their ability to inhibit inos-mediated relaxation in rat aorta

Transcription

1 Cardiovascular Research 56 (00) locate/ cardiores The molecular site of action of KATP channel inhibitors determines q their ability to inhibit inos-mediated relaxation in rat aorta * Andrew J. Wilson, Lucie H. Clapp Centre for Clinical Pharmacology and Therapeutics, Department of Medicine, University College London, 5University Street, London WCE 6JJ, UK Received 5 March 00; accepted 7 May 00 Abstract Objective: ATP-sensitive potassium (K ATP) 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 K ATP 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, 400W, as well as by PNU-37883A, Ba, 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: K ATP 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. 00 Elsevier Science B.V. All rights reserved. Keywords: Endotoxins; K-ATP channel; Nitric oxide; Smooth muscle. Introduction ly reverse vasorelaxation induced by LPS [,3]. NO is thought to elicit smooth muscle relaxation primarily Application of bacterial endotoxin (lipopolysaccharide; through stimulating soluble guanylyl cyclase (sgc) and LPS) to animals or to isolated blood vessels simulates elevating cyclic GMP (cgmp) [4]. This is consistent with many of the vascular defects associated with endotoxic the observation that inhibitors of sgc markedly attenuate shock, including hypotension and diminished responsive- relaxation to NO-donors and to LPS [3]. ness to vasoconstrictor agents []. Excess production of The downstream targets of NO are less well defined, nitric oxide (NO) is a likely mediator, since LPS causes although both the large conductance, Ca -activated K widespread expression of the inducible isoform of NO (BK Ca) channel [5,6] and the ATP-sensitive K (K ATP) synthase (inos) and inhibitors of this enzyme substantial- 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 q This work has previously been presented at the British Pharmaco- that following exposure to LPS, where NO levels are logical Society Autumn and Winter Meetings, 00. *Corresponding author. Tel.: ; fax: elevated, abnormal activation of K channels would result address: andrew.wilson@ucl.ac.uk (A.J. Wilson). Time for primary review days /0/$ see front matter 00 Elsevier Science B.V. All rights reserved. PII: S (0) Downloaded from on 30 January 08

2 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) Indeed in rat aorta, partial reversal ( 0%) of LPS-induced Rings were contracted twice with phenylephrine ( mm; hyporeactivity to the a-adrenergic agonist, phenylephrine PE), separated by a 0-min washout period. For LPS was observed following treatment with iberiotoxin and experiments, tissues were incubated with Salmonella typh- charybdotoxin, both highly selective inhibitors of the BKCa osa LPS ( mg ml ; 4 h), followed by denudation of the channel [9,0]. Moreover, responses to LPS in the same endothelium. Successful removal was confirmed by the tissue could be fully reversed by the non-selective K lack of response to the endothelium-dependent vasorelaxchannel inhibitor, tetraethylammonium (TEA), suggesting ant, acetylcholine (5 mm). Subsequently, rings were the involvement of additional K channels []. contracted with PE ( mm) and allowed to plateau before Results from in vivo studies provide good evidence that addition of K channel inhibitors; KCa channels KATP channels contribute to LPS-induced hypotension, iberiotoxin (50 nm), charybdotoxin (00 nm) and apamin since infusion of the classical KATP channel inhibitor, (00 nm); KATP channels glibenclamide (0 mm), tolglibenclamide, causes transient restoration of blood pres- butamide (0.5 mm), PNU-37883A ( mm) and PNUsure in rat, pig and canine models of endotoxic shock ( mm); voltage-gated (K V) channels 4-amino- [ 4]. In contrast, glibenclamide, does not reverse pyridine (5 mm); non-selective K channel inhibitor vascular hyporeactivity in rat aorta in vitro [9], perhaps a TEA (0 mm). These inhibitors, or modulators of the surprising result given that KATP channels are a major NO/ cgmp pathway were pre-incubated for 0 min. In the determinant of membrane potential in this tissue [5]. case of N-(3(aminomethyl)benzyl)acetamidine (400W; 0 However, application of extracellular L-arginine, the sub- mm), this was added with PE because it is a relatively strate for inos, does activate KATP channels in coronary slow inhibitor of inos []. Concentration response arterial cells exposed to LPS for 6 h [6]. curves to authentic NO, the S-nitrosothiol, S-nitroso-D,L- KATP channels are formed from pore-forming (Kir6.x) acetylpenicillamine (SNAP), the KATP channel opener, and sulphonylurea receptor (SUR) subunits [7]. One levcromakalim, or the adenylate cyclase activator, forpossible explanation for the disparate effects of gliben- 0 5 skolin, were constructed (ranging from 0 to 0 M) clamide is that LPS alters the communication between in the absence and presence of LPS. Concentration rethese two subunits. Indeed, such a situation exists follow- sponse curves were also constructed to L-arginine (hydroing metabolic inhibition, which renders KATP channels chloride salt; mm). insensitive to glibenclamide [8]. Therefore, the present study was designed to compare the effectiveness of SUR-.. Nitrite determination binding (glibenclamide, tolbutamide or the pinacidil derivative, PNU-99963) and pore-blocking (PNU-37883A, Endothelium-denuded aortic rings ( mm in length) Ba, and 4-aminopyridine) KATP channel inhibitors were incubated in -well dishes containing Dulbecco s [9,0], against a range of vasodilators in control and modified Eagle s medium (DMEM) at 37 8C for 8 h in a LPS-treated rat aorta. CO (5%) incubator in the absence and presence of LPS ( mg ml ) and the appropriate inhibitor. Nitrite (NO ) produced by the aortic strips was determined using the. Methods Griess reaction [] and normalised to protein levels using the Lowry method. Male Sprague Dawley rats (75 5 g) were humanely killed by cervical dislocation. This procedure conforms to.3. Chemicals the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Authentic NO solutions were prepared as previously Publication No. 85-3, revised 996). The thoracic aorta described. Under these conditions, the NO stock solution was removed and placed into physiological salt solution was estimated to be.75 mm [3]. All drugs were (PSS) containing in mm: NaCl, KCl 5, MgCl, prepared and diluted in water, with the exception of NaHCO3 5, NaHPO4 0.5, KHPO4 0.5, glucose 0, forskolin, levcromakalim, SNAP, H- [,,4]-oxadiazolo- CaCl.8, phenol red 0.03, ph 7.4 with 95% O / 5% CO [4,3-a]-quinoxalin--one (ODQ), tolbutamide, PNU The vessels were cleaned of adipose and connective tissue, and PNU-37883A, which were initially dissolved in diand cut into rings approximately mm in length. methyl sulphoxide (DMSO). Glibenclamide was prepared in 50% (v/v) DMSO/polyethylene glycol. The final con-.. Tension studies centration of DMSO in the bath did not exceed 0.%, which had no effect on PE contractions. 400W and ODQ were purchased from Alexis Corporation (Bingham, Not- tingham, UK), iberiotoxin, apamin and charybdotoxin from Alomone (Jerusalem, Israel). PNU-37883A and PNU were gifts from Pharmacia Upjohn (Kalamazoo, USA) and levcromakalim a gift from GlaxoSmithKline 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 g resting tension, after which tension was adjusted to g. Downloaded from on 30 January 08

3 56 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) (Harlow, Essex, UK). All other chemicals were from Sigma Aldrich (Poole, Dorset, UK)..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 mean6s.e.m. of n observations. The concentration of agent causing a 50% inhibition of the maximal relaxation (E max), 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, Results 3.. Effects of K channel inhibitors on L-arginineinduced relaxation Treatment of rat aortic rings with LPS ( mg ml ;4h) caused a 0% reduction in the magnitude of the PE contraction from to g(p,0.00, n ). Application of increasing concentrations ( Fig.. Representative recordings of L-arginine-induced effects on mm) of L-arginine to LPS-treated, aortic rings produced a phenylephrine-induced contractions in LPS-treated ( mg ml, 4 h) aortic rings, in the absence (A) and presence (B) of ODQ (3 mm). concentration-dependent relaxation of PE-induced tone, with an Emax of % (n58, Figs. A and A). Pre-treatment of tissues with the inos inhibitor, 400W and higher concentrations of tolbutamide ( mm) all (0 mm), or the sgc inhibitor, ODQ (3 mm), severely significantly inhibited relaxation, reducing Emax from attenuated Emax to 3.465% (n54, P,0.00) and 4.863% % (n57) to % (P,0.0, n57) for (n53; P,0.00), respectively (Figs. B and A). In the PNU-37883A, % (P,0.00, n55) for 4-AP, absence of