Differential Effect of Sumatriptan on Cerebellar 5-HT1B Receptors
in Rat Brain
Rama Krishna Devaki
Sumatriptan is a novel and highly effective drug against migraine and cluster
headache attacks. This antimigraine drug is known to exert its effect through
the modulation of serotonin (5-HT) mediated neurotransmission. 5-HT1B
receptors in rats and 5-HT1D receptors in humans have been thought
to be likely targets of sumatriptan. In the present study the effect of varying
dose and duration of sumatriptan on the density of 5-HT1B receptors
in the rat cerebellum were studied to understand its mechanism of action. Sprague
Dawley rats were administered with different doses of sumatriptan (0.2-16 mg
kg-1 b.wt. i.p.) for seven days and 2, 4 and 8 mg kg-1
of sumatriptan for 7, 14 and 21 days. The radioligand binding assays were performed
in cerebellar membranes using [3H]5-HT. Treatment with different
doses of sumatriptan for seven days showed a significant (p<0.0001) downregulation
of 5-HT1B receptors in a dose dependent manner. A significant decrease
in the density of 5-HT1B receptors was observed with 0.2 mg (45%),
0.5 mg (72%) and 1 mg (70%) of sumatriptan treatment, with a significant decrease
(p<0.001) in Kd values. No further decrease in either the density
or in the Kd values was observed with increasing doses of Sumatriptan
from 2 to 16 mg kg-1 b.wt. The magnitude of decrease in the receptor
density was more significant with a lower dose (2 mg) of sumatriptan for a prolonged
period of exposure (21 days). However, such change was not observed with higher
doses (4 and 8 mg kg-1). Furthermore, sumatriptan showed a higher
affinity for 5-HT1B receptors with a Ki value of 9.4±0.9
nM, when compared to other agonists and antagonists. Taken together, these findings
suggest that desensitization of 5-HT1B receptor is dose and time
dependent, which may be an important factor underlying the mechanism of action
of sumatriptan as an antimigraine drug.
Received: November 15, 2013;
Accepted: November 23, 2013;
Published: March 04, 2014
Neurogenic inflammation within the meninges has been shown as an important
event in the pathogenesis of migraine and headache (Millson,
2004) and evidence shows that 5-HT1B/1D ligands may be useful
in the treatment of migraine (Peroutka, 1990; Burstein
and Jakubowski, 2004). Sumatriptan is a novel and highly effective drug
against migraine and cluster headache attacks. It shows a remarkable pharmacological
profile in animals. 5-HT1D receptors, pharmacologically defined functional
sites in human brain, exhibit high affinity for sumatriptan (Weinshank
et al., 1992). So far 5-HT1B receptors in rats and 5-HT1D
receptors in guinea pigs and humans have been thought to be the likely target
of sumatriptan (Moskowitz, 1992). The amino acid sequence
within the transmembrane domains of 5-HT1D receptors is found to
be 96% identical with that of 5-HT1B receptors (Hoyer
et al., 1994). 5-HT1F receptors in guinea pig brain have
also been shown to have high affinity for sumatriptan (Waeber
and Moskowitz, 1995).
Drugs acting via 5-HT1B/1D receptors are effectively used to treat
migraine. However, the antinociceptive effects of many such agents have not
been understood. Sumatriptan may act on the cerebral arteries by blocking neurogenic
inflammation and nociceptive activity within trigemino-vascular afferents (Burstein
and Jakubowski, 2004). This action has been argued to be due to the activation
of 5-HT1B/1D receptors, which prevents protein extravasation induced
by trigeminal ganglion (Reuter et al., 2004).
