Extrinsic and intrinsic nerves predominantly control the motility of the gastrointestinal tract . Since perioperative drugs, including anesthetics, can stimulate these nerves to increase or decrease the release of excitatory or inhibitory neurotransmitter, or can act on the receptors on smooth muscle directly, they affect gastrointestinal motility . Various neurotransmitters, hormones, and peptides contribute to the regulation of esophageal motility and function of the lower esophageal sphincter (LES) , which is a specialized smooth muscle situated at the esophagogastric junction. However, the mechanism of esophageal motility disorders during anesthesia has not been elucidated.
Ketamine, a short-acting intravenous anesthetic, has been widely used. In recent years, there has been considerable interest in the pharmacological action of ketamine on vascular [3,4], tracheal , and intestinal  smooth muscles. In the cardiovascular system, ketamine exerts a direct vasodilating effect , and in the respiratory tract it causes a reduction in airway resistance in asthmatic patients  and has a spasmolytic effect . These vasodilating and bronchodilating effects of ketamine have been explained as being due mainly to interference of transmembrane influx of Ca2+ through the voltage-dependent Ca2+ channel [3,5] and to the inhibition of guanine nucleotide-binding proteins (G proteins) or phospholipase C , which result in the decrease in the synthesis of inositol 1,4,5-triphosphate (IP3).
Little is known about the effects of anesthetics on the gastrointestinal smooth muscle . Intracellular second messengers, such as 3 prime,5 prime-cyclic adenosine monophosphate (cAMP), 3 prime,5 prime-cyclic guanosine monophosphate (cGMP) or IP3, as well as Ca2+ mobilization, are responsible for the physiological regulation of intestinal smooth muscle contractility. By measuring the content of cyclic nucleotides in the absence or presence of ketamine and by measuring ketamine-induced relaxation as a percentage of maximal papaverine-induced relaxation in the absence or presence of various channel and messenger antagonists, we tested the hypothesis that ketamine relaxes isolated rabbit LES strips by affecting specific intracellular messengers.
The experimental protocol was approved by the Kobe University Animal Use Committee. Twenty adult male Japanese White rabbits weighing between 2 and 3 kg were anesthetized with thiamylal sodium (50 mg/kg intravenously) and killed by exsanguination. The lower part of the esophagus and stomach were immediately isolated. The sphincteric region was opened along the longitudinal axis, and the LES was excised by sharp cutting in a circular direction, making strips about 2 mm wide and 5 mm long. The mucosa was removed.
The strips were vertically fixed between two hooks under a resting tension of 1.0 g, and the hook anchoring the upper end was connected to a forcedisplacement transducer. Changes in the isometric tension of circular muscle were recorded. The strips were suspended in a thermostatically controlled (37.0 +/- 0.5 degrees C) 20-mL organ bath containing Krebs-Ringer solution (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl (2), 25 mM NaHCO3, 1.18 mM KH2 PO4, 1.19 mM MgSO4, 11 mM glucose). The bath fluid was aerated with a mixture of 95% O2 and 5% CO2 to keep the pH within 7.35-7.45. Before starting the experiments, the strips were allowed to equilibrate for 60-90 min in the Krebs solution, which was replaced every 15 min.
In this series of experiments, drugs were applied directly to the organ bath by micropipette. Ketamine was administered cumulatively, and a dose-response curve was obtained. Then, maximum relaxation was obtained by the application of 10 (-4) M papaverine. An antagonist was introduced and was preincubated for the settled time, remaining in the bath for 10-3 M ketamine-induced relaxation. As the available antagonists in this study, atropine (3 x 10-6 M; n = 6), propranolol (3 x 10 (-6) M; n = 6), phentolamine (3 x 10-6 M; n = 6), indomethacin (3 x 10-6 M; n = 6), diphenhydramine (3 x 10-6 M; n = 8), verapamil (10-6 M; n = 10), [Ac-Tyr1, D-Phe2]-growth hormone-releasing factor 1-29 amide (vasoactive intestinal peptide [VIP] antagonist) (10-6 M; n = 4), and tetrodotoxin (TTX) (3 x 10-7 M; n = 4) were pretreated for 10 min. Nicotinic acid (10-4 M; n = 8), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) (3 x 10-5 M; n = 10), methylene blue (10-5 M; n = 6), and NG-nitro-L-arginine (L-NNA) (10-5 M; n = 6) were pretreated for 20 min. The pharmacological action of papaverine in intestinal smooth muscle has been reported to result from inhibition of phosphodiesterase activity . Cholera toxin (CTX) activates the stimulatory G protein of adenylate cyclase (AC). Nicotinic acid is an inhibitor of AC, and because nicotinic acid may act on nicotinic receptors on smooth muscle or on presynaptic ganglia and may induce contraction, atropine was added together with nicotinic acid. H-89 is a specific inhibitor of cAMP-dependent protein kinase . Methylene blue and L-NNA are inhibitors of guanylate cyclase and nitric oxide (NO) synthase, respectively.
