Cell permeable exogenous ceramide reduces infarct size in spontaneously hypertensive rats supporting in vitro studies that have implicated ceramide in induction of tolerance to ischemia.

Previous work in primary cell culture has shown that TNF-alpha and ceramide are involved in the signaling that induces tolerance to brain ischemia (Ginis et al., 1999; Liu et al., 2000). To validate the in vitro studies, the authors administered cell permeable analogs of ceramides intracisternally or intravenously to examine their effect on neuroprotection after focal cerebral ischemia. Permanent middle cerebral artery occlusion (MCAO) was performed in spontaneously hypertensive rats. Infarct volumes were assessed at 24 hours after surgery. D-erythro-N-acetylsphingosine (C2-ceramide) or its vehicle was infused intracisternally for 1 hour before MCAO. In a second set of studies, D-erythro-N-octanoylsphingosine (C8-ceramide) or its vehicle was injected intravenously 48 or 24 hours before MCAO to mimic preconditioning (PC) and was also injected 5 minutes after MCAO. C2-ceramide infusion significantly reduced infarct volumes by approximately 14% (P < 0.05). C8-ceramide injection reduced infarct volumes approximately 17% compared with controls. This effect was constant and significant compared with controls over the time periods examined (P < 0.01). This work supports findings in primary brain cell cultures that implicate ceramide as a downstream signal that is proximate to development of tolerance to brain ischemia. Because the degree of protection represents approximately 50% of the maximal infarct reduction observed in this model, there are probably additional signaling pathways that subserve tolerance.

Lipopolysaccharide-induced ischemic tolerance is associated with increased levels of ceramide in brain and in plasma.

Intravenous administration of lipopolysaccharide (LPS) (0.9 mg/kg) has been shown to induce ischemic tolerance in spontaneously hypertensive rats (SHR). TNF-alpha is believed to play a crucial role in preconditioning as its inhibition with TNF-alpha-binding protein abolished tolerance. Our recent studies (Liu et al., Am. J. Physiol. 278 C144, 2000) have demonstrated that ceramide, a downstream messenger in TNF-alpha signaling, is a mediator of hypoxia-induced tolerance in neuronal cells. To test the hypothesis that ceramide contributes to LPS-induced tolerance in vivo, SHR were injected intravenously with either LPS or saline and the levels of ceramide in brain and in plasma were determined by reversed phase HPLC. LPS injection resulted in a significant increase of ceramide in plasma with a maximum at 24 h (8.32+/-1.14 pmol/microl (LPS) vs. 2.65+/-0.62 pmol/microl (saline)). LPS also induced ceramide upregulation in brain cortex, which started between 6 and 12 h and remained elevated up to 48 h after LPS injection. Fluorescent NBD-C6 ceramide was able to cross blood-brain barrier and was found in brain vessels, perivascular cells and in brain parenchyma 30 min after intravenous injection. These findings demonstrate that LPS preconditioning leads to elevation of ceramide in brain and plasma and, in conjunction with previous work, suggests that ceramide plays a role in LPS-induced protection against brain ischemic injury in vivo.

Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro.

Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells. We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells. In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80. The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells. Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines. This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies. Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells. Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus. These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

The protective effect of ceramide in immature rat brain hypoxia-ischemia involves up-regulation of bcl-2 and reduction of TUNEL-positive cells.

Preconditioning brain with tumor necrosis factor alpha (TNF-alpha) can induce tolerance to experimental hypoxia and stroke and ceramide is a downstream messenger in the TNF-alpha signaling pathway. A hypoxic-ischemic (HI) insult in the immature rat injures brain primarily through apoptosis. Apoptosis is regulated by Bcl-2 family proteins. The authors explored whether ceramide protects against HI in the immature rat, and whether Bcl-2 family protein expression is involved. Hypoxia-ischemia was produced in seven-day-old rats by ligating the right carotid artery, followed by 2 hours of 8% oxygen exposure. Thirty minutes after HI, C2-ceramide (150 microg/kg) was injected intraventricularly. Infarct volume was measured 5 days later. C2-ceramide reduced HI-induced brain damage by 45% to 65% compared with HI/dimethyl sulfoxide (DMSO) (vehicle control) or HI only groups. In separate experiments, brains of sham-operated control and HI only animals and animals subjected to HI plus C2-ceramide or DMSO infusion were sampled 6 hours, 24 hours, and 5 days after treatments and analyzed for Bcl-2, Bcl-xl, and Bax expression (Western blotting), and apoptosis (TUNEL assay). Augmented Bcl-2 and Bcl-xl levels in the C2-ceramide treated group were associated with a significant decrease in TUNEL-positive cells. The results support a protective role for ceramide in neonatal HI.

