AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 285: H2524-H2530, 2003. First published August 7, 2003; doi:10.1152/ajpheart.00530.2003
0363-6135/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/6/H2524    most recent
00530.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ichinose, F.
Right arrow Articles by Zapol, W. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ichinose, F.
Right arrow Articles by Zapol, W. M.

A selective inducible NOS dimerization inhibitor prevents systemic, cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice

Fumito Ichinose,1,2 Ryuji Hataishi,1 Justina C. Wu,3 Noriko Kawai,1,2 Ana Clara Tude Rodrigues,3 Cornell Mallari,4 Joe M. Post,4 John F. Parkinson,4 Michael H. Picard,3 Kenneth D. Bloch,2 and Warren M. Zapol1

1Department of Anesthesia and Critical Care, 2Cardiovascular Research Center, and 3Cardiac Ultrasound Laboratory, Cardiology Division, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114-2620; and 4Berlex Biosciences, Richmond, California 94804-0099

Submitted 6 June 2003 ; accepted in final form 4 August 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 
Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-L-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.

nitric oxide synthase; inflammation; inhibitors

CARDIOVASCULAR DYSFUNCTION in sepsis contributes to the high mortality rate for this syndrome (22). Nitric oxide (NO) is produced by NO synthases (NOS1, NOS2, and NOS3), and induction of NOS2 has been implicated in the pathogenesis of endotoxin-induced hypotension and vascular hyporeactivity (28). However, treatment of septic animals with inhibitors of all three NOS isoforms such as NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) significantly decreases cardiac output and oxygen delivery (21), further impairs renal (24) and liver (16) function, and increases mortality rate (1, 16, 24). These observations may relate to the deleterious effects of inhibition of NOS1 and/or NOS3 by nonselective NOS inhibitors in the setting of systemic sepsis (21).

In contrast, selective NOS2 inhibitors may protect against sepsis-induced end-organ damage without inhibiting the ability of NOS1 and NOS3 to participate in cardiovascular homeostasis (3, 9). NOS2 inhibitors with variable selectivity have been examined in endotoxin-induced cardiovascular dysfunction. Although less selective NOS2 inhibitors (e.g., aminoguanidine) showed variable effects depending on species and model (20, 25), recent studies with more selective NOS2 inhibitors [e.g., L-N6-(1-iminoethyl)lysine hydrochloride (27) and 1400W (32)] were more encouraging. Studying NOS2-deficient mice, we reported the key roles played by NOS2 in the endotoxin-induced impairment of myocardial function and hypoxic pulmonary vasoconstriction (HPV; Refs. 29, 30).

NOS isoforms are only active for NO formation as stable homodimers (18). Pyrimidylimidazole-based NOS dimerization inhibitors provide a novel approach to selective pharmacological NOS2 inhibition (4, 19). Here we hypothesized that the NOS2 dimerization inhibitor BBS-2 would be highly selective for NOS2 in vivo and would prevent endotoxin-induced systemic, cardiac, and pulmonary hemodynamic dysfunction. Specifically, we investigated the effects of BBS-2 on baseline hemodynamics and endotoxin-induced systemic hypotension, myocardial dysfunction, and impairment of HPV in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 
After we received institutional approval by the Massachusetts General Hospital Subcommittee on Research Animal Care, we studied male C57BL/6J wild-type mice and B6.129P2-NOS2tmlLau NOS2-deficient mice with an age range of 2–5 mo and body wt 18–29 g (Jackson Laboratory; Bar Harbor, ME) and Lewis rats with body wt 250–350 g.

NOS2 dimerization inhibitor. Stock solution of BBS-2 (compound 4 in Ref. 4) was prepared at 30 mg/ml in HCl-acidified sterile water (pH ~3–4) and stored at room temperature. HCl-acidified sterile water was used as vehicle. BBS-2 and vehicle were injected (10 µl/g ip) after appropriate dilution of stock solutions.

Serial measurement of blood pressure by tail cuff. Blood pressure was measured in mice with a tail-cuff pressure-recording device (Kent Scientific; Torrington, CT) before and 4 and 7 h after challenge with endotoxin or saline. At time 0, 14 mice received an injection of Escherichia coli 0111:B4 endotoxin (10 mg/kg ip, Sigma; Detroit, MI), and 1 h later they were injected with BBS-2 or vehicle (n = 9 and 5, respectively). An additional 4 mice received an intraperitoneal injection of saline at time 0 and were injected with BBS-2 1 h later. After the measurement of blood pressure 7 h after challenge, whole blood was sampled to analyze plasma nitrate and nitrite (NOx) levels.

