Skip Navigation

Cardiovascular Research 1998 37(3):780-790; doi:10.1016/S0008-6363(97)00237-X
© 1998 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Prior, H.M
Right arrow Articles by Yates, M.S
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Prior, H.M
Right arrow Articles by Yates, M.S
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1998, European Society of Cardiology

K+-induced dilation of a small renal artery: no role for inward rectifier K+ channels

H.M Prior, N Webster, K Quinn, D.J Beech* and M.S Yates

Department of Pharmacology, University of Leeds, Leeds, LS2 9JT, UK

* Corresponding author. Tel.: (+44-113) 2334310; Fax: (+44-113) 2334331; E-mail: d.j.beech@leeds.ac.uk

Received 22 January 1997; accepted 2 September 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: To investigate the mechanism of K+-induced vasodilation in a small artery from the kidney, with a particular emphasis on the role of inward rectifier K+ channels. Methods: Lumen diameter and isometric tension recordings have been made from rabbit renal arcuate artery using pressurised- and wire-myography respectively. In addition, conventional whole-cell and amphotericin-perforated patch whole-cell recordings have been made from single smooth muscle cells isolated from the vessel. Results: Arcuate arteries dilated when the extracellular K+ concentration was raised to 8–10 mM from either zero or a normal physiological level of about 6 mM. The effect was not endothelium-dependent. Application of 0.01–1 mM Ba2+ to block inward rectifier K+ channels had no significant effect on K+-induced vasodilation in the arcuate artery, but under the same experimental conditions K+-induced dilation of the rat posterior cerebral artery was abolished by Ba2+. In the presence of 60 mM extracellular K+, inward rectifier K+-current was detectable in some single smooth muscle cells isolated from arcuate arteries but on average the current density was low (–1.44 pA pF–1 at –60 mV). K+-induced vasodilation of the arcuate artery was abolished by 10 µM ouabain and the half-effective concentration of K+ which induced vasodilation was 0.9–1.5 mM. Conclusions: The observations suggest that an increase in the extracellular K+ concentration (up to about 10 mM) dilates the rabbit renal arcuate artery and that the primary mechanism underlying the effect may be stimulation of Na+–K+ ATPase in the smooth muscle cell membrane. Inward rectifier K+ channels have a low average density in smooth muscle cells isolated from arcuate arteries and play no significant role in K+-induced vasodilation.

KEYWORDS Vasodilation; Arcuate artery; Rabbit; Kidney; K+ channel; Na+–K+ ATPase


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
K+-induced vasodilation has been observed in intact blood-perfused vascular beds [1], isolated vessels [2, 3]and following topical application of K+ to arterioles in situ [4]. This phenomenon may be important in the kidney because hyperkalaemia, produced by infusion of potassium chloride, increases renal blood flow and glomerular filtration rate [5, 6]. The response would, in conjunction with depressed tubular absorption, help to alleviate hyperkalaemia and its associated toxicity. In addition, K+-induced vasodilation may be of functional consequence during hypoglycaemia and hypoxia when extracellular K+ levels are known to rise to concentrations of at least 10 mM [7, 8].

Stimulation of Na+–K+ ATPase has been suggested as a mechanism for K+-induced vasodilation because the effect can be inhibited by ouabain [3, 9]. An additional mechanism for K+-induced dilation has also been proposed by Edwards and Hirst [10]and this centres around a Ba2+-sensitive inward rectifier K+ channel which is expressed in the smooth muscle cells of some arteries and arterioles [11, 12]. This channel, like other K+ channels, increases in conductance when extracellular K+ levels are raised, and because the channels are open at negative membrane potentials, the increase in conductance may induce hyperpolarisation (despite a positive shift of the K+-equilibrium potential). In rat posterior cerebral artery there is evidence that both Na+–K+ ATPase and inward rectifier K+ channel mechanisms contribute to K+-induced dilation, the former being important for K+ concentrations in the 0–6 mM range and the latter in the 7–15 mM range [13].

Inward rectifier K+-current has been identified in small cerebral and coronary arteries, and submucosal arterioles in the ileum, but appears absent from other types of smooth muscle tissue [10, 14–16]. For this reason, it has been suggested that inward rectifier K+ channels may have an expression profile restricted, within the smooth muscles, to small arteries and arterioles. This hypothesis is supported by a recent study of pig coronary arteries which found inward rectifier K+ channel expression at a much higher density in small (diameter <0.3 mm) compared with large arteries (diameter >1 mm) [12].

We have been investigating the functional roles of K+ channels in small arteries of the kidney and are using the arcuate artery as the focus of our studies. To determine if inward rectifier K+ channels are expressed in this artery, as might be expected by analogy with coronary and cerebral vessels, we began by looking for Ba2+-sensitive K+-induced vasodilation and subsequently searched for the channels using patch-clamp recording. The findings do not support a significant role for inward rectifier K+ channels in K+-induced dilation. As a consequence, we also investigated the hypothesis that stimulation of Na+–K+ ATPase underlies K+-induced dilation in the arcuate artery.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of arteries
Male Dutch rabbits (1.0–2.5 kg) were killed by i.v. administration of heparinised (1000 U ml–1) sodium pentobarbitone (70 mg kg–1). Kidneys were removed and placed in cold (on ice) 1.5-Ca2+ bath solution. The kidneys were decapsulated and divided longitudinally into 4 slices from which sections of arcuate artery were dissected, isolated and cleared of adhering tubules and connective tissue. Male Wistar rats (200–250 g) were killed by CO2 inhalation prior to removal of posterior cerebral arteries. The care and use of animals was carried out according to the Code of Practice as set out by The (UK) Animals (Scientific Procedures) Act 1986.

