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Molecular Mechanisms of the Inhibitory Effects of Clonidine on Vascular Adenosine Triphosphate–Sensitive Potassium Channels

Kawahito, Shinji MD, PhD*; Kawano, Takashi MD, PhD; Kitahata, Hiroshi MD, PhD; Oto, Jun MD, PhD§; Takahashi, Akira MD, PhD; Takaishi, Kazumi DDS, PhD; Harada, Nagakatsu PhD; Nakagawa, Tadahiko MS; Kinoshita, Hiroyuki MD, PhD#; Azma, Toshiharu MD, PhD**; Nakaya, Yutaka MD, PhD; Oshita, Shuzo MD, PhD*

doi: 10.1213/ANE.0b013e3182321142
Anesthetic Pharmacology: Research Reports
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BACKGROUND: We investigated the effects of the imidazoline-derived α2-adrenoceptor agonist clonidine on vascular adenosine triphosphate–sensitive potassium (KATP) channel activity in rat vascular smooth muscle cells and recombinant vascular KATP channels transiently expressed in COS-7 cells.

METHODS: Using the patch-clamp method, we investigated the effects of clonidine on the following: (1) native vascular KATP channels; (2) recombinant KATP channels with different combinations of various types of inwardly rectifying potassium channel (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor (SUR1, 2A, 2B) subunits; (3) SUR-deficient channels derived from a truncated isoform of the Kir6.2 subunit (Kir6.2ΔC36 channels); and (4) mutant Kir6.2ΔC36 channels with diminished sensitivity to ATP (Kir6.2ΔC36-K185Q channels).

RESULTS: Clonidine (≥3 × 10−8 M) inhibited native KATP channel activity in cell-attached configurations with a half-maximal inhibitory concentration value of 1.21 × 10−6 M and in inside-out configurations with a half-maximal inhibitory concentration value of 0.89 × 10−6 M. With similar potency, clonidine (10−6 or 10−3 M) also inhibited the activities of various recombinant SUR/Kir6.0 KATP channels, the Kir6.2ΔC36 channel, and the Kir6.2ΔC36-K185Q channel.

CONCLUSIONS: Clinically relevant concentrations of clonidine inhibit KATP channel activity in vascular smooth muscle cells. This inhibition seems to be the result of its effect on the Kir6.0 subunit and not on the SUR subunit.

Published ahead of print October 14, 2011 Supplemental Digital Content is available in the text.

Author affiliations are provided at the end of the article.

Supported in part by a Grant-in-Aid for Scientific Research (C 15591636) from the Japan Society for the Promotion of Science, Tokyo, Japan.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Shinji Kawahito, MD, PhD, Department of Anesthesiology, Tokushima University Hospital, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. Address e-mail to kawahito@pb4.so-net.ne.jp.

Accepted July 28, 2011

Published ahead of print October 14, 2011

Alpha2-adrenoceptor agonists are used in anesthesia and intensive care for their sedative, amnestic, analgesic, and anesthetic properties.1 In addition, perioperative administration of α2-adrenoceptor agonists attenuates hyperadrenergic states2,3 and thus provides hemodynamic stability in the event of stressful perioperative events.4 Clonidine is the prototypical α2 agonist and is frequently used not only as a premedicant or an adjunct to general anesthesia but also for pain control by intrathecal and epidural administration.5 Although safe at usual dosages, clonidine must be used with caution when given in the injectable form: an overdose can produce significant vasospasm and hypertensive emergency.68

All of the clinically available α2 agonists possess an imidazoline ring and therefore interact with the imidazoline receptor. It has been reported that structurally related imidazoline compounds, including phentolamine, clonidine, and cibenzoline inhibit adenosine triphosphate–sensitive potassium (KATP) channel activity in cardiac myocytes.911 These reports suggest that clonidine, like other IV anesthetics and imidazoline compounds, may similarly inhibit KATP channel activity in cardiac myocytes. However, little is known of the effects of clonidine on the vascular KATP channel.

