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Cardiovascular Research 2002 56(1):154-163; doi:10.1016/S0008-6363(02)00504-7
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

The molecular site of action of KATP channel inhibitors determines their ability to inhibit iNOS-mediated relaxation in rat aorta

Andrew J Wilson* and Lucie H Clapp

Centre for Clinical Pharmacology and Therapeutics, Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK

* Corresponding author. Tel.: +44-20-7679-6203; fax: +44-20-7691-2838 andrew.wilson{at}ucl.ac.uk

{star} This work has previously been presented at the British Pharmacological Society Autumn and Winter Meetings, 2001.

Received 5 March 2002; accepted 27 May 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: ATP-sensitive potassium (KATP) channels are important modulators of vascular tone. Abnormal activation of these channels via over production of nitric oxide (NO) has been implicated in endotoxin-induced hypotension. However, based on studies with the sulphonylurea KATP channel inhibitor, glibenclamide, there is little evidence to support their role in mediating vasorelaxation to endotoxin in isolated blood vessels. In the present study, we investigated whether NO derived from inducible NO synthase (iNOS) modulates KATP channel function in rat aorta. Methods: Using standard organ bath techniques, the effects of structurally unrelated KATP channel inhibitors on the vasorelaxant responses to L-arginine (iNOS substrate), NO, levcromakalim (KATP channel opener) and forskolin were investigated in endothelium-denuded aortic rings exposed to endotoxin (lipopolysaccharide) for 4 h. Results: Relaxation evoked by L-arginine was unaffected by glibenclamide and the pinacidil-derived inhibitor, PNU-99963, but was significantly attenuated by the iNOS inhibitor, 1400W, as well as by PNU-37883A, Ba2+, 4-aminopyridine and tetraethylammonium, all known inhibitors of the KATP channel pore. In addition, endotoxin potentiated responses to levcromakalim and markedly reduced the efficacy of glibenclamide, and to a much lesser extent, PNU-37883A. Forskolin responses were unaffected by glibenclamide or PNU-37883A under control conditions, but were significantly potentiated following endotoxin treatment, an effect reversed by PNU-37883A, but not glibenclamide. Conclusion: KATP channels contribute to iNOS-mediated relaxation. However, the ability of sulphonylurea receptor-binding agents, but not those binding directly to the pore, to inhibit KATP channels, is greatly diminished in the presence of endotoxin.

KEYWORDS Endotoxins; K-ATP channel; Nitric oxide; Smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Application of bacterial endotoxin (lipopolysaccharide; LPS) to animals or to isolated blood vessels simulates many of the vascular defects associated with endotoxic shock, including hypotension and diminished responsiveness to vasoconstrictor agents [1]. Excess production of nitric oxide (NO) is a likely mediator, since LPS causes widespread expression of the inducible isoform of NO synthase (iNOS) and inhibitors of this enzyme substantially reverse vasorelaxation induced by LPS [2,3]. NO is thought to elicit smooth muscle relaxation primarily through stimulating soluble guanylyl cyclase (sGC) and elevating cyclic GMP (cGMP) [4]. This is consistent with the observation that inhibitors of sGC markedly attenuate relaxation to NO-donors and to LPS [3].

The downstream targets of NO are less well defined, although both the large conductance, Ca2+-activated K+ (BKCa) channel [5,6] and the ATP-sensitive K+ (KATP) channel [7] can be activated by NO and/or cGMP. Because K+ channels are major determinants of membrane potential and vascular tone [8], it is reasonable to suppose that following exposure to LPS, where NO levels are elevated, abnormal activation of K+ channels would result. Indeed in rat aorta, partial reversal (~20%) of LPS-induced hyporeactivity to the {alpha}-adrenergic agonist, phenylephrine was observed following treatment with iberiotoxin and charybdotoxin, both highly selective inhibitors of the BKCa channel [9,10]. Moreover, responses to LPS in the same tissue could be fully reversed by the non-selective K+ channel inhibitor, tetraethylammonium (TEA), suggesting the involvement of additional K+ channels [11].

