| [Download PDF]
|Year : 2005 | Volume
| Issue : 2 | Page : 120--125
Investigations of Sapindus trifoliatus in dopaminergic and serotonergic systems: Putative antimigraine mechanisms
DK Arulmozhi1, A Veeranjaneyulu2, SL Bodhankar3, SK Arora2,
1 New Chemical Entity Research, Lupin Research Park, Village Nande, Taluk Mulshi, Pune-411 042 and Department of Pharmacology, Bharati Vidyapeeth, Poona College of Pharmacy, Pune-411 038, India
2 New Chemical Entity Research, Lupin Research Park, Village Nande, Taluk Mulshi, Pune-411 042, India
3 Department of Pharmacology, Bharati Vidyapeeth, Poona College of Pharmacy, Pune-411 038, India
New Chemical Entity Research, Lupin Research Park, Village Nande, Taluk Mulshi, Pune-411 042
OBJECTIVE: To evaluate the potential dopaminergic and serotonergic receptor-mediated modulatory effect of the aqueous extract of Sapindus trifoliatus [(ST), (family: Sapindaceae)], a traditional phytomedicine used in the treatment of hemicrania (migraine), using animal models and receptor assays.
MATERIALS AND METHODS: ST (at 20 and 100 mg/kg, i.p. doses) was evaluated for its effect on apomorphine-induced climbing behavior, 5-hydroxytryptophan (l-5-HTP)-induced serotonin syndrome, and MK-801-induced hyperactivity in mice. The radioligand binding studies for various receptors and enzymes were carried out (outsourced) using standard procedures at 250 µg/ml concentration of ST.
RESULTS: ST significantly inhibited the apomorphine-induced climbing behavior, the l-5-HTP-induced serotonin syndrome and MK-801-induced hyperactivity in mice. In the receptor radioligand binding studies, ST exhibited affinity towards dopamine D2, 5-HT2A receptors.
CONCLUSION: The results of the behavioral studies in mice indicate that ST modulated D2 and 5-HT2A receptor-mediated paradigms. The radioligand binding studies supported these observations, suggesting the possible involvement of dopaminergic and serotonergic mechanisms in the antimigraine activity of ST.
|How to cite this article:|
Arulmozhi D K, Veeranjaneyulu A, Bodhankar S L, Arora S K. Investigations of Sapindus trifoliatus in dopaminergic and serotonergic systems: Putative antimigraine mechanisms.Indian J Pharmacol 2005;37:120-125
|How to cite this URL:|
Arulmozhi D K, Veeranjaneyulu A, Bodhankar S L, Arora S K. Investigations of Sapindus trifoliatus in dopaminergic and serotonergic systems: Putative antimigraine mechanisms. Indian J Pharmacol [serial online] 2005 [cited 2022 Jan 18 ];37:120-125
Available from: https://www.ijp-online.com/text.asp?2005/37/2/120/15114
Migraine is characterized by attacks of intense pulsatile and throbbing headache, typically unilateral in nature. A minority of patients experience a more specific sensory disturbance prior to the headache, called ′aura′ which usually has visual disturbances. Migraine affects a substantial fraction (10-20 %) of world population (more women than men). Attacks are episodic and, by their very nature resolve with time. Associated symptoms, such as nausea, vomiting and heightened sensitivity to light (photophobia), and sound (phonophobia) may occur during the headache phase. The headache phase may be preceded by symptoms such as mood fluctuations and gastrointestinal disturbances.
The diagnosis of migraine remains subjective since it relies on self-reports of various symptoms. Although a wide variety of antimigraine agents such as antiinflammatory drugs, ergots, 5-HT1 receptor agonists (triptans), 5-HT2 receptor antagonists and antiemetics,, are available, there are no objective guidelines concerning the selection of a particular therapeutic approach in a given individual. The molecular pathophysiology of migraine is not fully understood, thus making it very difficult to select the most efficacious therapeutic agent. Some of the available drugs have side effects and triptans are contraindicated in coronary artery disease. Hence, there is a real need for better prophylactic/abortive agents. At present the mode of action of prophylactic treatment is mostly unknown.
Although most recent studies have focused on the role of serotonin in migraine, the dopaminergic system is also implicated in the pathogenesis of migraine. Clinical and pharmacological evidence suggests that hypersensitivity of the dopaminergic system could be involved in the pathogenesis of migraine.
