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NEUROPHARMACOLOGY
7-Subunit
aw Rud
Department of Experimental Physiology, Medical University of Biaystok, Biaystok, Poland (U.B., M.G., R.R., B.M.); and Department of Pharmacology and Toxicology, University of Bonn, Bonn, Germany (M.G.)
Received for publication
May 6, 2008
Accepted
June 18, 2008.
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Abstract |
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7-nicotinic acetylcholine receptors (nAChRs) in a cannabinoid (CB) receptor-independent manner. The present study aimed at demonstrating that this mechanism can be activated in vivo. In anesthetized and vagotomized pithed rats treated with atropine, we determined the tachycardic response to electrical stimulation of preganglionic sympathetic nerves via the pithing rod or to i.v. nicotine (0.7 µmol/kg) activating nAChRs on the cardiac postganglionic sympathetic neurons. MAEA (3 and 10 µmol/kg) inhibited the electrically induced tachycardia (maximally by 15–20%; abolished by the CB1 receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; 3 µmol/kg) in pentobarbitone-anesthetized pithed rats, but not in urethane-anesthetized pithed rats, which, thus, are suitable to study the CB1 receptor-independent inhibition of nicotine-evoked tachycardia. The subunit-nonselective nAChR antagonist hexamethonium (100 µmol/kg) and the selective 7-subunit antagonist methyllycaconitine (MLA; 3 and 10 µmol/kg) decreased the nicotine-induced tachycardia by 100 and 40%, respectively (maximal effects), suggesting that nAChRs containing the 7-subunit account for 40% of the nicotine-induced tachycardia. MAEA (3 µmol/kg) produced an AM 251-insensitive inhibition (maximum again by 40%) of the nicotine-induced tachycardia. Simultaneous or sequential coadministration of MLA and MAEA inhibited the nicotine-induced tachycardia to the same extent (maximally by 40%) as each of the drugs alone. In conclusion, according to nonadditivity of the effects, MAEA mediates in vivo inhibition by the same receptors as MLA, namely 7-subunit-containing nAChRs, although at an allosteric instead of the orthosteric site.
9-tetrahydrocannabinol and synthetic cannabinoid receptor agonists exert their neuronal effects mainly via cannabinoid (CB)1 receptors (for review, see Pacher et al., 2006
). However, independently of cannabinoid receptors, they also induce effects at other sites of action, such as various ion channels, including transmitter-gated channels (for review, see Oz, 2006), e.g., they activate TRPV1 receptors (Zygmunt et al., 1999; Malinowska et al., 2001a), they inhibit currents through TASK-1 K+ channels (Maingret et al., 2001) and T-type Ca2+ channels (Chemin et al., 2001), and they potentiate the function of glycine receptors (Hejazi et al., 2006).
Within the cysteine loop family of transmitter-gated ion channels, to which the glycine receptor belongs, the effects of cannabinoids on 5-HT3 receptors have been studied most extensively. Experiments on human embryonic kidney 293 cells (Barann et al., 2002) or Xenopus oocytes (Oz et al., 2002) expressing recombinant 5-HT3A receptors or on the rat nodose ganglion endowed with native 5-HT3 receptors (Fan, 1995) revealed that cannabinoid receptor agonists inhibit 5-HT-induced currents. The cannabinoid receptor-independent inhibitory effect of cannabinoids on 5-HT3 receptor function is also relevant in vivo, as demonstrated in the models of the von Bezold-Jarisch reflex (Godlewski et al., 2003) and cocaine hyperlocomotion (Przegali
ski et al., 2005).
Another member of the cysteine loop superfamily of transmitter-gated ion channels is the 7-nicotinic acetylcholine receptor (nAChR), which, like the 5-HT3A receptor, can form functional homopentameric receptors, which can undergo allosteric modulation (for review, see Bertrand and Gopalakrishnan, 2007; Kalamida et al., 2007). In agreement with the close relationship between both receptors, the 7-nAChR resembles the 5-HT3A receptor in that AEA (Oz et al., 2003) and MAEA (Oz et al., 2004) noncompetitively inhibit the nicotine-induced current through recombinant 7-nAChRs expressed in Xenopus oocytes, the inhibition by AEA being even 10 times more potent than on recombinant 5-HT3A receptors. Clinically important observations were that the nicotine-induced current through the recombinant 7-nAChR was potentiated by ethanol (Oz et al., 2005) and the volatile anesthetic isoflurane (Jackson et al., 2008).