Measurements of left ventricular function. Echocardiographic measurements were obtained before and 7 h after challenge with endotoxin (100 mg/kg) or saline. Invasive measurements of systemic and left ventricular (LV) pressure were obtained 7 h after endotoxin challenge. Of the 16 mice challenged with endotoxin, 6 received BBS-2 (30 mg/kg), 5 received L-NAME (100 mg/kg), and 5 received vehicle 1 h later. Of the 9 mice challenged with saline, 4 received BBS-2 (30 mg/kg) and 5 received vehicle 1 h later. Pilot experiments established that a higher dose of endotoxin (100 mg/kg) was required to evoke consistent myocardial depression within 7 h than was required to cause hypotension or impair HPV (10 mg/kg). The dose of BBS-2 was adjusted to 30 mg/kg ip for these experiments to ensure complete inhibition of NOS2 dimerization. The dose of L-NAME was chosen based on a previous study (26).

Echocardiographic measurements. Echocardiography was performed using a 13-MHz ultrasound probe (Sequoia, Acuson; Mountain View, CA) in sedated mice (ketamine, 50 µg/g ip) as previously described (30). Fractional shortening (FS) was calculated from M-mode echocardiogram as described previously (23).

Invasive hemodynamics. Invasive measurements of LV pressure were performed with a 1.4-Fr high-fidelity pressure catheter (Millar Instruments; Houston, TX) in anesthetized mice (fentanyl, 250 ng/g ip, and ketamine, 50 µg/g ip) as previously described (30). The maximum and minimum first derivative of the developed LV pressure (dP/dtmax and dP/dtmin, respectively) were calculated by differentiation of the digitized analog LV pressure tracing. The time constant of LV isovolumic relaxation ({tau}) was calculated using the method of Weiss et al. (31).

Measurement of HPV in mice. Measurements of HPV were performed on anesthetized mice 22 h after challenge with endotoxin (10 mg/kg) or saline as previously described (12, 29). Of the 10 mice challenged with endotoxin, 5 received BBS-2 (10 mg/kg ip) and 5 received vehicle 1 and 8 h later. Of the 9 mice challenged with saline, 4 received BBS-2 (10 mg/kg) and 5 received vehicle 1 and 8 h later. Two doses of BBS-2 were given due to a relatively short elimination half-life in rodents (~1 to 1.5 h; J. F. Parkinson and B. Subramanyam, unpublished observations). Changes in left lung pulmonary vascular resistance (LPVR) in response to left mainstem bronchus occlusion (LMBO) were estimated by changing cardiac output via inferior vena cava occlusion while continuously measuring left pulmonary artery blood flow and pulmonary artery pressure as previously described (12).

Pulmonary NOS2 mRNA. Lung was harvested from BBS-2-treated (10 mg/kg, n = 3) and vehicle-treated (n = 3) mice 7 h after endotoxin challenge (10 mg/kg). Lung tissue mRNA was extracted by the guanidine isothiocyanate-cesium chloride method. RNA (10 µg) was fractionated in formaldehydeagarose gels and transferred to nylon membranes. Membranes were hybridized with a 32P-labeled 0.3-kb mouse NOS2 cDNA probe, washed, and exposed to X-ray films as described previously (30).

Low-temperature partially denaturing SDS-PAGE and immunoblot analysis. Lung and heart were harvested from BBS-2-treated mice (30 mg/kg, n = 3) and vehicle-treated mice (n = 3) 7 h after endotoxin challenge (100 mg/kg). NOS2 dimerization was analyzed by low-temperature PAGE as described previously (6, 33). Briefly, homogenates of lung and heart were mixed with sample buffer (250 mM Tris, pH 6.8, 8% SDS, 4% 2-mercaptoethanol, 0.004% bromphenol blue, 40% glycerol) and loaded on gels without boiling. After SDS-PAGE, proteins were electroblotted to nitrocellulose membranes and incubated with a polyclonal anti-NOS2 antibody (Biomol Research Laboratories; Plymouth Meeting, PA) at a 1:500 dilution. Bound antibodies were detected using horseradish peroxidase-labeled protein A and visualized using chemiluminescence.

Plasma NOx concentration. Plasma NOx concentrations were determined by Greiss reagent detection at 540 nm using nitrite standards in nine endotoxin-challenged BBS-2-treated, five endotoxin-challenged vehicle-treated, and four saline-challenged wild-type mice. In addition to samples obtained from wild-type mice, plasma was sampled from NOS2-deficient mice 7 h after endotoxin challenge (n = 5).

