Modulation of calcium conductance by opioid and anti-opioid peptides

In the central nervous system, opening of voltage-gated Ca2+ channels triggers the release of neurotransmitters. Numerous membrane receptors, particularly those belonging to the superfamily of G-protein coupled receptors modulate, in most cases inhibit the activity of these channels. In the present review, we describe the modulation of calcium channels by opioid and anti-opioid peptides. Following a brief presentation of the opioid system, we describe the characteristics of the modulation of calcium channels by opioids. Recent major advances concerning neuropeptide FF (NPFF), taken as an example of anti-opioid systems, are reviewed. Results from our laboratory demonstrating the anti-opioid activity of NPFF, in the modulation of Ca2+ channels in isolated neurones, are described.     

Opioid agonist modulation of cytoplasmic free Ca2+ level in concanavalin A-stimulated mouse lymphocytes.

In this study the influence of mu-, delta-, and kappa-selective opioid agonists (DAMGO, DSLET, and dynorphin A (1-13)) on cytoplasmic free Ca2+ ([Ca2+]i) level in normal and concanavalin-A (Con A)-activated mouse lymphocytes was investigated. [Ca2+]i was measured using the fluorescent dye FURA-2AM. The opioid peptides at 10-12-10-7 M induced some increase in [Ca2+]i in non-activated lymphocytes. However, DAMGO and DSLET (10-13-10-7 M) considerably inhibited a Con A-induced increase in [Ca2+]i. The inhibiting effect of both peptides was higher after 20-min preincubation compare to 2-h preincubation. The effect of the kappa-agonist dynorphin A (1-13) was significantly different depending on the duration of cell pretreatment and the concentration of the peptide used. After preincubation for 20 min at low concentrations (10-12-10-11 M) it slightly stimulated, while at higher (10-10-10-7 M) concentrations it inhibited lymphocyte response to Con A. After preincubation for 2 h, pronounced stimulation of mitogen-induced Ca2+ flux was observed at peptide concentration 10-9 M. The effects of opioids were antagonized by naloxone. These data indicate that functionally active opioid receptors expressed on lymphocytes could be involved in early stages of mitogen activation.     

Calcium-binding protein 1 is an inhibitor of agonist-evoked, inositol 1,4,5-trisphosphate-mediated calcium signaling

Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+]i responses to agonist stimulation. Measurement of [Ca2+] within the ER of permeabilized PC12 cells demonstrated that LCaBP1 directly inhibited InsP3-mediated Ca2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.     

Kappa opioid receptor endocytosis by dynorphin peptides

Internalization and downregulation are important steps in the modulation of receptor function. Recent work with the beta2 adrenergic and opioid receptors have implicated these processes in receptor-mediated activation of mitogen-activated protein kinase (MAPK). We have used CHO cells expressing epitope-tagged rat kappa opioid receptors (rKORs) and prodynorphin-derived peptides to characterize the agonist-mediated endocytosis of rKORs and activation of MAPK. Kappa receptor-selective peptides induced receptor internalization and downregulation whereas nonpeptide agonists did not. An examination of the ability of dynorphin A-17-related peptides (lacking C-terminal amino acids) to promote KOR internalization, inhibition of adenylyl cyclase, and MAPK phosphorylation revealed that the N-terminal seven residues play an important role in eliciting these responses. Both dynorphin peptides and nonpeptide agonists induced rapid and robust phosphorylation of MAPKs. Taken together, these results point to a difference in the ability of dynorphin peptides and nonpeptide ligands to promote rKOR endocytosis and support the view that rKOR internalization is not required for MAPK activation.     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

Knockout B lymphoma cell lines as biochemical tools to explore multiple signalling pathways

Studies on B lymphocyte signalling pathways using B lymphocytes from genetically modified mice have the disadvantages of primary cell polyclonality and finite life span. B lymphoma cell lines have been generated from mice with targeted mutations in the oct-2, OBF-1, vav-1 and btk genes, as a model system that lacks these limitations and possesses additional potential for experimental manipulation. To assess their utility, activation of the B cell receptor using anti- micro, the Toll-like receptor-4 using lipopolysaccharide and the interleukin-4 receptor were assessed in these cell lines. Differential tyrosine phosphorylation of intracellular proteins was measured in the wild-type controls compared to the corresponding mutant cell lines after B cell receptor stimulation. Intracellular calcium (Ca2+i) was mobilized in the control cell lines but not in the OBF-1 and Vav1-deficient cells, while Xid B cell lines (btk mutant) showed a reduced Ca2+ mobilization. Extracellular signal-regulated kinase 1/2 phosphorylation in response to anti- micro or lipopolysaccharide stimulation was significantly reduced in Vav1-deficient cells. Interleukin-4 stimulation of wild-type cells resulted in a 2-3-fold increase in Stat-6 phosphorylation. These results indicate that the cell lines mimic the biochemical responses of the corresponding primary B cells. They therefore represent a useful model system to investigate the regulation and roles of these and other gene products in B cell signal transduction and activation.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Kappa opiate agonists inhibit Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. Involvement of a GTP-binding protein

