barbiturate to affect the synthesis or action of nitric oxide, which probably ... (10-9-10-7 M) were obtained in rings precontracted with phenylephrine. (10-6 M), first ...
Proc. West. Pharmacol. Soc. 41: 147-148 (1998)
Thiopental Inhibits Nitric Oxide Synthesis C. CASTILLO*1, B. ESCALANTE2, R.A. BOBADILLA1, E. MERA1 & E.F. CASTILLO1 1
Escuela Superior de Medicina del I.P.N. & 2Departamento de Farmacologia CINVESTAV-IPN, Mexico, D.F., Mexico
Among other factors, endothelial-dependent relaxation may be affected by drugs [1]. Thus, barbiturates, and specially thiopental, can provoke damage and functional alterations of the endothelium [2-7]. For example, it is well known that thiopental interferes with the synthesis of endothelium-derived hyperpolarizing factor [8]. However, there is not enough information about the ability of this barbiturate to affect the synthesis or action of nitric oxide, which probably is the main endothelium-derived vasodilator. We have reported [9] that pretreatment with high concentrations of this barbiturate (up to 3.1 mg/ml) inhibits endothelium-dependent relaxation in rat aortic rings (a vascular preparation in which nitric oxide seems to be the main endothelium dependent vasodilator). The aim of this work was to evaluate if thiopental in concentrations close to those which are found in plasma after an anesthetic dose (10-100 µg/ml) could affect endothelium-dependent relaxation and nitrite production (an indirect measure of nitric oxide production) elicited by acetylcholine, histamine and A23187 in rat aortic rings. In addition, we evaluated if thiopental directly affected nitric oxide synthase activity in rat kidney homogenates. METHODS: Male Wistar rats (250 to 300g) were used. After the animals were killed the thoracic aorta and the kidney were immediately excised and placed in cold buffer Endothelium-dependent relaxation. The isolated arteries were freed from surrounding connective tissue and cut into rings (4-5 mm long) and placed in organ baths under a resting tension of 4 g in Krebsbicarbonate solution of the following composition (mM): NaCl 118; KCl 4.7; KH2PO4 1.2; MgSO4.7H2O 1.2; CaCl2.2H2O 2.5; NaHCO3 25; dextrose 11.7; and calcium disodium EDTA 0.026. Tissue baths were maintained at 37°C, pH 7.4, and bubbled with 95% O2 containing 5% CO2. Rings were mounted on two stainless steel hooks to fix them to the bottom of the chamber, and to a Grass FTO3 force displacement transducer connected with a 7D Grass Polygraph, to record the isometric tension developed by aortic rings. Endothelial integrity was pharmacologically assessed by means of acetylcholine (10-6 M). Aortic rings with damaged endothelium were not relaxed by acetylcholine. Concentration-response curves for acetylcholine (10-9-10-5 M), histamine (10-8-10-4 M), A23187 (10-8-10-6 M) and sodium nitroprusside (10-9-10-7 M) were obtained in rings precontracted with phenylephrine. (10-6 M), first in the absence and then 30 min after pretreatment with thiopental (10, 31 or 100 µg/ml) or its solvent (sodium carbonate 6 mg/ml, giving a final concentration in the incubation medium of 60 µg/ml). In all the experiments, each ring served as its own control. Assay of nitrites. The detailed procedure for measuring nitrites has been reported [10,11]. In brief the production of nitrites by aortic rings was measured using a variant of Griess reagent (20 µl 50% trichloroacetic acid, 10 µl 2% procainamide and 10 µl N,N-naphthylenediamine
1% in water) to form a stable azo-dye, which was measured by spectrophotometry at 548 nm. Nitric oxide synthase activity. Nitric oxide synthase activity was measured as 14C labeled citrulline production in rat kidney homogenates.
Figure 1. Effects of pretreatment with thiopental on the endothelium-dependent relaxation elicited by acetylcholine in rat aortic rings. Symbols are the means ± SEM (n=6-8). *p < 0.05.
RESULTS: The barbiturate inhibited, in a concentrationdependent fashion, vascular relaxation elicited by either acetylcholine (Figure 1), histamine, or A23187 (data not shown) in rat aortic rings precontracted with phenylephrine (10-6 M). The inhibitory effect was reversible as it disappeared after arterial preparations were washed (data not shown). On the other hand, relaxation provoked by sodium nitroprusside was not affected by the pretreatment with thiopental (data not shown). Thiopental also inhibited basal and acetylcholine- (10-5 M) or A23187- (10-6 M) stimulated nitrite production in the aortic preparations (data not shown). Finally, the barbiturate decreased 14C labeled citrulline production in rat kidney homogenates (10-5 M; Figure 2), the magnitude of the inhibition being similar to that observed with L-NAME (data not shown).
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fect of sodium nitroprusside disproves the possibility that the barbiturate affects the activity of guanylate cyclase in the vascular smooth muscle. Therefore, a direct interference with the activity of the nitric oxide synthase emerged as a possible explanation of the inhibitory effect of the barbiturate. The ability of thiopental to decrease basal and acetylcholine or A23187-elicited nitrite production in aortic preparations and 14C labeled citrulline production in rat kidney homogenates provided strong support to the contention that the barbiturate inhibits the activity of the endothelial nitric oxide synthase. Interestingly, the potency of thiopental as an inhibitor of the synthase was similar to that of L-NAME, an extensively used blocker of the NO synthesis [14] ACKNOWLEDGMENTS: This study was supported by a grant from the DEPI-IPN.
Figure 2. Effect of thiopental on the activity of nitric oxide synthase in rat kidney homogenates. The activity of the enzyme was measured as 14 C-labeled citrulline production. Values are means ± SEM of 6 experiments. *p < 0.05.
DISCUSSION: Thiopental inhibited the endotheliumand receptor-dependent relaxation elicited by both acetylcholine and histamine [12,13] in rat aortic rings, results which suggested an interference of the barbiturate with the synthesis or action of NO, as this compound seems to be the main endothelial relaxing factor in the rat aorta. Evidence that muscarinic and histaminergic endothelial receptors are not the target of the inhibitory effect of the barbiturate was provided by the finding that this drug also inhibited the endothelium-dependent but receptorindependent vasodilator effect of A23187. On the other hand, the fact that thiopental did not modify the endothelium-independent but cyclic GMP-dependent relaxant ef-
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