RESUMEN
BACKGROUND: and purpose: Phenazopyridine (PAP) is an over-the-counter drug widely used to provide symptomatic relief of bladder pain in conditions such as cystitis or bladder pain syndrome (BPS). Whereas the analgesic effect of PAP has been attributed to a local effect on the mucosa of the lower urinary tract (LUT), the molecular targets of PAP remain unknown. We investigated the effect of PAP on pain-related Transient Receptor Potential (TRP) channels expressed in sensory neurons that innervate the bladder wall. EXPERIMENTAL APPROACH: The effects of PAP on the relevant TRP channels (TRPV1, TRPA1, TRPM8, TRPM3) expressed in HEK293 or CHO cells was investigated using Fura-2-based calcium measurements and whole-cell patch-clamp recordings. Activity of PAP on TRPM8 was further analysed using Fura-2-based calcium imaging on sensory neurons isolated from lumbosacral dorsal root ganglia (DRG) of mice. KEY RESULTS: PAP rapidly and reversibly inhibits responses of TRPM8 expressed in HEK293 cells to cold and menthol, with IC50 values between 2 and 10 µM. It acts by shifting the voltage dependence of channel activation towards positive potentials, opposite to the effect of menthol. PAP also inhibits TRPM8-mediated, menthol-evoked calcium responses in lumbosacral DRG neurons. At a concentration of 10 µM, PAP did not significantly affect TRPA1, TRPV1, or TRPM3. CONCLUSION AND IMPLICATIONS: PAP inhibits TRPM8 in a concentration range consistent with PAP levels in the urine of treated patients. Since TRPM8 is expressed in bladder afferent neurons and upregulated in patients with painful bladder disorders, TRPM8 inhibition may underlie the analgesic activity of PAP.
Asunto(s)
Canales Catiónicos TRPM , Canales de Potencial de Receptor Transitorio , Animales , Cricetinae , Humanos , Ratones , Calcio/metabolismo , Cricetulus , Fura-2/farmacología , Ganglios Espinales/metabolismo , Células HEK293 , Mentol/farmacología , Dolor , Fenazopiridina/farmacología , Células Receptoras Sensoriales/metabolismo , Canal Catiónico TRPA1 , Vejiga Urinaria/metabolismoRESUMEN
AIMS: Lung type 2 alveolar cells, by secreting surfactant to lower surface tension, contribute to enhance lung compliance. Stretching, as a result of lung expansion, triggers type 1 alveolar cell to release ATP, which in turn stimulates Ca2+-dependent surfactant secretion by neighboring type 2 cells. In this report, we studied ATP-triggered Ca2+ signaling in human alveolar type 2 A549 cells. MAIN METHODS: Ca2+ signaling was examined using microfluorimetric measurement with fura-2 as fluorescent dye. KEY FINDINGS: Ca2+ oscillations triggered by ATP relied on inositol 1,4,5-trisphosphate-induced Ca2+ release and store-operated Ca2+ entry. Pathological conditions such as influenza virus infection and diabetes reportedly inhibit sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that a very mild inhibition of SERCA by cyclopiazonic acid (CPA) sufficed to decrease Ca2+ oscillation frequency and the percentage of cells exhibiting Ca2+ oscillations. Ochratoxin A (OTA), an activator of SERCA, could prevent the suppressive effects by CPA. Inhibition of SERCA by hydrogen peroxide also suppressed Ca2+ oscillations. Interestingly, hydrogen peroxide-induced inhibition was prevented by OTA but aggravated by CDN1163, an allosteric activator of SERCA. CDN1163 also had an untoward effect of releasing intracellular Ca2+. SIGNIFICANCE: Different modes of activation of SERCA may determine the outcome of rescue of Ca2+ oscillations in case of SERCA inhibition in alveolar type 2 cells.
