Pivotal role of mitochondrial Na⁺₋Ca²âº exchange in antigen receptor mediated Ca²âº signalling in DT40 and A20 B lymphocytes.

Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) increases upon activation of antigen-receptor in lymphocytes. Mitochondria have been suggested to regulate the [Ca(2+)](i) response, but the molecular mechanisms and the roles are poorly understood. To clarify them, we carried out a combination study of mathematical simulations and knockout or knockdown of NCLX, a gene candidate for the mitochondrial Na(+)-Ca(2+) exchanger (NCX(mit)), in B lymphocytes. A mathematical model of Ca(2+) dynamics in B lymphocytes demonstrated that NCX(mit) inhibition reduces basal Ca(2+) content of endoplasmic reticulum (ER) and suppresses B-cell antigen receptor (BCR)-mediated [Ca(2+)](i) rise. The predictions were validated in DT40 B lymphocytes of heterozygous NCLX knockout (NCLX(+/-)). In NCLX(+/-) cells, mitochondrial Ca(2+) efflux via NCX(mit) was strongly decelerated, suggesting NCLX is a gene responsible for NCX(mit) in B lymphocytes. Consistent with the predictions, ER Ca(2+) content declined and [Ca(2+)](i) hardly rose upon BCR activation in NCLX(+/-) cells. ER Ca(2+) uptake was reduced to ∼58% of the wild-type (WT), while it was comparable to WT when mitochondrial respiration was disturbed. Essentially the same results were obtained by a pharmacological inhibition or knockdown of NCLX by siRNA in A20 B lymphocytes. Unexpectedly, ER Ca(2+) leak was augmented and co-localization of mitochondria with ER was lower in NCLX(+/-) and NCLX silenced cells. Taken together, we concluded that NCLX is a key Ca(2+) provider to ER, and that NCLX-mediated Ca(2+) recycling between mitochondria and ER is pivotal in B cell responses to antigen.

Inhibitory effects of purified antibody against α-1 repeat (117-137) on Na(+)-Ca(2+) exchange and L-type Ca(2+) currents in rat cardiomyocytes.

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).

Stimulation of Na+-Ca2+ exchange by purified antibody against alpha-2 repeat of Na+-Ca2+ exchanger in rat cardiomyocytes.

AIM: The aim of the present study was to investigate the effect of the antibody against alpha-2 repeat on Na+-Ca2+ exchanger (NCX) current (I(Na/Ca)). To evaluate the functional specificity of this antibody, its effects on L-type Ca2+ current (I(Ca,L)), voltage-gated Na+ current (I(Na)) and delayed rectifier K+ current (I(K)) were also observed. METHODS: The whole-cell patch-clamp technique was used in this study. RESULTS: The antibody against alpha-2 repeat augmented both the outward and inward Na+-Ca2+ exchanger current concentration-dependently, with EC(50) values of 27.9 nmol/L and 24.7 nmol/L, respectively. Meanwhile, the antibody could also increase I(Ca,L) in a concentration-dependent manner with the EC(50) of 33.6 nmol/L. Effects of the antibody on I(Na) and I(K) were not observed in the present study. CONCLUSION: The present results suggest that antibody against alpha-2 repeat is a stimulating antibody to NCX and could also increase I(Ca,L) in a concentration-dependent manner, but did not have an obvious effect on I(Na) and I(K).

Asymmetric distribution of functional sodium-calcium exchanger in primary osteoblasts.

To understand calcium translocation in osteoblasts, we have determined the location of sodium-calcium (Na-Ca) exchanger (NCX) in relation to actin and alpha-tubulin in primary cultures of avian osteoblasts. Osteoblasts derived from the periosteal surface of tibias from growing chickens were cultured for 8 days in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Lysates immunoblotted with antibodies raised against the canine cardiac Na-Ca antibodies revealed a 70 kDa exchanger protein. Cross-reactivity of the anti-NCX antibody was confirmed by enriching for NCX in protein samples derived from plasma membrane vesicles by affinity chromatography using the exchanger inhibitory peptide. Fractions enriched for the exchanger were eluted from the column and subjected to immunoblotting with the anti-NCX antibody, revealing an intense single band at 70 kDa. Examination of live cells loaded with Calcium Green-1 AM ester by confocal microscopy demonstrated sodium-dependent calcium uptake, confirming the presence of functional NCX in intact cells. Immunolocalization studies of osteoblasts stained with anti-NCX antibodies revealed asymmetric localization of the exchanger in cultured osteoblasts, residing almost entirely within two 0.5-microm optical sections along the substrate adherent side of the cells. Since NCX is known to be a low-affinity, high-capacity calcium translocating molecule and also appears to be asymmetrically positioned, it is likely to play a key role in bone formation.