Ex vivo NOS3 selectivity assay. Lewis rats received BBS-2 at 30 mg/kg sc or vehicle twice daily for 10 days (8 animals/group). On the day of ex vivo evaluation, the aorta was harvested and cut into eight rings while care was taken not to damage the endothelium. Aortic rings were mounted on a tension transducer device and immersed in organ baths of physiological salt solution that contained (in mM) 138.3 NaCl, 4.7 KCl, 0.6 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25 NaHCO3, 0.26 CaNa2EDTA, and 11.1 glucose. Phenylephrine (PE)-induced contraction was allowed to reach steady state before addition of ACh. Concentration-response curves for ACh were constructed by sequential addition of ACh in increasing concentrations.

Statistical analysis. Differences between groups were determined by two-way ANOVA. When significance was detected by ANOVA, a post hoc Student-Newman-Keuls test was employed (Statistica for Windows, StatSoft; Tulsa, OK). A P value <0.05 indicated a significant difference. All data are expressed as means ± SD.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURE
 REFERENCES
 
BBS-2 does not improve mortality rate in endotoxemic mice. After endotoxin challenge, animals manifested weakness, lethargy, piloerection, and diarrhea. No mice died within 7 h after challenge with 10 mg/kg endotoxin whether or not they were treated with BBS-2. At 22 h after administration of 10 mg/kg endotoxin, mortality rate was similar between mice treated with BBS-2 (4 of 20) and vehicle (5 of 18). At 7 h after challenge with 100 mg/kg endotoxin, mortality rate was identical between mice treated with BBS-2 and vehicle (2 of 9 in each group) and tended to be greater in L-NAME-treated mice (7 of 14).

Treatment with BBS-2 prevents endotoxin-induced hypotension. In vehicle-treated mice, challenge with 10 mg/kg endotoxin resulted in a progressive and severe decline in systolic blood pressure at 4 and7h(P < 0.05 vs. baseline at both time points; Fig. 1A). Treatment 1 h after endotoxin administration with 10 mg/kg BBS-2 completely prevented systemic hypotension (P < 0.001 vs. endotoxin plus vehicle at 7 h).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. A: effects of treatment with BBS-2 (10 mg/kg) or vehicle on tail-cuff systolic blood pressure measurements at baseline and 4 and 7 h after mice were challenged with saline or endotoxin (10 mg/kg). Endotoxin-challenged mice were subsequently treated with BBS-2 ({blacktriangledown}, n = 9) or vehicle ({bullet}, n = 5). Saline-challenged mice were treated with BBS-2 ({triangleup}, n = 4). *P < 0.05 vs. corresponding baseline blood pressure values; #P < 0.01 vs. blood pressure measurements of endotoxin-challenged BBS-2-treated mice at 7 h after endotoxin challenge. B: left ventricular (LV) fractional shortening before and 7 h after mice were challenged with endotoxin (100 mg/kg) followed by treatment with BBS-2 (30 mg/kg, {blacktriangledown}, n = 6), vehicle ({bullet}, n = 5), or NG-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg, {blacksquare}, n = 5). Additional saline-challenged mice were treated with BBS-2 ({triangleup}, n = 4). *P < 0.05 vs. corresponding baseline and saline-challenged BBS-2-treated mice at 7 h after challenge; #P < 0.05 vs. endotoxin-challenged BBS-2-treated mice; {dagger}P < 0.01 vs. endotoxin-challenged vehicle-treated mice. C: percent increase of lung pulmonary vascular resistance (LPVR) in response to left mainstem bronchus occlusion in mice challenged with saline (endotoxin –) or endotoxin (endotoxin +) 22 h earlier. Some mice received BBS-2 or vehicle at 1 and 8 h after challenge. *P < 0.05 vs. saline-challenged mice; #P < 0.01 vs. endotoxin-challenged BBS-2-treated mice.

 

