High Intracellular Calcium Levels Research Articles

Palliative Treatment of Esophageal Cancer Using Calcium Electroporation.

Calcium electroporation (CaEP) is a novel cancer therapy wherein high intracellular calcium levels, facilitated by reversible electroporation, trigger tumor necrosis. This study aimed to establish safety with CaEP within esophageal cancer. Patients with non-curable esophageal cancer were included at Copenhagen University Hospital Rigshospitalet in 2021 and 2022. In an outpatient setting, calcium gluconate was injected intratumorally followed by reversible electroporation applied with an endoscopic electrode. The primary endpoint was the prevalence of adverse events, followed by palliation of dysphagia. All patients were evaluated with CT and upper endoscopies up to two months after treatment. The trial was registered at ClinicalTrials.gov (NCT04958044). Eight patients were treated. One serious adverse event (anemia, requiring a single blood transfusion) and three adverse events (mild retrosternal pain (two) and oral thrush (one)) were registered. Initially, six patients suffered from dysphagia: two reported dysphagia relief and four reported no change. From the imaging evaluation, one patient had a partial response, three patients had no response, and four patients had progression. Six months after treatment, the patient who responded well was still in good condition and without the need for further oncological treatment. CaEP was conducted in eight patients with only a few side effects. This study opens the way for larger studies evaluating tumor regression and symptom palliation.

Induced pluripotent stem cell-derived motor neurons from amyotrophic lateral sclerosis (ALS) patients carrying different superoxide dismutase 1 mutations recapitulate pathological features of ALS.

Background:Investigations of the pathogenic mechanisms in motor neurons (MNs) derived from amyotrophic lateral sclerosis (ALS) disease-specific induced pluripotent stem (iPS) cell lines could improve understanding of the issues affecting MNs. Therefore, in this study we explored mutant superoxide dismutase 1 (SOD1) protein expression in MNs derived from the iPS cell lines of ALS patients carrying different SOD1 mutations.Methods:We generated induced pluripotent stem cell (iPSC) lines from two familial ALS (FALS) patients with SOD1-V14M and SOD1-C111Y mutations, and then differentiated them into MNs. We investigated levels of the SOD1 protein in iPSCs and MNs, the intracellular Ca2+ levels in MNs, and the lactate dehydrogenase (LDH) activity in the process of differentiation into the MNs derived from the controls and ALS patients’ iPSCs.Results:The iPSCs from the two FALS patients were capable of differentiation into MNs carrying different SOD1 mutations and differentially expressed MN markers. We detected high SOD1 protein expression and high intracellular calcium levels in both the MN and iPSCs that were derived from the two SOD1 mutant patients. However, at no time did we observe stronger LDH activity in the patient lines compared with the control lines.Conclusions:MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis, providing a cell-based disease model to further elucidate disease pathogenesis and explore gene repair coupled with cell-replacement therapy. Incremental mutant expressions of SOD1 in MNs may have disrupted MN function, either causing or contributing to the intracellular calcium disturbances, which could lead to the occurrence and development of the disease.

A mechanism for learning with sleep spindles.

Spindles are ubiquitous oscillations during non-rapid eye movement (NREM) sleep. A growing body of evidence points to a possible link with learning and memory, and the underlying mechanisms are now starting to be unveiled. Specifically, spindles are associated with increased dendritic activity and high intracellular calcium levels, a situation favourable to plasticity, as well as with control of spiking output by feed-forward inhibition. During spindles, thalamocortical networks become unresponsive to inputs, thus potentially preventing interference between memory-related internal information processing and extrinsic signals. At the system level, spindles are co-modulated with other major NREM oscillations, including hippocampal sharp wave-ripples (SWRs) and neocortical slow waves, both previously shown to be associated with learning and memory. The sequential occurrence of reactivation at the time of SWRs followed by neuronal plasticity-promoting spindles is a possible mechanism to explain NREM sleep-dependent consolidation of memories. This article is part of the Theo Murphy meeting issue 'Memory reactivation: replaying events past, present and future'.

