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Am J Physiol Heart Circ Physiol 290: H1617-H1623, 2006. First published December 3, 2005; doi:10.1152/ajpheart.00746.2005
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Sodium azide dilates coronary arterioles via activation of inward rectifier K+ channels and Na+-K+-ATPase

Erion Qamirani,1 Habib M. Razavi,1 Xin Wu,1 Michael J. Davis,1 Lih Kuo,1 and Travis W. Hein2

1Department of Medical Physiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College Station, Texas; and 2Department of Surgery, Scott & White Memorial Hospital, College of Medicine, The Texas A&M University System Health Science Center, Temple, Texas

Submitted 14 July 2005 ; accepted in final form 18 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sodium azide (NaN3), a potent vasodilator, causes severe hypotension on accidental exposure. Although NaN3 has been shown to increase coronary blood flow, the direct effect of NaN3 on coronary resistance vessels and the mechanism of the NaN3-induced response remain to be established. To address these issues without confounding influences from systemic parameters, subepicardial coronary arterioles were isolated from porcine hearts for in vitro study. Arterioles developed basal tone at 60 cmH2O intraluminal pressure and dilated acutely, in a concentration-dependent manner, to NaN3 (0.1 µM to 50 µM). The NaN3 response was not altered by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester or endothelial removal. Neither inhibition of phosphoinositol 3-kinase and tyrosine kinases nor blockade of ATP-sensitive, Ca2+-activated, and voltage-dependent K+ channels affected NaN3-induced dilation. However, the vasomotor action of NaN3 was significantly attenuated in a similar manner by the inward rectifier K+ (KIR) channel inhibitor Ba2+, the Na+-K+ ATPase inhibitor ouabain, or the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Ba2+, in combination with either ouabain or ODQ, nearly abolished the vasodilatory response. However, there was no additive inhibition by combining ouabain and ODQ. The NaN3-mediated vasodilation was also attenuated by morin, an inhibitor of phosphatidylinositolphosphate (PIP) kinase, which can regulate KIR channel activity. With the use of whole cell patch-clamp methods, NaN3 acutely enhanced Ba2+-sensitive KIR current in isolated coronary arteriolar smooth muscle cells. Collectively, this study demonstrates that NaN3, at clinically toxic concentrations, dilates coronary resistance vessels via activation of both KIR channels and guanylyl cyclase/Na+-K+-ATPase in the vascular smooth muscle. The KIR channels appear to be modulated by PIP kinase.

vasodilation; ion channel; physiology

SODIUM AZIDE (NaN3) is a highly water-soluble substance that is devoid of taste and odor (4). NaN3 is mainly used as a preservative in aqueous laboratory reagents, as well as a gas-generating agent in automobile air bags (4, 38). Other commercial applications of NaN3 include its use as a pesticide, herbicide, nematocide, fungicide, bactericide, and as a necessary component in the manufacturing of rubber, latex, and lead azide (4). Because the industrial production and use of NaN3 have increased over the past century, culminating in 1996, it has become evident that human exposure to this compound (whether accidental at work sites or intentional in attempted suicide) is associated with high mortality rates. An extensive review of the literature recently by Chang and Lamm (4) pointed out that the main reported symptom of NaN3 poisoning is hypotension, which can lead to more severe symptoms, such as decreased mental status, arrhythmia, metabolic acidosis, and cardiorespiratory arrest. It was initially believed that the mechanism of NaN3-induced toxicity involved activation of central nervous system pathways (36). Recent evidence (37), however, suggests that the hypotensive side effect of NaN3 poisoning could be due to the direct vasodilator properties of this compound. In this earlier study (37), it was found that NaN3 relaxed preconstricted aortic rings in an endothelium-independent manner. This finding clearly implicates that NaN3 has a direct influence on vasomotor activity. Although evidence from this recent study (37) suggests that hyperpolarization of the vascular smooth muscle is involved in the vasodilator response to NaN3, the precise signaling mechanism has not been clearly established.

