Endothelin 1 Stimulates Na+,K+-ATPase and Na+-K+-Cl− Cotransport Through ETA Receptors and Protein Kinase C-Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET-1 and ET-3) on ⁸⁶Rb⁺ uptake as a measure of K⁺ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K⁺ uptake (EC₅₀ = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ET_A receptor antagonist BQ 123 (cyclo-d -Trp-d -Asp-Pro-d -Val-Leu). Neither the selective ET_B agonists IRL 1620 [N-succinyl-(Glu⁹,-Ala^11,15)-ET-1] and sarafotoxin S6c, nor the ET_B receptor antagonist IRL 1038 [(Cys¹¹,Cys¹⁵)-ET-1] had any effect on K⁺ uptake. Ouabain (inhibitor of Na⁺,K⁺-ATPase) and bumetanide (inhibitor of Na⁺-K⁺-Cl^? cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K⁺ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport. ET-1-but not PMA-stimulated K⁺ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na⁺/H⁺ exchange system), suggesting a linkage of Na⁺/H⁺ exchange with ET-1-stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity that is not mediated by PKC.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies

Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca²⁺ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca²⁺ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca²⁺ indicator dye. The uptake of Ca²⁺ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca²⁺. At 100 nm Ca²⁺, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H⁺-ATPase, or by NH₄Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca²⁺ increased as the Ca²⁺ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca²⁺ concentrations. At 700 nm Ca²⁺, the rate and extent of uptake were lower in the absence of K⁺ than in the presence of K⁺. The inhibitors of Ca²⁺-activated K⁺-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K⁺-dependent Ca²⁺ uptake at 700 nm Ca²⁺. Thus the uptake of Ca²⁺ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H⁺-gradient and (ii) the K⁺ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca²⁺ and contribute to regulation of cytosolic Ca²⁺ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999     

Endothelin contraction in pig coronary: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca²⁺ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC₅₀ = 6.5 ± 1 nM and a maximum contraction which was 98.6 ± 9% of the contraction produced by 60 mM KCl. BQ123 (5 µM), an ET_A antagonist, reversed 78 ± 3% of the ET-1 contraction (50 nM). IRL1620, a selective ET_B agonist, produced 23 ± 3% of the total ET-1 contraction with an EC₅₀ = 12.7 ± 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ET_B antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ET_A receptors, and the other 20% was mediated by ET_B receptors. To assess the Ca²⁺ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca²⁺ channel blocker nitrendipine, and in Ca²⁺ free medium containing 0.2 mM EGTA. In Ca²⁺ containing medium the contraction elicited by ET-1 was 98.6 ± 9% of the KCl contraction, however, in the presence 10 µM nitrendipine the ET-1 induced contraction was 54 ± 7% of the KCl contraction, and in Ca²⁺-free medium it was 13 ± 2%. Similarly, the IRL 1620 contractions in Ca²⁺ containing medium, in the presence of nitrendipine and in Ca²⁺-free medium were 22.4 ± 3%, 12 ± 3% and 11 ± 2% of the KCl response respectively. Thus, both ET_A and ET_B contractions utilize extracellular Ca²⁺ pools via L-type Ca²⁺ channels and other undefined route(s), as well as intracellular Ca²⁺ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ET_A receptors, with ET_B receptors using similar Ca²⁺ mobilization pathways, but the ET_B receptors appear to use the intracellular Ca²⁺ stores to a greater extent.     

Dopamine Stimulates K+ Efflux in the Chick Retina via D1 Receptors Independently of Adenylyl Cyclase Activation

Abstract: Dopamine (DA) stimulated K⁺ efflux (assessed as ⁸⁶Rb⁺ efflux) in retinal suspensions of posthatched chicken. This effect was dose dependent (EC₅₀= 22 μM), was mimicked by the D₁-selective antagonist SKF-38393, and reversed by the D₁-selective antagonist SCH-23390, indicating an involvement of D₁ receptors. Analogues of cyclic AMP (CAMP) did not mimic the DA action. Moreover, DA failed to affect cAMP levels, suggesting that adenylyl cyclase (AC) was not involved. In contrast, forskolin (FSK) stimulated both K⁺ efflux and cAMP accumulation in the retina (EC₅₀ of 10 μM for both effects). The FSK-elicited K⁺ efflux was not mimicked by 1,9-dideoxy-FSK (an analogue of FSK that does not activate AC), suggesting that FSK stimulated K⁺ efflux through the activation of AC. Both DA and FSK inhibited Na⁺,K⁺-ATPase activity in the retina. However, the DA-elicited K* efflux was independent of this inhibition, whereas the FSK effect on K⁺ efflux was largely due to the inhibitory action of the diterpene of the ion pump. A possible role of protein kinase C (PKC) in the DA action was explored. The PKC activator 4β-phorbol 12-myristate 13-acetate (4β-PMA) potently (EC₅₀= 4 nM) stimulated K⁺ efflux. This action was not mimicked by the inactive isomer 4α-PMA. When added together, DA and 4β-PMA behaved in an additive manner, suggesting separate mechanisms of action for these two drugs. Moreover, DA failed to stimulate retinal phosphoinositide hydrolysis, a well-known pathway leading to PKC activation. These data suggest that DA acting through D₁ receptors and independently of AC can modulate its target cell excitability in the chick retina by stimulating K⁺ efflux pathways. The mechanism of the DA action remains to be clarified.     

