RESUMO
Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.
Assuntos
Hormônios Peptídicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cálcio/metabolismo , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Grelina , Humanos , Ligantes , Dados de Sequência Molecular , Hormônios Peptídicos/genética , Hormônios Peptídicos/farmacologia , Hipófise/citologia , Hipófise/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , TransfecçãoRESUMO
Several studies have shown that melanin-concentrating hormone (MCH) is an orexigenic peptide in rat. In the present study, a structure-activity relationship with MCH analogs was performed in rat, both in vitro and in vivo. On rat recombinant SLC-1 receptor, both cAMP inhibition and [(125)I]S36057 binding were measured. In vivo, these analogs were injected intracerebroventricularly in rats and their effects were evaluated upon food intake. First, data obtained with the rat recombinant receptor were highly correlated with those obtained from its human counterpart. Second, agonist potencies in the cAMP assay were also highly correlated with binding affinities. These peptides could be classified into several groups according to their potency at the SLC-1 receptor (from subnanomolar activity to complete inactivity). Indeed, there was a strong correlation between their effects upon food intake and the results obtained at the rat SLC-1 receptor. The present report describes for the first time the rat SLC-1 receptor pharmacology and clearly establishes the relevance of the SLC-1 receptor in feeding behavior.
Assuntos
Comportamento Alimentar/efeitos dos fármacos , Hormônios Hipotalâmicos/farmacologia , Melaninas/farmacologia , Hormônios Hipofisários/farmacologia , Receptores de Somatostatina/efeitos dos fármacos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Clonagem Molecular , AMP Cíclico/metabolismo , Injeções Intraventriculares , Masculino , Oligopeptídeos/farmacologia , Poli A/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Using a genomics-based approach for screening orphan G-protein-coupled receptors, we have identified and cloned a novel high-affinity, melanin-concentrating hormone (MCH) receptor. This receptor, named S643b, displays the greatest overall identity (32%) with the previously reported human SLC-1 receptor (MCH1) and to a lesser extent with the somatostatin receptor subtypes. The gene encoding the S643b receptor spans more than 23 kilobase pairs (kb) and was mapped, by radiation hybrid experiments, on chromosome 6q14.3-q15. Comparison of the S643b cDNA with human genomic sequence reveals that the 340-amino-acid receptor is encoded by five exons. Its tissue distribution, as determined by Northern blot and reverse transcription-polymerase chain reaction analysis, indicates that a 4-kb transcript is predominantly expressed in the brain. When expressed in Chinese hamster ovary (CHO) cells, the S643b receptor displays a strong, dose-dependent, transient elevation of intracellular calcium in response to MCH (EC(50) = 9.5 nM). During the present study, we isolated a splice variant, designated S643a, encoding for a receptor that was not activated by MCH in a cellular calcium mobilization assay. Comparative pharmacological studies using CHO cells stably expressing either SLC-1 or S643b receptors demonstrated that similar structural features of MCH are required to stimulate intracellular Ca(2+) mobilization at both receptors. The identification and localization of this new MCH receptor (MCH2) provides further insight into the physiological implication of MCH in modulating behavioral responses, including food intake.
Assuntos
Cromossomos Humanos Par 6 , Receptores do Hormônio Hipofisário/genética , Receptores de Somatostatina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/análise , Humanos , Dados de Sequência Molecular , Peptídeos/farmacologia , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G , Receptores do Hormônio Hipofisário/metabolismo , Receptores de Somatostatina/química , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Shortened, more stable and weakly hydrophobic analogues of melanin-concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH(6 - 17), we found that: (1) substitution of Tyr(13) by a Phe residue; (2) addition of a 3-iodo-Tyr residue at the N-terminus; and (3) addition of a hydrophilic spacer 8-amino-3,6-dioxyoctanoyl between the 3-iodo-Tyr and MCH(6 - 17) (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]-GTPgammaS binding at membranes from HEK293 cells stably expressing the human MCH receptor. Specific binding of [125I]-S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg(-1)) than [125I]-[3-iodo Tyr(13)]-MCH. However, the K(D) for [125I]-S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]-[3-iodo Tyr(13)]-MCH (0.46 nM). Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (K(i)=0.053 nM). Finally, [125I]-S36057 also potently labelled the MCH receptor in membranes from whole rat brain (K(D) 0.044 nM, B(max)=11 fmol mg(-1)). In conclusion, [125I]-S36057 is a more potent and more stable radioligand than [125I]-[3-iodo Tyr(13)]-MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.
