Anesth Analg 2002;94:174-178
© 2002 International Anesthesia Research Society
OBSTETRIC ANESTHESIA
The Role of K+ Channels on the Inhibitor Effect of Sevoflurane in Pregnant Rat Myometrium
Haluk Kafali, MD*,
Tijen Kaya, PhD
,
Sinan Gürsoy, MD*,
Ihsan Bagcivan, MD,
Baris Karadas, MD, and
Yusuf Sarioglu, PhD
Departments of *Anesthesiology and Pharmacology, Cumhuriyet University School of Medicine, Sivas, Turkey
Address correspondence and reprint requests to Dr. Haluk Kafali, Department of Anesthesiology, Cumhuriyet University School of Medicine, 58140 Sivas, Turkey. Address e-mail to haluk.kafali{at}isnet.net.tr
 |
Abstract
|
|---|
Volatile anesthetics and K+ channel openers inhibit spontaneous contractions in myometrial smooth muscle. Volatile anesthetics modulate K+ channel activity. We investigated the role of two K+ channel blockers on the effect of sevoflurane in pregnant rat myometrium. Term pregnant rat uteri were excised, and cross-sectional myometrial strips were mounted for isometric force recording. Sevoflurane inhibited the amplitude and frequency of spontaneous myometrial contractions in a concentration-dependent manner. The maximal inhibition measured in amplitude and frequency of spontaneous myometrial contractions with sevoflurane (at 3 minimum alveolar anesthetic concentration) was 44.32% and 33.32% of control contractions, respectively. Tetraethylammonium (TEA) and glibenclamide, K+ channel blockers, increased spontaneous myometrial contractions in a concentration-dependent manner. Sevoflurane responses were repeated at concentrations with no effect on spontaneous contractility of TEA, a Ca2+-activated K+ channel blocker, and glibenclamide, an adenosine triphosphate-sensitive K+ channel blocker, in myometrial strips. TEA (3.10-4 M) caused a significant reduction in sevoflurane-induced inhibitor responses, but glibenclamide (10-6 M) did not. Sevoflurane-induced maximal inhibition (at 3 minimum alveolar anesthetic concentration) on amplitude and frequency of spontaneous myometrial contractions in the presence of TEA (3.10-4 M) was 31.85% and 22.33% of control contractions, respectively (P < 0.05). These results suggest that the in vitroapplication of sevoflurane inhibited the amplitude and frequency of spontaneous myometrial contractions in pregnant rats in a concentration-dependent manner. Such inhibition was reduced by TEA. The inhibition of myometrial smooth muscle induced by sevoflurane seems to be mediated, at least in part, via activation of Ca2+-activated K+ channels, because inhibition was reduced by TEA.
IMPLICATIONS: In this study, we found that sevoflurane causes significantly decreased myometrial contractile activity in pregnant rats. The inhibition of myometrial smooth muscle induced by sevoflurane seems to be mediated, at least in part, via activation of Ca2+-activated K+ channels, because inhibition was reduced by tetraethylammonium.
|
Introduction
|
|---|
Volatile anesthetics, including sevoflurane, isoflurane, and halothane, modulate smooth muscle contractility via a variety of mechanisms.
The cardiovascular effects of sevoflurane have been studied in animals and humans. Mild decreases in systemic arterial blood pressure during maintenance of sevoflurane anesthesia have been reported clinically. Dose-related depression of left ventricular function or cardiac output has been reported in humans and in vivo animal studies with sevoflurane anesthesia (1,2). Topical application of isoflurane or sevoflurane dilated large and small arterioles both in a concentration-dependent manner. Such vasodilation was inhibited completely by glibenclamide (3).
Emergent or elective surgical procedures under general anesthesia may be required during pregnancy for some patients. For this reason, the effects of volatile anesthetics on uterine contractions are very important. The unexpected relaxation or contraction of myometrium can be harmful to the fetus and endanger continuing pregnancy (4,5). There are no data investigating the in vitroeffects of sevoflurane on myometrial smooth muscle.
