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Am J Physiol Heart Circ Physiol 293: H1682-H1688, 2007. First published June 8, 2007; doi:10.1152/ajpheart.00389.2007
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Elucidation of the temporal relationship between endothelial-derived NO and EDHF in mesenteric vessels

Louise S. Harrington,1,2 Martin J. Carrier,2,* Nicola Gallagher,3 Derek Gilroy,4 Chris J. Garland,3 and Jane A. Mitchell1,*

1Cardiothoracic Pharmacology, Unit of Critical Care Medicine, The National Heart and Lung Institute, Imperial College, London; 2Cardiac, Vascular, and Inflammation Research, William Harvey Institute, London; 3Department of Pharmacy and Pharmacology, University of Bath; and 4Division of Medicine, Centre for Clinical Pharmacology, Rayne Institute, University College London, United Kingdom

Submitted 29 March 2007 ; accepted in final form 4 June 2007


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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Although the endothelium co-generates both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), the relative contribution from each vasodilator is not clear. In studies where the endothelium is stimulated acutely, EDHF responses predominate in small arteries. However, the temporal relationship between endothelial-derived NO and EDHF over more prolonged periods is unclear but of major physiological importance. Here we have used a classical pharmacological approach to show that EDHF is released transiently compared with NO. Acetylcholine (3 x 10–6 mol/l) dilated second- and/or third-order mesenteric arteries for prolonged periods of up to 1 h, an effect that was reversed fully and immediately by the subsequent addition of L-NAME (10–3 mol/l) but not TRAM-34 (10–6 mol/l) plus apamin (5 x 10–7 mol/l). When vessels were pretreated with L-NAME, acetylcholine induced relatively transient dilator responses (declining over ~5 min), and vessels were sensitive to TRAM-34 plus apamin. When measured in parallel, the dilator effects of acetylcholine outlasted the smooth muscle hyperpolarization. However, in the presence of L-NAME, vasodilatation and hyperpolarization followed an identical time course. In vessels from NOSIII–/– mice, acetylcholine induced small but detectable dilator responses that were transient in duration and blocked by TRAM-34 plus apamin. EDHF responses in these mouse arteries were inhibited by an intracellular calcium blocker, TMB-8, and the phospholipase A2 inhibitor AACOCF3, suggesting a role for lipid metabolites. These data show for the first time that EDHF is released transiently, whereas endothelial-derived NO is released in a sustained manner.

arteries; vasodilation; mediators; hyperpolarization; nitric oxide; endothelium-derived hyperpolarizing factor

THE ENDOTHELIUM-DERIVED relaxing factor, described in 1980 by Furchgott and Zawadozki (18), is now known to be nitric oxide (NO) (22, 23), formed from the amino acid L-arginine (32). In 1988, reports emerged of a distinct factor released by the endothelium that relaxed blood vessels via a process of hyperpolarization (endothelium-derived hyperpolarizing factor; EDHF) (2, 3, 15). Since this time, EDHF biology has developed at a rapid pace (9). However, there is still considerable debate about the identity of EDHF. Potential candidates include CYP450 metabolites (17), K+ (16), hydrogen peroxide (26), and K+ and/or electrical communications through gap junctions (39) and possibly C-type natriuretic peptide (CNP) (13).

Much of the evidence supporting a role for EDHF in endothelium-dependent dilation has been derived pharmacologically, for example, by removing other dilatory pathways (NO and prostacyclin), removing the endothelium, removing the ability to hyperpolarize (with high [K+]), or blocking K+ channels with agents such as TRAM-34 plus apamin. Usually, experiments are conducted where the vessels are stimulated acutely and the responses monitored for short periods of time (seconds or minutes). The temporal nature of EDHF is not well described, although a limited number of papers from the late 1980s and early 1990s show that EDHF is either transient (15, 24, 37) or sustained (7, 14), depending on the vessel type studied. In contrast, there is clear consensus that NO is released continually by activated endothelium (27, 34) via the enzyme nitric oxide synthase (NOS)III (20).

In small arteries, particularly of the mesenteric circulation, present literature suggests that EDHF and not NO is the predominant pathway controlling endothelium-dependent vasodilation (6, 19, 36). However, without comprehensive data describing the relative duration of response, it is difficult to conclude that EDHF and not NO is the predominant pathway.

