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(Stroke. 2005;36:1526.)
© 2005 American Heart Association, Inc.

Original Contributions

Possible Role for K⁺ in Endothelium-Derived Hyperpolarizing Factor–Linked Dilatation in Rat Middle Cerebral Artery

From the Department of Pharmacy and Pharmacology, the University of Bath, Claverton Down, United Kingdom.

Correspondence to Christopher J. Garland, Department of Pharmacy and Pharmacology, the University of Bath, Claverton Down, Bath, BA2 7AY, UK. E-mail c.j.garland{at}bath.ac.uk

	Abstract

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Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF) and K⁺ are vasodilators in the cerebral circulation. Recently, K⁺ has been suggested to contribute to EDHF-mediated responses in peripheral vessels. The EDHF response to the protease-activated receptor 2 ligand SLIGRL was characterized in cerebral arteries and used to assess whether K⁺ contributes as an EDHF.

Methods— Rat middle cerebral arteries were mounted in either a wire or pressure myograph. Concentration-response curves to SLIGRL and K⁺ were constructed in the presence and absence of a variety of blocking agents. In some experiments, changes in tension and smooth muscle cell membrane potential were recorded simultaneously.

Results— SLIGRL (0.02 to 20 µmol/L) stimulated concentration and endothelium-dependent relaxation. In the presence of N^G-nitro-L-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IK_Ca, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (1µmol/L), reflecting activation of EDHF. Combined inhibition of K_IR with Ba²⁺ (30µmol/L) and Na⁺/K⁺-ATPase with ouabain (1 µmol/L) markedly attenuated the relaxation to EDHF. Raising extracellular [K⁺] to 15 mmol/L also stimulated smooth muscle relaxation and hyperpolarization, which was also attenuated by combined application of Ba²⁺ and ouabain.

Conclusions— SLIGRL evokes EDHF-mediated relaxation in the rat middle cerebral artery, underpinned by hyperpolarization of the smooth muscle. The profile of blockade of EDHF-mediated hyperpolarization and relaxation supports a pivotal role for IK_Ca channels. Furthermore, similar inhibition of responses to EDHF and exogenous K⁺ with Ba²⁺ and ouabain suggests that K⁺ may contribute as an EDHF in the middle cerebral artery.

Key Words: endothelium • endothelium-dependent hyperpolarization factor • PAR-2 receptor • potassium channels

	Introduction

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It is clear that endothelium-derived hyperpolarizing factor (EDHF) is functionally important in the cerebral vasculature because it is in the peripheral circulation.^1–4 In common with peripheral arteries, the importance of EDHF appears to increase as vessel size decreases.^5,6 However, cerebrovascular smooth muscle does display a number of unique characteristics, so it is perhaps not surprising that the EDHF response, at least with certain agonists, also seems to have some unique features.

A defining feature of the EDHF response in the peripheral circulation is that block requires the simultaneous application of inhibitors for intermediate and small conductance calcium-activated K⁺ channels (IK_Ca and SK_Ca). In general, agonists used to evoke EDHF responses (eg, acetylcholine [ACh] and bradykinin) increase endothelial cell intracellular [Ca²⁺] after activation of G-protein–coupled receptors (G_q/11). This activates IK_Ca and SK_Ca channels, which are located on the endothelium, and leads to subsequent smooth muscle hyperpolarization and relaxation attributed to EDHF.^7–9 However, this profile seems to be slightly different in cerebral arteries in that inhibition of IK_Ca alone appears sufficient to block EDHF-mediated relaxation evoked by luminal UTP.¹⁰

A significant drawback in defining and interpreting such mechanisms is the small number of studies reporting direct measurements of hyperpolarization. To date, this is limited to responses in rabbit and rat middle cerebral arteries to ACh and ATP, respectively, and in human pial arteries to substance P,^3,6,11 in which smooth muscle hyperpolarization ranged between 15 and 25 mV. Furthermore, a recent study¹⁰ used a voltage-sensitive dye in pressurized rat middle cerebral arteries to indirectly demonstrate endothelial cell hyperpolarization to UTP. This response was blocked by charybdotoxin and mimicked by application of the IK_Ca activator 1-ethyl-2-benzimidazolinone (1-EBIO), thus implicating a primary role for these channels in the EDHF, at least to UTP. This suggestion was supported by an inability to inhibit dilatation with either apamin or iberiotoxin but block with charybdotoxin or the more selective IK_Ca blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34).

