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ORIGINAL ARTICLE

The Effect of Hydroxylamine on K_ATP Channels in Vascular Smooth Muscle and Underlying Mechanisms

Departments of Physiology (G.T., R.W.) and Pharmacology (L.W.), College of Medicine, University of Saskatchewan, Saskatoon, Canada

Received for publication November 5, 2004.

Accepted for publication February 16, 2005.

Abstract

Hydroxylamine (HA) is a putative intermediate in the conversion of L-arginine to nitric oxide (NO). HA was reported to cause the relaxation of precontracted aorta strips; however, the ionic mechanisms of HA-induced vasorelaxation were not yet known. In the present study, the whole-cell patch-clamp technique was used to examine the effects of HA on ATP-sensitive K⁺ (K_ATP) currents and membrane potentials in vascular smooth muscle cells from rat mesenteric arteries and underlying mechanisms. It was found that bath-applied HA reversibly enhanced K_ATP currents in a concentration-dependent fashion with an EC₅₀ of 54 ± 3.4 µM and hyperpolarized the cell membrane from –48 ± 5.2 to –65 ± 7.5 mV (n = 6, p < 0.01). The increase in K_ATP currents induced by HA was suppressed by superoxide dismutase (–380 ± 45 to –160 ± 20 pA, n = 4, p < 0.01) and N-acetyl-L-cysteine (–385 ± 55 to –150 ± 16 pA, n = 5, p < 0.01), indicating the involvement of different free radicals, including superoxide anion. Hypoxanthine/xanthine oxidase increased not only basal K_ATP currents, but also HA-enhanced K_ATP currents (from –355 ± 40 to –480 ± 62 pA, n = 6, p < 0.05). Sodium nitroprusside, a spontaneous NO donor, and a membrane-permeable cGMP analog (8-bromo-cGMP) were without effects on HA-enhanced K_ATP currents or basal K_ATP currents. Our results indicate that HA augmented K_ATP channel activity and hyperpolarized cell membrane, possibly via increased free radical generation.

In the present study, it was hypothesized that the reactive oxygen species generated from the metabolism of HA to NO may activate K_ATP channels and lead to the hyperpolarization of cell membrane. The whole-cell patch-clamp recording technique was used to examine the effects of HA on K_ATP channel currents in freshly isolated single vascular smooth muscle cells (VSMC) from rat mesenteric artery. Whether HA activated K_ATP channels via enhanced production of free radicals was further investigated.

Materials and Methods

Single VSMC Preparation. Single mesenteric artery VSMC were isolated according to our previously published method with modifications. In brief, male Sprague-Dawley rats (120–150 g) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg b.wt.). Small mesenteric arteries below the second branch off the main mesenteric artery were dissected and kept in ice-cold physiological salt solution (PSS) that contained 137 mM NaCl, 5.6 mM KCl, 0.44 mM NaH₂PO₄, 0.42 mM Na₂HPO₄, 4.17 mM NaHCO₃, 1 mM MgCl₂, 2.6 mM CaCl₂, 10 mM HEPES, and 5 mM glucose (pH adjusted to 7.4 with NaOH). Connective tissues were gently removed under a dissecting microscope with surgical tweezers. The freshly isolated tissues were cut into 5-mm pieces and then incubated for 40 min at 37°C in Ca²⁺-free PSS containing 1 mg/ml albumin, 0.5 mg/ml papain, and 1 mg/ml dithiothreitol and for another 30 min in the nominally Ca²⁺-free PSS containing 1 mg/ml albumin, 0.8 mg/ml collagenase, and 0.8 mg/ml hyaluronidase. Single cells released by gentle triturating through a Pasteur pipette exhibited long and spindle shapes under a microscope. Cells were stored in Ca²⁺-free PSS at 4°C and used within the same day of isolation.

Electrophysiological Recording of Membrane Potential and K_ATP Channel Currents. The whole-cell patch-clamp technique was used to record K_ATP channel currents. In brief, two or three drops of cell suspension were added to the recording chamber inside a Petri dish that was mounted on the stage of an Olympus IX70 inverted phase-contrast microscope (Olympus, Tokyo, Japan). Cells were left to stick to the glass coverslip in the recording chamber for 5 to 10 min before an experiment was started. Pipettes were pulled from soft microhematocrit capillary tubes (Fisher Scientific Co., Nepean, ON) with a tip resistance of 2 to 4 M when filled with the pipette solution. Currents were recorded with an Axopatch 200-B amplifier (Axon Instruments Inc., Union City, CA) and controlled by a Digidata 1200 interface and pCLAMP software (version 6.0; Axon Instruments Inc.). Membrane currents were filtered at 1 kHz with a four-pole Bessel filter, digitized, and stored. At the beginning of each experiment, junction potential between pipette and bath solutions was electronically adjusted to zero.

