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Cardiovascular Research 2007 74(1):151-158; doi:10.1016/j.cardiores.2006.12.022
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Copyright © 2007, European Society of Cardiology

Deletion of neuronal NOS prevents impaired vasodilation in septic mouse skeletal muscle

Darcy Lidingtona,b, Fuyan Lia,b and Karel Tymla,b,c,*

aThe Centre for Critical Illness Research, Lawson Health Research Institute, London, Canada
bDepartment of Medical Biophysics, University of Western Ontario, London, Canada
cDepartment of Physiology and Pharmacology, University of Western Ontario, London, Canada

* Corresponding author. The Centre for Critical Illness Research, Victoria Research Laboratory, 6th Floor, 800 Commissioners Road East, London, Canada, N6C 2V5. Tel.: +1 519 685 8300x55076; fax: +1 519 685 8341. Email address: ktyml{at}lhsc.on.ca

Received 16 June 2006; revised 11 December 2006; accepted 28 December 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Sepsis-stimulated nitric oxide (NO) production impairs arteriolar responsiveness in skeletal muscle. Using wild type (WT), eNOS–/–, iNOS–/– and nNOS–/– mice, we aimed to determine the key nitric oxide synthase (NOS) isoenzyme(s) responsible for the arteriolar hyporesponsiveness to acetylcholine (ACh) in septic skeletal muscle.

Methods: Sepsis was induced by the cecal ligation and perforation procedure (24 h model). We measured the post-ACh increase in red blood cell velocity (VRBC) in a capillary fed by the stimulated arteriole as an index of vasodilation. NOS activity and protein expression in the muscle were measured by standard procedures.

Results: In all non-septic mice, ACh increased VRBC by ~150% from baseline. Sepsis impaired this response in WT, eNOS–/– and iNOS–/– mice, but not in nNOS–/– mice. Accordingly, pharmacological inhibition of nNOS with 7-nitroindazole reversed this impairment in WT mice. cNOS (eNOS+nNOS) activity was elevated in septic WT mice; Western blots indicated that this occurred through a post-translational mechanism. iNOS protein activity/expression was negligible. ACh caused dilation via endothelial-derived relaxing factor (EDRF) in WT mice and via endothelial-derived hyperpolarizing factor (EDHF) in eNOS–/– mice. Although exogenous NO reduced EDHF-mediated dilation in eNOS–/– mice, NOS inhibition did not reverse the sepsis-impaired dilation in these mice.

Conclusions: In our 24-h mouse model of sepsis, NO in skeletal muscle is primarily derived from nNOS. Sepsis impairs both EDRF- and EDHF-mediated dilation in response to ACh. Both genetic deletion and inhibition of nNOS protect against this impairment when the dilation occurs via the EDRF but not EDHF pathway.

KEYWORDS Sepsis; Nitric oxide synthases; Impaired vasodilation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Sepsis, a systemic inflammatory response to a local infectious insult, is characterized by several microcirculatory deficits, including decreased systemic vascular resistance and a maldistribution of blood flow [1,2]. Sepsis impairs arteriolar responses to vasoactive stimuli [3,4], which may be the underlying cause of the microcirculatory dysfunction. Since enhanced generation of nitric oxide (NO) has been implicated in the sepsis-mediated attenuation of vascular responsiveness [3–5], clinical interventions that aim to inhibit NO synthesis have been proposed as therapeutic strategies [6]. However, since NO has important non-pathological signaling functions (including endothelial-derived relaxing factor (EDRF)-mediated vasodilation in response to acetylcholine), it is critical that these interventions limit the excessive (and putatively detrimental) production of NO, while preserving its important physiological signaling functions [7].

In general, inflammation is associated with an upregulation of inducible NOS (iNOS) protein expression and subsequent enhanced production of NO [8]. Not surprisingly, in a mouse model of sepsis, upregulation of iNOS expression was observed in the early stages (within 6 h; [9]), and its gene deletion prevented the attenuation of angiotension II-mediated vasoconstriction in this early septic phase [9]. However, the induction of iNOS may be transient in nature, peaking at 6–12 h following the onset of sepsis [10]. As a result, investigations by our laboratory group and others suggest that in rodent models of sepsis, iNOS plays a relatively minor role, if any, in the later stages (i.e., after 24 h) [3,10,11].