The vasodilatory action of NaN3 could extend to the coronary microcirculation. In vivo studies have shown that NaN3 can rapidly increase coronary blood flow in animal models (11, 37), which could possibly promote exposure of the underlying cardiac tissue to the toxicological levels of metabolic inhibition (4, 5, 20). However, the direct impact of NaN3 on vascular tone in the coronary microcirculation is unknown. Therefore, the goals of the present study were to determine the extent to which NaN3 influences vasomotor tone of isolated porcine coronary arterioles and to elucidate the underlying signaling pathways.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Isolation and cannulation of microvessels. The procedures were approved by the Laboratory Animal Care Committee at Texas A&M University and Scott & White Hospital and have been previously described in detail (22). Pigs (12 to 15 kg; 8 to 12 wk old of either sex; Milberger Farms, Kurten, TX; n = 64) were sedated with an intramuscular injection of Telazol (tiletamine and zolazepam, 1:1; 4.4 mg/kg) and xylazine (2.2 mg/kg), anesthetized with pentobarbital sodium (20 mg/kg iv), intubated, and ventilated with room air. The heart was removed and immediately placed in iced (5°C) saline. Subepicardial arteriolar branches (~1 mm in length; 60–100 µm inner diameter in situ) were dissected from the surrounding cardiac tissue. Vessels were then cannulated with glass micropipettes, pressurized to 60 cmH2O intraluminal pressure, and bathed in physiological salt solution (PSS) containing (in mM) 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS, at 37°C. The inner diameters of coronary arterioles were measured by using video microscopic techniques and recorded with a PowerLab data acquisition system (ADInstruments, Colorado Springs, CO) (22).

Experimental protocols. To determine the effect of NaN3 on coronary arteriolar tone, vessel diameter was measured under cumulative addition of NaN3 (0.1 µM to 50 µM) to the vessel bath. The concentrations of NaN3 used in the present study are consistent with the exposure levels leading to human hypotension (~1 to 10 µM) (1, 4). In some vessels, the role of endothelium in the NaN3-induced response was determined after endothelial removal by perfusion of the nonionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 0.4%), as previously described (17). Only vessels that exhibited normal basal tone, that showed no vasodilation to the endothelium-dependent vasodilator bradykinin (1 nM) (17), and that showed unaltered vasodilation to sodium nitroprusside (0.1 nM to 100 µM) after endothelial removal were accepted for data analysis.

In other vessels, the contributions of nitric oxide (NO), guanylyl cyclase, K+ channels, Na+-K+-ATPase, phosphoinositide kinases, and tyrosine kinases to the NaN3-induced response were evaluated in a series of experiments by incubation of vessels with their cognate inhibitors for 30 min. Specifically, the roles of NO and guanylyl cyclase were examined in the presence of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 µM) (17) and soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 0.1 µM) (15), respectively. The roles of inward rectifier K+ (KIR), ATP-sensitive K+ (KATP), and voltage-dependent K+ (KV) channels were assessed in the presence of their respective inhibitors: Ba2+ (30 µM) (2, 31), glibenclamide (5 µM) (2, 15), and 4-aminopyridine (1 mM) (13). The involvement of Ca2+-activated K+ (KCa) channels was determined by incubating the vessels with various KCa channel subtype inhibitors, e.g., iberiotoxin (for large-conductance KCa; 100 nM) (17), charybdotoxin (for large- and intermediate-conductance KCa; 10 nM) (26), and apamin (for small-conductance KCa; 100 nM) (2, 10). The roles of Na+-K+-ATPase, phosphatidylinositolphosphate (PIP) kinase, phosphoinositol (PI) 3-kinase, and tyrosine kinase were examined in the presence of their respective inhibitors: ouabain (1.5 µM) (21), morin (20 µM) (6), wortmannin (100 nM) (27), and genistein (5 µM) (24, 29). To examine the specificity of pharmacological inhibitors in the NaN3-induced effect, vasodilations to NO donor sodium nitroprusside and KATP channel opener pinacidil in the presence of these inhibitors were studied in some vessels.

Cell isolation and electrophysiology. The procedure for cell isolation has been previously reported in detail (39). Briefly, arterioles (<100 µm inner diameter) were freshly dissected from the pig heart and placed in a Silastic-coated Plexiglas chamber containing PSS with albumin at 4°C. Dissected segments of arterioles were transferred to a tube of low-Ca2+ PSS at room temperature for 10 min. The solution was aspirated and replaced with low-Ca2+ saline solution containing 26 U/ml papain and 1 mg/ml dithioerythritol. The vessels were incubated for 30 min at 37°C with occasional agitation, after which fragments were transferred to low-Ca2+ saline solution containing 1.95 furanacryloyl-Leu-Gly-Pro-Ala (FALGPA) U/ml collagenase, 1 mg/ml soybean trypsin inhibitor, and 75 U/ml elastase (Calbiochem) for 25 min at 37°C. After further digestion, the remaining fragments were rinsed two times in low-Ca2+ saline solution and gently triturated by using a fire-polished Pasteur pipette to release single smooth muscle cells.