In the present study, we have therefore investigated the relative temporal relationship between NO and EDHF-dependent responses in small arteries from the mesenteric bed. In doing so, we show that EDHF is released transiently, whereas NO is released in a more sustained manner. When studied in this way, endothelial-derived NO can be seen as having a predominant role over EDHF in dilator responses in these tissues.


   METHODS
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 METHODS
RESULTS
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Isometric wire myography. Male Wistar rats (250–280 g) were killed by lethal exposure to CO2 followed by cervical dislocation. Homozygous NOSIII–/– mice with a C57BL6 background (21), a gift from Dr. D. Gilroy (University College London), were killed by exposure to CO2, followed by exsanguination. All animals were maintained and killed in accordance with the United Kingdom Guidelines on the Operation of the Animals (Scientific Procedures) Act 1986 (The Stationery Office, UK). Procedures were approved by the institutional Ethical Review Board and regulated under United Kingdom Home Office Project license no. 70/5552.

The entire mesenteric bed was removed and placed into physiological salt solution (PSS; composition in mmol/l: NaCl 119, KCl 4.7, CaCl2 2.5, MgSO4 1.17, NaHCO3 25, KH2PO4 1.18, EDTA 0.027, and glucose 5.5). The mesentery was pinned flat on a dissecting dish containing PSS to allow second-order arteries to be cleaned of fat and connective tissue; these arteries were stored in fresh PSS solution at room temperature until use.

Isometric wire myograph recordings. With the use of 40-µm-diameter tungsten wire, segments of 2-mm length of artery were mounted in a four-channel Mulvany-Halpern myograph (model no. 610M, Danish Myo Technology). The vessels were equilibrated to 37°C, and the PSS was bubbled with 95% O2 and 5% CO2 for 30 min. The tension of the vessel was normalized to a tension equivalent to that generated at 90% of the diameter of the vessel at 100 mmHg, using standard procedures as described previously (31). Changes in arterial tone were recorded via a PowerLab/800 recording unit (ADI Instruments) and analyzed using the Chart 4.2 acquisition system (ADI Instruments).

To assess the viability of the vessels, they were challenged three times with high-potassium solution (KPSS; composition in mmol/l: KCl 123.7, CaCl2 2.5, MgSO4 1.17, NaHCO3 25, KH2PO4 1.18, EDTA 0.027, and glucose 5.5).

Effect of inhibitors on sustained endothelium-dependent vasodilator responses. Tissues were precontracted with an EC80 of 9,11-dideoxy-11{alpha},9{alpha}-epoxymethanoprostaglandin F2{alpha} (U46619 [GenBank] ; 3 x 10–7 mol/l), and once a plateau was reached, 3 x 10–6 mol/l acetylcholine was added to the organ bath. In some experiments, an EC80 of methoxamine was used to achieve precontraction.

Following 10 min of vasodilatation, 10–3 mol/l L-NAME was added and the direct action on dilated arteries noted. In some experiments, the endothelial layer lining the lumen of the vessels was removed physically using a human hair. Complete endothelial removal was documented by an absence in the ability of acetylcholine to induce relaxation of vessels contracted with an EC80 of U46619. [GenBank] Complete endothelium-independent vasodilatation of these arteries was achieved with 3 x 10–6 mol/l sodium nitroprusside (SNP), after which L-NAME was again added. In some experiments, 1-[(2-chlorophenyl)dipenylmethyl]-1H-pyrazole (TRAM-34; 10–6 mol/l) plus apamin (5 x 10–7 mol/l) were added in the above protocols instead of L-NAME.

Effect of inhibitors added before dilator agents. In separate experiments, L-NAME (10–3 mol/l) was added to the tissues 30 min before the addition of U46619 [GenBank] (3 x 10–7 mol/l). Once a plateau was reached, 3 x 10–6 mol/l acetylcholine was added to the organ bath. The tissue responses were monitored, and dilator responses to acetylcholine were measured as percent contraction at 30 s and 3 and 10 min from the start of dilation. In some tissues, other drugs were administered with L-NAME including 10–5 mol/l 8-(diethylamino)octyl-3,4,5-trimethyoxybenzoate hydrochloride (TMB-8), 3 x 10–5 mol/l arachidonate trifluoromethylketone (AACOCF3), 20 µg/ml oxyhemoglobin, 10–6 mol/l TRAM-34 plus 5 x 10–7 mol/l apamin or 10–5 mol/l indomethacin. In separate experiments, responses to L-NAME or TRAM-34 plus apamin in second- and third-order mesenteric arteries from the rat were compared.