2.2 Myography recordings
2.2.1 Pressure myograph
Segments of renal arcuate artery or posterior cerebral artery (unpressurised lumen diameters of 140–190 µm and 150–180 µm respectively) were mounted in an 8 ml chamber (Living Systems Instrumentation, Burlington, Vermont), cannulated at both ends with glass cannulae (outer tip diameter 130–170 µm) and secured using monofilament nylon (25 µm). The glass cannulae were filled with Krebs solution, which was maintained in the cannulae throughout all experiments. The distal cannula was closed and the proximal cannula was connected to a pressure-servo unit (Living Systems Instrumentation) (i.e. the artery was pressurised but not perfused). The myograph was placed on the stage of an inverted trinocular microscope (Nikon) with attached video camera (Sony). The lumen diameter was continuously measured with a video dimension analyzer [13](Living Systems Instrumentation) and presented using a chart recorder (Kipp and Zonen) or with Origin 3.5 software after on-line digital capturing at 0.1 Hz (Picolog, Pico Technology, Cambridge).

Arteries were continuously superfused with Krebs solution at a rate of 25 ml min–1 in a non-recirculating system. The solution was at 37°C and gassed with 95% O2, 5% CO2. Chemical agents were added via the superfusate and arteries were pressurised to 60 mmHg. Viability of arcuate arteries and posterior cerebral arteries was evaluated by their ability to constrict in response to 10 µM phenylephrine and 10 µM 5-hydroxytryptamine respectively. A functional endothelium was judged to be present if there was dilation in response to 10 µM acetylcholine. In some experiments, the endothelium was removed, prior to pressurisation of the artery, by injecting air through the lumen for a few seconds. The artery was then equilibrated in Krebs solution for 10 min and confirmed as endothelium-denuded with undamaged smooth muscle cells if there was no dilation in response to 10 µM acetylcholine but pronounced dilation in response to 10 µM sodium nitroprusside. All vessels were equilibrated in Krebs solution for 2 h during which most arteries developed myogenic tone. If the development of myogenic tone resulted in less than a 50 µm decrease in lumen diameter, phenylephrine (0.1–1 µM) was added to the superfusate to induce additional constriction. At the end of each experiment, vessels were superfused with a Ca2+-free solution containing 0.5 mM EGTA to determine vessel diameter during maximal relaxation.

2.2.2 Wire myograph
A 1-mm long segment of renal arcuate artery was mounted on two 40-µm wires in a Mulvany myograph (JP Trading, Denmark). Arteries were left to equilibrate for 1 h in Krebs solution at 37°C and continuously gassed with 95% O2, 5% CO2 before being stretched to an internal circumference equivalent to 90% of that produced under an intramural pressure of 100 mmHg [17]. Arteries were then equilibrated for a further 1.5 h before the bath solution was changed to the K+-free Krebs solution. Subsequently, a concentration–contraction curve was constructed for phenylephrine so that a concentration of phenylephrine could be chosen which produced about 70% of the maximal response. This concentration of phenylephrine (usually about 3 µM) was used to preconstrict the artery before K+ was added to the solution to observe K+-induced relaxation. Changes in wall tension were measured and sampled at 0.1 Hz using the on-line data capture system (Picolog). At the end of each experiment, Krebs solution containing 3 mM EGTA was introduced to determine the tension when the smooth muscle cells were maximally relaxed.

2.2.3 Measurement of responses
In all cases, the arterial diameter or wall tension observed in Ca2+-free (<10–7 M) solution is referred to as 100% dilation/relaxation. The arterial diameter or tension just before the application of a substance is taken as 0% dilation/constriction or relaxation/contraction. Percentage dilation/relaxation or constriction/contraction was calculated according to: 100x(d2–d1)/(d0–d1), where d0, d1 and d2 are the diameter/tension values in Ca2+-free solution (d0), in Ca2+-containing solution immediately prior to applying the vasodilator/constrictor substance (d1) and in Ca2+-containing solution once the response to the vasodilator/constrictor substance had reached a maximum (d2). In some experiments, the rate of development of myogenic tone was calculated from the decrease in lumen diameter divided by the time during which tone developed.

2.3 Patch-clamp recording
2.3.1 Cell isolation
Arcuate arteries were isolated as described above (Section 2.1) except after dissection they were placed in modified Hanks solution. Twenty 5 mm-long segments of artery were then transferred into modified Kraftbrühe (KB) medium containing 0.2 mg ml–1 collagenase (Sigma type 1A) and 0.24 mg ml–1 protease (Sigma type E) and incubated for 56 min at 37°C. Arteries were subsequently transferred back into enzyme-free KB medium at room temperature and single cells were isolated by gentle trituration of the arterial segments using a fire-polished Pasteur pipette. Cells were stored in the fridge at 2°C and used within 8 h. Single cells were allowed to settle to the base of a modified 35 mm culture dish on the stage of an inverted microscope (Zeiss) for at least 15 min at 23–25°C prior to making recordings.