The identity of EDHF is the subject of considerable controversy.¹² Although studies in cerebral arteries are much more restricted than peripheral arteries, it has been suggested that EDHF-evoked relaxation in guinea pig and rat middle cerebral arteries may involve a metabolite of arachidonic acid.^1,13 However, it was not possible in either study to conclude whether such a metabolite acts simply as a component of the EDHF pathway or as the final effector. An additional possibility is that at least part of the EDHF response reflects the opening of endothelial K_Ca channels, causing efflux of K⁺ sufficient to increase concentrations in the myoendothelial space and hyperpolarize and relax the underlying smooth muscle as a consequence. This mechanism has been suggested to operate in some peripheral resistance arteries⁹ and provides an attractive mechanism within the cerebral circulation. Cerebral arteries are very sensitive to extracellular [K⁺], which can accumulate secondary to neuronal activity, for example, and lead to pronounced vasodilatation in vitro and in vivo. The K⁺-induced vasodilatation is mediated mainly by the opening of K_IR channels, and possibly stimulation of Na⁺/K⁺-ATPase,^14,15 and may be altered by disease.¹⁶ Clearly, an ability to control arterial diameter by K⁺ released within the wall of cerebral arteries provides an additional dimension for controlling blood flow.

Our aims were 2-fold. We made measurements of membrane potential change to demonstrate directly the importance of the EDHF pathway in the middle cerebral artery after stimulation of protease-activated receptor 2 (PAR-2), or "trypsin receptor," with SLIGRL. These responses are of physiological relevance during inflammation in the cerebral vasculature¹⁷ and circumvent the weak EDHF response in the middle cerebral artery with mediators such as ACh, ADP, and bradykinin. Second, using wire and pressure myography, we investigated the possibility that K⁺ may contribute to the EDHF response in this artery.

	Methods

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Male Wistar rats (200 to 300 g; Charles River Laboratories, Wilmington, Mass) were humanely killed and the brain rapidly removed and stored immediately in ice-cold physiological salt solution (PSS) for a maximum of 30 minutes.

Wire Myography
A segment (1.5 to 2 mm) of the middle cerebral artery (internal diameter of 150 µm) was mounted in a Mulvany–Halpern myograph (model 400A; Danish Myotechnology) in Krebs solution containing (in mmol/L): 118.0 NaCl, 25 NaCO₃, 3.6 KCl, 1.2 MgSO_4·7H₂O, 1.2 KH₂PO₄, 11.0 glucose, 2.5 CaCl₂, and gassed with 95% O₂ and 5% CO₂ at 37°C. Vessels were allowed to equilibrate for 20 minutes and then tensioned at 0.5 mN/mm. Vessel viability was assessed by the addition of exogenous K⁺, and only vessels developing tension 3 mN were used. In some experiments, endothelial cells were removed by gently rubbing the luminal surface with a human hair. Smooth muscle tension and membrane potential were measured simultaneously as described previously.¹⁸ Briefly, individual smooth cells were impaled with a glass electrode (filled with 2 mol/L KCl; tip resistance 80 to 120 M).

