Tumor necrosis factor- impairs contraction but not relaxation in carotid arteries from iNOS-deficient mice

Departments of Internal Medicine and Pharmacology, and Cardiovascular Center, University of Iowa College of Medicine, Iowa City, Iowa 52242

	ABSTRACT

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We used mice deficient in expression of inducible NO synthase (iNOS /) to directly examine the role of iNOS in impaired vasoconstrictor responses following tumor necrosis factor- (TNF-). In iNOS +/+ mice, contraction of carotid arteries in response to prostaglandin F₂ (PGF₂) was impaired following TNF- (100 µg/kg ip)(n = 10, P < 0.01). In contrast to responses in wild-type mice, contraction to low concentrations of PGF₂ were normal, but maximum contraction to PGF₂ was impaired in arteries from iNOS / mice treated with TNF- [0.35 ± .0.02 g (n = 8) following vehicle and 0.25 ± 0.02 g (n = 7) following TNF- (P < 0.05)]. Aminoguanidine, a relatively selective inhibitor of iNOS, partially restored contraction to PGF₂ in vessels from iNOS +/+ mice but had no effect in iNOS / mice injected with TNF-, suggesting that a mechanism(s) other than iNOS contributes to impaired responses. In contrast to contractile responses, relaxation of the carotid artery in response to acetylcholine and nitroprusside was not altered following TNF- in iNOS +/+ or iNOS /mice. Responses of carotid arteries from iNOS / mice and effects of aminoguanidine suggest that both iNOS-dependent and iNOS-independent mechanisms contribute to impaired contractile responses following TNF-.

carotid artery; vasoconstriction; aminoguanidine; inducible nitric oxide synthase

	INTRODUCTION

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ENDOTOXIN (lipopolysaccharide, LPS) is an inflammatory stimulus that alters vascular function, including impairment of vasoconstrictor responses (6, 18, 19, 20, 33, 36) and possibly endothelium-dependent relaxation (27, 28). LPS stimulates production of several cytokines (9, 15, 24, 26, 31) and inducible enzymes, such as the inducible forms of NO synthase (iNOS) (18, 19, 33) and cyclooxygenase (COX-2) (14, 23, 29). Using iNOS-deficient mice, we previously reported that iNOS is expressed in carotid arteries and mediates impaired contractile responses following LPS (19). To further define mechanisms governing iNOS expression and its role in vascular function, the current study was designed to evaluate effects of tumor necrosis factor- (TNF-), a pro-inflammatory cytokine, which appears to play a central role in responses to LPS.

Although TNF- is thought to be a primary mediator of inflammatory responses (8, 40), the mechanism(s) that produces vascular effects is not clear. Pharmacological evidence suggests that proinflammatory and cardiovascular effects of TNF- may be both iNOS dependent and iNOS independent (2, 3, 16, 30, 38, 43). Therefore, to provide direct evidence for the role of iNOS in responses to TNF-, we used iNOS-deficient mice to examine the hypothesis that iNOS mediates altered vascular function following administration of TNF-. Vascular function was evaluated by examining both vasoconstrictor responses and vasorelaxation.

	METHODS

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Animal preparation. Mice with targeted disruption of exons 1-4 of the iNOS gene (iNOS /) were obtained initially from Dr. John Mudgett (Merck International) and then mated with C57BL/6 wild-type (+/+) mice to produce heterozygous (+/) iNOS-deficient mice. These heterozygote mice were then mated to provide iNOS-deficient mice (/) and wild-type littermates (+/+), which were used as controls. Some C57BL/J6 mice were used as additional wild-type controls. Vascular responses in vessels from wild-type C57BL/J6 mice and wild-type (iNOS +/+) offspring of the heterozygous iNOS-deficient mice were similar, and thus data from all wild-type mice were pooled for comparison to iNOS-deficient mice. Genotyping was accomplished by PCR of DNA from tail biopsies (19). In addition, RT-PCR of liver and carotid arteries confirmed the lack of expression of exons 1-4 of the iNOS gene in iNOS-deficient mice in this study.

