Neuroscience Letters 590 (2015) 1–5
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Research article
Harmane: An atypical neurotransmitter? Haya Abu Ghazaleh a,b,∗ , Maggie D. Lalies c , David J. Nutt d , Alan L. Hudson c a
Psychopharmacology Unit, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK General Education Department, American University of the Middle East, P.O. BOX 220 Dasman, 15453, Kuwait c Pharmacology Department, University of Alberta, Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada d Centre for Neuropsychopharmacology, Imperial College London, Burlington Danes Building, London W12 0NN, UK b
h i g h l i g h t s • • • •
[3 H]harmane accumulation was overt in rat brain cortex at 37 ◦ C. Uptake was unaltered by monoamine uptake blockers or imidazoline receptor ligands. [3 H]harmane transport was temperature-insensitive and Na+ -independent. The release of preloaded [3 H]harmane was not evoked by 25 mM K+ from rat brain.
a r t i c l e
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Article history: Received 29 September 2014 Received in revised form 15 January 2015 Accepted 22 January 2015 Available online 24 January 2015 Keywords: Harmane Monoamine -carboline Uptake Release
a b s t r a c t Harmane is an active component of clonidine displacing substance and a candidate endogenous ligand for imidazoline binding sites. The neurochemistry of tritiated harmane was investigated in the present study examining its uptake and release properties in the rat brain central nervous system (CNS) in vitro. At physiological temperature, [3 H]harmane was shown to be taken up in rat brain cortex. Further investigations demonstrated that treatment with monoamine uptake blockers (citalopram, nomifensine and nisoxetine) did not alter [3 H]harmane uptake implicating that the route of [3 H]harmane transport was distinct from the monoamine uptake systems. Furthermore, imidazoline ligands (rilmenidine, efaroxan, 2-BFI and idazoxan) showed no prominent effect on [3 H]harmane uptake suggesting the lack of involvement of imidazoline binding sites. Subsequent analyses showed that disruption of the Na+ gradient using ouabain or choline chloride did not block [3 H]harmane uptake suggesting a Na+ -independent transport mechanism. Moreover, higher temperatures (50 ◦ C) failed to impede [3 H]harmane uptake implying a non-physiological transporter. The failure of potassium to evoke the release of preloaded [3 H]harmane from rat brain cortex indicates that the properties of this putative endogenous ligand for imidazoline binding sites do not resemble that of a conventional neurotransmitter. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The family of -carbolines has drawn great interest over the years due to their natural existence in plants and animals and their diverse effects on biological systems. These compounds are formed endogenously via a Pictet–Spengler reaction between indolealkylamines and aldehydes [1]. The aromatic -carbolines, harmane and norharmane, are formed via the oxidation of their tetrahydro--carboline (THC) precursor by heme peroxidases
∗ Corresponding author at: General Education Department, American University of the Middle East, P.O. BOX 220 Dasman, 15453, Kuwait. Tel.: +965 6665 3037; fax: +965 5753379. E-mail address: [email protected] (H. Abu Ghazaleh). http://dx.doi.org/10.1016/j.neulet.2015.01.057 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
[2]. Furthermore, the metabolic fate of these -carbolines were shown to be driven by subtypes of cytochrome P450 enzymes [3]. -Carbolines are reported to interact with different classes of receptors, namely serotonin, dopamine, benzodiazepine, opiate, nicotine, histamine as well as a novel class of receptors/binding sites termed the imidazoline binding sites (I-BS) [4–8]. The existence of a heterogenous family of binding sites that bind imidazoline compounds has drawn great attention in identifying an endogenous ligand. The first potential candidate discovered in rat and bovine brain was clonidine-displacing substance (CDS) [9]. CDS was shown to displace [3 H]clonidine with high affinity in bovine cerebral cortex [9]. Further evaluations showed that CDS not only interacts with ␣2 -adrenoceptors (␣2 -AR) but in addition displays affinity at I-BS [10]. Soon after the discovery of CDS, agmatine was postulated to be an endogenous substrate at I-BS. It shared similar
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Table 1 Effect of monoamine uptake blockers on [3 H]harmane uptake in rat brain cortex. Monoamine uptake blockers
Nisoxetine (1 M) Nomifensine (10 M) Citalopram (10 M)
[3 H]Harmane uptake (fmol/mg/20 mins) Control
+Treatment
P value
880.8 ± 193 1070 ± 339 801.3 ± 257
1369 ± 376 1128 ± 329 703.4 ± 177
0.271 0.904 0.76
Specific uptake was defined as uptake at 37 ◦ C minus uptake at 0 ◦ C. The above data represent the mean ± s.e.mean (vertical bars) of 5–7 independent experiments each performed in triplicate. Statistical analyses were carried out using Student’s unpaired t-test (control vs. treatment). Significance was set at P < 0.05.
binding properties to CDS, binding to ␣2 -AR and the two subclasses of I-BS (I1 and I2 ), albeit weakly [11]. However, CDS and agmatine are two separate entities as their molecular weights [11] and distribution [12] differ considerably. Work from our laboratory has identified the active components of CDS from bovine lung to be harmane and harmalan [13], exhibiting marked nanomolar affinity at I1 -BS and I2 -BS [14]. Harmane has been shown to elicit numerous effects similar to those of I-BS selective ligands. In rat brain, harmane was reported to regulate blood pressure in a similar pattern of response to I1 -BS ligands [15,16]. More recently, harmane has been shown to augment the antinociceptive effect induced by nicotine and ethanol in the tail flick test, and such an effect was reproduced by I1 -BS and I2 -BS selective ligands [17]. Harmane has also been reported to exhibit a number of physiological and psychological effects attributable to I2 -BS involvement; such as monoamine turnover [18], hyperphagia [19], mood [20], drug discrimination and withdrawal [21]. Lastly, harmane displays insulin secretagogue properties in pancreatic -cells which is a common functional trait of the atypical I3 -BS [22,23]. Taken together the above findings it is evident that harmane is a functional ligand at all three I-BS and as such a potential endogenous candidate at I-BS. The aim of the current study was to determine whether harmane displays properties that resemble that of classical neurotransmitters examining its potential uptake and release in rat brain in vitro.
