5-HT2A receptor-stimulated phosphoinositide hydrolysis in the stimulus effects of hallucinogens
Introduction
The serotonergic receptor system appears to play a prominent role in mediating the discriminative effects of both indoleamine [e.g., d-lysergic acid diethylamide (LSD)] and phenethylamine [e.g., (−)2,5-dimethoxy-4-methylamphetamine (DOM)] hallucinogens. The initial finding that serotonergic antagonists block the stimulus effects of the phenethylamine hallucinogen mescaline Winter, 1975, Browne and Ho, 1975 was later expanded to include other hallucinogens including LSD, DOM and N,N-dimethyltryptamine (DMT) Kuhn et al., 1997, Winter, 1978, Glennon et al., 1983. Of the seven different families of serotonin receptors, activation of the 5-HT2 receptor family appears to be the primary site of action of the indoleamine/phenethylamine hallucinogens. Affinity at the 5-HT2 receptor for a series of phenethylamines strongly correlates with both the potency of these compounds to substitute for DOM as a discriminative cue (Glennon et al., 1984), as well as their potency as hallucinogens Titeler et al., 1988, Sadzot et al., 1989. Based upon antagonist correlation analysis, Fiorella et al. (1995a) concluded that the 5-HT2A receptor rather than the 5-HT2C receptor mediates the stimulus effects of LSD. Similarly, the discriminative stimulus effects of the phenethylamine DOI are blocked by MDL 100,907, a selective 5-HT2A antagonists, but not by the selective 5-HT2C antagonist SB 200,646 (Schreiber et al., 1994). In addition, behavioral tolerance to the stimulus effects of DOI is associated with a down-regulation of the 5-HT2A and not the 5-HT2C receptor (Smith et al., 1999).
Although the data indicate that the discriminative stimulus effects of indoleamine and phenethylamine hallucinogens involve activation of the 5-HT2A receptor, the resulting biochemical changes responsible for the interceptive state induced by these hallucinogens are unknown. The 5-HT2A receptor is coupled to stimulation of phospholipase C through an activation of the Gq/11 GTP-binding protein. This stimulation results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) with the subsequent generation of inositol 1,4,5-trisphosphate (IP3), which regulates the release of calcium from internal stores, and sn(1,2) diacylglycerol (DAG), which is involved in the activation of protein kinase C. The objective of the present study was to determine whether 5-HT2A receptor-mediated stimulation of phosphoinositide hydrolysis is a necessary component of the discriminative state produced by hallucinogens. To address this question, generation of inositol phosphates was measured in the presence of various indoleamine, phenethylamine and β-carboline hallucinogens, as well as some nonhallucinogenic congeners. For these studies, PC12 cells expressing the rat 5-HT2A receptor cDNA were used to preclude the confounding stimulation of phospholipase C by other members of the 5-HT2 receptor family.
Section snippets
Material
[3H]Myo-inositol was purchased from American Radiochemical (St. Louis, MO) and [3H]ketanserin was obtained from DuPont/NEN (Boston, MA). DOM, LSD, psilocybin, DMT, N,N-diethyltryptamine (DET) and 2-bromo-lysergic acid diethylamide (BOL) were generously provided by the National Institute on Drug Abuse (Rockville, MD). Quipazine, lisuride, and 5-methoxy-N,N-dimethyltryptamine (MDMT) were purchased from RBI (Natick, MA). Harmane and harmaline were purchased from Sigma (St. Louis, MO). 6-Fluoro-N,N
Results
The binding of [3H]ketanserin to PC12-5-HT2A cells was best fitted by a one-site model (Hill coefficient 1.003±0.03) with a Bmax of 326±70.6 fmol/mg and pKD of 8.89±0.072 (n=3; Fig. 1). This affinity is comparable to values observed for the binding of [3H]ketanserin in the rat cerebral cortex Conn and Sanders-Bush, 1986, Johnson et al., 1993, Helsley et al., 1998a, as well as in other clonal cells expressing the cDNA for the 5-HT2A receptor Pritchett et al., 1988, Teitler et al., 1990, Berg et
Discussion
Hallucinogens can serve as discriminative stimuli in nonverbal animals (see Winter et al., 1999). Although interactions mediating stimulus control may not be completely identical to those responsible for the induction of hallucinations, nonessential interactions can be identified by using a series of compounds for behavioral and biochemical studies. Previous studies have shown that activation of the 5-HT2A receptor is necessary for the stimulus effects of both the indoleamine LSD and the
Acknowledgements
This study was supported by U.S. Public Health Service grant DA03385. M. Doat is supported by National Research Service Award (F31 MH12696) and by a grant from the Schering-Plough Research Foundation. The expert technical assistance of Barbara Hughes is greatly appreciated.
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