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Departments of Dental Anesthesiology and Anesthesiology and Resuscitology, Okayama University Hospital of Medicine and Dentistry, and Departments of Dental Anesthesiology and Oral Physiology, Okayama University Graduate School of Medicine and Dentistry
Address correspondence and reprint requests to Atsushi Kohjitani, DDS, PhD, Department of Dental Anesthesiology, Okayama University Hospital of Medicine and Dentistry, 2–5–1 Shikata-cho, Okayama 700–8525, Japan. Address e-mail to atsushik{at}md.okayama-u.ac.jp.
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Abstract |
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Introduction |
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Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) is a prominent inhibitory NANC smooth muscle relaxant, which stimulates smooth muscle cell soluble guanylate cyclase to generate 3',5'-cyclic guanosine monophosphate (cGMP). A functional role for the l-arginine—NO pathway in mediating NANC LES relaxation has been demonstrated (4). In vascular smooth muscle, it has been shown that NO reacts rapidly with superoxide anions, leading to the production of peroxynitrite (5) and the loss of its vasodilating activity (6), indicating that superoxide anions contribute to the instability of endothelium-derived relaxing factor (EDRF) (6). In addition, the peripheral nitrergic transmitters have been shown to be stable NO-releasing molecules because various superoxide generators reduced relaxation in response to exogenous NO, but they had no effect on nitrergic-stimulated relaxation (7). One possible hypothesis for the stability of the nitrergic transmitters is that they are protected by high levels of Cu/Zn superoxide dismutase (Cu/Zn SOD), as inhibition of Cu/Zn SOD by diethyldithiocarbamic acid (DETCA) increased susceptibility to destruction by superoxide anions (8). Although the role of Cu/Zn SOD in the LES has not been fully elucidated, it appears to play a role in modulating normal esophageal motor function (9).
In the central nervous system, excitatory amino acid receptors of the N-methyl-d-aspartate (NMDA) subtype play a pivotal role in glutamatergic neurotransmission and regulate the release of several neurotransmitters, including acetylcholine and norepinephrine. In peripheral tissues, including the gastrointestinal tract, excitatory amino acid receptors have also been described. Excitatory amino acid receptors in the myenteric plexus of the guinea pig ileum were shown to have similar pharmacologic properties to the NMDA receptor subtype (10,11). These peripheral receptors are activated by glutamate and NMDA to induce muscle contraction, become inactive in the presence of Mg2+ and tetrodotoxin, and are blocked by a competitive antagonist, dl-2-amino-5-phosphonovaleric acid (AP5) (10,12), and a noncompetitive antagonist, 5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK801) (13). However, limited data are available regarding the role of excitatory amino acids and their receptors in the myenteric plexus in the regulation of intestinal motility.
We previously reported that NANC LES relaxation was mediated by endogenously released NO (14), and that the IV anesthetics ketamine and midazolam inhibit NANC relaxation via NO—cGMP pathway modulation (14). We also reported that the inhibitory mechanisms for ketamine involved the generation of superoxide anions (15). However, the roles of the peripheral NMDA receptors distributed in the myenteric plexus in mediating contractile and relaxant responses, and/or NO-mediated responses, have not yet been elucidated. In the current study, we therefore investigated the role of peripheral NMDA receptors in mediating NANC LES relaxation.
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Methods |
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The LES strips were fixed vertically between two hooks and the hook anchoring the upper end was connected to a force-displacement transducer. The resting tension was adjusted to 1.0 g. The strips were suspended in a thermostatically controlled (37.0°C ± 0.5°C) 20 mL organ bath containing Krebs-Ringer solution. The bath fluid was aerated with a mixture of 95% O2 and 5% CO2 to maintain the pH between 7.35 and 7.45. Before starting the experiments, the strips were allowed to equilibrate for 60 min in the Krebs solution.
First, the effect of MK801, a noncompetitive NMDA receptor antagonist, on NANC relaxation was examined. NANC relaxation was induced by 30 mM KCl in the presence of atropine (3 x 10–6 M) and guanethidine (3 x 10–6 M). Atropine and guanethidine were pretreated for at least 10 min. MK801 was applied with washout between each change in concentration. NANC relaxation in the absence of MK801 was taken as 100%, and the concentration-response relationship was obtained.