Cryotolerance of Stallion Spermatozoa Relies on Aquaglyceroporins rather than Orthodox Aquaporins.

Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen–thawed samples. In the presence of AC, different parameters were altered (p < 0.05), but not in a concentration- or time-depending manner. PHL was found to decrease sperm motility, viability, acrosome integrity, and the percentages of spermatozoa with low membrane lipid disorder, high mitochondrial membrane potential (MMP) and high intracellular levels of calcium and superoxides (p < 0.05). Finally, the sperm motility, viability, acrosome integrity, the percentages of spermatozoa with low membrane lipid disorder, high MMP and high intracellular calcium levels were higher (p < 0.05) in PDO treatments than in the control. The sperm response to AC, PHL and PDO indicates that GLPs, rather than orthodox AQPs, play a crucial role during stallion sperm cryopreservation. Furthermore, post-thaw sperm quality was higher in PDO treatments than in the control, suggesting that this molecule is a potential permeable cryoprotectant.

Lead exposure-induced cognitive impairment through RyR-modulating intracellular calcium signaling in aged rats

Lead is widely distributed in the environment and has become a global public health issue. It is well known that lead exposure induces not only neurodevelopmental toxicity but also neurodegenerative diseases, with learning and memory impairment in the later stage. However, the molecular mechanisms remain elusive. The present study investigated the effects of early life and lifetime lead exposure on cognition and identified the molecular mechanisms involved in aged rats. The results herein demonstrated that the lead concentration in peripheral blood and brain tissues in aged rats was significantly increased in a lead dose-dependent manner. High-dose lead exposure caused cognitive functional impairment in aged rats, concomitant with a longer escape latency and a lower frequency of crossing the platform via Morris water maze testing compared to those in the control and low-dose lead exposure groups. Importantly, neuron functional defects were still observed even in early life lead exposure during the prenatal and weaning periods in aged rats. The neurotoxicity induced by lead exposure was morphologically evidenced by a recessed nuclear membrane, a swollen endoplasmic reticulum, and mitochondria in the neurons. Mechanistically, the exposure of aged rats to lead resulted in increasing free calcium concentration, reactive oxygen species, and apoptosis in the hippocampal neurons. Lead exposure increased RyR3 expression and decreased the levels of p-CaMKIIα/CaMKIIα and p-CREB/CREB in the hippocampus of aged rats. These findings indicated that early life lead exposure-induced cognition disorder was irreversible in aged rats. Lead-induced neurotoxicity might be related to the upregulation of RyR3 expression and high levels of intracellular free calcium with increasing lead concentration in injured neurons.

Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils

Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.

What happens to the unsuccessful spermatozoa?

To study apoptosis as a functional pathway in mature spermatozoa and apoptosis correlated to the acrosome reaction via the intracellular calcium concentration, semen samples from 27 healthy human donors were treated with inducers of apoptosis (betulinic acid, thapsigargin), inducers of the acrosome reaction (thapsigargin, calcium ionophore) or hydrogen peroxide to produce reactive oxygen species with and without prior incubation with a calcium chelator. Computer-assisted sperm analysis, flow cytometry, and transmission electron microscopy were performed to analyze changes in the acrosomal status and in apoptotic features. Betulinic acid, thapsigargin, and the calcium ionophore treatment resulted in an increased number of sperm cells with caspase 9 and caspase 3 activation, disrupted mitochondrial membrane potential, and a reacted acrosome. Sperm motility was decreased in all cases. Transmission electron analyses showed ultra-morphological changes, such as membrane integrity, membrane blebbing, the formation of head vacuoles, defects of the nuclear envelope, nuclear fragmentation, and the acrosome reaction. Acrosome reaction and apoptotic features decreased due to the reduction in intracellular calcium by the calcium chelator NP-EGTA, AM. Therefore, apoptotic cell death in acrosome-reacted sperm cells mediated by high intracellular calcium levels is possible.

An erythroid-specific ATP2B4 enhancer mediates red blood cell hydration and malaria susceptibility.

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.

Multiple Kinases Involved in the Nicotinic Modulation of Gamma Oscillations in the Rat Hippocampal CA3 Area.