Asunto(s)
Células Epiteliales Alveolares , Diabetes Mellitus Tipo 2 , Células A549 , Adenosina Trifosfato/metabolismo , Células Epiteliales Alveolares/metabolismo , Aminoquinolinas , Benzamidas , Calcio/metabolismo , Señalización del Calcio/fisiología , Colorantes Fluorescentes , Fura-2/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Inositol 1,4,5-Trifosfato/farmacología , Ocratoxinas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , TensoactivosRESUMEN
Chlorella is a green alga consumed as dietary food supplement in pulverized form. In addition to its high nutritional value, it is reported as an excellent detoxifying agent. The pulverized Chlorella is partially soluble in water and insoluble portion has been reported for removal of mercury, cadmium and radioactive strontium from body. Chlorella contains a variety of metal-binding functional groups such as carboxyl, amino, phosphoryl, hydroxyl and carbonyl groups, which has high affinity towards various metal ions. The present study was envisaged to evaluate the chelating effect of water soluble fraction of Chlorella powder (AqCH) on metal ions. Fura-2 fluorescence ratio (F340/F380) was measured by fluorescence spectrometer (FS) after the exposure of chloride salt of metals viz., strontium, cobalt, barium, cesium, thallium and mercury to lymphocytes. Pretreatment of AqCH (0.1-20 mg mL-1) was given to evaluate the attenuating effect on fura-2 fluorescence ratio induced by metal ions. The intracellular levels of these metal ions were analyzed by atomic absorption spectrophotometer (AAS) and fluorescence microscopy (FM). Pretreatment with AqCH significantly attenuated the metal induced fluorescence ratio in dose-dependent manner. The results of AAS and FM were found in coherence with fura-2 fluorescence ratio which emphasized that AqCH significantly prevented the metal ions internalization. The present study suggests AqCH chelates with these metal ions and prevents its interaction with cells thereby reducing the intracellular mobilization of Ca2+.
Asunto(s)
Quelantes/farmacología , Chlorella/química , Fura-2/farmacología , Linfocitos/efectos de los fármacos , Metales Pesados/farmacología , Células Cultivadas , Quelantes/química , Fura-2/química , Humanos , Metales Pesados/química , Polvos/químicaRESUMEN
The fluorescent dye fura-2 AM was employed to record activation of Ca2+ entry in response to a decrease in Ca2+ concentration in the endoplasmic reticulum. Using whole-cell voltage clamp technique, we revealed Ca2+ currents with an amplitude of 0.46±0.13 pA/pF that passed through selective channels with current-voltage characteristics similar to those of classical store-operated CRAC channels. These currents were sensitive to 2-APB (50 µM), an inhibitor of store-operated channels. The data suggest that store-operated calcium entry is a characteristic feature of mature ventricular cardiomyocytes. Pathological alterations in store-operated Ca2+ entry can be implicated in the development of heart diseases.
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Canales de Calcio Activados por la Liberación de Calcio/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Transporte Iónico/fisiología , Miocitos Cardíacos/fisiología , Animales , Células Cultivadas , Fura-2/análogos & derivados , Fura-2/farmacología , Ventrículos Cardíacos/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Función VentricularRESUMEN
BACKGROUND: Atrial tachyarrhythmia (AT) is a common complication in patients who have undergone a Fontan operation. In this study, we investigated whether abnormal Ca2+ handling contributes to the Fontan operation-related atrial arrhythmogenic substrate. METHODS: Mongrel dogs were randomly assigned to sham and Fontan groups. The Fontan operation model was developed by performing an atriopulmonary anastomosis. After 14 days, an electrophysiological study was performed to evaluate the AT vulnerability. Ca2+ handling properties were measured by loading atrial cardiomyocytes (CMs) with fura-2 AM. The L-type Ca2+ (ICa-L) and Na+-Ca2+ exchanger (INCX) currents of the CMs were recorded by the whole-cell patch-clamp technique. The key Ca2+ handling proteins expression was assessed by Western blotting. RESULTS: The AT inducibility was higher in the Fontan group than in the sham group (85.71 vs. 14.29%, P < 0.05). The Fontan operation resulted in decreased Ca2+ transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca2+ content, but in enhanced diastolic intracellular Ca2+ concentration and SR Ca2+ leak in the atrial CMs. The spontaneous CaT events, triggered ectopic activity and INCX density were increased, but ICa-L density was reduced in CMs from the Fontan atria (all P < 0.05). Additionally, the Fontan operation resulted in decreased SR Ca2+ ATPase expression and Cav1.2 expression, but in increased NCX1 and Ser2814-phosphorylated ryanodine receptor 2. The calmodulin-dependent protein kinase II expression and function were markedly enhanced in the Fontan atria. CONCLUSION: The Fontan operation caused atrial CM Ca2+ handling abnormalities that produced arrhythmogenic-triggered activity and increased vulnerability to AT in experimental Fontan dogs.