In the current-clamp mode, the membrane potential of single VSMC was measured using the nystatin-perforated patch-recording technique as the current was held at 0 pA. A stable recording of membrane potential was achieved at least 2 min after the penetration of cell membrane. The bath solution contained 140 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl₂, 10 mM HEPES, 2 mM EGTA, and 5 mM glucose (pH adjusted to 7.4 with NaOH). The pipette solution used in the nystatin-perforated whole-cell recording contained 140 mM KCl, 1 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, 5 mM glucose, and 250 µg/ml nystatin. Because nystatin may destabilize the cell, the appearance of nystatin at the tip of the electrode was avoided by dipping the pipette tip into a nystatin-free solution and backfilling the remainder of the pipette with a nystatin-containing solution.

In the voltage-clamp mode, K_ATP channel currents of single VSMC were mostly recorded at a membrane potential of –60 mV with symmetrical 140 mM K⁺. In some experiments, test pulses were made with a 10-mV increment from –80 to +70 mV at a holding potential of –60 mV with extracellular 5.4 mM K⁺ or from –150 to +50 mV at a holding potential of –20 mV with extracellular 40 mM K₊. A 600-ms test pulse to different membrane potentials was applied every 10 s. In other experiments, the voltage ramps ranging from –150 to +100 mV were applied with a holding potential of –60 mV. A 600-ms ramp pulse was used every 10 s. The bath solution for recording the whole-cell K_ATP currents contained 140 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl₂, 10 mM HEPES, 1 mM EGTA, and 5 mM glucose (pH adjusted to 7.4 with NaOH). The pipette solution contained 140 mM KCl, 1 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, 5 mM glucose, 0.3 mM Na₂ATP, and 0.5 mM MgGDP (pH adjusted to 7.2 with KOH). The K⁺ concentration of bath solutions was increased, in some experiments, to 40 or 140 mM by the removal of equimolar NaCl. The cells were superfused continuously with the bath solution at a rate of approximately 2 ml/min. A complete solution change in the recording chamber was accomplished within 30 s. The absence of Ca²⁺ in the bath and pipette solutions, the presence of EGTA in the pipette solution, and the recording made at a negative membrane potential (–60 mV) would minimize K_Ca and K_V currents. All electrophysiological experiments were conducted at room temperature (20–22°C).

Chemicals and Data Analysis. Pinacidil, nystatin, GDP, ATP, 8-bromo-cGMP (8-Br-cGMP), N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), HA, sodium nitroprusside (SNP), and adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, were purchased from Sigma-Aldrich (St. Louis, MO); glibenclamide (Glib) was purchased from Sigma/RBI (Natick, MA); and iberiotoxin was purchased from Alomone Labs (Jerusalem, Israel). Stock solutions of pinacidil and glibenclamide were made in dimethyl sulfoxide and diluted to the desired concentrations immediately before use. Dimethyl sulfoxide alone was without effect at the concentration used (up to 0.3%). Na₂ATP, MgGDP, and nystatin were directly dissolved in the pipette solution to achieve the desired concentrations on the day of experiments.

All data were expressed as means ± S.E.M. and analyzed using Student's t test or analysis of variance in conjunction with Newman-Keuls test, where applicable. Group differences were considered statistically significant at p < 0.05.

