RESUMO
The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.
Assuntos
AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Substâncias de Crescimento/farmacologia , Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Tamanho Celular , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
The neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and leukemia inhibitory factor (LIF) were investigated in human neuroblastoma SH-SY5Y cells. Effects on differentiation were assessed through monitoring morphological changes and Western blot analysis of the expression of neuronal marker proteins. In contrast to PACAP-38, which induced a 5.5-fold increase in the number of neurite-bearing cells, LIF had no significant effect on cell morphology compared to control cells over the 4-day time course. Cells co-treated with PACAP-38+LIF showed a similar increase in neurite-bearing cells compared to those treated with PACAP-38 alone. Cell morphology was similar for PACAP-38-treated and PACAP-38+LIF-co-treated cells, with the formation of bipolar neuron-like cells with long thin neurites, topped by growth cone-like structures and varicosities. SH-SY5Y cells express tyrosine hydroxylase (TH) but only low levels of the neuronal marker proteins: Bcl-2, GAP-43 and choline acetyltransferase (ChAT). Treatment of cells with PACAP-38 induced the expression of Bcl-2, GAP-43, and ChAT but did not appear to alter the expression of TH. LIF failed to induce the expression of GAP-43 and had little effect on the expression of TH, but did induce the expression of Bcl-2 and upregulated the expression of ChAT. Co-treatment with LIF had no effect on PACAP-38-induced expression of Bcl-2, GAP-43, and ChAT. Cells differentiated for 4 days with PACAP-38 or treated with LIF also displayed increased resistance to hypoxic conditions and to treatment with H2O2 and TNFalpha. The increased resistance to hypoxic conditions for PACAP-differentiated cells was blocked by the p38 MAP kinase inhibitor, SB203580, but not by the MEK1 inhibitor, PD98059. Additionally, cell proliferation assays show that LIF, but not PACAP-38, stimulates proliferation of SH-SY5Y cells, and this observed increase by LIF is not attenuated by co-treatment with PACAP. Further investigation of the intracellular signaling pathways mediating the neurotrophic effects of PACAP on SH-SY5Y cells indicate that neither phospholipase C activation nor Ca2+/calmodulin-dependent kinase II (CAMKII) are involved.
Assuntos
Fator Inibidor de Leucemia/farmacologia , Fatores de Crescimento Neural/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Biomarcadores/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular , Humanos , Neuroblastoma , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
The VPAC(2) receptor is a seven transmembrane spanning G protein-coupled receptor for two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). It has a distinct tissue-specific, developmental and inducible expression that underlies an important neuroendocrine role. Here, we report the characterisation of the gene that encodes the mouse VPAC(2) receptor (Vipr2), localisation of the transcriptional start site and functional analysis of the promoter region. The Vipr2 gene contains 12 introns within its protein-coding region and spans 68.6 kb. Comparison of the 5' untranslated region sequences for cloned 5'-RACE products amplified from different tissues showed they all were contained within the same exon, with the longest extending 111 bp upstream of the ATG start site. Functional analysis of the 3.2-kb 5'-flanking region using sequentially deleted sequences cloned into a luciferase gene reporter vector revealed that this region is active as a promoter in mouse AtT20 D16:16 and rat GH4C1 cell lines. The core promoter is located within a 180-bp GC-rich region proximal to the ATG start codon and contains potential binding sites for Sp1 and AP2, but no TATA-box. Further upstream, in two out of three mice strains examined, we have discovered a 496-bp polymorphic DNA sequence that bears a significant identity to mouse LINE-1 DNA. Comparison of the promoter activity between luciferase reporter gene constructs derived from the BALB/c (which contains this sequence) and C57BL/6J (which lacks this sequence) Vipr2 promoter regions has shown three-fold difference in luciferase gene activity when expressed in mouse AtT20 D16:16 and alphaT3-1 cells, but not when expressed in the rat GH4C1 cells or in COS 7 cells. Our results suggest that the mouse Vipr2 gene may be differentially active in different mouse strains, depending on the presence of this LINE-1-like sequence in the promoter region.
