RESUMO
The successful integration of nanoparticles into biomedical applications requires modulation of their surface properties so that the interaction with biological systems is regulated to minimize toxicity for biological function. In the present work, we have engineered bioactive surfaces on gold (Au) and silver (Ag) nanoparticles and subsequently evaluated their interaction with mouse skin fibroblasts and macrophages. The Au and Ag nanoparticles were synthesized using tyrosine, tryptophan, isonicotinylhydrazide, epigallocatechin gallate, and curcumin as reducing and stabilizing agents. The nanoparticles thus prepared showed surface corona and exhibited free radical scavenging and enzyme activities with limited cytotoxicity and genotoxicity. We have thus developed avenues for engineering the surface of nanoparticles for biological applications.
Assuntos
Dano ao DNA , Fibroblastos/citologia , Sequestradores de Radicais Livres/farmacologia , Ouro/química , Macrófagos/citologia , Nanopartículas Metálicas/administração & dosagem , Prata/química , Animais , Sobrevivência Celular , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Nanopartículas Metálicas/química , Camundongos , Peroxidase/metabolismo , Propriedades de SuperfícieRESUMO
Nanotechnology has the potential to circumvent several drawbacks of conventional therapeutic formulations. In fact, significant strides have been made towards the application of engineered nanomaterials for the treatment of cancer with high specificity, sensitivity and efficacy. Tailor-made nanomaterials functionalized with specific ligands can target cancer cells in a predictable manner and deliver encapsulated payloads effectively. Moreover, nanomaterials can also be designed for increased drug loading, improved half-life in the body, controlled release, and selective distribution by modifying their composition, size, morphology, and surface chemistry. To date, polymeric nanomaterials, metallic nanoparticles, carbon-based materials, liposomes, and dendrimers have been developed as smart drug delivery systems for cancer treatment, demonstrating enhanced pharmacokinetic and pharmacodynamic profiles over conventional formulations due to their nanoscale size and unique physicochemical characteristics. The data present in the literature suggest that nanotechnology will provide next-generation platforms for cancer management and anticancer therapy. Therefore, in this critical review, we summarize a range of nanomaterials which are currently being employed for anticancer therapies and discuss the fundamental role of their physicochemical properties in cancer management. We further elaborate on the topical progress made to date toward nanomaterial engineering for cancer therapy, including current strategies for drug targeting and release for efficient cancer administration. We also discuss issues of nanotoxicity, which is an often-neglected feature of nanotechnology. Finally, we attempt to summarize the current challenges in nanotherapeutics and provide an outlook on the future of this important field.
RESUMO
Manufacturing nanoparticles with controlled physicochemical properties using environment-friendly routes have potential to open new prospects for a variety of applications. Accordingly, several approaches have been established for manufacturing metal nanoparticles. Many of these approaches entail the use of hazardous chemicals and could be toxic to the environment, and cannot be used readily for biomedical applications. In the present work, we report a single step bio-friendly approach to formulate gold (Au), silver (Ag), and Au-Ag alloy nanoparticles with desired surface corona and composition using isonicotinylhydrazide (INH) as a reducing agent. INH also functioned as a stabilizing agent by enabling a surface corona around the nanoparticles. Remarkably, within a single step INH could also provide a handle in regulating the composition of Au and Ag in bimetallic systems without any additional chemical modification. The physicochemical and surface properties of the different nanoparticles thus obtained have been examined by analytical, spectroscopic and microscopic techniques. Cell cytotoxicity (release of lactate dehydrogenase), cell viability and intracellular reactive oxygen species (ROS) assays confirmed that the Au, Ag, and Au-Ag bimetallic nanoparticles prepared with INH are biocompatible. Finally, the presence of organic surface corona of INH on the nanoparticles was found to impart nanozyme activity and antimycobacterial sensitivity to the nanoparticles.
