Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.     

Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor) or amiloride (Na(+)/H(+) exchange channel inhibitor). Suppressing amiloride-sensitive Na(+)/H(+) exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na(+)-K(+)-2Cl(-) channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na(+)/Ca(2+) exchanger extrudes Na(+) in exchange for Ca(2+), thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na(+)/Ca(2+) exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na(+)-K(+)-2Cl(-) channels. The Na(+)/Ca(2+) exchanger then extrudes Na(+) and increases endothelial Ca(2+). The increase in endothelial Ca(2+) causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

Genistein inhibits osmotic activation of Na(+) /H( +) exchange in human platelets

The osmotic shrinkage is an important activator of the Na(+)/H( *) exchanger. The intracellular signaling mechanisms by which shrinkage changes intracellular pH have not been fully elucidated. In human platelets, the removal of calcium did not prevent the osmotic activation of the exchanger. The increase of pH(i) after an hyperosmotic stress was reduced by W-7 (63 micromol l(-1)), and by ML-7 (25 micromol l(-1)), inhibitors of responses mediated by calmodulin or by myosin light chain kinase, but the high concentrations needed suggested that non-specific effects could be involved. Although the exchanger was quiescent during preincubation in hypertonic sodium free solutions, some steps of the signal transduction chain that links the shrinkage to the exchanger activation suffers a modification. Therefore, upon exposure to isotonic sodium-containing media, the rate of recovery from acid loads was increased. The presence of genistein (100 micromol l( -1)) during the preincubation inhibited this activation of Na(+)/H( +) exchanger. We propose that shrinkage induce activation of tyrosine kinases, which in turn leads to the activation of Na(+)/H(+) exchanger and contributes to the restoration of cell volume in human platelets.     

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.     

Osmotic stress and muscle tissue volume response of a freshwater bivalve

The freshwater bivalve, Corbicula fluminea, when submitted to hyperosmotic solutions, behaves as a hyperosmoconformer; we have observed an increase in osmolality and ions in its extracellular fluid. Osmotic and ionic changes in its watery environment represent a challenge for the tissues of this mollusk. Thus we evaluated, in vitro, muscle tissue volume variations (based on wet weight change) under anisosmotic salines, as well the possible regulatory mechanisms involved in the processes. This tissue did not exhibit complete volume regulation under anisosmotic saline solutions, but showed less variation than would be predicted by Van't Hoff's law, and tissue volume remained essentially stable throughout 90 min of exposure. To minimize tissue swelling in hyposmotic situations, C. fluminea muscle mobilizes organic osmolytes (ninhydrin positive substances) and inorganic ions (K(+) and Cl(-)). While under hyperosmotic stimulus, apparently only inorganic osmolytes (Na(+) and Cl(-)) are mobilized by the tissue. Our results indicate ionic accumulation by the Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)/H(+) coupled to Cl(-)/HCO(3)(-) exchangers. Exposure of the muscle tissue to Ca(2+)-free anisosmotic saline did not result in a detectable inhibition of the mechanisms described above. The Ca(2+) gradient that derives from the absence of this ion, even apparently enhances the regulatory mechanisms. These responses of this freshwater mollusk in hyperosmotic solutions, and the muscle tissue under anisosmotic (hypo and hyperosmotic) saline solutions, have not been previously characterized in the manner and approach as reported here. Specifically, we analyze both organic and inorganic osmolytes mobilized under hyposmotic stress, and can infer the participation of Na(+) and Cl(-) pathways stimulated by hyperosmotic stress. From the perspective gained in this study, tissue volume responses may be used as models for toxicological investigations.     

alpha-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na(+)/Ca(2+) exchange in mouse ventricle

Mechanisms underlying the negative inotropic response to alpha-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by approximately 40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca(2+) concentration ([Ca(2+)](o)) conditions but was significantly smaller under high-[Ca(2+)](o) conditions and was not observed under low-[Na(+)](o) conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca(2+) channel, Ca(2+) release channel, Na(+)-K(+) pump, or Na(+)/H(+) exchanger, respectively. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca(2+) and Na(+)/Ca(2+) exchanger currents but had no effect on the inwardly rectifying K(+), transient outward K(+), or Na(+)-K(+)-pump currents. These results suggest that the sustained negative inotropic response to alpha-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca(2+) efflux through the Na(+)/Ca(2+) exchanger.     

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.     

Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger

Na(+) cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na(+)/Ca(2+) exchanger, bumetanide for the Na(+)-K(+)-2Cl(-) cotransporter, and amiloride for Na(+)/H(+) cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na(+)/Ca(2+) exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na(+)/H(+) cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na(+)-K(+)-2Cl(-) cotransporter and Na(+)/Ca(2+) exchanger functions are key to cerebral vascular oxygen responses.     

