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Cardiovascular Research 1999 41(3):754-764; doi:10.1016/S0008-6363(98)00249-1
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Cytokine-induced venodilatation in humans in vivo: eNOS masquerading as iNOS

Kiran Bhagata,1, Aroon D Hingoranila, Miriam Palaciosb, Ian G Charlesb and Patrick Vallancea,*

aCentre for Clinical Pharmacology & Therapeutics, Wolfson Institute for Biomedical Research, University College London, 5 University St, London WC1E 6JJ, UK
bMolecular Biology Laboratory, Wolfson Institute for Biomedical Research, University College London, 5 University St, London WC1E 6JJ, UK

* Corresponding author. Tel.: +44-171-209-6351; Fax: +44-171-813-2846; E-mail: patrick.vallance@ucl.ac.uk

Received 15 May 1998; accepted 22 July 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Venodilatation is a feature of endotoxaemia and sepsis. We have tested directly the hypothesis that three cytokines (IL-1β, TNF{alpha} and IL-6) generated during endotoxaemia affect venous tone in humans in vivo by increasing NO generation and explored whether the NO comes from the iNOS or eNOS isoform. Design and intervention: Cytokines were given into a superficial vein in very low doses sufficient only to produce changes in the study vessel. The effects of cytokines on the response to noradrenaline were examined. Results: IL-1β increased basal NO-induced dilatation in the study vein, and this was sufficient to attenuate the constrictor response to exogenous noradrenaline or sympathetic stimulation. The effects were maximal at 6 h and both NG-monomethyl-L-arginine and aminoguanidine caused significant reversal of the IL-1β effects. However, no induction of iNOS mRNA was detected in the tissue samples. Instead, mRNA encoding eNOS and GTP cyclohydrolase-1 was detected in all vessels. Conclusion: The simplest explanation of these results is that IL-1β induces expression of GTP cyclohydrolase-1 which leads to increased generation of BH4 and activation of eNOS. This study identifies IL-1β as a key cytokine causing physiologically significant venodilatation in humans by increasing NO generation and suggests that this can occur even in the absence of iNOS expression.

KEYWORDS Nitric oxide; Veins; Septic shock; GTP cyclohydrolase 1; Human; Cytokines; Tetrahydrobiopterin; Inducible nitric oxide synthase


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Systemic sepsis and endotoxaemia are associated with abnormal dilatation of arteries and veins, blunted vascular responses to activation of the sympathetic nervous system, and hyporesponsiveness to vasoconstrictors [1–3]. Studies in animals suggest that the vasodilatation is dependent upon generation of pro-inflammatory cytokines which induce de novo expression of the inflammatory isoform of nitric oxide synthase (iNOS) throughout the vessel wall and thereby increase synthesis of the vasodilator nitric oxide (NO). Whilst there is no direct evidence for expression of functionally active iNOS in the vasculature in human sepsis, production of NO appears to be increased and an inhibitor of NOS (NG-monomethyl-L-arginine; L-NMMA) reverses the hypotension of septic shock [4]. However, it is not known whether the substantial venodilatation which occurs in sepsis [3]and certain other inflammatory conditions is also mediated by NO, or which of the many cytokines generated during infection are responsible for the changes in vascular reactivity.

Studies of human vessels in vitro suggest that veins, unlike arteries, do not increase NO generation in response to endotoxin or cytokines [5], but it is possible that the vessels respond differently in vivo. Therefore, we developed an in vivo model to explore the mechanisms of septic vasodilatation in volunteers [6, 7]. In this model, drugs, endotoxin or cytokines are given into a single superficial hand vein in very low doses sufficient only to produce changes in the study vessel – to create a single septic, or inflamed, vessel without initiating a systemic cytokine cascade. We have used this approach to determine the functional consequences of exposing a human blood vessel to individual cytokines in vivo and sought to identify the molecular mechanisms underlying the changes seen.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 In vivo pharmacology
Studies were approved by the local ethics committee and all studies conformed with the latest Declaration of Helsinki [8]. Studies were performed on male and female subjects aged 19–40. Subjects were included who stated that they were healthy and taking no medication, and who gave their informed, written consent.

