RESUMO
In our previous investigations we have shown that platelets and their precursors express nicotinic α7 acetylcholine receptors (nAChRα7) that are involved in platelet function and in vitro differentiation of the megakaryoblastic cell line MEG-01. In this study, we were interested in the expression analysis of additional nAChR and the effects of nicotine in an ex vivo model using megakaryocytic cells differentiated from cord blood derived CD34(+) cells (CBMK) and an in vivo model using blood samples from smokers. CBMK were differentiated with thrombopoietin (TPO) for up to 17 days. Quantitative real-time PCR (QRT-PCR), Western blot analysis and flow cytometry were used to investigate nAChR expression (nAChRα7, nAChRα4, nAChRß2) and nicotine effects. In blood samples of 15 nonsmokers and 16 smokers platelet parameters (count, mean platelet volume--MPV and platelet distribution width--PDW) were determined as indicators for changes of in vivo megakaryopoiesis. Platelet function was determined by the use of whole blood aggregometry and flow cytometry. The functional role of nAChR was evaluated using specific antagonists in aggregometry. CHRNA7, CHRNA4 and CHRNB2 gene transcripts and the corresponding proteins could be identified in CBMK during all stages of differentiation. Platelets contain nAChRα7 and nAChRß2 but not nAChRα4. Nicotine had no effect on TPO-induced differentiation of CBMK. There was no significant difference in all platelet parameters of the smokers compared to the nonsmokers. In line with this, cholinergic gene transcripts as well as the encoded proteins were equally expressed in both the study groups. Despite our observation of nAChR expression in megakaryopoiesis and platelets, we were not able to detect effects of nicotine in our ex vivo and in vivo models. Thus, the functional role of the nAChR in these cells remains open.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Nicotina/farmacologia , Receptores Nicotínicos/sangue , Adulto , Plaquetas/citologia , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Masculino , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Células Progenitoras de Megacariócitos/metabolismo , Megacariócitos/citologia , Projetos Piloto , Ativação PlaquetáriaRESUMO
Since many years acetylsalicylic acid (ASA) is known for its antithrombotic, antiphlogistic and analgesic effects caused by irreversible acetylation of cyclooxygenase. ASA also inhibits capsaicin- and heat-induced responses in cultured dorsal root ganglia (DRG) neurons, suggesting TRPV1 (transient receptor potential channel of the vanilloid receptor family, subtype 1) to be an additional target of ASA. We now studied the effect of ASA on heterologously expressed rat TRPV1 using calcium microfluorimetry. Capsaicin dose-dependently increased intracellular calcium with an EC50 of 0.29 µM in rTRPV1 expressing HEK293 cells. During repetitive stimulation the second response to capsaicin was reduced (53.4 ± 8.3% compared to vehicle control; p<0.005; Student's unpaired t-test) by 1µM ASA, a concentration much below the one needed to inhibit cyclooxygenase (IC50 of 35 µM in thromboxane B2 production assay). In contrast, calcium transients induced by a single stimulus of 0.3 or 1 µM capsaicin were not significantly reduced by 0.3 or 1 µM ASA. These data suggest that ASA increases the tachyphylaxis of rTRPV1 channel activation. Mechanisms are unknown and may be direct by e.g. stabilization of the desensitized state or indirect via inhibition of intracellular signaling pathways e.g. of the mitogen-activated protein kinase family (MAPK/ERK).
Assuntos
Analgésicos/farmacologia , Aspirina/farmacologia , Capsaicina/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Canais de Cátion TRPV/metabolismo , Taquifilaxia , Acetilação , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/metabolismo , Ciclo-Oxigenase 1/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Ratos , Proteínas Recombinantes de Fusão/genética , Canais de Cátion TRPV/genética , Tromboxano B2/biossínteseRESUMO
