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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2003 Apr 11;138(7):1285–1294. doi: 10.1038/sj.bjp.0705168

Effect of tumour necrosis factor-α and interleukin 1β on endothelium-dependent relaxation in rat mesenteric resistance arteries in vitro

R Wimalasundera 1, S Fexby 1, L Regan 2, S A McG Thom 1, A D Hughes 1,*
PMCID: PMC1573778  PMID: 12711629

Abstract

  1. Pre-eclampsia is associated with elevated proinflammatory cytokine levels and endothelial dysfunction. This study examined the effect of two cytokines, tumour necrosis factor-α (TNF) and interleukin-1β (IL-1) on endothelium-dependent relaxation in response to acetylcholine (ACH), bradykinin (BK) and histamine (HIS) in rat mesenteric small arteries in vitro.

  2. Rat mesenteric arteries were mounted in an isometric myograph. Tone was induced with phenylephrine (PE) or a depolarizing solution containing 80 mM KCl (K80). Relaxation was measured in response to ACH, BK, HIS and sodium nitroprusside (SNP), an endothelium-independent relaxant. Inhibition of NO synthase by a combination of Nω-monomethyl-L-arginine (L-NMMA) and Nω-nitro-L-arginine methyl ester (L-NAME) significantly inhibited relaxation in response to ACH and BK. Addition of an inhibitor of cyclooxygenase, indomethacin, had no additional effect when added to L-NMMA and L-NAME. Inhibition of endothelium-derived hyperpolarizing factor (EDHF) by K80 partially reduced responses to ACH and BK. Inhibition of HIS-induced relaxation was more marked with K80. L-NMMA and L-NAME largely abolished the remaining relaxation to ACH, BK and HIS in arteries contracted with K80.

  3. Preincubation with TNF for 30 min caused an inhibition of relaxation in response to ACH and BK in arteries contracted with PE. Responses to HIS and SNP were not affected by TNF under these conditions. TNF also inhibited ACH-induced relaxation in arteries contracted with K80. IL-1 had no effect on responses to ACH and the combination of TNF and IL-1 was not more effective than TNF alone.

  4. The inhibitory effect of TNF on ACH-induced relaxation was abolished by coincubation with superoxide dismutase (SOD) and was not seen if NO synthase was inhibited by L-NMMA and L-NAME.

  5. TNF inhibits the NO-dependent component of endothelium-dependent relaxation in response to ACH and BK, but does not inhibit the EDHF-dependent component. This effect may be attributable to the ability of TNF to increase levels of superoxide anions (O2−) and the ability of O2− to inactivate NO. This mechanism could contribute to the endothelial dysfunction seen in situations where TNF is elevated, such as pre-eclampsia.

Keywords: Tumour necrosis factor-α, interleukin-1, endothelium, vasodilation, superoxide, nitric oxide

Introduction

Pre-eclampsia is a common medical disorder of pregnancy affecting 5–7% of pregnancies and is a major cause of maternal and perinatal mortality and morbidity. Pre-eclampsia is characterized by a rise in blood pressure in the second half of pregnancy, associated with proteinuria, thrombocytopenia, abnormal liver function tests and increased peripheral vascular resistance (Higgins & de Swiet, 2001). The cause of the increased vascular resistance in pre-eclampsia remains uncertain, but it has been proposed that endothelial dysfunction could be a contributory factor. Loss of endothelium-dependent relaxation in response to acetylcholine (ACH) (McCarthy et al., 1993; Oguogho et al., 1996; Pascoal et al., 1998), bradykinin (BK) (Knock & Poston, 1996; Pascoal et al., 1998; Ashworth et al., 1999), histamine (HIS) (Oguogho et al., 1996; Suzuki et al., 2000) and flow (Cockell & Poston, 1997) has been reported in a range of human arteries from pre-eclamptic women studied in vitro, but the cause of this endothelial dysfunction is unknown.

