Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails.

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 Å resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed ß-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

Extracellular ATP induces fast and transient non-selective cationic currents and cytosolic Ca2+ changes in human umbilical artery smooth muscle cells.

Ionotropic purinergic receptors (P2X) are expressed in endothelial and smooth muscle cells of blood vessels. ATP acting on smooth muscle P2X receptors is able to induce vasoconstriction in different kind of vessels. However, to our knowledge, there are no reports that directly show the activity of these purinergic receptors in native human vascular smooth muscle cells. In this work, we describe for the first time an ATP-induced current in freshly isolated human umbilical artery (HUA) smooth muscle cells. The current was measured by patch-clamp technique in whole-cell condition on cells clamped at -50 mV. At 100 µM of ATP the current showed a rapid activation and desensitization, and was carried by both Na(+) and Ca(2+). The current was completely blocked by suramin (300 µM) and partially blocked by 100 µM of Zn(2+) without affecting the kinetic of desensitization. All these properties suggest that the ATP-induced ionic currents are mediated through P2X(1)-like receptors. Moreover, we show that ATP transiently increased cytosolic Ca(2+) in "in situ" smooth muscle cells of intact HUA segments and that this response is dependent of extracellular and intracellular Ca(2+). These data expand the knowledge of purinergic receptors properties in vascular smooth muscle cells and the probable role of ATP as a paracrine modulator of contractile tone in a human artery which is fundamental for feto-placental blood flow.

Arginine-rich peptides destabilize the plasma membrane, consistent with a pore formation translocation mechanism of cell-penetrating peptides.

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.

Genistein effects on Ca2+ handling in human umbilical artery: inhibition of sarcoplasmic reticulum Ca2+ release and of voltage-operated Ca2+ channels.

Isoflavones are a group of natural phytoestrogens including the compound genistein. Health beneficial effects have been attributed to the consumption of this compound, but the fact that it has estrogen-like activity has raised doubts regarding its potential risk in infants, newborns, or in the fetus and placenta during pregnancy. This work is aimed at studying genistein effects on Ca2+ handling by smooth muscle cells of the human umbilical artery (HUA). Using fluorometric techniques, we found that in these cells genistein reduces the intracellular Ca2+ peak produced by serotonin. The same result could be demonstrated in absence of extracellular Ca2+, suggesting that the isoflavone reduces Ca2+ release from the sarcoplasmic reticulum. Force measurement experiments strengthen these results, since genistein reduced the peak force attained by intact HUA rings stimulated by serotonin in a Ca2+-free solution. Moreover, genistein induced the relaxation of HUA rings precontracted either with serotonin or a depolarizing high-extracellular K+ solution, hinting at a reduction of extracellular Ca2+ entry to the cell. This was confirmed by whole-cell patch-clamp experiments where it was shown that the isoflavone inhibits ionic currents through voltage-operated Ca2+ channels. In summary, we show that genistein inhibits two mechanisms that could increase intracellular Ca2+ in human umbilical smooth muscle cells, behaving in this way as a potential vasorelaxing substance of fetal vessels. Taking into account that genistein is able to cross the placental barrier, these data show that isoflavones may have important implications in the regulation of feto-maternal blood flow in pregnant women who consume soy-derived products as part of their meals.

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis.

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43-67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.

Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle.

The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.

Non-selective cationic channels (NSCC) in smooth muscle cells from human umbilical arteries.

The aim of our work was to investigate the presence of non-selective cation channels (NSCC) in freshly isolated smooth muscle cells from the human umbilical artery (HUA), one of the vessels involved in fetal-placental circulation. We studied the electrophysiological properties of NSCC using the patch-clamp technique in whole-cell configuration, and their possible role in the contractile state of intact vessels' rings. Recording with a high intracellular Cs(+) solution and a near physiological extracellular saline solution, we found a Gd(3+)-sensitive current (IC(50) = 1.05 microM) with a linear current-voltage relationship showing a reversal potential (E(rev)) of -2.1 +/- 1.2 mV (n =15 cells). La(3+) (100 microM) and Mg(2+) (5 mM) also blocked this current. In such conditions, inward currents were carried by Na(+) and Ca(2+); hence, a Na(+)-free solution inhibited only inward current (-67.3 +/- 11.4%, at -40 mV, n = 7, p < 0.05) and a Ca(2+)-Na(+)-free solution decreased the current even further with respect to values obtained in Na(+)-free solution (-69.8 +/- 8.8% at -40 mV, n = 9, p < 0.05). The permeability ratios (P(X)/P(Cs(+))) for monovalent and divalent cations were 1, 0.9, 0.7, 0.7, 0.7, and 0.5 where X = Cs(+), Na(+), Li(+), Ca(2+), Ba(2+) and Tris(+), respectively. In intact tissue, a 0 Ca(2+) extracellular solution, Gd(3+) (100-250 microM), La(3+) (200 microM) and Mg(2+) (5 mM) induced vasorelaxation in non-stimulated HUA rings.