Assuntos
Regiões Promotoras Genéticas , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Elementos Nucleotídeos Longos e Dispersos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Polimorfismo Genético , Ratos , Homologia de Sequência do Ácido Nucleico , SomatotrofosRESUMO
The vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 (VPAC(2)) receptor was shown to induce both [(3)H]inositol phosphate ([(3)H]InsP)and cAMP production in transfected COS7 cells and in GH(3) cells where it is natively expressed. Neither cholera toxin nor forskolin could elicit an equivalent [(3)H]InsP response, suggesting independent coupling of the two pathways. The VPAC(2) receptor-mediated [(3)H]InsP response was partially inhibited by pertussis toxin (Ptx) and by the G beta gamma-sequestering C-terminal fragment of GRK2 (GRK2-ct) in COS7 and GH(3) cells, whereas responses of control receptors were unaffected. Blockers of receptor-activated Ca(2+) influx pathways (Co(2+) and SKF 96365) also partially inhibited VPAC(2) receptor-mediated [(3)H]InsP responses. This inhibition was not present in the component of the response remaining after Ptx treatment. A range of blockers of voltage-sensitive Ca(2+) channels were ineffective, consistent with the reported lack of these channels in COS7 cells. The data suggest that the VPAC(2) receptor may couple to phospholipase C through both Ptx-insensitive and Ptx-sensitive G proteins (G(q/11) and G(i/o), respectively) to generate [(3)H]InsP. In addition to G beta gamma, G(i/o) activation appears to require receptor-activated Ca(2+) entry. This is consistent with the possibility that not only G alpha(q/11)-responsive and G beta gamma-responsive isoforms of phospholipase C but also Ca(2+)-responsive forms may contribute to the overall [(3)H]InsP response.
Assuntos
Ativação Enzimática/fisiologia , Receptores do Hormônio Hipofisário/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Fosfolipases Tipo C/metabolismo , Toxina Adenilato Ciclase , Animais , Células COS , Cálcio/metabolismo , Linhagem Celular , Proteínas de Ligação ao GTP/fisiologia , Fosfatos de Inositol/biossíntese , Toxina Pertussis , Isoformas de Proteínas/fisiologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Transdução de Sinais , Fatores de Virulência de Bordetella/farmacologiaRESUMO
GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores LHRH/efeitos dos fármacos , Animais , Northern Blotting , Linhagem Celular , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio do Crescimento/metabolismo , Fosfatos de Inositol/biossíntese , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Prolactina/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores LHRH/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção/genéticaRESUMO
Two subtypes of receptors for serotonin (5-hydroxytryptamine; 5-HT) are known to stimulate inositol (1,4,5)-trisphosphate production, the 5-HT1c and 5-HT2 receptors. In this study we investigated the ability of 5-HT1c receptors, transiently expressed in COS 7 cells, to functionally interact with protein kinase C-alpha, the indigenous (phorbol ester-responsive) isoform of the enzyme in those cells. Serotonin caused translocation of the [3H]phorbol 12,13-dibutyrate (PDBu) binding site of PKC-alpha from the cytosolic to the membrane fraction in a Ca(2+)-dependent manner which was prevented by the 5-HT1c receptor antagonist mianserin. The lipid activators of PKC, PDBu and 1,2-dioctanoyl-sn-glycerol (DOG) also caused translocation, but through a mechanism apparently quite independent of Ca2+.
Assuntos
Proteína Quinase C/fisiologia , Receptores de Serotonina/metabolismo , Animais , Cálcio/fisiologia , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , DNA/genética , Expressão Gênica , Técnicas In Vitro , Dibutirato de 12,13-Forbol/farmacologia , Receptores de Serotonina/genética , Transdução de Sinais , TransfecçãoRESUMO
We have cloned and sequenced a novel cDNA (RPR7) encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP). RPR7 was identified by PCR of rat pituitary cDNA, and full-length clones were isolated from a rat olfactory bulb cDNA library. When expressed in COS cells, RPR7 was functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP) in response to stimulation by PACAP-38, PACAP-27, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The order of potency of these ligands was PACAP-38-PACAP-27 > VIP > PHI, suggesting that the receptor corresponds to the pharmacologically characterised PACAP Type I receptor.