Mechanisms of secretion-associated shrinkage and volume recovery in cultured rabbit parietal cells

We have previously shown that stimulation of acid secretion in parietal cells causes rapid initial cell shrinkage, followed by Na(+)/H(+) exchange-mediated regulatory volume increase (RVI). The factors leading to the initial cell shrinkage are unknown. We therefore monitored volume changes in cultured rabbit parietal cells by confocal measurement of the cytoplasmic calcein concentration. Although blocking the presumably apically located K(+) channel KCNQ1 with chromanol 293b reduced both the forskolin- and carbachol-induced cell shrinkage, inhibition of Ca(2+)-sensitive K(+) channels with charybdotoxin strongly inhibited the cell volume decrease after carbachol, but not after forskolin stimulation. The cell shrinkage induced by both secretagogues was partially inhibited by blocking H(+)-K(+)-ATPase with SCH28080 and completely absent after incubation with NPPB, which inhibits parietal cell anion conductances involved in acid secretion. The subsequent RVI was strongly inhibited with the Na(+)/H(+) exchanger 1 (NHE1)-specific concentration of HOE642 and completely by 500 muM dimethyl-amiloride (DMA), which also inhibits NHE4. None of the above substances induced volume changes under baseline conditions. Our results indicate that cell volume decrease associated with acid secretion is dependent on the activation of K(+) and Cl(-) channels by the respective secretagogues. K(+), Cl(-), and water secretion into the secretory canaliculi is thus one likely mechanism of stimulation-associated cell shrinkage in cultured parietal cells. The observed RVI is predominantly mediated by NHE1.     

Saliva secretion and ionic composition of saliva in the cockroach Periplaneta americana after serotonin and dopamine stimulation,and effects of ouabain and bumetamide

Isolated salivary glands of Periplaneta americana were used to measure secretion rates and, by quantitative capillary electrophoresis, Na(+), K(+), and Cl(-) concentrations in saliva collected during dopamine (1 micro M) and serotonin (1 micro M) stimulation in the absence and presence of ouabain (100 micro M) or bumetanide (10 micro M). Dopamine stimulated secretion of a NaCl-rich hyposmotic saliva containing (mM): Na(+) 95 +/- 2; K(+) 38 +/- 1; Cl(-) 145 +/- 3. Saliva collected during serotonin stimulation had a similar composition. Bumetanide decreased secretion rates induced by dopamine and serotonin; secreted saliva had lower Na(+), K(+) and Cl(-) concentrations and osmolarity. Ouabain caused increased secretion rates on a serotonin background. Saliva secreted during dopamine but not serotonin stimulation in the presence of ouabain had lower K(+) and higher Na(+) and Cl(-) concentrations, and was isosmotic. We concluded: The Na(+)-K(+)-2Cl(-) cotransporter is of cardinal importance for electrolyte and fluid secretion. The Na(+)/K(+)-ATPase contributes to apical Na(+) outward transport and Na(+) and K(+) cycling across the basolateral membrane in acinar P-cells. The salivary ducts modify the primary saliva by Na(+) reabsorption and K(+) secretion, whereby Na(+) reabsorption is energized by the basolateral Na(+)/K(+)-ATPase which imports also some of the K(+) needed for apical K(+) extrusion.     

Modification of sarcolemmal Na+-K+-ATPase and Na+/Ca2+ exchanger expression in heart failure by blockade of renin-angiotensin system

The activities of both sarcolemmal (SL) Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger, which maintain the intracellular cation homeostasis, have been shown to be depressed in heart failure due to myocardial infarction (MI). Because the renin-angiotensin system (RAS) is activated in heart failure, this study tested the hypothesis that attenuation of cardiac SL changes in congestive heart failure (CHF) by angiotensin-converting enzyme (ACE) inhibitors is associated with prevention of alterations in gene expression for SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger. CHF in rats due to MI was induced by occluding the coronary artery, and 3 wk later the animals were treated with an ACE inhibitor, imidapril (1 mg.kg(-1).day(-1)), for 4 wk. Heart dysfunction and cardiac hypertrophy in the infarcted animals were associated with depressed SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities. Protein content and mRNA levels for Na(+)/Ca(2+) exchanger as well as Na(+)-K(+)-ATPase alpha(1)-, alpha(2)- and beta(1)-isoforms were depressed, whereas those for alpha(3)-isoform were increased in the failing heart. These changes in SL activities, protein content, and gene expression were attenuated by treating the infarcted animals with imidapril. The beneficial effects of imidapril treatment on heart function and cardiac hypertrophy as well as SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the infarcted animals were simulated by enalapril, an ACE inhibitor, and losartan, an angiotensin receptor antagonist. These results suggest that blockade of RAS in CHF improves SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the failing heart by preventing changes in gene expression for SL proteins.     

