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Antimigraine drug interactions with 5-hydroxytryptamine1A receptors.

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Antimigraine Drug
Interactions with
Receptors
Bradley C. Hiner, MD, Heidi L. Roth,
and Stephen J. Peroutka, MD, PhD
The interactions of four antimigraine drugs with 5hydroxytryptaminelA and beta-adrenergic receptors
were analyzed in rat brain membranes. Methysergide,
cyproheptadine, ( - )propranolol, and pizotifen display
similar affinities (Icsovdues, 83 to 280 nM) for 5hydroxytryptamine,A receptors labeled by 13H)8-hydroxy-N,N-dipropyl-2-aminotetralin.Cyproheptadine,
pizotifen, and methysergide are essentially inactive at
beta-adrenergic receptors labeled by K3Hldihydroalprenolol (ICs0 values, 8,700 to 87,000 nM). By contrast,
( - )propranolol is an extremely potent beta-adrenergic
agent (ICs0, 3 n ~ )These
.
data indicate that the 5-hydroxytryptamine,A receptor is a common site of action for
( - )propranolol and other antimigraine agents.
Hiner BC, Roth HL, Peroutka SJ: Antimigraine
drug interactions with 5-hydroxytryptaminelA
receptors. Ann Neurol 19:5 11-5 13, 1986
A variety of drugs are effective in the treatment of
migraine. Initially, drugs such as methysergide, cyproheptadine, and pizotifen were the most commonly
used agents. Their clinical efficacy is attributed to an
ability to modulate the effects of 5-hydroxytryptamine
(5-HT) in the central nervous system [4, 8, 131. More
recently, ( - )propranolol and other beta-adrenergic receptor antagonists have been shown to be effective
against migraine. T h e efficacy of these agents has been
attributed to a reduction in vasodilation as well as to a
decrease in the physiological manifestations of anxiety
14, 8}. However, it has also been suggested that the
antimigraine effects of ( - )propranolol may be secondary to an action other than, or in addition to, betareceptor blockade [4, 133. In the present study, the
interactions of methysergide, cyproheptadine, pizotifen, and ( - )propranolol were analyzed at both 5HTlA receptors (a recently defined subtype of the 5HT receptor [5]) and beta-adrenergic receptors in
neuronal rat brain membranes.
From the Department of Neurology, Stanford University Medical
Center, Stanford, CA.
Received Aug 5, 1985, and in revised form Sept 16. Accepted for
publication Sept 28, 1985.
Address reprint requests to Dr Peroutka, Department of Neurology, Stanford University Medical Center, Stanford, C A 94305.
Materials and Methods
Adult rat brains were obtained from Pel-Freeze Biological:;
(Rogers, AK) and stored at - 20°C until use. On the day of
study, the brains were defrosted and the frontal cortex was
dissected. Tissues were homogenized in 20 vol of 50 mM
Tris-hydrochloride buffer (pH 7.7 at 25°C) using a Brink-.
mann Polytron and then centrifuged in an IEC B20A centrifuge at 47,000 g for 10 minutes. The supernatant was
discarded and the pellet was resuspended in the same
volume of Tris-hydrochloride buffer and incubated at 37°C
for 10 minutes prior to a second centrifugation at 49,000 g
for 10 minutes. The final pellet was resuspended in 80 vol of
Tris-hydrochloride buffer containing 10 p , pargyline,
~
4 mM
calcium chloride, and 0.1% ascorbic acid. The suspensions
were used immediately in the binding assay.
Binding assays for drug displacement studies consisted of
0.1 ml of a tritiated ligand (final concentrations: 0.15 to 0.20
nM [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin ['H-8OH-DPAT]; 1.5 to 1.8 nM [3H]dihydroalprenoloI ['HDHA]), 0.1 ml of a buffer or displacing drug, and 0.8 ml of a
tissue suspension. Following incubation at 25°C for 30 minutes, the assays were rapidly filtered under vacuum through
Whatman GF/B filters with two 5-ml washes using 50 mM
Tris-hydrochloride buffer. Radioactivity was measured by
liquid scintillation spectroscopy in 5 ml of Aquasol (New
England Nuclear, Boston, MA) at 545% efficiency. Specific
binding was defined using 10 WM 5-HT for 'H-8-OH-DPAT
binding and 1 p , (~? )propranolol for 'H-DHA binding.