Assuntos
Ligas/química , Fibroblastos/metabolismo , Ouro/química , Hidrazinas/química , Teste de Materiais , Nanopartículas Metálicas/química , Prata/química , Animais , Células Cultivadas , Fibroblastos/citologia , Camundongos , OxirreduçãoRESUMO
PURPOSE: The outflow facility for aqueous humor across the trabecular meshwork (TM) is enhanced by agents that oppose the actomyosin contraction of its resident cells. Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complex of Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC, leading to an increase in actomyosin contraction. In this study, we examined the effects of Rho kinase (ROCK) inhibitors on the relative dephosphorylation of the two sites of MYPT1 using human TM cells (GTM3). METHODS: Dephosphorylation of MYPT1 at Thr853 and Thr696 was determined by western blot analysis following exposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983. Consequent dephosphorylation of MLC and decreases in actomyosin contraction were assessed by western blot analysis and collagen gel contraction assay, respectively. Changes in the cell-matrix adhesion were measured in real time by electric cell-substrate impedance sensing and also assessed by staining for paxillin, vinculin, and focal adhesion kinase (FAK). RESULTS: Both ROCK inhibitors produced a concentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1 in adherent GTM3 cells. IC50 values for Y-39983 were 15 nM and 177 nM for dephosphorylation at Thr853 and Thr696, respectively. Corresponding values for Y-27632 were 658 nM and 2270 nM. Analysis of the same samples showed a decrease in MLC phosphorylation with IC50 values of 14 nM and 1065 nM for Y-39983 and Y-27632, respectively. Consistent with these changes, both inhibitors opposed contraction of collagen gels induced by TM cells. Exposure of cells to the inhibitors led to a decrease in the electrical cell-substrate resistance, with the effect of Y-39983 being more pronounced than Y-27632. Treatment with these ROCK inhibitors also showed a loss of stress fibers and a concomitant decrease in tyrosine phosphorylation of paxillin and FAK. CONCLUSIONS: Y-39983 and Y-27632 oppose ROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with a corresponding decrease in MLC phosphorylation. A relatively low effect of both ROCK inhibitors at Thr696 suggests a role for other Ser/Thr kinases at this site. Y-39983 was several-fold more potent when compared with Y-27632 at inhibiting the phosphorylation of MYPT1 at either Thr853 or Thr696 commensurate with its greater potency at inhibiting the activity of human ROCK-I and ROCK-II enzymes.
Assuntos
Actomiosina/metabolismo , Junções Célula-Matriz/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfoproteínas/metabolismo , Fibras de Estresse/efeitos dos fármacos , Malha Trabecular , Amidas/farmacologia , Humor Aquoso/fisiologia , Western Blotting , Linhagem Celular Transformada , Colágeno/análise , Proteína-Tirosina Quinases de Adesão Focal/análise , Proteína-Tirosina Quinases de Adesão Focal/biossíntese , Géis/análise , Humanos , Concentração Inibidora 50 , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Paxilina/análise , Paxilina/biossíntese , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Malha Trabecular/citologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Vinculina/análise , Vinculina/biossínteseRESUMO
Increased contractility of the actin cytoskeleton by phosphorylation of the regulatory myosin light chain (MLC) results in a loss of barrier integrity in corneal endothelial cells. This study has investigated the effect of extracellular ATP, which may influence both Ca2+ and cAMP signalling, on MLC phosphorylation and barrier integrity in cultured bovine corneal endothelial cells (BCEC) known to express A2B and P2Y purinergic receptors, and ecto-nucleotidases. Extracellular ATP (100 microM) promoted MLC dephosphorylation (pMLC=61.8% at 18 min; n=9). Pre-exposure to ARL-67156, an ecto-nucleotidase inhibitor, prevented ATP-induced dephosphorylation. Other P2Y agonists, UTP and ATPgammaS, also induced MLC dephosphorylation but to a lesser degree compared to ATP. Thrombin (2 U/ml), which activate Rho kinase through PAR-1 receptors in the endothelium, induced MLC phosphorylation (pMLC=129.2%; n=14). This phosphorylation was completely abolished by concomitant exposure to ATP. When cells were pretreated with adenosine (100 microM; A2B agonist) or forskolin (10 microM), thrombin-induced phosphorylation was suppressed. ATP also led to a significant increase in cAMP (> 3-fold compared to 10 microM adenosine). Thrombin-induced increase in trans-endothelial flux of horseradish peroxidase (44 kDa) and disruption of the cortical actin were suppressed by ATP. These findings indicate that in BCEC (1) ATP induces elevated cAMP through its metabolite adenosine leading to MLC dephosphorylation, (2) Stimulation of P2Y2 receptors also leads to activation of MLCP since UTP- and ATPgammaS caused MLC dephosphorylation, and (3) ATP is antagonistic to thrombin since the latter inhibits MLCP through increased activity of Rho kinase. These findings further emphasize the role of contractility of the actin cytoskeleton in regulating the barrier integrity of corneal endothelium.
