Activation of Barium-Sensitive Inward Rectifier Potassium Channels Mediates Remote Dilation of Coronary Arterioles
Background Conducted vasodilation seems to be critical for the functional distribution of blood flow in the skeletal muscle microcirculation. However, this vasoregulatory phenomenon has not been documented in the coronary microcirculation, and its underlying mechanism remains elusive. Because potassium ions are potent metabolic vasodilators in the heart, by activating vascular inward rectifier K+ (Kir) channels, we tested the hypothesis that coronary arterioles exhibit remote vasodilation through activation of this type of channel.
Methods and Results Porcine coronary arterioles were isolated, cannulated, and pressurized for in vitro study. Vessels dilated concentration-dependently to extraluminal KCl (5 to 20 mmol/L), bradykinin, adenosine, pinacidil, and sodium nitroprusside. A Kir channel blocker, BaCl2 (30 μmol/L), inhibited vasodilatory responses to KCl and bradykinin but not to adenosine, pinacidil, or nitroprusside. In a flow chamber, localized administration of bradykinin, adenosine, and KCl to the downstream end of the arterioles caused ≈80% dilation at the site of drug application (local site) and also produced 30% to 60% dilation at the upstream end of arterioles (remote site). Nitroprusside produced a similar dilation at the local site but failed to initiate remote vasodilation. In the presence of Ba2+, adenosine still dilated the local site, but the local dilations to bradykinin and KCl and the remote dilations to adenosine, bradykinin, and KCl were inhibited.
Conclusions We demonstrated that some modes of local vasodilation can be conducted to remote sites in coronary arterioles and that local and remote dilations can occur through different vasodilatory mechanisms. Activation of Kir channels seems critical for some agonist-induced local vasodilations and also for the initiation and/or transmission of signals causing remote vasodilation.
Received June 28, 2001; revision received August 16, 2001; accepted August 16, 2001.
In the skeletal muscle microcirculation, it has been shown that vasomotor responses elicited at one location (direct local response) can result in changes in vessel diameter at remote sites (conducted response).1 Conducted vasomotor responses have been considered a result of changes in membrane potential through cell-cell electrical conduction along the vessel wall.2 Although this conducted response has been described in a number of tissues and has been suggested to play an important role in the integrative coordination of blood flow distribution during increased metabolic demand,3 it remains unclear whether this vasomotor response also exists in the heart. In addition, the underlying mechanisms involved in the propagation of vasodilation have not been elucidated.
A possible mechanism for remote dilations in microvessels could involve K+ channels because they play a central role in the maintenance and regulation of the membrane potential in smooth muscle cells.4 In excitable cells, the release of K+ during metabolic activation can contribute to flow recruitment through membrane hyperpolarization and vasodilation. Recent studies indicate that the activation of inward rectifier K+ (Kir) channels is responsible for coronary arterial dilation elicited by K+.5 Moreover, the conductance of Kir channels can be further increased by membrane hyperpolarization.4 Thus, it is possible that the activation of Kir channels mediates the conducted vasodilation of coronary microvessels. In this regard, the goal of the present study was to document that remote vasodilatory signals are present in coronary arterioles and to define the role of Kir channels in this response.
