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Am J Physiol Heart Circ Physiol 275: H1078-H1083, 1998;
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Vol. 275, Issue 3, H1078-H1083, September 1998

Effects of antisense oligonucleotide to iNOS on hemodynamic and vascular changes induced by LPS

Azizul M. Hoque, Andreas Papapetropoulos, Richard C. Venema, John D. Catravas, and Leslie C. Fuchs

Vascular Biology Center and Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Lipopolysaccharide (LPS) causes impaired vascular contractility proposed to be mediated by induction of nitric oxide synthase (iNOS). Antisense (AS) oligonucleotide inhibits the translation of target mRNA into functional proteins. We hypothesize that in vivo pretreatment with AS oligonucleotide targeted to iNOS mRNA can prevent LPS-induced hyporeactivity to norepinephrine (NE). Three groups of conscious male Wistar rats received one of the following: saline, AS, or mismatch (MM) oligonucleotide at 0.4 mg/kg iv at 12 and 24 h before LPS (5 mg/kg iv). The fourth group received saline only. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded before and 6 h after LPS or saline administration. Aorta, lung lavage, and lung tissue were collected for determination of iNOS protein expression and NOS activity. Small mesenteric arteries (approx 250 µm) were isolated, denuded of endothelium, and maintained at a constant intraluminal pressure of 40 mmHg for study in vitro. LPS produced significant tachycardia that was not altered by AS or MM oligonucleotide. AS, but not MM oligonucleotide, reduced the accumulation of cGMP, the increase in conversion of L-[3H]arginine to L-[3H]citrulline, and iNOS protein expression in tissue from LPS-treated rats. Small mesenteric arterial contraction to NE was significantly impaired in vessels from LPS-treated rats and was restored by AS, but not MM, oligonucleotide. In a rat model of septic shock, AS oligonucleotide to iNOS mRNA inhibits NOS activity and iNOS protein expression and prevents the vascular hyporeactivity to NE, which may contribute to hypotension in shock.

septic shock; inducible nitric oxide synthase; antisense oligonucleotide; mesenteric small arteries

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

VASCULAR HYPOREACTIVITY to catecholamines and other vasoconstrictor agents are characteristic features of human septic shock that contribute to the associated high mortality rate (9). Bacterial lipopolysaccharide (LPS), or endotoxin, has been used to produce a rat model of septic shock that exhibits significant vascular hyporeactivity to catecholamines both in vivo and in vitro (13, 24). LPS increases nitric oxide (NO) production via induction of a calcium-independent isoform of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (17). Inhibitors of NOS activity prevent the vascular effects of endotoxin, indicating a role for NO in LPS-induced cardiovascular dysfunction (14, 15, 12, 21, 24, 32).

Antisense (AS) oligonucleotides selectively inhibit the translation of target mRNA into functional proteins in vitro and in vivo (7, 20, 29, 33). Because of their ability to inhibit gene expression in a sequence-specific manner, AS oligonucleotides may become a novel class of therapeutic agents for the treatment of a number of diseases (30). Because enhanced production of NO appears to contribute to the pathogenesis of endotoxin shock, a highly selective AS oligonucleotide to iNOS may be a successful therapeutic strategy. By inhibiting induction of iNOS, AS oligonucleotide may prevent the vascular effect of high levels of NO, which appears to contribute to vascular hyporeactivity to norepinephrine (NE). This hyporeactivity to NE is thought to contribute to mortality from septic shock. In the present study, an AS oligonucleotide targeted to iNOS mRNA has been used to determine whether inhibition of the induction of iNOS in vivo can prevent the LPS-induced hyporeactivity to NE in isolated rat small mesenteric arteries.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