Thirty iNOS +/+ (12 wild-type littermates and 18 C57BL/6) and 30 iNOS / mice were randomly assigned to receive injections of recombinant mouse TNF- (100 µg/kg, ip) or vehicle (saline). Carotid arteries were obtained 15 h after injection of TNF- or vehicle. Preliminary experiments were performed with TNF- (10 µg to 10 mg/kg) to establish a sublethal dose that produced impaired contractile function. Mice used in this study were 8-12 wk old. There were no differences in body weights between groups (means ± SE = 23 ± 1 g) at the time of the study. Data for male and female mice were analyzed separately. No significant differences were found between genders, and thus all data presented are the results of a pooled analysis.

Vascular function. Fifteen hours after treatment with vehicle or TNF-, mice were anesthetized with pentobarbital (150 mg/kg ip). The carotid arteries were removed and immediately placed in cold, oxygenated Krebs buffer with the following ionic composition (in mmol/l): 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, and 11 glucose. Loose connective tissue covering the adventitia was removed, and each carotid artery was cut into two rings (3-4 mm in length). Carotid rings were mounted between two stirrup-shaped support hooks and suspended in organ baths containing 25 ml of Krebs solution maintained at 37°C and bubbled with a mixture of 95% O₂ and 5% CO₂. One stirrup was connected to a stationary bracket, and the other was connected to a force transducer to measure isometric tension. Optimal resting tension was determined by evaluation of vasoconstriction in response to prostaglandin F₂ (PGF₂) at various tensions. Resting tension was increased stepwise to reach a final tension of 0.25 g, and the rings were allowed to equilibrate for 30 min. We have used this method previously to study mouse carotid arteries (13, 19).

We examined contraction of carotid rings in response to KCl (100 mM) and PGF₂ (3-100 µM). Vasorelaxation was evaluated by measuring responses to acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent) following submaximal precontraction using PGF₂. We have shown previously that relaxation of the carotid artery in response to acetylcholine is mediated by the endothelial isoform of NO synthase (eNOS) (13).

To provide pharmacological evidence that iNOS may contribute to impaired contraction following treatment with TNF-, some vessels were exposed to aminoguanidine (300 µM). This agent is reported to be a relatively specific inhibitor of iNOS at this concentration (17, 25). Vessels were incubated in organ chambers in the presence of aminoguanidine for 1 h prior to and during the administration of vasoconstrictor agents. The inhibitor was re-administered following each rinse with Krebs.

To determine whether COX enzymes contribute to impaired contraction following TNF-, some vessels were treated with indomethacin (1 or 10 µM). Vessels were incubated in organ baths in the presence of indomethacin for 1 h prior to administration of vasoconstrictor agents. We have shown previously that 10 µM indomethacin is efficacious in studies of vascular responses (9, 32).

RT-PCR. Total RNA was extracted from carotid arteries following the method of Chomczynski and Sacchi (7) as described previously (19). RNA (0.25-1 µg) was reverse-transcribed to produce cDNA using random hexamers as primers. For the PCR reaction, 2 µl of RT product were used. To ensure that mRNA could be detected, if present, all samples were run in duplicate with primers for iNOS and for a housekeeping gene, -actin. The forward primer for iNOS was 5'-GGCTTGCCCCTGGAAGTTTCTCTTCAAAGTC-3' (187-217, M84373 in GenBank). The reverse primer for iNOS was 5'-AAGGAGCCATAATACTGGTTGATG-3' (). The expected length of amplification product for iNOS was 441 bp. The 5' primer for -actin was 5'-GAGAAGATGACCCAGATCATG-3', and the 3' primer was 5'-GCCATCTCTTGCTCGAAGTC-3', as modified from Cheng et al. (5). The expected length of amplification product for -actin was 350 bp.

Drugs. Acetylcholine, aminoguanidine, sodium nitroprusside, KCl, and indomethacin were obtained from Sigma Chemical (St. Louis, MO). PGF₂ was obtained from Upjohn (Kalamazoo, MI). Mouse recombinant TNF- was obtained from Calbiochem (La Jolla, CA). The solution of TNF- (dissolved in PBS with 0.1% BSA at Calbiochem) was injected undiluted, and endotoxin level was certified to be 0.004 ng/µg protein. Indomethacin was dissolved in ethanol and diluted in normal saline. All other drugs were dissolved and diluted in normal saline. All concentrations are expressed as final concentration in the organ bath.