The procedures followed were in accordance with approved guidelines set by the Laboratory Animal Care Committee at Texas A&M University. Pigs were anesthetized with phenobarbital (20 mg/kg) and ventilated. The heart was removed and placed on iced saline. To eliminate confounding influences from hemodynamic, neurohumoral, and metabolic factors associated with in vivo preparations, individual coronary arterioles (≈1.5 mm in length; 50 to 110 μm in diameter in situ) were dissected out for in vitro study, as previously described.6 Vessels were cannulated with glass micropipettes and pressurized to 60 cm H2O intraluminal pressure. In one group of vessels, the vasodilatory responses were studied in the presence of a global increase in the extraluminal agonist concentration (KCl, 5 to 20 mmol/L; bradykinin, 10−12 to 10−9 mol/L; adenosine, 10−10 to 10−5 mol/L; pinacidil, 10−8 to 3×10−6 mol/L; and sodium nitroprusside, 10−9 to 10−5 mol/L). Changes in the concentration of extraluminal KCl were balanced by respective opposite changes in the NaCl concentration in physiological salt solution to maintain constant osmolarity. The role of the Kir channel in these dilations was assessed in the presence of 30 μmol/L BaCl2 (a specific inhibitory concentration for Kir channels).4,5,7
In another group of vessels, the physiological salt solution, at 37°C, was continuously suffused across the vessels at a rate of 1 to 2 mL/min. Agonists were applied to the vessel wall at the downstream end (relative to the suffusion flow) of the cannulated arteriole through glass micropipettes attached to a pneumatic microinjector, as described in our previous study (Narishige Microinjector model IM-200).8 This assured that the agonists only acted on the site of vessels subjected to microapplication. Dye studies verified that the suffusion flow was sufficient to eliminate convection of drug to the upstream end of the vessel. The agonists tested for conducted responses were bradykinin (1 nmol/L), adenosine (10 μmol/L), nitroprusside (10 μmol/L), and KCl (10 mmol/L). The inner diameters of coronary arterioles were measured using videomicroscopic techniques incorporated with the MacLab data acquisition system.6 The vasodilatory response was measured at the site of agonist application (ie, local dilation) and at the other end of the vessel, typically at least 800 μm upstream from the local site (ie, remote dilation). To determine the role of Kir channels in local and remote vasodilations, the vasomotor responses were reexamined after suffusing the vessel with physiological salt solution containing 30 μmol/L BaCl2 for 30 minutes. In some preparations, the role of ATP-sensitive K+ (KATP) channels in remote vasodilation was assessed in the presence of its selective inhibitor glibenclamide (5 μmol/L). All drugs were dissolved in physiological salt solution and were obtained from Sigma.
Statistical comparisons of vasomotor responses to drugs before and after treatment with Ba2+ were performed with either ANOVA with repeated measures (tested with Fisher protected least significant difference multiple range test) or paired Student’s t test when appropriate. Significance was accepted at P<0.05.
In the present study, isolated coronary arterioles developed basal tone within 40 minutes at a 37°C bath temperature and 60 cm H2O intraluminal pressure. The level of basal tone (83±4 μm) developed in these vessels was similar and corresponded to 66±1% of maximum passive diameter (126±7 μm; measured in the presence of 0.1 mmol/L nitroprusside at the end of the experiment). Only vessels exhibiting basal tone were used for further study.
Role of Kir Channels in Coronary Arteriolar Dilations to KCl, Bradykinin, Adenosine, Pinacidil, and Sodium Nitroprusside
Moderate increases in KCl concentrations (5 to 20 mmol/L) in the vessel bath produced dilation of coronary arterioles. Ba2+ (30 μmol/L) significantly inhibited this vasodilatory response (Figure 1A). Bradykinin (Figure 1B), adenosine (Figure 1C), pinacidil (Figure 1D), and sodium nitroprusside (Figure 1E) also produced concentration-dependent dilation of coronary arterioles; however, adenosine-, pinacidil-, and nitroprusside-induced vasodilations were insensitive to Ba2+.
Role of Kir Channels in Remote Arteriolar Dilations
The local and remote dilations of coronary arterioles to bradykinin, adenosine, sodium nitroprusside, and KCl are shown in Figure 2. Direct application of bradykinin (1 nmol/L), adenosine (10 μmol/L), nitroprusside (10 μmol/L), and KCl (10 mmol/L) produced ≈80% of maximal dilation at the local site. Unlike nitroprusside, the dilations to bradykinin, adenosine, and KCl were also seen at the remote sites (ie, at 800 μm upstream of local sites). Remote dilation started 5±1 seconds after the initiation of local dilation.
Without altering basal tone, Ba2+ inhibited the local dilation of coronary arterioles caused by bradykinin, whereas local dilations to adenosine and nitroprusside were not affected (Figure 2A). Remote vasodilations elicited by bradykinin and adenosine were abolished by Ba2+ (Figures 2A and 2B). Ba2+ also inhibited remote vasodilation in response to KCl (n=3; data not shown). However, the local and remote vasodilations elicited by KCl were not affected by glibenclamide (Figures 2C and 2D). The efficacy of glibenclamide was verified by its ability to abolish vasodilation to the KATP channel opener pinacidil (1 μmol/L; 76±7% versus 5±3% dilation in the absence and presence of glibenclamide, respectively; n=3).
In the present study, we found that isolated coronary arterioles possess the needed machinery to initiate and transmit vasomotor signals in response to specific agonist stimulation. Our data suggest that the activation of Kir channels is involved in the initiation and/or transmission of a vasodilatory signal to the remote sites.
