Journal of the American College of Nutrition, Vol. 23, No. 5, 518S-520S (2004)
Published by the American College of Nutrition
In Vitro Application of Endotoxin to Thoracic Aortas from Magnesium-Deficient Rats Enhances Vascular Hyporeactivity to Phenylephrine
Atsushi Miyamoto, DVM, PhD,
Hiroshi Moriki, DVM,
Shigeru Ishiguro, DVM, PhD and
Akira Nishio, DVM, PhD
Department of Veterinary Pharmacology, Faculty of Agriculture, Kagoshima University, Kagoshima, JAPAN
Address reprint requests to: Atsushi Miyamoto, DVM, PhD, Department of Veterinary Pharmacology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, JAPAN. E-mail: miyamoto{at}vet.agri.kagoshima-u.ac.jp
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ABSTRACT
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Objective: Endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is well described in rat aortas, but has not been investigated in those from magnesium (Mg)-deficient rats in vitro.
Methods: Segments of thoracic aorta from control and Mg-deficient rats were incubated in culture medium for 6 hours in the presence or absence of bacterial lipopolysaccharide (LPS; 0.001–10 µg/mL). Contractions to PE were measured with or without an inducible nitric oxide synthase (iNOS) inhibitor (1400W; 0.1 and 1 µM), a guanylate cyclase inhibitor (ODQ; 0.1 and 1 µM), or a potassium channel inhibitor (TEA; 1 and 10 mM).
Results: LPS induced hyporeactivity in a concentration-dependent manner under relatively low concentrations (0.001–0.1 µg/mL), however, there was no significant difference at 0.1, 1 and 10 µg/mL. LPS-induced hyporeactivity was not significantly affected by endothelium-denudation. The hyporeactivity was enhanced in thoracic aortas from Mg-deficient rats by LPS (0.01, 0.1 and 1 µg/mL). LPS (1 µg/mL) induced hyporeactivity was reversed with 1400W, ODQ or TEA in both aortas in a concentration-dependent manner, however the degree of reversal was weaker in the Mg-deficient rat aorta than in the control rat one. iNOS mRNA level was increased by LPS (0.1 µg/mL) and the increment was significantly high in Mg-deficient rat thoracic aorta.
Conclusions: From these results it is clearly demonstrated that LPS-induced vascular hyporeactivity to PE is enhanced in thoracic aorta from Mg-deficient rats, and it is suggested that LPS-induced NO production might contribute to the enhancement via stimulation of NO-cyclic GMP-potassium channel pathway.
Key words: endotoxin, vascular hyporeactivity, iNOS, Mg-deficiency, phenylephrine
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INTRODUCTION
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Endotoxin-induced vascular hyporeactivity to the 
1-agonist phenylephrine (PE) is well described in rat aortas. It is also known that magnesium (Mg) deficiency aggravates bacterial lipopolysaccharide (LPS) lethality [1].
We have recently reported that the degree of vascular hyporeactivity to PE is greater in Mg-deficient rats and serum levels of inflammatory cytokines, such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-, are significantly higher in Mg-deficient rats than in control rats after 6 hour administration of LPS [2]. However, it has not been examined whether in vitro application of endotoxin to thoracic aortas from Mg-deficient rats enhances vascular hyporeactivity to PE, therefore, the present study assessed whether endotoxin challenge in vitro enhances (1) vascular hyporeactivity to PE, and (2) expression of inducible nitric oxide synthase (iNOS) mRNA in thoracic aortas isolated from Mg-deficient rats, and also examined what kind of inhibitors, such as an iNOS inhibitor, reverse this hyporeactivity.
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MATERIALS AND METHODS
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LPS (Escherichia coli 011:0.001–10 µg/mL; Sigma, USA) was used as endotoxin. Adult male Wistar rats (7–9 weeks old) were fed a Mg-deficient diet (Mg: 0.001%) for 20 days. A control group received a normal diet (Mg: 0.07%). The composition of the purified diet has been shown in a previous paper [3].
Thoracic aorta segments isolated from each group were preincubated in Dulbecco’s modified Eagle’s medium (DMEM) in the presence of penicillin (100 µg/mL) and streptomycin (100 µg/mL) for 1 hour then incubated in DMEM in the presence or absence of LPS for 6 hours. This procedure was done under sterile conditions.