Assuntos
Trifosfato de Adenosina/farmacologia , Endotélio Corneano/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Trombina/antagonistas & inibidores , Actinas/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/antagonistas & inibidores , Animais , Bovinos , Células Cultivadas , AMP Cíclico/biossíntese , Endotélio Corneano/metabolismo , Fosforilação/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2Y2 , Trombina/farmacologiaRESUMO
PURPOSE: Phosphorylation of the regulatory light chain of myosin II (referred to as myosin light chain or MLC) leads to a loss of barrier integrity in cellular monolayers by an increase in the contractility of the cortical actin cytoskeleton. This effect has been examined in corneal endothelial (CE) cells. METHODS: Experiments were performed using cultured bovine CE cells (BCEC). MLC phosphorylation was induced by a thrombin-mediated activation of the proteinase-activated receptor-1 (PAR-1). Expression of MLC kinase (MLCK), a Ca2+/calmodulin-dependent protein kinase that phosphorylates MLC at its Ser-19 and Thr-18 residues, was determined by RT-PCR and Western blotting. Expression of PAR-1, RhoA, and Rho kinase-1 (effector of RhoA) was ascertained by RT-PCR. MLC phosphorylation was assessed by urea-glycerol gel electrophoresis followed by immunoblotting. The effects of Rho kinase-1 and PKC were characterized by using their selective inhibitors, Y-27632 and chelerythrine, respectively. Reorganization of the cytoskeleton was evaluated by the phalloidin staining of actin. [Ca2+]i was measured using Fura-2. The barrier integrity was assayed as permeability of BCEC monolayers to horseradish peroxidase (HRP; 44 kDa). RESULTS: RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA. Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK. Exposure to thrombin induced an increase in [Ca2+]i with the peak unaffected by an absence of extracellular Ca2+. Pre-exposure to thrombin (2 U ml(-1); 2 min) led to mono- and di-phosphorylation of MLC. Under both basal conditions and in the presence of thrombin, MLC phosphorylation was prevented by chelerythrine (10 microm) and Y-27632 (<25 microm). Thrombin led to inter-endothelial gaps secondary to the disruption of the cortical actin cytoskeleton, which under resting conditions was organized as a perijunctional actomyosin ring (PAMR). These responses were blocked by pre-treatment with Y-27632. Thrombin also increased permeability to HRP, which was abolished by pre-treatment with Y-27632. CONCLUSIONS: Thrombin induces MLC phosphorylation in BCEC. The consequent increase in the contractility of the actin cytoskeleton produces a centripetal force resulting in inter-endothelial gaps and a breakdown of barrier integrity. These responses are PKC- and Rho kinase-dependent. [Ca2+]i increase, as well as sensitivity of the thrombin response to PKC and Rho kinase inhibitors, are consistent with the expression of PAR-1 receptors in BCEC. Thrombin-induced hyperpermeability is a model to investigate barrier dysfunction induced by MLC phosphorylation.
