4alpha]]-butano nate N-methyl-D-glucamine) (bicyclo system 1S-endo), YM-022 ((R)-1-[2,3-dihydro-1-(2'-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-++ +benzodiazepin-3-yl]-3-(3-methylphenyl)urea) and JMV-180 (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-O-CH2-CH2-C6H5) exhibited the same profile for inhibition of [Leu11]gastrin-(5-17) or CCK-8-induced [14C]aminopyrine accumulation in rabbit gastric glands. These results suggested that [Leu11]gastrin-(5-17) and CCK-8 induced [14C]aminopyrine accumulation by the same mechanism. [Leu11]gastrin-(5-17)- or CCK-8-induced [14C]aminopyrine accumulation was inhibited by about 40% by the histamine H2 receptor blocker cimetidine. These results are consistent with there being cooperativity between [Leu11]gastrin-(5-17) (or CCK-8) and histamine in the acid secretory pathway. Similarly, the CCK(B)/gastrin receptor antagonists were tested against histamine-induced [14C]aminopyrine accumulation and surprisingly, only compound L-365,260 appeared active and even more potent than cimetidine.
Assuntos
Ácido Gástrico/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Gastrinas/farmacologia , Sincalida/farmacologia , Sequência de Aminoácidos , Aminopirina/metabolismo , Animais , Mucosa Gástrica/metabolismo , Histamina/farmacologia , Técnicas In Vitro , Dados de Sequência Molecular , Coelhos , Receptores da Colecistocinina/efeitos dos fármacos , Receptores da Colecistocinina/metabolismoRESUMO
alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.
Assuntos
Bombesina/análogos & derivados , Bombesina/metabolismo , Receptores da Bombesina/metabolismo , Células 3T3 , Amilases/efeitos dos fármacos , Amilases/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Bombesina/química , Bombesina/farmacologia , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fosfatos de Inositol/metabolismo , Radioisótopos do Iodo , Masculino , Camundongos , Pâncreas/citologia , Pâncreas/metabolismo , Ratos , Ratos WistarRESUMO
We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and cholecystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCKB receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCKB receptors were measured by inhibition of [125I]Bolton Hunter-CCK-8 (3-[125I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTP gamma S (guanosine 5'-O-(3-thio)triphosphate) or aluminum tetrafluoride (AlF4-). Activation of the G proteins by GTP gamma S or AlF4- led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCKB receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCKB receptors and their related intracellular events.
Assuntos
Gastrinas/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Receptores da Colecistocinina/metabolismo , Sincalida/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Cobaias , Humanos , Masculino , Dados de Sequência Molecular , Linfócitos T/metabolismoRESUMO
BACKGROUND AND PURPOSE: Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca(2+)]i) in beta cells, in the absence of any co-stimulating factor. EXPERIMENTAL APPROACH: Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca(2+)]i were measured using the ratiometric fluorescent Ca(2+) indicator Fura-2. Ca(2+) channel currents were recorded with the whole-cell patch-clamp technique. KEY RESULTS: Quercetin concentration-dependently increased insulin secretion and elevated [Ca(2+)]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 µmol·L(-1)), but were nearly abolished by the L-type Ca(2+) channel antagonist nifedipine (1 µmol·L(-1)). Similar to the L-type Ca(2+) channel agonist Bay K 8644, quercetin enhanced the L-type Ca(2+) current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca(2+)]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 µmol·L(-1)), with the two drugs having cumulative effects on [Ca(2+)]i. CONCLUSIONS AND IMPLICATIONS: Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca(2+) influx through an interaction with L-type Ca(2+) channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Quercetina/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Células Cultivadas , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Nifedipino/farmacologia , RatosRESUMO
BACKGROUND AND PURPOSE: Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic ß-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting ß-cells. Using the INS-1 ß-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on ß-cell dysfunctions induced by hydrogen peroxide (H(2)O(2)). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison. EXPERIMENTAL APPROACH: Insulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay. KEY RESULTS Quercetin (20 µmol·L(-1)) potentiated both glucose (8.3 mmol·L(-1))- and glibenclamide (0.01 µmol·L(-1))-induced insulin secretion and ERK1/2 phosphorylation. The ERK1/2 (but not the protein kinase A) signalling pathway played a crucial role in the potentiation of glucose-induced insulin secretion by quercetin. In addition, quercetin (20 µmol·L(-1)), protected ß-cell function and viability against oxidative damage induced by 50 µmol·L(-1) H(2)O(2) and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective. CONCLUSION AND IMPLICATIONS: Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected ß-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing ß-cell dysfunction associated with diabetes deserves further investigation.
Assuntos
Antioxidantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Quercetina/farmacologia , Acetilcisteína/farmacologia , Animais , Linhagem Celular , Glucose/metabolismo , Glibureto/farmacologia , Peróxido de Hidrogênio/toxicidade , Hipoglicemiantes/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologiaRESUMO
The aim of this study was to analyze the role of cholecystokinin (CCK(B)) receptor in human lymphoblastic Jurkat T cells. We investigated the trophic effect resulting from activation of such a receptor by using the reporter gene strategy. For this purpose, we transiently transfected Jurkat T cells with the reporter plasmid p[(TRE)3-tk-Luc] and found that CCK-8 was able to dose-dependently induce luciferase expression related to activator protein-1 (AP-1) activation with a maximal response identical to that obtained with compounds known to activate AP-1 complex (quantitatively, the same level of induction was obtained with 1 nM 12-O-tetradecanoylphorbol-13-acetate, 100 microM diacylglycerol, or 4 nM epidermal growth factor). The involvement of the CCK(B) receptor in such a stimulation was demonstrated by the inhibiting effect of the selective CCK(B) receptor antagonist PD-135,158. This effect was confirmed in COS-7 cells transfected with the cDNA of CCK(B) receptor cloned from Jurkat T cells. To better understand the AP-1-dependent luciferase expression in Jurkat T cells, we tested two specific inhibitors of serine/threonine phosphatases-1 and -2A: okadaic acid and calyculin A. These compounds strongly increased the phorbol-12-myristate-13-acetate response, whereas we have not observed a contribution of phosphatase inhibitors on a CCK-8-induced luciferase activity. To confirm that CCK(B) receptors are involved in AP-1 response, we investigated the CCK-8 effect on interleukin-2 expression, a natural endogenous gene regulated by several factors, including AP-1. In Jurkat T cells activated by phorbol-12-myristate-13-acetate and phytohemagglutinin, CCK-8 induced IL-2 expression. This induction was abolished by PD-135,158. Our results indicate that CCK-8 exerts a trophic effect in Jurkat T cells through stimulation of CCK(B) receptors by modulation of expression of AP-1-regulated genes.