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1.
Curr Protoc Pharmacol ; Chapter 4: Unit 4.13, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-21965069

RESUMO

Cholecystokinin (CCK) is a peptide that acts as a peripheral hormone and as a central neurotransmitter. To date, two distinct receptors have been identified for CCK using structural and operational criteria; CCK1 and CCK2 (formerly CCKA and CCKB, respectively). In addition, there is the gastrin receptor found in the stomach which has a high structural similarity to the CCK2 receptor, but which displays a different pharmacology. This unit presents two methods for the quantification of selective agonists and antagonists at CCK1 receptors and an assay for CCK2 receptors. In the first tissue, the guinea-pig ileum longitudinal muscle with myenteric plexus, both CCK1 and CCK2 receptors are present in the same preparation. Each receptor is distinguished in this assay using selective agonists and antagonists against the unwanted receptor subtype. The second preparation, the guinea-pig gallbladder, is a classical preparation for studying CCK1 receptor-active compounds in the absence of CCK2 sites.


Assuntos
Quimiocinas CC/fisiologia , Colecistocinina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Fatores de Transcrição/fisiologia , Animais , Quimiocinas CC/agonistas , Colecistocinina/agonistas , Proteínas de Ligação a DNA/agonistas , Relação Dose-Resposta a Droga , Complexos Endossomais de Distribuição Requeridos para Transporte/agonistas , Cobaias , Masculino , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/fisiologia , Técnicas de Cultura de Órgãos/métodos , Receptores da Colecistocinina/agonistas , Receptores da Colecistocinina/fisiologia , Sincalida/análogos & derivados , Sincalida/farmacologia , Fatores de Transcrição/agonistas
2.
J Med Chem ; 43(20): 3596-613, 2000 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-11020274

RESUMO

A series of 5-phenyl-3-ureidobenzodiazepine-2,4-diones was synthesized and evaluated as cholecystokinin-B (CCK-B) receptor antagonists. Structure-activity relationship (SAR) studies revealed the importance of the N-1 substituent for potent and selective CCK-B affinity. Addition of substituents at the urea side chain provided in some cases more potent compounds. Moreover the introduction of bulky substituents such as adamantylmethyl at N-1 and resolution of the racemic ureas resulted in our lead compound GV150013.


Assuntos
Ansiolíticos/síntese química , Benzodiazepinas/síntese química , Receptores da Colecistocinina/antagonistas & inibidores , Animais , Ansiolíticos/química , Ansiolíticos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Callithrix , Córtex Cerebral/metabolismo , Cristalografia por Raios X , Cobaias , Células HeLa , Humanos , Técnicas In Vitro , Membranas , Camundongos , Modelos Moleculares , Pâncreas/metabolismo , Ensaio Radioligante , Ratos , Receptor de Colecistocinina A , Receptor de Colecistocinina B , Receptores da Colecistocinina/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade
3.
Pharm Acta Helv ; 74(2-3): 221-9, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10812962

RESUMO

Glutamic acid is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Specific receptors bind glutamate and some of these when activated open an integral ion channel and are thus known as ionotropic receptors. Within the ionotropic family of glutamate receptors, three major subtypes have been identified using classical specific agonist activation, selective competitive antagonists together with their structural heterogeneity. These receptors have thus been named N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors. The NMDA receptor has sites in addition to its agonist-binding site and these seem to either positively or negatively modulate the agonist effect. The NMDA receptor also is unique in that another amino acid, glycine, acts as a co-agonist with glutamate. Changes in glutamate transmission have been associated with a number of CNS pathologies; these include, acute stroke, chronic neurodegeneration, chronic pain, depression, drug dependency, epilepsy, Parkinson's Disease and schizophrenia.


Assuntos
Agonistas de Aminoácidos Excitatórios/farmacologia , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Animais , Humanos , Receptores de Glutamato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
4.
Blood Press ; 8(1): 57-64, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10412884

