Vascular effects of LPS in mice deficient in expression of the gene for inducible nitric oxide synthase

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

	ABSTRACT

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The inducible isoform of nitric oxide synthase (iNOS) is expressed after systemic administration of lipopolysaccharide (LPS). The importance of expression of iNOS in blood vessels is poorly defined. Because nitric oxide from iNOS may alter vasomotor function, we examined effects of LPS on vasomotor function in carotid arteries from iNOS-deficient mice. We studied contraction of the carotid artery from wild-type and iNOS-deficient mice in vitro 12 h after injection of LPS (20 mg/kg ip). Contractile responses to PGF₂ (3-30 µM) and thromboxane A₂ analog (U-46619; 3-100 nM) were evaluated using vascular rings from mice treated with vehicle or LPS. Maximum force of contraction generated by rings in response to PGF₂ was 0.39 ± 0.02 and 0.25 ± 0.01 (SE) g (n = 14) in vehicle and LPS-treated wild-type mice, respectively (P < 0.001 vs. vehicle). Thus LPS reduced constrictor responses in wild-type mice. Thiocitrulline and aminoguanidine (inhibitors of iNOS) improved contractile responses from LPS-treated wild-type vessels. Indomethacin also improved constrictor responses in arteries from wild-type mice injected with LPS. In contrast, contraction of the carotid arteries in response to PGF₂ and U-46619 was not impaired in LPS-treated iNOS-deficient mice, and contraction was not altered by inhibitors of iNOS. Expression of iNOS mRNA was confirmed using RT-PCR in carotid arteries from wild-type mice after injection of LPS but not vehicle. PCR products for iNOS were not observed in iNOS-deficient mice. These findings provide the first direct evidence that iNOS mediates impairment of vascular contraction after treatment with LPS.

carotid artery; acetylcholine; vasoconstriction; reverse transcriptase-polymerase chain reaction; lipopolysaccharide

	INTRODUCTION

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NITRIC OXIDE (NO) plays a major role in modulation of vasomotor function. There are three isoforms of the enzyme NO synthase (NOS), which generates NO from L-arginine (19, 23). Endothelial NOS (eNOS) and neuronal NOS are expressed constitutively and can influence vascular tone. Under normal conditions, the inducible form of NOS (iNOS) is not expressed in blood vessels (13, 14, 24, 26, 27). Exposure to lipopolysaccharide (LPS) and some proinflammatory cytokines, however, can stimulate expression of iNOS in blood vessels (13, 16, 18, 26, 27).

Although iNOS mRNA and protein have been detected in blood vessels after treatment with LPS, the functional significance of this expression is not completely clear. Indirect approaches, using almost exclusively pharmacological inhibitors, suggest that induction of iNOS in blood vessels results in impaired responses to vasoconstrictor stimuli (1, 9, 13, 16, 27), presumably from generation of large amounts of NO. A major limitation in this approach, however, is that inhibitors of iNOS are not selective for a single isoform of NOS. For example, aminoguanidine, which is used commonly as a relatively selective inhibitor of iNOS (4, 13, 14, 27), can also inhibit eNOS (7, 31). In addition, aminoguanidine may inhibit other enzyme systems including cyclooxygenase (COX) (32). Furthermore, some evidence suggests that vascular effects attributed to iNOS are actually mediated by eNOS (2).

The use of iNOS-deficient mice in which the gene that encodes iNOS has been disrupted provides a novel approach for studying the role of iNOS in blood vessels. In this study, we used iNOS-deficient mice to test the hypothesis that expression of iNOS mediates impairment of vasoconstrictor responses in carotid arteries after LPS.

	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) (20) and mated with C57BL/6 wild-type (+/+) mice to produce heterozygous (+/) iNOS-deficient mice. These heterozygotes were then mated to provide iNOS-deficient mice (/) and wild-type littermates (+/+) that were used as controls. Genotyping was accomplished by PCR of DNA from tail biopsies. 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.

Mice used in this study were 8-12 wk old. There were no differences in body weights between groups [mean = 21 ± 1 (SE) 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. We studied wild-type littermates (+/+) of the iNOS-deficient mice and C57Bl/J6 mice as wild-type controls. Contraction and relaxation of carotid arteries were similar in vessels from wild-type C57Bl/J6 mice and wild-type offspring of the heterozygous iNOS-deficient mice. Thus data of all wild-type mice were pooled for comparisons to iNOS-deficient mice.