Epidemiological evidence points to an immune aetiology of pre-eclampsia. There is increasing evidence that normal pregnancy is a proinflammatory state and that pre-eclampsia may be an exaggeration of this response (Redman et al., 1999). In pre-eclampsia, there is increased release of some pro-inflammatory cytokines such as tumour necrosis factor-α (TNF) (Kupferminc et al., 1994; Visser et al., 1994; Vince et al., 1995; Hamai et al., 1997; Conrad et al., 1998), interleukin-6 (Conrad et al., 1998; Benyo et al., 2001), interleukin-2 (Hamai et al., 1997) and interleukin-12 (Daniel et al., 1998). Plasma levels of other cytokines, such as interleukin-1β (IL-1) have not been found to be elevated (Kupferminc et al., 1994; Conrad et al., 1998), although placental expression of IL-1 has been reported to be increased (Rinehart et al., 1999), possibly as a result of placental hypoxia (Benyo et al., 2001).

Cytokines such as TNF and IL-1 are known to have a variety of procoagulant and proinflammatory effects on the vascular endothelium (reviewed in Mantovani et al., 1998). Moreover, elevated levels of cytokines may also contribute to the increased oxidative stress that has been reported to exist in pre-eclampsia (reviewed in Poston & Chappell, 2001). The aim of this study was to determine the effect of TNF and IL-1 on endothelium-dependent relaxation in response to vasodilators and to explore the possible role of superoxide (O2−) in mediating any effects of these cytokines.

Methods

Male Wistar rats (∼250 g) were killed by cervical dislocation following CO2 narcosis and the mesenteries removed. Small arteries (third order, internal diameter=346±65 μm (mean±s.d.), n=168) were mounted in parallel as ring segments in separate chambers of an isometric dual-channel myograph (Mulvany & Halpern, 1977), allowing one segment to act as time control for the other. Only one pair of arteries from an individual animal was used for each individual relaxant. All studies (unless otherwise specified) were conducted in physiological saline solution (PSS) comprising (mM): NaCl, 118; KCl, 4.7; CaCl2·6H2O, 2.5; MgSO4·7H2O, 1.17; NaHCO3, 25.0; NaH2PO4·2H2O, 1.0; Na2EDTA, 0.03; and glucose 5.5, maintained at 37°C and bubbled with 95% O2 and 5% CO2.

After mounting, the vessels were allowed to equilibrate for 1 h and then set to a ‘normalized' internal circumference estimated to be 90% of the circumference they would maintain if relaxed and exposed to 100 mmHg transmural pressure (0.9L100). This was calculated for each individual vessel on the basis of the passive length–tension characteristics of the artery and the Laplace relation (Mulvany & Halpern, 1977). This procedure optimized active force generation by these vessels.

Before beginning the studies, vessel viability was assessed by exposing arteries for ∼2 min to high potassium solution (PSS with equimolar substitution of 118 mM KCl for NaCl). This was repeated until stable responses were achieved (usually three exposures). The artery was then contracted with high potassium solution containing noradrenaline (10 μM) to assess maximum contraction. After washout, the functional integrity of the endothelium was established by adding ACH (10 μM) to a vessel contracted by noradrenaline (10 μM) in PSS. Vessels that failed to reproducibly produce tension equivalent to more than 100 mmHg effective pressure (calculated by Laplace's law) in response to high potassium solution containing noradrenaline, or relax completely to ACH were discarded.

Role of NO, cyclooxygenase and EDHF in responses to relaxants

Initial studies were designed to investigate the contribution of NO, cyclooxygenase products and endothelium-derived hyperpolarizing factor (EDHF) to dilatation induced by ACH (1 nM–10 μM), HIS (10 nM–10 μM) and BK (1 nM–10 μM) in rat mesenteric small arteries. Sodium nitroprusside (SNP; 1 nM–10 μM) was used as an endothelium-independent relaxant in these studies. Preliminary experiments (not shown) confirmed that relaxation in response to ACH, HIS and BK were completely abolished by physical removal of the endothelium as described previously (Prieto et al., 1997). Responses to substance P were also examined in this preliminary study, but maximal relaxation rarely exceeded 10%; so further studies were not conducted using this peptide.

