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CARDIOVASCULAR

K_v Channels Contribute to Nitric Oxide- and Atrial Natriuretic Peptide-Induced Relaxation of a Rat Conduit Artery

Department of Chemical Pharmacology (Y.T., K.K.) and Department of Pharmacology (K.T., K.O., K.S.), Toho University School of Pharmaceutical Sciences, Funabashi, Japan; and Department of Anesthesiology, Division of Molecular Medicine, (G.T., M.E., M.S., K.N., E.S., L.T.), Department of Molecular & Medical Pharmacology (L.T.), Department of Physiology (E.S.), Cardiovascular Research Laboratory (E.S., L.T.), and Brain Research Institute (E.S., L.T.), David Geffen School of Medicine at UCLA, Los Angeles, California

Received September 27, 2005; accepted January 3, 2006.

	Abstract

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The role of K⁺ channels in nitric oxide (NO)-induced vasorelaxation has been largely investigated in resistance vessels where iberiotoxin-sensitive MaxiK channels play a predominant role. However, the nature of the K⁺ channel(s) involved in the relaxation triggered by NO-releasing compounds [nitroglycerin, NTG; NOR 3 [(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide]] or atrial natriuretic peptide (ANP) in the conduit vessel aorta has remained elusive. We now demonstrate that, in rat aorta, the relaxation due to these vasorelaxants is not affected by the MaxiK channel blocker iberiotoxin (10^–7–10^–6 M) as was the control vascular bed used (mesenteric artery). The inability of iberiotoxin to prevent NO/ANP-induced aortic relaxations was not due to lower expression of MaxiK in aorta or due to the predominance of iberiotoxin-resistant channels in this conduit vessel. Aortic relaxations were strongly diminished by 4-aminopyridine (4-AP) (5 x 10^–3 M) or by tetraethylammonium (>2 x 10^–3 M) at concentrations known to inhibit voltage-dependent K⁺ (K_v) 2-type channels but not by other K⁺ channel inhibitors, glibenclamide, apamin, charybdotoxin, tertiapin, or E-4031 N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl-]carbonyl]phenyl]methanesulfonamide dihydrochloride). Consistent with a role of K_v2-type channels, K_v currents in A7r5 aortic myocytes were stimulated by NTG and inhibited by 5 x 10^–3 M 4-AP. Furthermore, immunocytochemistry, immunoblot, and real-time polymerase chain reaction analyses confirmed the presence of K_v2.1 channels in aorta. K_v2.1 transcripts were 100-fold more abundant than K_v2.2. Our results support low-affinity 4-AP-sensitive K_v channels, assembled at least partially by K_v2.1 subunit, as downstream effectors of NO/ANP-signaling cascade regulating aortic vasorelaxation and further demonstrate vessel-specific K⁺ channel involvement in NO/ANP-induced relaxation.

Electrophysiological and pharmacomechanical studies in a variety of vascular tissues have pointed out to a pivotal role of plasmalemmal K⁺ channels in PKG-induced relaxation, mainly the large conductance voltage-dependent and Ca²⁺-activated K⁺ (MaxiK, BK) channel. MaxiK channels have been found to be downstream effectors of cGMP-PKG pathway in various nonconduit arteries like cerebral (Robertson et al., 1993; Price and Hellermann, 1997), coronary (Bychkov et al., 1998; Khan et al., 1998), pulmonary (Archer et al., 1994; Bialecki and Stinson-Fisher, 1995; Zhao et al., 1997), and mesenteric arteries (Khan et al., 1993; Tanaka et al., 1998). In addition, Alioua et al. (1998) have demonstrated that activation of MaxiK channels by PKG in vivo involves a direct phosphorylation of the channel protein. Consistent with this idea, Nara et al. (2000) showed that mutations of serine residues of the MaxiK pore-forming -subunit suppressed NO- or ANP-induced enhancement of the channel activity.

Other K⁺ channels found to play a role in NO-PKG relaxation pathway are the ATP-sensitive K⁺ channel (K_ATP) in pial arteries (Armstead, 1996) and voltage-dependent K⁺ channels (K_v) in pulmonary arteries (Zhao et al., 1997). Increased K⁺ channel activity by PKG resulted in membrane hyperpolarization limiting sarcolemmal Ca²⁺ influx through L-type Ca²⁺ channels, decreasing cytosolic Ca²⁺ concentration leading to vasorelaxation. Additional mechanisms found to contribute to NO-PKG relaxation are: 1) activation of the plasmalemmal Ca²⁺ extrusion pump; 2) stimulation of Ca²⁺-ATPase activity to produce enhanced loading of Ca²⁺ into intracellular Ca²⁺ stores; 3) inhibition of agonist-stimulated Ca²⁺ release from sarcoplasmic reticulum; and 4) decrease in the sensitivity of contractile elements to Ca²⁺ (for review, see Tanaka et al., 2004). The contribution of each of these mechanisms may be determined by the relative expression and/or colocalization of different proteins in each vascular bed.

Tissue-specific patterns of PKG-K⁺ channel coupling have begun to emerge. For example, MaxiK but not K_ATP channels significantly contribute to the nitrocompound-induced vascular relaxation in rabbit mesenteric, canine coronary, and rat and guinea pig pulmonary arteries (Khan et al., 1993, 1998; Bialecki and Stinson-Fisher, 1995; Zhao et al., 1997). In contrast to smaller arteries, in conduit arteries, such as rabbit and guinea pig aorta (Bialecki and Stinson-Fisher, 1995; Ishibashi et al., 1995) and carotid artery (Bialecki and Stinson-Fisher, 1995; Plane et al., 1998), the role of MaxiK channel is absent or minimal as assessed by the channel blocker iberiotoxin (IbTX) (Galvez et al., 1990) and, when present, it seems to depend on the NO donor (Bialecki and Stinson-Fisher, 1995; Ishibashi et al., 1995). Moreover, the identification of K⁺ channels involved in NO/ANP-PKG-mediated vascular relaxation in conduit arteries has not been accomplished yet. In this regard, glibenclamide (blocker of K_ATP channels) (Ashcroft and Gribble 2000), noxiustoxin (blocker of voltage-dependent K⁺ channels of the K_v1 family K_v1.2, 1.3, and 1.7) (Grissmer et al., 1994; Kalman et al., 1998), and leiurotoxin I (blocker of small conductance K_Ca channels, SK1 and SK2) (Strobaek et al., 2000) are ineffective blockers of NO donor-induced relaxation in aorta and carotid artery (Bialecki and Stinson-Fisher, 1995), discarding a possible role of K_ATP, K_v1 family, and SK1/SK2 channels in these large vessels.

In this work, we investigated the nature of the K⁺ channel involved in NO/ANP-PKG relaxant pathway in a rat conduit artery. Our results show that activation of IbTX-sensitive MaxiK channels, which significantly contribute to the mesenteric artery relaxations, is not the main mechanism by which NO and ANP produce relaxation of rat aorta, although the channel -subunit protein is expressed to similar levels in both arteries. The lack of IbTX effect in NO/ANP-induced relaxations cannot be explained by a MaxiK current resistant to IbTX but rather to the involvement of a K⁺ conductance sensitive to high concentrations of 4-aminopyridine (4-AP) (IC₅₀ 3.5 x 10^–3 M) and to relatively high concentrations of tetraethylammonium (TEA) (IC₅₀ 3 x 10^–3 M). This pharmacomechanical profile, together with electrophysiological, molecular biology, and immunochemical experiments, supports the view that, in rat aorta, a K_v2-type channel built at least partially by K_v2.1 subunit plays a significant role in NO/ANP-induced relaxations.

