An inhibitor of Na/Ca exchange blocks activation of insect olfactory receptors

Earlier we showed that the Na⁺/Ca²⁺ exchanger inhibitor, KB-R7943, potently blocks the odor-evoked activity of lobster olfactory receptor neurons. Here we extend that finding to recombinant mosquito olfactory receptors stably expressed in HEK cells. Using whole-cell and outside-out patch clamping and calcium imaging, we demonstrate that KB-R7943 blocks both the odorant-gated current and the odorant-evoked calcium signal from two different OR complexes from the malaria vector mosquito, Anopheles gambiae, AgOr48 + AgOrco and AgOr65 + AgOrco. Both heteromeric and homomeric (Orco alone) OR complexes were susceptible to KB-R7943 blockade when activated by VUAA1, an agonist that targets the Orco channel subunit, suggesting the Orco subunit may be the target of the drug’s action. KB-R7943 represents a valuable tool to further investigate the functional properties of arthropod olfactory receptors and raises the interesting specter that activation of these ionotropic receptors is directly or indirectly linked to a Na⁺/Ca²⁺ exchanger, thereby providing a template for drug design potentially allowing improved control of insect pests and disease vectors.     

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na/Ca exchange

Fluctuating extracellular Ca²⁺ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca²⁺ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O₂ consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca²⁺, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na⁺ and the subsequent activation of mitochondrial Na⁺/Ca²⁺ and Na⁺/H⁺ exchange. The mitochondrial Na⁺/Ca²⁺ exchanger (_mNCX) activated by Na⁺ influx reduces Ca²⁺ and increases Na⁺ levels in the mitochondrial matrix. The excess of Na⁺ activates the mitochondrial Na⁺/H⁺ exchanger (NHE) increasing the outward pumping of protons, electron transport and O₂ consumption. Reduction in extracellular Na⁺ and inhibition of Na⁺ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the _mNCX, L-type voltage gated Ca²⁺ channels or the release of Ca²⁺ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca²⁺ exchange is critical for response development.     

Searching for a Role of NCX/NCKX Exchangers in Neurodegeneration

Control of intracellular calcium signaling is essential for neuronal development and function. Maintenance of Ca²⁺ homeostasis depends on the functioning of specific transport systems that remove calcium from the cytosol. Na⁺/Ca²⁺ exchange is the main calcium export mechanism across the plasma membrane that restores resting levels of calcium in neurons after stimulation. Two families of Na⁺/Ca²⁺ exchangers exist, one of which requires the co-transport of K⁺ and Ca²⁺ in exchange for Na⁺ ions. The malfunctioning of Na⁺/Ca²⁺ exchangers has been related to the development of pathological conditions in the regulation of neuronal death after hypoxia–anoxia, brain trauma, and nerve injury. In addition, the Na⁺/Ca²⁺ exchanger function has been associated with impaired Ca²⁺ homeostasis during aging of the brain, as well as with a role in Alzheimer’s disease by regulating β-amyloid toxicity. In this review, we summarize the current knowledge about the Na⁺/Ca²⁺ exchanger families and their implications in neurodegenerative disorders.     

Na+-dependent Ca2+ Extrusion Governs Response Recovery in Frog Olfactory Receptor Cells

To study the mechanism by which Ca²⁺, which enters during the odor response, is extruded during response recovery, recordings were made from isolated frog olfactory receptor cells using the suction pipette technique, while superfusing the olfactory cilia with solutions of modified ionic composition. When external Na⁺ was substituted with another cation, the response to odor was greatly prolonged. This prolongation of the response was similar irrespective of whether Na⁺ was replaced with Li⁺, which permeates the cyclic nucleotide-gated conductance, or choline, which does not. The prolonged current was greatly reduced by exposure to 300 μM niflumic acid, a blocker of the calcium-activated chloride channel, indicating that it is carried by this conductance, and abolished if Ca²⁺ was omitted from the external solution, demonstrating that Ca²⁺ influx is required for its generation. When the cilia were exposed to Na⁺-free solution after odor stimulation, the recovery of the response to a second stimulus from the adaptation induced by the first was greatly reduced. We conclude that a Na⁺-dependent Ca²⁺ extrusion mechanism is present in frog olfactory cilia and that it serves as the main mechanism that returns cytoplasmic Ca²⁺ concentration to basal levels after stimulation and mediates the normally rapid recovery of the odor response and the restoration of sensitivity after adaptation.     

Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter

Summary In rabbit ileum, Ca²⁺/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca²⁺/CaM stimulate linked Na⁺ and Cl^– absorption. The role of Ca²⁺/CaM-dependent phosphorylation in regulation of the brush-border Na⁺/H⁺ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na⁺/H⁺ antiporter and Ca²⁺/CaM-dependent protein kinase(s) and its phosphoprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca²⁺, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca²⁺/CaM and ATP inhibited Na⁺/H⁺ exchange by 45±13%. This effect was specific since Ca²⁺/CaM and ATP did not alter diffusive Na⁺ uptake, Na⁺-dependent glucose entry, or Na⁺ or glucose equilibrium volumes. The inhibition of the Na⁺/H⁺ exchanger by Ca²⁺/CaM/ATP was due to an effect on theV _max and not on theK _m for Na⁺. In the presence of CaM and ATP, Ca²⁺ caused a concentration-dependent inhibition of Na⁺ uptake, with an effect 50% of maximum occurring at 120nm. This Ca²⁺ concentration dependence was similar to the Ca²⁺ concentration dependence of Ca²⁺/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca²⁺/CaM/ATP-inhibition of Na⁺/H⁺ exchange was reversed by W₁₃, a Ca²⁺/CaM antagonist, but not by a hydrophobic control, W₁₂, or by H-7, a protein kinase C antagonist. we conclude that Ca²⁺, acting through CaM, regulates ileal brush-border Na⁺/H⁺ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.     

Signal transduction mechanism for the stimulation of the sarcolemmal Na+−Ca2+ exchanger by insulin

The signal transduction pathway for insulin-mediated activation of sarcolemmal Na⁺–Ca²⁺ exchange was examined. Insulin stimulated Na⁺–Ca²⁺ exchanger activity in a dose-dependent manner, with the EC50 being about 0.7 U/l. The insulin effect was blocked by the protein kinase inhibitor, staurosporine, indicating possible involvement of a protein kinase in insulin action. Also, the relationship between the insulin effect and activation of a G protein, was examined by testing the effects of 5 guanylyl imidodiphosphate (Gpp(NH))p) on Na⁺–Ca²⁺ exchange in, the presence and absence of insulin. When exchanger activity was assayed at a calcium concentration of 40 M, insulin alone had no effect whereas ATP and Gpp(NH)p increased exchanger activity. However, insulin responsiveness was restored in vesicles preloaded with either ATP or Gpp(NH)p, suggesting that insulin may act through a combination of G protein coupling and protein phosphorylation to enhance Na⁺–Ca²⁺ exchanger activity. We conclude that calcium overload in the diabetic heart may involve a defect in acute activation of the exchanger by insulin.     

Sodium-calcium exchange in renal epithelial cells: Dependence on cell sodium and competitive inhibition by magnesium

Summary Kinetic properties of Na⁺–Ca²⁺ exchange in a renal epithelial cell line (LLC-MK₂) were assessed by measuring cytosolic free Ca²⁺ with fura-2 and⁴⁵Ca²⁺ influx. Replacing external Na⁺ with K⁺ produced relatively small increases in free Ca²⁺ and⁴⁵Ca²⁺ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na⁺ and strongly potentiated the effect of replacing external Na⁺ with K⁺ on free Ca²⁺ and⁴⁵Ca²⁺ uptake.⁴⁵Ca²⁺ influx in 140mm K⁺ or N-methyl-d-glucamine minus influx in 140mm Na⁺ was used to quantify Na⁺–Ca²⁺ exchange activity of Na⁺-loaded cells. The dependence of exchange on cell Na⁺ was sigmoidal; theK _0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na⁺ partly account for the small increase in Ca²⁺ influx when all external Na⁺ is replaced by K⁺. Besides raising cell Na⁺ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg²⁺ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na⁺. Potassium also increased theV _max of exchange by 86% and had no effect on theK _m for Ca²⁺. The exchanger does not cause detectable²²Na⁺–Mg²⁺ exchange and does not appear to require K⁺ or transport⁸⁶Rb⁺. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na⁺–Ca²⁺ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.     

Hypoxia‐induced increase in intracellular calcium concentration in endothelial cells: Role of the Na+‐glucose cotransporter

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet‐activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca²⁺]_i) which then activates the phospholipase A₂ (PLA₂). The link between the decrease in ATP and the increase in [Ca²⁺]_i was not known and is investigated in this work. We first showed that the presence of extracellular Na⁺ was necessary to observe the hypoxia‐induced increase in [Ca²⁺]_i and the activation of PLA₂. This increase was not due to the release of Ca²⁺ from intracellular stores, since thapsigargin did not inhibit this process. The Na⁺/Ca²⁺ exchanger was involved since dichlorobenzamil inhibited the [Ca²⁺]_i and the PLA₂ activation. The glycolysis was activated, but the intracellular pH (pH_i) in hypoxic cells did not differ from control cells. Finally, the hypoxia‐induced increase in [Ca²⁺]_i and PLA₂ activation were inhibited by phlorizin, an inhibitor of the Na⁺‐glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na⁺ through the activated Na⁺‐glucose cotransport followed by the activation of the Na⁺/Ca²⁺ exchanger, resulting in a net influx of Ca²⁺. J. Cell. Biochem. 84: 115–131, 2002. © 2001 Wiley‐Liss, Inc.     