LPS, L-arginine produced no relaxation (not % (P,0.005, n55) for Ba, and % shown, see Ref. []). (P,0.00, n54) for tolbutamide (Fig. D). Increasing the The effects of a variety of K channel inhibitors were Ba concentration to 300 mm had no additional effect assessed. Selective inhibitors of either BKCa channels (not shown). Glibenclamide did reduce contractions to PE (iberiotoxin 50 nm, or charybotoxin; 00 nm), or small- from to g (P,0.00, n53) in the conductance Ca -activated K channels (apamin; 00 presence of LPS, although it had no effect under control nm) did not significantly reduce relaxation evoked by conditions. Conversely, PNU-37883A, did not significantly L-arginine (Fig. B). However, L-arginine responses were alter the PE contraction in either LPS-treated or control highly sensitive to inhibition by 0 mm TEA (E max, tissues, causing a.6.6% (n58) and.6.3% (n vs % for control; n57, P,0.00). ) decrease in tone, respectively. Inhibitors of KATP channels were also assessed for effects on L-arginine responses (Fig. ). Inhibitors were 3.. Effects of K channel inhibitors on nitrite (NO ) classified according to their primary site of action, either accumulation the SUR (Fig. C), or the pore (Fig. D). The sulphonylurea agents glibenclamide (0 mm) and tolbutamide It is feasible that some of the effects of K channel (500 mm) had no significant effect on relaxation induced inhibitors on tension could be related to non-specific by L-arginine, nor did the pinacidil derivative, PNU actions, such as inhibition of inos induction or activity. ( mm). In contrast, pore-binding inhibitors, PNU-37883A To assess this, NO production was measured by determin- ( mm), Ba (00 mm), 4-aminopyridine (4-AP; 5 mm) ing NO levels in the supernatant of rat aortic rings Downloaded from on 30 January 08

4 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) Fig.. Concentration-dependent effects of L-arginine on phenylephrine ( mm) 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, 400W (A) and in absence and presence of the K channel inhibitors, iberiotoxin (IbTx), charybdotoxin (ChTx), apamin and TEA (B). Mean effects of structurally dissimilar K ATP channel inhibitors on relaxation to L-arginine are shown for SUR-binding (C) and pore-binding (D) inhibitors. Data are expressed as mean6s.e.m. (n53 7). incubated with LPS for several hours. LPS significantly mm) (n57). In contrast, both tolbutamide ( mm) and increased NO in the culture medium by mm TEA (0 mm) significantly inhibited NO production mg protein (P,0.00, n57, Table ). This increase was (n57, P,0.00). In separate experiments, levcromakalim fully inhibited by pre-incubation with 400W (0 mm), but caused a small, but insignificant (P50.39, n56) reducwas not significantly affected by ODQ (3 mm), gliben- tion in nitrite accumulation in LPS-treated tissues. clamide (0 mm), PNU-37883A ( mm) or Ba (00 Table 3.3. Characterisation of relaxation to authentic NO Effects of K channel inhibitors on nitrite accumulation in endothelium denuded rat aortic rings pretreated with LPS Bath application of increasing concentrations of authen- Compound DNO (mm mg protein ) n tic NO to endothelium-denuded, aortic rings produced a (compared to control) rapid concentration-dependent inhibition of PE contrac- LPS tions. Relaxation typically peaked within 0 5 s, with 0 400W (0 mm) * 7 mm NO inducing almost complete reversal of PE-induced ODQ (3 mm) tone (E max, 98.96%, pec 50, ; n534). These Glibenclamide (0 mm) PNU-37883A ( mm) values were essentially unchanged if tissues were pre- Ba (00 mm) incubated with LPS (n53; Fig. 3A). The effects of NO Tolbutamide ( mm) * 7 were abolished by the NO scavenger oxyhaemoglobin (30 TEA (0 mm) * 7 mm), reducing Emax to.363% (P,0.00, n55) and *P, % (P,0.00, n55) for control and LPS-treated Downloaded from on 30 January 08

