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Cardiovascular Research 2006 69(1):236-244; doi:10.1016/j.cardiores.2005.09.003
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Copyright © 2005, European Society of Cardiology

Reduced arteriolar conducted vasoconstriction in septic mouse cremaster muscle is mediated by nNOS-derived NO

Rebecca L. McKinnona,b, Darcy Lidingtona,d, Michael Bolona,b, Yves Ouellettec, Gerald M. Kiddera,b and Karel Tymla,b,d,*

aLawson Health Research Institute, The Centre for Critical Illness Research, Victoria Research Laboratory, 6th Floor, 800 Commissioners Road East London, Ontario, Canada, N6C 2V5
bDepartment of Physiology and Pharmacology, University of Western Ontario, London, Canada
cDepartment of Pediatrics, Mayo Clinic College of Medicine, Rochester, MN, United States
dDepartment of Medical Biophysics, University of Western Ontario, London, Canada

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

Received 25 January 2005; revised 31 August 2005; accepted 3 September 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Increased nitric oxide (NO) production in sepsis precipitates microcirculatory dysfunction. We aimed (i) to determine if NO is the key water-soluble factor in the recently discovered sepsis-induced deficit in arteriolar conducted vasoconstriction, (ii) to identify which nitric oxide synthase (NOS) isoforms account for this deficit, and (iii) to examine the potential role of connexin37 (Cx37, a hypothesized signaling target of NO) in arteriolar conduction.

Methods: Using intravital microscopy and the cecal ligation and perforation 24-h model of sepsis, arterioles in the cremaster muscle of male C57BL/6 wild-type (WT), iNOS–/–, eNOS–/–, nNOS–/– and Cx37–/– mice were locally stimulated with KCl to initiate conducted vasoconstriction. We used the ratio of conducted constriction (500 µm upstream) to local constriction as an index of conduction (CR500). NOS enzymatic activity and protein expression were determined in control and septic cremaster muscles.

Results: Sepsis reduced CR500 in WT mice [from 0.77 ± 0.05 to 0.20 ± 0.02 (means ± SE) independent of the site of stimulation along the arteriole], in iNOS–/– and eNOS–/– mice, but not in nNOS–/– mice. The nNOS inhibitor 7-nitroindazole or NO scavenger HbO2 restored CR500 in septic WT mice, but blockade of soluble guanylate cyclase had no effect. Sepsis increased cNOS (eNOS+nNOS) activity in WT mice (from 340 ± 40 to 490 ± 30 pmol/mg/h) and in eNOS–/–, but not in nNOS–/– mice (iNOS activity was negligible in all mice). Sepsis did not alter nNOS protein expression in WT mice. CR500 in non-septic Cx37–/– mice (0.15 ± 0.1) was similar to that observed in septic WT mice.

Conclusion: Increased nNOS activity and the resultant increased NO production in the septic mouse cremaster muscle are the key factors responsible for the deficit in conducted vasoconstriction along the arteriole. Deletion of Cx37 results in reduced CR500, which is consistent with the hypothesis that Cx37 in the arteriole could be a target of NO signaling.

KEYWORDS Cell communication; Nitric oxide; Sepsis; Connexin37


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The blood flow in the microcirculation is regulated to meet the ongoing metabolic needs of the tissue. This regulation is achieved by coordinated vasodilation or vasoconstriction of resistance vessels over a relatively long vessel length. Vascular cell communication (e.g., the arteriolar conducted response) is now believed to play a key role in the coordination of microvascular blood flow control, by enhancing the arteriole's ability to mount the resistance to blood flow [1,2].

In general, little is known about the effect of pathophysiology on vascular cell communication. Recently we showed that sepsis, which is a systemic inflammatory response to a local infection [3], reduces the arteriolar conducted vasoconstriction in the mouse cremaster muscle [4]. Sepsis also reduces arteriolar reactivity, and together these deficits could lead to a maldistribution of blood flow within the microvasculature, poor oxygen delivery to the tissue [5–7], and subsequently to organ failure. Since superfusion of the septic tissue with physiological saline solution can restore the conducted vasoconstriction [4], a number of water-soluble factors associated with sepsis could account for the conduction deficit (e.g., lipopolysaccharide, cytokines, oxidants, nitric oxide [8]). Among these, nitric oxide (NO) could be the key/sole factor, since the NO donor SNAP completely mimics the conduction deficit in control non-septic tissue [4].

