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Am J Physiol Heart Circ Physiol 294: H1724-H1735, 2008. First published February 8, 2008; doi:10.1152/ajpheart.01072.2007
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Lysozyme, a mediator of sepsis that produces vasodilation by hydrogen peroxide signaling in an arterial preparation

Steven N. Mink,1,2 Krika Kasian,1 Luis E. Santos Martinez,4 Hans Jacobs,3 Ratna Bose,2 Zhao-Qin Cheng,1 and R. Bruce Light1

Departments of 1Medicine, 2Pharmacology and Therapeutics, and 3Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada; and 4Instituto Nacional de Cardiologia Ignacio Chavez, Mexico City, Mexico

Submitted 15 September 2007 ; accepted in final form 6 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In septic shock, systemic vasodilation and myocardial depression contribute to the systemic hypotension observed. Both components can be attributed to the effects of mediators that are released as part of the inflammatory response. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression that develops in a canine model of septic shock. Lzm-S binds to the endocardial endothelium, resulting in the production of nitric oxide (NO), which, in turn, activates the myocardial soluble guanylate cyclase (sGC) pathway. In the present study, we determined whether Lzm-S might also play a role in the systemic vasodilation that occurs in septic shock. In a phenylephrine-contracted canine carotid artery ring preparation, we found that both canine and human Lzm-S, at concentrations similar to those found in sepsis, produced vasorelaxation. This decrease in force could not be prevented by inhibitors of NO synthase, prostaglandin synthesis, or potassium channel inhibitors and was not dependent on the presence of the vascular endothelium. However, inhibitors of the sGC pathway prevented the vasodilatory activity of Lzm-S. In addition, Aspergillus niger catalase, which breaks down H2O2, as well as hydroxyl radical scavengers, which included hydroquinone and mannitol, prevented the effect of Lzm-S. Electrochemical sensors corroborated that Lzm-S caused H2O2 release from the carotid artery preparation. In conclusion, these results support the notion that when Lzm-S interacts with the arterial vasculature, this interaction results in the formation of H2O2, which, in turn, activates the sGC pathway to cause relaxation. Lzm-S may contribute to the vasodilation that occurs in septic shock.

reactive oxygen species; catalase; compound I; hydroxyl radical; septic shock; hypotension

SEPTIC SHOCK is a clinical syndrome that results from an activated systemic host inflammatory response to infection leading to cardiovascular collapse (1). Widespread systemic vasodilation and a decrease in myocardial contractility contribute to the hypotension observed in this condition (1, 39, 40, 42). Both components can be attributed to the effects of mediators that are released as part of the inflammatory response. The recruitment of leukocytes into sites of inflammation leads to interaction with the vascular endothelium and promotes the release of a wide range of proinflammatory cytokines, such as IL-6 and TNF-{alpha} among other classes of mediators (20, 42). Many of these mediators are associated with the production of nitric oxide (NO) by the activation of endothelial and inducible NO synthases (NOSs). NO is thought to play a prominent role in the vasodilation that accompanies septic shock (16).

We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression that develops in canine models of septic shock (21, 33–35). We showed that the mechanism by which Lzm-S caused myocardial depression was related to the binding of its catalytic site to the endocardial endothelium (EE) (33). This interaction resulted in the production of NO, the effect of which could be inhibited by the nonspecific NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). From the EE, NO then diffuses to adjacent myocytes to increase cGMP. In turn, cGMP depresses contraction by the activation of PKG, a known effector of myocardial depression (28).

Since Lzm-S causes the production of NO by an interaction with the EE, we hypothesized that Lzm-S may also contribute to the systemic vasodilation that occurs in septic shock by binding to the endothelium of the systemic vasculature. In the present study, we used a phenylephrine-contracted carotid artery ring preparation to examine the extent to which Lzm-S may contribute to a reduction in vasculature tone in septic shock. We examined whether the production of NO by the endothelium or whether other mechanisms contributed to the vasodilatory response of Lzm-S.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
These experiments were approved by the University Animal Care Committee and conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, 1996) (38).

Carotid artery ring preparation. Internal carotid artery segments (4 cm length) were removed from mongrel dogs (15–25 kg) previously anesthetized with pentobarbital (45 mg/kg) (37, 51). Arteries were placed in cold HEPES-buffered physiological solution [containing (in mmol/l) 118 NaCl, 2.5 CaCl2, 1.2 MgCl2, 1.4 KH2PO4, 4.9 KCl, 25 HEPES, and 11 glucose] bubbled with 100% O2. Arteries were dissected free from the surrounding tissues and cut to obtain rings of ~4 mm in length and 4 mm in outer diameter. Rings were placed into a 10-ml organ bath set at 37°C with a pH of 7.35. Rings were suspended by means of 2 stainless steel triangles in which the ring was stretched to optimal length (to ~4 g). The rationale for using the carotid vasculature rather than other systemic vessels was that a greater number of vasculature rings could be obtained from the carotid artery. In a subgroup of experiments, it was also determined whether Lzm-S caused similar findings in a superior mesenteric artery ring preparation in which the superior mesenteric artery was removed from the animal.

In all of the protocols described below, the carotid rings were preconstricted with phenylephrine (10–5 mol/l) (37, 51). Measurements were determined at ~20 min postphenylephrine instillation when a stable plateau had been reached. After Lzm-S or placebo treatment was added to the preparation, measurements were obtained at 5, 15, and 30 min postinstillation. In initial experiments, the degree to which canine Lzm-S produced vasodilation in the precontracted carotid artery preparation was determined. The plasma concentration of Lzm-S previously determined in our sepsis model was ~10–9–10–6 mol/l (34). The three concentrations of canine Lzm-S used in the individual experiments were 6 x 10–7, 9 x 10–7, and 1.2 x 10–6 mol/l, respectively. The results obtained with Lzm-S were compared with those determined over an identical interval in which an equivalent amount of buffer solution was placed into the tissue bath. Canine Lzm-S was purified as previously described from the spleens of nonseptic dogs by ARVYS Proteins (Stamford, CT) (34). The purity of the preparation was determined by the finding of a single molecule species on MS/MS mass spectroscopy, which was performed by the WM Keck Foundation Biotechnology Resource Laboratory of Yale University (New Haven, CT). The turbidimetric method of Shugar (45) was used to confirm the presence of Lzm-S enzymatic activity in the preparation. Aliquots of a suspension of 25 mg of heat-killed Micrococcus lysodeikticus were placed in 100 ml of phosphate buffer (pH 6.4) with 0.1% NaCl and 0.3 ml Lzm-S. Lzm-S activity was measured in a spectrophotometer as the change in percent transmission at 450 nm for an interval of 1 min following the addition of the Lzm-S sample to the bacterial suspension.