Phytomedicine has offered an alternative source of therapy for migraine sufferers, and provided some additional information about the pathogenesis of migraine., Feverfew (Tanacetam parthenium) and butter-bur (Petasites hybridus) are some of the plants that have been used for centuries for relief of migraine.,
Sapindus trifoliatus (ST) Linn. family Sapindaceae is a medium-sized deciduous tree largely found in south India. The pericarp has a high content of saponins and sugars. The saponin moiety is characterized as hederagenin group of glycosides. The pericarp has been reported to possess various medicinal properties. It is regarded as a tonic, stomachic, spermicidal and useful in the treatment of hemicrania (migraine) etc. A thick watery solution of the pericarp is used for the relief of hemicrania, hysteria or epilepsy. However, there have been no systematic pharmacological studies reported on ST for its antimigraine use.
Since ST is used in folklore medicine in the treatment of hemicrania, an investigation was undertaken to explore the possible mechanism. In the present study, the lyophilized aqueous extract of ST was used in animal studies to predict dopamine and serotonin antagonist activities; along with in vitro binding affinity studies in various receptors.
Materials and Methods
Apomorphine, pargyline, haloperidol and l-5-HTP were obtained from Sigma Chemical Co (ST. Louis, USA). MK-801 was obtained from RBI (USA) and methysergide was procured from Tocris (Avonmouth, UK). Except haloperidol, which was dissolved in 0.01% lactic acid solution, all other drugs and chemicals were dissolved in normal saline. All the drugs were administered intraperitoneally.
Plant material and extraction procedure
Pharmacognostically identified dried pericarp of fruits of Sapindus trifoliatus Linn, family Sapindaceae were collected from the local market. Aqueous extract of ST was prepared as reported. Briefly, 100 grams of the pericarp was soaked in 400 ml of distilled water for 16 h. The percolate was then decanted, centrifuged and filtered through Whatman (No.1) filter paper to obtain a clear extract (300 ml). The extraction process was repeated again with the same volume of distilled water. The percolates were pooled and lyophilized to give a brown colored powder (68% yield). Acid hydrolysis of the extract yielded only one glycone, which was identified as hederagenin. Therefore, estimation of the saponins present in the extract was calculated as hederagenin. The content of hederagenin in the extract was estimated by boiling it with 50% methanolic hydrochloric acid. The entire mixture was evaporated to dryness and reconstituted in methanol. HPLC estimation was carried out using Kromasil C-18 column (5 µm, 250 x 4.6 mm) with gradient elution (0.1% formic acid and acetonitrile in the ratio of 80: 20 v/v) and evaporative light-scattering detector. The concentration of hederagenin was found to be between 5.61-6.97 % by weight of the extract.
Adult male Swiss albino mice (18-22 g) were obtained from the Research Animal Facility of Poona College of Pharmacy (PCP), Pune, India. On arrival, the animals were placed at random and allocated to treatment groups (10 animals per treatment) in polypropylene cages with paddy husk bedding. They were housed at a temperature of 24+2 oC and relative humidity of 30-70% with a 12:12 light:dark cycle. All had free access to water filtered through Aquaguard ® and standard pelleted laboratory animal diet. The experimental procedures and protocols used in the present study were reviewed and approved by the Institutional Animal Care and Use Committee of PCP, Pune, India, and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Forests and Environment, Government of India. Experiments were carried out between 1000 and 1700 h.
Apomorphine-induced climbing behavior
The method of Chung et al  was followed with minor modifications. Each mouse was placed in a cylindrical wire mesh cage (height 13 cm, diameter 14 cm and mesh size 3 mm) for 1 h prior to the experiments. Saline (10 ml/kg, i.p.) or ST (20 or 100 mg/kg, i.p.) or haloperidol (1 mg/kg, i.p.) was administered 30 min prior to apomorphine (2.5 mg/kg, i.p.). Climbing behavior was assessed at 5-min intervals up to 20 min, starting 10 min after apomorphine, using the following scoring system. 0-no paws on the cage, 1-two paws on the cage, 2-four paws on the cage. The score recorded for each animal was based on the position of the animal at the moment it was first observed. The total time spent on the cage was also recorded for each animal. An observer unaware of the specific treatments recorded the observations.