Standard patch-clamp techniques as devised by Hamill et al. (12) were used to assess the role of vascular smooth muscle KIR channels in the NaN3 response. Suspensions of freshly dispersed smooth muscle cells were studied in a recording chamber, suffused at a rate of 1.5 ml/min with high-K+ bath solution, on the stage of an inverted microscope (Zeiss IM-405, Thornwood, NY). The bath solution had the following composition (in mM): 140 KCl, 10 HEPES, 0.5 MgCl2, 2.1 CaCl2 and 2.0 EGTA (pH adjusted to 7.4 with NaOH). Micropipettes for whole cell recordings were pulled from 1.5-mm (outer diameter) glass tubing (Corning 8161; Warner Instruments, Hamden, CT) on a programmable puller and were fire-polished. Pipette resistances ranged from 1 to 3 M{Omega}. For conventional whole cell recordings, pipettes were filled with high-K+ pipette solution containing (in mM) 140 KCl, 10 HEPES, 1.0 Mg-ATP, 1.1 CaCl2, and 10 EGTA (pH adjusted to 7.2 with NaOH). An EPC-7 amplifier (HEKA, Darmstadt-Eberstadt) was used to record current, and a Burleigh micromanipulator (Burleigh Instruments, Fishers, NY) was used for fine control of the micropipettes. Analog-to-digital conversions were made by using a Digidata interface (Axon Instruments, Union City, CA) and stored on a Pentium computer for subsequent analysis. Data were sampled at 5–10 kHz and filtered at 1–2 kHz with the use of an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Series resistances varied from 2 to 9 M{Omega}. Series resistance compensation was used in whole cell recordings to improve voltage control. Current records were analyzed with the use of pCLAMP (version 9, Axon Instruments). All experiments were performed at 22°C. Raw current values were normalized to cell capacitance (an index of cell size) and were expressed as current density (in pA/pF) for comparisons. Cell capacitance ranged from 4 to 12 pF.

Chemicals. Glibenclamide, wortmannin, and genistein were initially dissolved in dimethyl sulfoxide, whereas morin, ODQ, and pinacidil were dissolved in absolute ethanol. Subsequent concentrations of these drugs, and all other drugs, were dissolved in PSS. The final concentrations of dimethyl sulfoxide and ethanol in the tissue bath were 0.03% and 0.1%, respectively. Vehicle control studies indicated that these final concentrations of solvent had no effect on arteriolar function. All drugs were obtained from Sigma.

Data analysis. Data are presented as means ± SE. At the end of each experiment, the vessel was relaxed with sodium nitroprusside (100 µM) in an ethylenediaminetetraacetic acid (1 mM)-Ca2+-free PSS after the washout of pharmacological inhibitors to obtain its maximal diameter at 60 cmH2O intraluminal pressure (16). All diameter changes in response to agonists were normalized to this maximal dilation and expressed as a percentage. In each set of interventions, the vessels had their own control, with each vessel being from a different heart. From some hearts, two vessels were studied with different interventions performed on each vessel. The control vessels were pooled when comparing a series of experiments examining the effect of antagonists on vasodilation. Statistical comparisons of vasomotor responses, resting vascular tone, and current density before and after various treatments were performed by using paired Student’s t-tests or analysis of variance for repeated measures with Bonferonni multiple-range tests when appropriate. A value of P <0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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In the present study, isolated coronary arterioles developed basal tone within 40 min; only vessels exhibiting spontaneous tone were used for further study. The level of resting vascular tone was 63 ± 1% of maximal passive diameter (95 ± 3 µm; range 65–119 µm). NaN3 (50 µM) produced a robust dilation of an isolated arteriole from the baseline diameter of 52 to 76 µm (Fig. 1A). The diameter gradually returned to the baseline level after the vessel bath was replaced with PSS (Fig. 1A). Further study showed that NaN3 produced concentration-dependent dilation of coronary arterioles (Fig. 1B). In general, the dilation of arterioles to each concentration of NaN3 was developed within seconds, and the highest concentration (50 µM) of NaN3 elicited nearly 60% of maximal dilation (Fig. 1B). Neither the inhibition of NO synthase by L-NAME (10 µM) nor the removal of endothelium altered the NaN3-induced vasodilation (Fig. 1B). We have previously demonstrated that the concentration of L-NAME used in this study effectively inhibits agonist- (14) and flow-stimulated (17) NO-mediated dilation of coronary arterioles. Endothelial denudation was verified by complete block of bradykinin-induced dilation (control, 82 ± 5% of maximal dilation; and denudation, 4 ± 2% of maximal dilation; n = 5). Vascular smooth muscle function remained intact in these denuded vessels because vasodilation to sodium nitroprusside (0.1 nM to 100 µM) was not altered (data not shown).