Hypoxia affects tumor cell invasiveness in vitro: the role of hypoxia-activated ligand HAL1/13 (Ku86 autoantigen).

We have recently identified and characterized a new adhesion ligand, HAL1/13 (hypoxia-activated ligand), which mediates the increase in leukocyte adhesion to endothelium under hypoxic conditions (J. Immunol. 155 (1995) 802-810). The HAL1/13 antigen was cloned and found to be identical to p86 subunit of Ku autoantigen. In this study we demonstrate that exposure of neuroblastoma and breast carcinoma cells to hypoxia results in upregulation of HAL1/13 surface expression, coincident with an increased ability of these tumor cells to invade endothelial monolayers, which could be partially attenuated by the anti-HAL1/13 antibody. Hypoxia also potentiated neuroblastoma and breast carcinoma cell transmigration through Matrigel filters. Anti-HAL1/13 antibody inhibited haptotactic locomotion of hypoxic tumor cells on laminin.

Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia.

Ischemic stroke is a leading cause of death and disability in developed countries. Yet, in spite of substantial research and development efforts, no specific therapy for stroke is available. Several mechanism for neuroprotection have been explored including ion channels, excitatory amino acids and oxygen radicals yet none has culminated in an effective therapeutic effect. The review article on "inflammation and stroke" summarizes key data in support for the possibility that inflammatory cells and mediators are important contributing and confounding factors in ischemic brain injury. In particular, the role of cytokines, endothelial cells and leukocyte adhesion molecules, nitric oxide and cyclooxygenase (COX-2) products are discussed. Furthermore, the potential role for certain cytokines in modulation of brain vulnerability to ischemia is also reviewed. The data suggest that novel therapeutic strategies may evolve from detailed research on some specific inflammatory factors that act in spatial and temporal relationships with traditionally recognized neurotoxic factors. The dual nature of some mediators in reformatting of brain cells for resistance or sensitivity to injury demonstrate the delicate balance needed in interventions based on anti-inflammatory strategies.

Hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF-alpha and ceramide.

Brief "preconditioning" ischemia induces ischemic tolerance (IT) and protects the animal brain from subsequent otherwise lethal ischemia. Identification of the signaling steps most proximal to the development of the IT will allow induction of the resistance to ischemia shortly after the onset of stroke. Animal studies demonstrate a key role of tumor necrosis factor-alpha (TNF-alpha) in induction of IT. The sphingolipid ceramide is known as a second messenger in many of the multiple effects of TNF-alpha. We hypothesized that ceramide could mediate IT. We demonstrate that preconditioning of rat cortical neurons with mild hypoxia protects them from hypoxia and O(2)-glucose deprivation injury 24 h later (50% protection). TNF-alpha pretreatment could be substituted for hypoxic preconditioning (HP). HP was attenuated by TNF-alpha-neutralizing antibody. HP and TNF-alpha pretreatment cause a two- to threefold increase of intracellular ceramide levels, which coincides with the state of tolerance. Fumonisin B(1), an inhibitor of ceramide synthase, attenuated ceramide upregulation and HP. C-2 ceramide added to the cultures right before the hypoxic insult mimicked the effect of HP. Ceramide did not induce apoptosis. These results suggest that HP is mediated via ceramide synthesis triggered by TNF-alpha.

Tumor necrosis factor and reactive oxygen species cooperative cytotoxicity is mediated via inhibition of NF-kappaB.

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) plays a key role in pathogenesis of brain injury. However, TNFalpha exhibits no cytotoxicity in primary cultures of brain cells. This discrepancy suggests that other pathogenic stimuli that exist in the setting of brain injury precipitate TNFalpha cytotoxicity. The hypothesis was tested that reactive oxygen species (ROS), that are released early after brain injury, act synergistically with TNFalpha in causing cell death. MATERIALS AND METHODS: Cultured human and rat brain capillary endothelial cells (RBEC), and cortical astrocytes were treated with TNFalpha alone or together with different doses of H2O2, and apoptotic cell death and DNA fragmentation were measured by means of 3'-OH-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Hoechst fluorescence assay, respectively. The effect of H2O2 on TNFalpha-induced activation of nuclear factor kappa B (NF-kappaB) was measured by Western blots of cytoplasmic and nuclear extracts of RBEC using anti-inhibitor of NF-kappaB (IkappaB) and anti-p65 subunit of NF-kappaB antibodies. Nuclear translocation of NF-kappaB was investigated by immunofluorescent staining of RBEC with anti-p65 antibodies. RESULTS: TNFalpha alone had no cytotoxic effect in brain endothelial cells and astrocytes at concentrations up to 100 ng/ml. Co-treatment with 5-10 microM of H2O2 caused a two-fold increase in the number of apoptotic cells 24 hr later. Similar doses (1-3 microM) of H2O2 initiated early DNA fragmentation. H2O2 inhibited TNFalpha-induced accumulation of p65 in the nucleus, although it had no effect on degradation of the IkappaB in cytoplasm. Immunostaining confirmed that H2O2 inhibited p65 transport to the nucleus. CONCLUSIONS: Reactive oxygen species could act synergistically with TNFalpha in causing cytotoxicity via inhibition of a cytoprotective branch of TNFalpha signaling pathways, which starts with NF-kappaB activation.