The aim of the present study has been to characterize the regulation by opiates of 45Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. We have demonstrated that K+-induced depolarization, in the presence of the Ca2+ channel agonist Bay K8644, stimulated Ca2+ influx (3-4-fold) via the dihydropyridine class of voltage-dependent Ca2+ channels. While mu and delta opiates had no effect, kappa opiate agonists (e.g. U50488, dynorphin) profoundly depressed the stimulated Ca2+ influx (86% inhibition at 100 microM U50488). The kappa agonist action was stereospecific and could be reversed by the opiate antagonist naloxone. The inhibition produced by kappa agonists was greatly diminished following pertussis toxin treatment, and this effect was accompanied by toxin-induced ADP-ribosylation of a 40-41-kDa protein. This suggests that kappa opiate receptors are negatively coupled to voltage-dependent Ca2+ channels, via a pertussis toxin-sensitive GTP-binding protein. Basal 45Ca2+ uptake, stimulated by adenylate cyclase activators (forskolin and cholera toxin), was potently inhibited by kappa opiates suggesting that, under conditions of neurohormonal stimulation of adenylate cyclase, kappa receptors are coupled to Ca2+ channels indirectly via the adenylate cyclase complex. In addition, cAMP-independent coupling pathways may also be involved.     

Anti-opioid activities of NPFF1 receptors in a SH-SY5Y model

In order to elucidate the mechanisms of the neuronal anti-opioid activity of Neuropeptide FF, we have transfected the SH-SY5Y neuroblastoma cell line, which expresses mu- and delta-opioid receptors, with the human NPFF1 receptor. The SH1-C7 clone expresses high affinity NPFF1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca2+ currents and enhances the intracellular Ca2+ release triggered by muscarinic receptors activation. In contrast, preincubation of cells with NPVF decreases the response to opioids on both calcium signaling, thus reproducing the cellular anti-opioid activity described in neurons. SH1-C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.     

Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing

Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2.     

Multiple opiate receptors on neurons of the mammalian central nervous system. In vivo and in vitro studies

Experiments were performed in rat spinal cord cells in vivo and on hippocampal pyramidal cells in vitro. These investigations suggest that acute and chronic treatment renders the neurons subsensitive to opiate alkaloids without altering their sensitivity to opioid peptides. The experiments performed in the dorsal horn of the spinal cord provide evidence that in this structure mu- and delta-receptors may also be localized on the same cell. The evidence for the existence of distinct types of opiate receptors as originally proposed by (1) and suggested by the differing pattern of opiate and opioid peptide activity in various assay systems has been substantiated by investigations involving the selective development of tolerance and the protection of a particular receptor subtype by chemical manipulation. Furthermore, they have been characterized by the use of low concentrations of radiolabelled agonists and antagonists and through the ability of GTP to influence differentially their binding to the opiate receptor (for refs. see: 2). Recently autoradiographic techniques were able to provide direct evidence by mu- and delta-receptors in the mammalian brain (3; 4; 5; 6; and cits. therein). The presence of multiple opiate receptors located on the same cell is suggested by the present study.     

Effect ot thyroliberin on rat brain opiate receptors

The ability of thyroliberin to interact with opiate receptors of the rat midbrain and hypothalamus has been studied. It was shown by competitive displacement analysis that thyroliberin did not replace labeled opioid peptides in opiate receptor binding sites when added in vitro at concentrations of up to 10(-5) M. The specific binding of opioid peptides was increased by 10-20% in the presence of 10(-7)-10(-6) M thyroliberin. This effect was, probably, due to the rise in the affinity of high-affinity opiate receptors. At the same time the affinity of low-affinity binding sites was decreased. It is suggested that the antagonistic properties of thyroliberin are mediated by the modulation of the binding characteristics of enkephalin-low-affinity opiate receptors.     

Alternate coupling of receptors to Gs and Gi in pancreatic and submandibular gland cells.

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.     

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.     

Platelet-activating factor induces an increase in intracellular calcium and expression of regulatory genes in human B lymphoblastoid cells.