General procedures. Male Wistar rats (200-250 g) were anesthetized with pentobarbital sodium (40 mg/kg ip), and femoral venous and arterial catheters (polyethylene-10) were implanted in each rat. Arterial pressure (AP) was measured via the femoral catheter, and heart rate (HR) was triggered from the pulse pressure and recorded on a Grass polygraph in conscious, unrestrained rats at least 48 h after surgery. Drugs were administered into the femoral vein. Rats were divided into four groups (n = 6 per group). Three groups received LPS (5 mg/kg iv) after one of the following pretreatments: 1) AS oligonucleotide to iNOS (5'-CAG-GGG-CAA-GCC-ATG-TC-3'), 0.4 mg/kg iv in a volume of 200 µl saline, 12 and 24 h before LPS; 2) mismatch (MM) oligonucleotide (5'-CAC-CGC-CAT-GGC-ATC-TG-3'), 0.4 mg/kg iv in a volume of 200 µl saline, 12 and 24 h before LPS; and 3) saline (200 µl) 12 and 24 h before LPS. The fourth group received three injections of saline (200 µl) only. AP and HR were monitored continuously for 30 min before and 6 h after LPS or saline injection.

At the end of the experiment, rats were heparinized (500 U iv) and euthanized with an overdose of pentobarbital sodium (50 mg/kg iv). Assays of NOS activity were used to assess LPS-induced production of iNOS and to determine the ability of AS oligonucleotide to inhibit iNOS production. Lung lavage was obtained by injecting saline (4 ml) into the lungs through a tracheal cannula. The saline was immediately withdrawn and chilled before exposure to reporter rat aortic smooth muscle cells (RASMC) for measurements of cGMP. The lungs and aorta were then removed and frozen in liquid nitrogen for determination of NOS activity or iNOS protein expression, respectively, as described later. A section of small intestine with the mesentery intact was removed and placed in chilled, oxygenated (20% O2 and 5% CO2) modified Krebs-Ringer bicarbonate solution (mM composition: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.1 dextrose). All procedures followed were in accordance with institutional guidelines.

Vascular reactivity. A section of small mesenteric artery (200-300 µm diameter and 1-2 mm long) was isolated microscopically from the surrounding tissue and placed in a microvessel bath containing oxygenated Krebs-Ringer solution. The endothelium was removed by gently sliding the vessel over a glass micropipette. One end of the vessel was then tied onto the micropipette (100 µm diameter tip) with 10-0 ophthalmic suture, and 0.3 ml of air was infused slowly through the vessel lumen via a syringe connected to the pipette. The vessel lumen was then slowly perfused with Krebs-Ringer solution, and the other end of the vessel was tied to a second micropipette. The lumen of the vessel was filled with Krebs-Ringer solution through the micropipette and maintained at a constant pressure of 40 mmHg. The tissue bath was transferred to the stage of an Olympus inverted light microscope coupled to a monitor and video dimension analyzer (Living Systems Instrumentation, Burlington, VT). Small mesenteric artery intraluminal diameter was continuously monitored on a TV monitor and recorded on a Grass polygraph.

Oxygenated (20% O2 and 5% CO2) Krebs-Ringer solution was maintained at 37°C and continuously circulated through the tissue bath. The vessel was allowed to equilibrate for 1 h. Denudation of the endothelium was confirmed by the absence of relaxation to the endothelium-dependent vasodilator acetylcholine (ACh, 10-6 M) following preconstriction to 30-50% of baseline diameter (in vitro diameter at 40 mmHg intraluminal pressure) with the thromboxane A2 analog U-46619. The vessel was then washed with fresh Krebs-Ringer solution until it returned to baseline diameter. Vessels were allowed to reequilibrate for at least 15 min before we performed a cumulative dose-response curve to NE (10-8 to 3 × 10-5 M).

cGMP production in RASMC induced by lung lavage cells. The lung lavage was centrifuged at 2,000 rpm for 5 min at 4°C. The pellet was resuspended in 5 ml of Earle's balanced salt solution (EBSS, pH 7.4, 37°C) and centrifuged a second time. The pellet was then resuspended in EBSS (6 ml), and 300-µl samples were placed in a 2-cm2 tissue culture well containing untreated RASMC. Lavage samples were incubated in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM) and treated with L-arginine (1 mM), the substrate for NOS, or Nomega -nitro-L-arginine methyl ester (L-NAME; 1 mM), an inhibitor of NOS activity, for 15 min. The medium was then rapidly aspirated, and 0.5 ml of 0.1 N HCl was added to each well to stop enzymatic reactions and extract cGMP. Thirty minutes later, the HCl extract was collected and directly analyzed by radioimmunoassay using the automated Gammaflow system with a monoclonal antibody for cGMP. All results were normalized per milligram of lavage cell protein and per milligram of reporter RASMC protein.