In addition, the effect of human Lzm-S on the production of vasodilation in the precontracted carotid artery ring preparation was examined. The objective was to determine whether human Lzm-S produced changes in vasomotor tone comparable with those found with canine Lzm-S. Human Lzm-S was derived from neutrophils and was purchased from EMD Biosciences (San Diego, CA). The two concentrations of human Lzm-S used in the individual experiments were 3.3 x 10–7 and 6.7 x 10–7 mol/l, respectively. Although the degree to which vasodilation might occur was variable among the different batches of Lzm-S bought from EMD, it should be noted that the same batch was always used in all of the Lzm-S-treated muscles in a given experiment.

A number of control experiments were also performed to further corroborate that the vasodilation caused by Lzm-S was specific for this molecule. In one experiment, it was examined whether denatured human Lzm-S would produce arterial vasodilation. The technique was as described by Hancock and Hsu (18), in which an irreversibly denatured but soluble form of Lzm-S was prepared in which a solution of 3.5 mM Lzm-S was made 20% (wt/wt) in polyethylene glycol (PEG) 1000 and kept in a water bath of 81°C for 16 h. Thereafter, the preparation was kept frozen until use. The concentration of denatured Lzm-S placed into the organ bath was 6.7 x 10–7 mol/l, which was the same as that used for the nondenatured preparation.

Furthermore, the effect of a related protein, {alpha}-lactalbumin (from human milk), on vasorelaxation in the carotid artery preparation was studied to see if vasodilation was specific for the Lzm-S molecule. Lactalbumin is a sequence homolog of Lzm-S (15). Lactalbumin is the regulatory subunit in the heterodimeric enzyme lactose synthase of the milk gland. Both {alpha}-lactalbumin and Lzm-S belong to the glycosyl hydrolase 22 family. The concentration of {alpha}-lactalbumin used in this experiment was also 6.7 x 10–7 mol/l.

In addition, to determine whether heavy metals might be a factor involved in the vasodilatory response of Lzm-S, human Lzm-S was treated with Chelex 100 (200–400 mesh sodium form, Bio-Rad Laboratories, Hercules, CA) (4). The batch method was used in a manner similar to that described by Buettner (4), in which Lzm-S mixed with 0.5 ml HEPES was bathed with Chelex resin, after which the resin was removed by centrifugation. The result obtained with the Chelex preparation was compared with that found with the nontreated preparation. A final set of control experiments included those in which pH and ionized calcium were determined before and after human Lzm-S was placed into the carotid artery bath to assess whether changes in these variables (i.e., decreases in calcium and pH, respectively) could contribute to the vasodilation observed after Lzm-S.

Experiments to determine whether NO production, the endothelium, or prostanoids contribute to Lzm-S-induced carotid artery vasodilation. In one set of experiments, it was determined whether the production of NO by the carotid artery endothelium was responsible for the vasodilation caused by Lzm-S. Human Lzm-S was used in all of the following experiments since the human preparation appeared to produce a more consistently potent vasodilatory response compared with the canine preparation (see RESULTS). In these experiments, after contraction was produced by phenylephrine, L-NMMA was added to the tissue bath at 10–3 mol/l (AG Scientific, San Diego,CA) for ~30 min to allow for the maximal inhibitory effect (47). Immediately after the instillation of L-NMMA, a vasoconstrictive response was noted that usually stabilized over the 30-min interval. After stabilization was achieved, human Lzm-S at a concentration of 6.7 x 10–7 mol/l was added to the preparation. Measurements were expressed relative to the steady-state contraction achieved at ~30 min after L-NMMA. Measurements of force were determined at 5, 15, and 30 min after Lzm-S instillation, and these results were compared with those obtained in a L-NMMA-treated group in which HEPES buffer solution rather than Lzm-S was placed into the organ bath.

In similarly designed protocols, it was then determined whether pretreatment with indomethacin (10–6 mol/l) to prevent prostaglandin release (22) and whether pretreatment with the traditional competitive inhibitors of the catalytic site of Lzm-S [i.e., N,N'-diacetylchitobiose (chitobiose; 10–3 mol/l) or N,N',N''-triacetylchiotriose (chitotriose) (10–3 mol/l) (Sigma)] (35, 41) would prevent the vasodilatory effect of Lzm-S.

After the above experiments had been completed, we examined whether the carotid artery endothelium was necessary for Lzm-S-induced vasorelaxation. In these experiments, the endothelium was mechanically denuded from the carotid artery by means of a cotton-tipped applicator (5, 6, 11, 32). The vasomotor response to carbachol (10–3 mol/l) was the criterion by which it was determined whether the endothelium was successfully removed. Carbachol produces vasodilation in the intact preparation, whereas in the denuded preparation, vasoconstriction is predominantly observed. After the denudation of the endothelium was confirmed, the preparation was washed to remove excess carbachol. After stabilization of the preparation, phenylephrine was then added to produce contraction. At the three predetermined intervals, the effect of Lzm-S was compared between the endothelium-denuded and intact preparations.

Since L-NMMA, indomethacin, and competitive inhibitors of the catalytic site of Lzm-S (i.e., N,N'-diacetylchitobiose and N,N',N''-triacetylchitotriose) as well as removal of the endothelium did not inhibit Lzm-S-induced vasodilation (see RESULTS), this represented the rationale for subsequent screening experiments that were performed to examine whether other pathways may be involved. Among others, these experiments involved the inhibition of bradykinin β1 and β2 receptors by B9340 (19), inhibition of adenyl cyclase by SQ-22,536 (27), inhibition of potassium channels by a high-KCl bath (30 mmol), 20-hydroxyeicosatetraenoic acid (46), tetraethylammonium acetate, 4-aminopyridine, charybdotoxin, and iberiotoxin (9), inhibition of β-receptor activation by propranolol, inhibition of the muscarinic receptor by atropine, and inhibition of the histamine receptor by dipheniramine hydrochloride. Since none of these inhibitors prevented the vasodilatory response of Lzm-S, other pathways were considered.