RESUMO

The aim of this study was to investigate the extracellular matrix gene expression in the hypertrophied left ventricular tissue of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, at early and mature ages. Interestingly, with age, a marked increase (+85% and +187% at 25 and 30 weeks of age, respectively, p < 0.01, vs 5 weeks) in matrix metalloproteinase-1 (MMP-1) mRNA levels in SHR and a progressive decrease (-50%, -70%, -78%, -70% at 10, 15, 25 and 30 weeks, respectively, p < 0.01, vs 5 weeks) in WKY were seen. Moreover, mRNA levels were significantly lower in SHR at 5 weeks. The analysis of mRNA expression for the tissue inhibitor of metalloproteinase-1 (TIMP-1) showed a significant increase in WKY (+44% and +44%, vs 15 and 25 weeks, respectively, p < 0.05), whereas there were no significant changes in SHR with development. At 30 weeks TIMP-1 mRNA levels were significantly reduced in SHR. Temporal trends of procollagen alpha1(I) and procollagen alpha1(III) mRNA levels were similar in both strains, but lower levels for procollagen alpha1(III) were found in SHR at 5 and 30 weeks. Although no significant differences were measured between the strains, mRNA levels for fibronectin were found decreased in WKY and increased in SHR with age. The results of the present study suggest an altered balance between collagen deposition and collagen degradation with development in this model of left ventricular hypertrophy and hypertension.


Assuntos
Matriz Extracelular/genética , Hipertensão/metabolismo , Envelhecimento/fisiologia , Animais , Colagenases/análise , Colagenases/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica , Hipertensão/genética , Hipertrofia Ventricular Esquerda/metabolismo , Masculino , Metaloproteinase 1 da Matriz , Pró-Colágeno/análise , Pró-Colágeno/genética , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
5.
J Pharmacol Exp Ther ; 290(1): 158-69, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10381772

RESUMO

Central sensitization is a condition of enhanced excitability of spinal cord neurons that contributes to the exaggerated pain sensation associated with chronic tissue or nerve injury. N-methyl-D-aspartate (NMDA) receptors are thought to play a key role in central sensitization. We have tested this hypothesis by characterizing in vitro and in vivo a novel antagonist of the NMDA receptor acting on its glycine site, GV196771A. GV196771A exhibited an elevated affinity for the NMDA glycine binding site in rat cerebral cortex membranes (pKi = 7.56). Moreover, GV196771A competitively and potently antagonized the activation of NMDA receptors produced by glycine in the presence of NMDA in primary cultures of cortical, spinal, and hippocampal neurons (pKB = 7.46, 8. 04, and 7.86, respectively). In isolated baby rat spinal cords, 10 microM GV196771A depressed wind-up, an electrical correlate of central sensitization. The antihyperalgesic properties of GV196771A were studied in a model of chronic constriction injury (CCI) of the rat sciatic nerve and in the mice formalin test. In the CCI model GV196771A (3 mg/kg twice a day p.o.), administered before and then for 10 days after nerve ligature, blocked the development of thermal hyperalgesia. Moreover, GV196771A (1-10 mg/kg p.o.) reversed the hyperalgesia when tested after the establishment of the CCI-induced hyperalgesia. In the formalin test GV196771A (0.1-10 mg/kg p.o.) dose-dependently reduced the duration of the licking time of the late phase. These antihyperalgesic properties were not accompanied by development of tolerance. These observations strengthen the view that NMDA receptors play a key role in the events underlying plastic phenomena, including hyperalgesia. Moreover, antagonists of the NMDA glycine site receptor could represent a new analgesic class, effective in conditions not sensitive to classical opioids.


Assuntos
Analgésicos não Narcóticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hiperalgesia/tratamento farmacológico , Indóis/farmacologia , Pirróis/farmacologia , Receptores de Glicina/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Comportamento Animal/efeitos dos fármacos , Ligação Competitiva , Córtex Cerebral/metabolismo , Tolerância a Medicamentos , Eletrofisiologia , Embrião de Mamíferos , Técnicas In Vitro , Masculino , Camundongos , Medição da Dor/efeitos dos fármacos , Técnicas de Patch-Clamp , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos
6.
Neuropharmacology ; 38(5): 625-33, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10340300