L-[3H]arginine to L-[3H]citrulline conversion. Frozen lung samples were homogenized on ice in the following buffer: tris(hydroxymethyl)aminomethane (Tris) · HCl 50 mM, EDTA 0.1 mM, EGTA 0.1 mM, dithiothreitol (DTT) 0.5 mM, pepstatin A 10 µg/ml, leupeptin 10 µg/ml, bestatin 10 µg/ml, and phenylmethylsulfonyl fluoride l mM (pH 7.4). Conversion of L-[3H]arginine to L-[3H]citrulline was then measured in the homogenate (5). Tissue homogenate (100 µl) was incubated in the presence of Larginine (100 µM), L-[3H]arginine (~300,000 counts/min), L-citrulline (1 mM), NADPH (1 mM), calmodulin (20 U), tetrahydrobiopterin (30 µM), flavin adenine dinucleotide (5 µM), and flavin mononucleotide (5 µM) at 37°C for 45 mm in Tris · HCl homogenization buffer in the absence of calcium. The reaction was then stopped by the addition of HEPES buffer (pH 5.5) containing EDTA (2 mM) and EGTA (2 mM). The reaction mixture was applied to AG-50 Dowex resin (Na+ form) columns equilibrated in stop buffer. The eluted L-[3H]citrulline activity was measured by scintillation counting (Beckman LS 3801, CA). Performing reactions in a calcium-free medium and in the presence of calcium chelators allowed for the selective determination of L-[3H]citrulline formation by iNOS. Conversion was normalized per milligram of protein in the sample.

Protein determination. Protein content of lung lavage cells, RASMC, and lung tissue was measured by the Bradford method (4). Sample aliqouts were combined with the protein binding dye (Bio-Rad Laboratories, Richmond, CA), and optical density was determined at 630 nm using a multiwell plate reader (Dynatech Laboratories, Chantilly, VA). Bovine serum albumin (fraction V, Sigma) was used as a standard.

Immunoblotting. Frozen aortas were pulverized in a prechilled tissue pulverizer and then suspended in ice-cold ristocetin-induced platelet aggregation buffer (2 ml) containing 20 mM Tris · HCl, pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1% phenylmethylsulfonyl fluoride 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 5 µg/ml aprotinin. Samples were sonicated with three 2-s bursts of a Virtis Virsonic 60 sonicator, and the protein concentration of each sample was determined by Bio-Rad DC (Detergent Compatible) Protein Assay. A 70-µg aliquot of each sample was suspended in sodium dodecyl sulfate (SDS) sample buffer (0.5 Tris · HCl, pH 6.8, 30% glycerol, 6% SDS, 14% 2-mercaptoethanol, 30 mM EDTA, and 0.0025% bromophenol blue) and subjected to SDS polyacrylamide gel electrophoresis on 7.5% polyacrilamide gels. Proteins were transferred to nitrocellulose membranes by electroblotting, and membranes were probed with rabbit polyclonal anti-iNOS antibody (1:1,000) purchased from Transduction Laboratories (Lexington, KY). Bound antibody was visualized by the enhanced chemiluminescense detection system (Amersham) following secondary probing of the membrane with peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad). Relative amounts of iNOS protein in each gel lane were quantitated by densitometric scanning of autoradiograms.

