RESUMO
Cyclic 3',5'-adenosine monophosphate and cyclic 3',5'-guanosine monophosphate are intracellular (second) messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. These enzymes are involved in a broad array of signal transduction pathways mediated by the cyclic nucleotide monophosphates and their kinases, which control multiple aspects of cell function through the phosphorylation of protein substrates. We review the findings and working hypotheses on the role of the cyclic nucleotides and their kinases in the control of electrical activity of the endocrine pituitary cells and the plasma membrane channels involved in this process.
Assuntos
AMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Células Endócrinas/fisiologia , Canais Iônicos/fisiologia , Hipófise/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Adenilil Ciclases/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Colforsina/farmacologia , Células Endócrinas/efeitos dos fármacos , Guanilato Ciclase/fisiologia , Humanos , Canais Iônicos/efeitos dos fármacos , Modelos Animais , Modelos Biológicos , Hipófise/efeitos dos fármacos , Hipófise/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
All secretory anterior pituitary cells fire action potentials spontaneously and exhibit a high resting cation conductance, but the channels involved in the background permeability have not been identified. In cultured lactotrophs and immortalized GH(3) cells, replacement of extracellular Na(+) with large organic cations, but not blockade of voltage-gated Na(+) influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at -50 mV, which was close to the resting membrane potential in these cells, replacement of bath Na(+) with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH(3) cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH(3) cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, Ca(2+) influx and prolactin release in a concentration-dependent manner. Gd(3+), a common Ca(2+) channel blocker, and flufenamic acid, an inhibitor of non-selective cation channels, also inhibited electrical activity, Ca(2+) influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials with consequent contribution to Ca(2+) influx and hormone release in lactotrophs and GH(3) cells.
Assuntos
Canais Iônicos/metabolismo , Lactotrofos/metabolismo , Prolactina/metabolismo , Canais de Cátion TRPC/metabolismo , Potenciais de Ação , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Lactotrofos/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , Canais de Cátion TRPC/efeitos dos fármacos , Canais de Cátion TRPC/genética , Fatores de TempoRESUMO
The founder of physiology studies in the Balkans and the pioneer of research on hypothermia, Ivan Djaja (Jean Giaja) was born 1884 in L'Havre. Giaja gained his PhD at the Sorbonne in 1909. In 1910 he established the first Chair of Physiology in the Balkans and organized the first Serbian Institute for Physiology at the School of Philosophy of the University of Belgrade. He led this Institute for more than 40 subsequent years. His most notable papers were in the field of thermoregulation and bioenergetics. Djaja became member of the Serbian and Croatian academies of science and doctor honoris causa of Sorbonne. In 1952 for the seminal work on the behavior of deep cooled warm blooded animals he became associate member of the National Medical Academy in Paris. In 1955 the French Academy of Sciences elected him as associate member in place of deceased Sir Alexander Fleming. Djaja died in 1957 during a congress held in his honor. He left more than 200 scientific and other papers and the golden DaVincian credo "Nulla dies sine experimento". His legacy was continued by several generations of researchers, the most prominent among them being Stefan Gelineo, Radoslav Andjus and Vojislav Petrovic.
Assuntos
Pesquisa Biomédica/história , Regulação da Temperatura Corporal , Fisiologia/história , Universidades/história , Animais , História do Século XX , Humanos , SérviaRESUMO
Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.