Various agents were used to inhibit the putative mediators responsible for endothelium-dependent relaxation. NO synthase (NOS) was inhibited by using Nω-monomethyl-L-arginine (L- NMMA; 100 μM) and Nω-nitro-L-arginine methyl ester (L-NAME; 10 μM). Since arginine analogues may not fully inhibit NO synthesis in some preparations (Vanheel & Van de Voorde, 2000), the effectiveness of these concentrations of inhibitor was examined by comparing the inhibition seen to ACH in the presence of L-NMMA and L-NAME in the presence and absence of oxyhaemoglobin (10 μM), an NO scavenger (Simonsen et al., 1999). Preincubation with oxyhaemoglobin, L-NMMA and L-NAME did not significantly inhibit further relaxation in response to ACH compared with that when the NO synthase inhibitors were present alone (−log EC50 and maximum relaxation to ACH in the presence of L-NMMA & L-NAME=−7.06±0.11 and 96.9±2.1% compared with −7.21±0.18 and 93.1±4.5% (mean±s.e.m., n=4), respectively, in oxyhaemoglobin, and L-NMMA & L-NAME). It was concluded therefore that the concentrations of L-NMMA and L-NAME used were adequate to inhibit NO synthesis in this preparation. Indomethacin (INDO; 5 μM) was used as an inhibitor of cyclooxygenase. The relaxant effects of EDHF were inhibited by contracting vessels with a depolarizing potassium solution (K80) comprising PSS with equimolar substitution of 80 mmol KCl for NaCl.

Concentration–response curves to relaxants were constructed following establishment of stable contraction with phenylephrine (PE; 10 μM), or K80. These contractile stimulants induced maximal or near-maximal tone. The sequence of addition of relaxants was conducted using a Latin square design to overcome possible time- or order-dependent effects. For all studies, one of the pair of arteries (test vessel) was exposed to inhibitors, while the other acted as control. NO synthase inhibitors were added 30 min prior to addition of PE or K80 and remained in the bath throughout the experiment.

Effects of TNF and IL-1 on endothelium-dependent and endothelium-independent relaxation

In order to assess the effects of TNF and IL-1 on endothelium-dependent relaxation, the test vessel was incubated in TNF (1 nM), IL-1 (100 pM) or vehicle (distilled H2O) for 30 min prior to contraction with PE (10 μM), or K80 in some experiments. When arteries were exposed to TNF and NO synthase inhibitors, all inhibitors were present in the bath for 30 min prior to induction of tone. The concentrations of TNF and IL-1 used had no effect on resting tone and did not affect contraction in response to PE or K80 (data not shown). Cumulative concentration–response curves to relaxants were then constructed as described above with the arteries remaining exposed to the cytokine/vehicle. Separate sets of vessels were used for each relaxant to avoid confounding effects resulting from prolonged exposure to TNF or IL-1.

The possibility that the effect of TNF involved release of O2− was investigated by assessing whether superoxide dismutase (SOD; 150 IU ml−1) could influence the effects of TNF on ACH-induced relaxation. The test vessel was incubated in SOD for 10 min prior to incubation with TNF or vehicle for 30 min. The effect of TNF on relaxation in response to ACH in the continued presence or absence of SOD was then compared.

Data analysis and statistics

All data are presented as means±s.e.m.'s of (n) observations. Relaxation was calculated as per cent reduction in PE- or K80-induced contraction (there was negligible basal tone in these preparations). Cumulative concentration–response data were fitted to a logistic function by nonlinear regression using GraphPad Prism 3.02 (GraphPad Software Inc., Institute for Scientific Information, San Diego, U.S.A.) to estimate log EC50 values. log EC50 values were not calculated if maximum responses (Emax) were less than 20%. Data were compared statistically using a paired or unpaired two-tailed Student's t-test as appropriate, and P<0.05 was considered significant.

Drugs and reagents

All drugs and reagents were obtained from Sigma (Poole, Dorset, U.K.). Aliquots of human TNF (1 μM) and human IL-1 (100 nM) were prepared in sterile buffered saline containing 0.1% bovine serum albumin, stored at –20°C and defrosted immediately prior to use. Other drugs were made up in distilled water.