Pressure Myography
A segment of middle cerebral artery was carefully dissected free of adherent tissue in chilled 3-[N-morpholino]propane sulfonic acid (MOPS) buffer (4°C) containing (in mmol/L): 145 NaCl, 4.7 KCl, 2.0 CaCl₂, 1.17 MgSO₄, 2.0 MOPS, 1.2 NaH₂PO₄, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 2.75 NaOH (the pH of the solution was adjusted to 7.39 to 7.41 at 37°C). A segment of artery (circa 3-mm long; diameter 150 to 250 µm) was cut and cannulated at each end with a glass pipette and positioned in a temperature-regulated chamber (Warner Instruments). To avoid luminal flow, the upstream and downstream pressures (generated by gravity) were equalized throughout an experiment. During equilibration for 30 minutes at 50 mm Hg and 37°C, arteries developed spontaneous myogenic tone. The pressure was then raised to 70 to 80 mm Hg and held at this level for the remainder of the experiment. In some experiments, an air bubble was perfused through the lumen of the artery to disrupt the endothelium. Arteries were visualized using a microscope (FV500-SU; Olympus) and images stored for offline analysis of artery outer diameter (MetaMorph; Universal Instruments).

Solutions and Drugs
Exogenous K⁺ was added as an isotonic solution and expressed as the final bath concentration. BaCl, indomethacin, N^G-nitro-L-arginine methyl ester (L-NAME), ouabain, and papaverine were all obtained from Sigma. Apamin, charybdotoxin, and iberiotoxin were obtained from Latoxan. SLIGRL was obtained from Auspep. TRAM-34 was a gift from Dr H. Wulff (University of California, Davis) and U46619 was from Calbiochem.

Statistical Analysis
Results are expressed as the mean±SEM of n animals. Relaxation and vasodilatation are expressed as the peak percentage reduction of the total vascular tone (from the myogenic tone to the tension/diameter after addition of papaverine; 150 µmol/L). Graphs were drawn and statistical comparisons made using either Student’s t test or 1-way ANOVA with Bonferroni’s post hoc test (Prism; Graphpad).

	Results

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Endothelium-Dependent Relaxation and Hyperpolarization to SLIGRL
Using a wire myograph, segments of middle cerebral artery developed spontaneous myogenic tone equivalent to 15% (1.3±0.1 mN) of maximum tension (8 to 10 mN, with 55 mmol/L KCl). Under these basal conditions, the smooth muscle cells had a resting potential of –49.5±2.5 mV (n=10), and 20 µmol/L SLIGRL evoked a hyperpolarization of 15.1±3.4 mV (n=7) associated with 81.3±5.7% relaxation (Figure 1A). To obtain clear concentration-dependent relaxation under control conditions, tension was increased with U46619 (10 nmol/L), which contracted the vessels by an additional 2.5±0.3 mN (n=6; Figure 2A). Addition of the NO synthase inhibitor L-NAME (100 µmol/L) increased resting tension by 2.8±0.4 mN and evoked depolarization of 12.8±0.7 mV (n=8; No. U46619). This effect was associated with a rightward shift in the concentration-relaxation curve to SLIGRL (Figure 2A), with no reduction in the maximum relaxation (Figure 2A). The cyclooxygenase inhibitor indomethacin (10 µmol/L) had no further effect. Removal of the endothelium abolished SLIGRL-induced relaxation (20 µmol/L; 2.5±3.5%; n=7). The relaxation to 20 µmol/L SLIGRL (80.9±6.7%) was associated with smooth muscle hyperpolarization (19.6±3.1 mV; n=4; Figures 1B and 3).

View larger version (16K): [in this window] [in a new window]   Figure 1. Traces of simultaneous smooth muscle cell tension and membrane potential in rat middle cerebral arteries mounted under isometric conditions. A, Relaxation and hyperpolarization to 20 µmol/L SLIGRL (left panels) and 15 mmol/L K+ (right panels) from resting myogenic tone. B, The addition of L-NAME increased myogenic tone and depolarized arteries but did not inhibit the relaxation and hyperpolarization to 20 µmol/L SLIGRL (left panels). The additional presence of TRAM-34 (1µmol/L; right panels) fully inhibited the response to SLIGRL yet had no effect on the relaxation and hyperpolarization to K+.