TNF-alpha pretreatment prevents subsequent activation of cultured brain cells with TNF-alpha and hypoxia via ceramide.

We have developed a cellular model in which cultured astrocytes and brain capillary endothelial cells preconditioned with tumor necrosis factor-alpha (TNF-alpha) fail to upregulate intercellular adhesion molecule-1 (ICAM-1) protein (80% inhibition) and mRNA (30% inhibition) when challenged with TNF-alpha or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-alpha, its levels were measured at various times after the TNF-alpha preconditioning. We present evidence for the first time that, in normal brain cells, TNF-alpha pretreatment causes a biphasic increase of ceramide levels: an early peak at 15-20 min, when ceramide levels increased 1.9-fold in astrocytes and 2.7-fold in rat brain capillary endothelial cells, and a delayed 2- to 3-fold ceramide increase that occurs 18-24 h after addition of TNF-alpha. The following findings indicate that the delayed ceramide accumulation results in cell unresponsiveness to TNF-alpha: 1) coincident timing of the ceramide peak and the tolerance period, 2) mimicking of preconditioning by addition of exogenous ceramide, and 3) attenuation of preconditioning by fumonisin B1, an inhibitor of ceramide synthesis. In contrast to observations in transformed cell lines, the delayed ceramide increase was transient and did not induce apoptosis in brain cells.

Dual role of tumor necrosis factor alpha in brain injury.

Brain injury (ischemia, trauma) is among the leading cause of mortality and disability in the western world. It induces increased production of tumor necrosis factor (TNF alpha) by brain resident cells. There is conflicting evidence on the role of this response in the injured brain, showing its potential effect in both processes of repair and of damage. This review presents data from clinical and experimental studies on the stimulation of TNF alpha production in brain injury and on the deleterious consequence of this acute response. Its inhibition by pharmacologic agents, neutralizing antibodies or soluble receptors has protective effects. In contrast, there are reports (from in-vitro studies or knock-out mice) on the beneficial effects of TNF alpha. To reconcile these apparently conflicting reports, the exact timing and extent of TNF alpha activation must be taken into account, as well as the presence of other mediators such as reactive oxygen species. It is suggested that the appropriate context of mediators, at any given time after brain injury may well determine whether the effect of TNF alpha is protective or toxic.

Protection from apoptosis in human neutrophils is determined by the surface of adhesion.

Recent work suggests that various neutrophil agonists affect the rate of apoptosis in these cells. On the basis of these observations, we hypothesized that signals triggered in neutrophils via their adhesion receptors might also modify their life span. This hypothesis has been tested using human neutrophils adherent to tissue culture plastic, either untreated or coated with extracellular matrix (ECM) proteins or with monolayers of human umbilical vein endothelial cells. To detect and quantitate apoptotic changes in adherent cells, we developed a microtiter plate assay using a cell-permeable DNA-binding fluorescent dye, Hoechst 33342. Use of this assay demonstrated that 1) the number of apoptotic cells among neutrophils adherent to plastic after 6-20 h of incubation was significantly lower than that among neutrophils adherent to the ECM proteins fibronectin or laminin; 2) adhesion to interleukin-1-activated endothelial cells delayed apoptosis, whereas adhesion to nonactivated endothelium accelerated neutrophil death; and 3) monoclonal antibodies directed against intercellular adhesion molecule 1 or against the common beta 2-chain of the leukocyte integrins abolished the protective effect of interleukin-1-activated endothelial cells on apoptosis of adherent neutrophils. These results suggest that the life span of adherent neutrophils. depends on the activating signals triggered by the surface of adhesion.

Characterization of a hypoxia-responsive adhesion molecule for leukocytes on human endothelial cells.

