RESUMO
Exocytosis of the sperm's single secretory granule, or acrosome, is a regulated exocytosis triggered by components of the egg's investments. In addition to external calcium, sperm exocytosis (termed the acrosome reaction) requires cAMP synthesized endogenously and calcium mobilized from the acrosome through IP3-sensitive channels. The relevant cAMP target is Epac. In the first part of this paper, we present a novel tool (the TAT-cAMP sponge) to investigate cAMP-related signaling pathways in response to progesterone as acrosome reaction trigger. The TAT-cAMP sponge consists of the cAMP-binding sites of protein kinase A regulatory subunit RIß fused to the protein transduction domain TAT of the human immunodeficiency virus-1. The sponge permeated into sperm, sequestered endogenous cAMP, and blocked exocytosis. Progesterone increased the population of sperm with Rap1-GTP, Rab3-GTP, and Rab27-GTP in the acrosomal region; pretreatment with the TAT-cAMP sponge prevented the activation of all three GTPases. In the second part of this manuscript, we show that phospholipase Cε (PLCε) is required for the acrosome reaction downstream of Rap1 and upstream of intra-acrosomal calcium mobilization. Last, we present direct evidence that cAMP, Epac, Rap1, and PLCε are necessary for calcium mobilization from sperm's secretory granule. In summary, we describe here a pathway that connects cAMP to calcium mobilization from the acrosome during sperm exocytosis. Never before had direct evidence for each step of the cascade been put together in the same study.
Assuntos
Acrossomo/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Espermatozoides/metabolismo , AMP Cíclico/fisiologia , Exocitose/genética , Exocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/fisiologia , Masculino , Fosfoinositídeo Fosfolipase C/metabolismo , Fosfoinositídeo Fosfolipase C/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transfecção , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/fisiologiaRESUMO
Oestradiol (E(2)) accelerates oviductal transport of oocytes in cycling rats through a nongenomic pathway that involves the cAMP-PKA signalling cascade. Here we examined the role of the inositol triphosphate (IP3) and mitogen-activated protein kinase (MAPK) signalling cascades in this nongenomic pathway. Oestrous rats were injected with E(2) s.c. and intrabursally (i.b) with the selective inhibitors of phospholipase C (PLC) ET-18-OCH(3) or MAPK PD98059. The number of eggs in the oviduct assessed 24 h later showed that ET-18-OCH(3) blocked E(2)-induced egg transport acceleration, whereas PD98059 had no effect. Other oestrous rats were treated with E(2) s.c. and 1, 3 or 6 h later oviducts were excised and the levels of IP3 and phosphorylated MAPK p44/42 (activated) were determined by radioreceptor assay and western blot, respectively. Oestradiol administration increased IP3 level at 1 and 6 h after treatment, whereas activated MAPK p44/42 level was unchanged. Finally, we explored whether cAMP-PKA and PLC-IP3 signalling cascades are coupled. Inhibition of adenylyl cyclase by i.b. injection of SQ 22536 blocked the increase of IP3 levels induced by E(2), while inhibition of PLC by ET-18-OCH(3) had no effect on E(2)-induced PKA activity. Furthermore, activation of adenylyl cyclase by Forskolin increased oviductal IP3 levels. Thus, activation of PLC-IP3 by E(2) requires previous stimulation of cAMP-PKA. We conclude that the nongenomic pathway utilised by E(2) to accelerate oviductal transport of oocytes in cycling rats involves successive activation of the cAMP-PKA and PLC-IP3 signalling cascades and does not require activation of MAPK. These findings clearly illustrate a non-genomic pathway triggered by E(2) that regulates a complex physiologic process accomplished by an entire organ.
Assuntos
Estradiol/farmacologia , Tubas Uterinas/metabolismo , Fosfatos de Inositol/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transporte do Óvulo/fisiologia , Transdução de Sinais/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Estro , Feminino , Flavonoides/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fosfatidilcolinas/farmacologia , Éteres Fosfolipídicos , Ratos , Ratos Sprague-Dawley , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
We assessed the ability of thymulin, a zinc-dependent nonapeptide produced by the thymic epithelial cells, to influence the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from dispersed anterior pituitary (AP) cells from young, adult, and senescent female rats. Perifusion of young and senescent AP cells with thymulin doses of 10(-6) to 10(-5) M gave a significant stimulatory response for LH but not FSH. Gonadotropin release was always lower in the senescent cells. AP cells from both age groups incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both gonadotropins, with a maximal stimulation at 10(-7) M. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with the secretagogue gonadotropin-releasing hormone (GnRH) revealed a synergistic effect on LH release and an additive effect on the release of FSH. The calcium chelator EGTA blocked the gonadotropin-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF, and forskolin significantly increased the thymulin-stimulated release of gonadotropins. The inositol phosphate enhancer LiCl potentiated the action of thymulin on gonadotropins. It is concluded that the gonadotropin-releasing activity documented here for thymulin is an age- and receptor-dependent effect mediated in part by calcium, cAMP, and inositol phosphates.