Figure 1
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  		Fig. 1. Effect of acute exposure to NaN3 on vascular tone of isolated coronary arterioles. A: NaN3 (50 µM) produced a sustained dilation of coronary arterioles for 5 min. Diameter returned to baseline level after washout. B. Coronary arterioles were exposed to cumulative extraluminal concentrations of NaN3 (2–3 min for each concentration). Vasodilatory responses to NaN3 were evaluated before and after endothelial denudation (n = 5 vessels) or incubation with nitric oxide synthase inhibitor NG-nitro-L-arginine methylester (L-NAME, 10 µM, n = 5 vessels). Data are expressed as means ± SE.

 
In the presence of Ba2+, a specific KIR channel inhibitor at the concentration used (30 µM) (25), the NaN3-induced vasodilation was significantly attenuated (Fig. 2A). Ouabain, a Na+-K+-ATPase inhibitor, caused a slight constriction of resting arterioles (control, 63 ± 1% of maximal diameter; and ouabain, 58 ± 2% of maximal diameter; P < 0.05) and significantly attenuated the vasodilation to NaN3 (Fig. 2A). In the presence of both Ba2+ and ouabain, the vasodilation to NaN3 was almost completely blocked (Fig. 2A). In another group of vessels, inhibition of soluble guanylyl cyclase by ODQ significantly attenuated the dilation to NaN3 in a similar manner as by either Ba2+ or ouabain alone (Fig. 2B). The vasodilatory response was further reduced in the presence of ODQ with Ba2+ (Fig. 2B) but not with ouabain (data not shown). By contrast, blockade of large-conductance KCa channels with iberiotoxin (Fig. 2C), intermediate-conductance and small-conductance KCa channels with charybdotoxin plus apamin (Fig. 2C), KV channels with 4-aminopyridine (Fig. 2D), or KATP channels with glibenclamide (Fig. 2D) had no effect on NaN3-induced vasodilation. The only K+ channel inhibitor that significantly affected resting basal tone was 4-aminopyridine (control, 59 ± 1% of maximal diameter; and 4-aminopyridine, 54 ± 2% of maximal diameter; P < 0.05). Because the dilation of coronary arterioles to pinacidil was not altered by ouabain or by Ba2+ in combination with ODQ or ouabain (Fig. 3), it is likely that these inhibitors specifically blocked the vasodilation to NaN3.


Figure 2
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  		Fig. 2. Roles of K+ channels, Na+-K+-ATPase, and guanylyl cyclase in NaN3-induced dilation of isolated coronary arterioles. Vasodilatory responses to NaN3 were evaluated before and after incubation with inward rectifier K+ (KIR) channel inhibitor Ba2+ (30 µM, n = 5 vessels) and Na+-K+-ATPase inhibitor ouabain (1.5 µM, n = 5 vessels) alone or in combination (n = 5 vessels) (A); guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.1 µM, n = 4 vessels) alone or in combination with Ba2+ (n = 4 vessels) (B); large-conductance Ca2+-activated K+ (KCa) channel inhibitor iberiotoxin (100 nM, n = 5 vessels) or intermediate-conductance and small-conductance KCa channel inhibitors charybdotoxin (10 nM) and apamin (100 nM) (n = 4 vessels) (C); and voltage-dependent K+ channel inhibitor 4-aminopyridine (1 mM, n = 4 vessels) or the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide (5 µM, n = 5 vessels) (D). Data are expressed as means ± SE. *P < 0.05 vs. control; {dagger}P < 0.05 vs. Ba2+ + ouabain or ODQ + Ba2+.