l-5-HTP-induced serotonin syndrome
The syndrome was measured after placing a mouse in a Perspex cage for a 30-min habituation period. The animal was then injected with pargyline (75 mg/kg, i.p.) in order to prevent the rapid degradation of l-5-HTP. Thirty min later, ST (20 or 100 mg/kg, i.p.) or methysergide (10 mg/kg, i.p.) was administered. After 30 min the animal received l-5-HTP (50 mg/kg, i.p.) and was returned to test cage. After 20 min, the serotonin syndrome was assessed every 10 min through 50 min. Five behavioral parameters, such as tremor, hind limb extension, forepaw treading, head weaving and head twitch were monitored using the following scoring system: 0-absent, 1-moderate, 2-marked., A trained observer unaware of the specific treatments recorded the findings.
Mouse locomotor activity was measured using a computerized animal activity meter (Opto Varimex Auto Track System, Columbus Instruments, OH, USA), made of clear perspex box (40 x 40 x 40 cm). An array of 15 infrared emitters (spaced at 2.65 cm intervals; beam wave length 940 nm) measured the animal′s activity along the single axis of motion. To minimize the variation among the individuals, mice were placed in pairs in the locomotor activity boxes for a 30 min period. After this time, the animals were injected with ST (20 or 100 mg/kg, i.p.) or haloperidol (0.1 mg/kg, i.p.) or vehicle and then returned to the test boxes. The recording was begun immediately. After a 30 min period, the animals were injected with MK-801 (0.3 mg/kg, i.p.) and the recording was continued immediately. Activity was measured for a further 90 min. The perspex boxes allowed for continuous visual monitoring of the animals in addition to the automated measurement of locomotor activity. The photocell counts that were measured every minute were grouped into 10 min time intervals.,
Receptor radioligand binding studies
The receptor radioligand binding studies were carried out by NovaScreen Biosciences Corporation, USA. The studies were done as per the standard protocols,, and the details of the receptor source ligands and reference compounds used are listed in [Table:1].
Results are expressed as mean+SEM. Comparisons between the groups were made by Student′s ′t ′ test or analysis of variance (ANOVA) followed by Dunnetts′ post test as per suitability. A P value of < 0.05 was considered as significant.
ST (20 and 100 mg/kg, i.p.) and haloperidol (1 mg/kg, i.p.) significantly (P< 0.05-0.001) inhibited apomorphine-induced climbing behavior in mice [Figure:1] and [Figure:2].
l-5-HTP-induced behavioral syndrome
ST (100 mg/kg, i.p.) significantly (P< 0.001) reduced all the five behavioral syndromes induced by l-5-HTP, however ST 20 mg/kg and methysergide (10 mg/kg, i.p.) inhibited only l-5-HTP-induced head twitches (P< 0.05) [Figure:3].
ST (20 and 100 mg/kg) and haloperidol (0.1 mg/kg) significantly (P< 0.001) inhibited the MK-801-induced hyperactivity. Immediately after the administration of ST, a significant reduction in spontaneous locomotor activity was also observed [Figure:4].
Receptor ligand binding studies
The radioligand binding data of ST on selected receptors are shown in [Table:1].
In receptor radioligand-binding studies, ST exhibited an affinity towards various receptors and enzymes that are relevant targets in migraine [Table:1] at an optimal concentration of 250 µg/ml.
The antimigraine gold standard sumatriptan was originally identified as a selective agonist at 5-HT1-like receptor mediating contraction of dog/rabbit saphenous vein. Sumatriptan was soon proven to be beneficial in the treatment of acute migraine headache, a finding that generated considerable scientific interest towards the identification of the selective serotonin receptor involved in the contraction of the cerebrovascular smooth muscle. While it was reported to act on the 5-HT1-like receptor causing contraction of the dog/rabbit saphenous vein, it was pointed out that there were strong pharmacological similarities between the 5-HT1B and/or 5-HT1D receptors and 5-HT1 receptor mediating cerebral vasoconstriction in the cat and human cerebral arteries. Indeed, now there is overwhelming evidence that the 5-HT1B receptor mediates the contraction of vascular smooth muscles. In line with the above, ST has been studied for its potential affinities towards 5-HT1B/1D receptors in rabbit saphenous vein. However, ST failed to exhibit an affinity towards acute migraine targets viz. α-adrenoceptors and 5-HT1B receptors in rabbit aorta and rabbit saphenous vein respectively. Since ST has been used in the treatment of hemicrania in folklore medicine and the role of 5-HT1B and α-adrenoceptors was ruled out in functional studies, the possible effects of ST through 5-HT2A and dopamine D2 receptor mediation were studied in the present investigation.