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Fibrilación Atrial/etiología , Calcio/metabolismo , Procedimiento de Fontan/efectos adversos , Miocardio/metabolismo , Animales , Perros , Fura-2/análogos & derivados , Fura-2/farmacología , Atrios Cardíacos , Modelos Animales , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Purpose: The purpose of this study was to determine if histamine receptors interact with the epidermal growth factor receptor (EGFR) in cultured rat conjunctival goblet cells. Methods: Goblet cells from rat conjunctiva were grown in organ culture. First-passage goblet cells were used in all experiments. Phosphorylated (active) and total EGFR, AKT, and extracellular signal-regulated kinase (ERK)1/2 were measured by Western blot analysis. Cells were preincubated with the EGFR antagonist AG1478 for 30 minutes or small interfering RNA specific to the EGFR for 3 days prior to stimulation with histamine or agonists specific for histamine receptor subtypes for 2 hours. Goblet cell secretion was measured using an enzyme-linked lectin assay. Goblet cells were incubated for 1 hour with the calcium indicator molecule fura-2/AM, and intracellular [Ca2+] ([Ca2+]i) was determined. Data were collected in real time and presented as the actual [Ca2+]i with time and as the change in peak [Ca2+]i. Results: Histamine increased the phosphorylation of the EGFR. Mucin secretion and increase in [Ca2+]i stimulated by histamine, and agonists specific for each histamine receptor subtype were blocked by inhibition of the EGFR. Increase in [Ca2+]i stimulated by histamine and specific agonists for each histamine receptor was also inhibited by TAPI-1, a matrix metalloproteinase (MMP) inhibitor. The histamine-stimulated increase in activation of AKT, but not ERK1/2, was blocked by AG1478. Conclusions: In conjunctival goblet cells, histamine, using all four receptor subtypes, transactivates the EGFR via an MMP. This in turn phosphorylates AKT to increase [Ca2+]i and stimulate mucin secretion.
Asunto(s)
Conjuntiva/metabolismo , Receptores ErbB/metabolismo , Células Caliciformes/metabolismo , Mucinas/metabolismo , Receptores Histamínicos/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Células Cultivadas , Colorantes Fluorescentes/farmacología , Fura-2/análogos & derivados , Fura-2/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Técnicas de Cultivo de Órganos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Calcium dysregulation, membrane leakage and Aß amyloid growth are hallmarks of Alzheimer's disease. Here we show that Ca2+ ions inhibit membrane damage due to amyloid channels but enhance membrane disruption associated with fibers growing on the lipid surface. The similarities with IAPP suggest that this may represent a mechanism common to all proteinopathies.
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Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Fragmentos de Péptidos/metabolismo , Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Animales , Membrana Celular/efectos de los fármacos , Fluoresceínas/farmacología , Colorantes Fluorescentes/farmacología , Fura-2/farmacología , Cinética , Ratones , Fragmentos de Péptidos/química , Porosidad/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacosRESUMEN
Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary.