 

Figure 3
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  		Fig. 3. Effect of pharmacological inhibitors on pinacidil-induced dilation of isolated coronary arterioles. Vasodilation to KATP channel opener pinacidil was evaluated before and after incubation with ouabain (1.5 µM, n = 6 vessels) alone or Ba2+ (30 µM) in combination with ODQ (0.1 µM, n = 6 vessels) or ouabain (1.5 µM, n = 3 vessels). Data are expressed as means ± SE.

 
To directly test the involvement of smooth muscle KIR channels in NaN3-induced dilation, patch-clamp studies were performed by using freshly isolated vascular smooth muscle cells from arterioles of comparable size to those used in functional studies. KIR currents were elicited by voltage ramp or stepwise hyperpolarization or depolarization from a holding potential of –50 mV. With physiological concentrations of K+ in the bath, KIR currents were too small to be reliably recorded, as reported in other vascular smooth muscle preparations (25, 30, 32). Therefore, as in other studies (30, 32), recordings were typically made in 140 mM K+ (bath) to shift the reversal potential of the current to near 0 mV and amplify their magnitude at negative potentials. Basal KIR current, which rectified in the inward direction, was recorded as shown in Fig. 4A, and this KIR current was inhibited by 30 µM Ba2+ (Fig. 4A), as reported previously (25). The instantaneous current-voltage (I-V) relationship for coronary arteriolar smooth muscle KIR current was obtained by using a voltage ramp as shown in Fig. 4B. Note that larger K+ current was evoked at positive potentials, presumably reflecting the activation of KV and KCa channels. Figure 4C shows the effect of NaN3, with and without Ba2+, on the I-V relationship for a representative cell. Figure 4D summarizes the results from 10–14 cells (7 pigs) in terms of the magnitude of current at the test potential of –125 mV. NaN3 significantly increased the magnitude of Ba2+-sensitive KIR current.


Figure 4
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  		Fig. 4. A and B: KIR currents for a single pig coronary vascular smooth muscle cell were elicited by 200-ms voltage steps from –140 to +40 mV in 20 mV increments (A) or 200-ms voltage ramps from –140 to +60 mV (B) before and after 2-min application of Ba2+ (30 µM). C: KIR currents elicited by voltage ramps before and after 2-min application of NaN3 (50 µM) or NaN3 + Ba2+. D: average KIR current in cells after application of Ba2+ (n = 10 cells), NaN3 (n = 14 cells), or NaN3 + Ba2+ (n = 10 cells). All values were normalized to value of control current at test potential of –125 mV from a holding potential of –50 mV. Data are expressed as means ± SE. *P < 0.05 vs. control; {dagger}P < 0.05 vs. NaN3.

 
In another series of studies, the roles of phosphoinositide and tyrosine kinases, all of which are known to be involved in K+ channel regulation, in NaN3-induced vasodilation were examined. The PIP kinase inhibitor morin (6) significantly attenuated NaN3-induced vasodilation (Fig. 5), suggesting the involvement of this phosphoinositide kinase. However, vasodilation to NaN3 was not altered by the PI3-kinase inhibitor wortmannin (27) or a general tyrosine kinase inhibitor genistein (24, 29) (Fig. 5). It is noted that morin, wortmannin, and genistein did not alter resting vascular tone. Kuo’s laboratory (31) has previously shown that vasodilation in response to moderate increases in KCl concentration (5–20 mM) is mediated by KIR channel activation. To verify whether the attenuation of NaN3-induced dilation by morin is mediated via KIR channel blockade, the effect of this inhibitor on KCl-induced vasodilation was determined. Morin significantly attenuated the vasodilation in response to KCl (Fig. 6A). Subsequent administration of Ba2+ to the morin-treated vessels did not further reduce vasodilations to NaN3 (Fig. 5) or KCl (Fig. 6A). Morin did not exert a general impairment of vasodilator function because dilation to sodium nitroprusside in the presence of this inhibitor was not altered (Fig. 6B). On the other hand, ouabain and ODQ significantly reduced the vasodilation to sodium nitroprusside (Fig. 6B).


Figure 5
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  		Fig. 5. Effects of tyrosine kinase and phosphoinositide blockade on NaN3-induced dilation of isolated coronary arterioles. Vasodilatory responses to NaN3 were evaluated before and after incubation with tyrosine kinase inhibitor genistein (5 µM, n = 5 vessels), phosphoinositol 3-kinase inhibitor wortmannin (100 nM, n = 5 vessels), or phosphatidylinositolphosphate kinase inhibitor morin (20 µM, n = 10 vessels) alone or in combination with Ba2+ (30 µM, n = 5 vessels). Data are expressed as means ± SE. *P < 0.05 vs. control.