BACKGROUND AND PURPOSE: Catechol containing compounds have anti-inflammatory properties, yet for catecholamines these properties are modest. Since we have previously demonstrated that the synthetic dopamine derivative N-octanoyl dopamine (NOD) has superior anti-inflammatory properties compared to dopamine, we tested NOD in more detail and sought to elucidate the molecular entities and underlying mechanism by which NOD down-regulates inflammation. EXPERIMENTAL APPROACH: Genome wide gene expression profiling of human umbilical vein endothelial cells (HUVECs) was performed after stimulation with TNF-α or in the combination with NOD. Confirmation of these differences, NFκB activation and the molecular entities that were required for the anti-inflammatory properties were assessed in subsequent experiments. KEY RESULTS: Down regulation of inflammatory genes by NOD occurred predominantly for κB regulated genes, however not all κB regulated genes were affected. These findings were explained by inhibition of RelA phosphorylation at Ser276. Leukocyte adherence to TNF-α stimulated HUVECs was inhibited by NOD and was reflected by a diminished expression of adhesion molecules on HUVECs. NOD induced HO-1 expression, but this was not required for inhibition of NFκB. The anti-inflammatory effect of NOD seems to involve the redox active catechol structure, although the redox active para-dihydroxy benzene containing compounds also displayed anti-inflammatory effects, provided that they were sufficiently hydrophobic. CONCLUSIONS AND IMPLICATIONS: The present study highlighted important mechanisms and molecular entities by which dihydroxy benzene compounds exert their potential anti-inflammatory action. Since NOD does not have hemodynamic properties, NOD seems to be a promising candidate drug for the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Dopamina/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Serina/metabolismo , Fator de Transcrição RelA/química , Fator de Transcrição RelA/metabolismo , Anti-Inflamatórios não Esteroides/química , Adesão Celular/efeitos dos fármacos , Dopamina/química , Dopamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Dopamine (DA) is a co-agonist for platelet activation; yet, donor DA treatment is associated with improved transplantation outcome in renal and heart recipients. Recently, N-octanoyl-dopamine (NOD) was developed which displays superior effects compared to DA in terms of graft protecting properties. Whereas DA is a known platelet co-agonist, the effect of NOD on platelet function is unknown. This is a hypothesis generating study with the aim to assess the effects and molecular mechanisms of NOD and NOD-like compounds on platelet function. The influence of DA, NOD, and NOD-like compounds on platelet responses to classical agonists (adenosine 5'-diphosphate (ADP), U46619) was investigated in six healthy donors by applying whole blood aggregometry (Multiplate®) and flow cytometry for Pac-1, CD62P, and CD63 expression. Changes in platelet cAMP concentrations were assessed by ELISA. While DA showed synergy in platelet activation by ADP and U46619, NOD caused significant inhibition of platelet function both in whole blood aggregometry and flow cytometry. The inhibitory effect of NOD was not mediated via cAMP levels. The nonredox-active NOD-analog N-octanoyl-tyramine had no effects on platelet function. Acetylated NOD conferred to NOD by intracellular esterases showed similar inhibitory effects as NOD. In contrast to DA, NOD is a potent inhibitor of platelet function most likely through intracellular redox-active processes. This adds to the overall protective effect of NOD on pre-transplantation injury and makes NOD an attractive candidate compound for donor or organ conditioning prior to transplantation.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Dopamina/análogos & derivados , Degranulação Celular/efeitos dos fármacos , AMP Cíclico/biossíntese , Dopamina/farmacologia , Humanos , Agregação Plaquetária/efeitos dos fármacos , Receptores de Fibrinogênio/agonistas , Receptores de Fibrinogênio/metabolismoRESUMO
Besides thrombopoietin several additional factors (i.e neurotransmitters and receptors) are known to be involved in the regulation of megakaryopoiesis at different stages. Recently, we identified functional α7 nicotinic acetylcholine receptors (nAChRα7) on platelets and megakaryocytic precursors. In platelets nAChRα7 form functional Ca(2+) channels and are involved in fibrinogen receptor activation and aggregation. Here, we investigated the impact of nAChRα7 on the differentiation of the human megakaryoblastic cell line MEG-01. In vitro differentiation of MEG-01 cells was induced by the phorbol ester TPA for 5 days in the absence or presence of nicotine or the nAChRα7-selective antagonist methyllycaconitine (MLA), and this was monitored by the expression of the megakaryocytic antigens CD41 and CD61. In the presence of the cholinergic drugs (nicotine or MLA) CD41 and CD61 expression was significantly reduced, both at RNA and protein level. We postulate that the nAChRα7 receptor is involved in megakaryopoietic signal transduction and gene regulation. This could affect the generation of platelets in vivo and contribute to the development of novel therapeutic drugs that regulate platelet formation.