2.3.2 Ionic current recordings and analysis
Recordings were made at room temperature (23–25°C) using an Axopatch 1D patch-clamp amplifier (Axon Instruments, Inc.) and either the amphotericin B perforated-patch whole-cell recording technique [18]or the conventional whole-cell configuration of the patch-clamp technique. Current signals were filtered at 1 kHz (–3 dB, Bessel) and then digitised at 2 kHz by a 1401-plus CED analogue-to-digital converter (Cambridge Electronic Design Ltd.; CED) and stored on a 486 computer. Voltage-clamp commands and data-sampling were controlled by CED software. Patch pipettes were made from borosilicate glass (Clark Electromedical Instruments: outside and inside diameters of 1 and 0.58 mm respectively) and had resistances of 2–4 M{Omega} after fire-polishing and when filled with pipette solution. Series resistance and cell capacitance values were determined from capacity current elicited by a square voltage step from –70 to –65 mV. This current was filtered at 10 kHz and digitised at 20 kHz. For amphotericin B perforated-patch whole-cell recordings, average series resistance and cell capacitance values were 8.14±0.67 M{Omega} and 14.18±0.93 pF (n=23). The solution in the recording chamber was exchanged using a gravity-flow perfusion system with multiple input reservoirs. The bath volume was 100 µl and the flow rate through the bath was about 2 ml min–1; solutions were fully exchanged in less than 1 min. Amphotericin recordings were made under sodium light.

2.4 Analysis of data
All statistical comparisons were made using non-paired or paired Student's t-tests and differences were taken to be statistically significant if P<0.05. Results are expressed as mean±s.e.mean. The value of ‘n’ indicates number of arteries or number of cells. Data presentation and mathematical fitting of functions to data using a least-squares method were performed by the program Origin (version 3.5; MicroCal Inc, Northampton, MA, USA). The K+ concentration-effect curve was fitted to a Hill equation assuming a one-site model.

2.5 Salts, reagents and solutions
Acetylcholine iodide, BaCl2 (Ba2+), EDTA (ethylenediaminetetra-acetic disodium salt: dihydrate) EGTA (ethylene glycol-bis(β-amino-ethyl ether) N,N'-tetra-acetic acid), HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), 5-hydroxytryptamine, ouabain octahydrate, sodium nitroprusside and L-phenylephrine hydrochloride were from Sigma. Pentobarbitone sodium (Sagatal) was from Rhone Merieux Ltd (UK) and heparin from Leo Laboratories Ltd. (UK).

Krebs solution contained (mM) NaCl 119, KCl 4.7, MgSO4·7H2O 1.17, NaHCO3 24, CaCl2 1.6, KH2PO4 1.18, EDTA 0.023, D-glucose 5. (Ca2+-free Krebs solution was the same as Krebs solution except CaCl2 was omitted and 0.5 mM EGTA was added. K+-free Krebs solution was the same as Krebs solution but with KCl omitted). Modified Hanks solution contained (mM) NaCl 137, KCl 5.4, CaCl2 0.01, NaH2PO4 0.34, K2HPO4 0.44, glucose 8 and HEPES 5. KB medium contained (mM) KCl 85, K2PO4 30, MgSO4 5, sodium pyruvate 5, glucose 8, taurine 20, creatine 5 and β-hydroxybutyrate 5 (with 0.1% fatty acid-free bovine serum albumin). The 1.5-Ca2+ bath solution (mM) NaCl 130, KCl 5, CaCl2 1.5, MgCl2 1.2, HEPES 10, glucose 8. The 60-K+, 1.5-Ca2+ bath solution contained (mM) NaCl 80, KCl 60, CaCl2 1.5, MgCl2 1.2, HEPES 10, glucose 8. The Quayle 60-K+ bath solution contained (mM) NaCl 80, KCl 60, CaCl2 0.1, MgCl2 1, HEPES 10. The Quayle recording pipette solution [19]contained (mM) KCl 107, KOH 33, EGTA 10, MgCl2 1, CaCl2 1, HEPES 10, Na2ATP 3, Na2ADP 0.1. The 0.1 mM or 10 mM EGTA pipette solution contained (mM) NaCl 5, KCl 130, MgCl2 2, EGTA 0.1 or 10, HEPES 10. When the latter solution was used for conventional whole-cell recording 3 mM Na2ATP was always added, and in some experiments 1 mM NaGTP was also added. When the solution was used for amphotericin B perforated-patch whole-cell recording 240 µg ml–1 amphotericin B was added from a 60 mg ml–1 stock solution prepared in dimethylsulphoxide. Solutions were titrated to pH 7.2 with NaOH (Quayle recording pipette solution), pH 7.4 with KOH (KB medium and pipette solutions) or pH 7.4 with NaOH (all other solutions except Krebs solution). Recording pipette solutions were passed through a 0.2 µm filter prior to recording (and before adding amphotericin B). Pipette solutions containing ATP, or ATP and GTP, were frozen after preparation and stored on ice on the day of recording.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
During the 2-h equilibration period for experiments with the pressure myograph, lumen diameter decreased from 224±9 µm to 168±11 µm (n=39). In seventeen of these arteries the development of tone occurred rapidly (10±1 µm min–1) after an initial quiescent period of 59±4 min, and in the remaining arteries tone developed gradually, reaching a maximum after 108±5 min. Application of the Ca2+-free solution at the end of each experiment resulted in a mean lumen diameter of 230±9 µm (n=39).

3.1 K+-induced dilation in the arcuate artery
Raising the K+ concentration of the superfusate from 5.87 mM to 10 mM caused dilation of about half of the arcuate arteries studied (Fig. 1). The effect occurred whether tone in the artery was purely myogenic or potentiated by application of phenylephrine (0.1–1 µM). When there was only myogenic tone there was a 60±10% dilation in response to K+ (n=4), and in the presence of phenylephrine the dilation was 49±6% (n=10). In eight of the arteries the dilation was sustained for at least 5 min (Fig. 1), and in the remaining six arteries it was transient (Fig. 5A).