Results from in vivo studies provide good evidence that KATP channels contribute to LPS-induced hypotension, since infusion of the classical KATP channel inhibitor, glibenclamide, causes transient restoration of blood pressure in rat, pig and canine models of endotoxic shock [12–14]. In contrast, glibenclamide, does not reverse vascular hyporeactivity in rat aorta in vitro [9], perhaps a surprising result given that KATP channels are a major determinant of membrane potential in this tissue [15]. However, application of extracellular L-arginine, the substrate for iNOS, does activate KATP channels in coronary arterial cells exposed to LPS for 6 h [16].

KATP channels are formed from pore-forming (Kir6.x) and sulphonylurea receptor (SUR) subunits [17]. One possible explanation for the disparate effects of glibenclamide is that LPS alters the communication between these two subunits. Indeed, such a situation exists following metabolic inhibition, which renders KATP channels insensitive to glibenclamide [18]. Therefore, the present study was designed to compare the effectiveness of ‘SUR-binding’ (glibenclamide, tolbutamide or the pinacidil derivative, PNU-99963) and ‘pore-blocking’ (PNU-37883A, Ba2+, and 4-aminopyridine) KATP channel inhibitors [19,20], against a range of vasodilators in control and LPS-treated rat aorta.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Male Sprague–Dawley rats (175–225 g) were humanely killed by cervical dislocation. This procedure conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The thoracic aorta was removed and placed into physiological salt solution (PSS) containing in mM: NaCl 112, KCl 5, MgCl2 1, NaHCO3 25, NaH2PO4 0.5, KH2PO4 0.5, glucose 10, CaCl2 1.8, phenol red 0.03, pH 7.4 with 95% O2/5% CO2. The vessels were cleaned of adipose and connective tissue, and cut into rings approximately 2 mm in length.

2.1 Tension studies
Aortic rings were mounted in organ baths containing continuously gassed PSS. Isometric tension was measured using a Grass transducer (FT-03; Grass Instrument Company, USA) and tissues equilibrated for 30 min under 2 g resting tension, after which tension was adjusted to 1 g. Rings were contracted twice with phenylephrine (1 µM; PE), separated by a 20-min washout period. For LPS experiments, tissues were incubated with Salmonella typhosa LPS (1 µg ml–1; 4 h), followed by denudation of the endothelium. Successful removal was confirmed by the lack of response to the endothelium-dependent vasorelaxant, acetylcholine (5 µM). Subsequently, rings were contracted with PE (1 µM) and allowed to plateau before addition of K+ channel inhibitors; KCa channels—iberiotoxin (50 nM), charybdotoxin (100 nM) and apamin (100 nM); KATP channels—glibenclamide (10 µM), tolbutamide (0.5–1 mM), PNU-37883A (1 µM) and PNU-99963 (1 µM); voltage-gated (KV) channels—4-aminopyridine (5 mM); non-selective K+ channel inhibitor—TEA (10 mM). These inhibitors, or modulators of the NO/cGMP pathway were pre-incubated for 20 min. In the case of N-(3(aminomethyl)benzyl)acetamidine (1400W; 10 µM), this was added with PE because it is a relatively slow inhibitor of iNOS [21]. Concentration–response curves to authentic NO, the S-nitrosothiol, S-nitroso-D,L-acetylpenicillamine (SNAP), the KATP channel opener, levcromakalim, or the adenylate cyclase activator, forskolin, were constructed (ranging from 10–10 to 10–5 M) in the absence and presence of LPS. Concentration–response curves were also constructed to L-arginine (hydrochloride salt; 0.3–300 µM).