Drugs and chemicals. Phosphorothioate AS oligonucleotide (5'-CAG GGG CAA GCC ATG TC-3') to rat inducible vascular smooth muscle NOS (GENBANK RATVSMNOS) and its phosphothiorate MM control oligonucleotide (5'-CAC CGC CAT GGC ATC TG-3') were purchased from Molecular Research Laboratory, Durham, NC. The AS oligonucleotide to iNOS mRNA is located at bases -3 to 14 with base 1 being the translation start site for iNOS as described by Geng et al. (8). AS and MM oligonucleotides were dissolved in 0.9% NaCl solution immediately before administration. NE, IBMX, and E. coli lipopolysaccharide (serotype 0127:B8) were obtained from Sigma Chemical, St. Louis, MO. L-[3H]arginine was obtained from New England Nuclear.

Data and statistical analysis. Data are presented as means ± SE. Statistical comparisons between groups were performed using analysis of variance (ANOVA). Significance levels were determined by Student's modified t-test with Bonferroni correction for multiple comparisons. Differences among means were considered significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The effect of AS or MM oligonucleotides on the LPS-induced changes in mean arterial pressure (MAP) and HR is shown in Fig. 1, A and B, respectively. In the saline + saline group, MAP and HR did not significantly differ from baseline. In the saline + LPS group, LPS produced an immediate decrease in MAP, which returned to baseline within 30 mm, and did not differ from baseline for up to 6 h. Tachycardia was also observed in this group and was sustained for 6 h. In AS or MM oligonucleotide-pretreated groups, LPS produced an initial pressor response, followed by a return of MAP to baseline. Pretreatment with AS or MM oligonucleotide did not prevent the LPS-induced tachycardia.


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  		Fig. 1.   Time course of percent change (%Delta ) in mean arterial pressure (MAP) (A) and heart rate (HR) (B) in rats receiving saline (S), lipopolysaccharide (LPS), antisense (AS), or mismatch (MM) oligonucleotides. MAP (in mmHg) before injection of S or LPS was 111 ± 2 in S group, 102 ± 3 in LPS group, 114 ± 6 in AS group, and 103 ± 3 in MM group. HR (beats/min) before injection of S or LPS was 390 ± 7 in S group, 401 ± 16 in LPS group, 412 ± 5 in AS group, and 403 ± 4 in MM group. Values represent means ± SE; n = 6 rats per group. * P < 0.05 vs. S + LPS.

The effect of AS and MM oligonucleotides on LPS-induced cGMP accumulation was determined with lavage cells in the presence of reporter RASMC. The results are illustrated in Fig. 2. In the saline + saline group, there was no accumulation of cGMP. cGMP accumulation in the presence of IBMX was significantly increased in the saline + LPS group and was further enhanced by the addition of L-arginine. cGMP accumulation was inhibited in the AS oligonucleotide + LPS group but not in the MM oligonucleotide + LPS group compared with the saline + LPS group. In the presence of L-NAME, cGMP accumulation was abolished in all groups, indicating that this effect was mediated by iNOS.


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  		Fig. 2.   cGMP accumulation in 3-isobutyl-1-methylxanthine (IBMX)-, L-arginine (L-Arg)-, or NG-nitro-L-arginine methyl ester (L-NAME)-treated rat aortic smooth muscle cellls (RASMC) in presence of lung lavage cells collected from rats receiving S, LPS, AS, or MM oligonucleotides. Values represent means ± SE; n = 6 rats per group. * P < 0.05 vs. S + S; # P < 0.05 vs. S + LPS.

Conversion of L-[3H]arginine to L-[3H]citrulline was measured in lung homogenates and is illustrated in Fig. 3, where the saline + saline group is 14.6 ± 1.1 pmol/mg protein per 40 min and represents 100%. A significant increase in conversion of L-[3H]arginine to L-[3H]citrulline was observed in the saline + LPS compared with the saline + saline group. Pretreatment with AS, but not MM, oligonucleotide inhibited the LPS-induced increase in L-[3H]arginine-to-L-[3H]citrulline conversion. The effect of LPS on expression of iNOS protein in aorta is shown in Fig. 4. As observed in Fig. 4A, AS oligonucleotide largely reduced iNOS expression in LPS-treated rats. The average optical density of the iNOS obtained from two-rat aorta per group is shown in Fig. 4B.


