Antagonist properties of d-LSD at 5-hydroxytryptamine2 receptors.

The hallucinogenic agent d-lysergic acid diethylamide (d-LSD) interacts with a number of serotonin (5-hydroxytryptamine [5-HT]) receptor subtypes in the central nervous system. It has been hypothesized that hallucinosis is produced by agonist activity at 5-HT2 receptors. There exist, however, numerous data from radioligand binding, cellular, smooth muscle, and behavioral studies that suggest that d-LSD is a potent 5-HT2 antagonist. These data are reviewed in this report. In addition, d-LSD displays agonist activity at 5-HT1A and 5-HT1C receptor subtypes, as determined in biochemical studies. At the present time, agonist interactions at 5-HT1C receptors, as opposed to 5-HT2 receptors, appears to be a more likely "common mechanism of action" of hallucinogenic agents.

d-Lysergic acid diethylamide differentially affects the dual actions of 5-hydroxytryptamine on cortical neurons.

5-Hydroxytryptamine (5-HT; 10(-4) M) produces an initial depolarization, followed by a long-lasting hyperpolarization, when focally applied to pyramidal neurons in the somatosensory cortex of the rat. Application of the selective 5-HT1A agonist, 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT; 10(-6) M) or d-lysergic acid diethylamide (d-LSD; 10(-6) M), produced only a hyperpolarizing response which was larger than the response to 5-HT. Application of 8-OH-DPAT (10(-6) M) and 5-HT (10(-4) M) together produced an initial depolarizing response, similar to the response with 5-HT alone, followed by a hyperpolarizing response which was 23 +/- 3% larger than with 5-HT alone. By contrast, the application of d-LSD (10(-6) M) and 5-HT (10(-4) M) together produced either no depolarization (7 of 13 cells) or a significantly smaller depolarizing response (36 +/- 4% of the response to 5-HT alone), as well as a hyperpolarizing response which was 33 +/- 4% larger than with 5-HT alone. Therefore, d-LSD displayed a unique pharmacological ability to both mimic and block the effect of 5-HT on single neurons in somatosensory cortex of the rat.

5-Hydroxytryptamine receptor subtype messenger RNAs in human dorsal root ganglia: a polymerase chain reaction study.

Although serotonin has been shown to play an important role in peripheral pain mechanisms, the specific subtypes of serotonin receptors involved in pain and hyperalgesia remain poorly understood. To date, no previous study has attempted to determine the presence of any serotonin receptor subtype in human dorsal root ganglia. In this study, the presence of messenger RNA for eight human serotonin receptor subtypes in lumbar dorsal root ganglia was detected using the method of polymerase chain reaction. Dorsal root ganglia were excised post mortem from four patients. Oligonucleotide primers were chosen based on unique regions of complimentary DNA sequence for eight cloned human serotonin receptor subtypes (i.e. 5-HT1A, 5-HT1D alpha, 5-HT1D beta, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2C and 5-HT7). The presence of 5-HT1D alpha, 5-HT1D beta, 5-HT1E, 5-HT1F, 5-HT2A and 5-HT7 receptor subtype messenger RNA was detected in dorsal root ganglia from three of the four subjects. 5-HT1A receptor subtype messenger RNA was detected in one of the four subjects. No 5-HT2C receptor subtype messenger RNA could be detected. Findings from this study may direct further efforts to determine the role of serotonin receptors in the peripheral nervous system.

5-Hydroxytryptamine receptor subtype messenger RNAs in rat peripheral sensory and sympathetic ganglia: a polymerase chain reaction study.

While serotonin has been shown to play an important role in peripheral pain mechanisms, the specific subtypes of receptors involved and their differential distribution between the sensory and sympathetic nervous system remains poorly understood. In this study, the presence of messenger RNA for rat serotonin receptor subtypes in peripheral sensory and sympathetic ganglia was detected using the method of polymerase chain reaction. Lumbar dorsal root ganglia, superior cervical sympathetic ganglia and lumbar sympathetic ganglia were excised from anesthetized Sprague-Dawley rats. Oligonucleotide primers were chosen based on unique regions of complementary DNA sequence for each of the 12 cloned rat serotonin receptor subtypes (i.e. 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7) and high stringency conditions were used during polymerase chain reaction. Within lumbar dorsal root ganglia, the presence of the 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2C, 5-HT3 and 5-HT7 receptor subtype messenger RNAs was detected. Within superior cervical ganglia, the presence of messenger RNA for 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT3, 5-HT6, and 5-HT7 receptor subtypes was detected. Lumbar sympathetic ganglia displayed banding identical to the superior cervical ganglia with the exception of the 5-HT6 receptor which was not detected in the lumbar sympathetic ganglia. The polymerase chain reaction product from each positively-detected receptor subtype was subcloned and sequenced and found to correspond to published complementary DNA sequences. Findings from this study may direct further efforts to determine the role of 5-hydroxytryptamine receptors in the peripheral nervous system.