Mice were randomly assigned to receive either vehicle or LPS. Carotid arteries and liver were obtained 12 h after injection of LPS (20 mg/kg ip) or vehicle (saline). Mice had free access to food and water throughout the study. Preliminary experiments demonstrated that mice became severely hypothermic after injection of LPS (mean 31.6°C without heat lamp vs. 35.2°C with heat lamp). Therefore, mice were placed under a heat lamp after injection of LPS.

Studies of vessels in vitro. Twelve hours after treatment with vehicle or LPS, mice were anesthetized with pentobarbital sodium (150 mg/kg ip). The carotid arteries were removed and immediately placed in oxygenated Krebs buffer with the following ionic composition (in mM): 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). Each carotid ring was 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% O₂-5% 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 preliminary evaluation of vasoconstriction in response to 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. Organ baths were drained and refilled with fresh Krebs every 20-30 min throughout the study. We have used this method to study mouse arteries previously (3, 8).

We examined contraction of carotid rings in response to PGF₂ (3-30 µM) and the thromboxane A₂ analog U-46619 (3-100 nM). A 45-min recovery period was allowed between application of vasoconstrictors.

To provide pharmacological evidence that iNOS may contribute to impaired contraction after treatment with LPS (similar to the approach used previously), some vessels were exposed to aminoguanidine (100 or 300 µM) or thiocitrulline (30 µM). These agents are reported to be relatively specific inhibitors of iNOS (10, 14, 21). Vessels were incubated in organ chambers in the presence of inhibitors for 1 h before the administration of vasoconstrictor agents. The inhibitors were readministered after each rinse with Krebs solution.

To determine if COX enzymes contribute to impaired contraction after LPS, some vessels were exposed to indomethacin (1 µM). As with inhibitors of iNOS, vessels were incubated in organ baths in the presence of indomethacin for 1 h before administration of vasoconstrictors.

To determine whether disruption of the iNOS gene alters vascular function, vasorelaxation was evaluated in vehicle-treated groups by measuring relaxation in response to acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent) after submaximal precontraction using PGF₂ or U-46619.

RT-PCR. Total RNA was extracted from liver samples and carotid arteries following the method of Chomczynski and Sacchi (6). Both carotid arteries were snap-frozen in liquid nitrogen and ground with a mortar and pestle. Carotid and liver samples were then homogenized in TRI Reagent (Molecular Research Center), and RNA was extracted. Glycogen (0.05 µg/µl) was added to carotid extractions to facilitate RNA precipitation.

RNA (2-4 µg) was reverse-transcribed to produce cDNA using random hexamers as primers. Two microliters of RT product were used for the PCR reaction. To ensure that mRNA could be detected, if present, all samples were run in duplicate with primers for iNOS and primers for a housekeeping gene, -actin. A plasmid containing cDNA for mouse iNOS was used as a positive control for PCR. Perfect Match (Stratagene) was added to tubes containing iNOS primers to improve primer annealing, but not to tubes with -actin primers. The PCR product was analyzed by electrophoresis utilizing a 2% agarose gel containing ethidium bromide. The gel was photographed using a ultraviolet transilluminator (Fisher) and the NIH Image 1.52 program. We have used this method previously (26).

The forward primer for iNOS (located in exon 2) was 5'-GGCTTGCCCCTGGAAGTTTCTCTTCAAA-GTC-3' (no. 187-217, M84373 in Genbank). The reverse primer for iNOS (beyond the exon 4/5 boundary) was 5'-AAGGAGCCATAATACTGGTTGATG-3' (no. 603-628). The expected length of amplification product was 441 bp. The 5'-primer for -actin was 5'-GAGAAGATGACC-CAGATCATG-3', and the 3'-primer was 5'-GCCATCTCTTGCTCGAAGTC-3', as modified from Cheng et al. (5). The expected length of amplification product was 350 bp.

