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the Department of Pharmacy and Pharmacology, the University of Bath, Claverton Down, United Kingdom.
Abstract
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 NG-nitro-L-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IKCa, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (1μmol/L), reflecting activation of EDHF. Combined inhibition of KIR 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 IKCa 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
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 (IKCa and SKCa). In general, agonists used to evoke EDHF responses (eg, acetylcholine and bradykinin) increase endothelial cell intracellular [Ca2+] after activation of G-protein–coupled receptors (Gq/11). This activates IKCa and SKCa 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 IKCa 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 IKCa 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 IKCa 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 KCa 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 KIR 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
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 MgSO4·7H2O, 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, NG-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
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).
Effect of KCa Blockers on SLIGRL Responses
Simultaneous measurement of changes in membrane potential and tension showed that the IKCa 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 (Rmax 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 KIR 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).
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 KIR and the Na+/K+-ATPase, together with other mechanisms for dilatation.
Discussion
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 IKCa 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 IKCa, TRAM-34, indicating a primary role for IKCa, 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 IKCa channels. In peripheral arteries, IKCa 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 IKCa 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 KIR 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 IKCa 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.
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