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Anesth Analg 2006;102:682-689 © 2006 International Anesthesia Research Society doi: 10.1213/01.ane.0000196521.62806.43
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Abstract |
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In this in vitro study we examined the effects of diazepam on a phenylephrine-induced contraction in rat aorta and determined the associated cellular mechanism focusing on the endothelium-derived vasodilators. The concentration-response curves for phenylephrine and potassium chloride were generated in the presence or absence of diazepam. Phenylephrine concentration-response curves were generated from the endothelium-intact rings pretreated independently with NW-nitro-l-arginine methyl ester, PK 11195, tetraethylammonium, and indomethacin in the presence or absence of diazepam. Diazepam (7 x 10–7 M) attenuated the phenylephrine-induced contraction in the endothelium-intact rings, whereas a large dose (5 x 10–6 M) of diazepam attenuated the phenylephrine-induced contraction in the aortic rings with or without the endothelium. A pretreatment with the NW-nitro-l-arginine methyl ester completely abolished the diazepam (7 x 10–7 M)-induced attenuation of the phenylephrine concentration-response curve, as well as the diazepam (5 x 10–6 M)-induced attenuation of the maximal contractile response to phenylephrine. The NW-nitro-l-arginine methyl ester (10–4 M)-induced contraction was enhanced in the rings pretreated with diazepam (5 x 10–6 M). These results indicate that a supraclinical concentration of diazepam attenuates phenylephrine-induced contraction by increasing endothelial nitric oxide activity and directly affecting vascular smooth muscle.
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Introduction |
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Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepine-2-one) is a highly lipid-soluble and water-insoluble benzodiazepine (1) that is often used as a preoperative medication, anesthetic, and in the treatment of seizure. When diazepam is administered to induce anesthesia, it decreases the arterial blood pressure in humans by inhibiting sympathetic activity (2) and depressing baroreflex function (3). In addition, benzodiazepine causes the relaxation of vascular smooth muscle (4) and relaxes airway smooth muscle (5). In previous in vitro studies, diazepam was reported to inhibit phenylephrine-induced calcium oscillations (6) and produce vasodilation in a phenylephrine-precontracted rat aorta (7).
Pretreatment with the nitric oxide (NO) synthase (NOS) inhibitor, NW-nitro-l-arginine methyl ester (L-NAME) was reported to decrease diazepam-induced relaxation in an endothelium-denuded rat aorta (7). The vascular endothelium releases the endothelium-derived relaxing factor (8), which relaxes vascular smooth muscle by forming 3',5'-cyclic guanosine monophosphate via the activation of guanylate cyclase. This previous result (7) suggests that NO released from muscle stimulated with inducible NOS (iNOS) plays a role in diazepam-induced relaxation. However, the detailed effects of diazepam on a phenylephrine-induced contraction in an endothelium-intact blood vessel have not been investigated. Therefore, the aims of this in vitro study were to examine the effects of diazepam on a phenylephrine-induced contraction in a rat aorta and to determine the associated cellular mechanism with particular focus on the endothelium-derived vasodilators (NO, endothelium-derived hyperpolarizing factor [EDHF], prostacyclin) (8–10).
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Methods |
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All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Gyeongsang National University Hospital. Male Sprague Dawley rats weighing 250–350 g each were anesthetized by the intraperitoneal administration of pentobarbital sodium (50 mg/kg). The descending thoracic aorta was dissected free, and the surrounding connective tissue and fat were removed under microscopic guidance while the blood vessels were bathed in Krebs solution of the following composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.4 mM CaCl2, 25 mM NaHCO3, 11 mM glucose, and 0.03 mM EDTA. The aorta was then cut into 2.5-mm rings, which were suspended on Grass isometric transducers (FT-03, Grass Instrument, Quincy, MA) with a 2.0 g resting tension in 10 mL temperature-controlled baths (37°C) containing the Krebs solution that was continuously gassed with 95% O2 and 5% CO2. The rings were equilibrated at a 2.0 g resting tension for 120 min, during which time the bathing solution was changed every 15 min. In some aortic rings, the endothelium was intentionally removed by inserting a 25-gauge needle tip into the lumen of the rings and gently rubbing the ring for a few seconds. Only one concentration-response curve elicited by phenylephrine and KCl was made for each ring in the experiments. The contractile response induced by isotonic 60 mM KCl was measured in all the aortic rings. Once the isotonic 60 mM KCl-induced contraction had stabilized, acetylcholine (10–6 M) was added to assess the integrity of the endothelium. The endothelial integrity was confirmed by an observation of >30% relaxation by acetylcholine (10–6 M).