Assuntos
Oligopeptídeos/metabolismo , Receptores do Hormônio Hipofisário/agonistas , Receptores do Hormônio Hipofisário/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Hormônios Hipotalâmicos/química , Hormônios Hipotalâmicos/metabolismo , Hormônios Hipotalâmicos/farmacologia , Radioisótopos do Iodo , Cinética , Ligantes , Melaninas/química , Melaninas/metabolismo , Melaninas/farmacologia , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Hormônios Hipofisários/química , Hormônios Hipofisários/metabolismo , Hormônios Hipofisários/farmacologia , Ligação Proteica , Ensaio Radioligante , Especificidade por Substrato , TermodinâmicaRESUMO
Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5'-O-(3-[(35)S]thiotriphosphate ([(35)S]- GTPgammaS) binding. The dodecapeptide MCH-(6-17) (MCH ring between Cys(7) and Cys(16), with a single extra amino acid at the N terminus (Arg(6)) and at the C terminus (Trp(17))) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [(35)S]- GTPgammaS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met(8), Arg(11), and Tyr(13), were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys(7) and Cys(16) were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [(35)S]- GTPgammaS binding, these compounds behaved as weak antagonists (K(B) 1-4 microm). Finally, substitution in MCH-(6-17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K(B) = 0.1-0.2 microm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.
Assuntos
Hormônios Hipotalâmicos/metabolismo , Melaninas/metabolismo , Hormônios Hipofisários/metabolismo , Receptores de Somatostatina/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , AMP Cíclico/metabolismo , Dissulfetos , Relação Dose-Resposta a Droga , Deleção de Genes , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/farmacologia , Ligação Proteica , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Saponinas/farmacologia , Relação Estrutura-Atividade , Temperatura , TransfecçãoRESUMO
A modulation of the expression of immediate-early genes (IEGs) such as c-fos is likely involved in the long-term influence of dopaminergic ligands on the activity of basal ganglia neurons. The roles of individual dopamine receptor types in this regard remain unclear, and the present study employed primary cultures of rat striatal neurons to examine the potential modulation of c-fos expression by D(3) receptors. Neurons were treated with the preferential D(3) receptor agonists, (+/-)-7-OH-DPAT and PD 128,907, which showed, respectively, 16-fold and 56-fold selectivity for recombinant rat D(3) vs. D(2) receptors (K(i) values, rD(2)/rD(3) = 59.5/3.7 nM and 251/4.5 nM, respectively). At concentrations of 3 and 30 nM, respectively, (+/-)-7-OH-DPAT and PD 128,907 significantly increased the expression of c-fos mRNA. The action of (+/-)-7-OH-DPAT was expressed stereospecifically; its (+)-isomer (K(i) values, D(3)/D(2) = 1.6/56.7 elicited a 26% +/- 7.6% increase in c-fos expression whereas its (-)-isomer (K(i) values, D(3)/D(2) = 59/1,060 nM) was ineffective. Further, stimulation of c-fos mRNA expression by PD 128,907 (20 nM) was markedly and significantly (P < 0.05) attenuated (-91.8% +/- 5.3%) by 30 nM of the selective D(3) receptor antagonist, (+)-S 14297 (K(i) values, D(3)/D(2) = 11/401 nM). In contrast, the action of PD 128,907 was significantly less affected (-24.5% +/- 7.8%) by 30 nM of its less active distomer, (-)-S 17777 (K(i) values, D(3)/D(2) = 294/3,191 nM). An examination of the concentration-response relationship revealed that (+/-)-7-OH-DPAT and PD 128,907 both produced bell-shaped dose-response curves for c-fos induction. The sequential activation of D(2) receptors-which inhibit striatal c-fos expression (Simpson and Morris [1995] Neuroscience 68:97-106)-by higher concentrations of (+/-)-7-OH-DPAT and PD 128,907 is presumably involved in the inflexion at higher doses. In conclusion, the present data demonstrate that activation of D(3) receptors results in a selective induction of c-fos mRNA expression in cultured striatal neurons. These data show that neuronal D(3) receptors control the expression of IEGs and suggest that D(3) receptors may mediate long-term adapative changes in the activity of neurons in the basal ganglia.