Assuntos
Cisteína/química , Receptores Purinérgicos P2X4/química , Animais , Sítios de Ligação , Sequência Conservada , Cisteína/genética , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Ratos , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismoRESUMO
Overproduction of the beta-amyloid fragment 1-42 (A beta(1-42)) is thought to contribute to synaptic dysfunction and neuronal death in Alzheimer's disease. Mounting evidence suggests that purinergic receptors play critical roles in synaptic plasticity and neuronal survival, but the potential involvement of these receptors in A beta(1-42)-induced synaptic dysfunction and neuronal death has not been addressed. Here we report that A beta(1-42) promoted accumulation of the calcium-permeable purinergic receptor P2X4 in neurons. We also report evidence that A beta(1-42) induced a caspase-3-mediated cleavage of the receptor that slowed channel closure times and prevented agonist-induced internalization of the receptor. Molecular interference to reduce the expression of P2X4 in primary rodent neurons attenuated A beta(1-42)-induced neuronal death while induced expression of P2X4 in a neuronal cell line that does not normally express P2-receptors enhanced the toxic effect of A beta(1-42). Together these findings suggest that A beta(1-42)-induced synaptic dysfunction and neuronal death may involve perturbations in P2X4 purinergic receptors.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Caspase 3/metabolismo , Morte Celular/fisiologia , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Purinas/toxicidade , Receptores Purinérgicos P2/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Hipocampo/citologia , Hipocampo/patologia , Humanos , Dados de Sequência Molecular , Neurônios/citologia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X4 , Sinapses/metabolismoRESUMO
The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Here, we studied the expression and role of HCN channels in pacemaking activity, Ca(2+) signalling, and prolactin secretion in GH(3) immortalised pituitary cells. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. A hyperpolarisation of the membrane potential below - 60 mV elicited a slowly activating voltage-dependent inward current (I(h)) in the majority of tested cells, with a half-maximal activation voltage of -89.9 +/- 4.2 mV and with a time constant of 1.4 +/- 0.2 s at -120 mV. The bath application of 1 mM Cs(+), a commonly used inorganic blocker of I(h), and 100 microM ZD7288, a specific organic blocker of I(h), inhibited I(h) by 90 +/- 4.1% and 84.3 +/- 1.8%, respectively. Receptor- and nonreceptor-mediated activation of adenylyl and soluble guanylyl cyclase and the addition of a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, 8-Br-cAMP, did not affect I(h). Inhibition of basal adenylyl cyclase activity, but not basal soluble guanylyl cyclase activity, led to a reduction in the peak amplitude and a leftward shift in the activation curve of I(h) by 23.7 mV. The inhibition of the current was reversed by stimulation of adenylyl cyclase with forskolin and by the addition of 8-Br-cAMP, but not 8-Br-cGMP. Application of Cs(+) had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and I(h)-independent effects on spontaneous electrical activity, Ca(2+) signalling, and prolactin release. These results indicate that HCN channels in GH(3) cells are under tonic activation by basal level of cAMP and are not critical for spontaneous firing of action potentials.
Assuntos
Canais Iônicos/metabolismo , Potenciais da Membrana/fisiologia , Hipófise/metabolismo , Prolactina/metabolismo , Adenilil Ciclases/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/classificação , Canais Iônicos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Hipófise/citologia , Canais de Potássio , RNA Mensageiro/análise , Ratos , Transdução de Sinais/fisiologiaRESUMO
Activation of high-conductance Ca(2+)-activated K(+) (BK) channels normally limits action potential duration and the associated voltage-gated Ca(2+) entry by facilitating membrane repolarization. Here we report that BK channel activation in rat pituitary somatotrophs prolongs membrane depolarization, leading to the generation of plateau-bursting activity and facilitated Ca(2+) entry. Such a paradoxical role of BK channels is determined by their rapid activation by domain Ca(2+), which truncates the action potential amplitude and thereby limits the participation of delayed rectifying K(+) channels during membrane repolarization. Conversely, pituitary gonadotrophs express relatively few BK channels and fire single spikes with a low capacity to promote Ca(2+) entry, whereas an elevation in BK current expression in a gonadotroph model system leads to the generation of plateau-bursting activity and high-amplitude Ca(2+) transients.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Adeno-Hipófise/metabolismo , Canais de Potássio Cálcio-Ativados , Canais de Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/farmacocinética , Canais de Cálcio/metabolismo , Compartimento Celular/fisiologia , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retroalimentação/fisiologia , Feminino , Gonadotropinas/metabolismo , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Indóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Modelos Biológicos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Bloqueadores dos Canais de Potássio , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.
