When stimulated by certain agonists or fluid shear stress, the vascular endothelium mediates relaxation of the underlying smooth muscle by releasing factors such as nitric oxide (NO) and prostacyclin. Such stimuli also evoke hyperpolarization of smooth muscle cells that is independent of NO and prostacyclin, a response first described in guinea pig and porcine arteries exposed to acetylcholine and bradykinin (Beny & Brumet, 1988, Chen et al., 1988). These findings led to the hypothesis that a third and distinct factor, named endothelium-derived hyperpolarizing factor (EDHF), travels across the myoendothelial space to modulate the membrane potential of smooth muscle cells and thereby control vascular tone. Despite extensive research, a universal EDHF has still not yet been identified.
Smooth muscle hyperpolarizations evoked by EDHF cause marked reductions in vascular tone because the voltage-dependent Ca2+ channels responsible for maintaining contraction are highly sensitive to membrane potential (Griffith, 2004). Results of many studies combine to suggest that EDHF-mediated hyperpolarizations of smooth muscle cells involve the opening of potassium (K+) channels and an increase in potassium efflux (Quignard et al., 2000). This is evidenced by an associated efflux of rubidium ions, an inverse relationship between ACh-induced relaxations and the prevailing extracellular K+ concentration, and inhibition of the EDHF response by non-selective inhibitors of potassium channels such as tetraethylammonium and tetrabutylammonium (Griffith, 2004; Quignard et al., 2000).
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Due the elusive nature of EDHF, no consensus has been reached on the type of K+ channels that EDHF acts on. In various tissues, charybdotoxin (a non-selective inhibitor of intermediate and large conductance calcium-activated K+ channels, IKca and BKca , and certain voltage dependent K+ channel subtypes, Kv), alone or in combination with apamin (a selective inhibitor of small conductance calcium-activated K+ channels, SKca) attenuates EDHF-mediated relaxations (Zygmunt & Hogestatt, 1996; Parsons et al., 1996). Iberiotoxin, which selectively blocks BKca channels, was ineffective against EDHF-mediated relaxations, even in combination with apamin, making it highly unlikely that BKca is involved in the EDHF response (Zygmunt & Hogestatt, 1996). Since charybdotoxin also inhibits delayed rectifier channels, Kv, it was proposed by the current article (Zygmunt et al., 1997) that these channels may play a role in the EDHF-mediated response. By using the whole-cell configuration of the patch-clamp technique along with tension studies, Zygmunt et al. tried to obtain more information about the K+ channel(s) that EDHF acts on.
Methods and Drugs
Tissue bath experiments
Tension studies with ring segments of rat hepatic artery (isolated from female Sprague-Dawley rats) were conducted to determine the type of K+ channels involved in the EDHF-mediated vascular relaxation. The tissue bath experiments were conducted based on the knowledge that the EDHF-mediated relaxations in the rat hepatic artery are abolished by a combination of apamin plus charybdotoxin, but not apamin plus iberiotoxin (Zygmunt & Hogestatt., 1996). The ineffectiveness of iberiotoxin, along with at least partial inhibition by charybdotoxin, collectively suggested that the charybdotoxin-sensitive channel was of the voltage-sensitive type, Kv (see above). Thus, to test whether Kv channels are involved in the EDHF-mediated response, a series of potent and specific Kv inhibitors were used (in addition to charybdotoxin), all of which were administered alone or in combination with apamin. These Kv inhibitors included ciclazindol, margatoxin, agitocin-2, kaliotoxin and beta-dendrotoxin. 4-aminopyridine, a selective Kv inhibitor previously found to inhibit endothelium-dependent hyperpolarization in the guinea pig artery (Quignard et al., 2000), was also used. Relaxations were studied in preparations contracted with phenylephrine (an Î±-adrenergic receptor agonist). When stable contractions were obtained, acetylcholine was added cumulatively to determine the concentration-response relationship. The experiments were conducted in the presence of 0.3mM N-nitro-L-arginine (L-NOARG) and 10ÂµM indomethacin, under which conditions acetylcholine-induced relaxations in the tissue are mediated by EDHF (Zygmunt et al., 1997).
To determine the presence of delayed rectifier K+ channels (Kv) on smooth muscle cells, charybdotoxin, apamin, ciclazindol and 4-aminoperidine were also tested electrophysiologically. For the single-cell electrophysiology studies, the whole cell configuration of patch-clamp technique was employed. Hepatic arteries were removed from male Sprague-Dawley rats and the smooth muscles cells of the media layer were isolated with a collagenase/pronase enzyme solution. Individual smooth muscle cells were placed in calcium-free bath (external) solution. Amphotericin B was used to produce "perforated patches". Whole cell currents (Itotal) in the hepatic arterial cells were generated by stepping from a holding potential -90mV to test potentials of -30, -10, 10, 30 and 50mV for 500ms. The effects of the compounds were investigated by adding the appropriate amount of each agent to the main reservoir containing the external solution to ensure that responses were obtained under steady-state conditions.