Statistical analysis. All data are expressed as means ± SE. Between group differences were determined by ANOVA followed by Tukey's post hoc test, where appropriate, to evaluate significant differences between means. P < 0.05 was considered to be statistically significant. Tension is expressed as grams of isometric force generated by contraction. Relaxation to acetylcholine and sodium nitroprusside are expressed as percent relaxation from submaximal precontraction to PGF₂.

	RESULTS

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Effect of TNF- on contraction of carotid arteries. PGF₂ produced concentration-dependent contractions of carotid artery segments (Fig. 1). Contraction was similar in carotid arteries from iNOS +/+ and iNOS / mice injected with vehicle (Fig. 1). Contraction of the carotid rings from iNOS +/+ mice injected with TNF- was impaired in response to PGF₂ (Fig. 1A).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Contraction of carotid artery from wild-type (iNOS+/+, n = 10) (A) and iNOS-deficient (iNOS /, n = 9) (B) mice in response to PGF2 following tumor necrosis factor- (TNF-) or vehicle. iNOS, inducible NO synthase. Data are means ± SE. *P < 0.05 vs. vehicle.

In contrast to results in iNOS +/+ mice, no significant depression of contractile response was observed in response to low concentrations of PGF₂ (3-30 µM) in vessels from iNOS / mice following TNF-. Maximum contraction to PGF₂ (100 µM), however, was impaired in arteries from iNOS / mice (Fig. 1B).

We also evaluated responses of the carotid artery to a high concentration of KCl (100 mM) and found that contraction was similar in iNOS +/+ and iNOS / mice following TNF- or vehicle. Maximum force of contraction to KCl was 0.12 ± 0.01 g in vessels from both iNOS +/+ and iNOS / mice injected with vehicle and was 0.10 ± 0.01 and 0.11 ± 0.01 g following TNF- in vessels from iNOS / and iNOS +/+ mice, respectively (n = 11-15, P > 0.05).

Effects of pharmacological inhibitors. Contraction of vessels from TNF--treated iNOS +/+ mice in response to PGF₂ was improved following incubation with aminoguanidine (300 µM) (Fig. 2A). In contrast to effects in iNOS +/+ mice, aminoguanidine had no effect on contractile responses of carotid arteries from iNOS / mice (Fig. 2B), and contraction to the highest concentration of PGF₂ (100 µM) remained impaired in the presence of the inhibitor of iNOS.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of the iNOS inhibitor, aminoguanidine (AG, 300 µM), on contractile responses of carotid arteries from iNOS +/+ (A) and iNOS / (B) mice to PGF2 following TNF- or vehicle (n = 8). Responses to PGF2 were significantly reduced in both iNOS +/+ and iNOS / mice following TNF- (*P < 0.05). Responses to PGF2 were improved significantly in only iNOS +/+ mice treated with TNF- (**P < 0.05).

Addition of indomethacin (1 or 10 µM) to organ baths prior to PGF₂ produced no significant improvement of contractile responses in carotid arteries from either iNOS +/+ or iNOS / mice following injection with TNF- (data not shown). The higher concentration of indomethacin tended to reduce contractile responses in vessels from both iNOS / and iNOS +/+ mice, but these effects were not statistically significant. Maximum contractions to PGF₂ in vessels from iNOS / mice were 0.25 ± 0.03 g following vehicle, 0.27 ± 0.04 g following TNF-, 0.23 ± 0.03 g following vehicle + indomethacin (10 µM), and 0.21 ± 0.05 g following TNF- + indomethacin. Thus indomethacin fails to improve contraction following TNF-.

Endothelium-dependent relaxation. Endothelium-dependent relaxation of blood vessels may be impaired during inflammation. Therefore, we evaluated effects of acetylcholine on carotid arteries. Following TNF- or vehicle, arteries from iNOS +/+ and iNOS / mice both relaxed by 85% or more (expressed as % precontraction to PGF₂) in response to the highest concentration of acetylcholine (1 µM) (Fig. 3). Relaxation of carotid arteries in response to nitroprusside was also similar ( 90%) in iNOS +/+ and iNOS / mice with or without TNF- (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Relaxation of the carotid artery from iNOS +/+ mice (A) and iNOS / mice (B) in response to acetylcholine following vehicle or TNF-. Values are means ± SE; n = 8-10 for each group.

RT-PCR. RT-PCR yielded no iNOS products from liver or carotid arteries in iNOS +/+ mice injected with vehicle (n = 3, data not shown). In contrast, iNOS cDNA was present following RT-PCR in carotid arteries from iNOS +/+ mice injected with TNF- (Fig. 4). No iNOS PCR products were detected in liver or carotid arteries from iNOS-deficient mice treated with vehicle (n = 3) or TNF- (n = 4) (data not shown). These findings indicate expression of iNOS mRNA in carotid arteries from iNOS +/+ mice treated with TNF-.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Agarose gel with RT-PCR products corresponding to in vivo expression of mRNA for iNOS and -actin from carotid arteries from 4 different iNOS +/+ mice. Lane 1: positive control showing the 441-bp amplification product derived from iNOS primers and a plasmid containing mouse iNOS cDNA. Lanes 2-5: results of RT-PCR using primers for mouse iNOS and RNA from 2 mice injected with TNF- (lanes 2 and 3) and 2 mice injected with vehicle (lanes 4 and 5). Lanes 7-10: positive bands for -actin (from RNA used in lanes 2-5, respectively), which verifies the integrity of RNA recovered from carotid arteries.

	DISCUSSION

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A major new finding in this study is that TNF- is a sufficient stimulus to induce expression of iNOS and to produce impairment of contractile function in carotid arteries. Results from the study of iNOS / mice provide direct evidence that impairment of contractile responses of the carotid artery following TNF- is mediated by both iNOS-dependent and iNOS-independent mechanisms. We also evaluated relaxation of carotid arteries in these studies. In contrast to effects on contractile function, TNF- produced no change in vasorelaxation.

Previously, we used iNOS / mice to demonstrate that iNOS mediates impaired contractile responses of carotid arteries following LPS (19). The new finding in the present study is that TNF-, one of many cytokines produced in response to LPS, alters contractile responses. Furthermore, our data suggest that iNOS is not the sole mediator of impaired contractile function following TNF-.

Effects of TNF- in wild-type mice. Reports regarding vascular effects of TNF- have varied. In some studies, TNF- has been shown to impair contractile responses of arteries (2, 33, 38). In contrast, other studies found augmented vasoconstrictor responses following treatment with TNF- (21, 35, 41). Some studies suggest that TNF- stimulates expression of iNOS, which contributes to altered vascular function (2, 3, 30, 38), whereas others conclude that iNOS is not the cause of altered function after TNF- (16, 43). One possible explanation for contrasting conclusions is that more than one mechanism may contribute to impaired vascular function following TNF- and that both iNOS-dependent and iNOS-independent mechanisms may mediate vascular dysfunction.

We found that contractile responses to PGF₂ were impaired in carotid arteries from wild-type mice after injection with TNF-. These data in normal mice are consistent with several previous studies in other species (2, 34, 38). We also found no impairment of contractile responses to KCl in arteries from any group following injections with TNF-. Thus, in contrast to responses to PGF₂, vasoconstriction in response to a high concentration of KCl (100 mM, which may produce maximum depolarization of vascular muscle) is not impaired. These data are consistent with others who reported no changes in vasoconstriction to KCl after TNF- (2, 41) or LPS (36) but differ from one study that reported impairment of responses to KCl following TNF- (37). The explanation for why LPS or TNF- may have selective effects on responses to receptor-mediated vasoconstrictors (present study, 36, 41) but not responses to KCl is not clear.

Previous reports suggest that TNF- plays a role in the expression of iNOS following treatment with LPS, but studies that have directly examined effects of TNF- on iNOS expression in blood vessels or other tissues are limited. iNOS is induced by TNF- in cultured macrophages and vascular smooth muscle cells (4, 11). There is a correlation between increased levels of circulating TNF- and increased plasma levels of nitrite and nitrate (breakdown products of NO) (22). One recent study in septic humans reported 223-fold increases in TNF- in arteries concurrent with increased activity of iNOS (1). It has been suggested, however, that although expression of TNF- and iNOS each occur following LPS, they are not causally related, because antibodies to TNF- failed to inhibit iNOS expression in liver (12). In contrast, antibodies to TNF- block induction of iNOS in lung homogenates following LPS (38) but fail to inhibit increases in plasma nitrates following LPS (43). Following treatment with LPS, TNF- receptors are required for iNOS induction in liver but not in spleen (30). Thus regulation of iNOS expression by TNF- may be tissue specific. We performed RT-PCR on RNA extracted from carotid arteries to determine whether iNOS is expressed in these vessels following treatment with TNF-. These data, together with functional data discussed below, indicate that TNF- stimulates expression of iNOS mRNA in carotid arteries.