5 58 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) Fig. 3. Mean concentration response curves to authentic NO (A) in the presence and absence of LPS ( mg ml ; 4 h) and ODQ or oxyhaemoglobin (OxyHb, n55 34). (B) The effects of the K channel inhibitors, 4-aminopyridine (4-AP), tetraethylammonium ions (TEA) and barium (Ba ) on NO responses in the presence of LPS (n58). Responses to SNAP were assessed in control (C) and LPS-treated ( mg ml ; 4 h; D) aortic rings in the absence and presence of glibenclamide and PNU-37883A (n54). Data are expressed as mean6s.e.m of n observations. tissues, respectively. Similarly, ODQ (0 mm) severely in E max, although there was a small, but significant shift to attenuated (P,0.00) responses to NO under both con- the right in the pec50 value for Ba (Table ; P,0.05, ditions (n55, Fig. 3A), though unlike responses to L- n58). Glibenclamide (0 mm) also had no effect on arginine, relaxation was not fully inhibited at the higher relaxation induced by NO at any concentration (n58, NO (. mm) concentrations. In LPS-treated tissues, the Table ). NG NO synthase inhibitor, L- -nitro-arginine methyl ester (L-NAME, 300 mm, n55) had no significant effect on NO responses (pec in control and in 3.4. Effects of KATP channel inhibitors on relaxation LPS, n55). However, both ODQ and L-NAME increased induced by SNAP PE contractions by % (n55) and % (n55), respectively. Concentration response curves were constructed using To assess the contribution of K channels to NO the S-nitrosothiol, SNAP ( mm). Under control generated exogenously, concentration response curves to conditions, SNAP produced full relaxation of PE-con- NO in tissues treated with LPS were constructed in the tracted tissues with a pec50 of (n54) which was absence and presence of a variety of K Ca, KATP or KV essentially unaffected by LPS treatment (pec 50, , channel inhibitors. Table shows that inhibitors of K n54). Under control conditions, neither glibenclamide, nor Ca channels, ChTx, IbTx and apamin, had no apparent effect PNU-37883A had any significant effect on the concenon relaxations to NO. In contrast to the substantial tration response curve to SNAP (Fig. 3C). However, inhibitory effect of TEA on L-arginine-induced relaxations, following incubation with LPS, PNU-37883A significantly this agent had essentially no effect on responses to NO inhibited relaxation shifting the pec50 to (P, (Table, Fig. 3B). Moreover, the effect of Ba (00 mm) 0.05, n54), although glibenclamide was still without effect and 4-AP (5 mm) was weak (Fig. 3B), with no reduction (Fig. 3D). Downloaded from on 30 January 08

6 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) Table The effects of K channel inhibitors on relaxation induced by authentic NO in endothelium-denuded, LPS-treated rat aortic rings; comparison of pec 50 values and maximal relaxation (E ) obtained in the absence (control) and presence of inhibitors max Compound pec (M) E (%) n 50 max Control With inhibitor Control With inhibitor Nitric oxide IbTx (50 nm) ChTx (00 nm) Apamin (00 nm) TEA (0 mm) Ba (00 mm) * Glib (0 mm) AP (5 mm) Values are mean6s.e.m. of 4 8 observations. *P, Effects of LPS on responses to levcromakalim LPS significantly increased Emax from to % (P,0.00, n5). Levcromakalim ( mm) induced concentration- To elucidate whether the potentiation of levcromakalimdependent relaxation of PE-contracted aortic rings with a induced relaxation by LPS was mediated by inos, rings pec50 of for control tissues, which was sig- were pre-treated with 400W (0 mm). Under control nificantly potentiated (P,0.00, Fig. 4A) to in conditions, relaxations to levcromakalim were essentially the presence of LPS (n5). Furthermore, incubation with insensitive to 400W, with Emax and pec50 being effec- 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; n5) and in a separate series of experiments in the absence and presence of 400W (0 mm, 45 min, n54) (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 (n55). Data are presented as mean6s.e.m. Downloaded from on 30 January 08

7 60 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) tively unchanged (Fig. 4B). In contrast, following LPS ness of glibenclamide in the presence of LPS resulted from incubation, 400W caused a significant rightward shift in activation of the inos/ cgmp-dependent pathway, tissues the levcromakalim concentration response curve, from were incubated with 400W or ODQ. In the presence of to (P,0.00, n54) (Fig. 4B). The ODQ or 400W, glibenclamide fully inhibited the maximal latter was not significantly different from that obtained relaxation to levcromakalim by % (n53) and under control conditions in the absence of LPS % (n53) for ODQ and 400W, respectively. To determine whether potentiation was mediated directly by NO or indirectly via the cgmp pathway, the effect of 3.7. Forskolin-induced relaxation under control and LPS the sgc inhibitor ODQ (3 mm) was investigated. Under conditions control conditions, ODQ had a small potentiating effect on the concentration response curve and increased Emax from To determine whether LPS potentiated responses to to % (P,0.05, n54, Fig. 5). Under other vasodilators, experiments were carried out using LPS conditions, pre-incubation with ODQ now caused a forskolin. Under control conditions, forskolin induced full significant rightward shift in the levcromakalim response relaxation (E max, %, n55), although it was from a pec of to (n54, P,0.0) significantly (P,0.00) more potent in the presence of 50 but did not suppress E max (Fig. 5). Thus, ODQ did not LPS, with over a log-fold shift in potency (pec 50, reverse the effects of LPS on levcromakalim responses to and for control and LPS, respectivethe same extent as that observed with 400W. ly; n55, Fig. 6A). Application of PNU-37883A had no significant effect on forskolin responses in control tissues 3.6. Effects of K channel inhibitors on relaxation to (Fig. 6B), although in the presence of LPS, it caused a ATP levcromakalim significant rightward shift in pec50 to (P, 0.005, n55, Fig. 6C), which was now not significantly Both PNU-37883A ( mm) and glibenclamide (0 mm) different (P50.07) from control responses. In contrast, effectively abolished responses to levcromakalim under glibenclamide had a negligible effect on pec50 under both control conditions (Fig. 4C). Interestingly, following expo- conditions (Fig. 6B,C). sure to LPS, the inhibitory effects of both agents were diminished (P,0.005, n55, Fig. 4D). Glibenclamide, inhibited the maximal response to levcromakalim by 4. Discussion % in control tissues, while in the presence of LPS, it only inhibited the maximal response by The major finding of the present study is that K ATP % (n55). PNU-37883A also failed to complete- channels appear to mediate inos-induced relaxation in rat ly abolish relaxation in the presence of LPS, although it aorta. This is based on the finding that PNU-37883A, a appeared a more effective inhibitor. Under control con- putative smooth-muscle selective KATP channel inhibitor ditions, PNU-37883A inhibited the maximum relaxation to [4] and Ba, a non-selective K channel inhibitor, levcromakalim by %, while in LPS-treated tissues, it inhibited the maximal response by % (n55). To determine whether the relative lack of effective- Fig. 5. The effects of ODQ on the response to levcromakalim in control and LPS-treated rat aortic rings (n53). Data are presented as mean6s.e.m. ATP 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 K ATP channels mediate hypotension induced by LPS in vivo [], glibenclamide does not reverse LPS-induced hyporeactivity to phenylephrine in vitro [9,4]. 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 Downloaded from on 30 January 08

8 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) 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 mean6s.e.m (n55). reduction in PE-induced tone, as noted previously [9,4]. The reason for this is unclear, although glibenclamide is known to antagonise prostanoid-induced contractions [6], and we have previously found that inhibition of prostaglandin synthesis with a selective cyclooxygenase inhibitor reduces PE contractions in the presence of LPS [7]. However, glibenclamide has been shown to partially reverse LPS-induced vascular hyporeactivity [8] and membrane hyperpolarisation [4,5] 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 00 mm [8]. At these higher concentrations, additional mechanisms may be involved in the observed effects on blood pressure, such as inos inhibition [8]. Alternatively, glibenclamide may directly inhibit the KATP channel pore, via a low-affinity sulphonylurea binding site [9]. This is consistent with our finding that PNU-37883A, a structurally unrelated KATP channel inhibitor that binds directly to the pore [0,30], was an effective inhibitor of L-arginine-induced relaxation, as were other inhibitors of the channel pore, Ba 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 LPStreated rat mesenteric artery [30]. The effects of PNU A 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 [4]. Moreover, PNU-37883A did not significantly affect either the magnitude of PE contractions nor forskolin responses in control tissues (see also Ref. [3]) 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 highaffinity glibenclamide binding [3]. 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 Downloaded from on 30 January 08