Figure 1
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 K+-induced vasodilation in a rabbit renal arcuate artery preconstricted by application of phenylephrine. The artery was pressurised at 60 mmHg and the lumen diameter is plotted against time. The K+ concentration in the superfusate was raised from 5.87 mM to 10 mM as indicated. The broken line in this Fig. and in Figs. 2 and 3 and 5GoGo marks the diameter of the vessel in Ca2+-free Krebs solution.

 

Figure 5
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effect of 10 µM ouabain on K+-induced dilation of a segment of rabbit renal arcuate artery in a pressurised myograph induced by elevating the K+ concentration in the Krebs solution from (A) 5.87 mM to 10 mM, and (B) 0 to 10 mM. Also shown in (A) is a constriction in response to 20 mM K+. Changes in the K+ concentration were compensated by equimolar changes in Na+ concentration. Dilation is represented in a downwards direction. Changes in the K+ concentration and addition of ouabain are indicated by solid bars.

 
K+-induced dilation was observed in all experiments and was pronounced and always sustained if arteries were initially incubated in K+-free superfusate for 10 min before applying 10 mM K+ (Fig. 2C). In two arteries with only myogenic tone the addition of 10 mM K+ induced dilations of 87% and 83%, and in the presence of phenylephrine the dilation was 73±5% (n=5). The effect was endothelium-independent because each of five endothelium-denuded arteries dilated (62.2±8.1% in 10 mM K+) when K+ was added in 1 mM increments to the K+-free superfusate (an example is shown in Fig. 6A).


Figure 2
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of Ba2+ on K+-induced dilation in segments of rabbit renal arcuate artery. (A) Diameter measurement using the pressurised myograph. Effect of 50 µM Ba2+ on dilation induced by elevating the K+ concentration from 5.87 mM to 8 mM. (B) Isometric tension recording using the wire myograph. Effect of 1 mM Ba2+ on relaxation induced by elevating the K+ concentration from 5.87 mM to 10 mM. (C) Effect of 10–100 µM Ba2+ on dilation induced by adding K+ (10 mM) to the K+-free Krebs solution. Dilation is represented in a downwards direction. Changes in K+ concentration and addition of Ba2+ are indicated by solid bars.

 

Figure 6
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Dilation or relaxation of arcuate artery induced by adding K+ to the K+-free Krebs solution in (A) the pressurised myograph and (B, C) the wire myograph. In (A) the artery was endothelium-denuded and constricted with phenylephrine. Lumen diameter is plotted against time. In (B) tension is plotted against time and the arrow indicates the time at which 3 µM phenylephrine was introduced to the recording chamber. (C) K+ concentration-effect curve obtained in experiments with the wire myograph. The smooth curve is the fitted Hill equation and the mid-point of this curve occurred at 1.47 mM K+. Percentage of maximum tension is plotted on the y-axis, and the bath K+ concentration is plotted on the x-axis. Points with error bars were obtained from 4–8 experiments whilst other points represent 2 experiments. The dotted line marks 5.87 mM K+ concentration. Zero on the ordinate is the diameter/tension level in low-Ca2+ solution.

 
3.2 Action of Ba2+ on K+-induced dilation
To investigate if inward rectifier K+ channels mediate K+-induced dilation, we exposed vessels to Ba2+ which blocks the inward rectifier K+ channels suggested to mediate K+-induced dilation of coronary and cerebral arteries [11, 12, 19].

Ba2+ added to the superfusate did not inhibit dilation of arcuate arteries induced by raising the K+ concentration above 5.87 mM (Fig. 2A). On average K+-induced dilation was reduced from 53±8% to 42±7% by 10 µM Ba2+ (n=7), and from to 47±3% to 37±6% by 50 µM Ba2+ (3 determinations for each of 2 arteries); changes which were not statistically different. The 5-min preincubation in 10 µM Ba2+, prior to elevating the K+ level, induced a 55% constriction in one artery and small dilations in the other six arteries (13±4%). It was also observed that Ba2+, even at high concentrations, did not block K+-induced relaxation of arcuate arteries mounted for isometric tension recording on a wire myograph (Fig. 2B). Relaxations were induced by raising the K+ level from 5.87 mM to 8, 9 or 10 mM, depending on which gave the largest response, and then Ba2+ was tested against relaxations induced by the single chosen K+ level. Mean relaxations were 27.0±7.6%, 30.4±9.3% and 36.7±8.5% in the presence of 0, 0.1 and 1 mM Ba2+ (6 vessels). Ba2+ (1 mM) did not itself directly affect the level of tone in any of the 6 vessels.

Ba2+ (10–100 µM) had no effect on dilations induced by adding 10 mM K+ to the K+-free Krebs solution (Fig. 2C). The mean K+-induced dilation in the absence of Ba2+ was 77±5%, compared with 81±6%, 83±5% and 83±4% after 10-min incubations in the presence of 10, 30 and 100 µM Ba2+ respectively (n=4 for each). Ba2+ (100 µM) had essentially no effect on arterial diameter in the absence of K+, causing a constriction averaging 6±6% (n=4).