Based on pharmacologic and biophysical properties, a few K+ channel types have been identified in myometrial smooth muscle (6–9). The most common K+ channel in myometrial smooth muscle is the Ca2+-sensitive K+ channel. Another distinct K+ channel in myometrial smooth muscle is the adenosine triphosphate (ATP)-sensitive K+ channel (KATP). KCa and KATP channel openers inhibit contractile activity in pregnant human myometrium (10).
Even though K+ channel openers inhibit contractile activity in pregnant myometrium, the direct effect that volatile anesthetics have on K+ channels in myometrial smooth muscle is not well understood. Therefore, we designed this study to determine the role of K+ channel blockers on the effect of sevoflurane in myometrial smooth muscle isolated from pregnant rats. Investigating the effect and the mechanism of sevoflurane on myometrium will increase the safety of sevoflurane use in pregnant women.
|
Methods
|
|---|
Sixteen Wistar albino rats (at 20–22 days’ gestation), each weighing 250 to 350 g, were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and the experiments were approved by the Cumhuriyet University–Medical Faculty, Animal Care Committee. The rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg) and killed by exsanguination. The uterine horns were rapidly excised and carefully cleaned of surrounding connective tissues and opened longitudinally along the mesenteric border. Fetuses and placentas were gently removed from the late-stage pregnant rats and nonuterine tissues were dissected away and discarded. The myometrial tissue was cut into longitudinal strips approximately 10 x 2 x 2 mm, following the muscle orientation, and were mounted vertically in a 10-mL organ bath containing modified Krebs solution (composition in millimoles per liter: sodium chloride 125, potassium chloride 2.4, calcium chloride 1.8, magnesium chloride 0.5, sodium bicarbonate 23.9, and glucose 11.0) gassed with 95% oxygen and 5% carbon dioxide at 37°C (pH = 7.4).
The myometrial strips were allowed to equilibrate at 1 g of tension for 60 min before the addition of the experimental drug, and washed every 15 min. The myometrial tension was recorded isometrically with a Grass FT03 force-displacement transducer. The recorder was calibrated as 1 g tension represents 1 cm vertical displacement. The paper speed was set at 5 mm/min. Myometrial contractions started within 10 min after they were mounted into the organ bath and stabilized in 60 min. Preliminary time-control experiments with no further drug additions showed that strips exhibit stable uterine activity for at least 4 h after preparation in this manner. After the establishment of spontaneous myometrial contractions in the modified Krebs solution, the effect of increasing concentrations (0.5, 1, 2, 3 minimum alveolar anesthetic concentration [MAC]) of sevoflurane (Sevotec 5; Ohmeda, Madison, WI), in the absence and presence of glibenclamide (10-6 M), tetraethylammonium (TEA) (3.10-4 M), were investigated. In addition, the effect of increasing concentrations of TEA (10-4–10-3 M) alone and glibenclamide (10-6–10-5 M) alone on spontaneous myometrial contractions was investigated. Sixteen rats were used in these experiments. Five myometrial strips were obtained from each rat. The concentration-dependent effect of sevoflurane was evaluated by treating strips with TEA alone, glibenclamide alone, sevoflurane alone, TEA (3.10-4 M) with sevoflurane, and glibenclamide (10-6 M) with sevoflurane.
Sevoflurane was delivered via calibrated agent-specific vaporizers in line with the O2/CO2 equilibration gas mixture aerating the modified Krebs solution. The gas mixture was bubbled continuously in the bathing solution. The anesthetic concentration in the resulting gas mixture was monitored with a calibrated infrared multianesthetic gas analyzer (Model 1104; Criticare Systems, Inc.,Waukesha, WI). We confirmed that 15 min was sufficient for the Krebs solution to be equilibrated with the anesthetics. Therefore, the Krebs solution was equilibrated with the anesthetics for 15 min before induction into the chamber, which was covered with several pieces of glass plate to prevent the gas from escaping into the atmosphere. The MAC value of sevoflurane in pregnant rats at 37°C was calculated as 2.3%. This concentration in the gas phase corresponds to a concentration of approximately 0.25 mmol/L in the bathing solution. The concentrations tested were equivalent to 0.5, 1, 2, and 3 MAC. A 15-min period of equilibration was allowed between each anesthetic concentration and recording of mechanical variables. Spontaneous myometrial contractions just before the introduction of sevoflurane was taken as 100%, and the effect of sevoflurane was expressed as the percent change in amplitude and frequency of spontaneous contractions 15 min after introduction of sevoflurane in each myometrial strip. All experiments were performed in a paired manner. Thus, each myometrial strip served as its own control.
