scholarly journals Ca2+ release and Ca2+ entry induced by rapid cytosolic alkalinization in Jurkat T-lymphocytes

1994 ◽  
Vol 301 (1) ◽  
pp. 83-88 ◽  
Author(s):  
A H Guse ◽  
E Roth ◽  
F Emmrich
Keyword(s):  
T Cells ◽  
Intracellular Ph ◽  
Cell Receptor ◽  
K Channel ◽  
Cytosolic Ph ◽  
Intact Cells ◽  
Inositol Polyphosphates ◽  
Simultaneous Formation ◽  
Permeabilized Cells ◽  
Release System

4-Aminopyridine (4-AP), a compound usually known as a K(+)-channel inhibitor, induced rapid cytosolic alkalinization from pH 7.15 to pH 7.4, and subsequently Ca2+ mobilization in the T-lymphocyte cell line Jurkat. Other weak bases, such as NH4Cl or triethanolamine, induced a smaller and/or slower increase in cytosolic pH, resulting in a lower or no detectable Ca2+ signal. In the presence of extracellular Ca2+, 4-AP mediated a rapid and sustained increase in the free cytosolic Ca2+ concentration similar to that obtained by T-cell receptor-mediated stimulation. In the absence of extracellular Ca2+, 4-AP transiently released Ca2+ from an intracellular store that is most likely identical with the agonist- and Ins(1,4,5)P3-sensitive Ca2+ pool of Jurkat T-cells. As possible mechanisms for Ca2+ release from this particular pool as induced by 4-AP we examined (i) formation of Ins(1,4,5)P3 and (ii) sensitization of the Ins(1,4,5)P3-receptor/Ca(2+)release system by increasing intracellular pH. Although 4-AP did not induce formation of inositol polyphosphates, as demonstrated by h.p.l.c. analysis, in permeabilized cells the dose-response curve for Ins(1,4,5)P3 was shifted to the left by changing the intracellular pH from 7.2 to 7.4. This indicated that sensitization of the Ins(1,4,5)P3-receptor/Ca(2+)-release system was responsible for the effects of 4-AP seen in intact cells. In conclusion, 4-AP appears a novel tool for depletion of the agonist-sensitive Ca2+ pool of T-cells without simultaneous formation of Ins(1,4,5)P3, thereby inducing capacitative Ca2+ entry in these cells.

scholarly journals Agonist-induced inhibition of phosphatidylserine synthesis is secondary to the emptying of intracellular Ca2+ stores in Jurkat T-cells

1992 ◽  
Vol 288 (3) ◽  
pp. 785-789 ◽  
Author(s):  
C Pelassy ◽  
J P Breittmayer ◽  
C Aussel
Keyword(s):  
T Cells ◽  
Exchange System ◽  
Jurkat T Cells ◽  
Experimental Conditions ◽  
Intact Cells ◽  
Base Exchange ◽  
Permeabilized Cells ◽  
Atpase Inhibitors ◽  
Storage Organelle ◽  
Intracellular Compartments

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging

1998 ◽  
Vol 275 (4) ◽  
pp. C1158-C1166 ◽  
Author(s):  
Bing Siang Gan ◽  
Eric Krump ◽  
Lamara D. Shrode ◽  
Sergio Grinstein
Keyword(s):  
Intracellular Ph ◽  
Purinergic Receptors ◽  
Dynamic Range ◽  
Spectroscopic Properties ◽  
Hypotonic Solution ◽  
Cytosolic Ph ◽  
Intact Cells ◽  
Hypotonic Stress ◽  
Derivatives Of ◽  
Functional Responsiveness

Although used extensively for the measurement of intracellular pH, derivatives of fluorescein such as 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have suboptimal sensitivity and can generate toxic photoproducts. These limitations can be overcome using the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine), which has improved spectroscopic properties. However, the use of pyranine has been limited by the difficulties encountered in delivering this highly hydrophilic dye to the cell interior. We describe a strategy for intracellular delivery of pyranine based on the reversible activation of purinergic P2x7 receptors, which allow permeation of the dye into otherwise intact cells. When loaded into J774 or RAW cells by this method, pyranine is not only more sensitive than BCECF (the dynamic range is ∼7-fold greater), but is retained better and is less toxic. Pyranine was distributed throughout the cytosol but was not detectable in endomembrane compartments. Repeated illumination resulted in blebbing and loss of functional responsiveness of cells loaded with BCECF, whereas comparably irradiated cells loaded with pyranine remained healthy and responsive. Pyranine can also be loaded into cells not expressing P2x7 receptors by brief exposure to a hypotonic solution. The properties of cells labeled by this method are similar to those loaded via purinergic receptors and compare favorably with those of BCECF-loaded cells. Pyranine thus provides a useful alternative to fluorescein derivatives for the measurement of intracellular pH, particularly when using the high excitation intensities required for microscopic digital imaging.

scholarly journals The Ca2+-activated K+ channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

2007 ◽  
Vol 292 (4) ◽  
pp. C1431-C1439 ◽  
Author(s):  
Stella A. Nicolaou ◽  
Lisa Neumeier ◽  
YouQing Peng ◽  
Daniel C. Devor ◽  
Laura Conforti
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Immunological Synapse ◽  
Cell Receptor ◽  
K Channel ◽  
Activated T Cells ◽  
Barr Virus