To investigate if Ba2+ does inhibit inward rectifier K+ channels under our experimental conditions we recorded K+-induced dilation in the rat posterior cerebral artery because this is reported to be Ba2+-sensitive and may be mediated primarily by a Ba2+-sensitive inward rectifier [11, 13]. Furthermore, this cerebral artery is a similar size to the arcuate artery, having a lumen diameter of 231±9 µm at 60 mmHg in Ca2+-free solution (n=8), and thus provides a useful comparison. All posterior cerebral arteries had myogenic tone but raising the K+ concentration from 5.87 mM to 10 mM dilated only 3 out of 8 arteries, and in each case the response was sustained for at least 5 min. In the three responsive arteries, 50 µM Ba2+ almost abolished K+-induced dilation, reducing dilation from 52±15% to 4±1% (Fig. 3); the effect of 10 µM Ba2+ was also investigated in one of these arteries and this also abolished K+-induced dilation (not shown). Ba2+ (50 µM) on its own induced a small constriction of 17±8% (n=3).


Figure 3
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effect of 50 µM Ba2+ on dilation induced by elevating the K+ concentration from 5.87 mM to 10 mM in a segment of rat posterior cerebral artery in a pressurised myograph. Dilation is represented in a downwards direction. Changes in the K+ concentration and addition of BaCl2 are indicated by solid bars.

 
3.3 Patch-clamp recordings of inward rectifier K+-current
The myography experiments described above suggest that Ba2+-sensitive inward rectifier K+ channels are expressed in smooth muscle cells of the posterior cerebral artery but not the arcuate artery. To investigate directly if a Ba2+-sensitive inward rectifier is absent from the arcuate artery we made patch-clamp recordings from smooth muscle cells freshly isolated from about 20 arcuate arteries dissected from left and right kidneys on each experimental day. Inward rectifier K+-current was searched for in a total of 135 cells using amphotericin B perforated-patch recording (19 cells) or conventional whole-cell recording (116 cells) with the 0.1 mM EGTA, 10 mM EGTA or the Quayle pipette solutions. No differences were detected between the results under the different conditions and data have been grouped together for clarity of presentation. All measurements were made in the 60 mM K+ bath solution, giving a calculated K+-equilibrium potential of –22 mV.

An obvious inwardly rectifying K+-current was detected in some cells (Fig. 4A) and in these cells it was possible to determine the external Ba2+-sensitivity of the channels (Fig. 4B,C). Block was voltage-dependent; for example, the concentration of Ba2+ required for 50% inhibition increased by almost 10 times on depolarisation from –80 mV to –30 mV (Fig. 4C). From this analysis it was evident that 100 µM Ba2+ was sufficient to produce strong block of the inward rectifier even when the cells were quite depolarised at –40 mV or –30 mV, and 1 mM Ba2+ abolished the current.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Detection of inward rectifier K+-current in a fraction of smooth muscle cells isolated from the arcuate artery preparation. (A), (B) and (C) are data from conventional whole-cell recordings using the 0.1 mM EGTA pipette solution and the 60 mM K+ bath solution. (A) Inwardly rectifying current in response to a ramp protocol before and during bath-application of 1 mM Ba2+. The holding potential was –20 mV and a ramp voltage change from 0 mV to –60 mV was applied over a period of 100 ms every 10 s. (B) Percentage (%) of current remaining in the presence of bath-applied Ba2+ (mean±s.e.mean). The plot is for 8 selected cells which had an inwardly rectifying current. Current remaining in the presence of 1 mM Ba2+ was subtracted. Block was determined by fitting single exponential functions to currents during 1-s square test voltage steps from a holding potential of –20 mV. The smooth curves are fitted Hill equations with slopes of 0.63 (–80 mV) and 0.77 (–40 mV). (C) Voltage-dependence of the concentration of Ba2+ (µM) required for 50% inhibition of current, determined as in (B) for the 8 selected cells. (D). Frequency distribution for the occurrence of inward rectifier current, defined as current at –60 mV which was blocked by 100 µM Ba2+. N is the number of current amplitudes (cells) in each 20-pA bin.

 
The bulk of cells recorded from did not have an inwardly rectifying current. In order to express the frequency of detection of the current we have defined it as current which was blocked by 100 µM Ba2+ at –60 mV and which showed at least some inward rectification. In all experiments the holding potential was –20 mV and current amplitude was measured at the end of a 0.1-s or 1-s square voltage-step to –60 mV, or at –60 mV during a ramp change in voltage (0.4 or 0.5 V s–1). Resulting current amplitudes were allocated into 20-pA bins (Fig. 4D). Thus, the majority of cells had no inward rectifier and only 7 out of 135 cells had an inward rectifier of 100 pA or more. Expressed as a mean±s.e.mean current amplitudes the data give –20.5±5.1 pA for every cell (n=135) and –64.3±14.0 pA if only Ba2+-responsive cells are included (n=43). The mean capacitance for the cells was 14.2±0.9 pF (n=23), giving current densities of –1.44 pA pF–1 and –4.53 pA pF–1 for the two groups (n=135 and n=43 respectively).

3.4 Action of ouabain on K+-induced dilation
Because K+-induced dilation of the arcuate artery was not mediated by inward rectifier K+ channels we investigated the possibility that stimulation of a Na+–K+ ATPase might explain the effect by using ouabain, which inhibits the pump [20].

Ouabain (10 µM) inhibited K+-induced dilation whether the initial K+ concentration was 5.87 mM or zero mM. Dilation in response to raising the K+ concentration from 5.87 mM to 10 mM was reduced from 58±9% to 1±2% (n=7) (Fig. 5A). In three arteries, addition of 10 mM K+ to the K+-free superfusate caused dilation in the absence of ouabain (75±9%) but constriction in the presence of ouabain (23±14%) (Fig. 5B). Application of 10 µM ouabain (prior to raising the level of K+) caused constriction in some arteries which averaged 24±10% in 5.87 mM K+ (n=7), and 22±8% in K+-free solution (n=3).