Measurement of membrane potential. Smooth muscle cells of whole mesenteric arteries were impaled with sharp glass electrodes filled with 2 M KCl, tip resistances ~80–100 M{Omega}, and membrane potential and tension were measured simultaneously (19). Second-order rat mesenteric arteries were incubated with or without 10–3 mol/l L-NAME for 20 min and precontracted with EC80 phenylephrine. Once plateau was reached, arteries were exposed to 10–6 mol/l acetylcholine, and the tension and membrane potential were measured continuously for up to 20 min. Recordings were taken until the membrane potential had returned to baseline levels, which in control arteries occurred between 12 and 20 min of the response to acetylcholine.

Data and statistical analysis. All traces recorded changes in the force produced by each artery (measured in mN). Data are given as the mean contraction ± SE as a percentage of precontraction (induced by EC80 U46619 [GenBank] ). They were analyzed statistically using either one-way or two-way ANOVA, using Prism 4.0. A P value <0.05 was considered statistically significant.

Drugs. All drugs were purchased from Sigma Chemical (Gillingham, Dorset, UK). Drugs were prepared each day, except for TRAM-34, apamin, U46619 [GenBank] , TMB-8, and AACOCF3, which were prepared in high-concentration "stock" solutions and stored at –20°C until use. All drugs were dissolved in aqueous solutions except for indomethacin and TRAM-34, which were dissolved in DMSO. The final concentration of DMSO in the bath was 0.1%, which had no effect on the tissue.

To ensure full oxidation of the oxyhemoglobin, sodium dithionite was added to bovine hemoglobin at a ratio of 1:4 in 1 ml of PSS. To remove unoxygenized hemoglobin, 0.1 g of cytodex beads was presoaked in 5 ml of PSS for at least 30 min and added to the hemoglobin, and the supernatant was removed as the oxygenized hemoglobin fraction.


   RESULTS
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Acetylcholine induces concentration-dependent vasodilator responses in rat mesenteric arteries with a maximal effect obtained with 3 x 10–6 mol/l (data not shown). When given as a single concentration, acetylcholine at 3 x 10–6 mol/l induced an immediate and sustained dilation that lasted for >1 h (n = 3; data not shown) in tissues precontracted with an EC80 of U46619. [GenBank] When L-NAME (10–3 mol/l) was added to inhibit NO formed by the endothelium, there was an immediate and complete reversal of the vasodilatation induced by acetylcholine (Fig. 1, A and B). The effects of L-NAME were attributed to inhibition of NOS, because they were reversed by the subsequent addition of L-arginine (Fig. 1, A and C). In contrast, when apamin (5 x 10–7 mol/l) plus TRAM-34 (10–6 mol/l) were added to tissues to inhibit EDHF responses, no effect on acetylcholine-induced dilation was noted (Fig. 1D). Similarly, indomethacin, TMB-8, or AACOCF3 did not affect vascular tone in tissues dilated with acetylcholine (n = 3–4; data not shown).


Figure 1
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  		Fig. 1. L-NAME reverses acetylcholine (ACh)-induced vasodilatation. Arteries were precontracted with an EC80 of U46619 (U4) and treated with 3 x 10–6 mol/l ACh. Following full and continuous dilation for 10 min, addition of 10–3 mol/l L-NAME completely reversed dilation as shown in A and B. A: representative trace. B: data are shown as means ± SE of %induced tone (n = 3). C: effects of L-NAME on ACh-induced vasodilation is reversed by addition of increasing concentrations of L-arginine (L-Arg; 10–3 to 3 x 10–2 mol/l). ***P < 0.0001 by 2-way ANOVA; n = 3. D: addition of 10–6 mol/l TRAM-34 (TRAM) plus 5 x 10–7 mol/l apamin had no effect on ACh-induced vasodilation (representative trace from 6 examples). L-NAME reversed ACh-induced vasodilation when precontracted with EC80 methoxamine (meth) as shown in E and F. E: representative trace. F: data are shown as means ± SE of %induced tone (n = 3).

