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Am J Physiol Heart Circ Physiol 286: H329-H339, 2004. First published September 11, 2003; doi:10.1152/ajpheart.00503.2003
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Inhibition of endothelium-dependent vasorelaxation by extracellular K+: a novel controlling signal for vascular contractility

Geun Hee Seol,1 Seung Cheol Ahn,2 Ji Aee Kim,1 Bernd Nilius,3 and Suk Hyo Suh1

1Department of Physiology and Medical Research Center, College of Medicine, Ewha Women's University, 2Department of Physiology, College of Medicine, Dankook University, Seoul 158-710, Republic of Korea; and 3Laboratorium voor Fysiologie, Campus Gasthuisberg, Katholieke Universiteit, Leuven, B-3000 Leuven, Belgium

Submitted 4 June 2003 ; accepted in final form 4 September 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2{alpha} or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from –32.8 ± 2.7 to –8.6 ± 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from –50 to –25 mV decreased [Ca2+]i significantly from 0.95 ± 0.03 to 0.88 ± 0.03 µM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 ± 0.04 µM by depolarizing the holding potential from –25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 µM). These results indicate that an increase of [K+]o in the physiological range (6–12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.

endothelial cell; intracellular calcium

EXTRACELLULAR K+ concentration ([K+]o) changes in the vicinity of excitable and nonexcitable cells under various conditions. In endothelial and vascular smooth muscle cells, an increase in intracellular Ca2+ concentration ([Ca2+]i) by various stimuli stimulates Ca2+-activated K+ channels (5, 7, 29). This activation, in turn, induces an efflux of intracellular K+. In brain tissues, activated neuronal cells release K+, which enters surrounding astrocytes. As a result, K+ is further released from activated neuronal cells and astrocytes (28, 32, 39). Thus, when intracellular K+ is released, [K+]o can substantially increase. Also under pathological conditions, e.g., during subarachnoid hemorrhage or various cell injuries, [K+]o can increase.

The contractility of vascular smooth muscle is changed when [K+]o is increased. Most arteries contract in response to an increase in [K+]o to >30 mM. However, an increase to 6–15 mM relaxes vascular smooth muscle. This relaxation is well known as K+-induced relaxation, which is prominent in the vascular smooth muscles of resistant arteries and considered as an important regulator of resistant artery contractility (22, 25, 32). Compared with resistant arteries, K+-induced relaxations are relatively small or negligible in large arteries such as the aorta. The following mechanism of K+-induced relaxation has been determined (10, 11, 25, 33). An increase of [K+]o activates the Na+-K+ pump and increases the conductance of the inwardly rectifying K+ (IRK) channel, which causes a hyperpolarization of the membrane potential (25, 33). This hyperpolarization inhibits voltage-gated Ca2+ channels and relaxes the vascular smooth muscle. However, nothing is known about the effects of extracellular K+ on endothelial cells, which play a pivotal role in regulation of vascular smooth muscle contractility.

Here, we show that an increase of [K+]o within a millimolar range affects the contractility of vascular smooth muscle by modulating Ca2+ mobilization in endothelial cells. Increased [K+]o inhibits [Ca2+]i increase in endothelial cells and, thereby, EDR. We refer to this phenomenon as K+-induced inhibition and describe mechanisms involved in the process.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Contraction Measurement on Isolated Aortic Rings

Five- to 6-mo-old mice of either gender were anesthetized by injection of pentobarbital sodium (50 mg/kg body wt ip) and killed by cervical dislocation. The thoracic aorta was dissected out and cut into ~3.0-mm rings. A homemade myograph was used to record mechanical responses from the aortic ring segments (37). 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 using a peristaltic pump. The composition (in mM) of the Krebs buffer was 118.3 NaCl, 4.7 KCl, 1.2 MgCl2, 1.22 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose, pH 7.4. Optimal resting tension (0.8–1 g) was applied. Rings were precontracted with 3 µM prostaglandin F2{alpha} (PGF2{alpha}), and EDR was induced by 3 µM acetylcholine (ACh). When EDR had reached a maximum, [K+]o was increased from 6 to 12 mM in one step or in increments of 2 mM.