Experiments to determine whether activation of soluble guanylate cyclase to generate cGMP contributes to Lzm-S-induced vasodilation in the carotid artery preparation. Since the generation of cGMP, a known second messenger of vasodilation (5, 6), could be involved in the response of Lzm-S, inhibitors of the soluble guanylate cyclase (sGC)-cGMP-PKG activation that included LY-83583 (6-anilino-5,8-quinolinequinone, 10–4 mol/L, EMD), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10–4 mol/l, EMD), methylene blue (5.5 x 10–5 mol/l), and PKG inhibitor (guanosine 3',5' cyclic monophosphorothioate, β-phenyl-1,N2-etheno-8-bromo Rp-isomer, sodium salt, 10–6 mol/l, EMD) were examined (5, 6). In respective experiments, the carotid ring was incubated with one of these agents for ~30 min prior to phenylephrine-induced contraction. After a stable phenylephrine response was achieved, the effect of human Lzm-S (6.7 x 10–7 mol/l) on inhibition of the vasodilatory response was determined at 5, 15, and 30 min postinstillation.

Experiments to determine whether Lzm-S-induced vasodilation in the carotid artery preparation was due to generation of ROS. In precontracted isolated bovine intrapulmonary arterial rings, Burke and Wolin (5) showed that H2O2 produces a concentration-dependent relaxation by a mechanism that is independent of the endothelium. H2O2-induced relaxation could be inhibited by prior treatment with the H2O2 metabolizing enzyme Aspergillus niger catalase and by ethanol, both of which reduce the formation of compound I (see DISCUSSION). In the present study, in one set of experiments, ethanol (range: 10–8–2 x 10–7 mol/l) was added to the organ bath to see if it prevented the vasodilatory activity of Lzm-S. To also determine that ethanol does not directly inhibit guanylate cyclase or its downstream mediators of relaxation, in a subset of these experiments, after ethanol was found to inhibit the effect of Lzm-S, nitroprusside (10–4 mol/l) was added to the preparation to see if vasodilation was restored. In addition, the results obtained with ethanol were contrasted with those obtained when another alcohol, n-butanol (10–7 mol/l), was administered to assess whether inhibition of the vasodilatory effect of Lzm-S was specific for ethanol.

In other sets of experiments, we determined whether prior incubation of the carotid artery preparation with the peroxide metabolizing enzyme catalase from A. niger (10–6 mol/l, Sigma) and whether the addition of hydroxyl radical (OH·) scavengers (36) mannitol (2 x 10–2 mol/l) and hydroxyquinone (10–3–10–4 mol/l) would inhibit Lzm-S-induced vasorelaxation (5, 36). Since ROS appeared to contribute to the vasodilatory activity of Lzm-S, it was subsequently assessed whether Lzm-S-induced vasodilation could be prevented by diethyldithiocarbamic acid (DETCA), which inhibits Zn2+,Cu2+-SOD (11), since superoxide generation could be important in the effect of Lzm-S; by the iron chelator deferoxamine methanesulfonate salt (10–2 mol/l) (8), which would prevent the conversion of the H2O2 reaction to OH· and OH– (by the Fenton reaction); and by N-acetylcysteine, ascorbate, and reduced glutathione to see if these antioxidants (10–3–10–6 mol/l) would prevent Lzm-S-induced vasodilation.

Finally, H2O2 (range: 10–6–10–4 mol/l) was administered to the carotid artery preparation to see if it would produce vasodilation in a manner similar to that found for Lzm-S. As part of this protocol, moreover, since high concentrations of H2O2 might injure the carotid artery preparation, the viability of the preparation after H2O2 administration was determined by its ability to contract in a high-KCl (90 mmol/l) bath.

Direct H2O2 determinations. To corroborate that Lzm-S caused H2O2 release in the carotid artery preparation, real-time H2O2 release was measured with a H2O2 electrochemical sensor (ISO-HPO-100) and an Apollo 4000 Analyzer (World Precision Instruments, Sarasota, FL), as described by Kramarenko et al. (23). A small piece of carotid artery of ~5 mm length was cut longitudinally. The H2O2 electrode had a sharp tip, which was used to pierce the endothelial side of the artery. The probe and carotid artery section were placed into a 2-ml vial, which was filled with HBSS that contained (in mmol/l) 137 NaCl, 10 Tris·HCl, 1 MgCl2, 5 KCl, 0.9 CaCl2, 0.8 MgSO4, 0.33 KH2PO4, and 0.1 L-arginine (31). The pH was titrated to pH 7.4 with sodium hydroxide. A photosensitizing agent, hematoporphyrin (10–6 mol/l) (48), was placed into the bath. Low-flow oxygen (<0.5 l/M) was bubbled through a small polyethylene catheter that was also placed into the vial. The vial that contained the H2O2 electrode and carotid artery section was then positioned into a larger water jacket container that was maintained at 37°C. The H2O2 sensor was calibrated by placement of stock solutions of H2O2 into the buffer solution.

Determinations of H2O2 release were obtained from respective carotid artery sections under three conditions: 1) after Lzm-S treatment (100 µl of the 1.6 x 10–6 mol/l solution); 2) after Lzm-S treatment in which the carotid artery section was pretreated with A. niger catalase (10–5 mol/l, Sigma) for 30 min; and 3) after treatment with buffer solution as a time control. Relatively high concentrations of Lzm-S and catalase were used in this experiment, since it was difficult to otherwise ensure that mixing occurred in the vial. H2O2 release was measured over the 1,000-s interval after placement of the treatment, since evaporation of the buffer solution would occur if longer intervals were used. The maximal H2O2 response observed over this interval was recorded.