KATP channels are inhibited by intracellular ATP, which is expressed in a wide variety of tissues and believed to link cellular metabolic status and excitability.1216 In vascular smooth muscle cells, KATP channels regulate the membrane potential, which controls calcium entry through voltage-dependent calcium channels, and thereby contractility through changes in intracellular calcium.16,17 Moreover, physiologic studies have suggested that activation of KATP channels in vascular smooth muscle causes vasodilation as a physiologic response to certain neurotransmitters and during hypoxia, and as a pharmacologic response during therapy with KATP channel openers.15,17 Miki et al.14 reported that episodic coronary artery vasospasm and hypertension developed in the absence of KATP channels, and 1 clinical study suggests that prophylactic administration of the KATP channel opener nicorandil is useful for perioperative prevention of cardiac complications.18

The KATP channel is a heterooctamer composed of 2 subunits, an inwardly rectifying K+ channel (Kir) 6.0 family subunit (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR) subunit (SUR1, SUR2A, or SUR2B).19 The SUR subunit acts as a regulatory subunit, whereas the Kir subunit forms the ATP-sensitive channel pore, and different combinations of Kir and SUR subunits generate various tissue-specific KATP channel subtypes.19 Furthermore, different KATP channel activators and inhibitors show different tissue specificities, whereas different types of KATP channels exhibit different pharmacologic properties, which mainly depend on the SUR subunit.16,19

A previous mutagenesis study of ours showed that the major effects of various IV anesthetics on KATP channel activity are mediated by the Kir6.2 subunit of the channel.2023 However, tissue expression and the site of action of clonidine of the Kir6.2 subunit have not been investigated in detail. In the present study, we used patch-clamp techniques to examine the electrophysiologic effects of clinically relevant concentrations of clonidine on native vascular and recombinant KATP channels and the molecular mechanisms underlying such effects.

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METHODS

Native Vascular Smooth Muscle Cells

The A10 vascular smooth muscle cell line, derived from fetal rat thoracic aorta, was obtained from the American Type Culture Collection (Manassas, VA). The cells were incubated in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD), 3.7 mg/mL NaHCO3, and 100 μg/mL gentamicin at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed initially at 48 hours and then every 2 to 3 days. When the cells had formed a confluent monolayer after 7 to 9 days, they were made quiescent by incubation in serum-free medium for 24 hours. They were then harvested by the addition of 0.05% trypsin and 0.1% fetal bovine serum. Passages 5 to 12 were used for experimental purposes, and cultured A10 cells were stimulated with clonidine (10−9–10−3 M).

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Molecular Biology and Transfection

Details of the experimental design using recombinant KATP channels were similar to those of our previous studies.2023 In brief, KATP-deficient COS-7 cells (African green monkey kidney cells) were transiently cotransfected with 2 KATP channel subunits, SUR and Kir subunits, which comprise specific tissue-type KATP channels, with Lipofectamine and Opti-MEM1 reagents source. A truncated form of human Kir6.2 lacking the last 36 amino acids at the C terminus (Kir6.2ΔC36) was obtained by polymerase chain reaction (PCR) amplification. Mutagenesis cDNA of Kir6.2ΔC36 was performed with a site-directed mutagenesis kit (Invitrogen Corp., Carlsbad, CA). All DNA products were sequenced with a BigDye terminator cycle sequencing kit and an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA) to confirm the sequence. For electrophysiologic recordings, COS-7 cells were plated onto glass coverslips in dishes, and Kir and SUR subunits were cotransfected with green fluorescent protein cDNA (pEGFP) as a reporter gene. After transfection, cells were cultured for 48 to 72 hours before being subjected to electrophysiologic recording.

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Electrophysiologic Measurements

Cell-attached and inside-out patch configurations were used to record the current through single channels via a patch-clamp amplifier, as described previously.24,25 For the cell-attached configuration, the bath solution consisted of 140 mM KCl, 10 mM HEPES, 5.5 mM dextrose, and 1 mM EGTA. The pipette solution contained 140 mM KCl, 10 mM HEPES, and 5.5 mM dextrose. For the inside-out configuration, the bath solution (intracellular solution) consisted of 140 mM KCl, 10 mM HEPES, 5.5 mM dextrose, 1 mM MgCl2, 1 mM EGTA, 0.5 mM magnesium adenosine diphosphate, and 0.5 mM magnesium ATP. The pipette solution (extracellular medium) was of the same composition as that used in cell-attached experiments. The pH of all solutions was adjusted to 7.3 to 7.4 with potassium hydroxide. Recordings were made at 36°C ± 0.5°C. Patch pipettes were pulled with an electrode puller (PP-830; Narishige Co., Ltd., Tokyo, Japan). The resistance of pipettes filled with internal solution and immersed in Tyrode's solution was 5 to 7 MΩ. The sampling frequency of the single-channel data was 5 kHz with a low-pass filter (1 kHz).