Second, NMDA was cumulatively added in the absence and presence of MK801 (3 x 10–5 M) to examine the direct effects of NMDA on resting LES strips. Maximal relaxation induced by 3 x 10–5 M N-ethylethanamine:1,1-diethyl-2-hydroxy-2-nitrosohydrazine (diethylamine NONOate) was taken as 100%, and the concentration-response relationship was obtained.
Third, the effects of Mg2+-depleted Krebs-Ringer solution on MK801-induced inhibition of NANC relaxation were examined. NANC relaxation in the absence of MK801 was taken as 100%, and the concentration-response relationship was obtained. The curve was compared with that observed in normal Krebs-Ringer solution.
The effects of SOD and catalase on MK801-induced inhibition of NANC relaxation were examined. SOD (100 U/mL) or catalase (100 U/mL) was pretreated for 15 min, and the reversal of MK801-induced inhibition of NANC relaxation was examined. NANC relaxation in the absence of MK801 was taken as 100%, and concentration-response relationships were obtained.
For the cGMP radioimmunoassay, various concentrations of MK801 (10–5, 3 x 10–5 and 10–4 M) were applied to LES strips for 10 min in the presence of atropine (3 x 10–6 M) and guanethidine (3 x 10–6 M), and the strips were then frozen in liquid nitrogen 2 min after the application of KCl in both normal and Mg2+-depleted Krebs-Ringer solution. In the control study using other strips from the same animal, an equivalent volume of KCl was applied in the presence of atropine and guanethidine and the absence of MK801.
The effects of increasing concentrations of NMDA (10–4, 10–3 and 3 x 10–3 M) on cGMP production in the absence or presence of NG-nitro-l-arginine (L-NNA; 3 x 10–5 M for 15 min) were similarly examined. The strips were frozen in liquid nitrogen 3 min after the application of NMDA, when maximal relaxation was obtained. The strips were then homogenized in a 6% volume-to-volume ratio of trichloroacetic acid, and centrifuged at 3000 rpm for 10 min. The supernatant fractions were subjected to ether extraction and subsequent succinylation, while the pellets were analyzed for their protein content. The cGMP in each sample was radioimmunoassayed using a Yamasa assay kit® (Yamasa Shoyu, Chiba, Japan).
Concentration-response relationships of pyrogallol, a superoxide generator, ketamine, and MK801 on NANC relaxation were obtained, taking the control NANC relaxation as 100%. Thereafter, DETCA (3 mM) was applied for 60 min to inactivate Cu/Zn SOD (8). After washing out the DETCA, the LES strips were left under resting tension for about 60 to 80 min. NANC relaxation was measured again and taken as 100%, and the concentration-response relationships of pyrogallol, ketamine, and MK801 on NANC relaxation were obtained. Paired data sets derived from the non-DETCA-treated and DETCA-treated strips were obtained.
The results were expressed as the mean ± sd. One-way analysis of variance was used to compare differences in the effects of drugs or Mg2+-depleted Krebs-Ringer solution on NANC relaxation or cGMP production. A Bonferroni test or Scheffé's F-test (if the number of strips in each group was not equal) was used as a post hoc comparison to test statistical significance between control and drug-treated values or between groups. For all statistical analyses, a P value of <0.05 was regarded as significant.
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Results |
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NMDA induced a concentration-dependent relaxation (Fig. 2A, open circles), accompanied by an increase in cGMP production (Fig. 2B, open bars). The increase in cGMP production was significant at 3 x 10–3 M (P < 0.05) compared with the control value. NMDA-induced relaxation was antagonized by MK801 (Fig. 2A, closed circles), and the effect was significant at 10–3 M (P < 0.01) and 3 x 10–3 M (P < 0.05) NMDA. NMDA-induced cGMP production was inhibited in the presence of 3 x 10–5 M L-NNA (Fig. 2B, hatched bars). The inhibition of cGMP production was significant at 10–3 M (P < 0.05) and 3 x 10–3 M (P < 0.05) NMDA, and there were significant differences between the groups (P < 0.001) at both concentrations.
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In Mg2+-depleted Krebs-Ringer solution, NANC relaxation was significantly augmented to 223% ± 174% (mean ± sd, P < 0.05) as compared with that obtained in normal Krebs-Ringer solution, along with increased cGMP production (Fig. 1B, closed bar of the control; P < 0.01). MK801 inhibited NANC relaxation in a concentration-dependent manner (Fig. 1A, closed circles), and the concentration-response curve was shifted to the right (EC50 = 7.4 x 10–5 M) compared with that observed in normal Krebs-Ringer solution. Significant differences were found between the normal and Mg2+-depleted solution groups at 3 x 10–5 M (P < 0.01) and 10–4 M (P < 0.01) MK801. A decrease in cGMP production was also observed as the concentration of MK801 increased (Fig. 1B, closed bars). The decrease in cGMP was significant at 3 x 10–5 M (P < 0.01) and 10–4 M (P < 0.01) compared with the control value in Mg2+-free solution.