2. Methods and materials 2.1. Preparation of brain tissue slices Male Wistar rats (250–275 g) were killed by stunning followed by decapitation and brains were immediately removed. Brain tissue slices were prepared according to the methods of Abu Ghazaleh et al. (2004) [24].
2.2. Uptake studies Uptake was evaluated by incubating cortical tissue slices in Krebs buffer containing 50 nM [3 H]harmane, [3 H]serotonin (5-HT), [3 H]dopamine (DA) or [3 H]noradrenaline (NA) at physiological temperature (37 ◦ C) for 20 min in the presence or absence of monoamine uptake blockers, I-BS ligands or omission of Na+ from buffer medium with choline chloride/50 M ouabain. Non-specific uptake was determined on ice (0 ◦ C). Reaction was terminated by rapid filtration and tissue was washed in ice-cold Krebs buffer. Tritium tissue content was determined by scintillation counting and tissue protein contents were estimated using the Bradford method with bovine serum albumin as a standard [25].
2.3. Neurotransmitter release assays Brain cortical tissue slices were preincubated with 100 nM [3 H]harmane, [3 H]DA, [3 H]NA or [3 H]5-HT for 20 min at 37 ◦ C. Washed slices were loaded into a multi-chamber Brandel superfusion apparatus and perfused at 37 ◦ C with oxygenated Krebs solution (0.4 ml/min) containing 0.5 mM ascorbate and 0.35 mM pargyline. Perfusate samples were collected at 4 min intervals. Stimulation of [3 H]radioligand release was evoked with 25 mM KCl at t = 12 (S1 ), 40 (S2 ), and 68 (S3 ) minutes for a period of 2 min. The tritium content of the perfusate samples as well as that remaining in the tissue slices were determined by scintillation counting. Fractional efflux of the [3 H]radioligands was calculated by dividing the tritium overflow in each fraction perfusate by the amount of radioactivity in the tissue at the start of each particular collection. Data are expressed as fractional release to allow for neurotransmitter depletion. In these experiments, efforts were made to maximise usage of animal tissue and minimise animal suffering. All animal euthanasia were carried out by Schedule 1 in compliance with the U.K. Animals (scientific procedures) Act 1986 and associated guidelines.
Table 2 Effect of imidazoline binding site ligands on [3 H]harmane uptake in rat brain cortex. Imidazoline binding site ligands
[3 H]harmane uptake (fmol/mg/20 min) Control
+Treatment
P value
I1 -BS ligands Rilmenidine (10 M) Rilmenidine (100 M) Efaroxan (10 M) Efaroxan (100 M)
405.8 405.8 409.9 409.9.
± ± ± ±
75.7 75.7 62.3 62.3
402.8 405.86 415.5 401.2
± ± ± ±
57.7 30.1 52.6 54.2
0.975 1.00 0.947 0.919
I2 -BS ligands Idazoxan (10 M) Idazoxan (100 M) 2-BFI (10 M) 2-BFI (100 M)
330.2 330.2 282.8 282.8
± ± ± ±
36.1 36.1 40.9 40.9
349 356.6 267.8 238.6
± ± ± ±
44.5 40.8 46.9 40.9
0.747 0.638 0.814 0.462
Specific uptake was defined as uptake at 37 ◦ C minus uptake at 0 ◦ C. The above data represent the mean ± s.e.mean (vertical bars) of 4–6 independent experiments each performed in triplicate. Statistical analyses were carried out using Student’s one-way ANOVA followed by Dunnett’s t-test (control vs. treatment). Significance was set at P < 0.05.
2.4. Data analyses Results from uptake and release studies were analysed using GraphPad Prism version 6.0f for windows. Data were expressed as means ± s.e.mean from 3 to 11 independent experiments. Student’s unpaired t-test and one-way analysis of variance (ANOVA) were used to analyse statistical significance set at P < 0.05. 2.5. Materials [3 H]5-HT (specific activity of 30.0 Ci/mmol) and [3 H]DA (specific activity of 59.3 Ci/mmol) were purchased from PerkinElmer life sciences, Inc. [3 H]NA (specific activity 33.0 Ci/mmol) was obtained from Amersham Biosciences, UK. [3 H]Harmane (specific activity of 50.0 Ci/mmol) and 2-BFI hydrochloride were purchased from Tocris, UK. Citalopram hydrobromide, nisoxetine, nomifensine maleate salt, harmane hydrochloride, pargyline hydrochloride, idazoxan hydrochloride, ouabain octahydrate, rilmenidine hemifumarate salt, and bovine serum albumin (BSA) were obtained from Sigma–Aldrich, UK. Efaroxan hydrochloride was a gift from Reckitt and Colman, UK. Coomasie blue reagent was obtained from Pierce Wariner, USA. 3. Results 3.1. Uptake of
[3 H]harmane
Percent of Specific [3H]Harmane Uptake
H. Abu Ghazaleh et al. / Neuroscience Letters 590 (2015) 1–5
3
100 90 80 70 60 50 40 30 20 10 0 No treatment
Ouabain Choline Chloride
Fig. 1. Characterisation of Na+ dependency on [3 H]harmane uptake in rat brain cortex. Percent specific uptake was defined as uptake at 37 ◦ C or 50 ◦ C minus uptake at 0 ◦ C divided by total radioactivity. The above data represent the mean ± s.e.mean (vertical bars) of 4 independent experiments, each performed in triplicate. Statistical analyses were carried out using one-way ANOVA followed by Dunnett’s post hoc test.
uptake inhibition [26], did not hinder [3 H]harmane uptake in rat brain (P < 0.109) (Fig. 1). 3.5. Characterisation of membrane transport of [3 H]harmane into rat brain
into rat brain
The present study demonstrates that [3 H]harmane uptake occurred in rat cortical tissue slices in vitro (448.9 ± 111.6 fmol/mg protein/20 min). [3 H]Harmane uptake was greater than that of DA (260 ± 43.9 fmol/mg protein/20 min) and NA (291.4 ± 56.1 fmol/mg protein/20 min) and was markedly lower than 5-HT uptake (5703 ± 891.5 fmol/mg protein/20 min). 3.2. Effect of monoamine uptake blockers on [3 H]harmane uptake into rat brain [3 H]harmane
Subsequent studies examined the route of uptake in rat brain cortex was using selective reuptake inhibitors of 5HT, DA and NA. The presence of 10 M citalopram (5-HT), 10 M nomifensine (DA) or 1 M nisoxetine (NA) did not significantly affect [3 H]harmane uptake in rat brain cortex in vitro (Table 1). The selected concentrations of the monoamine uptake blockers were significantly effective in the uptake inhibition of their corresponding monoamines (data not shown).