Studies using pharmacological approaches to elucidate which NOS isoform(s) account for sepsis-induced vascular impairment have yielded limited insights, mainly because of the relative non-specificity of the inhibitors applied [3,10,12]. For example, aminoguanidine (AG), which is considered to be a selective inhibitor of iNOS, inhibits endothelial NOS (eNOS) and neuronal NOS (nNOS) at higher concentrations [3]. Further, AG also has antioxidant properties [13], and thus it could mitigate some of the effects of sepsis through an antioxidant mechanism [9]. Therefore, the observation that AG reverses the impairment of ACh-mediated vasodilation in a 24 h model of sepsis [3], where iNOS expression is absent, could be explained by mechanisms that involve the inhibition eNOS or nNOS, or perhaps even mechanisms that do not involve NO.

With these facts in mind, the primary aim of this study was to determine the NOS isoenzyme responsible for impairment of acetylcholine (ACh) responses after 24 h of sepsis. To this end, we used genetically modified mice with isoenzyme-specific NOS gene deletion. Based on recent reports indicating that nNOS could be the critical NO-producing isoenzyme in the later stages of sepsis [3,10], we hypothesized that nNOS-derived NO mediates sepsis-induced vascular hyporesponsiveness to ACh.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animal preparation
This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All experimental protocols were approved by the Council on Animal Care at the University of Western Ontario. Male wild type (WT), nNOS–/–, eNOS–/– and iNOS–/– mice of C57BL/6 background (18–25 g body weight) were obtained from The Jackson Laboratory (Bar Harbor, ME). Septic mice were prepared using the surgical cecal ligation and perforation (CLP) procedure (24 h model), as previously described [14]. Fluid resuscitation was provided by subcutaneous injections of saline (1 ml) containing the analgesic buprenorphine (4 µg/ml), given every 6 h post-CLP. To remain consistent with our previous investigations employing rats and mice [3,14], sepsis was defined here as the outcome of the CLP procedure (including laparotomy) and fluid resuscitation; control mice were not exposed to any surgical procedure or fluid resuscitation over the 24 h period. We recently reported that CLP, but not sham operated mice have elevated plasma lactate levels, necrosis of the cecum, and the presence of purulent peritoneal fluid [11]. We therefore used these parameters as markers of a septic state. Intravital experiments designed to assess vascular responsiveness in vivo were conducted at 24 h post-CLP. Following these experiments, a blood sample was drawn from the carotid artery to measure plasma lactate (Yellow Spring Instruments analyzer; Yellow Springs, OH). The abdominal cavity was visually inspected post-mortem.

2.2 Intravital microscopy
Mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (4 mg/kg) and placed on a heated intravital microscope stage. In a subset of WT and NOS knockout mice, the left internal carotid artery was cannulated so that arterial blood pressure could be monitored. The extensor digitorum longus (EDL) muscle was surgically exposed to permit the stimulation of a terminal arteriole on the muscle surface, as previously described [3]. Briefly, the muscle was covered by a plastic coverslip containing a 3-mm diameter hole filled with degassed heavy mineral oil. This allowed a micropipette to have access to the arteriole through the oil layer. The micropipette was filled with acetylcholine chloride dissolved in a physiological saline solution (PSS; in mM: 138.9 NaCl, 2.25 KCl, 2.25 KH2PO4 and 1.4 Na2HPO4, at pH 6.8). To stimulate the arteriole, an air pulse (30 psi; 10–20 ms; Picospritzer, General Valve; Fairfield, NJ) produced a droplet (~50–60 µm diameter) of the ACh solution within the oil layer; the droplet was then lowered onto the arteriole as described by Dietrich [15]. In several experiments, 5–30 min prior to ACh application, arterioles were pretreated with a similarly produced ~100 µm droplet of atropine sulfate (Atr), N{omega}-nitro-L-arginine (L-NNA), 7-nitroindazole (7-NI), 17-octadecynoic acid (ODYA), or S-nitroso-N-acetylpenicillamine (SNAP; all reagents from Sigma, St. Louis, MO). We estimate that agents in the micropipette were diluted ~100-fold before they reached the arteriolar wall [16]. The concentrations of agents reported in the present study are the micropipette concentrations. They were selected based on effective concentrations reported in the literature and on our previous experiments.