The first series of these experiments was aimed at assessing the effect of diazepam on the contractile response induced by the 
1-adrenoceptor agonist, phenylephrine, in the endothelium-intact and endothelium-denuded rings. Diazepam was added directly to the organ bath 20 min before the cumulative phenylephrine-induced contraction. The effect of diazepam on the concentration-response curves for phenylephrine (10–9 to 10–5 M) was assessed by comparing the contractile response in the presence or absence of diazepam (7 x 10–7, 5 x 10–6 M). In addition, the effect of the vehicle for diazepam (2 mL contains 0.79 mL propylene glycol, 0.16 mL ethanol, 0.03 mL benzyl alcohol and 1.02 mL distilled water), at an equivalent dose to diazepam (5 x 10–6 M), was also assessed in the endothelium-intact rings as a control.
The second series of experiments was designed to investigate the involvement of the endothelium-derived vasodilators (NO, EDHF, prostacyclin) in the diazepam-induced attenuation of the contractile response induced by phenylephrine. The effect of diazepam on the concentration-response curve for phenylephrine in the endothelium-intact rings pretreated independently with the NOS inhibitor, L-NAME (10–4 M), the calcium activated potassium channel blocker, 5 x 10–3 M tetraethylammonium (TEA), and the cyclooxygenase inhibitor, indomethacin (3 x 10–5 M), was assessed by comparing the contractile response in the presence or absence of diazepam (7 x 10–7, 5 x 10–6 M). There was a 30-min incubation period for each inhibitor plus diazepam or the inhibitor alone before the phenylephrine-induced contraction.
In the third series of experiments, the effect of diazepam on the contractile response induced by KCl in the endothelium-denuded rings was assessed by comparing the KCl dose-response curves obtained in the presence or absence of diazepam (7 x 10–7, 5 x 10–6 M). The diazepam was added directly to the organ bath 20 min before the KCl-induced contraction.
The fourth series of experiments was designed to confirm the participation of NO in the diazepam-induced attenuation of the contractile response induced by phenylephrine in the endothelium-intact rings. Diazepam was added directly to the organ bath 20 min before the phenylephrine (10–5 M)-induced maximal contraction in the endothelium-intact rings. L-NAME (10–4 M) was added directly to the organ bath after the phenylephrine-induced maximal contraction reached a stable tension in the presence or absence of diazepam (5 x 10–6 M). The effect of diazepam on the L-NAME-induced contraction in the endothelium-intact rings in the stable maximal contractile state induced by phenylephrine (10–5 M) was assessed by comparing the contractile response in the presence or absence of diazepam (5 x 10–6 M).
Finally, the role of the peripheral benzodiazepine receptor in the diazepam-induced attenuation of the contractile response induced by phenylephrine was determined by examining the phenylephrine concentration-response curve 30 min after the peripheral benzodiazepine receptor antagonist, PK 11195 (10–6 M), had been added directly to the organ bath, either alone or in combination with diazepam (5 x 10–6 M).
All the drugs were of the highest purity commercially available: phenylephrine HCl, L-NAME, PK 11195, indomethacin, TEA (Sigma Chemical, St. Louis, MO), diazepam (Myungin Pharmaceutical CO, Ltd., Seoul, Republic of Korea). All the drug concentrations are expressed as the final molar concentration in the organ bath. PK 11195 was initially dissolved in dimethyl sulfoxide and subsequently diluted in distilled water (final dimethyl sulfoxide concentration: 0.01%). Indomethacin was initially dissolved in 95% ethanol and then diluted in distilled water (final ethanol concentration: 0.28%). Unless stated otherwise, all other drugs were dissolved and diluted in distilled water.