Neuronal synchronization at gamma band frequency (20–80 Hz, γ oscillations) is closely associated with higher brain function, such as learning, memory and attention. Nicotinic acetylcholine receptors (nAChRs) are highly expressed in the hippocampus, and modulate hippocampal γ oscillations, but the intracellular mechanism underlying such modulation remains elusive. We explored multiple kinases by which nicotine can modulate γ oscillations induced by kainate in rat hippocampal area CA3 in vitro. We found that inhibitors of cyclic AMP dependent kinase (protein kinase A, PKA), protein kinase C (PKC), N-methyl-D-aspartate receptor (NMDA) receptors, Phosphoinositide 3-kinase (PI3K) and extracellular signal-related kinases (ERK), each individually could prevent the γ oscillation-enhancing effect of 1 μM nicotine, whereas none of them affected baseline γ oscillation strength. Inhibition of the serine/threonine kinase Akt increased baseline γ oscillations and partially blocked its nicotinic enhancement. We propose that the PKA-NMDAR-PI3K-ERK pathway modifies cellular properties required for the nicotinic enhancement of γ oscillations, dependent on a PKC-ERK mediated pathway. These signaling pathways provide clues for restoring γ oscillations in pathological conditions affecting cognition. The suppression of γ oscillations at 100 μM nicotine was only dependent on PKA-NMDAR activation and may be due to very high intracellular calcium levels.

An Automated Perifusion System for Modifying Cell Culture Conditions over Time.

BackgroundCells are continuously exposed to changes in their environment. Endocrine systems, in particular, communicate by rhythms and feedback loops. In this study, we developed an automated system to produce such conditions for cultured cells in a precisely timed manner. We utilized a programmable pair of syringe pumps for inflow and a peristaltic pump for outflow to create rhythmic pulses at 5-min intervals in solutions that mimic the endogenous patterns of insulin produced by pancreatic islets as a test case.ResultsThis perifusion system was first tested by measuring trypan blue absorbance, which was intermittently added and washed out at 3:3 and 2:3 min (in:out). Absorbance corresponded with patterns of trypan blue delivery. We then created patterns of forced oscillations in islets by intermittently switching between solutions containing 28 millimolar (mM) glucose (producing high levels of intracellular calcium ([Ca2+]i) and insulin secretion) and 28 mM glucose + calcium-channel blocker nifedipine (producing low levels of [Ca2+]i and insulin secretion). Forced perifusion effects were monitored by fura-2 AM fluorescence measurements of [Ca2+]i. Islets showed uniform oscillations in [Ca2+]i at time intervals consistent with the perifusion pattern, mimicking endogenous pulsatility.ConclusionsThis study highlights a valuable method to modify the environment of the cell culture over a period of hours to days.

On the importance of Bloom number of gelatin to the development of biodegradable in situ gelling copolymers for intracameral drug delivery

To overcome the drawbacks associated with conventional antiglaucoma eye drops, this work demonstrated the feasibility of an effective alternative strategy to administer pilocarpine directly via intracameral injections of drug-containing biodegradable in situ gelling GN copolymers composed of gelatin and poly(N-isopropylacrylamide). Specifically, this study aims to understand the importance of Bloom number of gelatin, a physicochemical parameter, to the development of GN carriers for intracameral drug delivery in glaucoma therapy. Our results showed that both imino acid and triple-helix contents increased with increasing Bloom index from 75⿿100 to 300. The drug encapsulation efficiency in response to temperature-triggered phase transition in GN copolymers was affected by the Bloom index of gelatin. In addition, the differences in protein secondary structure significantly influenced the degradation rates of GN carriers, which were highly correlated with drug release profiles. The increase in released pilocarpine concentration led to a high intracellular calcium level in rabbit ciliary smooth muscle cell cultures, indicating a beneficial pharmacological response to a drug. Irrespective of Bloom number of gelatin, all carrier materials exhibited excellent in vitro and in vivo biocompatibility with corneal endothelium. In a glaucomatous rabbit model, intracameral injections of pilocarpine-containing GN synthesized from gelatins with various Bloom numbers had different abilities to improve ocular hypertension and induce pupillary constriction, indicating distinct antiglaucoma efficacies due to in vivo drug release. It is concluded that the effects on pharmacological treatment using GN carriers for intracameral pilocarpine administration demonstrate a strong dependence on the Bloom number of gelatin.