Assuntos
Aconitina/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Trombopoese/efeitos dos fármacos , Aconitina/farmacologia , Western Blotting , Diferenciação Celular/genética , Linhagem Celular , Colinérgicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina beta3/biossíntese , Integrina beta3/genética , Células Progenitoras de Megacariócitos/citologia , Células Progenitoras de Megacariócitos/metabolismo , Nicotina/farmacologia , Ésteres de Forbol/farmacologia , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7RESUMO
OBJECTIVE: Nicotinic acetylcholine receptors, especially α7 (nAChRα7), form Ca(2+) channels and are expressed on a variety of neuronal and nonneuronal cells. Also, megakaryocytic cells have been shown to contain components of a nonneuronal cholinergic system, including acetylcholine and acetylcholine esterase. However, the corresponding nAChRs and their role in platelet function have not been demonstrated until now. Our previous platelet transcriptome data indicated the presence of nAChR gene transcripts. METHODS AND RESULTS: Here, we present evidence that human platelets and megakaryocytic precursor cells express nAChRα7 subunits, as revealed by mRNA and protein expression. The subunits form functional Ca(2+) channels, as demonstrated by Ca(2+) entry in platelets induced by the nAChRα7-selective agonist PNU-282987. PNU-282987 also enhanced fibrinogen receptor activation induced by classical platelet agonists (the thromboxane A(2) analog U46619 and ADP). Furthermore, agonist-induced platelet aggregation was significantly inhibited by the nAChRα7-selective antagonists α-bungarotoxin and methyllycaconitine. CONCLUSIONS: Ca(2+) influx via nAChRα7 channels represents a novel pathway for human platelets with significant impact on platelet function. Because platelets were suggested to contain acetylcholine, we conclude that on activation, stored acetylcholine is released, which activates nAChRα7 channels and thereby contributes to maintaining intracellular Ca(2+) levels and supporting platelet activation.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , Ativação Plaquetária , Receptores Nicotínicos/sangue , Aconitina/análogos & derivados , Aconitina/farmacologia , Benzamidas/farmacologia , Plaquetas/efeitos dos fármacos , Compostos Bicíclicos com Pontes/farmacologia , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Linhagem Celular , Humanos , Megacariócitos/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , RNA Mensageiro/sangue , Receptores de Fibrinogênio/sangue , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
BACKGROUND: Neuro-hormonal and hemostatic mechanisms are important in a wide range of psychological and cardiovascular diseases. The use of psychoactive drugs in mental illnesses is often involved with hematologic side effects including impaired platelet function. Subsequently, the risk for the development of cardiovascular diseases may be higher in these patients. Interestingly, platelets that play a key role in cardiovascular complications contain quite a number of neuronal receptors which are involved in psychotic disorders. It has been widely discussed whether psychoactive drugs used in the therapy of psychotic disorders have a direct effect on platelet function and whether the effects are transmitted through the corresponding receptors on the platelet surface. MATERIAL AND METHODS: In this study, we tested several psychoactive drugs regarding their impact on whole blood platelet aggregation. RESULTS: Antidopaminergics preferentially inhibited ADP-induced aggregation whereas anticholinergics mainly inhibited U46619-induced aggregation. Because platelets respond selectively to different psychoactive drugs we assume that corresponding receptors have a functional aspect on platelets and that receptor blockade affects platelet aggregation through different mechanisms. CONCLUSION: The knowledge about the effects of psychoactive drugs on platelet function may help to characterize neuronal receptors on platelets and may contribute to a better understanding of altered platelet function during therapy with psychoactive drugs.
RESUMO
Human blood platelets are anucleate cells that contain minute amounts of translational active mRNA. Investigation of the gene expression profile by microarray analysis has become an excellent tool for better understanding of normal and pathological platelet function. Its use, however, is often limited by the low yield of megakaryocytic-derived mRNA, the possible contamination with leukocytes during platelet preparation and the small platelet volume in clinical settings, especially in pediatric patients. In this chapter, we present a protocol for the isolation of leukocyte-depleted platelet samples in clinical settings and an optimized procedure for transcript profiling, using the Agilent oligo microarray technology. In particular, we discuss the special aspects of platelet purification when working with blood sample volumes not exceeding 3-5 ml, which is typical in pediatric patients and we furthermore provide detailed information for transcript profiling of extremely small amounts of platelet RNA.
Assuntos
Plaquetas/metabolismo , Perfilação da Expressão Gênica/métodos , Genoma Humano , Megacariócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/biossíntese , Coleta de Amostras Sanguíneas/métodos , HumanosRESUMO
Monoamines such as serotonin and epinephrine are known to be involved in platelet activation and aggregation. Dopamine is another monoamine identified in platelets, but published data about its effect on platelets and the receptors involved are controversial. In the present study, we investigated the dopamine agonism in platelets and the receptors involved in these pathways. Platelet-rich plasma (PRP) of healthy individuals was treated with agonists (ADP, epinephrine, dopamine) and various dopamine receptor and transporter antagonists such as SCH-23390, raclopride, clozapine, methylphenidate, and cocaine. Platelet activation was investigated by flow cytometry (CD62P and CD63 surface expression), optical aggregometry, and microaggregate adhesion to collagen IV in a flow chamber system. In our study, dopamine on its own had no effect on platelet activation in the different assays. However, when used in combination with ADP (10 muM), dopamine in a range of 1 to 100 muM significantly potentiated platelet microaggregate formation and adhesion to collagen under low shear flow conditions. Specific antagonists for D2-like receptors (L-741,626, raclopride, and clozapine) completely diminished the dopamine effect at selective concentrations, but not the effect of epinephrine. Neither the D1-like receptor antagonist SCH-23390 nor dopamine transporter antagonists (methylphenidate, cocaine) showed inhibitory effects on the dopamine agonism. Thus, dopamine is an ADP-dependent platelet agonist which acts via D2-like but not D1-like receptors or adrenergic receptors. Because many psychopharmacological drugs are directed to D2-like receptors, platelet dysfunction in patients being treated with such drugs may be linked to these mechanisms.