AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 289: H2020-H2029, 2005. First published July 1, 2005; doi:10.1152/ajpheart.00908.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/5/H2020    most recent
00908.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, M. Y.
Right arrow Articles by Suh, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, M. Y.
Right arrow Articles by Suh, S. H.

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

Moon Young Kim, Geun Hee Seol, Guo Hua Liang, Ji Aee Kim, and Suk Hyo Suh

Department of Physiology and Medical Research Institute, College of Medicine, Ewha Women's University, Seoul, Republic of Korea

Submitted 2 September 2004 ; accepted in final form 24 June 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 µM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 µM) or Ni2+ (>100 µM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 µM but did at 30 µM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 µM), Ni2+ (300 µM), or KB-R7943 (30 µM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.

extracellular potassium; sodium-potassium pump; forward mode of sodium/calcium exchanger; endothelial cells; intracellular calcium

THE NA+-K+ PUMP and the Na+/Ca2+ exchanger (NCX) have an important role in modulating vascular contractility. Na+-K+ pump activation relaxes vascular smooth muscle by hyperpolarizing the membrane potential (19, 24), and Na+-K+ pump inhibition contracts vascular smooth muscle through activating the reverse mode of NCX by Na+ accumulation in the myoplasm (9, 18).

In endothelial cells, the roles of the Na+-K+ pump and NCX are unclear and contradictory. It was reported that Na+-K+ pump inhibition affects the synthesis or release of endothelium-derived relaxing factor(s) rather than its effector pathway (29). On the other hand, we previously reported that Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells and thereby endothelium-dependent relaxation (EDR) (26). NCX is thought to contribute to endothelium-dependent control of vascular contractility (6, 25, 28). These results support the involvement of the reverse mode of NCX in Ca2+ mobilization in endothelial cells. However, little is known about the role of NCX operating in the forward mode.

The contribution of NCX to Ca2+ mobilization is likely to vary, depending on the ability of a physiological stimulus to alter intracellular Na+ concentration ([Na+]i) (16). Because Na+ gradients across cell membrane are mainly achieved by the action of the Na+-K+ pump, we hypothesized that Na+-K+ pump activation would activate the forward mode of NCX.

The present study was specifically designed to elucidate the roles of the Na+-K+ pump and the forward mode of NCX in endothelium-dependent control of vascular contractility. We provide evidence for the concomitant activation of the Na+-K+ pump and the forward mode of NCX during EDR.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Contraction measurement on isolated aortic rings. Five- to six-month-old mice of either gender were anesthetized by pentobarbital sodium (50 mg/kg body wt ip) and killed by cervical dislocation. Thoracic aorta was dissected out and cut into rings approximately 2.0–3.0 mm long. A homemade myograph (14) was used to record the mechanical responses of these aortic ring segments. Each aortic ring was threaded with two strands of tungsten wire (120-µm diameter): one anchored in the organ bath chamber (1 ml) and the other connected to a mechanotransducer (model FT-03, Grass). The chamber was perfused at a flow rate of 2.5 ml/min with oxygenated (95% O2-5% CO2) Krebs-Ringer bicarbonate solution by using a peristaltic pump. The composition (in mM) of this solution was 118.3 NaCl, 4.7 KCl, 1.2 MgCl2, 1.22 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, 11.1 glucose, pH 7.4. An optimal resting tension of 0.8–1 g was applied. Rings were precontracted with PGF2{alpha} or norepinephrine (NE). EDR was induced by 3 µM acetylcholine (ACh). When EDR had reached a maximum, extracellular K+ concentration ([K+]o) was increased from 6 to 12 mM to activate the Na+-K+ pump.

Mouse aorta endothelial cell culture, intracellular Ca2+ concentration measurement, and current clamping. We isolated endothelial cells from mouse aorta (MAEC) using the primary explant technique, which is described in detail elsewhere (17, 27). The cells were used up to passage 2 for functional studies. Membrane potential and intracellular Ca2+ concentration ([Ca2+]i) (using fura-2 AM) were measured as previously described (26). The external solution (HEPES-buffered solution) contained (in mM) 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. The osmolarity of the solution, as measured with a vapor pressure osmometer (Fiske), was 320 ± 5 mosM.

RT-PCR. Total RNA was extracted from MAECs that were prepared by using TRIzol reagent (Molecular Research Center; Cincinnati, OH). First-strand cDNA was generated from total RNA using BcaBEST polymerase (Takara Shuzo). The specific oligonucleotide primers used for the PCR were 5'-CTTCTTCTTTCCCATCTGCGTT-3' (sense) and 5'-GACTCTGACATTGCTAAGGTGC-3' (antisense). The size of the expected fragments was 783 bp. PCR was performed using Bca-optimized Taq polymerase (Takara Shuzo), and the conditionings used were as follows: initial denaturation for 1 min at 94°C, 35 amplification cycles (1 min at 94°C, 1.5 min at 55°C, and 1.5 min at 72°C for each cycle), a final extension of 10 min at 72°C, and rapid cooling to 4°C. PCR products were visualized on a 1% agarose gel using a 100-bp DNA ladder marker (New England BioLabs; Beverly, MA) as a standard.

