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Thiobarbiturates suppress depolarization-induced contraction of vascular smooth muscle without suppression of calcium influx
British Journal of Anaesthesia 1996;77:503–507
LABORATORY INVESTIGATIONS
Thiobarbiturates suppress depolarization-induced contraction of vascular smooth muscle without suppression of calcium influx R. KITAMURA, M. KAKUYAMA, K. NAKAMURA, I. MIYAWAKI AND K. MORI
Summary We have studied the effects of barbiturates on vascular smooth muscle tension and cytosolic calcium concentrations ([Ca2;]i) in endotheliumdenuded rat aortic rings, preloaded with fluo-3. Changes in [Ca2;]i were estimated by the fluorescence intensity of the calcium-bound form of fluo-3. In aortic rings under basal conditions, thiobarbiturates (thiopentone and thiamylal 100–300 mol litre91) increased [Ca2;]i, concomitantly with an increase in tension, although oxybarbiturates (pentobarbitone and secobarbitone up to 300 mol litre91) had no effect. Thiopentone (300 mol litre91)-induced increases in tension and fluorescence intensity were mean 25.1 (SD 3.2) % and 55.0 (6.0) %, respectively, of those induced by KCl 30 mmol litre91 (n:8, each). In KCl (30 mmol litre91)-precontracted aortic rings, thiopentone decreased tension without reduction of [Ca2;]i, whereas pentobarbitone decreased tension and [Ca2;]i. KCl (30 mmol litre91)-induced contraction was suppressed by pretreatment with all barbiturates (100–300 mol litre91); thiopentone 300 mol litre91 suppressed contraction to 64.8 (2.5) % (n:6) and pentobarbitone 300 mol litre91 to 57.5 (2.2) % (n:9). However, the increase in [Ca2;]i was suppressed by oxybarbiturates (pentobarbitone 300 mol litre91 to 77.9 (5.2) %; n:9), but not altered by thiobarbiturates. These results suggest that thiobarbiturates and oxybarbiturates affect vascular smooth muscle differently and that the affected site in thiobarbiturate-induced vasodilatation is distal to regulation of [Ca2;]i. (Br. J. Anaesth. 1996;77:503– 507) Key words Muscle vascular, responses. Anaesthetics i.v., thiopentone. Anaesthetics i.v., thiamylal. Hypnotics barbiturate, pentobarbitone. Hypnotics barbiturate, secobarbitone. Ions, calcium.
Thiobarbiturates have been used for induction of general anaesthesia and also for brain protection in cerebral ischaemia. Although the reduction in arterial pressure after i.v. injection of thiobarbiturates may be caused mainly by suppression of sympathetic tone and cardiac contractility, barbiturates have been shown to exert direct effects on vascular smooth muscle of various origins1–9. Previous authors have suggested that the direct vascular effect of pentobarbitone is a result of its Ca2; channel blocking action10.
Except for our previous reports11–13, however, there have been no investigations of the mechanism of thiobarbiturate-induced vasodilatation. In this study, we used a fluorescent indicator of cytosolic calcium concentration ([Ca2;]i) to determine the effect of barbiturates on [Ca2;]i under basal conditions and on depolarization-induced calcium influx. We demonstrated different effects of oxybarbiturates and thiobarbiturates on [Ca2;]i and calcium-contraction coupling in vascular smooth muscle.
Materials and methods The study was approved by the Kyoto University Animal Use Committee. We studied 20 male Wistar rats (250–350 g) anaesthetized with pentobarbitone 50 mg kg91 i.p. and killed by exsanguination from the common carotid arteries. The descending part of the thoracic aorta was isolated from each rat and dissected into 8–10 rings (2-mm wide). The rings were rinsed and bathed in Krebs’ bicarbonate solution at 25 ⬚C for more than 20 min until used. The rings were then bathed in Krebs’ bicarbonate solution containing fluo 3-AM 10 mol litre91 14 and 0.02% vol/vol Cremophor EL for 0.5–1 h at 25 ⬚C. Cremophor EL is a non-cytotoxic detergent which was added to increase the solubility of fluo 3-AM15. After rinsing, the endothelium was removed from the luminal surface by rubbing with cotton sticks in order to exclude the influence of endothelial [Ca2;]i on fluorescence intensity. The vascular rings were then mounted under a resting tension of 1 g weight, in Krebs’ bicarbonate solution, maintained at 37 ⬚C, and aerated with a mixture of 5% carbon dioxide in oxygen. They were then contracted with phenylephrine 0.1 mol litre91 and exposed to acetylcholine 10 mol litre91 which induced less than 10% of the phenylephrine-induced precontraction in all rings used16. Isometric tension and fluorescence intensity were recorded continuously using an Amplifier Case 7903 force-displacement transducer (San-ei, Tokyo, Japan) and a CAF-110 fluorometer (Japan Spectroscopic, Tokyo, Japan)17. The arterial rings were illuminated with a 75-W xenon lamp at an excitation wavelength of 490 nm, and the emitted fluorescence at 540 nm was considered to be an indicator of RIE KITAMURA, MD, MASAHIRO KAKUYAMA, MD, KUMI NAKAMURA, MD, IKUKO MIYAWAKI, MD, KENJIRO MORI, MD, FRCA, Department of Anesthesia, Kyoto University Hospital, Kyoto 606-01, Japan. Accepted for publication: June 4, 1996. Correspondence to R. K.
