RESUMEN
Platelets are small (1-2µm in diameter), circulating anuclear cell fragments with important roles in hemostasis and thrombosis that provide an excellent platform for studying the role of membrane components in cellular communication. Platelets use several forms of communication including exocytosis of three distinct granule populations, formation of bioactive lipid mediators, and shape change (allowing for adhesion). This work explores the role of stereochemistry and concentration of exogenous phosphatidylserine (PS) on platelet exocytosis and adhesion. PS, most commonly found in the phosphatidyl-l-serine (l-PS) form, is exposed on the outer leaflet of the cell membrane after the platelet is activated. Knowledge about the impact of exogenous phosphatidylserine on cell-to-cell communication is limited (particularly concentration and stereochemistry effects). This study found that platelets incubated in l-PS or phosphatidyl-d-serine (d-PS) are enriched to the same extent with their respective incubated PS. All levels of l-PS enrichment also showed an increase in platelet cholesterol, but only the 50µM d-PS incubation showed an increase in cholesterol. The uptake of d-PS induced the secretion of granules and manufactured platelet activating factor (PAF) in otherwise unstimulated platelets. The uptake of l-PS had a greater impact on platelet stimulation by decreasing both the amount of δ-granule secretion and the amount of PAF that was manufactured.
Asunto(s)
Plaquetas/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Fosfatidilserinas/farmacología , Factor de Activación Plaquetaria/antagonistas & inhibidores , Activación Plaquetaria/efectos de los fármacos , Animales , Plaquetas/química , Plaquetas/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Factor de Activación Plaquetaria/biosíntesis , Cultivo Primario de Células , EstereoisomerismoRESUMEN
Exocytosis is a highly regulated intercellular communication process involving various membrane proteins, lipids, and cytoskeleton restructuring. These components help control granule fusion with the cell membrane, creating a pore through which granular contents are released into the extracellular environment. Platelets are an ideal model system for studying exocytosis due to their lack of a nucleus, resulting in decreased membrane regulation in response to cellular changes. In addition, platelets contain fewer granules than most other exocytosing cells, allowing straightforward measurement of individual granule release with carbon-fiber microelectrode amperometry. This technique monitors the concentration of serotonin, an electroactive molecule found in the dense-body granules of platelets, released as a function of time, with 50 µs time resolution, revealing biophysical characteristics of the fundamental exocytotic process. Variations in fusion pore formation and closure cause deviations from the classic current versus time spike profile and may influence diffusion of serotonin molecules from the site of granule fusion. Physiologically, the delivery of smaller packets of chemical messengers or the prolonged delivery of chemical messengers may represent how cells/organisms tune biological response. The goals of this work are twofold: 1) to categorize secretion features that deviate from the traditional mode of secretion and 2) to examine how changing the cholesterol composition of the platelet membrane results in changes in the pore formation process. Results herein indicate that the expected traditional mode of release is actually in the minority of granule content release events. In addition, results indicate that as the cholesterol content of the plasma membrane is increased, pore opening is less continuous.
Asunto(s)
Plaquetas/metabolismo , Colesterol/metabolismo , Animales , Plaquetas/citología , Membrana Celular/metabolismo , Difusión , Porosidad , Conejos , Serotonina/metabolismoRESUMEN
BACKGROUND: Asthma is a chronic inflammatory disease characterized by narrowed airways, bronchial hyper-responsiveness, mucus hyper-secretion, and airway remodeling. Mast cell (MC) infiltration into airway smooth muscle (ASM) is a defining feature of asthma, and ASM regulates the inflammatory response by secreting chemokines, including CXCL10 and CCL5. Single cell analysis offers a unique approach to study specific cellular signaling interactions within large and complex signaling networks such as the inflammatory microenvironment in asthma. METHODS: Carbon-fiber microelectrode amperometry was used to study the effects of ASM-secreted chemokines on mouse peritoneal MC degranulation. RESULTS: MC degranulation in response to CXCL10 and CCL5 was monitored at the single cell level. Relative to IgE-mediated degranulation, CXCL10- and CCL5-stimulated MCs released a decreased amount of serotonin per granule with fewer release events per cell. Decreased serotonin release per granule was correlated with increased spike half-width and rise-time values. CONCLUSIONS: MCs are directly activated by ASM-associated chemokines. CXCL10 and CCL5 induce less robust MC degranulation compared to IgE- and A23187-stimulation. The kinetics of MC degranulation are signaling pathway-dependent, suggesting a biophysical mechanism of regulated degranulation that incorporates control over granule trafficking, transport, and docking machinery. GENERAL SIGNIFICANCE: The biophysical mechanisms, including variations in number of exocytotic release events, serotonin released per granule, and the membrane kinetics of exocytosis that underlie MC degranulation in response to CXCL10 and CCL5 were characterized at the single cell level. These findings clarify the function of ASM-derived chemokines as instigators of MC degranulation relative to classical mechanisms of MC stimulation.