	Materials and Methods

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Male Wistar or Sprague-Dawley rats were used. Food and water were available ad libitum to all animals. This study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Toho University School of Pharmaceutical Sciences, accredited by the Ministry of Education, Science, Sports and Culture, Japan. Procedures at UCLA (Los Angeles, CA) received institutional approval.

Tension Measurements. A section of the thoracic aorta between the aortic arch and the diaphragm was removed from male rats (200–300 g) and placed in normal Tyrode's solution: 158.3 mM NaCl, 4.0 mM KCl, 2.0 mM CaCl₂, 1.05 mM MgCl₂, 0.42 mM NaH₂PO₄, 10.0 mM NaHCO₃, and 5.6 mM glucose. The aorta was cleaned of loosely adhering fat and connective tissue and cut into ring segments of approximately 2 mm in length. For preparation of mesenteric artery ring segments, a section of intestine between the pylorus and the colon and its arcade of supplying blood vessels were isolated. Superior mesenteric arteries with an average outer diameter of 500 to 700 µm were isolated. Arteries were cleared of connective tissue and cut into ring segments of approximately 1 mm in length. Endothelium of both thoracic aortic and mesenteric artery rings was removed by rubbing gently the intimal surface with cotton strings moistened with Tyrode's solution.

Arterial rings were mounted using stainless steel hooks under a resting tension of 1.0 (aorta) and 0.75 g (mesenteric artery). A 5-ml organ bath was used (UC-5 or UC-5TD; UFER Medical Instrument, Kyoto, Japan). The bathing solution was normal Tyrode's solution gassed with 95% O₂, 5% CO₂ mixture and maintained at 36.5 ± 0.5°C, pH 7.4. The changes in tension were isometrically recorded with a force-displacement transducer [UL-10GR (Mineber, Tokyo, Japan) or T7-8-240 (Orientec, Tokyo, Japan)] connected to a minipolygraph (Signal Conditioner: model MSC-2; Labo Support, Co., Ltd., Suita-shi, Osaka, Japan). After a 40- to 60-min equilibration period, the rings were contracted with isotonic high-KCl (80 x 10^–3 M) Tyrode's solution of the following composition: 82.3 mM NaCl, 80.0 mM KCl, 2.0 mM CaCl₂, 1.05 mM MgCl₂, 0.42 mM NaH₂PO₄, 10.0 mM NaHCO₃, and 5.6 mM glucose. After replacing the high-KCl Tyrode's solution with normal Tyrode's solution, full recovery was obtained. A lack of acetylcholine-induced (10^–5 M) relaxation confirmed the absence of functional endothelial cells. The bath solution was then exchanged with a fresh one, and the rings were left to re-equilibrate for another 30 min before starting the experiments. Arterial preparations were then contracted with phenylephrine (3 x 10^–7 M for aorta or 3 x 10^–6 M for mesenteric artery) or high-KCl (80 x 10^–3 M).

Data were collected and analyzed using a MacLab/400 and Chart (version 3.5) software (ADInstruments Japan, Tokyo, Japan). The percentage of relaxation was calculated considering the maximal tension level obtained by phenylephrine or high-KCl just before administration of vasorelaxants as 0% relaxation and the full recovery to basal tension before application to phenylephrine or high-KCl as 100% relaxation. Data were plotted as a function of drug concentrations and fitted to the equation,

(1)

where E is percentage relaxation at a given drug concentration, E_max is the maximal relaxation, A is the concentration of the drug, n_H is the slope function, and EC₅₀ is the effective drug concentration that produces a 50% response. The curve fitting was carried out using Prism, version 2.01 (GraphPad Software, Inc., San Diego, CA).

The IC₅₀ (the drug concentration required to inhibit the tension development by 50%) values and the maximal relaxant responses were also determined from the concentration-response relationship of individual results. IC₅₀ values were expressed as pIC₅₀ (negative logarithm of IC₅₀) for statistical analysis.

Evaluation of K⁺ Channel Blockers and Inhibitors of Soluble Guanylyl Cyclase. Possible involvement of K⁺ channels in the vascular relaxations was examined in the absence and presence of several K⁺ channel blockers. IbTX (10^–7 M) (MaxiK channel blocker) and TEA (1–2 x 10^–3 M, MaxiK channel blocker; 5–10 x 10^–3 M, K_v channel blocker) were added to the bath solution 60 and 20 min before administration of the vasorelaxants. When examining the effect of higher concentrations (10^–6 M) of IbTX, preparations were treated for 90 min before applying vasorelaxants. Pretreatment periods for other K⁺ channel blockers before application of vasorelaxants are as follows: glibenclamide (10^–6 M), a blocker for K_ATP channel (Ashcroft and Gribble, 2000), 60 min; apamin (10^–7 M), a blocker for small Ca²⁺-activated K⁺ channels (SK2 and SK3) (Köhler et al., 1996), 20 min; charybdotoxin (10^–7 M), a blocker for MaxiK and voltage-dependent K⁺ channels K_v1.2 and K_v1.3 (Grissmer et al., 1994; Wallner et al., 1995), 20 min; and 4-AP (10^–3–10^–2 M), a blocker for K_v channels (Mathie et al., 1998), 20 min. In some experiments, the effect of 4-AP was examined by its cumulative applications after the vascular relaxation reached the maximal level. 1H-[1,2,4]-Oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) (10^–5 M) and methylene blue (10^–5 M) were added to the bath solution 90 min before cumulative addition of the vasorelaxants.

Membrane Preparations. All procedures were performed at 4°C, and all solutions contained protease inhibitors. Aorta and superior mesenteric artery were dissected from male rats and cleaned of loosely adhering fat and connective tissue. A section of the arteries was reserved for immunocytochemistry. Three to five arteries were opened longitudinally, and the internal surface was gently rubbed with a moistened filter paper to remove the endothelium. This procedure was performed as quickly as possible in 100% O₂-gassed ice-cold Krebs' solution: 126.9 mM NaCl, 5.9 mM KCl, 2.36 mM CaCl₂, 1.18 mM MgCl₂, 10.8 mM HEPES, and 11.8 mM glucose, pH 7.4, supplemented with protease inhibitors (100 µM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 8 µg/ml calpain inhibitor I, 8 µg/ml calpain inhibitor II, 0.2 x 10^–3 M Pefabloc SC, and 0.1 mg/ml benzamidine). Tissues were then quickly frozen in liquid nitrogen and stored at –80°C for later use. Frozen arteries were homogenized in 20 mM HEPES-KOH, 1 mM EDTA, and 250 mM sucrose, pH 7.4. The homogenate was centrifuged at 1000g for 15 min, and the supernatant was subsequently centrifuged at 100,000g for 30 min. Crude membranes were suspended in 10 mM HEPES-sucrose solution: 10 mM HEPES-KOH and 250 mM sucrose, pH 7.4. The protein content was determined using the Bradford method (Pierce Chemical, Rockford, IL). Membranes were then supplemented with Tris-SDS solution (62.5 x 10^–3 M Tris-HCl, 2% SDS, 10% glycerol, 0.01% bromphenol blue, and 42 mM DTT) and stored at –70°C until use.