2.2 Nitrite determination
Endothelium-denuded aortic rings (2 mm in length) were incubated in 12-well dishes containing Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C for 18 h in a CO2 (5%) incubator in the absence and presence of LPS (1 µg ml–1) and the appropriate inhibitor. Nitrite (NO2–) produced by the aortic strips was determined using the Griess reaction [22] and normalised to protein levels using the Lowry method.

2.3 Chemicals
Authentic NO solutions were prepared as previously described. Under these conditions, the NO stock solution was estimated to be ~1.75 mM [23]. All drugs were prepared and diluted in water, with the exception of forskolin, levcromakalim, SNAP, 1H- [1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), tolbutamide, PNU-99963 and PNU-37883A, which were initially dissolved in dimethyl sulphoxide (DMSO). Glibenclamide was prepared in 50% (v/v) DMSO/polyethylene glycol. The final concentration of DMSO in the bath did not exceed 0.2%, which had no effect on PE contractions. 1400W and ODQ were purchased from Alexis Corporation (Bingham, Nottingham, UK), iberiotoxin, apamin and charybdotoxin from Alomone (Jerusalem, Israel). PNU-37883A and PNU-99963 were gifts from Pharmacia–Upjohn (Kalamazoo, USA) and levcromakalim a gift from GlaxoSmithKline (Harlow, Essex, UK). All other chemicals were from Sigma–Aldrich (Poole, Dorset, UK).

2.4 Data and statistical analysis
Responses to vasorelaxants are expressed as % relaxation of the PE contraction observed immediately prior to their application, and presented as mean±S.E.M. of n observations. The concentration of agent causing a 50% inhibition of the maximal relaxation (Emax), is expressed as the mean pEC50 value, being derived from each experiment using the sigmoidal-curve fitting routine in Origin 6.0 (Microcal Software, Northampton, MA, USA). Statistical analysis was carried out using Student’s t-test, one-way, or two-way repeated measures, ANOVA, with corrections made for comparisons against control groups (Bonferroni t-test) or pair-wise comparisons (Student–Newman–Keuls method). Differences were considered statistically significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effects of K+ channel inhibitors on L-arginine-induced relaxation
Treatment of rat aortic rings with LPS (1 µg ml–1; 4 h) caused a 20% reduction in the magnitude of the PE contraction from 2.35±0.1 to 1.89±0.1 g (P<0.001, n=46–63). Application of increasing concentrations (0.3–300 µM) of L-arginine to LPS-treated, aortic rings produced a concentration-dependent relaxation of PE-induced tone, with an Emax of 65.6±3% (n=28, Figs. 1A and 2AGo). Pre-treatment of tissues with the iNOS inhibitor, 1400W (10 µM), or the sGC inhibitor, ODQ (3 µM), severely attenuated Emax to 31.4±5% (n=4, P<0.001) and 4.8±3% (n=3; P<0.001), respectively (Figs. 1B and 2AGo). In the absence of LPS, L-arginine produced no relaxation (not shown, see Ref. [11]).


Figure 1
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  		Fig. 1 Representative recordings of L-arginine-induced effects on phenylephrine-induced contractions in LPS-treated (1 µg ml–1, 4 h) aortic rings, in the absence (A) and presence (B) of ODQ (3 µM).

 

Figure 2
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  		Fig. 2 Concentration-dependent effects of L-arginine on phenylephrine (1 µM) contractions in endothelium-denuded rat aortic rings treated with LPS. Responses obtained in the absence and presence of the soluble guanylyl cyclase inhibitor, ODQ and iNOS inhibitor, 1400W (A) and in absence and presence of the K+ channel inhibitors, iberiotoxin (IbTx), charybdotoxin (ChTx), apamin and TEA (B). Mean effects of structurally dissimilar KATP channel inhibitors on relaxation to L-arginine are shown for SUR-binding (C) and pore-binding (D) inhibitors. Data are expressed as mean±S.E.M. (n=3–7).

