RESUMO
While the involvement of thermosensitive transient receptor potential channels (TRPs) in dry eye disease (DED) has been known for years, their expression in the meibomian gland (MG) has never been investigated. This study aims to show their expression and involvement in the lipogenesis of the MG, providing a possible new drug target in the treatment of DED. Our RT-PCR, Western blot and immunofluorescence analysis showed the expression of TRPV1, TRPV3, TRPV4 and TRPM8 in the MG at the gene and the protein level. RT-PCR also showed gene expression of TRPV2 but not TRPA1. Calcium imaging and planar patch-clamping performed on an immortalized human meibomian gland epithelial cell line (hMGECs) demonstrated increasing whole-cell currents after the application of capsaicin (TRPV1) or icilin (TRPM8). Decreasing whole-cell currents could be registered after the application of AMG9810 (TRPV1) or AMTB (TRPM8). Oil red O staining on hMGECs showed an increase in lipid expression after TRPV1 activation and a decrease after TRPM8 activation. We conclude that thermo-TRPs are expressed at the gene and the protein level in MGs. Moreover, TRPV1 and TRPM8's functional expression and their contribution to their lipid expression could be demonstrated. Therefore, TRPs are potential drug targets and their clinical relevance in the therapy of meibomian gland dysfunction requires further investigation.
Assuntos
Disfunção da Glândula Tarsal , Glândulas Tarsais , Humanos , Lipogênese/genética , Western Blotting , Capsaicina/farmacologiaRESUMO
In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca2+ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca2+ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca2+ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca2+ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk.
Assuntos
Receptor de Fator de Crescimento Neural , Neoplasias da Retina , Retinoblastoma , Canais de Cátion TRPM , Humanos , Linhagem Celular , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Proteínas de Membrana/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Receptor CB1 de Canabinoide/metabolismoRESUMO
Tear film hyperosmolarity induces dry eye syndrome (DES) through transient receptor potential vanilloid type 1 (TRPV1) activation. L-carnitine is a viable therapeutic agent since it protects against this hypertonicity-induced response. Here, we investigated whether L-carnitine inhibits TRPV1 activation by blocking heat- or capsaicin-induced increases in Ca2+ influx or hyperosmotic stress-induced cell volume shrinkage in a human corneal epithelial cell line (HCE-T). Single-cell fluorescence imaging of calcein/AM-loaded cells or fura-2/AM-labeled cells was used to evaluate cell volume changes and intracellular calcium levels, respectively. Planar patch-clamp technique was used to measure whole-cell currents. TRPV1 activation via either capsaicin (20 µmol/L), hyperosmolarity (≈450 mosmol/L) or an increase in ambient bath temperature to 43 °C induced intracellular calcium transients and augmented whole-cell currents, whereas hypertonicity induced cell volume shrinkage. In contrast, either capsazepine (10 µmol/L) or L-carnitine (1-3 mmol/L) reduced all these responses. Taken together, L-carnitine and capsazepine suppress hypertonicity-induced TRPV1 activation by blocking cell volume shrinkage.
Assuntos
Antineoplásicos , Carnitina , Humanos , Carnitina/farmacologia , Carnitina/metabolismo , Capsaicina/farmacologia , Capsaicina/metabolismo , Cálcio/metabolismo , Antineoplásicos/metabolismo , Células Epiteliais/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
The ruminal epithelium absorbs large quantities of NH4+ and Ca2+. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm2 with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH4+, K+, and Na+ and highly sensitive to GSK1016790A, while effects of butyrate- were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate- (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate- stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4).
Assuntos
Butiratos , Canais de Cátion TRPV , Animais , Transporte Biológico/fisiologia , Butiratos/metabolismo , Bovinos , Epitélio/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Canais de Cátion TRPV/metabolismoRESUMO
Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models, such as WERI-RB1. In addition, chemotherapy-resistant RB subclones, such as the etoposide-resistant WERI-ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide-sensitive WERI-RB1 and its etoposide-resistant subclone, WERI-ETOR, by proteomic analysis. Subsequently, quantitative proteomics data served for correlation analysis with known drug perturbation profiles. Methodically, WERI-RB1 and WERI-ETOR were cultured, and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode. The raw SWATH (sequential window acquisition of all theoretical mass spectra) files were processed using neural networks in a library-free mode along with machine-learning algorithms. Pathway-enrichment analysis was performed using the REACTOME-pathway resource, and correlated to the molecular signature database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug-connectivity analysis using the L1000 database was carried out to associate the mechanism of action (MOA) for different anticancer reagents to WERI-RB1/WERI-ETOR signatures. A total of 4756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q < 0.05 & log2FC |>2|, 22 higher in WERI-ETOR). Pathway analysis revealed the "retinoid metabolism and transport" pathway as an enriched metabolic pathway in WERI-ETOR cells, while the "sphingolipid de novo biosynthesis" pathway was identified in the WERI-RB1 cell line. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI-ETOR cells as well as ATPase inhibitors, acetylcholine receptor antagonists, and vascular endothelial growth factor receptor (VEGFR) inhibitors in the WERI-RB1 cell line. In this study, WERI-RB1 and WERI-ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. Analysis of the global proteome identified activation of "sphingolipid de novo biosynthesis" in WERI-RB1, and revealed future potential treatment options for etoposide resistance in RB.