Decreased oxygen tension is one important component of ischemic episodes, which are characterized by leukocyte accumulation at the site of infarction. We have reported recently that cultured endothelial and muscle RD cells transferred from pO2 of 120 torr to pO2 of 50 torr for 3 h dramatically increased their adhesiveness for lymphocytes and neutrophils. In the present study, we identify and characterize a new endothelial cell adhesion molecule that mediates hypoxia-induced adhesion to leukocytes. We have generated a mAb, HAL 1/13, that recognizes a cell surface epitope common to both RD and endothelial cells and inhibits lymphocyte and neutrophil adhesion to muscle and endothelial cell monolayers under normoxic conditions in a dose-dependent manner. The contribution of the HAL 1/13 ligand to leukocyte adhesion significantly increased (more than twofold) when the muscle or endothelial cells had been exposed previously to a hypoxic environment. HAL 1/13 mAb completely abolished the enhancing effect of hypoxia on RD and endothelial cell adhesiveness for leukocytes. HAL 1/13 mAb immunoprecipitated a protein with Mr approximately 85,000 Da from cell surface-iodinated muscle and endothelial cells. The Mr, CD cluster dendrogram analysis, and lack of induction by IL-1 of HAL 1/13 distinguish it from most other known endothelial cell adhesion molecules. We suggest that this hypoxia-sensitive adhesion receptor on mesenchymal cells participates in neutrophil adhesion and consequent diapedesis during hypoxia-associated pathologic conditions.

Quantitative estimation of the degree of cell spreading on different surfaces and cell monolayers using a fluorescent plate scanner.

A simple, rapid, and quantitative method for estimation of the degree of spreading of fluorescently labeled cells adherent to an artificial or biological surface has been designed, based on the principle that the medium between adherent cells and the surface of adhesion absorbs fluorescent light emitted by the adherent cells. Using the Beer-Lambert equation for extinction coefficient of monochromatic light, we have calculated the factor by which fluorescence signal actually emitted from cells adherent to the bottom of the microtiter well would differ from the fluorescence of these cells detected by a bottom-scanning plate reader. The physical nature of cell-cell ligand-receptor bridges depends on many parameters, such as the type of the receptor(s), the intensity and direction of a stress force, blockade of the receptors with monoclonal antibodies (MAb), and transfer of adhesion mediation to different ligand-receptor pairs. When employed in various adhesion models, this new technique has demonstrated quantitative changes of cellular spreading of human neutrophils adherent to different extracellular matrix protein-coated surfaces and to endothelial cells after neutrophil/endothelial cell activation or in the presence of MAb directed against neutrophil integrins. These measurements of spreading of adherent cells appear more sensitive than visual observation of cellular morphology on biological surfaces or cells.

Oxygen tension regulates neutrophil adhesion to human endothelial cells via an LFA-1-dependent mechanism.

Extravasation of leukocytes at the sites of ischemia-reperfusion is thought to exacerbate the tissue injury. It has been proposed that leukocyte accumulation is a secondary effect of the ischemic damage, mediated by inflammatory cytokines. We have recently demonstrated that physiologically low levels of oxygen tension alone can have a direct effect on the adhesive characteristics of mesenchymal cells for lymphocytes. We now report that decrease of oxygen tension in the environment induces the adhesion of neutrophils to human endothelial cells in culture. Adhesion of human neutrophils to human umbilical vein, bovine aortic, and mouse microvascular endothelial cell monolayers, which had been incubated at pO2 of 50 torr for 3 hours, increased 2.5-fold, 2-, and 1.5-fold, respectively. The effects of decreased oxygen concentration on adhesion were not mediated by a soluble factor elaborated by the hypoxic cells. Low oxygen tension upregulates a saturable, endothelial cell-associated adhesion mechanism, capable of withstanding centrifugation forces greater than 160g. Hypoxia-induced adhesion was inhibited by LFA-1-specific (CD11a/CD18 integrin) antibodies, but not by antibodies directed against the ICAM-1 ligand for the LFA-1 receptor. These studies demonstrate that decreases in oxygen tension alone increase the adhesive properties of endothelial cells for leukocytes. In addition, they provide evidence for the existence of a new ligand for the LFA-1 molecule on endothelial cells which can be affected by hypoxic environments.

Hypoxia induces lymphocyte adhesion to human mesenchymal cells via an LFA-1-dependent mechanism.