Immunoblots. Arterial membrane proteins (2 µg) were separated by 7.5% SDS-polyacrylamide gels under reducing conditions and electrotransferred to nitrocellulose paper. Blots were blocked with Tris-buffered saline (TBS: 50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, and 0.1% Tween 20, pH 7.4) containing 5% nonfat dry milk for 1 h at room temperature. Blots were incubated with 1.2 µg/ml affinity-purified rabbit polyclonal anti-MaxiK channel _(883–896)-subunit antibody against residues 883 to 896 (VNDTNVQ-FLDQDDD) of the human MaxiK channel -subunit (hSlo) (GenBank accession number U11058 [GenBank] ), with 3.3 µg/ml affinity-purified rabbit polyclonal anti-K_v2.1 (Alomone Labs Ltd., Jerusalem, Israel) or with 0.33 µg/ml monoclonal antibody against smooth muscle -actin (Sigma, St. Louis, MO) in 1% nonfat milk/TBS for 12 h at 4°C, washed with TBS three times for 10 min each, and then incubated with horseradish peroxidase-conjugated anti-rabbit (0.125 µg/ml for MaxiK) or anti-mouse secondary antibody (0.125 µg/ml for smooth muscle -actin) (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) for 1 h. After washing, blots were treated for 1 min with Chemiluminescence Reagent Plus (PerkinElmer Life and Analytical Sciences, Inc., Wellesley, MA) and autoradiographed on Kodak BioMax film (Eastman Kodak, Rochester, NY). The specificity of the antibodies was tested by preadsorbing them with the antigenic peptides (80 µg of peptide/µg of anti-MaxiK channel _(883–896)-subunit antibody and 0.6 µg of antigen/µg of anti-K_v2.1 antibody), which blocked the corresponding signals. Bands corresponding to the immunoreactive proteins were quantified using GS670 Imaging Densitometer (Bio-Rad, Hercules, CA). Results are expressed as arbitrary densitometric units.

Immunocytochemistry. Fresh artery segments were fixed by immersion for 2 h in 0.1 M phosphate-buffered saline, pH 7.4, supplemented with 4% paraformaldehyde and 2% picric acid. Transverse cryostat sections (10 µm) were incubated for 12 h at 4°C with 4.8 µg/ml affinity-purified anti-MaxiK _(883–896) antibody or with 3.3 µg/ml anti-K_v2.1 antibody in 1% normal goat serum and 0.2% Triton X-100 in phosphate-buffered saline (Sigma). After washing, the tissue sections were incubated for 1 h at room temperature with Cy5 or fluorescein isothiocyanate-donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). In the control sections, antibody immunoreactivity was blocked by preadsorbing the antibody with an excess amount of the corresponding antigenic peptides. Concentrations were the same as for immunoblots. Immunofluorescence was measured in five to seven randomly selected visual fields from each tissue section using Image-Pro plus software (Media Cybernetics, Inc., Silver Spring, MD). Results are expressed as average pixel intensity.

Real-Time PCR. Approximately 20 ng of polyA⁺ RNA from rat aorta (endothelium-removed) was converted to single-stranded cDNA by priming with an oligo(dT) primer. To quantify the relative abundance of each gene, we used real-time PCR (iCycler iQ Real-time PCR; Bio-Rad) with SYBR Green I (stock solution x 10,000 diluted at 1:80,000) as the fluorescent probe (Molecular Probes, Eugene, OR) and Platinum Quantitative PCR SuperMix-UDG (Invitrogen, Carlsbad, CA). Gene-specific primers for K_v2.1 (GenBank accession number NM_013186 [GenBank] ) were as follows: upstream primer corresponding to nucleotides 1828 to 1848 (5'-AGTCCTCTGGCATCCCTCTCC-3') and downstream primer corresponding to nucleotides 2093 to 2073 (5'-CTAAGAGGATCGTGATACAAG-3'). For K_v2.2 (GenBank accession number NM_054000 [GenBank] ), the upstream primer corresponding to nucleotides 2313 to 2333 was 5'-AGGGAGGCTGCACTGGATTAT-3', and the downstream primer corresponding to nucleotides 2564 to 2585 was 5'-TAGAAGAATGGTACTCATGTG-3'. As control, we used the reaction mixture without the cDNA. Reaction conditions were as follows: 5 min at 95°C to activate the "hot start" Platinum TaqDNA polymerase followed by 45 cycles at 95°C for 45 s, 58°C for 45 s, and 72°C for 45 s. As required for accurate measurements, melting curves showed the presence of a single peak. Standard curves for K_v2.1 and K_v2.2 were constructed using known amounts of the respective amplicon subcloned into pcDNA3.

The quality of the cDNA was confirmed in separate RT-PCR reactions by the lack of detectable genomic DNA using primers flanking an intronic region of -actin (GenBank accession number V01217 [GenBank] ). The upstream primer matches nucleotides 2383 to 2402 (5'-GGCTACAGCTTCACCACCAC-3'), and the downstream primer corresponds to nucleotides 3071 to 3091 (5'-TACTCCTGCTTGCTGATCCAC-3'). -Actin cDNA should give a product of 494 nt, and -actin genomic DNA would give a product of 708 nt.

Cell Isolation and A7r5 Cell Culture. Freshly dissociated aortic myocytes were used to record MaxiK currents. Nominally Ca²⁺-free Krebs' solution (119 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄, 22 mM NaHCO₃, 8 mM HEPES, and 5.5 mM glucose) was used for cell isolation. After removing connective tissues, the dissected vessel was cut into small pieces. Tissue pieces were transferred to nominally Ca²⁺-free Krebs' solution containing papain (1.5 mg/ml), dithiothreitol (10^–3 M), and bovine serum albumin (2 mg/ml) and incubated for 1 h at 4°C followed by 30 min at 37°C. Tissues then were immediately transferred to a solution containing collagenase F (1 mg/ml), collagenase H (0.3 mg/ml), and bovine serum albumin (2 mg/ml) and incubated for 10 min at 37°C. The digested tissues were subsequently washed with ice-cold Ca²⁺-free solution and suspended with a wide-bore Pasteur pipette. The cell suspension was kept at 4°C until use.

The rat aortic smooth muscle cell line (passage 5) A7r5 (American Type Culture Collection, Manassas, VA) was used to record K_v currents. Cells were maintained at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were split usually once a week and cultured on glass coverslips for 1 to 2 days prior to electrophysiological recordings.

Electrophysiology. Whole-cell currents were measured at room temperature. Pipette resistances were 2 to 3 M. Data were filtered at one-fifth the sampling frequency, which was 5 kHz. To record MaxiK currents, the bathing solution contained 135 mM sodium methanesulfonate, 5 mM potassium methanesulfonate, 2 mM MgCl₂, 0.1 mM CaCl₂, 10 mM HEPES, and 5.5 mM glucose, pH 7.4. The pipette solution contained 140 mM potassium methanesulfonate, 2 mM MgCl₂, 0.1 mM CaCl₂, 0.146 mM EGTA, 10 mM HEPES, and 10 mM glucose, pH 7.4 (calculated free Ca²⁺ concentration was 100 nM).