Assuntos
Endotélio Corneano/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/metabolismo , Trombina/farmacologia , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Citoplasma/metabolismo , Citoesqueleto/efeitos dos fármacos , Endotélio Corneano/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Cadeias Leves de Miosina/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Dephosphorylation of the myosin II regulatory light chain (MLC) promotes barrier integrity of cellular monolayers through relaxation of the actin cytoskeleton. This study has investigated the influence of adenosine (ADO) on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC). METHODS: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis and immunoblotting. Elevation of cAMP in response to agonists of A2b receptors (subtype of P1 purinergic receptors) was confirmed by phosphorylation of the cAMP response element binding protein (CREB), which was determined by Western blotting. Activation of MAP kinases (i.e. activated ERK1 and ERK2) was assessed by Western blotting to examine their influence on MLC phosphorylation. Transepithelial electrical resistance (TER) of cells grown on porous filters was measured to assess the altered barrier integrity. RESULTS: Exposure to ADO (200 microm; 30 min) and N-ethyl (carboxamido) adenosine (NECA; 50 microm; 30 min), known agonists of A2b receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 microm; 30 min), a direct activator of adenylate cyclase. Exposure to ADO, NECA, and FSK led to dephosphorylation of MLC by 51, 40, and 47%, respectively. ADO-induced dephosphorylation was dose-dependent with as much as 31% dephosphorylation at 1 microm ADO. CGS-21680, a selective A2a agonist, neither induced MLC dephosphorylation nor CREB phosphorylation. ADO phosphorylated MAP kinases which could be prevented by exposure to the MAP kinase-specific inhibitor, U0126 (10 microM). NECA and FSK also induced ERK1 and ERK2 activation similar to ADO. Exposure to U0126 inhibited MLC phosphorylation under basal conditions by 17%. ADO-induced MLC dephosphorylation was enhanced by a simultaneous exposure to U0126 (25% increase in dephosphorylation). Exposure to ADO caused an increase in TER from 17 to 22 ohms cm2. CONCLUSIONS: (1) CREB phosphorylation in response to ADO and NECA, which indicates activation of the cAMP-PKA axis, suggests expression of A2b receptors in BCEC. (2) ERK1 and ERK2, activated by cAMP and A2b receptors, promote MLC phosphorylation. However, the net result of cAMP elevation is MLC dephosphorylation, presumably because the competing pathways involving inactivation of MLCK and/or ROCK are dominant (Rho-associated coiled coil-containing protein kinase or Rho kinase). (3) Consistent with MLC dephosphorylation, exposure to ADO increases TER, which suggests increased barrier integrity.
Assuntos
Adenosina/farmacologia , Endotélio Corneano/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/metabolismo , Animais , Bovinos , Células Cultivadas , AMP Cíclico/fisiologia , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Corneano/citologia , Endotélio Corneano/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Cadeias Leves de Miosina/efeitos dos fármacos , Miosina Tipo II/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacosRESUMO
PURPOSE: Hyposmolar perfusion of intact trabecular meshwork (TM) induces a decrease in its hydraulic conductivity (Lp). However, exposure to agents that elevate intracellular cAMP in TM cells increases Lp. Since volume of TM cells could directly influence porosity of the TM and hence Lp, this study has investigated changes in volume in response to acute hyposmotic shock (i.e. regulatory volume decrease or RVD) and elevated cAMP in cultured TM cells. METHODS: Bovine trabecular meshwork cells (BTMC), grown on glass coverslips and loaded with the fluorescent dye MQAE, were used to measure rapid changes in cell volume using the principle of dynamic fluorescence quenching. Activation of volume-regulated anion channels (VRAC) was assessed by measuring volume-sensitive Cl(-) currents (I(Cl,swell)) in the whole cell configuration of the patch clamp technique and by determining the swelling-induced enhancement in I(-) permeability using the halide-sensitivity of MQAE. Expressions of ClC (chloride channels of the ClC gene family), P-glycoprotein (Pgp), and cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels were examined by RT-PCR. Elevation of cAMP in response to forskolin was confirmed by determining the phosphorylation of cAMP response element-binding protein and activating transcription factor-1 (CREB, ATF-1), which form the downstream targets of protein kinase A. RESULTS: As a response to hyposmotic shock, there was an acute increase in cell volume but there was no robust RVD. Patch clamp experiments showed activation of a characteristic Cl(-) current in response to cell swelling. This Cl(-) current was inhibited by NPPB (100microM) and fluoxetine (50microM), both of which are known blockers of VRAC. Experiments, which used the halide-sensitivity of MQAE, also indicated a 9-fold increase in I(-) influx upon cell swelling (8.9+/-4.6; n=9), consistent with activation of a VRAC-like Cl(-) current. To examine whether RVD is limited by K(+) conductance, the swollen cells were exposed to gramicidin, which is known to induce cation channel activity. Such a maneuver led to secondary swelling with [Na(+)](o)=140mM but a rapid shrinkage [Na(+)](o)=8mM indicating that the RVD is limited by cationic conductance necessary for K(+) efflux. Exposure to forskolin, which resulted in CREB and ATF-1 phosphorylation, caused a reversible decrease in cell volume (14.5+/-5%; n=20) under isosmotic and hyposmotic conditions. RT-PCR analysis confirmed expression of ClC-2, ClC-5, and Pgp Cl(-) channels in bovine TM cells. However, ClC-3 and CFTR were not expressed. CONCLUSIONS: TM cells respond to acute hyposmotic shock in an osmometric manner, but their RVD is limited by K(+) conductance. The lack of CFTR expression and decrease in cell volume in response to forskolin concomitant with hyposmolarity suggest that elevated cAMP activates a K(+) conductance. Thus, the altered resistance to aqueous outflow in response to hyposmotic perfusion of the TM and elevated cAMP may be attributed to persistent cell swelling and cell shrinkage, respectively.
