RESUMO
BACKGROUND: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store-operated Ca(2+) entry (SOCE). OBJECTIVES: To assess hTRPC phosphotyrosine content upon platelet stimulation. METHODS: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. RESULTS: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of alpha-actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr-evoked alpha-actinin tyrosine phosphorylation was increased by inhibiting the alpha-actinin phosphatase, SHP-1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca(2+) sensor STIM1. CONCLUSIONS: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.
Assuntos
Actinina/metabolismo , Plaquetas/metabolismo , Sinalização do Cálcio , Ativação Plaquetária , Canais de Cátion TRPC/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Western Blotting , Sinalização do Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Imunoprecipitação/métodos , Técnicas In Vitro , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinolinas/farmacologia , Molécula 1 de Interação Estromal , Trombina/metabolismo , Fatores de Tempo , Tirosina/metabolismoRESUMO
BACKGROUND AND PURPOSE: Recently, we demonstrated that a pericellular Ca(2+) recycling system potentiates agonist-evoked Ca(2+) signalling and granule secretion in human platelets and hypothesized a role for the membrane complex (MC) in orchestrating the accumulation of Ca(2+) in the pericellular region. Previous work has demonstrated that treatment with high concentrations of nicergoline may disrupt the MC through an ability to trigger a re-organization of the dense tubular system. Experiments were therefore performed to assess whether nicergoline-induced changes in platelet ultrastructure affects thrombin-evoked Ca(2+) fluxes and dense granule secretion. EXPERIMENTAL APPROACH: Thrombin-evoked Ca(2+) fluxes were monitored in Fura-2- or Fluo-5N-loaded human platelets, or using platelet suspensions containing Fluo-4 or Rhod-5N K(+) salts. Fluorescence microscopy was utilized to monitor microtubule structure and intracellular Ca(2+) store distribution in TubulinTracker- and Fluo-5N-loaded platelets respectively. Dense granule secretion was monitored using luciferin-luciferase. KEY RESULTS: Nicergoline treatment inhibited thrombin-evoked Ca(2+) signalling and induced alterations in the microtubule structure and the distribution of intracellular Ca(2+) stores in platelets. Nicergoline altered the generation and spreading of thrombin-induced pericellular Ca(2+) signals and almost completely prevented dense granule secretion. Stabilization of microtubules using taxol reversed most effects of nicergoline on platelet Ca(2+) signalling and partially reversed its effects on dense granule secretion. CONCLUSIONS AND IMPLICATIONS: Nicergoline-induced alterations to platelet ultrastructure disrupt platelet Ca(2+) signalling in a manner that would be predicted if the MC had been disrupted. These data suggest that nicergoline may be a useful prototype for the discovery of novel MC-disrupting anti-thrombotics.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Sinalização do Cálcio/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Nicergolina/farmacologia , Plaquetas/metabolismo , Cálcio/metabolismo , Humanos , Microscopia de Fluorescência , Microtúbulos/ultraestrutura , Nicergolina/antagonistas & inibidores , Paclitaxel/farmacologia , Vesículas Secretórias/efeitos dos fármacos , Trombina/antagonistas & inibidores , Trombina/farmacologiaRESUMO
BACKGROUND: Most Ca(2+) signaling research in platelets has relied solely on monitoring the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). Changes in [Ca(2+)](cyt) constitute the net effect of Ca(2+) fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca(2+) sequestration into the stores and Ca(2+) removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca(2+) signaling difficult and subject to error. OBJECTIVES: To validate the use of the low-affinity Ca(2+) indicator Fluo-5N to monitor the concentration of Ca(2+) in the intracellular stores ([Ca(2+)](st)) of human platelets as a first step in developing assays for a systems-level analysis of platelet Ca(2+) signaling. METHODS: Fluo-5N-loaded and Fura-2-loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca(2+)](cyt) and [Ca(2+)](st). RESULTS: Fluo-5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca(2+) stores and to physiologic agonists. Ca(2+) reuptake inhibitors and blockers of Ca(2+) release channels had the expected effects on Fura-2 and Fluo-5N fluorescence. Agonist-evoked Ca(2+) release was reversed by Ca(2+) addition to the medium, and required intact Ca(2+) reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca(2+) -ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non-SERCA Ca(2+) reuptake mechanism. Evidence for a role for Ca(2+) -induced Ca(2+) release in agonist-evoked responses was obtained. CONCLUSIONS: Our data provide a validation of the use of Fluo-5N as a method for monitoring changes in [Ca(2+)](st) in human platelets.