Endothelial Cell Culture

Mouse aorta endothelial cells. We isolated endothelial cells from mouse aorta using the "primary explant technique," which is described in detail elsewhere (24, 38). Cells were grown in growth medium (100 ml) composed of 80 ml of DMEM, 10 ml of fetal calf serum, 7.5 mg of endothelial cell growth supplement (catalog no. E-2759, Sigma), 200 µl of heparin (10 U/ml final concentration), 2 ml of penicillin-streptomycin (100 U/ml final concentration), 1 ml of L-glutamine, and 1 ml of minimal essential amino acids. After 4–7 days, the aortic pieces were removed. Cells were passaged as described by Suh et al. (38) and Voets et al. (42). The cells were used up to passage 2 for functional studies.

Human umbilical vein endothelial cells. Two types of human umbilical vein endothelial cells [HUVEC: EA.hy926 (EA) (9) and CRL-1730 cells] were used in this experiment. EA cells were grown in DMEM containing 20% fetal calf serum + 10% hypoxanthine-aminopterin-thymidine 50x supplement (Life Technologies). CRL-1730 cells were purchased from the American Type Culture Collection and grown in American Type Culture Collection medium (Ham's F-12K medium containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mg/ml heparin, 0.03–0.05 mg/ml endothelial cell growth supplement, and 10% FBS).

Cell culture was maintained at 37°C in a fully humidified 95% air-5% CO2 atmosphere. The cells were detached by exposure to trypsin, reseeded on gelatin-coated coverslips, and maintained in culture for 2–4 days before use. Measurements were performed on nonconfluent cells.

Electrophysiology

Electrophysiological methods and Ca2+ measurements have been previously described in detail (30). Membrane potential was monitored in current-clamp mode or controlled in voltage-clamp mode with an EPC-9 (HEKA Elektronik, Lambrecht, Germany) using a nystatin-perforated patch (100 mg/ml). Whole cell currents were measured using ruptured patches. Voltages were monitored in voltage-clamp mode with an EPC-9 (sampling rate 1 ms, 8-pole Bessel filter, 2.9 kHz). The holding potential for the whole cell experiment was 0 or –60 mV. We applied a voltage ramp from –100 or –150 to +100 mV every 10 s with duration of 650 ms. Currents were recorded at a sampling rate of 1–4 kHz.

Ca2+ Measurement

Cells were loaded with fura-2 AM, and [Ca2+]i was measured using a microfluorometer consisting of an inverted microscope (DM IRB, Leica, Germany) and a filter scan power illuminator system (Photon Technology International). Fura-2 AM (2 µM) was added to the bath, and the cells were incubated for 25 min at 37°C. The cells were illuminated alternatively at wavelengths of 340 and 380 nm through a chopper wheel (frequency = 50 Hz). Fluorescence was measured at 510 nm, and autofluorescence was subtracted from the signals. The free Ca2+ concentration was calculated from the ratio of the fluorescence signals emitted at each excitation wavelength. The calibration procedure was identical to that described previously (30, 31).

Solutions

The standard external 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. The standard pipette solution contained (in mM) 40 KCl, 100 potassium-aspartate, 1 MgCl2, 0.1 EGTA, 4 Na2ATP, and 10 HEPES, with pH adjusted to 7.2 with KOH; osmolarity was 290 mosM.

Chemicals

ATP, ACh, chlorozoxazone (CZ), norepinephrine bitartrate, histamine, ouabain, PGF2{alpha}, NG-nitro-L-arginine methyl ester (L-NAME), tetraethylammonium chloride (TEA), and zoxazolamine (ZOX) were purchased from Sigma; 1-ethyl-2-benzimidazolinone (1-EBIO) from Tocris, nystatin from ICN Biomedicals, and fura-2 AM from Molecular Probes. 1-EBIO, fura-2 AM, ZOX, and nystatin were applied from a stock solution in DMSO. CZ was applied from a stock solution in methanol. The final concentration of DMSO and methanol was <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 the Ewha Women's University. All experiments were performed at 37°C. Pooled data are means ± SE, and significant differences were detected using Student's t-test (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of Extracellular K+ on EDR