Differential interactions of dimethyltryptamine (DMT) with 5-HT1A and 5-HT2 receptors.

The interactions of the indolealkylamine N,N-dimethyltryptamine (DMT) with 5-hydroxytryptamine1A (5-HT1A) and 5-HT2 receptors in rat brain were analyzed using radioligand binding techniques and biochemical functional assays. The affinity of DMT for 5-HT1A sites labeled by [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT) was decreased in the presence of 10(-4) M GTP, suggesting agonist activity of DMT at this receptor. Adenylate cyclase studies in rat hippocampi showed that DMT inhibited forskolin-stimulated cyclase activity, a 5-HT1A agonist effect. DMT displayed full agonist activity with an EC50 of 4 x 10(-6) M in the cyclase assay. In contrast to the agonist actions of DMT at 5-HT1A receptors, DMT appeared to have antagonistic properties at 5-HT2 receptors. The ability of DMT to compete for [3H]-ketanserin-labeled 5-HT2 receptors was not affected by the presence of 10(-4) M GTP, suggesting antagonist activity of DMT at 5-HT2 receptors. In addition, DMT antagonized 5-HT2-receptor-mediated phosphatidylinositol (PI) turnover in rat cortex at concentrations above 10(-7) M, with 70% of the 5-HT-induced PI response inhibited at 10(-4) M DMT. Micromolar concentrations of DMT produced a slight PI stimulation that was not blocked by the 5-HT2 antagonist ketanserin. These studies suggest that DMT has opposing actions on 5-HT receptor subtypes, displaying agonist activity at 5-HT1A receptors and antagonist activity at 5-HT2 receptors.

Hallucinogenic drug interactions with neurotransmitter receptor binding sites in human cortex.

The binding affinities of four hallucinogenic agents were analyzed at nine neurotransmitter binding sites in human cortex. d-Lysergic acid diethylamide (d-LSD), N,N-dimethyltryptamine (DMT), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and 1-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane (DOB) display highest affinity for the recently identified "DOB binding site" labeled by 77Br-R(-)DOB. The phenalkylamines, DOI and DOB, display subnanomolar affinity for the 77Br-R(-)DOB-labeled site, whereas the indolealkylamines, d-LSD and DMT, display nanomolar affinity for this site. d-LSD was the most potent of the four hallucinogens at six of the other eight sites analyzed in this study. All four hallucinogens also display high affinity for the 5-hydroxytryptamine2 (5-HT2) receptor subtype, with potencies ranging from 4 to 360 nM. Marked differences in relative affinities were observed between the indolealkylamines and the phenalkylamines at the 5-HT1A, 5-HT1D, and DOB binding sites. These rank-order differences in affinities are likely to account for the differing effects of these agents in various biochemical and physiological assays.

Ring-substituted amphetamine interactions with neurotransmitter receptor binding sites in human cortex.

The binding affinities of 3 ring-substituted amphetamine compounds were determined at 9 neurotransmitter binding sites in human cortex. (+/-)-3,4-Methylenedioxyamphetamine (MDA), (+/-)-3,4-methylenedioxyethamphetamine (MDE), and (+/-)-3,4-methylenedioxymethamphetamine (MDMA or 'Ecstasy') all display highest affinity (approximately 1 microM) for the recently identified 'DOB binding site' labeled by [77Br]R(-)4-bromo-2,5-dimethoxyphenylisopropylamine [( 77Br]R(-)DOB). MDA displays moderate affinity (4-5 microM) for the 5-hydroxytryptamine1A (5-HT1A), 5-HT1D, and alpha 2-adrenergic sites in human cortex. MDE and MDMA display lower affinity or are inactive at all other sites tested in the present study. These observations are discussed in relation to the novel psychoactive effects of the ring-substituted amphetamines.

Molecular structural basis of ligand selectivity for 5-HT2 versus 5-HT1C cortical receptors.