Next, we tested the hypothesis that iNOS mediates impaired contractile responses following TNF- in wild-type mice. Previous studies have used pharmacological inhibitors of NOS to evaluate the involvement of NO in impaired contractile function following TNF-. Inhibitors of NOS [N^G-nitro-L-arginine methyl ester (L-NAME) and N^G-nitroarginine (L-NNA)] restore vasoconstrictor responses toward normal (2, 38). These inhibitors, however, do not differentiate between iNOS and other isoforms of NOS. Therefore, we chose aminoguanidine, an inhibitor that is relatively selective for iNOS at the concentration that we used (17, 25), to evaluate impaired contractile function following TNF-. Aminoguanidine improved contractile responses to PGF₂ of the carotid artery from iNOS +/+ mice that were treated with TNF-. Thus aminoguanidine provides pharmacological evidence that iNOS mediates impaired contractility following TNF-.

Effects of TNF- in iNOS / mice. To provide direct evidence that iNOS mediates vascular dysfunction, we injected TNF- into iNOS / mice. We found that contraction of the carotid artery in response to low concentrations of PGF₂ was not impaired following TNF- in iNOS / mice. An unexpected result was that maximum contractile responses to PGF₂ were diminished in iNOS / mice following TNF-. The effect of TNF- differs from that observed previously with LPS, in which we observed no impairment of contraction to PGF₂ in vessels from iNOS / mice following LPS (19).

Interestingly, in wild-type mice, aminoguanidine restored contraction to low concentrations of PGF₂ to normal, whereas maximal contraction remained impaired despite the presence of the iNOS inhibitor. At the same concentrations of PGF₂, we observed no impairment of responses in iNOS / mice. Thus findings with both a pharmacological inhibitor and genetically altered mice suggest that impaired contractile responses of the carotid artery following treatment with TNF- are mediated by iNOS-dependent and iNOS-independent mechanisms.

Other investigators have suggested that iNOS is not the sole mechanism that produces vascular alterations following LPS. Wu et al. (42) provide evidence that an unknown factor (not NO or carbon monoxide) activates guanylyl cyclase in rat aorta following endotoxin. In other studies, NOS inhibitors, L-NNA and L-NAME, did not prevent inhibition of contraction following LPS, which suggests an NO-independent mechanism(s) (34, 39). In addition, LPS produced increased oxidation and tyrosine nitration in aorta from iNOS / mice, which suggests that vascular effects of LPS are not limited to effects of iNOS (44). Thus our data, which suggest that not all effects of TNF- on vascular function are mediated by iNOS, are consistent with the concept that not all effects of LPS are mediated by iNOS.

We considered two possible explanations that could account for iNOS-independent impairment of contractile responses after TNF-. One mechanism is that activity of the COX pathway may contribute to impairment. No improvement of contraction, however, was observed following indomethacin. A second possibility was that aminoguanidine may have nonspecific effects, but no improvement of contraction was observed after aminoguanidine in iNOS / mice. These findings suggest that COX activity does not mediate impaired vasoconstrictor responses to high concentrations of PGF₂ and that nonspecific effects do not account for the response to aminoguanidine in iNOS / mice.

Effects of TNF- on vasorelaxation. Relaxation of the carotid artery in response to acetylcholine and nitroprusside was not altered following treatment with TNF-. These findings suggest that eNOS-mediated responses and the response of vascular muscle to NO were not altered following TNF-. Results of the present study do not exclude the possibility that higher doses of TNF- or different duration of incubation may alter endothelial function.

In summary, our results indicate that TNF-, a proinflammatory cytokine thought to play an important role in endotoxic shock (12, 15, 24), is a sufficient stimulus to induce expression of iNOS and produce impaired contraction in carotid arteries from wild-type mice. Results in iNOS / mice indicate that both iNOS-dependent and iNOS-independent mechanisms mediate impaired vasoconstrictor responses following TNF-. The failure of indomethacin to improve contractile responses of the carotid artery in iNOS / mice suggests that impaired vasoconstrictor responses are not due to activity of COX.