Blockade of store-operated calcium entry alleviates high glucose-induced neurotoxicity via inhibiting apoptosis in rat neurons

Altered store-operated calcium entry (SOCE) has been suggested to be involved in many diabetic complications. However, the association of altered SOCE and diabetic neuronal damage remains unclear. This study aimed to investigate the effects of altered SOCE on primary cultured rat neuron injury induced by high glucose. Our data demonstrated that high glucose increased rat neuron injury and upregulated the expression of store-operated calcium channel (SOC). Inhibition of SOCE by a pharmacological inhibitor and siRNA knockdown of stromal interaction molecule 1 weakened the intracellular calcium overload, restored mitochondrial membrane potential, downregulated cytochrome C release and inhibited cell apoptosis. As well, treatment with the calcium chelator BAPTA-AM prevented cell apoptosis by ameliorating the high glucose-increased intracellular calcium level. These findings suggest that SOCE blockade may alleviate high glucose-induced neuronal damage by inhibiting apoptosis. SOCE might be a promising therapeutic target in diabetic neurotoxicity.

PINK1 and Parkin cooperatively protect neurons against constitutively active TRP channel-induced retinal degeneration in Drosophila.

Calcium has an important role in regulating numerous cellular activities. However, extremely high levels of intracellular calcium can lead to neurotoxicity, a process commonly associated with degenerative diseases. Despite the clear role of calcium cytotoxicity in mediating neuronal cell death in this context, the pathological mechanisms remain controversial. We used a well-established Drosophila model of retinal degeneration, which involves the constitutively active TRPP365 channels, to study calcium-induced neurotoxicity. We found that the disruption of mitochondrial function was associated with the degenerative process. Further, increasing autophagy flux prevented cell death in TrpP365 mutant flies, and this depended on the PINK1/Parkin pathway. In addition, the retinal degeneration process was also suppressed by the coexpression of PINK1 and Parkin. Our results provide genetic evidence that mitochondrial dysfunction has a key role in the pathology of cellular calcium neurotoxicity. In addition, the results demonstrated that maintaining mitochondrial homeostasis via PINK1/Parkin-dependent mitochondrial quality control can potentially alleviate cell death in a wide range of neurodegenerative diseases.

Interrelationship between Changes in the Amyloid β 42/40 Ratio and Presenilin 1 Conformation.

The ratio of the longer (i.e., Aβ42/Aβ43) to shorter (i.e. Aβ40) species is a critical factor determining amyloid fibril formation, neurotoxicity and progression of the amyloid pathology in Alzheimer's disease. The relative levels of the different Aβ species are affected by activity and conformation of the γ-secretase complex catalytic component - presenilin 1 (PS1). The enzyme exists in a dynamic equilibrium of the conformational states, with so-called "close" conformation associated with the shift of the γ-secretase cleavage towards the production of longer, neurotoxic Aβ species. In the current study, fluorescence lifetime imaging microscopy, spectral Förster resonance energy transfer, calcium imaging and cytotoxicity assays were utilized to explore reciprocal link between the Aβ42 and Aβ40 peptides present at various ratios and PS1 conformation in primary neurons. We report that exposure to Aβ peptides at a relatively high ratio of Aβ42/40 causes conformational change within the PS1 subdomain architecture towards the pathogenic "closed" state. Mechanistically, the Aβ42/40 peptides present at the relatively high ratio increase intracellular calcium levels, which were shown to trigger pathogenic PS1 conformation. This indicates that there is a reciprocal crosstalk between the extracellular Aβ peptides and PS1 conformation within a neuron, with Aβ40 showing some protective effect. The pathogenic shift within the PS1 domain architecture may further shift the production of Aβ peptides towards the longer, neurotoxic Aβ species. These findings link elevated calcium, Aβ42 and PS1/γ-secretase conformation, and offer possible mechanistic explanation of the impending exacerbation of the amyloid pathology.