To record K_v currents, the bath solution contained 136 mM NaCl, 5.4 mM KCl, 2 mM MgCl₂, 10 mM glucose, 10 mM HEPES, 0.02 mM glibenclamide, and 1 mM TEA, pH 7.4. The pipette solution was 135 mM KCl, 10 mM EGTA, 10 mM HEPES, 5 mM glucose, 1 mM MgATP, and 1 mM Na₂GTP, pH 7.2. To minimize MaxiK currents, the blocker 10^–3 M TEA ± 10^–7 M IbTX was included in the bath solution, Ca²⁺ was excluded in both bath and pipette solutions, and a high-Ca²⁺ buffer capacity, 10 mM EGTA, was used in the pipette solution. The calculated free Ca²⁺ of the pipette solution is <10^–7 M (assuming a contaminant Ca²⁺ of 5 x 10^–6 M); under these conditions, MaxiK + complexes are only activated at very high potentials of >150 mV. Thus, K_v currents were elicited up to +70 mV, and mean values were obtained at +50 mV. Bath solutions containing 5 and 10 x 10^–3 M 4-AP were readjusted to pH 7.4 before the experiment. These concentrations of 4-AP do not inhibit MaxiK channels (Wallner et al., 1995). Glibenclamide was used to abolish K_ATP currents. Data acquisition and analysis were performed using pCLAMP (Axon Instruments, Inc., Union City, CA) or a home-made acquisition system and software.

Drugs. The followings drugs were used: l-phenylephrine hydrochloride (Wako Pure Chemical Industries, Osaka, Japan); NTG (Nihon Kayaku, Tokyo, Japan); NOR 3 [(±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (FK409; Dojindo, Kumamoto, Japan); IbTX, charybdotoxin, apamin, and ANP (Peptide Institute, Minohshi, Osaka, Japan); ODQ (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA); methylene blue (Tokyo Kasei, Tokyo, Japan); glibenclamide (Sigma); acetylcholine (Daiichi, Tokyo, Japan); and phentolamine mesylate (Ciba-Geigy, Takarazuka-shi, Hyogo, Japan). Cromakalim was kindly donated from Nissan Chemical Industries (Tokyo, Japan). All other chemicals used in the present study were commercially available and of reagent grade.

Cromakalim was dissolved in 70% ethanol at 10^–2 M and diluted further with distilled water to the desired concentrations. Glibenclamide and ODQ were dissolved in dimethyl sulfoxide at 10^–3 and 10^–2 M. All other drugs were prepared as aqueous solutions and diluted with distilled water. Final ethanol and dimethyl sulfoxide concentrations in the bath medium did not exceed more than 1 and 0.1%, respectively, which did not affect the vascular responses. All drugs are expressed in molar concentrations in the bathing solution.

Statistical Analysis. The data are presented as mean value ± S.E.M., and n refers to the number of experiments. The significance of the difference between mean values was evaluated by paired or unpaired Student's t test, unpaired Student's t test with Welch's correction if necessary, and one-way analysis of variance followed by Tukey's multiple comparison test. A P value less than 0.05 was considered statistically significant.

	Results

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IbTX, a MaxiK Channel Blocker, Does Not Antagonize the Relaxation Induced by NO-Releasing Compounds in Rat Aorta but Does Inhibit It in Mesenteric Artery. We first determined whether NTG-induced relaxation could be prevented by IbTX, a MaxiK channel blocker, in rat aorta versus mesenteric artery. Figure 1 shows typical mechanical recordings in de-endothelialized aorta (A and B) and mesenteric artery (C and D) treated with NTG in the absence (–) or presence (+) of IbTX (10^–7 M). Experiments were performed under the continuous presence of phenylephrine (3 x 10^–7 M for aorta, 3 x 10^–6 M for mesenteric artery) (line). Prior to treatment with any other drug, phenylephrine induced a sustained contraction in both arteries. Application of NTG (10^–10–10^–5 M) produced a concentration-dependent relaxation in both types of blood vessels (Fig. 1, A, aorta, and C, mesenteric artery). However, IbTX appeared to only prevent NTG-induced relaxation in mesenteric artery.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Typical traces showing the inhibitory effects of IbTX on the endothelium-independent vascular relaxations in response to NTG in rat aorta (A and B) and mesenteric artery (C and D). Ring preparations without endothelium from rat aorta and mesenteric artery were precontracted with phenylephrine (aorta, 3 x 10–7 M; mesenteric artery, 3 x 10–6 M). NTG (open dots below numbers) was cumulatively applied to the bath solution. IbTX (10–7 M) was applied to the bath solution 60 min before cumulative application of NTG (shown as filled squares) (B and D). IbTX (10–7 M), when applied during the sustained relaxation by NTG, did not counteract the relaxation in aorta (A) but appreciably reversed the relaxation in mesenteric artery (C). Numbers show the negative logarithm of NTG concentrations in the bath solution. Phentolamine: 3 x 10–7 M, aorta; 3 x 10–6 M, mesenteric artery.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Concentration-response relationships for the vascular relaxations in response to nitroglycerin (A and B), NOR 3 (C and D), and ANP (E and F) in aorta and mesenteric artery in the absence and presence of IbTX. Rat aorta and mesenteric artery were precontracted with phenylephrine (aorta, 3 x 10–7 M; mesenteric artery, 3 x 10–6 M). Muscle preparations were treated with IbTX (10–7 M) for 60 min before cumulative application of vasorelaxants. Vascular relaxations are expressed as percentage inhibition of phenylephrine-induced contractions just before application of relaxants and are plotted as a function of compound concentration (negative logarithm of compound concentrations). Data are shown as mean values ± S.E.M. of three to nine experiments. *, P < 0.05; **, P < 0.01, significant differences from control (–IbTX) values.

The distinct inhibitory action of IbTX in aorta versus mesenteric artery was also evident if the toxin was applied after vasorelaxation with NTG (Fig. 1, A and C); IbTX (10^–7 M) counteracted the relaxant effect of NTG in mesenteric artery but did not in aorta. Maximal relaxation values attained with NTG were similar in the absence (102.3%, n = 2) or presence (98.3%, n = 2) of the toxin in aorta. In contrast, in mesenteric artery, 10^–7 M IbTX applied after the complete relaxation with NTG (Fig. 1C, filled square) was able to produce contraction, inhibiting the NTG-induced relaxation by 44.3 ± 4.6% (n = 3; P < 0.01). As a control, this concentration of IbTX (10^–7 M) did not affect the vascular relaxation induced by phentolamine, an -adrenoceptor antagonist (Fig. 1, C and D). Relaxations by 3 x 10^–6 M phentolamine achieved similar values with (103.3 ± 1.4%; n = 5) or without IbTX (108.1 ± 3.9%; n = 14) (P > 0.05).

Vascular relaxation induced by NTG is mediated via NO, which is an intracellular byproduct of NTG (Waldman and Murad, 1987). Therefore, we decided to explore whether the differential inhibitory action of IbTX on NTG-induced relaxation in rat aorta versus mesenteric artery could be mimicked by an NO donor, NOR 3 (also named FK409). Similar to NTG, NOR 3 (10^–9–10^–6 M) produced a concentration-dependent relaxation in both endothelium-denuded aorta (Fig. 2C, open circles) and mesenteric artery (Fig. 2D, open circles). As in the case of NTG-induced relaxation, IbTX (10^–7 M) did not significantly affect the NOR 3-induced relaxation in aorta but it significantly inhibited the relaxant response to NOR 3 (<10^–6 M) in mesenteric artery (Fig. 2, C versus D, filled circles). The effects of IbTX on pIC₅₀ and E_max for NOR 3 in both arteries are shown in Table 1. These results demonstrate that rat aorta and mesenteric arteries use different mechanisms to relax in response to NO donors.