Assuntos
AMP Cíclico/fisiologia , Malha Trabecular/citologia , Animais , Humor Aquoso/fisiologia , Bovinos , Permeabilidade da Membrana Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas , Canais de Cloreto/fisiologia , Fluorescência , Gramicidina/farmacologia , Pressão Osmótica , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Compostos de Quinolínio/farmacologia , Malha Trabecular/metabolismo , Malha Trabecular/fisiologiaRESUMO
Light scattering is an empirical technique employed to measure rapid changes in cell volume. This study describes a new configuration for the method of light scattering and its corroboration by measurements of cell height (as a measure of cell volume). Corneal endothelial cells cultured on glass cover-slips were mounted in a perfusion chamber on the stage of an inverted microscope. A beam of light was focused on the cells from above the stage at an angle of 40 degrees to the plane of the stage. The scattered light intensity (SLI), captured by the objective and referred to as forward light scatter (FLS), increased and decreased in response to hyposmotic and hyperosmotic shocks, respectively. The rapid increase and decrease in SLI corresponded to cell swelling and shrinkage, respectively. Subsequently, SLI decreased and increased as expected for a regulatory volume decrease (RVD) and increase (RVI), respectively. These data are in agreement with measurements of cell height, demonstrating that the method of light scatter in FLS mode is useful for monitoring rapid changes in cell volume of cultured cells. Changes in SLI caused by gramicidin were consistent with cell volume changes induced by equilibration of NaCl and KCl concentrations across the cell membrane. Similarly, an additional decrease in SLI was recorded during RVD upon increasing K+ conductance by valinomycin. Decreasing K+ conductance of the cell membrane with Ba2+ changed the time course of SLI consistent with the effect of the K+ channel blocker on RVD. Bumetanide and dihydro-ouabain inhibited increases in SLI during RVI. In conclusion, FLS is a valid method for qualitative analysis of cell volume changes with a high time resolution.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/fisiologia , Luz , Animais , Bovinos , Tamanho Celular/fisiologia , Projetos de Pesquisa/normas , Espalhamento de RadiaçãoRESUMO
In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin-luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing-type chamber. We developed a LL pulse protocol to determine the rate of ATP release (R(ATP)) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure R(ATP) as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 microl of A6 cells released ATP at a rate of 66 +/- 8 fmol min(-1). A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H(2)O)(-1) elevated R(ATP) rapidly to a peak value of 1.89 +/- 0.11 pmol min(-1) (R(ATP)(peak)) followed by a plateau phase reaching 0.51 +/- 0.07 pmol min(-1) (R(ATP)(plat)). Both R(ATP)(peak) and R(ATP)(plat) values increased with the degree of dilution. The magnitude of R(ATP)(plat) remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 +/- 0.08 pmol min(-1) was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H(2)O)(-1). This R(ATP) value, induced in the absence of cell swelling, is comparable to R(ATP)(plat). Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose-response curves for peak [Ca(2+)](i) increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with R(ATP)(peak). The link between the ATP release and the fast [Ca(2+)](i) transient was also demonstrated by the depression of both phenomena by Cl(-) removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin-sensitive rise of [Ca(2+)](i) during the hyposmotic shock.