BBS-2 prevents endotoxin-induced myocardial dysfunction. BBS-2 (30 mg/kg) had no adverse effects on hemodynamics or LV function in saline-challenged mice (Table 1). Seven hours after administration of 100 mg/kg endotoxin, FS, dP/dtmax, and dP/dtmin were markedly impaired in endotoxin-challenged vehicle-treated mice compared with saline-challenged mice (all P < 0.05; Table 1). Similarly, mean systemic arterial pressure (SAP) and LV end-systolic pressure (LVESP) were lower and {tau} was greater in endotoxin-challenged vehicle-treated mice than in saline-challenged mice 7 h after challenge (all P < 0.05; Table 1). BBS-2 completely prevented the endotoxin-induced decreases in dP/dtmax, LVESP, and SAP and attenuated the endotoxin-induced reduction in FS (P < 0.05; Fig. 1B). BBS-2 also prevented the endotoxin-induced prolongation of {tau} 7 h after endotoxin-challenge (P < 0.05; Table 1). Treatment with L-NAME prevented the endotoxin-induced decrease in SAP (P < 0.05 vs. endotoxin-challenged vehicle-treated mice; Table 1), whereas dP/dtmax and FS were further impaired by L-NAME (P < 0.05 for both vs. endotoxin-challenged vehicle-treated mice; Table 1 and Fig. 1B). Measures of diastolic function, LVEDP, dP/dtmin, and {tau} were markedly impaired by treatment with L-NAME after endotoxin challenge (for all three measurements, P < 0.01 vs. saline-challenged mice and endotoxin-challenged BBS-2-treated mice; P < 0.05 vs. endotoxin-challenged vehicle-treated mice; Table 1).


View this table:
[in this window]
[in a new window]
  		Table 1. Invasive hemodynamic measurements at 7 h after saline or 100 mg/kg endotoxin challenge

 

BBS-2 treatment prevents endotoxin-induced impairment of murine HPV. In saline-challenged mice, LMBO increased LPVR (101 ± 38%) without affecting SAP or pulmonary artery pressure (Table 2 and Fig. 1C). BBS-2 had no adverse effects on baseline pulmonary hemodynamics or their response to LMBO in saline-challenged mice. In contrast, LMBO did not increase the LPVR in mice 22 h after endotoxin challenge and treatment with vehicle (23 ± 22%; P < 0.01 vs. saline-challenged mice). In endotoxin-challenged BBS-2-treated mice, the LMBO-induced increase in LPVR was preserved (85 ± 16%; P < 0.01 vs. endotoxin-challenged vehicle-treated mice; Fig. 1C).


View this table:
[in this window]
[in a new window]
  		Table 2. Hemodynamic data during hypoxic pulmonary vasoconstriction studies at 22 h after 10 mg/kg endotoxin challenge

 

Effects of BBS-2 on NOS2 gene expression, NOS2 dimerization, and plasma NOx concentration. Lungs of saline-challenged mice had undetectable levels of NOS2 mRNA. In contrast, 7 h after endotoxin challenge, pulmonary NOS2 mRNA levels were markedly increased (Fig. 2A). Similarly, the plasma NOx level in saline-challenged mice was 41 ± 6 µM and increased >10-fold 7 h after endotoxin challenge (461 ± 240 µM; P < 0.001; Fig. 2C). Administration of BBS-2 did not affect the endotoxin-induced increase in pulmonary NOS2 gene expression. However, BBS-2 prevented cardiac and pulmonary NOS2 protein dimerization (Fig. 2B) and the increase of plasma NOx concentration at 7 h after endotoxin challenge (91 ± 33 µM; P < 0.001 vs. endotoxin-challenged mice). Despite only partially denaturing conditions, we were able to readily detect NOS2 homodimers in tissues from endotoxin-challenged mice but not in tissues from endotoxin-challenged mice treated with BBS-2 (Fig. 2B). Endotoxin did not increase plasma NOx levels in NOS2-deficient mice at 7 h (56 ± 17 µM; P < 0.001 vs. endotoxin-challenged wild-type mice).



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 2. A: pulmonary nitric oxide synthase 2 (NOS2) mRNA levels in wild-type mice 7 h after challenge with saline (endotoxin –) or endotoxin (endotoxin +) and 6 h after treatment with BBS-2 or vehicle. NOS2 mRNA was detected in RNA extracted from mouse lungs using RNA blot hybridization and a murine NOS2 cDNA probe. A photograph of ethidium bromide-stained 28S ribosomal RNA is shown to confirm equal loading of RNA on gels. Treatment with BBS-2 did not affect endotoxin-induced increases in pulmonary NOS2 mRNA levels. B: effects of BBS-2 (30 mg/kg) on cardiac and pulmonary NOS2 homodimerization in mice 7 h after endotoxin (100 mg/kg) challenge. An immunoblot for NOS2 protein in heart and lung homogenates after low-temperature gel electrophoresis is shown. SDS-stable NOS2 homodimers (Di-NOS2) are seen in heart and lung homogenates from endotoxemic animals that received vehicle but not in those from mice treated with BBS-2. NOS2 monomers are observed in all samples as expected under partially denaturing conditions. A representative blot from three independent experiments is shown. C: plasma nitrate and nitrite (NOx) levels in wild-type mice (NOS2 +/+) 7 h after challenge with saline (n = 4, endotoxin –) or endotoxin (n = 14, endotoxin +). One hour after endotoxin challenge, 9 mice received BBS-2 (BBS-2 +) while 5 mice received vehicle (BBS-2 –). Additional samples were obtained from 5 NOS2-deficient mice (NOS2 –/–) 7 h after endotoxin challenge. *P < 0.001 vs. saline-challenged wild-type mice, endotoxin-challenged BBS-2-treated wild-type mice, and endotoxin-challenged NOS2-deficient mice.