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  		Fig. 3.   Conversion of L-[3H]Arg to L-[3H]citrulline illustrated as percentage of control in lung homogenates from rats receiving S, LPS, AS, or MM oligonucleotides. Values represent means ± SE; n = 6 rats per group. * P < 0.05 vs. S + S; # P < 0.05 vs. S + LPS.


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  		Fig. 4.   Expression of inducible nitric oxide synthase (iNOS) protein in aorta of rats receiving S, LPS, AS, or MM oligonucleotides (A). iNOS protein was quantitated by immunoblotting and densitometry. Values for each condition represent average optical density of iNOS obtained from 2 rat aortas per group (B).

Typical sections of small intestine from which small mesenteric arteries were isolated in all four groups of rats are shown in Fig. 5. Significant hemorrhaging occurred in the saline + LPS compared with the saline + saline group. Hemorrhaging was prevented by pretreatment with AS but not MM oligonucleotide.


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  		Fig. 5.   Typical sections of small intestine from which small mesenteric arteries were isolated from rats receiving S, LPS, LPS + AS, or LPS + MM oligonucleotides.

The effect of AS and MM oligonucleotides on LPS-induced vascular hyporeactivity to NE is shown in Fig. 6. Baseline diameters of small mesenteric arteries did not differ significantly between groups. In endothelium-denuded vessels from rats in the saline + LPS group, the dose-response curve to NE was significantly shifted to the right, and the maximum contractile response was decreased compared with the saline + saline group. Pretreatment with AS oligonucleotide completely prevented the LPS-induced rightward shift of the dose-response curve as well as the decrease in the maximum contraction to NE. However, pretreatment with MM oligonucleotide did not prevent the LPS-induced hyporeactivity to NE. The pD2 values for NE in each group are shown in Table 1. LPS alone significantly lowered the pD2 value compared with the saline + saline group. Pretreatment with AS, but not MM, oligonucleotide prevented the LPS-induced decrease in pD2 value.


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  		Fig. 6.   Dose-response curves to norepinephrine (NE) in small mesenteric arteries from rats receiving S, LPS, AS, or MM oligonucleotides. Intraluminal diameter (in µm) in isolated vessels at 40 mmHg intraluminal pressure was 262 ± 6 in S group, 254 ± 24 in LPS group, 246 ± 16 in AS group, and 250 ± 10 in MM group. Values represent means ± SE; n = 6 rats per group. * P < 0.05 vs. S + S; # P < 0.05 vs. S + LPS.

                              
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  		Table 1.   Effects of AS and MM oligonucleotide on LPS-induced hyporeactivity to NE

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

LPS-induced sepsis is characterized by sustained production of NO by iNOS and vascular hyporesponsiveness to catecholamines in vivo and in vitro (13, 17, 24). A reduced response to NE has been observed in the rat mesenteric vascular bed and aorta and pig pulmonary artery following in vivo administration of LPS (18, 21, 28). Lipoteichoic acid from Staphylococcus aureus was found to depress contraction to NE in human gastroepiploic arteries (26). Studies utilizing inhibitors of NOS activity indicate that NO mediates the LPS-induced hyporesponsiveness to catecholamines (14, 15, 21, 24, 32). The present study has demonstrated that LPS produces hyporesponsiveness to NE in isolated small mesenteric arteries and that in vivo pretreatment with AS oligonucleotide targeted to iNOS mRNA prevents the LPS-induced vascular hyporeactvity.