Precontracted endothelium-intact aortic rings were relaxed by ACh application (Fig. 1, A1 and B). In contrast, endothelium-intact aortic rings pretreated with the nitric oxide (NO) synthase inhibitor L-NAME (30 µM; Fig. 1A2) or endothelium-denuded aortic rings (Fig. 1C) showed no relaxation on administration of ACh, indicating that the ACh-induced relaxation was endothelium dependent. Then the effect of extracellular K+ on EDR was examined. ACh-induced relaxation was abolished by increasing [K+]o from 6 to 12 mM (K+-induced inhibition of EDR), and this reduced EDR was increased by reducing [K+]o from 12 to 6 mM (Fig. 1A1). This pattern could be repeated several times (Fig. 1A1). K+-induced inhibition of EDR was not dependent on contractile agents. When an aortic ring was contracted with norepinephrine, EDR was abolished by increasing [K+]o from 6 to 12 mM (Fig. 1B). Because a change in [K+]o modified the contractility of vascular smooth muscle, we examined whether an increase of [K+]o within a few millimolars could induce a change in contractility (Fig. 1, D and E). No significant change of tension was observed in vascular smooth muscle when [K+]o was increased from 6 to 15 mM. However, when [K+]o was increased to 18 mM, the aortic ring started to contract, with a response of 15.7 ± 3.5% of that observed at 50 mM K+ (n = 8). In addition, we examined whether an increase of [K+]o within a few millimolars could change the contractile response induced by PGF2{alpha} and ACh. No change of contraction could be evoked by increasing [K+]o in the L-NAME-pretreated aorta (Fig. 1A2) or in the denuded aorta (Fig. 1C). These contractions were measured in bicarbonate-buffered solution. In HEPES-buffered solution, K+-induced inhibition of EDR was observed (data not shown).



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  		Fig. 1. K+-induced inhibition of endothelium-dependent relaxation (EDR). A1: PGF2{alpha} was used to contract the aortic ring, and EDR was evoked by application of ACh. ACh-induced relaxation was inhibited by increasing extracellular K+ concentration ([K+]o, K+-induced inhibition of EDR). K+-induced inhibition of EDR was transient (arrow). A2: aorta used in A1 was pretreated with NG-nitro-L-arginine methyl ester (L-NAME). ACh or increase in [K+]o did not change tension produced by PGF2{alpha}. B: norepinephrine (NE) was used as a contractile agent. K+ induced inhibition of EDR in aorta contracted with NE. C: ACh or increase in [K+]o did not change tension produced by NE in denuded aorta. D and E: effect of [K+]o on contractility of vascular smooth muscle. No significant increase of tension was observed when [K+]o was increased to 15 mM.

 

The K+-induced inhibition of EDR was concentration dependent (Fig. 2). ACh-evoked relaxation of 84.4 ± 2.0% with 6 mM extracellular K+ was reduced to 65.7 ± 6.2, 29.2 ± 5.1, and –9.2 ± 2.5% by increasing [K+]o to 8, 10, and 12 mM, respectively (n = 8). The reduced EDR recovered to 67.5 ± 3.4% by return of [K+]o to 6 mM (Fig. 2, A and B). However, when [K+]o was maintained at 12 mM, ACh evoked EDR as much as it did when [K+]o was 6 mM (data not shown). Similar to K+, Cs+ also inhibited EDR. ACh-evoked EDR of 79.7 ± 1.5% with 0 mM extracellular Cs+ was reduced to 49.7 ± 9.4, 20.9 ± 12.2, and –6.3 ± 2.9% by increasing extracellular Cs+ concentration ([Cs+]o) to 2, 4, and 6 mM, respectively (n = 5). The reduced EDR recovered to 61.5 ± 4.3% by removal of Cs+ (Fig. 2, C and D).