The values are expressed as mean ± sd. The contractile responses to phenylephrine are expressed as percentages of their own maximum contraction to isotonic 60 mM KCl. The logarithm of the drug concentration (ED50) eliciting 50% of the maximum contractile response was calculated using nonliner regression analysis by fitting the concentration-response relation for phenylephrine to a sigmoidal curve using commercially available software (Prism version 2.0; GraphPad Software, San Diego, CA). The data were fitted to a sigmoid dose-response curve using the following algorithm Y = Bottom + (Top – Bottom)/(1 + 10
((LogED50 – X) x Hill Slope)). The phenylephrine-induced maximal contractile responses were calculated as the percentage of their own maximum contraction to isotonic 60 mM KCl. The L-NAME (10–4 M)-induced maximal contraction was calculated as the percentage change from the phenylephrine (10–5 M)-induced maximal contraction obtained in the absence or presence of diazepam (5 x 10–6 M). Statistical analysis for the comparison of ED50 and the contractions (maximal contraction or contractions at each KCl concentration) between the no-drug and diazepam-treated groups was performed using Student's t-test. A P value < 0.05 was considered significant. N refers to the number of rats whose descending thoracic aortic rings were used in each experiment. Each group contained at least two rings from the same rat.
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Results |
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In the endothelium-intact rings, diazepam (7 x 10–7, 5 x 10–6 M) increased (P < 0.05) the ED50 (no drug: –7.29 ± 0.19, 7 x 10–7 M diazepam: –7.02 ± 0.20, 5 x 10–6 M diazepam: –6.82 ± 0.28) for phenylephrine, and a large dose (5 x 10–6 M) of diazepam decreased (P < 0.05) the phenylephrine-induced maximal contraction compared with the rings not treated with diazepam (Fig. 1A). In the endothelium-denuded rings, a small dose (7 x 10–7 M) of diazepam had no effect, whereas a large dose (5 x 10–6 M) of diazepam increased (P < 0.05) the ED50 (no drug: –8.46 ± 0.31, 5 x 10–6 M diazepam: –8.08 ± 0.18) for phenylephrine compared with the rings not treated with diazepam (Fig. 1B). In the endothelium-intact rings, the vehicle for diazepam had no significant effect on the phenylephrine concentration-response curve (ED50: no drug: –7.17 ± 0.18, vehicle: –7.14 ± 0.25).
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Diazepam (7 x 10–7 M) had no effect on phenylephrine-induced contraction in the endothelium-intact rings pretreated with L-NAME. Moreover, a large dose (5 x 10–6 M) of diazepam had no effect on the phenylephrine-induced maximal contraction and increased (P < 0.05) the ED50 (10–4 M L-NAME: –7.72 ± 0.21, 10–4 M L-NAME + 5 x 10–6 M diazepam: –7.45 ± 0.22) for phenylephrine compared with the rings not treated with diazepam (Fig. 2).
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In the endothelium-intact rings pretreated with TEA, diazepam (5 x 10–6 M) increased (P < 0.05) the ED50 (5 x 10–3 M TEA: –7.79 ± 0.34, 5 x 10–3 M TEA + 5 x 10–6 M diazepam: –7.16 ± 0.18) for phenylephrine and decreased the phenylephrine-induced maximal contraction compared with the rings not treated with diazepam (Fig. 3).
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In the endothelium-intact rings pretreated with indomethacin, diazepam decreased (P < 0.05) the phenylephrine-induced maximal contraction compared with the rings not treated with diazepam (Fig. 4).
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Diazepam (7 x 10–7, 5 x 10–6 M) had no significant effect on the KCl-induced contraction compared with the endothelium-denuded rings not treated with diazepam.
In the endothelium-intact rings that developed a stable phenylephrine (10–5 M)-induced maximal contraction in the presence or absence of diazepam (5 x 10–6 M), L-NAME (10–4 M) induced a larger contractile response (P < 0.05) in the diazepam (5 x 10–6 M)-pretreated ring than in the ring not treated with diazepam (Fig. 5).