Assuntos
Corpo Estriado/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/farmacologia , Animais , Benzopiranos/farmacologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Técnicas de Cultura de Células , Corpo Estriado/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Embrião de Mamíferos , Furanos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxazinas/farmacologia , RNA Mensageiro/análise , Ratos , Receptores de Dopamina D3 , Tetra-Hidronaftalenos/farmacologiaRESUMO
A new series of benzopyrano[3,4-c]pyrrole derivatives were synthesized and evaluated for their interaction with dopamine D3 versus D2 receptors. Amongst these compounds, 4x (S 33084) was found to be a potent and selective dopamine D3 receptor antagonist.
Assuntos
Benzopiranos/síntese química , Benzopiranos/farmacologia , Antagonistas de Dopamina/síntese química , Antagonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , 8-Hidroxi-2-(di-n-propilamino)tetralina/análogos & derivados , 8-Hidroxi-2-(di-n-propilamino)tetralina/química , Animais , Células CHO/metabolismo , Cricetinae , Agonistas de Dopamina/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Radioisótopos do Iodo , Pirróis/química , Pirrolidinas/química , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3 , Relação Estrutura-Atividade , Sulpirida/metabolismo , Tetra-Hidronaftalenos/química , TransfecçãoRESUMO
The two forms of angiotensin II (Ang II) receptors, AT1 and AT2 subtypes, have been demonstrated in many other cells beside the anterior pituitary cells. Attempting to investigate the subtype(s) of Ang II receptors implicated in the multiple transduction mechanisms involved in Ang II stimulation of prolactin (PRL) release by lactotropes, we studied the effect of selective nonpeptidergic Ang II antagonists on the PRL release, adenylate cyclase (AC), and phospholipase C activities. In intact cells, the AT1 antagonist DuP753 blocked Ang II-induced PRL release, reversed in a dose dependent manner Ang II-evoked inositol phosphates production, and inhibited completely the PLC and protein kinase C (PKC) dependent cAMP accumulation induced by Ang II. In membrane preparations, the Ang II receptors were negatively coupled to AC. The AT1 antagonist blocked in a dose dependent manner the inhibitory effect of Ang II on cAMP production. In intact cells, the negative coupling of Ang II receptor with AC was observed only when PKC was down regulated by long term 12-O-tetradecanolylphorbol-13-acetate pretreatment. Ang II was able to inhibit vasoactive intestinal peptide-induced cAMP accumulation, a response which was also prevented by DuP753. The different coupling of Ang II receptor described above implicated only the AT1 type receptor since the AT2 antagonists (PD123177 and PD123319) were ineffective at any doses tested (10(-8) to 10(-5) M). The obtained results indicate that the regulation of PRL secretion involves the AT1 receptor subtype and that this receptor might be coupled to multiple effectors.
Assuntos
Angiotensina II/farmacologia , Angiotensina I/metabolismo , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Receptores de Angiotensina/fisiologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Antagonistas de Receptores de Angiotensina , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Feminino , Fosfatos de Inositol/biossíntese , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
This study examined the mechanism underlying the influence of clozapine upon serotoninergic transmission in the rat. In vitro, clozapine manifested weak affinity at 5-HT1A receptors (pKi = 6.5) as compared to the agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (9.0), but high affinity at alpha 1-adrenoceptors (8.2) as compared to the alpha 1-adrenoceptor antagonist, prazosin (9.7). Ex vivo, clozapine (inhibitory dose (ID)50 = 0.7 mg/kg s.c.) mimicked prazosin (0.5) in potently occupying central alpha 1-adrenoceptors whereas, as compared to 8-OH-DPAT (0.2), it failed to occupy 5-HT1A receptors (> 10.0). The firing of serotoninergic neurones in the dorsal raphe nucleus was abolished by 8-OH-DPAT, clozapine and prazosin with ID50 values of 0.006, 0.09 and 0.07 mg/kg i.v., respectively. At comparable doses, they reduced striatal turnover of 5-HT. While the 5-HT1A receptors antagonists, (-)-tertatolol (2.0 mg/kg i.v.) and spiperone (0.63 mg/kg i.v.), blocked the action of 8-OH-DPAT upon dorsal raphe nucleus firing, they failed to modify the effect of clozapine and prazosin. In contrast, the alpha 1-adrenoceptor agonist, cirazoline (0.005 mg/kg i.v.) prevented the actions of clozapine and prazosin, but not that of 8-OH-DPAT. It is concluded that clozapine only weakly interacts with 5-HT1A receptors and that its potent alpha 1-adrenoceptor antagonist properties underlie inhibition of serotoninergic transmission.