The neuronal nicotinic acetylcholine receptors in general and nAChRs containing the 7-subunit in particular mediate multiple pre- and postsynaptic effects in both the central and the peripheral nervous system (for review, see Skok, 2002; Wang et al., 2002; Kalamida et al., 2007), e.g., in the brain, they are involved in the regulation of transmitter release, cell excitability, and in physiological functions such as sleep, anxiety, processing of pain, and cognitive functions; in the peripheral nervous system, they mediate synaptic transmission in ganglia of the autonomic nervous system and, thus, participate in the regulation of cardiovascular and gastrointestinal functions. In view of the important physiological role of the 7-subunit-containing nAChRs (for review, see Kalamida et al., 2007), it was highly desirable to examine whether the inhibitory effect of AEA or its stable analog MAEA observed in transfected Xenopus oocytes plays a role in vivo and, thus, may modulate, e.g., autonomic neurotransmission.
To obtain an answer to this open question, the first aim of the present investigation was to prove the suitability of a simple and reproducible method to study the function of the 7 subunit-containing nAChR under in vivo conditions. Such receptors are expressed in autonomic ganglia and on postganglionic sympathetic nerve fibers of the rat (Franceschini et al., 2000; Skok, 2002; Wang et al., 2002; Del Signore, 2004; Lips et al., 2006; Mozayan and Lee, 2007), including those innervating the heart (Franceschini et al., 2000; Del Signore et al., 2004). The receptors under consideration comprise not only homopentameric 7-nAChRs, but also heteropentamers containing the 7 plus another subunit such as 5 and 10 (Girod et al., 1999; Lips et al., 2006). Taking these aspects into account, we sought to prove that the nicotine (NIC)-stimulated tachycardia in the pithed rat reflects to a certain percentage the norepinephrine release from the cardiac sympathetic nerve fibers in response to activation of the 7-subunit-containing nAChRs. If so, this preparation could be used as an appropriate model for the in vivo determination of the biological significance of these receptors. The second and main aim was to examine by means of this technique whether MAEA is actually capable of inhibiting this receptor function in a cannabinoid receptor-independent manner under in vivo conditions.
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Materials and Methods |
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ystok (Poland). They were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) as adopted and promulgated by the U.S. National Institutes of Health. Preparation of Rats and Experimental Protocol. Male Wistar rats weighing 180 to 300 g were anesthetized with either 300 µmol/kg i.p. pentobarbitone sodium or 1.25 g/kg i.p. urethane. Then, they received injections i.p. with 2 µmol/kg atropine or i.v. with 3 µmol/kg atropine. After cannulation of the trachea, the animals were pithed through the orbit with a stainless steel rod inserted into the spinal canal and then artificially ventilated (10 ml/kg; 60 strokes/min) using a rodent respiratory system Ugo Basile (Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten, Germany). In some of the experiments, the pithing rod (enameled except for a 1-cm section 7 cm from the tip with the uncovered segment situated at vertebrae C7–T1) was used as an electrode for electrical stimulation of the preganglionic sympathetic nerve axons leaving the spinal canal; for stimulation, an electrical field was generated between the rod and an indifferent electrode placed ventrally by means of Stimulator T (Hugo Sachs Elektronik-Harvard Apparatus GmbH). Both vagal nerves were cut. Heart rate (HR) was derived from the electrocardiogram recorded via s.c. electrodes. Diastolic blood pressure (DBP) was measured from the right carotid artery via a pressure transducer DTX (Spectramed, Bromma, Sweden). Body temperature was kept constant at 37 ± 1°C using an electric heating pad (Bio-Sys-Tech, Bialystok, Poland) and monitored by a rectal probe transducer. The transducers were connected to the monitor Trendoscope 8031 (S&W Vickers Ltd., Bialystok, Poland). The left femoral vein was cannulated for i.v. administration of drugs in a volume of 0.5 ml/kg. Animals were allowed to stabilize for 20 to 30 min after the end of preparation. The proper experiments started at the end of this period.