The concentration dependence of the action of ouabain (0.01–100 µM) was investigated in experiments where dilation was induced in the arcuate artery by adding 3 mM K+ to the K+-free superfusate. Fifty percent inhibition of the dilation occurred with 0.39±0.12 µM ouabain and the dilation was inhibited by at least 95% in the presence of 10 µM ouabain (n=3, data not shown).

3.5 Concentrations of K+ that induce dilation in arcuate artery
The concentration dependence of the dilatory action of K+ was investigated to make a comparison with the K+-sensitivity of Na+–K+ ATPase [21, 22]and for comparison with the K+ concentration–dilation curve in the rat posterior cerebral artery, which is biphasic [13]. In the latter study it was suggested that low concentrations of K+ dilate the artery by stimulating Na+–K+ ATPase and higher concentrations by increasing the conductance of inward rectifier K+ channels.

The K+ concentration-effect curve was constructed by adding K+ in 1 mM increments to the K+-free superfusate whilst recording lumen diameter from a pressurised arcuate artery. Low concentrations of K+ induced strong dilation but dilations continued to occur at concentrations up to 8 mM K+ (Fig. 6A). The mid-point of the concentration-effect curve occurred at 0.9 mM K+ (n=5, data not shown). K+ concentration-effect curves were also constructed by measuring isometric tension in arcuate arteries mounted on a Mulvany wire myograph, a method which permits the measurement of relaxant effects against a high level of initial tone (Fig. 6B). Again relaxation occurred at low concentrations of K+ but relaxations were still clear on raising the K+ concentration from 4 mM to 10 mM (mid-point at 1.47 mM K+; Fig. 6C)


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study we observed that an increase in the extracellular K+ concentration from a normal physiological level of about 6 mM up to 10 mM causes dilation of a small artery from the rabbit kidney. The effect is endothelium-independent and may occur as a result of enhanced Na+–K+ ATPase activity in the smooth muscle cell plasma membrane. Ba2+-sensitive inward rectifier K+ channels could only be detected at a low average density in the single smooth muscle cell preparation from arcuate arteries and these channels play no significant role in the mechanism underlying K+-induced dilation.

To put the average inward rectifier K+-current density in arcuate artery smooth muscle cells into perspective a comparison can be made with measurements from coronary artery smooth muscle cells where the inward rectifier current density at –140 mV was –0.8 pA pF–1 in cells from the left anterior descending coronary artery (LAD) and –20.5 pA pF–1 in cells from 4th order branches of the LAD [12]. Our arcuate artery value is –1.44 pA pF–1 at –60 mV, and this can be estimated to be equivalent to –4.56 pA pF–1 at –140 mV by using the data from Fig. 2 of Quayle et al. [12]. On this basis, inward rectifier current density in arcuate artery cells is 5.7 times larger than that for LAD cells and 4.5 times smaller than that for 4th order LAD cells. The arcuate artery cells, therefore, appear to be intermediate. However, the possibility that two populations of smooth muscle cells have been isolated from arcuate arteries should also be considered. On this premise the arcuate artery preparation has 30% of cells with a mean inward rectifier current density of –14.35 pA pF–1 (–140 mV) and 70% of cells with no detectable inward rectifier. The value of –14.35 pA pF–1 approaches that of the 4th order LAD. We routinely removed side-branches from arcuate arteries before isolating single smooth muscle cells but microscopic inspection of the arteries entering the isolation procedure (not shown) has revealed that it is unrealistic to presume that smooth muscle cells from smaller arteries can be excluded from the preparation. Depending on the relative yield of cells from arcuate arteries and smaller vessels it is conceivable that, when we have detected inward rectifier K+-current, this was from smooth muscle cells which do not affect diameter or tension measurements from the intact arcuate artery. Although this implies that inward rectifier K+ channels are expressed in arterial branches down-stream of the arcuate arteries we have no direct test of this hypothesis. An alternative explanation could be that inward rectifier K+ channels are induced or activated during the cell isolation procedure or by a signal transduction pathway. Indeed, the channels detected may be of a different type to those described in other arterial preparations because the Ba2+-sensitivity was lower; apparent Kd (at –60 mV) of 12.6 µM (Fig. 4) compared with 2.2 µM [19]. However, we have not been able to alter the frequency of detection or amplitude of inward rectifier K+-current by including ATP or GTP in the conventional whole-cell recording pipette (unpublished), and inward rectifier current was detected at a similar frequency in amphotericin compared with conventional whole-cell recordings (26% cf. 33%). Therefore, we have no evidence to support the hypothesis that inward rectifier K+ channel activity is in some way induced in the single cell preparation.

Although inward rectifier K+ channels are expressed on average at a low density in arcuate artery smooth muscle cells the artery is, nevertheless, able to dilate in response to elevated K+ concentrations. The relative absence of inward rectifier K+ channels appears only to restrict the concentration range over which K+ can be an effective dilator. In the rat posterior cerebral artery, which expresses inward rectifier K+ channels, K+-induced dilation continues to occur even at K+ concentrations as high as 15 mM [13]. In the arcuate artery, however, we have only observed dilatory effects of K+ at concentrations up to 10 mM; at higher concentrations the spasmogenic (depolarising) action of K+ begins to dominate. Indeed, the apparent absence of a dilatory action of 10 mM K+ in some arcuate arteries and the occasional transient responses to 10 mM K+ may be explained if this concentration puts the artery near a balancing point between the dilatory and spasmogenic actions of K+.