Channel currents were recorded with a patch-clamp amplifier (CEZ 2200; Nihon Kohden, Tokyo, Japan) and stored on a personal computer (Aptiva; International Business Machines Corp., Armonk, NY) with an analog-to-digital converter (DigiData 1200; Axon Instruments, Foster City, CA). pClamp version 7 software (Axon Instruments) was used for data acquisition and analysis. The open probability (Po) was determined from current amplitude histograms and was calculated as follows:

where tj is the time spent at current levels corresponding to j = 0, 1, 2, N channels in the open state, Td is the duration of the recording, and N is the number of channels active in the patch. Recordings of 2 to 3 minutes were analyzed to determine Po. Channel activity was expressed as NPo. Changes in channel activity in the presence of drugs were calculated as the relative channel activity, i.e., the ratio between values obtained before and after drug treatment. When the concentration-dependent effect of clonidine was studied, increasing concentrations of clonidine were injected into the cell bath with a glass syringe (total volume injected was approximately 10 to 20 μL).

Drug concentrations needed to induce half maximal inhibition of the channels (IC50) and the Hill coefficient were calculated as follows:

where y is the relative NPo, [D] is the concentration of drug, Ki is the IC50, and H is the Hill coefficient.

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Reverse Transcription–PCR Study

Total RNA was extracted from A10 cells with TRIzol reagent (Invitrogen) and the obtained RNA was reverse transcribed (RT) with the PrimeScript® RT reagent kit (Takara, Kyoto, Japan). PCR reactions, performed by using gene-specific primers for each of the α2-adrenegic receptor variants26 and β-actin (internal standard),27 were designed to amplify a 311-bp fragment of α2A, a 455-bp fragment of α2B, a 426-bp fragment of α2C, and a 510-bp fragment of β-actin cDNA. PCR reactions were also performed by using an Ex-Taq DNA polymerase (Takara) with the following cycling variables: 94°C for 2 minutes, 35 cycles of 94°C for 30 seconds, 54.5°C (58.5°C for α2A and α2C) for 30 seconds, and 72°C for 50 seconds with a final extension at 72°C for 7 minutes. The PCR products were then electrophoresed on a 1.8% agarose gel.

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Immunoblotting

A10 cell extracts were prepared by homogenizing cells for 30 minutes on ice in a RIPA buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet-P40, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The cells were then passed through a 21-gauge needle followed by centrifugation at 13,500g for 30 minutes at 4°C. The supernatant was used as a protein sample of the A10 cell extracts. Protein concentration was measured with the BCA™ protein assay kit (Pierce Biotechnology, Rockford, IL). A 20-μg aliquot of protein was denatured by boiling for 2 minutes in a loading buffer (4% SDS, 250 mM Tris-HCl, pH 6.8, 1% β-mercaptoethanol, 1% bromophenol blue, and 20% glycerol). The proteins were then subjected to SDS–polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). The membranes were blocked for 1 hour using 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 and then incubated with either an anti–α2A-adrenergic receptor rabbit polyclonal antibody (1:500; Sigma, St. Louis, MO) or anti–β-actin rabbit polyclonal antibody (1:1000; Cell Signaling Technology, Inc., Danvers, MA). The attached antibodies were visualized with an ECL Plus detection kit (Amersham Pharmacia Biotech, Aylesbury, UK) using goat antirabbit immunoglobulin G horseradish peroxidase conjugate secondary antibody (1:5000; Biosource International, Camarillo, CA).

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Drugs

The following pharmacologic agents were used: clonidine, dimethyl sulfoxide (DMSO), glibenclamide, and pinacidil (Sigma-Aldrich Japan, Tokyo, Japan). Drugs were dissolved in distilled water with volumes of <15 μL of the solution being added to the organ chamber. Stock solutions of glibenclamide and pinacidil were prepared in DMSO. The final concentration of DMSO in the bath solution never exceeded 0.01%, and at a 2-fold–higher concentration, this agent was shown to not affect KATP channel currents. Drug concentrations are expressed as final molar (M) concentrations.