The characteristics of the contractions analyzed immediately before and after the addition of sevoflurane and K+ channel blockers included mean amplitude (in grams) and frequency (number per 1000 s) of each contraction for 1000-s intervals. Data were presented as means ± SE and were analyzed by two-way analysis of variance with repeated measured analysis of variance followed by Newman-Keuls test, and t-test when appropriate. A P value < 0.05 was considered significant.
Modified Krebs solution, purchased from Sigma (St. Louis, MO), was prepared fresh daily in our laboratory. Chemicals used in the current experiments were glibenclamide and TEA, purchased from Sigma. Glibenclamide and TEA were dissolved in distilled water.
|
Results
|
|---|
At the start of each experiment for experimental procedures and data analysis, the amplitude of spontaneous myometrial contractions was considered as a reference response (RR = 2.13 ± 0.36 g, number of myometrial strips = 80, number of animals = 16). At the end of each experiment, spontaneous contractions after washing myometrial strips were measured again (RR = 1.98 ± 0.22 g, number of myometrial strips = 80, number of animals = 16). Changes in the amplitude and frequency of myometrial contractions were expressed as a percent of the initial reference response (% RR).
TEA (10-4–10-3 M) and glibenclamide (10-6–10-5 M) increased myometrial contractions in a concentration-dependent manner (Fig. 1). Sevoflurane (0.5–3 MAC) inhibited the amplitude and frequency of spontaneous contractions in myometrial strips isolated from pregnant rats in a concentration-dependent manner (number of myometrial strips = 16, number of animals = 16) (Fig. 2). Sevoflurane, at 1, 2, and 3 MAC, significantly decreased the amplitude and the frequency of spontaneous myometrial contractions. (P < 0.05) (Fig. 3). The maximal decrease in amplitude of contractions measured at 3 MAC was 44.32% of control contractions. The maximal decrease in frequency of contractions measured at 3 MAC was 33.32% of control contractions (Fig. 3).
View larger version (57K):
[in this window]
[in a new window]
|
Figure 1. The effect of tetraethylammonium (TEA) and glibenclamide on spontaneous contractile activity of isolated pregnant rat myometrium.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Figure 2. The effect of sevoflurane on spontaneous contractile activity of isolated pregnant rat myometrium.
|
|
View larger version (56K):
[in this window]
[in a new window]
|
Figure 3. The effect of sevoflurane on the amplitude and frequency of spontaneous contractions in myometrial strips isolated from pregnant rats. Data were expressed as the means ± SE of 16 experiments. *Significantly different from the control (P < 0.05).
|
|
In an attempt to examine whether blockade of the K+ channels interferes with sevoflurane-induced relaxation of myometrial smooth muscles, the effect of sevoflurane was determined before (control) and after treatment of the myometrial strips with TEA and glibenclamide (number of myometrial strips = 16, number of animals = 16). At concentrations with no effect on spontaneous contractility, glibenclamide (10-6 M) and TEA (3.10-4 M) were added to the bath 20 min before the addition of sevoflurane in a concentration-dependent manner. The effect of pretreatment with glibenclamide, a blocker of the ATP-sensitive K+ channel, and TEA, a blocker of the Ca2+-activated K+ channel on sevoflurane-induced inhibitor responses, is shown in Figure 4. Pretreatment of the myometrial strips with glibenclamide (10-6 M) produced no significant effect on sevoflurane-induced inhibitor responses in pregnant rat myometrial smooth muscle (P > 0.05) (Figs. 5, 6). Sevoflurane-induced inhibition on the amplitude and frequency of spontaneous myometrial contractions in the presence of TEA (3.10-4 M) was significantly decreased at 1, 2, and 3 MAC (Figs. 5, 6). In the presence of TEA, the maximal inhibition in the amplitude and frequency of spontaneous myometrial contractions by sevoflurane measured at 3 MAC was 31.85% and 22.33% of control contractions, respectively (P < 0.05) (Figs. 5, 6). After the administration of all concentrations of drugs, we washed the myometrial strips and obtained initial levels of spontaneous contractions.
