Neuroscience Letters 302 (2001) 9±12
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5-HT7 receptor mRNA expression in human trigeminal ganglia Jose A. TerroÂn 1, Isabelle Bouchelet, Edith Hamel* Department of Neurology and Neurosurgery, Montreal Neurological Institute, room 751, 3801 University street, Montreal, Quebec, Canada H3A 2B4 Received 9 September 2000; received in revised form 4 December 2000; accepted 30 January 2001
Abstract The present study investigated, by using the method of reverse transcriptase polymerase chain reaction, whether the 5-HT7 receptor mRNA is expressed in human trigeminal ganglia. Trigeminal ganglia were excised post mortem from ®ve human subjects. Oligonucleotide primers were selected based upon unique regions of complementary DNA sequence for the cloned human 5-HT7 receptor. Sequence analysis revealed the presence of a single message that matched the sequence of the cloned human 5-HT7 receptor. The present results may direct future efforts to determine the potential excitatory role of the 5-HT7 receptor in the neuropathological events that develop in migraine headache. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: 5-HT7 receptor mRNA; Migraine; Neurogenic in¯ammation; Pain; Human Trigeminal ganglia
The complex neurological alterations implicated in the pathogenesis of migraine are still poorly understood. At present, according to a neurogenic hypothesis, it is believed that a primary generator, the identity of which has not yet been determined, triggers activation of trigeminovascular ®bers in meningeal blood vessels with the subsequent development of an in¯ammatory process that ultimately results in headache [17]. In view of the evidence supporting a primary role of serotonin (5-hydroxytryptamine; 5-HT) in the pathophysiology of migraine (see [14] for review), it seems reasonable to hypothesize that the mechanism(s) leading to trigeminovascular activation may involve a 5-HT-mediated neuronal excitation. Among the multiple receptor subtypes for 5HT, excitatory responses have been associated with 5-HT3 and 5-HT1A receptors [6,12,13]. However, 5-HT3 antagonists are not effective antimigraine therapies [11] and prophylactic activity of 5-HT receptor antagonists does not correlate with agonism or antagonism at the 5-HT1A receptor [18]. Evidence is accumulating, on the other hand, to suggest that the 5-HT7 receptor may be involved in pain, hyperalgesia and neurogenic in¯ammation by mediating excitatory * Corresponding author. Tel.: 11-514-398-8931; fax: 11-514398-8106. E-mail address:
[email protected] (E. Hamel). 1 Present address: Departamento de FarmacologIÂa, CINVESTAV-IPN, Apartado postal 14±740, MeÂxico D.F. 07360 MEXICO.
responses in neuronal systems [4,10,19,26]. Indeed, it is now recognized that excitatory responses previously believed to be mediated by the 5-HT1A receptor may well correspond to activation of the 5-HT7 receptor (see [9,27] for reviews). Based on this information, the hypothesis that the 5-HT7 receptor might be related to the neurogenic mechanisms of migraine seems attractive. As an initial step in this direction, the present study investigated whether the 5-HT7 receptor mRNA is expressed in human trigeminal ganglia. Human trigeminal ganglia (n 5) were obtained at autopsy (post-mortem delay of 6±12 h) from the Royal Victoria Hospital (MontreÂal, QC, Canada) from both male and female subjects who died from diseases not related to the central nervous system. Samples of parieto-occipital cortex were dissected out for use as control tissue [1]. Tissues were kept at 2808C until use. All tissues were obtained with approval from the Institutional Research Ethics Committee. Total RNA was isolated from tissues with TRIzol Reagent (Gibco BRL, Gaithersburg, MD, USA), as previously described [5], and residual genomic DNA removed with RQ1-DNase (12.5 U, Promega, Madison, WI, USA) for 30 min at 378C. The RNA was then primed with random hexamers (200 ng/mg RNA, Gibco BRL) and reverse transcribed for 60 min at 428C with 1±3 U of avian myeloblastosis virus reverse transcriptase (AMV RT,
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 61 7- 2
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J.A. TerroÂn et al. / Neuroscience Letters 302 (2001) 9±12