Since the muscle was epi-illuminated, the resulting optical image of the arteriolar wall was of poor quality and did not permit accurate measurement of arteriolar diameter response to ACh. Instead, we employed a video flying spot technique to measure the velocity of red blood cells (VRBC) in a capillary fed by the arteriole [17], and used the change in RBC velocity ({delta}VRBC) as an index of the diameter response. We have previously shown that acute arteriolar diameter changes are essentially entirely responsible for the ensuing acute changes in capillary VRBC [3,16,18]. We computed {delta}VRBC (%)=100%x(VRBC PEAK–VRBC BASELINE)/VRBC BASELINE, where VRBC PEAK was the maximal post-ACh velocity and VRBC BASELINE was a 2-min average pre-ACh velocity.

2.3 Immunoblotting and NOS activity assay
All procedures for NOS immunoblots and activity assays have been previously described [11,12]. Briefly, EDL muscle samples were homogenized (1:5 wt/vol) in buffer containing 20 mM Tris–HCl, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM PMSF, 20 µg/ml leupeptin and 1% Triton-X-100 and then centrifuged (10,000 rpm for 20 min at 4 °C).

For immunoblots, the homogenate was mixed 1:1 with a standard SDS-glycerol buffer (125 mM Tris–HCl, 20% glycerol, 4% SDS), denatured at 95 °C for 5–10 min, resolved on a 7.5% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with nonfat skimmed milk and incubated with mouse monoclonal anti-nNOS, anti-eNOS or anti-iNOS antibodies (1:1000 dilution for 2 h; all antibodies were obtained from Transduction Laboratories through Bio/Can Scientific, Mississauga, ON), then washed, and further incubated with a peroxidase-labelled rabbit polyclonal anti-mouse IgG (1:1000 for 1 h; Santa Cruz Biotechnology, Santa Cruz, CA). A similar procedure was employed for the detection of GAPDH (mouse monoclonal; 1:5000 dilution; Helena Biosciences, UK), which was used as a loading control. Blots were visualized using an enhanced chemiluminescence kit (LUMIGLO, KPL laboratories; Gaithersburg, MA) and radiographic film (Kodak BIOMAX MR; Rochester, NY).

NOS activity measurements are a standard procedure in our laboratory and were completed without modification as described previously [12]. The NOS enzyme assay yielded measurements of the calcium-dependent constitutive cNOS activity (eNOS+nNOS) and the calcium-independent iNOS activity in the presence of excess cofactors. Homogenate protein concentrations were determined using the BioRad (Hercules, CA) DC protein assay.

2.4 Statistics
All data are presented as mean±SEM; unless stated otherwise, n indicates the number of arterioles or EDL muscles. In experiments involving stimulation with ACh, we used 1–3 arterioles per muscle. Each arteriole (with or without pretreatment) was stimulated only once. Data were statistically analyzed using an unpaired Student's t-test or an ANOVA followed by t-tests with Bonferroni correction, where appropriate. We considered P<0.05 to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Baseline measurements in control and septic mice
As shown in Table 1, plasma lactate levels were elevated in all CLP mice. Additionally, visual post-mortem inspection revealed that all CLP mice had a necrotic cecum, with purulent peritoneal fluid. These markers of sepsis were not observed in any of the control non-septic mice. The overall survival rate for septic mice at 24 h was 91%, a value similar to our previous study [11]. Measurements of mean arterial pressure (MAP) and baseline pre-ACh VRBC in WT, nNOS–/–, eNOS–/– and iNOS–/– mice are also shown in Table 1. Our fluid resuscitation 24 h model of sepsis was not associated with reductions in MAP or alterations in baseline VRBC. Although genetic deletion of eNOS elevated MAP compared to WT mice, the MAP of septic eNOS–/– was not different from non-septic eNOS–/– mice or septic mice from any of the other groups.


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  		Table 1 Baseline measurements in control and septic mice