Assuntos
Expressão Gênica , Canais Iônicos/genética , Adeno-Hipófise/metabolismo , Animais , Cálcio/farmacologia , Canais de Cálcio/genética , Células Cultivadas , Eletrofisiologia , Feminino , Gonadotropinas Hipofisárias/metabolismo , Hormônio do Crescimento/metabolismo , Humanos , Ativação do Canal Iônico/fisiologia , Canais de Potássio/genética , Prolactina/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Sódio/genética , Tetrodotoxina/farmacologiaRESUMO
In excitable cells, voltage-gated calcium influx provides an effective mechanism for the activation of exocytosis. In this study, we demonstrate that although rat anterior pituitary lactotrophs, somatotrophs, and gonadotrophs exhibited spontaneous and extracellular calcium-dependent electrical activity, voltage-gated calcium influx triggered secretion only in lactotrophs and somatotrophs. The lack of action potential-driven secretion in gonadotrophs was not due to the proportion of spontaneously firing cells or spike frequency. Gonadotrophs exhibited calcium signals during prolonged depolarization comparable with signals observed in somatotrophs and lactotrophs. The secretory vesicles in all three cell types also had a similar sensitivity to voltage-gated calcium influx. However, the pattern of action potential calcium influx differed among three cell types. Spontaneous activity in gonadotrophs was characterized by high amplitude, sharp spikes that had a limited capacity to promote calcium influx, whereas lactotrophs and somatotrophs fired plateau-bursting action potentials that generated high amplitude calcium signals. Furthermore, a shift in the pattern of firing from sharp spikes to plateau-like spikes in gonadotrophs triggered luteinizing hormone secretion. These results indicate that the cell type-specific action potential secretion coupling in pituitary cells is determined by the capacity of their plasma membrane oscillator to generate threshold calcium signals.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Hormônios Hipofisários/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Potenciais de Ação , Animais , Agonistas dos Canais de Cálcio/farmacologia , Células Cultivadas , Eletrofisiologia , Feminino , Hormônio do Crescimento/metabolismo , Hormônios/metabolismo , Imuno-Histoquímica , Hormônio Luteinizante/metabolismo , Hipófise/citologia , Potássio/metabolismo , Prolactina/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Pituitary cells secrete ATP, which acts as an autocrine and/or paracrine extracellular messenger on two families of purinergic receptors: G-protein-coupled P2Y receptors (P2YRs) and ion-conducting P2X receptors (P2XRs). Lactotrophs and GH(3)-immortalized cells express the P2Y(2)R subtype. Several P2XR subtypes are expressed in pituitary cells. Gonadotrophs and somatotrophs express P2X(2a)R and P2X(2b)R, which occur as heteromeric channels. Lactotrophs and GH(3) cells express one or more ion-conducting subtypes from among P2X(3)R, P2X(4)R and P2X(7)R in homomeric form. Thyrotrophs and corticotrophs also express P2XRs, but their identification requires further study. Pituitary cells express purinergic P1 receptors, which are activated by adenosine. The A(1)R subtype of these receptors is expressed in melanotrophs and GH(3) cells. In this review, we briefly discuss the expression and coupling of A(1)R and P2Y(2)R, and focus on the expression and Ca(2+) signaling of P2XRs.
Assuntos
Trifosfato de Adenosina/metabolismo , Adeno-Hipófise/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Comunicação Autócrina , Sinalização do Cálcio , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Comunicação Parácrina , Adeno-Hipófise/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genéticaRESUMO
Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.