Statistics. Differences in variables among groups were determined by two-way ANOVA (between-within ANOVA), one-way ANOVA, and analysis of covariance. Student-Newman-Keuls multiple-comparison test was included to determine statistical differences among treatment groups when the ANOVA was used. In the statistical analysis, moreover, results were compared in which force (in g) rather than percentage declines were used in the analysis. In the design of the experiment, of the 8–10 rings obtained from a carotid artery of a given dog, each ring was used for a different subset of experiments in a specific study. Results are given as means ± SD.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
An example of the vasodilatory effect of canine Lzm-S is shown in Fig. 1A, whereas that of human Lzm-S is shown in Fig. 1B. The carotid artery preparation was precontracted with phenylephrine. After the instillation of either canine or human Lzm-S, there was a precipitous fall in force that occurred within a few minutes after Lzm-S was administered. Measurements were determined at 5, 15, and 30 min after Lzm-S instillation. The results obtained with Lzm-S were compared with those found in a time control group in which HEPES buffer solution was instilled.


Figure 1
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  		Fig. 1. In the examples shown, the carotid artery was preconstricted with phenylephrine. Canine lysozyme (Lzm-S) was placed into the organ bath in A, whereas human Lzm-S was placed into the organ bath in B. There was a decrease in force to ~40% of the phenylephrine value with both preparations over the 30-min interval. On the other hand, there was just a slight decrease in force in the placebo-treated carotid artery ring.

 
In Fig. 2A, the mean changes in force over the course of the canine study are shown. At a concentration of 1.2 x 10–6 mol/l, canine Lzm-S caused a decrease in contraction of ~50% at 30 min compared with at the pre-Lzm-S value, whereas there were smaller effects at lesser concentrations. For the human Lzm-S experiment, phenylephrine also caused a similar increase in contraction among the three groups. As shown in Fig. 2B, at a concentration of 3.3 x 10–7 mol/l, Lzm-S resulted in a minimal vasodilatory effect. For the higher concentration, however, there was a marked reduction in force in which Lzm-S nearly eliminated the increase in force induced by phenylephrine. Since human Lzm-S appeared to produce a large degree of vasorelaxation at 6.7 x 10–7 mol/l, this concentration of Lzm-S was used in the experiments described below. A similar degree of vasodilation was also caused by human Lzm-S in the superior mesenteric artery ring preparation (see Fig. 3).


Figure 2
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  		Fig. 2. In the graphs shown, mean values are shown for the canine Lzm-S experiment in A and for the human Lzm-S experiment in B. The different concentrations of Lym-s used in the 2 experiments are shown. Both Lzm-S preparations caused significant vasorelaxation in the carotid artery preparation.

 

Figure 3
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  		Fig. 3. In the superior mesenteric artery ring preparation, Lzm-S caused a decrease in force compared with the buffer-treated time control group. This decrease in force was comparable with that observed in the carotid artery ring preparation.

 
In control experiments, Lzm-S was denatured as described by Hancock and Hsu (18). The degree to which denatured-Lzm-S caused vasorelaxation was significantly less than that found with native Lzm-S (see Fig. 4) and was not different from that observed in the PEG control group and time control group.


Figure 4
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  		Fig. 4. Denatured Lzm-S did not produce vasorelaxation compared with native Lzm-S. The decline in force with denatured Lzm-s was comparable with that found in the polyethylene glycol (PEG)-treated control group and the time control group.

 
In addition, another related protein, lactalbumin (6.7 x 10–7 mol/l), did not cause vasodilation compared with the time control group. In the lactalbumin experiment, the phenylephrine response in the lactalbumin group was similar to that found in time control group (4.1 ± 0.7 vs. 4.4 ± 0.6 g). In the lactalbumin-treated preparation, mean results (n = 5) showed that lactalbumin caused a decrease in contraction to 88 ± 8% of the phenylephrine plateau at 5 min, 75 ± 3% at 15 min (P < 0.05 vs. the phenylephrine plateau), and 66 ± 8% at 30 min (P < 0.05 vs. the phenylephrine plateau). These results were not different from those found in the time control group (n = 5), where the corresponding values were 87 ± 6% of the phenylephrine plateau at 5 min, 70 ± 7% at 15 min (P < 0.05 vs. the phenylephrine plateau), and 56 ± 8% at 30 min (P < 0.05 vs. the phenylephrine plateau) [note, also, that in the statistical analysis, the results were compared in which force (in g) rather than percentage declines were used in the analysis].

Chelex treatment also did not prevent Lzm-S -induced vasodilation. There were no differences in the phenylephrine responses (4.3 ± 1.2 vs. 4.0 ± 1.4 g) or reductions in force between the Chelex-treated (n = 5) and nontreated (n = 6) groups. As a percentage of the phenylephrine plateau, the reductions in force at the three measurement intervals in the Chelex-treated group were 47 ± 24% at 5 min, 6 ± 24% at 15 min, and –24 ± 36% at 30 min (the minus value indicates that contraction was below resting force) (all P < 0.05 vs phenylephrine plateau). The corresponding values in the non-Chelex-treated group were 27 ± 46% (P < 0.05 vs. the phenylephrine plateau), –5 ± 55%, and –24 ± 56% (P < 0.05 vs. the phenylephrine plateau). Finally, there was little change in pH or ionized calcium when Lzm-S was placed into the organ bath [pH 7.36 ± 0.04 vs. 7.34 ± 0.04 and calcium 1.63 ± 0.03 vs. 1.7 ± 0.03 mmol/l (before Lzm-S vs. 30 min after Lzm-S)].

