Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.     

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.     

Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts

CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders induced sustained [Ca(2+)](i) increases in CD38(-) target cells. Co-culture on CD38(+) feeders enhanced the proliferation of CD38(-) target cells over control values and significantly shortened the S phase of cell cycle. These results demonstrate a paracrine process based on Cx43-mediated release of NAD(+), its CD38-catalyzed conversion to extracellular cADPR, and influx of this nucleotide into responsive cells to increase [Ca(2+)](i) and stimulate cell proliferation.     

Regulation of calcium signaling by the second messenger cyclic adenosine diphosphoribose (cADPR)

Ca2+ ions are involved in the regulation of many diverse functions in animal and plant cells, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation. The intracellular Ca2+ concentration can be increased by different molecular mechanisms, such as Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ stores. Release from intracellular Ca2+ stores is accomplished by the small molecular compounds D-myo-inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review will focus on the effects of cADPR in different cells and tissues, the mechanisms of cADPR-mediated Ca2+ release and Ca2+ entry, extracellular effects of cADPR, and the role of cADPR in a cell system studied in detail, human T-lymphocytes.     

Extracellular NAD(+) induces calcium signaling and apoptosis in human osteoblastic cells

ADP-ribosyl cyclase/CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD(+) (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing second messenger cyclic ADP ribose (cADPR). Furthermore, CD38 mediates cADPR influx inside a number of cells, thereby inducing Ca(2+) mobilization. Intracellularly, cADPR releases Ca(2+) from ryanodine-sensitive pools, thus activating several Ca(2+)-dependent functions. Among these, the inhibition of osteoclastic-mediated bone resorption has been demonstrated. We found that HOBIT human osteoblastic cells display ADP-ribosyl cyclase activity and we examined the effects of CD38 stimulation on osteoblasts function. Extracellular NAD(+) induced elevation of cytosolic calcium due to both Ca(2+) influx from the extracellular medium and Ca(2+) release from ryanodine-sensitive intracellular stores. Culturing these cells in the presence of NAD(+) caused a complete growth arrest with a time-dependent decrease of cell number and the appearance of apoptotic nuclei. The first changes could be observed after 24 h of treatment and became fully evident after 72-96 h. We propose a role of extracellular NAD(+) in bone homeostatic control.     

Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose

Cyclic ADP ribose (cADPR) is a calcium-mobilizing metabolite that regulates intracellular calcium release and extracellular calcium influx. Although the role of cADPR in modulating calcium mobilization has been extensively examined, its potential role in regulating immunologic responses is less well understood. We previously reported that cADPR, produced by the ADP-ribosyl cyclase, CD38, controls calcium influx and chemotaxis of murine neutrophils responding to fMLF, a peptide agonist for two chemoattractant receptor subtypes, formyl peptide receptor and formyl peptide receptor-like 1. In this study, we examine whether cADPR is required for chemotaxis of human monocytes and neutrophils to a diverse array of chemoattractants. We found that a cADPR antagonist and a CD38 substrate analogue inhibited the chemotaxis of human phagocytic cells to a number of formyl peptide receptor-like 1-specific ligands but had no effect on the chemotactic response of these cells to ligands selective for formyl peptide receptor. In addition, we show that the cADPR antagonist blocks the chemotaxis of human monocytes to CXCR4, CCR1, and CCR5 ligands. In all cases, we found that cADPR modulates intracellular free calcium levels in cells activated by chemokines that induce extracellular calcium influx in the apparent absence of significant intracellular calcium release. Thus, cADPR regulates calcium signaling of a discrete subset of chemoattractant receptors expressed by human leukocytes. Since many of the chemoattractant receptors regulated by cADPR bind to ligands that are associated with clinical pathology, cADPR and CD38 represent novel drug targets with potential application in chronic inflammatory and neurodegenerative disease.     

CD38 signaling in B lymphocytes is controlled by its ectodomain but occurs independently of enzymatically generated ADP-ribose or cyclic ADP-ribose

CD38 is a type II transmembrane glycoprotein that is expressed by many cell types including lymphocytes. Signaling through CD38 on B lymphocytes can mediate B cell activation, proliferation, and cytokine secretion. Additionally, coligation of CD38 and the B cell Ag receptor can greatly augment B cell Ag receptor responses. Interestingly, the extracellular domain of CD38 catalyzes the conversion of NAD+ into nicotinamide, ADP-ribose (ADPR), and cyclic ADPR (cADPR). cADPR can induce intracellular calcium release in an inositol trisphosphate-independent manner and has been hypothesized to regulate CD38-mediated signaling. We demonstrate that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme activity. In contrast, independent point mutations in the extracellular domain of CD38 dramatically impaired signal transduction. However, no correlation could be found between CD38-mediated signaling and the capacity of CD38 to catalyze an enzyme reaction and produce cADPR, ADPR, and/or nicotinamide. Instead, we propose that CD38 signaling and coreceptor activity in vitro are regulated by conformational changes induced in the extracellular domain upon ligand/substrate binding, rather than on actual turnover or generation of products.     

Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes

The role that extracellular calcium plays in activating resting cloned cytotoxic T lymphocytes (CTL) to proliferate and to produce lymphokines was examined. In these cells, stimulation with interleukin 2 (IL-2) induced a proliferative response without a concomitant production of macrophage-activating factor (MAF), whereas stimulation with antigen or lectin (in the absence of IL-2) induced MAF production but not proliferation. In the case of IL-2-induced proliferation, extracellular calcium was required to initiate proliferation as well as to prevent cellular arrest later in the G2 + M phase of the cell cycle. In MAF production extracellular calcium was required both to activate the phosphatidylinositol signal-transducing mechanism and to mobilize intracellular calcium in antigen- or lectin-stimulated cytotoxic T lymphocytes. Further, extracellular calcium was required for only 8 of the 18 hr of stimulation time which was needed to achieve maximal MAF production, indicating that both calcium-dependent and -independent events exist in the signal pathway. Additional experiments with calcium ionophores and activators of protein kinase C indicated that although both intracellular calcium mobilization and de novo protein phosphorylation are involved in MAF production, an optimal increase in the level of intracellular calcium by itself is insufficient to induce the production of this lymphokine.     

Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.     

Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway Wei W,Graeff R,Yue J简

Mobilization of intracellular Ca²⁺ stores is involved in many diverse cell functions, including: cell proliferation; differentiation; fertilization; muscle contraction; secretion of neurotransmitters, hormones and enzymes; and lymphocyte activation and proliferation. Cyclic adenosine diphosphate ribose (cADPR) is an endogenous Ca²⁺ mobilizing nucleotide present in many cell types and species, from plants to animals. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide. The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. It has been shown that many extracellular stimuli can induce cADPR production that leads to calcium release or influx, establishing cADPR as a second messenger. cADPR has been linked to a wide variety of cellular processes, but the molecular mechanisms regarding cADPR signaling remain elusive. The aim of this review is to summarize the CD38/cADPR/Ca²⁺ signaling pathway, focusing on the recent advances involving the mechanism and physiological functions of cADPR-mediated Ca²⁺ mobilization.     

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP): novel regulators of Ca2+-signaling and cell function

Ca2+ ions are involved in the regulation of many diverse functions in animal and plant cells, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation. The intracellular Ca2+ concentration can be increased by different molecular mechanisms, such as Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ stores. Release from intracellular Ca2+ stores is accomplished by the small molecular compounds D-myo-inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review concentrates on (i) receptor-mediated formation of cADPR by ADP-ribosyl cyclases, (ii) intracellular and extracellular effects of cADPR in a variety of cell types, and (iii) cADPR in the nucleus. Though our understanding of the role of NAADP is still unclear in many aspects, important recent findings are reviewed, e.g. Ca2+ release activity and binding studies in mammalian cell types.     

Generation of Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate by CD38 for Ca2+ Signaling in Interleukin-8-treated Lymphokine-activated Killer Cells

We have previously demonstrated that cyclic ADP-ribose (cADPR) is a calcium signaling messenger in interleukin 8 (IL-8)-induced lymphokine-activated killer (LAK) cells. In this study we examined the possibility that IL-8 activates CD38 to produce another messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), in LAK cells, and we showed that IL-8 induced NAADP formation after cADPR production. These calcium signaling messengers were not produced when LAK cells prepared from CD38 knock-out mice were treated with IL-8, indicating that the synthesis of both NAADP and cADPR is catalyzed by CD38 in LAK cells. Application of cADPR to LAK cells induced NAADP production, whereas NAADP failed to increase intracellular cADPR levels, confirming that the production of cADPR precedes that of NAADP in IL-8-treated LAK cells. Moreover, NAADP increased intracellular Ca²⁺ signaling as well as cell migration, which was completely blocked by bafilomycin A1, suggesting that NAADP is generated in lysosome-related organelles after cADPR production. IL-8 or exogenous cADPR, but not NAADP, increased intracellular cAMP levels. cGMP analog, 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate, increased both cADPR and NAADP production, whereas the cAMP analog, 8-(4-chlorophenylthio)-cAMP, increased only NAADP production, suggesting that cAMP is essential for IL-8-induced NAADP formation. Furthermore, activation of Rap1, a downstream molecule of Epac, was required for IL-8-induced NAADP formation in LAK cells. Taken together, our data suggest that IL-8-induced NAADP production is mediated by CD38 activation through the actions of cAMP/Epac/protein kinase A/Rap1 in LAK cells and that NAADP plays a key role in Ca²⁺ signaling of IL-8-induced LAK cell migration.     