SOD (100 U/mL) shifted the concentration-response relationship of MK801-mediated inhibition of NANC relaxation to the right (EC50 = 3.4 x 10–5 M; Fig. 3, open squares), whereas catalase (100 U/mL) did not (Fig. 3, closed circles). The recovery of MK801-induced inhibition by SOD was significant at 3 x 10–5 M (P < 0.01). The inhibition of Cu/Zn SOD activity by DETCA shifted the concentration-response relationships of pyrogallol-, ketamine-, and MK801-mediated inhibition of NANC relaxation to the left (Fig. 4 A-C). The inhibition was significant at 10–6 M (P < 0.05) and 10–5 M (P < 0.05) for pyrogallol, 10–5 M (P < 0.05) and 10–4 M (P < 0.05) for ketamine, and 3 x 10–5 M (P < 0.05) for MK801. There was no difference between the control NANC relaxations obtained before and after the application of DETCA.
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Discussion |
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Because the Mg2+ ion also acts as a noncompetitive blocker of the ion channel component of NMDA receptors (16), depletion of Mg2+ ions increases Ca2+ permeability and may therefore affect NOS function. In the present study, significantly augmented NANC relaxation accompanied by increased cGMP production was observed in Mg2+-depleted Krebs-Ringer solution, compared with that observed in normal Krebs-Ringer solution. Mg2+-depletion shifted the concentration-response relationship of MK801-mediated inhibition of NANC relaxation to the right, suggesting that NANC relaxation becomes less susceptible to inhibition by the NMDA antagonist as a result of decreased occupancy of Mg2+ ions within the ion channel component. Although we cannot exclude the possibility that Mg2+-depletion affected other physiological cellular functions, these findings suggest a role for Mg2+ ions in modulating NMDA receptor-mediated activation of NOS.
SOD, but not catalase, shifted the concentration-response relationship of MK801-mediated inhibition of NANC relaxation to the right. These findings imply that superoxide production is involved in MK801-induced inhibition of NANC relaxation. The inactivation of Cu/Zn SOD by DETCA shifted the concentration-response relationship of MK801 to the left, suggesting that increased susceptibility to destruction by superoxide anions attenuated the relaxant effect of endogenous NO. These findings suggest that superoxide production, which is supposed to be stimulated by KCl in the presence of MK801, is involved in the inhibition of NANC relaxation. DETCA also shifted the curve of pyrogallol, a superoxide generator, to the left, which supports our notion. Similarly, the inactivation of Cu/Zn SOD shifted the curve of ketamine to the left, is consistent with our previous results that ketamine inhibits NANC relaxation, in part, through extracellular superoxide production (15).
Taking these findings together, binding of agonists to NMDA receptors is coupled with NOS activation, as reported for the central nervous system (17). The NMDA receptors in the myenteric plexus possibly have an important role in mediating nitrergic smooth muscle relaxation by triggering the NO—cGMP pathway. In situations when NMDA receptors are occupied by an antagonist, such as MK801 or ketamine, nitrergic smooth muscle relaxation is inhibited, in part, by superoxide production, which eliminates the relaxant effect of the nitrergic transmitter. There are observations with regard to the relevance of glutamate, NMDA, or its receptors in NANC smooth muscle responses in the gut, using awake dogs (18) and volunteers (19). However, the site of action of NMDA antagonists is unclear because these observations have been made in in vivo conditions using systemic application of a specific NMDA antagonist. Our in vitro observation demonstrated a role for peripheral NMDA receptors in mediating nitrergic relaxation, independent of vagal afferents. However, the following issues remain unclear. First, NMDA induced direct relaxation of LES strips in the present study. This finding is inconsistent with previous studies showing that l-glutamate or NMDA induced contraction of guinea pig ileal smooth muscle, which was supposed to be the result of stimulation of cholinergic interneurons in the myenteric neurons (10). We speculate that the response to excitatory amino acids may involve different sites within the same species or may differ among species. The second issue is the mechanism of superoxide production when the strips are stimulated by KCl in the presence of an NMDA antagonist. Superoxide production has been reported to be triggered by NMDA and inhibited by SOD or MK801 in cultured cerebellar granule cells (20), and furthermore nNOS was responsible for catalyzing superoxide formation at smaller concentrations or in the absence of l-arginine in a Ca2+/calmodulin-dependent manner (21). Therefore, peripheral NMDA receptors may modulate l-arginine availability. Third, we cannot exclude the possibility that ketamine inhibits NANC relaxation via mechanisms other than antagonism of NMDA receptors, such as activation of adenylate cyclase or inhibition of extracellular Ca2+ influx (22).