Determination of mouse aorta cGMP levels. Mouse aortic rings, prepared as described above, were allowed to equilibrate in a chamber of a myograph, and one ring was mounted to record contractile response in the same chamber. The chamber was perfused at a flow rate of 2.5 ml/min with oxygenated (95% O2-5% CO2) Krebs-Ringer bicarbonate solution by using a peristaltic pump. Rings were precontracted with NE, and EDR was induced by 3 µM ACh. When the mounted ring showed an expected response, the other rings were frozen in liquid nitrogen, and cGMP levels were measured using cGMP assay kits (EIA kit, R&D Systems) following the manufacture's protocol. Briefly, frozen tissue samples in liquid nitrogen were ground to a fine powder using a stainless steel mortar. After the liquid nitrogen had evaporated, the frozen tissue was homogenized in 300 µl of 0.1 M HCl to stop the action of phosphodiesterase. Centrifugation was done at 10,000 rpm at 4°C, and the supernatant was then collected for quantitative immunoassay of cGMP levels. After incubation with p-nitrophenyl phosphate substrate, a microplate autoreader (Emax ED 927, Molecular Devices Instruments) was used to measure the intensity of the bound yellow color at 405 nm. Total protein content in aorta homogenates was determined using a bicinchoninic acid protein assay kit (Pierce).

Chemicals. ACh, ATP, monensin, NE, NG-nitro-L-arginine methyl ester (L-NAME), ouabain, and PGF2{alpha} were purchased from Sigma; KB-R7943 from Tocris, 2',4'-dichlorobenzamil (DCB) from Biomol, fura-2 AM from Molecular Probes, and Nystatin from ICN Biomedicals. Fura-2 AM and nystatin were applied from stock solutions in DMSO. KB-R7943, monensin, and DCB were applied from stock solutions in ethanol. Final concentrations of DMSO and ethanol were both <0.05%.

The mice were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996), and the experimental and animal care protocol was approved by the Animal Care and Use Committee of Ewha Women's University. All experiments were performed at 37°C. Pooled data are presented as means ± SE, and significant differences were detected using the Student's t-test (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Presence of NCX in MAECs. The Na+ ionophore monensin relaxed precontracted endothelium-intact aortic rings in a concentration-dependent manner (Fig. 1, A, B, and D). Monensin induced 25.2 ± 4.3% relaxation at 0.1 µM, and this relaxation was completely inhibited by KB-R7943 (Fig. 1A). In contrast, endothelium-denuded aortic rings (Fig. 1C) or endothelium-intact aortic rings pretreated with the nitric oxide (NO) synthase inhibitor L-NAME (30 µM) (data not shown) showed no relaxation on administration of monensin (0.1 µM), indicating that the KB-R7943-sensitive, monensin-induced relaxation was EDR. On the other hand, monensin relaxed not only precontracted endothelium-intact aortic rings but also endothelium-denuded aorta at high concentrations (>1 µM). Monensin (1 µM) induced 56.5 ± 4.8 and 10.3 ± 3.1% relaxation in endothelium-intact and -denuded aortic rings, respectively (Fig. 1, B–D), and this relaxation was significantly greater in endothelium-intact aortic rings (Fig. 1D). Monensin-induced relaxation of endothelium-denuded aortic rings was not inhibited by KB-R7943 (data not shown). We then examined the effects of monensin-induced Na+ loading and extracellular Na+ depletion (Na+ replaced by Li+) on [Ca2+]i in MAEC (Fig. 1, E and F). With monensin application or extracellular Na+ depletion, [Ca2+]i was increased from 0.15 ± 0.02 to 0.23 ± 0.03 µM or from 0.16 ± 0.02 to 0.24 ± 0.02 µM, respectively (n = 10), and these increases were inhibited by KB-R7943 (data not shown). In addition, extracellular Na+ depletion increased [Ca2+]i in ATP-stimulated MAECs (Fig. 1G). Increased [Ca2+]i by ATP application was further increased by extracellular Na+ depletion, and this Na+ depletion-induced [Ca2+]i increase was inhibited by KB-R7943.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 1. Activation of the reverse mode of Na+/Ca2+ exchanger (NCX) by monensin-induced Na+ loading and extracellular Na+ depletion (Na+ replaced by Li+). A–D: monensin (M) relaxed endothelium-intact (E+; A, B, and D) and endothelium-denuded aortic rings (E–; C and D) in a concentration-dependent manner. The magnitude of the relaxation at each experiment was expressed as a percentage of the initial PGF2{alpha}-induced contraction. The magnitude of monensin-induced relaxation was compared with that of ACh (3 µM)-induced endothelium-dependent relaxation (EDR; D). In endothelium-intact rings, monensin-induced relaxations were 29.2 ± 4.7% (0.1 µM) and 58.6 ± 3.3% (1 µM) of ACh-induced relaxation, respectively. E–G: monensin-induced Na+ loading or extracellular Na+ depletion significantly increased [Ca2+]i (E and F) and extracellular Na+ depletion augmented ATP-induced intracellular Ca2+ concentration ([Ca2+]i) increase (G) in mouse aortic endothelial cells (MAECs). C, control; M, monensin. H: RT-PCR studies of MAECs for NCX1. M, 100-bp ladder size standard. **Statistical significance (P < 0.01).