 
The effects of a variety of K+ channel inhibitors were assessed. Selective inhibitors of either BKCa channels (iberiotoxin 50 nM, or charybotoxin; 100 nM), or small-conductance Ca2+-activated K+ channels (apamin; 100 nM) did not significantly reduce relaxation evoked by L-arginine (Fig. 2B). However, L-arginine responses were highly sensitive to inhibition by 10 mM TEA (Emax, 18.3±3.1 vs. 63.9±2.7% for control; n=7, P<0.001).

Inhibitors of KATP channels were also assessed for effects on L-arginine responses (Fig. 2). Inhibitors were classified according to their primary site of action, either the SUR (Fig. 2C), or the pore (Fig. 2D). The sulphonylurea agents glibenclamide (10 µM) and tolbutamide (500 µM) had no significant effect on relaxation induced by L-arginine, nor did the pinacidil derivative, PNU-99963 (1 µM). In contrast, pore-binding inhibitors, PNU-37883A (1 µM), Ba2+ (100 µM), 4-aminopyridine (4-AP; 5 mM) and higher concentrations of tolbutamide (1 mM) all significantly inhibited relaxation, reducing Emax from 71.8±5.1% (n=7) to 44.1±4.3% (P<0.01, n=7) for PNU-37883A, 36.2±3.2% (P<0.001, n=5) for 4-AP, 32.1±8.0% (P<0.005, n=5) for Ba2+, and 24.9±4.9% (P<0.001, n=4) for tolbutamide (Fig. 2D). Increasing the Ba2+ concentration to 300 µM had no additional effect (not shown). Glibenclamide did reduce contractions to PE from 2.18±0.16 to 1.89±0.15 g (P<0.001, n=13) in the presence of LPS, although it had no effect under control conditions. Conversely, PNU-37883A, did not significantly alter the PE contraction in either LPS-treated or control tissues, causing a 1.1±1.6% (n=18) and 2.1±1.3% (n=12) decrease in tone, respectively.

3.2 Effects of K+ channel inhibitors on nitrite (NO2–) accumulation
It is feasible that some of the effects of K+ channel inhibitors on tension could be related to non-specific actions, such as inhibition of iNOS induction or activity. To assess this, NO production was measured by determining NO2– levels in the supernatant of rat aortic rings incubated with LPS for several hours. LPS significantly increased NO2– in the culture medium by 1.07±0.1 µM mg protein–1 (P<0.001, n=7, Table 1). This increase was fully inhibited by pre-incubation with 1400W (10 µM), but was not significantly affected by ODQ (3 µM), glibenclamide (10 µM), PNU-37883A (1 µM) or Ba2+ (100 µM) (n=7). In contrast, both tolbutamide (1 mM) and TEA (10 mM) significantly inhibited NO2– production (n=7, P<0.001). In separate experiments, levcromakalim caused a small, but insignificant (P=0.391, n=6) reduction in nitrite accumulation in LPS-treated tissues.


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  		Table 1 Effects of K+ channel inhibitors on nitrite accumulation in endothelium denuded rat aortic rings pretreated with LPS

 
3.3 Characterisation of relaxation to authentic NO
Bath application of increasing concentrations of authentic NO to endothelium-denuded, aortic rings produced a rapid concentration-dependent inhibition of PE contractions. Relaxation typically peaked within 10–15 s, with 10 µM NO inducing almost complete reversal of PE-induced tone (Emax, 98.9±1%, pEC50, 6.95±0.05; n=34). These values were essentially unchanged if tissues were pre-incubated with LPS (n=32; Fig. 3A). The effects of NO were abolished by the NO scavenger oxyhaemoglobin (30 µM), reducing Emax to 2.3±3% (P<0.001, n=5) and 4.0±1% (P<0.001, n=5) for control and LPS-treated tissues, respectively. Similarly, ODQ (10 µM) severely attenuated (P<0.001) responses to NO under both conditions (n=5, Fig. 3A), though unlike responses to L-arginine, relaxation was not fully inhibited at the higher NO (>1 µM) concentrations. In LPS-treated tissues, the NO synthase inhibitor, L-NG-nitro-arginine methyl ester (L-NAME, 300 µM, n=5) had no significant effect on NO responses (pEC50 7.18±0.13 in control and 6.88±0.09 in LPS, n=5). However, both ODQ and L-NAME increased PE contractions by 10.5±3.6% (n=5) and 15.4±1.8% (n=5), respectively.