RESUMO

Changes in the levels of mRNA for the NR1 subunit of the glutamate NMDA receptor and in NMDA-sensitive glutamate binding were investigated in consecutive sections of the prefrontal cortex and striatum of control and Parkinson's disease (PD) post-mortem brain using in-situ hybridisation and receptor autoradiography. Both markers of NMDA receptors were found to be relatively unaffected when measured by microdensitometry in the prefrontal cortex of control and PD brains. At a cellular level, a subpopulation of small and medium neurons in the superficial layers of the prefrontal cortex of the PD group showed a decreased expression of NMDA NR1 mRNA, with the maximal decrease in cortical layer IV. In the striatum, levels of glutamate binding to the NMDA receptor detected by receptor autoradiodgraphy were significantly reduced in the PD group, while no change could be detected at a macroscopical level in NMDA NR1 mRNA expression. Consequently, we suggest that the important decrease in agonist binding to the NMDA receptor observed in this study in the caudate and putamen of PD brains, in the absence of any major change in NMDA NR1 mRNA levels might reflect the degeneration of pre-synaptic NMDA receptors located on nigro-striatal projections particularly affected by the disease. Small changes observed at a cellular level in subsets of neurons of both prefrontal cortex and striatum will be discussed at the light of neurochemical changes characteristics of PD.


Assuntos
Corpo Estriado/química , Doença de Parkinson/metabolismo , Córtex Pré-Frontal/química , RNA Mensageiro/análise , Receptores de N-Metil-D-Aspartato/química , Idoso , Feminino , Humanos , Masculino , Receptores de N-Metil-D-Aspartato/genética
7.
Br J Pharmacol ; 124(5): 865-72, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9692770

RESUMO

1. Cumulative concentration-response curves (CRC) to prostaglandin E1 (PGE1), PGE2, PGD2 and PGF2alpha (0.01-30 microM) and to the thromboxane A2 (TXA2) receptor agonist U-46619 (0.01-30 microM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation. 2. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF2alpha > U-46619 > PGE2 whereas weak contractile responses were obtained with PGD2 and PGE1. Any of the agonists tested was able to induce a clear plateau of response even at 30 microM. 3. The selective TXA2 antagonist, GR 32191B (vapiprost), antagonized U-46619-induced contractions with an apparent pK(B) value of 8.27+/-0.12 (n = 4 for each antagonist concentration). GR 32191B (0.3 microM) did not antagonize the contractile responses to PGF2alpha and it was a non-surmountable antagonist of PGE2 (apparent pK(B) of 7.09+/-0.04; n = 5). The EP receptor antagonist AH 6809 at 10 microM shifted to the right the CRC to U-46619 (apparent pK(B) value of 5.88+/-0.04; n = 4). 4. Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 microM) and atropine (1 microM). U-46619 (0.01-3 microM) potentiated the twitch contraction in a dose-dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pK(B) of 8.54+/-0.14 (n = 4 for each antagonist concentration). PGF2alpha in the range 0.01-10 microM (n = 7), but not PGE2 and PGE1 (n = 3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5+/-0.3% of KCl 100 mM-induced contraction) but this potentiation was unaffected by 0.3 microM GR 32191B (n = 5). 5. Cumulative additions of U-46619 (0.01-30 microM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 microM; n = 3). Moreover, pretreatment of the tissue with 0.3 microM U-46619 did not potentiate the smooth muscle response to 7 microM bethanecol (n = 2). 6. We concluded that TXA2 can induce direct contraction of human isolated urinary bladder through the classical TXA2 receptor. Prostanoid receptors, fully activated by PGE2 and PGF2alpha are also present. All these receptors are probably located post-junctionally. The rank order of agonist potency and the fact that GR 32191B, but not AH6809, antagonized responses to PGE2 seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejunctional TXA2 and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.


Assuntos
Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Tromboxanos/agonistas , Receptores de Tromboxanos/antagonistas & inibidores , Tromboxano A2/fisiologia , Bexiga Urinária/efeitos dos fármacos , Xantonas , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Alprostadil/farmacologia , Compostos de Bifenilo/farmacologia , Dinoprosta/farmacologia , Dinoprostona/farmacologia , Estimulação Elétrica , Ácidos Heptanoicos/farmacologia , Humanos , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Antagonistas de Prostaglandina/farmacologia , Prostaglandina D2/farmacologia , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2 , Tromboxano A2/farmacologia , Bexiga Urinária/fisiologia , Xantenos/farmacologia
8.
Neurosci Lett ; 249(1): 45-8, 1998 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-9672385

RESUMO

The distribution of the mRNA coding for the two N-terminal splice variants of the N-methyl-D-aspartate receptor 1 (NMDAR1) subunit of the glutamate NMDA receptor was studied on whole-hemisphere human and macaca fascicularis brain sections by in-situ hybridisation. Synthetic oligonucleotides directed against NMDAR1a and NMDAR1b variants showed a specific distribution that was similar in human and monkey brain, with the NMDAR1a isoform present in the majority of the NMDA receptors, and the NMDAR1b variant present at high levels only in the cortex and dentate gyrus of the hippocampus. The distribution of the mRNAs for the NMDAR1pan and NMDAR1a subunit reported in this study support previous findings in rodent brain, while the restricted distribution of the NMDAR1b variant found in human and monkey suggests some important differences in the composition of the NMDA receptor in rodents and primates.


Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/anatomia & histologia , Feminino , Humanos , Hibridização In Situ , Macaca fascicularis , Masculino , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
9.
Neurochem Int ; 32(4): 345-51, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9596557

RESUMO

Glutamate-induced changes in intracellular free Ca++ concentration ([Ca++]i) were recorded in resting and electrically-stimulated primary cultures of rat cerebral cortical cells, employing the Ca++ indicator Fura 2. A brief (10 min) exposure to glutamate led to a concentration-dependent basal [Ca++]i increase, measured 30 min after glutamate removal. In order to unmask more subtle modifications in [Ca++]i movements associated with neurosecretion, the glutamate effect was also studied in electrically-stimulated cells. The application of trains (10 s) of electrical pulses (intensity 30 mA, duration 1 ms) induced frequency-related Na+- and Ca++-dependent [Ca++]i transients. A 5 min treatment with 50 microM glutamate reduced to 48% the electrically-evoked [Ca++]i transients, evaluated 30 min after glutamate challenge. The neuroprotective effect of sodium 4,6-dichloro-3-[(E)-3-(N-phenyl)propenamide]indole-2-carboxylate (GV150526A), a new indole derivative with high affinity and selectivity for the glycine site of the NMDA receptor-channel complex, was compared with that of DL-2-amino-5-phosphonopentanoic acid (AP5), ifenprodil, 7-chlorokynurenic acid and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX) on glutamate-induced [Ca++]i changes in resting and electrically-stimulated cells. In both experimental conditions, GV150526A showed to be the most potent compound. Moreover, GV150526A and 7-chlorokynurenic acid were 2-3 times more active in stimulated neurons than in resting neurons, indicating a major involvement of the glycine site in the protection of the cells kept in an active state.


Assuntos
Cálcio/metabolismo , Córtex Cerebral/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/toxicidade , Indóis/farmacologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Interações Medicamentosas , Estimulação Elétrica , Piperidinas/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
10.
Brain Res Mol Brain Res ; 54(1): 13-23, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9526033

RESUMO

The distributions of [3H]MK-801 binding and the NMDA NR1 subunit mRNA were studied using receptor autoradiography and in-situ hybridization in rat and human brain whole-hemisphere coronal sections. Receptor protein detected by radioligand autoradiography and the mRNA for the key subunit of the receptor presented similar distributions in the forebrain, with a few areas showing an imbalance between the levels of mRNA and receptor protein. Human frontal cortex showed a relative abundance of NMDAR1 mRNA as compared to [3H]MK-801 binding. The same area in rat brain did not show any difference in the two distributions. In comparison, the rat claustrum presented a relative excess of NMDAR1 mRNA which was not detected in human sections. Human caudate nucleus exhibited relatively high levels of [3H]MK-801 binding that were unmatched in rat caudate. The hippocampi of either species presented similar levels of [3H]MK-801 binding and NMDAR1 mRNA, but when the two signals were measured in specific subfields of the hippocampal formation, the differential distribution of the two signals reflected the anatomy of hippocampal connections assuming a preferential dendritic distribution for MK-801 binding. Interestingly, rat and human hippocampi also showed some important species-dependent difference in the relative distribution of the receptor protein and mRNA. The data presented show an overall good correlation between the mRNA for the key subunit of the NMDA receptor and the functional receptor detected with radioligand binding and highlight the presence of local differences in their ratio. This may reflect different splicing of the mRNA for the NMDAR1 subunit in specific brain areas of rat and human. The species-dependent differences in the relative distribution of the mRNA for the key subunit of the NMDA receptor and that of a marker of functional receptors also highlights important differences in the NMDA function in rat and human brain.