504 [Ca2;]i14. In our preliminary study, thiopentone 300 mol litre91 decreased the transmittance of light at wavelengths of 340 nm and 380 nm to 91.8% and 94.0%, respectively, and thiamylal to 86.5% and 96.3%. However, transmittance of 490 nm light was not significantly altered by the barbiturates tested (up to 1 mmol litre91). In the relaxation study, vascular rings were precontracted with KCl 30 mmol litre91. When the tension and fluorescence intensity had stabilized after KCl exposure, the rings were exposed to cumulative concentrations of thiopentone or pentobarbitone (30– 300 mol litre91). The time-control was run in an identical manner but without exposure to barbiturates. The stabilized tension and fluorescence intensity after exposure to each barbiturate concentration were expressed as percentages, taking the levels before exposure to KCl as 0% and those after exposure and just before the start of barbiturate exposure as 100%. In the contraction study, all rings were first exposed to KCl 30 mmol litre91 for 5 min and then washed out. The change induced by this first exposure to KCl was taken as 100%, and the changes in tension and fluorescence intensity in the following study were expressed as values relative to this. The rings were then exposed to one of the barbiturates for 10 min or, after 5 min of pretreatment with a barbiturate, exposed to KCl 30 mmol litre91 again for another 5 min. The barbiturates used were thiopentone, thiamylal, pentobarbitone and secobarbitone (30–300 mol litre91). After washout, the rings were exposed to KCl 30 mmol litre91 as a post-barbiturate control. The time-control was run in an identical manner but without barbiturates. Krebs’ bicarbonate solution contained (mmol litre91): NaCl 127.5, NaHCO3 15.5, glucose 10.0, KCl 4.6, KH2PO4 1.2, MgSO4 1.2 and CaCl2 2.5. Drugs used were fluo 3-AM (Dojindo Laboratories, Kumamoto, Japan), Cremophor EL (Sigma, St Louis, MO, USA), L-phenylephrine hydrochloride (Sigma), acetylcholine (Daiichi Pharmaceutical Co., Tokyo, Japan), sodium thiopentone (Tanabe Pharmaceutical, Osaka, Japan), sodium thiamylal (Kyorin Pharmaceutical, Tokyo, Japan), sodium secobarbitone (Yoshitomi Pharmaceutical, Osaka, Japan) and sodium pentobarbitone (Nacalai Tesque, Kyoto, Japan). Data in the relaxation study were analysed by Student’s t test for unpaired data. All other data were analysed using analysis of variance and Scheffe’s test. Differences were considered significant at P:0.05. In the relaxation study, vascular rings were exposed to KCl 30 mmol litre91 twice, and cumulative concentrations of thiopentone or pentobarbitone. The order of the experiments for the two barbiturates was randomized. In the contraction study, after the first exposure to KCl and washout, the rings were exposed to one of the barbiturates for 10 min, or exposed to KCl again for another 5 min after 5 min of pretreatment with a barbiturate. The timecontrols were run in an identical manner, but without barbiturates.
Results KCl-precontracted aortic rings were relaxed by thiopentone and pentobarbitone 100–300 mol litre91 in a concentration-dependent manner (P:0.05–0.01)
British Journal of Anaesthesia
Figure 1 Representative recordings of fluorescence intensity (as an indicator of [Ca2;]i) and tension in rat aortic rings, which had been precontracted with KCl 30 mmol litre91 and then exposed to thiopentone (A) or pentobarbitone (B) in cumulative concentrations (30–300 mol litre91).