Pretreatment with L-NMMA, mechanical removal of the endothelium, indomethacin, and inhibitors of the catalytic site of Lzm-S did not prevent carotid artery vasodilation. It was initially determined whether pretreatment with L-NMMA would prevent the vasodilatory effect of human Lzm-S in the carotid artery preparation. An example from these experiments is shown in Fig. 5A. In the L-NMMA-treated group, phenylephrine caused a mean increase in active force that averaged 6.2 ± 2.4 g, and this contraction increased further to 8.1 ± 3.5 g with L-NMMA treatment. In the non-L-NMMA-treated group, phenylephrine caused a mean increase in contraction of 7.1 ± 2.1 g from resting force, and this contraction remained stable at 6.7 ± 2.1 g over the 30 min prior to Lzm-S administration. Despite the additional increase in force observed with L-NMMA, Lzm-S produced a marked degree of relaxation that occurred to a similar extent in both the L-NMMA-treated and non-treated groups. In the L-NMMA-treated group (n = 8) (relative to the value obtained after L-NMMA treatment), Lzm-S caused a decrease in contraction to 37 ± 22% at 5 min, 13 ± 16% at 15 min, and –0.3 ± 1% at 30 min (all P < 0.01 vs. the pre-Lzm-S plateau). In the non-L-NMMA-treated group (n = 12), relative to the value found pre-Lzm-S treatment, Lzm-S caused a decrease in contraction to 23 ± 24% at 5 min, 8 ± 27% at 15 min, and 2 ± 34% at 30 min (all P < 0.01 vs. the pre-Lzm-S plateau).


Figure 5
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  		Fig. 5. Examples in which treatment with the nonspecific nitric oxide (NO) inhibitor NG-monomethyl-L-arginine (L-NMMA; A) and removal of the endothelium by mechanical denudation (B), respectively, did not inhibit Lzm-S-induced vasodilation in the carotid artery ring preparation.

 
In the experiments in which the endothelium was mechanically removed, there was also no inhibitory effect on the vasodilatory response of Lzm-S in the carotid artery preparation. An example is shown in Fig. 5B. There were no differences in the phenylephrine response in the endothelium-denuded and intact preparations (7.1 ± 0.5 vs. 6.7 ± 0.7 g). In the denuded preparation (n = 4), the mean results showed that Lzm-S caused a decrease in contraction to 79 ± 18% of the phenylephrine plateau at 5 min, 58 ± 21% at 15 min (P < 0.05 vs. the phenylephrine plateau), and 54 ± 20% at 30 min (P < 0.05 vs. the phenylephrine plateau). In the intact preparation (n = 4), Lzm-S caused a decrease in contraction to 64 ± 25% of the phenylephrine plateau at 5 min, 53 ± 33% at 15 min (P < 0.05 vs. the phenylephrine plateau), and 76 ± 21% at 30 min (P < 0.05 vs. the phenylephrine plateau).

A similar lack of effect was seen with indomethacin (10–6 mol/l) treatment (see Fig. 6). In addition, it was found that the competitive inhibitors of the catalytic site of Lzm-S, namely, chitobiose and chitotriose, did not block the vasodilatory response Lzm-S. The mean results are shown in Table 1.


Figure 6
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  		Fig. 6. A: pretreatment of the carotid artery ring preparation with indomethacin (Indo) did not prevent Lzm-S-induced vasodilation. B: mean data.

 

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  		Table 1. Failure of chitobiose and chitotriose to block Lzm-S-induced vasodilation

 
Inhibitors of the sGC-cGMP-PKG pathway prevented Lzm-S-induced vasorelaxtion in the carotid artery preparation. Since generation of cGMP, a known second messenger of vasodilation, could be involved in the response of Lzm-S, compounds that would inhibit activation of the sGC-cGMP-PKG pathway were tested. These compounds included methylene blue (5.5 x 10–5 mol/l), LY-83583 (10–4 mol/l), ODQ (10–4 mol/l), and a PKG inhibitor (10–4 mol/l). All of these agents were found to inhibit the effect of Lzm-S. An example for methylene blue is shown in Fig. 7. The mean results of the four inhibitors are shown in Fig. 8.


Figure 7
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  		Fig. 7. In the example shown, incubation with methylene blue inhibited the effect of Lzm-S on relaxation in the carotid artery ring preparation.

 

Figure 8
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  		Fig. 8. Inhibitors of the soluble guanylyl cyclase (sGC)-cGMP-PKG pathway prevented Lzm-S-induced vasodilation in the carotid artery ring preparation. These inhibitors included methylene blue (A), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; B), PKG inhibitor (C), and LY-83583 (D).

 
Agents that modify H2O2 metabolism also inhibited the carotid artery vasodilation caused by Lzm-S. Based on the work of Wolin and colleagues (5, 6, 11), we determined whether agents known to alter H2O2 metabolism might inhibit Lzm-S-induced vasorelaxation in the carotid artery preparation. The H2O2 metabolizing enzyme A. niger catalase induced a contraction that was independent of phenylphrine treatment, so that administration of phenylephrine was not required to produce a contraction in these experiments (see Fig. 9A and DISCUSSION). A. niger catalase almost completely inhibited the vasodilation caused by Lzm-S (see Fig. 9, A and B). Ethanol, which decreases compound I, was also markedly effective in the prevention of Lzm-S-induced vasodilation at the various concentrations used (see Fig. 10 and DISCUSSION). Furthermore, in a subset of these experiments, it was shown that ethanol does not directly inhibit guanylate cyclase or its downstream mediators of relaxation. In these experiments, after ethanol (10–7 mol/l) was found to inhibit the effect of Lzm-S, nitroprusside (10–4 mol/l) was added to the preparation. Nitroprusside produced a marked decrease in force from 2.5 ± 1 g at 30 min after Lzm-S to –4.3 ± 2.3 g at 5 min after nitroprusside (n = 6, P < 0.01). Moreover, in ethanol-treated control groups, there was little change in force over time when HEPES buffer was added to the preparation. At an ethanol concentration of 2 x 10–7 mol/l, force measured 10 ± 2.6 g after phenylephrine and did not change further when HEPES buffer was added (10.7 ± 2.8 g at 5 min, 11 ± 2.8 g at 15 min, and 11 ± 2.6 g at 30 min).


Figure 9
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  		Fig. 9. A: pretreatment with Aspergillus niger catalase inhibited the effect of Lzm-S on vasodilation in the carotid artery ring preparation. B: mean results.

 

Figure 10
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  		Fig. 10. A: ethanol (1 x 10–7 mol/l) inhibited the effect of Lzm-S on relaxation in the carotid artery ring preparation. B: mean results for the various ethanol concentrations.