Results

Basal K_ATP Currents in Rat Mesenteric Artery VSMC. K_ATP channels in VSMC are activated by GDP, and a low concentration of ATP facilitates channel opening (Zhang and Bolton, 1995). Cell capacitance of the isolated rat mesenteric artery VSMC was 11.2 ± 0.7 pF (n = 54). The current densities of K_ATP currents were significantly higher with the inclusion of 0.3 mM Na₂ATP and 0.5 mM MgGDP in the pipette solution than that without the inclusion of ATP and GDP (at +40 mV; n = 8 for each group) (Fig. 1A). With ATP and GDP in the pipette solution, basal K_ATP currents in VSMC were increased from –11 ± 6 to –156 ± 19 pA by elevating KCl concentrations of the bath solution from 5 to 140 mM (at –60 mV; n = 4, p < 0.01) (Fig. 1, B and C). High-K⁺-enhanced K_ATP currents were not sensitive to externally applied Ba²⁺ at 10 µM (–156 ± 19 versus –142 ± 15 pA at –60 mV; n = 4, p > 0.05). Pinacidil, a K_ATP channel opener, further increased K_ATP channel current to –286 ± 37 pA at 10 µM (n = 4, p < 0.01), and glibenclamide inhibited K_ATP channel currents to –76 ± 15 pA at 10 µM (n = 4, p < 0.01) (Fig. 1, B and C). The current-voltage relationship curves showed that the reversal potentials were shifted from –78 ± 2.1 mV (n = 4) in 5.4 mM [K⁺]_o to –28 ± 1.2 mV (n = 5) in 40 mM [K⁺]_o, quite close to the calculated K⁺ electrochemical equilibrium potentials (E_K) of –80.1 and –32.6 mV, respectively (Fig. 1D), indicating that the recorded current is K₊-selective. With 40 mM [K⁺]_o, inward K_ATP currents were enhanced by increased K⁺ driving force. The inward currents were also stimulated and suppressed by pinacidil and glibenclamide (Fig. 1D), respectively. All of these results demonstrated that the recorded membrane currents under our recording conditions were mainly conducted by K_ATP channels.

View larger version (29K): [in this window] [in a new window]   Fig. 1. The pharmacological properties and reversal potentials of basal KATP currents in VSMC dialyzed with 0.3 mM ATP and 0.5 mM GDP. A, summary of time-dependent increase of basal KATP current densities by the dialysis of 0.3 mM ATP and 0.5 mM GDP compared with the lack of dialysis of ATP and GDP. Holding potential (HP), –60 mV; testing potential (TP), +40 mV (n = 8 for each group). B, representative original recording of basal KATP currents activated by 10 µM pinacidil (Pina) and then inhibited by 10 µM Glib with symmetrical 140 mM K+. Membrane potential (MP), –60 mV. The current amplitude with slow activation is measured at the maximal value of different treatments for 0.5 to 1 min. The dashed line indicates zero current. C, summary of basal KATP currents activated by Pina and inhibited by Glib (n = 5 for each group). **, p < 0.01 (140 mM K+ versus 5.4 mM K+; 10 µM Pina versus 140 mM K+; 10 µM Glib versus 10 µM Pina). D, average current-voltage relationship curves of basal KATP currents with 5.4 or 40 mM [K+]o, showing that the reversal potentials were shifted rightward with a rise in [K+]o (n = 5). Inset, representative original traces of inward part of basal KATP currents activated by Pina and then inhibited by Glib with 40 mM [K+]o. The dashed line indicates zero current. TP, –80 mV; HP, –60 mV.

HA Stimulated K_ATP Currents and Hyperpolarized Cell Membrane in VSMC. Bath-applied HA at 0.5 mM with symmetrical 140 mM K⁺ increased K_ATP currents from –180 ± 32 to –380 ± 70 pA (n = 8, p < 0.01), which were inhibited by glibenclamide to –110 ± 13 pA (n = 8, p < 0.01) (Fig. 2, A and B). HA activated K_ATP currents in a concentration-dependent fashion with an EC₅₀ of 54 ± 3.4 µM (Fig. 2C). Bath-applied HA at 0.5 mM hyperpolarized cell membrane from –48 ± 5.2 to –65 ± 7.5 mV (n = 6, p < 0.01), which was inhibited by glibenclamide to –34 ± 3mV(n = 6, p < 0.01). With extracellular physiological K⁺ concentration ([K⁺]_o = 5.4 mM), the whole-cell K_ATP currents were increased by including 0.5 mM HA in the pipette solution in a time-dependent fashion (Fig. 3A). The inward currents (at –120 mV) were increased by 98 ± 5.4, 135 ± 6.2, and 160 ± 8.6% at 10, 15, and 20 min after HA dialysis, respectively (Fig. 3B). Outward K_ATP currents became noisier with the increase of depolarizing stimuli (Fig. 3A). To exclude the possibility of K_Ca channel contamination, 200 nM iberiotoxin (IbTX), a selective K_Ca channel blocker, was used, and it failed to prevent the HA-induced K_ATP current increase under conditions of Ca²⁺-free recording solutions (–195 ± 21 to –255 ± 30 pA at –120 mV; n = 5, p < 0.05) (Fig. 3B). The contamination of our results by Kv channels is unlikely, because at this negative membrane potential, the activation of Kv channels is impossible. After the elevation of [K⁺]_o to 40 mM, K_ATP currents were profoundly increased by HA with the testing potentials of –150 to +50 mV, especially the inward current component (Fig. 4A). The inward K_ATP currents were increased in a time-dependent fashion after HA dialysis (Fig. 4, B and C). However, HA-increased currents were not significantly inhibited by extracellularly applied Ba²⁺ at 10 µM(–657 ± 45 versus –624 ± 52 pA at –150 mV; n = 5) but were blocked by a high 0.5 mM concentration of Ba²⁺ from –624 ± 52 to –334 ± 22 pA at –150 mV (n = 5, p < 0.01).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effects of HA on KATP currents and membrane potentials in VSMC. A, representative original current recording showing that bath-applied HA enhanced KATP currents, and these currents were inhibited by Glib with symmetrical 140 mM K+. MP, –60 mV. The current amplitude with slow activation is measured at the maximal value of different treatments for 0.51 min. The dashed line indicates zero current. B, summary of the effects of HA and Glib on KATP currents. MP, –60 mV. **, p < 0.01 (0.5 mM HA versus control; 10 µM Glib versus 0.5 mM HA) (n = 8 for each group). C, concentration-dependent effect of HA on KATP channel currents with symmetrical 140 mM K+.MP, –60 mV (n = 5–7 for each group).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Hydroxylamine enhanced KATP currents in VSMC with extracellular 5.4 mM K+. A, original current recording showing intracellularly applied 0.5 mM HA enhanced IbTX-insensitive KATP currents. The ramp pulse was set from –150 to +50 mV with HP of –20 mV. The dashed line indicates zero current. B, effect of bath-applied IbTX (at 200 µM) on KATP currents at different time points after the dialysis of cells with 0.5 mM HA. The testing potential was set at –120 mV with HP of –20 mV (n = 5 for each group). *, p < 0.05 (20 min versus initial); **, p < 0.01 (10 min versus initial).