2.2 Assessment of venous reactivity
Subjects lay supine in a temperature-controlled laboratory (28–30°C). A congesting cuff was placed around the upper arm and inflated to 40 mmHg. Drugs or physiological saline were infused through a 23-gauge needle placed in a dorsal hand vein. The diameter of the vein was measured 5–10 mm downstream from the tip of the infusion needle by recording the linear displacement of a light-weight probe placed on the skin overlying the summit of the vessel when the pressure in the congesting cuff was lowered from 40 to 0 mmHg [6, 7]. In all studies saline was infused for at least 15 min until a stable baseline vein diameter was recorded. Under the conditions in which these experiments were undertaken the veins have no basal tone and are maximally relaxed [6, 7].

Dose–response curves to noradrenaline (10–640 pmol/min), or the sympathetic venoconstriction induced by taking a deep breath [9–12], were recorded before and for up to 48 h after instillation of cytokines. To explore dilator responses, vessels were partially (50%) preconstricted with a continuous infusion of noradrenaline (10–640 pmol/min) and the dilators were then co-infused with the noradrenaline. In some studies inhibitors of NOS were co-infused with constrictor or dilator agents. A total of 170 studies were performed in 50 volunteers.

2.3 Instillation of cytokines
To instil cytokines, a length of the vein under study was isolated from the circulation by means of two wedges placed 2–3 cm apart on the skin overlying the vessel as described previously [6, 7]. Cytokines [interleukin (IL) 1β, IL-6 or tumour necrosis factor (TNF{alpha})] were instilled for 1 h, either individually or together. At the end of the period of instillation, the vein was emptied, the wedges were removed and the vein was reconnected with the circulation for assessment of reactivity. This method of instillation produces local changes in the study vein but adjacent vessels remain unaffected [6, 7]. The volume of blood in the isolated vein is in the order of 1–2 ml and the calculated concentration of cytokine was in the order of 300–1000 pg/ml (TNF{alpha} and IL-1β given at 1 ng/ml) and 30–100 pg/ml (IL-6 given at 100 pg/ml).

2.4 Calculations and statistics
Changes in vein size were measured in arbitrary units and converted to millimetres following calibration of the transducer at the end of each experiment. The response of the resting vein to drugs is expressed as a reduction in diameter from that measured during infusion of saline alone. The response of the noradrenaline-preconstricted vein to drugs is expressed as percentage reversal of the induced constriction. Results are compared using the Students’ t-test for paired data or analysis of variance of the means as appropriate; p<0.05 is considered statistically significant.

2.5 Biopsy
Surgical removal of the vein was carried out under local anaesthesia (1% lignocaine). All samples were handled minimally and immediately frozen to –80°C. All biopsies were taken 3–4 h after instillation of cytokines, a time at which functional changes were already evident.

