RESUMEN
The aim of the present research was to evaluate if the endocannabinoid system (enzymes and receptors) could be modulated by light in rod outer segment (ROS) from bovine retina. First, we analyzed endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in purified ROS obtained from dark-adapted (DROS) or light-adapted (LROS) retinas. To this end, diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL), and lysophosphatidate phosphohydrolase (LPAP) enzymatic activities were analyzed using radioactive substrates. The protein content of these enzymes and of the receptors to which cannabinoids bind was determined by immunoblotting under light stimulus. Our results indicate that whereas DAGL and MAGL activities were stimulated in retinas exposed to light, no changes were observed in LPAP activity. Interestingly, the protein content of the main enzymes involved in 2-AG metabolism, phospholipase C ß1 (PLCß1), and DAGLα (synthesis), and MAGL (hydrolysis), was also modified by light. PLCß1 content was increased, while that of lipases was decreased. On the other hand, light produced an increase in the cannabinoid receptors CB1 and CB2 and a decrease in GPR55 protein levels. Taken together, our results indicate that the endocannabinoid system (enzymes and receptors) depends on the illumination state of the retina, suggesting that proteins related to phototransduction phenomena could be involved in the effects observed.
Asunto(s)
Endocannabinoides/metabolismo , Luz , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Bovinos , Lipoproteína Lipasa/metabolismo , Modelos Biológicos , Monoacilglicerol Lipasas/metabolismo , Fosfolipasa C beta/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
KEY POINTS: We investigated the excitation-contraction coupling mechanisms in small pulmonary veins (SPVs) in rat precision-cut lung slices. We found that SPVs contract strongly and reversibly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced vasoconstriction in SPVs was associated with the stimulation of purinergic P2Y2 receptors in vascular smooth muscle cell, activation of phospholipase C-ß and the generation of intracellular Ca2+ oscillations mediated by cyclic Ca2+ release events via the inositol 1,4,5-trisphosphate receptor. Active constriction of SPVs may play an important role in the development of pulmonary hypertension and pulmonary oedema. ABSTRACT: The small pulmonary veins (SPVs) may play a role in the development of pulmonary hypertension and pulmonary oedema via active changes in SPV diameter, mediated by vascular smooth muscle cell (VSMC) contraction. However, the excitation-contraction coupling mechanisms during vasoconstrictor stimulation remain poorly understood in these veins. We used rat precision-cut lung slices and phase-contrast and confocal microscopy to investigate dynamic changes in SPV cross-sectional luminal area and intracellular Ca2+ signalling in their VSMCs. We found that the SPV (â¼150 µm in diameter) contract strongly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced SPV contraction was fast, concentration-dependent, completely reversible upon ATP washout, and inhibited by purinergic receptor antagonists suramin and AR-C118925 but not by MRS2179. Immunofluorescence showed purinergic P2Y2 receptors expressed in SPV VSMCs. ATP-induced SPV contraction was inhibited by phospholipase Cß inhibitor U73122 and accompanied by intracellular Ca2+ oscillations in the VSMCs. These Ca2+ oscillations and SPV contraction were inhibited by the inositol 1,4,5-trisphosphate receptor inhibitor 2-APB but not by ryanodine. The results of the present study suggest that ATP-induced vasoconstriction in SPVs is associated with the activation of purinergic P2Y2 receptors in VSMCs and the generation of Ca2+ oscillations.
Asunto(s)
Calcio/fisiología , Contracción Muscular , Miocitos del Músculo Liso/fisiología , Venas Pulmonares/fisiología , Receptores Purinérgicos P2Y2/metabolismo , Vasoconstricción , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Estudios Transversales , Acoplamiento Excitación-Contracción , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miocitos del Músculo Liso/citología , Fosfolipasa C beta/metabolismo , Venas Pulmonares/citología , RatasRESUMEN
BACKGROUND: The angiotensin-I converting enzyme (ACE) plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II). More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet. AIM: Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration. RESULTS: We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC), and experimental results in CHO cells, demonstrated that the ß3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5) showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril) or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein. CONCLUSION: ACE activation regulates melanoma cell proliferation and migration.