The effect of temperature on [3 H]harmane accumulation was examined to determine whether an active transport system mediates [3 H]harmane influx. The current study showed that of [3 H]harmane uptake was not altered at 50 ◦ C and was not significantly different compared to uptake values at 37 ◦ C (P < 0.871) (Fig. 2). Parallel analyses showed that [3 H]NA uptake was significantly reduced (∼70%) at 50 ◦ C in comparison with uptake at 37 ◦ C (P < 0.0004) (data not shown). 3.6. Effect of 25 mM K+ on the efflux of [3 H]harmane from rat brain The synaptic release of [3 H]harmane was assessed using depolarizing concentration of extracellular K+ . Application of 25 mM K+ at t = 12, 40 and 68 did not induce the release of preloaded [3 H]harmane from rat cortical tissue slices in comparison to [3 H]NA, [3 H]DA and [3 H]5-HT in vitro (Fig. 3).
Further analyses investigated the effect of I-BS ligands on [3 H]harmane uptake in rat brain in vitro. The I1 -BS ligands, rilmenidine and efaroxan, as well as the I2 -BS compounds, 2-BFI and idazoxan, did not affect [3 H]harmane uptake in rat brain cortex (Table 2). 3.4. Characterisation of Na+ dependency of [3 H]harmane uptake into rat brain Further studies examined whether [3 H]harmane uptake, like that of monoamine transporters, is Na+ -dependent. Cationic replacement of Na+ ions with choline chloride did not significantly alter [3 H]harmane uptake (P < 0.885) (Fig. 1). Furthermore, inhibition of the Na+ –K+ ATPase pump using 50 M ouabain, a concentration shown to be effective in monoamine and agmatine
Percent of Specific [3H]Harmane Uptake
100
3.3. Effect of imidazoline binding site ligands on [3 H]harmane uptake into rat brain
90 80 70 60 50 40 30 20 10 0 37°C (control)
50°C
Fig. 2. Effect of temperature on [3 H]harmane uptake in rat brain cortex. Percent specific uptake was defined as uptake at 37 ◦ C or 50 ◦ C minus uptake at 0 ◦ C divided by total radioactivity. The above data represent the mean ± s.e.mean (vertical bars) of 4 independent experiments, each performed in triplicate.
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Fractional Release Value
0.25
Harmane NA 5-HT DA
0.20 0.15 0.10 0.05 0.00
S1
S2
S3
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 Time (minutes) Fig. 3. Release of [3 H]harmane and [3 H]monoamines in rat brain cortex. Tritium efflux was evoked using 25 mM KCl applied at t = 12, 40 and 68 min for a 2 min stimulation period. S1 , S2 and S3 denote first, second and third stimulation periods. Data points expressed as mean ± s.e.mean (vertical bars) of 3–8 separate experiments each performed in triplicate.
4. Discussion Harmane has been proposed as a potential endogenous ligand for I-BS due to its high binding affinity at these sites [14], as well as being identified as one of the primary bioactive components of CDS [13]. Moreover, harmane has been shown to elicit numerous functional and behavioural effects attributed to I-BS stimulation [27]. However, whether harmane can be classified as a neurotransmitter remains to be elucidated. The present data illustrates that [3 H]harmane was taken up in rat brain cortex at physiological temperature. Harmane accumulation in rat brain cortex comes as no surprise as previous reports have demonstrates that harmane labelled a single highaffinity binding site in the rat cerebral cortex [28]. Further characterisation showed that [3 H]harmane uptake occurred by a mechanism independent of the 5-HT transport system as uptake was not affected by citalopram. THC have shown to competitively inhibit the high-affinity uptake of 5-HT in rat brain synaptosomes [29] and in human blood platelets [30]; inferring that THC bind to a site on the 5-HT transporter. Radioligand binding studies showed that several -carbolines, including harmane, competitively inhibited [3 H]citalopram binding in rat cortical membranes [31]. The reported uptake of [3 H]6-methoxy-1-4tetrahydro--carboline and [14 C]tetrahydronorharmane in rabbit blood platelets and in rat hypothalamic slices, respectively, were proposed to occur via the 5-HT uptake system [32,33]. Thus in light of the current literature suggesting the close association of -carbolines and the 5-HT system it was anticipated that harmane would display a similar profile to its precursors. However, this was not observed in the current study. Further analyses examined the potential involvement of the DA and NA uptake systems in mediating [3 H]harmane uptake in rat brain. The dopaminergic and to a lesser extent noradrenergic systems were proposed to play a role in mediating the uptake of particular THC [30]. DA uptake in rat striatal synaptosomes [29] and in human blood platelets [30] was shown to be inhibited in a competitive manner by THC, suggesting that THC modulate DA uptake through its interaction with the dopamine transporter. Similarly, as the case with DA, THC inhibited NA uptake in rat brain synaptosomes [30]. However, in the present study the DA and NA selective reuptake blockers were ineffective in blocking [3 H]harmane uptake. This shows that harmane uptake occurs independently of the DA and NA transport systems.
