RESUMEN
Pancreatic alterations such as inflammation and insulin resistance accompany hypothyroidism. Molecular iodine (I2) exerts antioxidant and differentiation actions in several tissues, and the pancreas is an iodine-uptake tissue. We analyzed the effect of two oral I2 doses on pancreatic disorders in a model of hypothyroidism for 30 days. Adult female rabbits were divided into the following groups: control, moderate oral dose of I2 (0.2 mg/kg, M-I2), high oral dose of I2 (2.0 mg/kg, H-I2), oral dose of methimazole (MMI; 10 mg/kg), MMI + M-I2,, and MMI + H-I2. Moderate or high I2 supplementation did not modify circulating metabolites or pancreatic morphology. The MMI group showed reductions of circulating thyroxine (T4) and triiodothyronine (T3), moderate glucose increments, and significant increases in cholesterol and low-density lipoproteins. Acinar fibrosis, high insulin content, lipoperoxidation, and overexpression of GLUT4 were observed in the pancreas of this group. M-I2 supplementation normalized the T4 and cholesterol, but T3 remained low. Pancreatic alterations were prevented, and nuclear factor erythroid-2-related factor-2 (Nrf2), antioxidant enzymes, and peroxisome proliferator-activated receptor gamma (PPARG) maintained their basal values. In MMI + H-I2, hypothyroidism was avoided, but pancreatic alterations and low PPARG expression remained. In conclusion, M-I2 supplementation reestablishes thyronine synthesis and diminishes pancreatic alterations, possibly related to Nrf2 and PPARG activation.
Asunto(s)
Hipotiroidismo , Yodo , Animales , Conejos , Femenino , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Factor 2 Relacionado con NF-E2 , PPAR gamma , Hipotiroidismo/tratamiento farmacológico , Hipotiroidismo/metabolismo , Triyodotironina/metabolismo , Tiroxina/metabolismo , ColesterolRESUMEN
The venoms of Conus snails contain neuroactive peptides named conotoxins (CTXs). Some CTXs are nicotinic acetylcholine receptor (nAChRs) antagonists. nAChRs modulate the release of neurotransmitters and are implicated in several pathophysiologies. One venom peptide from Conus archon, a vermivorous species from the Mexican Pacific, was purified by RP-HPLC and its activity on human α7, α3ß2, and α7ß2 nAChRs was assessed by the two-electrode voltage clamp technique. At 36.3 µM the purified peptide (F27-1, renamed tentatively ArchIIIA) slowly reversibly inhibited the ACh-induced response of the hα7 subtype by 44.52 ± 5.83%, while it had low or no significant effect on the response of the hα3ß2 and hα7ß2 subtypes; the EC50 of the inhibiting effect was 45.7 µM on the hα7 subtype. This peptide has 15 amino acid residues and a monoisotopic mass of 1654.6 Da (CCSALCSRYHCLPCC), with three disulfide bridges and a free C-terminus. This sequence with a CC-C-C-CC arrangement (framework III) belongs to the M superfamily of conotoxins, corresponding to the mini-M´s (M-1-M-3) conotoxins; due to its size and inter-Cys spacings it is an M-2 conotoxin. This toxin is a novel mini-M conotoxin affecting ligand-gated ion channels, like the maxi-M CTX ψ-conotoxins and α-MIIIJ conotoxin (nAChRs blockers). This peptide seems to be homologous to the reg3b conotoxin (from Conus regius) with an identity of 93.3%, differing only in the third residue in the sequence, serine for threonine, both uncharged polar residues. We obtained, in silico, a probable 3D structure, which is consistent with its effect on neuronal subtypes.
Asunto(s)
Conotoxinas , Caracol Conus , Antagonistas Nicotínicos , Receptores Nicotínicos , Animales , Conotoxinas/química , Conotoxinas/farmacología , Caracol Conus/química , Humanos , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacología , Péptidos/metabolismo , Receptores Nicotínicos/metabolismoRESUMEN
Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 µM, and it does not affect the hα3ß2 subtype at 6.5 µM.