 
Inasmuch as the above results suggested the presence of NCX, we next performed RT-PCR using mRNA of freshly isolated MAECs to examine whether the cells contain a molecular candidate for NCX. As a complete sequence for mouse NCX has been published, we performed RT-PCR using primers based on this NCX sequence. Figure 1H shows that an abundant RT-PCR product was found at the expected molecular size using a PCR primer set, indicating that NCX1 mRNA is expressed in MAECs.

[Na+]i-dependent mechanism? In a previous study (26), we reported that EDR and agonist-induced [Ca2+]i increase in various endothelial cells were inhibited by increasing extracellular concentrations of K+ or Cs+ within a millimolar range (K+-induced inhibition), and this K+-induced inhibition was inhibited by the Na+-K+ pump inhibitor ouabain. These data suggested that Na+-K+ pump activation evoked K+-induced inhibition. Because Na+-K+ pump activation reduces [Na+]i, a decrease in [Na+]i might be an initial step in K+-induced inhibition. Thus we tested the effect of monensin-induced Na+ loading on K+-induced inhibition.

Precontracted endothelium-intact aortic rings were relaxed by ACh application. ACh-induced EDR was abolished by increasing [K+]o from 6 to 12 mM in a reversible manner (K+-induced inhibition of EDR) (Fig. 2A). This K+-induced inhibition of EDR was almost completely abolished by monensin (0.1 µM) pretreatment for 30 min (Fig. 2B). In addition, 1 µM monensin completely inhibited K+-induced inhibition of EDR (Fig. 2, C and D). We then examined the effect of monensin-induced Na+ loading on K+-induced inhibition of [Ca2+]i increase. ATP increased [Ca2+]i, and this increased [Ca2+]i reduced by increasing [K+]o from 6 to 12 mM in a reversible manner (K+-induced inhibition of [Ca2+]i increase) (Fig. 2E). This K+-induced inhibition of [Ca2+]i increase was abolished by pretreating with monensin (10 µM) for 5 min (Fig. 2F). These results suggest that intracellular Na+ depletion by Na+-K+ pump activation evokes K+-induced inhibition.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 2. Inhibition of K+-induced inhibition by monensin-induced Na+ loading. PGF2{alpha} was used to contract aortic rings, and EDR was induced by ACh application. A: K+-induced inhibition of EDR. [K+]o, extracellular K+ concentration. B: aortas used in A were pretreated with monensin. Note that this pretreatment almost completely abolished K+-induced inhibition of EDR. C and D: monensin (M) inhibited K+-induced inhibition of EDR. K12, [K+]o of 12 mM. The magnitude of the relaxation at each experiment was expressed as a percentage of the initial PGF2{alpha}-induced contraction. E: extracellular K+ inhibited ATP-induced Ca2+ transients in MAECs (K+-induced inhibition of [Ca2+]i increase). F: MAECs used in E were pretreated with monensin. Note that the pretreatment completely abolished K+-induced inhibition of [Ca2+]i increase. ***Statistical significance (P < 0.001).