Asunto(s)
Angiotensina II/metabolismo , Núcleo Celular/metabolismo , Melanoma/enzimología , Peptidil-Dipeptidasa A/metabolismo , Fosfolipasa C beta/metabolismo , Vinculina/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Cricetulus , Humanos , Lisinopril/farmacología , Melanoma/genética , Melanoma/metabolismo , Peptidil-Dipeptidasa A/genética , Transporte de ProteínasRESUMEN
The basic mechanisms that lead obesity are not fully understood; however, several peptides undoubtedly play a role in regulating body weight. Obesity, a highly complex metabolic disorder, involves central mechanisms that control food intake and energy expenditure. Previous studies have shown that central or peripheral oxytocin administration induces anorexia. Recently, in an apparent discrepancy, rodents that were deficient in oxytocin or the oxytocin receptor were shown to develop late-onset obesity without changing their total food intake, which indicates the physiological importance of oxytocin to body metabolism. Oxytocin is synthesized not only within magnocellular and parvocellular neurons but also in several organs, including the ovary, uterus, placenta, testis, thymus, kidney, heart, blood vessels, and skin. The presence of oxytocin receptors in neurons, the myometrium and myoepithelial cells is well recognized; however, this receptor has also been identified in other tissues, including the pancreas and adipose tissue. The oxytocin receptor is a typical class I G protein-coupled receptor that is primarily linked to phospholipase C-ß via Gq proteins but can also be coupled to other G proteins, leading to different functional effects. In this review, we summarize the present knowledge of the effects of oxytocin on controlling energy metabolism, focusing primarily on the role of oxytocin on appetite regulation, thermoregulation, and metabolic homeostasis.
Asunto(s)
Tejido Adiposo/metabolismo , Metabolismo Energético , Oxitocina/fisiología , Fosfolipasa C beta/metabolismo , Receptores de Oxitocina/metabolismo , Tejido Adiposo/efectos de los fármacos , Regulación del Apetito/fisiología , Regulación de la Temperatura Corporal , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/fisiología , Homeostasis , Humanos , Leptina/metabolismo , Leptina/farmacología , Neuronas/citología , Neuronas/metabolismo , Obesidad , Oxitocina/farmacología , Fosfolipasa C beta/genética , Receptores de Oxitocina/genéticaRESUMEN
The retinal pigment epithelium (RPE) forms the outer blood-retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial-mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR-1 induces the time- and dose-dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho-MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y-27632, whereas MLCK inhibitor ML-7 and PLC-ß inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC-ß-mediated calcium increase. Additionally, thrombin-induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP(3)/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin-induced RPE cell transformation subsequent to BRB dysfunction.
Asunto(s)
Actinas/metabolismo , Barrera Hematorretinal/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Epitelio Pigmentado de la Retina , Fibras de Estrés/metabolismo , Trombina/farmacología , Quinasas Asociadas a rho/metabolismo , Animales , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C beta/metabolismo , Fosforilación , Ratas , Ratas Long-Evans , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Taurine (TAU) supplementation increases insulin secretion in response to high glucose concentrations in rodent islets. This effect is probably due to an increase in Ca2+ handling by the islet cells. Here, we investigated the possible involvement of the cholinergic/phospholipase C (PLC) and protein kinase (PK) A pathways in this process. Adult mice were fed with 2% TAU in drinking water for 30 d. The mice were killed and pancreatic islets isolated by the collagenase method. Islets from TAU-supplemented mice showed higher insulin secretion in the presence of 8.3 mm-glucose, 100 µm-carbachol (Cch) and 1 mm-3-isobutyl-1-methyl-xanthine (IBMX), respectively. The increase in insulin secretion in response to Cch in TAU islets was accompanied by a higher intracellular Ca2+ mobilisation and PLCß2 protein expression. The Ca2+ uptake was higher in TAU islets in the presence of 8.3 mm-glucose, but similar when the islets were challenged by glucose plus IBMX. TAU islets also showed an increase in the expression of PKAα protein. This protein may play a role in cation accumulation, since the amount of Ca2+ in these islets was significantly reduced by the PKA inhibitors: N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) and PK inhibitor-(6-22)-amide (PKI). In conclusion, TAU supplementation increases insulin secretion in response to glucose, favouring both influx and internal mobilisation of Ca2+, and these effects seem to involve the activation of both PLC-inositol-1,4,5-trisphosphate and cAMP-PKA pathways.