Drugs. Acetylcholine, sodium nitroprusside, indomethacin, and LPS were obtained from Sigma Chemical (St. Louis, MO). PGF₂ was obtained from Upjohn (Kalamazoo, MI). U-46619 was obtained from Cayman Chemical (Ann Arbor, MI). Aminoguanidine and thiocitrulline were obtained from Calbiochem (La Jolla, CA). Stock solutions of U-46619 were prepared in ethanol, and subsequent dilutions were made in normal saline. All other drugs were dissolved and diluted in normal saline. All of the concentrations were expressed as a final concentration of each drug in the organ bath.

Statistical analysis. All data are expressed as means ± SE. Within-group differences were determined by one-way 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 was expressed as grams of isometric force generated by contraction. Relaxation responses to acetylcholine and sodium nitroprusside were expressed as percent relaxation from maximal precontraction to PGF₂ and U-46619, respectively.

	RESULTS

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Effect of LPS on responses of carotid arteries from wild-type mice. PGF₂ (Fig. 1) and U-46619 (data not shown) produced concentration-dependent contractions of carotid artery segments. Contractile responses were similar in carotid arteries from wild-type and iNOS-deficient mice that were injected with vehicle (Figs. 1 and 2). Contraction of the carotid rings from wild-type mice injected with LPS was inhibited compared with rings obtained from mice injected with vehicle (Figs. 1 and 2). Maximum force of contraction elicited by PGF₂ (30 µM) was 0.39 ± 0.02 vs. 0.25 ± 0.01 (SE) g, respectively, in vehicle and LPS-treated vessels (P < 0.001) (Fig. 2). Similarly, maximum force of contraction in response to U-46619 (100 nM) was 0.45 ± 0.02 and 0.33 ± 0.02 g, respectively, in vehicle- and LPS-treated groups (data not shown) (P < 0.05). In contrast, injection of iNOS-deficient mice with LPS caused no impairment of constrictor responses in carotid arteries (Figs. 1 and 2). The lack of impaired constrictor responses in vessels from the iNOS-deficient mice after LPS represents the major new finding of this study.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Contractile responses of rings of carotid artery to PGF2. A: responses of carotid rings from wild-type mice. B: responses of vessels from inducible nitric oxide synthase (iNOS)-deficient mice. Responses of rings from mice injected with vehicle are shown on left, and responses of rings from lipopolysaccharide (LPS)-injected mice are shown on right. In wild-type mice, constrictor responses are impaired after exposure to LPS.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Contraction of rings of carotid artery from wild-type mice (A) (n = 12) and iNOS-deficient mice (B) (n = 12) in response to PGF2. Data are expressed as means ± SE. * P < 0.05 vs. vehicle.

Effect of iNOS inhibitors and indomethacin. Contraction of vessels from LPS-treated wild-type mice in response to PGF₂ was improved after incubation with aminoguanidine (300 µM) (Fig. 3A). A higher concentration of aminoguanidine (1.0 mM) had similar but not additional effects (data not shown). Similar results were obtained with another inhibitor of iNOS, thiocitrulline (Fig. 3B). In wild-type mice treated with vehicle, contraction of the carotid artery in response to PGF₂ was not affected by aminoguanidine (n = 4). In these mice, 10, 30, and 100 µM PGF₂ contracted the carotid artery by 0.14 ± 0.04, 0.28 ± 0.03, and 0.35 ± 0.03 g, respectively, in the absence of aminoguanidine and 0.13 ± 0.03, 0.24 ± 0.02, and 0.32 ± 0.01 g, respectively, in the presence of aminoguanidine.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Effects of iNOS inhibitors aminoguanidine (300 µM) (A) and thiocitrulline (100 µM) (B) on contractile responses to PGF2. All vessles are from mice injected with LPS. Solid circles indicate responses of vessels without iNOS inhibitors, and open circles indicate responses of vessels with iNOS inhibitors (n = 4). * P < 0.05.

Indomethacin also improved constrictor responses in carotid arteries from LPS-treated wild-type mice. Addition of indomethacin (1 µM) to organ baths before PGF₂ (30 and 100 µM) caused constrictor responses to increase from 0.19 ± 0.02 to 0.24 ± 0.02 g (n = 4, P < 0.07) and 0.21 ± 0.02 to 0.28 ± 0.02 g (n = 4, P < 0.05), respectively. In response to lower doses of PGF₂ (3 and 10 µM), indomethacin tended to improve vasoconstrictor responses, but the differences were not statistically significant.