 
3.2 Sepsis attenuates the microvascular response to ACh in an nNOS-dependent manner
The data in Fig. 1 characterize the capillary VRBC response to ACh deposition on the terminal arteriole in control WT mice. The response was dose-dependent and inhibited by pretreatment with the muscarinic receptor blocker atropine (30 µM, 5 min). Atropine pretreatment did not alter the baseline pre-ACh VRBC when compared with that of control WT mice in Table 1 (data not shown). Application of the PSS vehicle did not stimulate a change in VRBC (Fig. 1, bar 1). Fig. 2A shows a typical VRBC response to ACh (10 mM droplet) in a control and septic WT mouse. Here, VRBC increased by more than 100% in the control mouse but only by 20–30% in the septic mouse. Fig. 2B summarizes the {delta}VRBC responses following 10 mM ACh application in control and septic WT, nNOS–/–, eNOS–/– and iNOS–/– mice. Sepsis significantly attenuated the {delta}VRBC response in WT, eNOS–/– and iNOS–/– mice, but not in nNOS–/– mice. Fig. 2C shows that in septic WT mice, pretreatment of arterioles with the nNOS specific inhibitor 7-NI (10 µM, 10 min) restored the response to ACh to the control level (Fig. 2C, bar 4 vs. 1); 7-NI pretreatment did not affect the response to ACh in control WT mice (bar 2 vs. 1). Pretreatment with 7-NI did not alter the baseline pre-ACh VRBC in control and septic mice when compared with the corresponding VRBC BASELINE values in Table 1 (data not shown). Taken together, Fig. 2B and C demonstrate that genetic deletion and pharmacological inhibition of nNOS prevent/reverse the sepsis-induced deficit in VRBC response to ACh.


Figure 1
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  		Fig. 1 Changes in capillary red blood cell velocity ({delta}VRBC) elicited by ACh stimulation of the feeding arteriole in the extensor digitorum longus (EDL) muscle of control wild type mice. ACh (0–10 mM droplet applied via micropipette) increased VRBC dose-dependently (bars 1–5). The response to 10 mM ACh was attenuated by 5 min pretreatment of the arteriole with the muscarinic receptor blocker atropine (Atr, 30 µM, bar 6). *P<0.05 compared with bar 1; #P<0.05 compared with bar 5; n=5–13 arterioles from 3–10 mice.

 

Figure 2
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  		Fig. 2 Sepsis attenuates the VRBC response to ACh by an nNOS-dependent mechanism. Panel A: ACh (10 mM droplet) was applied to an EDL muscle arteriole in a wild type (WT) mouse at time=0 s. Shown are representative VRBC tracings for control (open circles) and septic mice (24 h cecal ligation and perforation (CLP) model; closed circles). Panel B: In WT mice, sepsis significantly attenuated the VRBC increase elicited by 10 mM ACh (bar 2 vs. 1). Deletion of nNOS prevented this attenuating effect, while deletion of eNOS or iNOS had no effect. There were no differences in baseline VRBC among the groups. *P<0.05 compared to the respective control; n=9–16 arterioles from 5–8 mice. Panel C: In septic WT mice, pretreatment of arterioles with the nNOS specific inhibitor 7-NI (10 µM, 10 min) restored the response to ACh, to the level of control mice (bar 4 vs. 1). 7-NI pretreatment did not affect the response to ACh in non-septic WT mice (bar 2 vs. 1). *P<0.05 compared to bar 1; n=6–18 arterioles from 5–6 mice.

 
3.3 Sepsis attenuates both EDRF and EDHF responses
Fig. 3A shows that the {delta}VRBC response was markedly attenuated following pretreatment with the non-specific NOS inhibitor L-NNA (100 µM, 30 min), indicating that the eNOS-mediated EDRF mechanism dominates ACh-induced dilation in WT mice [19]. L-NNA pretreatment did not alter the baseline pre-ACh VRBC when compared with that of control WT mice in Table 1 (data not shown). In contrast to WT mice, pretreatment with L-NNA did not affect the {delta}VRBC response in eNOS–/– mice (Fig. 3B, bar 2); this was an expected result [19]. Pretreatment with the putative endothelial-derived hyperpolarizing factor (EDHF) inhibitor ODYA (100 µM, 30 min), however, significantly reduced the response in eNOS–/– mice (Fig. 3B, bar 3), indicating that EDHF-mediated dilation dominates responses to ACh in eNOS–/– mice [19]. Neither L-NNA nor ODYA altered the baseline pre-ACh VRBC when compared with that of control eNOS–/– mice in Table 1 (data not shown).