Assuntos
Neoplasias da Retina , Retinoblastoma , Linhagem Celular Tumoral , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Humanos , Proteômica , Neoplasias da Retina/metabolismo , Retinoblastoma/tratamento farmacológico , Retinoblastoma/genética , Retinoblastoma/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Esfingolipídeos , Inibidores da Topoisomerase , Ubiquitina-Proteína Ligases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.
Assuntos
Carnitina/uso terapêutico , Transdiferenciação Celular/efeitos dos fármacos , Ceratócitos da Córnea/efeitos dos fármacos , Substância Própria/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Carnitina/farmacologia , Células Cultivadas , Substância Própria/citologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Miofibroblastos , Canais de Cátion TRPV/efeitos dos fármacosRESUMO
There are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.
Assuntos
Antioxidantes/uso terapêutico , Ácido Ascórbico/uso terapêutico , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Canais de Potencial de Receptor Transitório/metabolismo , Antineoplásicos Fitogênicos , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Etoposídeo , Humanos , Estresse Oxidativo , Neoplasias da Retina/metabolismo , Retinoblastoma/metabolismoRESUMO
Optimal vision requires an ocular surface with vital epithelial coverage and a stable tear film. A multitude of endogenous and external factors may alter this delicate balance. Studies over the last decade have established that Ca2+ is an important factor in the control of ocular surface epithelial function and has led to the identification of critical components. In particular, transient receptor potential channels (TRPs) have been identified as important components in corneal and conjunctival cells. These channels encompass a group of ion channels of numerous cell types. TRPs are nonselective cation channels which are Ca2+ permeable. Most TRPs serve as thermosensitive molecular sensors (thermo-TRPs). Our Ca2+ imaging and patch-clamp studies indicate that the activity of two TRP isoforms, TRPV1 and TRPM8, can be modulated by changes in external temperature osmolarity and some endogenous substrates. These TRPs are expressed on both non-neuronal and neuronal corneal cells. Furthermore, these interactions affect control of cytokines either promoting or inhibiting inflammation, one of the key findings in dry eye syndrome. Taken together, these results not only lead to a better understanding of the pathophysiology in pain and inflammation reactions on the surface of the eye, but these studies may also offer new avenues to understand the pathophysiology of the disease and support the development of novel therapeutic targets in dry eye syndrome.
Assuntos
Canais de Potencial de Receptor Transitório , Cálcio , Túnica Conjuntiva , Córnea , LágrimasRESUMO
Transient receptor potential (TRP) channels sense and transduce environmental stimuli into Ca(2+) transients that in turn induce responses essential for cell function and adaptation. These non-selective channels with variable Ca(2+) selectivity are grouped into seven different subfamilies containing 28 subtypes based on differences in amino acid sequence homology. Many of these subtypes are expressed in the eye on both neuronal and non-neuronal cells where they affect a host of stress-induced regulatory responses essential for normal vision maintenance. This article reviews our current knowledge about the expression, function and regulation of TRPs in different eye tissues. We also describe how under certain conditions TRP activation can induce responses that are maladaptive to ocular function. Furthermore, the possibility of an association between TRP mutations and disease is considered. These findings contribute to evidence suggesting that drug targeting TRP channels may be of therapeutic benefit in a clinical setting. We point out issues that must be more extensively addressed before it will be possible to decide with certainty that this is a realistic endeavor. Another possible upshot of future studies is that disease process progression can be better evaluated by profiling changes in tissue specific functional TRP subtype activity as well as their gene and protein expression.