Assuntos
Maleato de Dizocilpina/metabolismo , Prosencéfalo/metabolismo , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Autorradiografia , Sítios de Ligação , Córtex Cerebral/metabolismo , Feminino , Hipocampo/metabolismo , Humanos , Hibridização In Situ , Masculino , Especificidade de Órgãos/genética , Prosencéfalo/anatomia & histologia , Ratos , Receptores de N-Metil-D-Aspartato/genética , Trítio
11.
Alcohol ; 14(4): 327-32, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9209547

RESUMO

Naive adult male Wistar rats free to choose between water or 10% ethanol (v/v) spontaneously became water-preferring (WP) rats, as they drank mainly water (approximately 35 ml per day), or alcohol-drinking (ED) rats, as they also drank a significant amount of ethanol (approximately 14 ml per day). The selective CCKA receptor antagonist L-364,718 at doses selective for the CCKA receptor (5 micrograms/kg, IP) halved the consumption of alcohol of the ED rats without modifying their total liquid in-take. In contrast, the CCKB antagonists L-365,260 or GV150013 were without effect when used at doses selective for the CCKB receptor. These data indicate that the CCK system could be involved in the modulation of alcohol intake. In particular, they suggest that CCKA receptors could play a role in the ethanol preference.


Assuntos
Consumo de Bebidas Alcoólicas/tratamento farmacológico , Compostos de Fenilureia , Receptores da Colecistocinina/antagonistas & inibidores , Animais , Benzodiazepinonas/farmacologia , Peso Corporal/efeitos dos fármacos , Devazepida , Ingestão de Líquidos/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Masculino , Ratos , Ratos Wistar
14.
J Med Chem ; 40(6): 841-50, 1997 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-9083472

RESUMO

A series of indole-2-carboxylates bearing suitable chains at the C-3 position of the indole nucleus was synthesized and evaluated in terms of in vitro affinity using [3H]glycine binding assay and in vivo potency by inhibition of convulsions induced by N-methyl-D-aspartate (NMDA) in mice. 3-[2-[(Phenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxyl ic acid (8) was an antagonist at the strychnine-insensitive glycine binding site (noncompetitive inhibition of the binding of [3H]TCP, pA2 = 8.1) displaying nanomolar affinity for the glycine binding site (pKi = 8.5), coupled with high glutamate receptor selectivity (> 1000-fold relative to the affinity at the NMDA, AMPA, and kainate binding sites). This indole derivative inhibited convulsions induced by NMDA in mice, when administered by both iv and po routes (ED50 = 0.06 and 6 mg/kg, respectively). The effect of the substituents on the terminal phenyl ring of the C-3 side chain was investigated. QSAR analysis suggested that the pKi value decreases with lipophilicity and steric bulk of substituents and increases with the electron donor resonance effect of the groups present in the para position of the terminal phenyl ring. According to these results the terminal phenyl ring of the C-3 side chain should lie in a nonhydrophobic pocket of limited size, refining the proposed pharmacophore model of the glycine binding site associated with the NMDA receptor.


Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicinérgicos/farmacologia , Glicina/antagonistas & inibidores , Indóis/farmacologia , Animais , Sítios de Ligação , Ligação Competitiva , Ácidos Carboxílicos , Córtex Cerebral/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/síntese química , Antagonistas de Aminoácidos Excitatórios/química , Antagonistas de Aminoácidos Excitatórios/metabolismo , Glicina/metabolismo , Glicinérgicos/síntese química , Glicinérgicos/química , Glicinérgicos/metabolismo , Indóis/síntese química , Indóis/química , Indóis/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , N-Metilaspartato/farmacologia , Ratos , Receptores de Glutamato/metabolismo , Relação Estrutura-Atividade , Estricnina/farmacologia
15.
Br J Pharmacol ; 119(5): 819-28, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8922727

RESUMO

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.


Assuntos
2-Amino-5-fosfonovalerato/análogos & derivados , Encéfalo/metabolismo , Antagonistas de Aminoácidos Excitatórios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 2-Amino-5-fosfonovalerato/metabolismo , Animais , Autorradiografia , Sítios de Ligação , Masculino , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Trítio
16.
Trends Pharmacol Sci ; 17(6): 220-2, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8763199

RESUMO

The co-agonism between glutamate and glycine at the NMDA receptor raises uncertainty about the estimation of the values of dissociation constants for agonists and antagonists and of efficacy for agonists. In this article, Mauro Corsi, Paolo Fina and David Trist discuss how to analyse the interaction of the two agonists with the NMDA receptor by applying an operational receptor model. Data simulation indicates that co-agonism can affect the potency as well as the efficacy of agonists. Moreover, the interaction of antagonists with the NMDA receptor can also be affected, leading to altered estimation of the antagonist dissociation constants.