2.6 mRNA extraction, reverse transcription and competitive PCR
Reverse transcription was carried out on poly A+ mRNA extracted from hand vein biopsies using Fast Track reagents (Invitrogen, Carslbad CA) according to the manufacturer’s instructions. Poly A+ mRNA was resuspended in 20 µl Tris (pH 7.5). Since mRNA rather than total RNA was extracted from very small biopsy specimens, it was not feasible to measure the amount of extracted RNA by conventional methods such as spectrophotometry. For this reason, for each vein sample, competitive PCR was conducted for the constitutively expressed gene glyceraldehyde-3 phosphate dehydrogenase to allow a correction for between-sample differences in the efficiency of RNA extraction. A constant volume of poly A+ RNA and cDNA was used in all cases. Duplicate 2-µl aliquots of poly A+ mRNA were reverse transcribed in a final volume of 20 µl and then pooled. Each reaction contained 150 ng of random hexanucleotides, 0.5 mM each of dATP, dTTP, dGTP and dCTP, 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol and 200 U SuperScript II RNAse H– reverse transcriptase (Life Technologies, Paisley, UK). The reactions were incubated at 42°C for 55 min followed by denaturation at 70°C for 15 min before cooling to 4°C. PCR was conducted in a volume of 25 µl [containing 200 µM dNTPs, 1.5 mM MgCl2, 10 pmol of specific forward and reverse primers and 1 U of recombinant Taq DNA polymerase (Life Technologies, Paisley UK)] for 30 cycles on a Biometra-TRIO thermocycler with denaturing at 95°C for 35 s, annealing at 56°C for 1 min and extension at 72°C for 2 min. Each reaction contained 1 µl of human hand vein cDNA and 1 µl of an appropriate gene-specific competitor DNA (in the concentration range 10–2 amol/µl to 10–6 amol/µl) for eNOS, iNOS and GTP cyclohydrolase-1 and (1 amol/µl to 10–4 amol/µl) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Preliminary experiments confirmed that amplification was in the exponential phase of the amplification curve and that targets and respective mimics amplified with similar efficiency in all cases. Competitors were non-homologous dsDNA fragments synthesised by PCR with a BamHI/EcoRI fragment of the v-erbB gene as a DNA template. Composite oligonucleotide primers were used comprising a v-erbB-specific sequence at the 3' end and endothelial NOS (eNOS), inducible NOS (iNOS), GTP cyclohydrolase I or GAPDH-specific sequences (as appropriate) at the 5' end. The conditions for the PCR and for the purification and quantitation of the synthesised competitor fragments was as detailed in the MIMIC construction kit (Clontech, Palo Alto, CA). The specificity of the gene-specific PCR primers was confirmed in reactions containing poly A+ mRNA from human placenta (for eNOS) and from the human pancreatic adenocarcinoma line CAPAN-1 stimulated with cytokines (for iNOS and GTP cyclohydrolase-1). The identity of the PCR products was confirmed by DNA sequencing. All gene-specific sense primers were labelled at their 5' end with the fluorophore FAM in order to allow PCR product detection and sizing on the ABI 377 DNA sequencer using Genescan analysis software (Perkin-Elmer/Applied Biosystems, Foster City CA). To avoid amplification of any contaminating DNA, primers were designed so as to span introns. The sequences of the gene-specific primers and the predicted sizes of target and competitor PCR products are as follows:

Human eNOS:
5'-CAGTGTCCAACATGCTGCTGGAAATTG-3' (sense).
5'-TAAAGGTCTTCTTCCTGGTGATGCC-3' (antisense).
Target: 486 bp. competitor 608 bp.
Human iNOS:
5'-CAGTACGTTTGGCAATGGAGACTGC-3' (sense).
5'-GGTCACATTGGAGGTGTAGAGCTTG-3' (antisense).
Target 340 bp, competitor 556 bp.
Human GTP cyclohydrolase I:
5'-TTGGTTATCTTCCTAACAAG-3' (sense).
5'-GTGCTGGTCACAGTTTTGCT-3' (antisense).
Target 226 bp, competitor 440 bp.
Human GAPDH:
5'-AAGGTGAAGGTCGGAGTCAACG-3' (sense).
5'-GGCAGAGATGATGACCCTTTTGGC-3' (antisense).
Target 363 bp, competitor 432 bp.