Platelet-activating factor (PAF) has recently been demonstrated to be metabolized by B lymphocytes and to cause enhancement of Ig synthesis by Ig-secreting B lymphoblastoid cell lines. We have now examined some of the early activation events triggered by PAF binding to three Ig-secreting B cell lines, LA350 (IgM secreting), HSCE- (IgG secreting), and U266 (IgE secreting). After addition of 10(-7) to 10(-11) M PAF, but not equimolar concentrations of the inactive metabolite lyso-PAF, all three cell lines demonstrated rapid dose-dependent increases in free cytosolic Ca2+ concentrations ([Ca2+]i). The increases in [Ca2+]i resulted from both the release of Ca2+ from internal stores as well as transmembrane Ca2+ uptake. Addition of PAF triggered the rapid hydrolysis of phosphatidylinositol bisphosphate and accumulation of inositol phosphates. PAF also increased expression of the cell cycle-active genes c-fos and EGR2 in a dose-dependent fashion. The stimulated increases in [Ca2+]i and phosphatidylinositol bisphosphate hydrolysis and the increases in gene expression were all inhibited by the specific PAF receptor antagonist Web 2086. The LA350 cell line (which expresses surface IgM) was also shown to increase [Ca2+]i after addition of anti-IgM antibodies. Sequential addition of PAF or anti-IgM antibody in either order failed to reveal any evidence for heterologous desensitization. Furthermore, the PAF receptor antagonist did not affect anti-IgM induced changes in [Ca2+]i. These data provide evidence for the presence of functional PAF receptors on B lymphoblastoid cells and indicate a potential role for PAF in the regulation of B cell activation.     

Beta-endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes

Lymphocytes stimulated by mitogens or antigens exhibit an enhanced calcium uptake early in the proliferation or activation response. Modulation of this calcium uptake results in alterations of proliferation and immunocompetence. beta-endorphin and other opioids affect several parameters of lymphocyte competence. Limited data are available concerning the mechanism(s) of these effects. This study examines whether a possible opioid mechanism is the modification of the early calcium influx into stimulated lymphocytes. The time course of both concanavalin A (Con A) and phytohemagglutinin (PHA)-stimulated 45Ca2+ uptake into thymocytes was characterized to determine the optimal time for testing the effects of opioids. beta-Endorphin 1-31 significantly enhanced Con A-stimulated 45Ca2+ uptake into rat thymocytes. This peptide had no significant effect on PHA-stimulated 45Ca2+ uptake or on basal thymocyte 45Ca2+ flux. The beta h-endorphin stimulatory effect was titratable in the range of 0.1 nM to 10 microM. Naloxone did not reverse the enhancement. Met-enkephalinamide and other opioid agonists did not duplicate the stimulatory effect. Thus, the beta h-endorphin 1-31 enhancement of Con A-stimulated 45Ca2+ uptake by rat thymocytes does not operate via classical opioid receptor mechanisms. beta h-endorphin 1-31 appears to be acting on a subset of T cells that are responsive to Con A but not to PHA.     

Mu-opioid agonist inhibition of kappa-opioid receptor-stimulated extracellular signal-regulated kinase phosphorylation is dynamin-dependent in C6 glioma cells

In previous studies we found that mu-opioids, acting via mu-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the kappa-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with mu-opioid agonists for 1 h results in the inhibition of kappa-opioid mitogenic signaling. The mu-selective agonist endomorphin-1 attenuates kappa-opioid-stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal-regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin-1 and its GTPase-defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal-regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect kappa-opioid phosphorylation of extracellular signal-regulated kinase. However, it does block the inhibitory action on kappa-opioid signaling mediated by the kappa-opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of mu- and kappa-opioids and provide new insight into the role of opioid receptor trafficking in signaling.     

Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.

Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn-negative and syk-negative B cell lines were established. Syk-deficient B cells abolished the tyrosine phosphorylation of phospholipase C-gamma 2, resulting in the loss of both inositol 1,4,5-trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn-deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.     

The role of cytoplasmic free calcium concentration in B-cell tolerance

Calcium is an important factor in the immune response. Extracellular calcium is required for antibody production by B lymphocytes. Several investigators have demonstrated that crosslinking of receptors on B lymphocytes by anti-mu antibody induces an increase in intracellular calcium. There are few data on the role of intracellular calcium mobilization or calcium influx in tolerance induction in B cells. We studied changes in free intracellular calcium concentration ([Ca+2]i) induced by exposure of dinitrophenyl (DNP)-specific B cells to the tolerance-inducing conjugate DNP-murine IgG2a (DNP-MGG). Splenic B cells enriched for DNP-specific cells and DNP-specific continuous B-cell lines were used for the studies. Exposure of B cells to the tolerogen DNP-MGG, the antigen DNP-keyhole limpet hemocyanin (DNP-KLH), or the antigen DNP-Ficoll induced an increase in free [Ca+2]i which was due to both mobilization of Ca+2 from endoplasmic reticulum (ER) and influx of extracellular Ca+2. This increase was DNP specific since no significant change was seen with carriers alone and no change was seen in cells that were not DNP specific. The DNP-MGG and DNP-Ficoll induced the same amount of Ca+2 release from ER but the release induced by DNP-KLH was higher. When B cells, which were made tolerant by in vitro incubation with DNP-MGG, were incubated with antigens, a mobilization of Ca+2 from endoplasmic reticulum occurred that was the same as that of nontolerant B cells. Since Ca+2 mobilization is associated with Ig receptor-dependent early B-cell activation, it is likely that the tolerant B cell can still receive an activation signal through the Ig receptors.