Assuntos
Oftalmopatias , Fenômenos Fisiológicos Oculares , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Oftalmopatias/metabolismo , Oftalmopatias/fisiopatologia , Humanos , Canais de Potencial de Receptor Transitório/genéticaRESUMO
BACKGROUND/AIMS: Ocular surface health depends on conjunctival epithelial (HCjE) layer integrity since it protects against pathogenic infiltration and contributes to tissue hydration maintenance. As the same increases in tear film hyperosmolarity described in dry eye disease can increase corneal epithelial transient receptor potential vanilloid type-1 (TRPV1) channel activity, we evaluated its involvement in mediating an osmoprotective effect by L-carnitine against such stress. METHODS: Using siRNA gene silencing, Ca(2+) imaging, planar patch-clamping and relative cell volume measurements, we determined if the protective effects of this osmolyte stem from its interaction with TRPV1. RESULTS: TRPV1 activation by capsaicin (CAP) and an increase in osmolarity to ≈ 450 mOsM both induced increases in Ca(2+) levels. In contrast, blocking TRPV1 activation with capsazepine (CPZ) fully reversed this response. Similarly, L-carnitine (1 mM) also reduced underlying whole-cell currents. In calcein-AM loaded cells, hypertonic-induced relative cell volume shrinkage was fully blocked during exposure to L-carnitine. On the other hand, in TRPV1 gene-silenced cells, this protective effect by L-carnitine was obviated. CONCLUSION: The described L-carnitine osmoprotective effect is elicited through suppression of hypertonic-induced TRPV1 activation leading to increases in L-carnitine uptake through a described Na(+)-dependent L-carnitine transporter.
Assuntos
Carnitina/farmacologia , Túnica Conjuntiva/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Túnica Conjuntiva/citologia , Células Epiteliais/efeitos dos fármacos , Inativação Gênica , Humanos , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , Canais de Cátion TRPV/genéticaRESUMO
We here provide a brief summary of the characteristics of transient receptor potential channels (TRPs) identified in corneal tissue layers and cells. In general, TRPs are nonselective cation channels which are Ca(2+) permeable. Most TRPs serve as thermosensitive molecular sensors (thermo-TRPs). Based on their functional importance, the possibilities are described for drug-targeting TRP activity in a clinical setting. TRPs are expressed in various tissues of the eye including both human corneal epithelial and endothelial layers as well as stromal fibroblasts and stromal nerve fibers. TRP vanilloid type 1 (TRPV1) heat receptor, also known as capsaicin receptor, along with TRP melastatin type 8 (TRPM8) cold receptor, which is also known as menthol receptor, are prototypes of the thermo-TRP family. The TRPV1 functional channel is the most investigated TRP channel in these tissues, owing to its contribution to maintaining tissue homeostasis as well as eliciting wound healing responses to injury. Other thermo-TRP family members identified in these tissues are TRPV2, 3 and 4. Finally, there is the TRP ankyrin type 1 (TRPA1) cold receptor. All of these thermo-TRPs can be activated within specific temperature ranges and transduce such inputs into chemical and electrical signals. Although several recent studies have begun to unravel complex roles for thermo-TRPs such as TRPV1 in corneal layers and resident cells, additional studies are needed to further elucidate their roles in health and disease.
Assuntos
Canais de Cálcio/metabolismo , Córnea/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Temperatura Corporal/fisiologia , Humanos , Canal de Cátion TRPA1RESUMO
Corneal wound healing in mice subsequent to an alkali burn results in dysregulated inflammation and opacification. Transient receptor potential vanilloid subtype 1 (TRPV1) channel activation in all tissue layers by endogenous ligands contributes to this sight compromising outcome since in TRPV1 knockout mice wound healing results instead in tissue transparency restoration. However, it is not known if primary human stromal fibroblasts exhibit such expression even though functional TRPV1 expression is evident in an immortalized human corneal epithelial cell line. In primary human corneal fibroblasts (HCF), TRPV1 gene expression and localization were identified based on the results of quantitative RT-PCR and immunocytochemistry, respectively. Western blot analysis identified a 100 kD protein corresponding to TRPV1 protein expression in a positive control. Single-cell fluorescence imaging detected in fura2-AM loaded cells Ca(2+) transients that rose 1.8-fold above the baseline induced by a selective TRPV1 agonist, capsaicin (CAP), which were blocked by a TRPV1 antagonist, capsazepine (CPZ) or exposure to a Ca(2+) free medium. The whole-cell mode of the planar patch-clamp technique identified TRPV1-induced currents that rose 1.76-fold between -60 and +130 mV. CAP-induced time dependent changes in the phosphorylation status of mitogen activated protein kinase (MAPK) signaling mediators that led to a 2.5-fold increase in IL-6 release after 24 h. This rise did not occur either in TRPV1 siRNA gene silenced cells or during exposure to SB203580 (10 µM), a selective p38 MAPK inhibitor. Taken together, identification of functional TRPV1 expression in HCF suggests that in vivo its activation by injury contributes to corneal opacification and inflammation during wound healing. These undesirable effects may result in part from increases in IL-6 expression mediated by p-p38 MAPK signaling.