 

BBS-2 does not affect NOS3-dependent aortic relaxation. Contractions to 10–5 M PE were 2.2 ± 0.4 and 2.2 ± 0.5 g for aortic rings obtained from vehicle- and BBS-treated animals, respectively. ACh dose-response curves in aortic rings preconstricted with 10–5 M PE did not differ between BBS-2- and vehicle-treated rats (Fig. 3). These results confirm that BBS-2 did not affect agonist-stimulated NOS3-dependent aortic relaxation ex vivo.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. ACh-induced vasodilation dose-response curves in aortic rings from vehicle- and BBS-2-treated rats preconstricted with 10–5 M phenylephrine (PE). No difference in relaxation to ACh was observed at all concentrations tested (10–9 to 10–5 M) between BBS-2 (n = 8) and vehicle (n = 8)-treated rats.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURE
 REFERENCES
 
The present study demonstrates that systemic administration of a highly selective NOS2 dimerization inhibitor protected mice from endotoxin-induced systemic hypotension and impairment of HPV. BBS-2 also largely prevented endotoxin-induced myocardial dysfunction. These protective effects of BBS-2 were associated with inhibition of the endotoxin-induced increase in plasma NOx levels. Endotoxin-induced formation of NOS2 homodimers was blocked by BBS-2, whereas the increase in NOS2 gene expression was not altered. These observations are consistent with the pharmacological action of BBS-2, which prevents NOS2 dimerization (4).

The observation that BBS-2 treatment did not improve survival in endotoxemia confirms previous studies in NOS2-deficient mice (15, 30). Although the lack of a survival advantage for BBS-2 treatment in our model is not unexpected, it has been shown that NOS2 deficiency does promote survival in models of sepsis associated with sustained bacteremia (1, 11). It is possible that BBS-2 treatment might improve survival rates in different models of sepsis.

BBS-2 inhibits NOS2 enzyme activity in cytokine-stimulated A-172 cells (IC50 = 0.49 nM) and has a high affinity for NOS2 monomers (Kd = 0.1 nM; Ref. 4). In cell-based NOS dimerization assays, the IC50 values of BBS-2 for NOS3 and NOS1 are >1,500- and 600-fold greater than that for NOS2 (J. F. Parkinson and G. B. Phillips, unpublished observations). The present results demonstrate that the high selectivity of BBS-2 in vitro translates into selective effects in vivo by showing the lack of effects of BBS-2 on baseline pulmonary and systemic hemodynamics (see Fig. 1 and Tables 1 and 2). To further examine possible inhibitory effects of BBS-2 on NOS3 dimerization, we administered BBS-2 to rats for 10 days and assessed NOS3-dependent vascular reactivity ex vivo. Agonist-stimulated (PE and ACh) vascular reactivity was indistinguishable in aortic rings from BBS-2- and vehicle-treated rats (see Fig. 3). Because NOS3 turnover has been reported to occur with a half-life of 43 h (7), a 10-day treatment period with BBS-2 should have afforded sufficient time for the inhibition of NOS3 dimerization by BBS-2. Taken together, these findings suggest that BBS-2 has no measurable effects on NOS3 activity in vivo or ex vivo at doses that are highly inhibitory for NOS2 activity.

Excessive production of NO by sepsis-induced NOS2 has been implicated in the refractory systemic hypotension of sepsis. A variety of selective (32) and nonselective (27) NOS2 inhibitors and congenital NOS2 deficiency (17) have been shown to attenuate endotoxin-induced hypotension. Our observation that BBS-2 maintains the SAP in conscious mice 4 and 7 h after endotoxin challenge supports the hypothesis that augmented NOS2 activity contributes to the evolution of sepsis-induced systemic hypotension.

HPV is markedly impaired in sepsis and may be associated with marked systemic hypoxemia. Although the mechanisms responsible for sepsis-induced attenuation of HPV remain incompletely elucidated, NOS2-deficient mice were protected from endotoxin-induced impairment of HPV (29). The present study using BBS-2 supports the hypothesis that NOS2-derived NO plays an important role in septic pulmonary vascular dysfunction and represents a potential therapeutic target for preventing the impairment of HPV associated with sepsis.