Recent structural and functional insights into the family of sodium calcium exchangers

Maintenance of calcium homeostasis is necessary for the development and survival of all animals. Calcium ions modulate excitability and bind effectors capable of initiating many processes such as muscular contraction and neurotransmission. However, excessive amounts of calcium in the cytosol or within intracellular calcium stores can trigger apoptotic pathways in cells that have been implicated in cardiac and neuronal pathologies. Accordingly, it is critical for cells to rapidly and effectively regulate calcium levels. The Na⁺/Ca²⁺ exchangers (NCX), Na⁺/Ca²⁺/K⁺ exchangers (NCKX), and Ca²⁺/Cation exchangers (CCX) are the three classes of sodium calcium antiporters found in animals. These exchanger proteins utilize an electrochemical gradient to extrude calcium. Although they have been studied for decades, much is still unknown about these proteins. In this review, we examine current knowledge about the structure, function, and physiology and also discuss their implication in various developmental disorders. Finally, we highlight recent data characterizing the family of sodium calcium exchangers in the model system, Caenorhabditis elegans, and propose that C. elegans may be an ideal model to complement other systems and help fill gaps in our knowledge of sodium calcium exchange biology. genesis 52:93–109. © 2013 Wiley Periodicals, Inc.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

线粒体和细胞内钙自稳平衡

线粒体对胞浆钙信号调节作用的研究已经历较长时间.近年,随着研究方法和技术的不断改进,发现在绝大多数生理条件下,线粒体都能参与胞内钙通信过程.线粒体可感受其周围钙微区的存在从而摄取钙,又可以通过钠-钙交换和大分子孔道将钙释放出来,因此可以调节胞浆钙信号的时空特性,影响相关的细胞功能.但是,由于技术上的局限性,目前的研究仍然存在模糊不清和自相矛盾之处,有待于进一步研究.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Analysis of the Na+/Ca2+ Exchanger Gene Family within the Phylum Nematoda

Na⁺/Ca²⁺ exchangers are low affinity, high capacity transporters that rapidly transport calcium at the plasma membrane, mitochondrion, endoplasmic (and sarcoplasmic) reticulum, and the nucleus. Na⁺/Ca²⁺ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na⁺/Ca²⁺ exchangers are divided into three groups based upon stoichiometry: Na⁺/Ca²⁺ exchangers (NCX), Na⁺/Ca²⁺/K⁺ exchangers (NCKX), and Ca²⁺/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na⁺/Ca²⁺ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na⁺/Ca²⁺ exchangers across the phylum Nematoda. In this analysis we identify Na⁺/Ca²⁺ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na⁺/Ca²⁺ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.     

Iron-Induced Inhibition of Na+, K+-ATPase and Na+/Ca2+ Exchanger in Synaptosomes: Protection by the Pyridoindole Stobadine

The effect of oxidative stress, induced by Fe²⁺-EDTA system, on Na⁺,K⁺-ATPase, Na⁺/Ca²⁺ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 M FeSO₄-EDTA per mg of protein at 37°C for 30 min. The oxidative insult reduced Na⁺,K⁺-ATPase activity by 50.7 ± 5.0 % and Na⁺/Ca²⁺ exchanger activity measured in potassium and choline media by 47.1 ± 7.2 % and 46.7 ± 8.6 %, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na⁺/Ca²⁺ exchanger activity. The Na⁺,K⁺-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na⁺/Ca²⁺ exchanger and the Na⁺,K⁺-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.     

Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes

We have investigated the possible role of plasma membrane oxidoreductases in the Ca²⁺ export mechanisms in rat brain synaptic membranes. Ca²⁺ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca²⁺ pump and by a low affinity/high capacity ATP-independent Na⁺-Ca²⁺ exchanger. Both Ca²⁺ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP>NAD>NADPH>NADH with IC₅₀ values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca²⁺ pump and with IC₅₀ values between 8 and 10 mM for the Na⁺-Ca²⁺ exchanger. Oxidizing agents such as DCIP and ferricyanide inhibited the ATP-driven Ca²⁺ efflux mechanism but not the Na⁺-Ca²⁺ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca²⁺ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na⁺-Ca²⁺ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by eithercAMP-dependent protein kinase, Ca²⁺ calmodulin-dependent protein kinases, or protein kinase C.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - EGTA ethylenglycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - DCIP dichlorophenol-indophenol     