In the present investigation, ST exhibited pharmacological actions mediated through dopamine and serotonin receptors in the central nervous system. The current data is in conformity with the ligand binding studies, where ST (250 µg/ml) exhibited affinity towards dopamine D2 (104.54%) and serotonin (5HT2A) (47.53%) receptors.
Apomorphine-induced climbing response is reported to be mediated through the activation of both D1 and D2 receptors, and D1 and D2 antagonists were proved to be efficacious in this model.
The prophylactic antimigraine methysergide, a serotonin (5HT2) antagonist, reportedly inhibited the components of l-5-HTP-induced behavioral syndromes, especially the head twitch response.
The locomotor stimulant effects of MK-801 (a non-competitive NMDA channel blocker) have been correlated to increased dopamine and serotonin release in the brain., Dopamine (5HT2) and serotonin antagonists are reported to antagonize MK-801-induced hyperactivity in mice. Recent pharmacological data have demonstrated that MK-801 increases 5-HT turnover in several brain regions including the cortex, hippocampus, nucleus accumbens, amygdala and striatum. In addition to direct glutamatergic-serotonergic interactions, dopaminergic-serotonergic interactions might be involved in the alteration of serotonin turnover produced by NMDA antagonists. The serotonergic system modulated through 5-HT2A receptors could play a permissive role in the regulation of forebrain dopaminergic systems via glutamate., The ability of MK-801 to increase dopamine and serotonin release in brain favors the notion that increased monoaminergic neurotransmission underlies the locomotor stimulant effect.
ST, a phytomedicine used for the treatment of hemicrania, exhibited dopamine D2 and serotonin (5HT2) antagonism by inhibiting the behavioral effects induced by dopamine and serotonin agonists in the aforementioned animal models. ST exhibited less affinity towards 5-HT2A receptors than dopamine D2 receptors in the ligand binding studies, which may be related to the higher dose required for the significant inhibition of 5-HT-induced syndromes [Figure:3].
Clinical and pharmacological evidence suggests that the involvement of dopamine in migraine and most prodromal and accompanying symptoms could be related to dopaminergic activation. Furthermore, migraine patients show a higher incidence of dopaminergic symptoms following acute dopamine agonist administration, when compared with normal volunteers. Migraine associated with nausea and vomiting is often controlled with dopamine receptor antagonist metoclopromide.
A growing body of biological, pharmacological and genetic evidence suggests the role of dopamine in the pathophysiology of certain types of migraine. Most migraine symptoms can be induced by dopaminergic stimulation. Moreover, there is dopamine receptor hypersensitivity in migraineurs, as demonstrated by the induction of yawning, nausea, vomiting, hypotension and other symptoms of migraine attack by dopaminergic agonists at doses that do not affect non-migraineurs. Conversely, dopamine receptor antagonists are effective therapeutic agents in migraine. Recent genetic data suggest that molecular variations within dopamine receptor genes play a modifying role in the pathophysiology of migraine with aura., In a recent study, dopamine D2 antagonist prochlorperazine showed efficacy in hyperalgesic mice, claimed to be an in vivo model for migraine.
It has been well established that serotonin is implicated in the pathophysiology of migraine and several drugs with potent serotonin (5HT2) receptor blocking activity (methysergide, pizotifen, cyproheptadine and mianserin) have been recognized as being clinically effective in migraine prophylaxis. The 5-HT2A receptor mediated actions of serotonin relevant to migraine include increased cranial capillary permeability and platelet aggregation. It is also reported that activation of 5-HT2A receptor leads to an enhancement of NO production in the trigeminovascular pathway. NO may trigger migraine attack by inducing cerebral vasodilation and sensitizing the perivascular nociceptors and central nociceptive neurons in the trigeminovascular systems. An up-regulation of 5-HT2A receptor has been demonstrated to be correlated with the frequency of migraine attacks. Interestingly, at a concentration of 250 µg/ml, ST has an 84% inhibition of NOS (constitutive neuronal) in radioligand binding studies [Table:1]. Nitroglycerin-induced hyperalgesia is considered to be a model of migraine headaches. It is evident from the literature that nitroglycerin produces dose-dependent headache in normal volunteers and migraine sufferers. Furthermore, we have recently reported the antinociceptive activity of ST in nitroglycerin-induced hyperalgesic rats. In a double-blind study, selective serotonin (5HT2) receptor antagonist ritanserin was highly effective in reducing Pain Total Index and analgesic consumption in chronic headache.