After incubation, each aortic ring was mounted in an organ bath containing 5 mL physiological salt solution. Optimal resting tension was 10 mN. Relative iNOS mRNA levels of thoracic aorta segments from each group were determined using real-time RT-PCR [4].
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RESULTS AND DISCUSSION
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LPS-Induced Hyporeactivity to PE
As shown in Fig. 1, PE (10–9–10–5 M) induced contraction and ACh (10–8–10–5M) induced relaxation in a concentration-dependent manner, respectively, in thoracic aortic rings isolated from control rats. LPS induced hyporeactivity in a concentration dependent manner under relatively low concentrations (0.001–0.1 µg/mL), however, there was no significant difference at 0.1, 1 and 10 µg/mL. LPS did not affect ACh-induced relaxation.
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Fig. 1. Effects of LPS on phenylephrine-induced contraction and acetylcholine-induced relaxation in thoracic aorta isolated from control and Mg-deficient rats.
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The inhibitory effect of LPS on PE-induced contraction was significantly enhanced in Mg-deficient rats than in control rats. These results suggest that the effect of LPS may be involved in smooth muscle cells, but not in endothelial cells.
To confirm this suggestion, we used aortic rings without endothelial cells. Endothelium-denudation was done by gently rubbing the intimal surface with cotton swab wetted with physiological salt solution. The inhibitory effect of 1 µg/mL LPS on PE-induced contractions did not differ significantly between arteries with and without endothelial cells. ACh-induced relaxation was completely abolished by endothelium-denudation in both group rats.
These results suggest that NO in smooth muscle cells, but not in endothelial cells, may be involved in LPS-induced hyporeactivity. Involvement of iNOS in LPS-induced hyporeactivity to PE.
1400W is known as a selective iNOS inhibitor. 1400W (1 µM) almost recovered the PE-induced contraction inhibited with LPS (1 µg/mL) to the control level in both group rats. However, at a concentration of 0.1 µM 1400W, the degree of reversal was significantly weaker in Mg-deficient rats than in control rats (Fig. 2).
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Fig. 2. Effects of 1400 W, ODQ and TEA on LPS (1 µg/mL)-induced hyporeactivity in thoracic aortas isolated from control and Mg-deficient rats. *Tension: mN/mg wet tissue.
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Involvement of cGMP in LPS-Induced Hyporeactivity to PE
ODQ is known as a selective soluble guanylate cyclase inhibitor. ODQ (1 µM) almost recovered the PE-induced contraction inhibited with LPS (1 µg/mL) to the control level in both group rats. ACh-induced relaxation was abolished by use of ODQ, however, at concentration of 0.1 µM ODQ, the degree of reversal was significantly weaker in Mg-deficient rats than in control rats (Fig. 2).
Involvement of Potassium-Channel in LPS-Induced Hyporeactivity to PE
Tetraethylammonium (TEA) is known as a non-selective potassium-channel inhibitor. TEA (1 and 10 mM) concentration-dependently recovered the PE-induced contraction inhibited with LPS (1 µg/mL) in both groups of rats, however, the degree of reversal was significantly weaker in Mg-deficient rats than in control rats (Fig. 2).
Expression of iNOS mRNA in Isolated Thoracic Aorta
The incubation of aortic segment with LPS (0.1 µg/mL) increased the expression of iNOS mRNA to 7 times of that of no treatment in the control rats.
In the Mg-deficient rat, the level of iNOS mRNA was 12 times of that of no treatment in the control rats. The incubation of aortic segment isolated from the Mg-deficient rats with LPS (0.1 µg/mL) increased to 50 times of that of no treatment in control rat.
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CONCLUSION
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From these results, it is clearly demonstrated that LPS-induced vascular hyporeactivity to PE in vitro is enhanced in the thoracic aorta of Mg-deficient rats. LPS-induced NO production might contribute to the enhancement of vascular hyporeactivity to PE in Mg-deficient rats via stimulation of NO-cyclic GMP-potassium-channel pathway.
Received August 5, 2004.
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REFERENCES
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ABSTRACT
INTRODUCTION