(figs 1, 2). [Ca2;]i, as measured by fluorescence intensity, was decreased by pentobarbitone 300 mol litre91 (P:0.05), but not by thiopentone (figs 1, 2). Mean thiopentone- and pentobarbitone 300 mol litre91-induced reductions in tension were 59.4 (SD 2.8) % and 40.7 (4.4) %, respectively, and pentobarbitone 300 mol litre91-induced reduction in [Ca2;]i was 931.2 (17.0) % (n:4, each). The tension and fluorescence intensity of aortic rings under basal conditions were increased by 10 min exposure to thiopentone and thiamylal 100–300 mol litre91 (figs 3, 4), but not to pentobarbitone or secobarbitone (up to 300 mol litre91). The increase in tension and fluorescience intensity induced by thiopentone 300 mol litre91 was 25.1 (3.2) % and 55.0 (6.0) % of KCl 30 mmol litre91-induced changes, respectively, and those for thiamylal 300 mol litre91 were 29.1 (8.9) % and 48.5 (5.5) %, respectively. Although the addition of KCl 30 mmol litre91 to barbiturate-treated rings increased their tension and fluorescence intensity, the total increase in tension induced by KCl and barbiturates was less than in the barbiturate-untreated controls (P:0.05–0.01) (fig. 4). The changes in tension in the presence of thiopentone, thiamylal, pentobarbitone and secobarbitone 300 mol litre91 were 64.8 (2.5)% (n:6), 70.8 (4.2) % (n:6), 57.5 (2.2) % (n:9) and 36.6 (3.4) % (n:6), respectively. KCl-induced increase in fluorescence intensity was decreased by pentobarbitone and secobarbitone 300 mol litre91 to 77.9 (5.2) % (n:9) and 67.3 (8.7) % (n:6), respectively (P:0.05). In contrast, the total
Thiobarbiturates and contraction of vascular smooth muscle
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Figure 2 Changes in fluorescence intensity and tension of aortic rings precontracted with KCl 30 mmol litre91. :Without barbiturates; ●:exposed to thiopentone or pentobarbitone at cumulative concentrations (30–300 mol litre91). Levels before exposure to KCl 30 mmol litre91 were taken as 0% and those just before exposure to barbiturates as 100%. n:4, each. *P:0.05, **P:0.01 compared with untreated control.
Figure 3 Representative recordings of fluorescence intensity and tension of aortic rings exposed twice to KCl 30 mmol litre91 in the absence (first exposure) and presence of thiopentone 100 mol litre91 (second exposure) (A). Recordings of rings exposed to thiopentone but not to KCl are also shown (B).
increase in fluorescence intensity in rings treated with up to 300 mol litre91 of thiopentone or thiamylal was not significantly different from controls. Changes in tension and fluorescence intensity induced by the post-barbiturate and KCl-exposure were not significantly different from the prebarbiturate control values (data not shown), indicating that the effects of the barbiturates were reversible.
Discussion One might criticize the fact that we used pentobarbitone anaesthesia before exsanguination of rats, arguing that pentobarbitone injected i.p. would have affected the result. However, our in vitro results suggested that this was unlikely, because the vascular effects of barbiturates at up to 300 mol litre91 added to the bathing solution were abolished completely
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British Journal of Anaesthesia
Figure 4 Changes in fluorescence intensity (open bars) and tension (hatched bars) induced by KCl 30 mmol litre91 and barbiturates 30–300 mol litre91. Solid bars:changes induced by barbiturates alone. Changes induced by the first KCl-exposure in the absence of barbiturates (pre-barbiturate controls) were taken as 100%. Values in parentheses indicate the number of rings studied. *P:0.05, **P:0.01 compared with values without barbiturates (time-controls).
after washout and the anaesthetic concentration of pentobarbitone was less than 300 mol litre91. Also, in preliminary studies we used rats anaesthetized with both ketamine and pentobarbitone and the results obtained were similar. Our previous studies demonstrated that thiobarbiturates, but not oxybarbiturates, exerted vasoconstrictive effects on rat aortae and canine peripheral arteries under basal conditions7–9. This study confirmed these findings and further demonstrated that the thiobarbiturate-induced contractions in aortic rings under basal conditions were associated with increased [Ca2;]i. In contrast, in KCl-precontracted aortic rings, the thiopentone-induced decrease in tension was not associated with a reduction in [Ca2;]i. Moreover, although the KCl-induced contraction was smaller in thiobarbiturate-treated than in untreated rings, the KCl-induced increase in [Ca2;]i did not differ between the two treatments. Previous studies by Sanchez-Ferrer and colleagues6 and ourselves11–13 have shown that thiobarbiturates suppressed contractions induced by an influx of extracellular Ca2; and also those caused by the release of Ca2; from intracellular stores in vascular smooth muscle, including those mediated by inositol triphosphate (IP3) and calcium-induced calcium release (CICR). These results suggest two possibilities; either thiobarbiturates suppress Ca2; mobilization by non-specific means or they affect some site distal to the regulation of [Ca2;]i. The former possibility was clearly not supported by the present finding that thiobarbiturates, at concentrations of up to 300 mol litre91, did not decrease [Ca2;]i in the presence of high K. Furthermore, the latter possibility was supported by previous reports showing that thiobarbiturates suppressed Ca2;-induced contractions of chemically skinned muscle fibres from canine mesenteric artery11 12.
Figure 5 Chemical structures of thiopentone, thiamylal, pentobarbitone and secobarbitone. R1:-CH2CH3; R2:- CH2CH:CH2.