Assuntos
Ceratócitos da Córnea/metabolismo , Regulação da Expressão Gênica/fisiologia , Canais de Cátion TRPV/genética , Western Blotting , Capsaicina/farmacologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Interleucina-6/metabolismo , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Human corneal endothelial cells (HCEC) maintain appropriate tissue hydration and transparency by eliciting net ion transport coupled to fluid egress from the stroma into the anterior chamber. Such activity offsets tissue swelling caused by stromal imbibition of fluid. As corneal endothelial (HCE) transport function is modulated by temperature changes, we probed for thermosensitive transient receptor potential melastatin 8 (TRPM8) functional activity in immortalized human corneal endothelial cells (HCEC-12) and freshly isolated human corneal endothelial cells (HCEC) as a control. This channel is either activated upon lowering to 28 °C or by menthol, eucalyptol and icilin. RT-PCR and quantitative real-time PCR (qPCR) verified TRPM8 gene expression. Ca(2+) transients induced by either menthol (500 µmol/l), eucalyptol (3 mmol/l), or icilin (2-60 µmol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La(3+), 100 µmol/l) as well as the TRPM8 blockers BCTC (10 µmol/l) and capsazepine (CPZ, 10 µmol/l) suppressed icilin-induced Ca(2+) increases. In and outward currents induced by application of menthol (500 µmol/l) or icilin (50 µmol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to ≈ 18 °C led to Ca(2+) increases that were inhibited by a TRPM8 blocker BCTC (10 µmol/l). Other thermosensitive TRP pathways whose heterogeneous Ca(2+) response patterns are suggestive of other Ca(2+) handling pathways were also detected upon strong cooling (≈10 °C). Taken together, functional TRPM8 expression in HCEC-12 and freshly dissociated HCEC suggests that HCE function can adapt to thermal variations through activation of this channel subtype.
Assuntos
Endotélio Corneano/metabolismo , Regulação da Expressão Gênica , Temperatura Alta , RNA/genética , Canais de Cátion TRPM/genética , Sensação Térmica/genética , Cálcio/metabolismo , Linhagem Celular , Endotélio Corneano/citologia , Humanos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Canais de Cátion TRPM/biossínteseRESUMO
The G protein-coupled receptor, MAS, is the receptor of the endogenous ligand, Angiotensin (Ang)-(1-7). It is a promising drug target since the Ang-(1-7)/MAS axis is protective in the cardiovascular system. Therefore, a characterization of MAS signalling is important for developing novel therapeutics for cardiovascular diseases. In this paper, we show that Ang-(1-7) increases intracellular calcium in transiently MAS-transfected HEK293 cells. The calcium influx induced by the activation of MAS is dependent on plasma membrane Ca2+ channels, phospholipase C, and protein kinase C. Specifically, we could demonstrate that MAS employs non-selective, transient receptor potential channels (TRPs) for calcium entry.
Assuntos
Proto-Oncogene Mas , Proteínas Proto-Oncogênicas , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Cálcio , Células HEK293RESUMO
Transient receptor potential vanilloid (TRPV) channels respond to polymodal stresses to induce pain, inflammation and tissue fibrosis. In this study, we probed for their functional expression in human conjunctival epithelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome experience the same type of symptomology induced by TRPV channel activation in other ocular tissues. TRPV gene and protein expression were determined by RT-PCR and immunohistochemistry in HCjE cells and human conjunctivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca(2+) transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 localization was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5-20 µM) -induced Ca(2+) transients were blocked by capsazepine (CPZ) (10 µM), the TRPV4 activator 4α-PDD (10 µM) -induced Ca(2+) increases were reduced by ruthenium-red (RuR) (20 µM). Different heating (<40°C or >43°C) led to Ca(2+) increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca(2+) transients and nonselective cation channel currents. In conclusion, conjunctiva express TRPV1, TRPV2, and TRPV4 channels which may provide novel drug targets for dry eye therapeutics. Their usage may have fewer side effects than those currently encountered with less selective drugs.