In view of the modulatory role of ST on dopamine D2 and serotonin (5HT2) receptors, there is a strong possibility of the involvement of these receptors in its antimigraine activity. The present data adds value to the reported ethnomedical usage of ST in the treatment of hemicrania.
We wish to thank Dr. Vikas Kumar and Dr. N. Sridhar for critical comments on the manuscript. Thanks also to Dr. V. Srivastava and Mr. S. K. Joshi for their constant help in the extraction of ST. We hereby acknowledge NovaScreen Biosciences Corporation, USA for carrying out the radioligand binding studies.
|1||Stewart WF, Lipton RB. Migraine headache: Epidemiology and health care utilization. Cephalalgia 1993;13:41-6.|
|2||Classification and diagnostic criteria for headache disorders, cranial neurologies and facial pains. Headache Classification Committee of the International Headache Society. Cephalalgia 1988;8:1-96.|
|3||Caviness VS, O' Brien P. Current Concept. Headache. N Engl J Med 1980; 302:446-50.|
|4||Raskin NH. Pharmacology of migraine. Annu Rev Pharmacol Toxicol 1981; 21:463-78.|
|5||Wilkinson M. Treatment of acute migraine attack-current status. Cephalalgia 1983; 3:61-7.|
|6||Goadsby PJ. How do the currently used prophylactic agents work in migraine? Cephalalgia 1997;17:85-92.|
|7||Peroutka SJ. Dopamine and migraine. Neurology 1997; 49:650-6.|
|8||Del Zompo M, Cherchi A, Palmas MA, Ponti M, Bocchetta A, Gessa GL, et al. Association between dopamine receptor genes and migraine without aura in a Sardinian sample. Neurology 1998;51:781-6.|
|9||Russo EB. Headache treatments by native peoples of the Eucadononian amazon: A preliminary cross disciplinary assessment. J Ethnopharmacol 1992; 36:193-206.|
|10||Gorji A. Pharmacological treatment of headache using traditional Persian medicine. Trends Pharmacol Sci 2003;24:331-4.|
|11||Groenwegen WA, Heptinstall S. A comparison of the effects of an extract of feverfew and parthenolide, a component of feverfew, on human platelet activity in vitro. J Pharm Pharmacol 1990;42:553-7.|
|12||Grossman W, Schmidramsl H. An extract of Petasites hybridus is effective in the prophylaxis of migraine. Altern Med Rev 2001;6:303-10.|
|13||Kritikar KR, Basu BD. Indian medicinal plants. Vol. 1. New Delhi: International Book Distributors; 1999.|
|14||Arulmozhi DK, Veeranjaneyulu A, Bodhankar SL, Arora SK. Investigations into the antinociceptive activity of Sapindus trifoliatus in various pain models. J Pharm Pharmacol 2004;56:655-61.|
|15||Gupta DR, Ahmed B. Emarginatosides B and C: Two new saponins from Sapindus emarginatus fruits. Indian J Chem 1991;29:268-70.|
|16||Chung IW, Moore NA, Oh WK, O' Neil MF, Ahn JS, Park JB, et al. Behavioral pharmacology of polygalasaponins indicates potential antipsychotic efficacy. Pharmacol Biochem Behav 2002;71:191-5.|
|17||Pengsuparp T, Indra B, Nakagawasai O, Tadano T, Mimaki Y, Sashida Y, et al. Pharmacological studies of geissochizine methyl ether, isolated from Uncaria sinensis Oliv., in the central nervous system. Eur J Pharmacol 2001;425:211-8.|
|18||O' Neil MF, Shaw G. Comparison of dopamine receptor antagonists on hyperlocomotion induced by cocaine, amphetamine, MK-801 and the dopamine D1 agonist C-APB in mice. Psychopharmacology 1999;145:237-50.|
|19||Bylund DB, Toews ML. Radioligand binding methods: Practical guide and tips. Am J Physiol 1993;265:421-9.|
|20||Bylund DB, Murrin LC. Radioligand saturation binding experiments over large concentration ranges. Life Sci 2000;67:2897-911.|