In contrast with the thiobarbiturates, relaxation induced by pentobarbitone was accompanied by a decrease in [Ca2;]i. In oxybarbiturate-pretreated rings, KCl-induced changes in [Ca2;]i were smaller than in control rings. These findings concur with those of previous authors10 and ourselves using these compounds12 13. Therefore we speculate that oxybarbiturates suppress depolarization-induced calcium influx, although the possibility that they also affect some site distal to calcium mobilization cannot be excluded. Except for substitution of an oxygen atom for sulphur on the second carbon atom, thiopentone has the same chemical structure as pentobarbitone, and thiamylal the same as secobarbitone (fig. 5). Thus we postulate that thiobarbiturates and oxybarbiturates,
Thiobarbiturates and contraction of vascular smooth muscle at concentrations of up to 300 mol litre91, have different effects on smooth muscle from rat aorta; only thiobarbiturates increased [Ca2;]i to constrict rat aorta under basal conditions, whereas only oxybarbiturates decreased [Ca2;]i in KCl-contracted rat aorta. In addition to their effects on [Ca2;]i, thiobarbiturates seemed to affect some site distal to the regulation of [Ca2;]i, such as calmodulin, the phosphorylation or dephosphorylation process of myosin light chain (MLC), or the contractile machinery itself. The peak plasma concentration of thiobarbiturates after induction of anaesthesia is 100–300 mol litre91, and as more than 85% is bound to plasma proteins, the concentration of the unbound form is less than 50 mol litre91 18 19. Therefore, the effective concentrations of thiobarbiturates in this study were not much higher than those that are found clinically. In summary, we found that thiobarbiturates relaxed vascular smooth muscle isolated from rat aorta independently of [Ca2;]i, while oxybarbiturates reduced both tension and [Ca2;]i. In regulating vascular smooth muscle tone, thiobarbiturates seemed to affect sites distal to the regulation of [Ca2;]i.
References 1. Price ML, Price HL. Effects of general anesthetics on contractile responses of rabbit aortic strips. Anesthesiology 1962; 23: 16–20. 2. Altura BT, Altura BM. Barbiturates and aortic and venous smooth-muscle function. Anesthesiology 1975; 43: 432–444. 3. Clark SC, MacCannell KL. Vascular responses to anaesthetic agents. Canadian Anaesthetists Society Journal 1975; 22: 20–33. 4. Edney SM, Downes H. Contractor effect of barbiturates on smooth muscle. Archives Internationales de Pharmacodynamie et de Therapie 1975; 217: 180–196. 5. Fukuda S, Inomata I, Tsuji T, Takeshita H. Thiopental potentiation of isolated rabbit pulmonary artery contractions with alpha receptor agonists. Anesthesiology 1984; 60: 187– 192. 6. Sanchez-Ferrer CF, Martin J, Salaices M, Rico ML, Munoz-
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7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18. 19.
Blanco JL. Interference of pentobarbital and thiopental with the vascular contraction and noradrenaline release in human cerebral arteries. General Pharmacology 1985; 16: 469–473. Hatano Y, Nakamura K, Moriyama S, Mori K, Toda N. The contractile responses of isolated dog cerebral and extracerebral arteries to oxybarbiturates and thiobarbiturates. Anesthesiology 1989; 71: 80–86. Moriyama S, Nakamura K, Hatano Y, Harioka T, Mori K. Responses to barbiturates of isolated dog cerebral and mesenteric arteries contracted with KCl and prostaglandin F2␣. Acta Anaesthesiologica Scandinavica 1990; 34: 523–529. Nakamura K, Hatano Y, Nishiwada M, Yakushiji T, Park JW, Mori K. Effects of thiobarbiturates on smooth muscle reactivity in isolated aortas from spontaneously hypertensive rats. Anesthesia and Analgesia 1990; 70: 267–274. Altura BA, Turlapaty PDMV, Altura BM. Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle. Biochimica et Biophysica Acta 1980; 595: 309–312. Nakamura K, Hatano Y, Nishiwada M, Mori K. The vasodilator effect of thiamylal in dog mesenteric artery: contribution of intracellular action. Canadian Journal of Physiology and Pharmacology 1991; 69: 1163–1167. Yakushiji T, Nakamura K, Hatano Y, Mori K. Comparison of the vasodilator effects of thiopentone and pentobarbitone. Canadian Journal of Anaesthesia 1992; 39: 604–609. Nishiwada M, Nakamura K, Hatano Y, Mori K. The relaxing effects of barbiturates in vascular smooth muscle of rat aorta. Journal of Anesthesia 1991; 5: 380–387. Kao JPY, Harootunian AT, Tsien RY. Photochemically generated cytosolic pulses and their detection by fluo-3. Journal of Biological Chemistry 1989; 264: 8179–8184. Sato K, Ozaki H, Karaki H. Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator Fura 2. Journal of Pharmacology and Experimental Therapeutics 1988; 246: 294–300. Furchgott RF. Role of endothelium in responses of vascular smooth muscle. Circulation Research 1983; 53: 557–573. Kakuyama M, Hatano Y, Nakamura K, Toda H, Terasako K, Nishiwada M, Mori K. Halothane and enflurane constrict canine mesenteric arteries by releasing Ca2; from intracellular Ca2; stores. Anesthesiology 1994; 80: 1120–1127. Becker KE. Plasma levels of thiopental necessary for anesthesia. Anesthesiology 1978; 49: 192–196. Burch PG, Stanski DR. The role of metabolism and protein binding in thiopental anesthesia. Anesthesiology 1983; 58: 146–152.