Asunto(s)
Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Fosfolipasa C beta/metabolismo , Taurina/administración & dosificación , 1-Metil-3-Isobutilxantina/farmacología , Animales , Carbacol/farmacología , Células Cultivadas , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Citoplasma , Suplementos Dietéticos , Secreción de Insulina , Ratones , Ésteres del Forbol/farmacología , Fosfolipasa C beta/genética , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Taurina/farmacologíaRESUMEN
The retinal pigment epithelium (RPE) plays an essential role in the maintenance and normal functioning of the neural retina. Alterations in RPE function are involved in several ocular pathologies involving the breakdown of the blood-retina barrier (BRB), which exposes RPE to serum components, thrombin among them. Our previous work has shown that thrombin stimulates the proliferation of RPE cells. We here analyzed the molecular pathways leading to this outcome, in order to support thrombin involvement in proliferative vitreoretinopathy (PVR), a major cause of retinal surgery failure. We demonstrated that thrombin activation of PAR-1 promotes cyclin D1 expression at the transcriptional level by stimulating c-Fos expression, mediated by PI3K, MAPK ERK1/2, and conventional PKC activity. Our results show that ERK activation is necessary but not sufficient for the induction of cyclin D1 expression and proliferation, since the inhibition of PI3K or cPKC prevents this outcome. Analysis of thrombin-activated PAR-1 downstream effectors demonstrated that c-Fos expression by the sustained activation of ERK and c-fos transcription triggers the expression and nuclear translocation of cyclin D1, a key regulator of cell cycle G1/S phase progression leading to proliferation. Evidence here provided contributes to the understanding of the mechanisms involved in proliferative eye diseases and enhances the possibility of controlling pathologies such as proliferative PVR, which eventually lead to blindness.
Asunto(s)
Proliferación Celular , Ciclina D1/metabolismo , Células Epiteliales/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Trombina/metabolismo , Vitreorretinopatía Proliferativa/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina D1/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C beta/metabolismo , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Receptor PAR-1/agonistas , Receptor PAR-1/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Factores de Tiempo , Regulación hacia Arriba , Vitreorretinopatía Proliferativa/patologíaRESUMEN
Focal adhesions (FAs) are specialized regions of cell attachment to the extracellular matrix. Previous works have suggested that bradykinin (BK) can modulate cell-matrix interaction. In the present study, we used a physiological cellular model to evaluate the potential role of BK in modulating FAs and stress fibers. We performed a quantitative morphometric analysis of FAs in primary cultured rat renal papillary collecting duct cells, which included size, axial ratio (shape), and average length. After 1, 5, or 10 min of incubation with BK, cultured cells were immunostained and analyzed by confocal microscopy. Although the shape of FAs was not altered, BK induced a decrease in the number of vinculin-stained FAs per cell, and a decrease in both their size and their average length, but not in talin-containing FAs, thus suggesting that BK could be inducing a restructuring of FAs. BK also induced a remodeling of the actin filament assemblies rather than their dissipation. Since we have previously demonstrated that BK stimulates activation of PLCbeta in rat renal papillae, we attempted to determine whether BK can modulate FA restructuring by this mechanism, by pretreating cultured cells with the PLCbeta inhibitor U73122. The present study, performed under physiological conditions with cells that were not genetically manipulated, provides new experimental evidence supporting the notion that the intrarenal hormone BK modulates FAs and actin cytoskeleton organization through a mechanism that involves the activation of PLCbeta. We propose this finding as a novel mechanism for BK modulation of tubular collecting duct function.