9 6 A.J. Wilson, L.H. Clapp / Cardiovascular Research 56 (00) potentiates levcromakalim-induced relaxation. The inos cromakalim responses might potentiate NO release by pathway probably mediates this since both 400W, and to a activating L-arginine uptake, although lack of any signifilesser extent, ODQ, reversed potentiation. Similar findings cant effect on nitrite accumulation would suggest this not have been reported in ex vivo models of LPS-induced to be the case. hyporeactivity [0,4]. Potentiation can also be observed In summary, our results suggest that in rat aorta, in the absence of LPS with NO donors and the cgmp activation of the inos pathway by LPS results in a analogue, 8-bromo-cGMP [34]. This implies that cgmp significant alteration in the pharmacology of both acmodulates the effects of KATP channel openers, possibly tivators and inhibitors of KATP channels. We found that via phosphorylation of the channel or closely associated inhibitors that bind directly to the KATP channel pore, were proteins. The processes responsible for the potentiation of significantly more effective at inhibiting relaxation induced control levcromakalim responses by ODQ cannot easily be by L-arginine, SNAP, levcromakalim and forskolin than the explained and remain to be determined. Potentiation of classical sulphonylurea-based inhibitor, glibenclamide. The forskolin responses by LPS was also observed, an effect molecular mechanisms responsible for these changes are that was substantially reversed by PNU-37883A. This presently unknown, although we suggest that LPS via suggests some synergy between the camp and cgmp NO/ cgmp may uncouple/ alter the communication bepathways in activating KATP channels, although mecha- tween the pore and the SUR. nisms 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 Acknowledgements be mediated by BKCa channels, since iberiotoxin and charybdotoxin, caused a small reduction in relaxation. This LHC is a MRC Senior Research Fellow in Basic is consistent with previous findings showing BK channel Science. This work was supported by the Medical Re- Ca inhibitors to partially reverse vascular hyporeactivity search Council (G7/ 80). The authors would like to [9,0]. Electrophysiological data also support a role for thank Professor Martin Feelisch for help in the preparation BK channels, where application of L-arginine to LPS- of authentic NO solutions, Jonathan Alis for technical Ca treated cultured coronary vascular smooth muscle cells assistance and Dr Stephen Humphrey (Pharmacia Upjohn, resulted in persistent activation of BK channels [35]. USA) for helpful comments on the manuscript. Ca 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. References In the presence of LPS, responses to NO in endo- [] Landry DW, Oliver JA. The pathogenesis of vasodilatory shock. N thelium-denuded tissues, were only weakly affected by Engl J Med 00;345: either Ba or 4-AP. Interestingly, TEA which had large [] Kilbourn RG, Jubran A, Gross SS et al. Reversal of endotoxineffects on L-arginine responses, was without effect. Thus, G mediated shock by N -methyl-l-arginine, an inhibitor of nitric oxide activation of K channels do not appear to be the primary synthesis. Biochem Biophys Res Commun 990;7:3 38. [3] Thiemermann C. Nitric oxide and septic shock. Gen Pharmacol mechanism of relaxation for authentic NO, even though 997;9: like L-arginine, effects were virtually abolished by ODQ. [4] Lincoln TM, Dey N, Sellak H. cgmp-dependent protein kinase The reason for these differences could be related to a signaling mechanisms in smooth muscle: from the regulation of tone number of factors, such as the type of NO species to gene expression. J Appl Physiol 00;9: generated, the site and rate of NO/ cgmp production. [5] Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric Results with SNAP would support this idea, since unlike oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature (Lond) 994;368: authentic NO, relaxation in the presence of LPS could be [6] Mistry DK, Garland CJ. Nitric oxide (NO)-induced activation of inhibited by PNU-37883A. It is possible that the effects of large conductance Ca -dependent K channels (BK Ca) in smooth K channel inhibitors on tension may be related to muscle cells isolated from the rat mesenteric artery. 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