Insulin preincubation effects on rat vessel contractile responses: role of the endothelium.

The effect of contractions elicited with ET1 and AVP after preincubating rat aortic and tail artery rings with a hyperinsulinemic dose (3 nM) of insulin were studied. Insulin preincubation (120 min), in the presence of 0.1 mM L-NAME, depressed contraction of aortic rings to 0.01 microM ET1 (132 +/- 6 vs. 161 +/- 9 mg/mm2 in control, n = 25; p < 0.05) and to 1 microM AVP (84 +/- 7 vs. 110 +/- 9 mg/mm2 in control, n = 16; p < 0.05), but did not modify 45Ca influx to the cell. Insulin-induced relaxation was inhibited by indomethacin 10 microM, an antagonist of prostaglandin synthesis, and also by blockade of insulin receptors with 30 microM genistein. A short insulin preincubation (15 min) did not modify ET1 contractions. In rat tail artery, insulin preincubation (120 min) increased the force developed by ET1 (847 +/- 45 vs. 596 +/- 99 mgF/mgW in controls, n = 14) by stimulating TXA2 release and/or actions. In summary, the present results suggest that endothelial factors are involved in both the vasoconstrictor and vasodilator effects of insulin on rat vessels.

Insulin preincubation effects on rat vessel contractile responses: role of the sarcoplasmic reticulum.

In the present work, we studied the possible mechanisms involved in the insulin-induced acceleration of ET1 contractions. We observed a shortening of the half-life needed to achieve maximal developed force (t(1/2)) at 10(-7) M ET1 in rat aortic rings preincubated for 120 min with 3 nM insulin (control 380 +/- 15 s vs. 319 +/- 8 s with insulin, n = 28, p < 0.05). A tyrosine kinase linked receptor was involved in this effect because it was abolished by 30 microM genistein. Endothelium denudation and 10 microM indomethacin treatment did not effect this insulin effect, suggesting its independence of endothelial-derived factors. The effect was still present when the only source of Ca2+ was intracellular (t(1/2) values in the absence of external Ca2+: control 467 +/- 68 s vs. 213 +/- 28 s with insulin, n = 16, p < 0.05), but was blunted if the sarcoplasmic reticulum (SR) Ca2+ source was suppressed by exposure to 10 microM thapsigargin or 10 microM ryanodine. Preincubation with insulin did not potentiate either SR 45Ca2+ uptake or contractions evoked by caffeine-sensitive SR Ca2+ release. Since 30 microM cheleritrine abolished insulin-induced acceleration of ET1 contractions, we propose that the hormone might enhance a signal pathway related to PKC in order to produce a faster Ca2+ release from the SR.

Mechanical properties of human saphenous veins from normotensive and hypertensive patients.

BACKGROUND: Different reactivities of saphenous vein grafts in hypertensive and normotensive patients could lead to differences in the postoperative patency of the grafts. METHODS: In saphenous vein rings isolated from remnants of aorta-coronary grafts obtained from hypertensive and normotensive patients we studied the length-tension relationship; response to high levels of potassium, norepinephrine, and epinephrine; and relaxation in response to calcium deprivation. RESULTS: The rings from hypertensive patients were stiffer and developed more force (grams force/grams weight) than the rings from normotensive subjects to 80 mmol/L potassium (59+/-16 versus 25+/-5, p < 0.05) and to 1 micromol/L norepinephrine (61+/-8 versus 36+/-7, p < 0.05), but not to 10 micromol/L epinephrine (57+/-11 and 54+/-11; not significant). The rings from hypertensive patients relaxed more slowly than those of the normotensive subjects in a calcium-free medium (time to half-relaxation of 976+/-180 versus 548+/-81 seconds; p < 0.05). CONCLUSIONS: The saphenous vein from hypertensive patients is less distensible, slower to relax, and more reactive to at least two agonists. These differences could influence the graft's patency and the clinical outcome.

Short-term streptozotocin-induced diabetes induces blood pressure decrease associated with reduced aortic (45)Ca(2+) uptake and selective depression of the sustained noradrenergic contraction.