 

Figure 6
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  		Fig. 6. Effect of morin on KCl- and sodium nitroprusside-induced dilations of isolated coronary arterioles. A: vasodilation to KCl was evaluated before and after incubation with morin (20 µM, n = 9) alone or in combination with Ba2+ (30 µM, n = 5). B: vasodilation to sodium nitroprusside (SNP) was evaluated before and after incubation with morin (20 µM, n = 6), ouabain (1.5 µM, n = 5), or ODQ (0.1 µM, n = 4). Data are expressed as means ± SE. *P < 0.05 vs. control; {dagger}P < 0.05 vs. ouabain.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that NaN3 evokes endothelium-independent dilation of isolated porcine coronary arterioles. The vasodilatory response is mediated by activation of both Ba2+-sensitive KIR channels and guanylyl cyclase/Na+-K+-ATPase signaling pathway in the smooth muscle. These conclusions are based on functional data showing that the NaN3-induced dilation was insensitive to endothelial removal but was reduced by Ba2+, ouabain, or ODQ and was nearly abolished by Ba2+ in combination with either ouabain or ODQ. In addition, direct evidence for KIR channel involvement was provided by electrophysiological data showing that NaN3 enhanced Ba2+-sensitive KIR current in coronary arteriolar smooth muscle cells.

Previous studies have shown that intracoronary administration of NaN3 in vivo (37) or in isolated heart (11) elicits an increase in coronary blood flow. Although these findings suggested that NaN3 might act as a vasodilator of coronary resistance vessels, its vasomotor action was not unequivocally determined due to the possible confounding effects from cardiomyocytes. Using the isolated vessel approach, we demonstrated that NaN3 exhibits direct vasomotor activity in the coronary microvessels. Because the arteriolar network is the major site of vascular resistance, the dilation of coronary arterioles to NaN3 is expected to have a significant impact on coronary blood flow. It is likely that this acute vasodilatory response and accompanying blood flow increase could elevate the amount of NaN3 delivered to the myocardial tissue. As a result, prolonged exposure to elevated levels of NaN3 could lead to cardiac myocyte death by inhibiting mitochondrial respiration and increasing intracellular Ca2+ levels (5, 20). This idea is supported by previous reports (4) that lower doses of NaN3 trigger vasodilation, whereas higher doses inhibit mitochondrial cytochrome oxidase.

Several studies (23, 35, 40) in conduit blood vessels have shown that the vascular smooth muscle relaxation in response to NaN3 occurs through the activation of soluble guanylyl cyclase and elevation of cellular cGMP levels. Our results are consistent with a cGMP-mediated response in the microcirculation because the soluble guanylyl cyclase inhibitor ODQ attenuated the NaN3-induced dilation of coronary arterioles. Interestingly, recent evidence (37) suggests that the NaN3-induced cGMP-dependent vasodilation is mediated by smooth muscle hyperpolarization because a high K+ (100 mM)-depolarizing solution blocked the response in rat aortic rings. However, the specific signaling pathways contributing to the hyperpolarization in response to NaN3 remain unknown. Our results indicate that NaN3-induced vasodilation is mediated in part by activating electrogenic Na+-K+-ATPase. Indeed, similar to the effect of ODQ, the Na+-K+-ATPase inhibitor ouabain partially reduced the coronary arteriolar dilation to NaN3. Because subsequent administration of ODQ to ouabain-treated vessels did not further reduce the response, it appears that these two signaling pathways are arranged in series. Further support for this signaling cascade was provided by the ability of ouabain and ODQ to inhibit vasodilation in response to guanylyl cyclase activation by the NO donor sodium nitroprusside. As shown in some vascular beds, guanylyl cyclase activation is capable of increasing Na+-K+-ATPase activity (8, 28, 34). Therefore, it is likely that stimulation of guanylyl cyclase by NaN3 leads to the activation of Na+-K+-ATPase for vasodilation of coronary arterioles. Although we did not determine the precise mechanism triggering guanylyl cyclase, previous evidence (35, 37) suggests that catalase-dependent conversion of NaN3 to NO in the presence of oxygen may activate this enzyme.