Asunto(s)
Conotoxinas/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Receptores Nicotínicos/fisiología , Secuencia de Aminoácidos , Animales , Caracol Conus , Femenino , México , Oocitos/efectos de los fármacos , Oocitos/fisiología , Péptidos/química , Xenopus laevisRESUMEN
PURPOSE: Specific proteolytic cleavages of the hormone prolactin (PRL) generate vasoinhibins, a family of peptides (including 16-kDa PRL) that are able to inhibit the pathologic increase in retinal vasopermeability (RVP) associated with diabetes. Here the authors tested the ability of an adenoassociated virus type 2 (AAV2) vasoinhibin vector to inhibit vascular endothelial growth factor (VEGF)- and diabetes-induced RVP. METHODS: AAV2 vectors encoding vasoinhibin, PRL, or soluble VEGF receptor 1 (soluble FMS-like tyrosine kinase-1 [sFlt-1]) were injected intravitreally into the eyes of rats. Four weeks later, either VEGF was injected intravitreally or diabetes was induced with streptozotocin. Tracer accumulation was evaluated as an index of RVP using fluorescein angiography or the Evans blue dye method. RT-PCR verified transgene expression in the retina, and the intravitreal injection of an AAV2 vector encoding green fluorescent protein revealed transduced cells in the retinal ganglion cell layer. In addition, Western blot analysis of AAV2-transduced HEK293 cells confirmed the expression and secretion of the vector-encoded proteins. RESULTS: The AAV2-vasoinhibin vector prevented the increase in tracer accumulation that occurs 24 hours after the intravitreal injection of VEGF. Diabetes induced a significant increase in tracer accumulation compared with nondiabetic controls. This increase was blocked by the AAV2-vasoinhibin vector and reduced by the AAV2-sFlt-1 vector. The AAV2-PRL vector had no effect. CONCLUSIONS: These results show that an AAV2-vasoinhibin vector prevents pathologic RVP and suggest it could have therapeutic value in patients with diabetic retinopathy.
Asunto(s)
Permeabilidad Capilar/genética , Proteínas de Ciclo Celular/genética , Dependovirus/genética , Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/terapia , Terapia Genética/métodos , Albúminas/farmacocinética , Animales , Permeabilidad Capilar/efectos de los fármacos , Colorantes/farmacocinética , Dextranos/farmacocinética , Diabetes Mellitus Experimental/genética , Retinopatía Diabética/genética , Modelos Animales de Enfermedad , Azul de Evans/farmacocinética , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Inyecciones Intravítreas , Masculino , Plásmidos/genética , Ratas , Ratas Wistar , Hemorragia Retiniana/inducido químicamente , Hemorragia Retiniana/genética , Hemorragia Retiniana/terapia , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
It is now apparent that regulation of blood vessel growth contributes to the classical actions of hormones on development, growth, and reproduction. Endothelial cells are ideally positioned to respond to hormones, which act in concert with locally produced chemical mediators to regulate their growth, motility, function, and survival. Hormones affect angiogenesis either directly through actions on endothelial cells or indirectly by regulating proangiogenic factors like vascular endothelial growth factor. Importantly, the local microenvironment of endothelial cells can determine the outcome of hormone action on angiogenesis. Members of the growth hormone/prolactin/placental lactogen, the renin-angiotensin, and the kallikrein-kinin systems that exert stimulatory effects on angiogenesis can acquire antiangiogenic properties after undergoing proteolytic cleavage. In view of the opposing effects of hormonal fragments and precursor molecules, the regulation of the proteases responsible for specific protein cleavage represents an efficient mechanism for balancing angiogenesis. This review presents an overview of the actions on angiogenesis of the above-mentioned peptide hormonal families and addresses how specific proteolysis alters the final outcome of these actions in the context of health and disease.
Asunto(s)
Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Hormonas Peptídicas/fisiología , Animales , Femenino , Humanos , Péptido Hidrolasas/metabolismo , Embarazo , Reproducción/fisiologíaRESUMEN
Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.
Asunto(s)
Catepsina D/metabolismo , Adenohipófisis/metabolismo , Prolactina/metabolismo , Vesículas Secretoras/metabolismo , Animales , Western Blotting , Catepsina D/genética , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Fragmentos de Péptidos/metabolismo , Adenohipófisis/citología , Adenohipófisis/ultraestructura , Prolactina/química , Ratas , Ratas Wistar , Vesículas Secretoras/ultraestructuraRESUMEN
Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17beta-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) alpha(1)A-, alpha(1)B-, alpha(1)D-, alpha(2)A-, alpha(2)C-, and beta(1)-AR, and showed for the first time that mRNAs for alpha(2)B-, beta(2)- and beta(3)-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17beta-estradiol, alpha(1)B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while alpha(1)B- and alpha(1)D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1-10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.