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  		Fig. 2. Concentration-dependent inhibition of EDR by extracellular K+ or Cs+. As indicated by a–e in A and C, the magnitude of relaxation at each treatment was expressed as a percentage of initial PGF2{alpha}-induced contraction (B and D). [K+]o or extracellular Cs+ concentration ([Cs+]o) was increased from 6 to 12 mM or from 0 to 6 mM, respectively, in increments of 2 mM. 6 K, 8 K, 10 K, and 12 K, 6, 8, 10, and 12 mM K+; 0 Cs, 2 Cs, 4 Cs, and 6 Cs, 0, 2, 4, and 6 mM Cs+.

 

These results indicate that a millimolar increase in [K+]o or [Cs+]o inhibits EDR in a concentration-dependent and reversible manner without changing vascular smooth muscle contractility. Inasmuch as EDR is evoked by vasorelaxing substances released from endothelial cells and the substances are released by an increase in [Ca2+]i, we examined the effects of extracellular K+ on [Ca2+]i increase in endothelial cells.

Effects of Extracellular K+ on [Ca2+]i

In unclamped MAEC, ATP increased [Ca2+]i from 0.11 ± 0.02 to 0.92 ± 0.10 µM (n = 8). This increased [Ca2+]i was reduced to 0.22 ± 0.05 µM by increasing [K+]o from 6 to 12 mM in a reversible manner (Fig. 3, A and D). The same result was observed in voltage-clamped cells (holding potential = 0 mV; Fig. 3, C and E). ATP increased [Ca2+]i from 0.13 ± 0.03 to 1.11 ± 0.13 µM, and this elevated [Ca2+]i was reduced to 0.27 ± 0.05 µM by increasing [K+]o from 6 to 12 mM (K+-induced inhibition of [Ca2+]i increase, n = 8). Similar to K+, Cs+ also inhibited the increase in [Ca2+]i (Fig. 3B). The K+-induced inhibition of [Ca2+]i increase was not dependent on agonists. When endothelial cells were stimulated by ACh (Fig. 3C) or histamine (Fig. 4C), the increase in [Ca2+]i was reduced by raising [K+]o from 6 to 12 mM.



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  		Fig. 3. Extracellular K+ or Cs+ inhibited intracellular Ca2+ concentration ([Ca2+]i) increase in mouse aorta endothelial cells (MAEC). A and D: ATP-induced elevation in [Ca2+]i was reduced by increasing [K+]o in unclamped cells. B: similar to the effect of K+, increasing [Cs+]o inhibited [Ca2+]i increase. C and E: effect of [K+]o was independent of membrane potential (Vm). Increase in [K+]o (12 mM K+) inhibited [Ca2+]i]i increase in voltage-clamped cells. HP, holding potential.

 


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  		Fig. 4. Extracellular K+ inhibited [Ca2+]i increase in human umbilical vein endothelial cells (HUVEC: EA and CRL-1730 cells). In addition to MAEC, [Ca2+]i increase in HUVEC was also inhibited by increasing [K+]o. K+-induced inhibition of [Ca2+]i increase was transient and magnitude and duration of inhibition were dependent on rate of [K+]o increase in EA (A) and CRL-1730 cells (B). C: K+ induced inhibition of [Ca2+]i increase in EA cells voltage clamped at –60 mV.

 

The K+-induced inhibition of [Ca2+]i increase was observed in MAEC and two types of HUVEC (EA and CRL-1730 cells; Fig. 4). The agonist-induced increase in [Ca2+]i was reduced concentration dependently by increasing [K+]o from 0 to 1, 3, and 6 mM (Fig. 4A) or from 6 to 9 and 12 mM (Fig. 4B). The K+-induced inhibition of [Ca2+]i increase in HUVEC was also independent of membrane potential. When membrane potential was clamped at 0 or –60 mV, [Ca2+]i was reduced by increasing [K+]o from 6 to 12 mM (Fig. 4C). The K+-induced inhibition was transient, and the duration of its inhibition was concentration dependent (Fig. 4, A and B). The reduced [Ca2+]i increase was reversed with time, although [K+]o was maintained at the elevated level.

These results indicate that an increase of [K+]o or [Cs+]o within a few millimolars reversibly inhibits [Ca2+]i increase in a concentration-dependent manner and, thereby, likely inhibits release of endothelium-derived relaxing factor(s) [EDRF(s)] from endothelial cells.