Summary of Results
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Tissue Bath experiments
Effects of apamin, charybdotoxin and ciclazindol
It has been found previously that charybdotoxin (0.3uM) when combined with apamin (0.3uM) completely abolished the L-NOARG/Indomethacine-resistant relaxation induced by acetylcholine (Zygmunt & Hogestatt, 1996). However, in the current experiment, neither 1uM apamin nor 1uM charybdotoxin by themselves produced any inhibitory effect on the EDHF-mediated relaxations. Ciclazindol (10uM) alone inhibited the EDHF-mediated response by shifting the acetylcholine concentration-response curve to the right by 1 log-molar unit, while keeping efficacy of acetylcholine the same. Furthermore, the combination of ciclazindol (10uM) plus apamin (0.3uM) completely abolished the EDHF-mediated relaxations.
Effects of margatoxin, agitoxin, beta-dendrotoxin and kaliotoxin
Each of agitoxin, beta-dendrotoxin, kaliotoxin administered alone (0.3uM) or in combination with apamin (0.3uM) had no effect on EDHF-mediated relaxations induced by acetylcholine. Although margatoxin (0.3uM) by itself produced no inhibition on EDHF-mediated relaxations, the combination of this toxin (0.3uM) with apamin (0.3uM) significantly inhibited the EDHF-mediated response by shifting the acetylcholine concentration-response curve to the right.
Effects of 4-aminopyridine
In the smooth cells of the rat hepatic artery, stepping from a holding potential of -90mV to test potential between -80mV and +50mV revealed a slowly-activating and slowly-inactivating outward current (Itotal), with an activation threshold of -30mV. When cells were held at -10mV (to inactivate voltage-dependent K+ channels, Kv), stepping to the same series of test potentials generated a non-inactivating current of much smaller amplitude. This suggests that Itotal consisted mainly of a current mediated by voltage-sensitive delayed rectifier K+ channels (IKV). This idea is further confirmed by the marked and reversible inhibition of Itotal by 4-aminoperidine (3mM), a potent inhibitor of delayed rectifier channels. After exposure to 4-aminopyridine, the resulting current-voltage relationship curve is less steep. At a step-depolarization of +50mV, 4-aminopyridine decreased Itotal by 67% compared to control.
Effects of apamin, charybdotoxin and ciclazindol
Apamin (0.3uM), charybdotoxin (0.3uM) and ciclazindol (10uM) by themselves had no effect on Itotal. Even when administered in combination with apamin (0.3uM), neither charybdotoxin nor ciclazindol inhibited Itotal. However, a ten times higher concentration of ciclazindol (0.1mM) caused a marked inhibition of this current (reduction of Itotal by 50% at a test potential of +50mV), an effect that was not further enhanced by simultaneous administration of apamin (0.3uM).
The objective of the current study was to determine the type(s) of K+ channels involved in the EDHF-mediated relaxation of the rat hepatic artery. The Kv channels were particularly of interest based on earlier observations (see above). This has never been an easy effort as it still remains controversial as to the identity of the K+ channels opened by EDHF. In some tissues such as the rat and rabbit mesenteric arteries, EDHF-mediated responses are virtually abolished by apamin, providing evidence that the small conductance Ca2+-sensitive potassium channel (SKca) is the K+ channel involved (Parsons et al., 1996; Murphy & Brayden, 1995). On the other hand, EDHF-mediated relaxations of the rabbit carotid artery are not affected by apamin but are inhibited by charybdotoxin, the inhibitor of large conductance calcium-sensitive potassium channels (Lischke et al., 1995).
In the current study with rat hepatic arteries, EDHF-mediated relaxations were not affected by either apamin or charybdotoxin, even when the concentration of each agent was raised to three times of that previously tested (Zygmunt & Hogestatt, 1996). However, the EDHF-mediated response was completely abolished when the two agents were combined, an effect previously observed in the rat hepatic and guinea pig basilar arteries (Zygmunt & Hogestatt 1996, Corriu et al, 1996). This may suggest a synergistic relationship between apamin and charybdotoxin. Alternately, the results could indicate that EDHF simultaneously activates both SKCA and BKca, and that simultaneous inhibition of both channels is necessary to inhibit EDHF-mediated responses (Zygmunt et al., 1997).
Since charybdotoxin inhibits not only BKca, but also Kv1.2 and Kv1.3 (Chandy & Gutman, 1995), antagonism of EDHF-mediated relaxations by this toxin (albeit in combination with apamin) could indicate that a delayed rectifier-like channel is a target for EDHF.