View larger version (22K): [in this window] [in a new window]   Fig. 4. HA enhanced KATP currents in VSMC with extracellular 40 mM K+. A, original current recordings on the effect of HA dialysis on KATP currents. The dashed line indicates zero current. HP, –20 mV; TP, –150 to +50 mV. B, representative time-dependent effect of HA dialysis on KATP currents with HP of –20 mV and TP of –150 mV. Inset, original current traces of HA-increased KATP currents 1, 2, and 4 min after HA dialysis. The dashed line indicates zero current. C, summary of HA-increased KATP currents at initial and 5 min after the dialysis of 0.5 mM HA. HP, –20 mV; TP, –150 mV. **, p < 0.01 (n = 6 for each group).

Effects of Free Radical Generating System and Scavengers on K_ATP Channels in VSMC. To determine the involvement of free radicals in HA-induced effects, a free radical generation system, hypoxanthine (HX)/xanthine oxidase (XO), was applied to VSMC. With 5.4 mM K⁺ in the bath solution, basal K_ATP currents recorded by a ramp pulse were increased by HX/XO (100 µM/20 mU/ml) by 118% (at –120 mV), which was blocked by SOD by 60% (Fig. 5, A and B), although HX alone at 100 µM had no effect on K_ATP currents. With symmetrical 140 mM K⁺ solutions, the combined application of HX at 100 µM and XO at 20 mU/ml enhanced HA-elicited K_ATP currents at –60 mV from –355 ± 40 to –480 ± 62 pA (n = 6, p < 0.05), which were blocked by 500 U/ml SOD to –150 ± 20 pA (n = 6, p < 0.01) (Fig. 5, C and D). On the other hand, the bath-applied HA at 0.5 mM enhanced K_ATP currents with symmetrical 140 mM K⁺ solutions from –250 ± 26 to –380 ± 45 pA (n = 4, p < 0.05), which were inhibited by SOD to –160 ± 20 pA (n = 4, p < 0.01) and further inhibited by glibenclamide to –45 ± 3 pA (n = 4, p < 0.01) (Fig. 6, A and B). To confirm the inhibition of HA-enhanced K_ATP currents by SOD, another free radical scavenger, NAC, was applied. K_ATP currents enhanced by the bath-applied HA were inhibited reversibly by 300 and 600 µM NAC by 48 ± 5% (n = 5, p < 0.01) and 61 ± 9% (n = 5, p < 0.01), respectively, and also inhibited by SOD by 43 ± 6% (n = 5, p < 0.05) (Fig. 6, C and D).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Effects of HX and XO on KATP currents in VSMC. A, original current recordings of the effects of HX/XO (b) and SOD (c) on basal KATP currents (a) with extracellular 5.4 mM K+. The ramp pulse was set from –150 to +100 mV with HP of –20 mV. The dashed line indicates zero current. B, time-dependent effects of HX/XO (b) and SOD (c) on basal KATP currents (a). HP, –20 mV; TP, –120 mV. C, original current traces showing the effects of 100 µM HX with 20 mU/ml XO and 500 U/ml SOD on HA-enhanced KATP currents with symmetrical 140 mM K+. MP was held at –60 mV. The current amplitude with slow activation was measured at the maximal value of different treatments for 0.5 to 1 min. The dashed line indicates zero current. D, summary of the augmentation and suppression of HA-enhanced KATP currents by HX/XO and SOD, respectively. MP, –60 mV (n = 5–6 for each group). *, p < 0.05 (HX + XO versus 0.5 mM HA); **, p < 0.01 (0.5 mM HA versus 140 mM K+; 500 U/ml SOD versus HX + XO).