Asunto(s)
Astrocitos/metabolismo , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/farmacología , Túbulos Renales Proximales/metabolismo , Miocitos Cardíacos/metabolismo , Neuronas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Compuestos de Anilina/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Células Cultivadas , Fura-2/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/enzimología , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Potasio/metabolismo , Xantenos/farmacologíaRESUMEN
OBJECTIVE: Magnolol, a polyphenol compound from herbal medicines, was shown to alter physiology in various cell models. However, the effect of magnolol on Ca2+ homeostasis and its related physiology in oral cancer cells is unclear. This study examined whether magnolol altered Ca2+ signaling and cell viability in OC2 human oral cancer cells. METHODS: Cytosolic Ca2+ concentrations ([Ca2+]i) in suspended cells were measured by using the fluorescent Ca2+-sensitive dye fura-2. Cell viability was examined by 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. RESULTS: Magnolol at concentrations of 20-100⯵M induced [Ca2+]i rises. Ca2+ removal reduced the signal by approximately 50%. Magnolol (100⯵M) induced Mn2+ influx suggesting of Ca2+ entry. Magnolol-induced Ca2+ entry was partially suppressed by protein kinase C (PKC) regulators, and inhibitors of store-operated Ca2+ channels. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished magnolol-evoked [Ca2+]i rises. Conversely, treatment with magnolol abolished BHQ-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 partially inhibited magnolol-induced [Ca2+]i rises. Magnolol at 20-100⯵M decreased cell viability, which was not reversed by pretreatment with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). CONCLUSIONS: Together, in OC2 cells, magnolol induced [Ca2+]i rises by evoking partially PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. Magnolol also caused Ca2+-independent cell death. Therefore, magnolol-induced cytotoxicity may not be involved in activation mechanisms associated with intracellular Ca2+ mobilization in oral cancer cells.
Asunto(s)
Compuestos de Bifenilo/farmacología , Calcio/metabolismo , Homeostasis/efectos de los fármacos , Lignanos/farmacología , Neoplasias de la Boca/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Fura-2/farmacología , Humanos , Manganeso/metabolismo , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Sales de Tetrazolio , Fosfolipasas de Tipo C/efectos de los fármacosRESUMEN
In this work we investigated how modifications of the Ca2+ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca2+ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of LECs in cataractogenesis.
Asunto(s)
Señalización del Calcio , Catarata/metabolismo , Células Epiteliales/metabolismo , Cápsula del Cristalino/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Catarata/patología , Células Epiteliales/patología , Femenino , Fura-2/farmacología , Humanos , Cápsula del Cristalino/patología , Masculino , Persona de Mediana EdadRESUMEN
The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic ß-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and ß-cell survival. We have previously demonstrated loss of ß-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1ß (IL-1ß) combined with or without cycloheximide or actinomycin D. IL-1ß treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1ß led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1ß on SERCA2b protein stability. Similarly, IL-1ß-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas ß-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1ß, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered ß-cell calcium homeostasis and reduced ß-cell survival in diabetes.
