DMA增加正常大鼠心肌细胞钙瞬变和收缩

实验观察了钠氢交换或钠钙交换抑制剂 5 (N ,N 二甲基 )氨氯吡咪 (DMA)对正常和心肌肥厚大鼠分离心室肌细胞钙瞬变和细胞收缩的影响。通过负载荧光染料Fura 2 /Am ,应用离子影像分析系统 (IonImagingSystem)同步测定离体大鼠心肌细胞钙瞬变和细胞长度。结果表明 :DMA 10 μmol/L分别使钙瞬变和细胞缩短从对照组的 2 0 9.6 0± 5 4.96和 3.0 7± 0 .97μm增加到 2 38.5 0± 80 .41和 4.0 7± 1.0 2 μm (P <0 .0 5 ,n =7)。应用特异性反向钠钙交换阻断剂KB R7943可完全阻断DMA的激动作用。DMA还可使尼卡地平抑制L 型钙通道后的钙瞬变和细胞收缩增加。在肥厚心肌细胞 ,DMA表现出相同的药理作用 ,但对钙瞬变和细胞缩短的刺激作用更强。结果表明 :DMA可通过反向钠钙交换途径增加正常和肥厚大鼠心肌细胞钙瞬变和细胞收缩 ,且对肥厚心肌细胞的影响比对正常心肌细胞大。     

Molecular and cellular characterization of a water-channel protein,AQP-h3, specifically expressed in the frog ventral skin

The Ca2+ content of pancreatic juice is closely regulated by yet unknown mechanisms. One aim of the present study was to find whether rat pancreatic ducts have a Na+/Ca2+ exchanger, as found in some Ca2+ transporting epithelia. Another aim was to establish whether the exchanger is regulated by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger intralobular/extralobular ducts the exchanger was predominantly on the luminal membrane. Na+/Ca2+ exchange in ducts was monitored by changes in intracellular Ca2+ activity upon reversal of the Na+ gradient. Secretin (1 nM) and carbachol (1 mM) reduced Na+/Ca2+ exchange by 40% and 51%, respectively. Insulin (1 nM) increased Na+/Ca2+ exchange by 230% within 5 min. The present study shows that pancreatic ducts express the Na+/Ca2+ exchanger. Its distinct localization along the ductal tree and regulation by secretin, carbachol and insulin indicate that ducts might be involved in regulation of Ca2+ concentrations in pancreatic juice.     

Venom of Latrodectus mactans from Chile (Araneae, Theridiidae): effect on smooth muscle

The venoms of Latrodectus sp. have been reported to induce contraction probably mediated by adrenergic and cholinergic transmitters. We have demonstrated that the venom of Chilean Latrodectus mactans contains neurotoxins that induce a contraction partially independent of transmitters release. Transmembrane mobility of Na+ and Ca2+ ions and more specifically, the increase of cytoplasmic calcium concentration are responsible for tonic contraction in smooth muscle. Calcium may enter the cell by several ways, such as the voltage-dependent Ca2+ L-type channels and the Na+/Ca2+ exchanger. This study aimed to examine the participation of this exchanger in the tonic contraction of smooth muscle in vas deferent of rat induced by the venom of the Chilean spider L. mactans. Blockers of Na+ channels (amiloride) and Ca2+ L-type channels (nifedipine), and a stimulator of the exchanger (modified Tyrode, Na+ 80 mM) were used. Simultaneously, variations of the cytoplasmic concentration of Ca2+ were registered by microfluorimetry (Fura-2 indicator) in the presence of nifedipine. In presence of amiloride, dose-dependent inhibition of venom-induced contraction was observed, suggesting the participation of voltage-dependent Ca2+ L-type channels. The contraction was only partially inhibited by nifedipine and the Ca2+ cytoplasmic concentration increased, as assessed by the microfluorimetric registration. Finally, the venom-induced contraction increased in the presence of modified Tyrode, probably due to the action of the Na+/Ca2+ exchanger. Taken together, our results support the idea that the Na+/Ca2+ exchanger is active and may be, at least in part, responsible for the contraction induced by the venom of Chilean L. mactans.     

Effects of antisense oligonucleotides to the cardiac Na+/Ca2+ exchanger on calcium dynamics in cultured cardiac myocytes.