 
Contribution of the forward mode of NCX to K+-induced inhibition. [Ca2+]i in endothelial cells is increased during EDR. Because low [Na+]i and high [Ca2+]i might activate the forward mode of NCX, we tested the effect of NCX inhibitors on K+-induced inhibition. After evoking K+-induced inhibition of EDR, the NCX (forward and reverse mode) inhibitor Ni2+ was applied. Rings, recontracted by extracellular K+, were slightly relaxed by 100 µM Ni2+ and markedly relaxed by 300 µM Ni2+ (Fig. 3A). In contrast, 300 µM Ni2+ did not relax endothelium-intact aortic rings pretreated with L-NAME (30 µM) (Fig. 3B) or endothelium-denuded aortic rings (data not shown). These results suggest that the NCX inhibitor Ni2+ inhibits K+-induced inhibition of EDR. Pretreatment with Ni2+ for 5 min also inhibited K+-induced inhibition of EDR (Fig. 3, C–E). In control rings, EDR was almost completely inhibited by increasing [K+]o from 6 to 12 mM. EDR of 90.9 ± 4.1% was reduced to 7.7 ± 3.2% by increasing [K+]o (n = 6). In contrast, when the aortic rings were pretreated with 100 µM Ni2+, EDR was only partially inhibited by increasing [K+]o from 6 to 12 mM. EDR of 94.6 ± 5.1% was reduced to 60.2 ± 4.7% by increasing [K+]o. The inhibitory effect of Ni2+ on K+-induced inhibition was further augmented by increasing the Ni2+ concentration from 100 to 300 µM (Fig. 3D). These results support the suggestion that the NCX inhibitor Ni2+ inhibits K+-induced inhibition of EDR. We then examined the effect of Ni2+ on K+-induced inhibition of [Ca2+]i increase. Ni2+ restored the reduced [Ca2+]i by extracellular K+ (Fig. 3F), which suggests that Ni2+ inhibits K+-induced inhibition of [Ca2+]i increase.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 3. Inhibition of K+-induced inhibition by the NCX (forward and reverse mode) inhibitor Ni2+. A: Ni2+ inhibited K+-induced inhibition of EDR in a concentration-dependent manner. B: aortas used in A were pretreated with NG-nitro-L-arginine methyl ester (L-NAME). Note that Ni2+ did not relax the aortic ring. C: K+-induced inhibition in control rings. NE, norepinephrine. D and E: aortas used in C were pretreated with Ni2+. The magnitude of the relaxation at each experiment was expressed as a percentage of the initial NE-induced contraction, and K+-induced inhibition of EDR was significantly reduced by Ni2+ pretreatment (E). In addition, this pretreatment significantly potentiated EDR (E). F: Ni2+ also inhibited K+-induced inhibition of [Ca2+]i increase. Oxygenated (100% O2) HEPES-buffered solution was used in Ni2+ experiments. *P < 0.05, ***P < 0.001.

 
Like Ni2+, another NCX (forward and reverse mode) inhibitor, DCB, also inhibited K+-induced inhibition of EDR (Fig. 4). In control rings, EDR was almost completely inhibited by extracellular K+ (Fig. 4, A and D). EDR of 81.0 ± 1.6% was reduced to 3.1 ± 1.9% by increasing [K+]o from 6 to 12 mM (n = 6). The aortic rings were then pretreated with DCB for 10 min. DCB pretreatment inhibited K+-induced inhibition of EDR in a concentration-dependent manner. Pretreatment with 10 µM DCB partially inhibited K+-induced inhibition of EDR. EDR of 88.8 ± 1.0% was reduced to 50.1 ± 5.1% by increasing [K+]o from 6 to 12 mM (Fig. 4, B and D). Furthermore, pretreatment with 30 µM DCB almost completely abolished K+-induced inhibition of EDR (Fig. 4, C and D). EDR of 89.0 ± 1.7% was slightly reduced to 81.0 ± 1.2% by increasing [K+]o from 6 to 12 mM. In addition, pretreatment with Ni2+ or DCB potentiated EDR (Figs. 3E and 4D). EDRs of 90.9 ± 4.1% and 81.0 ± 1.6% were significantly increased to 94.6 ± 5.1% and 88.8 ± 1.0% by pretreating with Ni2+ and DCB, respectively. The potentiating effect of DCB on EDR was more evident, when a low concentration of ACh (0.3 µM) was used to induce EDR (Fig. 4, E–G). The EDR of 34.6 ± 4.9% was significantly increased to 68.3 ± 4.1% by pretreating with DCB (n = 6).



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4. Inhibition of K+-induced inhibition of EDR by the NCX (forward and reverse mode) inhibitor 2',4'-dichlorobenzamil (DCB). The magnitude of the relaxation at each experiment was expressed as a percentage of the initial NE-induced contraction. A: K+-induced relaxation in control. B–D: aortas used in A were pretreated with 10 µM DCB (B) or 30 µM DCB (C). K+-induced inhibition was significantly and concentration dependently reduced by these pretreatment (D). E–G: EDR induced by a low concentration of ACh (0.3 µM) in control rings (E) and after DCB pretreatment (F). The magnitude of EDR was significantly increased by pretreating with DCB (+DCB; G). **P < 0.01, ***P < 0.001.

 
KB-R7943 (the reverse mode inhibitor) did not inhibit K+-induced inhibition of EDR or [Ca2+]i increase at up to 10 µM (Fig. 5, B and E). When the aortic rings were pretreated with 10 µM KB-R7943, K+-induced inhibition of EDR was still evoked (Fig. 5B). In contrast, KB-R7943 completely abolished K+-induced inhibition of EDR at 30 µM (Fig. 5C). In addition, KB-R7943 inhibited K+-induced inhibition of [Ca2+]i increase at 30 µM (Fig. 5, E and F). A [Ca2+]i of 0.12 ± 0.03 µM in the resting state was increased to 1.06 ± 0.19 µM by ATP, and this was reduced to 0.26 ± 0.12 µM by increasing [K+]o from 6 to 12 mM. The reduced [Ca2+]i was reincreased to 0.63 ± 0.14 µM by 30 µM KB-R7943 (n = 8). These data indicate that Na+-K+ pump activation inhibits [Ca2+]i increase in endothelial cells and EDR by activating the forward mode of NCX. On the other hand, KB-R7943 inhibited EDR and ATP-induced Ca2+ transients in MAECs at high concentrations (>10 µM) (Fig. 5, A–D).