Substitution of a single residue, Asp575, renders the NCKX2 K+-dependent Na+/Ca2+ exchanger independent of K+

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na(+) gradient and the outward K(+) gradient to drive Ca(2+) extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K(+) dependence of NCKX-mediated Ca(2+) transport. Charge-removing replacement of Asp(575) by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K(+), whereas the charge-conservative substitution of Asp(575) to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp(575) is conserved in the NCKX1-5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K(+)-independent Na(+)/Ca(2+) exchanger.     

Stress response to high osmolarity in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.     

Lithium-calcium exchange is mediated by a distinct potassium-independent sodium-calcium exchanger

Sodium-calcium exchangers have long been considered inert with respect to monovalent cations such as lithium, choline, and N-methyl-d-glucamine. A key question that has remained unsolved is how despite this, Li(+) catalyzes calcium exchange in mammalian tissues. Here we report that a Na(+)/Ca(2+) exchanger, NCLX cloned from human cells (known as FLJ22233), is distinct from both known forms of the exchanger, NCX and NCKX in structure and kinetics. Surprisingly, NCLX catalyzes active Li(+)/Ca(2+) exchange, thereby explaining the exchange of these ions in mammalian tissues. The NCLX protein, detected as both 70- and 55-KDa polypeptides, is highly expressed in rat pancreas, skeletal muscle, and stomach. We demonstrate, moreover, that NCLX is a K(+)-independent exchanger that catalyzes Ca(2+) flux at a rate comparable with NCX1 but without promoting Na(+)/Ba(2+) exchange. The activity of NCLX is strongly inhibited by zinc, although it does not transport this cation. NCLX activity is only partially inhibited by the NCX inhibitor, KB-R7943. Our results provide a cogent explanation for a fundamental question. How can Li(+) promote Ca(2+) exchange whereas the known exchangers are inert to Li(+) ions? Identification of this novel member of the Na(+)/Ca(2+) superfamily, with distinct characteristics, including the ability to transport Li(+), may provide an explanation for this phenomenon.     

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells

The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 μg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.     

Regulation of cardiac Ca(2+) channel by extracellular Na(+)

Hyponatremia is a predictor of poor cardiovascular outcomes during acute myocardial infarction and in the setting of preexisting heart failure [1]. There are no definitive mechanisms as to how hyponatremia suppresses cardiac function. In this report we provide evidence for direct down-regulation of Ca(2+) channel current in response to low serum Na(+). In voltage-clamped rat ventricular myocytes or HEK 293 cells expressing the L-type Ca(2+) channel, a 15mM drop in extracellular Na(+) suppressed the Ca(2+) current by ～15%; with maximal suppression of ～30% when Na(+) levels were reduced to 100mM or less. The suppressive effects of low Na(+) on I(Ca), in part, depended on the substituting monovalent species (Li(+), Cs(+), TEA(+)), but were independent of phosphorylation state of the channel and possible influx of Ca(2+) on Na(+)/Ca(2+) exchanger. Acidification sensitized the Ca(2+) channel current to Na(+) withdrawal. Collectively our data suggest that Na(+) and H(+) may interact with regulatory site(s) at the outer recesses of the Ca(2+) channel pore thereby directly modulating the electro-diffusion of the permeating divalents (Ca(2+), Ba(2+)).     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation

Short-term exposure of coho salmon smolts (Oncorhynchus kisutch) to a gradual increase in salinity over 2 d (0 per thousand -32 per thousand ) resulted in a decrease in proton pump abundance, detected as changes in immunoreactivity with a polyclonal antibody against subunit A of bovine brain vacuolar H(+)-ATPase. N-ethylmaleimide (NEM)-sensitive H(+)-ATPase activities in gill homogenates remained unchanged over 8 d to coincide with a 3.5-fold increase in Na(+)/K(+)-ATPase activities. A transient increase in plasma [Na(+)] and [Cl(-)] levels over the 8-d period was preceded by a 10-fold increase in plasma cortisol levels, which peaked after 12 h. Long-term (1 mo) acclimation to seawater resulted in the loss of apical immunoreactivity for vH(+)-ATPase and band 3-like anion exchanger in the mitochondria-rich cells identified by high levels of Na(+)/K(+)-ATPase immunoreactivity. The polyclonal antibody Ab597 recognized a Na(+)/H(+) exchanger (NHE-2)-like protein in what appears to be an accessory cell (AC) type. Populations of these ACs were found associated with Na(+)/K(+)-ATPase rich chloride cells in both freshwater- and seawater-acclimated animals.