Results

ACH-induced relaxation

ACH (1 nM–10 μM) induced a concentration-dependent relaxation of rat mesenteric arteries contracted by PE (Figure 1a). L-NAME (10 μM) and L-NMMA (100 μM) inhibited Emax to ACH (P=0.03) and caused a small rightward shift in the concentration–response relation (Figure 1a, Table 1), although this was not statistically significant (P=0.07). Addition of INDO to L-NMMA and L-NAME had no additional effect on the effect of L-NMMA and L-NAME on relaxation in response to ACH (Figure 1a, Table 1).

Figure 1.

Figure 1

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Effect of NO synthase inhibition by L-NMMA and L-NAME in the presence or absence of INDO on relaxation in response to ACH in rat mesenteric small arteries contracted with (a) PE, (b) depolarizing potassium solution containing 80 mM KCl. Data are means±s.e.m. of (n) observations. *P<0.05 and **P<0.01 comparing maximum response between control and L-NMMA+L-NAME.

Table 1.

Effect of NO synthase inhibition by Nω-monomethyl-L-arginine (L-NMMA) and Nω-nitro-L-arginine methyl ester (L-NAME) in the presence or absence of indomethacin (INDO) on log EC50 and maximum response (Emax) to acetylcholine (ACH), bradykinin (BK), histamine (HIS) and sodium nitroprusside (SNP) in rat mesenteric small arteries contracted with phenylephrine (PE), or depolarizing potassium solution containing 80 mM KCl (K80)

    Control l-NAME+l-NMMA l-NAME+l-NMMA+INDO
ACH (PE) log EC50 −7.3±0.1 (13) −6.9±0.1 (7) −6.8±0.2 (6)
  Emax (%) 90±3 (13) 77±6 (7)* 69±13 (6)
ACH (K80) log EC50 −6.7±0.2 (11)† NC ND
  Emax (%) 48±8 (11)†† 8±2.2 (5)** ND
BK (PE) log EC50 −9.0±0.5 (9) NC NC
  Emax (%) 56±9 (9) 15±6 (5)* 17±11 (3)
BK (K80) log EC50 −6.0±0.2 (11)†† NC ND
  Emax (%) 39±5 (11) 5±1 (5)** ND
HIS (PE) log EC50 −6.5±0.1 (13) −6.4±0.1 (6) −6.6±0.5 (6)
  Emax (%) 71±8 (13) 58±9 (6) 46±17 (6)
HIS (K80) log EC50 −5.7±0.3 (11)†† NC ND
  Emax (%) 26±5 (11)†† 9±2 (5)* ND
SNP (PE) log EC50 −7.7±0.4 (13) −8.0±0.7 (7) −8.0±0.1 (6)
  Emax (%) 92±3 (13) 88±5 (7) 94±4* (6)
SNP (K80) log EC50 −6.8±0.1 (11)† −6.7±0.1 (5) ND
  Emax (%) 77±3 (11) 82±2 (5) ND
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Data are means±s.e.m. of (n) observations.

*P<0.05 and **P<0.01 comparing responses with control.

†P<0.05 and ††P<0.01 comparing PE- and K80-contracted arteries.

NC=not calculated (maximum relaxation<20%), ND=not done.

ACH also induced a concentration-dependent relaxation in arteries contracted by K80 (Figure 1b). The potency of ACH was significantly reduced in arteries contracted by K80 compared with PE-contracted arteries (P=0.01) and Emax was also reduced (P<0.001; Figure 1b, Table 1). L-NAME and L-NMMA almost completely abolished relaxation in response to ACH following K80 contraction (P=0.004; Figure 1b, Table 1).

BK-induced relaxation

BK (1 nM–10 μM) induced a concentration-dependent relaxation of rat mesenteric arteries contracted by PE. L-NAME and L-NMMA significantly inhibited Emax in response to BK (P=0.04) (Figure 2a, Table 1). Addition of INDO to L-NMMA and L-NAME had no additional effect on the impairment of relaxation to BK compared to vessels incubated with L-NMMA and L-NAME alone (Figure 2a, Table 1).