Figure 3
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  		Fig. 3 Mean concentration–response curves to authentic NO (A) in the presence and absence of LPS (1 µg ml–1; 4 h) and ODQ or oxyhaemoglobin (OxyHb, n=5–34). (B) The effects of the K+ channel inhibitors, 4-aminopyridine (4-AP), tetraethylammonium ions (TEA) and barium (Ba2+) on NO responses in the presence of LPS (n=8). Responses to SNAP were assessed in control (C) and LPS-treated (1 µg ml–1; 4 h; D) aortic rings in the absence and presence of glibenclamide and PNU-37883A (n=4). Data are expressed as mean±S.E.M of n observations.

 
To assess the contribution of K+ channels to NO generated exogenously, concentration–response curves to NO in tissues treated with LPS were constructed in the absence and presence of a variety of KCa, KATP or KV channel inhibitors. Table 1 shows that inhibitors of KCa channels, ChTx, IbTx and apamin, had no apparent effect on relaxations to NO. In contrast to the substantial inhibitory effect of TEA on L-arginine-induced relaxations, this agent had essentially no effect on responses to NO (Table 2, Fig. 3B). Moreover, the effect of Ba2+ (100 µM) and 4-AP (5 mM) was weak (Fig. 3B), with no reduction in Emax, although there was a small, but significant shift to the right in the pEC50 value for Ba2+ (Table 2; P<0.05, n=8). Glibenclamide (10 µM) also had no effect on relaxation induced by NO at any concentration (n=8, Table 2).


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  		Table 2 The effects of K+ channel inhibitors on relaxation induced by authentic NO in endothelium-denuded, LPS-treated rat aortic rings; comparison of pEC50 values and maximal relaxation (Emax) obtained in the absence (control) and presence of inhibitors

 
3.4 Effects of KATP channel inhibitors on relaxation induced by SNAP
Concentration–response curves were constructed using the S-nitrosothiol, SNAP (0.001–10 µM). Under control conditions, SNAP produced full relaxation of PE-contracted tissues with a pEC50 of 6.54±0.1 (n=4) which was essentially unaffected by LPS treatment (pEC50, 6.74±0.1, n=4). Under control conditions, neither glibenclamide, nor PNU-37883A had any significant effect on the concentration–response curve to SNAP (Fig. 3C). However, following incubation with LPS, PNU-37883A significantly inhibited relaxation shifting the pEC50 to 6.08±0.2 (P<0.05, n=4), although glibenclamide was still without effect (Fig. 3D).

3.5 Effects of LPS on responses to levcromakalim
Levcromakalim (0.001–10 µM) induced concentration-dependent relaxation of PE-contracted aortic rings with a pEC50 of 6.61±0.03 for control tissues, which was significantly potentiated (P<0.001, Fig. 4A) to 6.99±0.07 in the presence of LPS (n=12). Furthermore, incubation with LPS significantly increased Emax from 56.4±3.8 to 84.4±2.8% (P<0.001, n=12).


Figure 4
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  		Fig. 4 LPS potentiates relaxations to the KATP channel opener, levcromakalim. Effect of levcromakalim in endothelium-denuded rat aortic rings under control conditions and after treatment with LPS (A; n=12) and in a separate series of experiments in the absence and presence of 1400W (10 µM, 45 min, n=4) (B). The mean effects of LPS on the efficacy of glibenclamide (C) and PNU-37883A (D) against levcromakalim-induced relaxation in endothelium-denuded rat aortic rings are shown (n=5). Data are presented as mean±S.E.M.