Asunto(s)
Bronquios/inmunología , Degranulación de la Célula , Quimiocinas/fisiología , Mastocitos/fisiología , Músculo Liso/inmunología , Análisis de la Célula Individual/métodos , Animales , Carbono , Fibra de Carbono , Células Cultivadas , Quimiocina CCL5/fisiología , Quimiocina CXCL10/fisiología , Ratones , Ratones Endogámicos C57BL , Microelectrodos , Serotonina/metabolismoRESUMEN
Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.
Asunto(s)
Plaquetas/metabolismo , Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Agregación Plaquetaria , Animales , Plaquetas/ultraestructura , Carbono/química , Fibra de Carbono , Bovinos , Membrana Celular/química , Colesterol/química , Colesterol/metabolismo , Cromatografía Líquida de Alta Presión , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Exocitosis , Cinética , Lípidos de la Membrana/química , Proteínas de la Membrana/química , Ratones , Microelectrodos , Microscopía Electrónica de Transmisión , Conejos , Especificidad de la Especie , Espectrometría de Masas en TándemRESUMEN
Schistosomiasis is a parasitic flatworm disease that infects 200 million people worldwide. The drug praziquantel (PZQ) is the mainstay therapy but the target of this drug remains ambiguous. While PZQ paralyses and kills parasitic schistosomes, in free-living planarians PZQ caused an unusual axis duplication during regeneration to yield two-headed animals. Here, we show that PZQ activation of a neuronal Ca²âº channel modulates opposing dopaminergic and serotonergic pathways to regulate 'head' structure formation. Surprisingly, compounds with efficacy for either bioaminergic network in planarians also displayed antischistosomal activity, and reciprocally, agents first identified as antischistocidal compounds caused bipolar regeneration in the planarian bioassay. These divergent outcomes (death versus axis duplication) result from the same Ca²âº entry mechanism, and comprise unexpected Ca²âº phenologs with meaningful predictive value. Surprisingly, basic research into axis patterning mechanisms provides an unexpected route for discovering novel antischistosomal agents.
Asunto(s)
Tipificación del Cuerpo/efectos de los fármacos , Praziquantel/farmacología , Schistosoma/efectos de los fármacos , Esquistosomicidas/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Planarias/efectos de los fármacos , Interferencia de ARN , EsquistosomiasisRESUMEN
Asthma is a chronic respiratory disease initiated by a variety of factors, including allergens. During an asthma attack, the secretion of C-X-C-motif chemokine 10 (CXCL10) and chemokine ligand 5 (CCL5) causes the migration of immune cells, including platelets, into the lungs and airway. Platelets, which contain three classes of chemical messenger-filled granules, can secrete vasodilators (adenosine diphosphate and adenosine triphosphate), serotonin (a vasoconstrictor and a vasodilator, depending on the biological system), platelet-activating factor, N-formylmethionyl-leucyl-phenylalanine ((fMLP), a bacterial tripeptide that stimulates chemotaxis), and chemokines (CCL5, platelet factor 4 (PF4), and C-X-C-motif chemokine 12 (CXCL12)), amplifying the asthma response. The goal of this work was threefold: (1) to understand if and how the antibody immunoglobulin E (IgE), responsible for allergic reactions, affects platelet response to the common platelet activator thrombin; (2) to understand how allergen stimulation compares to thrombin stimulation; and (3) to monitor platelet response to fMLP and the chemokines CXCL10 and CCL5. Herein, high-pressure liquid chromatography with electrochemical detection and/or carbon-fiber microelectrode amperometry measured granular secretion events from platelets with and without IgE in the presence of the allergen 2,4,6-trinitrophenyl-conjugated ovalbumin (TNP-Ova), thrombin, CXCL10, or CCL5. Platelet adhesion and chemotaxis were measured using a microfluidic platform in the presence of CXCL10, CCL5, or TNP-OVA. Results indicate that IgE binding promotes δ-granule secretion in response to platelet stimulation by thrombin in bulk. Single-cell results on platelets with exogenous IgE exposure showed significant changes in the post-membrane-granule fusion behavior during chemical messenger delivery events after thrombin stimulation. In addition, TNP-Ova allergen stimulation of IgE-exposed platelets secreted serotonin to the same extent as thrombin platelet stimulation. Enhanced adhesion to endothelial cells was demonstrated by TNP-Ova stimulation. Finally, only after incubation with IgE did platelets secrete chemical messengers in response to stimulation with fMLP, CXCL10, and CCL5.
