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.     

Reverse Na(+)/Ca(2+)-exchange mediated Ca(2+)-entry and noradrenaline release in Na(+)-loaded peripheral sympathetic nerves

[(3)H]noradrenaline ([(3)H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2Hz, 1ms, 60V for 3min) in the presence of uptake blockers (cocaine, 3 x10(-5)M and corticosterone, 5 x10(-5)M). The [(3)H]NA-release was fully blocked by the combined application of the selective and irreversible 'N-type' voltage-sensitive Ca(2+)-channel (VSCC)-blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) and the 'non-selective' VSCC-blocker aminoglycoside antibiotic neomycin (3x10(-3)M). Na(+)-loading (Na(+)-pump inhibition by K(+)-free perfusion) was required to elicit further NA-release after blockade of VSCCs (omega-CgTx GVIA+neomycin). In K(+)-free solution, in the absence of functioning VSCCs (omega-CgTx GVIA+neomycin), the fast Na(+)-channel activator veratridine (10(-5)M) further potentiated the nerve-evoked release of [(3)H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by Ca(o)(2+)-removal. However, Li(+)-substitution was surprisingly ineffective. The non-selective K(+)-channel blocker 4-aminopyridine (4-AP, 10(-4)M) also further potentiated the nerve-evoked release of NA in K(+)-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li(+)-substitution and abolished by Ca(o)(2+)-removal. It is concluded that in Na(+)-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na(+)/Ca(2+)-exchange-mediated Ca(2+)-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na(+)-channel and the exchanger proteins are close to the vesicle docking sites.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Effect of Cd(2+) and Hg(2+) on the activity of Na(+)/K(+)-ATPase and Mg(2+)-ATPase adsorbed on polystyrene microtiter plates.

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4 degrees C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na(+)/K(+)-ATPase and Mg(2+)-ATPase, retained 70-80% activity after the adsorption. In addition, adsorption stabilizes Na(+)/K(+)-ATPase and Mg(2+)-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na(+)/K(+)-ATPase and Mg(2+)-ATPase and their sensitivity to and mechanism of Cd(2+)- or Hg(2+)-induced inhibition. The only exception is the "high affinity" Mg(2+)-ATPase moiety, whose affinity for ATP and sensitivity toward Cd(2+) were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Cloning, sequencing and functional expression in Escherichia coli of the gene for a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum

Cloning and sequencing of the gene encoding a P-type Na(+)-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, were conducted. The structural gene was composed of 2628 nucleotides. The deduced amino acid sequence (876 amino acid residues; Mr, 96,664) suggested that the enzyme possesses 10 membrane-spanning regions. When the amino acid sequences of the four putative membrane regions, M4, M5, M6 and M8, of BL77/1 ATPase were aligned with those of fungal Na(+)-ATPase, Na(+)/K(+)-ATPase, H(+)-ATPases and sarcoplasmic reticulum Ca(2+)-ATPase, it exhibited the highest homology with Ca(2+)-ATPase except M5 region. By the transformation of Escherichia coli with the expression vector (pQE30) containing the ATPase gene, the enzyme was functionally expressed in E. coli membranes.     

K(+)-site-directed pyridine derivative, AU-1421, activates hydrolysis of the K(+)-sensitive phosphoenzyme of sarcoplasmic reticulum Ca(2+)-ATPase and inactivates that of K(+)-transporting ATPases.

(Z)-5-Methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, AU-1421, interacted at 0 degree C with the K(+)-sensitive phosphoenzymes of three transport ATPases, Ca(2+)-, H+/K(+)- and Na+/K(+)-ATPase. In the case of Ca(2+)-ATPase, AU-1421 at about 80 microM stimulated 6-fold the rate of splitting of the phosphoenzyme, on which K+ simply functions as an accelerator from one side of the membrane. Probably AU-1421 also simply interacts with the K(+)-binding site of the phosphoenzyme that is easily accessible from the aqueous phase. In the cases of H(+)/K(+)- and Na(+)/K(+)-ATPases, AU-1421 stabilized the phosphoenzymes which accept K+ as the translocating ion. The rate constants of dephosphorylation for H(+)/K(+)-ATPase and Na(+)/K(+)-ATPase were decreased to half by AU-1421 at about 5 and 10 microM, respectively. Presumably after binding of AU-1421 to a K(+)-recognition site of the phosphoenzyme, local motion of the peptide region near the binding site that serves to move the bound ion into the ion-transport pathway (occlusion center) might be inhibited. Thus AU-1421 may be able to distinguish two modes of K+ action on the K(+)-sensitive phosphoenzymes.     

Dual abnormal effects of mutant MITF encoded by Mi(wh) allele on mouse mast cells: decreased but recognizable transactivation and inhibition of transactivation

Structural localization of a peptide region, KRQPRNPKTDKLVNE, in the catalytic subunit of (Na(+) + K(+))-ATPase was investigated using a specific antibody directed against this peptide in cultured African green monkey kidney CV-1 cells. Immunofluorescence staining of frozen cell sections shows that an anti-KRQPRNPKTDKLVNE antibody (SSA95) interacts with its antigenic site and binds to the extracellular side of the cell membrane. Indirect immunofluorescence and flow cytometric analyses confirmed the presence of this epitope on intact cell surfaces. These results suggest that the KRQPRNPKTDKLVNE region of the (Na(+) + K(+))-ATPase is expressed on the cellular membrane surface.     

Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Maize tonoplast PP(i)-dependent H(+)/Ca(2+) exchange: two K(s) for Ca(2+) and inhibition by thapsigargin

Maize root tonoplasts are able to accumulate Ca(2+) using the energy derived from the H(+) gradient formed during PP(i) hydrolysis. Oxalate increases 6- to 10-fold the amount of Ca(2+) accumulated by tonoplast. Two apparently different K(s) values for Ca(2+) with values of 0.36 and 4.70 microM were detected when oxalate was included in the medium and the free Ca(2+) concentration in the medium was buffered with the use of EGTA. Binding of Ca(2+) to the outer surface of tonoplasts inhibits the outflow of Ca(2+) previously accumulated by the tonoplast, half-maximal inhibition being observed in presence of 1 microM Ca(2+). Thapsigargin, a specific inhibitor of Ca(2+)-ATPase, inhibits the Ca(2+) uptake driven by H(+) gradient but does not inhibit the hydrolysis of PP(i) nor the formation of a H(+) gradient.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

The digitalis-like steroid hormones: new mechanisms of action and biological significance

Digitalis-like compounds (DLC) are a family of steroid hormones synthesized in and released from the adrenal gland. DLC, the structure of which resembles that of plant cardiac glycosides, bind to and inhibit the activity of the ubiquitous cell surface enzyme Na(+), K(+)-ATPase. However, there is a large body of evidence suggesting that the regulation of ion transport by Na(+), K(+)-ATPase is not the only physiological role of DLC. The binding of DLC to Na(+), K(+)-ATPase induces the activation of various signal transduction cascades that activate changes in intracellular Ca(++) homeostasis, and in specific gene expression. These, in turn, stimulate endocytosis and affect cell growth and proliferation. At the systemic level, DLC were shown to be involved in the regulation of major physiological parameters including water and salt homeostasis, cardiac contractility and rhythm, systemic blood pressure and behavior. Furthermore, the DLC system has been implicated in several pathological conditions, including cardiac arrhythmias, hypertension, cancer and depressive disorders. This review evaluates the evidence for the different aspects of DLC action and delineates open questions in the field.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules.

Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.     

Effects of palytoxin on cation occlusion and phosphorylation of the (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.     

K+-induced ion-exchanges trigger trypsin activation in pancreas acinar zymogen granules

Trypsin premature activation has been thought to be a key event in the initiation phase of acute pancreatitis. Here we test a hypothesis that a sustained increase of cytosolic Ca(2+) concentration ([Ca(2+)](C)) can trigger K(+) influx into pancreas acinar zymogen granules (ZGs) via a Ca(2+)-activated K(+) channel (K(Ca)), and this influx of K(+) then mobilizes bound-Ca(2+) by K(+)/Ca(2+) ion-exchange to increase free Ca(2+) concentration in the ZGs ([Ca(2+)](G)) and release bound-H(+) by K(+)/H(+) ion-exchange to decrease the pH in ZGs (pH(G)). Both the increase of [Ca(2+)](G) and the decrease of pH(G) will facilitate trypsinogen autoactivation and stabilize active trypsin inside ZGs that could lead to acute pancreatitis. The experimental results are consistent with our hypothesis, suggesting that K(+) induced ion-exchanges play a critical role in the initiation of trypsin premature activation in ZGs.     

The evaluation of the role of endogenous Ca-dependent regulators and protein kinases in activating and inhibiting ion-transport ATPases]

The data on hormonal regulation of ATP-driving ion pumps are contradictory depending on the object used: whether native cells or isolated membranes. To eliminate this contrariety, we studied the ion transporting ATPases in saponin-permeabilized cells in the presence of all endogenous regulators. In permeabilized erythrocytes we obtained the presence of Ca(2+)-dependent activation of Ca(2+)-ATPase by factor(s) not affected by calmodulin antagonist R24571. We obtained also Ca(2+)-dependent activation and inhibition of Na+,K(+)-ATPase. At a concentration of Mg(2+)-ions corresponding to the intracellular level (370 microM), the 0.5-0.7 microM Ca(2+)-activated Na+,K(+)-ATPase (up to 3-fold), whereas the 1-5 microM Ca2+ inhibited it. The cyclic AMP (10(-5) M) inhibited or eliminated Ca(2+)-dependent activation. The decrease in Mg(2+)-ion concentration to 50 microM eliminated the activation and strengthened the inhibition, which reached 100% at the 1-2 microM Ca2+ concentration. The washing of membranes with EGTA eliminated Ca2+ effects on Na+,K(+)-ATPase. These data suggest that the ion-transporting ATPases are activated or inhibited by Ca(2+)-dependent regulators whose activities may be changed by protein kinase catalysed phosphorylation.     

Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.     

The Na+/Ca2+ exchanger is active and working in the reverse mode in human umbilical artery smooth muscle cells

The data presented in this work suggest that in human umbilical artery (HUA) smooth muscle cells, the Na(+)/Ca(2+) exchanger (NCX) is active and working in the reverse mode. This supposition is based on the following results: (i) microfluorimetry in HUA smooth muscle cells in situ showed that a Ca(2+)-free extracellular solution diminished intracellular Ca(2+) ([Ca(2+)](i)), and KB-R7943 (5microM), a specific inhibitor of the Ca(2+) entry mode of the exchanger, also decreased [Ca(2+)](i) (40.6+/-4.5% of Ca(2+)-free effect); (ii) KB-R7943 produced the relaxation of HUA rings (-24.7+/-7.3gF/gW, n=8, p<0.05); (iii) stimulation of the NCX by lowering extracellular Na(+) increases basal [Ca(2+)](i) proportionally to Na(+) reduction (Delta fluorescence ratio=0.593+/-0.141 for Na(+)-free solution, n=8) and HUA rings' contraction (peak force=181.5+/-39.7 for 130mM reduction, n=8), both inhibited by KB-R7943 and a Ca(2+)-free extracellular solution. In conclusion, the NCX represents an important Ca(2+) entry route in HUA smooth muscle cells.