Asunto(s)
Estradiol/fisiología , Regulación de la Expresión Génica/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/metabolismo , Receptores Adrenérgicos alfa/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Animales , Línea Celular Transformada , Hormona Liberadora de Gonadotropina/biosíntesis , Hormona Liberadora de Gonadotropina/genética , Ratones , Neuronas/fisiología , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/metabolismo , Sistemas Neurosecretores/fisiología , Receptores Adrenérgicos alfa/genética , Receptores Adrenérgicos alfa/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Receptores de Kisspeptina-1RESUMEN
The neurotransmitter gamma-aminobutyric acid (GABA) is an important modulator of gonadotropin-releasing hormone (GnRH), and consequently of reproduction. GABA, acting via ionotropic GABAA receptors, exerts a biphasic effect on GnRH secretion in immortalized GnRH cells. The initial increase in GnRH secretion is triggered by a sharp rise in [Ca2+]i, while the progressive decline of GnRH levels that follows is paralleled by reduced levels of intracellular cAMP. The experiments described here were designed to explore the potential signaling pathways involved in this novel GABAA ionotropic inhibition of cAMP synthesis in GT1-7 cells. Using RT-PCR and real-time PCR, we found that GT1-7 cells express 8 of 9 known membrane adenylyl cyclase (AC) isoforms, including a large proportion of AC3 and AC9, as well as AC5 and AC6, all of which are negatively regulated by increases in [Ca2+]i. In contrast, isoforms of AC that are positively regulated by [Ca2+]i were barely detectable (AC1) or undetectable (AC8). Pharmacological activation of L-type voltage-operated calcium channels with BayK 8644 produced a decrease in [cAMP]i similar to that induced by GABA, while blocking these calcium channels with verapamil reversed the effect of GABA on cAMP synthesis. Furthermore, blocking calcineurin with deltamethrin, FK-506 or cyclosporin A blocked the inhibitory effect of GABA on [cAMP]i, supporting the involvement of AC9 in this effect. In addition, blocking Ca2+/calmodulin-dependent protein kinase II (CamKII) with KN-62 partially reversed the action of GABA, suggesting that AC3 may also be involved in this effect. Finally, GABA increased phosphatase activity in a calcium-dependent manner, an effect blocked by calcineurin inhibitors. Collectively, our results show that the ionotropic action of GABA via the activation of GABAA receptors can decrease AC activity in immortalized GnRH neurons, and that the effect of GABA appears to be mediated by a transient increase in [Ca2+]i followed by activation of calcineurin and CamKII, leading to dephosphorylation of AC9 and phosphorylation of AC3, respectively, and subsequently reducing the synthesis of cAMP.
Asunto(s)
Adenilil Ciclasas/metabolismo , Calcineurina/fisiología , AMP Cíclico/biosíntesis , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Inhibidores de la Calcineurina , Calcio/farmacología , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular Transformada , Isoenzimas/metabolismo , Neuronas/metabolismo , Fosfoproteínas Fosfatasas/metabolismoRESUMEN
Vasoinhibins are a family of peptides derived from prolactin, growth hormone and placental lactogen that act on endothelial cells to suppress vasodilation and angiogenesis and to promote apoptosis-mediated vascular regression. Some of the pathways by which vasoinhibins act have now been defined, and recent developments indicate that endogenous vasoinhibins exert tonic and essential actions on blood vessel growth, dilation and regression in vivo. By studying the pathways that can generate vasoinhibins, and the nature of their receptors and key biological mediators, it should be possible to clarify the role of vasoinhibins in controlling vascular function in health and disease.
Asunto(s)
Endotelio Vascular/fisiología , Hormona del Crecimiento/fisiología , Neovascularización Fisiológica , Lactógeno Placentario/fisiología , Prolactina/fisiología , Moduladores de la Angiogénesis/química , Moduladores de la Angiogénesis/metabolismo , Animales , Hormona del Crecimiento/química , Humanos , Modelos Biológicos , Modelos Moleculares , Lactógeno Placentario/química , Prolactina/química , Unión Proteica , Transducción de Señal , Vasoconstrictores/química , Vasoconstrictores/metabolismoRESUMEN
Antiangiogenic molecules derived from prolactin (PRL) are not a single entity, but rather a family of peptides with different molecular masses, all containing the N-terminal region of PRL. Cleavage of PRL by cathepsin-D or by matrix metalloproteases generates N-terminal fragments that act on endothelial cells to suppress vasodilation and angiogenesis and promote vascular regression. N-terminal PRL fragments have been identified in cartilage and retina, where angiogenesis is highly restricted. In vivo experiments demonstrate that these PRL fragments exert a tonic and essential suppression of retinal blood vessel growth and dilation. Similar PRL fragments have been detected in the pituitary gland, a highly vascularized organ where the control of vascular growth may differ from that in tissues where angiogenesis is highly restricted. We have previously proposed the name vasoinhibins to describe the collection of N-terminal PRL fragments having blood vessel-blocking activity, and here we discuss their promise as factors to control vascular function in health and disease.
