Because of the bronchodilating properties of the dissociative anesthetic drug ketamine [1,2] and magnesium (Mg2+) [3,4], they have been used to treat patients with status asthmaticus. However, the mechanism of the spasmolytic effect of these drugs in airway smooth muscle is still unclear.
Excitatory amino acids are key neurotransmitters in the brain , but they also participate in most of the neural circuits, including those implicated in cardio-respiratory and airway control [6-10]. The amino acid L-glutamate is the neurotransmitter at most excitatory synapses in the central nervous system . Glutamate receptors are classified functionally as ligand-gated ion channels (also called inotropic receptors) or as metabotropic (G protein-coupled) receptors. The ligand-gated ion channels are multisubunit complexes that form a cation channel that gates Na+ and Ca2+. The metabotropic receptors are composed of a single seven-transmembrane-spanning protein and couple via a GTP-binding protein to several effector mechanisms. The ligand-gated, cation-permeable channels are further classified according to the identity of the agonist that selectively activates each receptor subtype in: the N-methyl-D-aspartic acid (NMDA) receptor, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor, and the kainate receptor. Recently, NMDA receptors have been demonstrated in the lung [12,13] and the airway , where their activation triggers acute lung injury characterized by pulmonary edema and airway constriction.
The bronchodilators ketamine [15-18] and Mg2+[11,19] have NMDA receptor-antagonizing properties. Phencyclidine (PCP; "angel dust") and ketamine are believed to bind inside the open channel of the NMDA receptor . Hence, we designed the present study to investigate the effect of ketamine, Mg2+, Zn (2+), and MK-801, which are NMDA receptor antagonists , on the contraction of isolated guinea pig trachea induced by histamine.
After approval of the experimental protocol by our animal care and use committee, female guinea pigs weighing 250-400 g were killed with an overdose of sodium pentobarbital (75 mg/kg intraperitoneally), and the tracheas were removed and placed in ice-cold Tyrode's solution bubbled through with 95% O2/5% CO2. The trachea was then isolated from surrounding connective tissue and cut spirally into two strips 3 mm wide and 15 mm long. The composition of Tyrode's solution was (in mM): NaCl 138, KCl 2.7, MgCl2 1.05, NaH2 PO4 0.42, NaHCO3 11.9, glucose 5.5, and CaCl2 1.8. The ends of each tracheal strip were then fixed, via two small clips, to the bottom of the chamber and to a force displacement transducer for recording tension with a polygraph. The chamber (10-mL capacity) was filled with Tyrode's solution at 37[degree sign]C and bubbled through with 95% O (2/5)% CO2. The strip was then subjected to a load of 2 g for at least 2 h until a stable basal tone was obtained. In pilot studies of the dose-response curve to histamine, 10-5 M histamine produced 50% of the maximal response . When reproducible contractions were attained after 10-5 M histamine, the following sets of experiments were performed.
Experiment 1 was designed to determine the effects of several NMDA receptor antagonists on guinea pig tracheal strips contracted by histamine. The tracheal strips used in this set were randomly assigned to one of four groups: Mg2+, Zn2+, ketamine, or MK-801. These drugs were added to the bath cumulatively.
Protocol 1 measured the effect of Mg2+ on histamine-induced contractions (n = 8).
- Step 1 = histamine 10-5 M.
- Step 2 = histamine 10-5 M, then 2, 4, and 8 mM MgSO4 added at 10-min intervals.
- Step 3 = histamine 10-5 M, then 2, 4, and 8 mM Na2 SO4 added at 10-min intervals.
- Step 4 = histamine 10-5 M, then 2, 4, and 8 mM MgCl2 added at 10-min intervals.
Protocol 2 measured the effect of Zn2+ on histamine-induced contractions (n = 8).
- Step 1 = histamine 10-5 M.
- Step 2 = histamine 10-5 M, then 0.4, 0.8, and 1.6 mM of NaCl added at 5-min intervals.
- Step 3 = histamine 10-5 M, then 0.2, 0.4, and 0.8 mM of ZnCl2 added at 5-min intervals.
Protocol 3 measured the effect of ketamine on histamine-induced contractions (n = 8).
- Step 1 = histamine 10-5 M.
- Step 2 = histamine 10-5 M, then 0.5, 1, and 1.5 mM ketamine added at 10-min intervals.
Protocol 4 measured the effect of MK-801 on histamine-induced contractions (n = 8).