View larger version (35K): [in this window] [in a new window]   Fig. 6. Effects of free radical scavengers on HA-enhanced KATP currents with symmetric 140 mM K+. A, original current recording of the inhibitory effects of 500 U/ml SOD and 10 µM Glib on HA-enhanced KATP currents. MP was held at –60 mV. The current amplitude with slow activation is measured at the maximal value of different treatments for 0.5 to 1 min. The dashed line indicates zero current. B, summary of the inhibition of HA-enhanced KATP currents by SOD and Glib (n = 4 for each group). *, p < 0.05 (0.5 mM HA versus 140 mM K+); **, p < 0.01 (500 U/ml SOD versus 0.5 mM HA; 10 µM Glib versus 500 U/ml SOD). C, original current recording of the reversible inhibition of HA-enhanced KATP currents by NAC and SOD. MP, –60 mV. The current amplitude with slow activation is measured at the maximal value of different treatments for 0.5 to 1 min. The dashed line indicates zero current. D, summary of the inhibition of HA-enhanced KATP currents by NAC and SOD. *, p < 0.05 (500 U/ml SOD versus washout); **, p < 0.01 (300 µM NAC versus 0.5 mM HA; 600 µM NAC versus washout) (n = 5 for each group).

Effects of NO Donor and cGMP Analog on K_ATP Currents in VSMC. To examine whether the NO-sGC-cGMP signaling pathway mediated HA effects, NO donor and cGMP analog were used to test K_ATP currents. The NO donor SNP had no effect on HA-stimulated K_ATP currents with symmetrical 140 mM K⁺ at 0.5 mM (–293 ± 46 versus –284 ± 32 pA; n = 5, p > 0.05) (Fig. 7, A and B). With the same recording conditions, the membrane-permeable cGMP analog 8-Br-cGMP failed to affect HA-increased K_ATP currents (–232 ± 30 versus –248 ± 34 pA; n = 5, p > 0.05) (Fig. 7, C and D). Basal K_ATP currents were not affected by SNP (–182 ± 23 versus –200 ± 30 pA; n = 5, p > 0.05) or 8-Br-cGMP (–142 ± 21 versus –165 ± 23 pA; n = 5, p > 0.05). However, HA-increased K_ATP currents were inhibited completely by glibenclamide at 10 µM, indicating that HA-activated currents are K_ATP currents. Furthermore, when 100 µM Rp-cAMP was included in the pipette solution to inhibit the cAMP-dependent protein kinase pathway (Wellman et al., 1998), the stimulatory effect of bath-applied HA on K_ATP currents was significantly reduced from –175 ± 21 to –204 ± 26 pA (at the testing potential of –60 mV; n = 5, p > 0.05). This result suggests that the phosphorylation of K_ATP channels by the cAMP-dependent protein kinase pathway alter the sensitivity of K_ATP channels to HA modulation (Quayle et al., 1994).

View larger version (37K): [in this window] [in a new window]   Fig. 7. Effects of SNP and 8-Br-cGMP on HA-enhanced KATP currents with symmetrical 140 mM K+. A, original current recording of the effect of SNP and Glib on HA-enhanced KATP currents. MP was held at –60 mV. The current amplitude with slow activation is measured at the maximal value of different treatments for 0.5 to 1 min. The dashed line indicates zero current. B, summary of the effects of SNP and Glib on HA-enhanced KATP currents. **, p < 0.01 (0.5 mM HA versus 140 mM K+; 10 µM Glib versus 0.5 mM SNP) (n = 5 for each group). C, original current traces of the effect of 8-Br-cGMP and Glib on HA-enhanced KATP currents. MP, –60 mV. The current amplitude with slow activation is measured at the maximal value of different treatments for 0.5 to 1 min. The dashed line indicates zero current. D, summary of the effect of 8-Br-cGMP and Glib on HA-enhanced KATP currents. **, p < 0.001 (0.5 mM HA versus 140 mM K+; 10 µM Glib versus 0.5 mM 8-Br-cGMP) (n = 5 for each group).