AS oligonucleotides enter cells through endocytosis and are designed to inhibit translation of iNOS proteins (19, 23, 25, 31). The ability of intravenous administration of AS oligonucleotide to iNOS mRNA to significantly inhibit the LPS-induced increase in NOS activity and iNOS protein expression has been demonstrated in this study. Assays of NOS activity, including cGMP production and L-[3H]arginine-to-L-[3H]citrulline conversion, indicate that administration of AS oligonucleotide in vivo inhibits NOS activity, whereas administration of MM oligonucleotide has no effect. Additionally, in vivo administration of AS oligonucleotide inhibited iNOS protein expression, whereas MM oligonucleotide did not. The actions of AS oligonucleotide also include a complete restoration of contraction to NE in small mesenteric arteries, whereas MM oligonucleotide had no effect on the LPS-induced hyporeactivity to NE. Because the small mesenteric arteries were denuded of endothelium, potential LPS-induced alterations in endothelial NOS did not affect contraction to NE.

LPS produced tachycardia that was maintained throughout the experiment. Whereas this effect has been reported in other studies (22, 27), the mechanism is unknown. The results of our study demonstrate that tachycardia is not related to induction of iNOS. In isolated rat hearts, LPS did not alter the rate pressure product or the rate of ventricular contraction or relaxation (3). Another study showed that LPS had no effect on the force of cardiac contractility in isolated hearts (2). Therefore, tachycardia induced by LPS may not be mediated by a direct effect on the heart. It is possible that LPS exerts its effect through the central nervous system. LPS was shown to activate serotonergic and noradrenergic neurotransmission in the rat hippocampus (16). Additionally, endotoxin enhanced the release of NE by an action at peripheral nerve terminals in anesthetized rats (12).

In this study, LPS did not significantly alter arterial pressure at 6 h after administration. Hypotension occurring within 6 h is a well-characterized response to LPS in anesthetized rats (11, 22). However, studies have shown that LPS does not produce hypotension within 6 h in conscious rats (27). One study reported that tumor necrosis factor, which is thought to be a primary mediator of the response to endotoxin, produced hypotension 24 h after it was administered to conscious rats (27). LPS did produce a transient depressor response that was blocked by AS or MM oligonucleotide. However, the ability of AS and MM oligonucleotides to inhibit this response is not due to inhibition of production of iNOS, since this involves transcription of new mRNA and biosynthesis of new protein, which requires several hours. Because significant tachycardia was observed 1 h after administration of LPS, this response was initiated before induction of NOS and consequently was not inhibited by AS oligonucleotide.

The finding that AS oligonucleotide afforded only partial inhibition of LPS-induced production of iNOS cannot be explained from the results of this study. Problems that may exist with the use of AS oligonucleotide in vivo include degradation by nucleases in serum, delivery to specific cells, and incomplete inhibition of mRNA translation (1). The phosphothiorate AS oligonucleotide used in this study has a sulfur atom replacing one of the nonbridging oxygen atoms at each interbase phosphorus, creating a phosphothiorate linkage that is nuclease resistant (26). Therefore, degradation by nucleases in serum is unlikely. When administered intravenously, phosphothiorate oligonucleotide is distributed into most organs of rats (19). Presently, a pharmacokinetic analysis of AS oligonucleotide to iNOS mRNA has not been performed.

In conclusion, results of our study demonstrate that in vivo pretreatment with AS oligonucleotide to iNOS mRNA prevents the LPS-induced vascular hyporeactivity to NE. Further investigations are required to determine whether AS oligonucleotide to mRNA will prevent vascular hyporeactivity if administered after the onset of sepsis and to develop optimal dose regimens for this AS oligonucleotide.

    ACKNOWLEDGEMENTS

These studies were supported by National Heart, Lung, and Blood Institute Grant HL-52958.

    FOOTNOTES

Current address of A. Papapetropoulos: Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536-0812.

Address for reprint requests: L. C. Fuchs, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912.

Received 14 October 1997; accepted in final form 21 May 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

1.   Agrawal, S., J. Temsamani, W. Galbraith, and J. Tang. Pharmacokinetics of anitsense oligonucleotides. Clin. Pharmacokinet. 28: 7-16, 1995[Medline].

