Characterization of the interference of T cell activation by reserpine

It has been suggested that reserpine blocks expression of delayed hypersensitivity (DH) reactions by depleting tissue mast cells of serotonin, thereby preventing a T cell-dependent release of mast cell serotonin necessary to localize and to amplify the DH response. However, reserpine blocks expression of DH in mast cell-deficient mice. Recently, we showed that the ability of reserpine to interfere with the expression of contact sensitivity was independent of an effect on mast cells, but reflected an effort of the drug on effector T cell function. In the present study we evaluated the mechanisms by which reserpine abrogates the expression of T cell functions. By using human peripheral blood mononuclear cells or enriched T cell populations we found that the drug inhibited, in a dose-dependent fashion, the proliferation of T cells after mitogen stimulation. Reserpine also interfered with the mitogen-induced IL-2 production by these cells, but the IL-2 receptor expression, as measured by immunofluorescence, was unaffected. Despite this, in the continuous presence of reserpine, exogenous IL-2 did not bypass reserpine inhibition of PHA-induced proliferation. By using the fluorescent indicator quin-2 we have demonstrated that preincubation with reserpine prevented the increase of cytosolic free calcium, which accompanies PHA-induced proliferative responses of human T lymphocytes. These results identify the sites of action of reserpine in human T lymphocytes and are sufficient to explain its ability to block cell-mediated immune responses in vitro and in vivo.     

Action of calcium channel and beta-adrenergic blocking agents in bilayer lipid membranes

The action of beta-adrenergic blockers (propranolol, exprenolol, metoprolol, sotalol, atenolol, timolol) and calcium-channel blockers (verapamil, diltiazem) on the electrical properties and fluidity of bilayer lipid membranes (BLM and liposomes) has been investigated. When antibiotic ionophore substances were used as a probe, the electrical measurements showed that many of the drugs inhibited the cation transport across the membrane facilitated by the mobile carrier valinomycin, while having no significant effect on the cation transport through channels formed by gramicidin. The ability of the drugs to decrease the carrier-dependent membrane conductance was correlated to their partition into the lipid bilayer and the magnitude of transmembrane potential induced by them. In the TEMPO ESR spectral measurements, a number of beta-adrenergic and calcium blockers showed the fluidizing effect on liposomes composed of different lipids. The drug concentration required for a detectable change in TEMPO spectra parameter (f) was rather high (0.01 M verapamil), and the variation of pH from 6.5 to 3.0 did not affect the fluidizing effect of the drugs.     

Mechanisms of T cell activation by the calcium ionophore ionomycin

We have investigated signaling mechanisms that may underlie the T cell mitogenic properties of the Ca2+ ionophore ionomycin. Ionomycin induces highly purified resting human T cells to proliferate in the presence of monocytes with accompanying IL-2R expression and IL-2 synthesis. Treatment of T cells with ionomycin triggers the hydrolysis of phosphoinositides, as evidenced by the accumulation of the hydrolytic by-products phosphatidic acid and inositol phosphates. Ionomycin also induces the activation of protein kinase C (PKC), as demonstrated by the auto-phosphorylation of PKC and the phosphorylation of the PKC target proteins CD4 and CD8. Ionomycin synergizes with PMA in enhancing the activation of PKC. It is concluded that, in addition to its putative activation of Ca2+/calmodulin-dependent signaling pathways, ionomycin induces the hydrolysis of phosphoinositides and the activation of PKC in human T cells. The synergy of ionomycin with phorbol esters in triggering T cell activation may relate, at least in part, to enhanced activation of PKC.     

Different actions of calcium channel blocking agents on resting membrane conductance in developing skeletal muscle

The effects of Co2+, Mn2+, and La3+ (2 mM) and verapamil (5 x 10(-6) M) on membrane conductance (Gm) and resting potential (Em) were studied in chick skeletal muscle fibres developing in culture. Cobalt and manganese had no effect on Gm at any time during myogenesis but verapamil caused a decrease in Gm in immature myotubes. This effect diminished with time and was absent by 3 days after myoblast fusion. Lanthanum caused an increase in Gm at all stages of development. All the agents studied caused a significant depolarization of Em. It is concluded that there is no resting calcium conductance in developing skeletal muscle but that there may be a resting sodium conductance which declines with maturation. Lanthanum may increase Gm by displacing membrane-bound calcium and destabilizing membrane structure. All the agents studied were thought to induce depolarization by an inhibitory action on (Na+ + K+)-ATPase.     