 
To elucidate whether the potentiation of levcromakalim-induced relaxation by LPS was mediated by iNOS, rings were pre-treated with 1400W (10 µM). Under control conditions, relaxations to levcromakalim were essentially insensitive to 1400W, with Emax and pEC50 being effectively unchanged (Fig. 4B). In contrast, following LPS incubation, 1400W caused a significant rightward shift in the levcromakalim concentration–response curve, from 7.18±0.05 to 6.70±0.03 (P<0.001, n=4) (Fig. 4B). The latter was not significantly different from that obtained under control conditions in the absence of LPS.

To determine whether potentiation was mediated directly by NO or indirectly via the cGMP pathway, the effect of the sGC inhibitor ODQ (3 µM) was investigated. Under control conditions, ODQ had a small potentiating effect on the concentration–response curve and increased Emax from 62.1±2.8 to 77.6±3.2% (P<0.05, n=4, Fig. 5). Under LPS conditions, pre-incubation with ODQ now caused a significant rightward shift in the levcromakalim response from a pEC50 of 7.18±0.05 to 6.78±0.08 (n=4, P<0.01) but did not suppress Emax (Fig. 5). Thus, ODQ did not reverse the effects of LPS on levcromakalim responses to the same extent as that observed with 1400W.


Figure 5
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  		Fig. 5 The effects of ODQ on the response to levcromakalim in control and LPS-treated rat aortic rings (n=3). Data are presented as mean±S.E.M.

 
3.6 Effects of KATP channel inhibitors on relaxation to levcromakalim
Both PNU-37883A (1 µM) and glibenclamide (10 µM) effectively abolished responses to levcromakalim under control conditions (Fig. 4C). Interestingly, following exposure to LPS, the inhibitory effects of both agents were diminished (P<0.005, n=5, Fig. 4D). Glibenclamide, inhibited the maximal response to levcromakalim by 74.2±14.1% in control tissues, while in the presence of LPS, it only inhibited the maximal response by 39.1±12.9% (n=5). PNU-37883A also failed to completely abolish relaxation in the presence of LPS, although it appeared a more effective inhibitor. Under control conditions, PNU-37883A inhibited the maximum relaxation to levcromakalim by 87.3±3.5%, while in LPS-treated tissues, it inhibited the maximal response by 62.2±8.8% (n=5). To determine whether the relative lack of effectiveness of glibenclamide in the presence of LPS resulted from activation of the iNOS/cGMP-dependent pathway, tissues were incubated with 1400W or ODQ. In the presence of ODQ or 1400W, glibenclamide fully inhibited the maximal relaxation to levcromakalim by 97.0±3.1% (n=3) and 86.6±0.7% (n=3) for ODQ and 1400W, respectively.

3.7 Forskolin-induced relaxation under control and LPS conditions
To determine whether LPS potentiated responses to other vasodilators, experiments were carried out using forskolin. Under control conditions, forskolin induced full relaxation (Emax, 99.2±0.8%, n=5), although it was significantly (P<0.001) more potent in the presence of LPS, with over a log-fold shift in potency (pEC50, 6.68±0.17 and 7.92±0.15 for control and LPS, respectively; n=5, Fig. 6A). Application of PNU-37883A had no significant effect on forskolin responses in control tissues (Fig. 6B), although in the presence of LPS, it caused a significant rightward shift in pEC50 to 7.22±0.11 (P<0.005, n=5, Fig. 6C), which was now not significantly different (P=0.07) from control responses. In contrast, glibenclamide had a negligible effect on pEC50 under both conditions (Fig. 6B,C).