Figure 2.

Figure 2

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Effect of NO synthase inhibition by L-NMMA and L-NAME in the presence or absence of INDO on relaxation in response to BK in rat mesenteric small arteries contracted with (a) PE, (b) depolarizing potassium solution containing 80 mM KCl. Data are means±s.e.m. of (n) observations. *P<0.05 and **P<0.01 comparing maximum response between control and L-NMMA+L-NAME.

The potency of BK was significantly reduced in arteries contracted by K80 compared with PE-contracted arteries (P<0.001; Figure 2b, Table 1), and although Emax was reduced, this was not statistically significant. L-NAME and L-NMMA almost completely abolished relaxation in response to BK when vessels were contracted with K80 (Figure 2b, Table 1).

HIS-induced relaxation

HIS (1 nM–10 μM) induced a concentration-dependent relaxation of rat mesenteric arteries contracted by PE (Figure 3a, Table 1). L-NAME and L-NMMA caused a small and statistically insignificant inhibition of relaxation in response to HIS in vessels contracted by PE (Figure 3a, Table 1). Addition of INDO had no additional effect compared with L-NAME and L-NMMA (Figure 3a). Responses to HIS were markedly reduced following contraction with K80 with both Emax (P<0.001) and log EC50 (P=0.008) being affected (Figure 3b, Table 1). L-NAME and L-NMMA almost completely abolished the remaining small response to HIS (Figure 3b, Table 1).

Figure 3.

Figure 3

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Effect of NO synthase inhibition by L-NMMA and L-NAME in the presence or absence of INDO on relaxation in response to HIS in rat mesenteric small arteries contracted with (a) PE, (b) depolarizing potassium solution containing 80 mM KCl. Data are means±s.e.m. of (n) observations. **P<0.01 comparing maximum response between control and L-NMMA+L-NAME.

SNP-induced relaxation

SNP (1 nM–10 μM) induced a concentration-dependent relaxation of rat mesenteric arteries contracted by PE (Figure 4a, Table 1). L-NAME and L-NMMA had no significant effect on responses to SNP, although addition of INDO to the NO synthase inhibitors caused a small enhancement of Emax to SNP (P=0.03) (Figure 4a, Table 1). SNP was less potent following K80 contraction than PE contraction (P=0.04; Figure 4a and b, Table 1). L-NMMA and L-NAME had no significant effect on SNP-induced relaxation in arteries contracted with K80 (Figure 4b, Table 1).

Figure 4.

Figure 4

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Effect of NO synthase inhibition by L-NMMA and L-NAME in the presence or absence of INDO on relaxation in response to SNP in rat mesenteric small arteries contracted with (a) PE, (b) depolarizing potassium solution containing 80 mM KCl. Data are means±s.e.m. of (n) observations. †P<0.05 comparing maximum response between control and L-NMMA+L-NAME+INDO.

Effect of TNF and IL-1 on relaxation

In arteries contracted with PE, TNF induced impairment of relaxation to ACH, causing a significant shift in the ACH concentration–response curve to the right (P=0.03), but Emax was not significantly inhibited (Figure 5a, Table 2). TNF also inhibited BK-induced relaxation (Figure 5b, Table 2) causing a significant rightward shift in the concentration–response curve (P=0.02). In contrast, TNF did not significantly inhibit responses to HIS in arteries contracted by PE (Figure 6a, Table 2) and relaxation in response to SNP was also unaffected by TNF under these conditions (Figure 6b, Table 2). In arteries contracted with K80, preincubation with TNF markedly inhibited relaxation to ACH (Figure 7a, Table 2), but had no effect on responses to SNP (Figure 7b, Table 2).

Figure 5.

Figure 5

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Effect of TNF on relaxation in response to (a) ACH and (b) BK in rat mesenteric small arteries contracted with PE. Data are means±s.e.m. of (n) observations. ‡P<0.05 comparing log EC50 response between control and TNF-treated arteries.

Table 2.