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca2+ influx. KCa3.1 channels modulate Ca2+ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3ε, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3ε to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15–30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca2+ influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.

scholarly journals Increased intracellular cyclic AMP inhibits inositol phospholipid hydrolysis induced by perturbation of the T cell receptor/CD3 complex but not by G-protein stimulation. Association with protein kinase A-mediated phosphorylation of phospholipase C-γ 1

1992 ◽  
Vol 284 (1) ◽  
pp. 189-199 ◽  
Author(s):  
M A Alava ◽  
K E DeBell ◽  
A Conti ◽  
T Hoffman ◽  
E Bonvini
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
G Protein ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Inhibitory Effect ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Inositol Phospholipid

Modulation of inositol phospholipid (InsPL) hydrolysis in response to increasing intracellular concentrations of cyclic AMP (cAMP) was studied in a murine T helper type II (Th2) lymphocyte clone, 8-5-5. Intact 8-5-5 cells produced maximal amounts of cAMP in response to prostaglandin E2 (PGE2), cholera toxin (CTx) or 7 beta-deacetyl-7 beta-(gamma-N-methylpiperazino)butyryl forskolin (dmpb-forskolin). cAMP generation reached a plateau after 5 min of treatment with dmpb-forskolin (300 microM) or PGE2 (1 microM), but required 60 min of treatment with CTx (1 microgram/ml). Preincubation of 8-5-5 cells with 1 microM-PGE2 or 300 microM-dmpb-forskolin (10 min at 37 degrees C) or with 1 microgram of CTx/ml (60 min at 37 degrees C) completely inhibited InsPL hydrolysis induced by perturbation of the T cell receptor (TCR)/CD3 complex with the monoclonal antibody 145.2C11. Preincubation with the cAMP analogue 8-bromo-cyclic AMP (8-Br-cAMP) also inhibited InsPL hydrolysis. Tetanolysin-permeabilized 8-5-5 cells produced cAMP in response to PGE2, dmpb-forskolin and guanosine 5′-[gamma-thio]triphosphate (GTP[S]), a non-cell-permeating, non-hydrolysable analogue of GTP that directly activates G-proteins. No inhibition of TCR/CD3-induced InsPL hydrolysis was observed under these conditions. InsPL hydrolysis was also unaffected when permeabilized cells were incubated with up to 10 mM-8-Br-cAMP, suggesting that permeabilized cells lost (a) soluble effector molecule(s) involved in mediating the inhibitory effect observed in intact cells. Treatment of 8-5-5 cells with dmpb-forskolin or CTx prior to permeabilization resulted in inhibition of TCR/CD3-induced InsPL hydrolysis, but did not affect InsPL hydrolysis induced via G-protein stimulation with GTP[S]. Treatment of permeabilized 8-5-5 cells with purified cAMP-dependent protein kinase (PKA) resulted in inhibition of TCR/CD3- but not GTP[S]-induced InsPL hydrolysis. This effect was associated with phosphorylation of phospholipase (PLC)-gamma 1 in the absence of phosphorylation of components of the TCR/CD3 complex. These results suggest that PKA-mediated phosphorylation of PLC may regulate TCR/CD3-induced InsPL hydrolysis.

Immune checkpoint and checkpoint inhibitors in hepatocellular carcinoma

2018 ◽  
Vol 1 (1) ◽  
pp. 28-32
Author(s):  
Piyawat Komolmit
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Major Histocompatibility Complex ◽  
T Cell Receptor ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Cell Receptor ◽  
Immune Checkpoints ◽  
Histocompatibility Complex

การรักษามะเร็งด้วยแนวความคิดของการกระตุ้นให้ภูมิต้านทานของร่างกายไปทำลายเซลล์มะเร็งนั้น ปัจจุบันได้รับการพิสูจน์ชัดว่าวิธีการนี้สามารถหยุดยั้งการแพร่กระจายของเซลล์มะเร็ง โดยไม่ก่อให้เกิดภาวะแทรกซ้อนทางปฏิกิริยาภูมิต้านทานต่ออวัยวะส่วนอื่นที่รุนแรง สามารถนำมาใช้ทางคลินิกได้ ยุคของการรักษามะเร็งกำลังเปลี่ยนจากยุคของยาเคมีบำบัดเข้าสู่การรักษาด้วยภูมิต้านทาน หรือ immunotherapy ยากลุ่ม Immune checkpoint inhibitors โดยเฉพาะ PD-1 กับ CTLA-4 inhibitors จะเข้ามามีบทบาทในการรักษามะเร็งตับในระยะเวลาอันใกล้ จำเป็นแพทย์จะต้องมีความรู้ความเข้าใจในพื้นฐานของ immune checkpoints และยาที่ไปยับยั้งโมเลกุลเหล่านี้ Figure 1 เมื่อ T cells รับรู้แอนทิเจนผ่านทาง TCR/MHC จะมีปฏิกิริยาระหว่าง co-receptors หรือ immune checkpoints กับ ligands บน APCs หรือ เซลล์มะเร็ง ทั้งแบบกระตุ้น (co-stimulation) หรือยับยั้ง (co-inhibition) TCR = T cell receptor, MHC = major histocompatibility complex

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