It has been shown as early as in 1989 that sumatriptan has high affinity for
5-HT1D and 5-HT1B binding sites (Peroutka
and McCarthy, 1989). It selectively interacts with 5-HT1B sites,
which may be the basis for its apparent efficacy in the acute treatment of migraine
(Peroutka and McCarthy, 1989). Sumatriptan has been
shown to inhibit neurogenic inflammation dose dependently via 5-HT1D
receptors in guinea pigs and via 5-HT1B receptors in rats (Xian-Jie
et al., 1997). Blockade of dural extravasation is concomitant with
an action at rat 5-HT1D receptors, while inhibition of dural vasodilation
is concomitant with 5-HT1B receptors. Apart from mediating inhibition
of K+ evoked release of 5-HT (Pineyro et
al., 1995; Pineyro and Blier, 1996), sumatriptan
is also known to increase growth hormone (GH) levels as well as its response
to growth hormone releasing hormone (Mota et al.,
1995) and activates pituitary-adrenal axis (Facchinetti
et al., 1994). Study of the mechanism of action of sumatriptan via
5-HT1B/1D receptors would further our understanding of the pathophysiology
of migraine and the putative involvement of 5-HT receptors.
Although, migraine is inextricably bound with 5-HT receptors and its many subtypes,
its precise role continues to elude the scientists and there is still no clear
evidence suggesting either vascular or neurogenic hypothesis, unequivocally.
Therefore, the present study was designed to investigate the dose and duration
dependent effects of sumatriptan administration on the density of 5-HT1B
receptors in rat cerebellum.
MATERIALS AND METHODS
Chemicals: [3H]5-HT (s.a. 8.2 Ci mmol-1) was obtained
from M/s. Amersham Plc. UK. 8-hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT),
mianserin, 5-HT, N-3[(Tri-fluoromethyl) phenyl] piperazine (TFMPP) and pargyline
were obtained from M/s. Sigma Chemicals, MO, USA. Sumatriptan was a generous
gift from M/s. Torrent Pharmaceuticals, India. GF/B filters were obtained from
M/s. Millipore, UK.
Animals and administration of drugs: Adult male Sprague Dawley rats (n=6 for each treatment group), weighing 200-240 g, were procured from Central Animal Research Facility (CARF) of NIMHANS. Rats were housed four per cage with food and water ad libitum in a well-ventilated room, with 12 h light and dark cycles. Rats were injected, once in the morning, with different doses of sumatriptan (0.2, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 16.0 mg kg-1 b.wt., i.p.) for seven days. In another experiment, rats were injected (i.p.) once in the morning, with different doses (2, 4 and 8 mg kg-1 b.wt.) of sumatriptan, each dose for 7, 14 and 21 days. Control rats were similarly treated with saline and housed under identical conditions. Animals were sacrificed 24 h after the last dose and brains removed. The cerebellum was dissected from each rat brain and immediately used for the assay. All animal experimental procedures were performed in accordance with local guidelines for the Care and Use of Laboratory Animals and approved by the institutes Animal Ethics Committee.
Membrane preparation and receptor binding assay: Crude synaptic membranes
were isolated from the cerebellar tissues as described earlier (Subhash
et al., 1998) for the radioligand binding studies. The membrane pellet
was suspended in 50 mM Tris-HCl buffer (pH 7.4) containing 10 μM pargyline,
4 mM CaCl2 and 0.1% ascorbic acid. The protein content of the membrane
pellet was determined by Lowrys method (Lowry et
al., 1951). The saturation experiments for 5-HT1B receptors
were done with [3H]5-HT (0.2-2.0 nM) in presence of 100 nM 8-OH-DPAT
and 3000 nM mianserin, to block 5-HT1A and 5-HT2 receptors,
respectively, essentially following the procedure described earlier (14). Non-specific
binding of the radioligand was obtained using 100 nM TFMPP, a selective 5-HT1B
receptor agonist. Drug displacement studies were done by incubating cerebellar
membranes obtained from control rats with 2 nM of [3H] 5-HT, in presence
of 100 nM 8-OH-DPAT and 3000 nM mianserin and with different concentrations
of drugs (10-11-10-4 M). The binding data were analyzed
using LIGAND program (McPherson, 1983) to obtain the
equilibrium dissociation constant (Kd), the density of receptor (Bmax),
the Hill co-efficient (n), inhibitor constant (Ki) and the concentration
of the inhibitor at half of the maximal binding (IC50).