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Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/biosíntesis , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Apoptosis/fisiología , Calcio/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Retículo Endoplásmico/metabolismo , Activación Enzimática , Fura-2/análogos & derivados , Fura-2/farmacología , Humanos , Células Secretoras de Insulina/citología , Insulinoma/patología , Interleucina-1beta/farmacología , Masculino , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Estrés Oxidativo/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Ribonucleótidos/farmacología , S-Nitroso-N-Acetilpenicilamina/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Transducción de Señal/genética , omega-N-Metilarginina/farmacologíaRESUMEN
The subcellular distribution and early signalling events of P2X7 receptors were studied in mouse cerebellar granule neurons. Whole-cell patch-clamp recordings evidenced inwardly directed non-desensitizing currents following adenosine 5'-triphosphate (ATP; 600 µM) or 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 µM) administration to cells bathed in a medium with no-added divalent cations (Ca(2+) and Mg(2+)). Nucleotide-activated currents were inhibited by superfusion of 2.5 mM Ca(2+), 1.2 mM Mg(2+) or 100 nM Brilliant Blue G (BBG), hence indicating the expression of ionotropic P2X7 receptors. Fura-2 calcium imaging showed [Ca(2+)]i elevations in response to ATP or BzATP at the somas and at a small number of axodendritic regions of granule neurons. Differential sensitivity of these [Ca(2+)]i increases to three different P2X7 receptor antagonists (100 nM BBG, 10 µM 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester, KN-62, and 1 µM 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate, A-438079) revealed that P2X7 receptors are co-expressed with different P2Y receptors along the plasmalemma of granule neurons. Finally, experiments with the fluorescent dye YO-PRO-1 indicated that prolonged stimulation of P2X7 receptors does not lead to the opening of a membrane pore permeable to large cations. Altogether, our results emphasise the expression of functional P2X7 receptors at both the axodendritic and somatic levels in mouse cerebellar granule neurons, and favour the notion that P2X7 receptors might function in a subcellular localisation-specific manner: presynaptically, by controlling glutamate release, and on the cell somas, by supporting granule neuron survival against glutamate excytotoxicity.
Asunto(s)
Cerebelo/metabolismo , Neuronas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Células Cultivadas , Cerebelo/efectos de los fármacos , Fura-2/farmacología , Ácido Glutámico/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2X/farmacología , Piridinas/farmacología , Tetrazoles/farmacologíaRESUMEN
OBJECTIVE: Nitric oxide (NO) production and Ca(2+) homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca(2+) homeostasis in a highly complex and cell-specific manner and although Ca(2+) homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca(2+)]c homeostasis in PE/CA-PJ15 cells. DESIGN: Cells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca(2+) concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La(3+) were employed to perturb intracellular calcium levels. RESULTS: NO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca(2+)]c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca(2+) homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca(2+)]c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca(2+)]c was also reduced in the presence of La(3+) or thapsigargin. CONCLUSIONS: NO induces a concentration-dependent [Ca(2+)]c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca(2+) buffering in the presence of TG or La(3+). Further, we show that exogenous NO deregulates Ca(2+) homeostasis in PE/CA-PJ15 cells with fully energized mitochondria.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Óxido Nítrico/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Supervivencia Celular/efectos de los fármacos , Cisteína/análogos & derivados , Cisteína/farmacología , Citosol/química , Relación Dosis-Respuesta a Droga , Fura-2/farmacología , Homeostasis/efectos de los fármacos , Humanos , Mitocondrias , Oligomicinas/farmacología , Oxihemoglobinas/farmacología , S-Nitrosotioles/farmacología , Tapsigargina/farmacología , Células Tumorales CultivadasRESUMEN
PROBLEM: Inflammation and genital infections promote the increase in leukocytes, pro-inflammatory cytokines, and oxygen reactive species, impairing sperm functions such as motility, capacitation, and acrosome reaction. All these functions are primarily regulated by cytoplasmic concentration of Ca(2+) ([Ca(2+) ]cyto ). This study evaluated the effect of tumor necrosis factor (TNF)-α on the [Ca(2+) ]cyto and its regulation in human sperm. METHOD OF STUDY: Sperm loaded with fura-2 were incubated with or without TNF-α (0-500 pg/mL) from 0 to 120 min. After incubation, the basal [Ca(2+) ]cyto and membrane permeability to Ca(2+) were evaluated by spectrofluorometry, before and after Ca(2+) addition to the extracellular medium. RESULTS: Without TNF-α, the addition of Ca(2+) promotes an transitory increase in [Ca(2+) ]cyto in the spermatozoa, that returns in a few minutes to a basal level, indicating calcium regulation activation. TNF-α decreases the Ca(2+) permeation and increases the basal level of [Ca(2+) ]cyto after a Ca(2+) pulse (P < 0.04); affecting calcium regulation in a way that is time and concentration dependent. TNF-α effect was partially prevented by the addition of an antioxidant (butylated hydroxytoluene) (P < 0.03). CONCLUSION: Tumor necrosis factor-α decreases membrane permeability to Ca(2+) and affects Ca(2+) regulation in sperm cells in vitro, probably via lipid peroxidation, which may explain the decrease in sperm fertilizing capacity during inflammatory and infectious processes.