Asunto(s)
Inhibidores de la Angiogénesis/fisiología , Proteínas de Ciclo Celular/fisiología , Prolactina/química , Animales , Proteínas de Ciclo Celular/metabolismo , Humanos , Familia de Multigenes , Fenómenos Fisiológicos Oculares , Hipófisis/fisiología , Estructura Terciaria de ProteínaRESUMEN
Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+ -dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming alpha(1) subunit and other modulatory subunits, including the beta subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel beta subunits (Ca(v)betavar) found in these parasites. In particular, we examine the role these beta subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Ca(v)betavar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant beta subunits may thus represent a platyhelminth-specific gene family.
Asunto(s)
Antihelmínticos/farmacología , Canales de Calcio/efectos de los fármacos , Platelmintos/efectos de los fármacos , Praziquantel/farmacología , Secuencia de Aminoácidos , Animales , Canales de Calcio/fisiología , Activación del Canal Iónico/efectos de los fármacos , Datos de Secuencia Molecular , Platelmintos/metabolismo , Alineación de SecuenciaRESUMEN
PURPOSE: Disruption of the normally antiangiogenic environment of the retina leads to aberrant angiogenesis, the major cause of vision loss throughout the world. Prolactin (PRL), the hormone originally associated with milk production, can be proteolytically processed to 16K-PRL, a 16 kDa N-terminal PRL fragment with potent antiangiogenic and vasoconstrictive actions. This study was conducted to determine whether 16K-PRL is found naturally in the retina and plays a role in angiogenesis and vasodilation. METHODS: Expression of PRL mRNA in rat retina was determined by RT-PCR and in situ hybridization. Western blot was used to examine the expression of PRL and derived fragments in retinal homogenates. The role of PRL and 16K-PRL in the retina was studied by intravitreal injection of either antibodies against PRL or small interfering RNAs (siRNA), to suppress expression of retinal PRL mRNA. RESULTS: Rat retina expressed PRL mRNA in the outer nuclear, outer plexiform, inner nuclear, and ganglion cell layers. Both full-length PRL and N-terminal 16K-PRL were detected in retinal homogenates by polyclonal and monoclonal antibodies. The intravitreal injection of antibodies able to neutralize the actions of 16K-PRL increased the number of retinal blood vessels and capillary area by threefold. Furthermore, siRNA-mediated inhibition of PRL mRNA increased retinal neovascularization threefold and resulted in a significant increase in vasodilation. CONCLUSIONS: These results demonstrate that PRL is synthesized and cleaved to antiangiogenic 16K-PRL by retinal tissue and that these molecules play a key role in preventing angiogenesis in the healthy retina.
Asunto(s)
Inhibidores de la Angiogénesis/fisiología , Prolactina/fisiología , Retina/metabolismo , Neovascularización Retiniana/prevención & control , Inhibidores de la Angiogénesis/genética , Animales , Anticuerpos/farmacología , Western Blotting , Hibridación in Situ , Masculino , Peso Molecular , Fragmentos de Péptidos/fisiología , Prolactina/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vasodilatación/efectos de los fármacosRESUMEN
We review the role of iodothyronine deiodinases (IDs) in the evolution of vertebrate thyroidal systems within the larger context of biological metabolism of halogens. Since the beginning of life, the ubiquity of organohalogens in the biosphere has provided a major selective pressure for the evolution and conservation of cellular mechanisms specialized in halogen metabolism. Among naturally available halogens, iodine emerged as a critical component of unique developmental and metabolic messengers. Metabolism of iodinated compounds occurs in the three major domains of life, and invertebrate deuterostomes possess several biochemical traits and molecular homologs of vertebrate thyroidal systems, including ancestral homologs of IDs identified in urochordates. The finely tuned cellular regulation of iodometabolite uptake and disposal is a remarkable event in evolution and might have been decisive for the explosive diversification of ontogenetic strategies in vertebrates.