View larger version (30K): [in this window] [in a new window]   Figure 2. Summary of peak concentration-dependent responses to SLIGRL in rat middle cerebral arteries mounted under isometric conditions. A, Effect of L-NAME (100 µmol/L) alone and together with indomethacin (10µmol/L; Indo; n=4 to 7). B, Effect of apamin (50 nmol/L) alone and together with charybdotoxin (ChTX; 100 nmol/L) in the presence of L-NAME (n=3 to 7). C, Effect of apamin alone and together with TRAM-34 (1 µmol/L) in the presence of L-NAME (n=3 to 5). D, Effect of apamin alone and together with iberiotoxin (IbTX; 100 nmol/L) in the presence of L-NAME (n=4). *Significant difference (P<0.05) from control.

View larger version (13K): [in this window] [in a new window]   Figure 3. Summary of peak responses to 20 µmol/L SLIGRL and 15 mmol/L K+ in rat middle cerebral arteries mounted under isometric conditions. A, Relaxation and hyperpolarization to SLIGRL was almost abolished in the presence of TRAM-34 (1 µmol/L; n=4). B, The response to K+ was unaffected by TRAM-34 (n=4). *Significant difference (P<0.05) from L-NAME.

Effect of K_Ca Blockers on SLIGRL Responses
Simultaneous measurement of changes in membrane potential and tension showed that the IK_Ca blocker TRAM-34 (1 µmol/L) alone could abolish the hyperpolarization and relaxation evoked with SLIGRL (Figure 3). Relaxation to SLIGRL was also blocked in the presence of 100 nmol/L charybdotoxin (Figure 2B), but not with, 50 nmol/L (79.8±7.6%; n=3; Figure 2), or 100 nmol/L iberiotoxin, either alone (R_max 63.6±11.2%; n=4) or in combination with apamin (80.0±5.6%; n=4).

Endothelium-Independent Relaxation and Hyperpolarization to Exogenous K⁺
Against basal myogenic tone, raising the K⁺ concentration to 15 mmol/L evoked a hyperpolarization of 30.9±6.8 mV and 94.9±4.3% relaxation (n=4; Figure 1A). These responses were reduced in the presence of L-NAME to 18.9±3.4 mV and 89.2±2.5%, respectively (n=4). Relaxation responses to K⁺ were independent of the endothelium (77.0±17.5%; n=3) and unaltered by TRAM-34 (Figure 3).

Effect of Inhibiting the Na⁺/K⁺-ATPase and K_IR Channels on Relaxation to SLIGRL and K⁺
Relaxation to 20 µmol/L SLIGRL was attenuated by ouabain (1 µmol/L; 55.2±2.5; n=5) or Ba²⁺ (30 µmol/L; 57.6±17.8%; n=4; Figure 4A). In combination, this effect was additive, reducing relaxation to 29.1±5.5% (n=5; Figure 4A). A very similar profile of inhibition was obtained against relaxation to exogenous K⁺, in which ouabain and Ba²⁺ alone reduced the relaxation (41.9±13.3% and 41.6±8.5% at 13.8 mmol/L K⁺, respectively; n=4), an effect increased by combined application (relaxation of 25.7±3.8% at 13.8 mmol/L K⁺; n=4; Figure 4B).

View larger version (18K): [in this window] [in a new window]   Figure 4. Summary of concentration-dependent relaxation in rat middle cerebral arteries mounted under isometric conditions. A, The relaxation to SLIGRL was attenuated by either Ba2+ (30 µmol/L; n=4) or ouabain (1 µmol/L; n=5) alone, and to a greater extent, by the combined presence of these inhibitors (n=4). B, A very similar profile was observed against the relaxation to K+. *Significant difference (P<0.05) from L-NAME.