Assuntos
Ácido Glutâmico/farmacologia , Glicina/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Animais , Humanos , Transdução de Sinais
17.
Mol Pharmacol ; 49(3): 387-90, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8643076

RESUMO

Xenopus oocytes were injected with RNAs for the two inward-rectifier potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (rcKATP or CIR) in addition to RNA from the neuroblastoma cell line KAN-TS. Potassium currents were evoked by neuropeptide Y in oocytes injected with polyadenylated RNA or with cRNA from pools of a neuroblastoma (KAN-TS) cDNA library, and progressive subdivision of responding pools yielded a single cDNA. The encoded protein contains 381 amino acids, has the seven hydrophobic domains characteristic of G protein-coupled receptors, and is 31% identical to the Y1 receptor: potassium currents were induced by neuropeptide Y (EC50=60pm) and Y2-selective analogues. Coexpression with potassium channel subunits will be a generally useful method for the cloning of G protein-coupled receptors.


Assuntos
Neuropeptídeo Y/metabolismo , Canais de Potássio/genética , Receptores de Neuropeptídeo Y/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Neuroblastoma , Neuropeptídeo Y/farmacologia , Oócitos/fisiologia , Oócitos/ultraestrutura , Canais de Potássio/metabolismo , Canais de Potássio/fisiologia , RNA/genética , RNA Mensageiro/genética , Receptores de Neuropeptídeo Y/metabolismo , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Xenopus
18.
Br J Pharmacol ; 116(5): 2401-6, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8581275

RESUMO

1. The pharmacological activity of neuropeptide Y (NPY) and some analogues in inhibiting the twitch contractions induced by electrical stimulation (single pulses at 25 V, 0.15 Hz, 1 ms) in the prostatic portion of the rat isolated vas deferens was investigated. The rank order of agonist potency was: PYY > NPY2-36 > NPY >> NPY13-36 >> NPY18-36 >> [Leu31,Pro34]NPY = hPP, which is consistent with the activation of a Y2 receptor. 2. The putative Y1 and Y2 antagonist, benextramine (BXT), incubated at 100 microM for 10 or 60 min, was ineffective against PYY-induced inhibition of the twitch response, suggesting that the prejunctional Y2 receptor in this tissue is different from the postjunctional one reported in the literature to be sensitive to BXT blockade. 3. The putative NPY antagonist, PYX-2, incubated at 1 microM for 20 min, was completely ineffective in antagonizing PYY-induced inhibition of twitches. 4. The twitch response was totally inhibited by suramin (100 microM) but was little affected by prazosin (1 microM). Furthermore, NPY was without effect on the dose-response curve to ATP in resting conditions. Taken together, these results suggest that in our paradigm, NPY inhibits the release of a purinergic neurotransmitter which mediates contraction of the prostatic portion of the rat vas deferens.


Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Cistamina/análogos & derivados , Neuropeptídeo Y/análogos & derivados , Neuropeptídeo Y/antagonistas & inibidores , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Próstata/metabolismo , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Ducto Deferente/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cistamina/farmacologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Próstata/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Ratos , Ratos Sprague-Dawley , Suramina/farmacologia , Ducto Deferente/efeitos dos fármacos
19.
J Neurosci Methods ; 61(1-2): 201-12, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-8618420