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 5. Effect of the NCX (reverse mode) inhibitor KB-R7943 on K+-induced inhibition. A: K+-induced relaxation in control rings. B and C: aortas used in A were pretreated with 10 µM KB-R7943 (B) or 30 µM KB-R7943 (C). K+-induced inhibition was not inhibited by 10 µM KB-R7943 but was inhibited by 30 µM KB-R7943. D: KB-R7943 inhibited ATP-induced Ca2+ transients in MAECs at high concentrations (>10 µM). In addition, EDR was inhibited by pretreating with KB-R7943 (B and C). E and F: K+-induced inhibition of [Ca2+]i increase was not inhibited by 10 µM KB-R7943 but was inhibited by 30 µM KB-R7943. ***P < 0.001.

 
Concomitant activation of Na+-K+ pump and the forward mode of NCX. The above data suggests that the Na+-K+ pump and the forward mode of NCX are simultaneously activated by increasing [K+]o. Because activation of the Na+-K+ pump and the forward mode of NCX might affect the membrane potential, we examined the effect of ouabain and NCX blockers on the membrane potential. An increase in [K+]o from 6 to 12 mM depolarized the resting membrane potential from –29.8 ± 4.8 to –3.1 ± 3.8 mV in current-clamped MAECs (n = 14) (Figs. 6C and 7B), and this effect of extracellular K+ was inhibited by ouabain (100 µM) or Ni2+ (300 µM). Depolarized membrane potential was significantly hyperpolarized by ouabain or Ni2+ from –2.1 ± 5.5 to –9.6 ± 5.1 mV (n = 12) (Fig. 6, C and D) and from –3.9 ± 4.1 to –15.4 ± 3.2 mV (n = 11) (Fig. 7, B and C), respectively. In addition, KB-R7943 also hyperpolarized this depolarized potential (Fig. 7D). In contrast, when [K+]o was maintained at 6 mM, the membrane potential was significantly depolarized by ouabain (100 µM) from –27.8 ± 5.8 to –20.6 ± 6.0 mV (n = 9) (Fig. 6, A and B) and not changed by Ni2+ (300 µM, n = 8) or KB-R7943 (Fig. 7A). These data suggest that the Na+-K+ pump and the forward mode of NCX are simultaneously activated by extracellular K+.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 6. Effect of the Na+-K+ pump inhibitor ouabain on the depolarized membrane potential by extracellular K+. A and B: resting membrane potentials were depolarized by ouabain, when [K+]o was maintained at 6 mM. C and D: effect of extracellular K+ on the membrane potential was inhibited by the Na+-K+ inhibitor ouabain. The depolarized membrane potential by extracellular K+ was hyperpolarized by ouabain. **Statistical significance (P < 0001).

 


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7. Effects of the NCX inhibitors Ni2+ and KB-R7943 on the depolarized membrane potential (VM) by extracellular K+. A: resting membrane potentials were not changed by Ni2+ or KB-R7943, when [K+]o was maintained at 6 mM. B and C: effect of extracellular K+ on the membrane potential was inhibited by the NCX inhibitor Ni2+. The depolarized membrane potential was hyperpolarized by Ni2+. D: KB-R7943 also hyperpolarized the depolarized membrane potential. **Statistical significance (P < 001).

 
Effect of extracellular K+ on cGMP levels. When [Ca2+]i in endothelial cells is increased, endothelium-derived NO is released from endothelial cells. This released NO increases cGMP levels in vascular smooth muscle and thereby relaxes vascular smooth muscle. Thus we examined the effect of extracellular K+ on cGMP levels in aortic rings (Fig. 8). The cGMP level in aortic rings exposed to NE (1 µM) was 1.37 ± 0.37 pmol/mg protein. When aortic rings were exposed to NE and then to ACh (3 µM), this level was significantly increased to 107.42 ± 11.11 pmol/mg protein. After K+-induced inhibition of EDR was evoked, it fell significantly to 7.25 ± 1.36 pmol/mg protein (K+-induced inhibition of cGMP production). K+-induced inhibition of cGMP production was significantly inhibited by pretreating with ouabain (100 µM), Ni2+ (300 µM), or KB-R7943 (30 µM).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 8. Effect of extracellular K+ on cGMP concentration in mouse aortic smooth muscle. Increased cGMP concentrations were markedly reduced by increasing [K+]o. The inhibitory effect of extracellular K+ on cGMP concentration was reduced by pretreating with ouabain, Ni2+, or KB-R7943. ***Statistical significance (P < 0001).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provides new evidence concerning the roles of the Na+-K+ pump and the forward mode of NCX in endothelium-dependent vasorelaxation. In endothelial and vascular smooth muscle cells, an increase in [Ca2+]i by various stimuli stimulates Ca2+-activated K+ channels (5, 22). This activation in turn induces an efflux of intracellular K+ and may increase K+ concentration transiently in the extracellular space close to the cell membrane. It was reported that endothelial cell stimulation by ACh raises the K+ concentration in the myoendothelial space by 5.9 ± 1.0 mM (7). Therefore, when endothelial cells are stimulated, an increase in [K+]o might be enough to activate the Na+-K+ pump and the forward mode of NCX.