The present study was designed to explore the role of the Na+/Ca2+ exchanger on spontaneous beating of cultured cardiac myocytes. Antisense oligonucleotides (AS) based on the sequence of the cardiac Na+/Ca2+ exchanger were used to decrease expression of this Ca2+ transporting protein in cardiac myocytes. An application of AS (10 microM) caused an increase in beating rate of myocytes within 6-24 h. After 24 h of exposure, AS increased the beating rate from an average rate of 77 beats/min in control and sense-treated myocytes to 103 beats/min. Moreover, myocytes treated for 24 h with 10 microM AS exhibited an increase in diastolic [Ca2+]i levels. The antisense treatment also led to a approximately 20% decrease in expression of Na+/Ca2+ exchanger proteins within 6-24 h. Changes in mRNA levels following AS treatment could not be detected within 3- to 24-h periods. The results of these studies suggest that the Na+/Ca2+ exchanger plays a potentiating role in spontaneous the beating process by regulating [Ca2+]i dynamics and that even a small reduction in the levels of the exchanger protein has marked effects on the handling of [Ca2+]i during the cardiac cycle.     

A novel class of sodium/calcium exchanger inhibitor: design, synthesis, and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis, and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives as inhibitors of the sodium/calcium (Na(+)/Ca(2+)) exchanger are discussed. These studies, based on a lead compound 9a, which was identified in our library, involved systematic modification of three regions and revealed that (1) the 3,4-dihydro-2(1H)-quinazolinone having a tertiary amino alkyl side chain at the 3-position is essential for activity, (2) a nonsubstituted phenyl ring is most suitable for high activity, and (3) introduction of a 4-substituted piperidine moiety enhanced the activity, in particular 4-benzylpiperidin-1-yl showed strong inhibitory activity. Based on these SAR studies, a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger, 12g (SM-15811), was discovered. In particular, SM-15811 directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency. The activity was almost two orders more potent than the lead compound 9a and SM-15811 exerted a protective effect against myocardial ischemic reperfusion injury. These Na(+)/Ca(2+) inhibitors could have a therapeutic potential for the treatment of ischemic reperfusion injury.     

Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.     

Discovery of a novel potent Na+/Ca2+ exchanger inhibitor: design, synthesis and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis and structure-activity relationships for 3,4-dihydro-2(1H)-quinazolinone derivatives with the inhibitory activities of the Na(+)/Ca(2+) exchanger are discussed. These studies based on lead compound 1a lead to the discovery of a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger 4f (SM-15811), which directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency.     

Fowl (Gallus domesticus) sperm motility depends upon mitochondrial calcium cycling driven by extracellular sodium

A relationship between extracellular Ca(+2), fowl sperm phospholipase A2 activity, long-chain acylcarnitine content, and motility was demonstrated in previous work. Sperm motility appeared to depend upon Na+-dependent Ca(+2) cycling when sperm were incubated at body temperature without glucose. In the present work, motility decreased as a function of time when sperm were incubated in 2 mM Ca(+2) prepared with either buffered isotonic sucrose or LiCl. However, this effect was less pronounced in the case of LiCl. The sparing effect of Li+ was attributed to the mitochondrial Na+/Ca(+2) exchanger. Motile concentration decreased exponentially in response to micromolar concentrations of CGP 37157, a specific inhibitor of the mitochondrial Na+/Ca(+2) exchanger. KB-R7943 mesylate, an inhibitor of the reverse mode of the Na+/Ca(+2) exchanger, prevented re-initiation of motility when exogenous Ca(+2) was added to sperm rendered immotile by incubation with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a high-affinity Ca(+2) chelator. The presence of voltage-gated Ca(+2) channels was confirmed by the effect of nifedipine on motile concentration. Neither motile concentration nor straight line velocity was affected by either ouabain or orthovanadate, which inhibit Na+-K+ ATPase and Ca(+2)-ATPase, respectively. In summary, we infer that 1) fowl sperm motility is dependent upon extracellular Ca(+2) cycling through mitochondria; 2) such cycling is dependent upon extracellular Na+; and 3) fowl sperm conserve ATP by moving neither Na+ nor Ca(+2) by active transport. Understanding the relationship between mitochondrial Ca(+2) cycling and ATP production may be applicable to long-term semen storage.     

Functional expression of the human cardiac Na+/Ca2+ exchanger in Sf9 cells: rapid and specific Ni2+ transport

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.     

N(omega)-nitro-L-arginine decreases resting cytosolic [Ca2+] and enhances heat stress-induced increase in cytosolic [Ca2+] in human colon carcinoma T84 cells

N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on [Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting [Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, [Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in [Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased [Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting [Ca2+]i to 115+/-2 nM and greatly reduced the increase in [Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of [Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced [Ca2+]i in unheated cells, and heating failed to enhance [Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in [Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in [Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.     