All experiments were performed according to Malinowska et al. (2001b). They were initiated by the injection of pancuronium (0.8 µmol/kg i.v.) to avoid muscle twitches associated with the electrical stimulation. It was also administered to those rats, which did not undergo electrical stimulation to ascertain identical experimental conditions in all animals. One minute later, atropine (3 µmol/kg) was injected i.v. in rats that were anesthetized with urethane and in which HR was stimulated by isoprenaline (ISO) and NIC to avoid parasympathetic effects or to ascertain identical experimental conditions.
Increase in HR was induced by i.v. bolus injection of NIC (0.7 µmol/kg) or ISO (0.1–0.2 nmol/kg) or by electrical stimulation (1 Hz, 1 ms, 50 V for 10 s) of preganglionic sympathetic nerves. The different doses of ISO were suitable to establish an increase in HR similar to that induced by electrical stimulation and by NIC. The first period of stimulation leading, to an increase in HR (S1), was applied 5 min after the injection of pancuronium, i.e., 5 min after onset of the proper experiments. Tachycardic responses to five periods of electrical stimulation were recorded (S1, S2, S3, S4, S5), with the second to fifth periods (S2–S5) being established 15, 20, 30, and 40 min after onset of experiments. In the case of NIC- and ISO-evoked tachycardia, four stimulatory injections were administered (S1, S2, S3, S4) at 20-min intervals, i.e., the second to fourth ones (S2–S4) were applied 25, 45, and 65 min after onset of the experiments. In one series of experiments with isoprenaline as stimulant, the time schedule for experiments with electrical stimulation was applied. This refers to experiments in which the influence of MAEA on the isoprenaline-induced tachycardia was studied as a control of the inhibitory effect of MAEA on the electrically induced tachycardia.
The cannabinoid CB1 receptor antagonist AM 251 (3 µmol/kg) or its vehicle was injected simultaneously with pancuronium. Each dose of the cannabinoid CB1 receptor agonist MAEA (0.3, 1, 3, or 10 µmol/kg) and of the 7-subunit antagonist methyllycaconitine (MLA; 0.3, 1, 3, or 10 µmol/kg) and the nonselective nAChR antagonist hexamethonium (HEX; at the dose of 100 µmol/kg) or their respective vehicle were administered i.v. 5 (electrical stimulation) or 10 (stimulation with NIC and ISO) min before S2. In some experiments, MAEA was given 10 min before S3 (see Fig. 5b). The doses of AM 251 and HEX chosen were sufficient to achieve complete abolishment of the inhibitory effect of the CB1 receptor agonist CP 55940 1 µmol/kg on the electrically evoked neurogenic tachycardia in pithed rats (our own unpublished data) and complete ganglionic blockade in rats (e.g., Osei-Owusu and Scrogin, 2004), respectively. Because the influence of i.v. administration of MLA or MAEA on the peripheral 7-nAChRs has not yet been investigated, we had to find the appropriate doses to establish the maximal blockade of NIC responses obtainable with these drugs (Figs. 3 and 4).
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Drugs. Drugs were obtained from the following sources: (R)-(+)-methanandamide (in Tocrisolve 100), AM 251, and methyllycaconitine citrate (Tocris Cookson Inc., Bristol, UK); atropine (atropine sulfate salt), hexamethonium bromide, isoprenaline [(-)-isoproterenol (+)-bitartrate salt], nicotine [(-)-nicotine hydrogen tartrate salt], pancuronium bromide, and urethane (ethyl carbamate) (Sigma-Aldrich, Deisenhofen, Germany); and pentobarbitone sodium (Biowet, Pulawy, Poland). Drugs were dissolved in saline with the following exceptions: MAEA was purchased from Tocris as 10 mg/ml stock solution in soya oil/water (1:4) emulsion, which was diluted in saline before experiment. AM 251 was dissolved in saline containing dimethyl sulfoxide, ethanol, and Cremophor El (36:2:1:1). The solvents did not affect basal DBP and HR.