Vasorelaxation in the absence of LPS. Relaxation to acetylcholine was similar in vessels from vehicle-treated wild-type and iNOS-deficient mice (Fig. 4A). Sodium nitroprusside, an endothelium-independent vasodilator, also produced similar relaxation of carotid arteries from wild-type and iNOS-deficient mice (Fig. 4B). Thus disruption of the iNOS gene per se does not alter endothelium-dependent or -independent relaxation of the carotid artery.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Relaxation of carotid artery rings in response to acetylcholine (A) and sodium nitroprusside (B). Responses of vessels from wild-type mice are represented by squares; responses of vessels from iNOS-deficient mice are represented by circles. Values are means ± SE (n = 6).

RT-PCR. cDNA products were present in liver and carotid arteries from wild-type mice injected with LPS (Fig. 5). In contrast, no PCR products were detected in liver or carotid arteries from iNOS-deficient mice treated with LPS. These findings confirm the expression of iNOS mRNA in carotid arteries and liver from wild-type mice treated with LPS and the lack of iNOS mRNA expression in iNOS-deficient mice after treatment with LPS.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Agarose gel with RT-PCR products corresponding to in vivo expression of mRNA for iNOS and -actin. Lane 1 contained a negative reagent control. Primers for mouse iNOS produce positive amplification products of 441 bp in liver (lane 2) and carotid arteries (lane 4) from wild-type mice exposed to LPS and in positive control (lane 6). In contrast, no iNOS positive bands were observed in liver (lane 3) or carotid arteries (lane 5) from iNOS-deficient mice exposed to LPS. All 4 samples produced positive bands for housekeeping gene, -actin (lanes 8-11).

	DISCUSSION

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The major new finding in the present study is that administration of LPS in vivo does not cause impaired contraction in carotid arteries from iNOS-deficient mice. In contrast, contractile responses of carotid arteries from wild-type mice are impaired under the same conditions. Thus these findings provide the first direct evidence that iNOS is a critical mediator of vascular hypocontractilty that occurs after exposure to bacterial LPS.

Effects of LPS in wild-type mice. We (4, 24, 27) and others (9, 13, 14, 16, 25, 28, 29) have used pharmacological approaches to examine the effects of LPS on contractile responses of blood vessels, including vessels from humans. Administration of LPS impaired vasoconstrictor responses to several agonists including norepinephrine (9, 16, 24), phenylephrine (13, 14, 27, 28), and U-46619 (29). Similar to these previous studies, we found that carotid arteries from wild-type mice injected with LPS had impaired vasoconstrictor responses to PGF₂ and U-46619.

The gene that encodes iNOS is not thought to be expressed in normal vessels. However, pharmacological evidence suggests that expression of iNOS occurs after treatment with LPS and that expression of iNOS contributes to impaired contractile responses in vessels. Previous studies have demonstrated iNOS mRNA expression in rat aorta after LPS treatment (16, 27, 30). In the present study, iNOS PCR products were not found in carotid arteries from wild-type mice that were injected with vehicle. After LPS treatment, iNOS PCR products were found in carotid arteries and liver samples from wild-type mice. These findings are consistent with a possible role for iNOS in the functional impairment of vessels after LPS. Neither liver nor carotid arteries from iNOS-deficient mice contained iNOS PCR products after injection with vehicle or LPS.

Several investigators have reported that pharmacological inhibitors of NOS improve constrictor responses in LPS-treated vessels (9, 11, 12, 17, 27). These data suggest that the L-arginine/NO pathway contributes to impaired constrictor responses after LPS exposure. Several studies, including our own, have used inhibitors of NOS that have some selectivity for iNOS (4, 10, 21, 22, 27). In the present study, inhibitors of iNOS, aminoguanidine and thiocitrulline, improved constrictor responses in carotid arteries from wild-type mice that had been treated with LPS. These data are consistent with previous studies that found that aminoguanidine improved constrictor responses in arteries after LPS-induced impairment (14, 23, 27). Although these inhibitors are useful, the inhibitors are not completely selective. For example, in addition to inhibiting iNOS, aminoguanidine can impair vasorelaxation to acetylcholine, which is mediated by eNOS (5, 26), and can also inhibit COX enzymes (27). Non-arginine-based NOS inhibitors are potent inhibitors of COX activity in J774 macrophages (28). Thiocitrulline is also semiselective for iNOS and can inhibit eNOS as well (10). Thus, to obtain direct evidence that vascular hypocontractility after LPS is mediated by iNOS, we utilized the iNOS-deficient mice.