Aliquots of each PCR reaction were subjected to agarose gel electrophoresis and analysis by fluorescence detection in an ABI 377 DNA sequencer using Genescan software. Using this system when there is a molar equivalence of target and competitor PCR products (as estimated by fluorescence intensity measured as the area under the Genescan electropherogram peak) the amount of initial target cDNA is approximately equal to the amount of initial corresponding competitor. The investigator performing the molecular analysis was blinded to the treatment status of the vein. The lower limit of detection of cDNA was in the order of 10–5 amol.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
First we sought to explore the effects of inflammatory cytokines on the vasoconstrictor responses to noradrenaline. Three cytokines implicated in the systemic cardiovascular response to sepsis were studied-IL-1β, TNF{alpha}, and IL-6. Instillation of IL-1β (1 ng in 1 ml of saline for 1 h; n=5) into a single superficial blood vessel attenuated the response to noradrenaline (Fig. 1; panel A). This effect developed over several hours and reached a maximum 6 h after exposure of the vessel to the cytokine. By 24 h the response to noradrenaline was fully restored. Instillation of either TNF{alpha} (1 ng in 1 ml of saline for 1 h; n=5), or IL-6 (100 pg in 1 ml of saline for 1 h; n=5) alone produced no significant change in the response to noradrenaline (Table 1), but co-instillation of these cytokines with IL-1β for 1 h caused a prolonged (>24 h) attenuation of the response to noradrenaline induced by cytokines [Fig. 1, panels B (n=5) and C (n=5])]. The slowly developing hyporesponsiveness to noradrenaline was prevented by prior treatment of subjects with oral hydrocortisone given in a therapeutically relevant dose. Oral hydrocortisone (100 mg) taken 2 h before the administration of the cytokine mix (IL-1β, TNF{alpha}, and IL-6) prevented the development of hyporesponsiveness to noradrenaline. In subjects pre-treated with hydrocortisone, the constriction to the four doses of noradrenaline used were: 25±5%, 53±7%, 71±11%, 89±12% before the cytokine mix and 27±6%, 48±5%, 68±4% and 85±10% 1 h after instillation of cytokine (ns).


Figure 1
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  		Fig. 1 Dose–response curves were constructed to noradrenaline before ({circ}) and 1 h (bullet), 6 h ({blacksquare}), 24 h ({diamond}) after instillation of IL-1β alone (1 ng; n=5; panel A); IL-1β (1 ng) and TNF{alpha} (1 ng) together (n=5; panel B); and 1, 6, 24 and 48 h ({diamondsuit}) after instillation of the combination IL-1β (1 ng), TNF{alpha} (1 ng) and IL-6 (100 pg) (n=5; panel C). * Indicates p<0.05 compared to control.

 

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  		Table 1 Dose–response curves were constructed to noradrenaline before and 6 h after the vessel was exposed to TNF{alpha} (upper panel) or IL-6 (lower panel)

 
3.1 Effects of NOS inhibitors
In order to determine whether the hyporesponsiveness to noradrenaline was mediated by NO, we tested the effects of NG-monomethyl-L-arginine (L-NMMA; a non-isoform-selective NOS inhibitor [13]), and aminoguanidine (a compound that has been used widely as a selective inhibitor of iNOS [14]). Both L-NMMA (1 µmol/min; Fig. 2 panel A; n=5) and aminoguanidine (1 µmol/min; Fig. 2, panel B; n=5) reversed the IL-1β-induced attenuation in the response to noradrenaline. Infusion of the substrate for NOS (L-arginine; 1 µmol/min) reversed the effect of the inhibitors (Fig. 2, panels A and B).


Figure 2
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  		Fig. 2 Dose–responses curves to noradrenaline were constructed before ({circ}) and 6 h (bullet) after instillation of IL-1β. Then, a repeat dose–response curve was constructed in the presence of L-NMMA (1 µmol/min; {blacksquare}, panel A) or aminoguanidine (1 µmol/min; {blacksquare}, panel B) co-infused with noradrenaline. This was followed by a saline washout period of 15 min and a repeat dose–response curve to noradrenaline was constructed in the presence of L-arginine (1 µmol/min; {blacktriangledown}). * Indicates p<0.05 compared to the initial dose response to noradrenaline; + Indicates p<0.05 compared to the response in the presence of IL-1β alone.