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In the endothelium-intact rings pretreated with PK 11195 (10–6 M), diazepam (5 x 10–6 M) increased (P < 0.05) the ED50 (10–6 M PK 11195: –7.18 ± 0.28, 10–6 M PK 11195 + 5 x 10–6 M diazepam: –6.77 ± 0.27) for phenylephrine and decreased (P < 0.05) the phenylephrine-induced maximal contraction compared with the rings not treated with diazepam (Fig. 6A). In addition, diazepam (5 x 10–6 M) increased (P < 0.05) the ED50 (10–6 M PK 11195: -8.26 ± 0.21, 10–6 M PK 11195 + 5 x 10–6 M diazepam: -7.94 ± 0.28) for phenylephrine in the endothelium-denuded rings pretreated with PK 11195 (10–6 M) (Fig. 6B).
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Discussion |
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Previous in vitro studies focused on the effect of benzodiazepine on calcium homeostasis using either blood vessels (4,11) or vascular smooth muscle cells (6). Our results indicate that diazepam (5 x 10–6 M), at supraclinical concentrations, attenuates phenylephrine-induced contraction by increasing endothelial NO activity and directly affecting vascular smooth muscle. This diazepam-induced attenuation did not occur via peripheral benzodiazepine receptor activation.
Diazepam (7 x 10–7 M) attenuated the phenylephrine-induced contraction in the endothelium-intact rings but had no effect on the endothelium-denuded rings. In addition, larger doses of diazepam (5 x 10–6 M) produced a significant rightward shift in the phenylephrine concentration-response curve and attenuated the maximal contractile response in the endothelium-intact rings but had no effect on the maximal contraction and produced a significant rightward shift in the phenylephrine concentration response in the endothelium-denuded rings. Overall, these results suggest that the diazepam-induced attenuation was endothelium-dependent.
The most important endothelium-derived autacoids are NO and prostacyclin (9). In addition, EDHF causes endothelium-dependent relaxation in the rat aorta, which is resistant to the full blockade of NOS and cyclooxygenase (12). Previous studies (12,13) reported that NO inhibits the contractile agonist-induced contraction of rat aorta. The L-NAME pretreatment completely abolished the diazepam (7 x 10–7 M)-induced attenuation of the phenylephrine concentration-response curve as well as the diazepam (5 x 10–6 M)-induced attenuation of the maximal contractile response to phenylephrine. These results, along with previous reports (12,13), suggest that the diazepam-induced attenuation of the phenylephrine concentration-response curve is in part mediated via the modulation of the phenylephrine-induced contraction by NO. TEA (10–2 M) enhances the contractile response to norepinephrine in the endothelium-intact rings (12), suggesting that EDHF attenuates the contractile response to phenylephrine. Diazepam caused a significant rightward shift in the phenylephrine concentration-response curves in the endothelium-intact rings pretreated with TEA (5 x 10–3 M) and attenuated the maximal contraction. In the endothelium-intact rings pretreated with indomethacin, diazepam (5 x 10–6 M) attenuated the phenylephrine-induced maximal contraction. Indomethacin does not alter the maximal contraction to vasopressin in male rat aorta (13). Overall, the diazepam-induced attenuation of the phenylephrine concentration-response curve in rat aorta is not associated with increased endothelial prostacyclin production and EDHF-mediated vasodilation.