Effects of caffeine, verapamil, lithium, and KB-R7943 on mechanics and energetics of rat myocardial bigeminies

We examined the effects of pharmacological alteration of Ca2+ sources on mechanical and energetic properties of paired-pulse ("bigeminic") contractions. The fraction of heat release that is related to pressure development and pressure-independent heat release were measured during isovolumic contractions in arterially perfused rat ventricles. The heat released by regular and bigeminic contractions showed two brief pressure-independent components (H1 and H2) and a pressure-dependent component (H3). We used the ratio of active heat (Ha') to pressure-time integral (PtI) and the ratio of H3 to PtI to estimate the energetic cost of muscle contraction (overall economy) and pressure maintenance (contractile economy), respectively. Neither of these ratios was affected by stimulation pattern. Caffeine (an inhibitor of sarcoplasmic reticulum function) significantly decreased mechanical responses and increased the energetic cost of contraction (delta = 101 +/- 12.6%). Verapamil (an L-type Ca2+ channel blocker) decreased pressure maintenance of extrasystolic (delta = 43.4 +/- 3.7%) and postextrasystolic (delta = 37.5 +/- 3.5%) contractions without affecting postextrasystolic potentiation, suggesting that a verapamil-insensitive fraction is responsible for potentiation. The verapamil-insensitive fraction was further studied in the presence of lithium (45 mM) and KB-R7943 (5 microM), inhibitors of the Na+/Ca2+ exchanger. Both agents decreased all mechanical responses, including postextrasystolic potentiation (delta = 67.3 +/- 3.3%), without altering overall or contractile economies, suggesting an association of the verapamil-insensitive Ca2+ fraction to the sarcolemmal Na+/Ca2+ exchanger. The effect of the inhibitors of the Na+/Ca2+ exchanger on potentiation suggests an increased participation of extracellular Ca2+ (and, thus, a redistribution of the relative participation of the Ca2+ pools) during bigeminic contractions in rat myocardium.     

Presence of Na+/Ca2+ exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells.

The presence of a Na+/Ca2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca2+ was dependent on extracellular Na+ (Na+o); 45Ca2+ efflux was significantly decreased by replacing Na+o with N-methylglucamine (NMG), or Li+. Replacement of Na+o by NMG increased the resting intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated chromaffin cells. This could be reversed by adding Na+, suggesting that Na+/Ca2+ exchanger activity was involved in maintaining [Ca2+]i at its resting level. The initial rate of Na(+)-dependent [Ca2+]i recovery after Ca2+ loading by depolarization was dependent on the level of [Ca2+]i. There was an apparent linear relationship between the activity of the Na+/Ca2+ exchanger and [Ca2+]i both in the presence and absence of Na+o. When cells were treated with other stimuli, including 10 microM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca2+]i was significantly reduced upon replacing Na+o with NMG. Our data show that the Na+/Ca2+ exchanger is one of the major pathways for regulating [Ca2+]i in chromaffin cells in both resting and stimulated states.     

Amiloride and its analogs as tools in the study of ion transport

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.     

Effect of losartan on the Na+/Ca2+ exchanger in left ventricle of the insulin resistant and hypertensive hHTg rat

As the Na+/Ca2+ exchanger plays an important role in the regulation of myocyte contractility, it has been suggested that alterations in this system might be involved in the development of insulin resistance and/or diabetes-induced myocardial alterations. Moreover, gene expression and function of the Na+/Ca2+ exchanger in states of combined hypertension and insulin resistance is of a special interest. Thus, we used hereditary hypertriglyceridemic (hHTg) rat (a model of genetically induced insulin resistance and hypertension) to study the effect of losartan, the blocker of type 1 angiotensin receptors, on the Na+/Ca2+ exchanger in the rat heart. We found that gene expression, but not activity of the Na+/Ca2+ exchanger was decreased in the left ventricle of hHTg rats when compared to their normotensive mates. No changes were observed in the right ventricle. In addition, losartan decreased mRNA levels of the Na+/Ca2+ exchanger in the left, but not in the right ventricle of normotensive rats. In hHTg rats, losartan had no effect on the gene expression of this transporter. Our results point to different modulatory pathways of Na+/Ca2+ exchanger in normotensive and hHTg rats.     

A mechanism of Ca2+ release from Ca2+ stores coupling to the Na+/Ca2+ exchanger in cultured smooth muscle cells.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.     