Statistical analysis: Statistical analysis was performed using Sigma Stat software (SPSS, Inc., Chicago, IL). The differences in binding parameters between and among the groups were analyzed using one-way ANOVA and p-values were considered significant at p<0.05.
Acute sumatriptan exposure: The density of cerebellar 5-HT1B
receptors in saline treated rats was 60.3±3.8 fmol mg-1 protein
with a Kd of 0.96±0.02 nM. Seven days of treatment with different
doses of sumatriptan showed a significant (Fdf59 = 326.6, p<0.0001)
decrease in the density of 5-HT1B receptors in a dose dependent manner
(Table 1). A significant decrease in the density of these
receptors was seen with 0.2 mg (45%), 0.5 mg (72%) and 1.0 mg (70%) of sumatriptan
treatment (Fig. 1). No further decrease was observed with
increasing doses of sumatriptan from 2 to 16 mg. The affinity of [3H]5-HT
to 5-HT1B receptors was increased significantly with 0.5 and 1 mg
dose, as evident from the decreased Kd values. However, no further
change in the affinity was observed with further increase in the dose of sumatriptan
(2 to 16 mg kg-1 b.wt.).
|| In vivo effect of Sumatriptan administration for 7
days on cerebellar 5-HT1B receptors in rat brain
|@ f mole mg-1 protein, Values are mean and SD of
3-4 experiments, each assayed in duplicate. The density of 5-HT1B receptors
was estimated using [3H]5-HT, as described, in membranes obtained
from cerebellum of rats treated with different doses (0.2-16 mg kg-1
b.wt.) of sumatriptan for 7 days
||Scatchard plot of [3H]5-HT binding to 5-HT1B receptors
in cerebellar membranes of rats Administered (i.p) with 0.2, 0.5 and 1.0
mg kg-1 b.wt. of sumatriptan 7 days and control rats. Values
are mean of 3-4 experiments, each assayed in duplicate
||5-HT1B receptor density was estimated, as described, in cerebellar
membranes of rats treated with various doses (2, 4 and 8 mg kg-1
body wt, i.p.) of sumatriptan for 7, 14 and 21 days, Values are mean and
SD of 3-4 experiments, each assayed in duplicate, *the percentage of downregulation
of receptor with 2 mg kg-1 b.wt. dose, was significantly different
with duration (Fdf23 = 34.9; p<0.0001)
Chronic sumatriptan exposure: Rats were also treated with 2, 4 and 8
mg kg-1 b.wt. of sumatriptan for varied duration (7, 14 and 21 days).
When compared to control levels there was a significant decrease in the density
of 5-HT1B receptors with 2, 4 and 8 mg kg-1 doses, when
rats were administered for 7, 14 and 21 days (Fig. 2). However,
with 2.0 mg kg-1 dose, there was a significant (Fdf23
= 34.9; p<0.0001) up regulation of 5-HT1B receptors at 21 days
of exposure, when compared to 7 and 14 days of exposure (Table
2). There was however, no change in the receptor affinity.
|| Effect of various doses and durations of sumatriptan administration
on cerebellar 5-HT1B receptors in rat brain
|@ f moles mg-1 protein , $ nM, Values are mean
and SD of 3-4 experiments, each assayed in duplicate, The density of 5-HT1B
receptors was estimated using [3H]5-HT, as described, in the
cerebellar membranes obtained from rats treated with different doses (2,
4, 8 mg kg-1 b.wt.) of sumatriptan for 7, 14 and 21 days, *Significance
compared with 7 days (Fdf 23 = 34.9, p<0.0001), **% downregulation
compared to control levels
This trend, however, was not observed with higher doses (4 and 8 mg kg-1),
where there was no significant change in the receptor density after 14 and 21
days of sumatriptan treatment, when compared to 7 days. Drug displacement studies
revealed that sumatriptan has a high affinity towards 5-HT1B receptors
with a Ki of 9.4±0.9 nM and an IC50 of 28.7±1.4 nM. When compared
to sumatriptan the affinity for other agents was in the order of TFMPP (Ki =
29.5±2.1 nM)>mCPP (Ki = 108.0±15.1 nM)>pindolol (Ki = 148.0±17.6
nM)>5-CT (Ki = 272.0±2.6 nM).