Three distinct nitric oxide synthase (NOS) isoforms have been identified, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Any or all of these isoforms could produce NO in sepsis and be responsible for the conduction deficit. It has been shown that iNOS is induced in response to a variety of inflammatory mediators associated with sepsis and that iNOS accounts for the arteriolar hyporeactivity to angiotensin II in sepsis [9,10]. nNOS was reported to be upregulated in septic skeletal muscle [11] and nNOS-produced NO was proposed to reduce the arteriolar vasodilator response to acetylcholine [6]. eNOS has also been shown to be upregulated in sepsis [11], while eNOS-derived NO was suggested to mediate histamine-induced deficit in arteriolar conduction in the mouse cremaster muscle [12].

Since inhibition of a particular NOS isozyme(s) has been a clinical target in patients with septic shock [13], the main objective of the present study was to determine if NO is indeed the key factor in the sepsis-induced arteriolar conduction deficit, and to identify which NOS isozymes are involved in this deficit. Because NO has been shown to target the gap junction protein Cx37 to reduce intercellular coupling independently of cGMP [14], we also tested the effect of blockade of soluble guanylate cyclase on this deficit, and examined the role of Cx37 in the arteriolar conducted vasoconstriction in the mouse cremaster muscle.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Reagents
The 7-nitroindazole (7-NI), N-nitro-L-arginine methyl ester hydrochloride (L-NAME), hemoglobin, β-nicotinamide adenine dinucleotide phosphate reduced (NADPH), tetrahydrobiopterin, flavin adenine dinucleotide (FAD), NaCl, KCl, CaCl2, MgSO4, NaHCO3, Hb, EGTA, EDTA, dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), leupeptin, triton, diaminobenzidine (DAB) were purchased from Sigma Chemical Co. (St. Louis, MO). Calmodulin was purchased from Roche Diagnostics (Laval, QC). Mouse monoclonal nNOS and anti-GAPDH antibodies were obtained from Transduction Laboratories (Bio/Can Scientific; Mississauga, ON) and BD Biosciences (Mississauga, ON), respectively. A peroxidase-labeled anti-mouse IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 1-H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1 (ODQ) was from Calbiochem (San Diego, CA).

2.2 Preparation of septic mice
This investigation conformed with 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) and all experimental protocols were approved by the Animal Use Subcommittee of the Department of Animal Care and Veterinary Services at the University of Western Ontario. We used male wild type (WT), eNOS–/–, iNOS–/–, nNOS–/– and Cx37–/– mice of C57BL/6 background (18–25 g body weight). Wild type and NOS isozyme knockout mice were obtained from The Jackson Laboratory (Bar Harbor, ME), while Cx37–/– mice were obtained from Dr. David Paul, Harvard Medical School [15], and maintained in our facility as described previously [16]. Wild type and NOS knockout mice were made septic by the cecal ligation and perforation (CLP) surgical procedure (24 h model) as previously detailed by us [4]. A subcutaneous injection of saline (1 mL) containing the analgesic buprenorphine (4 µg/mL) was administered every 6 h post-CLP. Sepsis was defined as the outcome of the CLP procedure (including laparotomy) and fluid resuscitation. Control mice were not subjected to any surgery or resuscitation over the 24 h period. Following the intravital experiment, blood lactate obtained from carotid artery was measured (Yellow Springs Instruments analyzer), and the abdominal cavity was examined.

2.3 Mouse cremaster muscle preparation for intravital microscopy
To study conducted arteriolar responses, we used a mouse cremaster muscle preparation and stimulation protocol as detailed by us [4]. The cremaster muscle of anesthetized mice (ketamine 80 mg/kg plus xylazine 4 mg/kg) was isolated, spread over a glass support, and irrigated with physiological saline solution (PSS), 33–34 °C, pH 7.4, composed (in mmol/L) 131.9 NaCl, 4.7 KCl, 2.2 CaCl2, 1.2 MgSO4 and 20.0 NaHCO3 bubbled continuously with 5% CO2/95% N2 gas. The muscle was epi-illuminated with bright light via fiber optic light-guide, visualized with an intravital microscope, and the resultant field of view (0.65 x 0.48 mm) was video recorded.

