RESUMO
BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.
Assuntos
Neutrófilos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Cloreto de Amônio/farmacologia , Benzamidas/farmacologia , Bicarbonatos/farmacologia , Citocalasina B/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonamidas/farmacologiaRESUMO
It is well known that chemotactic agents active Na(+)/H(+) exchanger, increasing intracellular pH of neutrophils, but their effect on bicarbonate transporters have not been established yet. To study the effect of fMLP on the activity of Cl(-)/HCO(3)(-) exchange, the rate of pH recovery after acute Cl(-) readmission in cell subjected to an alkaline load by CO(2) washout in a Cl-free medium was measured. The activity of the exchanger was reduced to 72% of control when cells were pre-incubated for 5 min with 0.1 µM fMLP and reached 48% of control in steady state after acute exposure. After extracellular bicarbonate or TMA addition the rate recovery of intracellular pH was reduce at 72% and at 84%, respectively. The inhibitory effect on the intracellular pH recovery was not affected by blockers of Na(+)/H(+) exchange. We conclude from these studies that an increase of pH(i) produced for this chemotactic agent is facilitated by the simultaneous activation of Na(+)/H(+) exchange and inhibition of Cl(-)/HCO(3)(-) exchange in neutrophils.
Assuntos
Bicarbonatos/metabolismo , Cloretos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Receptores de Formil Peptídeo/agonistas , Células Cultivadas , Quimiotaxia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Humanos , Hidrogênio/metabolismo , Neutrófilos/metabolismo , Sódio/metabolismoRESUMO
The increase of extracellular glycine concentration prevents or mitigates a variety of pathological dysfunctional inflammatory responses. To eliminate the systemic effects of glycine as the reduction in the release of cytokines, this study was performed in isolated human neutrophils. The increase of the intracellular calcium concentration ([Ca2+](i)) and reactive oxygen species (ROS) release in cells incubated with glycine (0.1 to 10 mM) and stimulated with fMLP or PMA were compared with glycine-free controls. Glycine inhibited ROS production but increased [Ca2+](i) signal produced by fMLP. The inhibition of ROS production was observed even when glycine was added after the ROS release had reached maximal rate. The inhibitory effect was insensitive to strychnine and also obtained when PMA was used as stimulant. This study demonstrated that glycine impaired the activation of oxidative burst independently of glycine-gated chloride channel, presumably at the membrane level.
Assuntos
Glicina/farmacologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Separação Celular , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacos , Estricnina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Experimental and clinical investigations using hyperosmotic solutions for resuscitation of hemorrhagic shock demonstrated modulation of the inflammatory response. Decreased postinjury hyperinflammation has been attributed to a reduction in neutrophil-mediated tissue damage. This study shows that cytoskeletal disruption with cytochalasinB did not reverse or prevent the inhibitory effect of an osmolarity increase on the neutrophil cytotoxic response to a formyl peptide. In cytochalasin-primed neutrophils, the hyperosmolarity-dependent inhibition promptly reversed after returning to iso-osmotic levels. Paradoxically, an increase in osmolarity after stimulation produced an increase in the release of reactive oxygen species to the extracellular medium. The inhibitory effect of hyperosmotic NaCl can be reproduced by solutions of similar osmolarity containing N-methyl glucamine or sucrose, but solutions containing mannitol allowed an almost complete response to N-formyl methionyl leucyl phenylalanine. The effects on the release of reactive oxygen species to the extracellular media found with the OxyBURST-bovine serum albumin assay correlated with the changes of the intracellular calcium signal, indicating that the inhibition by hyperosmolarity occurs near the receptor level.
Assuntos
Citocalasina B/metabolismo , Neutrófilos/metabolismo , Concentração Osmolar , Cálcio/metabolismo , Fatores Quimiotáticos/metabolismo , Citoesqueleto/metabolismo , Humanos , Inflamação , Modelos Biológicos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Osmose , Espécies Reativas de Oxigênio , Transdução de Sinais , Cloreto de Sódio/farmacologia , Superóxidos/metabolismoRESUMO
The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback. This work shows that exogenous H2O2 produced a rapid influx of Mn2+ and an increase of intracellular calcium. The H2O2 was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca2+ to H2O2-treated cells suspended in a free Ca2+/EGTA buffer resulted in a great increase in [Ca2+]i reflecting influx of Ca2+ across the cell membrane. The increase of intracellular calcium was inhibited by Ni2+, La3+, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H2O2 by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry.
Assuntos
Cálcio/metabolismo , Peróxido de Hidrogênio/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Soluções Tampão , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Citocalasina B/farmacologia , Fura-2 , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lantânio/farmacologia , Manganês/metabolismo , Manitol/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Níquel/farmacologia , Osmose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , SoluçõesRESUMO
In treatment of hemorrhagic shock, small-volume infusion of 7.5% NaCl gives immediate hemodynamic improvement, but in vitro experiments suggest it depresses the hemostatic system. Since previous reports showed that hyperosmotic glycine solutions preserved the platelet function better than hyperosmotic NaCl solutions, we investigated whether glycine changes the intracellular calcium ([Ca]i) signal. Platelets were incubated in hyperosmotic solutions containing sodium glycine or glycine base and stimulated with 0.1 IU/ml thrombin. [Ca]i increases were compared with an isosmotic control. Platelets incubated in zero calcium/EGTA were used to study separately the effect of glycine on calcium mobilization from intracellular stores and extracellular calcium entry. When NaCl was replaced by sodium glycine, the [Ca]i increase produced by thrombin was enhanced, because the calcium entry increased without changes in the mobilization of stored calcium. The addition of 50 mmol/l glycine base to the HEPES-buffered media increases the thrombin-induced entry of calcium or manganese. This study demonstrates that hyperosmotic glycine solutions increase the entry of calcium. This effect contrasts with the impairment of the thrombin-induced calcium signals by NaCl. The addition of low amounts of glycine in resuscitation solutions would be useful to reduce dysfunctional inflammatory responses without the risk of bleeding; however, concentrated solutions could cause toxic effects.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Glicinérgicos/farmacologia , Glicina/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Citosol/fisiologia , Humanos , Soluções Hipertônicas/farmacologia , Manganês/metabolismo , Trombina/fisiologiaRESUMO
2-aminoethoxydiphenyl borate (2APB) blocks agonist-induced Ca2+ mobilization from intracellular stores and Ca2+ entry. To compare the sensitivity profiles of these two components of calcium signaling, human platelets were exposed to different concentrations of 2APB. The drug interferes with the Ca2+ mobilization from intracellular stores induced by thrombin with an IC50 of 52+/-2 microM but it has a more potent inhibitory effect on the Ca2+ entry (IC50=16+/-1 microM). This value was similar to the IC50 found when the immediate inhibition of the calcium entry was studied after Ca2+ mobilization was completed (IC50=18+/-5 microM). The calcium influx induced by thapsigargin showed an IC50 of 17+/-1 microM for the immediate inhibition. The sensitivity to bivalent ion entry to 2APB was confirmed by Mn2+ entry inhibition. Although the specificity of 2APB with respect to the intracellular signaling system has not been fully established, our results confirm that the direct inhibition of calcium entry through the store-operated channels requires lower concentrations than the inhibition of mobilization, suggesting an independent and different site of action.