Effect of tumour necrosis factor-α (TNF) on log EC50 and Emax to ACH, BK, HIS and SNP in rat mesenteric small arteries contracted with PE or depolarizing potassium solution containing 80 mM KCl (K80)

    Control TNF
ACH (PE) log EC50 −7.5±0.2 (8) −7.2±0.2 (8)*
  Emax (%) 95±2 (8) 86±4 (8)
ACH (K80) log EC50 −6.9±0.2 (8) NC
  Emax (%) 57±8 (8) 15±4 (8)**
BK (PE) log EC50 −8.4±0.4 (8) −7.8±0.4 (8)*
  Emax (%) 59±8 (8) 44±8 (8)
HIS (PE) log EC50 −5.8±0.1 (9) −6.0±0.1 (9)
  Emax (%) 84±4 (9) 87±2 (9)
SNP (PE) log EC50 −7.0±0.3 (8) −7.0±0.3 (8)
  Emax (%) 72±5 (8) 74±6 (8)
SNP (K80) log EC50 −7.1±0.1 (6) −7.2±0.1 (6)
  Emax (%) 83±4 (6) 84±4 (6)
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Data are means±s.e.m. of (n) observations.

*P<0.05 and **P<0.01 comparing responses with control.

NC=not calculated (maximum relaxation<20%).

Figure 6.

Figure 6

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Effect of TNF on relaxation in response to (a) HIS and (b) SNP in rat mesenteric small arteries contracted with PE. Data are means±s.e.m. of (n) observations.

Figure 7.

Figure 7

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Effect of TNF on relaxation in response to (a) ACH and (b) SNP in rat mesenteric small arteries contracted with depolarizing potassium solution containing 80 mM KCl. Data are means±s.e.m. of (n) observations. **P<0.01 comparing maximum response between control and TNF-treated arteries.

Preincubation with IL-1 (100 pM) for 30 min had no significant effect on relaxation in response to ACH in PE-contracted arteries (Figure 8a, Table 3). IL-1 co-incubated with TNF shifted the concentration – response curve to ACH to the right (Figure 8b, Table 3). This effect was similar to that seen with TNF alone. IL-1 had no significant effect on relaxation in response to SNP in arteries contracted with PE (Table 3). Similarly, the combination of IL-1 and TNF did not affect responses to SNP (Table 3).

Figure 8.

Figure 8

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Effect of (a) interleukin 1-β (IL-1), (b) IL-1 and TNF on relaxation in response to ACH in rat mesenteric small arteries contracted with PE. Data are means±s.e.m. of (n) observations. ‡P<0.05 comparing log EC50 response between control, and IL-1 and TNF-treated arteries.

Table 3.

Effect of interleukin 1β (IL-1) alone or in the presence of TNF on log EC50 and Emax to ACH and SNP in rat mesenteric small arteries contracted with PE

    Control IL-1 IL-1+TNF
  log EC50 −7.5±0.1 (12) −7.5±0.2 (5) −7.1±0.2 (8)**
ACH Emax (%) 90±5 (12) 74±8 (5) 84±7.7 (8)
  log EC50 −7.4±0.2 (12) −7.5±0.2 (4) −7.4±0.3 (8)
SNP Emax (%) 82±3 (12) 73±10 (4) 79±5 (8)
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Data are means±s.e.m. of (n) observations.

**

P<0.01 comparing responses with control.

Effect of SOD and NO synthase inhibitors on the effect of TNF

Addition of SOD did not significantly alter potency or efficacy of the concentration–response relation to ACH in arteries contracted with PE or K80. When arteries were exposed to TNF in the presence of SOD, TNF failed to inhibit responses to ACH in arteries contracted with PE or K80 (Figure 9a and b, Table 4). Furthermore, when arteries were preincubated with L-NAME and L-NMMA to inhibit NO synthase, prior to contraction with PE, responses to ACH were unaffected by TNF (Figure 9c, Table 4).

Figure 9.