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Statistical Analysis

All data are presented as mean ± SD. Differences between data sets were evaluated by repeated-measures 1-way analysis of variance followed by the Scheffé F test or the Student t test with Welch correction. P < 0.05 was considered statistically significant.

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RESULTS

Effects of Clonidine on KATP Channel Activity in Native Vascular Smooth Muscle Cells

To investigate whether clonidine affects KATP channel activity in vascular smooth muscle cells, we measured single KATP channel currents by the patch-clamp technique. Application of 10−4 M pinacidil, a selective KATP channel opener, to the bath solution significantly activated K+-selective channels (NPo 0.416 ± 0.132, P < 0.05 versus baseline, n = 14), as shown in Figure 1A, and this channel activity was completely blocked by 3 × 10−6 M glibenclamide, a specific KATP channel blocker (Fig. 1A, n = 14). The channel showed a single-channel conductance of 30.1 ± 5.0 pS (n = 6) as measured by the current-voltage relation between −80 and +60 mV membrane potential. These channel properties were consistent with those reported previously.16 Representative examples of the effects of clonidine on pinacidil-induced KATP channel activity in the cell-attached configuration are shown in Figure 1B. Application of 10−9, 10−6, or 10−3 M clonidine to the outside of the membrane surface inhibited pinacidil-induced KATP channel currents, with relative channel activity decreasing to 0.95 ± 0.04 (n = 10), 0.54 ± 0.18 (n = 10), and 0.09 ± 0.10 (n = 10), respectively.

Figure 1

Figure 1

Concentration-dependent effects of clonidine on pinacidil-induced KATP channel activity in the cell-attached and inside-out configurations are shown in Figure 2. Clonidine significantly inhibited KATP channel activity at concentrations of 3 × 10−8 M or larger in both the cell-attached and inside-out configurations. The IC50 values in the cell-attached and inside-out configurations were 1.21 × 10−6 M and 0.89 × 10−6 M, respectively, and the corresponding Hill coefficient in the cell-attached and inside-out configurations was 1.23 and 1.44. Clonidine did not alter the single-channel conductance in either the cell-attached or inside-out configuration (data not shown).

Figure 2

Figure 2

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Effects of Clonidine on Recombinant KATP Channel Activity

To determine the tissue-specific effects of clonidine on KATP channel activity, we used the inside-out patch-clamp configuration to investigate the effects of clonidine on the activities of various types of recombinant Kir6.0/SUR channels. Sarcolemmal KATP channels, SUR2B/Kir6.1 (vascular smooth muscle type), SUR2B/Kir6.2 (nonvascular smooth muscle type), and SUR2A/Kir6.2 (cardiac type) were heterologously expressed in COS-7 cells.19 Our previous studies showed that the single-channel characteristics of all types of expressed KATP channels are similar to those of native KATP channels.2023 Application of 10−6 M clonidine to the intracellular membrane surface inhibited pinacidil-induced SUR2B/Kir6.1, SUR2B/Kir6.2, and SUR2A/Kir6.2 channel currents with equivalent potency, as shown in Figure 3A; relative channel activity decreased to 0.58 ± 0.14 (n = 12), 0.52 ± 0.11 (n = 12), and 0.53 ± 0.07 (n = 12), respectively (Fig. 3B). The inhibitory effects of clonidine on recombinant KATP channel activity were reversible because the activity was restored after washout (Fig. 3A).

Figure 3

Figure 3

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Effects of Clonidine on Kir6.2ΔC36 Channel Activity

A C terminal–truncated pore-forming subunit of Kir6.2 (Kir6.2ΔC36), which lacks the 36 C terminal amino acids, is capable of forming a functional channel in the absence of SUR.28 This has proved to be a useful tool for discriminating the site of action of various agents on KATP channels. Clonidine at 10−3 M inhibited Kir6.2ΔC36 channel currents, with relative channel activity decreasing to 0.12 ± 0.09 (n = 10) as can be seen in Figure 4A. This result indicates that the Kir subunit, rather than SUR, is primarily responsible for the effects of clonidine on wild-type KATP channels.