Micropipettes were backfilled with 3.0 mol/L KCl in PSS and connected to a source of pressurized air (Picospritzer II, General Valve). To initiate the conducted vasoconstriction, unbranched arterioles (~50 µm in diameter, ~2 mm long, 1A or 2A branching order, one arteriole/mouse) were stimulated by pressure ejecting KCl onto the arteriole (average pulse duration ~60 ms at 50 psi pressure). Pulse duration was adjusted to yield a local constriction of ~50%. Subsequent stimulations were repeated at the same local site using the same pulse duration. During all stimulations, arterioles were superfused with PSS at 1 mL/min ensuring that the KCl puff was carried away from the 500 µm upstream arteriolar site. The luminal arteriolar diameter at the local and 500 µm upstream sites was measured off-line from the video screen (i.e., distance between the inner vessel walls with resolution ~ ± 1 µm) at three time points: pre-KCl (Dlocal, pre and D500, pre), maximal constriction at ~4 s post-KCl (i.e., minimal diameter, Dlocal, min and D500, min) and at 30 s post-KCl (details of time course of diameter response published by us [4]). The relative diameter changes for the local and upstream sites were {Delta}Dlocal(%)=100% x (Dlocal, pre–Dlocal, min)/Dlocal, pre and {Delta}D500(%)=100% x (D500, pre–D500, min)/D500, pre, respectively. The communication ratio, CR500={Delta}D500(%)/{Delta}Dlocal(%), was used as index of arteriolar conducted vasoconstriction (Fig. 1).


Figure 1
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  		Fig. 1 Effect of sepsis on arteriolar conducted vasoconstriction in wild type mouse cremaster muscle. Panel A: KCl-stimulated change in diameter was measured at the site of stimulation (Local) and 500 µm upstream site at the time of maximal constriction (4.4 ± 1.0, 4.8 ± 0.9, and 4.2 ± 0.7 s post-KCl, for control, laparotomy+fluid resuscitation and CLP groups, respectively). Sepsis (24 h following cecal ligation and perforation, CLP) was found to decrease constriction at the 500 µm site. The baseline pre-KCl diameters were 53 ± 3, 65 ± 5, and 59 ± 2 µm for control, laparotomy+fluid resuscitation and CLP arterioles (diameters not significantly different), while the KCl pulse durations were 71 ± 19, 66 ± 15, and 59 ± 14 ms, respectively. Panel B: The local and upstream changes in diameter were converted to CR500 value (details in METHODS), which was used in the present study as index of arteriolar conducted vasoconstriction. Sepsis reduced CR500. *P<0.05 compared with control; n=20, 5 and 20 mice in control, laparotomy+fluid resuscitation, and CLP groups, respectively.

 
2.4 Effects of 7-nitroindazole (7-NI), ODQ, and oxyhemoglobin (HbO2)
Baseline conducted vasoconstrictions were determined following post-surgical stabilization in both control and CLP mice. For 7-NI (nNOS inhibitor) or ODQ (antagonist of soluble guanylate cyclase), cremaster muscles in both groups of mice were irrigated with 0.5 mL of PSS containing 10 µmol/L of 7-NI or ODQ. A plastic cover was placed over the muscle for 1 h (7-NI) or 30 min (ODQ) and then KCl-induced conducted vasoconstriction was again determined. For HbO2 (prepared from Hb as described previously [17]), cremaster muscles were superperfused with PSS containing HbO2 (10 µmol/L) for 20 min (~2 mL total) and then KCl-induced conducted vasoconstriction was evaluated. We have previously shown that a small volume of the PSS vehicle (less than 7 mL) does not alter the effect of CLP on conducted vasoconstriction [4]. We selected 7-NI, ODQ and HbO2 concentrations based on effective concentrations reported in the literature and on preliminary experiments.

2.5 Controls for intravital experiments
We carried out two control experiments regarding the effect of sepsis on the arteriolar vasoconstrictor response to KCl. The first experiment addressed the spatial consistency of both the local response to KCl and the resulting conducted vasoconstriction. Referring to Fig. 2A, the arteriole was locally stimulated with KCl, and the local and 500 µm upstream diameter responses were determined. The pipette was then moved 500 µm upstream, and the diameter response was measured at the new local site and at a site 500 µm further upstream, following KCl stimulation of the same pulse duration.


Figure 2
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  		Fig. 2 Consistency of KCl-stimulated diameter responses along the arteriole in wild type mice. Panel A: Schematic diagram of the experiment. The arteriole was stimulated twice with the same KCl puff, at Site 1 (diameter changes measured at Sites 1 and 2), and then at Site 2 (diameter changes measured at Sites 2 and 3). Panel B: There were no differences between diameter responses to Stimulations 1 and 2 in control mice, n=5. Panel C: No differences between responses to Stimulations 1 and 2 were seen in CLP mice, n=5.