ANP-Induced Relaxation Is Resistant to IbTX in Aorta but Not in Mesenteric Artery. We have previously shown that, in rat mesenteric artery, IbTX substantially inhibits the relaxation induced by ANP (Tanaka et al., 1998); however, it is not known whether this also applies to aorta. In addition, NO-releasing agents and ANP use different guanylyl cyclases, soluble and membrane-bound, respectively; these cyclases may differentially deliver cGMP to its target(s) in aorta versus mesenteric artery. Thus, we tested whether ANP-induced relaxation could be inhibited by IbTX in aorta. In control experiments, ANP (10^–10–10^–7 M) produced a concentration-dependent relaxation in both aorta (Fig. 2E, open circles) and mesenteric artery (Fig. 2F, open circles). However, in aorta, preincubation with IbTX (10^–7 M) for 60 min did not affect the relaxation to ANP (Fig. 2E; Table 1), as was the case for NTG and NOR 3 (see Fig. 2, A and C) in this vessel. As control and confirming our previous work, Fig. 2F shows that, in mesenteric arteries, IbTX (10^–7 M) largely inhibited ANP-induced relaxation (Table 1). Thus, our results show that, similar to NO donors, ANP-induced relaxation is not inhibited by IbTX in aorta, whereas it is in mesenteric artery. They also indicate that activation of different guanylyl cyclases (soluble versus membrane-bound) cannot explain the differential response to IbTX of me-senteric artery versus aorta after NO/ANP-PKG pathway stimulation.

The Distinct Downstream Mechanism of NO Donors in Aorta versus Mesenteric Artery Cannot Be Attributed to Differences in Soluble Guanylyl Cyclase Activity. To test the possibility that the observed divergence in the role of MaxiK channels in NO donors-induced relaxations of aorta versus mesenteric artery is due to differences in the activity of soluble guanylyl cyclase between these tissues, we examined the effects of its inhibitor, ODQ. As shown in Table 2, aortic relaxations induced by both NTG and NOR 3 were almost abolished by the treatment with 10^–5 M ODQ. Likewise, both relaxant effects on mesenteric artery were also eliminated by the ODQ treatment (Table 2). NTG-induced relaxations in both arteries were also significantly inhibited by methylene blue (10^–5 M), another inhibitor of soluble guanylyl cyclase (Table 2). As expected, ANP-induced relaxations in both arteries were not affected by methylene blue (10^–5 M) (Table 2), which was unable to prevent the activity of membrane-bound guanylyl cyclase. These results show that the lack of MaxiK channel role in the relaxation of aortic vessel cannot be explained by a lower activity of soluble guanylyl cyclase in this type of smooth muscle.

Neither High Concentrations of IbTX Nor TEA Pharmacology Support a Role for MaxiK in the Aortic Smooth Muscle Relaxations by NO-Releasing Compounds and ANP. The access of IbTX to its binding site in the MaxiK channel pore-forming -subunit is greatly slowed down when channels are coassembled with the regulatory ₄-subunit, making the channels practically "insensitive" to nanomolar concentrations of IbTX within experimental times. To overcome the resistance to IbTX block of channels formed by + ₄-subunits, IbTX concentrations need to be increased to the micromolar range; under these conditions, almost complete blockade is observed within 10 min (Meera et al., 2000). Thus, to rule out the possibility that the lack of IbTX effect on the aortic vessel relaxations induced by NO and ANP is due to the expression of MaxiK channels assembled by - and ₄-like subunits, we examined the effects of 1 x 10^–6 M IbTX. None of the aortic relaxations was affected by this high concentration of IbTX (Table 3). These observations are against the possibility that the resistance to IbTX blockade of rat aortic NO/ANP relaxations is due to the expression of an IbTX-inaccessible + ₄ type of MaxiK channel.

To further test the contribution of MaxiK channel to aortic relaxations induced by NO-releasing compounds and ANP, we tested the effects of TEA. It is commonly accepted that blockade with 1 x 10^–3 M TEA is sufficient to almost fully inhibit a MaxiK-related event since TEA blocks MaxiK channel from the external side of the channel with a IC₅₀ value of 2 to 3 x 10^–4 M (Wallner et al., 1995), whereas K_v and K_ATP currents have a higher IC₅₀ (10 mM) in smooth muscle (Nelson and Quayle, 1995). Although, 1 x 10^–3 M TEA had a minor effect on aortic relaxations induced by NTG, NOR 3, and ANP (Table 3; see Fig. 6), this modest inhibitory action was clearly less effective than the one produced by 10^–7 M IbTX in the mesenteric artery (Fig. 2, B, D, and F) and might reflect the partial blockade of another class of K⁺ channel. Taken together, the mechanical results obtained with IbTX and TEA support the view that in aorta the main effectors of these cGMP-elevating (and relaxant) agents are non-MaxiK-type of K⁺ channels. The results could also reflect a much less expression of MaxiK in rat aorta compared with mesenteric artery or that MaxiK channels in aorta need much higher (>1 x 10^–6 M) concentrations of IbTX to be blocked. We can discard the last possibility, since patch-clamp experiments using freshly isolated aortic myocytes demonstrated that MaxiK currents were significantly blocked by 2 x 10^–7 M IbTX (n = 10 cells) (data not shown). Therefore, we investigated whether MaxiK protein expression levels in aorta versus mesenteric artery differed. In addition, we examined whether high-K⁺ or other K⁺ channel blockers could inhibit the relaxation by NO-releasing compounds.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Inhibitory effects of higher concentrations of 4-AP and TEA on the relaxant responses of rat aorta to NO donors (nitroglycerin and NOR 3) and ANP. A, B, and C, when the relaxant response to a single administration of nitroglycerin (10–5 M) (A), NOR 3 (10–6 M) (B), and ANP (10–7 M) (C) reached a steady-state level, 4-AP (10–3–10–2 M) was applied cumulatively to the bath solution. Data are mean values ± S.E.M. of six to eight experiments. **, P < 0.01, significant differences from the response before applying 4-AP. D, E, and F, concentration-response relationships for the relaxation of rat aorta in response to nitroglycerin (D), NOR 3 (E), and ANP (F) in the absence and presence of TEA (10–3–10–2 M). Rat aorta was precontracted with phenylephrine (3 x 10–7 M), and relaxants (nitroglycerin, NOR 3, and ANP) were applied cumulatively to the bath solution. Vascular relaxations are expressed as percentage inhibition of the tension development just before application of vasorelaxants. Data are mean values ± S.E.M. of four to 16 experiments. *, P < 0.05; **, P < 0.01, significant differences from control responses.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Western blot analysis and immunocytochemistry of MaxiK channel -subunit expression in rat aorta and mesenteric artery. A, Western blots of MaxiK channel -subunit in rat aorta (RA) and rat mesenteric artery (RMA). Equal amounts of RA and RMA protein (2 µg) and 5 µg of human coronary artery (HCA) were separated by 6% SDS-polyacrylamide gels under reducing conditions and electrophoretically transferred to nitrocellulose paper. Western blots of smooth muscle -actin in both arteries are also shown. B, mean densitometric values for MaxiK channel -subunit and smooth muscle -actin in aorta and mesenteric artery (left) and its ratio between these arteries (right). Results are expressed in arbitrary densitometric units (optical density) and show insignificant differences between aorta and mesenteric artery. Data are mean values ± S.E.M. of five experiments. C, MaxiK channel -subunit immunolabeling in aorta and mesenteric artery smooth muscle cells. Rat aorta and mesenteric artery were labeled using a polyclonal anti-MaxiK channel -subunit antibody. No staining of smooth muscle cells was detected when the antibody was preadsorbed with the corresponding antigen (control). Bar indicates 100 µm for all.