Curcumin suppresses T cell activation by blocking Ca2+ mobilization and nuclear factor of activated T cells (NFAT) activation

Curcumin is the active ingredient of the spice turmeric and has been shown to have a number of pharmacologic and therapeutic activities including antioxidant, anti-microbial, anti-inflammatory, and anti-carcinogenic properties. The anti-inflammatory effects of curcumin have primarily been attributed to its inhibitory effect on NF-κB activity due to redox regulation. In this study, we show that curcumin is an immunosuppressive phytochemical that blocks T cell-activation-induced Ca(2+) mobilization with IC(50) = ～12.5 μM and thereby prevents NFAT activation and NFAT-regulated cytokine expression. This finding provides a new mechanism for curcumin-mediated anti-inflammatory and immunosuppressive function. We also show that curcumin can synergize with CsA to enhance immunosuppressive activity because of different inhibitory mechanisms. Furthermore, because Ca(2+) is also the secondary messenger crucial for the TCR-induced NF-κB signaling pathway, our finding also provides another mechanism by which curcumin suppresses NF-κB activation.     

Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica(prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca²⁺]_i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca²⁺]_i was significantly curtailed in calcium-free buffer (0% Ca²⁺) as compared to that in 100% Ca²⁺ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca²⁺ release-activated Ca²⁺(CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca²⁺]_i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca²⁺]_i in 0% Ca²⁺ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP₃ production, completely abolished increases in [Ca²⁺]_i, induced by OFPC, suggesting that these polyphenols induce the production of IP₃ that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca²⁺]_i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca²⁺]_i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica (prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca2+]i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca2+]i was significantly curtailed in calcium-free buffer (0% Ca2+) as compared to that in 100% Ca2+ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca2+ release-activated Ca2+ (CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca2+]i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca2+]i in 0% Ca2+ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca2+-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP3 production, completely abolished increases in [Ca2+]i, induced by OFPC, suggesting that these polyphenols induce the production of IP3 that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca2+]i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca2+]i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Retrograde activation of store-operated calcium channel

Store-operated Ca2+ entry represents an important mechanism for refilling of a depleted intracellular-reticulum Ca2+ store following sustained activation of the IP3 receptor or ryanodine receptor RyR/Ca2+ release channel in the endoplasmic/sarcoplasmic reticulum (ER/SR). Recent studies have demonstrated the existence of store-operated Ca2+ channel (SOC) in muscle cells, whose activation process appears to be coupled to conformational changes of the RyR. Regulation of the plasma membrane (PM)-resided SOC by the SR-located RyR requires an integrity of the junctional membrane structure between SR and PM. Proteins that interact with RyR or influence the Ca2+ buffering capacity in the ER or SR lumen also participate in the activation process of SOC. Calsequestrin (CSQ) and calreticulin (CRT) are SR/ER-resident proteins, with highly negative charged regions at the carboxyl-terminal end that exhibit high buffering capacity for luminal Ca2+. CSQ and CRT not only modulate the intracellular Ca2+ release process but also might provide retrograde signals to regulate the function of SOC. The functional interplay between CSQ, RyR and SOC may serve essential roles of Ca2+ signaling in muscle contraction and development. A tight link between the expression of CRT and operation of SOC exist in certain cancer cells, where the reduced sensitivity to apoptosis may correlate with the altered function of SOC.     

The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium

A monoclonal antibody to the antigen-receptor on the T cell line Jurkat induces substantial increases in [Ca++]i. Ca++ ionophores can substitute for this antibody in activation by increasing [Ca++]i to levels comparable with those seen with the antigen-receptor antibody. Stimulation with either the antigen-receptor antibody or a Ca++ ionophore leads to the appearance of the same phosphoproteins. These results suggest that the antigen-receptor initiates T cell activation by increasing [Ca++]i.     