Assuntos
Clonagem Molecular , DNA/genética , Expressão Gênica , Receptores de Superfície Celular/genética , Receptores do Hormônio Hipofisário , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , AMP Cíclico/metabolismo , DNA/química , Masculino , Dados de Sequência Molecular , Neuropeptídeos/farmacologia , Bulbo Olfatório/química , Peptídeo PHI/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Receptores de Superfície Celular/química , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Homologia de Sequência , Transfecção , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
The VPAC(2) (vasoactive intestinal peptide (VIP)(2)) receptor is a seven-transmembrane spanning G protein-coupled receptor which responds similarly to VIP and pituitary adenylate cyclase activating polypeptide (PACAP) in stimulating cAMP production. Recently, we reported the localisation of the human VPAC(2) receptor gene (VIPR2) to chromosome 7q36.3 (Mackay, M. et al. (1996) Genomics 37, 345-353). Here, we describe the characterisation of the VIPR2 gene structure and promoter region. The VIPR2 gene is encoded by 13 exons, the initiator codon of the 438 amino acid open reading frame is located in exon 1 and the termination signal and a poly-adenylation signal sequence are located in exon 13. The 5' untranslated region extends 187 bp upstream of the initiator codon and is extremely GC-rich (80%). The poly-adenylation signal is located 2416 bp downstream of the stop codon. Intron sizes range from 68 bp (intron 11) to 45 kb (intron 4) and the human gene spans 117 kb.
Assuntos
Receptores de Peptídeo Intestinal Vasoativo/química , Receptores de Peptídeo Intestinal Vasoativo/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 7 , Clonagem Molecular , Sequência Consenso , Citosina , DNA Complementar/química , DNA Complementar/isolamento & purificação , Éxons , Proteínas de Ligação ao GTP/metabolismo , Guanina , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Receptores de Peptídeo Intestinal Vasoativo/isolamento & purificação , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full-length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP-38 and PACAP-27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.
Assuntos
Receptores de Peptídeo Intestinal Vasoativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Clonagem Molecular , DNA Complementar , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Sensory neuronal cell bodies in the leg of locust, Schistocerca gregaria, were visualized with antibodies to locust choline acetyltransferase and with antibodies to serotonin by the avidin-biotin peroxidase technique. Two groups of sensory cells react with the antibody to choline acetyltransferase: One group is associated with external mechanoreceptors (i.e., hair-plate hairs and campaniform sensilla) and the other with internal proprioceptors (i.e., chordotonal organs and multiterminal receptors). Sensory cells which react with the antibody to serotonin are associated only with internal proprioceptors being found in both chordotonal organs and multiterminal receptors. In the metathoracic femoral chordotonal organ indirect evidence suggests that some sensory cells are reactive to both antibodies. Some multiterminal receptors react with anti-choline-acetyltransferase, while others react with antiserotonin. These results support the conclusion that most insect sensory neurones are cholinergic but some are serotoninergic.
Assuntos
Colina O-Acetiltransferase/análise , Gafanhotos/análise , Neurônios Aferentes/análise , Serotonina/análise , Animais , Feminino , Gafanhotos/enzimologia , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Perna (Membro)/inervação , Masculino , Neurônios Aferentes/enzimologia , PropriocepçãoRESUMO
The distribution of rat vasoactive intestinal peptide2 (VIP2) receptor messenger RNA in the brain and the pituitary gland was examined by in situ hybridization and by ribonuclease protection assay. labelled cells were found chiefly in the suprachiasmatic nucleus, the central nucleus of the amygdala and the thalamus (the lateral geniculate nucleus, and the paraventricular, mediodorsal and ventral nuclei of the thalamus). The distribution of the VIP2 receptor overlaps only in part with that of the VIP1 receptor, for example in the hippocampus, where VIP2 receptor messenger RNA was found in the pyramidal cells of the CA1-CA3 subfields and in the granule cells of the dentate gyrus. Small numbers of neurons containing high concentrations of VIP2 receptor messenger RNA were present in the brainstem in the principal sensory trigeminal nucleus and in the substantia gelatinosa of the spinal cord, suggesting a role for the VIP2 receptor in the processing of sensory information. The presence of the VIP2 receptor in the suprachiasmatic nucleus suggests that it is this receptor subtype which is involved in the control of circadian rhythms.