LABORATORY INVESTIGATIONS
Thiobarbiturates suppress depolarization-induced contraction of vascular smooth muscle without suppression of calcium influx R. KITAMURA, M. KAKUYAMA, K. NAKAMURA, I. MIYAWAKI AND K. MORI
Summary We have studied the effects of barbiturates on vascular smooth muscle tension and cytosolic calcium concentrations ([Ca2;]i) in endotheliumdenuded rat aortic rings, preloaded with fluo-3. Changes in [Ca2;]i were estimated by the fluorescence intensity of the calcium-bound form of fluo-3. In aortic rings under basal conditions, thiobarbiturates (thiopentone and thiamylal 100–300 mol litre91) increased [Ca2;]i, concomitantly with an increase in tension, although oxybarbiturates (pentobarbitone and secobarbitone up to 300 mol litre91) had no effect. Thiopentone (300 mol litre91)-induced increases in tension and fluorescence intensity were mean 25.1 (SD 3.2) % and 55.0 (6.0) %, respectively, of those induced by KCl 30 mmol litre91 (n:8, each). In KCl (30 mmol litre91)-precontracted aortic rings, thiopentone decreased tension without reduction of [Ca2;]i, whereas pentobarbitone decreased tension and [Ca2;]i. KCl (30 mmol litre91)-induced contraction was suppressed by pretreatment with all barbiturates (100–300 mol litre91); thiopentone 300 mol litre91 suppressed contraction to 64.8 (2.5) % (n:6) and pentobarbitone 300 mol litre91 to 57.5 (2.2) % (n:9). However, the increase in [Ca2;]i was suppressed by oxybarbiturates (pentobarbitone 300 mol litre91 to 77.9 (5.2) %; n:9), but not altered by thiobarbiturates. These results suggest that thiobarbiturates and oxybarbiturates affect vascular smooth muscle differently and that the affected site in thiobarbiturate-induced vasodilatation is distal to regulation of [Ca2;]i. (Br. J. Anaesth. 1996;77:503– 507) Key words Muscle vascular, responses. Anaesthetics i.v., thiopentone. Anaesthetics i.v., thiamylal. Hypnotics barbiturate, pentobarbitone. Hypnotics barbiturate, secobarbitone. Ions, calcium.
Thiobarbiturates have been used for induction of general anaesthesia and also for brain protection in cerebral ischaemia. Although the reduction in arterial pressure after i.v. injection of thiobarbiturates may be caused mainly by suppression of sympathetic tone and cardiac contractility, barbiturates have been shown to exert direct effects on vascular smooth muscle of various origins1–9. Previous authors have suggested that the direct vascular effect of pentobarbitone is a result of its Ca2; channel blocking action10.
Except for our previous reports11–13, however, there have been no investigations of the mechanism of thiobarbiturate-induced vasodilatation. In this study, we used a fluorescent indicator of cytosolic calcium concentration ([Ca2;]i) to determine the effect of barbiturates on [Ca2;]i under basal conditions and on depolarization-induced calcium influx. We demonstrated different effects of oxybarbiturates and thiobarbiturates on [Ca2;]i and calcium-contraction coupling in vascular smooth muscle.
Materials and methods The study was approved by the Kyoto University Animal Use Committee. We studied 20 male Wistar rats (250–350 g) anaesthetized with pentobarbitone 50 mg kg91 i.p. and killed by exsanguination from the common carotid arteries. The descending part of the thoracic aorta was isolated from each rat and dissected into 8–10 rings (2-mm wide). The rings were rinsed and bathed in Krebs’ bicarbonate solution at 25 ⬚C for more than 20 min until used. The rings were then bathed in Krebs’ bicarbonate solution containing fluo 3-AM 10 mol litre91 14 and 0.02% vol/vol Cremophor EL for 0.5–1 h at 25 ⬚C. Cremophor EL is a non-cytotoxic detergent which was added to increase the solubility of fluo 3-AM15. After rinsing, the endothelium was removed from the luminal surface by rubbing with cotton sticks in order to exclude the influence of endothelial [Ca2;]i on fluorescence intensity. The vascular rings were then mounted under a resting tension of 1 g weight, in Krebs’ bicarbonate solution, maintained at 37 ⬚C, and aerated with a mixture of 5% carbon dioxide in oxygen. They were then contracted with phenylephrine 0.1 mol litre91 and exposed to acetylcholine 10 mol litre91 which induced less than 10% of the phenylephrine-induced precontraction in all rings used16. Isometric tension and fluorescence intensity were recorded continuously using an Amplifier Case 7903 force-displacement transducer (San-ei, Tokyo, Japan) and a CAF-110 fluorometer (Japan Spectroscopic, Tokyo, Japan)17. The arterial rings were illuminated with a 75-W xenon lamp at an excitation wavelength of 490 nm, and the emitted fluorescence at 540 nm was considered to be an indicator of RIE KITAMURA, MD, MASAHIRO KAKUYAMA, MD, KUMI NAKAMURA, MD, IKUKO MIYAWAKI, MD, KENJIRO MORI, MD, FRCA, Department of Anesthesia, Kyoto University Hospital, Kyoto 606-01, Japan. Accepted for publication: June 4, 1996. Correspondence to R. K.