To test the hypothesis that diabetes can selectively affect the intracellular and extracellular components of the noradrenergic vascular response in rats, we studied changes in blood pressure, in vitro vascular contraction and (45) Ca(2+) uptake in experimental diabetes induced by injection of 60 mg/kg of streptozotocin (STZ). One week after induction of diabetes mean blood pressure decreased significantly from 106 +/- 3 mmHg to 89 +/- 2 mmHg. After incubation in Ca(2+) =1.6 mM, contraction of STZ aortic rings to 10(- 7) M of norepinephrine was preserved in its intracellular component (Control: 231 +/- 28, STZ: 274 +/- 22 mgForce/mgTissue, NS) but depressed in its extracellular component (Control: 277 +/- 24, STZ: 133 +/- 33 mgForce/mgTissue, P<0.05). Uptake of (45) Ca(2+) in the same rings was depressed in both components. Norepinephrine contractions due to extracellular Ca(2+) (prior depletion of norepinephrine-sensitive Ca(2+) stores) unexpectedly exhibited a initial component whose magnitude in control rings was similar to the response due to intracellular Ca(2+) (extra: 503 +/- 65 mg, intra: 411 +/- 30 mgForce/mgTissue), and was not depressed in STZ preparations (399 +/- 62 mgForce/mgTissue). The sustained contraction to norepinephrine in extracellular Ca(2+) was significantly reduced in STZ aortas (1163 +/- 92 vs. 528 +/- 95 mgForce/mgTissue). We conclude that: 1) Short-term streptozotocin-induced diabetes features reduced blood pressure along with deficient aortic (45) Ca uptake and contraction to norepinephrine, and 2) Only the sustained phase of the norepinephrine contraction, dependent on extracellular Ca(2+), was depressed in the diabetic rats and could possibly be associated with the observed fall in blood pressure.

Sensory adaptation in frog vestibular organs.

Adaptation, i.e., the decrease with time in sensory units' afferent discharge to a constant stimulus, appears to be a common feature of the receptors belonging to acoustico-lateralis system: However, the mechanisms underlying this process are still a matter of debate. The present experiments demonstrate that sensory adaptation to both mechanical and electrical stimuli can be nearly suppressed after perilymphatic ouabain administration. This clearly indicates that the K+ homeostatic mechanisms [Valli et al., (1990) J. Physiol. (London) 430, 585-594] which control the K+ concentration gradient at both ends of vestibular hair cells play a predominant role in this process. The possible importance of different K+-dependent mechanisms in hair cell adaptation is discussed.

Evidence for L-glutamate release in frog vestibular organs.

The present study was devised in order to ascertain whether L-glutamate (Glu) is the neurotransmitter at the primary afferent synapse in frog vestibular organs. To this end different groups of frog isolated semicircular canals were stimulated by means of solutions slightly enriched in K+ (5 mM K(+)-rich solutions are sufficient to produce a strong, long-lasting, transmitter release from the basal pole of sensory cells) both in normal conditions and after low-Ca(2+)-high-Mg2+ impairment of the synaptic transmission. The concentration of Glu in the surrounding medium, determined by means of a bioluminescence-enzymatic method, was evaluated in two different experimental conditions: a) when the canals (5 canals placed inside little net bags) were immersed in a 5 mM K(+)-stimulating solution; b) during the superfusion of the canals (25 canals placed into a little perfusion chamber) with a 5 mM K(+)-stimulating solution. The net bag experiments demonstrated that K(+)-rich solutions can provoke an outflow of Glu from canal organs only if the crista ampullaris is present and functioning. Glu fluctuations were in fact suppressed by employing canals deprived of the ampulla or after low-Ca2(+)-high-Mg2+ synaptic blockade. The superfusion experiments demonstrated that the time course of 5 mM K(+)-induced release of Glu from the sensory organ strictly parallels the time course of 5 mM K(+)-induced EPSPs and spike discharge in afferent axons. These results strongly support the hypothesis that Glu is, or is released with, the afferent transmitter in frog inner ear sensory organs.

Cardiac and vascular effects of diltiazem, dobutamine and amrinone, drugs used after myocardial revascularization.