Figure 6
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  		Fig. 6 The effects of LPS on the concentration–response curve to the adenylyl cyclase activator, forskolin (A). The effects of glibenclamide and PNU-37883A are shown in the absence (B) and presence (C) of LPS. Data are expressed as mean±S.E.M (n=5).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The major finding of the present study is that KATP channels appear to mediate iNOS-induced relaxation in rat aorta. This is based on the finding that PNU-37883A, a putative smooth-muscle selective KATP channel inhibitor [24] and Ba2+, a non-selective KATP channel inhibitor, substantially inhibited relaxation to the iNOS substrate, L-arginine, while having essentially no effect on LPS-induced NO production. Interestingly, the effects of K+ channel inhibitors on relaxant responses to L-arginine and SNAP could not be mimicked by exogenous application of authentic NO, which induced relaxation that was largely insensitive to a wide range of K+ channel inhibitors. Furthermore, LPS altered KATP channel pharmacology, such that the relaxant effects of levcromakalim were potentiated and inhibitors binding primarily to SUR were significantly less effective at inhibiting responses to L-arginine and levcromakalim. Moreover, KATP channels appeared to contribute to forskolin-induced relaxation only in the presence of LPS, where again responses were sensitive to PNU-37883A, but not glibenclamide.

Although several studies have concluded that KATP channels mediate hypotension induced by LPS in vivo [1], glibenclamide does not reverse LPS-induced hyporeactivity to phenylephrine in vitro [9,14]. This latter finding is analogous to our results where, neither glibenclamide, nor PNU-99963, inhibited L-arginine-induced relaxation. In fact, we found that glibenclamide actually caused a small reduction in PE-induced tone, as noted previously [9,14]. The reason for this is unclear, although glibenclamide is known to antagonise prostanoid-induced contractions [26], and we have previously found that inhibition of prostaglandin synthesis with a selective cyclooxygenase 2 inhibitor reduces PE contractions in the presence of LPS [27]. However, glibenclamide has been shown to partially reverse LPS-induced vascular hyporeactivity [28] and membrane hyperpolarisation [14,25] ex vivo, although the effects in the former study were concluded to result from inhibition of iNOS induction. However, the lack of effect of glibenclamide on LPS-induced nitrite accumulation does not support a role for inhibition of iNOS in our studies.

One possible explanation for these seemingly contradictory results in vivo versus in vitro relates to the concentration of glibenclamide used, which in vivo has been estimated to exceed 100 µM [28]. At these higher concentrations, additional mechanisms may be involved in the observed effects on blood pressure, such as iNOS inhibition [28]. Alternatively, glibenclamide may directly inhibit the KATP channel pore, via a low-affinity sulphonylurea binding site [29]. This is consistent with our finding that PNU-37883A, a structurally unrelated KATP channel inhibitor that binds directly to the pore [20,30], was an effective inhibitor of L-arginine-induced relaxation, as were other inhibitors of the channel pore, Ba2+ and 4-AP [8]. Differences could also be due to the types of vessel studied in vitro, compared with those responsible for controlling blood pressure. However, we think this a less likely conclusion since we have observed similar differential pharmacological responses to KATP inhibitors in LPS-treated rat mesenteric artery [30]. The effects of PNU-37883A are unlikely to be related to inhibition of other K+ channels since at the concentration used, this agent has no effect on KV or inward rectifier K+ channels [24]. Moreover, PNU-37883A did not significantly affect either the magnitude of PE contractions nor forskolin responses in control tissues (see also Ref. [31]) suggesting that it does not interfere with the contractile process.

Therefore, we believe that the most likely explanation for the striking difference between the effects of glibenclamide and PNU-37883A is due to their different sites of action (SUR vs. pore). It has been previously shown in rat aorta that cytoskeletal disruption results in a loss of high-affinity glibenclamide binding [32]. Given that immunohistochemical studies show that exposure of cultured aortic smooth muscle cells to NO causes actin depolymerisation [33], we hypothesise that LPS may cause NO-dependent disruption of the cytoskeleton, which would render SUR agents ineffective, whilst leaving pore-blockers relatively unaffected. Consistent with this, we observed that in the presence of LPS, glibenclamide was significantly less effective than PNU-37883A at inhibiting SNAP, levcromakalim and forskolin relaxation.