Agmatine has been proposed as an endogenous candidate ligand at I-BS and compounds selective for these binding sites attenuated the level of agmatine uptake into rat brain synaptosomes [26]. However, in the current study compounds selective for I1 -BS and I2 -BS did not alter the level of [3 H]harmane uptake; suggesting that harmane could be taken up into the rat CNS via a novel site. The accumulation of harmane in rat brain could be due to the involvement of the GABAergic system. -carbolines have been reported to interact with benzodiazepine receptors [7]. Competition studies demonstrated that certain -carbolines displaced [3 H]flunitrazepam from the benzodiazepine binding sites in the GABA receptor complex [34]. Alternatively, it is possible that the uptake of harmane could be driven by the acetylcholinergic system as harmane was reported to displace [3 H]quinuclidinyl benzilate from muscarinic receptors [35]. Unlike the monoamine transporters, the uptake of [3 H]harmane occurred via a Na+ -independent system, a feature shared by neutral amino acid transporters [36] and the previously proposed endogenous I-BS ligand agmatine [26]. Furthermore, the uptake of [3 H]harmane was not affected at higher temperatures (50 ◦ C) inferring a non-enzymatic pump is used to drive the transport of harmane, and so most likely be due to passive transport. High temperatures (>40 ◦ C) results in denaturation of the transporter protein leading to loss of function and increase in membrane leakiness. Lastly, we examined the potential release of [3 H]harmane the rat CNS and found that it did not fulfil all the criteria to define it as a neurotransmitter/neuromodulator since depolarizing concentrations of K+ failed to induce its release from rat brain cortex. However, not all neurotransmitters relay their actions across nerve terminals via exocytotic means. The neurotransmitter nitric oxide (NO) cannot undergo vesicular storage due to its high permeability and as such cannot be released by exocytosis [37]. Synthesis of NO close to its site of action enables it to modulate neuronal release of neurotransmitters or enzymatic activity. Therefore, it is possible that harmane shares similar neurotransmitter properties to that of NO. Due to its lipophilic property, harmane may not be able to undergo vesicular storage and subsequent release; and is instead synthesized close to its site of action when needed. The observed accumulation of [3 H]harmane in rat brain cortex may simply be due to passive diffusion. This is not uncommon as the uptake of certain endogenous ligands, such as anandamide, are not coupled to Na+ ion gradient [38] and its transport is insensitive to temperature changes [39]. A carrier protein has been suggested to mediate anandamide transport [40]. However, most of the find-
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ings deduce that anandamide uptake occurs by simple diffusion [40]. Alternatively, it is possible that harmane possess its own novel transport system in the CNS or perhaps an alternative neurotransmitter system may be involved. 5. Conclusion In conclusion, the present study demonstrated that harmane accumulation was evident in rat brain cortex. Its transport into brain cells occurs through a non- Na+ -driven mechanism and is independent of the monoamine transport systems as well as lack of involvement of I-BS. Lastly, the failure of depolarising concentrations of K+ to induce harmane release implicates that this -carboline does not fulfil the criteria of a classical neurotransmitter. However, needless to say not all neurotransmitters follow the same conventional means of uptake and release and it is possible that harmane may be a unique endogenous ligand with a distinctive transport system of which is yet to be fully explored. Acknowledgment We thank the Wellcome Trust for their financial support. References [1] W.M. McIsaac, D. Taylor, K.E. Walker, B.T. Ho, 6-Methoxy-1-4-tetrahydro-carboline – a serotonin elevator, J. Neurochem. 19 (1972) 1203–1206. [2] T. Herraiz, J. Galisteo, Naturally-occurring tetrahydro--carboline alkaloids derived from tryptophan are oxidized to bioactive -carboline alkaloids by heme peroxidases, Biochem. Biophys. Res. Commun. 451 (2014) 42–47. [3] T. Herraiz, H. Guillén, V.J. Arán, Oxidative metabolism of the bioactive and naturally occurring beta-carboline alkaloids, norharman and harman, by human cytochrome P450 enzymes, Chem. Res. Toxicol. 21 (2008) 2172–2180. [4] A.L. Hudson, R. Price, R.J. Tyacke, M.D. Lalies, C.A. Parker, D.J. Nutt, Harmane, norharmane and tetrahydro -carboline have high affinity for rat imidazoline binding sites, Br. J. Pharmacol. 126 (1999) 2P. [5] O. Arib, P. Rat, R. Molimard, A. Chait, P. Faure, R. de Beaurepaire, Electrophysiological characterization of harmane-induced activation of mesolimbic dopamine neurons, Eur. J. Pharmacol. 629 (2010) 47–52. [6] R.A. Glennon, M. Dukat, B. Grella, S. Hong, L. Costantino, M. Teitler, et al., Binding of beta-carbolines and related agents at serotonin (5-HT(2) and 5-HT(1A)), dopamine (D(2)) and benzodiazepine receptors, Drug Alcohol Depend. 60 (2000) 121–132. [7] M.M. Airaksinen, E. Mikkonen, Affinity of beta-carbolines on rat brain benzodiazepine and opiate binding sites, Med. Biol. 58 (1980) 341–344. [8] M. Nasehi, E. Mashaghi, F. Khakpai, M.-R. Zarrindast, Suggesting a possible role of CA1 histaminergic system in harmane-induced amnesia, Neurosci. Lett. 556 (2013) 5–9. [9] D. Atlas, Y. Burstein, Isolation and partial purification of a clonidine-displacing endogenous brain substance, Eur. J. Biochem. 144 (1984) 287–293. [10] S. Regunathan, D.J. Reis, Imidazoline receptors and their endogenous ligands, Annu. Rev. Pharmacol. Toxicol. 36 (1996) 511–544. [11] G. Li, S. Regunathan, C.J. Barrow, J. Eshraghi, R. Cooper, D.J. Reis, Agmatine: an endogenous clonidine-displacing substance in the brain, Science. 263 (1994) 966–969. [12] W. Raasch, S. Regunathan, G. Li, D.J. Reis, Agmatine is widely and unequally distributed in rat organs, Ann. N. Y. Acad. Sci. 763 (1995) 330–334. [13] C.A. Parker, N.J. Anderson, E.S.J. Robinson, R. Price, R.J. Tyacke, S.M. Husbands, et al., Harmane and harmalan are bioactive components of classical clonidine-displacing substance, Biochemistry 43 (2004) 16385–16392. [14] S.M. Husbands, R.A. Glennon, S. Gorgerat, R. Gough, R. Tyacke, J. Crosby, et al., beta-carboline binding to imidazoline receptors, Drug Alcohol Depend. 64 (2001) 203–208. [15] M.L. Parkin, S.J. Godwin, G.A. Head, Importance of imidazoline-preferring receptors in the cardiovascular actions of chronically administered moxonidine, rilmenidine and clonidine in conscious rabbits, J. Hypertens. 21 (2003) 167–178. [16] I.F. Musgrave, E. Badoer, Harmane produces hypotension following microinjection into the RVLM: possible role of I(1)-imidazoline receptors, Br. J. Pharmacol. 129 (2000) 1057–1059.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Harmane: An atypical neurotransmitter? Haya Abu Ghazaleh a,b,∗ , Maggie D. Lalies c , David J. Nutt d , Alan L. Hudson c a
Psychopharmacology Unit, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK General Education Department, American University of the Middle East, P.O. BOX 220 Dasman, 15453, Kuwait c Pharmacology Department, University of Alberta, Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada d Centre for Neuropsychopharmacology, Imperial College London, Burlington Danes Building, London W12 0NN, UK b
h i g h l i g h t s • • • •
[3 H]harmane accumulation was overt in rat brain cortex at 37 ◦ C. Uptake was unaltered by monoamine uptake blockers or imidazoline receptor ligands. [3 H]harmane transport was temperature-insensitive and Na+ -independent. The release of preloaded [3 H]harmane was not evoked by 25 mM K+ from rat brain.