|21||Zhu M, Bowery NG, Greengrass PM, Phillipson JD. Application of radioligand receptor binding assays in the search for CNS active principles from Chinese medicinal plants. J Ethnopharmacol 1996;54:153-64.|
|22||Arulmozhi DK, Sridhar N, Bodhankar SL, Veeranjaneyulu A, Arora SK. In vitro pharmacological investigations of Sapindus trifoliatus in various migraine targets. J Ethnopharmacol 2004 ; 95:239-45|
|23||Moore NA, Axton MS. The role of multiple dopamine receptors in apomorphine and N-n-propylnorapomorphine-induced climbing and hypothermia. Eur J Pharmacol 1990;178:195-201.|
|24||Vasse M, Chagraoui A, Protais P. Climbing and stereotyped behavior in mice require stimulation of D1-dopamine receptors. Eur J Pharmacol 1998;148:221-9.|
|25||Yamamato T, Ueki S. The role of central serotonergic mechanism on head twitch and backward locomotion induced by hallucinogenic drugs. Pharmacol Biochem Behav 1981;14:89-95.|
|26||Liljequist S, Ossowska K, Grabowska-Anden M, Anden ME. Effect of NMDA receptor antagonist MK-801, on locomotor activity and metabolism of dopamine in various brain areas of mice. Eur J Pharmacol 1991;195:55-61.|
|27||Yan QS, Reith ME, Johe PC, Dailey JW. Dizocilpine (MK-801) increases not only dopamine but also serotonin and norepinephrine transmission in the nucleus accumbens as measured by microdialysis in freely moving rats. Brain Res 1997;765:149-58.|
|28||Loscher W, Annies R, Honach D. The N-methyl-D aspartate receptor antagonist MK-801 induces increase in dopamine and serotonin metabolism in several brain regions of rats. Neurosci Lett 1991;128:191-4.|
|29||Carlsson ML. The selective 5-HT2A receptor antagonist MDL100907 counteracts the psychomotor stimulation ensuing manipulations with monominergic, glutamatergic or muscarinic neurotransmission in the mouse-implication for psychosis. J Neurol Transm Gen Sect 1995;100:225-37.|
|30||Schmidt CJ, Fadayel GM. Regional effects of MK-801 on dopamine release: effects of competitive NMDA or 5-HT2A receptor blockade. J Pharmacol Exp Ther 1996;277:1541-9.|
|31||Emilien G, Maloteaux JM, Geurts M, Hoogenberg K, Cragg S. Dopamine receptors-physiological understanding to therapeutic intervention potential. Pharmacol Ther 1999;84:133-56.|
|32||Dahlof CG, Hargreaves RJ. Pathophysiology and pharmacology of migraine. Is there a place for antiemetics in future treatment strategies? Cephalalgia 1998;18:593-604.|
|33||Ophoff RA, Van den Maagdenberg AM, Roon KI, Ferrari MD, Frants RR. The impact of pharmacogenetics for migraine. Eur J Pharmacol 2001;9:1-10.|
|34||Cerbo R, Barbanti P, Buzzi MG, Fabbrini G, Brusa L, Roberti C, et al. Dopamine hypersensitivity in migraine: role of the apomorphine test. Clin Neuropharmacol 1997;20:36-41.|
|35||Galeotti N, Ghelardini C, Graziol I, Uslenghi C. Indomethacin, caffeine and prochlorperazine alone and in combined revert hyperalgesia in in vivo models of migraine. Pharmacol Res 2002;46:245-50.|
|36||Mylecharane EJ. 5-HT2 receptor antagonist and migraine therapy. J Neurol 1991;238 Suppl 1;45-52.|
|37||Srikiatkhachorn A, Suwattanasophon C, Ruangpattanatawee U, Phansuwan-Pujito P. 5-HT2A receptor activation and nitric oxide synthesis: A possible mechanism determining migraine attacks. Headache 2002;42:566-74.|
|38||Olesen J, Iversen HK, Thomson LL. Nitric oxide supersensitivity. A possible molecular mechanism of migraine pain. Neuroreport 1993;4:1027-30.|
|39||Nappi G, Sandrini G, Granella F, Ruiz L, Cerutti G, Facchinetti F, et al. A new 5-HT2 antagonist (ritanserin) in the treatment of chronic headache with depression. A double-blind study vs amitryptyline. Headache 1990;30:439-44.|