A molecular structural criterion of ligand selectivity for the 5-HT2 versus 5-HT1C receptor was hypothesized on the basis of radioligand binding data. Despite the large number of compounds which have been tested at both receptors, analysis of published data led to the identification of only five agents which are greater than 10-fold selective for the 5-HT2 versus the 5-HT1C receptor. Comparison of the two-dimensional structures revealed that, although these five compounds represent three distinct structural classes, they share a common structural feature located in the region hypothesized to be involved in receptor binding: a carbonyl or carboxyl oxygen interposed spatially between an aromatic ring and nitrogen atom. This structural feature was used to predict the relative selectivity of compounds that had not previously been analyzed at both the 5-HT2 and 5-HT1C receptors. All six drugs tested which contain the identified reactive carbonyl or carboxyl group were found to be selective for the 5-HT2 versus the 5-HT1C receptor with selectivity ratios ranging from 26 to 380. By contrast, three agents which are structurally similar but do not contain the reactive carbonyl or carboxyl group displayed equally high affinity for both receptor binding sites. Since the physiological roles of the 5-HT2 and 5-HT1C receptor are markedly different, it would be of potential clinical and scientific value to utilize this molecular structural feature to further identify chemical compounds which would selectively interact with only one of the two receptors.

Motor pool organization of the external gastrocnemius muscle in the turtle, Pseudemys (Trachemys) scripta elegans.

The spinal cord of the adult turtle, Pseudemys (Trachemys) scripta elegans, is now considered a promising model for the study of the segmental motor system in the generalized tetrapod. To facilitate such studies we have examined the location, soma geometry, soma size, and number of motoneurons innervating the external gastrocnemius (EG) muscle in this species, as this muscle is ideally suited to the study of interrelations between the neuronal and muscular components of the segmental motor system. Motoneurons were retrogradely labeled following application of horseradish peroxidase to the EG muscle nerve. In both horizontal and transverse planes, labeled motoneurons innervating the EG muscle were concentrated in the S1 lumbosacral segment, and extended rostrally and caudally as far as the exists of the D10 and S2 spinal nerves, respectively. In the transverse plane, motoneurons were arranged in a longitudinal column which occupied the dorsolateral quadrant of the ventral horn. EG motoneurons are fusiform in shape and present their largest dimension in the transverse plane with their long axis oriented in the ventromedial to dorsolateral plane. The soma diameters of EG motoneurons were normally distributed, reflecting the absence of separate fusimotor innervation in reptilian species. In individual turtles, there was a two- to threefold range in soma diameter while soma surface area extended over a seven- to tenfold range. Based on cell counts from five animals, the EG motor pool was composed of approximately 75 motoneurons. Taken together, the results of this study provide valuable information for interpreting the results of future studies on the segmental motor system of this species under both normal and pathophysiological conditions.

Antagonism of 5-hydroxytryptamine2 receptor-mediated phosphatidylinositol turnover by d-lysergic acid diethylamide.

The interactions of the indolealkylamine hallucinogen d-lysergic acid diethylamide (d-LSD) and two phenalkylamine hallucinogens, 2,5-dimethoxy-4-bromoamphetamine (DOB) and 2,5-dimethoxy-4-iodoamphetamine (DOI), with 5-hydroxytryptamine2 (5-HT2) receptors were analyzed in rat cortex using both radioligand binding techniques and biochemical measurements of phosphatidylinositol (PI) turnover. 5-HT2 binding sites were labeled by [3H]ketanserin. DOB and DOI displayed decreased affinity for 5-HT2 sites in the presence of 10(-4) M GTP, whereas the ability of d-LSD to compete for these sites was not affected by the presence of 10(-4) M GTP. Moreover, the Hill slope of the d-LSD competition curve was unity in both the absence and presence of 10(-4) M GTP. These findings suggest that d-LSD is an antagonist at 5-HT2 receptors. PI turnover studies in rat cortex showed that at 10(-5) M concentrations d-LSD, DOB and DOI display partial agonist activity in comparison to 10(-5) 5-HT. Stimulation of PI turnover by 5-HT, DOB and DOI was inhibited by the 5-HT2 antagonist ketanserin (10(-6) M). The d-LSD PI signal was not affected by the presence of ketanserin. In addition, nanomolar concentrations of d-LSD did not stimulate PI turnover in rat cortex. Moreover, nanomolar concentrations of d-LSD are able to significantly antagonize the stimulatory effect of 10(-5) M 5-HT on PI turnover. These data suggest that d-LSD acts as an antagonist at 5-HT2 receptors in rat cortex. At high concentrations (greater than 1 microM) d-LSD stimulates low-level PI turnover via a non-5-HT2 receptor-mediated mechanism.

Evidence for distinct 5-hydroxytryptamine2 binding site subtypes in cortical membrane preparations.

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.

Periodicity and directionality in the propagation of epileptiform discharges across neocortex.