MaxiK channel expression levels in aorta versus mesenteric artery were also examined in tissue sections with confocal microscopy (Fig. 3C). The specificity of the antibody was assessed by preadsorbing anti-_(883–896) antibody with excess antigenic peptide, a maneuver that practically abolished the fluorescent signal, as shown in Fig. 3C (control). In agreement with Western blot analysis, MaxiK channel signals were similar in both aorta and mesenteric artery smooth muscle cells with pixel intensity (PI) values of 120.3 ± 23.8 PI (n = 4) for aorta and 137.9 ± 30.5 PI (n = 4) for mesenteric artery (P > 0.05). These biochemical studies clearly indicate that MaxiK channel poreforming -subunit is expressed at a similar level in both aortic and mesenteric artery smooth muscle cells. Thus, the lack of IbTX sensitivity of the aortic relaxations by NO-releasing compounds and ANP cannot be explained by a diminished expression of this channel -subunit.

K⁺ Channel Activation as a Predominant Mechanism Responsible for Aortic Relaxation by NTG, NOR 3, and ANP. The contribution of K⁺ channels to drug-induced relaxations can be assessed by minimizing K⁺ efflux with high extracellular K⁺, which produces cell membrane depolarization and vessel contraction. Figure 4, A, B, and C, demonstrates that relaxations of rat aorta induced by NO donors (NTG and NOR 3) and ANP are strongly diminished when the muscles are contracted with high-KCl (80 x 10^–3 M) (Table 3). Furthermore, the attenuation of the relaxant effects in high-KCl-contracted muscle was much more pronounced in aorta compared with mesenteric artery (Fig. 4, D and E; Table 3). These findings indicate that indeed K⁺ channel activation plays a predominant role in the relaxant effects of NO donors and ANP on rat aortic smooth muscle, although the contribution of MaxiK channel is apparently lacking. The nature of the K⁺ channel was established with the aid of a pharmacological screening supported with electrophysiological, molecular, and biochemical approaches.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Concentration-response relationships for the relaxations of rat aorta and mesenteric artery in response to nitroglycerin, NOR 3, or ANP in preparations contracted with phenylephrine or high-KCl. Tissue was precontracted with phenylephrine (aorta, 3 x 10–7 M; mesenteric artery, 3 x 10–6 M) or high-KCl (80 x 10–3 M). Nitroglycerin (A and D), NOR 3 (B), and ANP (C and E) were applied cumulatively to the bath solution. Vascular relaxations are expressed as percentage inhibition of the tension development just before application of vasorelaxants and are plotted as a function of vasorelaxants concentration. Data are mean values ± S.E.M. of three to nine experiments. *, P < 0.05; **, P < 0.01, significant differences between two groups. Note that the extent of NTG- and ANP-induced relaxations in high-KCl-precontracted mesenteric artery (D and E) were close to those in the vessels treated with 10–7 M iberiotoxin (cf. Fig. 2, B and F; 19.5 versus 26.8% for NTG relaxation; 40.6 versus 41.8% for ANP relaxation).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Concentration-response relationships for the relaxations of rat aorta in response to nitroglycerin in the absence and presence of various types of K+ channel blockers. A, glibenclamide. B, apamin. C, charybdotoxin. D, 4-AP. E, E-4031. F, tertiapin. Rat aorta was precontracted with phenylephrine (3 x 10–7 M), and nitroglycerin was applied cumulatively to the bath solution. Vascular relaxations are expressed as percentage inhibition of the tension development just before application of nitroglycerin. Data are mean values ± S.E.M. of three to four experiments.

Higher Concentrations of 4-AP and TEA Counteract the Aortic Relaxations to NTG, NOR 3, and ANP. To test the possibility that K_v2.1, K_v2.2, K_v4, rEag2, or rElk K⁺ channels mediate the NO- and ANP-induced aortic relaxation, we tested the effect of higher concentrations (10^–3–10^–2 M) of 4-AP and TEA. We found that the aortic relaxations induced by NTG, NOR 3, and ANP were not significantly inhibited by 10^–3 M 4-AP (n = 3–6 for all relaxations). In contrast, 5 x 10^–3 and 10^–2 M significantly reduced the relaxations induced by all of the test compounds (Fig. 6, A–C). IC₅₀ values of 4-AP for aortic relaxations were around 3.5 x 10^–3 M (2.9 x 10^–3 M for NTG, 3.6 x 10^–3 M for NOR 3, and 3.9 x 10^–3 M for ANP). Likewise, intermediate concentrations of TEA (5 x 10^–3 and 10^–2 M) reduced to a large extent the relaxations induced by all of the test compounds (Fig. 6, D–F). IC₅₀ values were around 3 x 10^–3 M (4.1 x 10^–3 M for NTG, 10^–7 M; 2.2 x 10^–3 M for NOR 3, 3 x 10^–8 M; and 1.9 x 10^–3 M for ANP, 10^–8 M).

Because K_v4.3 and rElk1 (Kcnh4) channels are insensitive to external TEA (1 x 10^–1 M) (Engeland et al., 1998; Song et al., 2001) and NO relaxation in aorta is sensitive to intermediate concentrations of TEA (IC₅₀, 3 x 10^–3 M) (Fig. 6, D–F), it is unlikely that these classes of K⁺ channels are effectors of the NO cascade. Likewise, because rEag2 (Kcnh5) channels are insensitive to 10^–2 M 4-AP (Ludwig et al., 2000) and NO relaxation in aorta is prevented by 10^–2 M 4-AP (Fig. 6, A–C), this channel family can also be ruled out.

The reported IC₅₀ values for 4-AP (0.5–20 x 10^–3 M) (Kirsch and Drewe, 1993; Mathie et al., 1998) and TEA (5 x 10^–3 M) (Taglialatela et al., 1991) against K_v2.1 are in reasonable agreement with the ones found to inhibit NO-induced aortic relaxation in the present study (IC_50-4AP = 3.5 x 10^–3 M and IC_50-TEA = 3 x 10^–3 M). However, K_v2.2 is also sensitive to both 4-AP and TEA within similar ranges (IC₅₀-4AP = 1.5 x 10^–3 M and IC_50-TEA = 3–8 x 10^–3 M) (Yamane et al., 1995; Mathie et al., 1998). Thus, the pharmacomechanical profile, in particular the low-4-AP affinity, indicates that channels composed of K_v2.1 or K_v2.2 or both may be responsible for NO/ANP-induced aortic vessel relaxations. This prompted us to examine whether NO donors could stimulate K_v currents with low-4-AP affinity in aortic myocytes.