For the radioimmunoassay of cAMP and cGMP, ketamine was applied to LES strips, and soon after maximal relaxation was obtained, the strips were frozen in liquid nitrogen. In the control study, using another strip from the same animal, an equivalent volume of distilled water was applied. The strips were then homogenized in 6% (v/v) trichloroacetic acid. The homogenate was centrifuged at 3000 rpm for 15 min, and the supernatant fractions were subjected to ether extraction and subsequent succinylation, and the pellet was analyzed for protein content. cAMP and cGMP in each sample was radioimmunoassayed using a Yamasa assay kit (Yamasa Shoyu Co., Chiba, Japan). Levels of cAMP or cGMP in tissues were expressed as picomoles per milligram of protein.
The following drugs were used: DL-propranolol hydrochloride, indomethacin, diphenhydramine hydrochloride, phentolamine hydrochloride, methylene blue, dibutyryl cGMP (DBcGMP, 10-4 M), and nicotinic acid from Nacalai Tesque (Kyoto, Japan); ketamine hydrochloride, atropine sulfate, verapamil hydrochloride, papaverine hydrochloride, L-NNA, dibutyryl cAMP (DBcAMP, 10-4 M), and TTX from Sigma Chemical (St. Louis, MO); H-89 from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA); VIP antagonist from Peninsula Laboratories (Belmont, CA); and CTX (1 micro g/mL) from Research Biochemicals International (Natick, MA). Indomethacin was dissolved in ethanol, and H-89 was dissolved in same dose of ethanol and distilled water. The final concentrations of ethanol in the bath were <0.1% and <0.2%, respectively. In a preliminary study, 0.2% ethanol did not induce any effect on isometric tension of the muscle. Other drugs were dissolved in distilled water and handled in siliconized glassware. All other chemicals used were of reagent grade.
The results were expressed as mean +/- SD. Analysis of variance with a Bonferroni test for post hoc comparisons was used to test for statistical significance both between control versus drug-treated values and between groups. For all statistical tests, a P value of 0.05 or less taken as significant.
Ketamine induced dose-dependent relaxation of the LES strips in doses from 10-5 to 10-3 M (Figure 1). The effects of ketamine were expressed as percent relaxation of 10-4 M papaverine-induced relaxation in Figure 1. Pretreatment with atropine, propranolol, phentolamine, TTX, and VIP antagonist did not alter the 10-3 M ketamine-induced relaxation. However, indomethacin and diphenhydramine enhanced this response (Table 1). The application of atropine, propranolol, phentolamine, TTX, diphenhydramine, indomethacin, and VIP antagonist did not affect the resting tension of the LES.
Ketamine increased the content of cAMP in the LES dose-dependently (Figure 2A). The content of cGMP in the LES was significantly decreased in the presence of 10-5 M ketamine (Figure 2B). Subsequently, we examined the effects of several inhibitors on 10-3 M ketamine-induced relaxation (Figure 3). Pretreatment with nicotinic acid along with atropine antagonized this response. Pretreatment with H-89 significantly reduced the response. However, pretreatment with methylene blue or L-NNA did not alter it. Nicotinic acid, H-89, methylene blue, and L-NNA did not affect the resting tension of the muscle. The application of both CTX and DBcAMP induced relaxation; however, that of DBcGMP induced little relaxation of LES strips (Figure 4). Relaxation induced by DBcAMP was about 26% of papaverine-induced relaxation, and both CTX-and DBcAMP-induced relaxation were significantly more than that induced by DBcGMP (Figure 4).
In the presence of verapamil, ketamine-induced relaxation was significantly attenuated (Figure 3). The resting tension of the muscle remained unchanged with the application of verapamil.
Several mechanisms have been proposed to explain LES relaxation, which is known to be induced by several neurotransmitters, drugs, or hormones, or by electrical field stimulation . Increased cyclic nucleotide content has been reported to accompany relaxation . Relaxation of the LES is obtained by stimulation of the vagus nerve in vivo  and by electrical field stimulation in vitro . This stimulation-induced relaxation, both in vivo and in vitro, is mediated by intrinsic neurons that are part of the nonadrenergic noncholinergic (NANC) inhibitory system . The neurotransmitter of NANC innervation has not been demonstrated. However, as NANC-mediated relaxation has been inhibited by L-NNA or NO, NO has been proposed as a NANC neurotransmitter . Other investigators have hypothesized that NO is not the direct inhibitory neurotransmitter, but rather initiates the release of other inhibitory neurotransmitters or relaxant substances . LES relaxation can also be achieved by VIP, one of the gut-brain peptides that acts as a representative inhibitory neurotransmitter in LES, along with an increase in the content of IP3 as well as cAMP in cat LES . Other than VIP, neuropeptide Y and calcitonin gene-related peptides are associated as possible inhibitory neurotransmitters, which may participate in the regulation of LES function. Since pretreatment with a VIP antagonist did not affect ketamine-induced relaxation, VIP-mediated relaxation would not be involved.
