RESUMEN
Apolipoprotein E (ApoE) isoforms exert intricate effects on cellular physiology beyond lipid transport and metabolism. ApoEs influence the onset of Alzheimer's disease (AD) in an isoform-dependent manner: ApoE4 increases AD risk, while ApoE2 decreases it. Previously we demonstrated that syndecans, a transmembrane proteoglycan family with increased expression in AD, trigger the aggregation and modulate the cellular uptake of amyloid beta (Aß). Utilizing our previously established syndecan-overexpressing cellular assays, we now explore how the interplay of ApoEs with syndecans contributes to key events, namely uptake and aggregation, in Aß pathology. The interaction of ApoEs with syndecans indicates isoform-specific characteristics arising beyond the frequently studied ApoE-heparan sulfate interactions. Syndecans, and among them the neuronal syndecan-3, increased the cellular uptake of ApoEs, especially ApoE2 and ApoE3, while ApoEs exerted opposing effects on syndecan-3-mediated Aß uptake and aggregation. ApoE2 increased the cellular internalization of monomeric Aß, hence preventing its extracellular aggregation, while ApoE4 decreased it, thus helping the buildup of extracellular plaques. The contrary effects of ApoE2 and ApoE4 remained once Aß aggregated: while ApoE2 reduced the uptake of Aß aggregates, ApoE4 facilitated it. Fibrillation studies also revealed ApoE4's tendency to form fibrillar aggregates. Our results uncover yet unknown details of ApoE cellular biology and deepen our molecular understanding of the ApoE-dependent mechanism of Aß pathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Apolipoproteína E2/metabolismo , Apolipoproteína E4/metabolismo , Apolipoproteínas E/metabolismo , Agregado de Proteínas , Sindecano-3/metabolismo , Línea Celular Tumoral , Humanos , Isoformas de ProteínasRESUMEN
Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na(+) and Ca(2+) influx via TRPV1 channels, which leads to mitochondrial Ca(2+) accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist-induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid-sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau-type Ca(2+) signals, mitochondrial Ca(2+) accumulation and Na(+)- and Ca(2+)-dependent membrane disorganization. Moreover, long-term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment.
Asunto(s)
Neoplasias de la Mama/patología , Capsaicina/farmacología , Diterpenos/farmacología , Células Epiteliales/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Neoplasias de la Próstata/patología , Células Receptoras Sensoriales/efectos de los fármacos , Canales Catiónicos TRPV/agonistas , Animales , Apoptosis/fisiología , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/biosíntesis , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/fisiología , Ganglio del Trigémino/metabolismoRESUMEN
This review aims to create an overview of the currently available results of site-directed mutagenesis studies on transient receptor potential vanilloid type 1 (TRPV1) receptor. Systematization of the vast number of data on the functionally important amino acid mutations of TRPV1 may provide a clearer picture of this field, and may promote a better understanding of the relationship between the structure and function of TRPV1. The review summarizes information on 112 unique mutated sites along the TRPV1, exchanged to multiple different residues in many cases. These mutations influence the effect or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, calmodulin, PIP2, ATP, and lipid binding. The main goal of this paper is to publish the above mentioned data in a form that facilitates in silico molecular modelling of the receptor by promoting easier establishment of boundary conditions. The better understanding of the structure-function relationship of TRPV1 may promote discovery of new, promising, more effective and safe drugs for treatment of neurogenic inflammation and pain-related diseases and may offer new opportunities for therapeutic interventions.