Alterations in Na+/H+ exchanger and Na+/HCO3- cotransporter immunoreactivities within the gerbil hippocampus following seizure.

In this study, a chronological and comparative analysis of the immunoreactivities of Na(+)/H(+) exchanger 1 (NHE1), Na(+)/HCO(3)(-) cotransporter (NBC) and Na(+)/Ca(2+) exchanger (NCE) was conducted in order to identify the effects of spontaneous seizure on their protein expression levels using the gerbil model. The distribution of NHE1 and NBC immunoreactivity in the hippocampus of seizure-resistant (SR) gerbils was similar to that observed in the pre-seizure group of seizure-sensitive (SS) gerbils. From 30 min to 3 h after the onset of the seizure, both NHE1 and NBC immunoreactivities were elevated in the hippocampus, as compared to the pre-seizure group of SS gerbils. At 6 h postictal, these immunoreactivities in the hippocampus had reduced to the pre-seizure level. However, NCE immunoreactivity within the hippocampus was unaltered. These findings suggest that the changes in both NHE1 and NBC immunoreactivity within the hippocampus following seizure may affect tissue excitability and play a role in the reduction of the seizure activity in the gerbil.

Immunolocalization of the Na(+)-Ca2+ exchanger in mammalian myelinated axons.

Previous studies on the pathophysiology of white matter anoxic injury have revealed that the Na(+)-Ca2+ exchanger is an important mediator of Ca2+ overload. To date, however, the localization of this key Ca2+ transporter in myelinated axons has not been demonstrated. The present study uses immunofluorescence labeling with a monoclonal antibody (R3F1) to the canine cardiac type I Na(+)-Ca2+ exchanger to localize exchanger protein to rat peripheral and central myelinated axons. The indirect immunofluorescence labeling technique was used to study paraformaldehyde fixed frozen cryostat sections of sciatic nerve, optic nerve and spinal cord. Examination of sciatic nerve sections with both conventional and confocal microscopy revealed a staining pattern which suggested both a glial and axonal localization of the exchanger. In the rat optic nerve, positive label was associated with cell bodies and their processes, likely glia, and with numerous finer processes arranged in parallel, running longitudinally. These finer processes likely represent axonal profiles. A similar staining pattern was observed in lateral and dorsal columns from spinal cord. Immunoelectron microscopy of dorsal root axons revealed gold particles associated with the paranodal and internodal myelin, in the axoplasm, and close to the nodal/paranodal axon membrane. The high density of Na(+)-Ca2+ exchanger demonstrated in central and peripheral myelinated mammalian axons supports the importance of this transporter in Ca2+ regulation in these tissues.

Oxidative stress-induced up-regulation of the chloride channel and Na+/Ca2+ exchanger during cataractogenesis in diabetic rats.

We have determined the abundance of the chloride channel, ClC-3, and Na(+)/Ca(2+) exchanger proteins in isolated rat lens cortex fiber cells by immunofluorescence method using polyclonal anti-ClC-3 antibodies and monoclonal antibodies against the canine cardiac Na(+)/Ca(2+) exchanger protein. These proteins were also quantified in the lens cortex of streptozotocin-injected rats by Western blots. Also, mRNA for ClC-3 was determined by Northern blot analysis. The isolated rat lens cortical fibers expressed basal levels of ClC-3 and Na(+)/Ca(2+) exchanger proteins. As compared to controls, the ClC-3 protein in the lens cortex of diabetic rats (blood glucose>400 mg%) increased by 2.5-fold in 7 days and 4.5-fold in 14 days. However, the ClC-3 protein decreased to near-normal values in 40 days. The changes in ClC-3 mRNA closely followed the protein levels. Similarly, as compared to controls, on Day 7, the Na(+)/Ca(2+) exchanger protein in the diabetic rat lens cortex increased by 3.5-fold and on Day14 by 5.5-fold. Subsequently, it decreased to control levels on Day 40. Treatment with the antioxidant, Trolox (2 mg/kg body weight), prevented the initial increase in ClC-3 and Na(+)/Ca(2+) exchanger proteins. The up-regulation of ClC-3 and Na(+)/Ca(2+) exchanger proteins during the early stages of diabetes and its prevention by antioxidants suggests that the proteins regulating ion transport may have a pathophysiological role in the development of diabetic cataracts.