Asunto(s)
Evolución Molecular , Yoduro Peroxidasa/metabolismo , Yodo/metabolismo , Glándula Tiroides/enzimología , Animales , Humanos , FilogeniaRESUMEN
Substrate availability has been thought to be a major regulator of the outer-ring deiodinating pathway (ORD) in fish. However, current information strongly suggests that while fish iodothyronine deiodinase type 2 (D2) responds to iodothyronines in the same manner as its mammalian counterpart, fish deiodinase type 1 (D1) exhibits a distinct response. Furthermore, 3,5-T2, generally considered to be an inactive product of iodothyronine metabolism, has recently been described as bioactive, but its effects upon D1 and D2 are not yet known. We examined the effect that short-term immersion in T4, T3, and 3,5-T2 (0.1 microM; 12 or 24 h) exerts on both D1 and D2 activities and on the levels of expression of D1 and D2 mRNAs in killifish liver. In agreement with previous reports in teleosts, no iodothyronine exerted a significant effect on D1 enzymatic activity. However, all three iodothyronines significantly decreased D2 activity. Furthermore, at 24 h post-immersion T4, T3, and 3,5-T2 inhibited both D1 and D2 transcription. Together, the present results confirm the differential effect of iodothyronines upon the hepatic ORD pathway in fish and show that this effect can occur at a transcriptional level. Furthermore, we provide the first evidence that 3,5-T2 can affect both activity and transcription of hepatic deiodinases in teleosts.
Asunto(s)
Diyodotironinas/farmacología , Fundulidae/metabolismo , Yoduro Peroxidasa/metabolismo , Hígado/enzimología , Glándula Tiroides/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Yoduro Peroxidasa/genética , Masculino , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Tiroxina/farmacología , Triyodotironina/farmacología , Yodotironina Deyodinasa Tipo IIRESUMEN
Diminished oxygen concentration within growing tumors may stimulate neovascularization by inducing both up-regulation of angiogenic factors and down-regulation of antiangiogenic agents. A potentially important molecule in the growth of pituitary adenomas is prolactin (PRL), which can be cleaved by cathepsin-D to yield a 16-kDa form (16K-PRL) with potent antiangiogenic effects. We examined the expression of PRL in cultured GH4C1 pituitary adenoma cells after exposure to hypoxia (0.1% oxygen) for periods of 12 to 36 hours. In contrast to increased expression of the angiogenic factor vascular endothelial growth factor in hypoxic cells, PRL mRNA and levels of intracellular and secreted PRL were significantly reduced under hypoxia. The reduction was not attributable to a general suppression of either transcription or protein synthesis. Although 16K-PRL was not evident in conditioned medium at physiologic pH, lowering the pH to mimic the acidic tumor microenvironment resulted in generation of 16K-PRL, which was sharply reduced in medium drawn from hypoxic cells. Production of 16K-PRL was blocked by the cathepsin-D inhibitor pepstatin-A, and the reduced 16K-PRL formation in hypoxic-conditioned medium correlated with a decrease in secretion of cathepsin-D and its precursor, procathepsin-D. Thus, hypoxia acts upon GH4C1 cells to increase vascular endothelial growth factor expression, decrease PRL synthesis, and suppress conversion of PRL to 16K-PRL via inhibition of cathepsin-D proteolysis. These mechanisms may act in concert to stimulate angiogenesis in prolactinomas.
Asunto(s)
Adenoma/metabolismo , Catepsina D/metabolismo , Neoplasias Hipofisarias/metabolismo , Prolactina/metabolismo , Animales , Catepsina D/antagonistas & inhibidores , Hipoxia de la Célula , Línea Celular Tumoral , Medios de Cultivo Condicionados/metabolismo , Pepstatinas/farmacología , Prolactina/genética , Inhibidores de Proteasas/farmacología , ARN Mensajero/metabolismo , Ratas , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Activity of the magnocellular neurons that synthesize vasopressin and oxytocin in the paraventricular and supraoptic nuclei of the hypothalamus can be modulated by local release of neuromediators within the nuclei. Among the bioactive peptides that may play autocrine or paracrine roles in this system is prolactin (PRL). Paraventricular and supraoptic neurons express PRL mRNA and contain and secrete PRL-like proteins of 23 and 14 kDa. We investigated the localization of PRL receptors in vasopressinergic and oxytocinergic magnocellular neurons using dual-label immunofluorescence. The results demonstrate that both vasopressin- and oxytocin-immunoreactive cells of the paraventricular and supraoptic nuclei contain the PRL receptor. In addition, we investigated the possible regulation of vasopressin secretion by PRL using hypothalamo-neurohypophyseal explants in culture. The results show that PRL and a 16 kDa N-terminal fragment of the hormone that is analogous to the neurohypophyseal 14-kDa PRL fragment stimulate the release of vasopressin. Together, these findings support the hypothesis that vasopressinergic and oxytocinergic neurons of the magnocellular secretory system are regulated directly by various isoforms of PRL via autocrine/paracrine mechanisms.