Functional linkage of NMDA receptors and nNOS in the central nervous system has been clearly established. EDRF, which was shown to be identical to NO, is released after NMDA receptor activation by glutamate (23). Evidence for a specific function of NO in the brain was derived from demonstrations that glutamate stimulates the conversion of l-arginine to l-citrulline and the production of cGMP and that NOS inhibitors block both NOS activity and cGMP production (17). Therefore, NMDA transmission activates NO formation in the brain under presumed physiological conditions. With regard to the effect of anesthetics on NO production, Gonzales et al. (24) have reported that ketamine, MK801 and NG-monomethyl-l-arginine, a NOS inhibitor, inhibited NMDA-stimulated cGMP production using cortical neurons. Although the role of peripheral excitatory amino acids in the regulation of gastrointestinal motility under physiological and pathological conditions remains unclear, functional linkage of peripheral NMDA receptors in the myenteric plexus and the NO—cGMP pathway leading to smooth muscle relaxation could provide a key for elucidating the roles of endogenous NO in regulating gastrointestinal motility in the near future.
To elicit NANC relaxation of smooth muscle, electrical field stimulation is generally used. In our previous study, however, electrical field stimulation under various conditions never induced relaxation but did induce contractions that were sensitive to tetrodotoxin in this preparation (25). The transient relaxation induced by KCl is considered to be a truly neural response because the relaxation component is abolished by pretreatment with tetrodotoxin or by extracellular Ca2+ depletion (14). The KCl-induced relaxation in the presence of atropine and guanethidine is inhibited by pretreatment with L-NNA or methylene blue, indicating that this relaxation is nitrergic (14). Also, the relaxation is reproducible by repeated exposure to KCl elicited under the same condition, given complete washout was done and muscle strips were allowed to have a 15-min interval until the next KCl stimulation, while resting tension of muscle strips returns to the baseline.
Ketamine is not classified as either a LES tone increasing or tone decreasing drug (26). The present findings, which are limited to rabbit LES, suggest the possibility that anesthetics may affect gastrointestinal motility by modulating nitrergic relaxation through NMDA receptors in clinical situations. The effects of ketamine alone or in combination with other anesthetics on gastrointestinal functions in total IV anesthesia or sedation have been investigated. The lack of effect on gastric emptying in healthy volunteers at an analgesic dose (0.5 mg/kg IM) (27), and on the frequency, amplitude or rhythmical activity of the internal anal sphincter in children (1 mg/kg IV or 7–10 mg/kg IM) (28) are preferable effects of ketamine. However, propofol-ketamine combined anesthesia (1.4 mg/kg, continuous infusion) appeared to prolong the gastro-cecal transit time compared with propofol-based or isoflurane-based anesthesia (29). Increasing doses of ketamine (0.5, 0.8, and 1.2 mg/kg, continuous infusion) in sedation with the ketamine-propofol combination increased the incidence of postoperative nausea and vomiting (PONV) (30). According to these studies, although it is difficult to elucidate whether these clinical findings occurred through peripheral NMDA receptor antagonism, ketamine appears to decrease upper gastrointestinal motility and increase the frequency of PONV at larger doses. Further clinical investigation may reveal the role of peripheral NMDA receptors in modulating gastrointestinal motility.
In conclusion, the present findings suggest that the peripheral NMDA receptors in the myenteric plexus mediate NANC LES relaxation. NANC relaxation is partly modulated by the extracellular production of superoxide anions, thus eliminating the relaxant effect of endogenous NO. Ketamine possibly inhibits NANC relaxation via antagonism of the peripheral NMDA receptors.
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Footnotes |
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Accepted for publication June 22, 2005.
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P < 0.05, significant difference from the respective control value. *P < 0.05, significant difference between the groups.