Hemodynamic care during postoperative management of myocardial revascularization should include vasorelaxing drugs to insure adequate graft and coronary flow, and stimulation of stroke volume to maintain vascular perfusion pressure. We tested the cardiac (inotropic and lusitropic) and vascular (relaxant) effects of diltiazem (0.1 nM to 0.1 mM), dobutamine (10 microM to 10 mM) and amrinone (10 microM to 1 mM) on isolated rat atria and thoracic aorta, and also on isolated human saphenous vein (HSV) and human mammary artery (HMA). Dobutamine produced a maximal positive inotropic effect (+dF/dt max = 29 +/- 7%) at its ED50 for aortic relaxation (88 +/- 7 microM). Conversely, at their ED50 for aortic relaxation diltiazem depressed myocardial contractility and amrinone did not exhibit myocardial effects. In HSV and HMA contracted with 80 mM potassium, diltiazem and dobutamine (but not amrinone) had a vasorelaxant activity similar to that in rat aorta. Norepinephrine-contracted human vessels were significantly more sensitive than potassium-contracted vessels to the relaxant effect of amrinone (ED50 HMA = 15 +/- 5 microM, ED50 HSV = 72 +/- 31 microM, P < 0.05). We conclude that at concentrations still devoid of myocardial effects dobutamine and amrinone are effective dilators in graft segment vessels and rat aorta contracted by membrane depolarization. If the difference between aortic and myocardial tissue still holds in human tissues, at the appropriate concentrations these drugs should be expected to improve cardiac performance while still contributing to the maintenance of graft patency.

Osmolar changes and neural activity in frog vestibular organs.

The effects of hypotonic and hypertonic solutions (the normal value was 240 mOsm) on posterior canal resting and evoked discharge were studied in isolated labyrinth preparations. Hypotonic solutions (60-180 mOsm) were obtained by reducing the perilymphatic NaCl content. Hypertonic solutions (300-420 mOsm) were obtained by adding to normal perilymphatic solutions suitable amounts of NaCl, glucose, sucrose, glycerol, mannitol and urea. The results demonstrated that any kind of receptor activity was inhibited by hypotonic solutions. On the contrary, hypertonic solutions produced different effects on resting and evoked activity. The resting discharge was, with the exception of urea, constantly increased whereas the evoked responses were constantly decreased by all the hypertonic solutions tested. The possible effects of media with changed osmolarity in Meniere's patients is also discussed.

[Effect of insulin on contractile response and calcium binding in rat aorta]. / Efectos de la insulina sobre la respuesta contráctil y la captación de calcio en aorta de rata.

UNLABELLED: Insulin affects physiological mechanisms involved in blood pressure regulation, and at the cellular level modifies endothelial and vascular smooth muscle functions underlying the changes in peripheral resistance. We describe the effects of preincubation with insulin (40 microU/ml for 1-2 hs) on the contractile reactivity of intact rat aortic rings and on 45Ca2+ uptake of EGTA-hyperpermeabilized rat aortic segments. Preincubation with insulin did not affect either contractions induced by 1 microM of NA, or their relaxation induced by 10 mM of caffeine. The contractile response to 1 microM of Ang-II (which in rat aorta is endothelium-independent) was stimulated by insulin preincubation resulting in increases of both maximal developed force and velocity of its spontaneous relaxation. The difference in 45Ca2+ uptake between insulin-treated and insulin-untreated aortic segments was greater at 5 minutes than it was at 30 minutes. CONCLUSIONS: Insulin preincubation affects directly the mechanical response of Ang-II stimulated aortic smooth muscle; we suggest that the modification of SR function is one of the mechanisms involved in insulin regulation of cytosolic Ca2+.

[Insulin, vascular reactivity and hypertension]. / Insulina, reactividad vascular e hipertensión arterial.

Several epidemiological studies have shown that there is a relationship between hyperinsulinemia, insulin resistance and arterial hypertension. Insulin produces sympathetic nervous system stimulation, enhances renal sodium retention and it directly modifies vascular mechanisms involved in both contraction and relaxation of the vascular smooth muscle. These actions of insulin could lead either to elevation or reduction of blood pressure. The absence of vasodilation due to insulin resistance and/or the enhancement of the hypertensive effects due to hyperinsulinemia could be the link between insulin and hypertension.

[Structural and functional aspects of the human saphenous vein used as aorto-coronary graft in myocardial revascularization]. / Aspectos estructurales y funcionales de la vena safena humana utilizada como puente aorto-coronario en la cirugia de revascularización miocárdica.

The human saphenous vein (HSV) is currently used as a graft in coronary revascularization as well as in some other vascular beds, namely those of the inferior limbs. Since a significant proportion of HSV grafts develop stenosis, many studies have focused on the factors that could promote graft failure. This article reviews the results on structural and functional features that might be concurrent in the production of saphenous vein graft stenosis. The reactivity of HSV to several physiological agonists is analyzed, including those derived from the endothelium with contractile or relaxing properties, since these are relevant inducers of graft spasm and/or modifiers of the expression of graft factors involved in either tissue growth or thrombotic-atherosclerotic processes. Mechanisms that regulate vascular smooth muscle contractile state, in particular the activity of K+ channels of the plasma membrane, are described.

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.