 
In other experiments where tissues were contracted with methoxamine (10–5 mol/l) instead of U46619 [GenBank] , acetylcholine caused similar sustained dilator responses that were reversed by the addition of L-NAME (n = 4; Fig. 1, E and F). Furthermore, although the results shown in this study are from tissues of male rats or mice, we also found that acetylcholine induced sustained dilator responses in mesenteric tissues from female animals and, again, that this response was reversed when L-NAME was added into the organ bath (n = 4; data not shown).

Temporal relationship between NO and EDHF. The above experiments suggest that at any time point after established dilation (i.e., 2–5 min), endothelial-derived NO and not EDHF mediates endothelial-dependent dilator responses to acetylcholine. To study this further, we performed experiments where vessels were pretreated with L-NAME. In these experiments, the ability of acetylcholine to cause sustained endothelial-dependent dilator responses was inhibited and instead resulted in a transient response (Fig. 2, A and D). By contrast, pretreatment of vessels with apamin plus TRAM-34 did not reduce the duration of the dilator response of vessels to acetylcholine (Fig. 2, B and D). The transient dilator response induced by acetylcholine in the presence of L-NAME was not due to any residual uninhibited NOS activity because no further reduction in response was seen when oxyhemoglobin, which scavenges NO, was added (n = 6; data not shown). The ability of acetylcholine to cause transient dilation in the presence of L-NAME was blocked by co-incubation with apamin plus TRAM-34 (Fig. 2, C and D). In separate experiments where vasomotor responses were measured simultaneously with membrane potential, we found that the vasodilator response induced by acetylcholine outlasted the more transient hyperpolarization it induced (Fig. 3A). Moreover, when experiments were performed in the presence of L-NAME, the vasodilator response and the hyperpolarization induced by acetylcholine occurred simultaneously (Fig. 3B). In tissue from NOSIII–/– mice, a small, but detectable, vasodilatation was noted when acetylcholine was added to preconstricted mesenteric vessels (see Fig. 4, C and D). In line with observations made when NOSIII was blocked pharmacologically with L-NAME, as described above, the responses in vessels from NOSIII–/– mice were transient and blocked by apamin plus TRAM-34 (see Fig. 4D). Vasodilator responses to acetylcholine in precontracted vessels from wild-type mice were, as in the case of tissues from the rat, sustained (Fig. 4, A and B).


Figure 2
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  		Fig. 2. Effects of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) inhibition on the duration of ACh-induced dilator responses. Mesenteric arteries were incubated for 30 min with 10–3 mol/l L-NAME (A), 10–6 mol/l TRAM-34 plus 5 x 10–7 mol/l apamin (B), and L-NAME and TRAM-34 plus apamin (C) followed by precontraction with EC80 U46619, and the effects of 3 x 10–6 mol/l ACh were measured. D: measurements were taken 30 s and 3, 10, and 20 min following addition of ACh, and data are represented as means %U46619-induced tone ± SE. ***Significance by 2-way ANOVA compared with control: P < 0.001 (n = 5–6).

 

Figure 3
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  		Fig. 3. Effects of ACh on membrane potential and tension recorded in parallel in control arteries (A) and arteries treated with 10–4 mol/l L-NAME (B). Arteries incubated with or without L-NAME were precontracted with EC80 phenylephrine, and the effect of a single concentration of ACh (10–6 mol/l) was measured. *Final reading taken between 12 and 20 min of ACh response. Tension, represented as %induced tone, and membrane potential, measured as mV, were recorded continuously and are shown as means ± SE at 30 s and 3, 10, and 20 min (n = 4).

 

Figure 4
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  		Fig. 4. The small and transient ACh-induced vasodilatory response in nitric oxide synthase (NOS)III–/– mouse mesenteric arteries is eradicated by treatment with TRAM-34 plus apamin. Arteries were precontracted with EC80 U46619, and the dilatory response to 3 x 10–6 mol/l ACh was recorded over 4 min. Responses from wild-type mice (NOSIII+/+) are shown in A and B. A: example trace. B: measurements taken at 10 and 30 s and 1, 2, and 4 min of the initial response to Ach. The effects in NOSIII–/– mice are shown in C and D. C: original trace. D: measurements of Ach response with or without 10–6 mol/l TRAM-34 plus 5 x 10–7 mol/l apamin. In each type of tissue, full dilation was achieved by addition of 3 x 10–6 mol/l sodium nitroprusside (SNP). Responses to ACh were plotted as mean %induced tone ± SE. ***Significance by 2-way ANOVA: P < 0.001 (n = 4–6 for each group).