Vascular dysfunction is thought to play a major role in cardiovascular responses to sepsis. In arteries from humans with sepsis, levels of both TNF- and iNOS are markedly increased (1). Thus results of these experiments have implications for understanding of mechanisms that mediate vascular dysfunction in sepsis.

Perspectives

iNOS is thought to be a major mediator of cardiovascular dysfunction during sepsis. Levels of proinflammatory cytokines, including TNF-, are increased markedly in blood vessels during sepsis and are thought to be key mediators of cardiovascular dysfunction. Results of the current study using gene-targeted mice suggest that TNF- activates both iNOS-dependent and iNOS-independent mechanisms to produce impaired vascular function. Future studies may help better define mechanisms responsible for iNOS-independent effects of TNF-. Potential mechanisms include increased production of reactive oxygen species (including superoxide) by vascular oxidases such as NAD(P)H oxidase.

	ACKNOWLEDGEMENTS

We thank Dr. Sean P. Didion for critical reading of this manuscript.

	FOOTNOTES

These studies were supported by National Institutes of Health Grants NS-24621 and HL-38901. C. A. Gunnett is a National Research Service Award Fellow supported by National Heart, Lung, and Blood Institute Grant HL-09880. F. M. Faraci is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: F. M. Faraci, E315-GH Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242-1081 (E-mail: Frank-Faraci{at}uiowa.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.

Received 19 May 2000; accepted in final form 12 July 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

1.   Annane, D, Sanquer S, Sebille V, Faye A, Djuranovic D, Raphael JC, Gajdos P, and Bellissant E. Compartmentalised inducible nitric-oxide synthase activity in septic shock. Lancet 355: 1143-1148, 2000[ISI][Medline].

2.   Baudry, N, and Vicaut E. Role of nitric oxide in effects of tumor necrosis factor- on microcirculation in rat. J Appl Physiol 75: 2392-2399, 1993[Abstract/Free Full Text].

3.   Brian, JE, and Faraci FM. Tumor necrosis factor--induced dilatation of cerebral arterioles. Stroke 29: 509-515, 1998[Abstract/Free Full Text].

4.   Busse, R, and Mulsch A. Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett 275: 87-90, 1990[ISI][Medline].

5.   Cheng, HF, Becker BN, Burns KD, and Harris RC. Angiotensin II upregulates type-I angiotensin II receptors in renal proximal tubule. J Clin Invest 95: 2012-2019, 1995.

6.   Cheng, X, Wang YX, and Pang CCY Reversal by L- and D-enantiomers of N^G-nitroarginine of endotoxin-induced hypotension and vascular hyporesponsiveness. J Cardiovasc Pharmacol 28: 75-81, 1996[ISI][Medline].

7.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

8.   DeForge, LE, Nguyen DT, Kunkel SL, and Remick DG. Regulation of the pathophysiology of tumor factor. J Lab Clin Med 116: 429-438, 1990[ISI][Medline].

9.  Didion SP, Sigmund CD, and Faraci FM. Impaired endothelial function in transgenic mice expressing both human renin and human angiotensinogen. Stroke 31: 760-764, 765, 2000.

10.   Dinarello, CA. Interleukin-1 and its biologically related cytokines. Adv Immunol 44: 153, 1989[ISI][Medline].

11.   Drapier, JC, Wietzerbin J, and Hibbs JB, Jr. Interferon- and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages. Eur J Immunol 18: 1587-1592, 1988[ISI][Medline].

12.   Evans, T, Carpenter A, Silva A, and Cohen J. Differential effects of monoclonal antibodies to tumor necrosis factor alpha and gamma interferon on induction of hepatic nitric oxide synthase in experimental gram-negative sepsis. Infect Immun 60: 4133-4139, 1992[Abstract/Free Full Text].

13.   Faraci, FM, Sigmund CD, Shesely EG, Maeda N, and Heistad DD. Responses of carotid artery in mice deficient in expression of the gene for endothelial NO synthase. Am J Physiol Heart Circ Physiol 274: H564-H570, 1998[Abstract/Free Full Text].