Asunto(s)
Sistema Hipotálamo-Hipofisario/metabolismo , Prolactina/metabolismo , Vasopresinas/metabolismo , Animales , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/fisiología , Células Cultivadas , Femenino , Sistema Hipotálamo-Hipofisario/citología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Hipotálamo Anterior/citología , Hipotálamo Anterior/efectos de los fármacos , Hipotálamo Anterior/metabolismo , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/fisiología , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Fragmentos de Péptidos/farmacología , Prolactina/farmacología , Ratas , Ratas Wistar , Receptores de Prolactina/metabolismoRESUMEN
The presence of a type 1 deiodinase (D1) in the liver of teleosts has been a controversial issue. Recently we characterized the deiodinase activity in rainbow trout and killifish liver and found that the liver of both species co-expresses the two enzymes (D1 and D2) that catalyze the outer ring-deiodinating pathway. We here report the cloning and characterization of an mRNA from the liver of the killifish Fundulus heteroclitus that encodes a D1 (FhD1). The cDNA amplified by RT-PCR from F. heteroclitus liver is 1314 nt long and encodes a protein of 248 aa. It contains a TGA codon in its open reading frame and a selenocysteine insertion sequence in its 3(') untranslated region, consistent with the structure of a selenoenzyme mRNA. The deduced peptide sequence is 73% identical to that encoded by the tilapia D1 cDNA cloned from kidney and 46% identical to the D1s reported in other vertebrates. Northern blot analysis shows that FhD1 mRNA is expressed in F. heteroclitus liver, consistent with prior biochemical evidence for hepatic D1 activity. Furthermore, heterologous expression of the FhD1 cDNA resulted in a protein with properties similar to the D1-like activity in F. heteroclitus liver. The cloned enzyme, like the native species, is relatively insensitive to inhibition by PTU, but mutation of Ser-159 in FhD1 to the Pro residue found in D2 and D3 isoforms increased the sensitivity to PTU. Our results show that, under basal conditions, killifish liver indeed expresses a D1 enzyme that is homologous to mammalian D1s, establishing this as a useful model in which to study the regulation of D1 and D2 concurrently.
Asunto(s)
Fundulidae/metabolismo , Yoduro Peroxidasa/genética , Hígado/enzimología , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Elementos Transponibles de ADN/genética , ADN Complementario/química , Femenino , Fundulidae/genética , Expresión Génica , Yoduro Peroxidasa/química , Masculino , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oocitos/enzimología , Propiltiouracilo/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Transfección , Xenopus laevisRESUMEN
Recently, we reported the cloning of a cDNA fragment from Fundulus heteroclitus liver encoding the open reading frame of type 2 deiodinase (FhD2). We here report the cloning of 14 kb of genomic sequence from F. heteroclitus that includes the previously reported coding region of the F. heteroclitus Dio2 gene (FhDio2), the 5(') and 3(') untranslated regions, and flanking regions and introns. This FhDio2 gene comprises two exons divided by a 4.8-kb intron. The position of the intron is similar to that of introns in other Dio2 genes. The analysis of approximately 1.3 kb of genomic sequence upstream of the mRNA start site revealed that, in contrast to mammalian Dio2 genes, there were no apparent TATA or CRE sequences. Nevertheless, a putative Sp1 site was found, similar to that in other F. heteroclitus TATA-less promoters. We have also cloned the complete FhD2 cDNA, which spans 4652 bp and contains a sequence adjacent to its poly(A) tail that is highly similar to the selenocysteine insertion sequence (SECIS) found in human D2 cDNA. The expression of a construct containing the FhD2 ORF plus the native SECIS resulted in a protein with deiodinase activity similar to that of the native FhD2. Analysis of the regulation of this gene, combined with ongoing studies of the F. heteroclitus D1 gene, will allow us to elucidate the functions of the colocalized deiodinases in teleost liver.