- Step 1 = histamine 10-5 M.
- Step 2 = histamine 10-5 M, then 1.5, 3, and 6 x 10-5 M MK-801 added at 10-min intervals.
In all four protocols, the tension of the strip in Step 1 was recorded for 20 or 30 min after the peak response to histamine to determine the spontaneous relaxing curve of the strip. In the other steps, only the peak response to histamine and the tensions obtained 5 or 10 min after the addition of each dose of salts or drugs were measured and recorded. In all four protocols, each salt, ketamine, or MK-801 was added immediately after the peak response to histamine.
Experiment 2 was designed to examine whether NMDA could reverse the spasmolytic effect of ketamine on histamine-induced contractions. Once the full response to 10 (-5) M histamine (peak and spontaneous relaxing curve) was recorded, the same dose of histamine was repeated, and the relaxing effect of 0.5-1.5 mM ketamine alone (n = 6) or in combination with 0.1 mM NMDA, added immediately before ketamine (n = 6), was studied. In three different tracheal strips, the effect of NMDA on the relaxant effect of ketamine was studied in the presence of 10-4 M glycine.
Experiment 3 was designed to test whether the non-competitive NMDA receptor antagonist MK-801  potentiates the relaxant effects of ketamine. Once the full response to 10-5 M histamine (peak and spontaneous relaxing curve) was recorded, the same dose of histamine was repeated, and the relaxing effect of 0.5-2 mM ketamine alone (n = 6) or in combination with 0.75 (n = 6) or 1.5 (n = 6) x 10 (-5) M MK-801 was studied.
Pentobarbital was obtained from Abbott Laboratories (North Chicago, IL). All salts used in the Tyrode's solution were purchased from Fisher Scientific (Pittsburgh, PA). Histamine, MgSO4, MgCl2, Na2 SO4, ZnCl2, racemic ketamine hydrochloride, NMDA, and glycine were purchased from Sigma Chemical Company (St. Louis, MO). MK-801 was a gift from Merck & Co (Rahway, NJ). All drugs, including histamine, were dissolved in double-distilled/deionized water and were added into the organ bath in a volume <or=to100 [micro sign]L. Drugs concentrations are given as the final concentrations in the organ bath.
The net relaxing effect of each drug was obtained by subtracting the spontaneous relaxation after histamine alone and is expressed (mean +/- SEM) as the percentage of the peak tension induced by histamine alone. Comparison of mean values was performed by using analysis of variance followed by Student's t-tests for paired or unpaired samples and the Bonferroni adjustment. Differences were considered significant at P < 0.05.
All NMDA receptor antagonists tested decreased the histamine-induced contractions in a dose-dependent manner. When MgSO4 was added to provide concentrations of 2, 4, and 8 mM in the organ bath after the peak response to 10-5 M histamine, there was a decrease in the strip tension to 94.5% +/- 1.7%, 80.5% +/- 1.7%, and 58.9% +/- 1.4%, respectively, of that developed by histamine alone. The relaxant effect of MgSO4 was caused by Mg2+ because Na2 SO4, giving the same concentration of SO42- in the organ bath solution, had no effect on the contraction after the administration of histamine (Figure 1). We also found that ZnCl2 was more potent than MgSO4 in relaxing the tracheae contracted by histamine. At 0.2, 0.4, and 0.8 mM ZnCl2, the tension of the strips after histamine decreased to 92.4% +/- 1.1%, 79.6% +/- 2.3%, and 62.5% +/- 4.1%, respectively, of the tension developed by histamine alone. In this case, the relaxant effect of ZnCl2 was also caused by Zn2+ because the amount of NaCl that provides the same concentrations of Cl- in the organ bath solution did not alter the contraction induced by histamine (Figure 2). At 0.5, 1.0, and 1.5 mM, Ketamine decreased the contraction after histamine to 93.3% +/- 3.1%, 70.2% +/- 6.9%, and 35.3% +/- 7.9% of the response to histamine alone. Similarly, when 1.5, 3, and 6 x 10-5 M MK-801 were added after the full response to histamine, there was a decrease in the strips tension to 92.1% +/- 4.6%, 86.3% +/- 1.7%, and 76.1 +/- 2.0% of the tension that developed after histamine alone (Figure 3).
The synthetic analog of aspartic acid, NMDA, had no effect on either the basal tone of the strips or the relaxant effect of ketamine (Figure 4), even in the presence of 10-4 M glycine (results not shown).