The increase of [K+]o or [Cs+]o may depolarize membrane potential, decrease K+ efflux, and activate the Na+-K+ pump. Therefore, we examined whether these changes might be involved in K+-induced inhibition of EDR and [Ca2+]i increase.

A Membrane Potential-Dependent Mechanism?

In current-clamped EA cells, ATP increased [Ca2+]i and hyperpolarized the membrane potential simultaneously (Fig. 5A). Both effects were reversed by increasing [K+]o from 6 to 12 mM. An increase of [K+]o depolarized the membrane potential rapidly from –43 to –25 mV without changing [Ca2+]i. This depolarization was followed by a gradual decrease of [Ca2+]i and a further depolarization. These data suggest that the initial rapid depolarization initiates [Ca2+]i reduction and that further depolarization decreases the driving force for Ca2+ influx. To further confirm this mechanism, we examined the effect of membrane potential on [Ca2+]i (Fig. 5, E–G). Inasmuch as the increase in [K+]o from 6 to 12 mM depolarized the resting membrane potential from –32.8 ± 2.7 to –8.6 ± 4.9 mV (n = 8) in current-clamped unstimulated EA cells (Fig. 5, C and D), we examined the effect of 25-mV depolarization on [Ca2+]i. The endothelial cells were clamped at –50 mV initially, and [Ca2+]i was increased by the application of ATP. When the increased [Ca2+]i reached a steady state, the holding potential was shifted to –25 and 0 mV. In the cells voltage clamped at –50 mV, ATP increased [Ca2+]i from 0.10 ± 0.01 to 0.95 ± 0.03 µM, which was significantly decreased to 0.88 ± 0.03 µM by the shift of the holding potential to –25 mV (n = 11, P < 0.01), and [Ca2+]i was further decreased to 0.47 ± 0.04 µM by the shift of the holding potential from –25 to 0 mV (n = 11, P < 0.001; Fig. 5, E–G). The decrease in [Ca2+]i as a result of depolarization from –25 to 0 mV was significantly greater than the decrease due to depolarization from –50 to –25 mV (n = 11, P < 0.001). These results suggest that the amount of [Ca2+]i decrease due to depolarization is augmented by depolarization of the membrane potential. Similar responses in [Ca2+]i were recorded in voltage-clamped MAEC by depolarization of the membrane potential.



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  		Fig. 5. Effects of depolarization on [Ca2+]i increase in EA cells. A: ATP-induced hyperpolarization and [Ca2+]i increase were reversed by increasing [K+]o. B: relation between membrane potential and [Ca2+]i during period a in A. Increasing [K+]o depolarized membrane potential without changing [Ca2+]i, gradually decreased [Ca2+]i, and resulted in further depolarization. C and D: resting membrane potential of endothelial cells was significantly depolarized by increasing [K+]o. E–G: significant decrease in [Ca2+]i by depolarization from –50 to –25 mV (**P < 0.01) or from –25 to 0 mV (***P < 0.001).

 

A K+ Efflux-Dependent Mechanism?

In voltage-clamped EA cells, ATP increased [Ca2+]i and activated an outwardly rectifying current simultaneously (Fig. 6). This current is identified as the large-conductance Ca2+-activated K+ (BKCa) current, inasmuch as it was activated by intracellular Ca2+ and blocked by 50 nM iberiotoxin (data not shown). The increased [Ca2+]i was reduced and the K+ current disappeared when [K+]o was increased from 6 to 12 mM (Fig. 6, A–C and E). An increase of [K+]o decreased the outward current without changing [Ca2+]i at the initial stage, and [Ca2+]i simultaneously decreased with the decrease of the current in the late stage. These data suggest that the initial decrease of K+ current initiates the decrease in [Ca2+]i. To further confirm this mechanism, we examined the effect of K+ channel blockers and activators on EDR and [Ca2+]i (Figs. 7 and 8).