Indeed, in the present study, a current typical Kv channels was observed in the smooth muscles cells of the rat hepatic artery. The recorded current could be inactivated using a holding potential of -10mV and was inhibited by 4-aminopyridine. However, the involvement of Kv channels in the overall process of EDHF-mediated relaxation remains questionable because 4-aminoperidine along with other potent Kv inhibitors (agitoxin, beta-dendrotoxin and kaliotoxin) failed to inhibit the EDHF-mediated responses in the tissue bath experiments.
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Ciclazindol, in contrast to other Kv inhibitors tested in the current experiment, reduced EDHF-mediated relaxations. In addition, the combination of ciclazindol and apamin completely abolished the relaxations. Ciclazindol has been shown to inhibit delayed rectifier currents in smooth muscle cells of rat portal vein and pulmonary artery (Walker et al., 1996). In the current experiment, Ciclazindol also inhibited IKV in rat hepatic artery smooth muscle cell, but a ten times higher concentration was required than that necessary to antagonise EDHF-mediated relaxations. Furthermore, the effect of ciclazindol on IKV was not enhanced by apamin. These findings derived from both mechanical and electrophysiological experiments, do not support the view that Kv is the target for EDHF (Zygmunt et al, 1997).
EDHF - a controversy
Endothelium causes relaxation of the underlying smooth muscle cells by releasing at least three factors: nitric oxide (NO), prostacyclin (PGI2), and an unidentified endothelium-derived hyperpolarizing factor (EDHF). The roles of PGI2 and NO in mediating endothelium-dependent vasorelaxation are well-established. NO activates guanylyl cyclise and elevates intracellular cyclic guanosine monophosphate (cGMP) in the smooth muscle cells (Katzung, 2000). cGMP in turn facilitates dephosphorylation of myosin light chains, thus preventing the interaction of myosin with actin. cGMP also decreases intracellular Ca2+ (a major modulator of the activation of myosin light chain kinase) by enhancing sacroplasmic reticulum Ca2+ uptake. PGI2 is known to elevate intracellular levels of cyclic adenosine monophosphate (cAMP), which increases the rate of inactivation of myosin light chain kinase, the enzyme responsible for triggering the interaction between actin and myosin during muscle contraction (Katzung, 2000).
The endothelium-dependent relaxation that remains after NO and prostacyclin productions have been blocked by L-NOARG and indomethacin, respectively, confirms the release of EDHF as a distinct vasorelaxant factor. It is also well-established that EDHF mediates vasorelaxation by hyperpolarizing the underlying smooth muscle cells. As Ca2+ channels that maintain contraction are highly sensitive to membrane potential, smooth muscle hyperpolarizations evoked by EDHF result in closing of the voltage-dependent Ca2+ channels and thereby decrease in vascular tone (Griffith, 2004). Measurements in smooth muscle cells during the EDHF-mediated response (i.e. in the presence of L-NOARG and indomethacin) thus show a substantial and rapid decrease in the concentration of intracellular Ca2+ (Griffith, 2004).
That being said, the cellular mechanisms, let alone the identity, of EDHF remain poorly defined. The mechanisms by which EDHF elicits its effects seem to vary with tissue types and species. For instance, the Kv inhibitor 4-aminopyridine (4-AP) has been shown to inhibit EDHF-mediated relaxations in various tissues such as the guinea pig coronary artery (Quignard et al., 2000), yet in the current experiment, 4-AP had no effect in the rat hepatic artery (Zygmunt et al., 1997). In addition, the characterization of the K+ channels involved in the EDHF-mediated relaxation has not been elucidated. Many studies with peripheral arteries seem to suggest that the subtypes of K+ channels involved in the EDHF response also depend on tissue source and species (Dong et al., 1998). Thus, despite the controversy, it is reasonable to assume that the variable actions of EDHF in different tissues may be due to the different subtypes of K+ channels involved.
Location of K+ Channels
The involvement of K+ channels in the EDHF-mediated relaxation raises the critical question of where exactly these channels are located. In the current study (1997), Zygmunt et al. assumes the targets of charybdotoxin and apamin are located on the smooth muscle cells, based on the finding that the increase in endothelial intracellular calcium induced by acetylcholine was not affected by the two toxins (Yamanaka et al., 1998). It was argued that if K+ channels were located on endothelial cells, depolarization as a result of the inhibition of these K+ channels (by charybdotoxin and/or apamin) would decrease electrical driving force, and thereby decrease Ca2+ influx (Yamanaka et al., 1998). However, an increasing number of studies have demonstrated the presence/expression of various types of K+ channels on endothelial cells including BKca, SKca and Kv (Griffith, 2004). These findings are crucial in understanding the mechanism of the EDHF-mediated response. Of particular concern to the current study is the possibility that the observed drug effects may have resulted from an action on the endothelial cells, thus affecting release or transfer of EDHF, rather than on the smooth muscle cells.