Promega) in a ®nal volume of 50 ml. The cDNA was ampli®ed with primers selective for the human 5-HT7 receptor. Oligonucleotides were those previously reported to be based on the nucleotide sequence for the human 5-HT7 receptor [7] and their sequences were: sense, GGAACAGATCAACTACGGCAGAGT (base position 240) and antisense, GGTGGTGGCTGCTTTCTGTTCTCGCTTAAA (base position, 1011). This set of primers encompasses an 825 base pair fragment. The reaction mixture contained 3 ml of cDNA samples, 0.2 mM of each deoxynucleotide triphosphate, 5% of dimethylsulfoxide, 2 mM MgCl2, 0.5 mM of each primer and 1 U of Taq DNA polymerase (Promega) in a buffer containing 50 mM KCl, 10 mM Tris±HCl (pH 9.0) and 0.1% Triton X-100 for a total volume of 50 ml. The polymerase chain reaction (PCR) ampli®cation was performed using a MJ research thermal cycler under the following conditions: denaturation at 958C for 3 min followed by a hot start at 858C for 2 min; annealing at 568C for 40 s, extension at 728C for 60 s and denaturation for 40 s (40 cycles); and ®nal extension at 728C for 5 min. In each experiment, RNA from human cortex, which is known to express the 5-HT7 receptor mRNA [1], was run in parallel as a positive control. The PCR products were electrophoretically separated in a 1.2% agarose gel in Tris/borate/EDTA containing 0.5 mg/ml ethidium bromide (ICN Biomedicals, Ohio, USA) and a 100 bp DNA ladder to con®rm the molecular size of the products. Control reactions lacking the reverse transcriptase enzyme were run in parallel for all samples to monitor for any potential contamination of RNA samples with genomic DNA. A representative PCR product was puri®ed using the QIAquick PCR Puri®cation Kit (Qiagen, Germany) and subjected to automatic ¯uorescence sequencing by the Sanger dideoxynucleotide chain termination method (W.N. Keck Foundation Biotechnology Resource Laboratory, Yale University, CT, USA). The sequence was checked with BLAST program of the NCBI database to reveal the percent of homology with the corresponding fragments of the published cloned human 5-HT7 receptor gene [1]. PCR products of the expected size (i.e. ~825 base pair) for the 5-HT7 receptor were consistently ampli®ed in all the samples of human trigeminal ganglia, as determined by gel electrophoresis (Fig. 1). Contamination with genomic DNA was excluded from all RNA preparations as no PCR product could be ampli®ed in samples that were not reverse transcribed (-RT in Fig. 1). As depicted in Fig. 1, PCR products corresponding to the 5-HT7 receptor were also obtained from the cerebral cortex RNA, as previously reported [1]. Sequence analysis of a selected PCR product con®rmed its identity with the published sequence of the corresponding human 5-HT7 receptor gene. Accumulating evidence suggest that the 5-HT7 receptor may play a role in sensory processing, hyperalgesia i.e. a lowering of nociceptive threshold, and neurogenic in¯ammation. Thus, high expression levels of 5-HT7 transcripts
were detected centrally in areas implicated in sensory and pain processing, including medial geniculate nucleus, superior and inferior colliculi, central gray and spinal trigeminal nuclei [31]. In the periphery, the 5-HT7 receptor mRNA was found strongly expressed in sensory neuronal groups, such as the dorsal root ganglia, superior cervical ganglia and lumbar sympathetic ganglia [20,21]. Signi®cantly, the 5HT7 receptor was recently reported to mediate increases of the hyperpolarization-activated cation current (IH) in speci®c populations of rat dorsal root ganglion cells [4]. This observation led to the suggestion that 5-HT may modulate excitability, neurotransmitter release and ®ring patterns in certain subpopulations of sensory neurons via (5-HT7 receptor mediated) changes in IH [4]. Since sumatriptan and other acute antimigraine drugs inhibit dural in¯ammation induced by chemical or electrical stimulation of the trigeminal ganglion in rodents [3,24], previous studies focused on the molecular characterization of sumatriptan-sensitive 5-HT1 receptor subtypes in human trigeminal ganglia. Thus, PCR studies in this tissue reported expression of mRNAs encoding for 5-HT1Da(5HT1D), 5-HT1Db(5-HT1B) and 5-HT1F receptors [2,22]. Based on the use of selective agonists, it has been proposed that activation of 5-HT1D and 5-HT1F receptors [15,16] inhibit neurogenic in¯ammation and that those agents would be clinically effective in aborting migraine headaches. Now, the present ®ndings show that the 5-HT7 receptor mRNA is expressed in human trigeminal ganglia thus raising the possibility that an excitatory mechanism triggered by 5-HT operates in the trigeminovascular system. Interestingly, a role for the 5-HT7 receptor in peripheral neurogenic in¯ammation has already been proposed as a high concentration of sumatriptan, which displays low to moderate af®nity at the 5-HT7 receptor [1,23,25,31], signi®cantly potentiated capsaicin-induced plasma extravasation in the rat knee joint [19]. Importantly, as mentioned above, the 5-HT7 receptor mRNA was detected in lumbar dorsal root ganglia i.e. a tissue innervating the knee joint [19,20], and activation of the 5-HT7
Fig. 1. Expression of 5-HT7 receptor mRNA in human trigeminal ganglia and parieto-occipital cortex (CX) as visualized by gel electrophoresis of PCR-ampli®ed products obtained using selective oligonucleotide primers. Bands of the expected size i.e. ~825 base pair (bp), were obtained in all tissues. Samples were subjected (1) or not (2) to reverse transcriptase (RT) treatment before PCR ampli®cation to monitor for genomic DNA contamination. Numbers indicate the samples of trigeminal ganglia.