2.   Baydoun, A. R., R. D. Foale, and G. E. Mann. Bacterial endotoxin rapidly stimulates prolonged endothelium-dependent vasodilation in the rat isolated perfused heart. Br. J. Pharmacol. 109: 987-991, 1993[Medline].

3.   Bensard, D. D., A. Banerjee, R. C. McIntyre, Jr., R. L. Berens, and A. H. Harken. Endotoxin disrupts beta-adrenergic signal transductin in the heart. Arch. Surg. 129: 198-204, 1994[Abstract].

4.   Bradford, M. M. A rapid and sensitive method for the quantification of protein dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

5.   Bredt, D. S., and S. H. Snyder. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA 87: 682-685, 1990[Abstract/Free Full Text].

6.   Brooker, G., W. Terasaki, and M. Price. Gammaflow: a completely automated radioimmunoassay system. Science 94: 270-276, 1976.

7.   Cooney, M., G. Czernuszewicz, E. H. Postel, S. J. Flint, and M. B. Hogan. Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science 241: 456-459, 1988[Abstract/Free Full Text].

8.   Geng, Y., M. Almqvist, and G. K. Hansson. cDNA cloning and expression of inducible nitric oxide synthase from rat vascular smooth muscle cells. Biochim. Biophys. Acta 1218: 421-424, 1994[Medline].

9.   Groeneveld, A. B. J., W. Bronsveld, and T. G. Thijs. Hemodynamic determinants of mortality in human septic shock. Surgery 99: 140-153, 1986[Medline].

10.   Gross, S. S., and R. Levi. Tetrahydrobiopterin synthesis: an absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J. Biol. Chem. 267: 25722-25729, 1992[Abstract/Free Full Text].

11.   Hoque, A. M., N. Marczin, J. D. Catravas, and L. C. Fuchs. Anesthesia with sodium pentobarbital enhances lipopolysaccharide-induced cardiovascular dysfunction in rats. Shock 6: 365-370, 1996[Medline].

12.   Jones, S. B., P. Kotsonia, and H. Majewski. Endotoxin enhances norepinephrine release in the rat by peripheral mechanisms. Shock 2: 370-375, 1994[Medline].

13.   Julou-Schaeffer, G., G. A. Gray, I. Fleming, C. Schott, J. R. Parratt, and J. C. Stoclet. Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1038-H1043, 1990[Abstract/Free Full Text].

14.   Kilbourn, R. G., A. Jubran, S. S. Gross, O. W. Griffith, R. Levi, J. Adams, and R. F. Lodato. Reversal of endotoxin-mediated shock by N G-methyl-L-arginine, an inhibitor of nitric oxide synthesis. Biochem. Biophys. Res. Commun. 172: 1132-1138, 1992.

15.   Knowles, R. G., M. Salter, S. L. Brooks, and S. Moncada. Anti-inflammatory glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in the lung, liver, and aorta of the rat. Biochem. Biophys. Res. Commun. 172: 1042-1048, 1990[Medline].

16.   Linthorst, A. C., C. Flachskamm, F. Holsboer, and J. M. Reul. Activation of serotonergic and noradrenergic neurotransmission in the rat hippocampus after peripheral administration of bacterial endotoxin: involvement of the cyclo-oxygenase pathway. Neuroscience 72: 989-997, 1996[Medline].

17.   Marczin, N., A. Papapetropoulos, and J. D. Catravas. Tyrosine kinase inhibitors suppress endotoxin- and IL-1-induced NO synthesis in aortic smooth muscle cells. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H1014-H1018, 1993[Abstract/Free Full Text].

18.   Mitolo-Chieppa, D., M. Serio, M. A. Potenza, M. Montagnani, G. Mansi, S. Pece, E. Jirillo, and J. C. Stoclet. Hyporeactivity of mesenteric vascular bed in endotoxin-treated rats. Eur. J. Pharmacol. 309: 175-82, 1996[Medline].