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Results |
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Electrical stimulation (1 Hz, 1 ms, 50 V for 10 s) of preganglionic sympathetic nerves or i.v. bolus injection of NIC (0.7 µmol/kg) or ISO (0.1–0.2 nmol/kg) increased HR by approximately 60 to 80 beats/min (for results in control experiments, see Table 1). The degree of the tachycardic response did not substantially change upon repeated electrical (S2–S5) or chemical (S2–S4) stimulation; in other words, in control groups, the ratios S2/S1, S3/S1, S4/S1, and S5/S1 remained either very close to unity, or, in very few cases, they slightly decreased with time (Table 1). AM 251 did not affect increases in HR induced electrically or by injection of NIC or ISO (Table 1). It should be noted that under the present conditions, electrical or chemical stimulation did not induce a substantial increase in blood pressure.
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In urethane-anesthetized rats, MAEA (3 µmol/kg), given alone or in combination with AM 251 (3 µmol/kg), did not modify the electrically stimulated increase in HR (Fig. 1d). Because the main aim of the present study was to examine cannabinoid receptor-independent effect of cannabinoids, all further experiments were performed in urethane-anesthetized rats, which represent an ideal model for such investigations.
Inhibition by HEX and MLA of the NIC-Evoked Tachycardia. The subunit-nonselective nAChR antagonist HEX at the very high dose of 100 µmol/kg abolished the nicotine-evoked increase in HR (Fig. 2), indicating that the latter is mediated exclusively by nicotinic receptors irrespective of their subunit composition. The 7-subunit antagonist MLA (1–10 µmol/kg) inhibited the NIC-induced increase in HR (Figs. 2 and 3) in a dose-dependent manner (Fig. 3). The maximal effect, inhibition by approximately 40%, was reached already at MLA (3 µmol/kg) because MLA (10 µmol/kg) did not produce a more pronounced inhibition (Fig. 3). Figure 2 shows the time dependence of the inhibitory effect of HEX (100 µmol/kg) and MLA (1 µmol/kg). In the case of both antagonists, the inhibition was already fully established 10 min after their application, and this effect persisted until the end of the experiments (i.e., 50 min after their administration).
Inhibition by MAEA of NIC-Evoked Tachycardia. The CB1 receptor agonist MAEA (1 and 3 µmol/kg) resembled MLA in that it inhibited the NIC-evoked tachycardia with the same maximum, i.e., a decrease in evoked tachycardia by 40% (Fig. 4). This maximum was already obtained with the CB1 receptor agonist dose of 1 µmol/kg (Fig. 4). In contrast to the rapid development of the MLA-induced inhibition, the inhibitory effect of MAEA (3 µmol/kg) increased gradually (Fig. 2). Thus, NIC-evoked tachycardia was not yet inhibited 10 min after injection, but the inhibition was fully developed after 30 min and persisted until the end of the experiment (Fig. 2). This time course of action was basically confirmed by another series of experiments in which very weak inhibition of NIC-evoked tachycardia was observed 10 min after injection of MAEA, and the full effect of approximately 40% inhibition was observed 30 min after injection (Fig. 5b). The inhibitory effect of MAEA (1 µmol/kg) was not antagonized by AM 251 (3 µmol/kg; Fig. 4), supporting the view that CB1 receptors are not involved in the inhibitory effect of MAEA on NIC-evoked tachycardia.
To exclude that the MAEA-induced inhibition of NIC-evoked tachycardia is mediated by a postsynaptic site of action, a control series of experiments with ISO as a stimulant of heart rate was carried out. The ISO-evoked tachycardia was not affected by MAEA (3 µmol/kg), excluding the involvement of a postsynaptic cardiac site of action (Fig. 2).
Lack of Enhanced Inhibition of NIC-Evoked Tachycardia by Additive Administration of MAEA and MLA. Two experimental approaches were designed to examine whether MAEA acts as an inhibitor of NIC-evoked tachycardia at the same site as MLA. The first one was characterized by additive administration of the drug (Fig. 5a), and the second one was characterized by sequential injection (Fig. 5b).