In view of the potential pathophysiological and therapeutic implications, presynaptic
5-HT receptors have been extensively studied by molecular biological and pharmacological
techniques. Studies have established that the terminal auto receptors controlling
5-HT release in the rat brain are of 5-HT1B types. The 5-HT1B
receptors were originally thought to exist only in rodents. Later they have
been characterized and found to be identical with 5-HT1Dβ receptors
found in higher species (Hoyer et al., 1994).
5-HT1D receptors (5-HT1Dα)) have 63% overall structural
homology and 77% amino acid sequence homology with 5-HT1B (5-HT1Dβ)
receptors. Rat brain has very low levels of 5-HT1D mRNA. Similar
to 5-HT1A receptors, mRNA for both 5-HT1B and 5-HT1D
subtypes are found in Dorsal Raphe Nucleus (DRN), suggesting that non-5HT1A
receptors like 5-HT1B receptors, with very small amount of
5-HT1D component, exist in DRN (Stamford et
al., 2000). Both 5-HT1Dα and 5-HT1Dβ
receptors have also been shown to exhibit high affinity for sumatriptan (Weinshank
et al., 1992). In the rat dura mater, 5-HT1Dα and
5-HT1Dβ receptors mediate the inhibitory effects of sumatriptan,
suggesting the role of 5-HT1B receptors in the mechanism of action
of sumatriptan. This finding was further supported by the report that showed
lack of sumatriptan activity in the 5-HT1B receptors knockout mice
(Yu et al., 1996). The 5-HT1B receptors
in rats and 5-HT1D receptors in guinea pigs have been thought to
be the relevant targets of sumatriptan within the dura mater (Matsubara
et al., 1991). Pre junctional action of 5-HT1 receptors
on trigemino vascular axons but not the post junctional action, like constriction
of vascular smooth muscle, mediates the effect of sumatriptan (Moskowitz
et al., 1991). Inhibitory 5-HT1B receptors have been shown
to be present in cat brain and 5-HT1B receptors mediated inhibition
has been suggested to be the most potent action in the inhibition of trigemino
vascular nociceptive traffic (Goadsby and Classey, 2003).
Since in rats 5-HT1B receptors are the targets of sumatriptan, the
dose and duration dependent in vivo effects of sumatriptan on the density
of cerebellar 5-HT1B receptors were studied in rat brain. The present
study demonstrated a dose dependent down regulation of 5-HT1B receptors
in the cerebellum at lower doses of sumatriptan. A dose of 1 mg was sufficient
to down regulate nearly 70% of the receptors. Interestingly, no further down
regulation of 5-HT1B receptors was observed with increasing dose.
However, with increasing duration of exposure, a significant reduction in the
down regulation of 5-HT1B receptors (from 65% to 50%) was seen at
a dose of 2 mg. However, this trend was not seen at 4 and 8 mg kg-1
b.wt. doses. The significant decrease in Kd values is concomitant with significant
decrease in the density of 5-HT1B receptors at 0.5 and 1.0 mg kg-1
dose of sumatriptan. However, the affinity was same as that of control with
further increase in dosage. It was observed that the affinity of [3H5-HT
to 5-HT1B receptors increases with increase in downregulation.
As there was no further increase in downregulation with increasing dosage, the affinity was not altered. These findings suggest that administration of lower doses (2 mg) for a moderate period (2 weeks) would be critical for a significant downregulation of cerebellar 5-HT1B receptors, which may be involved in the therapeutic action of sumatriptan and higher doses for a longer period may be required for the prophylactic activity.