Figure 4

Figure 4

We next used site-directed mutagenesis of the Kir6.2ΔC36 channel to examine whether the site at which clonidine mediates KATP channel inhibition is identical to that involved in ATP block. We used a double-mutant form of Kir6.2 (Kir6.2ΔC36-K185Q). The inhibitory potency of ATP in this mutant was significantly reduced, whereas 10−3 M clonidine inhibited Kir6.2ΔC36-K185Q currents as effectively as it inhibited Kir6.2ΔC36 currents (Fig. 4B). These findings indicate that the clonidine binding site is not identical to that of ATP at the amino acid level because the mutation of K185Q markedly reduced sensitivity to ATP but had no significant effect on inhibition by clonidine.

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RT-PCR and Immunoblotting

We performed RT-PCR experiments (for checking mRNA expression) on A10 cells (Fig. 5A) and immunoblotting experiments (for checking protein expression) on A10 and COS-7 cells (Fig. 5B). Expression of the α2A-adrenergic receptor in A10 and COS-7 cells could be confirmed, but not expressions of the α2B- and α2C-adrenergic receptors.

Figure 5

Figure 5

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DISCUSSION

The major finding of the current study is that clonidine, a representative imidazoline-derived α2-adrenergic agonist inhibited vascular KATP channel activity in patch-clamp experiments. Clonidine (≥3 × 10−8 M) inhibited native vascular KATP channel activity in a concentration-dependent manner in both the cell-attached and inside-out configurations. These results indicate that clonidine inhibits vascular KATP channel activity at clinical concentrations. Clonidine also inhibited the activity of various types of recombinant SUR/Kir6.0 KATP channels, the Kir6.2ΔC36 channel, and the Kir6.2ΔC36-K185Q channel with equivalent potency. Thus, this inhibition seems to occur through its effect on the Kir6.0 subunit and not on the SUR subunit.

α2-Adrenergic agonists, of which clonidine is the prototype, were introduced as centrally acting antihypertensive agents and were later found to be useful adjuncts in anesthesia because they have desirable central effects beyond antihypertension, such as sedation, analgesia, and sympatholysis. It has been shown in the laboratory and clinically that these agents are able to enhance the effects of general anesthetic drugs and potent analgesics.14 Several adrenergic drugs, including clonidine, have been used for pain control. Specifically, intrathecal and epidural administration of clonidine is being used with increasing frequency for the management of chronic pain and is considered safe, effective, and nonaddictive.5 α2-Adrenergic agonists produce clinical effects after binding to α2-adrenergic receptors, of which there are 3 subtypes (α2A, α2B, and α2C). For example, the α2B subtype mediates the short-term hypertensive response to α2 agonists,29 whereas the α2A subtype is responsible for anesthetic and sympatholytic responses.

Clonidine is safe and has few serious adverse effects when administered at usual dosages, although an overdose can produce significant toxicity in both adults and children. This toxicity causes depression of the central nervous system as well as bradycardia and hypotension and may also lead to hypertension and vasoconstriction because of its predominant peripheral action.68 Lee et al.10 reported that imidazoline compounds inhibit KATP channel activity in guinea pig ventricular myocytes and in pancreatic β cells. Although little is known about the effects of clonidine on vascular KATP channels, we hypothesize that, in case of an overdose, the inhibitory effects of clonidine on vascular KATP channel activity may cause hypertensive crisis and coronary spasms.

Patch-clamp studies of vascular KATP channels showed that these channels are targets of a wide variety of vasodilators and constrictors, which act through multiple cellular signaling pathways, such as those of protein kinase A and protein kinase C.17 In the present study, however, clonidine inhibited native vascular KATP channel activity with similar potency in both cell-attached and inside-out configurations (Fig. 2). To exclude the effects of adrenergic receptors, we performed additional RT-PCR experiments to check mRNA expression of A10 cells and immunoblotting experiments to check protein expression of A10 and COS-7 cells. Because no α2-adrenergic receptors have been cloned from monkeys, an RT-PCR assay for these receptors could not be developed.