We tried to examine the intracellular effects of ketamine on LES smooth muscle. Ketamine induced dose-dependent relaxation and the content of cAMP in LES strips was increased dose-dependently. As ketamine-induced relaxation was antagonized by nicotinic acid with atropine, activation of AC and subsequent accumulation of cAMP may play a role in mediating relaxation. Then, to determine the roles of AC activation or increase in intracellular concentration of cAMP ([cAMP]i) on LES contractility, we tested CTX and DBcAMP. The former increases [cAMP]i via activating the stimulatory G protein of AC, the latter increases [cAMP]i by permeating the cell membrane and by remaining intact in the presence of phosphodiesterase. Just as the application of both CTX and DBcAMP actually induced LES relaxation, the increase in [cAMP]i mediated LES relaxation. The inability of phentolamine, propranolol, and atropine to alter the relaxant effect indicates that the mechanisms involved in adrenoceptors and cholinoceptors are not involved in the relaxation of LES smooth muscle produced by ketamine. The inability of TTX to affect ketamine-induced relaxation indicates that no neural mechanisms are associated. Little relaxation of LES strips was observed by an application of DBcGMP, and the inability of L-NNA and methylene blue to affect ketamine-induced relaxation shows that the NO-cGMP pathway would not be concerned. These findings suggest that ketamine induces relaxation of LES smooth muscle at least by modulating the activity of the AC-cAMP system, and that cAMP is a possible intracellular messenger mediating LES relaxation. Ketamine's site of action would exist somewhere between receptor activation and the activation of AC, perhaps stimulatory G protein.
Change in intracellular concentration of Ca2+ ([Ca2+]i) is one of the principal factors in regulating the contraction-relaxation cycle of several smooth muscles. The L-type Ca2+ channel is one of the voltage-dependent channels in vascular smooth muscle and is a primary factor involved in the maintenance of smooth muscle tone by modulating [Ca2+]i. Hormones and/or neurotransmitters , intracellular second messengers, including Ca2+ and cAMP-dependent protein kinase , or G protein  depend on the activation or inactivation of L-type Ca2+ channels. In guinea pig ileum, ketamine inhibits histamine-induced contraction by decreasing the influx of Ca2+ through the L-type Ca2+ channel . Ketamine-induced relaxation of the LES was significantly attenuated with the blockade of the L-type Ca2+ channel by verapamil, thereby indicating that inhibition of extracellular Ca2+ entry by ketamine appears to play a role. Intracellular Ca2+ mobilization may also affect the contractility of LES smooth muscle, a factor we have not examined in this study.
Drugs that increase cAMP levels, such as isoproterenol, have been known to activate cAMP-dependent protein kinase . In addition, it is generally accepted that the activation of cAMP-dependent protein kinase could at least mediate the relaxation of vascular and airway smooth muscle through the dephosphorylation of some contractile proteins, such as myosin, by the phosphorylation of myosin light chain kinase. Our results show that ketamine-induced relaxation was significantly attenuated by H-89. The possibility that the effects on contractile proteins through the activation of cAMP-dependent protein kinase cannot be excluded.
Pretreatment with indomethacin or diphenhydramine potentiated the ketamine-induced relaxation. However, the mechanism of these potentiations are unclear. Metabolites of arachidonic acid have a wide range of physiological actions on the motility, blood flow, and secretion of smooth muscle in different animal species, and the mechanisms of their actions on LES have as yet not been elucidated. Among them, prostaglandin E1 has been reported to act on specific receptors in the opossum LES smooth muscle and stimulate the formation of cAMP . If this is applicable to rabbit LES, cAMP formation would be inhibited in the presence of indomethacin. As ketamine-induced relaxation was potentiated in such a condition, other mechanisms that relax LES smooth muscle might be involved. Histamine induces contraction of intestinal smooth muscle by IP3-induced release of Ca2+ from intracellular stores . Similar to vascular smooth muscle , ketamine may inhibit phospholipase C in LES smooth muscle. Inhibition of IP3 accumulation in the presence of diphenhydramine has been reported in airway smooth muscle , thereby indicating that the potentiation of ketamine-induced relaxation by diphenhydramine may be due to the inhibition of phosphatidylinositol turnover.
In conclusion, ketamine relaxes rabbit LES strips by activating AC and a subsequent increase in [cAMP]i as well as by affecting extracellular Ca2+ mobilization. Dephosphorylation of contractile protein might be responsible for the resultant relaxation.
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