Assuntos
Cálcio/metabolismo , Células Epiteliais/metabolismo , Canais de Potencial de Receptor Transitório/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Differences in transient receptor potential (TRP) and cannabinoid receptor type 1 (CB1) expression levels can serve as prognostic factors for retinoblastoma (RB) tumor progression. We hypothesized in RB tissue that such differences are also indicators of whether or not they are sensitive to etoposide. Accordingly, we compared in malignant etoposide-sensitive and etoposide-resistant WERI-Rb1 cells TRPV1, TRPM8 and TRPA1 subtype and CB1 gene expression pattern levels and accompanying functional activity using quantitative real-time RT-PCR, immunohistochemistry, immunofluorescence microscopy, calcium imaging as well as patch-clamp technology. Gene expression patterns were evaluated in enucleated human RB tissues (n = 4). Both etoposide-resistant and etoposide-sensitive WERI-Rb1 cells expressed all of the aforementioned channels based on responses to known activators and thermal challenges. However, TRPA1 was absent in the etoposide-resistant counterpart. Even though both types of RB cells express TRPV1 as well as TRPM8 and CB1, the capsaicin (50 µM) (CAP)-induced Ca(2+) rise caused by TRPV1 activation was prompt and transient only in etoposide-resistant RB cells (n = 8). In this cell type, the inability of CB1 activation (10 µM WIN) to suppress Ca(2+) responses to CAP (50 µM; n = 4) may be attributable to the absence of TRPA1 gene expression. Therefore, using genetic approaches to upregulate TRPA1 expression could provide a means to induce etoposide sensitivity and suppress RB cell tumorigenesis.
Assuntos
Cálcio/metabolismo , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias da Retina/metabolismo , Retinoblastoma/metabolismo , Canais de Potencial de Receptor Transitório/genética , Capsaicina/farmacologia , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor CB1 de Canabinoide/genética , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológicoRESUMO
The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.
RESUMO
Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-TRPV4 are all heat-activated non-selective cation channels that are modestly permeable to Ca(2+). TRPV1, TRPV3, and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca(2+)](i) transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT-PCR and TRPV2 transcripts were also detected. In fura2-loaded HCEC, a TRPV1-3 selective agonist, 100 µM 2-aminoethoxydiphenyl borate (2-APB), induced intracellular Ca(2+) transients and an increase in non-selective cation outward currents that were suppressed by ruthenium-red (RuR) (10-20 µM), a non-selective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 to over 40 °C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine CPZ (10 µM) or another related blocker, lanthanum chloride (La(3+)) (100 µM) suppressed these temperature-induced Ca(2+) increases. Planar patch-clamp technique was used to characterize the currents underlying Ca(2+) transients. Increasing the temperature to over 40 °C induced reversible rises in non-selective cation currents. Moreover, a hypotonic challenge (25%) increased non-selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermosensitive TRPV3 activity TRPV1, TRPV2, and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Ativação do Canal Iônico , Canais de Cátion TRPV/metabolismo , Sensação Térmica , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Transformada , Forma Celular , Tamanho Celular , Células Epiteliais/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Humanos , Soluções Hipotônicas , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Moduladores de Transporte de Membrana/farmacologia , Microscopia de Fluorescência , Pressão Osmótica , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética , Fatores de TempoRESUMO
The transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-and Mg(2+) permeable cation channel that might be a cellular osmosensor since it is activated upon hypotonic cell swelling. TRPV4 is also thermosensitive and responds to moderate heat (from 24 to 27 °C) as well as to phorbol esters (4α-PDD) and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. The resulting Ca(2+) influx occurring in response to swelling induces regulatory volume decrease (RVD) behavior. As regulation of cell volume is essential for corneal endothelial function, we determined whether human corneal endothelial cells have functional TRPV4 channel activity. RT-PCR identified TRPV4 gene expression in the HCEC-12 cell line as well as two clonal daughter cell lines (HCEC-H9C1, HCEC-B4G12). [Ca(2+)](i) transients were monitored in fura-2 loaded cells. Nonselective cation channel currents were recorded in the whole-cell mode of the planar patch-clamp technique. TRPV4 mRNA was found in HCEC-12 and the clonal daughter cell lines. TRPV4 channel agonists (4α-PDD and GSK1016790A; both 5 µmol/l) as well as moderate heat (<40 °C) elicited [Ca(2+)](i) transients. Hypotonicity increased [Ca(2+)](i) and nonselective cation channel currents in HCEC-12 cells. There is functional TRPV4 expression in HCEC-12 and in its clonal daughter cell lines based on Ca(2+) transients and underlying currents induced by known activators of this channel.