1. The horizontal propagation of epileptiform discharges has been studied in slices of neocortex treated with high concentrations of bicuculline methiodide, an antagonist of the inhibitory transmitter gamma-aminobutyric acid (GABA). The cortical areas examined were: primary somatosensory (SmI) and motor (MI), and primary (area 17) and secondary (area 18) visual areas of rats, and area 17 of cats. In all of these areas an electrical stimulus evoked single, all-or-none paroxysmal field potentials (PFPs) that propagated across the entire width of the slice without decrement. 2. The velocity of PFP propagation was approximately 0.06-0.09 m/s when averaged over cortical distances of several millimeters. PFP propagation occurred equally well in both directions across a slice. 3. Measurement of PFP propagation at higher spatial resolution (100-180 micron intervals) revealed that velocity was not homogeneous within rat SmI, rat area 18 and cat area 17, but instead varied manyfold as horizontal position changed. In these areas of cortex, propagation patterns were spatially periodic; power spectra reveal that the dominant spatial frequencies were centered about 1 mm-1, with negligible contributions above 2 mm-1. Occasionally PFP propagation was discontinuous, skipping over a small region of cortex and arriving distally before propagating into the more proximal region. 4. In those cortices with periodic propagation patterns, PFP velocity was also strongly direction-dependent. Propagation patterns measured in opposite directions across the same strip of cortex displayed similar periodicities, but in many slices they were negatively correlated, i.e., the propagation pattern in one direction was antiphasic compared to that in the other direction. 5. In contrast, propagation velocity across the center of area 17 of the rat was relatively constant and not directional. Near the boundaries of areas 17 and 18, however, PFP velocity changed abruptly and became periodic within area 18. Similarly, velocity within rat MI was more constant and less directional than in the adjacent SmI. 6. The patterns of PFP propagation velocity are often spatially periodic, directionally asymmetric, and depend upon cortical area. We suggest that the periodic patterns reflect systematic variations in the length or density of horizontal excitatory connections. Alternatively, or concurrently, periodicities could arise from the patchy distributions of intrinsic connections that have been observed anatomically in many areas of neocortex.

Differential binding of ergot compounds to human versus rat 5-HT2 cortical receptors.

Eleven ergot compounds were competed against [3H]-ketanserin-labelled 5-HT2 receptors in rat and human cortex. Four of the ergots are selective for rat 5-HT2 receptors--mesulergine, methysergide, nicergoline and metergoline, whereas pergolide, d-lysergic acid, ergonovine, ergotamine, d-lysergic acid diethylamide, lisuride and dihydroergotamine display selectivity for human 5-HT2 receptors. Rat-selective compounds contain a methyl substitution on the indole nitrogen, whereas human-selective compounds contain a hydrogen. This structural feature may allow the two groups of ergots to differentiate between the two species of receptors.

Halothane selectively inhibits bradykinin-induced synovial plasma extravasation.

BACKGROUND: In vitro studies demonstrate that halothane, but not isoflurane, inhibits bradykinin-induced calcium currents and prostacyclin release in cultured endothelial cells. Because bradykinin is an important endogenous mediator of inflammation, we assessed the effects of halothane, isoflurane, and pentobarbital on plasma extravasation, a component of tissue inflammation induced by bradykinin, in rats. METHODS: We anesthetized 23 rats with halothane (0.8 or 1.3 minimum alveolar concentration [MAC]), isoflurane (1.3 MAC), or pentobarbital (total of 85 mg/kg intraperitoneally). Their tracheas were intubated and their lungs mechanically ventilated. After intravenous administration of Evans blue dye, we perfused normal saline followed by bradykinin or platelet-activating factor, another inflammatory mediator, intraarticularly via needles placed in the knee joint. We collected perfusate and estimated extravasation by measuring dye in the perfusate using spectrophotometry. RESULTS: Bradykinin increased plasma extravasation eight- to ninefold above baseline in both pentobarbital- and isoflurane-anesthetized rats. In contrast, bradykinin-induced plasma extravasation at 0.8 MAC and 1.3 MAC of halothane was approximately 40% (P < 0.01) and 15% (P < 0.001), respectively, of that in pentobarbital- and isoflurane-anesthetized rats. Baseline plasma extravasation was lower in rats anesthetized with either concentration of halothane compared with pentobarbital or isoflurane (all P < 0.001). Platelet-activating factor-induced plasma extravasation was similar for all anesthetics. CONCLUSION: Halothane, but not isoflurane or pentobarbital, inhibits both baseline and bradykinin-induced peripheral plasma extravasation, demonstrating that volatile anesthetics differentially modulate this important component of inflammation.