The blockade of the tracheal NMDA receptor with MK-801 did not perturb the relaxant effect of ketamine on histamine-induced contraction. The response to ketamine did not change in the presence of 0.75 or 1.5 x 10-5 M MK-801 (Figure 5).
The principal finding of the present study is that ketamine relaxes the guinea pig tracheal strips contracted by histamine through a mechanism not involving the NMDA receptor. The existence of functional NMDA receptors in the lung and airway has been demonstrated in experiments showing that NMDA induced acute injury of perfused rat lung characterized by high-permeability pulmonary edema and increased peak airway pressure [12-14]. This lung injury was attenuated or prevented by the competitive NMDA receptor antagonists APV and AP7, by the NMDA channel blocker MK-801, and by Mg2+. In guinea pig tracheal segments, NMDA increased the resting tone more than methacholine, and this action was attenuated by MK-801 . In our study, we focused on histamine as the contracting agonist because of the modulatory effects of histamine on responses to NMDA. Histamine can potentiate NMDA receptor activity at doses up to 5 x 10-4 M [24,25]. In the present study, we found that Mg2+, Zn2+, and MK-801 attenuated the tracheal contraction induced by histamine, results consistent with previous observations indicating that the NMDA receptors in the airway may regulate basal airway tone [12,13]. In addition, the fact that, at high concentration, these NMDA receptor antagonists partially reduced the histamine-induced contraction suggests the predominant involvement of other mechanisms in the action of histamine on airway tone.
The NMDA receptor is a pentamer composed of at least two types of receptor subunits: NMDAR1 and NMDAR2. This receptor channel has five modulatory sites: the primary transmitter recognition site at which glutamate and NMDA bind; the glycine binding site; the cation-binding site for Mg2+; the cation-binding site for Zn2+; and the PCP site [26-29]. The glutamate and glycine binding sites are located in the large extracellular N-terminal domain of the receptor, whereas the Zn2+, PCP (ketamine, MK-801), and Mg2+ binding sites are located inside the channel, with the PCP and Mg2+ sites close to the intracellular end of the channel . An important feature of this receptor channel is its high permeability to Ca2+, so that when the receptor is stimulated, it allows the entry of Ca (2+) into the cell. The increase of intracellular Ca2+ is the principal mechanism that initiates contraction in smooth muscles. L-glutamate and NMDA increase uptake of Ca2+ into rat cortical slices and increase the Ca2+ concentration in rat cortical synaptosomes. These effects were blocked by ketamine . Similar inhibition of the NMDA receptor-mediated increase in intracellular Ca2+ in mouse hippocampal neurons by MK-801 has been described . Thus, one may speculate that if ketamine has a similar effect on the Ca2+ influx through the NMDA receptor in the airway, the blockade of these receptors with ketamine will result in relaxation of the airway smooth muscle. However, in our study, we found that although ketamine did relax the tracheal muscle, this effect was not affected by co-exposing the tissue to the agonist NMDA or the receptor blocker MK-801.
Another characteristic property of native NMDA receptors is the coactivation by glutamate and glycine. NMDA receptors are activated by the natural amino acid L-glutamate, provided that the coagonist glycine is also present [32,33]. Kinetic analysis of NMDA receptor activation indicates that two molecules of glutamate and two molecules of glycine must bind to a receptor to activate the ion-channel gating. However, the binding of glutamate decreases the NMDA receptor affinity for glycine . This negative allosteric coupling between binding of glutamate and binding of glycine produces desensitization of the receptor; that is, a decline in the response to NMDA that is reversed by increasing the concentration of glycine in the extracellular fluid. Because there is no evidence of the effect of glycine on the NMDA receptor of the airway or to exclude this glycine-sensitive desensitization as a mechanism for the lack of action of NMDA in reversing the effect of ketamine, we examined the effect of NMDA and ketamine on the tension of strips contracted by histamine in the presence of 10-4 M glycine, at which concentration the binding of glycine to the NMDA receptor greatly exceeds the rate of dissociation of glycine. However, even in the presence of excess glycine, the agonist NMDA failed to reverse the action of ketamine.
In conclusion, the results of these studies provide new information demonstrating that although NMDA receptors in the airway seem to modulate the airway basal tone, they are not involved in the mechanism by which ketamine relaxes the airway smooth muscle.
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