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Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Reacción Acrosómica , Adulto , Membrana Celular/metabolismo , Células Cultivadas , Fertilización , Fura-2/farmacología , Homeostasis , Humanos , Masculino , Capacitación Espermática/efectos de los fármacos , Espermatozoides/metabolismo , Adulto JovenRESUMEN
PURPOSE: Recent evidence suggests that the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli and signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder. This led us to establish a method of co-culturing dorsal root ganglion sensory neurons and bladder urothelial cells. MATERIALS AND METHODS: Sensory neurons and urothelial cells obtained from dorsal root ganglia and bladders dissected from adult female Sprague-Dawley® rats were isolated by enzyme treatment and mechanical dissociation. They were plated together or separately on collagen coated substrate and cultured in keratinocyte medium for 48 to 72 hours. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing. RESULTS: Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to that in neuronal monocultures. The growth promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor K252a but upstream inhibition of nerve growth factor signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to adenosine triphosphate (100 µM) and activation of TRPV4 (4α-PDD, 10 µM) but not TRPV1 (capsaicin, 1 µM), TRPV3 (farnesyl pyrophosphate, 1 µM) or TRPA1 (mustard oil, 100 µM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate. CONCLUSIONS: Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Results suggest that neuronal properties are maintained in the presence of urothelium and neurite growth is potentiated by a nerve growth factor independent mechanism.
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Ganglios Espinales/metabolismo , Células Receptoras Sensoriales/metabolismo , Urotelio/citología , Adenosina Trifosfato/farmacología , Análisis de Varianza , Animales , Capsaicina/farmacología , Carbazoles/farmacología , Técnicas de Cocultivo , Dronabinol/farmacología , Femenino , Fura-2/farmacología , Inmunohistoquímica , Alcaloides Indólicos/farmacología , Indoles/farmacología , Mentol/farmacología , Modelos Animales , Factor de Crecimiento Nervioso/farmacología , Faloidina/farmacología , Fosfatos de Poliisoprenilo/farmacología , Ratas , Ratas Sprague-Dawley , Sesquiterpenos/farmacología , Canales Catiónicos TRPV/biosíntesisRESUMEN
Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1-6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2-4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.
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Regulación de la Expresión Génica , Neuronas/metabolismo , Canales Catiónicos TRPC/biosíntesis , Canales Catiónicos TRPC/fisiología , Canales de Potencial de Receptor Transitorio/biosíntesis , Canales de Potencial de Receptor Transitorio/fisiología , Acroleína/análogos & derivados , Acroleína/farmacología , Animales , Exones , Femenino , Fura-2/farmacología , Ganglios Espinales/metabolismo , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Isotiocianatos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Lectinas de Plantas/farmacología , Unión Proteica , Ratas , Canal Catiónico TRPA1RESUMEN
INTRODUCTION: Transmembrane ionic signaling regulates many cellular processes in both physiological and pathologic settings. In this study, the biophysical properties of voltage-dependent Na(+) channels in odontoblasts derived from human dental pulp (HOB cells) were investigated together with the effect of bradykinin on intracellular Ca(2+) signaling and expression of Ca(2+)-activated K(+) channels. METHODS: Ionic channel activity was characterized by using whole-cell patch-clamp recording and fura-2 fluorescence. RESULTS: Mean resting membrane potential in the HOB cells was -38 mV. Depolarizing steps from a holding potential of -80 mV activated transient voltage-dependent inward currents with rapid activation/inactivation properties. At a holding potential of -50 mV, no inward current was recorded. Fast-activation kinetics exhibited dependence on membrane potential, whereas fast-inactivation kinetics did not. Steady-state inactivation was described by a Boltzmann function with a half-maximal inactivation potential of -70 mV, indicating that whereas the channels were completely inactivated at physiological resting membrane potential, they could be activated when the cells were hyperpolarized. Inward currents disappeared in Na(+)-free extracellular solution. Bradykinin activated intracellular Ca(2+)-releasing and influx pathways. When the HOB cells were clamped at a holding potential of -50 mV, outward currents were recorded at positive potentials, indicating sensitivity to inhibitors of intermediate-conductance Ca(2+)-activated K(+) channels. CONCLUSIONS: Human odontoblasts expressed voltage-dependent Na(+) channels, bradykinin receptors, and Ca(2+)-activated K(+) channels, which play an important role in driving cellular functions by channel-receptor signal interaction and membrane potential regulation.