Figure 9

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Effect of (a) SOD on inhibition of acetylcholine-induced relaxation by TNF in rat mesenteric small arteries contracted with PE, (b) SOD on inhibition of ACH-induced relaxation by TNF in rat mesenteric small arteries contracted with depolarizing potassium solution containing 80 mM KCl, (c) L-NMMA and L-NAME on inhibition of ACH-induced relaxation by TNF in rat mesenteric small arteries contracted with PE. Data are means±s.e.m. of (n) observations.

Table 4.

Effect of TNF on log EC50 and Emax to ACH in rat mesenteric small arteries contracted with PE in the presence of superoxide dismutase (SOD) or L-NMMA and L-NAME

    Control+SOD TNF+SOD
  log EC50 −7.4±0.2 (6) −7.4±0.2 (6)
ACH (PE) Emax (%) 90±4 (6) 90±5 (6)
  log EC50 −6.9±0.2 (3) −6.9±0.2 (3)
ACH (K80) Emax (%) 52±8 (3) 52±8 (3)
    CONTROL+l-NAME+l-NMMA TNF+l-NAME+l-NMMA
  log EC50 −7.2±0.1 (6) −7.1±0.2 (6)
ACH (PE) Emax (%) 93±4 (6) 92±4 (6)
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Data are means±s.e.m. of (n) observations.

Discussion

The main findings of this study were that exposure of rat mesenteric arteries to TNF for a brief period (30 min) significantly impaired endothelium-dependent relaxation to ACH and BK without inhibiting responses to HIS or SNP. The inhibitory effect of TNF on ACH-induced relaxation was more marked when arteries were contracted by a high potassium solution to block the contribution from EDHF. The effect of TNF could be inhibited by preincubation with SOD and was not seen when NO synthase was inhibited. Exposure to another proinflammatory cytokine, which is not elevated in pre-eclampsia, IL-1, had no effect on endothelium-dependent relaxation to ACH and combination of IL-1 with TNF had effects that were similar to that induced by TNF alone.

Previous studies have reported a range of effects of TNF and IL-1 on vascular and endothelial responses. Greenberg et al. (1993) reported generally similar findings to ours in bovine intralobar pulmonary vessels, where 30 min exposure to TNF inhibited NO-dependent relaxation to ACH, BK and HIS without affecting responses to nitroprusside. The responses to all these agents in this preparation appear to have been attributable to release of NO, since they were all inhibited by inhibitors of NO synthase. Aoki et al. (1989) reported that 2 h perfusion of cat carotid artery with TNF inhibited responses to ACH, but not NaNO2; however, these effects were not seen after perfusion for only 1 h. In contrast, longer exposure (7–15 h) of rabbit carotid or porcine coronary arteries to IL-1 or TNF (the latter in combination with interferon-γ and lipopolysaccharide) reduced endothelium-dependent relaxations to ACH and substance P, but not to the calcium ionophore, A23187 (Kessler et al., 1997). Kessler et al. (1997) also reported that brief (15 min) exposure to IL-1 had no effect on responses to ACH, consistent with our findings. Prolonged exposure to cytokines is also associated with reduced contraction, as has been reported by many investigators (Beasley et al., 1989; Robert et al., 1992,1993; Thorin-Trescases et al., 1995; Kessler et al., 1997) and this may involve activation of inducible NOS (iNOS) (Busse & Mulsch, 1990), or NO-independent effects on guanylate cyclase or other systems in smooth muscle (Beasley & McGuiggin, 1994; Thorin-Trescases et al., 1995; Kessler et al., 1997). In vivo, a 30-min infusion of TNF in rats was reported to depress ACH-induced relaxation in isolated pulmonary arteries and aorta (Wang et al., 1994). Similarly, a 5-day infusion of TNF into pregnant rats has also been reported to cause impaired endothelium-dependent relaxation to ACH in aortic strips isolated from pregnant rats (Davis et al., 2002). It is noteworthy that these studies have been performed using larger conduit-type arteries where EDHF may play a smaller role in mediating responses to ACH and other endothelium-dependent agents.