Additional evidence for the involvement of KATP channels comes from our observation that LPS significantly potentiates levcromakalim-induced relaxation. The iNOS pathway probably mediates this since both 1400W, and to a lesser extent, ODQ, reversed potentiation. Similar findings have been reported in ex vivo models of LPS-induced hyporeactivity [10,14]. Potentiation can also be observed in the absence of LPS with NO donors and the cGMP analogue, 8-bromo-cGMP [34]. This implies that cGMP modulates the effects of KATP channel openers, possibly via phosphorylation of the channel or closely associated proteins. The processes responsible for the potentiation of control levcromakalim responses by ODQ cannot easily be explained and remain to be determined. Potentiation of forskolin responses by LPS was also observed, an effect that was substantially reversed by PNU-37883A. This suggests some synergy between the cAMP and cGMP pathways in activating KATP channels, although mechanisms remain unclear.

Relaxation to L-arginine was completely abolished by ODQ, suggesting that effects are mediated by cGMP. A small proportion of the L-arginine-induced relaxation could be mediated by BKCa channels, since iberiotoxin and charybdotoxin, caused a small reduction in relaxation. This is consistent with previous findings showing BKCa channel inhibitors to partially reverse vascular hyporeactivity [9,10]. Electrophysiological data also support a role for BKCa channels, where application of L-arginine to LPS-treated cultured coronary vascular smooth muscle cells resulted in persistent activation of BKCa channels [35]. Interestingly, only transient increases in BKCa current were seen with an NO donor, suggesting that the iNOS pathway may produce different biological responses from exogenously NO.

In the presence of LPS, responses to NO in endothelium-denuded tissues, were only weakly affected by either Ba2+ or 4-AP. Interestingly, TEA which had large effects on L-arginine responses, was without effect. Thus, activation of K+ channels do not appear to be the primary mechanism of relaxation for authentic NO, even though like L-arginine, effects were virtually abolished by ODQ. The reason for these differences could be related to a number of factors, such as the type of NO species generated, the site and rate of NO/cGMP production. Results with SNAP would support this idea, since unlike authentic NO, relaxation in the presence of LPS could be inhibited by PNU-37883A. It is possible that the effects of K+ channel inhibitors on tension may be related to inhibition on L-arginine transport since it can be activated by membrane hyperpolarisation and inhibited by depolarisation [36]. However, apart from tolbutamide and TEA, the other KATP channel inhibitors had no significant effect on LPS-induced nitrite accumulation. Thus, inhibition of L-arginine uptake might underlie part of the inhibitory action of TEA and tolbutamide on L-arginine-induced relaxation. This might explain why both tolbutamide and TEA inhibited L-arginine-induced relaxation to a greater extent than either PNU-37883A or Ba2+. Similarly, levcromakalim responses might potentiate NO release by activating L-arginine uptake, although lack of any significant effect on nitrite accumulation would suggest this not to be the case.

In summary, our results suggest that in rat aorta, activation of the iNOS pathway by LPS results in a significant alteration in the pharmacology of both activators and inhibitors of KATP channels. We found that inhibitors that bind directly to the KATP channel pore, were significantly more effective at inhibiting relaxation induced by L-arginine, SNAP, levcromakalim and forskolin than the classical sulphonylurea-based inhibitor, glibenclamide. The molecular mechanisms responsible for these changes are presently unknown, although we suggest that LPS via NO/cGMP may uncouple/alter the communication between the pore and the SUR.

Time for primary review 21 days.


   Acknowledgements
 
LHC is a MRC Senior Research Fellow in Basic Science. This work was supported by the Medical Research Council (G117/180). The authors would like to thank Professor Martin Feelisch for help in the preparation of authentic NO solutions, Jonathan Alis for technical assistance and Dr Stephen Humphrey (Pharmacia–Upjohn, USA) for helpful comments on the manuscript.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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