Cardiac sarcolemmal Na+-Ca2+ exchange and Na+-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na⁺-Ca²⁺ exchange, Na⁺-K⁺ ATPase and Ca²⁺-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na⁺-K⁺ ATPase and Ca²⁺-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg²⁺ ATPase activity was not depressed throughout the experimental periods. Both Na⁺-Ca²⁺ exchange and ATP-dependent Ca²⁺-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na⁺-K⁺ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of _--subunit of Na⁺-K⁺ ATPase and Na⁺-Ca²⁺ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na⁺-K⁺ ATPase _-- subunit mRNA was noted in diabetic heart. A significant increase in the Na⁺-Ca²⁺ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na⁺-K⁺ ATPase, Na⁺Ca²⁺ exchange and Ca²⁺-pump activities may lead to the occurrence of intracellular Ca²⁺ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

Ca2+-independent form of protein kinase C may regulate Na+ transport across frog skin

Summary Activators of protein kinase C (PKC) stimulate Na^– transport (J _Na) across frog skin. We have examined the effect of Ca²⁺ on PKC stimulation ofJ _Na. Both the phorbol ester 12-O-tetradecanoylglycerol (DiC₈) were used as PKC activators. Blocking Ca²⁺ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na⁺ channels, since the stimulations produced by blocking Ca²⁺ entry and adding cyclic AMP were simply additive.The Ca²⁺ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca²⁺ on PKC or an indirect action on the final receptor site (the Na⁺ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca²⁺, and in fact was inhibited at millimolar concentrations of Ca²⁺.We conclude that the effects of Ca²⁺ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca²⁺ may have stimulated Na⁺ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na⁺ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca²⁺ independent and seems related to thenPKC or PKC observed in other systems.     

K+-evoked taurine efflux from cerebellar astrocytes: On the roles of Ca2+ and Na+

The ionic requirements for K⁺-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca²⁺ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K⁺-induced efflux. The Ca²⁺-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K⁺. In addition, verapamil did not antagonize 50 mM K⁺-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K⁺-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K⁺ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K⁺-evoked release, replacing Na⁺ with choline abolished the taurine efflux seen in response to K⁺ stimulation. Together, these findings suggest that neuronal N- and L-type Ca²⁺- and voltage-dependent Na⁺-channels are not involved in the influx of Ca²⁺ which appears to be necessary for K⁺-evoked taurine efflux, and that in addition to Ca²⁺, extracellular Na⁺ is also required.     

The modulation of the calcium pump of human red cells by Na+ and K+

1. The sidedness of Ca²⁺-pump activation by Na⁺ and K⁺ was studied by atomic absorption spectrophotometry in human erythrocyte ghosts, which had been prepared in dextran solutions and resealed to alkali cations. 2. When ghosts were incubated in an all-choline medium, the increase in Na_i⁺ elicited an inhibitory-stimulatory effect on Ca²⁺ extrusion. By contrast, only a stimulatory action was induced when choline was replaced by Na₀⁺. 3. A dual effect on active Ca²⁺ efflux was also produced by increasing K_i⁺ or K₀⁺. The biphasic response to the latter, however, was absent from high-K⁺ ghosts. Furthermore, the stimulation obtained at high K₀⁺ was additive to that elicited by K_i⁺. 4. The results suggest that Na⁺ and K⁺ stimulate the Ca²⁺ pump of human red cells through two different mechanisms. The first one appears to be an electric coupling between Ca²⁺ efflux and the external activating cation. The other seems associated with the molecular reactions of the Ca²⁺-pump protein.     

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule

Summary The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K⁺ and Ca²⁺ transport and respiration (QO₂) of proximal tubule suspensions. Hg²⁺, Cu²⁺, and Au³⁺ (10^–4 m) each caused a rapid net K⁺ efflux and a delayed inhibition of QO₂. The Hg²⁺-induced net K⁺ release represented passive K⁺ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg²⁺ and Ag⁺ promoted a net Ca²⁺ uptake that was nearly coincident with the onset of the net K⁺ efflux. A delayed inhibition of ouabainsensitive QO₂ and nystatin-stimulated QO₂, indicative of Na⁺, K⁺-ATPase inhibition, was observed after 30 sec of exposure to Hg²⁺. More prolonged treatment (2 min) of the tubules with Hg²⁺ resulted in a 40% reduction in the CCCP-uncoupled QO₂, indicating delayed injury to the mitochondria. The net K⁺ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmaleimide (10^–4 m) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10^–4 m). In addition, both DTT and GSH immediately reversed the Ag⁺-induced net Ca²⁺ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.     