Endothelium-dependent relaxation to a particular agent may be mediated by a number of factors, including NO (Moncada et al., 1991), cyclooxygenase products such as prostacyclin (PGI2) (Vane et al., 1987) and EDHF (Triggle & Ding, 2002). The identity of EDHF remains uncertain, but it is accepted that its action involves an increase in K conductance and can therefore be inhibited by abolishing the electrochemical gradient for K+ions (Chen & Suzuki, 1989). This was conducted in these studies by using a solution containing 80 mM potassium to induce tone. This technique has been reported previously not to affect responses to NO (Fukao et al., 1995; Chen & Cheung, 1997); however, we observed that SNP was less potent in K80- than PE-contracted arteries and it is possible that responses to NO may also be affected by depolarization with high potassium solutions in our preparation.

Our data indicate that, in rat mesenteric arteries, the endothelium-dependent actions of ACH, BK and HIS involve both NO and EDHF, apparently acting in a synergistic manner, but that their individual contribution to relaxation by a particular agent may differ. Responses to ACH and BK were less affected by inhibition of EHDF than responses to HIS, whereas the effect of NO synthase inhibition was more marked for ACH and BK than HIS. These observations regarding the importance of EDHF in mediating responses to HIS are in keeping with a previous study of pressurized rat mesenteric arteries (Lagaud et al., 1999). Cyclooxygenase products do not appear to play a major role in responses to ACH, BK or HIS as previously reported for ACH-induced relaxation in this preparation (Wu et al., 1994; Lacy et al., 2000), although bradykinin-dependent contraction in pressurized rat mesenteric arteries has been reported to be inhibited by INDO (Fasciolo et al., 1990).

Unlike ACH and BK, responses to HIS were not inhibited by TNF. This could be explained by TNF selectively inhibiting NO, as opposed to EDHF. This proposal is supported by the inability of TNF to inhibit ACH-induced relaxation when NO synthase was inhibited, or when superoxide oxide formation was reduced by SOD. Contractile responses to PE or K80, or relaxation to SNP were unaffected by TNF and this suggests that changes in smooth muscle contractility, or responsiveness to cyclic guanosine monophosphate (cGMP) do not play a part in the effect of TNF after a brief duration of exposure as used in this study.

TNF may affect endothelial function through a number of mechanisms (reviewed in Madge & Pober, 2001). In particular, it has been reported to downregulate endothelial nitric oxide synthase (eNOS) mRNA expression (Yoshizumi et al., 1993) and protein (de Frutos et al., 1999). However, given the relatively short exposure times used in this study, a mechanism involving altered levels of protein expression does not appear to be particularly likely. Our observations with SOD suggest that generation of O2− may underlie the inhibitory effects of TNF on NO-dependent relaxation seen in our study. TNF has previously been reported to induce production of O2− in cultured endothelial cells (Murphy et al., 1992), probably via activation of a phagocyte-type NADPH oxidase complex (Li et al., 2002).

Although extrapolation of animal data to clinical scenarios must be made with caution, it is interesting that the magnitude of the impairment of response to ACH and BK was similar to that previously reported in subcutaneous arteries from pre-eclamptic women compared to normal pregnant women (McCarthy et al., 1993; Knock & Poston, 1996). Inhibitory effects of TNF on endothelium-dependent relaxation have been observed in vivo in humans. Infusion of TNF into the brachial artery has been reported to impair forearm vasodilation to ACH (Nakamura et al., 2000), and anti-TNF therapy with etanercept, a recombinant TNF receptor that binds to, and functionally inactivates TNF, improves endothelium-dependent relaxation in patients with heart failure (Fichtlscherer et al., 2001). It is possible that the elevated TNF levels, resulting from the excessive maternal immune response or placental hypoxia could contribute to the endothelial dysfunction seen in pre-eclampsia through increased production of O2−. Such a mechanism could explain a recent study reporting that antioxidant treatment with vitamin E and vitamin C has a beneficial effect on the incidence of pre-eclampsia (Chappell et al., 1999).

Acknowledgments

R.W. was supported by a British Heart Foundation Fellowship and S.F. is supported by a British Heart Foundation Ph.D. Studentship. We are grateful to Dr R. Garcha for technical assistance with some of the experiments.

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