Modulation of Ca2+ signaling by Na+/Ca2+ exchangers in mast cells.

Mast cells rely on Ca(2+) signaling to initiate activation programs leading to release of proinflammatory mediators. The interplay between Ca(2+) release from internal stores and Ca(2+) entry through store-operated Ca(2+) channels has been extensively studied. Using rat basophilic leukemia (RBL) mast cells and murine bone marrow-derived mast cells, we examine the role of Na(+)/Ca(2+) exchangers. Calcium imaging experiments and patch clamp current recordings revealed both K(+)-independent and K(+)-dependent components of Na(+)/Ca(2+) exchange. Northern blot analysis indicated the predominant expression of the K(+)-dependent sodium-calcium exchanger NCKX3. Transcripts of the exchangers NCX3 and NCKX1 were additionally detected in RBL cells with RT-PCR. The Ca(2+) clearance via Na(+)/Ca(2+) exchange represented approximately 50% of the total clearance when Ca(2+) signals reached levels > or =200 nM. Ca(2+) signaling and store-operated Ca(2+) entry were strongly reduced by inverting the direction of Na(+)/Ca(2+) exchange, indicating that Na(+)/Ca(2+) exchangers normally extrude Ca(2+) ions from cytosol and prevent the Ca(2+)-dependent inactivation of store-operated Ca(2+) channels. Working in the Ca(2+) efflux mode, Na(+)/Ca(2+) exchangers such as NCKX3 and NCX3 might, therefore, play a role in the Ag-induced mast cell activation by controlling the sustained phase of Ca(2+) mobilization.

Characterization of the 70 kDa polypeptide of the Na/Ca exchanger.

The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1-21; NCX1, residues 393-406; and Exon F, residues 622-644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger.

Novel subcellular and molecular tools to study Ca(2+) transport mechanisms during the elusive moulting stages of crustaceans: flow cytometry and polyclonal antibodies.

Our understanding of calcium homeostasis during the crustacean moulting cycle derives from research on intermoult animals that has been extrapolated to other stages. In terms of transepithelial Ca(2+) flux, the more interesting stages are those surrounding ecdysis since crustaceans experience a sizeable negative calcium balance in immediate premoult and a significant positive calcium balance in immediate postmoult. These stages are elusive in the sense that larger species such as lobsters are rarely captured at this time, and smaller species such as blue crabs and crayfish are seldom synchronized in their moulting cycle. The reductionist approaches employed in cellular physiology, such as vesicle techniques, employ pooling of fresh tissues from many organisms. Examination of the elusive moulting stages requires more sensitive approaches that can utilize tissue from an individual crustacean to characterize Ca(2+) pumps (Sarco/Endoplasmic Reticulum Ca(2+)-ATPase, SERCA; Plasma Membrane Ca(2+)-ATPase, PMCA) and the Na(+)/Ca(2+) eXchanger (NCX). An emerging subcellular approach described in this paper is to use flow cytometry as a technique to monitor Ca(2+) uptake into Fluo-3-loaded membrane vesicles. This paper illustrates the utility of this technique for measuring ATP-dependent Ca(2+) uptake into hepatopancreatic basolateral membrane vesicles. Obstacles to progress in molecular studies have not been limited by synchronization of moulting since tissue can be snap-frozen and collected from many animals over time. Here, the problem has been the lack of specific antibodies that hybridize with the Ca(2+) transporters of interest so that they can be localized within epithelia. In this paper, we introduce polyclonal antibodies raised in rabbits against crayfish SERCA, PMCA and NCX. Immunocytochemistry of SERCA in muscle, PMCA in antennal gland and NCX in heart confirms the specificity of the antibodies.