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Señalización del Calcio/fisiología , Pulpa Dental/citología , Odontoblastos/metabolismo , Canales de Potasio Calcio-Activados/fisiología , Canales de Sodio Activados por Voltaje/fisiología , Potenciales de Acción/fisiología , Análisis de Varianza , Bradiquinina/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular Transformada , Fura-2/química , Fura-2/farmacología , Humanos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Canales de Potasio Calcio-Activados/biosíntesis , Canales de Potasio de Rectificación Interna/fisiología , Receptores de Bradiquinina/metabolismo , Factores de TiempoRESUMEN
The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 µM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.
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Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Homeostasis/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Fura-2/farmacología , Humanos , Indoles/farmacología , Masculino , Tapsigargina/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2-based ratiometric Ca(2+)-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 µM, 100 µM and 300 µM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca(2+) imaging protocols revealed that HiK(+) (30 mM)-induced intracellular free Ca(2+) ([Ca(2+)](i)) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 µM, 100 µM and 300 µM LEV (respectively), without any significant effect on basal levels of [Ca(2+)](i). This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca(2+), providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.
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Potenciales de Acción , Piracetam/análogos & derivados , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Analgésicos/farmacología , Animales , Anticonvulsivantes/farmacología , Calcio/química , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología/métodos , Epilepsia/tratamiento farmacológico , Colorantes Fluorescentes/farmacología , Fura-2/farmacología , Ganglios Espinales/metabolismo , Levetiracetam , Neuronas/metabolismo , Dolor , Técnicas de Placa-Clamp/métodos , Piracetam/farmacología , RatasRESUMEN
Acid-sensing ion channel 1 (ASIC1) is a newly characterized contributor to store-operated Ca(2+) entry (SOCE) in pulmonary vascular smooth muscle (VSM). Since SOCE is implicated in elevated basal VSM intracellular Ca(2+) concentration ([Ca(2+)](i)) and augmented vasoconstriction in chronic hypoxia (CH)-induced pulmonary hypertension, we hypothesized that ASIC1 contributes to these responses. To test this hypothesis, we examined effects of the specific pharmacologic ASIC1a inhibitor, psalmotoxin 1 (PcTX1), on vasoconstrictor and vessel wall [Ca(2+)](i) responses to UTP and KCl (depolarizing stimulus) in fura-2-loaded, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) Wistar rats. PcTX1 had no effect on basal vessel wall [Ca(2+)](i), but attenuated vasoconstriction and increases in vessel wall [Ca(2+)](i) to UTP in arteries from control and CH rats; normalizing responses between groups. In contrast, responses to the depolarizing stimulus, KCl, were unaffected by CH exposure or PcTX1. Upon examining potential Ca(2+) influx mechanisms, we found that PcTX1 prevented augmented SOCE following CH. Exposure to CH resulted in a significant increase in pulmonary arterial ASIC1 protein. This study supports a novel role of ASIC1 in elevated receptor-stimulated vasoconstriction following CH which is likely mediated through increased ASIC1 expression and SOCE.