Assuntos
Encéfalo/metabolismo , Hipófise/metabolismo , RNA Mensageiro/biossíntese , Receptores de Peptídeo Intestinal Vasoativo/biossíntese , Animais , Encéfalo/citologia , Hibridização In Situ , Masculino , Neurônios/metabolismo , Hipófise/anatomia & histologia , Hipófise/citologia , Ratos , Ratos Wistar , Ribonucleases/metabolismo , Medula Espinal/metabolismoRESUMO
1 The VPAC2 and PAC1 receptors are closely related members of the Group II G protein-coupled receptor family. At the VPAC2 receptor, VIP is equipotent to PACAP-38 in stimulating cyclic AMP production, whereas at the PAC1 receptor PACAP-38 is many fold more potent than VIP. In this study, domains which confer this selectivity were investigated by constructing four chimaeric receptors in which segments of the VPAC2 receptor were exchanged with the corresponding segment from the PAC1 receptor. 2 When expressed in COS 7 cells all the chimaeric receptors bound the common ligand [125I]PACAP-27 and produced cyclic AMP in response to agonists. 3 Relative selectivity for agonists was determined primarily by the amino terminal extracellular domain of the PAC1 receptor and the VPAC2 receptor. The interchange of other domains had little effect on the potency of PACAP-38 or PACAP-27. 4 For chimaeric constructs with a PAC1 receptor amino terminal domain, the substitution of increasing portions of the VPAC2 receptor decreased the potency of VIP yet increased that of helodermin. 5 This suggests that the interaction of VIP/helodermin but not PACAP with the PAC1 receptor may be influenced (and differentially so) by additional receptor domains.
Assuntos
Receptores do Hormônio Hipofisário/agonistas , Receptores de Peptídeo Intestinal Vasoativo/agonistas , Proteínas Recombinantes de Fusão/agonistas , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , AMP Cíclico/biossíntese , Primers do DNA , Dados de Sequência Molecular , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
To investigate the phospholipase D (PLD) responses of the VIP/PACAP receptors, VPAC1 and VPAC2, and the PACAP-specific PAC1 receptors (short and "hop" intracellular loop 3 (i3) splice variants), stable CHO cell lines expressing similar levels of each wildtype receptor were generated (except for the VPAC1 receptor clone which showed considerably lower expression and lesser responses in signalling assays). All clones caused activation of PLD in response to agonists, as monitored by [3H]phosphatidylbutanol production. The PLD responses of the PAC1 "hop", but not the "null" receptor, were sensitive to the ARF inhibitor, brefeldin A (BFA) (as were VPAC1 and VPAC2 responses). Chimeric constructs of VPAC2 receptors containing i3 of either PAC1 hop or PAC1 null receptors were transiently expressed in COS 7 cells and PLD responses were measured. Only the PLD response of the hop construct was sensitive to BFA. This suggests that i3 motifs in certain Group II GPCRs may play a key role in determining their linkage to ARF-dependent PLD activation.