The Extracellular K+ Concentration Dependence of Outward Currents through Kir2.1 Channels Is Regulated by Extracellular Na+ and Ca2+

It has been known for more than three decades that outward Kir currents (I_K1) increase with increasing extracellular K⁺ concentration ([K⁺]_o). Although this increase in I_K1 can have significant impacts under pathophysiological cardiac conditions, where [K⁺]_o can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K⁺]_o dependence of Kir2.1-mediated outward I_K1 was due to [K⁺]_o-dependent inhibition of outward I_K1 by extracellular Na⁺ and Ca²⁺. This could be accounted for by Na⁺/Ca²⁺ inhibition of I_K1 through screening of local negative surface charges. Consistent with this, extracellular Na⁺ and Ca²⁺ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I_K1 in a similar [K⁺]_o-dependent manner as Na⁺/Ca²⁺. Site-directed mutagenesis studies identified Asp¹¹⁴ and Glu¹⁵³ as the source of surface charges. Reducing K⁺ activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na⁺ and Ca²⁺, suggesting that in addition to exerting a surface electrostatic effect, Na⁺ and Ca²⁺ might inhibit outward I_K1 by inhibiting K⁺ activation. This study identified interactions of K⁺ with Na⁺ and Ca²⁺ that are important for the [K⁺]_o dependence of Kir2.1-mediated outward I_K1.     

Endothelin receptor dimers evaluated by FRET, ligand binding, and calcium mobilization

Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ET_A) and B (ET_B) receptors, which may form homo- and heterodimers with unknown function. Here, we investigated ET-receptor dimerization using fluorescence resonance energy transfer (FRET) between receptors tagged with CFP (donor) and receptors tagged with tetracysteine-FlAsH (fluorescein arsenical hairpin) (acceptor) expressed in HEK293 cells. FRET efficiencies were 15%, 22%, and 27% for ET_A/ET_A, ET_B/ET_B and ET_A/ET_B, respectively, and dimerization was further supported by coimmunoprecipitation. For all dimer pairs, the natural but nonselective ligand ET-1 rapidly (≤30 s) reduced FRET by >50%, but did not detectably reduce coimmunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca²⁺ ([Ca²⁺]_i) lasting 1-2 min for both homodimer pairs, and these ET-1 actions on FRET and [Ca²⁺]_i elevation were blocked by the appropriate subtype-selective antagonist. In contrast, ET_A/ET_B heterodimers mediated a sustained [Ca²⁺]_i increase lasting >10 min, and required a combination of ET_A and ET_B antagonists to block the observed FRET and [Ca²⁺]_i responses. The sensitive CFP/FlAsH FRET assay used here provides new insights into endothelin-receptor dimer function, and represents a unique approach to characterize G-protein-coupled receptor oligomers, including their pharmacology.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis in the succulent xerophyte Zygophyllum xanthoxylum

The inward‐rectifying K⁺ channel AKT1 constitutes an important pathway for K⁺ acquisition in plant roots. In glycophytes, excessive accumulation of Na⁺ is accompanied by K⁺ deficiency under salt stress. However, in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits excellent adaptability to adverse environments, K⁺ concentration remains at a relatively constant level despite increased levels of Na⁺ under salinity and drought conditions. In this study, the contribution of ZxAKT1 to maintaining K⁺ and Na⁺ homeostasis in Z. xanthoxylum was investigated. Expression of ZxAKT1 rescued the K⁺‐uptake‐defective phenotype of yeast strain CY162, suppressed the salt‐sensitive phenotype of yeast strain G19, and complemented the low‐K⁺‐sensitive phenotype of Arabidopsis akt1 mutant, indicating that ZxAKT1 functions as an inward‐rectifying K⁺ channel. ZxAKT1 was predominantly expressed in roots, and was induced under high concentrations of either KCl or NaCl. By using RNA interference technique, we found that ZxAKT1‐silenced plants exhibited stunted growth compared to wild‐type Z. xanthoxylum. Further experiments showed that ZxAKT1‐silenced plants exhibited a significant decline in net uptake of K⁺ and Na⁺, resulting in decreased concentrations of K⁺ and Na⁺, as compared to wild‐type Z. xanthoxylum grown under 50 mm NaCl. Compared with wild‐type, the expression levels of genes encoding several transporters/channels related to K⁺/Na⁺ homeostasis, including ZxSKOR, ZxNHX, ZxSOS1 and ZxHKT1;1, were reduced in various tissues of a ZxAKT1‐silenced line. These findings suggest that ZxAKT1 not only plays a crucial role in K⁺ uptake but also functions in modulating Na⁺ uptake and transport systems in Z. xanthoxylum, thereby affecting its normal growth.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K⁺-dependent Na⁺/Ca²⁺-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K⁺-dependent Na⁺/Ca²⁺-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca²⁺ signal dynamics in olfactory and other neurons. Synaptic Ca²⁺ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca²⁺ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca²⁺ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca²⁺ signalling by activating a signal damping and termination pathway.     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.