Differences of Ca2+ handling properties in identified central olfactory neurons of the antennal lobe

Information processing in neurons depends on highly localized Ca²⁺ signals. The spatial and temporal dynamics of these signals are determined by a variety of cellular parameters including the calcium influx, calcium buffering and calcium extrusion. Our long-term goal is to better understand how intracellular Ca²⁺ dynamics are controlled and contribute to information processing in defined interneurons of the insect olfactory system. The latter has served as an excellent model to study general mechanisms of olfaction. Using patch-clamp recordings and fast optical imaging in combination with the ‘added buffer approach’, we analyzed the Ca²⁺ handling properties of different identified neuron types in Periplaneta americana's olfactory system. Our focus was on two types of local interneurons (LNs) with significant differences in intrinsic electrophysiological properties: (1) spiking LNs that generate ‘normal’ Na⁺ driven action potentials and (2) non-spiking LNs that do not express voltage-activated Na⁺ channels. We found that the distinct electrophysiological properties from different types of central olfactory interneurons are strongly correlated with their cell specific calcium handling properties: non-spiking LNs, in which Ca²⁺ is the only cation that enters the cell to contribute to membrane depolarization, had the highest endogenous Ca²⁺ binding ratio and Ca²⁺ extrusion rate.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

Regulatory Processes on the Cytoplasmic Surface of the Na+/Ca2+ Exchanger from Lobster Exoskeletal Muscle

A partially purified preparation of the lobster muscle Na⁺/Ca²⁺ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca²⁺ efflux from ⁴⁵Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na⁺ (Na _ev )-dependent Ca²⁺ efflux depended directly upon the extravesicular Ca²⁺ concentration ([Ca²⁺] _ev ) with a half-maximal activation at [Ca²⁺] _ev = 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca²⁺, as in most other species. In contrast, at low [Na⁺] _ev , the Ca _ev -binding site (i.e., on the cytoplasmic surface) for Ca²⁺ transported via Ca²⁺/Ca²⁺ exchange was half-maximally activated by about 7.5 μm Ca²⁺. Mild proteolysis of the Na⁺/Ca²⁺ exchanger by α-chymotrypsin also upregulated the Na _ev -dependent Ca²⁺ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca²⁺. Proteolytic digestion in the presence of 1.9 μm free ev Ca²⁺, however, induced only a 1.6-fold augmentation of Ca²⁺ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca²⁺ also inhibited proteolytic degradation of the Na⁺/Ca²⁺ exchanger measured by immunoblotting. These data suggest that Ca²⁺, bound to a high affinity binding site, protects against the activation of the Na⁺/Ca²⁺ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na⁺/Ca²⁺ exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids. Received: 20 October 1999/Revised: 13 January 2000     

Cardiomyopathy associated with noninsulin-dependent diabetes

Summary Cardiovascular disease represents the major cause of morbidity and mortality in noninsulin-dependent diabetic patients. While it was once thought that atherosclerotic vascular disease was responsible for all of these adverse effects, recent studies support the notion that one of the major adverse complications of diabetes is the development of a diabetic cardiomyopathy characterized by defects in both diastolic and systolic function. Contributing to the development of the cardiomyopathy is a shift in myosin isozyme content in favor of the least active V₃ form. Also defective in the noninsulin-dependent diabetic heart is regulation of calcium homeostasis. While transport of calcium by the sarcolemmal and sarcoplasmic reticular calcium pumps are minimally affected by noninsulin-dependent diabetes, significant impairment occurs in sarcolemmal Na⁺-Ca²⁺ exchanger activity. This defect limits the ability of the diabetic heart to extrude calcium, contributing to an elevation in [Ca²⁺]_i. Also promoting the accumulation of calcium by the diabetic cell is a decrease in Na⁺, K⁺ ATPase activity, which is known to increase [Ca²⁺]_i secondary to a rise in [Na⁺]_i. In addition, calcium influx via the calcium channel is stimulated. Although the molecular mechanisms underlying these defects are presently unknown, the possibility that they may be related to aberrations in glucose or lipid metabolism are considered. The evidence suggests that classical theories of glucose toxicity, such as excessive polyol production or glycosylation, appear to be insignificant factors in heart. Also insignificant are defects in lipid metabolism leading to accumulation of toxic lipid amphiphiles or triacylglycerol. Rather, the major defects involve membrane changes, such as phosphatidylethanolamine N-methylation and protein phosphorylation, which can be attributed to the state of insulin resistance.