a r t i c l e
i n f o
Article history: Received 29 September 2014 Received in revised form 15 January 2015 Accepted 22 January 2015 Available online 24 January 2015 Keywords: Harmane Monoamine -carboline Uptake Release
a b s t r a c t Harmane is an active component of clonidine displacing substance and a candidate endogenous ligand for imidazoline binding sites. The neurochemistry of tritiated harmane was investigated in the present study examining its uptake and release properties in the rat brain central nervous system (CNS) in vitro. At physiological temperature, [3 H]harmane was shown to be taken up in rat brain cortex. Further investigations demonstrated that treatment with monoamine uptake blockers (citalopram, nomifensine and nisoxetine) did not alter [3 H]harmane uptake implicating that the route of [3 H]harmane transport was distinct from the monoamine uptake systems. Furthermore, imidazoline ligands (rilmenidine, efaroxan, 2-BFI and idazoxan) showed no prominent effect on [3 H]harmane uptake suggesting the lack of involvement of imidazoline binding sites. Subsequent analyses showed that disruption of the Na+ gradient using ouabain or choline chloride did not block [3 H]harmane uptake suggesting a Na+ -independent transport mechanism. Moreover, higher temperatures (50 ◦ C) failed to impede [3 H]harmane uptake implying a non-physiological transporter. The failure of potassium to evoke the release of preloaded [3 H]harmane from rat brain cortex indicates that the properties of this putative endogenous ligand for imidazoline binding sites do not resemble that of a conventional neurotransmitter. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The family of -carbolines has drawn great interest over the years due to their natural existence in plants and animals and their diverse effects on biological systems. These compounds are formed endogenously via a Pictet–Spengler reaction between indolealkylamines and aldehydes [1]. The aromatic -carbolines, harmane and norharmane, are formed via the oxidation of their tetrahydro--carboline (THC) precursor by heme peroxidases
∗ Corresponding author at: General Education Department, American University of the Middle East, P.O. BOX 220 Dasman, 15453, Kuwait. Tel.: +965 6665 3037; fax: +965 5753379. E-mail address: [email protected] (H. Abu Ghazaleh). http://dx.doi.org/10.1016/j.neulet.2015.01.057 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
[2]. Furthermore, the metabolic fate of these -carbolines were shown to be driven by subtypes of cytochrome P450 enzymes [3]. -Carbolines are reported to interact with different classes of receptors, namely serotonin, dopamine, benzodiazepine, opiate, nicotine, histamine as well as a novel class of receptors/binding sites termed the imidazoline binding sites (I-BS) [4–8]. The existence of a heterogenous family of binding sites that bind imidazoline compounds has drawn great attention in identifying an endogenous ligand. The first potential candidate discovered in rat and bovine brain was clonidine-displacing substance (CDS) [9]. CDS was shown to displace [3 H]clonidine with high affinity in bovine cerebral cortex [9]. Further evaluations showed that CDS not only interacts with ␣2 -adrenoceptors (␣2 -AR) but in addition displays affinity at I-BS [10]. Soon after the discovery of CDS, agmatine was postulated to be an endogenous substrate at I-BS. It shared similar
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Table 1 Effect of monoamine uptake blockers on [3 H]harmane uptake in rat brain cortex. Monoamine uptake blockers
Nisoxetine (1 M) Nomifensine (10 M) Citalopram (10 M)
[3 H]Harmane uptake (fmol/mg/20 mins) Control
+Treatment
P value
880.8 ± 193 1070 ± 339 801.3 ± 257
1369 ± 376 1128 ± 329 703.4 ± 177
0.271 0.904 0.76
Specific uptake was defined as uptake at 37 ◦ C minus uptake at 0 ◦ C. The above data represent the mean ± s.e.mean (vertical bars) of 5–7 independent experiments each performed in triplicate. Statistical analyses were carried out using Student’s unpaired t-test (control vs. treatment). Significance was set at P < 0.05.
binding properties to CDS, binding to ␣2 -AR and the two subclasses of I-BS (I1 and I2 ), albeit weakly [11]. However, CDS and agmatine are two separate entities as their molecular weights [11] and distribution [12] differ considerably. Work from our laboratory has identified the active components of CDS from bovine lung to be harmane and harmalan [13], exhibiting marked nanomolar affinity at I1 -BS and I2 -BS [14]. Harmane has been shown to elicit numerous effects similar to those of I-BS selective ligands. In rat brain, harmane was reported to regulate blood pressure in a similar pattern of response to I1 -BS ligands [15,16]. More recently, harmane has been shown to augment the antinociceptive effect induced by nicotine and ethanol in the tail flick test, and such an effect was reproduced by I1 -BS and I2 -BS selective ligands [17]. Harmane has also been reported to exhibit a number of physiological and psychological effects attributable to I2 -BS involvement; such as monoamine turnover [18], hyperphagia [19], mood [20], drug discrimination and withdrawal [21]. Lastly, harmane displays insulin secretagogue properties in pancreatic -cells which is a common functional trait of the atypical I3 -BS [22,23]. Taken together the above findings it is evident that harmane is a functional ligand at all three I-BS and as such a potential endogenous candidate at I-BS. The aim of the current study was to determine whether harmane displays properties that resemble that of classical neurotransmitters examining its potential uptake and release in rat brain in vitro.