 
Characterization of the EDHF response in mesenteric vessels. In the presence of L-NAME, the transient dilator response induced by acetylcholine (EDHF) was not reduced when cyclooxygenase was blocked by indomethacin (10–5 mol/l, n = 4; Fig. 5A), but it was significantly reduced when either the phospholipase A2 (PLA2) inhibitor AACOCF3 (3 x 10–5 mol/l) or the intracellular calcium antagonist TMB-8 (10–5 mol/l) was added together with L-NAME as pretreatments (Fig. 5, B or C, respectively).


Figure 5
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  		Fig. 5. The transient ACh vasodilatory response in rat mesenteric arteries following L-NAME incubation is further reduced by TMB-8 and AACOCF3 but not indomethacin. Original traces are shown. Mesenteric arteries were treated for 30 min with L-NAME (10–3 mol/l) plus indomethacin (Indo, 10–5 mol/l) (A), AACOCF3 (B; 3 x 10–5 mol/l), and TMB-8 (C; 10–5 mol/l), following which arteries were precontracted with EC80 U46619, and the duration of the dilatory response to 3 x 10–6 mol/l ACh was recorded. D: measurements taken at 30 s and 3 and 10 min of the ACh response are represented as mean %U46619-induced tone ± SE. *Significance by 2-way ANOVA: P < 0.05 (n = 3–5).

 
In vessels where endothelium had been removed, acetylcholine had no dilator effect on U46619 [GenBank] -contracted vessels. In these tissues, the NO donor SNP (3 x 10–6 mol/l) induced full and sustained dilator responses that were not modified by the subsequent addition of L-NAME (Fig. 6).


Figure 6
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  		Fig. 6. L-NAME does not reverse endothelium-independent dilation by SNP. A: original trace in which the loss of endothelium was confirmed in arteries precontracted with EC80 U46619 by a lack of effect by 3 x 10–6 mol/l ACh. Full endothelium-independent dilation was induced by 3 x 10–6 mol/l SNP for 10 min, where addition of 10 mol/l L-NAME had no effect. B: data are presented as mean %U46619-induced tone ± SE.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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The major finding of this study is that EDHF and NO are released with different patterns of duration; EDHF is released transiently, whereas NO is released in a sustained fashion. These observations are important, because when duration of responses was considered, the majority of endothelium-dependent dilation could be attributed to the release of NO. These data challenge the notion that in small mesenteric arteries of the rat, endothelial-derived NO is a "redundant" pathway and that EDHF predominates. Specifically, we show that when short periods are considered, NO and EDHF contribute equally to dilator responses, and when longer periods are considered, NO is the principle dilator pathway.

In vessels prepared with an intact endothelium and precontracted with the thromboxane mimetic U46619 [GenBank] , acetylcholine induced a profound and sustained dilator response that lasted for upward of 1 h. These responses were immediately and fully reversed when L-NAME was added at any point after the response had reached plateau. However, when apamin and TRAM-34 were added to block EDHF responses, the dilation induced by acetylcholine was not affected. This suggested to us that NO release by the endothelium was responsible for the sustained dilation we observed. This observation is consistent with what we know about endothelial-derived NO from larger vessels or from cultured endothelial cells (27). Furthermore, the release of NO from the endothelium in vivo is also occurring in a sustained manner, since the administration of NOS inhibitors produces an immediate increase in blood pressure (34), akin to the reversal of dilator response we see in our study.

There have been suggestions that the relative roles of EDHF vs. NO in the endothelium-dependent dilator effects of acetylcholine differ according to the contractile agent used. For example, where U46619 [GenBank] is used to contract the tissue, some studies show that the EDHF component is reduced (8, 18,). In the present study, we show that the relative temporal pattern of EDHF and NO is similar when vessels are contracted with U46619 [GenBank] or {alpha}-adrenergic agonists (methoxamine or phenylephrine), thereby ruling out any specific contribution of the individual contractile pathway to our results.