Assuntos
Cálcio/metabolismo , GMP Cíclico/biossíntese , Guanilato Ciclase/metabolismo , Adeno-Hipófise/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Células Cultivadas , Hormônio Liberador da Corticotropina/farmacologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Guanidinas/farmacologia , Immunoblotting , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Adeno-Hipófise/citologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hormônio Liberador de Tireotropina/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. In this paper we studied the interactions between NO and intracellular Ca(2+) in the control of sGC in rat anterior pituitary cells. Experiments were done in cultured cells, which expressed neuronal and endothelial NO synthases, and in cells with elevated NO levels induced by the expression of inducible NO synthase and by the addition of several NO donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In contrast, depolarization of cells by high K(+) and Bay K 8644, an L-type Ca(2+) channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca(2+)-deficient medium. This inhibition was independent from the pattern of Ca(2+) signaling (oscillatory versus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca(2+). These results indicate that inactivation of sGC by intracellular Ca(2+) serves as a negative feedback to break the stimulatory action of NO on enzyme activity in intact pituitary cells.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Guanilato Ciclase/metabolismo , Óxido Nítrico/metabolismo , Adeno-Hipófise/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Feminino , Guanidinas/farmacologia , Isoenzimas/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitroprussiato/farmacologia , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/enzimologia , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Solubilidade , Alcaloides de Vinca/farmacologiaRESUMO
Purinergic G protein-coupled receptors (P2YR) and ion-conducting receptor-channels (P2XR) are present in the pituitary. However, their identification, expression within pituitary cell subpopulations, and the ability to elevate intracellular Ca2+ concentration ([Ca2+]i) in response to ATP stimulation were incompletely characterized. Here we show that mixed populations of rat anterior pituitary cells express messenger RNA transcripts for P2Y2R, P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R. The transcripts and functional P2Y2R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs, and their activation by ATP led to an extracellular Ca2+-independent rise in [Ca2+]i in about 40% of cells tested. Lactotrophs and GH3 cells, but not somatotrophs, also express transcripts for P2X7R, P2X3R, and P2X4R. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Coexpression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X2aR and P2X2bR, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. Consistent with the cell-specific expression of transcripts for P2X2R and P2X3R, the expression of their functional heteromers was not evident in pituitary cells. Receptors differed in their capacities to elevate and sustain Ca2+ influx-dependent rise in [Ca2+]i during the prolonged ATP stimulation. These results indicate that the purinergic system of anterior pituitary is extremely complex and provides an effective mechanism for generating a cell- and receptor-specific Ca2+ signaling pattern in response to a common agonist.
Assuntos
Adeno-Hipófise/metabolismo , Receptores Purinérgicos P2/genética , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Expressão Gênica , Gonadotropinas Hipofisárias/análise , Hormônio do Crescimento/análise , Adeno-Hipófise/química , Adeno-Hipófise/citologia , Prolactina/análise , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Transdução de Sinais , Transfecção , Uridina Trifosfato/farmacologiaRESUMO
Polychlorinated biphenyl (PCB)-based transformer fluids belong to a class of environmentally persistent mixtures with known toxic effects. Here, we studied the acute effects of Askarel (which contains Aroclor 1260) and two substitute transformer fluids (the silicone oil-based DC561 and the mineral oil-based ENOL C) on rat testicular steroidogenesis. Single intraperitoneal (ip; 10 mg/kg body weight) or bilateral intratesticular (itt; 25 microg/testis) injections of Askarel markedly decreased serum androgen levels 24 hr after administration. In acute testicular cultures from these animals, chorionic gonadotropin-stimulated progesterone and androgen productions were severely attenuated. When itt was injected or added in vitro, Askarel inhibited 3ss-hydroxysteroid dehydrogenase (3ssHSD), stimulated 17[alpha]-hydroxylase/lyase (P450c17), and did not affect 17ss-hydroxysteroid dehydrogenase in testicular postmitochondrial fractions. The ip-injected Askarel did not affect 3ssHSD, but inhibited P450c17, suggesting that a more intensive metabolism of peripherally injected Askarel reduces the circulating levels of active ingredients below the threshold needed for inhibition of 3ssHSD and generates a derivative that inhibits P450c17. In contrast to Askarel, itt-injection (25 microg/testis) of DC561 and ENOL C did not affect in vivo and in vitro steroidogenesis. These findings show the acute effects of Askarel, but not silicone and mineral oils, on testicular steroidogenesis.