Subsequent activation of the forward mode of NCX after Na+-K+ pump activation is the mechanism of K+-induced inhibition. In this study, we showed that Na+-K+ pump activation stimulates the forward mode of NCX and thereby inhibits intracellular Ca2+ mobilization and endothelium-dependent vasorelaxation. Therefore, we suggest that subsequent activation of the forward mode of NCX after Na+-K+ pump activation is the mechanism of K+-induced inhibition of EDR and [Ca2+]i increase. This subsequent activation of the forward mode of NCX after Na+-K+ pump activation is consistent with a previous finding that the NCX current reverses from an outward to an inward direction during Na+-K+ pump activation (3, 10). These findings revealed a dynamic interaction between the two Na+ transport mechanisms, the Na+-K+ pump and NCX.

A [K+]o increase affects the membrane potential in various ways. A [K+]o increase might hyperpolarize membrane potential by activating the Na+-K+ pump. On the other hand, the membrane potential might be depolarized by activating the forward mode of NCX. Na+-K+ pump activation decreases [Na+]i. Fujioka et al. (10) estimated Na+-K+ pump activation-induced [Na+]i changes by measuring the reversal potential of NCX current. They reported that Na+-K+ pump activation by extracellular K+ decreased [Na+]i from 20 to 6 mM. This decrease shifted the reversal potential of NCX current markedly to a more positive potential and thereby accelerated the forward mode of NCX (10). The present study concurs with this finding by showing that the NCX blockers hyperpolarize the membrane potential when the Na+-K+ pump is activated. In addition, a [K+]o increase might depolarize the membrane potential by changing the equilibrium potential of K+. According to the Nernst equation, the equilibrium potential of K+ is ~85 mV at 6 mM K+ and approximately –65 mV at 12 mM K+. Thus K+ equilibrium potential change is almost 20 mV on increasing [K+]o from 6 to 12 mM. In a previous study in our laboratory, we (1) found that there is an intermediate conductance Ca2+-activated K+ (IKCa) channel in MAECs. The reversal potential of IKCa current was shifted from –54.3 to –33.2 mV by increasing [K+]o from 6 to 12 mM (1). Thus the membrane potential might be depolarized by increasing [K+]o in cells like MAECs.

We found that the NCX inhibitors Ni2+ and DCB potentiated EDR. These results suggest that the forward mode of NCX operates to extrude Ca2+ from endothelial cells during EDR and thus reduces EDR. Therefore, EDR may be potentiated by inhibiting the forward mode of NCX. [Ca2+]i in endothelial cells is increased during EDR, and the increased intracellular Ca2+ stimulates Ca2+-activated K+ channel in endothelial cells. In our previous study, we found that IKCa current contributes to EDR (1). When endothelial cells are stimulated during EDR, K+ efflux through activated IKCa may increase the K+ concentration in extracellular space close to the cell membrane within a millimolar range (7), and this increase in [K+]o may inhibit EDR by activating the Na+-K+ pump and the forward mode of NCX.

K+-induced inhibition of EDR was not blocked by a low concentration of KB-R7943 (up to 10 µM) but was blocked by a high concentration (30 µM). In addition, K+-induced inhibition of [Ca2+]i increase was slightly inhibited by 10 µM KB-R7943 and markedly inhibited by 30 µM KB-R7943. These results suggest that 30 µM KB-R7943 is enough to abolish K+-induced inhibition by inhibiting the forward mode of NCX1 completely in MAECs. This suggestion is consistent with a previous report that suggested that KB-R7943 inhibits the forward mode at high concentration (IC50 ≥ 30 µM) (13).

We found that KB-R7943 at low concentrations (up to 10 µM) did not change ATP-induced Ca2+ transients in MAECs, which is consistent with the finding that KB-R7943 at 1 µM did not inhibit EDR (25). In addition, we found that the subtype of NCX in MAECs is NCX1. These data suggest that the reverse mode of NCX does not contribute to EDR under physiological conditions, since KB-R7943 inhibits the reverse mode of NCX1 at low concentration (IC50 = 1.2–2.4 µM) (13). The reverse mode of NCX may contribute EDR in pathophysiological conditions such as Na+-loaded conditions or hyponatremia (<100 mM) (25). On the other hand, KB-R7943 inhibited EDR at a high concentration (>10 µM). This inhibition can be explained by the finding that KB-R7943 (>10 µM) inhibited ATP-induced Ca2+ transients (Fig. 5D). It was reported that KB-R7943, at a high concentration (30 µM), affected other ion transporters such as Na+/H+ exchanger, Ca2+ channels, Ca2+ pumps, Na+-K+ pump, and Na+ channels (13, 23). Thus we suggest that KB-R7943 inhibits EDR by affecting these ion transporters on endothelial or smooth muscle cells at a high concentration.