Assuntos
Trifosfato de Adenosina/metabolismo , Células Epiteliais/metabolismo , Soluções Hipotônicas/farmacologia , Rim/metabolismo , Animais , Cálcio/metabolismo , Calibragem , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Rim/efeitos dos fármacos , Luciferases/metabolismo , Medições Luminescentes , Concentração Osmolar , Xenopus laevisRESUMO
Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (C(T)) that is inhibited by extracellular Mg(2+). Elevation of free cytosolic [Ca(2+)] ([Ca(2+)](i)) is known to increase C(T). Therefore, we examined [Ca(2+)](i) dynamics and their sensitivity to extracellular Mg(2+) during hyposmotic conditions. Fura-2-loaded A6 monolayers, cultured on permeable supports were subjected to a sudden reduction in osmolality at both the basolateral and apical membranes from 260 to 140 mosmol (kg H(2)O)(-1). Reduction of apical osmolality alone did not affect [Ca(2+)](i). In the absence of extracellular Mg(2+), the hyposmotic shock induced a biphasic rise in [Ca(2+)](i). The first phase peaked within 40 s and [Ca(2+)](i) increased from 245 +/- 12 to 606 +/- 24 nM. This phase was unaffected by removal of extracellular Ca(2+), but was abolished by activating P2Y receptors with basolateral ATP or by exposing the cells to the phospholipase C (PLC) inhibitor U73122 prior to the osmotic shock. Suramin also severely attenuated this first phase, suggesting that the first phase of the [Ca(2+)](i) rise followed swelling-induced ATP release. The PLC inhibitor, the ATP treatment or suramin did not affect a second rise of [Ca(2+)](i) to a maximum of 628 +/- 31 nM. The second phase depended on Ca(2+) in the basolateral perfusate and was largely suppressed by 2 mM basolateral Mg(2+). Acute exposure of the basolateral membrane to Mg(2+) during the upstroke of the second phase caused a rapid decline in [Ca(2+)](i). Basolateral Mg(2+) inhibited Ca(2+) entry in a dose-dependent manner with an inhibition constant (K(i)) of 0.60 mM. These results show that polarized A6 epithelia respond to hyposmotic shock by Ca(2+) release from inositol trisphosphate-sensitive stores, followed by basolateral Ca(2+) influx through a Mg(2+)-sensitive pathway. The second phase of the [Ca(2+)](i) response is independent of the initial intracellular Ca(2+) release and therefore constitutes non-capacitative Ca(2+) entry.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Magnésio/farmacologia , Trifosfato de Adenosina/farmacologia , Algoritmos , Cálcio/metabolismo , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletrofisiologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Potenciais da Membrana/fisiologia , Pressão Osmótica , Técnicas de Patch-Clamp , Suramina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
Swelling induced enhancement of anion permeability was investigated using the halide-sensitive fluorescent dye SPQ in cultured bovine corneal endothelial cells (BCEC). Rates of anion influx were quantified in terms of the rate of change of SPQ fluorescence during exposure to short duration pulses of Cl-, I-or NO3-while the cells were being perfused with I-, NO3-or Cl-Ringer, respectively. Since SPQ fluorescence is quenched to different extents by these anions, their influx or efflux causes significant changes in fluorescence. The ratio of the maximum rate of change of fluorescence during the pulse period under hyposmotic conditions to that under isosmotic conditions, referred to as the enhancement ratio (ER), was calculated as a measure of the increase in anion permeability. When cells were perfused with NO3-Ringer, exposure to I-pulses yielded an ER=9.0+/-2.6 for 110+/-5 mosmhyposmotic shock. This was higher than with Cl-/I-(6.4+/-0.7) or NO3-/Cl-(3.2+/-0.8) anion-pairs for the same level of shocks. In all cases, the enhancement occurred within approximately 100 seconds after swelling but decreased with continued progress of regulatory volume decrease (RVD). ER returned to approximately 1 within 4 minutes after returning to isosmotic conditions. The membrane potential (Em) depolarized immediately after hyposmotic shock. When cells were depolarized prior to the shocks by high [K+], changes in Emwere relatively small. ER, for the NO3-/I-anion-pair, was significantly reduced by DIDS (100% at 500 microm), NPPB ( approximately 80% at 100 microm) and tamoxifen (approximately 85% at 12 microm). Tamoxifen and NPPB also inhibited swelling induced depolarization. Increasing cationic conductance with Gramicidin D at approximately 2 minutes following hyposmotic shock induced NPPB-inhibitable secondary swelling or accelerated RVD under normal or low Na+conditions, respectively. These results demonstrate that BCEC express swelling activated Cl-channels, which facilitate RVD by enhancing anionic permeability and also by providing a favorable electrical gradient for K+efflux.