 
The effects of the NOS inhibitors on classical eNOS-mediated responsiveness were also studied. In healthy human dorsal hand veins there is no basal generation of NO [15]and, in the absence of IL-1β treatment, neither L-NMMA (1 µmol/min; n=5; Fig. 3, panel A) nor aminoguanidine (1 µmol/min; n=5 – data not shown) had any constrictor action nor did they alter the response to noradrenaline. The dose–response to the endothelium-dependent dilator bradykinin (2–8 pmol/min; n=5) was almost abolished by infusion of L-NMMA (1 µmol/min); >80% inhibition of bradykinin-induced dilatation; p<0.05; whereas aminoguanidine (1 µmol/min; n=5) had no effect on bradykinin-induced venodilatation, consistent with its putative selectivity for iNOS (Fig. 3, panel B). Mean dilatation to bradykinin (2, 6 and 8 pmol/min) was 23±6%, 46±4% and 86±7% and after L-NMMA was 6±3%, 5±3% and 6±5% and after aminoguanidine was 27±7%, 49±3% and 80±5%.


Figure 3
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  		Fig. 3 Traces showing effects of L-NMMA on vessel tone in control vessel (upper trace) and vein treated with IL-1β (lower trace). The constrictor effects of L-NMMA (1 µmol/min) in cytokine-treated vessels were reversed by L-arginine (1 µmol/min). Congesting cuff deflation and re-inflation is marked by a solid circle. This trace is representative of five similar experiments. The mean constriction to L-NMMA in control vessels was 4±3% and in the IL-1 β was 44±4%. (b) Representative traces showing the effects of co-infusing L-NMMA (1 µmol/min) and bradykinin (2, 4, 8 pmol/min, each dose for 5 min) (upper trace) or aminoguanidine (1 µmol/min) and bradykinin (2, 4, 8 pmol/min, each dose for 5 min) (lower trace). Congesting cuff deflation and re-inflation is marked by a solid circle. This trace is representative of five similar experiments (full data in Section 3.1). (c) Two adjacent vessels were studied simultaneously at a distension pressure of 40 mmHg. The sympathetic nervous system was activated by asking subjects to take and hold a deep breath [5]. This manoeuvre (arrows) produced transient constriction in both vessels. Incubation of one vessel (lower trace) with IL-1β for 1 h virtually abolished sympathetically mediated constriction assessed 6 h later. Sympathetic constriction was restored by infusion of L-NMMA (1 µmol/min). The trace is a representation of three similar experiments.

 
3.2 Functional significance of NO generated
The potential pathophysiological significance of the increase in NO synthesis induced by IL-1β is illustrated in Fig. 3, panel C. Instillation of cytokines virtually abolished endogenous venoconstriction due to activation of the sympathetic nervous system [10, 12, 16]. Activation of the sympathetic nervous system produced simultaneous transient venoconstriction in two adjacent superficial veins (15±6% constriction). After IL-1β, the constrictor response to deep breath was abolished in the treated (3±5%) but not in the control vein (17±7%). Local infusion of L-NMMA did not alter control responses to sympathetic activation (15±6%), but restored the ability of the sympathetic nervous system to cause venoconstriction in vessels exposed to IL-1β 19±5%).

3.3 Molecular basis of increased NO generation
To explore further the mechanism of the functional induction of NOS activity, we studied gene expression in the vessel wall. Messenger RNA was extracted from segments of hand vein excised from eight healthy male volunteers; three had received a local instillation of IL-1β 4 h prior to biopsy, and five were untreated (see Table 2). Biopsies were taken at a time when functional changes were already evident and using competitive RT–PCR, the lower limit of detection of cDNA was of the order of 10–5 amol. Messenger RNA encoding eNOS was present in all samples, but there was no detectable iNOS mRNA in cytokine-treated veins and we did not detect mRNA encoding for the neuronal form of NOS (nNOS; data not shown). Instead, mRNA encoding the enzyme GTP cyclohydrolase 1 was detected exclusively in veins which had been exposed to cytokines, with a level of expression approximately equal to that of eNOS mRNA (Table 2 and Fig. 4). Expression of mRNA for GAPDH was similar in all samples, indicating that RNA extraction efficiency was similar in all cases.