The L-NAME-induced contractile response was significantly enhanced in endothelium-intact rings that developed a stable phenylephrine-induced maximal contraction in the presence of diazepam (5 x 10–6 M). Galindo et al. (7) reported that L-NAME pretreatment in the endothelium-denuded aortic rings attenuates the diazepam-induced relaxation, whereas endothelial denudation has no effect on diazepam-induced relaxation. This suggests that diazepam could stimulate the release of muscle NO via iNOS (7). In contrast, the diazepam-induced attenuation of phenylephrine concentration-response curve observed in the current study was more prominent in the endothelium-intact rings than in the endothelium-denuded rings, and L-NAME pretreatment in the endothelium-intact rings decreased the diazepam-induced attenuation, suggesting that diazepam-induced attenuation is associated with an endothelium-dependent mechanism involving enhanced NO activity. This might have been a result of the experimental method used to determine the cumulative phenylephrine concentration-response curves in the presence or absence of diazepam and their own maximum contraction to isotonic 60 mM KCl as the reference values in each ring. In contrast, cumulative diazepam dose-response curves and a 10–6 M phenylephrine-induced precontraction, as reference values in all the rings with or without an endothelium, were used by Galindo et al. (7). The use of phenylephrine ED50 (the concentration of phenylephrine required to generate a half-maximal contraction) for precontraction in each ring with or without endothelium is believed to be reasonable for examining the effect of endothelium-denudation on diazepam-induced relaxation because phenylephrine sensitivity resulting from the inhibition of the basal NO release was enhanced in the endothelium-denuded rings (14,15). In addition, it is unclear if diazepam-induced relaxation reported in reference (7) would be involved in the stimulated release of NO directly from the muscle by iNOS because iNOS expression appears 24 hours after endothelium-denudation in a rat aorta (16). Diazepam inhibits the formation of the superoxide anion (O2 –), which inactivates NO (17). Further study will be needed to determine the effect of diazepam on the endothelial NOS activity and superoxide anion production in the rat aorta.
Pretreatment with the peripheral benzodiazepine receptor antagonist, PK 11195 (18), did not significantly alter the diazepam (5 x 10–6 M)-induced attenuation of the phenylephrine concentration-response curve in aortic rings with or without an endothelium. The existence of a peripheral benzodiazepine receptor in rat aortic smooth muscle has been demonstrated (19). Previous studies reported that PK 11195 has no effect on diazepam-induced relaxation in phenylephrine-precontracted endothelium-denuded rat aorta (7) or airway smooth muscles (5). In accordance with previous studies (5,7), this inhibitory effect appears to be caused by the direct action on the rat aorta but not via the peripheral benzodiazepine receptor.
Diazepam (5 x 10–6 M) produced a significant rightward shift in the phenylephrine concentration-response curve in the endothelium-denuded rings, whereas diazepam (5 x 10–6 M) had no effect on the KCl-induced contraction. The benzodiazepine-induced relaxation observed in previous studies (4,5,11,14) appears to be associated with a decrease in the intracellular Ca2+ concentration. We speculate that an endothelium-independent mechanism appears to be associated with the diazepam (5 x 10–6 M)-induced attenuation through a decrease in the intracellular Ca2+ concentration on the vascular smooth muscle.
Diazepam at 7 x 10–7 M, which is the concentration in a clinical setting with 96.8% protein binding (20), attenuated the phenylephrine-induced contraction. Diazepam is highly lipid-soluble (pH = 7.4, octanol; water partition coefficient = 840) (1). With the enhanced unidirectional brain uptake resulting from increased lipophilicity, the effective unbound fraction in the brain capillaries is substantially larger than that estimated in vitro (21). Small changes in the amount or binding capacity of a protein in certain pathological conditions (liver disease, hemodilution, hypoalbuminemia) could result in an increase in the free fraction of diazepam. This suggests that the 5 x 10–6 M diazepam required for an inhibitory effect on phenylephrine-induced contraction might be the concentration encountered in a clinical setting such as an overdose of diazepam.
Diazepam reduces arterial blood pressure (22) and increases the coronary blood flow in humans (23). Any clinical implication of diazepam on the regional hemodynamics must be tempered by the fact that the aorta was used in this in vitro experiment, whereas the resistance vessels with the arterioles of a diameter of 100-300 µm control the major organ blood flow (24). Proceeding from the larger to smaller arteries and arterioles, the relative importance of EDHF in the control of blood flow increases whereas that of NO decreases (25). Even with these limitations, the vasorelaxant effect of diazepam in this in vitro study might help explain the hypotension or vasodilation observed in previous in vivo studies (22,23).
In conclusion, a clinical dose (7 x 10–7 M) of diazepam attenuates the phenylephrine-induced contractions via an endothelium-dependent mechanism. This endothelium-dependent mechanism is associated with increased NO activity. Diazepam-induced attenuation is independent of the peripheral benzodiazepine receptor activation.
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Footnotes |
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Supported, in part, by a research grant from Gyeongsang National University Hospital.
Accepted for publication October 17, 2005.
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