Assuntos
Fosfolipase D/metabolismo , Receptores do Hormônio Hipofisário/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Células COS , Cricetinae , AMP Cíclico/biossíntese , DNA Recombinante/genética , Ativação Enzimática/efeitos dos fármacos , Glicerofosfolipídeos/biossíntese , Fosfatos de Inositol/biossíntese , Dados de Sequência Molecular , Ratos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/química , Receptores do Hormônio Hipofisário/genética , Receptores de Peptídeo Intestinal Vasoativo/química , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Receptores do Hormônio Hipofisário/química , Receptores do Hormônio Hipofisário/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/genética , Relação Estrutura-AtividadeRESUMO
The extracellularly recorded electrophysiological activity of single multireceptive dorsal horn neurons was markedly increased by ionophoretic administration of vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase activating polypeptide (PACAP)-38. Some cells responded selectively to PACAP-38 (suggesting mediation by a PACAP receptor), whereas others responded to both VIP and PACAP-38 (suggesting a VIP1 and/or VIP2 receptor). Most non-nociceptive cells were unaffected by PACAP-38 and all were unaffected by VIP. The selectivity of VIP/PACAP receptor antagonists was established on cloned rat VIP1, VIP2 and PACAP receptors in vitro before their utilization to indicate the likely involvement of VIP1, and possibly PACAP receptors, in VIP- and PACAP-38-mediated responses of dorsal horn neurons. The VIP/PACAP receptor antagonists inhibited responses of multireceptive cells to sustained innocuous (brush) and noxious (mustard oil) stimuli, with a selectivity suggesting the involvement of VIP1 and PACAP receptors, although the participation by VIP2 receptors cannot be excluded. These data implicate both VIP and PACAP in regulating the basal responsiveness of multireceptive dorsal horn neurons to sensory stimuli.
Assuntos
Neurônios Aferentes/fisiologia , Neurônios/fisiologia , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Receptores do Hormônio Hipofisário/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Medula Espinal/fisiologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Células COS , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Ratos Wistar , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/efeitos dos fármacos , Receptores de Peptídeo Intestinal Vasoativo/efeitos dos fármacos , Receptores Tipo II de Peptídeo Intestinal Vasoativo , Proteínas Recombinantes/metabolismo , Medula Espinal/efeitos dos fármacos , Transfecção , Peptídeo Intestinal Vasoativo/análogos & derivadosRESUMO
Immunohistochemistry of the locust central nervous system with antibody to choline acetyltransferase (ChAT) purified from the same species shows: first, there are relatively few immunoreactive cell bodies in the CNS; and second, sensory neuropiles, such as the ventral association centre and the ventral VAC (vVAC), the anterior ring tract, the tritocerebrum and the antennal lobe, are immunoreactive. That ChAT is contained in sensory neurones is suggested by immunoreactivity found in peripheral neurone cell bodies. These results indicate that acetylcholine serves primarily as a sensory transmitter in the locust.
Assuntos
Sistema Nervoso Central/enzimologia , Colina O-Acetiltransferase/análise , Gafanhotos/enzimologia , Animais , Gânglios/enzimologia , Histocitoquímica , Técnicas Imunoenzimáticas , Interneurônios/enzimologia , Neurônios Aferentes/enzimologiaRESUMO
Immunocytochemistry of the locust central nervous system shows that most segmental nerves, in particular those of the legs, contain afferent fibres that react with antibody to 5-hydroxytryptamine (5-HT). Adsorption controls indicate that the antigen is 5-HT or a closely related compound. This is supported by the finding of significant amounts of 5-HT in leg nerves using reverse phase high-performance liquid chromatography (HPLC) and electrochemical detection. On the other hand 5-HT was not detectable in locust antennal and cercal nerves with either immunocytochemistry or with HPLC. These results strongly support that some populations of sensory neurones in the locust contain 5-HT.