Dilatation to SLIGRL and K⁺ in Pressurized Arteries
Artery diameter (184±7 µm; n=16) reduced to 129±9 µm (32±3% tone; n=9) as myogenic tone developed. L-NAME further decreased the diameter to 116±6 µm (37±3% tone; n=16), whereas TRAM-34 did nothing further (38±5%; n=4). All dilatation responses to either K⁺ or SLIGRL were synchronized along the entire arterial segment, suggesting effective electrical coupling. L-NAME did not reduce the peak dilatation to SLIGRL (20 µmol/L; 82.0±9.6 and 72.4±3.4; n=5 and n=4, respectively) but, as with the wire myograph, caused a rightward shift in the concentration-response curve (Figure 5A) and reduced the duration of dilatation. L-NAME and TRAM-34, or removal of the endothelium, abolished dilatation to SLIGRL (20 µmol/L; 9.8±3.7%, n=4; 5.6±4.1%, n=3; Figure 5A). Dilatation to K⁺ was not affected by these manipulations (Figure 5B). In general, K⁺ (but not SLIGRL) dilatation was less in pressurized arteries than in the wire myograph and tended to be transient or to oscillate. The addition of Ba²⁺ fully blocked the dilatation to 15 mmol/L K⁺ (Figure 6B), whereas ouabain slowed the dilatation and prevented transient peaks. However, Ba²⁺ plus ouabain was required for significant inhibition of SLIGRL responses (Figure 6A), possibly reflecting a complex underlying pattern of activation of K_IR and the Na⁺/K⁺-ATPase, together with other mechanisms for dilatation.

View larger version (16K): [in this window] [in a new window]   Figure 5. Summary of peak responses to SLIGRL and K+ in rat middle cerebral arteries mounted under isobaric conditions. A, Concentration-dependent dilatation to SLIGRL was fully inhibited by the combined presence of L-NAME (100 µmol/L) and TRAM-34 (1 µmol/L; n=4) or damaging the endothelium (–EC; n=3). B, The dilatation to 15 mmol/L K+ was unaffected by these treatments. *Significant difference (P<0.05) from L-NAME.

View larger version (15K): [in this window] [in a new window]   Figure 6. Summary of peak responses to SLIGRL and K+ in rat middle cerebral arteries mounted under isobaric conditions. A, Concentration-dependent dilatation to SLIGRL was attenuated by either Ba2+ (30 µmol/L; n=6 to 8) or significantly by the combination of Ba2+ and ouabain (10 µmol/L; n=9 to 12). B, Peak dilatation to 15 mmol/L K+ was fully blocked by Ba2+ (n=6), whereas ouabain slowed the dilatation and prevented any transient dilatations (n=3). *Significant difference (P<0.05) from control.

	Discussion

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We show for the first time that PAR-2 receptor stimulation in cerebral arteries, of physiological relevance during inflammation, stimulates marked EDHF-linked dilatation. By direct measurement of smooth muscle membrane potential, we show that PAR-2 stimulation (SLIGRL) causes EDHF hyperpolarization in the middle cerebral artery, which was sensitive to block of IK_Ca channels alone, extending previous observations with other agonists. We also provide evidence that K⁺ may contribute as an EDHF in this artery on the basis of similarities between EDHF and exogenous K⁺ responses.