Effects of ApoE on intracellular calcium levels and apoptosis of neurons after mechanical injury

Objective: The current study aimed to explore the effects of apolipoprotein e (ApoE) on intracellular calcium ([Ca2+]i) and apoptosis of neurons after mechanical injury in vitro. Methods: A neuron mechanical injury model was established after primary neurons obtained from APOE knockout and wild-type (WT) mice, and four experimental groups were generated: Group-ApoE4, Group-ApoE3, Group-ApoE(−) and Group-WT. Recombinant ApoE4 and ApoE3 were added to Group-ApoE4 and Group-ApoE3 respectively, and Group-ApoE(−) and Group-WT were control groups. Intracellular calcium was labeled by fluo-3/AM and examined using laser scanning confocal microscope and flow cytometry, and the apoptosis of neurons was also evaluated. Results: The intracellular calcium levels and apoptosis rates of mice neurons were significantly higher in Group-ApoE4 than in Group-ApoE3 and Group-WT after mechanical injury. However, without mechanical injury on neurons, no significant differences in intracellular calcium levels and apoptosis rates were found among all four experimental groups. The effects of ApoE4 on intracellular calcium levels and apoptosis rates of injured neurons were partly decreased by EGTA treatment. Conclusion: Compared with ApoE3-treatment and WT neurons, ApoE4 caused higher intracellular calcium levels and apoptosis rates of neurons after mechanical injury. This suggested APOE polymorphisms may affect neuron apoptosis after mechanical injury through different influences on intracellular calcium levels.

Myocardial protection using del nido cardioplegia solution in adult reoperative aortic valve surgery.

The immediate postischemic period is marked by elevated intracellular calcium levels, which can lead to irreversible myocyte injury. Del Nido cardioplegia was developed for use in the pediatric population to address the inability of immature myocardium to tolerate high levels of intracellular calcium following cardiac surgery. Our aim in this study is to determine if this solution can be used safely and effectively in an adult, reoperative population. All patients undergoing isolated reoperative aortic valve replacement at our institution from 2010 to 2012 were retrospectively reviewed. Demographics, comorbidities, operative variables, postoperative complications, and patient outcomes were collected. Patients were divided into two groups based on cardioplegia strategy used: whole blood cardioplegia (WB, n = 61) and del Nido cardioplegia (DN, n = 52). Mean age in the study was 73.4 ± 14.3 years and 86 patients were male (76.1%). Eighty-four patients had undergone prior coronary artery bypass graft (CABG) (74.3%). Patients in the DN group required significantly lower total volume of cardioplegia (1147.6 ± 447.2 mL DN vs. 1985.4 ± 691.1 mL WB, p < 0.001) and retrograde cardioplegia dose (279.3 ± 445.1 mL DN vs. 1341.2 ± 690.8 mL WB, p < 0.001). There were no differences in cross-clamp time, bypass time, postoperative complication rate, or patient outcomes between groups. Del Nido cardioplegia use in an adult, reoperative aortic valve population offers equivalent postoperative outcomes when compared with whole blood cardioplegia. In addition, use of del Nido solution requires lower total and retrograde cardioplegia volumes in order to achieve adequate myocardial protection.