14.   Feng, L, Xia Y, Garcia GE, Hwang D, and Wilson CB. Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide. J Clin Invest 95: 1669-1675, 1995.

15.   Feuerstein, G, Hallenbeck JM, Vanatta B, Rabinovici R, Perera PY, and Vogel SN. Effect of gram-negative endotoxin on levels of serum corticosterone, TNF, circulating blood cells and the survival of rats. Circ Shock 30: 265-278, 1990[ISI][Medline].

16.   Greenberg, SS, Xie J, Joseph KO, Kolls J, and Summer W. In vivo administration of endotoxin and tumor necrosis factor- produce different effects on constitutive and inducible nitric oxide synthase activity in rat neutrophils and aorta ex vivo. Proc Soc Exp Biol Med 208: 199-208, 1995[Abstract].

17.   Griffiths, MJD, Messent M, MacAllister RJ, and Evans TW. Aminoguanidine selectively inhibits inducible nitric oxide synthase. Br J Pharmacol 110: 963-968, 1993[ISI][Medline].

18.   Griffiths, MJD, Liu S, Curzen NP, Messent M, and Evans TW. In vitro treatment with endotoxin induces nitric oxide synthase in rat main pulmonary artery. Am J Physiol Lung Cell Mol Physiol 268: L509-L518, 1995[Abstract/Free Full Text].

19.   Gunnett, CA, Chu Y, Heistad DD, Loihl A, and Faraci FM. Vascular effects of LPS in mice deficient in expression of the gene for inducible nitric oxide synthase. Am J Physiol Heart Circ Physiol 275: H416-H421, 1998[Abstract/Free Full Text].

20.   Hom, GJ, Grant SK, Wolfe G, Bach TJ, MacIntyre DE, and Hutchinson NI. Lipopolysaccharide-induced hypotension and vascular hyporeactivity in the rat: tissue analysis of nitric oxide synthase mRNA and protein expression in the presence and absence of dexamethasone, N^G-monomethyl-L-arginine or indomethacin. J Pharmacol Exp Ther 272: 452-459, 1995[Abstract/Free Full Text].

21.   Johnson, A, and Ferro TJ. TNF- augments pulmonary vasoconstriction via the inhibition of nitrovasodilator activity. J Appl Physiol 73: 2483-2492, 1992[Abstract/Free Full Text].

22.   Leu, RW, Leu NR, Shannon BJ, and Fast DJ. IFN-gamma differentially modulates the susceptibility of L1210 and P815 tumor targets for macrophage-mediated cytotoxicity. Role of macrophage-target interaction coupled to nitric oxide generation, but independent of tumor necrosis factor production. J Immunol 147: 1816-1822, 1991[Abstract].

23.   Martin-Sanz, P, Callejas NA, Casado M, Diaz-Guerra MJ, and Bosca L. Expression of cyclooxygenase-2 in foetal rat hepatocytes stimulated with lipopolysaccharide and pro-inflammatory cytokines. Br J Pharmacol 125: 1313-1319, 1998[ISI][Medline].

24.   Michie, HR, Manogue KR, Spriggs DR, Revhaug A, O'Dwyer S, Dinarello CA, Cerami A, Wolff SM, and Wilmore DW. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 318: 1481-1486, 1988[Abstract].

25.   Misko, TP, Moore WM, Kasten TP, Nickols GA, Corbett JA, Tilton RG, McDaniel ML, Williamson JR, and Currie MG. Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur J Pharmacol 233: 119-125, 1993[ISI][Medline].

26.   Nathan, CF. Secretory products of macrophages. J Clin Invest 79: 319-326, 1987.

27.   Parker, JL, Myers PR, Zhong Q, Kim K, and Adams HR. Inhibition of endothelium-dependent vasodilation by Escherichia coli endotoxemia. Shock 2: 451-458, 1994[ISI][Medline].

28.   Peters, TS, and Lewis SJ. Lipopolysaccharide inhibits acetylcholine- and nitric oxide-mediated vasodilation in vivo. J Pharmacol Exp Ther 279: 918-925, 1996[Abstract/Free Full Text].

29.   Quan, N, Whiteside M, and Herkenham M. Cyclooxygenase 2 mRNA expression in rat brain after peripheral injection of lipopolysaccharide. Brain Res 802: 189-197, 1998[ISI][Medline].