2. Methods and materials 2.1. Preparation of brain tissue slices Male Wistar rats (250–275 g) were killed by stunning followed by decapitation and brains were immediately removed. Brain tissue slices were prepared according to the methods of Abu Ghazaleh et al. (2004) [24].
2.2. Uptake studies Uptake was evaluated by incubating cortical tissue slices in Krebs buffer containing 50 nM [3 H]harmane, [3 H]serotonin (5-HT), [3 H]dopamine (DA) or [3 H]noradrenaline (NA) at physiological temperature (37 ◦ C) for 20 min in the presence or absence of monoamine uptake blockers, I-BS ligands or omission of Na+ from buffer medium with choline chloride/50 M ouabain. Non-specific uptake was determined on ice (0 ◦ C). Reaction was terminated by rapid filtration and tissue was washed in ice-cold Krebs buffer. Tritium tissue content was determined by scintillation counting and tissue protein contents were estimated using the Bradford method with bovine serum albumin as a standard [25].
2.3. Neurotransmitter release assays Brain cortical tissue slices were preincubated with 100 nM [3 H]harmane, [3 H]DA, [3 H]NA or [3 H]5-HT for 20 min at 37 ◦ C. Washed slices were loaded into a multi-chamber Brandel superfusion apparatus and perfused at 37 ◦ C with oxygenated Krebs solution (0.4 ml/min) containing 0.5 mM ascorbate and 0.35 mM pargyline. Perfusate samples were collected at 4 min intervals. Stimulation of [3 H]radioligand release was evoked with 25 mM KCl at t = 12 (S1 ), 40 (S2 ), and 68 (S3 ) minutes for a period of 2 min. The tritium content of the perfusate samples as well as that remaining in the tissue slices were determined by scintillation counting. Fractional efflux of the [3 H]radioligands was calculated by dividing the tritium overflow in each fraction perfusate by the amount of radioactivity in the tissue at the start of each particular collection. Data are expressed as fractional release to allow for neurotransmitter depletion. In these experiments, efforts were made to maximise usage of animal tissue and minimise animal suffering. All animal euthanasia were carried out by Schedule 1 in compliance with the U.K. Animals (scientific procedures) Act 1986 and associated guidelines.
Table 2 Effect of imidazoline binding site ligands on [3 H]harmane uptake in rat brain cortex. Imidazoline binding site ligands
[3 H]harmane uptake (fmol/mg/20 min) Control
+Treatment
P value
I1 -BS ligands Rilmenidine (10 M) Rilmenidine (100 M) Efaroxan (10 M) Efaroxan (100 M)
405.8 405.8 409.9 409.9.
± ± ± ±
75.7 75.7 62.3 62.3
402.8 405.86 415.5 401.2
± ± ± ±
57.7 30.1 52.6 54.2
0.975 1.00 0.947 0.919
I2 -BS ligands Idazoxan (10 M) Idazoxan (100 M) 2-BFI (10 M) 2-BFI (100 M)
330.2 330.2 282.8 282.8
± ± ± ±
36.1 36.1 40.9 40.9
349 356.6 267.8 238.6
± ± ± ±
44.5 40.8 46.9 40.9
0.747 0.638 0.814 0.462
Specific uptake was defined as uptake at 37 ◦ C minus uptake at 0 ◦ C. The above data represent the mean ± s.e.mean (vertical bars) of 4–6 independent experiments each performed in triplicate. Statistical analyses were carried out using Student’s one-way ANOVA followed by Dunnett’s t-test (control vs. treatment). Significance was set at P < 0.05.
2.4. Data analyses Results from uptake and release studies were analysed using GraphPad Prism version 6.0f for windows. Data were expressed as means ± s.e.mean from 3 to 11 independent experiments. Student’s unpaired t-test and one-way analysis of variance (ANOVA) were used to analyse statistical significance set at P < 0.05. 2.5. Materials [3 H]5-HT (specific activity of 30.0 Ci/mmol) and [3 H]DA (specific activity of 59.3 Ci/mmol) were purchased from PerkinElmer life sciences, Inc. [3 H]NA (specific activity 33.0 Ci/mmol) was obtained from Amersham Biosciences, UK. [3 H]Harmane (specific activity of 50.0 Ci/mmol) and 2-BFI hydrochloride were purchased from Tocris, UK. Citalopram hydrobromide, nisoxetine, nomifensine maleate salt, harmane hydrochloride, pargyline hydrochloride, idazoxan hydrochloride, ouabain octahydrate, rilmenidine hemifumarate salt, and bovine serum albumin (BSA) were obtained from Sigma–Aldrich, UK. Efaroxan hydrochloride was a gift from Reckitt and Colman, UK. Coomasie blue reagent was obtained from Pierce Wariner, USA. 3. Results 3.1. Uptake of
[3 H]harmane
Percent of Specific [3H]Harmane Uptake
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3
100 90 80 70 60 50 40 30 20 10 0 No treatment
Ouabain Choline Chloride
Fig. 1. Characterisation of Na+ dependency on [3 H]harmane uptake in rat brain cortex. Percent specific uptake was defined as uptake at 37 ◦ C or 50 ◦ C minus uptake at 0 ◦ C divided by total radioactivity. The above data represent the mean ± s.e.mean (vertical bars) of 4 independent experiments, each performed in triplicate. Statistical analyses were carried out using one-way ANOVA followed by Dunnett’s post hoc test.
uptake inhibition [26], did not hinder [3 H]harmane uptake in rat brain (P < 0.109) (Fig. 1). 3.5. Characterisation of membrane transport of [3 H]harmane into rat brain
into rat brain
The present study demonstrates that [3 H]harmane uptake occurred in rat cortical tissue slices in vitro (448.9 ± 111.6 fmol/mg protein/20 min). [3 H]Harmane uptake was greater than that of DA (260 ± 43.9 fmol/mg protein/20 min) and NA (291.4 ± 56.1 fmol/mg protein/20 min) and was markedly lower than 5-HT uptake (5703 ± 891.5 fmol/mg protein/20 min). 3.2. Effect of monoamine uptake blockers on [3 H]harmane uptake into rat brain [3 H]harmane
Subsequent studies examined the route of uptake in rat brain cortex was using selective reuptake inhibitors of 5HT, DA and NA. The presence of 10 M citalopram (5-HT), 10 M nomifensine (DA) or 1 M nisoxetine (NA) did not significantly affect [3 H]harmane uptake in rat brain cortex in vitro (Table 1). The selected concentrations of the monoamine uptake blockers were significantly effective in the uptake inhibition of their corresponding monoamines (data not shown).