In this study, we could confirm the expression of α2A-adrenergic receptors in A10 and COS-7 cells, but not the expressions of α2B- and α2C-adrenergic receptors. It is therefore suggested that the inhibitory effect of clonidine on KATP channel activity may be attributable to direct binding to these channels rather than to modulation of signaling pathways or adrenergic receptors. Because α2-adrenergic receptor antagonists such as atipamezole were not used in our experiments, we cannot completely exclude the effects of adrenergic receptors. However, our conjectures seem to be supported by the results of experiments performed by Proks and Ashcroft9 or Mukai et al.11

In the present study, however, the inhibitory potency of the effect of clonidine on recombinant SUR/Kir6.0 channel activities was not influenced by the type of SUR subunit (Fig. 3), which suggests that the Kir6.0 rather than the SUR subunit is primarily responsible for the effects of clonidine on KATP channels. This is supported by the fact that clonidine inhibited the Kir6.2ΔC36 channel with the same potency as that of the recombinant SUR/Kir6.0 channels (Fig. 4A). Our results also indicate that the clonidine binding site is not identical to that of ATP at the amino acid level because the mutation of K185Q markedly reduced ATP sensitivity but showed no significant effect on clonidine inhibition (Fig. 4B). Although the precise clonidine binding site remains unclear, electrophysiologic studies have shown that other imidazoline compounds, including phentolamine and cibenzoline, block recombinant KATP channels via the Kir6.2 subunit.9,11 It is therefore possible that inhibitory action via Kir6.0 is a common feature of imidazoline compounds.

It has further been reported that the KATP channel is critical for the regulation of vascular tonus and that genetic disruption of KATP channel expression in mice causes sudden death associated with arrhythmia (atrioventricular block) caused by spontaneous cardiac ischemia, a phenotype resembling that of Prinzmetal (or variant) angina, a syndrome in humans of sudden coronary vasoconstriction without underlying atherosclerosis.14,30 Miki et al.14 reported that Kir6.1-knockout mice develop spontaneous bouts of coronary vasospasms and ST elevation, which often proves fatal because of conduction block. These mice showed the key features of vasospastic or Prinzmetal angina. Vascular smooth muscle cells from Kir6.1-knockout mice were found to have no detectable KATP currents and to lack all vasodilatory responses to KATP channel agonists. However, Chutkow et al.30 showed that SUR2-knockout mice also develop a Prinzmetal phenotype and show loss of KATP channels in vascular muscle. The findings of these 2 studies indicate that the KATP channels of vascular smooth muscle consist of one or another splice variant of SUR2 as well as Kir6.1. This particular combination of subunits thus represents a promising target for the development of novel antianginal compounds.

Clonidine concentrations exerting an inhibitory effect on vascular KATP channel activity in vitro are in the range of those used clinically and in animal models.31 The lower range (10−7–10−6 M) found to be effective in this study is similar to the clinically effective range in cerebrospinal fluid,32 whereas concentrations of 10−5 to 10−4 M are higher than those reached under clinical conditions in humans. However, caution must be exercised in the extrapolation of these in vitro results to in vivo conditions across species.

In conclusion, this electrophysiologic patch-clamp study demonstrated that clinically relevant concentrations of clonidine can inhibit vascular smooth muscle KATP channel activity via the Kir6.0 subunit.

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AUTHOR AFFILIATIONS

From the *Department of Anesthesiology, Tokushima University Hospital, Tokushima; †Department of Anesthesiology and Critical Care Medicine, Kochi Medical School, Kochi; ‡Department of Dental Anesthesiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima; §Department of Emergency and Critical Care Medicine, Tokushima University Hospital, Tokushima; Departments of ‖Preventive Environment and Nutrition, and ¶Nutrition and Metabolism, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima; #Department of Anesthesiology, Wakayama Medical University, Wakayama; and **Department of Anesthesiology, Saitama Medical University, Saitama, Japan.

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DISCLOSURES

Name: Shinji Kawahito, MD, PhD.

Conflicts of Interest: None.

Contribution: Study design, Data analysis, Manuscript preparation.

Name: Takashi Kawano, MD, PhD.

Conflicts of Interest: None.

Contribution: Study design, Data analysis.

Name: Hiroshi Kitahata, MD, PhD.

Conflicts of Interest: None.

Contribution: Conduct of study, Manuscript preparation.

Name: Jun Oto, MD, PhD.

Conflicts of Interest: None.

Contribution: Study design, Data analysis.

Name: Akira Takahashi, MD, PhD.

Conflicts of Interest: None.

Contribution: Conduct of study.

Name: Kazumi Takaishi, DDS, PhD.