The Kir channel is highly and selectively expressed in the coronary microvessels,9 suggesting its potential role in coronary flow regulation. Vascular Kir channels are very sensitive to Ba2+. It has been shown at single channel and at intact tissue levels that a low concentration (30 μmol/L) of Ba2+ specifically inhibits vascular Kir channel activity.4 In addition, both functional and electrophysiological studies in rat septal coronary arteries indicate that the activation of Kir channels, as assessed by a low concentration of Ba2+, is responsible for the dilation of coronary blood vessels to a moderate increase in extracellular K+.5 This is in agreement with our present finding in porcine coronary subepicardial arterioles that KCl-induced dilation is sensitive to Ba2+. Interestingly, the K+-induced vasodilation has been suggested to be one of the mechanisms involved in the metabolic regulation of coronary blood flow because the increased K+ concentration during metabolic activation10 or stress11 is sufficient to evoke coronary vasodilation.
It is generally believed that conducted vasomotor responses in arterioles rely on passive electrotonic spread of the change in membrane potential through gap junctions.2 However, the decay of the conducted signal is too slow to be explained by simple decay of an electrical response. Some mode of regenerative process seems inevitable. Therefore, the present brief set of experiments was performed with the idea that there must be additional modes of cell-cell signaling for remote vasodilation. One possibility for regenerative cell-cell signaling could involve K+. Release of this ion could initiate the serial opening of K+ channels that would transmit a hyperpolarizing signal for remote vasodilation. It is likely that a small increase in K+ concentration at the local interstitium during membrane hyperpolarization, via opening of K+ channels, could subsequently lead to the opening of Kir channels at nearby vascular cells. It is known that Kir channels are more active at negative membrane potentials9; as such, the initial efflux of K+ via opened potassium channels would preferentially activate Kir channels. The sequential opening of nearby Kir channels by increased K+ could be a vehicle for transmitting the vasodilatory signals to the remote site. This contention is supported by a recent finding in small resistance arteries that membrane hyperpolarization is capable of elevating K+ concentration within the extracellular space of gap junctions to vasoactive levels.7 Our results also support this idea because inhibition of Kir channels by Ba2+ blocked the remote vasodilations elicited by KCl, bradykinin, and adenosine, indicating that activation of Kir channels, presumably by K+, is essential for the initiation and/or transmission of signals for remote vasodilation.
Although Ba2+ has been shown to be a selective inhibitor for Kir channels in various preparations,4,5,7 the specificity of Ba2+ in blocking Kir channels in our vessels was critical to the data interpretation and conclusion. In electrophysiological studies using the patch-clamp technique, we previously found that the activation of Kir channels by KCl in vascular smooth muscle cells isolated from porcine coronary arterioles was specifically blocked by Ba2+ (30 μmol/L). In addition, Ba2+ has no effect on vasodilations to adenosine, pinacidil, and sodium nitroprusside, but it inhibited vasodilation to KCl (Figure 1), indicating that the inhibitory effect of Ba2+ is specific for Kir channels in our vessel preparations.
Interestingly, our data on bradykinin- and adenosine-induced dilations show that the vasodilatory mechanism at the remote location can be dissociated from that occurring at the local site. In our previous study, for example, we showed that coronary arteriolar dilation to bradykinin, through membrane hyperpolarization, is mediated by the release of cytochrome P-450 metabolites of arachidonic acid.12 It is likely that bradykinin-induced vasodilation is a result of releasing endothelium-derived hyperpolarizing factor because the metabolic products from cytochrome P-450 enzyme have been considered potential candidates for this factor. In the present study, we showed that bradykinin-induced coronary arteriolar dilation is sensitive to Ba2+ (Figure 1B), indicating that activation of Kir channels is involved in the vasodilation evoked by cytochrome P-450 metabolites. In contrast, the dilation of coronary arterioles to adenosine has been shown to be mediated by the activation of KATP channels.13 These channels are insensitive to Ba2+ because vasodilations to global increases in adenosine (Figure 1C) and the selective KATP channel opener pinacidil (Figure 1D) were not affected by Ba2+.
Although different agonists elicit different vasodilatory mechanisms, it seems that the initial activation of K+ channels associated with membrane hyperpolarization at the local site is capable of initiating and transmitting vasodilatory signals to the remote area through Kir channel activation (Figure 2). In contrast to the K+ channel-mediated vasodilation in response to bradykinin and adenosine stimulation, the coronary arteriolar dilation to sodium nitroprusside is insensitive to Ba2+ (Figure 1E) and is not affected by KATP channel inhibition or by nonspecific K+ channel blockers.13 Nitroprusside also produces little14 or no15 change in the membrane potential of coronary vascular cells. Thus, it is expected that this agent would not produce remote vasodilation in coronary microvessels, as shown in the present study. In addition, we performed some studies in vessels treated with a nitric oxide synthase inhibitor, NG-monomethyl-l-argine (L-NMMA). The concentration of L-NMMA (10 μmol/L) used was sufficient to block nitric oxide–mediated vasodilation in our previous study6 but failed to affect remote vasodilations to bradykinin and adenosine (n=3; data not shown). These findings are consistent with the nitroprusside data and suggest that nitrovasodilators are not involved in these remote responses.