Assuntos
Canais de Cloreto/fisiologia , Endotélio Corneano/metabolismo , Homeostase/fisiologia , Animais , Bovinos , Técnicas de Cultura de Células , Permeabilidade da Membrana Celular/fisiologia , Tamanho Celular/fisiologia , Canais de Cloreto/efeitos dos fármacos , Cloretos/farmacocinética , Endotélio Corneano/citologia , Iodetos/farmacocinética , Potenciais da Membrana/fisiologia , Pressão OsmóticaRESUMO
PURPOSE: To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport. METHODS: BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2). RESULTS: Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response. CONCLUSIONS: Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused primarily by depolarization of Em, which drives HCO3- influx through the basolateral electrogenic Na+:nHCO3- cotransporter. In contrast with cultured BCECs, AE2 may be present in fresh cells.
Assuntos
Antiporters/metabolismo , Bicarbonatos/metabolismo , Cloretos/metabolismo , Endotélio Corneano/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Transporte Biológico , Western Blotting , Bovinos , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato , Endotélio Corneano/citologia , Endotélio Corneano/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Potenciais da Membrana/efeitos dos fármacos , Ácido Niflúmico/farmacologia , Nitrobenzoatos/farmacologia , ortoaminobenzoatos/farmacologiaRESUMO
PURPOSE: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. RESULTS: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. CONCLUSION: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.
Assuntos
Cálcio/metabolismo , Endotélio Corneano/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Corneano/citologia , Endotélio Corneano/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Fura-2/análogos & derivados , Indóis/farmacologia , Manganês/metabolismo , Tionucleotídeos/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.
Assuntos
Canais de Cloreto/metabolismo , Corantes Fluorescentes , Compostos de Quinolínio , Animais , Fenômenos Biofísicos , Biofísica , Bovinos , Permeabilidade da Membrana Celular , Tamanho Celular , Células Cultivadas , Endotélio Corneano/citologia , Endotélio Corneano/metabolismo , Técnicas In Vitro , Iodetos , Potenciais da Membrana , Modelos Biológicos , Nitratos , Pressão OsmóticaRESUMO
PURPOSE: Permeability (Pdc) to sodium fluorescein (F) is a characteristic of the barrier function of the corneal epithelium. The repeatability of several in vivo fluorophotometric methods used to measure permeability in humans remains largely undocumented. This study examines the repeatability of a method based on topical instillation of a single drop of F for the quantitative assessment of Pdc. METHODS: Nine healthy subjects with no history of ocular disease provided 1 (n = 1), 2 (n = 1), or 3 (n = 7) repeated measurements of each eye at successive visits. After making 3 baseline fluorescence scans centrally through the tear film and cornea, 2 microliters of 0.35% F were instilled and 10 fluorescence scans were obtained at approximately 2-minute intervals immediately after instillation. Subsequently, the eyes were rinsed three times with nonpreserved saline and four additional scans were performed. RESULTS: Pdc was calculated by dividing the baseline-corrected postrinse stromal fluorescence by the time integral of the tear film fluorescence calculated over the 20-minute exposure period. After applying a logarithmic transformation to the Pdc estimates, a mixed-model analysis was used to assess measurement repeatability. On the Pdc scale, there is an estimated 95% chance that a second measurement could be as much as 2.88 times higher or 0.35 times lower than a first measurement. CONCLUSIONS: This substantial variability between repeated measurements indicates that the single-drop procedure is unreliable for monitoring individual patient changes. However, with careful sample size planning, this technique can be used in population-based research to compare differences in treatment effects between groups of subjects.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Córnea/metabolismo , Adulto , Córnea/citologia , Epitélio/metabolismo , Fluoresceína , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Fluorofotometria/métodos , Humanos , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