Physiological implications: role of NCX in EDR. In this study, we found that the magnitude of EDR is significantly increased by the NCX (forward and reverse mode) inhibitors Ni2+ or DCB. This finding indicates that the forward mode of NCX reduces EDR in physiological conditions. In physiological conditions and in the absence of Na+ loading, NCX seems to contribute to Ca2+ extrusion during cell activation to prevent Ca2+ overload (11). Because K+ is released to the extracellular space through Ca2+-activated K+ channels during endothelial cell activation, we speculate a dynamic interaction between Ca2+-activated K+ channels, the Na+-K+ pump, and NCX. During cell activation, the increased intracellular Ca2+ stimulates Ca2+-activated K+ channel, which releases K+ to the extracellular space. This released K+ then activates Na+-K+ pump and the forward mode of NCX subsequently, which prevents a further increase in [Ca2+]i. Through this mechanism, endothelial cells may inhibit an excessive increase in [Ca2+]i and sensitively control NO release. This control in NO release may be important, because NO is converted to toxic species such as peroxynitrite on reacting with oxygen radicals (4, 12, 15, 20, 21). This mechanism may enable endothelial cells to release an appropriate amount of NO by regulating increases in [Ca2+]i. On the other hand, as shown in this study, the reverse mode of NCX increases [Ca2+]i and evokes EDR in Na+-loaded conditions (6, 25, 28). In pathophysiological situations such as under oxidative stress, excessive Na+ entry via redox-activated cation channels has been observed (2, 8). The resulting profound Na+ loading is expected to activate the reverse mode of NCX. Therefore, we suggest Na+-Ca2+ exchange is a physiological and pathophysiological link between Na+ homeostasis and Ca2+-associated cell signaling.