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  		Table 2 Tabular summary of competitive RT–PCR results

 

Figure 4
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  		Fig. 4 Competitive RT–PCR of hand vein cDNA from a subject who received a local instillation of IL-1β (subject G; see Table 2). A constant volume (1 µl) of extracted hand vein cDNA was amplified by PCR in a final volume of 25 µl (see Section 2). Amplifications were performed in the presence of varying quantities of the appropriate competitor fragment in the range 10–2 amol to 10–6 amol (for eNOS, iNOS and GTP cyclohydrolase-I) and 1 amol to 10–4 amol for the housekeeping gene GAPDH. Ten µl of the respective PCR products were electrophoresed on a 2% agarose gel stained with ethidium bromide (Lane M is a 100-bp size standard and lane C the negative control).

 
3.4 Effects of BH4 on eNOS
Induction of GTP cyclohydrolase I would be expected to increase BH4 generation. To determine the effects of an increased concentration of BH4, we infused it into preconstricted healthy control hand veins. BH4 (250 nmol/min for 20 min; n=6) caused a rapid vasodilatation (Fig. 5) and, in contrast to the dilatation induced by bradykinin, the response to BH4 was fully reversed by infusion of either L-NMMA (data not shown) or aminoguanidine (1 µmol/min; Fig. 5).


Figure 5
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  		Fig. 5 Panel A: Infusion of BH4 (250 nmol/min for 20 min) into a control vein causes sustained vasodilatation (hatched line; n=6). Co-infusion of aminoguanidine reverses the dilatation (solid line; n=6). Panel B: Original trace showing effects of BH4 infusion (upper trace) and reversal by aminoguanidine (lower trace). Congesting cuff deflation and re-inflation is marked by solid circle.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Venodilatation is a prominent feature of sepsis and endotoxaemia and leads to pooling of blood, reduced venous pressure, postural hypotension and decreased cardiac output [2, 3]. The venodilatation occurs despite enhanced sympathetic nervous system activity and increased circulating levels of endogenous constrictors. The underlying mechanism is unknown. The results of the present study in healthy volunteers demonstrate that the pro-inflammatory cytokine IL-1β, which is synthesised during a systemic inflammatory response [17], induces a slowly-developing basal NO-mediated dilatation in human veins in vivo. The NO generated is sufficient to cause significant hyporesponsiveness to the constrictor actions of noradrenaline and almost abolishes the ability of the sympathetic nervous system to venoconstrict. These effects of IL-1β are prevented by a therapeutic dose of a glucocorticoid and reversed by inhibitors of NOS, however, the NO generated appears to come from constitutively expressed eNOS rather than de novo induction of iNOS.

4.1 IL-1β induces significant venous hyporesponsiveness
We have shown previously that endotoxin itself does not directly initiate NO generation in the hand veins [6]and it is likely that the systemic haemodynamic effects of endotoxin are dependent upon cytokine generation. Three cytokines implicated in the systemic response to sepsis were studied: IL-1β, IL-6 and TNF{alpha}. The vein model makes it possible to study the effects of each cytokine separately and the results show that whilst IL-1β is necessary to initiate the NO-mediated venodilatation in vivo, TNF{alpha} and IL-6 act synergistically to prolong the effect. Neither saline or IL-6 or TNF{alpha} alone altered the response to noradrenaline at any time point in this or a previous study [7], indicating that the effects of IL-1β were specific and not due to a non-specific ‘time’ effect. It is possible that the IL-1β stimulated local generation of other cytokines in the vein segment and that this is why induction of NO generation occurred readily in this in vivo model but not in similar studies performed in human vessels or cells in vitro [5, 18, 19]. Additional cytokines are also synthesised during a systemic inflammatory response and it would be interesting to know how these would affect the response to IL-1β in this model.