Ion coupled fluid transport by the corneal endothelium is stimulated by adenosine through a cAMP dependent mechanism. This study examines if anion conductance is enhanced by cAMP and, hence by adenosine. Cl- fluxes, measured by changes in fluorescence of the Cl- sensitive dye SPQ, following removal or re-addition of Cl- Ringer, could be accelerated by 20 microM forskolin or 10 microM adenosine. The cAMP cocktail (20 microM forskolin + 100 microM IBMX + 100 microM cpt-cAMP) had no effect on resting [Cl-]i. However, when Cl- influx was inhibited by 100 microM furosemide, net Cl- efflux was observed in response to the cAMP cocktail. Exposure to the cAMP cocktail alone depolarized the resting membrane potential. Conversely, the cAMP cocktail caused a relative hyperpolarization in cells which had been previously depolarized beyond the equilibrium potential for Cl- (ECl-), by application of 1 microM Gramicidin D. cAMP dependent changes in membrane potential could be inhibited by 50 microM NPPB, but not by 200 microM DPC, 100 microM H2DIDS or 50 microM glibenclamide. Taken together, these results are consistent with NPPB-sensitive, cAMP activated Cl- channels. To examine if these channels are permeable to HCO3-, changes in pHi in response to the cAMP cocktail were measured in acidified and depolarized cells in the absence of Na+. The cAMP cocktail caused an increase in pHi only when HCO3- was present, consistent with HCO3- influx. In control HCO3- Ringer, the cAMP cocktail caused a transient decrease in pHi, which could not be accounted for by inhibition of Na+:nHCO3- cotransport or stimulation of Cl-/HCO3- exchange. These results are consistent with conductive HCO3- efflux through cAMP activated channels. We conclude that cultured bovine corneal endothelial cells possess cAMP activated anion channels.
Assuntos
Canais de Cloreto/efeitos dos fármacos , AMP Cíclico/farmacologia , Endotélio Corneano/efeitos dos fármacos , Animais , Bicarbonatos/metabolismo , Bovinos , Células Cultivadas , Canais de Cloreto/fisiologia , Cloro/metabolismo , AMP Cíclico/agonistas , Endotélio Corneano/citologia , Endotélio Corneano/fisiologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Nitrobenzoatos/farmacologiaRESUMO
The intracellular fluorescence of 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ), a Cl(-) -sensitive fluorescent dye, is quenched by intracellular organic anions and proteins of unknown identity. The concentration of these intracellular quenchers (ICQs), however, is dependent on cell volume. In the absence of Cl-, changes in the observed SPQ fluorescence may therefore reflect changes in cell volume. This concept has been applied to determine relative changes in cell volume of cultured corneal endothelium in response to anisosmotic shocks, using NO3- as the Cl- substituent. SPQ fluorescence increased with decreasing osmolarity and vice versa. A 20 mosM hypertonic shock was needed to detect a change in SPQ fluorescence with a signal-to-noise ratio of >25. Assuming dynamic quenching by ICQs, we applied an extension of the Stern-Volmer equation to develop a simple relationship between the measured SPQ fluorescence and relative changes in cell volume. For large hyposmotic shocks, regulatory volume decrease (RVD) was observed. The rate of RVD could be enhanced by exposure to 0.5 microM gramicidin in low-Na+ Ringer solution (i.e., K+-NO3- efflux), indicating that K+ conductance is rate limiting for RVD. These results demonstrate the principle of using fluorescence quenching to measure changes in cell volume in real time. Because SPQ is sensitive to Cl-, its usefulness as a quenching probe is limited. However, a structure-activity study can be expected to yield useful Cl(-)-insensitive analogs.