In the first approach (Fig. 5a), the effects of injection of rats with MLA or MAEA alone, 1 µmol/kg each 10 min before S2, were compared with that of simultaneous administration of the drugs at the same dose and time schedule. The drugs given alone reduced the tachycardia evoked by the third stimulus by 30 to 40%, i.e., they led to maximal inhibition within the 30 min that elapsed until S3. Injection with both drugs simultaneously did not result in a more pronounced inhibition of the NIC-evoked tachycardia (Fig. 5a).
The second approach to the question of whether or not the effects of MLA and MAEA are additive was also based on experiments with three groups of rats that, in this case, received the drugs at 3 µmol/kg (Fig. 5b). The rats in group 1 received injections with MLA 10 min before S2 as usual; this led within 10 min to the 40% maximal inhibition that persisted until S4, i.e., the end of the experiments. In group 2, rats received MAEA 10 min before S3, leading to the slow development of the 40% maximal inhibition within 30 min that is characteristic for this drug (compare Fig. 2). In group 3, rats received injections with MLA the first 10 min before S2; this produced the same 40% maximal inhibition within 10 min as in the group 1 rats. Additional administration of MAEA to group 3 rats 10 min before S3 did not cause further enhancement of inhibition, and the line representing the inhibition by MLA alone and that for sequential administration of MLA and MAEA are identical (Fig. 5b).
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Discussion |
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7-subunit-containing nAChRs under in vivo conditions. This problem was based on the observation by Oz et al. (2003, 2004) that the endocannabinoid AEA and its stable analog MAEA, but not synthetic cannabinoids, inhibit NIC-evoked currents through recombinant homopentameric 7-nAChRs expressed in Xenopus oocytes. The inhibition was independent of cannabinoid receptors, but an allosteric mechanism directly modifying 7-nAChRs function is probably involved (Oz, 2006). It is important to know whether this mechanism can be initiated in vivo because allosteric sites on a transmitter-gated ion channel may be considered as potential targets of new classes of therapeutic drugs. The latter may have more favorable properties than ligands at the orthosteric site, e.g. novel positive allosteric modulators of 7-nAChRs have been suggested to have significant advantages over indiscriminate and direct activation of nAChRs by nicotine/nicotinic agonists or by acetylcholinesterase inhibitors (Faghih et al., 2007). Likewise, in the case of GABAA receptors, benzodiazepines act therapeutically via allosteric sites, whereas ligands at the orthosteric site of the GABAA receptor play a much less important role.
To approach the main aim of the present study, it was necessary to establish a highly reproducible, if possibly simple, model for the examination of the function of nAChRs containing the 7-subunit under in vivo conditions. Our approach to this first aim, which, in fact, covered a major part of our study, was based on a report by Malinowska et al. (2001b). In that investigation, the electrically and NIC-evoked tachycardic response was determined in pentobarbitone-anesthetized and vagotomized pithed rats treated with atropine, and the operation of presynaptic CB1 receptors on postganglionic sympathetic nerve fibers in vivo was proven using synthetic cannabinoids to stimulate these receptors.
In a systematic approach to our aim, we studied first whether MAEA shares the property of exocannabinoids to act as an agonist at CB1 receptors in vivo. We applied the same experimental conditions as in the study of Malinowska et al. (2001b), and we found that MAEA reduced the tachycardia evoked by electrical stimulation of preganglionic sympathetic nerves in a dose-dependent manner, maximally by approximately 15 to 20%. This inhibitory effect appeared 5 min after its administration and lasted for only 10 min in the case of the highest dose (10 µmol/kg). It was mediated by presynaptic cannabinoid CB1 receptors located on sympathetic nerve terminals innervating the heart because it was abolished by the cannabinoid CB1 receptor antagonist AM 251 (3 µmol/kg) and because MAEA did not affect the ISO-evoked tachycardia.
The very short action of the extremely lipophilic MAEA as a CB1 receptor agonist is probably not caused by rapid metabolism of the compound but, analogous to the pharmacokinetics of the very lipophilic i.v. anesthetics, by redistribution. The orthosteric recognition sites of the CB1 receptor on the extracellular surface of the cell membrane of the cardiac sympathetic nerve terminals are easily accessible for CB1 receptor ligands from the aqueous phase, leading to a fast onset of the CB1 receptor-mediated inhibition of noradrenaline release.