High Concentrations of 4-AP Inhibit K_v Currents following Stimulation by NTG in Aortic Myocytes. Whole-cell K_v currents from A7r5 aortic myocytes were recorded in the presence of MaxiK blockers (10^–3 M TEA ± 10^–7 M IbTX) in the bath solution and 10 mM EGTA in the patch pipette. Figure 7A shows typical K_v currents elicited from a holding potential of –70 mV with 200-ms test pulses from –80 to +70 mV in 10-mV increments. Resembling the mechanical results, currents were clearly stimulated by 4 x 10^–7 M NTG and were insensitive to 10^–3 M 4-AP but inhibited by 5 x 10^–3 and 10^–2 M 4-AP. Figure 7B shows the corresponding I-V curves, whereas Fig. 7C displays the mean values. At +50 mV, NTG stimulated K_v currents from 18.3 ± 3.7 to 29.5 ± 6.1 pA/pF (n = 8 of 10 cells, P < 0.01). Similar results were obtained with (n = 4) or without (n = 4) IbTX. 4-AP at 10^–3 M had no appreciable effect on NTG-stimulated K_v currents (n = 5 cells, four in the continuous presence of NTG); in contrast, 5 x 10^–3 M 4-AP inhibited K_v currents by 40% from 29.3 ± 9.8 to 18.7 ± 7.3 pA/pF (n = 4 cells in the continuous presence of NTG, P < 0.01). Increasing concentrations of 4-AP (10^–2 M) further decreased K_v currents (n = 2). Together the pharmacomechanical and electrophysiological studies support the view that one mechanism involved in NTG-induced relaxation in rat aorta is via activation of low-affinity 4-AP-sensitive K_v channels of the K_v2 class.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Kv currents are stimulated by NTG and inhibited by high concentrations of 4-AP in A7r5 rat aortic myocytes. A, original current traces showing the stimulation of Kv currents by NTG, lack of effect of 1 x 10–3 M 4-AP, and corresponding inhibition by 4-AP at 5 x 10–3 M. Test potentials were from –80 mV to +70 mV with a holding potential of –70 mV. B, corresponding Idensity-V relationship of traces in A. Idensity (pA/pF) was obtained by dividing each current value by the cell capacitance. C, mean values were obtained at +50 mV in each condition. Data are mean values ± S.E.M. of two to eight experiments. **, P < 0.01, significant differences between two groups.

View larger version (39K): [in this window] [in a new window]   Fig. 8. Kv2.1 channel protein and its mRNA is abundant in rat aortic smooth muscle. A, Western blot analysis of rat brain (Br) and rat aorta (RA) membranes (10 µg of protein/lane) showing similar expression. The antibody labeled a band of 100 kDa. Preincubation with the antigenic peptide completely blocked the Kv2.1 channel signal (n = 4 and 7 rats). B, confocal image of Kv2.1 expressed in rat aortic smooth muscle cells. Preincubation of the antibody with the antigenic peptide practically blocked the signal (control) (n = 6). Bar indicates 100 µm for both. C, real-time PCR of Kv2.1 and Kv2.2 in rat aortic smooth muscle. Mean fluorescence (F) per nanogram of mRNA versus cycle number of PCR. Data are mean values ± S.E.M. of five experiments with two different mRNA preparations obtained from two rats each. D, bar graph comparing the abundance of Kv2.1 versus Kv2.2 transcripts in rat aorta. *, P < 0.05, significant differences between two groups. Inset, standard regression lines to measure threshold cycle (Tcycle) as a function of amplicon concentrations (log [ampliconKv2.1] or log [ampliconKv2.2]). E, qualitative RT-PCR showing little expression of Kv2.2 versus Kv2.1 transcripts (products of 270 nt) at the end of 30 cycles of PCR (94°C for 1 min, 54°C for 1 min, and 72°C for 1 min). To check the possible contamination of genomic DNA in mRNA samples, RT-PCR was also applied to -actin, and a single band corresponding to the expected size of product (500 nt) eliminates the possibility of contamination of genomic DNA (700 nt). The right two lanes (H2O) represent controls for Kv2.1 and Kv2.2. mRNA employed for the reverse transcription was 208 ng for Kv2.1 and Kv2.2 and 55 ng for -actin. Qualitatively, the same results were obtained from n = 4 different mRNA preparations.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we identify for the first time the nature of the K⁺ channel involved in the endothelium-independent relaxations induced by NO-releasing compounds and ANP of a conduit artery. Our results show that, in aorta but not in mesenteric artery (small vessel control), the contribution of IbTX-sensitive MaxiK channels to the relaxations induced by NO-releasing compounds and ANP is apparently negligible or absent. Instead, pharmacomechanical, electrical, molecular, and biochemical evidence supports a major role of low-affinity 4-AP-sensitive K_v2 channel type typified as containing K_v2.1 subunit in the aortic smooth muscle relaxations induced by NO donors and ANP.

The lack or minimal contribution of IbTX-sensitive MaxiK channel to NO/ANP-induced relaxation of rat aorta is in agreement with previous findings in conduit vessels of other species (rabbit and guinea pig) like aorta and carotid artery (Bialecki and Stinson-Fisher, 1995; Ishibashi et al., 1995; Plane et al., 1998). In contrast, in smaller vessels where functional abnormalities can be closely related to circulatory dysfunctions, the role of MaxiK channels seems to be significant in NO-induced relaxation; for instance, in rat (this work) and rabbit mesenteric arteries (Khan et al., 1993), canine coronary (Khan et al., 1998) and rat pulmonary (Zhao et al., 1997) arteries, the blockade of MaxiK channels, decrease NO-induced relaxation. Thus, the contribution of MaxiK channel activation to the relaxations in response to cGMP-elevating agents, including nitrates and ANP, is seemingly negligible or very small if it exists in the large conduit arteries but significant in smaller arteries. This inference needs to be confirmed in other animal species.

Our mechanical results indicate that activation of IbTX-sensitive MaxiK channel does not play a pivotal role in aortic relaxations by NO donors and ANP (Figs. 1 and 2). Nevertheless, these relaxations seem to be mainly mediated via the opening of K⁺ channels, since they were dramatically diminished in the preparations contracted with high-KCl (Fig. 4). Several explanations are possible to account for the apparent lack of IbTX sensitivity in the aortic relaxations.

1) Lowered Activity of Soluble Guanylyl Cyclase in the Aortic Vessel. This possibility can be ruled out because a) ODQ and methylene blue greatly diminished the relaxations induced by NO donors (NTG and NOR 3) in both arteries (Table 2) and b) selective inhibition by IbTX of the relaxation in mesenteric artery versus aorta was also observed in the relaxation due to particulate guanylyl cyclase activation by ANP (Fig. 2).

2) Lower Expression Level of MaxiK Channel Protein in Aortic Myocytes. MaxiK (-subunit) protein expression levels were practically the same in both aortic and mesenteric myocytes, and thus, this possibility cannot account for the observed phenomenon (Fig. 3).

3) Slowdown of IbTX Association with MaxiK -Subunit. ₄-Type subunit renders MaxiK -subunit resistant to nanomolar IbTX and charybdotoxin, possibly via its extracellular loop. Therefore, high concentrations and/or long-exposure times are necessary to determine the role of the toxin-resistant MaxiK channel formed by + ₄-subunits (Meera et al., 2000). Aortic relaxations induced by NO donors and ANP were not diminished by IbTX, even when the muscle was treated with 10^–6 M IbTX for 90 min (Table 3). Therefore, the possible expression of ₄-type of subunit in rat aorta is not likely to account for the apparent lack of MaxiK role in the relaxations by NO donors and ANP in this vessel.