The sympathetic nervous system is a major determinant of venous tone in humans [10, 12, 16]and the functional antagonism produced by IL-1β-induced NO generation would cause profound venodilatation. The finding that L-NMMA restored the ability of noradrenaline and of the sympathetic nervous system to constrict IL-1β-treated vessels, but did not alter sympathetic constrictions, basal vein size or the response to noradrenaline in healthy vessels, explains the observation that systemic administration of L-NMMA does not alter venous pressure in healthy volunteers [20]but increases it in patients with septic shock [4].

4.2 New gene transcription underlies the effect
In order to explore the molecular basis for the NO generation, a segment of the study vein was removed from eight subjects. As expected, mRNA encoding the constitutive endothelial isoform of NOS (eNOS) and mRNA encoding the ‘house-keeping’ enzyme GAPDH was present in all of the biopsies and we did not detect mRNA for nNOS. Contrary to previous reports in cell culture systems there was no decrease in mRNA for eNOS after cytokine treatment in this in vivo system. Following cytokine treatment no mRNA encoding iNOS was detected, even though the biopsies were taken at a time when hyporesponsiveness to noradrenaline was already evident and the lower limit of detection for cDNA was of the order of 10–5 amol (10–23 mol). Instead mRNA encoding GTP cyclohydrolase-1 was detected in veins treated with cytokines but not in control vessels, suggesting that the cytokines had induced expression of this enzyme. It has been shown previously that expression of GTP cyclohydrolase I is transcriptionally regulated in certain cells and tissues in vitro and that induction can be prevented by glucocorticoids [21].

Expression of GTP cyclohydrolase 1 is rate limiting for the formation of BH4, a co-factor for all NOS isoforms [21, 22]and to determine the effects of an elevated local concentration of BH4 we infused it into healthy control veins which had not been treated with cytokines. BH4 caused rapid vasodilatation that was reversed by L-NMMA indicating that the dilatation was due to NO generated from a constitutive NOS present in healthy veins (eNOS). Similar to the results of our experiments in vivo, cytokine-stimulated NO generation in human umbilical vein endothelial cells maintained in culture is associated with increased eNOS-like activity and expression of GTP cyclohydrolase I [23–25], with no evidence of iNOS mRNA or protein (our unpublished observations). It has been shown that BH4 will lead to increased NO generation from eNOS for each given increment in calcium [26]. Thus it would appear that eNOS might be activated by keeping calcium constant and increasing BH4 or alternatively by keeping BH4 constant and increasing calcium. It may be that in the healthy vein, a low level of BH4 is generated through the salvage pathway that by-passes GTP cyclohydrolase 1 [27]and that this is sufficient to render eNOS active provided calcium levels increase. In the cytokine-treated vein, the elevated BH4 would activate eNOS even in the presence of low basal concentrations of calcium. Further studies will be required to test this hypothesis directly.

4.3 Effects of aminoguanidine
Aminoguanidine reversed the hyporesponsiveness induced by IL-1β. This compound has been widely used as a selective inhibitor of iNOS in studies in animals [14]and humans [28]and, consistent with its proposed selectivity, does not inhibit the eNOS-mediated vasorelaxant responses to bradykinin in human veins in vitro [29]or in vivo (this study). Therefore, the finding that aminoguanidine reversed IL-1β-induced hyporesponsiveness in the hand veins, even though iNOS was not expressed, presents an apparent paradox. However, while aminoguanidine has little effect on the activity of BH4-deficient purified eNOS, it has been suggested that it becomes an effective inhibitor when BH4 is added to the enzyme preparation [30]. Thus aminoguanidine inhibits eNOS provided a sufficient concentration of BH4 is present, possibly because the BH4 acts as an allosteric regulator of the arginine binding site of NOS, altering its affinity for substrate-based inhibitors [31, 32]. Consistent with these observations, in the present study aminoguanidine fully reversed the rapid eNOS-mediated dilatation to local infusion of BH4. These findings further support the suggestion that the IL-1β-induced dilatation is due to induction of local BH4 synthesis and suggests that aminoguanidine should not be considered a selective inhibitor of iNOS; however further studies will be required to test this hypothesis directly and to establish the precise mechanism of these effects of aminoguanidine.

