Agonist and Antagonist Binding to Rabbit Cortical
5-HT2A Receptors: Opposite Effects of
Magnesiuma
V. J. ALOYO,b G. L. SALT, M. E. HOFFMAN,
AND J. A. HARVEY
Department of Pharmacology, Division of Behavioral
Neurobiology, Allegheny University of the Health Sciences,
Philadelphia, Pennsylvania 19129, USA
Serotonin 5-HT 2A and/or 5-HT 2C
receptors have been implicated in modulating the acquisition of
the classically conditioned nictitating membrane response in the
rabbit.1 Thus, we have undertaken the characterization
of the 5-HT 2A receptor in rabbit brain using the
agonist [125I]DOI, and the antagonists
[3H]ketanserin and [3H]MDL 100,907.
Autoradiographic studies using both [125I]DOI and
[3H]ketanserin demonstrate a high density of 5-HT
2A receptors in adult New Zealand rabbit cortex.
Therefore, binding studies were performed using membranes
prepared from the frontal cortex. (3H]Ketanserin (New
England Nuclear) and [3H]MDL 100,907 (Amersham)
binding assays were performed at 25° C in 20 mM Tris HCl pH
7.4. Nonspecific binding was defined by 1 µM methysergide
and I µM spiperone [3H]ketanserin and
[3H]MDL 100,907, respectively. Ketanserin binding (in
the presence of 100 nM prazosin) was consistent with 2 classes of
binding sites. The high affinity ketanserin site had a
Kd of approximately 1 nM, which is consistent with its
reported affinity at 5-HT 2A receptors. The GTP
analogue, Gpp(NH)p, at concentrations up to 100 mM had no effect
on ketanserin binding. In contrast, magnesium dose-dependently
inhibited ketanserin binding. The IC50 for DOI
displacement of bound [3H]ketanserin was decreased
approximately fourfold in the presence of 5 mM MgSO4.
More extensive studies were carried out with the highly selective
5-HT 2A receptor antagonist, MDL 100,907. Saturation
analysis demonstrated that the binding of MDL 100,907 was
consistent with a single class of sites with a Kd of
approximately 0.1 nM. As for ketanserin, Gpp(NH)p at
concentrations up to 100 mM had no effect, whereas magnesium
dose-dependently inhibited MDL 100,907 binding. MgSO4
(5 mM) decreased the affinity of [3H]MDL 100,907 by
approximately fourfold. The IC50 for DOI displacement
of bound [3H]MDL 100,907 was decreased in the presence
of 5 mM MgSO4. As expected, Mg++ shifts the
5HT 2A receptor toward the agonist high affinity state
as demonstrated by the increased affinity of the agonist, DOI.
Unexpectedly, Mg++ decreases the binding of the
putative neutral antagonists ketanserin and MDL 100,907,
suggesting that these compounds may actually be inverse agonists
at the 5-HT 2A receptor.
REFERENCE
1. HARVEY, J. A. 1996 Behav. Brain Res 73:
47-50.
a Supported in part by USPHS MERIT Award
Grant MH16841-26.
b Corresponding author Tel: 215.842,4750;
Fax: 215,843,1515
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