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.     

Cyclic ADP-ribose and the regulation of calcium-induced calcium release in eggs and cardiac myocytes

Cyclic ADP-ribose (cADPR) is a cyclic metabolite of NAD⁺ synthesised in cells and tissues expressing ADP-ribosyl cyclases. Although it was first discovered in sea-urchin egg extracts as a potent calcium mobilizing agent, subsequent studies have indicated that it may have a widespread action in the activation of calcium-release channels in such diverse systems as mammalian neurones, myocytes, blood cells, eggs, and plant microsomes. In this review we focus on recent work suggesting that cADPR enhances the sensitivity of ryanodine-sensitive calcium-release channels (RyRs) to activation by calcium, a phenomenon termed calcium-induced calcium release (CICR). Two roles for cADPR in calcium signaling are discussed. The first is as a classical second messenger where its levels are controlled by extracellular stimuli, and the second mode of cellular regulation is that the levels of intracellular cADPR may set the sensitivity of RyRs to activation by an influx of calcium in excitable cells. These two possible actions of cADPR are illustrated by considering the signal transduction events during the fertilization of the sea-urchin egg and the modulation of CICR during excitation-coupling in isolated guinea-pig ventricular myocytes, respectively.     

Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle

It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50?μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells.     

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells resuts in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated ⁴⁵Ca⁺², while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression. © 1995 Wiley-Liss, Inc.     

Mechanisms of ATP-induced calcium signaling and growth arrest in human prostate cancer cells

This study investigates the calcium mechanisms involved in growth arrest induced by extracellular ATP in DU-145 androgen-independent human prostate cancer cells. Exposure of DU-145 cells to 100 microM ATP produced an increase in cytoplasmic calcium concentration ([Ca(2+)](i)), due to a mobilization of calcium from the endoplasmic reticulum stores and to subsequent capacitative calcium entry (CCE). We have shown that this [Ca(2+)](i) increase occurs after stimulation by ATP of the phospholipase C (PLC) pathway. For the first time, we have identified the inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms expressed in this cell line and have demonstrated a participation of protein kinase C in CCE. Using fluorescence imaging, we have shown that a long-term treatment with ATP leads to a decrease in the intraluminal endoplasmic reticulum calcium concentration as well as in the amount of releasable Ca(2+). Modulating extracellular free calcium concentrations indicated that variations in [Ca(2+)](i) did not affect the ATP-induced growth arrest of DU-145 cells. However, treating cells with 1 nM thapsigargin (TG) to deplete intracellular calcium pools prevented the growth arrest induced by ATP. Altogether, these results indicate that growth arrest induced in DU-145 cells by extracellular ATP is not correlated with an increase in [Ca(2+)](i) but rather with a decrease in intracellular calcium pool content.     

Interleukin 2-induced lymphocyte proliferation is independent of increases in cytosolic-free calcium concentrations

Activation of lymphocytes by mitogenic lectins initiates a sequence of events that culminates in DNA synthesis and cell proliferation. The mitogenic effects of lectins on T lymphocytes leads to the production of a group of lymphokines including the interleukins. The binding of interleukin 2 (IL 2) to its receptor results in activation of the cell leading to DNA synthesis. An increase in cytosolic-free Ca++ ([Ca++]i) is associated with activation of lymphocytes by mitogenic lectins and also appears to be a prerequisite for induction of DNA synthesis and cell proliferation. We have determined whether the proliferative response triggered by IL 2 binding to its receptor is associated with or requires an increase in [Ca++]i. Using human and murine IL 2-sensitive cell lines, we have demonstrated that the IL 2-induced proliferative response, in contrast to that induced by mitogens such as phytohemagglutinin or concanavalin A, is not accompanied by an increase in [Ca++]i as monitored by the fluorescent indicator quin-2. Furthermore, IL 2-dependent triggering of lymphoblasts occurs in the presence of extremely low extracellular calcium concentrations that prevent transmembrane calcium flux. Activation of IL 2 receptor-bearing T cells, therefore, does not appear to be associated with or to require an increase in [Ca++]i as part of the activation and signaling process. The critical step requiring calcium flux in cell signaling by mitogenic lectins must therefore occur elsewhere in the activation cascade.