Asunto(s)
Mutación , Canales Catiónicos TRPV/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Ratas , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/genéticaRESUMEN
BACKGROUND: Excitotoxicity is a central pathological pathway in many neurological diseases with blood-brain barrier (BBB) dysfunction. Kainate, an exogenous excitotoxin, induces epilepsy and BBB damage in animal models, but the direct effect of kainate on brain endothelial cells has not been studied in detail. Our aim was to examine the direct effects of kainate on cultured cells of the BBB and to test three anti-inflammatory and antioxidant drugs used in clinical practice, simvastatin, edaravone and dexamethasone, to protect against kainate-induced changes. METHODS: Primary rat brain endothelial cell, pericyte and astroglia cultures were used to study cell viability by impedance measurement. BBB permeability was measured on a model made from the co-culture of the three cell types. The production of nitrogen monoxide and reactive oxygen species was followed by fluorescent probes. The mRNA expression of kainate receptors and nitric oxide synthases were studied by PCR. RESULTS: Kainate damaged brain endothelial cells and made the immunostaining of junctional proteins claudin-5 and zonula occludens-1 discontinuous at the cell border indicating the opening of the barrier. The permeability of the BBB model for marker molecules fluorescein and albumin and the production of nitric oxide in brain endothelial cells were increased by kainate. Simvastatin, edaravone and dexamethasone protected against the reduced cell viability, increased permeability and the morphological changes in cellular junctions caused by kainate. Dexamethasone attenuated the elevated nitric oxide production and decreased the inducible nitric oxide synthase (NOS2/iNOS) mRNA expression increased by kainate treatment. CONCLUSION: Kainate directly damaged cultured brain endothelial cells. Simvastatin, edaravone and dexamethasone protected the BBB model against kainate-induced changes. Our results confirmed the potential clinical usefulness of these drugs to attenuate BBB damage.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Edaravona/farmacología , Células Endoteliales/efectos de los fármacos , Simvastatina/farmacología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Células Endoteliales/metabolismo , Óxido Nítrico/metabolismo , Permeabilidad/efectos de los fármacos , RatasRESUMEN
Scientific evidence suggests that α-synuclein and tau have prion-like properties and that prion-like spreading and seeding of misfolded protein aggregates constitutes a central mechanism for neurodegeneration. Heparan sulfate proteoglycans (HSPGs) in the plasma membrane support this process by attaching misfolded protein fibrils. Despite of intense studies, contribution of specific HSPGs to seeding and spreading of α-synuclein and tau has not been explored yet. Here we report that members of the syndecan family of HSPGs mediate cellular uptake of α-synuclein and tau fibrils via a lipid-raft dependent and clathrin-independent endocytic route. Among syndecans, the neuron predominant syndecan-3 exhibits the highest affinity for both α-synuclein and tau. Syndecan-mediated internalization of α-synuclein and tau depends heavily on conformation as uptake via syndecans start to dominate once fibrils are formed. Overexpression of syndecans, on the other hand, reduces cellular uptake of monomeric α-synuclein and tau, yet exerts a fibril forming effect on both proteins. Data obtained from syndecan overexpressing cellular models presents syndecans, especially the neuron predominant syndecan-3, as important mediators of seeding and spreading of α-synuclein and tau and reveal how syndecans contribute to fundamental molecular events of α-synuclein and tau pathology.
Asunto(s)
Endocitosis , Sindecanos/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Humanos , Células K562 , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Sindecanos/química , alfa-Sinucleína/química , Proteínas tau/químicaRESUMEN
Intraneuronal accumulation of amyloid-ß(1-42) (Aß1-42) is one of the earliest signs of Alzheimer's disease (AD). Cell surface heparan sulfate proteoglycans (HSPGs) have profound influence on the cellular uptake of Aß1-42 by mediating its attachment and subsequent internalization into the cells. Colocalization of amyloid plaques with members of the syndecan family of HSPGs, along with the increased expression of syndecan-3 and -4 have already been reported in postmortem AD brains. Considering the growing evidence on the involvement of syndecans in the pathogenesis of AD, we analyzed the contribution of syndecans to cellular uptake and fibrillation of Aß1-42. Among syndecans, the neuron specific syndecan-3 isoform increased cellular uptake of Aß1-42 the most. Kinetics of Aß1-42 uptake also proved to be fairly different among SDC family members: syndecan-3 increased Aß1-42 uptake from the earliest time points, while other syndecans facilitated Aß1-42 internalization at a slower pace. Internalized Aß1-42 colocalized with syndecans and flotillins, highlighting the role of lipid-rafts in syndecan-mediated uptake. Syndecan-3 and 4 also triggered fibrillation of Aß1-42, further emphasizing the pathophysiological relevance of syndecans in plaque formation. Overall our data highlight syndecans, especially the neuron-specific syndecan-3 isoform, as important players in amyloid pathology and show that syndecans, regardless of cell type, facilitate key molecular events in neurodegeneration.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Endocitosis , Fragmentos de Péptidos/metabolismo , Sindecanos/metabolismo , Péptidos beta-Amiloides/ultraestructura , Línea Celular , Humanos , Células K562 , Cinética , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/ultraestructura , Dominios Proteicos , Sindecanos/químicaRESUMEN
Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Here, we examined the menthol-evoked Ca2+ signals in breast and prostate cancer cell lines. The effect of menthol (50-500µM) was predicted to be mediated by the transient receptor potential ion channel melastatin subtype 8 (TRPM8). However, the intensity of menthol-evoked Ca2+ signals did not correlate with the expression levels of TRPM8 in breast and prostate cancer cells indicating a TRPM8-independent signaling pathway. Menthol-evoked Ca2+ signals were analyzed in detail in Du 145 prostate cancer cells, as well as in CRISPR/Cas9 TRPM8-knockout Du 145 cells. Menthol (500µM) induced Ca2+ oscillations in both cell lines, thus independent of TRPM8, which were however dependent on the production of inositol trisphosphate. Results based on pharmacological tools point to an involvement of the purinergic pathway in menthol-evoked Ca2+ responses. Finally, menthol (50-500µM) decreased cell viability and induced oxidative stress independently of the presence of TRPM8 channels, despite that temperature-evoked TRPM8-mediated inward currents were significantly decreased in TRPM8-knockout Du 145 cells compared to wild type Du 145 cells.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Mentol/farmacología , Estrés Oxidativo/efectos de los fármacos , Canales Catiónicos TRPM/agonistas , Canales Catiónicos TRPM/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismoRESUMEN
There is convincing epidemiological and experimental evidence that capsaicin, a potent natural transient receptor potential cation channel vanilloid member 1 (TRPV1) agonist, has anticancer activity. However, capsaicin cannot be given systemically in large doses, because of its induction of acute pain and neurological inflammation. MRS1477, a dihydropyridine derivative acts as a positive allosteric modulator of TRPV1, if added together with capsaicin, but is ineffective, if given alone. Addition of MRS1477 evoked Ca2+ signals in MCF7 breast cancer cells, but not in primary breast epithelial cells. This indicates that MCF7 cells not only express functional TRPV1 channels, but also produce endogenous TRPV1 agonists. We investigated the effects of MRS1477 and capsaicin on cell viability, caspase-3 and -9 activities and reactive oxygen species production in MCF7 cells. The fraction of apoptotic cells was increased after 3 days incubation with capsaicin (10 µM) paralleled by increased reactive oxygen species production and caspase activity. These effects were even more pronounced, when cells were incubated with MRS1477 (2 µM) either alone or together with CAPS (10 µM). Capsazepine, a TRPV1 blocker, inhibited both the effect of capsaicin and MRS1477. Whole-cell patch clamp recordings revealed that capsaicin-evoked TRPV1-mediated current density levels were increased after 3 days incubation with MRS1477 (2 µM). However, the tumor growth in MCF7 tumor-bearing immunodeficient mice was not significantly decreased after treatment with MRS1477 (10 mg/ kg body weight, i.p., injection twice a week). In conclusion, in view of a putative in vivo treatment with MRS1477 or similar compounds further optimization is required.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Dihidropiridinas/farmacología , Terapia Molecular Dirigida , Canales Catiónicos TRPV/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Capsaicina/farmacología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Células MCF-7 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Transient Receptor Potential Vanilloid 1 (TRPV1) confers noxious heat and inflammatory pain signals in the peripheral nervous system. Clinical trial of resiniferatoxin from Euphorbia species is successfully aimed at TRPV1 in cancer pain management and heading toward new selective painkiller status that further validates this target for drug discovery efforts. Evodia species, used in traditional medicine for hundreds of years, are a recognised source of different TRPV1 agonists, but no antagonist has yet been reported. HYPOTHESIS/PURPOSE: In a search for painkiller leads, we noted for the first time a TRPV1 antagonist activity in the fresh fruits of Tetradium daniellii (Benn.) T.G. Hartley (syn. Evodia hupehensis Dode). METHODS: Through a combination of extraction and purification methods with functional TRPV1-specific Ca2+ uptake assays (bioactivity-guided fractionation/isolation/purification); we isolated a new painkiller candidate that is a distant structural homologue of capsiate exovanilloids and endovanilloids such as anandamide, but a putative competitive inhibitor of the TRPV1. Four additional inactive compounds (N-isobutyl-4,5-epoxy-2E-decadienamide, geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol) were also co-purified with pellitorine. Their structures were established by extensive 1D- and 2D-NMR spectroscopic analysis. RESULTS: 1H- and 13C NMR determination of the chemical structure revealed it to be pellitorine, (2E,4E)-N-(2-methylpropyl)deca-2,4-dienamide, which can compete structurally with algesics released in inflammation. In contrast to previous isolates from Evodia species, pellitorine blocked capsaicin-evoked Ca2+ uptake with an IC50 of 154⯵g/ml (0.69â¯mM/l). N-Isobutyl-4,5-epoxy-2E-decadienamide and geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol did not affect the TRPV1. CONCLUSION: This is the first evidence that pellitorine, an aliphatic alkylamide analogue of capsaicin, can serve as an antagonist of the TRPV1 and may inhibit exovanilloid-induced pain.