Although the nitric oxide pathway may not be involved in conducted vasodilation in the present study, the overall role of endothelium in this vasodilatory response remains unclear. Recent evidence suggests that there is a direct electrical coupling between vascular smooth muscle and endothelium in arterioles isolated from hamster retractor muscle.16 However, it is unclear whether this finding can be extrapolated to the coronary arterioles. Although we did not particularly focus on the function of endothelium in the present study, our data suggest that vascular smooth muscle is capable of initiating and transmitting conducted signals. This contention is supported by our observation that KCl (10 mmol/L) elicited remote vasodilation, although its local dilation is independent of endothelium (n=3; data not shown). Furthermore, the KCl-induced remote dilation was not affected by glibenclamide but was inhibited by Ba2+, suggesting the significant role of Kir but not KATP channels in this signal transmission. Collectively, without completely ruling out the role of endothelium, these data support the hypothesis that activation of Kir channels on the vascular smooth muscle is sufficient to initiate and transmit the remote vasodilation. It seems that an initial change in membrane potential in vascular smooth muscle during vasodilation is essential for the subsequent transmission of vasomotor signal to the remote sites through Kir channel activation.
In summary, these are the first data to demonstrate the presence of vascular communication in the coronary microcirculation and to suggest that Kir channels play a role in the remote dilation of coronary arterioles. It is speculated that this vasoregulatory mechanism could contribute to integrated flow regulation by coordinating the activities of parent and daughter vessels in the coronary vascular network during metabolic stresses, because a significant increase in the interstitial levels of bradykinin,17 adenosine,18 and K+10,11 are recognized under these conditions.
Supported by the following grants from the National Heart, Lung, and Blood Institute: HL-55524, HL-48179, and the K02HL03693 Research Career Award to Dr Kuo.
Duling BR, Berne RM. Propagated vasodilation in the microcirculation of the hamster cheek pouch. Circ Res. 1970; 26: 163–170.
Cohen KD, Berg BR, Sarelius IH. Remote arteriolar dilations in response to muscle contraction under capillaries. Am J Physiol. 1999; 278: H1916–H1923.
Quayle JM, Nelson MT, Standen NB. ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev. 1997; 77: 1165–1232.
Kuo L, Chilian WM, Davis MJ. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am J Physiol. 1991; 261: H1706–H1715.
Rivers RJ. Cumulative conducted vasodilation within a single arteriole and the maximum conducted response. Am J Physiol. 1997; 273: H310–H316.
Murray PA, Belloni FL, Sparks HV. The role of potassium in the metabolic control of coronary vascular resistance of the dog. Circ Res. 1979; 44: 767–780.
Kleber AG. Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global ischemia in isolated perfused guinea pig hearts. Circ Res. 1983; 52: 442–450.
Hein TW, Liao JC, Kuo L. oxLDL specifically impairs endothelium-dependent, NO-mediated dilation of coronary arterioles. Am J Physiol. 2000; 278: H175–H183.
Hein TW, Kuo L. cAMP-independent dilation of coronary arterioles to adenosine: role of nitric oxide, G proteins, and KATP channels. Circ Res. 1999; 85: 634–642.
Miura H, Liu Y, Gutterman DD. Human coronary arteriolar dilation to bradykinin depends on membrane hyperpolarization: contribution of nitric oxide and Ca2+-activated K+ channels. Circulation. 1999; 99: 3132–3138.
Chen GF, Cheung DW. Characterization of acetylcholine-induced membrane hyperpolarization in endothelial cells. Circ Res. 1992; 70: 257–263.
Emerson GG, Segal SS. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: role in vasomotor control. Circ Res. 2000; 87: 474–479.
Node K, Kitakaze M, Kosaka H, et al. Bradykinin mediation of Ca2+-activated K+ channels regulates coronary blood flow in ischemic myocardium. Circulation. 1997; 95: 1560–1567.
Berne RM. The role of adenosine in the regulation of coronary blood flow. Circ Res. 1980; 47: 807–813.