RESUMO

Selective and simultaneous voltammetric analysis of catechols and indoles in vivo and in vitro has until now been feasible only by means of 'slow' scanning methods (scan speed in tens of seconds) such as differential pulse (DPV) and differential normal pulse voltammetry in conjunction with electrically and/or chemically treated carbon-fiber micro-electrodes (mCFE). Faster electrochemical techniques, such as chronoamperometry and cyclic voltammetry (CV), allow more rapid (seconds or fractions of a second) and frequent measurements of these chemicals. However, these methods show poor sensitivity and selectivity in the presence of different electroactive compounds with similar oxidation potentials. In order to analyze whether the lack of sensitivity and selectivity of the fast voltammetric methods results from the rapidity of the measurement or from the use of untreated sensors, the methods of CV (scan speed: 1000 mV/s) and DPV (scan speed: 10 mV/s) have been applied with either untreated or electrically treated mCFE to analyze the in vitro oxidation potential and current values of DA and 5-HT. When associated with untreated mCFE, neither method was able to separate and selectively detect the two compounds dissolved together in an inert vehicle; the voltammogram recorded resulted in a single broad oxidation signal. In contrast, when these techniques were performed with electrically treated mCFE, oxidation signals for DA (peak A) and 5-HT (peak B) were monitored simultaneously at approximately + 65 mV and + 240 mV, with DPV respectively, and at + 120 mV and + 300 mV with CV, respectively. Additionally, CV with treated mCFE on anesthetized rats, simultaneously monitored two striatal signals at approximately + 100 mV and + 300 mV. The oxidation values (Em) and current levels (nA) of these peaks remained stable during control recordings. The current levels were selectively increased by peripheral injection of fluphenazine (DA antagonist) or of 5-hydroxytryptophan (precursor of serotonin). The chemical nature of these two peaks may therefore be considered catecholaminergic and indolaminergic, respectively. Hence, this report provides the first evidence for the feasibility of concomitant in vitro analysis of DA and 5-HT using a rapid scanning method such as CV. In addition, the values of current level (nA) obtained with CV-mCFE for DA and 5-HT are comparable to those monitored with DPV-mCFE, supporting the view that treatment of the sensor is a key point for increasing the selectivity and the sensitivity of these voltammetric techniques. The feasibility of using CV with electrically treated mCFE for fast in vivo analysis of catechol and indole activities is also demonstrated.


Assuntos
Catecolaminas/fisiologia , Indóis/metabolismo , Transdução de Sinais/fisiologia , Animais , Dopamina/metabolismo , Masculino , Ratos , Ratos Wistar , Serotonina/metabolismo
20.
Br J Pharmacol ; 115(1): 3-10, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7647980

RESUMO

1. In order to characterize the neuropeptide Y (NPY) Y1 receptors known to be present in rabbit isolated vas deferens and saphenous vein, the pharmacological activity of the selective NPY Y1 receptor agonists, [Leu31,Pro34] NPY and various other peptide agonists, together with the putative NPY antagonist, benextramine, were compared in the two tissues. 2. In rabbit isolated saphenous vein, cumulative dose-response curves to various NPY agonists were obtained. All the peptides tested caused contractions which developed quite slowly. The rank order of potency obtained was: PYY > NPY > [Leu31,Pro34] NPY = NPY2-36 > hPP >> NPY13-36 = NPY18-36. Incubation with benextramine (BXT) at 100 microM for 30 min irreversibly abolished the contractile response to [Leu31,Pro34] NPY but was ineffective against NPY18-36-induced contractions. 3. Cumulative dose-response curves to [Leu31,Pro34] NPY were performed in the same preparation before and after incubation with 100 microM BXT for 20 min in order to inactivate NPY Y1 receptors. The pKA (-logKA) estimation for [Leu31,Pro34] NPY was 7.60 +/- 0.30 using the operational model and 7.20 +/- 0.33 using the null method; the difference between the two methods was not statistically significant (P = 0.36). 4. Prostatic segments of rabbit vas deferens were electrically stimulated with single pulses. Immediately after stabilization of the contractile response, a cumulative dose-response curve to various NPY agonists was obtained in each tissue. The rank order of potency for twitch inhibition was: PYY> [Leu31,Pro34]NPY > NPY > hPP>NPY2- 36 >>NPY13-36>> NPY 18-36 which indicates the presence of a prejunctional NPY Y1 receptor. BXT at 100 microM incubated for 10 or 60 min did not antagonize the response to[Leu31,Pro34] NPY.5. We conclude that rabbit isolated saphenous vein contains a population of post-junctional NPY Y1 receptors irreversibly blocked by BXT, as well as a population of post-junctional NPY Y2 receptors,which are insensitive to BXT. In contrast, the rabbit isolated vas deferens express a pre-junctional NPYY1 receptor subtype which is not blocked by BXT. Tetramine disulphides such as BXT could be useful tools in classifying NPY receptors.


Assuntos
Cistamina/análogos & derivados , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Veia Safena/metabolismo , Ducto Deferente/metabolismo , Animais , Técnicas de Cultura , Cistamina/farmacologia , Humanos , Masculino , Neuropeptídeo Y/farmacologia , Peptídeos/farmacologia , Coelhos , Receptores de Neuropeptídeo Y/classificação , Receptores de Neuropeptídeo Y/metabolismo , Veia Safena/efeitos dos fármacos , Ducto Deferente/efeitos dos fármacos
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