A related question to the above comment is whether the endothelium is electrically coupled to the underlying smooth muscle cells. It has been suggested that EDHF, instead of being a freely transferable mediator of hyperpolarization, may simply be an electronic spread of hyperpolarization from the endothelium to the smooth muscle cells (Coleman et al, 2004). In deed, it has been shown that agonists that evoke EDHF-mediated relaxations such as acetylcholine initially induce a rapid change of the membrane potential towards the reversal potential of K+, ~ -90mV, followed by a slow return back to baseline (Griffith, 2004). More importantly, the electrical response that occurs on the smooth muscle closely parallels that in the endothelium during the EDHF-mediated relaxation. The initial hyperpolarization of the endothelium is believed to be driven by the opening of Ca2+-activated K+ channels (Kca) and/or voltage-dependent K+ channels (Kv), as the response can be attenuated by agents such as charybdotoxin, apamin and 4-amonopyridine (Coleman et al., 2004). Furthermore, it has been shown that selective intimal application of charybdotoxin and apamin blocks EDHF-mediated relaxations (Griffith, 2004). Thus, the endothelial actions of the toxins might be responsible for the inhibition of EDHF-mediated responses, which Zygmunt et al. might have neglected in the current study.
Drug action on the endothelial cells
An endothelial site for the initiation of the EDHF response suggests that drug effects may have resulted from an action on the endothelial cells rather than the smooth muscle cells. The results derived from mechanical and electrophysiological studies in the current experiment seem to agree with this point. Zygmunt et al. (1997) concluded that the EDHF-mediated relaxation in the rat hepatic artery is not mediated by the opening of either K v or BK ca channels on the smooth muscle cells. Although a current typical of K v channels was found in the smooth muscles, it does not necessarily equate to its involvement in the overall EDHF response. In deed, a ten times higher concentration of ciclazindol was required to abolish IKV in smooth muscle cells than that necessary to antagonize EDGF-mediated relaxations. Furthermore, the fact that Kv does not activate until the membrane has depolarized to a threshold of ~ -30mV means that this channel is unlikely to participate in the EDHF-mediated hyperpolarizations, which shift the membrane potential into the range of -50 to -70mV (Quignard et al., 2000).
Despite the absence of a ciclazindol-sensitive current in the smooth muscle cell, the Kv inhibitor did inhibit EDHF-mediated relaxations in the rat hepatic artery (Zygmunt et al, 1997). However, Zygmunt et al. fail to explain the inhibitory effects of ciclazindol on the overall EDHF-response, perhaps because they neglected the possible actions of the drug on the endothelium. In microelectrode studies with isolated endothelial cells of guinea pig coronary arteries, ciclazindol was found to inhibit endothelial hyperpolarizations produced by acetylcholine (Quignard et al., 2000). A study by Edwards et al. (1998) showed that the site of action of the combination of charybdotoxin plus apamin is likely to be the small and intermediate conductance Ca2+-sensitive K+ channels (SK ca and IK ca, respectively) present on endothelial cells. The inhibitory effect of ciclazindol was not increased by apamin or charybdotoxin, suggesting that ciclazindol acts on endothelial cells but independently to the targets of these two toxins, possibly a step proximal to the activation of SK ca and IK ca (Edwards et al., 1998). To identify the exact site of action of ciclazindol would be beyond the scope of this paper, yet this remains to be an area for more extensive research to elucidate the role of KV channels in the EDHF response.
Ionic mechanisms are perhaps ideally studied by recording the membrane currents under voltage clamp. Voltage-clamp studies of vascular tissues typically involve enzymatic isolation of either the smooth muscle or endothelial cells and recording from isolated cells using the patch-clamp technique. This is the case in the current experiment with isolated smooth muscle cells. Such cellular isolation overcomes the problems of spatial clamp control in syncytial tissue (Coleman et al., 2004). However, to record the ionic currents underlying the elusive and controversial EDHF, a preparation would be required in which the endothelial and smooth muscle cells remain in their normal functional relationship, especially in view of electronic spread as a potential mechanism of EDHF. Such a preparation needs to be amenable to voltage clamp, preferably without exposing the cells to digestive enzyme that could potentially disrupt mechanisms underlying EDHF. Yamamoto et al. (1999) demonstrated one such preparation in which electrical responses of individual smooth muscle and endothelial cells to Ach were recorded (by patch-clamp methods) in multicellular preparations where the two types of cells remained in close proximity. An approach similar to Yamamoto et al. (1999) would have been more appropriate and relevant for investigating the involvement of potassium channels in the EDHF response.
Ca2+ K+ IKca BKca Kv ÂµM Itotal IKV SKca PGI2
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