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  		Fig. 6. Effects of extracellular K+ on [Ca2+]i increase and membrane current in voltage-clamped cell. A–C: current and [Ca2+]i were measured simultaneously at a holding potential of 0 mV in nystatin-perforated mode. B: data points were obtained at +100 and –50 mV during repetitive ramps from –150 to +100 mV. C: membrane current changes at holding potential of 0 mV. D: relation between [Ca2+]i (A) and membrane current at holding potential (C) during the period indicated as a in A. Increasing [K+]o decreased membrane current without changing [Ca2+]i and simultaneously decreased [Ca2+]i and current. E: current-voltage relations obtained at points indicated as 1–3 in B.

 


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  		Fig. 7. Effect of K+ efflux inhibition on [Ca2+]i and EDR. A and B: Ca2+-activated K+ current was activated by clamping [Ca2+]i at 1 µM in MAEC. Increasing [K+]o shifted reversal potential and decreased K+ efflux at negative potentials (A). Tetraethylammonium (TEA) inhibited, but not completely, K+ current. C: TEA inhibited [Ca2+]i increase in voltage-clamped cell. D: TEA inhibited EDR. Magnitude of TEA-induced inhibition was similar to that induced by increasing [K+]o from 6 to 12 mM.

 


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  		Fig. 8. Effect of K+ channel activators [chlorozoxazone (CZ), 1-ethyl-2-benzimidazolinone (1-EBIO), and zoxazolamine (ZOX)] on K+-induced inhibition. A and B: channel activators reversed K+-induced inhibition of EDR. K+-induced inhibition of EDR was transient (arrow in A). C: channel activators reversed K+-induced inhibition of [Ca2+]i increase in voltage-clamped cell. D: channel activators slightly relaxed precontracted endothelium-denuded aorta.

 

When MAEC were loaded with 1 µM Ca2+ via the patch pipette, an outward current was developed (Fig. 7, A and B). The Ca2+-activated current was an intermediate-conductance K+ (IKCa) current, inasmuch as it was blocked by the IKCa current blockers charybdotoxin (50 nM) and clotrimazole (10 µM; data not shown). The reversal potential of the current obtained by ramp pulses from –100 to +100 mV was –70.9 ± 0.1 mV and shifted to –54.3 ± 0.7 mV when [K+]o was increased from 6 to 12 mM (n = 3; Fig. 7A). The increase of [K+]o decreased K+ efflux, especially at negative potentials. On the other hand, TEA decreased the current without changing the reversal potential (Fig. 7B). Similar to K+ or Cs+, TEA also inhibited the [Ca2+]i increase in voltage-clamped MAEC (Fig. 7C) and the EDR of mouse aorta (Fig. 7D). These data suggest that the decrease of K+ efflux inhibits the increase in [Ca2+]i and, thereby, EDR.

The IKCa current activators 1-EBIO, CZ, and ZOX reversed K+-induced inhibition of EDR and [Ca2+]i increase (Fig. 8). When the endothelium was removed, the IKCa current activators did not relax the precontracted aorta (Fig. 8D). On the other hand, the activators restored the decrease in EDR (Fig. 8, A and B), and the decrease in [Ca2+]i was also restored by the IKCa current activators in voltage-clamped MAEC (Fig. 8C). These data indicate that the increase of K+ efflux by the activators reversed K+-induced inhibition of EDR and [Ca2+]i increase. Therefore, we could conclude that K+-induced inhibition might be caused by the decrease of K+ efflux.

An Ouabain-Sensitive Mechanism?

The Na+-K+ pump is activated by an increase in [K+]o or [Cs+]o. Therefore, we examined whether the Na+-K+ pump might be involved in the K+-induced inhibition of EDR and [Ca2+]i increase. EDR of 86.2 ± 1.6% with 6 mM extracellular K+ was reduced to 2.2 ± 3.6% by increasing [K+]o to 12 mM, and ouabain restored the reduced EDR to 70.2 ± 2.9% (n = 6; Fig. 9, A and B), although ouabain itself inhibited EDR concentration dependently (Fig. 9). In addition, the K+-induced inhibition of [Ca2+]i increase was also relieved by ouabain (Fig. 9C). These results indicate that Na+-K+ pump activation is involved in the K+-induced inhibition of EDR and [Ca2+]i increase.