Conflicts of Interest: None.

Contribution: Conduct of additional study.

Name: Nagakatsu Harada, PhD.

Conflicts of Interest: None.

Contribution: Conduct of additional study.

Name: Tadahiko Nakagawa, MS.

Conflicts of Interest: None.

Contribution: Conduct of additional study.

Name: Hiroyuki Kinoshita, MD, PhD.

Conflicts of Interest: None.

Contribution: Conduct of additional study, Data analysis.

Name: Toshiharu Azma, MD, PhD.

Conflicts of Interest: None.

Contribution: Conduct of additional study, Data analysis.

Name: Yutaka Nakaya, MD, PhD.

Conflicts of Interest: None.

Contribution: Conduct of study.

Name: Shuzo Oshita, MD, PhD.

Conflicts of Interest: None.

Contribution: Manuscript preparation.

This manuscript was handled by: Marcel E. Durieux, MD, PhD.

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ACKNOWLEDGMENTS

We thank Susumu Seino, MD, PhD (Department of Cellular and Molecular Medicine, Chiba University, Chiba, Japan), for providing the cDNAs (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) and the expression vector (pCMV6C).

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REFERENCES

1. Kamibayashi T, Maze M. Clinical uses of α2-adrenergic agonists. Anesthesiology 2000;93:1345–9
2. Ghignone M, Calvillo O, Quintin L. Anesthesia and hypertension: the effect of clonidine on perioperative hemodynamics and isoflurane requirements. Anesthesiology 1987;67:3–10
3. Englelman E, Lipszyc M, Gilbart E, Van der Linden P, Bellens B, Van Romphey A, de Rood M. Effects of clonidine on anesthetic drug requirements and hemodynamic response during aortic surgery. Anesthesiology 1989;71:178–87
4. Aho M, Lehtinen AM, Erkola O, Kallio A, Korttila K. The effect of intravenously administered dexmedetomidine on perioperative hemodynamics and isoflurane requirements in patients undergoing abdominal hysterectomy. Anesthesiology 1991;74:997–1002
5. Eisenach JC, De Kock M, Klimscha W. α2-Adrenergic agonists for regional anesthesia: a clinical review of clonidine (1984–1995). Anesthesiology 1996;85:655–74
6. Marruecos L, Roglan A, Frati ME, Artigas A. Clonidine overdose. Crit Care Med 1983;11:959–60
7. Domino LE, Domino SE, Stockstill MS. Relationship between plasma concentrations of clonidine and mean arterial pressure during an accidental clonidine overdose. Br J Clin Pharmacol 1986;21:71–4
8. Frye CB, Vance MA. Hypertensive crisis and myocardial infarction following massive clonidine overdose. Ann Pharmacother 2000;34:611–5
9. Proks P, Ashcroft FM. Phentolamine block of KATP channels is mediated by Kir6.2. Proc Natl Acad Sci USA 1997;94:11716–20
10. Lee K, Groh WJ, Blair TA, Maylie JG, Adelman JP. Imidazoline compounds inhibit KATP channels in guinea pig ventricular myocytes. Eur J Pharmacol 1995;285:309–12
11. Mukai E, Ishida H, Horie M, Noma A, Seino Y, Takano M. The antiarrhythmic agent cibenzoline inhibits KATP channels by binding to Kir6.2. Biochem Biophys Res Commun 1998;251:477–81
12. Nichols CG, Shyng SL, Nestorowicz A, Glaser B, Clement JP IV, Gonzalez G, Aguilar-Bryan L, Permutt MA, Bryan J. Adenosine diphosphate as an intracellular regulator of insulin secretion. Science 1996;272:1785–7
13. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–36
14. Miki T, Suzuki M, Shibasaki T, Uemura H, Sato T, Yamaguchi K, Koseki H, Iwanaga T, Nakaya H, Seino S. Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1. Nat Med 2002;8:466–72
15. Daut J, Maier-Rudolph W, von Beckerath N, Mehrke G, Gunther K, Goedel-Meinen L. Hypoxic dilation of coronary arteries is mediated by ATP-sensitive potassium channels. Science 1990;247:1341–4