Phosphatidyl inositol-4,5-bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes.

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100-130% when membrane vesicles are incubated (15-20 s at 37 degrees C) with 1 mM MgATP and 1 microM Ca2+ (b) in the presence of 100 microM Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 microM Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.     

The mitochondrial Na(+)/Ca(2+) exchanger

Powered by the steep mitochondrial membrane potential Ca(2+) permeates into the mitochondria via the Ca(2+) uniporter and is then extruded by a mitochondrial Na(+)/Ca(2+) exchanger. This mitochondrial Ca(2+) shuttling regulates the rate of ATP production and participates in cellular Ca(2+) signaling. Despite the fact that the exchanger was functionally identified 40 years ago its molecular identity remained a mystery. Early studies on isolated mitochondria and intact cells characterized the functional properties of a mitochondrial Na(+)/Ca(2+) exchanger, and showed that it possess unique functional fingerprints such as Li(+)/Ca(2+) exchange and that it is displaying selective sensitivity to inhibitors. Purification of mitochondria proteins combined with functional reconstitution led to the isolation of a polypeptide candidate of the exchanger but failed to molecularly identify it. A turning point in the search for the exchanger molecule came with the recent cloning of the last member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX (Na(+)/Ca(2+)/Li(+) exchanger). NCLX is localized in the inner mitochondria membrane and its expression is linked to mitochondria Na(+)/Ca(2+) exchange matching the functional fingerprints of the putative mitochondrial Na(+)/Ca(2+) exchanger. Thus NCLX emerges as the long sought mitochondria Na(+)/Ca(2+) exchanger and provide a critical molecular handle to study mitochondrial Ca(2+) signaling and transport. Here we summarize some of the main topics related to the molecular properties of the Na(+)/Ca(2+) exchanger, beginning with the early days of its functional identification, its kinetic properties and regulation, and culminating in its molecular identification.     

Immunohistochemical evidence for the Na+/Ca2+ exchanger in squid olfactory neurons

The olfactory organs from the squid Lolliguncula brevis are composed of a pseudostratified epithelium containing five morphological subtypes of chemosensory neurons and ciliated support cells. Physiological recordings have been made from two of the subtypes and only the type 4 neuron has been studied in detail. Odour-stimulated increases in intracellular calcium and rapid activation of an electrogenic Na+/Ca2+ exchanger current in type 4 neurons suggest that the exchanger proteins are localized very close to the transduction machinery. Electrophysiological studies have shown that olfactory signal transduction takes place in the apical ciliary regions of olfactory neurons. Using polyclonal antiserum against squid Na+/Ca2+ proteins, we observed specific staining in the ciliary region of cells that resemble type 2, 3, 4 and 5 neurons. Staining was also observed in axon bundles, and in muscle tissue. Collectively, these data support the model that Na+/Ca2+ exchanger proteins are localized to transduction machinery in cilia of type 4 neurons and suggest that the other olfactory subtypes also use Ca2+ during chemosensory responses.     

Interdependence between sodium transport, external chloride, and sodium/calcium exchanger in the isolated skin of the Rana pipiens

The aim of this study was to analyze the relationship of the Na+/Ca2+ exchanger, cytosolic calcium, and chloride to the transepithelial transport of sodium in isolated frog skin. Sodium transport was measured as amiloride-inhibitable short circuit current (SCC). We studied the effect of variations in the concentrations of external chloride and of the manipulation of calcium on sensitive amiloride SCC. Modifications in the movement of Ca2+ were induced by an ionophore, A23187, and a Ca2+ channel blocker, nifedipine. Calcium ionophore A23187 (5 and 20 microM), in a normal Ringer's solution, increased SCC and transepithelial potential difference (PD). In contrast, nifedipine (20 microM) reduced SCC and PD. The role of the Na+/Ca2+ exchanger was studied using dichlorobenzamil (DCB, 50 microM) and quinacrine (1 mM), inhibitors of this exchanger. They selectively increased SCC and PD on the mucosal side of the skin, with no effect on the serosal side. This response occurred only in the presence of extracellular calcium. Replacement of NaCl by sodium methanesulfonate or the addition of furosemide (1 mM) at the serosal compartment, decreased basal SCC and PD and blocked the response to A23187 and the mucosal effect of DCB and quinacrine. These results suggest the presence of an Na+/Ca2+ exchanger located on the mucosal side of the frog skin, which participates in the transepithelial sodium transport. The action of this exchanger may be modulated by external chloride and calcium. J. Exp. Zool. 289:23-32, 2001.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.