View larger version (35K):
[in this window]
[in a new window]
|
Figure 4. The effect of sevoflurane on spontaneous contractile activity of isolated pregnant rat myometrium in the presence of glibenclamide (A) and tetraethylammonium (TEA) (B).
|
|
View larger version (23K):
[in this window]
[in a new window]
|
Figure 5. The effect of sevoflurane (alone), sevoflurane in the presence of 10-6 M glibenclamide, and sevoflurane in the presence of 3.10-4 M tetraethylammonium (TEA) on the amplitude of spontaneous contractions in myometrial strips isolated from pregnant rats. Glibenclamide (10-6) and TEA (3.10-4 M) were added to the bath 20 min before the addition of sevoflurane. Data were expressed as the means ± SE of eight experiments. *Significantly different from the control (P < 0.05). **Significantly different from the relaxation responses of sevoflurane (P < 0.05).
|
|
View larger version (23K):
[in this window]
[in a new window]
|
Figure 6. The effect of sevoflurane (alone), sevoflurane in the presence of 10-6 M glibenclamide, and sevoflurane in the presence of 3.10-4 M tetraethylammonium (TEA) on the frequency of spontaneous contractions in myometrial strips isolated from pregnant rats. Glibenclamide (10-6 M) and TEA (3.10-4 M) were added to the bath 20 min before the addition of sevoflurane. Data were expressed as the means ± SE of 16 experiments. *Significantly different from the control (P < 0.05). **Significantly different from the relaxation responses of sevoflurane (P < 0.05).
|
|
|
Discussion
|
|---|
Myometrial smooth muscle has spontaneous rhythmic contractions. The tension developed by spontaneously contracting uterine muscle has been used as a control in in vitrostudies (11,12). We found that sevoflurane depressed the spontaneous contractions of myometrial smooth muscle isolated from pregnant rats in a concentration-dependent manner. TEA (Ca2+-activated K+ channel blocker) caused a significant reduction in sevoflurane-induced inhibitor responses, but glibenclamide (ATP-sensitive K+ channel) did not. The result of this investigation correlates with the findings of previous studies that volatile anesthetics may exert an inhibitor effect on the active tension developed by spontaneously contracting myometrial smooth muscle. Previous studies have suggested that volatile anesthetics and K+ channel openers decrease uterine contractility (12,13). Laszlo et al. (14) showed that halothane, enflurane, isoflurane, and verapamil produce an inhibitor effect on contractions of longitudinal myometrial strips isolated from pregnant rats. Cheuk et al. (10) showed that apricalim and BRL 38227 relax the human myometrium by opening potassium channels. Iberiotoxin (IbTx), a potent blocker of Ca2+-activated K+ channels, caused phasic contractions in human myometrial strips and increased both the frequency and amplitude of spontaneous contractions in estrogen-primed rat myometrial strips (15). K+ channel openers cromokalim and pinacidil inhibit the spontaneous activity of the myometrium and vasopressin-induced contractions of myometrium and intramyometrial arteries (13). Our results confirm that sevoflurane inhibits spontaneous myometrial contractions in pregnant rats and that the effect is mediated by Ca2+-activated K+ channels because the effect was blocked by TEA.
A potential weakness of our model is that we studied full-thickness cross-sectional myometrial strips, rather than isolated circular or longitudinal layers. Some investigators have found differences in agonist sensitivity among the different uterine muscle layers of the rat, but at very large concentrations of agonists (16). The orientation of muscle fiber in our strips suggests that the contractions were primarily attributable to circular muscle, although the contribution of short segments of longitudinal muscle cannot be excluded.
The molecular mechanism by which gaseous anesthetics modify ion currents in cell membranes is not clear. The studies of anesthetics on artifical lipid membranes show that, as an anesthetic gas dissolves in the lipid, there is an increase in membrane fluidity suggestive of a reduction in the "orderlines" of the lipid structure. Such effect on the lipid fluidity may result in changes of the function of the channels floating in the membrane (17). An anesthetic-activated potassium current has been shown in identified "pacemaker" neuron in the right parietal ganglion of Lymnaea stagnalis (18,19). Benkusky et al. (20) have shown that multiple isoforms of the maxi-K channel are present in the mouse myometrium and are regulated differentially during gestation, which is a likely mechanism for modulation of myometrial excitability during pregnancy.