 
The second experiment assessed the ability of control and septic arterioles to constrict in response to increasing concentrations of KCl. Following post-surgical stabilization, the muscle was flooded every 10 min with 0.5 mL bolus of PSS containing KCl (20–60 mmol/L), and the diameter was measured at the time of maximum constriction. Before each bolus, the muscle was washed with ~1 mL PSS to allow the arteriole to return to control resting diameter. We kept the total volume of the stimulation bolus/PSS washout to a minimum (less than 7 mL) to prevent washout of the effect of sepsis [4].

Control experiments have been previously performed [4] to address the effect of diffusion of pipette-ejected KCl on the conducted vasoconstriction, and the effect of buprenorphine and fluid resuscitation on conducted vasoconstriction in septic mice. Due to the lack of spontaneous arteriolar tone in our preparation [4], the vasodilative ability of arterioles was confirmed by testing the effect of SNAP (50 µmol/L) in pre-constricted arterioles (superfusion with 10% O2), as detailed by us [4]. We also addressed the effect of laparotomy and fluid resuscitation procedures on conducted vasoconstriction.

2.6 NOS activity assay
Cremaster muscles were isolated and homogenized in 5 volumes (wt/vol) of homogenizing buffer (20 mM Tris–Cl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 20 µg/mL leupeptin and 1% triton x 100) and centrifuged (10,000 rpm for 20 min at 4 °C). The centrifugation was repeated on the supernatant. The NOS enzyme activity in the cremaster muscle homogenates was measured as described previously [18]. 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. Lysate protein concentrations were determined using the BioRad DC protein assay.

2.7 Western blotting
Tissue homogenate was mixed 1:1 with SDS glycerol, denatured at 95 °C for 5–10 min, resolved (25 µL/well) on a 7.5% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with nonfat skim milk, incubated with mouse monoclonal anti-nNOS at 1:1000 dilution for 2 h, washed, and further incubated with peroxidase-labeled anti-mouse IgG (1:1000, 1 h at room temperature). Blots were washed and banding was visualized using an enhanced chemiluminescence kit (LUMIGLO, KPL laboratories, Gaithersburg, MA) with Kodak BIOMAX MR imaging film (Rochester, NY). GAPDH (using anti-GAPDH antibody at 1:2000, 5 µL homogenate/well) was visualized using DAB and used as loading control.

2.8 Statistics
Data were presented as mean ± SE. In each mouse, one arteriole was used; n indicates the number of mice. Data were analyzed by Student's t-test, or by ANOVA followed by Bonferroni post-test. We considered P<0.05 as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Effect of sepsis on arteriolar conduction in wild type mice
Control mice, and mice with laparotomy+fluid resuscitation only, had normal lactate (0.6 ± 0.1 mmol/L in both groups) and normal cecum; survival was 100% for both groups. CLP mice had significantly higher lactate (1.8 ± 0.2 mmol/L), necrotic cecum with purulent peritoneal fluid and reduced survival (92%) at 24 h.

When compared to diameter responses in arterioles of control mice (or laparotomy+fluid resuscitated mice), arterioles at 24 h post-CLP had the same local diameter response to KCl, but a significantly smaller upstream diameter response, and hence reduced CR500 (Fig. 1A, B). Fig. 2 shows that the local and upstream responses were independent of the site of local stimulation along the arteriole. Thus, the upstream site showing attenuated conducted vasoconstriction retained its ability to constrict to a local stimulus during sepsis. Fig. 3 demonstrates that sepsis did not affect arteriolar vasoconstriction caused by increasing KCl concentrations.


Figure 3
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  		Fig. 3 Sepsis does not affect arteriolar constriction to increasing concentrations of KCl in wild type mice. Cremaster muscles were flooded with 0.5 mL bolus of PSS containing KCl (20–60 mmol/L), and the diameter change (% of pre-KCl baseline diameter) at a random location along the arteriole was measured at the time of maximal constriction. Baseline diameters were 69 ± 3, and 61 ± 4 µm for control and CLP arterioles (P>0.05), respectively, n=5/group.