RESUMEN
Opioids are typically used for the treatment of pain related to disease or surgery. In the body, they enter the bloodstream and interact with a variety of immune and neurological cells that express the µ-, δ-, and κ-opioid receptors. One blood-borne cell-like body that is not well understood in the context of opioid interactions is the platelet. The platelet is a highly sensitive anucleate cell-like fragment responsible for maintaining hemostasis through shape change and the secretion of chemical messengers. This research characterizes platelet opioid receptors, how specific receptor agonists impact platelet exocytosis, and the role of the κ-and µ-receptors in platelet function. Platelets were found to express all three opioid receptors, but upon stimulation with their respective agonist no activation was detected. Furthermore, exposure to the opioid agonists did not impact traditional platelet secretion stimulated by thrombin, a natural platelet activator. In addition, data collected from knockout mice suggest that the opioid agonists may be interacting nonspecifically with platelets. Dark-field images revealed differences in activated platelet shape between the κ- and µ-knockout platelets and the control platelets. Finally, κ-knockout platelets showed variations in their ability to adhere and aggregate compared to control platelets. Overall, these data show that platelet function is not likely to be heavily affected by blood-borne opioids.
RESUMEN
BACKGROUND: Current thermal ablation methods for atrial fibrillation, including radiofrequency and cryoablation, have a suboptimal success rate. To avoid pulmonary vein (PV) stenosis, ablation is performed outside of the PV, despite the importance of triggers inside the vein. We previously reported on the acute effects of a novel direct current electroporation approach with a balloon catheter to create lesions inside the PVs in addition to the antrum. In this study, we aimed to determine whether the effects created by this nonthermal ablation method were associated with irreversible lesions and whether PV stenosis or other adverse effects occurred after a survival period. METHODS AND RESULTS: Initial and survival studies were performed in 5 canines. At the initial study, the balloon catheter was inflated to contact the antrum and interior of the PV. Direct current energy was delivered between 2 electrodes on the catheter in ECG-gated 100 µs pulses. A total of 10 PVs were treated demonstrating significant acute local electrogram diminution (mean amplitude decrease of 61.2±19.8%). After the survival period (mean 27 days), computed tomography imaging showed no PV stenosis. On histologic evaluation, transmural, although not circumferential, lesions were seen in each treated vein. No PV stenosis or esophageal injury was present. CONCLUSIONS: Irreversible, transmural lesions can be created inside the PV without evidence of stenosis after a 27-day survival period using this balloon-based direct current ablation approach. These early data show promise for an ablation approach that could directly treat PV triggers in addition to traditional PV antrum ablation.
Asunto(s)
Fibrilación Atrial/terapia , Electroporación/métodos , Venas Pulmonares/diagnóstico por imagen , Animales , Fibrilación Atrial/fisiopatología , Modelos Animales de Enfermedad , Perros , Electrocardiografía , Estudios de Factibilidad , Estudios de Seguimiento , Resultado del TratamientoRESUMEN
As tissue-resident immune cells, mast cells are frequently found in close proximity to afferent neurons and are subjected to immunoactive mediators secreted by these neurons, including substance P (SP) and calcitonin gene-related peptide (CGRP). Neurogenic inflammation is thought to play an important role in the pathophysiology of many diseases. Unraveling the cellular mechanisms at the interface between the immune response and the peripheral nervous system is important for understanding how these diseases arise and progress. In this work, mast cell degranulation following direct exposure to CGRP and SP was studied both at the bulk and single-cell levels to characterize the mouse peritoneal mast cell response to neuropeptides and compare this response to well-studied mast cell activation pathways. Results show that mast cells secrete fewer chemical messenger-filled granules with increased IgE preincubation concentrations. The biophysical characteristics of mast cell degranulation in response to SP and CGRP is in many ways similar to calcium ionophore-induced mast cell degranulation; however, neuropeptide-stimulated mast cells secrete reduced chemical messenger content per secretion event, resulting in an overall relative decrease in secreted chemical messengers.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Inmunoglobulina E/administración & dosificación , Mastocitos/efectos de los fármacos , Neuropéptidos/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , MicroelectrodosRESUMEN
Mast cells play a significant role in both the innate and adaptive immune response; however, the tissue-bound nature of mast cells presents an experimental roadblock to performing physiologically relevant mast cell experiments. In this work, a heterogeneous cell culture containing primary culture murine peritoneal mast cells (MPMCs) was studied to characterize the time-dependence of mast cell response to allergen stimulation and the time- and concentration-dependence of the ability of the heterogeneous MPMC culture to uptake and degranulate exogenous serotonin using high performance liquid chromatography (HPLC) coupled to an electrochemical detector. Additionally, because mast cells play a central role in asthma, MPMCs were exposed to CXCL10 and CCL5, two important asthma-related inflammatory cytokines that have recently been shown to induce mast cell degranulation. MPMC response to both allergen exposure and cytokine exposure was evaluated for 5-HT secretion and bioactive lipid formation using ultraperformance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer (UPLC-MS/MS). In this work, MPMC response was shown to be highly regulated and responsive to subtle alterations in a complex environment through time- and concentration-dependent degranulation and bioactive lipid formation. These results highlight the importance of selecting an appropriate mast cell model when studying mast cell involvement in allergic response and inflammation.