Asunto(s)
Analgésicos/farmacología , Ácidos Grasos Insaturados/farmacología , Extractos Vegetales/farmacología , Alcamidas Poliinsaturadas/farmacología , Rutaceae/química , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Línea Celular , Evodia/química , Ácidos Grasos Insaturados/química , Frutas/química , Humanos , Ratones Endogámicos BALB C , Alcamidas Poliinsaturadas/químicaRESUMEN
There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron-blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1-calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell-regulatory calmodulin-protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain-modulating behavior of these important pharmacons.
Asunto(s)
Calmodulina/metabolismo , Dolor/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Canales Catiónicos TRPV/metabolismo , Sitios de Unión , Calmodulina/química , Calmodulina/genética , Capsaicina/metabolismo , Capsaicina/farmacología , Diterpenos/metabolismo , Diterpenos/farmacología , Humanos , Dolor/tratamiento farmacológico , Unión Proteica , Conformación Proteica , Mapas de Interacción de Proteínas/genética , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/genéticaRESUMEN
Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast.
Asunto(s)
Proteínas de Choque Térmico Pequeñas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Trehalosa/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Choque Térmico Pequeñas/genética , Calor , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Mutación , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Estrés Fisiológico , Trehalosa/genéticaRESUMEN
The B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mouse strain, widely used to study neuronal development and regeneration, expresses the yellow fluorescent protein (YFP) in the peripheral nerves and the central nervous system under the control of regulatory sequences of the Thy1 gene. The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction. We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system. We demonstrated that the stroma of subcutaneous tumours induced by the injection of 4T1 or MC26 carcinoma cells into BALB/c(Thy1-YFP) mice, carrying the same construct, indeed expressed the YFP transgene. In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein. Local inflammation induced by subcutaneous injection of complete Freund's adjuvant, as well as the experimental wound-healing milieu, also triggered YFP fluorescence in both the BALB/c(Thy1-YFP) and B6.Cg-Tg(Thy1-YFP)16Jrs/J mice, pointing to eventual overlapping pathways of wound-healing, inflammation and tumour growth.
Asunto(s)
Diagnóstico por Imagen/métodos , Inflamación/diagnóstico , Neoplasias Experimentales/diagnóstico , Cicatrización de Heridas , Animales , Inflamación/genética , Inflamación/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Imagen Molecular/métodos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transporte de Proteínas , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Activación Transcripcional , Cicatrización de Heridas/genéticaRESUMEN
Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co(2+) [half-maximal inhibitory concentration (IC(50)) = 13 µM] > Cd(2+) (I (50) = 38 µM) > Ni(2+) (IC(50) = 62 µM) > Cu(2+) (IC(50) = 200 µM). Zn(2+) proved to be a weak (IC(50) = 27 µM) and only partial inhibitor of the channel function, whereas Mg(2+), Mn(2+) and La(3+) did not exhibit any substantial effect. Co(2+), the most potent channel blocker, was able not only to compete with Ca(2+) but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co(2+) accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co(2+) applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked (45)Ca(2+) influx assays in cells expressing TRPV1. The IC(50) of Co(2+) on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca(2+).