504 [Ca2;]i14. In our preliminary study, thiopentone 300 mol litre91 decreased the transmittance of light at wavelengths of 340 nm and 380 nm to 91.8% and 94.0%, respectively, and thiamylal to 86.5% and 96.3%. However, transmittance of 490 nm light was not significantly altered by the barbiturates tested (up to 1 mmol litre91). In the relaxation study, vascular rings were precontracted with KCl 30 mmol litre91. When the tension and fluorescence intensity had stabilized after KCl exposure, the rings were exposed to cumulative concentrations of thiopentone or pentobarbitone (30– 300 mol litre91). The time-control was run in an identical manner but without exposure to barbiturates. The stabilized tension and fluorescence intensity after exposure to each barbiturate concentration were expressed as percentages, taking the levels before exposure to KCl as 0% and those after exposure and just before the start of barbiturate exposure as 100%. In the contraction study, all rings were first exposed to KCl 30 mmol litre91 for 5 min and then washed out. The change induced by this first exposure to KCl was taken as 100%, and the changes in tension and fluorescence intensity in the following study were expressed as values relative to this. The rings were then exposed to one of the barbiturates for 10 min or, after 5 min of pretreatment with a barbiturate, exposed to KCl 30 mmol litre91 again for another 5 min. The barbiturates used were thiopentone, thiamylal, pentobarbitone and secobarbitone (30–300 mol litre91). After washout, the rings were exposed to KCl 30 mmol litre91 as a post-barbiturate control. The time-control was run in an identical manner but without barbiturates. Krebs’ bicarbonate solution contained (mmol litre91): NaCl 127.5, NaHCO3 15.5, glucose 10.0, KCl 4.6, KH2PO4 1.2, MgSO4 1.2 and CaCl2 2.5. Drugs used were fluo 3-AM (Dojindo Laboratories, Kumamoto, Japan), Cremophor EL (Sigma, St Louis, MO, USA), L-phenylephrine hydrochloride (Sigma), acetylcholine (Daiichi Pharmaceutical Co., Tokyo, Japan), sodium thiopentone (Tanabe Pharmaceutical, Osaka, Japan), sodium thiamylal (Kyorin Pharmaceutical, Tokyo, Japan), sodium secobarbitone (Yoshitomi Pharmaceutical, Osaka, Japan) and sodium pentobarbitone (Nacalai Tesque, Kyoto, Japan). Data in the relaxation study were analysed by Student’s t test for unpaired data. All other data were analysed using analysis of variance and Scheffe’s test. Differences were considered significant at P:0.05. In the relaxation study, vascular rings were exposed to KCl 30 mmol litre91 twice, and cumulative concentrations of thiopentone or pentobarbitone. The order of the experiments for the two barbiturates was randomized. In the contraction study, after the first exposure to KCl and washout, the rings were exposed to one of the barbiturates for 10 min, or exposed to KCl again for another 5 min after 5 min of pretreatment with a barbiturate. The timecontrols were run in an identical manner, but without barbiturates.
Results KCl-precontracted aortic rings were relaxed by thiopentone and pentobarbitone 100–300 mol litre91 in a concentration-dependent manner (P:0.05–0.01)
British Journal of Anaesthesia
Figure 1 Representative recordings of fluorescence intensity (as an indicator of [Ca2;]i) and tension in rat aortic rings, which had been precontracted with KCl 30 mmol litre91 and then exposed to thiopentone (A) or pentobarbitone (B) in cumulative concentrations (30–300 mol litre91).
(figs 1, 2). [Ca2;]i, as measured by fluorescence intensity, was decreased by pentobarbitone 300 mol litre91 (P:0.05), but not by thiopentone (figs 1, 2). Mean thiopentone- and pentobarbitone 300 mol litre91-induced reductions in tension were 59.4 (SD 2.8) % and 40.7 (4.4) %, respectively, and pentobarbitone 300 mol litre91-induced reduction in [Ca2;]i was 931.2 (17.0) % (n:4, each). The tension and fluorescence intensity of aortic rings under basal conditions were increased by 10 min exposure to thiopentone and thiamylal 100–300 mol litre91 (figs 3, 4), but not to pentobarbitone or secobarbitone (up to 300 mol litre91). The increase in tension and fluorescience intensity induced by thiopentone 300 mol litre91 was 25.1 (3.2) % and 55.0 (6.0) % of KCl 30 mmol litre91-induced changes, respectively, and those for thiamylal 300 mol litre91 were 29.1 (8.9) % and 48.5 (5.5) %, respectively. Although the addition of KCl 30 mmol litre91 to barbiturate-treated rings increased their tension and fluorescence intensity, the total increase in tension induced by KCl and barbiturates was less than in the barbiturate-untreated controls (P:0.05–0.01) (fig. 4). The changes in tension in the presence of thiopentone, thiamylal, pentobarbitone and secobarbitone 300 mol litre91 were 64.8 (2.5)% (n:6), 70.8 (4.2) % (n:6), 57.5 (2.2) % (n:9) and 36.6 (3.4) % (n:6), respectively. KCl-induced increase in fluorescence intensity was decreased by pentobarbitone and secobarbitone 300 mol litre91 to 77.9 (5.2) % (n:9) and 67.3 (8.7) % (n:6), respectively (P:0.05). In contrast, the total
Thiobarbiturates and contraction of vascular smooth muscle
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Figure 2 Changes in fluorescence intensity and tension of aortic rings precontracted with KCl 30 mmol litre91. :Without barbiturates; ●:exposed to thiopentone or pentobarbitone at cumulative concentrations (30–300 mol litre91). Levels before exposure to KCl 30 mmol litre91 were taken as 0% and those just before exposure to barbiturates as 100%. n:4, each. *P:0.05, **P:0.01 compared with untreated control.