Assuntos
Clozapina/farmacologia , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Serotonina/fisiologia , Transmissão Sináptica/efeitos dos fármacos , 5-Hidroxitriptofano/metabolismo , Animais , Química Encefálica/efeitos dos fármacos , Eletrofisiologia , Masculino , Neurônios/efeitos dos fármacos , Núcleos da Rafe/citologia , Núcleos da Rafe/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Purinérgicos P1/efeitos dos fármacosRESUMO
Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decrease density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph cells.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Ácido Araquidônico/metabolismo , Canais de Cálcio/metabolismo , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Neoplasias Hipofisárias/patologia , Prolactinoma/patologia , Receptores de Dopamina D2/química , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
In vitro and in vivo release of pituitary hormones were studied in the presence of (hydroxyproline9)LHRH ((Hyp)LHRH), a newly characterized endogenous molecular form of LHRH. Results were compared to those obtained with LHRH itself. (Hyp)LHRH, as LHRH, stimulated both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a homothetic manner. The hydroxylated compound was, however, 24 times (in vitro) and 5 times (in vivo) less potent than LHRH. The lower activity of (Hyp)LHRH than of LHRH in the in vitro assay correlated well with a 28-fold lesser potency in a binding test using pituitary membrane preparations. The higher relative potency and the prolonged effect of (Hyp)LHRH in the in vivo test were related to a lesser susceptibility of the hydroxylated form to proteolytic degradation. Effects of LHRH and of (Hyp)LHRH were not additive, both peptides were equally able to desensitize gonadotrophs to a subsequent challenge by the other. Taken together, these observations suggest that both forms of LHRH act at the same receptor site. The lesser affinity of the hydroxylated compound is compensated to a certain extent by its higher resistance to enzymatic degradation. It is concluded that in spite of its lesser potency, (Hyp)LHRH may participate in the regulation of gonadotropins.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Luteinizante/metabolismo , Adeno-Hipófise/efeitos dos fármacos , Animais , Castração , Células Cultivadas , Ditiotreitol/farmacologia , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Luteinizante/sangue , Masculino , Adeno-Hipófise/metabolismo , Radioimunoensaio , Ratos , Ratos EndogâmicosRESUMO
Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene, are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decreased density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph cells.