 
3.2 Effect of isoenzyme-specific NOS gene knockout
Fig. 4 shows that conducted vasoconstriction was not significantly different in control wild type, iNOS–/–, eNOS–/– and nNOS–/– mice. However, conduction was reduced in septic WT, iNOS–/– and eNOS–/– mice. Notably, there was no difference in conduction between control and septic nNOS–/– mice. The nNOS inhibitor 7-NI restored conduction in WT septic mice (Fig. 5A). Thus, genetic deletion (Fig. 4) and pharmacological inhibition of nNOS (Fig. 5A) inhibited the sepsis-induced deficit in arteriolar conduction, establishing the central role of nNOS in this deficit.


Figure 4
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  		Fig. 4 Effect of isozyme-specific NOS gene knockout on arteriolar conducted vasoconstriction (CR500) in control and CLP mouse cremaster muscle. Deletion of nNOS prevented the effect of CLP on the conducted response, while deletion of eNOS or iNOS did not affect the CLP-induced reduction in conduction. There were no differences in baseline diameter among the groups. *P<0.05 compared with control, n=5–7/group.

 

Figure 5
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  		Fig. 5 Effects of 7-nitroindazole (7-NI; panel A) and HbO2 (panel B) on arteriolar conducted vasoconstriction in wild type mice. CR500 was determined immediately after the 30 min post-surgical equilibration period in control and CLP arterioles (open bars). The same arterioles were then treated with the nNOS inhibitor 7-NI (10 µmol/L) for 1 h or with HbO2 (10 µmol/L) for 20 min, after which CR500 was again determined. 7-NI and HbO2 restored arteriolar conduction in CLP mice (hatched and filled bars, respectively). There were no differences in baseline diameter between pre- and post-7-NI groups, in control (59 ± 6 vs. 59 ± 6 µm) and CLP (54 ± 5 vs. 53 ± 3 µm) mice, n=5 mice per group. There was no difference in baseline diameter between pre- and post-HbO2 groups in control mice (56 ± 5 vs. 61 ± 6 µm). HbO2 slightly but significantly reduced baseline diameter from 68 ± 1 to 63 ± 2 µm in CLP mice; n=5/group. In both panels, *P<0.05 compared with control.

 
3.3 nNOS activity and protein expression in control and septic cremaster muscles
Since exogenous NO applied to arterioles in control non-septic cremaster muscle reduces conduction to the level observed in sepsis [4], nNOS-derived NO could be responsible for the conduction deficit in WT septic mice. Therefore, we examined the effect of sepsis on nNOS activity and protein expression in the cremaster muscle of WT mice at 24 h post-CLP. Fig. 6A shows that (i) cNOS activity in septic muscle increased by 47% when compared to that in control muscle, and (ii) iNOS activity was negligible in both control and septic muscles (note that cNOS activity was measured with excess cofactors and therefore the resultant level of 300–500 pmol/mg/h could be higher than the level of actual cNOS activity in vivo). Based on cNOS activity measurements in cremaster muscles of eNOS–/– and nNOS–/– mice, Fig. 6B, C shows that nNOS rather than eNOS activity accounted for cNOS activities in control and septic WT mice. Despite increased activity, nNOS protein expression was not altered by sepsis (Fig. 7). Finally, Fig. 5B demonstrates that nNOS-derived NO could be involved in the sepsis-induced deficit in conduction, since the application of the NO scavenger HbO2 restored conduction in septic WT mice.


Figure 6
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  		Fig. 6 NOS enzyme activity in cremaster muscles of control and CLP wild type, eNOS–/– and nNOS–/– mice. In wild type and eNOS–/– mice (Panels A, B, respectively), CLP increased cNOS enzyme activity (i.e., total of eNOS and nNOS activities), while iNOS activity was negligible in all groups. Negligible cNOS activity in nNOS–/– group (Panel C) indicates that nNOS was the key isozyme in both control and CLP cremaster muscles. *P<0.05 compared with control, n=5/group.

 

Figure 7
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  		Fig. 7 nNOS protein expression in control and CLP wild type mice. Panel A: Representative immunoblots from control and CLP mouse cremaster muscles. GAPDH was used as loading control. Panel B: Densitometric ratios of nNOS to GAPDH immunoblots for control and CLP muscles, normalized to the average ratio in the control group. Sepsis did not alter nNOS protein expression in the cremaster muscle. n=4/group.

