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1.
J Immunol ; 191(12): 6273-80, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24227782

RESUMO

Adenosine, a purine nucleoside, is present at high concentrations in tumors, where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine's immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors, as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist), and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/protein kinase A isoform (PKAI) signaling pathway, as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI, thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.


Assuntos
Adenosina/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenosina/análogos & derivados , Cálcio/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Proteína Quinase Tipo I Dependente de AMP Cíclico/antagonistas & inibidores , Proteína Quinase Tipo I Dependente de AMP Cíclico/fisiologia , Feminino , Humanos , Vigilância Imunológica/fisiologia , Molécula 1 de Adesão Intercelular , Interleucina-2/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Transporte de Íons/efeitos dos fármacos , Canal de Potássio Kv1.3/fisiologia , Ativação Linfocitária , Masculino , Técnicas de Patch-Clamp , Fenetilaminas/farmacologia , Proteínas Serina-Treonina Quinases , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptor A2A de Adenosina/fisiologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Canais de Cátion TRPM/fisiologia , Triazóis/farmacologia
2.
Am J Physiol Cell Physiol ; 302(10): C1504-12, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22378744

RESUMO

The cAMP/PKA signaling system constitutes an inhibitory pathway in T cells and, although its biochemistry has been thoroughly investigated, its possible effects on ion channels are still not fully understood. K(V)1.3 channels play an important role in T-cell activation, and their inhibition suppresses T-cell function. It has been reported that PKA modulates K(V)1.3 activity. Two PKA isoforms are expressed in human T cells: PKAI and PKAII. PKAI has been shown to inhibit T-cell activation via suppression of the tyrosine kinase Lck. The aim of this study was to determine the PKA isoform modulating K(V)1.3 and the signaling pathway underneath. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a nonselective activator of PKA, inhibited K(V)1.3 currents both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI(6-22). Selective knockdown of PKAI, but not PKAII, with siRNAs abolished the response to 8-BrcAMP. Additional studies were performed to determine the signaling pathway mediating PKAI effect on K(V)1.3. Overexpression of a constitutively active mutant of Lck reduced the response of K(V)1.3 to 8-Br-cAMP. Moreover, knockdown of the scaffolding protein disc large 1 (Dlg1), which binds K(V)1.3 to Lck, abolished PKA modulation of K(V)1.3 channels. Immunohistochemistry studies showed that PKAI, but not PKAII, colocalizes with K(V)1.3 and Dlg1 indicating a close proximity between these proteins. These results indicate that PKAI selectively regulates K(V)1.3 channels in human T lymphocytes. This effect is mediated by Lck and Dlg1. We thus propose that the K(V)1.3/Dlg1/Lck complex is part of the membrane pathway that cAMP utilizes to regulate T-cell function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteína Quinase Tipo I Dependente de AMP Cíclico/fisiologia , Canal de Potássio Kv1.3/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Proteínas de Membrana/fisiologia , Linfócitos T/enzimologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Células Cultivadas , Proteína 1 Homóloga a Discs-Large , Humanos , Imunossupressores/farmacologia , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
3.
J Immunol ; 183(10): 6296-302, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19841189

RESUMO

The immunological synapse (IS), a highly organized structure that forms at the point of contact between a T cell and an APC, is essential for the proper development of signaling events, including the Ca(2+) response. Kv1.3 channels control Ca(2+) homeostasis in human T cells and move into the IS upon Ag presentation. However, the process involved in channel accumulation in the IS and the functional implications of this localization are not yet known. Here we define the movement of Kv1.3 into the IS and study whether Kv1.3 localization into the IS influences Ca(2+) signaling in Jurkat T cells. Crosslinking of the channel protein with an extracellular Ab limits Kv1.3 mobility and accumulation at the IS. Moreover, Kv1.3 recruitment to the IS does not involve the transport of newly synthesized channels and it does not occur through recycling of membrane channels. Kv1.3 localization in the IS modulates the Ca(2+) response. Blockade of Kv1.3 movement into the IS by crosslinking significantly increases the amplitude of the Ca(2+) response triggered by anti-CD3/anti-CD28-coated beads, which induce the formation of the IS. On the contrary, the Ca(2+) response induced by TCR stimulation without the formation of the IS with soluble anti-CD3/anti-CD28 Abs is unaltered. The results presented herein indicate that, upon Ag presentation, membrane-incorporated Kv1.3 channels move along the plasma membrane to localize in the IS. This localization is important to control the amplitude of the Ca(2+) response, and disruption of this process can account for alterations of downstream Ca(2+)-dependent signaling events.


Assuntos
Cálcio/metabolismo , Sinapses Imunológicas/imunologia , Canal de Potássio Kv1.3/metabolismo , Linfócitos T/imunologia , Anticorpos/farmacologia , Antígenos/imunologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Cálcio/imunologia , Humanos , Sinapses Imunológicas/metabolismo , Células Jurkat , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo
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