Inhibition of K+-induced dilation in the arcuate artery by ouabain suggests that the effect occurs because the increased extracellular K+ concentration enhances activity of a Na+–K+ ATPase in the smooth muscle cells of the arterial wall. The observation that K+-induced dilation was inhibited by 50% by about 0.4 µM ouabain is at least consistent with the hypothesis that an {alpha}2- or {alpha}3-isoform of Na+–K+ ATPase is involved in the effect because they have a ouabain affinity of 0.1–0.5 µM [23]. However, block of K+-induced vasodilation by ouabain does not prove that K+ caused dilation by stimulating the Na+–K+ ATPase. Block of Na+–K+ ATPase may have profound effects on arterial smooth muscle cells which non-specifically inhibit the actions of several vasodilators. Ouabain constricted some, although not all, arcuate arteries in which it inhibited K+-induced dilation. Furthermore, we have observed that ouabain inhibits dilation induced by the adenosine A2a receptor agonist CGS-21680 [24]and ouabain inhibits a component of the relaxation induced by the K+ channel opener drug cromakalim in mesenteric arteries [25]. Nevertheless, additional support for the Na+–K+ ATPase hypothesis of K+-induced dilation comes from the finding that K+ dilates the arcuate artery in the concentration range 0–10 mM, with 50% effect occurring at 0.9–1.5 mM. Na+–K+ ATPase is also stimulated by K+ in this concentration range, with 50% effect occurring at about 1 mM [21, 22].

If the Na+–K+ ATPase hypothesis is true for K+-induced dilation then stimulation of the pump by quite high K+ concentrations (5 to 10 mM) must be functionally important. This concept is initially surprising given that Na+–K+ ATPase is stimulated by low concentrations of K+ and might be expected to be maximally stimulated at a normal physiological level of K+ (about 6 mM). However, our recordings from the arcuate artery show that although there is an 80% reduction in tone when the K+ concentration is increased from zero to 5.87 mM the reduction in total tone becomes 88% once the K+ concentration reaches 10 mM (Fig. 6C). Therefore, starting from 5.87 mM K+ and increasing the level to 10 mM gives a 40% reduction of the tone present at 5.87 mM K+ (i.e. 8 units of a remaining 20 units are lost). This 40% change in arterial tone is similar to that recorded in the pressurised myograph in response to a change in K+ concentration from 5.87 to 10 mM (cf. 49% dilation of phenylephrine-constricted arteries).

Stimulation of Na+–K+ ATPase by K+ may induce relaxation because it causes hyperpolarisation. In rat and rabbit blood vessels, activation of Na+–K+ ATPase by the return of a K+-containing solution after exposure to a K+-free solution produces hyperpolarisation of 5 to 10 mV [26, 27]. This might be expected to reduce the opening probability of voltage-gated Ca2+ channels and thus reduce the intracellular Ca2+ concentration [28]. An additional mechanism could result from a lowering of the intracellular Na+ concentration when the Na+–K+ ATPase is stimulated. This would increase Na+–Ca2+ exchange and thus Ca2+-efflux. There is evidence for a functional Na+–Ca2+ exchange mechanism in small human subcutaneous arteries which have a lumen diameter of about 200 µm [29].

It can be concluded from this study that Ba2+-sensitive inward rectifier K+ channels are not expressed at a functionally important level in the arcuate artery of the kidney even though they are expressed at a substantial level in arteries of a similar size in the heart and brain (Fig. 3 and [12, 19]). Nevertheless, the small renal artery does dilate in response to raised extracellular K+ levels and this effect may be accounted for solely by enhanced Na+–K+ ATPase activity. The dilator effect of K+ in the kidney could be important not only as a protective mechanism against consequences of metabolic inhibition but also to increase renal blood flow and glomerular filtration rate in response to hyperkalaemia.

Time for primary review 20 days.