In either a wire or pressure myograph, SLIGRL and K⁺ elicited small vasodilator responses, demonstrating the presence of myogenic tone. This is consistent with other studies, in which spontaneous tone is commonly recorded in cerebral arteries.^19–22 Relaxation to either SLIGRL or K⁺ was associated with smooth muscle hyperpolarization of similar magnitude. Basal NO appears to suppress myogenic tone in this artery because inhibition of NO synthase caused significant vasoconstriction, associated with smooth muscle depolarization, consistent with previous reports.^10,23–25 Relaxation to SLIGRL was slightly inhibited by L-NAME, suggesting some link to NO release, and again consistent with the effect of other endothelium-dependent agonists (eg, UTP,²² ACh,¹ ADP,²⁶ and substance P³). However, the majority of the SLIGRL response was resistant to L-NAME, was endothelium-dependent, and was associated with hyperpolarization, all features consistent with EDHF responses. This contrasts with the rat basilar artery, in which the majority of the SLIGRL response was NO dependent,²⁷ a difference most probably explained by altered relative contributions of NO and EDHF because the latter assumes dominance as vessel size decreases.⁶ After inhibiting NO synthase, the residual response to SLIGRL was fully inhibited by either charybdotoxin or the more selective inhibitor of IK_Ca, TRAM-34, indicating a primary role for IK_Ca, and consistent with the observations of Marrelli et al,¹⁰ using luminal UTP to stimulate an EDHF response.

To characterize further the EDHF response to SLIGRL, we recorded smooth muscle cell membrane potential, showing that hyperpolarization immediately preceded relaxation, suggesting a causal role. Hyperpolarization was also blocked by TRAM-34 alone, reinforcing the importance of IK_Ca channels. In peripheral arteries, IK_Ca channels are restricted to the endothelium and may contribute to the EDHF response by releasing K⁺ into the myoendothelial space to serve as an EDHF on the adjacent smooth muscle.⁹ Although in arteries such as the mouse mesenteric,²⁸ guinea pig carotid, and porcine coronary artery,²⁹ K⁺ does not appear to contribute significantly in this way, there has been a steady growth in the number of reports supporting a role for K⁺ in the overall EDHF response in a range of arteries.^30–34 These include human arteries, in vivo and in vitro. So it is feasible that at least part of the EDHF response in the middle cerebral artery may reflect the opening of endothelial IK_Ca channels and efflux of K⁺. In man and other species, K⁺-induced vasodilatation in cerebral vessels is a well-documented phenomenon; for example, during active hyperemia, neurones release K⁺, which can relax arteries and arterioles nearby by activating smooth muscle K_IR channels and possibly Na⁺/K⁺-ATPase.^9,35,36 Together with the potential for this dilatation to spread upstream away from the site of K⁺ release,³⁷ this effect will readily improve tissue blood supply to address local increases in metabolic demand. That Ba²⁺ and ouabain together inhibit EDHF-mediated and K⁺-induced cerebral artery relaxations in pressure- and wire myograph–mounted arteries suggests that K⁺ could well contribute to the former.

This suggestion contrasts with a previous study by You et al, in which EDHF-induced dilatation to UTP in middle cerebral arteries was unaffected by 75 µmol/L Ba²⁺.³⁸ Furthermore, other laboratories have demonstrated that K⁺ relaxation in middle cerebral arteries is inhibited by Ba²⁺ alone.^15,39 Such discrepancies may reflect differences between agonists or other methodological considerations. For example, initial [K⁺]_o has marked influence on the relaxation mechanism. Raising external K⁺ from 0 evokes ouabain-sensitive relaxation in cerebral arteries while simply increasing K⁺ in the PSS (from 5.8 mmol/L) gives Ba²⁺ sensitive relaxation. In the present study, the PSS [K⁺]_o fell between these extremes. Whatever the precise explanation, our data suggest that K⁺ released from within the cerebral artery wall may contribute in the local regulation of blood flow.

In summary, smooth muscle relaxation to SLIGRL in the middle cerebral artery is endothelium dependent and predominantly attributable to EDHF. The profile of the EDHF response is qualitatively similar in pressurized or wire-mounted arteries and reflects activation of IK_Ca channels. In addition, the profile of EDHF-linked hyperpolarization and relaxation is very similar to the equivalent effects that follow an increase in extracellular K⁺, leading us to speculate that this ion may contribute as an EDHF in the cerebral circulation.

	Acknowledgments

 
This study was funded by the British Heart Foundation.

Received January 30, 2005; revision received February 18, 2005; accepted February 23, 2005.

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