 
3.4 Effect of ODQ and Cx37 knockout
Since exogenous NO inhibits, independently of cGMP, coupling of HeLa cells transfected with Cx37 [14] we questioned whether the sepsis-induced deficit in conduction in WT mice is sensitive to ODQ (antagonist of soluble guanylate cyclase), and whether Cx37 could be a target of NO in the mouse cremaster muscle arterioles. Fig. 8A shows that NO may not influence conducted vasoconstriction via cGMP-dependent pathway since treatment of septic arterioles with ODQ did not affect conduction in septic mice. The effectiveness of ODQ was confirmed in a control experiment where ODQ inhibited the SNAP-mediated dilation in pre-constricted arterioles (data not shown). Further, Fig. 8B shows that conduction in non-septic Cx37–/– mice was lower than that in non-septic WT mice, and that it was similar to that observed in septic WT mice (Fig. 1). To our knowledge, this is the first report that Cx37 plays a key role in the arteriolar conducted response.


Figure 8
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  		Fig. 8 Effect of ODQ (antagonist of soluble guanylate cyclase, Panel A) and of Cx37 absence (Panels B and C) on arteriolar conducted vasoconstriction. Panel A: CR500 was determined in wild type mice after post-surgical equilibration period in control and CLP arterioles (open bars). The same arterioles were then exposed to ODQ (10 µmol/L) for 30 min, after which CR500 was again determined. ODQ did not affect arteriolar conduction in control or CLP mice (filled bars). There was no difference in baseline diameter between pre- and post-ODQ groups, in control (60 ± 4 vs. 59 ± 4 µm) and CLP (62 ± 4 vs. 64 ± 4 µm) mice (n=5/group). Panels B and C: Using control non-septic mice, absence of Cx37 yielded a significantly smaller diameter change at 500 µm upstream site (panel B) and a lower CR500 value (panel C) when compared with those of control non-septic wild type mice (these data redrawn from Fig. 1). The baseline pre-KCl diameter in Cx37–/– group was 65 ± 4 µm. *P<0.05 compared with wild type group, n=20 and 5 mice in wild type and Cx37–/– groups, respectively.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
4.1 Methodological considerations for studying arteriolar conduction during pathophysiology
In general, little is known about the effect of pathophysiological conditions on conduction along the arteriolar wall. The paucity of data could be due to the methodological challenge of directly measuring intercellular electrical coupling along the arteriolar wall during a disease state. Since the conducted diameter response at the upstream site relies on both the longitudinal electrical coupling along the arteriole and the electromechanical coupling at this upstream site, appropriate controls are required to assess the effect of pathophysiology at the upstream site. Previous studies of arteriolar conducted response addressed the effect of inflammatory agents (angiotensin II, lipopolysaccharide, histamine) on the pharmaco/electromechanical coupling at the local stimulation site [19,2,12]. We show here for the first time that sepsis does not affect electromechanical coupling at either the local and or upstream sites (Fig. 2), and that the sensitivity of electromechanical coupling along the arteriole is unchanged with increasing concentrations of KCl (Fig. 3). Together, we interpret the results of Figs. 2 and 3Go to indicate that the reduced upstream constriction in sepsis (Fig. 1A) reflects attenuated intercellular coupling along the arteriole, rather than reduced arteriolar ability to constrict at the upstream site.

4.2 Effect of NO on vascular cell coupling
Although the effect of NO on vascular function is well documented [20], little is known about the effect of NO on vascular cell coupling. We showed that a NO donor applied to the mouse cremaster muscle arteriole reduces the conducted vasoconstriction along its length [4]. In septic cremaster muscle, partial restoration of conduction occurred after local treatment with the NOS inhibitor L-NAME [4]. Similarly, the inflammatory agent histamine reduced arteriolar conduction via eNOS-derived NO-dependent mechanism [12]. Here, NO-activated cGMP was proposed to lead to disruption of arteriolar wall gap junctions.