4) Possible Contribution of IbTX-Insensitive MaxiK Channel. Electrophysiological studies showed that MaxiK channel currents from rat aortic myocytes have a significant IbTX-sensitive component. If these IbTX-sensitive MaxiK channels could have been activated by NO donors and ANP and contributed to the resultant blood vessel relaxation, at least some portion of the relaxant response should have been diminished by IbTX (10^–7–10^–6 M). Because this was not the case, the role of IbTX-sensitive MaxiK in the relaxations to NO donors and ANP seems negligible or very minor if it exists. This view is also supported by the results with TEA. In particular, TEA blocks MaxiK from the external side with a IC₅₀ value of around 2 to 3 x 10^–4 M (Wallner et al., 1995), and TEA at 1 to 2 x 10^–3 M is expected to exhibit almost full blockade of MaxiK channel (80%) (Braun et al., 2000). However, aortic relaxations induced by NTG, NOR 3, and ANP were only partially inhibited, even with 1 to 2 x 10^–3 M TEA (Fig. 6, D, E, and F; Table 3), concentrations ten times higher than the IC₅₀ for MaxiK channels. Therefore, NO donor- and ANP-induced aortic relaxations cannot be attributable to MaxiK activation. Although aortic muscle seems to contain all of the molecular components of the cGMP-MaxiK-signaling cascade, their subcellular microlocalization may not allow for a functional coupling to support relaxation as observed in the mesenteric artery.

The non-MaxiK type of K⁺ channel responsible for NO- and ANP-induced relaxations of rat aorta seems to be a low-affinity 4-AP-sensitive K_v2 type. This is substantiated by an extensive pharmacological screening (Figs. 5 and 6) and by direct evidence in aortic myocytes of NTG-induced potentiation of a 4-AP-sensitive K_v current (Fig. 7) whose low affinity resembled the concentration required to inhibit NTG-induced relaxations (Fig. 6). Because the degree of inhibition of NTG-induced relaxation by 5 x 10^–3 M 4-AP is larger than for NTG-potentiated K_v currents, additional mechanisms may contribute to 4-AP effects (e.g., cell-cell coupling in tissue, regulation of other signaling pathways). The identification of K_v2.1 protein in aortic myocytes by immunochemical methods and its predominant transcript expression over K_v2.2 (Fig. 8) (assuming RNA to protein direct correlation) supports a key role of K_v2.1 subunit as one of the molecular constituents of the low-affinity 4-AP-sensitive K⁺ channel triggering NO-mediated relaxation of rat aortic smooth muscle. Whether K_v2.1 subunit protein forms heteromultimers with K_v2.2 or with other subunits has yet to be determined. Because the relaxations of rat aorta in response to NTG and NOR 3 were almost abolished by the inhibitor of soluble guanylyl cyclase ODQ (Table 2), cGMP-dependent rather than cGMP-independent pathway (e.g., NO-direct action) (Yuan et al., 1996; Zhao et al., 1997) is likely to be the primary mechanism for the relaxant response in rat aorta.

Judging from the inhibition caused by IbTX in mesenteric arteries, the relative contribution of MaxiK channel activation to the NTG- and ANP-induced vasorelaxations is 20% of the maximal response (Fig. 2). Mesenteric artery relaxations in response to NTG and ANP were also significantly suppressed by approximately 20% when arterial preparations were precontracted with high-KCl (80 x 10^–3 M) instead of phenylephrine (Fig. 4). Based on the latter finding with high-KCl, plasma membrane K⁺ channels would mediate approximately 20% of the NTG-induced relaxation of mesenteric artery. Thus, it is likely that the MaxiK channel is the main plasmalemmal K⁺ channel involved in cGMP-mediated relaxations of the rat mesenteric artery as opposed to other K⁺ channels like K_v, inward rectifiers, and K_ATP channels. Consistent with this view, NTG-induced relaxation of this artery preparation was not affected by glibenclamide (Table 4). Thus, the involvement of the K_ATP channel activation can be ruled out as a major mechanism underlying cGMP-mediated relaxation of the rat mesenteric artery. Interestingly, K_v2.1 protein has also been detected in mesenteric arteries (Xu et al., 1999), and its signals are similar as in aortic rings (data not shown). This underscores the possibility that the differential contribution of MaxiK versus K_v channels to NO/ANP-induced relaxations in distinct vessels depends on their local distribution within the microdomain defining NO-relaxing signaling. In rat pulmonary arteries, both MaxiK and K_v (of unknown nature) channels can mediate NO-induced relaxation (Yuan et al., 1996; Zhao et al., 1997), further highlighting the heterogeneity in vascular smooth muscle molecular architecture and its functional consequences.

Although several possibilities have been postulated as mechanisms responsible for the vascular relaxation induced by NO-releasing vasodilators and ANP, it is now widely recognized that the intracellular cGMP increase following the activation of soluble guanylyl cyclase by NO or of particulate guanylyl cyclase by ANP plays a central role in their vasorelaxant effects. This view would predict cGMP pathways as key components of the functional coupling between NTG (via NO) or ANP and MaxiK channel activation, leading to vascular relaxation of mesenteric artery. Results that support this view are as follows. 1) NTG- and NOR 3-induced relaxations were potently antagonized by ODQ or methylene blue, which inhibit soluble guanylyl cyclase, and 2) ANP-induced mesenteric relaxation was inhibited by IbTX (Fig. 2F), mimicked by a membrane-permeable cGMP analog, 8-Br-cGMP (Tanaka et al., 1998), and insensitive to methylene blue (Table 2). These findings indicate that NO- and ANP-induced relaxations of mesenteric arteries occur via an increase in cGMP contents, possibly activating PKG and enhancing MaxiK channel activity (Alioua et al., 1998).

In summary, we showed that the contribution of IbTX-sensitive MaxiK channel is irrelevant to NO donor- and ANP-induced relaxations in thoracic aorta but is significant in mesenteric artery isolated from the rat. NO aortic relaxations are largely mediated by a low-affinity 4-AP-sensitive K_v channel fingerprinted by K_v2.1 subunit. Function, expression, and/or microlocalization of voltage-gated K⁺ channels, including MaxiK channels, with signaling proteins might largely differ in large conduit arteries compared with small arteries.

	Footnotes

 
This study was partially supported by Grant-in-Aids for Scientific Research (C) (12672226 and 14572165) from Japan Society for the Promotion of Science (JSPS) (Y.T.) and by grants from the Uehara Memorial Foundation (Y.T.), the Ichiro Kanehara Foundation (Y.T.), the Pharmacological Research Foundation, Tokyo (Japan) (Y.T.), and the National Institutes of Health Grants HL47382 (L.T.), HL54970 (L.T.), HD046510 (E.S.), and P01 HL08011 (E.S. and L.T.).

doi:10.1124/jpet.105.096115.

ABBREVIATIONS: NTG, nitroglycerin; NO, nitric oxide; PKG, cGMP-dependent protein kinase; RT, reverse transcription; ANP, atrial natriuretic peptide; 4-AP, 4-aminopyridine; K_ATP, ATP-sensitive K⁺ channel; K_v, voltage-dependent K⁺ channel; MaxiK or BK, large-conductance, voltage-dependent and Ca²⁺-activated K⁺ channel; nt, nucleotides; NOR 3, (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide; 8-Br-cGMP, 8-bromo-cGMP; RA, rat aorta; RMA, rat mesenteric artery; E-4031, N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]-methanesulfonamide dihydrochloride; TEA, tetraethylammonium; ODQ, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one; TBS, Tris-buffered saline; PI, pixel intensity; HCA, human coronary artery.

¹ Current affiliation: Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, California.

² Current affiliation: Department of Pharmacology, Yamagata University School of Medicine, Yamagata, Japan.

Address correspondence to: Dr. Yoshio Tanaka, Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, 2-2-1 Miyama, Funabashi-City, Chiba 274-8510, Japan. E-mail: yotanaka{at}phar.toho-u.ac.jp

	References

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