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/sangue , Sinalização do Cálcio/efeitos dos fármacos , Corantes Fluorescentes , Fura-2 , Humanos , Hidroquinonas/farmacologia , Técnicas In Vitro , Receptores de Inositol 1,4,5-Trifosfato/sangue , Líquido Intracelular/metabolismo , Ionomicina/farmacologia , Microscopia Confocal , NADP/análogos & derivados , NADP/sangue , Nigericina/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Tapsigargina/farmacologia , Trombina/farmacologiaRESUMO
SUMMARY BACKGROUND: Platelets are reported to possess a protein kinase C (PKC)-dependent non-capacitative Ca(2+)entry (NCCE) pathway. The phorbol ester, phorbol, 12-myristate, 13-acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store-operated Ca(2+)entry in human platelets for Na(+)/Ca(2+) exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA-evoked Ca(2+)entry. OBJECTIVES: To investigate the roles of NCXs and dense granule secretion in PMA-evoked Ca(2+)signaling in human platelets. METHODS: Fura-2- or sodium-binding benzofuran isophthalate (SBFI)-loaded platelets were used to monitor cytosolic Ca(2+)or Na(+) concentrations. Dense granule secretion was monitored as ATP release using luciferin-luciferase. RESULTS: The NCX inhibitors KB-R7943 or SN-6, and removal of extracellular Na(+), significantly reduced PMA-evoked Ca(2+)entry. PMA-evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro-31-8220 and significantly slowed by KB-R7943. The P(2X1) antagonists Ro-0437626 or MRS-2159, or desensitization of P(2X1) receptors by prior treatment with alpha,beta-Methylene-ATP or omitting apyrase from the medium, reduced PMA-evoked Ca(2+)entry. Ro-0437626 or chelation of extracellular Ca(2+) slowed but did not abolish PMA-evoked ATP release, indicating that PMA-evoked dense granule secretion does not require P(2X1) receptor activation but is accelerated by P(2X1)-mediated Ca(2+)entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. CONCLUSION: PMA-evoked Ca(2+)entry results from an NCX3-dependent dense granule secretion and subsequent P(2X1) receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.
Assuntos
Comunicação Autócrina/fisiologia , Plaquetas/metabolismo , Sinalização do Cálcio , Receptores Purinérgicos P2X1/metabolismo , Trifosfato de Adenosina , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Ésteres de Forbol/farmacologia , Vesículas Secretórias , Trocador de Sódio e Cálcio/fisiologiaRESUMO
BACKGROUND: Platelets play a role in a number of inflammatory conditions including atherosclerosis; however, the mechanisms of platelet activation under these conditions are unclear. OBJECTIVES: To investigate the presence of the vanilloid receptor, TRPV1, which is stimulated by noxious stimuli and by inflammatory mediators, in human platelets. METHODS: Platelets loaded with fura-2 or sodium-binding benzofuran isophalate acetoxymethyl ester (SBFI) were used to monitor cytosolic calcium or sodium concentrations. 5-HT secretion was determined by fluorescence assay after conjugation with o-phthaldialdehyde. ATP secretion was determined using luciferin-luciferase. RESULTS: TRPV1 was identified by Western blotting using a specific anti-hTRPV1 antibody. The TRPV1 agonist, capsaicin, evoked both Ca(2+) influx and Ca(2+) release from intracellular stores, responses that were blocked in a dose-dependent manner by the TRPV1 antagonists, 5'-Iodo-resiniferatoxin (5'-Iodo-RTX) and AMG 9810. Capsaicin also increased platelet cytosolic [Na(+)]. Capsaicin-evoked Ca(2+) release was abolished in the absence of extracellular Na(+) or by the 5-HT(2A) receptor antagonist, ketanserin. Capsaicin evoked 5-HT release from platelets, a response abolished in the absence of extracellular Na(+) or by 5'-Iodo-RTX. Thus capsaicin-evoked Ca(2+) release appeared to be mediated by Na(+)-dependent 5-HT release. TRPV1-dependent 5-HT release also contributed to ADP- and thrombin-evoked Ca(2+) entry and release. 5'-Iodo-RTX reduced ADP- and thrombin-evoked Ca(2+) signals, effects not additive with those of ketanserin, and 5'-Iodo-RTX inhibited agonist-evoked 5-HT and ATP release. CONCLUSION: These results indicate that TRPV1 is present and functionally important in human platelets. The presence of this receptor may provide a link between inflammatory mediators and platelet activation in conditions such as atherosclerosis.