Figure 3
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  		Fig. 3 EDRF and EDHF primarily account for VRBC responses to ACh in WT and eNOS–/– mice, respectively. Panel A: In control WT mice, L-NNA (100 µM, 30 min) attenuated the {delta}VRBC response to ACh, indicating that the EDRF mechanism dominated the ACh-induced response in these mice. *P<0.05; n=12–13 arterioles from 5 mice. Panel B: In control eNOS–/– mice, L-NNA did not affect the {delta}VRBC response to ACh, while the EDHF inhibitor ODYA (100 µM, 30 min) attenuated this response. This result indicates that the EDHF mechanism dominated the ACh-induced response in eNOS–/– mice. *P<0.05 compared with bar 1; n=12, 5 and 12 arterioles from 5, 2 and 5 mice in bars 1–3, respectively. Panel C: In control eNOS–/– mice, the NO donor SNAP (50 µM, 5–6 min) attenuated the {delta}VRBC response to ACh to a similar degree as observed for sepsis (bar 2 vs. 3). However, inhibition of NOS with either L-NNA (100 µM, 30 min) or 7-NI (10 µM, 10 min) did not reverse the sepsis-induced deficit in {delta}VRBC response (bars 4 and 5). *P<0.05 compared with bar 1, n=9–11 arterioles from 5 mice in bars 1–4, n=5 arterioles from 2 mice in bar 5.

 
Since sepsis reduced {delta}VRBC responses in eNOS–/– mice (Fig. 2B, bar 6), we investigated whether nNOS-derived NO was responsible for the observed reduction. Pretreatment of control non-septic arterioles with NO donor SNAP (50 µM, 5–6 min) attenuated the {delta}VRBC response to ACh (Fig. 3C, bar 2). In this experiment, VRBC peaked at 1 min post-SNAP ({delta}VRBC=82±16%), and then it decayed to 169±18 µm/s at 5–6 min post-SNAP (i.e., a value not different from VRBC BASELINE in eNOS–/– mice (Table 1), before the subsequent ACh stimulation. Therefore, NO apparently has the ability to inhibit EDHF-mediated dilation in eNOS–/– mice. Surprisingly, inhibition of NO synthesis in septic eNOS–/– mice, with either L-NNA (100 µM, 30 min) or 7-NI (10 µM, 10 min) did not restore {delta}VRBC responses following ACh application (Fig. 3C, bars 4 and 5). Neither L-NNA nor 7-NI altered the baseline pre-ACh VRBC when compared with that of control eNOS–/– mice in Table 1 (data not shown).

3.4 NOS protein expression and activity in wild type mice
Sepsis did not alter nNOS or eNOS protein expression in the EDL muscle (Fig. 4). Consistent with our previous report utilizing a 24 h model of sepsis in mice [11], the expression of iNOS was not detected in EDL homogenates from control or septic animals (Fig. 4B). Activated mouse macrophage lysate served as a positive control for the iNOS antibody (data not shown).


Figure 4
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  		Fig. 4 NOS protein expression in EDL muscles of control and septic wild type mice. Panel A: Immunoblots for nNOS and eNOS from control and septic EDL muscle homogenates. iNOS expression was not detected in either group (not shown). GAPDH was used as loading control. Panel B: Densitometric ratios of NOS/GAPDH for control and septic muscles. Sepsis did not alter any of the NOS/GAPDH ratios, indicating that sepsis did not affect NOS protein expression in the EDL muscle. n=4 muscles form 4 mice for each group.

 
As illustrated in Fig. 5, sepsis increased calcium-dependent NOS activity (eNOS+nNOS) in EDL homogenates. Consistent with the western blots of Fig. 4, and our previous study involving septic mouse cremaster muscles [11], iNOS activity was negligible in both control and septic groups. NOS activities were minimal in EDL homogenates from nNOS–/– mice (4±2 pmol/mg/h, n=4), indicating that cNOS activity was dominated by nNOS.


Figure 5
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  		Fig. 5 NOS enzyme activity in EDL muscles of control and septic wild type mice. Sepsis increased cNOS enzyme activity (i.e., total of eNOS+nNOS activities), while iNOS activity was negligible in both groups. *P<0.05 compared with control, n=5 muscles from 5 mice for each group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The progressive dysfunction of the microcirculation is one of the prominent effects of sepsis, both in the clinical setting [20–22] and in animal models [2,23,24]. Enhanced generation of NO has been implicated in the diminished arteriolar responsiveness, and is believed to underlie the sepsis-induced microcirculatory deficits [3,4]. However, while clinical evidence supports a role for NO synthesis blockade (via inhibition of NOS) in the treatment of septic shock [6], at least one clinical study employing non-selective NOS inhibition was halted prematurely due to an increased risk of mortality [25]. Therefore, it appears critical to tailor the inhibition of NO generation, such that the excessive (and putatively detrimental) NO production is limited, while preserving its important signaling and bactericidal functions [7]. In order to achieve this objective, the pharmacological approach must target the correct NOS isoenzyme.