Elevated maxi-K+ ion channel current in glaucomatous lamina cribrosa cells

The connective tissue plates of the lamina cribrosa (LC) region are continuously exposed to a mechanically dynamic environment. To study how the LC cells respond to these mechanical forces, we measured the mechano-sensitive calcium dependent maxi-K+ ion channel current in the cell membrane of LC cells of glaucoma and normal subjects. Primary culture LC cells from 7 normal and 7 age matched glaucoma donors were studied. Perfusion of cells with hypotonic solution was used to stretch the cell membrane. Whole-cell patch-clamp technique was used to measure the basal (non stretched) and hypotonic stretch-induced changes in maxi-K+ ion channel activity in normal and glaucoma LC cells. The role of membrane-type Ca2+ entry channel inhibition (verapamil) and internal Ca2+ store re-uptake blockade (2-APB) on maxi-K+ activity was also examined. Basal and stretched-induced maxi-K+ current were significantly elevated in the glaucoma LC cells compared to normal controls (p < 0.05). In normal LC cells hypotonic stretch elevated the mean maxi-K+ current from 18.5 ± 5.7 pA/pF (at Vp = +100 mV) to 88.4 ± 12.4 pA/pF (P < 0.05), and from 39.5 ± 7.3 pA/pF to 133.1 ± 18.5 pA/pF in glaucoma LC cells (P < 0.02). Verapamil and 2-APB significantly reduced basal maxi-K+ current in glaucoma LC cells (33.1 ± 8.2 pA/pF to 17.9 ± 5.6 pA/pF; and 32.2 ± 8.3 pA/pF to 17.3 ± 5.4 pA/pF, P < 0.05, respectively) but not in normal LC cells (P > 0.05). Following hypotonic stretch, verapamil and 2-APB significantly (P < 0.05) reduced the maxi-K+ current in both normal and glaucoma LC cells. Baseline and hypotonic stretch induced Ca2+-dependent maxi-K+ channel activity are elevated in LC cells of glaucoma patients, which may result from the abnormally high levels of intracellular calcium in glaucoma LC cells.

A Case of Cocaine-Induced Myopathy

A Case of Cocaine-Induced Myopathy

Delayed pharyngeal repolarization promotes abnormal calcium buildup in aging muscle

In the pharynx of Caenorhabditis elegans, the accessory subunit MPS-4, homolog to human KCNE1, forms a complex with K+ channel EXP-2 that terminates the action potential. An aspartate residue critical for KCNE1 function, asp76, is conserved in MPS-4 (asp74). Here, we studied the effects of D74N-MPS-4 on the aging pharynx. Electrophysiological studies showed that D74N delays pharyngeal repolarization. Pharynxes of transgenic worms expressing D74N exhibited higher levels of intracellular calcium compared to normal pharynxes. Accordingly, loss of pharyngeal function was accelerated in aging D74N worms. The pharyngeal action potential resembles the action potential that controls the mechanical activity of human left ventricle. Hence, these findings argue that the hearts of patients affected by delayed repolarization, a condition known as long QT syndrome, may experience dysregulated calcium homeostasis.

Macrophage Recognition of Cells with Elevated Calcium Is Mediated by Carbohydrate Chains of CD43

Macrophages remove deteriorating cells (those undergoing apoptosis and oxidation) via poly-N-acetyllactosaminyl chains on CD43 caps, a major cell-surface glycoprotein. Unusually high intracellular calcium levels are also deteriorating for cells and tissue. Here we artificially elevated calcium levels in cells and examined the mechanism by which this elevation was resolved by macrophages. Results showed that treatment with the calcium ionophore A23187 and ionomycin induces capping of CD43 on Jurkat cells, which are subsequently recognized and phagocytosed by macrophages, indicating that macrophages regard cells with elevated calcium as targets for removal. Further tests showed that A23187- and ionomycin-treated Jurkat cells did not induce apoptotic changes such as DNA fragmentation or phosphatidylserine expression, indicating that these cells were removed despite still being viable. Jurkat cells pretreated with anti-CD43 antibody or those with poly-N-acetyllactosaminyl chains containing oligosaccharides inhibited macrophage binding, indicating that macrophages recognize the poly-N-acetyllactosaminyl chains on CD43. Binding was also inhibited by treating macrophages with anti-nucleolin antibody, indicating that recognition occurs through nucleolin, a cell-surface receptor. Further, nucleolin-transfected HEK293 cells bound A23187-treated cells, and this binding was inhibited by in the presence of oligosaccharides. Taken together, these results show that elevated calcium levels induce CD43 capping, and macrophages remove the cells if their nucleolin receptors can bind to the poly-N-acetyllactosaminyl chains of capped CD43.