A number of specific K+ channels types have been described in myometrium. More than one type has been detected in individual myometrial cells from a number of species, including rat and human (6–9). Calcium-activated K+ channels (KCa), also called "maxi-K" channels, are large (130–300 pS) voltage-dependent channels and are inhibited by TEA, barium (Ba2+), and charybdotoxin and IbTx (21). Blockage of KCa channels with IbTx depolarizes human myometrial cells and is associated with a nifedipine-sensitive increase in intracellular Ca2+, consistent with activation of voltage-sensitive L-type Ca2+ channels (15). Ibtx also increases contractile activity in rat and human myometrium, probably as a result of changes in intracellular Ca2+. TEA stimulates the mechanical activity of rat myometrium, most likely because of its ability to block KCa channels. These data suggest that KCa channels are involved in setting resting membrane potential in myometrium, as well as being potentially involved in regulating the shape and duration of action potentials. In this study, pretreatment of the myometrial strips with TEA, a blocker of the KCa channel, produced a significant decrease on sevoflurane-induced inhibition. These data suggest that the sevoflurane-induced inhibitor effect in pregnant myometrial smooth muscle may be through KCa channels.
KATP has been implicated in myometrial function, but has not been measured directly. The KATP channel activators aprikalim and levocromakalim depress oxytocin-stimulated human and rat uterine contractions, and partially inhibit the action of the uterine relaxant relaxin in vivo but not in vitro (22,23). The KATP channel inhibitor glibenclamide antagonized the action of the channel agonists. Glibenclamide has either a very small effect in nonpregnant myometrium (10) or no effect on uterine contractile activity in nonpregnant or pregnant myometrium (24). In our study, the inhibitor effect of sevoflurane in pregnant rat myometrial strips may not have been related to the activation KATP channels, because the sevoflurane-induced inhibitor effect was unaffected by glibenclamide. These findings suggest that the density of K+ channels in myometrium might be different and the effect of sevoflurane may be attributable to Ca2+-activated K+ channels rather than ATP-sensitive K+ channels.
In summary, this study showed that sevoflurane inhibits spontaneous myometrial contractions on pregnant rat uterus in a concentration-dependent manner and can be used safely in small concentrations in obstetric anesthesia. The inhibitor effect of sevoflurane could be reversed by TEA, which might have clinical implications. Further investigations are required to elucidate the clinical importance of these results.
|
References
|
|---|
-
Conzen PF, Vollmar B, Habazettl H, et al. Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg 1992; 74: 79–88.[Abstract/Free Full Text]
-
Frink EJ, Malan TP, Atlas M, et al. Clinical comparison of sevoflurane and isoflurane in healthy patients. Anesth Analg 1992; 74: 241–5.[Web of Science][Medline]
-
Iida H, Ohata H, Iida M, et al. Isoflurane and sevoflurane induce vasodilation of cerebral vessels via ATP-sensitive K+ channel activation. Anesthesiology 1998; 89: 954–60.[Web of Science][Medline]
-
Marshall BE, Longnecker DE. General anesthetics. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Goodman-Gilman A, eds. The pharmacological basis of therapeutics. New York: McGraw-Hill, 1996: 326–7.
-
Shin YK, Kim YD, Collea JV. The effect of propofol on isolated human pregnant uterine muscle. Anesthesiology 1998; 89: 105–9.[Web of Science][Medline]
-
Kihira M, Matsuzawa K, Tokuno H, et al. Effects of calmodulin antagonists on calcium-activated potassium channels in pregnant myometrium. Br J Pharmacol 1990; 100: 353–9.[Web of Science][Medline]
-
Anwer K, Toro L, Oberti C, et al. Calcium-activated potassium channels in pregnant rat myometrium: modulation by beta adrenergic agents. Am J Physiol 1992; 263: 1049–56.