Two isoforms of COX (COX-1 and COX-2) may be expressed in blood vessels. COX-1 is constitutively expressed in many cell types including endothelium (32). The inducible isoform (COX-2) may be expressed in addition to iNOS in response to LPS or during inflammation (22, 32). We tested whether activity of COX enzymes contributed to impaired contractile function by testing effects of indomethacin on carotid arteries. Similar to the effects of iNOS inhibitors, the COX inhibitor indomethacin caused partial restoration of vasoconstrictor function. Thus our data suggest that activity of COX enzymes contributes to the impairment of vasoconstrictor responses observed after LPS in wild-type mice. From these data, we cannot determine whether iNOS and COX enzymes are interactive or act independently.

iNOS-deficient mice. We considered the possibility that the disruption of the iNOS gene might alter vascular function in the absence of LPS treatment. Responses of carotid arteries from iNOS-deficient mice that were injected with vehicle were normal to vasoconstrictor (PGF₂ and U-46619) and vasodilator (acetylcholine and sodium nitroprusside) agents. Thus disruption of the gene for iNOS did not alter vasoconstrictor responses or endothelium-dependent or -independent vasorelaxation. In contrast, targeted deletion of the eNOS gene decreases vasorelaxation to acetylcholine and increases vasorelaxation to nitroprusside (8).

We evaluated effects of injection of LPS on vasoconstrictor responses in the iNOS-deficient mice. In contrast to impaired constrictor responses in vessels from wild-type mice, LPS treatment caused no impairment of constrictor responses in carotid arteries from the iNOS-deficient mice. These data indicate that constrictor responses are not impaired when iNOS is not expressed. iNOS inhibitors, aminoguanidine and thiocitrulline, had no significant effect on contractile responses of vessels from iNOS-deficient mice injected with LPS or vehicle. These results imply that the generation of NO by iNOS contributes to impaired vasoconstrictor responses.

MacMicking et al. (20) reported that LPS produced less hypotension in iNOS-deficient mice than in wild-type controls. The findings imply an alteration of vascular phenotype in iNOS-deficient mice, but because LPS can reduce blood pressure by effects on cardiac contractility (cardiac output) as well as by vascular resistance, the functional importance of expression of iNOS in blood vessels was not clear. Thus this study was designed specifically to evaluate the role of iNOS in vasomotor function. Although the carotid artery is not a microvessel, the use of large arteries (particularly the aorta) is very common in this field of research. Our finding that carotid arteries from iNOS-deficient mice maintain normal constrictor responses after LPS is consistent with minimal effects of LPS on blood pressure.

Our results in carotid arteries from iNOS-deficient mice indicate that iNOS mediates impaired constrictor responses in vessels after exposure to LPS. To our knowledge, this is the first direct evidence that iNOS mediates this response. After selective deletion of a gene, it is not uncommon for deficient animals to display no, or minimal, differences in phenotype. Such findings suggest the presence of redundant or compensatory mechanisms. Although expression of compensatory mechanisms can occur in mice with disrupted genes, we found no evidence for such compensation in these experiments using iNOS-deficient mice with regard to vasoconstrictor responses. Although these data do not preclude interactive effects of NO with COX-2 and/or other mechanisms that influence vascular tone, the data suggest that expression of iNOS is required to cause functional impairment.

	ACKNOWLEDGEMENTS

We thank Kristen Rummelhart, Kara Werner, and Robert Brooks for excellent technical assistance; Arlinda LaRose for secretarial support; and Drs. Sean Murphy and Kristy Lake for critical reading of this manuscript.

	FOOTNOTES

These studies were supported by National Institutes of Health Grants NS-24621, HL-38901, HL-16066, HL-14355, and AG-10269. C. A. Gunnett is an 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.

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. §1734 solely to indicate this fact.

Address for reprint requests: F. M. Faraci, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242-1081.

Received 30 January 1998; accepted in final form 16 April 1998.

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