Generally, 75 to 80% of total binding was specific for both
'H-8-OH-DPAT and 3H-DHA. Affinities (IC50 values)
were determined by log-logit analysis. All drugs were dissolved and diluted in assay buffer. Drugs were obtained from
the following sources: ?H-8-OH-DPAT (80 Cdmmol; Research Products International Corp, Mount Prospect, IL);
'H-DHA (103.8 Cdmmol; New England Nuclear, Boston, MA); methysergide, pizotifen (Sandoz Pharmaceuticals,
East Hanover, NJ); 5-HT, ( )propranolol, cyproheptadine
(Sigma Chemical Co, St. Louis, MO); and (-)propranolol
(Ayerst Laboratories Inc, New York, NY).
Results
Methysergide, cyproheptadine, pizotifen, and ( - )propranolol are potent inhibitors of 3H-8-OH-DPAT
binding to 5-HTIAreceptors. As shown in the Figure
(A), methysergide is the most potent agent, with displacement of 3H-8-OH-DPATfirst noted at nanomolar drug concentrations. Methysergide displaces 50%
of specific binding to the 5-HTIAsite at a concentration of 83 t 20 nM (SEM) (Table). At micromolar
concentrations of methysergide, essentially all specific
'H-8-OH-DPAT binding is displaced. Cyproheptadine is the second most potent agent. Displacement
begins at concentrations above 10 nM, and the drug
has an
value of 140 +- 4 nM. T h e beta-adrenergic
agent (-)propranolol has an ICso value of 230 ? 80
nM, while pizotifen is the weakest of the four agents
tested (ICsO,280 +- 60 nM). Thus, the ICso values for
the four antimigraine agents range from 83 to 280 nM
at the 5-HTIAsite labeled by 'H-8-OH-DPAT.
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A markedly different pharmacological pattern is
noted with drug interactions at the beta-adrenergic receptor labeled by 3H-DHA (Figure, B). ( - )Propranolo1 is the most potent agent, with an ICso value of 5.0
_t 2 nM versus 3H-DHA binding. Methysergide is the
second most potent antimigraine agent at the betaadrenergic receptor. However, methysergide is more
than three orders of magnitude less potent (ICso,
8,700 '-t 2,000 nM) than (-)propranolol at the betaadrenergic receptor. Pizotifen and cyproheptadine are
essentially inactive at the 'H-DHA site, with ICso
values of 15,000 nM and 87,000 nM, respectively.
Discussion
The major finding of the present study is that methysergide, cyproheptadine, pizotifen, and ( - )propranolo1 have similar affinities for the ~ - H T Ireceptor.
A
The
5-HTIAreceptor is a recently defined subtype of 5-HT
receptor in the central nervous system, which can be
directly labeled with 'H-8-OH-DPAT C53. The site is
distinct from 5-HTIB and 5-HT2 receptors that have
been defined in brain membranes 153 and does not
appear to be related to M or D serotonergic receptors
that have been defined in the periphery [141. 5 - H T 1 ~
sites are highly concentrated in the hippocampus [5]
and may also be present in intracranial blood vessels
512 Annals of Neurology
Vol 19 No 5
May 1086
Antimigraine drug interactions with 5 -h,ydroxytvyptaminelA
and beta-adrenergicreceptors labeled with 1' H)8-hydroxyN,N-dipropyl-2-aminotetralin (' H-8-OH-DPAT)(A) and
{'H)dihydroalprenolol (3H-DHA)( B ) . Drug displacement studies were pevformed as described in Materials and Methods. Data
shown are the results of a single experimentperformed in triplicate. Drugs studied are methysergide (O),cyproheptadine (+),
i- )propranolol(O).andpizotifn (H).
Cl2). No specific physiological role for the 5-HTlA
receptor has y e t been found.
The antimigraine effects of methysergide, cyproheptadine, and pizotifen have been attributed to their ahility to modulate 5-HT actions [4, 8, 131. By contrast,
the clinical efficacy of (-)propranolol is rarely attributed to antagonism of 5-HT effects C41. Instead, the
antimigraine effectiveness of ( - )propranolol has been
attributed to a variety of actions, including prevention
of intracranial vasodilation and blockade of the peripheral manifestations of anxiety 14, 81. The results of the
present study demonstrate that ( - )propranolol and
classic 5-HT antagonists are essentially equipotent at a
specific subtype of 5-HT receptor in the central nervous system. Therefore, the clinical efficacy of all four
agents may relate to this common site of action in the
central nervous system.
Antimigraine Drug Interactions
with 5 -Hydroxytryptamine I A and Beta-Adrenergic Receptors"
Drug
Me thysergide
Cyproheptadine
(-)Propranolol
Pizotifen
5-Hydroxytryptaminel*
83 ? 20
140 ? 4
230 k 80
280 5 60
gide, cyproheptadine, pizotifen, and ( - )propranolol at
a specific subtype of the central 5-HT receptor has not
previously been reported. Future studies are needed to
determine the exact role of the 5-HT,* receptor in the
pathogenesis of migraine.