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Metales Pesados/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Células 3T3 , Animales , Células COS , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/química , Cationes Bivalentes/química , Cationes Bivalentes/farmacología , Línea Celular , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Humanos , Metales Pesados/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratas , Relación Estructura-Actividad , Canales Catiónicos TRPV/metabolismoRESUMEN
BACKGROUND: Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca(2+)-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+)-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+)-cytotoxicity ([RTX]>15 microM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+)-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials.
Asunto(s)
Capsaicina/farmacología , Diterpenos/farmacología , Resistencia a Medicamentos , Queratinocitos/efectos de los fármacos , Canales Catiónicos TRPV/genética , Calcio/metabolismo , Muerte Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , ARN Mensajero/análisis , Piel/citología , Canales Catiónicos TRPV/análisisRESUMEN
Ca(2+)-loaded calmodulin normally inhibits multiple Ca(2+)-channels upon dangerous elevation of intracellular Ca(2+) and protects cells from Ca(2+)-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca(2+). Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca(2+)-uptake via the vanilloid inducible Ca(2+)-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca(2+) entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced (45)Ca(2+)-uptake at microM concentrations: calmidazolium (broad range) > or = trifluoperazine (narrow range) chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca(2+) or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca(2+)-uptake in intact TRPV1(+) cells, and suggests an extracellular site of inhibition. TRPV1(+), inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca(2+)-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca(2+)-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca(2+)-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca(2+)-channels but not affecting motoneurons.
Asunto(s)
Antidepresivos Tricíclicos/farmacología , Calcio/metabolismo , Calmodulina/antagonistas & inhibidores , Activación del Canal Iónico/efectos de los fármacos , Dolor/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Animales , Antipsicóticos/farmacología , Capsaicina/farmacología , Células Cultivadas , Quimioterapia Adyuvante , Clorpromazina/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Imidazoles/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Modelos Moleculares , Células 3T3 NIH , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Dolor/metabolismo , Ratas , Ratas Wistar , Fármacos del Sistema Sensorial/farmacologíaRESUMEN
Parallel with the heat/light-induced thylakoid microdomain reorganization and thermal stabilization of photosynthesis we observed an increase in the level of the highly saturated monoglucosyldiacylglycerol (MGlcDG) in Synechocystis cells. The unusually high microviscosity obtained in thylakoid MGlcDG liposomes by monitoring DPH anisotropy was in good agreement with its exceptionally high acyl chain saturation. The MGlcDG membranes remained stable even at extreme high temperatures. Strikingly, in monolayer experiments, out of the five thylakoid polar lipids tested, MGlcDG expressed the strongest interaction with the thylakoid-stabilizing small Hsp from Synechocystis, Hsp17. The preferential interaction of Hsp17 with non-bilayer phase forming lipids supports our notion that sHsps counteract the formation of thermally induced local non-bilayer structures [Proc. Natl. Acad. Sci. USA 99 (2002) 13504] and thus implicated in microdomain organization and in the preservation of functional integrity of thylakoid membranes challenged by heat stress in the light. We also suggest that the highly saturated MGlcDG functions as a "heat shock lipid" and is of potential importance in the development of acquired thermotolerance of heat/light-primed cyanobacterial thylakoids.
Asunto(s)
Cianobacterias/metabolismo , Anisotropía , Membrana Celular/metabolismo , Cromatografía Liquida , Ácidos Grasos/química , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Calor , Luz , Membrana Dobles de Lípidos/química , Lípidos/química , Liposomas/metabolismo , Espectrometría de Masas , Modelos Químicos , Chaperonas Moleculares/metabolismo , Fotosíntesis , Unión Proteica , Estructura Terciaria de Proteína , Synechocystis , Temperatura , Tilacoides/metabolismo , Factores de TiempoRESUMEN
We examined the expression and the function of the DnaK chaperone family in the photoautotrophic cyanobacterium, Synechocystis PCC 6803. Surprisingly, only one of the three dnaK genes was transcribed either under normal or heat shock conditions. Their predicted cochaperones (four dnaJs and one grpE) proved to be uninducible under our experimental conditions. Attempts to inactivate the active dnaK2 has failed, indicating that the gene is essential. The partial mutant displayed lower inducibility of chaperones (especially GroEL and HSP17) both at mRNA and protein levels upon heat shock. The mutant showed temperature sensitive phenotype, but was able to acquire thermotolerance.