4.4 Limitations of the study
The study was performed in human beings in vivo. This has obvious advantages, including clinical relevance, and in this study it was possible to detect cytokine-induced NO generation which has not been seen when cytokine-exposed human veins were studied in vitro [5]. However, the approach also carries certain necessary limitations. The constrictor responses to noradrenaline and sympathetic stimulation were impaired by cytokines but it is possible that the responses to other endogenous constrictors that we could not study in this model in humans (e.g. thromboxane or potassium) were unaffected. In addition, although a role for iNOS in the early phase of the IL-1β-induced response can be excluded, it is not feasible to repeatedly remove vessels in order to study later time points, at which iNOS mRNA might have appeared. Nonetheless, the functional responses were near maximal at the time the veins were removed and it can be concluded that expression of iNOS is not necessary to evoke a near maximal cytokine-induced NO-mediated dilatation in human veins in vivo. Finally, although our competitive PCR strategy which utilises DNA (rather than RNA) mimics does not allow for correction for differences in the efficiency of reverse transcription, the changes we have observed in the expression of GTP cyclohydrolase 1 were ‘all-or-nothing’ and could not be accounted for by differences in reverse transcription efficiency.

4.5 Clinical and therapeutic implications
The results of this study demonstrate that pro-inflammatory cytokines, particularly IL-1β, can induce functionally significant venous hyporesponsiveness to vasoconstrictors in humans and that the effect is mediated by NO. We suggest that this may be a fundamental mechanism underlying the venodilatation that occurs during a systemic inflammatory response or sepsis, and which is often treated by increasing the intravascular volume to maintain an adequate venous pressure. In animal models, and in many arteries, cytokines induce NO generation through transcriptional up-regulation of iNOS. However, the phenomenon we have observed in the hand veins is best explained by de novo gene transcription causing an increase in expression of GTP cyclohydrolase 1 and consequent activation of eNOS by BH4. An implication of the findings is that the venous endothelium is able to generate sufficient NO to cause profound vasodilatation even in the absence of expression of iNOS. The results and those of previous similar studies in cultured cells [23–25], suggest that drugs that inhibit GTP cyclohydrolase I might be of therapeutic use to reverse local or systemic inflammatory venous dilatation. The results of this study may also have relevance outside the venous system. Expression of GTP cyclohydrolase 1 leads to pterin synthesis and it has been known for many years that pterin synthesis is increased in a variety of inflammatory, infective and neoplastic conditions in humans. Indeed, plasma levels of neopterin have been used as a marker of the severity of inflammation in clinical practice [33–35]. It would be interesting to determine whether induction of GTP cyclohydrolase 1 in other cells also increases NO generation from constitutive isoforms of NOS.

Time for primary review 32 days.


   Acknowledgements
 
This work was supported by the British Heart foundation. KB is a BHF Research Fellow, AH is the BHF Gerry Turner Intermediate Fellow. We thank Weiming Xu and Lizhi Liu for their help in the construction of the PCR competitors, Mr M Adiseshiah for performing the vein biopsies and Professor Salvador Moncada for helpful discussions and critical review of the manuscript.


   Notes
 
1 These authors contributed equally to this work and should be considered as joint first authors. Back


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

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