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receptor protein was shown to increase IH in this tissue [4]. That a similar excitatory mechanism may have strong clinical implications in migraine is suggested by the fact that 20±30% of patients treated with sumatriptan experience lack of migraine relief and excessive injection site pain [30]. Interestingly, the maximum plasma concentration (Cmax) of sumatriptan after subcutaneous administration is high enough i.e. 69.5 ng/ml (~250 nM) [8], for the drug to interact with 5-HT7 receptors in sensory neurons [19]. Also of possible relevance to clinics is the observation that migraine prophylactic 5-HT receptor antagonists display high af®nity at the 5-HT7 receptor [27,28], which was preliminarily shown to correlate with their orally active doses [29]. On these bases, it is therefore tempting to hypothesize that, in addition to the suggested role of craniovascular 5-HT7 receptors in migraine [27±29], excitatory trigeminovascular 5-HT7 receptors might be involved in neurogenic in¯ammation. Certainly, functional studies with selective agonists and antagonists will be required to test this hypothesis. In conclusion, the present study has shown that the mRNA encoding for the 5-HT7 receptor is consistently expressed in human trigeminal ganglia. We expect this ®nding may direct novel research efforts aimed at elucidating the nature of 5-HT-mediated excitatory mechanisms within the trigeminovascular system and their possible relevance to migraine pathophysiology and prophylactic treatment. [1] Bard, J.A., Zgombick, J., Adham, N., Vaysse, P., Branchek, T.A. and Weinshank, R.L., Cloning of a novel human serotonin receptor (5-HT7) positively linked to adenylate cyclase, J. Biol. Chem., 268 (1993) 23422±23426. [2] Bouchelet, I., Cohen, Z., Case, B., Seguela, P. and Hamel, E., Differential expression of sumatriptan-sensitive 5-hydroxytryptamine receptors in human trigeminal ganglia and cerebral blood vessels, Mol. Pharmacol., 50 (1996) 219±223. [3] Buzzi, M.G. and Moskowitz, M.A., The antimigraine drug, sumatriptan (GR43175), selectively blocks neurogenic plasma extravasation from blood vessels in dura matter, Br. J. Pharmacol., 99 (1990) 202±206. [4] Cardenas, C.G., Del Mar, L.P., Vysokanov, A.V., Arnold, P.B., Cardenas, L.M., Surmeier, D.J. and Scroggs, R.S., Serotonergic modulation of hyperpolarization-activated current in isolated rat dorsal root ganglion neurons, J. Physiol., 518 (1999) 507±523. [5] Chomczynski, P.A., A reagent for the single-step isolation of RNA, DNA and proteins from cell and tissues samples, Biotechniques, 15 (1993) 532±535. [6] Cohen, M.L., Schenck, K.W. and Hemrick-Luecke, S.H., 5Hydroxytryptamine1A receptor activation enhances norepinephrine release from nerves in the rabbit saphenous vein, J. Pharmacol. Exp. Ther., 290 (1999) 1195±1201. [7] Cohen, Z., Bouchelet, I., Olivier, A., Villemure, J.G., Ball, R., Stanimirovic, D.B. and Hamel, E., Multiple microvascular and astroglial 5-hydroxytryptamine receptor subtypes in human brain: molecular and pharmacologic characterization, , J. Cereb. Blood Flow Metab., 19 (1999) 908±917. [8] Duquesnoy, C., Mamet, J.P., Sumner, D. and Fuseau, E., Comparative clinical pharmacokinetics of single doses of sumatriptan following subcutaneous, oral, rectal and intranasal administration, Eur, J. Pharm. Sci., 6 (1998) 99±104.
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