 
In contrast to ethanol, an unrelated alcohol, n-butanol, had no inhibitory effect on Lzm-S-induced vasorelaxation. n-Butanol caused an initial vasorelaxtion in which force decreased to 61 ± 17% of the phenylephrine plateau response after this alcohol was added (P < 0.001 vs. the plateau response). The addition of Lzm-S caused further vasodilation to 84 ± 9% of the n-butanol plateau at 5 min, to 62 ± 21% at 15 min (P < 0.05 vs. the plateau response), and to 23 ± 27% at 30 min (P < 0.05 vs. the plateau response). These findings were not different from those observed in a Lzm-S-treated group in which n-butanol treatment was not administered.

At concentrations that ranged between 10–4 to 10–6 mol/l, exogenous H2O2 caused a vasodilatory response. At 10–6 mol/l (n = 4), the decreases in contraction relative to baseline were 79 ± 7% at 5 min, 59 ± 13% at 15 min (P < 0.05 vs. baseline), and 47 ± 15% at 30 min postinstillation (P < 0.05 vs. baseline). At 10–5 mol/l (n = 6), decreases in contraction were 81 ± 6%, 64 ± 16%, and 55 ± 19% (all P < 0.05 vs. baseline), respectively. At 10–4 mol/l (n = 5), the changes in contraction were 103 ± 39%, 54 ± 6% (P < 0.05 vs. baseline), and 56 ± 10% (P < 0.05 vs. baseline), respectively. In experiments in which the viability of the preparation was determined, there were no differences in the increase in force among the various groups when KCl was added to the organ bath to cause contraction: 1.6 ± 0.25 g for the 10–6 mol/l concentration, 1.7 ± 0.6 g for the 10–5 mol/l concentration, 1.3 ± 0.8 g for the 10–4 mol/l concentration, and 2.0 ± 1.2 g for the non-H2O2-treated group (n = 4).

To determine whether OH· was involved in the effect of Lzm-S, we assessed whether OH· scavengers [mannitol and hydroxyquinone (36)] prevented Lzm-S-induced vasorelaxation. Both treatments had an inhibitory effect (see Fig. 11). To determine whether the osmolarity of mannitol contributed to this inhibitory property, subsequent experiments were performed with sorbitol. Sorbitol has a molecular weight similar to that of mannitol and was added to the preparation at a comparable concentration (10–2 mol/l). Sorbitol treatment did not inhibit Lzm-S-induced vasodilation. There were similar increases in contraction to phenylephrine in the sorbitol + Lzm-S-treated group (n = 5) versus the Lzm-S alone-treated group (n = 5) (3.5 ± 0.9 s. 3.5 ± 0.8 g). Decreases in relaxation as a percentage of the phenylephrine response were also similar at the three measurement intervals between the two groups: 78 ± 11%, 54 ± 19%, and 31 ± 27% vs. 78 ± 7%, 48 ± 14%, and 22 ± 17%, respectively (all P < 0.05 vs. the phenylephrine response).


Figure 11
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  		Fig. 11. Both hydroxyl radical scavengers [mannitol (A) and hydroquinone (B)] inhibited the vasodilatory effect of Lzm-S in the carotid artery ring preparation.

 
In screening experiments (data not shown), neither DETCA (10–2 mol/l) nor deferoxamine methanesulfonate salt (10–2 mol/l) prevented Lzm-S-induced vasodilation. Moreover, the antioxidants N-acetylcysteine, reduced glutathione, and sodium ascorbate also failed to inhibit the vasodilatory response of Lzm-S (see Table 2). Since, for these various substances, the findings were not different at the ranges of concentrations used, the results were pooled for each compound.


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  		Table 2. Failure of N-acetylcysteine, ascorbate, and reduced glutathione to prevent Lzm-S-induced vasorelaxation

 
Direct H2O2 determinations. Experiments were performed in which Lzm-S was added to the carotid artery preparation to measure the real-time release of H2O2. An example of these experiments is shown in Fig. 12. As shown in Fig. 12A, Lzm-S caused an increase in H2O2 release that could be inhibited by incubation with A. niger catalase (Fig. 12B). In buffer time control experiments (Fig. 12C), there was little change in the recording over the interval of the study. The mean results are shown in Fig. 13.


Figure 12
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  		Fig. 12. In the examples shown, real-time H2O2 generation was measured by an electrochemical sensor in the canine carotid artery ring prepartion. In A, Lzm-S caused an increase in H2O2 generation, whereas in B, pretreatment with A. niger catalase prevented H2O2 generation. In the buffer-treated preparation (C), there was no change in H2O2 generation over the course of the experiment. H2O2 release was measured over the 1,000-s interval after placement of the treatment, since evaporation of the buffer solution would occur if longer intervals were used. The maximal H2O2 response observed over this interval was recorded.

 

Figure 13
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  		Fig. 13. Mean data are shown in which real-time H2O2 generation was measured by an electrochemical sensor in the canine carotid artery ring preparation. Lzm-S caused an increase in H2O2 generation, whereas A. niger catalase prevented H2O2 generation. There was little change in H2O2 production over the course of experiment with buffer treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Lzm-S is a newly discovered inflammatory mediator that has been shown to cause myocardial depression in an Esherichia coli experimental model of sepsis (21, 33–35). In the present study, we examined the extent to which Lzm-S may contribute to the systemic vasodilation that is also observed in this model. The concentrations of Lzm-S used in this study were comparable with those previously found to occur after 4–6 h of E. coli bacteremia in dogs (34). In the present study, the results showed that both canine and human Lzm-S produced significant vasorelaxation in a phenylephrine-contracted carotid artery ring preparation. Canine Lzm-S was prepared from the spleen, whereas human Lzm-S was made from neutrophils. Thus, Lzm-S prepared from these different sources produced vasodilation in the carotid artery preparation. On the other hand, denatured Lzm-S and a related protein, lactalbumin, did not produce vasodilation, so this effect was specific for the native Lzm-S molecule.