In urethane-anesthetized rats, MAEA failed to modify the electrically evoked neurogenic tachycardia. Likewise, it has previously been demonstrated, that urethane reduces the inhibitory effects of presynaptic 2-adrenoceptors on sympathetically evoked tachycardia and pressor responses in pithed rats (Armstrong et al., 1982). The failure to identify the CB1 receptor-mediated effect of MAEA in urethane-anesthetized rats suggests that urethane anesthesia represents an ideal condition to study CB1 receptor-independent effects of MAEA.
In urethane-anesthetized pithed rats, the NIC-evoked tachycardia was abolished by the high dose of the subunit-nonselective nAChRs antagonist HEX, indicating that in our model, NIC acted exclusively via nAChRs. The selective, competitive 7-subunit antagonist MLA (Alkondon et al., 1992; Turek et al., 1995) also reduced the NIC-elicited tachycardia but with a maximum of approximately 40% only, suggesting that nAChRs containing the 7 subunit account for approximately 40% of the nAChRs involved in this tachycardia. As in the case of HEX, MLA reached this maximum already, with the first stimulation 10 min after its administration, and lasted for at least 50 min (until the end of the experiments). The relatively fast onset of action is in line with the contention that MLA acts at the orthosteric sites of the 7-subunit of the relevant nAChRs. These sites are located on the outer surface of the cell membrane, which is easily accessible within and from the extracellular fluid. These receptors may be localized as follows: 1) on the cell bodies of the postganglionic sympathetic nerves; 2) on postganglionic sympathetic nerve endings; and/or 3) in the adrenal medulla. In fact, norepinephrine release in response to activation of 7-subunit-containing nAChRs was demonstrated in vitro in rat intracardiac nerve fibers and superior cervical ganglia (Del Signore et al., 2004; Mozayan and Lee, 2007). However, we can exclude that catecholamines released from the adrenal medulla contributed significantly to the NIC-induced tachycardia because the increase in HR elicited by NIC did not differ between adrenalectomized rats and animals with intact adrenals (Malinowska et al., 2001b). Moreover, 7-nAChRs are not expressed in rat adrenal chromaffin cells (Sala et al., 2008).
Next, we studied the influence of MAEA on the NIC-evoked tachycardia mediated by nAChRs containing the 7-subunit in urethane-anesthetized pithed rats. MAEA decreased the NIC- (but not ISO-) induced increase in HR, with a maximum comparable with that elicited by MLA, i.e., by approximately 40%. We can exclude the involvement of CB1 receptors in the effect of MAEA not only by the experimental conditions, i.e., the use of urethane-anesthetized pithed rats, but also by the failure of the CB1 receptor antagonist AM 251 to block the effect of MAEA.
A remarkable feature of the inhibition by MAEA of the NIC-evoked tachycardia was its slow onset. Thus, in Xenopus oocytes, AEA and MAEA had to be present in the superfusion fluid for at least 20 min to establish the effect (Oz et al., 2003, 2004). In addition, in our in vivo experiments, the MAEA-induced reduction of the NIC-stimulated tachycardia developed slowly. This inhibition was not yet, or only weakly, established 10 min after injection, but 30 min had to elapse before the inhibition became manifest.