   Acknowledgements
 
Support from the Wellcome Trust (grant 042071/Z/94) is gratefully acknowledged. K. Quinn is supported by a British Heart Foundation Ph.D. Studentship (FS/95045).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Katz L.N., Linder E. The action of excess Na, Ca and K on the coronary vessels. Am J Physiol (1938) 124:155–160.[Free Full Text]
  2. Bonaccorsi A., Hermsmeyer K., Aprigliano O., Smith C.B., Bohr D.F. Mechanism of potassium relaxation in arterial muscle. Blood Vessels (1977) 14:261–276.[Web of Science][Medline]
  3. Webb R.C., Bohr D.F. Potassium-induced relaxation as an indicator of Na+, K+-ATPase activity in vascular smooth muscle. Blood Vessels (1978) 15:198–207.[Web of Science][Medline]
  4. Duling B.R. Effects of potassium ion on the microcirculation of the hamster. Circulation Res (1975) 37:325–332.[Abstract/Free Full Text]
  5. Scott J., Emanuel D., Haddy F. Effect of potassium on renal vascular resistance and urine flow rate. Am J Physiol (1959) 197:305–308.[Abstract/Free Full Text]
  6. Budtz-Olsen O.E., Clark R.C., Cross R.B., French T.J. Changes in renal haemodynamics and electrolyte excretion during acute hyperkalaemia in conscious adrenalectomized sheep. Quat J Exp Physiol Cog Med Sci (1975) 60:207–221.
  7. Smojen G.G. Extracellular potassium in the mammalian central nervous system. Ann Rev Physiol (1979) 14:15–177.
  8. Sieber F.E., Wilson D.A., Hanley D.F., Traystman R.J. Extracellular potassium activity and cerebral blood flow during moderate hypoglycaemia in anesthetized dogs. Am J Physiol (1993) 264:H1774–H1780.[Web of Science][Medline]
  9. Henderickx H., Casteels R. Electrogenic sodium pump in arterial smooth muscle cells. Pfluegers Arch (1974) 346:299–306.[CrossRef][Web of Science][Medline]
  10. Edwards F.R., Hirst G.D.S. Inward rectification in submucosal arterioles of guinea-pig ileum. J Physiol (1988) 404:437–454.[Abstract/Free Full Text]
  11. Nelson M.T., Quayle J.M. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol (1995) 268:C799–C822.[Web of Science][Medline]
  12. Quayle J.M., Dart C., Standen N.B. The properties and distribution of inward rectifier potassium currents in pig coronary arterial smooth muscle. J Physiol (1996) 494:715–726.[Abstract/Free Full Text]
  13. McCarron J.G., Halpern W. Potassium dilates rat cerebral arteries by two independent mechanisms. Am J Physiol (1990) 259:H902–H908.[Web of Science][Medline]
  14. Hirst G.D.S., Silverberg G.D., Van Helden D.F. The action potential and underlying ionic currents in proximal rat middle cerebral arterioles. J Physiol (1986) 371:289–304.[Abstract/Free Full Text]
  15. Bolton TB, Beech DJ. Smooth muscle potassium channels: their electrophysiology and function. In: Weston AH, Hamilton TC, editors. K Channel Modulators — Pharmacological, Molecular and Clinical Aspects. Blackwell Scientific, 1992:144–148.
  16. Bonev A.D., Robertson B.E., Nelson M.T. Inward rectifier K+ currents from rat coronary artery smooth muscle cells. Biophys J (1994) 66:A327.
  17. Mulvany M.J., Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res (1977) 41:19–26.[Free Full Text]
  18. Rae J., Cooper K., Gates P., Watsky M. Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods (1991) 57:15–26.[Medline]
  19. Quayle J.M., McCarron J.G., Brayden J.E., Nelson M.T. Inward rectifier K+ currents in smooth muscle cells from rat resistance-sized cerebral arteries. Am J Physiol (1993) 265:C1363–C1370.[Web of Science][Medline]
  20. Sweadner K.J. Isozymes of the Na+/K+-ATPase. Biochim Biophys Acta (1989) 988:185–220.[Medline]
  21. Glynn I.M., Lew V.L., Luthi U. Reversal of the ouabain-sensitive potassium entry mechanism in red cells with and without reversal of the entire pump cycle. J Physiol (1970) 207:371–391.[Abstract/Free Full Text]
  22. Logan J.G. The extrusion of sodium ions from presynaptic nerve endings of rat cerebral cortex. J Neurochem (1980) 35:349–353.[CrossRef][Web of Science][Medline]
  23. Fallows D., Kent R.B., Nelson D.L., Emanuel J.R., Levenson R., Housman D.E. Chromosome-mediated transfer of the murine Na,K-ATPase alpha subunit confers ouabain resistance. Mol Cell Physiol (1987) 7:2985–2987.
  24. Prior H.M., Yates M.S., Beech D.J. Inhibition by K+ of adenosine receptor (A2a)-mediated dilation of rabbit renal arcuate artery in vitro. J Physiol (1996) 493:21P.
  25. Hong K.W., Park M.G., Shin Y.W., Rhim B.Y., Ko K.H. Effect of ouabain on relaxation induced by cromakalim in human and canine mesenteric arteries. Eur J Pharmacol (1993) 231:1–6.[CrossRef][Web of Science][Medline]
  26. Casteels R., Kitamura K., Kuriyama H., Suzuki H. The membrane properties of the smooth muscle cells of the rabbit main pulmonary artery. J Physiol (1977) 271:41–61.[Abstract/Free Full Text]
  27. Hermsmeyer K. Sodium pump hyperpolarization–relaxation in rat caudal artery. Fed Proc (1983) 42:246–252.[Web of Science][Medline]
  28. Nelson M.T., Patlak J.B., Worley J.F., Standen N.B. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol (1990) 259:C3–C18.[Web of Science][Medline]
  29. Woolfson R.G., Hilton P.J., Poston L. Effects of ouabain and low sodium on contractility of human resistance arteries. Hypertension (1990) 15:583–590.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
B. Eichhorn, G. Muller, A. Leuner, T. Sawamura, U. Ravens, and H. Morawietz
Impaired vascular function in small resistance arteries of LOX-1 overexpressing mice on high-fat diet
Cardiovasc Res, June 1, 2009; 82(3): 493 - 502.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
K. K Bradley, J. H Jaggar, A. D Bonev, T. J Heppner, E. R. Flynn, M. T Nelson, and B. Horowitz
Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells
J. Physiol., March 15, 1999; 515(3): 639 - 651.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
H M Prior, M S Yates, and D J Beech
Functions of large conductance Ca2+-activated (BKCa), delayed rectifier (KV) and background K+ channels in the control of membrane potential in rabbit renal arcuate artery
J. Physiol., August 15, 1998; 511(1): 159 - 169.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Prior, H.M
Right arrow Articles by Yates, M.S
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Prior, H.M
Right arrow Articles by Yates, M.S
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?