RESUMEN
Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl(-)) and cation currents and is required for Ca(2+)-dependent PS. It is defective in blood cells from patients with Scott syndrome, a rare bleeding disorder. We examined if Cl(-) currents and PS are related, whether both processes are Ca(2+) dependent, and whether Ca(2+)-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca(2+) increase by ionomycin activated Ano6 Cl(-) currents and PS in normal lymphocytes, but not in B-lymphocytes from two different patients with Scott syndrome. Fas ligand (FasL) did not increase intracellular Ca(2+), but activated Cl(-) currents in normal but not in Scott lymphocytes. Whole-cell currents were inhibited by Cl(-) channel blockers and by siRNA knockdown of Ano6. In contrast, intrinsic mitochondrial apoptosis by ABT-737 did not induce Cl(-) currents in lymphocytes. PS was not inhibited by blockers of Ano6 or removal of Cl(-) ions. Remarkably, Ca(2+)-independent scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis was unchanged in Scott cells. We conclude that: (i) Ano6 Cl(-) currents are activated by increase in cytosolic Ca(2+), or Ca(2+) independent by stimulation of Fas receptors; (ii) Ca(2+)-dependent PS induced by Ano6 does not require Cl(-) currents; (iii) Ca(2+)-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca(2+)-dependent PS, but not by increasing intracellular Ca(2+).
Asunto(s)
Calcio/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Anoctaminas , Apoptosis/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/fisiología , Compuestos de Bifenilo/farmacología , Trastornos de la Coagulación Sanguínea/fisiopatología , Ionóforos de Calcio/farmacología , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/metabolismo , Proteína Ligando Fas/farmacología , Células HEK293 , Humanos , Transporte Iónico/efectos de los fármacos , Ionomicina/farmacología , Células Jurkat , Nitrofenoles/farmacología , Técnicas de Placa-Clamp , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Proteínas de Transferencia de Fosfolípidos/genética , Piperazinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Platelets in a thrombus interact with (anti)coagulation factors and support blood coagulation. In the concept of cell-based control of coagulation, three different roles of platelets can be distinguished: control of thrombin generation, support of fibrin formation, and regulation of fibrin clot retraction. Here, we postulate that different populations of platelets with distinct surface properties are involved in these coagulant functions. Platelets with elevated Ca(2+) and exposed phosphatidylserine control thrombin and fibrin generation, while platelets with activated α(IIb) ß(3) regulate clot retraction. We review how coagulation factor binding depends on the platelet activation state. Furthermore, we discuss the ligands, platelet receptors and downstream intracellular signaling pathways implicated in these coagulant functions. These insights lead to an adapted model of platelet-based coagulation.
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Activación Plaquetaria , Trombosis/sangre , Animales , Plaquetas/clasificación , Calcio/sangre , Retracción del Coagulo , Fibrina/metabolismo , Humanos , Fosfatidilserinas/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal , Trombina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) has been proposed to relax vascular smooth muscle cells (VSMC) via cAMP and can promote dissociation of endothelin-1 (ET-1) from ET(A) receptors. The latter is not mimicked by other stimuli of adenylate cyclases. Therefore, we evaluated the involvement of G-protein ßγ subunits (Gßγ) in the arterial effects of CGRP receptor stimulation. EXPERIMENTAL APPROACH: To test the hypothesis that instead of α subunits of G-proteins (Gαs), Gßγ mediates the effects of CGRP receptor activation, we used (i) rat isolated mesenteric resistance arteries (MRA), (ii) pharmacological modulators of cyclic nucleotides; and (iii) low molecular weight inhibitors of the functions of Gßγ, gallein and M119. To validate these tools with respect to CGRP receptor function, we performed organ bath studies with rat isolated MRA, radioligand binding on membranes from CHO cells expressing human CGRP receptors and cAMP production assays in rat cultured VSMC. KEY RESULTS: In isolated arteries contracted with K(+) or ET-1, IBMX (PDE inhibitor) increased sodium nitroprusside (SNP)- and isoprenaline (ISO)- but not CGRP-induced relaxations. While fluorescein (negative control) was without effects, gallein increased binding of [(125) I]-CGRP in the absence and presence of GTPγS. Gallein also increased CGRP-induced cAMP production in VSMC. Despite these stimulating effects, gallein and M119 selectively inhibited the relaxing and anti-endothelinergic effects of CGRP in isolated arteries while not altering contractile responses to K(+) or ET-1 or relaxing responses to ISO or SNP. CONCLUSION AND IMPLICATIONS: Activated CGRP receptors induce cyclic nucleotide-independent relaxation of VSMC and terminate arterial effects of ET-1 via Gßγ.