The present study shows for the first time that, in a 24 h model of sepsis, the impaired arteriolar response to ACh (assessed by capillary {delta}VRBC) in the EDL muscle of wild type mice is prevented by the genetic deletion and rescued by pharmacological inhibition of nNOS. Our results are consistent with the reported critical role of nNOS in the attenuated arteriolar conducted vasoconstriction in the septic mouse cremaster muscle [11]. The comparability of these two different muscles in the same septic model suggests that enhanced nNOS activity in the later phase of normotensive sepsis may be a global phenomenon common to skeletal muscle vascular beds. This phenomenon may also occur across species [10].

Consistent with these observations, NOS enzyme activity measurements confirmed that cNOS (eNOS+nNOS) dominates NO production in the septic EDL muscle of WT mice, with minimal activity attributable to iNOS (Fig. 5). Since cNOS enzymatic activity was negligible in EDL homogenates from nNOS–/– mice, our cNOS measurements in WT mice largely represent the nNOS activity. Although this activity could dominate the overall NO production within the EDL muscle, NO produced locally via the remaining NOS isoenzymes within cells of the microvascular wall could also affect microvascular function. However, regarding the particular impairment of arteriolar function in septic muscle studied here, iNOS and eNOS appear to play a minimal role in this impairment (Fig. 2B, bars 5–8).

Western blots of EDL muscle homogenates demonstrated that sepsis did not alter nNOS protein expression at 24 h post-CLP (iNOS protein was not detected; Fig. 4). Since the increase in NOS activity (Fig. 5) cannot be attributed to altered nNOS protein expression, a non-genomic, post-translational mechanism must operate here. Possible mechanisms could include altered nNOS phosphorylation status [26], increased interaction with heat shock protein 90 [27,28], or reduced interaction with either protein inhibitor (PIN) or caveolin-3 [29]. Although beyond the scope of the present study, the determination of the underlying processes that lead to increased nNOS activity in sepsis could provide for the development of treatment strategies in the clinical setting.

In WT mice, the arteriolar response to ACh was primarily dependent on eNOS/EDRF. Since NO can inhibit eNOS [30], we propose that sepsis-stimulated nNOS-derived NO attenuated eNOS signaling in EDL arterioles. Alternatively, it is possible that NO could attenuate ACh responses via inhibition of Gi and Gq family of G proteins [31], signaling mechanisms that would be upstream of eNOS activation.

Compared to WT mice, eNOS–/– mice had a similar response to ACh (Fig. 2B, bar 1 vs. 5), although the response depended on EDHF rather than EDRF. To our knowledge, we show for the first time that EDHF-mediated dilation in response to ACh is attenuated by sepsis (Fig. 2B, bar 6). Since EDHF is proposed to rely on several mechanisms and mediators (reviewed extensively in [32]), EDHF-mediated dilation could be inhibited by several mechanisms. EDHF-mediated ACh responses were attenuated by the application of exogenous NO (Fig. 3C bar 2), however, inhibition of NO generation with L-NNA or 7-NI did not rescue arteriolar responsiveness in septic eNOS–/– mice. Thus, while enhanced NO generation has the potential to inhibit EDHF responses in sepsis, other processes independent of NO (e.g., signaling via sepsis-induced inflammatory cytokines) may prevent any beneficial effects of NOS inhibition (Fig. 3C, bars 4 and 5).

From a clinical perspective, our findings have two important implications: (i) specific inhibition of the nNOS isoenzyme can restore eNOS signaling and vasodilatory responses in sepsis; and (ii), that the inhibition of NO synthesis would only be beneficial if arteriolar vasodilation is predominantly mediated by EDRF, rather than EDHF. It is not currently known to what extent EDRF and EDHF mediate dilatory responses in humans.

In summary, the present study shows that sepsis impairs ACh-stimulated, EDRF-mediated blood flow responses in the mouse EDL muscle in nNOS-dependent manner. Although EDHF-mediated responses may also be inhibited by NO, this impairment persists despite NOS inhibition. Our data indicate that isoenzyme-specific inhibition of NO production restores EDRF-, but not EDHF-mediated arteriolar blood flow responses to ACh in sepsis.


   Acknowledgements
 
We thank Mr. N. Gocan for stimulating discussion, and the Heart and Stroke Foundation of Ontario (operating grant to K.T.) and the Canadian Institutes of Health Research (operating grant to K.T., and salary award to D.L.) for support.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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