Based on our previous cardiac experiments (21, 33–35), we initially considered that the release of mediators from the endothelium, particularly NO production, was the mechanism by which Lzm-S caused this vasodilatory effect. Nevertheless, when we tested for the role of NO by pretreatment of the carotid ring with L-NMMA, we did not find that Lzm-S-induced vasorelaxation was inhibited, although it is not possible to exclude that NO contributes marginally to the effect of Lzm-S. We further tested for commonly known pathways of vasodilation that included blockade of the prostaglandin pathway with indomethacin, blockade of bradykinin β1 and β2 receptors by B9340, inhibition of adenyl cyclase by SQ-22,536, and inhibition of potassium channels as well as inhibition by the β-adrenergic receptor antagonist propranolol and the muscarinic receptor antagonist atropine (9, 19, 27, 46). None of these inhibitors prevented the effect of Lzm-S. In addition, when we removed the carotid artery endothelium, we found that the vasodilatory effect of Lzm-S was still present. Thus, the release of mediators from the endothelium or the aforementioned pathways did not contribute to the vasorelaxation caused by Lzm-S.

We also determined whether inhibitors of the catalytic site of Lzm-S, namely, chitobiose and chitotriose, would prevent the vasodilatory effect of Lzm-S (21, 35, 41). In the heart, we found that the binding of Lzm-S to a N-glycoprotein on the endocardial endothelium increases the production of NO (33). Lzm-S specifically binds to the mannose-β(1-4) N-acetylglucosamine (GlcNAc)-β(1-4)GlcNAc moiety in the tri-mannosyl core structure of high mannose/hybrid and tri-antennary carbohydrate classes (21). The mannose-β(1-4) GlcNAc-β(1-4)GlcNAc moiety is structurally similar to chitobiose and chitotriose compounds, and these two agents have been shown to competitively inhibit the myocardial depressant effect of Lzm-S in in vivo and in vitro preparations (21, 33–35). Thus, the characteristics that govern Lzm-S-induced vasodilation in terms of signaling pathway and binding are distinctly different from those that mediate myocardial depression, although the mechanisms for these differences are not clear.

In terms of the signaling involved, the present results indicate that activation of the sGC-cGMP-PKG pathway was a requirement for the vasodilatory effect of Lzm-S, since many inhibitors of this pathway prevented relaxation. Although this pathway represented the apparent second messenger system for vasorelaxation, the mechanism by which it was activated was not initially clear, since none of the commonly noted pathways, usually related to the production of cGMP, was able to block the effect of Lzm-S. We therefore looked for other possibilities. After many screening experiments had been performed, we found that ethanol was able to inhibit the vasodilatory activity of Lzm-S. Wolin and colleagues (5, 6) reported that H2O2 was a mediator of vasorelaxation in a precontracted pulmonary artery preparation and that A. niger catalase and ethanol inhibited the vasodilatory effect of H2O2. They further concluded that H2O2 was able to generate compound I, a species of catalase that is formed in the metabolism of H2O2 by endogenous catalase. In turn, compound I activates sGC to increase cGMP, resulting in vasodilation. A. niger catalase would therefore decrease compound I by scavenging H2O2 and preventing the activation of sGC. Ethanol is an agent that is known to modulate H2O2 metabolism through endogenous catalase (6), resulting in a decrease in compound I. This effect would also cause a reduction in cGMP. In the present study, we hypothesized that the binding of Lzm-S to the carotid artery preparation could generate H2O2. We further hypothesized that this generation may be inhibited by both A. niger catalase and ethanol. The results showed that both of these agents blocked the vasodilatory activity of Lzm-S in the carotid artery preparation. Furthermore, in the experiments in which the H2O2 sensor was used to measure real-time results, the findings showed that Lzm-S caused the generation of H2O2 and that this effect could be prevented by A. niger catalase.

Since mechanical denudation of the endothelium of the carotid artery did not inhibit the vasodilatory activity of Lzm-S, the binding of Lzm-S to a receptor on smooth muscle cells and/or on the surrounding adventitia appears responsible for the vasorelaxation that occurred. Although we favor the binding of Lzm-S to a receptor on smooth muscle cells as the mechanism involved, other investigators have shown that the adventitia/perivascular adipose tissue may also contribute to vasorelaxation and that this effect may occur by the release of H2O2 (14, 17). H2O2 could be generated either extracellularly or intracellularly, thereby resulting in an increase in cGMP. cGMP through PKG would then cause vasodilation by altering myofilament responsiveness to calcium or by inhibiting calcium entry into the cell (28). The question of the tissue and receptor to which Lzm-S binds needs to be addressed in future studies. Furthermore, we washed the carotid artery preparation multiple times (at least 6 times) before experiments were performed. Since these were nonseptic animals, little inflammatory response would be expected to be present in the vessel wall. This would make it unlikely that leukocytes adherent to the carotid artery preparation contributed to the generation of H2O2 that was observed.

Our results also show that the administration of antioxidants (see Table 2 and RESULTS) did not prevent Lzm-S-induced vasodilation at any of the concentrations used. We believe the explanation is as follows. From our results, we speculate that the important mechanism by which Lzm-S causes vasorelaxation is through metabolism of H2O2 by endogenous catalase to form compound I (5, 6). Compound I then causes vasorelaxation by increasing cGMP (5). Although other pathways of metabolism, such as by peroxidases, may also contribute to this metabolism of H2O2, as long as there is some formation of compound I by endogenous catalase, vasorelaxation will still occur. Wolin and coworkers (5) showed in an in vitro preparation that in the presence of H2O2 generation, liver bovine catalase caused an increase in cGMP attributable to the formation of compound I, whereas horseradish peroxidase and the peroxidase-metabolizing enzyme A. niger catalase did not produce this effect. The biochemical mechanisms responsible for this difference in the metabolism of H2O2 among the various species of catalase/peroxidases in terms of their ability to generate cGMP through compound I are not clear. However, since the antioxidants described in Table 2 were not direct inhibitors of compound I formation by endogenous catalase, we therefore speculate they did not prevent the vasodilatory activity of Lzm-S. On the other hand, agents such as ethanol and A. niger catalase, which were more potent in this regard, prevented vasodilation.