19.  Murray, J. A. H. Editor. Antisense Research and Applications. New York: Wiley-Liss, 1992.

20.   Owens, M. J., J. J. Mulchahey, J. W. Kasckow, P. M. Plotsky, and C. B. Nemeroff. Exposure to an antisense oligonucleotide decreases corticotropin-releasing factor receptor binding in rat pituitary cultures. J. Neurochem. 64: 2358-2361, 1995[Medline].

21.   Paya, D., G. A. Gray, I. Fleming, and J. C. Stoclet. Effect of dexamethasone on the onset and persistence of vascular hyporeactivity induced by E. coli lipopolysaccharide in rats. Circ. Shock 41: 103-112, 1993[Medline].

22.   Schaefer, C. F., B. Biber, D. J. Brackett, C. C. Schmidt, L. Fagraeus, and M. F. Wilson. Choice of anesthetic alters the circulatory shock pattern as gauged by conscious rat endotoxemia. Acta Anaesthesiol. Scand. 31: 550-556, 1987[Medline].

23.   Stein, C. A., J. L. Tonkinson, and L. Yakubov. Phosphorothioate oligodeonucleotides: anti-sense inhibitors of gene expression. Pharmacol. Ther. 52: 365-384, 1991[Medline].

24.   Szabo, C., J. A. Mitchell, C. Thiemermann, and J. R. Vane. Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock. Br. J. Pharmacol. 108: 786-792, 1993[Medline].

25.   Thomae, K. R., D. A. Geller, T. R. Billiar, P. Davies, B. R. Pitt, R. L. Simmons, and D. K. Nakayama. Antisense oligonucleotide to inducible nitric oxide synthase inhibits nitric oxide synthesis in rat pulmonary artery smooth muscle cells in culture. Surgery 114: 272-277, 1993[Medline].

26.   Tsuneyoshi, I., Y. Kanmura, and N. Yoshimura. Lipoteichoic acid from Staphylococcus aureus depresses contractile function of human arteries in vitro due to the induction of nitric oxide synthase. Anesth. Analg. 82: 948-953, 1996[Abstract].

27.   Turner, C. R., K. M. Esser, B. B. Wheeldon, M. Slivjak, and B. F. Smith III. Cardiovascular and pulmonary effects of human recombinant tumor necrosis factor in conscious rat. Circ. Shock 28: 369-384, 1989[Medline].

28.   Villamore, E., F. Perez-Vizcaino, T. Ruiz, J. C. Leza, M. Moro, and J. Tamargo. Group B streptococcus and E. coli LPS-induced NO-dependent hyporesponsiveness to noradrenaline in isolated intrapulmonary arteries of neonatal piglets. Br. J. Pharmacol. 115: 261-266, 1995[Medline].

29.   Wahlestedt, C., E. M. Pich, G. F. Koob, F. Yee, and M. Heilig. Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science 259: 528-531, 1993[Abstract/Free Full Text].

30.   Wickstrom, B. Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. New York: Wiley-Liss, 1991.

31.   Yakubov, L. A., E. A. Deeva, V. F. Zarytova, E. M. Ivanova, A. S. Ryte, L. V. Yurchenko, and V. V. Vlassov. Mechanism of oligonucleotide uptake by cells: involvement of specific receptors? Proc. Natl. Acad. Sci. USA 86: 6454-6458, 1989[Abstract/Free Full Text].

32.   Yang, B. C., L. Y. Chen, T. G. Saldeen, and J. L. Mehta. Reperfusion injury in the endotoxin-treated rat heart: reevaluation of the role of nitric oxide. Br. J. Pharmacol. 120: 305-311, 1997[Medline].

33.   Zhou, L. W., S. P. Zhang, Z. H. Qin, and B. Weiss. In vivo administration of an oligodeoxynucleotide antisense to the D2 dopamine receptor messenger RNA inhibits D2 dopamine receptor-mediated behavior and the expression of D2 dopamine receptors in mouse striatum. J. Pharmacol. Exp. Ther. 268: 1015-1023, 1994[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 275(3):H1078-H1083
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