Figure 3 Representative recordings of fluorescence intensity and tension of aortic rings exposed twice to KCl 30 mmol litre91 in the absence (first exposure) and presence of thiopentone 100 mol litre91 (second exposure) (A). Recordings of rings exposed to thiopentone but not to KCl are also shown (B).
increase in fluorescence intensity in rings treated with up to 300 mol litre91 of thiopentone or thiamylal was not significantly different from controls. Changes in tension and fluorescence intensity induced by the post-barbiturate and KCl-exposure were not significantly different from the prebarbiturate control values (data not shown), indicating that the effects of the barbiturates were reversible.
Discussion One might criticize the fact that we used pentobarbitone anaesthesia before exsanguination of rats, arguing that pentobarbitone injected i.p. would have affected the result. However, our in vitro results suggested that this was unlikely, because the vascular effects of barbiturates at up to 300 mol litre91 added to the bathing solution were abolished completely
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British Journal of Anaesthesia
Figure 4 Changes in fluorescence intensity (open bars) and tension (hatched bars) induced by KCl 30 mmol litre91 and barbiturates 30–300 mol litre91. Solid bars:changes induced by barbiturates alone. Changes induced by the first KCl-exposure in the absence of barbiturates (pre-barbiturate controls) were taken as 100%. Values in parentheses indicate the number of rings studied. *P:0.05, **P:0.01 compared with values without barbiturates (time-controls).
after washout and the anaesthetic concentration of pentobarbitone was less than 300 mol litre91. Also, in preliminary studies we used rats anaesthetized with both ketamine and pentobarbitone and the results obtained were similar. Our previous studies demonstrated that thiobarbiturates, but not oxybarbiturates, exerted vasoconstrictive effects on rat aortae and canine peripheral arteries under basal conditions7–9. This study confirmed these findings and further demonstrated that the thiobarbiturate-induced contractions in aortic rings under basal conditions were associated with increased [Ca2;]i. In contrast, in KCl-precontracted aortic rings, the thiopentone-induced decrease in tension was not associated with a reduction in [Ca2;]i. Moreover, although the KCl-induced contraction was smaller in thiobarbiturate-treated than in untreated rings, the KCl-induced increase in [Ca2;]i did not differ between the two treatments. Previous studies by Sanchez-Ferrer and colleagues6 and ourselves11–13 have shown that thiobarbiturates suppressed contractions induced by an influx of extracellular Ca2; and also those caused by the release of Ca2; from intracellular stores in vascular smooth muscle, including those mediated by inositol triphosphate (IP3) and calcium-induced calcium release (CICR). These results suggest two possibilities; either thiobarbiturates suppress Ca2; mobilization by non-specific means or they affect some site distal to the regulation of [Ca2;]i. The former possibility was clearly not supported by the present finding that thiobarbiturates, at concentrations of up to 300 mol litre91, did not decrease [Ca2;]i in the presence of high K. Furthermore, the latter possibility was supported by previous reports showing that thiobarbiturates suppressed Ca2;-induced contractions of chemically skinned muscle fibres from canine mesenteric artery11 12.
Figure 5 Chemical structures of thiopentone, thiamylal, pentobarbitone and secobarbitone. R1:-CH2CH3; R2:- CH2CH:CH2.