NOS1 and NOS3 are important regulators of myocardial, systemic, and pulmonary vascular homeostasis (3, 26). Although activation of NOS3 has been implicated in the myocardial depression that is observed early after exposure to inflammatory cytokines (13, 14), treatment of sepsis with agents that can inhibit all three NOS isoforms has been observed to further aggravate systemic and pulmonary hemodynamic alterations. For example, administration of L-NMMA causes systemic vasoconstriction and hypertension (10), decreases cardiac output (8) and dP/dtmax (5), and promotes pulmonary hypertension (2). In the present study, L-NAME, which is another inhibitor of all three NOS isoforms, further impaired LV systolic function (as reflected by LVESP and dP/dtmax) and diastolic function (as reflected by LVEDP, dP/dtmin, and {tau}) in endotoxin-challenged mice (see Table 1). We have previously reported that a single bolus administration of L-NAME (100 mg/kg iv) increased SAP without altering cardiac output in healthy mice (26). It is of note that BBS-2 and L-NAME similarly prevented endotoxin-induced hypotension while exerting markedly different effects on LV function. These results confirm the deleterious effects of nonselective NOS inhibition in endotoxemia and suggest the possibility that NOS1 and/or NOS3 activity protects against the LV dysfunction of sepsis.

In summary, we have demonstrated that the highly selective NOS2 dimerization inhibitor BBS-2 protects mice from the cardiovascular dysfunction of sepsis: endotoxin-induced hypotension, myocardial dysfunction, and impairment of HPV. Early treatment by selective inhibition of NOS2 may prove to be a valuable treatment strategy to prevent septic shock.


   DISCLOSURE
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-42397, HL-57172, and HL-70896.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Marielle Scherrer-Crosbie for advice in murine echocardiography, Dr. Paul L. Huang for advice on tail-cuff pressure measurements, and Dr. Yuchiao Chang for statistical advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Ichinose, Dept. of Anesthesia and Critical Care, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114 (E-mail: ichinose{at}etherdome.mgh.harvard.edu).