Assuntos
Receptores de Dopamina D2/fisiologia , Transdução de Sinais/fisiologia , Ácido Araquidônico/metabolismo , Bromocriptina/uso terapêutico , Cálcio/metabolismo , Resistência a Medicamentos , Proteínas de Ligação ao GTP/fisiologia , Humanos , Neoplasias Hipofisárias/tratamento farmacológico , Prolactinoma/tratamento farmacológicoRESUMO
When applied to rat anterior pituitary cells, angiotensin-II (AII) exerted two opposite effects on adenylate cyclase (AC) activity: a pertussis toxin (PTX)-sensitive inhibition of the enzyme with a maximal effect of -42 +/- 2% in crude cell membrane preparations, and, in contrast, a non-PTX-sensitive stimulation of cAMP production (maximal effect = 38 +/- 3%) in intact cells. The apparent affinity of both effects was equal to 1.8 nM. The stimulation of cAMP formation parallels the stimulation of PRL release. Under the same conditions, dopamine (DA) inhibited both membrane AC activity and cAMP formation in intact cells by a PTX-sensitive mechanism. After separation of pituitary cell types by sedimentation at unit gravity, the effects of AII and DA on intracellular cAMP and membrane AC activity coincided in the same fractions (those enriched in PRL cells). The stimulatory effect of AII on cAMP formation was about 5 times weaker than that of peptides positively coupled to AC as vasoactive intestinal peptide in total as well as in PRL-enriched cells. Since the AII receptor is also coupled to phospholipase-C (PLC) in a non-PTX-sensitive manner, we investigated whether protein kinase-C (PKC) could indirectly account for the positive effect of AII on cAMP formation. 12-O-Tetradecanolylphorbol 13-acetate (TPA), a stimulator of PKC was indeed able to increase intracellular cAMP; this effect was not additive with that of AII. conversely, application of the PKC inhibitors H7 [1-(5-isoquinolylsulfonyl)2-methyl-piperazine] and staurosporine or desensitization of PKC by long exposure of the cells to TPA abolished the cAMP response to TPA as well as that to AII. In addition, thyreoliberin, another activator of the PLC pathway, was able to stimulate cAMP formation in a PKC-dependent manner. DA inhibition of intracellular cAMP was not affected by any PKC inhibition. We conclude that in lactotroph cells, 1) the AII inhibitory coupling to AC observed in membrane preparations does not exist in intact cells, at least under basal conditions; and 2) the AII intracellular cAMP stimulation observed is not accounted for by a direct coupling with AC; it is due to a cross-talk of the PLC pathway mediated by PKC, an effect that might be shared by other PLC-stimulating mediators and may participate in the regulation of PRL release.
Assuntos
Angiotensina II/farmacologia , AMP Cíclico/metabolismo , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Proteína Quinase C/farmacologia , Adenilil Ciclases/metabolismo , Animais , Células Cultivadas , Dopamina/farmacologia , Fosfatos de Inositol/metabolismo , Membranas Intracelulares/metabolismo , Adeno-Hipófise/citologia , Ratos , Hormônio Liberador de Tireotropina/farmacologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
In primary cultures of anterior pituitary cells, dopamine inhibited basal and thyrotropin-releasing hormone (TRH)-stimulated inositol monophosphate, bisphosphate, and trisphosphate production. This inhibition by dopamine can be resolved into two distinct components. One of the components was rapid and already present after 10 s. The other was slower, starting after 1 min, and was mimicked by nimodipine, a dihydropyridine calcium channel antagonist. The effects of dopamine and nimodipine were not additive on both basal and TRH-stimulated inositol phosphate production. Furthermore, the dopamine inhibition in the presence of TRH was much higher than the inhibition induced by nimodipine. It is thus likely that calcium entry through voltage-dependent calcium channels triggers a positive feedback on TRH stimulation of phospholipase C. However, depolarizing concentrations of K+ or BAY-K-8644, a voltage-dependent calcium channel agonist, had no effect on inositol monophosphate and bisphosphate accumulation. Ionomycin, even at a very high concentration (10 microM), had only a slight and transient effect on inositol phosphate formation. In addition, these agents did not affect the TRH dose-dependent stimulation of inositol phosphate production. These results suggest that the intracellular calcium concentrations that we measured under basal and TRH-stimulated conditions are sufficient to allow the maximal activity of phospholipase C which can be obtained under these two experimental conditions. In contrast, any decrease in the intracellular calcium concentration by a dihydropyridine antagonist, suppression of extracellular calcium, or inactivation of a voltage-dependent calcium channel by long term depolarization with K+ decreased the phospholipase C activities measured under basal and TRH-stimulated conditions. From these data it can be concluded that dopamine inhibits inositol phosphate production by two distinct mechanisms. The slow dopamine-induced inhibition of TRH-stimulated inositol phosphate production which is mimicked by nimodipine is likely because of an inhibition of a voltage-dependent calcium channel. This is substantiated further by the fact that ionomycin (10 microM) was able to reverse the nimodipine inhibitions as well as this slow component of dopamine inhibition. The nature of the rapid inhibition of TRH-stimulated inositol phosphate production induced by dopamine, but not by nimodipine, remains to be determined. It is suppressed in the absence of extracellular Ca2+. This may suggest that this inhibition is related to blockade of non-dihydropyridine-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)