Assuntos
Arocloros/efeitos adversos , Bifenilos Policlorados/efeitos adversos , Hormônios Testiculares/biossíntese , Testículo/fisiologia , Animais , Técnicas de Cultura , Infusões Parenterais , Masculino , Ratos , Ratos Wistar , Testículo/efeitos dos fármacosRESUMO
ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca(2+) and facilitate voltage-sensitive Ca(2+) entry in excitable cells. To study Ca(2+) signaling by P2XRs and its dependence on voltage-sensitive Ca(2+) influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca(2+)](i). P2XR subtypes differed in the peak amplitude of [Ca(2+)](i) response independently of the level of receptor expression, with the following order: P2X(1)R < P2X(3)R < P2X(4)R < P2X(2b)R < P2X(2a)R < P2X(7)R. During prolonged agonist stimulation, Ca(2+) signals desensitized with different rates: P2X(3)R > P2X(1)R > P2X(2b)R > P2X(4)R >> P2X(2a)R >> P2X(7)R. The pattern of [Ca(2+)](i) response for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca(2+)](i). The P2X(1)R, P2X(3)R, and P2X(4)R-derived [Ca(2+)](i) signals were predominantly dependent on activation of voltage-sensitive Ca(2+) influx, both voltage-sensitive and -insensitive Ca(2+) entry pathways equally contributed to [Ca(2+)](i) responses in P2X(2a)R- and P2X(2b)R-expressing cells, and P2X(7)R operated as a nonselective pore capable of conducting larger amounts of Ca(2+) independently on the status of voltage-gated Ca(2+) channels. Thus, Ca(2+) signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca(2+)](i) patterns in response to a common agonist.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Receptores Purinérgicos/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X3 , TransfecçãoRESUMO
In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/fisiologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/fisiologia , Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologiaRESUMO
Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.
Assuntos
Cálcio/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Eletrofisiologia , Modelos Biológicos , Potássio/metabolismo , Canais de Potássio/metabolismo , RatosRESUMO
Pituitary cells express purinergic receptor-channels (P2XR), the activation of which by ATP is associated with the facilitation of Ca2+ influx. Pharmacological, RT-PCR, and nucleotide sequence analyses confirm the presence of a wild type P2X2aR and a spliced isoform P2X2bR, which lacks a portion of carboxyl terminal amino acids. Wild type and spliced isoform receptors have a similar EC50 for ATP and time-course for activation, but the spliced isoform exhibits rapid and complete desensitization, whereas the wild type channel desensitizes slowly and incompletely. Deletion and insertion studies have revealed that a 6 residue sequence located in carboxyl tail (Arg371-Pro376) is required for sustained Ca2+ influx through wild type receptors. When co-expressed, the wild type and spliced channels form functional heteropolymeric channels. The patterns of Ca2+ signaling in the majority of pituitary cells expressing ATP-gated receptor-channels are highly comparable to those observed in cells co-transfected with P2X2aR and P2X2bR. ATP-induced [Ca2+]i response in pituitary cells is partially inhibited by nifedipine, a blocker of voltage-gated L-type Ca2+ channels, suggesting that P2X2R not only drive Ca2+ into the cell, but also activate voltage-gated Ca2+ entry. Our results indicate that ATP represents a paracrine and (or) autocrine factor in the regulation of Ca2+ signaling, and that its actions are mediated in part by heteropolymeric P2X2R.
Assuntos
Cálcio/metabolismo , Hipófise/metabolismo , Receptores Purinérgicos P2/biossíntese , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Eletrofisiologia , Dados de Sequência Molecular , Neurônios/metabolismo , Isoformas de Proteínas , Ratos , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , TransfecçãoRESUMO
Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissues. Here, we studied the acute in vivo and in vitro effects of a commercially used PCB product, Aroclor 1248 (A1248), a mixture of tri-, tetra-, and pentachloro congeners. Single intraperitoneal (i.p.) or bilateral intratesticular (i.t.) injections of A1248 decreased serum androgen levels in both groups 24 h after injection. Chorionic gonadotropin-stimulated androgen production by acute testicular cultures from both groups was also reduced, and progesterone production was attenuated in cultures from i.t.-treated animals. The capacity of the postmitochondrial fractions from testes of i.t.-treated animals to convert pregnenolone to progesterone and progesterone to testosterone was reduced as well. In vitro studies revealed that a 10- to 15-min exposure of postmitochondrial testicular fractions and intact interstitial cells from normal animals to A1248 in a subnanomolar concentration range was sufficient to attenuate the conversion of pregnenolone to progesterone and progesterone to testosterone. At micromolar concentrations, A1248 added in vitro also inhibited the conversion of Delta(4)-androstendione to testosterone without affecting the viability of interstitial cells. These results indicate that A1248 down-regulates the testicular androgenesis by an acute inhibition of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/lyase, and 17beta-hydroxysteroid dehydrogenase activities.