Discussion

The novel findings of this study are summarized as follows. 1) Bath-applied HA reversibly enhanced K_ATP currents in a concentration-dependent fashion with an EC₅₀ of 54 ± 3.4 µM and hyperpolarized cell membrane of rat mesenteric artery VSMC. 2) HA activated K_ATP channels with different [K⁺]_o. The HA-stimulated inward currents increased with the elevation of [K⁺]_o (140 > 40 >5.4 mM). 3) HA-induced K_ATP channel activation and hyperpolarization were reduced by free radical scavengers (SOD and NAC). 4) The free radical generating system (HX/XO) mimicked and augmented the effect of HA on K_ATP currents, indicating the activation of K_ATP channels by 5) SNP and 8-Br-cGMP had no effect on basal and HA-stimulated K_ATP currents. Thus, the activation of K_ATP channels by HA is probably caused by increased free radical generation.

HA Targeted on K_ATP Channels in VSMC from Rat Mesenteric Artery. Although inward rectifier K⁺ currents are known to be expressed in rat mesenteric artery VSMC (Bradley et al., 1999), the recorded K⁺ currents in our recording condition are conducted through K_ATP channels. The following lines of evidence support this notion. 1) The recorded K⁺ current was enhanced by the dialysis with GDP and ATP. The NDP-induced activation is a hallmark of vascular K_ATP channels in VSMC (Zhang and Bolton, 1995). 2) The recorded K⁺ current was activated by K_ATP channel openers, such as pinacidil, and inhibited by glibenclamide. Glibenclamide suppressed not only high K⁺-enhanced currents, but also GDP-activated basal currents. 3) The recorded K₊ current exhibited a weak inward rectification without the voltage dependence, whereas the classic inward rectifier current was activated by hyperpolarization with strong inward rectification (Quayle et al., 1993, 1994). 4) It has been reported that glibenclamide inhibited or blocked K_ATP channels at 10 µM but had no effect on Kir channels (Quayle et al., 1993). In our study, both basal K⁺ currents and HA-enhanced K⁺ currents were inhibited by 10 µM glibenclamide, supporting a K_ATP channel entity under the current investigation. 5) In cells dialyzed with 0.5 mM GDP and 0.3 mM ATP, high K⁺-increased currents and HA-enhanced currents were not reduced by 10 µM Ba²⁺, indicating that no Kir channel is activated in this recording condition (Bradley et al., 1999). Quayle et al. (1993) showed that Ba²⁺ at 10 µM blocked Kir currents but not K_ATP current; thus, Ba²⁺ was used to identify or separate Kir from K_ATP currents.

HA Evoked K_ATP Channel Activation and Membrane Hyperpolarization in VSMC. The HA-induced vasodilation of different vascular tissues has been reported (Rapoport and Murad, 1984; DeMaster et al., 1989; Thomas and Ramwell, 1989; Feelisch et al., 1994; Huang, 1998); however, the exact cellular mechanisms underlying the vasorelaxant effect of HA has been largely unclear. It was reported that HA increased the rate of ⁸⁶Rb outflow from perfused pancreatic islets, which was counteracted by glibenclamide, indicating that K_ATP channels were involved in HA-inhibited insulin release (Antoine et al., 1996). HA was also reported to activate voltage-dependent K₊ channels in crustacean skeletal muscle (Hermann and Erxleben, 2001). But a high 10 mM concentration of HA blocked the inactivating K⁺ channels (Shaker-B) expressed in Xenopus laevis oocytes by an unknown mechanism (Yool, 1994) and depolarized cell membrane by inhibiting K⁺ channels (Mongin et al., 1998). Our results demonstrated for the first time that HA enhanced K_ATP currents in VSMC and hyperpolarized the cell membrane. HA-induced hyperpolarization by K_ATP channel activation may close voltage-dependent L-type Ca²⁺ channels and then decrease intracellular free [Ca²⁺]_i, leading to vasorelaxation.