In small arteries of the mesenteric circulation, EDHF is thought to play a predominant role in vasodilator responses mediated by the endothelium (36). Our observations raised the following question: if L-NAME, but not TRAM-34 plus apamin, could fully reverse the dilator responses we see with acetylcholine in these vessels, where is the EDHF? Perhaps EDHF, like another important vasoactive hormone, prostacyclin, could be released by the endothelium in a transient manner (25, 27) under conditions where endothelial-derived NO is released in a sustained fashion (27). To test this possibility, we pretreated mesenteric arteries with L-NAME to block NOSIII pharmacologically, or we used vessels from NOSIII–/– mice and repeated our protocols where acetylcholine was added at a maximal concentration to preconstricted vessels. Under conditions where NOSIII was not functional, the vasodilator response to acetylcholine was transient. To further establish that this was due to EDHF, we compared tonic and membrane potential responses simultaneously in rat mesenteric vessels. In these experiments, precontraction was achieved using phenylephrine, and the vasodilator response to acetylcholine was, as expected, sustained and clearly outlasted the hyperpolarization. However, again, when L-NAME was added, the dilator effects of acetylcholine were transient and now had a superimposable time course to changes in membrane potential (hyperpolarization). These experiments clearly and definitively show, therefore, that EDHF is released transiently, whereas NO is released in a sustained manner. A recent study looking at membrane potential in endothelial cells showed that responses induced by acetylcholine were even shorter in duration when ouabain was added, implicating a role for Na+-K+-ATPase in the maintenance of response (5).

It has been reported that NO can cause hyperpolarization of vascular smooth muscle (12, 38) through its direct actions on ATP-sensitive K+ channels (4, 40). However, our data found only a small and statistically insignificantly different contribution of NO to hyperpolarization.

It is not the focus of the present study to address the actual nature of EDHF but rather to understand how it is released and what relative importance it has to NO in controlling vasomotor tone in mesenteric vessels. Once we had established that EDHF was released transiently, parallels were noted in its relationship with prostacyclin and NO, as mentioned above. Prostacyclin is formed by the consecutive actions of cyclooxygenase and prostacyclin synthase (29). However, the rate-limiting step for prostacyclin production is the liberation of substrate, arachidonic acid, from cell membranes by the actions of PLA2 (30). Activation of PLA2 in endothelial cells is dependent on the liberation of calcium from intracellular stores and can be blocked by TMB-8 (11). In the present study, we found that EDHF responses were significantly reduced when TMB-8 was included as a pretreatment together with L-NAME. A role for PLA2 in EDHF responses studied here was further supported by our findings that the PLA2 inhibitor AACOCF3, like TMB-8, reduced EDHF responses. Although these findings do not take us closer to determining the identity of EDHF in these tissues, they are consistent with the view of some investigators that it is a lipid metabolite (1, 10) and provide a mechanism that explains why its release is transient. PLA2 requires a large increase in intracellular calcium, which only occurs in the initial stages of cell activation (when intracellular stores are emptying). After this stage, elevated calcium is maintained in endothelial cells, from extracellular stores, but at a reduced level. In endothelial cells, NOSIII is calcium dependent (28, 33, 35) but will remain activated during the sustained phase of calcium elevation (25), which explains why NO release can occur in a sustained manner (27).

Our findings suggesting that EDHF is a metabolite of arachidonic acid are consistent with reports showing a role for lipid metabolites including epoxyeicosatrienoic acids (1, 10). However, there are a range of candidate EDHF molecules, including K+ (16), CNP (13), hydrogen peroxide (26), and direct communication of K+ and/or electrical communications through gap junctions (39). Our observations do not directly rule out any of these potential pathways but clarify the temporal relationship between "EDHFs" and NO in prolonged stimulation of the endothelium in mesenteric vessels.

In conclusion, our findings show that when the endothelium is stimulated continuously, NO is released in a sustained manner while EDHF is released transiently. This may be explained by EDHF requiring liberation of calcium from intracellular stores and activation of PLA2. These findings dispel the notion that NO has a minor role, compared with EDHF, in the control of vasodilator responses in the mesenteric circulation.


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This work was funded by a grant from the British Heart Foundation and the Biotechnology and Biological Sciences Research Council.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Mitchell, Cardiothoracic Pharmacology, Royal Brompton Hospital, Imperial College School of Medicine, Dovehouse St., London, SW3 6LY, UK (e-mail: j.a.mitchell{at}imperial.ac.uk)

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.

* M. J. Carrier and J. A. Mitchell contributed equally to this study. Back


   REFERENCES
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