The effect of temperature on [3 H]harmane accumulation was examined to determine whether an active transport system mediates [3 H]harmane influx. The current study showed that of [3 H]harmane uptake was not altered at 50 ◦ C and was not significantly different compared to uptake values at 37 ◦ C (P < 0.871) (Fig. 2). Parallel analyses showed that [3 H]NA uptake was significantly reduced (∼70%) at 50 ◦ C in comparison with uptake at 37 ◦ C (P < 0.0004) (data not shown). 3.6. Effect of 25 mM K+ on the efflux of [3 H]harmane from rat brain The synaptic release of [3 H]harmane was assessed using depolarizing concentration of extracellular K+ . Application of 25 mM K+ at t = 12, 40 and 68 did not induce the release of preloaded [3 H]harmane from rat cortical tissue slices in comparison to [3 H]NA, [3 H]DA and [3 H]5-HT in vitro (Fig. 3).
Further analyses investigated the effect of I-BS ligands on [3 H]harmane uptake in rat brain in vitro. The I1 -BS ligands, rilmenidine and efaroxan, as well as the I2 -BS compounds, 2-BFI and idazoxan, did not affect [3 H]harmane uptake in rat brain cortex (Table 2). 3.4. Characterisation of Na+ dependency of [3 H]harmane uptake into rat brain Further studies examined whether [3 H]harmane uptake, like that of monoamine transporters, is Na+ -dependent. Cationic replacement of Na+ ions with choline chloride did not significantly alter [3 H]harmane uptake (P < 0.885) (Fig. 1). Furthermore, inhibition of the Na+ –K+ ATPase pump using 50 M ouabain, a concentration shown to be effective in monoamine and agmatine
Percent of Specific [3H]Harmane Uptake
100
3.3. Effect of imidazoline binding site ligands on [3 H]harmane uptake into rat brain
90 80 70 60 50 40 30 20 10 0 37°C (control)
50°C
Fig. 2. Effect of temperature on [3 H]harmane uptake in rat brain cortex. Percent specific uptake was defined as uptake at 37 ◦ C or 50 ◦ C minus uptake at 0 ◦ C divided by total radioactivity. The above data represent the mean ± s.e.mean (vertical bars) of 4 independent experiments, each performed in triplicate.
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Fractional Release Value
0.25
Harmane NA 5-HT DA
0.20 0.15 0.10 0.05 0.00
S1
S2
S3
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 Time (minutes) Fig. 3. Release of [3 H]harmane and [3 H]monoamines in rat brain cortex. Tritium efflux was evoked using 25 mM KCl applied at t = 12, 40 and 68 min for a 2 min stimulation period. S1 , S2 and S3 denote first, second and third stimulation periods. Data points expressed as mean ± s.e.mean (vertical bars) of 3–8 separate experiments each performed in triplicate.
4. Discussion Harmane has been proposed as a potential endogenous ligand for I-BS due to its high binding affinity at these sites [14], as well as being identified as one of the primary bioactive components of CDS [13]. Moreover, harmane has been shown to elicit numerous functional and behavioural effects attributed to I-BS stimulation [27]. However, whether harmane can be classified as a neurotransmitter remains to be elucidated. The present data illustrates that [3 H]harmane was taken up in rat brain cortex at physiological temperature. Harmane accumulation in rat brain cortex comes as no surprise as previous reports have demonstrates that harmane labelled a single highaffinity binding site in the rat cerebral cortex [28]. Further characterisation showed that [3 H]harmane uptake occurred by a mechanism independent of the 5-HT transport system as uptake was not affected by citalopram. THC have shown to competitively inhibit the high-affinity uptake of 5-HT in rat brain synaptosomes [29] and in human blood platelets [30]; inferring that THC bind to a site on the 5-HT transporter. Radioligand binding studies showed that several -carbolines, including harmane, competitively inhibited [3 H]citalopram binding in rat cortical membranes [31]. The reported uptake of [3 H]6-methoxy-1-4tetrahydro--carboline and [14 C]tetrahydronorharmane in rabbit blood platelets and in rat hypothalamic slices, respectively, were proposed to occur via the 5-HT uptake system [32,33]. Thus in light of the current literature suggesting the close association of -carbolines and the 5-HT system it was anticipated that harmane would display a similar profile to its precursors. However, this was not observed in the current study. Further analyses examined the potential involvement of the DA and NA uptake systems in mediating [3 H]harmane uptake in rat brain. The dopaminergic and to a lesser extent noradrenergic systems were proposed to play a role in mediating the uptake of particular THC [30]. DA uptake in rat striatal synaptosomes [29] and in human blood platelets [30] was shown to be inhibited in a competitive manner by THC, suggesting that THC modulate DA uptake through its interaction with the dopamine transporter. Similarly, as the case with DA, THC inhibited NA uptake in rat brain synaptosomes [30]. However, in the present study the DA and NA selective reuptake blockers were ineffective in blocking [3 H]harmane uptake. This shows that harmane uptake occurs independently of the DA and NA transport systems.