Another hyperpolarizing mechanism, besides Na+-K+-ATPase activation, for NaN3-induced vasodilation is the opening of KIR channels. This contention is supported by the evidence that Ba2+, at a concentration shown to be specific for KIR channels (2, 31), inhibited the NaN3-induced vasodilatory response. In contrast, other K+ channel inhibitors (i.e., glibenclamide, iberiotoxin, charybdotoxin, apamin, and 4-aminopyridine) were ineffective in blocking the NaN3-induced vasodilation. We and other investigators have previously shown that the concentrations of specific inhibitors used in the present study are effective in blocking the cognate smooth muscle K+ channels (2, 15, 17, 31). Because the ODQ and ouabain combination had no additive inhibitory effect on vasodilation to NaN3 and the combination of Ba2+ with either ODQ or ouabain almost completely blocked NaN3-induced vasodilation, it appears that KIR channel and guanylyl cyclase/Na+-K+-ATPase activation are two independent pathways mediating vasodilation to NaN3.

Activation of the phosphoinositide kinases, PIP kinase and PI3 kinase, leads to the phosphorylation of phosphatidylinositol and the production of phosphatidylinositol-4,5-bisphosphate [Ptdins(4,5)P2] and phosphatidylinositol-3,4-bisphosphate [Ptdins(3,4)P2], respectively (9). Interestingly, KIR channels have been shown to be activated by Ptdins(4,5)P2 but not by Ptdins(3,4)P2 (33). In addition, the open probability of KIR channels can be increased by Ptdins(4,5)P2 via direct electrostatic interactions (19). Based on this information, it is expected that inhibition of Ptdins(4,5)P2 production by morin (6) should attenuate the NaN3 effect as well as the dilation to KIR channel activator KCl if the Ptdins(4,5)P2 signaling pathway leading to modulation of KIR channel activity is involved. Indeed, morin effectively inhibited both NaN3- and KCl-induced vasodilations as shown in the present study. The inability of subsequent application of Ba2+ to morin-treated vessels to further reduce the response to NaN3 and KCl further supports the idea that Ptdins(4,5)P2 activates KIR channels. In contrast, PI3-kinase signaling does not appear necessary for NaN3-induced vasodilation because this response remained intact in the presence of PI3-kinase inhibitor wortmannin.

A potential caveat of our findings is related to the action of morin, which has also been shown to affect tyrosine kinase, protein kinase B, and protein kinase C (3, 7) in addition to the inhibition of Ptdins(4,5)P2. Tyrosine kinases are also potential modulators of KIR channel activity because shear stress-induced KIR current is sensitive to the nonspecific tyrosine kinase inhibitor genistein (18). However, our results do not support the role of tyrosine kinase because genistein did not alter the NaN3-induced vasodilatory response. It is worth noting that the concentration of genistein (5 µM) employed in our study has been shown to effectively block both the flow-induced dilation and tyrosine phosphorylation of proteins in isolated porcine coronary arterioles (24). Protein kinase B and protein kinase C pathways also do not appear to be involved in the NaN3-induced vasodilation because their respective inhibitors, wortmannin (Fig. 5) and calphostin C (data not shown), did not alter this response. Therefore, the morin data do support the view that NaN3-induced dilation of coronary arterioles is mediated in part by increasing KIR channel activity through the Ptdins(4,5)P2 signaling pathway.

In summary, our data provide the first evidence that NaN3 can directly dilate porcine coronary arterioles and that the effect is mediated by the activation of both guanylyl cyclase/Na+-K+-ATPase and KIR channels in the smooth muscle. Furthermore, KIR channels appear to be regulated by phosphoinositide metabolism through PIP kinase. These findings implicate that NaN3 is a direct smooth muscle vasodilator in the coronary microcirculation, which may contribute to the development of severe cardiac dysfunction after prolonged exposure to this compound.


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 GRANTS
REFERENCES
 
This study was supported by an American Heart Association Scientist Development Grant (to T. W. Hein) and National Heart, Lung, and Blood Institute Grants HL-71761 (to L. Kuo) and HL-71796 (to M. J. Davis).


   ACKNOWLEDGMENTS
 
Present location of M. Davis: Dept. of Medical Pharmacology & Physiology, Univ. of Missouri School of Medicine, Columbia, MO.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. W. Hein, Dept. of Surgery, Scott & White Memorial Hospital, College of Medicine, The Texas A&M Univ. System Health Science Ctr., 702 Southwest H. K. Dodgen Loop, Temple, TX 76504 (e-mail: thein{at}tamu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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