After intra vascular administration of sumatriptan, rats Johnson
et al. (2001) have shown that sumatriptan has high affinity agonistic
activity at 5-HT1D/B receptors, though less than Zolmitriptan. This
weaker functional 5-HT1B receptor agonistic potency of sumatriptan
when compared to Zolmitriptan is however, not due to its low concentration in
the brain (Johnson et al., 2001). In a recent
study, a significant decrease (40%) in 5-HT1B/1D receptor mRNA in
trigeminal ganglion was reported after 14 days of treatment with sumatriptan
(7). Furthermore, the drug displacement study done in our laboratory has revealed
that sumatriptan has a high affinity (Ki = 9.4 nM) to 5-HT1B receptors
in rat cerebellum, when compared to TFMPP, a 5-HT1B agonist, mCPP,
a 5-HT2B/2C agonist, pindolol, a 5-HT1A/1B antagonist
and 5-CT, a 5-HT1A/1B/1D agonist.
In another study in rats, the dose relation and complete inhibition of plasma
protein extravasation in the duramater was found to be similar with both sumatriptan
and electriptan, when injected intra venously (Gupta et
al., 2000). This suggests that the effectiveness of this drug, which
displays both high affinity and selectivity to 5-HT1B/1D receptors,
may indicate the importance of this specific 5-HT1-receptor subtype
in the pathogenesis of migraine. Experiments with new 5-HT1B and
5-HT1D subtype selective ligands are required to confirm these findings.
It is note worthy that 5-HT1B receptors also sub serve the role of
heteroreceptors. Future studies are needed to replicate findings in various
brain regions using more specific radioligands.
Prevention of dural vasodilation involves activation of trigeminal Aδ-fibers, whereas prevention of dural extravasation involves activation of trigeminal C-fibers. So, the differential effect of sumatriptan may be due to different receptor subtypes involved and to their location and species studied. Taken together, the desensitization of 5-HT1B receptors in rat brain, which are identical to 5-HT1D receptors in human brain, may underlie the mechanism of antimigraine properties of sumatriptan, wherein the neurogenic inflammation on terminals of trigeminal neurons in dural vessels may be inhibited.
Authors are grateful to NIMHANS, Bangalore, India, for the research fellowship and Kamineni Institute of Medical Sciences for constant support and encouragement.
1: Millson, D.S., 2004. Migraine pathophysiology. Headache, 44: 735-739.
2: Peroutka, S.J., 1990. The pharmacology of current anti-migraine drugs. Headache: J. Head Face Pain, 30: 5-11.
3: Burstein, R. and M. Jakubowski, 2004. Analgesic triptan action in an animal model of intracranial pain: A race against the development of central sensitization. Ann. Neurol., 55: 27-36.
4: Weinshank, R.L., J.M. Zgombick, M.J. Macchi, T.A. Branchek and P.R. Hartig, 1992. Human serotonin 1D receptor is encoded by a subfamily of two distinct genes: 5-HT1Dα and 5-HT1Dβ. Proc. Natl. Acad. Sci., 89: 3630-3634.
Direct Link |
5: Moskowitz, M.A., 1992. Neurogenic versus vascular mechanisms of sumatriptan and ergot alkaloids in migraine. Trends Pharm. Sci., 13: 307-311.
CrossRef | Direct Link |
6: Waeber, C. and M.A. Moskowitz, 1995. [3H] sumatriptan labels both 5-HT1D and 5-HT1F receptor binding sites in the guinea pig brain: An autoradiographic study. Naunyn-Schmiedeberg's Arch. Pharmacol., 352: 263-275.
CrossRef | Direct Link |
7: Reuter, U., S. Salomone, G.W. Ickenstein and C. Waeber, 2004. Effects of chronic sumatriptan and zolmitriptan treatment on 5-HT1 receptor expression and function in rats. Cephalalgia, 24: 398-407.
CrossRef | Direct Link |
8: Peroutka, S.J. and B.G. McCarthy, 1989. Sumatriptan (GR 43175) interacts selectively with 5-HT 1B and 5-HT 1D binding sites. Eur. J. Pharm., 163: 133-136.
CrossRef | Direct Link |
9: Xian-Jie, Y., F.M. Cutrer, M.A. Moskowitz and C. Waeber, 1997. The 5-HT1D receptor antagonist GR-127-935 prevents inhibitory effects of sumatriptan but not CP-122-288 and 5-CT on neurogenic plasma extravasation with guinea pig dura mater. Neuropharmacology, 36: 83-91.