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 

  1. Aranow JS, Zhuang J, Wang H, Larkin V, Smith M, and Fink MP. A selective inhibitor of inducible nitric oxide synthase prolongs survival in a rat model of bacterial peritonitis: comparison with two nonselective strategies. Shock 5: 116–121, 1996.[Web of Science][Medline]
  2. Avontuur JA, Biewenga M, Buijk SL, Kanhai KJ, and Bruining HA. Pulmonary hypertension and reduced cardiac output during inhibition of nitric oxide synthesis in human septic shock. Shock 9: 451–454, 1998.[Web of Science][Medline]
  3. Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi ZA, Hobai IA, Lemmon CA, Burnett AL, O'Rourke B, Rodriguez ER, Huang PL, Lima JA, Berkowitz DE, and Hare JM. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature 416: 337–339, 2002.[Medline]
  4. Blasko E, Glaser CB, Devlin JJ, Xia W, Feldman RI, Polokoff MA, Phillips GB, Whitlow M, Auld DS, McMillan K, Ghosh S, Stuehr DJ, and Parkinson JF. Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors. J Biol Chem 277: 295–302, 2002.[Abstract/Free Full Text]
  5. Cotton JM, Kearney MT, MacCarthy PA, Grocott-Mason RM, McClean DR, Heymes C, Richardson PJ, and Shah AM. Effects of nitric oxide synthase inhibition on basal function and the force-frequency relationship in the normal and failing human heart in vivo. Circulation 104: 2318–2323, 2001.[Abstract/Free Full Text]
  6. Eissa NT, Yuan JW, Haggerty CM, Choo EK, Palmer CD, and Moss J. Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization. Proc Natl Acad Sci USA 95: 7625–7630, 1998.[Abstract/Free Full Text]
  7. Habisch HJ, Gorren AC, Liang H, Venema RC, Parkinson JF, Schmidt K, and Mayer B. Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells. Mol Pharmacol 63: 682–689, 2003.[Abstract/Free Full Text]
  8. Harrison RW, Thakkar RN, Senzaki H, Ekelund UE, Cho E, Kass DA, and Hare JM. Relative contribution of preload and afterload to the reduction in cardiac output caused by nitric oxide synthase inhibition with L-NG-methylarginine hydrochloride 546C88. Crit Care Med 28: 1263–1268, 2000.[Web of Science][Medline]
  9. Hata JS and Dellinger RP. Nitric oxide inhibition in the treatment of septic shock. Crit Care Med 23: 1621–1624, 1995.[Web of Science][Medline]
  10. Haynes WG, Noon JP, Walker BR, and Webb DJ. Inhibition of nitric oxide synthesis increases blood pressure in healthy humans. J Hypertens 11: 1375–1380, 1993.[Web of Science][Medline]
  11. Hollenberg SM, Broussard M, Osman J, and Parrillo JE. Increased microvascular reactivity and improved mortality in septic mice lacking inducible nitric oxide synthase. Circ Res 86: 774–778, 2000.[Abstract/Free Full Text]
  12. Ichinose F, Zapol WM, Sapirstein A, Ullrich R, Tager AM, Coggins K, Jones R, and Bloch KD. Attenuation of hypoxic pulmonary vasoconstriction by endotoxemia requires 5-lipoxygenase in mice. Circ Res 88: 832–838, 2001.[Abstract/Free Full Text]
  13. Kinugawa K, Takahashi T, Kohmoto O, Yao A, Aoyagi T, Momomura S, Hirata Y, and Serizawa T. Nitric oxide-mediated effects of interleukin-6 on [Ca2+]i and cell contraction in cultured chick ventricular myocytes. Circ Res 75: 285–295, 1994.[Abstract/Free Full Text]
  14. Kumar A, Brar R, Wang P, Dee L, Skorupa G, Khadour F, Schulz R, and Parrillo JE. Role of nitric oxide and cGMP in human septic serum-induced depression of cardiac myocyte contractility. Am J Physiol Regul Integr Comp Physiol 276: R265–R276, 1999.[Abstract/Free Full Text]
  15. Laubach VE, Shesely EG, Smithies O, and Sherman PA. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad Sci USA 92: 10688–10692, 1995.[Abstract/Free Full Text]
  16. Liaudet L, Rosselet A, Schaller MD, Markert M, Perret C, and Feihl F. Nonselective versus selective inhibition of inducible nitric oxide synthase in experimental endotoxic shock. J Infect Dis 177: 127–132, 1998.[Web of Science][Medline]
  17. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, and Hutchinson N. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81: 641–650, 1995.[Web of Science][Medline]
  18. Marletta MA. Nitric oxide synthase: aspects concerning structure and catalysis. Cell 78: 927–930, 1994.[Web of Science][Medline]
  19. McMillan K, Adler M, Auld DS, Baldwin JJ, Blasko E, Browne LJ, Chelsky D, Davey D, Dolle RE, Eagen KA, Erickson S, Feldman RI, Glaser CB, Mallari C, Morrissey MM, Ohlmeyer MH, Pan G, Parkinson JF, Phillips GB, Polokoff MA, Sigal NH, Vergona R, Whitlow M, Young TA, and Devlin JJ. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry. Proc Natl Acad Sci USA 97: 1506–1511, 2000.[Abstract/Free Full Text]
  20. Metcalf K, Jungersten L, and Lisander B. Effective inhibition of nitric oxide production by aminoguanidine does not reverse hypotension in endotoxaemic rats. Acta Anaesthesiol Scand 46: 17–23, 2002.[Web of Science][Medline]
  21. Mitaka C, Hirata Y, Ichikawa K, Uchida T, Yokoyama K, Nagura T, Tsunoda Y, and Amaha K. Effects of nitric oxide synthase inhibitor on hemodynamic change and O2 delivery in septic dogs. Am J Physiol Heart Circ Physiol 268: H2017–H2023, 1995.[Abstract/Free Full Text]