It is probable that the allosteric cannabinoid recognition site suggested to be involved in the MAEA-induced inhibition of 7-nAchR function is not easily accessible. Basic evidence for the involvement of such an allosteric site in this effect was obtained in electrophysiological experiments in vitro (for review, see Oz et al., 2004; Oz, 2006). Thus, the inhibitory effect of MAEA was shown to be noncompetitive, suggesting that the orthosteric acetylcholine and nicotine binding site was not targeted but that this compound, as with other highly lipophilic compounds, binds to topographically distinct sites (Oz, 2006). Further results of in vitro studies with chimeric 7-nAchR-5-HT3 receptors suggest that the site of this action is at C-terminal or transmembrane domains of the 7-nAchR (Oz et al., 2004). The slow development of the inhibitory effect of cannabinoids observed in in vitro studies and in our in vivo experiments was also found for the closely related 5-HT3A receptor. The suggestion that the allosteric recognition site is located in one of the transmembrane domains was also in the case of 5-HT3 receptors supported by the slow development of the effect both in vitro (Barann et al., 2002) and in vivo (Godlewski et al., 2003) and by further cell biological data (Barann et al., 2002). MAEA has to diffuse into and within the cell membrane until the equilibrium concentration, determined by the extremely high lipophilicity, has been reached in the biophase of the allosteric site. According to our data, the drug seems to have reached the equilibrium concentration in the lipid biophase of the allosteric binding site of the nicotine receptor relatively slowly after 30 min.
Two kinds of experiments were designed to prove that MAEA acts at the same macromolecule as MLA. In the first, one we demonstrated that coadministration of MLA and MAEA (1 µmol/kg each) before S2 inhibited the nicotine-induced tachycardia to the same extent (by approximately 30–40%) as each of the drugs alone given at the dose of 1 µmol/kg. If the two drugs would have produced their inhibitory effect via two different macromolecules/receptors, each of which accounted for approximately 30 to 40%, one would have expected a significantly more pronounced inhibition than by 30 to 40% in the case of combination of both drugs. In the second type of experiments, we examined the influence of MAEA (3 µmol/kg) on the inhibitory effect of MLA (3 µmol/kg). MAEA was administered 20 min after the injection of MLA (i.e., 10 min before S3 and S2, respectively). Again, MLA and MAEA given separately inhibited the NIC-induced tachycardia by approximately 40%. However, no additional effect was observed when MAEA was injected 20 min after the above-mentioned maximally effective dose of MLA (3 µmol/kg), which given alone, already completely blocked the fraction of NIC-induced tachycardia accounted for by 7-subunit-containing nAChRs. These findings strongly support the view that MAEA acts at the same receptor as MLA, i.e., the nAChR containing the 7-subunit, albeit at the allosteric instead of the orthosteric site.
Taken together, in the model of the anesthetized pithed rat, we identified two different inhibitory effects of MAEA on the neurogenic tachycardia, the first one dependent on presynaptic CB1 receptors (on postganglionic sympathetic nerve endings) and the second one independent of CB1 receptors but mediated by nAChRs containing the 7-subunit (located probably in sympathetic ganglia and/or presynaptically on postganglionic sympathetic nerve endings innervating the heart). The second one was stronger than the first one (maximal effects were approximately 40 and 15–20%, respectively), and the inhibitory effect seemed slower but lasted longer. The CB1 receptor-independent mechanism mediated by nAChR containing the 7-subunit might be of clinical significance. It might play a protective role in myocardial ischemia. Anandamide, which is released during experimental myocardial infarction, exerts cardioprotective effects (for review, see Pacher et al., 2006), probably also by inhibiting the function of peripheral nAChR containing the 7 subunit. The latter effect is mimicked by MAEA, as demonstrated by Oz et al. (2004) in vitro and by the present results in vivo.
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Acknowledgements |
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Footnotes |
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Parts of this work were previously presented as follows: Göthert M, Baranowskea U, Godlewski G, and Malinowska B (2007) Cannabinoid receptor-independent inhibition by methanandamide of alpha-nicotinic acetylcholine receptor-mediated responses in vivo. 37th Annual Meeting of the Society for Neuroscience; 2007 Nov 3–7; San Diego, CA. Society for Neuroscience, Washington, DC.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: AEA, anandamide; MAEA, methanandamide; CB, cannabinoid; nAChR, nicotinic acetylcholine receptor; NIC, nicotine; HR, heart rate; DBP, diastolic blood pressure; ISO, isoprenaline; AM 251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; MLA, methyllycaconitine; HEX, hexamethonium; CP 55940, (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)-cyclohexanol.
Address correspondence to: Dr. Barbara Malinowska, Department of Experimental Physiology, Medical University of Biaystok, 2A Mickiewicz Str., 15-222 Biaystok, Poland. E-mail: bmalin{at}umwb.edu.pl
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