Furthermore, the metabolism of H2O2 within the cell may be compartmentalized (3). Although H2O2 has often been believed to freely cross membranes, contrary to this, recent studies have pointed out that some membranes are rather poorly permeable to H2O2 (2, 29, 44). This implies that the transport of H2O2 may be regulated and that this regulation constitutes a major factor in the determination of cellular H2O2 concentration and metabolism. These differences in permeability could either be explained by changes in membrane lipid compositions or by diffusion-facilitating channel proteins or a combination of both (2, 29, 44). Catalase is predominantly found in the peroxisomes and to some extent in the cytosol, whereas glutathione peroxidase is predominantly found in the cytosol and to a lesser extent in the peroxisomes (25, 43). This compartmentalization might affect the enzymatic pathway by which H2O2 is metabolized and, therefore, the amount of cGMP that is generated. After Lzm-S treatment, colocalization of catalase with H2O2 would lead to the formation of compound I. Compound I then interacts with sGC, which is found in the cytosol. This activation of sGC, in turn, leads to increased formation of cGMP. On the other hand, such colocalization would not be found with the peroxidases and therefore the increased formation of cGMP does not occur. These issues need to be determined in future investigations.

The mechanism by which Lzm-S would generate H2O2 is not clear. Among the possible mechanisms to consider, we initially thought that H2O2 would be derived from superoxide with SOD, since this mechanism has been shown to activate sGC under other experimental conditions (11). However, we could not prevent the vasodilator effect of Lzm-S with the SOD inhibitor DETCA, so we do not think that this enzyme system was involved in the vasodilation observed. In other studies, Wentworth and colleagues (48–50) reported that proteins, such as antibodies, may generate H2O2 from singlet molecular oxygen and water; moreover, this generation may occur regardless of the antigen specificity of the antibody. They suggest that some proteins, particularly antibodies have the intrinsic ability to intercept singlet molecular oxygen and efficiently reduce it to O2bullet–, which then dismutates spontaneously into H2O2. This effect could also be involved in the Lzm-S-induced generation of H2O2. DeYulia et al. (12) found that H2O2 could be generated by a specific receptor-ligand interaction in cells and in cell-free systems, although the mechanism of this generation was not well defined. Buettner and co-workers (10, 23) showed that ascorbate reacts with singlet oxygen to generate H2O2, and it is conceivable that Lzm-S could react with singlet oxygen to produce a similar reaction. Recently, NADPH oxidase (Nox4) has been identified in the vasculature as a source of ROS that produces H2O2 (30). In addition, other isoforms, such as Nox1, Nox2, and Nox5, and perhaps even the duoxes, which directly produce H2O2, need to be considered in terms of the effect of Lzm-S (26).

Moreover, we showed that OH· scavengers mannitol and hydroquinone prevented the vasodilatory activity of Lzm-S. Wolin and colleagues (5) did not find that the OH· was a requirement for cGMP generation to cause vasodilation in their pulmonary artery preparation. However, Mittal and Murad (36) found that in addition to H2O2, OH· was required for the generation of cGMP in a rat liver preparation. Although the pathways have not been totally elucidated, and it is not clear why mannitol prevents vasodilation in some preparations while not in others, we think that OH· may promote the effect of Lzm-S by contributing to a relative increase in the activation of guanylate cyclase in the carotid artery preparation. Nevertheless, the prevention of Lzm-S-induced vasodilation by mannitol and hydroquinone was less effective compared with what was observed with A. niger catalase (60% vs. 90%), so that the generation of OH· may play a lesser role than that of the H2O2 molecule in producing the vasodilation caused by Lzm-S. Such results would again point out that the pathway leading to the formation of compound I by endogenous catalase seems to be the primary consideration for the vasodilatory activity of Lzm-S.

Furthermore, we could not prevent Lzm-S-induced vasodilation by the administration of the iron chelator deferoxamine methanesulfonate salt, an agent that would prevent the conversion of H2O2 to OH· and OH– by the Fenton reaction, so that it appears that the generation of OH· by this pathway was not the likely mechanism. Wentworth et al. (50) also proposed that proteins could generate OH· through a series of reactions that involved the generation of trihydroxide radical, resulting in the formation of 3O2 and OH·, and that this reaction may not require iron (50). In general, the mechanisms of H2O2 and OH· generation as a signaling molecule are complex and are still being ascertained, and further investigations will have to be performed to determine the mechanism by which these reactive species are formed by Lzm-S.

In the carotid artery organ bath preparation, we also showed that the exogenous administration of H2O2 produced vasodilation over concentrations that ranged between 10–6 and 10–4 mol/l. These results are similar to those found by Burke and Wolin (5) in a constricted pulmonary artery preparation over a similar range of H2O2 concentrations. Furthermore, when A. niger catalase was added to the carotid artery preparation, we found that this enzyme induced a contraction. We believe that this occurred because of scavenging of constituently generated H2O2 that would have otherwise maintained a low resting force.

Lzm-S is a newly discovered inflammatory mediator of cardiovascular collapse in septic shock (21, 33–35). Lzm-S appears to cause vasorelaxation by the activation of the sGC-cGMP-PKG pathway that occurs through the generation of H2O2 and the formation of compound I by endogenous catalase. This pathway has never been described as a mechanism for vasodilation in septic shock. Although this is new information, we recognize that there is still more information to be obtained, such as the specific binding site of Lzm-S, how it generates H2O2, and the extent to which Lzm-S binds to the systemic vasculature in septic models. Burgess et al. (7) showed that blood Lzm-S concentrations increase in patients with abdominal sepsis, so we think that the findings in our canine models are relevant to what occurs in the human condition. Furthermore, in our previous study (35), we showed that competitive inhibitors of the catalytic site of Lzm-S, particularly chitotriose and chitobiose, were useful in the treatment of myocardial depression in canine models of septic shock. However, in the present study, these agents could not prevent Lzm-S-induced vasorelaxation. A therapy that also targets against the vasodilator effect of Lzm-S may therefore be of further benefit in the treatment of cardiovascular collapse in septic shock.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Canadian Institutes of Health Research, Health Sciences Centre Foundation, and the Biology of Breathing Group, Manitoba Institutes of Child Health.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. N. Mink, Health Sciences Centre, GF-221, 820 Sherbrook St., Winnipeg, Manitoba, Canada R3A 1R9 (e-mail: minksn{at}cc.umanitoba.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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