-
Erulkar SD, Ludmir J, Ger B, et al. Expression of different potassium channels in cells isolated from human myometrium and leiomyomas. Am J Obstet Gynecol 1993; 168: 1628–39.[Web of Science][Medline]
-
Khan RN, Smith SK, Morrison JJ, et al. Properties of large-conductance K+ channels in human myometrium during pregnancy and labour. Proc R Soc Lond B Biol Sci 1993; 251: 9–15.[Medline]
-
Cheuk JMS, Hollingsworth M, Hughes SJ, et al. Inhibition of contractions of the isolated human myometrium by potassium channel openers. Am J Obstet Gynecol 1993; 168: 953–60.[Web of Science][Medline]
-
Leroy MJ, Tanguy G, Vial M, et al. The effect of vasoactive intestinal peptide (VIP) on the contractile activity of human uterine smooth muscle. Clin Exp Pharmacol Physiol 1991; 18: 205–15.[Web of Science][Medline]
-
McGaughey HS, Corey EL, Eastwood D, Thornton WN. Effect of synthetic anesthetics on the spontaneous motility of human uterine muscle in vitro. Obstet Gynecol 1962; 19: 233–40.
-
Kostrzewska A, Laudanski T, Batra S. Inhibition of contractile responses of human myometrium and intramyometrial arteries by potassium channel openers. Acta Obstet Gynecol Scand 1996; 75: 886–91.[Web of Science][Medline]
-
Laszlo A, Buljubasic N, Zsolnai B, et al. Interactive effects of volatile anesthetics, verapamil and ryanodine on contractility and calcium homeostasis isolated pregnant rat myometrium. Am J Obstet Gynecol 1992; 167: 804–10.[Web of Science][Medline]
-
Anwer K, Oberti C, Perez GJ, et al. Calcium-activated K+ channels as modulators of human myometrial contractile activity. Am J Physiol 1993; 265: 976–85.
-
Chernaeva L. Adrenergic sensitivity of circular and longitudinal muscles of pregnant rat myometrium. Methods Find Exp Clin Pharmacol 1984; 6: 73–8.[Web of Science][Medline]
-
Trevor AJ, Miller LD. Inhaled anesthetics. In: Katzung BG, ed. Basic and clinical pharmacology. Stamford, CT: Appleton & Lange, 1998: 412–9.
-
Franks NP, Lieb WR. Volatile general anaesthetics activate a novel neuronal K+ current. Nature (London) 1988; 333: 662–4.[Medline]
-
Franks NP, Lieb WR. Selective effects of volatile general anesthetics on identified neurons. Ann NY Acad Sci 1991; 625: 54–70.[Web of Science][Medline]
-
Benkusky NA, Fergus DJ, Zucchero TM, England SK. Regulation of the Ca2+ sensitive domains of the maxi K channel in the mouse myometrium during gestation. J Biol Chem 2000; 8275: 27712–9.[Abstract/Free Full Text]
-
Mironneau J. Ion channels and the control of uterine contractility. In: Garfield RE, Tabb TN, eds. Control of uterine contractility. Boca Raton, FL: CRC Press, 1993: 1–22.
-
Hollingsworth M, Amedee T, Edwards D, et al. The relaxation of BRL34915 in rat uterus. Br J Pharmacol 1987; 91: 803–13.[Web of Science][Medline]
-
Downing SJ, Hollingsworth M. Action of relaxin on uterine contractions: a review. J Reprod Fertil 1993; 99: 275–82.[Abstract/Free Full Text]
-
Heaton RC, Wray S, Eisner DA. Effects of metabolic inhibition and changes of intracellular pH on potassium permeability and contraction of rat uterus. J Physiol 1993; 465: 43–56.[Abstract/Free Full Text]
Accepted for publication August 17, 2001.
This article has been cited by other articles:
|
|
|
|
 
K. Y. Yoo, J. C. Lee, M. H. Yoon, M.-H. Shin, S. J. Kim, Y. H. Kim, T. B. Song, and J. Lee
The effects of volatile anesthetics on spontaneous contractility of isolated human pregnant uterine muscle: a comparison among sevoflurane, desflurane, isoflurane, and halothane.
Anesth. Analg.,
August 1, 2006;
103(2):
443 - 7, table of contents.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