Assuntos
Gânglios/metabolismo , Gafanhotos/metabolismo , Serotonina/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Eletroquímica/métodos , Gafanhotos/análise , Neurônios Aferentes/metabolismo , Serotonina/análiseRESUMO
The effect of 1,2-diacylglycerols on specific binding of [3H]phorbol 12,13-dibutyrate to cytosolic protein kinase C (PKC) was investigated in tissues reported to contain different proportions of PKC isoforms. In lung, frontal cerebral cortex and cerebellum cytosols (enriched in PKC alpha, beta and gamma, respectively) displacement of specific binding by phorbol 12,13-dibutyrate or diacylglycerols containing unsaturated acyl chains was of similar potency for each tissue. A range of 1,2-diacylglycerols containing saturated acyl chains exhibited varying affinities for [3H]phorbol 12,13-dibutyrate binding sites in each tissue; defining an optimal acyl chain length of around 14 carbons in each case. However, the affinities of saturated diglycerides were consistently lower in lung cytosol than in frontal cerebral cortex and cerebellum cytosols, with the greatest differences occurring at lower acyl chain lengths, especially with 1,2-dioctanoyl-sn-glycerol. Furthermore, a mixed micelle assay of PKC activity showed that 1,2-dioctanoyl-sn-glycerol displayed reduced potency at PKC alpha partially-purified from COS 7 cell cytosol compared to the mixture of PKC isoforms present in rat midbrain cytosol. Both low potency of 1,2-dioctanoyl-sn-glycerol as a displacer of [3H]phorbol 12,13 dibutyrate binding and the ability of arachidonic acid to act as an allosteric enhancer of binding, correlated with the proportional PKC alpha content of a range of tissues reported in the literature. In PKC enzyme activity assays, 1,2-dioctanoyl-sn-glycerol, but not phorbol 12,13-dibutyrate, was correspondingly a much poorer activator of PKC alpha from COS 7 cells than of the broad consensus of isoforms in rat midbrain. When alpha and beta isoforms were extensively-purified on DEAE-cellulose then hydroxyapatite, both the low affinity of 1,2-dioctanoyl-sn-glycerol for [3H]phorbol 12,13-dibutyrate binding sites and their allosteric regulation by arachidonic acid were confirmed to be characteristic of the alpha rather than the beta isoforms.
Assuntos
Diglicerídeos/metabolismo , Metabolismo dos Lipídeos , Dibutirato de 12,13-Forbol/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Ácidos Araquidônicos/metabolismo , Cerebelo/enzimologia , Córtex Cerebral/enzimologia , Citosol/enzimologia , Isoenzimas/imunologia , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Pulmão/enzimologia , Masculino , Dados de Sequência Molecular , Fosfatidilserinas/química , Proteína Quinase C/imunologia , Proteína Quinase C/isolamento & purificação , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been reported to have a number of neurotrophic effects. We have examined the expression of mRNA for PACAP and PACAP type 1 (PAC1) receptor in the mouse embryo by in situ hybridization and the effects of PACAP and VIP on the growth of mouse embryos in vitro. Although we were unable to detect gross effects of either peptide on the growth rates of embryos maintained in culture, mRNAs for both PAC1 receptor and PACAP peptide were present in the nervous system from day 9.5 of embryonic development. PAC1 receptor mRNA was most abundant in the neural tube and the rhombencephalon and was present also in the dorsal root and trigeminal ganglia and the sympathetic chain. The distribution of mRNA for the PACAP peptide overlapped in part with that of the receptor, but was more extensively distributed in the rhombencephalon and in the developing hypothalamus. Within the neural tube, PAC1 receptor mRNA was located in the roof and floor plates, while the distribution of PACAP peptide mRNA was more complex, being located in two columns of cells in the ventromedial neural tube (consistent with the position of developing autonomic motor neurons) and in cells in the dorsolateral neural tube. These data are concordant with a role for PACAP or a related peptide in neural development.
Assuntos
Química Encefálica/fisiologia , Encéfalo/embriologia , Neuropeptídeos/biossíntese , Neurotransmissores/biossíntese , RNA Mensageiro/biossíntese , Receptores do Hormônio Hipofisário/biossíntese , Animais , Autorradiografia , DNA/biossíntese , Feminino , Hibridização In Situ , Camundongos , Técnicas de Cultura de Órgãos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Gravidez , Sondas RNA , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato CiclaseRESUMO
The distribution of rat calcitonin receptor mRNA in the brain and pituitary gland were examined by in situ hybridization. Labelled cells were found in the nucleus accumbens, the preoptic and medial preoptic areas, the paraventricular nucleus of the hypothalamus, and the arcuate nucleus; a smaller number of cells were found in the brainstem and olfactory bulb. No strongly labelled cells were found in the pituitary gland.