Assuntos
Envelhecimento/fisiologia , Hormônio Foliculoestimulante/metabolismo , Hormônio Luteinizante/metabolismo , Adeno-Hipófise/metabolismo , Fator Tímico Circulante/fisiologia , Animais , Cálcio/fisiologia , Linhagem Celular , Células Cultivadas , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Liberador de Gonadotropina/fisiologia , Fosfatos de Inositol/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais/fisiologia , Fator Tímico Circulante/farmacologiaRESUMO
The role of the external third of helix VI of the angiotensin II (AII) AT1 receptor for the interaction with its ligand and for the subsequent signal transduction was investigated by individually replacing residues 252-256 by Ala, and residues 259 or 261 by Tyr, and permanently transfecting the resulting mutants to Chinese hamster ovary (CHO) cells. Binding experiments showed no great changes in affinity of any of the mutants for AII, [Sar1]-AII, or [Sar1, Leu8]-AII, but the affinity for the nonpeptide antagonist DuP753 was significantly decreased. The inositol phosphate response to AII was remarkably decreased in mutants V254A, H256A, and F259Y. These results indicate that AT1 residues Val254, His256, and Phe259 are not involved in ligand binding but participate in signal transduction. Based in these results and in others from the literature, it is suggested that, in addition to the His256 imidazole ring, the Phe259 aromatic ring interacts with the AII's Phe8, thus contributing to the signal-triggering mechanism.
Assuntos
Angiotensina II/metabolismo , Receptores de Angiotensina/fisiologia , Transdução de Sinais/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Ligação ao GTP/fisiologia , Histidina/química , Humanos , Fosfatos de Inositol/fisiologia , Ligantes , Mutagênese Sítio-Dirigida , Fenilalanina/química , Ligação Proteica , Ratos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Relação Estrutura-Atividade , Transfecção , Valina/químicaRESUMO
In previous in vivo studies we have reported that atrial natriuretic factor enhanced induced salivary secretion and increased isoproterenol-induced amylase release in the rat suggesting that, ANF effect could be mediated by phosphatidylinositol hydrolysis. In the present work, the effect of ANF on rat parotid tissue incubated in vitro was investigated with the aim to assess whether the phosphoinositol pathway was involved in ANF intracellular signaling in the parotid gland. Results showed that ANF induced a dose dependent increase in amylase fractional release, which was lower than that evoked by any concentration of isoproterenol. Furthermore 100 nM ANF enhanced isoproterenol-evoked amylase release. The effect of ANF was not affected in the presence of propranolol suggesting the noninvolvement of the beta adrenergic receptor, which is the main stimulus for the output of the enzyme in the parotid gland. However, ANF increased phosphatidylinositol hydrolysis, which implies an increase in intracellular calcium, which is necessary for the achievement of maximal response in amylase release. This effect was abolished in the presence of neomycin supporting ANF direct stimulation of phospholipase C. These results suggest the involvement of the C type natriuretic peptide receptor coupled to phospholipase C in ANF evoked amylase release and ANF enhancement of the isoproterenol-induced output of the enzyme.
Assuntos
Amilases/metabolismo , Fator Natriurético Atrial/fisiologia , Fosfatos de Inositol/fisiologia , Glândula Parótida/enzimologia , Agonistas Adrenérgicos beta/farmacologia , Amilases/efeitos dos fármacos , Animais , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Sinergismo Farmacológico , Isoproterenol/farmacologia , Masculino , Glândula Parótida/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Propranolol/farmacologia , Ratos , Ratos WistarRESUMO
The role of inositol trisphosphate as a chemical messenger in excitation-contraction coupling is discussed, both in terms of positive and negative results. The evidence presented includes experiments on the effect of inositol trisphosphate in intact and skinned fibers, in calcium release from isolated sarcoplasmic reticulum vesicles, in activation of single calcium release channels incorporated in planar bilayers, and biochemical experiments that have established the presence of all the intermediate steps involved in the metabolism of phosphoinositides, both in intact muscle and in isolated membranes. From these results, it is clear that a role for inositol triphosphate in skeletal muscle function is highly likely; whether this molecule is the physiological messenger in excitation-contraction coupling remains to be established.