NO-sGC-cGMP Signaling Pathway Did Not Mediate the Effect of HA on K_ATP Channels. Among the known endogenous K_ATP channel modulators is endogenous NO, which activated K_ATP channels in cell-attached patches via the activation of sGC in cultured VSMC from porcine coronary artery (Kubo et al., 1994). Bath-applied atrial natriuretic factor and isosorbide dinitrate, which are activators of particulate and soluble guanylyl cyclase, respectively, activated unitary K_ATP channel currents. These effects were abolished by methylene blue (an sGC inhibitor) but potentiated by 8-Br-cGMP, suggesting that the cGMP pathway mediated the effects of atrial natriuretic factor and isosorbide dinitrate (Kubo et al., 1994). At the tissue level, SNP elicited the dilation of pial arterioles from anesthetized piglets, which was blocked by cGMP-dependent protein kinase inhibitor (8-Br-cGMP, Rp-isomer) and sGC inhibitor (LY83583), indicating that NO primarily elicited its effects via cGMP production (Armstead, 1996). Furthermore, SNP- and 8-Br-cGMP-elicited dilation of newborn pig pial artery was blunted by the K_ATP channel antagonist glibenclamide, indicating that NO and cGMP might interact with K_ATP channels (Armstead, 1999). However, SNP- and HA-induced vasorelaxation of rat aortic rings was not affected by glibenclamide, disproving the involvement of K_ATP channels in NO-induced vasorelaxation (Huang, 1998). SNP did not increase whole-cell K_ATP currents with symmetrical 140 mM K⁺, indicating that the activation of the NO-sGC-cGMP pathway did not lead to K_ATP channel activation (Quayle et al., 1994; Wellman et al., 1998). Therefore, NO effects on K_ATP channels in different vascular beds are controversial without clear mechanisms.

Some studies have shown hyperpolarization of smooth muscle by NO via activation of K_ATP channels. SNP activated cGMP-dependent protein kinase and produced glibenclamide-sensitive membrane hyperpolarization in rabbit mesenteric arteries (Murphy and Brayden, 1995); however, other studies in rabbit cerebral and canine coronary arteries failed to demonstrate hyperpolarization induced by exogenous NO (8–30 µM) (Tare et al., 1990). SNP-induced hyperpolarization may result from the cross-activation of protein kinase A by cGMP (Quayle et al., 1994). Only a large amount of NO could produce a hyperpolarizing effect in VSMC from rat mesenteric artery (Zhao et al., 2000). S-Nitroso-N-acetyl-penicillamine at a high 400 µM concentration caused membrane hyperpolarization that was reversed by glibenclamide and completely blocked by treatment with Tiron, a scavenger of , suggesting that peroxynitrite (OONO^–) other than NO exerts the hyperpolarizing effect via the activation of K_ATP channels (Zhao et al., 2000).

Our results present evidence that HA directly activated whole-cell K_ATP channels and hyperpolarized cell membrane, whereas both SNP and 8-Br-cGMP had no effect on basal K_ATP currents and HA-stimulated K_ATP currents. These observations suggested that the activation of the NO-sGC-cGMP signaling pathway did not mediate K_ATP channel activity in rat mesenteric artery VSMC. It is tempting to speculate that HA activated K_ATP channels via another mechanism. The yield of free radicals, including by HA could be one such mechanism (Santoian et al., 1993; Market et al., 1994; Vetrovsky et al., 1996).

Free Radical Generation Mainly Underlies the Effect of HA on K_ATP Channels. The modulation of K⁺ channel activity by cellular oxidative stress has been recognized as a significant determinant of vascular tone. Under certain conditions, many extracellular ligands generated and/or required free radicals to transmit biological signals to intracellular milieu as second messengers. Different kinds of free radicals can modify various types of K⁺ channels in vascular tissues. At the tissue level, , H₂O₂, and OONO^– dilated cerebral vasculature, which was not mediated by sGC activation (Wei et al., 1996). Both H₂O₂ and OONO^– elicited dilation via activating K_ATP channels, whereas dilated cerebral arterioles by opening K_Ca channels (Wei et al., 1996). H₂O₂ induced a glibenclamide-sensitive dose-dependent dilation in cat cerebral arterioles and rat gracilis skeletal muscle arterioles (Wei et al., 1996; Cseko et al., 2004). OONO^– elicited vasodilation in several vascular beds, including coronary (Liu et al., 1994), renal, mesenteric (Benkusky et al., 1998), and cerebral arteries (Wei et al., 1996, 1998; Liu et al., 2002). Dilation of cerebral and coronary arteries to OONO^– is blocked by glibenclamide, suggesting a role of K_ATP channels (Wei et al., 1996, 1998; Liu et al., 2002).