We and others have previously reported that mesenchymal cells, including endothelial and muscle cells, sense oxygen tension and respond in a specific way during exposure to hypoxic environment. We have examined the interactions of human muscle and endothelial cells, which have been exposed to hypoxic environments, with T and B lymphoid cell lines and peripheral blood lymphocytes (PBL), not subjected to hypoxia. The adhesion of B lymphocyte cell line (JY) and the adhesion of T lymphocyte cell line (Jurkat) to muscle cell monolayers that had been incubated at PO2 of 50 Torr for 3 h increased more than four- and twofold, respectively. Hypoxia appears to upregulate a saturable muscle cell-associated adhesion mechanism, which is capable of withstanding distraction forces greater than 45 g, and is inhibitable by LFA-1-specific monoclonal antibodies (MAbs). Hypoxia also induced a reciprocal decrease in lymphocyte-muscle cell adhesion mechanisms inhibitable by VCAM-1- or VLA-4-specific MAbs. Cultured human endothelial cells when subjected to hypoxic conditions also increased their adhesion for lymphoid cells and cell lines. This induction of adhesion could again be attenuated by anti-LFA-1, but not by anti-ICAM-1 MAb, suggesting that hypoxia activates an adhesion molecule on human mesenchymal cells that is likely to be a new ligand for LFA-1. This report is the first demonstration of a direct induction of cell adhesion mechanisms by hypoxic environments.

Comparison of actin changes and calcium metabolism in plastic- and fibronectin-adherent human neutrophils.

Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.

Activation mechanisms of adherent human neutrophils.

The mechanism by which unstimulated human neutrophils initiate a respiratory burst on adherence to a surface has been examined. When neutrophils adhere to a plastic surface, they immediately generate a sustained burst of superoxide (O2-). However, this respiratory burst is not initiated by adherence alone, since neutrophils attached to fibronectin fail to mount a response. Adhesion to plastic is calcium (Ca2+) independent, but O2- production requires Ca2(+)-containing buffer in the initiation phase, that is, during adhesion and the early phase of O2- production. The Ca2(+)-dependent step was shown to involve protein kinase C (PK-C) in that the O2- production, but not adherence, was blocked with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and PK-C was found to translocate from the cytosol to the membrane on adhesion. Furthermore, it may be inferred that this translocation results in the generation of a Ca2+ independent form of PK-C, PK-M, since leupeptin, which inhibits the generation of PK-M, also blocked O2- production. This finding was corroborated by showing that after 5 minutes in a Ca2(+)-containing buffer, enough time to initiate O2- production and PK-C translocation, Ca2+ is no longer required for sustained O2- release. These results, in aggregate, demonstrate that neutrophils are activated by adhesion to plastic to generate O2-, a PK-C-dependent process that appears to involve a Ca2(+)-independent form of the kinase, PK-M. Why adherent neutrophils generate a respiratory burst on plastic and not fibronectin surfaces probably reflects activation of distinct receptors, whose nature must still be defined. Another issue to address is the priming effect of adhesion, since cells adherent to plastic- or fibronectin-coated surfaces have an enhanced O2- response to formylmethionyl-leucine-phenylalanine (FMLP) compared with neutrophils stimulated in suspension. This may relate to increased Ca2+ mobilization, an important mediator of priming for FMLP responses. Thus, adhesion as a priming event does not necessarily initiate cell effector function, and the further elucidation of the plastic and fibronectin models suggests a means of characterizing the crucial event that control neutrophil activation.

Defective control of cytoplasmic calcium concentration in T lymphocytes from old mice.

Cytoplasmic calcium concentration ([Ca]i) rises within minutes of exposure of T lymphocytes to a mitogen. T cells from old mice are defective in this reaction, a defect that could reflect either altered signal transduction or instead a more general age-associated change in intracellular calcium regulation. We therefore tested the ability of T cells from old mice to regulate their [Ca]i concentration after exposure to low concentrations of ionomycin, an agent that raises [Ca]i but bypasses receptor-mediated signal transduction mechanisms. Exposure of T cells to ionomycin leads to an abrupt increase in [Ca]i followed by stabilization at a dose-dependent plateau level that is affected by extracellular EGTA, by calmodulin inhibitors, and by modulators of protein kinase C. Plateau levels of [Ca]i after ionomycin challenge were consistently lower in T cells from old mice than in T cells from young mice. Flow cytometric experiments showed that while essentially all T cells from both old and young mice responded to ionomycin, they did so to an extent that depended on donor age. The age-dependent increase in resistance to ionomycin-induced changes in [Ca]i cannot be attributed to diminished membrane permeability to the ionomycin-calcium complex. The data suggest that aging may lead, in T lymphocytes, to a relative resistance to increases in [Ca]i, a resistance that in turn prevents cell activation.