The calcium feedback loop and T cell activation: How cytoskeleton networks control intracellular calcium flux

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca^2 +) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell–APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca^2 + signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca^2 + from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca^2 + release-activated Ca^2 + (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca^2 + across the plasma membrane in a process termed store-operated Ca^2 + entry (SOCE). Because CRAC channels are themselves inhibited by Ca^2 + ions, additional factors are suggested to enable the sustained Ca^2 + influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca^2 + evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca^2 + flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca^2 + signaling by controlling the spatial and temporal distribution of Ca^2 + sources and sinks, modulating TCR-dependent Ca^2 + signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Blocking of human T lymphocyte activation by channel antagonists

It has been established that early events in lymphocyte activation involve a rise in intracellular Ca++ as well as changes in the flux of other ions. Although a Ca++ channel has been postulated to participate in the early Ca++ rise, its presence in lymphocytes remains controversial. Also although yet undetected, electrophysiological data suggest the presence of a Ca++ activated K+ channel on human peripheral blood lymphocytes (HPBL). Here we report on the effect of specific channel blockers as an approach to the identification of these channels on HPBL. At 40 nM nifedipine, an inhibitor of voltage-gated Ca++ channels, fully inhibits the PHA-promoted activation of HPBL. This effect is concentration dependent with a half maximum effect at approximately 10 nM and is demonstrable whether the drug is added at the same time as or up to 18 h after the addition of the mitogen. This inhibition of activation is not seen if the lymphocytes are activated using IL-2 instead of PHA. Charybdotoxin a toxin which blocks a Ca++ activated K+ channel of muscle cells also blocks to almost 100 per cent the PHA-induced activation of HPBL. This inhibition can be demonstrated regardless of whether the blocker is added together with or up to 4 h after PHA. As opposed to nifedipine charybdotoxin shows no effect if added 18 h after the initiation of the activation process. When nifedipine and charybdotoxin were tested on mice splenocytes we found that nifedipine fully inhibits the LPS-promoted activation of these cells while charybdotoxin has no effect on their activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of calcium channel blocking drugs on the thermotropic behavior of dimyristoylphosphatidylcholine

The effects of four calcium channel blocking drugs, diltiazem, verapamil, nimodipine and nisoldipine, on the main phase transition of dimyristoylphosphatidylcholine have been studied by high resolution differential scanning calorimetry. In all cases, the phase transition temperature is lowered, though much more effectively by nimodipine and nisoldipine than by the other two drugs. Nimodipine and nisoldipine markedly reduce the enthalpy of transition while diltiazem and verapamil have no significant effect on the enthalpy within the drug concentration range studied. Analysis of the data in terms of ideal solution theory is presented. X-ray and neutron scattering studies indicate that nimodipine and verapamil differ significantly with respect to their location within a lipid bilayer, and this difference suggests a partial rationalization of the experimental results presented here.     

Use of calcium channel antagonists as magnetoprotective agents

Human polymorphonuclear leukocytes (PMNs) exhibited a time-dependent (0 to 60 min) increase in the release of lysozyme and lactate dehydrogenase (degranulation) when exposed to a static (direct current) magnetic field of 0.1 Tesla. When 1 X 10(6) PMNs were treated with the calcium channel antagonists diltiazem, nifedipine, and verapamil before exposure to a magnetic field, no significant change in degranulation was detected compared to control and sham-exposed PMNs that were similarly treated. Likewise, magnetic field-induced inhibition of cell migration was prevented with the addition of these antagonists. Such changes in degranulation and cell migration occurred in a dose-dependent manner. These results indicated that these agents protected PMNs exposed to a magnetic field, and the damage to the cells that is mediated by magnetic field-stimulated Ca2+ influx might be preventable. In this regard, pharmaceutical agents might prove useful in protection against injurious electromagnetic field exposure.