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  		Fig. 9. Inhibition of K+-induced inhibition by ouabain. A, B, and D: ouabain inhibited EDR concentration dependently (D), but ouabain restored reduced EDR when [K+]o was increased. Ouabain 10, 10 µM ouabain. C: ouabain reversed K+-induced inhibition of [Ca2+]i increase.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
We report here for the first time that a millimolar increase in [K+]o, which is in the normal physiological range of paracellular [K+]o variation, decreases [Ca2+]i in vascular endothelial cells and inhibits ACh-induced endothelium-dependent vasorelaxation. Inasmuch as endothelium-derived vasoactive factors are released by an increase in endothelial [Ca2+]i, [K+]o can affect this release by modulating [Ca2+]i, which in turn modifies the vascular contractility. Because K+-induced inhibition was observed in at least two kinds of endothelial cells, i.e., MAEC and HUVEC, K+-induced inhibition is likely a general mechanism of endothelium-dependent vasomotor control in different kinds of blood vessels. Therefore, inasmuch as [K+]o may change by a few millimolars is in various physiological and pathological conditions, we suggest that this mechanism provides a novel controlling signal for vascular contractility.

Inhibitory Mechanisms of Extracellular K+ on [Ca2+]i

Our results suggest that at least three mechanisms are involved in K+-induced inhibition: membrane potential-dependent, K+ efflux-dependent, and ouabain-sensitive mechanisms. It is well known that membrane potential affects [Ca2+]i by changing the driving force for Ca2+ influx in endothelial cells (19, 20, 44), which suggests that the decrease in [Ca2+]i due to depolarization is dependent on how much the membrane potential is depolarized. When [K+]o was increased from 6 to 12 mM, the membrane potential was depolarized ~25 mV, which was enough to decrease [Ca2+]i. In addition, the decrease in [Ca2+]i due to depolarization is augmented by depolarization of the membrane potential, and the effect of depolarization on [Ca2+]i is weak in hyperpolarized endothelial cells. When the endothelium is stimulated to produce EDR, endothelium-derived hyperpolarizing factor and Ca2+-activated K+ currents hyperpolarize endothelial and vascular smooth muscle cells. Therefore, we suggest that the contribution of depolarization to K+-induced inhibition might be minimal at the initial stage, when the membrane potential is hyperpolarized. However, at the later stage, when the membrane potential is depolarized by [Ca2+]i decrease, depolarization facilitates the decrease in [Ca2+]i (23, 29, 35).

K+ channel blockers decrease K+ efflux and depolarize membrane potential. Inasmuch as there is no effect on membrane potential in voltage-clamped cells, TEA might inhibit [Ca2+]i increase by inhibiting K+ efflux; this was further confirmed by the effect of K+ channel activators. In voltage-clamped cells, K+ channel activators reversed K+-induced inhibition. The mechanism whereby K+ efflux inhibition causes K+-induced inhibition is unclear and requires further investigation.

The effect of extracellular K+ might be dependent on types of K+ channels in endothelial cells. The increase of [K+]o depolarizes membrane potential and reduces K+ efflux in endothelial cells with Ca2+-activated K+ channels such as BKCa or IKCa. On the other hand, in endothelial cells with IRK channels, the increase of [K+]o hyperpolarizes membrane potential and augments K+ efflux. Therefore, the response of extracellular K+ might be different in endothelial cells with IRK channels. Inasmuch as some endothelial cells express IRK channels (13, 16, 18, 29, 36, 43), the effect of extracellular K+ on [Ca2+]i and EDR in the vessels with such endothelial cells requires further evaluation.