In conclusion, we show here that activation of the forward mode of NCX following Na+-K+ pump activation decreases [Ca2+]i within endothelial cells and thus inhibits EDR. We suggest a dynamic interaction between Ca2+-activated K+ channels, the Na+-K+ pump, and NCX, which enables endothelial cells to sensitively control Ca2+ influx, and therefore [Ca2+]i, and may be a mechanism by which endothelial cells sensitively modulate vasomotor activity.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Korea Research Foundation Grant (KRF-2003–041-E00022).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. H. Suh, Dept. of Physiology, College of Medicine, Ewha Women's Univ., 911–1 Mok-6-dong, Yang Chun-gu, Seoul, Republic of Korea 158–710 (e-mail: shsuh{at}ewha.ac.kr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ahn SC, Seol GH, Kim JA, and Suh SH. Characteristics and a functional implication of Ca2+-activated K+ current in mouse aortic endothelial cells. Pflügers Arch 447: 426–435, 2004.[CrossRef][ISI][Medline]
  2. Balzer M, Lintschinger B, and Groschner K. Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells. Cardiovasc Res 42: 543–549, 1999.[Abstract/Free Full Text]
  3. Bielen FV, Glitsch HG, and Verdonck F. Changes of the subsarcolemmal Na+ concentration in internally perfused cardiac cells. Biochim Biophys Acta 1065: 269–271, 1991.[Medline]
  4. Bouloumie A, Bauersachs J, Linz W, Scholkens BA, Wiemer G, Fleming I, and Busse R. Endothelial dysfunction coincides with an enhanced nitric oxide synthase expression and superoxide anion production. Hypertension 30: 934–941, 1997.[Abstract/Free Full Text]
  5. Burnham MP, Bychkov R, Feletou M, Richards GR, Vanhoutte PM, Weston AH, and Edwards G. Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium: relevance to EDHF. Br J Pharmacol 135: 1133–1143, 2002.[CrossRef][ISI][Medline]
  6. Domotor E, Abbott NJ, and Adam-Vizi V. Na+-Ca2+ exchange and its implications for calcium homeostasis in primary cultured rat brain microvascular endothelial cells. J Physiol 515: 147–155, 1999.[Abstract/Free Full Text]
  7. Edwards G, Dora KA, Gardener MJ, Garland CJ, and Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 396: 269–272, 1998.[CrossRef][Medline]
  8. Elliott SJ and Koliwad SK. Oxidant stress and endothelial membrane transport. Free Radic Biol Med 19: 649–658, 1995.[CrossRef][ISI][Medline]
  9. Fernandez-Alfonso MS, Sanchez-Ferrer CF, Hernandez MC, and Marin J. Na+/Ca2+ exchange mediation in the ouabain-induced contraction in human placental vessels. Gen Pharmacol 23: 439–444, 1992.[ISI][Medline]
  10. Fujioka Y, Matsuoka S, Ban T, and Noma A. Interaction of the Na+-K+ pump and Na+-Ca2+ exchange via [Na+]i in a restricted space of guinea-pig ventricular cells. J Physiol 509: 457–470, 1998.[Abstract/Free Full Text]
  11. Graier WF, Paltauf-Doburzynska J, Hill BJ, Fleischhacker E, Hoebel BG, Kostner GM, and Sturek M. Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane. J Physiol 506: 109–125, 1998.[Abstract/Free Full Text]
  12. Ischiropoulos H, Zhu L, and Beckman JS. Peroxynitrite formation from macrophage-derived nitric oxide. Arch Biochem Biophys 298: 446–451, 1992.[CrossRef][ISI][Medline]
  13. Iwamoto T, Watano T, and Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1. J Biol Chem 271: 22391–22397, 1996.[Abstract/Free Full Text]
  14. Kim MY, Liang GH, Kim JA, Park SH, Hah JS, and Suh SH. Contribution of Na+-K+ pump and KIR currents to extracellular pH-dependent changes of contractility in rat superior mesenteric artery. Am J Physiol Heart Circ Physiol 289: H792–H800, 2005.[Abstract/Free Full Text]
  15. Koppenol WH, Moreno JJ, Pryor WA, Ischiropoulos H, and Beckman JS. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem Res Toxicol 5: 834–842, 1992.[CrossRef][ISI][Medline]
  16. Li L and van Breemen C. Na+-Ca2+ exchange in intact endothelium of rabbit cardiac valve. Circ Res 76: 396–404, 1995.[Abstract/Free Full Text]
  17. Manolopoulos VG, Liu J, Unsworth BR, and Lelkes PI. Adenylyl cyclase isoforms are differentially expressed in primary cultures of endothelial cells and whole tissue homogenates from various rat tissues. Biochem Biophys Res Commun 208: 323–331, 1995.[CrossRef][ISI][Medline]
  18. Marin J, Fernandez-Alfonso MS, and Sanchez-Ferrer CF Sodium pump activity and contractile effect of ouabain in human placental veins. Eur J Pharmacol 201: 75–82, 1991.[CrossRef][ISI][Medline]
  19. McCarron JG and Halpern W. Potassium dilates rat cerebral arteries by two independent mechanisms. Am J Physiol Heart Circ Physiol 259: H902–H908, 1990.[Abstract/Free Full Text]
  20. Muijsers RB, Folkerts G, Henricks PA, Sadeghi-Hashjin G, and Nijkamp FP. Peroxynitrite: a two-faced metabolite of nitric oxide. Life Sci 60: 1833–1845, 1997.[CrossRef][ISI][Medline]
  21. Munzel T, Heitzer T, and Harrison DG. The physiology and pathophysiology of the nitric oxide/superoxide system. Herz 22: 158–172, 1997.[ISI][Medline]
  22. Nilius B and Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 81: 1415–1459, 2001.[Abstract/Free Full Text]
  23. Ouardouz M, Zamponi GW, Barr W, Kiedrowski L, and Stys PK. Protection of ischemic rat spinal cord white matter: dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels. Neuropharmacology 48: 566–575, 2005.[CrossRef][ISI][Medline]
  24. Paulson OB and Newman EA. Does the release of potassium from astrocyte endfeet regulate cerebral blood flow? Science 237: 896–898, 1987.[Abstract/Free Full Text]
  25. Schneider JC, El Kebir D, Chereau C, Mercier JC, Dall'Ava-Santucci J, and Dinh-Xuan AT. Involvement of Na+/Ca2+ exchanger in endothelial NO production and endothelium-dependent relaxation. Am J Physiol Heart Circ Physiol 283: H837–H844, 2002.[Abstract/Free Full Text]
  26. Seol GH, Ahn SC, Kim JA, Nilius B, and Suh SH. Inhibition of endothelium-dependent vasorelaxation by extracellular K+: a novel controlling signal for vascular contractility. Am J Physiol Heart Circ Physiol 286: H329–H339, 2004.[Abstract/Free Full Text]
  27. Suh SH, Vennekens R, Manolopoulos VG, Freichel M, Schweig U, Prenen J, Flockerzi V, Droogmans G, and Nilius B. Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling. Pflügers Arch 438: 612–620, 1999.[CrossRef][ISI][Medline]
  28. Teubl M, Groschner K, Kohlwein SD, Mayer B, and Schmidt K. Na+/Ca2+ exchange facilitates Ca2+-dependent activation of endothelial nitric-oxide synthase. J Biol Chem 274: 29529–29535, 1999.[Abstract/Free Full Text]
  29. Woolfson RG and Poston L. Effect of ouabain on endothelium-dependent relaxation of human resistance arteries. Hypertension 17: 619–625, 1991.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/5/H2020    most recent
00908.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, M. Y.
Right arrow Articles by Suh, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, M. Y.
Right arrow Articles by Suh, S. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.