Agmatine has been proposed as an endogenous candidate ligand at I-BS and compounds selective for these binding sites attenuated the level of agmatine uptake into rat brain synaptosomes [26]. However, in the current study compounds selective for I1 -BS and I2 -BS did not alter the level of [3 H]harmane uptake; suggesting that harmane could be taken up into the rat CNS via a novel site. The accumulation of harmane in rat brain could be due to the involvement of the GABAergic system. -carbolines have been reported to interact with benzodiazepine receptors [7]. Competition studies demonstrated that certain -carbolines displaced [3 H]flunitrazepam from the benzodiazepine binding sites in the GABA receptor complex [34]. Alternatively, it is possible that the uptake of harmane could be driven by the acetylcholinergic system as harmane was reported to displace [3 H]quinuclidinyl benzilate from muscarinic receptors [35]. Unlike the monoamine transporters, the uptake of [3 H]harmane occurred via a Na+ -independent system, a feature shared by neutral amino acid transporters [36] and the previously proposed endogenous I-BS ligand agmatine [26]. Furthermore, the uptake of [3 H]harmane was not affected at higher temperatures (50 ◦ C) inferring a non-enzymatic pump is used to drive the transport of harmane, and so most likely be due to passive transport. High temperatures (>40 ◦ C) results in denaturation of the transporter protein leading to loss of function and increase in membrane leakiness. Lastly, we examined the potential release of [3 H]harmane the rat CNS and found that it did not fulfil all the criteria to define it as a neurotransmitter/neuromodulator since depolarizing concentrations of K+ failed to induce its release from rat brain cortex. However, not all neurotransmitters relay their actions across nerve terminals via exocytotic means. The neurotransmitter nitric oxide (NO) cannot undergo vesicular storage due to its high permeability and as such cannot be released by exocytosis [37]. Synthesis of NO close to its site of action enables it to modulate neuronal release of neurotransmitters or enzymatic activity. Therefore, it is possible that harmane shares similar neurotransmitter properties to that of NO. Due to its lipophilic property, harmane may not be able to undergo vesicular storage and subsequent release; and is instead synthesized close to its site of action when needed. The observed accumulation of [3 H]harmane in rat brain cortex may simply be due to passive diffusion. This is not uncommon as the uptake of certain endogenous ligands, such as anandamide, are not coupled to Na+ ion gradient [38] and its transport is insensitive to temperature changes [39]. A carrier protein has been suggested to mediate anandamide transport [40]. However, most of the find-
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ings deduce that anandamide uptake occurs by simple diffusion [40]. Alternatively, it is possible that harmane possess its own novel transport system in the CNS or perhaps an alternative neurotransmitter system may be involved. 5. Conclusion In conclusion, the present study demonstrated that harmane accumulation was evident in rat brain cortex. Its transport into brain cells occurs through a non- Na+ -driven mechanism and is independent of the monoamine transport systems as well as lack of involvement of I-BS. Lastly, the failure of depolarising concentrations of K+ to induce harmane release implicates that this -carboline does not fulfil the criteria of a classical neurotransmitter. However, needless to say not all neurotransmitters follow the same conventional means of uptake and release and it is possible that harmane may be a unique endogenous ligand with a distinctive transport system of which is yet to be fully explored. Acknowledgment We thank the Wellcome Trust for their financial support. References [1] W.M. McIsaac, D. Taylor, K.E. Walker, B.T. Ho, 6-Methoxy-1-4-tetrahydro-carboline – a serotonin elevator, J. Neurochem. 19 (1972) 1203–1206. [2] T. Herraiz, J. Galisteo, Naturally-occurring tetrahydro--carboline alkaloids derived from tryptophan are oxidized to bioactive -carboline alkaloids by heme peroxidases, Biochem. Biophys. Res. Commun. 451 (2014) 42–47. [3] T. Herraiz, H. Guillén, V.J. Arán, Oxidative metabolism of the bioactive and naturally occurring beta-carboline alkaloids, norharman and harman, by human cytochrome P450 enzymes, Chem. Res. Toxicol. 21 (2008) 2172–2180. [4] A.L. Hudson, R. Price, R.J. Tyacke, M.D. Lalies, C.A. Parker, D.J. Nutt, Harmane, norharmane and tetrahydro -carboline have high affinity for rat imidazoline binding sites, Br. J. Pharmacol. 126 (1999) 2P. [5] O. Arib, P. Rat, R. Molimard, A. Chait, P. Faure, R. de Beaurepaire, Electrophysiological characterization of harmane-induced activation of mesolimbic dopamine neurons, Eur. J. Pharmacol. 629 (2010) 47–52. [6] R.A. Glennon, M. Dukat, B. Grella, S. Hong, L. Costantino, M. Teitler, et al., Binding of beta-carbolines and related agents at serotonin (5-HT(2) and 5-HT(1A)), dopamine (D(2)) and benzodiazepine receptors, Drug Alcohol Depend. 60 (2000) 121–132. [7] M.M. Airaksinen, E. Mikkonen, Affinity of beta-carbolines on rat brain benzodiazepine and opiate binding sites, Med. Biol. 58 (1980) 341–344. [8] M. Nasehi, E. Mashaghi, F. Khakpai, M.-R. Zarrindast, Suggesting a possible role of CA1 histaminergic system in harmane-induced amnesia, Neurosci. Lett. 556 (2013) 5–9. [9] D. Atlas, Y. Burstein, Isolation and partial purification of a clonidine-displacing endogenous brain substance, Eur. J. Biochem. 144 (1984) 287–293. [10] S. Regunathan, D.J. Reis, Imidazoline receptors and their endogenous ligands, Annu. Rev. Pharmacol. Toxicol. 36 (1996) 511–544. [11] G. Li, S. Regunathan, C.J. Barrow, J. Eshraghi, R. Cooper, D.J. Reis, Agmatine: an endogenous clonidine-displacing substance in the brain, Science. 263 (1994) 966–969. [12] W. Raasch, S. Regunathan, G. Li, D.J. Reis, Agmatine is widely and unequally distributed in rat organs, Ann. N. Y. Acad. Sci. 763 (1995) 330–334. [13] C.A. Parker, N.J. Anderson, E.S.J. Robinson, R. Price, R.J. Tyacke, S.M. Husbands, et al., Harmane and harmalan are bioactive components of classical clonidine-displacing substance, Biochemistry 43 (2004) 16385–16392. [14] S.M. Husbands, R.A. Glennon, S. Gorgerat, R. Gough, R. Tyacke, J. Crosby, et al., beta-carboline binding to imidazoline receptors, Drug Alcohol Depend. 64 (2001) 203–208. [15] M.L. Parkin, S.J. Godwin, G.A. Head, Importance of imidazoline-preferring receptors in the cardiovascular actions of chronically administered moxonidine, rilmenidine and clonidine in conscious rabbits, J. Hypertens. 21 (2003) 167–178. [16] I.F. Musgrave, E. Badoer, Harmane produces hypotension following microinjection into the RVLM: possible role of I(1)-imidazoline receptors, Br. J. Pharmacol. 129 (2000) 1057–1059.
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