16. Yokoshiki H, Sunagawa M, Seki T, Sperelakis N. ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. Am J Physiol 1998;274:C25–37
17. Brayden JE. Functional roles of KATP channels in vascular smooth muscle. Clin Exp Pharmacol Physiol 2002;29:312–6
18. Ito I, Hayashi Y, Kawai Y, Kamibayashi T, Matsumiya G, Takahashi T, Matsuda H, Mashimo T. Prophylactic effect of intravenous nicorandil on perioperative myocardial damage in patients undergoing off-pump coronary artery bypass surgery. J Cardiovasc Pharmacol 2004;44:501–6
19. Seino S. ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 1999;61:337–62
20. Kawano T, Oshita S, Takahashi A, Tsutsumi Y, Tomiyama Y, Kitahata H, Kuroda Y, Nakaya Y. Molecular mechanisms of the inhibitory effects of propofol and thiamylal on sarcolemmal adenosine triphosphate-sensitive potassium channels. Anesthesiology 2004;100:338–46
21. Kawano T, Oshita S, Takahashi A, Tsutsumi Y, Tomiyama Y, Kitahata H, Kuroda Y, Nakaya Y. Molecular mechanisms of the inhibitory effects of bupivacaine, levobupivacaine, and ropivacaine on sarcolemmal adenosine triphosphate-sensitive potassium channels in the cardiovascular system. Anesthesiology 2004;101:390–8
22. Kawano T, Oshita S, Takahashi A, Tsutsumi Y, Tanaka K, Tomiyama Y, Kitahata H, Nakaya Y. Molecular mechanisms underlying ketamine-mediated inhibition of sarcolemmal adenosine triphosphate-sensitive potassium channels. Anesthesiology 2005;102:93–101
23. Nakamura A, Kawahito S, Kawano T, Nazari H, Takahashi A, Kitahata H, Nakaya Y, Oshita S. Differential effects of etomidate and midazolam on vascular adenosine triphosphate-sensitive potassium channels: isometric tension and patch-clamp studies. Anesthesiology 2007;106:515–22
24. Tsutsumi Y, Oshita S, Kitahata H, Kuroda Y, Kawano T, Nakaya Y. Blockade of adenosine triphosphate-sensitive potassium channels by thiamylal in rat ventricular myocytes. Anesthesiology 2000;92:1154–9
25. Kawano T, Oshita S, Tsutsumi Y, Tomiyama Y, Kitahata H, Kuroda Y, Takahashi A, Nakaya Y. Clinically relevant concentrations of propofol have no effect on adenosine triphosphate-sensitive potassium channels in rat ventricular myocytes. Anesthesiology 2002;96:1472–7
26. Chan SL, Perrett CW, Morgan NG. Differential expression of alpha 2-adrenoceptor subtypes in purified rat pancreatic islet A- and B-cells. Cell Signal 1997;9:71–8
27. Yoshida M, Harada N, Yamamoto H, Taketani Y, Nakagawa T, Yin Y, Hattori A, Zenitani T, Hara S, Yonemoto H, Nakamura A, Nakano M, Mawatari K, Teshigawara K, Arai H, Hosaka T, Takahashi A, Yoshimoto K, Nakaya Y. Identification of cis-acting promoter sequences required for expression of the glycerol-3-phosphate acyltransferase 1 gene in mice. Biochim Biophys Acta 2009;1791:39–52
28. Tucker SJ, Gribble FM, Zhao C, Trapp S, Ashcroft FM. Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature 1997;387:179–83
29. Link RE, Desai K, Hein L, Stevens ME, Chruscinski A, Bernstein D, Barsh GS, Kobilka BK. Cardiovascular regulation in mice lacking α2-adrenergic receptor subtypes b and c. Science 1996;273:803–5
30. Chutkow WA, Pu J, Wheeler MT, Wada T, Makielski JC, Burant CF, McNally EM. Episodic coronary artery vasospasm and hypertension develop in the absence of SUR2 KATP channels. J Clin Invest 2002;110:203–8
31. Herbert MK, Roth-Goldbrunner S, Holzer P, Roewer N. Clonidine and dexmedetomidine potently inhibit peristalsis in the guinea pig ileum in vitro. Anesthesiology 2002;97:1491–9
32. Eisenach J, Detweiler D, Hood D. Hemodynamic and analgesic actions of epidurally administered clonidine. Anesthesiology 1993;78:277–87
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