We suggest that Na+-K+ pump activation might be the initial step in this mechanism, as supported by the following findings. First, K+-induced inhibition is evoked by an increase in [K+]o. Inasmuch as the activity of the Na+-K+ pump is dependent on [K+]o, increased [K+]o would activate the Na+-K+ pump. Second, Cs+, a well-known activator of the Na+-K+ pump (34), has the same inhibitory effect as K+. Finally, K+-induced inhibition is blocked by the Na+-K+ pump inhibitor ouabain. Thus Na+-K+ pump activation by extracellular K+ might reduce [Ca2+]i in endothelial cells and, thereby, inhibit EDR, which is contrary to the previous finding that Na+-K+ pump inhibition affected synthesis or release of EDRF(s), rather than its effector pathway (45). However, because there has been no other report of the effect of Na+-K+ pump inhibition or activation on endothelial cells, the role of the Na+-K+ pump in EDRF release from endothelial cells is unclear, and the mechanism whereby EDR is inhibited by Na+-K+ pump activation requires further evaluation. In contrast, many reports suggested that Na+-K+ pump inhibition affected the EDRF effector pathway. Ouabain inhibited sodium nitroprusside-induced relaxation (14, 15, 40) and endothelium-derived hyperpolarizing factor-mediated relaxation (6, 8, 41). The inhibited relaxation might be caused by the increased contractility of vascular smooth muscle, inasmuch as Na+-K+ pump inhibition increased [Ca2+]i via Na+/Ca2+ exchange (NCX) (2, 3) or promotion of Ca2+ influx through Ca2+-permeable channels (1, 46). Inasmuch as [Ca2+]i in endothelial cells may also be increased through these mechanisms, we speculate that NCX plays an important role in K+-induced inhibition. The increased intracellular Na+ concentration induced by Na+-K+ pump inhibition favors the reverse mode of NCX, which increases [Ca2+]i. On the other hand, the decreased intracellular Na+ concentration induced by Na+-K+ pump activation favors the forward mode of NCX, which decreases [Ca2+]i.

The inhibitory effect of extracellular K+ on [Ca2+]i was transient and dependent on the amount of [K+]o increase. The inhibitory effect of extracellular K+ on EDR was also transient. With the repetition of K+-induced inhibition on EDR, the K+-induced inhibition becomes transient (Fig. 1A-1 and Fig. 8A, arrow). These findings indicate that endothelial cells do not respond to [K+]o per se but to the change in [K+]o. Therefore, it might be suggested that the activities of the Na+-K+ pump may not be dependent on [K+]o per se but, rather, on changes in [K+]o.

Physiological Implication

Here we propose K+-induced inhibition as a novel mechanism for the control of vascular contractility. Through this mechanism, endothelial cells may inhibit an excessive increase in [Ca2+]i and sensitively control EDRF release. Inasmuch as Ca2+-activated K+ channels are well developed in various endothelial cells, any increase in [Ca2+]i would augment the driving force for Ca2+ entry, resulting in a further increase in [Ca2+]i. The efflux of K+ through the activated K+ channels might increase [K+]o. Edwards et al. (12) reported that endothelial cell stimulation by ACh raised K+ concentration in the myoendothelial space by 5.9 ± 1.0 mM. Although the increase of K+ on the luminal side of the endothelium might be less than that in the myoendothelial space because of blood flow, such an increase might be enough to produce K+-induced inhibition, which decreases [Ca2+]i. Therefore, we suggest that K+-induced inhibition is a negative-feedback mechanism on [Ca2+]i. Through this process, an excessive increase in [Ca2+]i might be prevented, and NO release would be appropriately controlled. It is well known that NO is changed to toxic substances such as peroxynitrite by reacting with oxygen radicals (4, 17, 21, 26, 27). Thus the control of the amount of NO release is important, and K+-induced inhibition may enable endothelial cells to release an appropriate amount of NO by regulating increases in [Ca2+]i.

In conclusion, we have shown that an increase in [K+]o within only a few millimolars depolarizes the membrane potential, decreases K+ efflux, and activates the Na+-K+ pump to decrease [Ca2+]i within endothelial cells and, thus, inhibits EDR. This possibly accounts for the fine tuning of Ca2+ influx and, therefore, [Ca2+]i during stimulation of endothelial cells and may be a basic mechanism by which endothelial cells modulate vasomotor activity.


   DISCLOSURES
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 ABSTRACT
 METHODS
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 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by Korea Health 21 Research and Development Project 01-PJ1-PG3-21400-0007 from the Ministry of Health and Welfare, Republic of Korea, and, in part, by Action "Levenslijn" from the Flemish Government (to B. Nilius).


   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 158-710, Republic of Korea (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
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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