  22. Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med 328: 1471–1477, 1993.[Free Full Text]
  23. Scherrer-Crosbie M, Steudel W, Hunziker PR, Liel-Cohen N, Ullrich R, Zapol WM, and Picard MH. Three-dimensional echocardiographic assessment of left ventricular wall motion abnormalities in mouse myocardial infarction. J Am Soc Echocardiogr 12: 834–840, 1999.[Web of Science][Medline]
  24. Schwartz D, Brasowski E, Raskin Y, Schwartz IF, Wolman Y, Blum M, Blantz RC, and Iaina A. The outcome of nonselective vs. selective nitric oxide synthase inhibition in lipopolysaccharide treated rats. J Nephrol 14: 110–114, 2001.[Web of Science][Medline]
  25. Scott JA and McCormack DG. Selective in vivo inhibition of inducible nitric oxide synthase in a rat model of sepsis. J Appl Physiol 86: 1739–1744, 1999.[Abstract/Free Full Text]
  26. Steudel W, Ichinose F, Huang PL, Hurford WE, Jones RC, Bevan JA, Fishman MC, and Zapol WM. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circ Res 81: 34–41, 1997.[Abstract/Free Full Text]
  27. Strunk V, Hahnenkamp K, Schneuing M, Fischer LG, and Rich GF. Selective iNOS inhibition prevents hypotension in septic rats while preserving endothelium-dependent vasodilation. Anesth Analg 92: 681–687, 2001.[Abstract/Free Full Text]
  28. Thiemermann C and Vane J. Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. Eur J Pharmacol 182: 591–595, 1990.[Web of Science][Medline]
  29. Ullrich R, Bloch KD, Ichinose F, Steudel W, and Zapol WM. Hypoxic pulmonary blood flow redistribution and arterial oxygenation in endotoxin-challenged NOS2-deficient mice. J Clin Invest 104: 1421–1429, 1999.[Web of Science][Medline]
  30. Ullrich R, Scherrer-Crosbie M, Bloch KD, Ichinose F, Nakajima H, Picard MH, Zapol WM, and Quezado ZM. Congenital deficiency of nitric oxide synthase 2 protects against endotoxin-induced myocardial dysfunction in mice. Circulation 102: 1440–1446, 2000.[Abstract/Free Full Text]
  31. Weiss JL, Frederiksen JW, and Weisfeldt ML. Hemodynamic determinants of the time-course of fall in canine left ventricular pressure. J Clin Invest 58: 751–760, 1976.[Web of Science][Medline]
  32. Wray GM, Millar CG, Hinds CJ, and Thiemermann C. Selective inhibition of the activity of inducible nitric oxide synthase prevents the circulatory failure, but not the organ injury/dysfunction, caused by endotoxin. Shock 9: 329–335, 1998.[Web of Science][Medline]
  33. Xie QW, Leung M, Fuortes M, Sassa S, and Nathan C. Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes. Proc Natl Acad Sci USA 93: 4891–4896, 1996.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. S. Buys, A. Cauwels, M. J. Raher, J. J. Passeri, I. Hobai, S. M. Cawley, K. M. Rauwerdink, H. Thibault, P. Y. Sips, R. Thoonen, et al.
sGC{alpha}1{beta}1 attenuates cardiac dysfunction and mortality in murine inflammatory shock models
Am J Physiol Heart Circ Physiol, August 1, 2009; 297(2): H654 - H663.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. G. Tankersley, H. C. Champion, E. Takimoto, K. Gabrielson, D. Bedja, V. Misra, H. El-Haddad, R. Rabold, and W. Mitzner
Exposure to inhaled particulate matter impairs cardiac function in senescent mice
Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2008; 295(1): R252 - R263.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. Liu, Y. Hotta, A. R. Graveline, O. V. Evgenov, E. S. Buys, K. D. Bloch, F. Ichinose, and W. M. Zapol
Congenital NOS2 deficiency prevents impairment of hypoxic pulmonary vasoconstriction in murine ventilator-induced lung injury
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1300 - L1305.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. S. Joshi, M. W. Julian, J. E. Huff, J. A. Bauer, Y. Xia, and E. D. Crouser
Calcineurin Regulates Myocardial Function during Acute Endotoxemia
Am. J. Respir. Crit. Care Med., May 1, 2006; 173(9): 999 - 1007.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
F. Spohr, A. J. M. Cornelissen, C. Busch, M. M. Gebhard, J. Motsch, E. O. Martin, and J. Weimann
Role of endogenous nitric oxide in endotoxin-induced alteration of hypoxic pulmonary vasoconstriction in mice
Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H823 - H831.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
R. Liu, O. V. Evgenov, and F. Ichinose
NOS3 deficiency augments hypoxic pulmonary vasoconstriction and enhances systemic oxygenation during one-lung ventilation in mice
J Appl Physiol, February 1, 2005; 98(2): 748 - 752.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
R. Ramasamy, Y. C. Hwang, Y. Liu, N. H. Son, N. Ma, J. Parkinson, R. Sciacca, A. Albala, N. Edwards, M. J. Szabolcs, et al.
Metabolic and Functional Protection by Selective Inhibition of Nitric Oxide Synthase 2 During Ischemia-Reperfusion in Isolated Perfused Hearts
Circulation, April 6, 2004; 109(13): 1668 - 1673.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/6/H2524    most recent
00530.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ichinose, F.
Right arrow Articles by Zapol, W. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ichinose, F.
Right arrow Articles by Zapol, W. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.