Expression of Na(+)/Ca(2+) exchanger isoforms (NCX1 and NCX3) and plasma membrane Ca(2+) ATPase during osteoblast differentiation.

The ability to deliver calcium to the osteoid is critical to osteoblast function as a regulator of bone calcification. There are two known transmembrane proteins capable of translocating calcium out of the osteoblast, the Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). In this study, we reveal the presence of the NCX3 isoform in primary osteoblasts and examine the expression of NCX1, NCX3, and PMCA1 during osteoblast differentiation. The predominant NCX isoform expressed by osteoblasts is NCX3. NCX1 also is expressed, but at low levels. Both NCX isoforms are expressed at nearly static levels throughout differentiation. In contrast, PMCA expression peaks at 8 days of culture, early in osteoblast differentiation, but declines thereafter. Immunocytochemical co-detection of NCX and PMCA reveal that NCX is positioned along surfaces of the osteoblast adjacent to osteoid, while PMCA is localized to plasma membrane sites distal to the osteoid. The expression pattern and spatial distribution of NCX support a role as a regulator of calcium efflux from osteoblasts required for calcification. The expression pattern and spatial distribution of PMCA makes its role in the mineralization process unlikely and suggests a role in calcium homeostasis following signaling events.

Localization of thiazide-sensitive Na(+)-Cl(-) cotransport and associated gene products in mouse DCT.

The mammalian distal nephron develops a complex assembly of specialized cell types to accomplish the fine adjustment of urinary electrolyte composition. The epithelia of the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD) show an axial structural heterogeneity that has been functionally elucidated by the localization of proteins involved in transepithelial ion transport. We compared the distribution of the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC), basolateral Na(+)/Ca(2+) exchanger (Na/Ca), cytosolic calcium-binding proteins calbindin D(28K) and parvalbumin, and the key enzyme for selective aldosterone actions, 11 beta-hydroxysteroid-dehydrogenase 2 (11HSD2), in the distal convolutions of the mouse. In the mouse, as opposed to the rat, we found no clear subsegmentation of the DCT into a proximal (DCT1) and a distal (DCT2) portion. The TSC was expressed along the entire DCT. Na/Ca and calbindin D(28K) were similarly expressed along most of the DCT, with minor exceptions in the initial portion of the DCT. Both were also present in the CNT. Parvalbumin was found in the entire DCT, with an occasional absence from short end portions of the DCT, and was not present in CNT. 11HSD2 was predominantly located in the CNT and CCD. Short end portions of DCT only occasionally showed the 11HSD2 signal. We also observed an overlap of 11HSD2 immunoreactivity and mRNA staining. Our observations will have implications in understanding the physiological effects of gene disruption and targeting experiments in the mouse.

Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.

The organization of Na(+) and Ca(2+) transport pathways along the mouse distal nephron is incompletely known. We revealed by immunohistochemistry a set of Ca(2+) and Na(+) transport proteins along the mouse distal convolution. The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) characterized the distal convoluted tubule (DCT). The amiloride-sensitive epithelial Na(+) channel (ENaC) colocalized with NCC in late DCT (DCT2) and extended to the downstream connecting tubule (CNT) and collecting duct (CD). In early DCT (DCT1), the basolateral Ca(2+)-extruding proteins [Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PCMA)] and the cytoplasmic Ca(2+)-binding protein calbindin D(28K) (CB) were found at very low levels, whereas the cytoplasmic Ca(2+)/Mg(2+)-binding protein parvalbumin was highly abundant. NCX, PMCA, and CB prevailed in DCT2 and CNT, where we located the apical epithelial Ca(2+) channel (ECaC1). Its subcellular localization changed from apical in DCT2 to exclusively cytoplasmic at the end of CNT. NCX and PMCA decreased in parallel with the fading of ECaC1 in the apical membrane. All three of them were undetectable in CD. These findings disclose DCT2 and CNT as major sites for transcellular Ca(2+) transport in the mouse distal nephron. Cellular colocalization of Ca(2+) and Na(+) transport pathways suggests their mutual interactions in transport regulation.