There is evidence that gap junctions play a key role in the conducted response [21,22]. Among the four known vascular connexins (Cx37, Cx40, Cx43, Cx45; [23]), Cx37 could mediate the effect of NO on conduction, since Cx37 was reported to be a primary connexin between endothelial cells along the mouse cremaster muscle arteriole [24]. Kameritsch et al. [14] showed that communication-deficient HeLa cells transfected with Cx37 exhibit cGMP-independent reduction in dye coupling in response to NO. Although cGMP-dependent phosphorylation of Cx43 was linked to reduced coupling in SKHep1 cells [25], coupling in HeLa cells transfected with Cx43 was insensitive to exogenous NO [14]. Our data are consistent with the reported NO-sensitive Cx37 function in vitro and with the prominent presence of Cx37 in the mouse arteriole in vivo. Cx37 was found to play a key role in conducted vasoconstriction (Fig. 8C) and, since ODQ had no effect (Fig. 8A), the effect of sepsis on conduction was cGMP-independent. Based on electrophysiological assessment of intercellular electrical resistance [26], our preliminary experiments with cultured microvascular endothelial cell monolayers (mouse skeletal muscle origin) supported this role of Cx37. Exogenous NO (DETA NONOate, 500 µM) significantly increased resistance in WT cells (from 1.3 ± 0.1 to 2.3 ± 0.1 M{Omega}, P<0.05, n=3 monolayers) but not in Cx37–/– cells (1.5 ± 0.1 versus 1.4 ± 0.2 M{Omega}, P>0.05, n=3). In other tissues, the inhibitory effect of NO on coupling has also been shown for hybrid bass retinal horizontal cells [27] and for rabbit retina cells [28]. Further work is required to elucidate the mechanism of NO-mediated reduction in arteriolar conducted response during sepsis.

4.3 Effect of sepsis on NOS expression in skeletal muscle
Rats injected with lipopolysaccharide (LPS) show increased eNOS and skeletal muscle nNOS protein levels, but negligible iNOS, in the diaphragm muscle at 24 h post-LPS [11]. In this model of sepsis, iNOS expression is only seen at 6–12 h post-LPS. Expression of nNOS at 24 h is accompanied by increased cNOS activity and by negligible iNOS activity. Our laboratory reported similar increases in nNOS protein expression and cNOS activity (and negligible iNOS protein and activity levels) in the rat hindlimb muscle at 24 h post-CLP [6]. The present study is consistent with these reports. Using eNOS–/– and nNOS–/– mice, Fig. 6 demonstrates that nNOS activity in the WT mouse cremaster muscle dominated over eNOS and iNOS activities at 24 h post-CLP. Consistent with these negligible eNOS and iNOS activities, genetic deletion of eNOS and iNOS had no effect on conducted vasoconstriction at 24 h post-CLP (Fig. 4).

In view of unchanged nNOS protein expression in septic cremaster muscle (Fig. 7), increased nNOS activity could occur through several mechanisms. In terms of increased protein–protein interaction, heat shock protein 90, which is upregulated during sepsis [29], increases purified nNOS activity by enhancing the binding of calmodulin [30]. The increased nNOS activity could also be due to reduced binding to caveolin-3 or protein inhibitor of nNOS (PIN), both of which cause enzymatic inhibition [31].

Our study implies that elevated levels of NO derived from other NOS isozymes (e.g., iNOS) in other tissues, or at times other than 24 h, could also compromise intercellular coupling during inflammation. For example, increased iNOS expression and activity have been observed in skeletal muscle at 6 h post-CLP [32] and in heart 48 h after the onset of infarction [33]. Thus, iNOS-derived NO could also reduce conducted response along the vasculature, as well as compromise gap junctional coupling between cardiomyocytes (an event linked to cardiac arrhythmia [34]).

In conclusion, we demonstrate that the conducted vasoconstriction is significantly reduced in a 24 h model of sepsis, independent of the site of local stimulation along the arteriole. This deficit in conduction is prevented by genetic deletion of the nNOS protein, rescued by pharmacological inhibition of nNOS, but not affected by blockade of soluble guanylate cyclase. We propose that increased nNOS activity and the resultant increased NO production in the cremaster muscle are the key factors responsible for the sepsis-induced reduction in intercellular coupling along the arteriolar wall. Furthermore, based on the observation that deletion of Cx37 results in attenuated conduction in control mice, we suggest that this connexin plays a key role in mediating the arteriolar conducted vasoconstriction and could be a potential target in the arteriole for NO signaling in sepsis.


   Acknowledgements
 
We thank Ms. F. Li and Mr. Kevin Barr for technical assistance, Heart and Stroke Foundation of Ontario (salary award to K.T.), Canadian Institutes of Health Research (salary award to D.L., and research grants to K.T. and G.M.K.), and Natural Sciences and Engineering Research Council of Canada (salary award to R.L.M.).


   Notes
 
Time for primary review 34 days


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

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