At the cellular level, knowledge about the modulation of K₊ channels by free radicals in single VSMC is still limited. produced by xanthine (X)/XO or high glucose reduced the whole-cell Kv current density in freshly isolated rat coronary VSMC, which was reversed partially by SOD (Liu et al., 2001). However, generated by X/XO did not significantly alter the open-state probability of K_Ca channels (Liu et al., 2002). H₂O₂ activated macroscopic and unitary BK_Ca channel currents in porcine coronary arteries via a phospholipase A₂-arachidonic acid signaling cascade (Barlow et al., 2000). In isolated coronary arteriole VSMC, the IbTX-sensitive whole-cell K⁺ current density was reduced by OONO^– generated from the mixture of SNP with X/XO. OONO^– greatly decreased the open-state probability of K_Ca channels in inside-out excision, contributing to the inhibition of K_Ca channel activity (Liu et al., 2002); however, electrophysiological evidence for the effects of free radicals on K_ATP channel activity is largely lacking in VSMC. Our study provides for the first time the electrophysiological evidence that HA activated K_ATP channels in single VSMC from rat mesenteric artery, which was mimicked or augmented by the free radical generating system HX/XO and reduced by free radical scavengers such as SOD and NAC. It should be noted that HA in the cytosol is converted into NO and , which are likely to form OONO^– (Liu et al., 1994; Pryor and Squadrito, 1995). Whether HA-induced K_ATP channel activation and vasodilation are linked to OONO^– generation remains to be investigated.

Although HX/XO is widely used as the free radical generating system, direct effects of HX/XO on K⁺ channels in single VSMC are rarely reported. When HX is oxidized by XO in the presence of O₂, an electron from the reaction of HX with XO is transferred to O₂ to form . The dismutation of generated H₂O₂ via cytosolic or mitochondrial SOD. Further oxidation of H₂O₂ leads to highly potent OH^– via the catalysis of transient metal such as ferrous iron (Graf, 1984). Thus, HX/XO may generate various reactive species such as , H₂O₂, and OH^–, which determine different effects of HX/XO, along with species- and tissue-specific differences in various vascular beds. Application of HX/XO together with FeCl₃ to pial artery in vivo resulted in attenuated vasodilatation induced by K_ATP channel agonists (cromakalim and calcitonin gene-related peptide), NO donors (SNP and S-nitroso-N-acetyl-penicillamine), and 8-Br-cGMP (Armstead, 1999). From these results, however, one cannot conclude that inhibits K_ATP channel in VSMC. Changes in the diameter of pial artery in vivo are influenced by many vasoactive substances with multiple mechanisms. Blocking a common downstream cellular event by HX/XO would not only inhibit the vasodilatory effect of K_ATP channel agonists, but also that of many other vasodilators that may not interact with K_ATP channels at all. The direct effect of HX/XO on the basal diameter of pial artery was not examined. Electrophysiological evidence for the effect of HX/XO on K_ATP channels in VSMC of pial artery was also unavailable. In our present study, direct electrophysiological recording of K_ATP channel currents was carried out on isolated VSMC from rat mesenteric artery. Both electrophysiological and pharmacological results in our study demonstrated that HX/XO reaction in fact activated K_ATP channels in single VSMC. This effect is probably mediated by because HX/XO-activated K_ATP currents were reduced by SOD.

In summary, HA-induced K_ATP channel activation and resultant hyperpolarization in VSMC may underlie HA-induced vasorelaxation via enhanced production of free radicals. These observations will lead to a better understanding of the physiological functions of HA and the underlying cellular and molecular mechanisms. Novel therapeutic approaches in dealing with K_ATP channel abnormality-related disorders may also be yielded from the observations of this study.

Footnotes

This work was supported by an operating grant from the Canadian Institutes of Health Research (CIHR) (to R.W.). R.W. and L.W. were supported by the Investigator Awards of CIHR. G.T. was supported by a Doctoral Research Award from the Heart and Stroke Foundation of Canada.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.008953.

ABBREVIATIONS: HA, hydroxylamine; sGC, soluble guanylyl cyclase; VSMC, vascular smooth muscle cell(s); PSS, physiological salt solution; 8-Br-cGMP, 8-bromo-cGMP; NAC, N-acetyl-L-cysteine; SOD, superoxide dismutase; SNP, sodium nitroprusside; Glib, glibenclamide; IbTX, iberiotoxin; HX, hypoxanthine; XO, xanthine oxidase; LY83583, 6-anilino-5,8-quinolinedione; X, xanthine; HP, holding potential; TP, testing potential; MP, maximum potential.

Address correspondence to: Dr. Rui Wang, FAHA, Department of Physiology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada. E-mail: rui.wang{at}lakeheadu.ca

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