10: Pineyro, G., N. Castanon, R. Hen and P. Blier, 1995. Regulation of [3H] 5-HT release in raphe, frontal cortex and hippocampus of 5-HT1B knock-out mice. Neuroreport, 7: 353-359.
11: Pineyro, G. and B. Blier, 1996. Regulation of [3H]5 HT release from rat midbrain raphe nuclei by 5 HT1D receptors effect of tetrodotoxin G protein inactivation and long-term antidepressant administration. J. Pharmacol. Exp. Ther., 276: 697-707.
12: Mota, A., A. Bento, A. Penalva, M. Pombo and C. Dieguez, 1995. Role of the serotonin receptor subtype 5-HT1D on basal and stimulated growth hormone secretion. J. Clin. Endocrinol. Metab., 80: 1973-1977.
Direct Link |
13: Facchinetti, F., R.E. Nappi, G. Sances, L. Fioroni, G. Nappi and A.R. Genazzani, 1994. The neuroendocrine effects of sumatriptan, a specific ligand for 5-HT 1-like receptors. Clin. Endocrinol., 40: 211-214.
14: Subhash, M.N., B.N. Srinivas, K.Y. Vinod and S. Jagadeesh, 1998. Inactivation of 5-HT1A and [3H] 5-HT binding sites by N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) in rat brain. Neurochem. Res., 23: 1321-1326.
CrossRef | Direct Link |
15: Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275.
PubMed | Direct Link |
16: McPherson, G.A., 1983. A partial computer based ligand approach to the analysis of radio ligand binding experiments. Comput. Programs Biomed., 17: 107-114.
17: Hoyer, D., D.E. Clarke, J.R. Fozard, P.R. Hartig and G.R. Martin et al., 1994. International union of pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacological Rev., 46: 157-203.
18: Stamford, J.A., C. Davidson, D.P. McLaughlin and S.E. Hopwood, 2000. Control of dorsal raphe 5-HT function by multiple 5-HT 1 autoreceptors: Parallel purposes or pointless plurality? Trends Neurosci., 23: 459-465.
19: Yu, X.J., C. Waeber, N. Castanon, K. Scearce and R. Hen et al., 1996. 5-carboxamide-tryptamine, CP-122-288 and dihydroergot-amine but not sumatriptan, CP-93-129 and serotonin-5-o-carboxymethyl-glycyl-tyrosinamide block dural plasma protein extravasation in knockout mice that lack 5-hydroxytryptamine 1B receptors. Mol. Pharamacol., 49: 761-765.
20: Matsubara, T., M.A. Moskowitz and B. Byun, 1991. CP-93,129, a potent and selective 5‐HT1B receptor agonist blocks neurogenic plasma extravasation within rat but not guinea‐pig dura mater. Br. J. Pharmacol., 104: 3-4.
CrossRef | Direct Link |
21: Moskowitz, M.A., M.G. Buzzi, T.C. Theoharides and V. Dimitriadou, 1991. Evidence that 5-HTreceptors on trigeminovascular axons mediates the anti-migraine effects of ergot and sumatriptan. Neurology, 41: 164-164.
22: Goadsby, P.J. and J.D. Classey, 2003. Evidence for serotonin (5-HT)1B, 5-HT 1D and 5-HT 1F receptor inhibitory effects on trigeminal neurons with craniovascular input. Neuroscience, 122: 491-498.
CrossRef | Direct Link |
23: Johnson, D.E., H. Rollema, A.W. Schmidt and A.D. McHarg, 2001. Serotonergic effects and extracellular brain levels of eletriptan, zolmitriptan and sumatriptan in rat brain. Eur. J. Pharmacol., 425: 203-210.
CrossRef | Direct Link |
24: Gupta, P., P. Butler, N.S. Sheperson and A.D. McHarg, 2000. The in vivo pharmacological profile of electriptan: A potent and novel 5-HT1B/1D receptor agonist. Eur. J. Pharmacol., 9: 73-81.