Beta-Adrenergic
8 , 7 0 0 % 700
87,000
2,000
5.0 2 2
15,000 2 100
%
"Radioligand binding studies were performed as described in Materials and Methods. IC5" values were derived from log-logit analysis of
drug displacement data. Values given are the means 2 SEM of three
to six experiments, each performed in triplicate.
5-HT appears to have a major role in the pathogenesis of headache. For example, vasomotor dysregulation has been documented during both the aura and
headache phases of migraine {S). Depending on the
vascular tone and the specific vessel under study, 5-HT
may produce either vasoconstriction or vasodilation
[3]. In addition, 5-HT may modulate pain sensation
during a migraine attack, since injection of the m i n e
into inflamed tissues lowers pain thresholds { 11).
Moreover, serotonergic neurons have been identified
in both cerebral blood vessels [ l l ] and central pain
pathways El].
Beta-adrenergic drugs have been demonstrated to
interact with central 5-HT receptors in a number of
systems. Behaviorally, beta-adrenergic antagonists inhibit 5-HT-induced hyperactivity in rats [ 2 , 6).
( - )Propranolol antagonizes both 5-HT-induced
vasodilation of pial vessels { 3 } and the effects of 5-HT
at the "auto;eceptor" in synaptosomal release studies
[9}. Beta-adrenergic antagonists also interact with 5HT receptors as defined by radioligand binding techniques. For example, ( - )propranolol is greater than
20-fold more potent than cyproheptadine at total 5HT1 binding sites labeled by 3H-5-HT [lo). At the 5HT2 receptor, methysergide is greater than 200-fold
more potent than (-)propranolol [7). By contrast, in
the present study, only a 3-fold range of potencies
exists for the four antimigraine drugs at the 5-HT1A
receptor. A potent and similar interaction of methyser-
This work was supported by Biomedical Training Grant #RR 535323, National Institutes of Health Grant #NS 12151-11, and the
John A. and George L. Hartford Foundation.
We thank Jean Peroutka for editorial assistance.
References
1. Basbaum AI, Fields HL: Endogenous pain control mechanisms:
review and hypothesis. Ann Neurol4:451-462, 1978
2. Costain DW, Green AR. Beta-adrenoreceptor antagonists inhibit the behavioural responses of rats to increased brain 5hydroxyrryptamine. Br J Pharmacol 64:193-200, 1978
3. Edvinsson L, Hardebo JE, MacKenzie ET, Stewart M: Dual
action of serotonin on pial arterioles in situ and the effect of
propranolol on the response. Blood Vessels 14:366-371, 1975
4. Fozard JR: Basic mechanisms of antimigraine drugs. Adv
Neurol 33295-307, 1982
5. Gozlan H , El Mesrikawy S, Pichat L, et al: Identification of
presynaptic serotonin autoreceptors using a new ligand: 'HPAT. Nature 305:140-143, 1983
6. Green AR, Grahame-Smith DG: (- )Propranold inhibits the
behavioural responses to increased 5-hydroxytryptamine in the
central nervous system. Nature 262:594-596, 1976
7. Green AR, Johnson P, Nimgaonkar VL: Interactions of betaadrenergic agonists and antagonists with the 5-hydroxytryptamine, (5-HTz) receptor. Neuropharmacology 22:657-660,
1983
8. Lance JW: Headache. Ann Neurol 10:1-10, 1981
9. Middlemiss DN: Stereoselective blockade at 3H-5-HT binding
sites and at the 5-HT autoreceptor by propranolol. Eur J Pharmacol 101:289-293, 1984
10. Middlemiss D N , Blakeborough L, Leather SR: Direct evidence
for an interaction of beta-adrenergic blockers with the 5-HT
receptor. Nature 267:289-290, 1977
11. Moskowitz MA: The neurobiology of vascular head pain. Ann
Neurol 16:157-168, 1984
12. Peroutka SJ, Kuhar MJ: Autoradiographic localization of 5-HT,
receptors to human and canine basilar arteries. Brain Res
310:193-196, 1984
13 Raskin NH: Pharmacology of migraine. Annu Rev Pharmacol
Toxicol 21:463-478, 1981
14 kchardson BP, Engel G, Donatsch P, Stadler PA: Identification
of serotonin M-receptor subtypes and their specific blockade by
a new class of drugs. Nature 316:126-131, 1985
Brief Communication: H i n e r et al: Antimigraine Drugs and 5-HT1,4 513
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