In contrast with the thiobarbiturates, relaxation induced by pentobarbitone was accompanied by a decrease in [Ca2;]i. In oxybarbiturate-pretreated rings, KCl-induced changes in [Ca2;]i were smaller than in control rings. These findings concur with those of previous authors10 and ourselves using these compounds12 13. Therefore we speculate that oxybarbiturates suppress depolarization-induced calcium influx, although the possibility that they also affect some site distal to calcium mobilization cannot be excluded. Except for substitution of an oxygen atom for sulphur on the second carbon atom, thiopentone has the same chemical structure as pentobarbitone, and thiamylal the same as secobarbitone (fig. 5). Thus we postulate that thiobarbiturates and oxybarbiturates,
Thiobarbiturates and contraction of vascular smooth muscle at concentrations of up to 300 mol litre91, have different effects on smooth muscle from rat aorta; only thiobarbiturates increased [Ca2;]i to constrict rat aorta under basal conditions, whereas only oxybarbiturates decreased [Ca2;]i in KCl-contracted rat aorta. In addition to their effects on [Ca2;]i, thiobarbiturates seemed to affect some site distal to the regulation of [Ca2;]i, such as calmodulin, the phosphorylation or dephosphorylation process of myosin light chain (MLC), or the contractile machinery itself. The peak plasma concentration of thiobarbiturates after induction of anaesthesia is 100–300 mol litre91, and as more than 85% is bound to plasma proteins, the concentration of the unbound form is less than 50 mol litre91 18 19. Therefore, the effective concentrations of thiobarbiturates in this study were not much higher than those that are found clinically. In summary, we found that thiobarbiturates relaxed vascular smooth muscle isolated from rat aorta independently of [Ca2;]i, while oxybarbiturates reduced both tension and [Ca2;]i. In regulating vascular smooth muscle tone, thiobarbiturates seemed to affect sites distal to the regulation of [Ca2;]i.
References 1. Price ML, Price HL. Effects of general anesthetics on contractile responses of rabbit aortic strips. Anesthesiology 1962; 23: 16–20. 2. Altura BT, Altura BM. Barbiturates and aortic and venous smooth-muscle function. Anesthesiology 1975; 43: 432–444. 3. Clark SC, MacCannell KL. Vascular responses to anaesthetic agents. Canadian Anaesthetists Society Journal 1975; 22: 20–33. 4. Edney SM, Downes H. Contractor effect of barbiturates on smooth muscle. Archives Internationales de Pharmacodynamie et de Therapie 1975; 217: 180–196. 5. Fukuda S, Inomata I, Tsuji T, Takeshita H. Thiopental potentiation of isolated rabbit pulmonary artery contractions with alpha receptor agonists. Anesthesiology 1984; 60: 187– 192. 6. Sanchez-Ferrer CF, Martin J, Salaices M, Rico ML, Munoz-
507
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18. 19.
Blanco JL. Interference of pentobarbital and thiopental with the vascular contraction and noradrenaline release in human cerebral arteries. General Pharmacology 1985; 16: 469–473. Hatano Y, Nakamura K, Moriyama S, Mori K, Toda N. The contractile responses of isolated dog cerebral and extracerebral arteries to oxybarbiturates and thiobarbiturates. Anesthesiology 1989; 71: 80–86. Moriyama S, Nakamura K, Hatano Y, Harioka T, Mori K. Responses to barbiturates of isolated dog cerebral and mesenteric arteries contracted with KCl and prostaglandin F2␣. Acta Anaesthesiologica Scandinavica 1990; 34: 523–529. Nakamura K, Hatano Y, Nishiwada M, Yakushiji T, Park JW, Mori K. Effects of thiobarbiturates on smooth muscle reactivity in isolated aortas from spontaneously hypertensive rats. Anesthesia and Analgesia 1990; 70: 267–274. Altura BA, Turlapaty PDMV, Altura BM. Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle. Biochimica et Biophysica Acta 1980; 595: 309–312. Nakamura K, Hatano Y, Nishiwada M, Mori K. The vasodilator effect of thiamylal in dog mesenteric artery: contribution of intracellular action. Canadian Journal of Physiology and Pharmacology 1991; 69: 1163–1167. Yakushiji T, Nakamura K, Hatano Y, Mori K. Comparison of the vasodilator effects of thiopentone and pentobarbitone. Canadian Journal of Anaesthesia 1992; 39: 604–609. Nishiwada M, Nakamura K, Hatano Y, Mori K. The relaxing effects of barbiturates in vascular smooth muscle of rat aorta. Journal of Anesthesia 1991; 5: 380–387. Kao JPY, Harootunian AT, Tsien RY. Photochemically generated cytosolic pulses and their detection by fluo-3. Journal of Biological Chemistry 1989; 264: 8179–8184. Sato K, Ozaki H, Karaki H. Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator Fura 2. Journal of Pharmacology and Experimental Therapeutics 1988; 246: 294–300. Furchgott RF. Role of endothelium in responses of vascular smooth muscle. Circulation Research 1983; 53: 557–573. Kakuyama M, Hatano Y, Nakamura K, Toda H, Terasako K, Nishiwada M, Mori K. Halothane and enflurane constrict canine mesenteric arteries by releasing Ca2; from intracellular Ca2; stores. Anesthesiology 1994; 80: 1120–1127. Becker KE. Plasma levels of thiopental necessary for anesthesia. Anesthesiology 1978; 49: 192–196. Burch PG, Stanski DR. The role of metabolism and protein binding in thiopental anesthesia. Anesthesiology 1983; 58: 146–152.