30.   Salkowski, CA, Detore G, McNally R, van Rooijen N, and Vogel SN. Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo. J Immunol 158: 905-912, 1997[Abstract].

31.   Shalaby, MR, Waage A, Aarden L, and Espevik T. Endotoxin, tumor necrosis factor-/cachectin and interleukin-1 induce interleukin-6 production in vivo. Clin Immunol Immunopathol 53: 488, 1989[ISI][Medline].

32.   Sobey, CG, Heistad DD, and Faraci FM. Potassium channels mediate dilatation of cerebral arterioles in response to arachidonate. Am J Physiol Heart Circ Physiol 275: H1606-H1612, 1998[Abstract/Free Full Text].

33.   Sobey, CG, Brooks RM, II, and Heistad DD. Evidence that expression of inducible nitric oxide synthase in response to endotoxin is augmented in atherosclerotic rabbits. Circ Res 77: 536-543, 1995[Abstract/Free Full Text].

34.   Stevens, T, Morris K, McMurtry IF, Zamora M, and Tucker A. Pulmonary and systemic vascular responsiveness to TNF- in conscious rats. J Appl Physiol 74: 1905-1910, 1993[Abstract/Free Full Text].

35.   Stevens, T, Janssen PL, and Tucker A. Acute and long-term TNF- administration increases pulmonary vascular reactivity in isolated rat lungs. J Appl Physiol 73: 708-712, 1992[Abstract/Free Full Text].

36.   Taguci, H, Heistad DD, Chu Y, Rios CD, Ooboshi H, and Faraci FM. Vascular expression of inducible nitric oxide synthase is associated with activation of Ca²⁺-dependent K⁺ channels. J Pharmacol Exp Ther 279: 1514-1519, 1996[Abstract/Free Full Text].

37.   Takahashi, K, Ando K, Ono A, Shimosawa T, Ogata E, and Fujita T. Tumor necrosis factor- induces vascular hyporesponsiveness in Sprague-Dawley rats. Life Sci 50: 1437-1444, 1992[ISI][Medline].

38.   Thiemermann, C, Wu CC, Szabo C, Perretti M, and Vane JR. Role of tumor necrosis factor in the induction of nitric oxide synthase in a rat model of endotoxin shock. Br J Pharmacol 110: 177-182, 1993[ISI][Medline].

39.   Thorin-Trescases, N, Hamilton CA, Reid JL, McPherson KL, Jardine E, Berg G, Bohr D, and Dominiczak AF. Inducible L-arginine/nitric oxide pathway in human internal mammary artery and saphenous vein. Am J Physiol Heart Circ Physiol 268: H1122-H1132, 1995[Abstract/Free Full Text].

40.   Tracey, KJ, Lowry SF, and Cerami A. Cachectin/TNF mediates the pathophysiological effects of bacterial endotoxin/lipopolysaccharide (LPS). In: Bacterial Endotoxins: Pathophysiological Effects, Clinical Significance and Pharmacological Control. New York: Liss, 1988, p. 77-88.

41.   Vicaut, E, Rasetti C, and Baudry N. Effects of tumor necrosis factor and interleukin-1 on the constriction induced by angiotensin II in rat aorta. J Appl Physiol 80: 1891-1897, 1996[Abstract/Free Full Text].

42.   Wu, CC, Szabo C, Chen SJ, Thiemermann C, and Vane JR. Activation of soluble guanylate cyclase by a factor other than nitric oxide or carbon monoxide contributes to the vascular hyporeactivity to vasoconstrictor agents in the aorta of rats treated with endotoxin. Biochem Biophys Res Commun 201: 436-442, 1994[ISI][Medline].

43.   Xie, J, Joseph KO, Bagby GJ, Giles TD, and Greenberg S. Dissociation of TNF- from endotoxin-induced nitric oxide and acute-phase hypotension. Am J Physiol Heart Circ Physiol 273: H164-H174, 1997[Abstract/Free Full Text].

44.   Zingarelli, B, Virag L, Szabo A, Cuzzocrea S, Salzman AL, and Szabo C. Oxidation, tyrosine nitration and cytostasis induction in the absence of inducible nitric oxide synthase. Int J Molec Med 1: 787-795, 1998.