Dual effect of the serotonin agonist, sumatriptan, on peripheral neurogenic inflammation.

BACKGROUND AND OBJECTIVES: Sumatriptan is a novel drug for migraine headache pain, which, on the basis of its mechanism of action, may have therapeutic potential in other pain states. Sumatriptan inhibits neurogenic inflammation in dural vessels by activating the 5-HTIB and 5-HTID inhibitory serotonin (5-hydroxytryptamine [5-HT]) receptor subtypes on terminals of trigeminal neurons. This study was designed to determine the role of sumatriptan in peripheral pain mechanisms by detecting whether 5-HTIB and 5-HTID receptors and the recently cloned excitatory 5-HT7 receptor, for which sumatriptan displays moderate binding affinity, are present in peripheral sensory neurons, and by determining the effect of sumatriptan on peripheral neurogenic inflammation. METHODS: A polymerase chain reaction (PCR) technique was used to detect mRNA for 5-HT receptors in rat lumbar dorsal root ganglia. Rat knee joint plasma extravasation was used to determine the effect of sumatriptan on peripheral neurogenic inflammation. RESULTS: The mRNA for the sumatriptan-activated receptors 5-HTIB, 5-HTID, and 5-HT7, was detected in lumbar dorsal root ganglia. In rat knee joint, capsaicin-activated C-fibers stimulated plasma extravasation to 273 +/- 62% of baseline. Low-concentration sumatriptan (50 nM) significantly inhibited capsaicin-induced plasma extravasation to 106 +/- 6% of baseline. High-concentration sumatriptan (1 microM) significantly enhanced capsaicin-induced plasma extravasation to 572 +/- 55% of baseline. CONCLUSIONS: Sumatriptan inhibits peripheral neurogenic inflammation, probably via 5-HT1B/1D receptors, and may be a novel therapy for inflammatory pain states. However, high concentrations (> 200 nM) may enhance neurogenic inflammation, possibly by activation of 5-HT7 receptors, which may explain lack of migraine relief and excessive injection site pain in 20-30% of patients treated with sumatriptan.

Effects of pulse duration on neuromuscular blockade monitoring: implications for supramaximal stimulation.

Questions have been raised concerning the reliability of surface electrodes in achieving supramaximal stimulation during the monitoring of neuromuscular blockade; needle electrodes are considered reliable in this respect. This study compares interelectrode impedances of needle and surface electrodes during neuromuscular blockade monitoring and suggests those characteristics of the stimulation pulse that can ensure reliable supramaximal stimulation with either type of electrode. Interelectrode voltage and current for surface and needle electrodes were measured by using 1.0-ms pulses at low, medium, and high stimulation levels on 22 surgical patients during anesthesia. Data were collected immediately after electrode application, and again at 10 minutes after application. Stimulation with surface electrodes produced an initial, transient surge of current, followed by a lower steady-state value. At high stimulation levels, the peak transient current was 87% higher than the steady-state current. Needle electrodes produced a constant high-current response. At high stimulation levels the transient impedance of the surface electrode and the impedance of the needle electrode were essentially equal (0.7 k omega and 0.75 k omega, respectively). The transient impedance was significantly lower (P less than 0.001) and was associated with less interpatient variation (P less than 0.001) and less sensitivity to the duration of electrode application than was the steady-state impedance of the surface electrode. These data suggest that high-current pulses with widths of less than 0.2 ms could provide reliable supramaximal stimulation with either type of electrode.

The clinical utility of pharmacological agents that act at serotonin receptors.

The past decade has seen important advances in the clinical utility of serotonergic agents. The putative novel anxiolytic effects of 5-HT1A partial agonists such as buspirone, the antidepressant effects of selective serotonin (5-HT) uptake blockers such as fluoxetine, and the unique and potent antiemetic effects of 5-HT3 antagonists in cancer chemotherapy are excellent examples of the clinical relevance of selective 5-HT receptor agents. The increasing ability to modulate serotonergic neurotransmission through distinct 5-HT receptor subtypes should greatly facilitate the analysis of 5-HT in both normal and abnormal human brain function.

5-Hydroxytryptamine-induced synovial plasma extravasation is mediated via 5-hydroxytryptamine2A receptors on sympathetic efferent terminals.

5-Hydroxytryptamine (5-HT) is known to act in peripheral tissues to produce pain and inflammation, yet the mechanisms underlying 5-HT-induced inflammation have not been well studied. The present study uses a rat knee joint model of inflammation (synovial plasma extravasation) and molecular biological techniques to determine the site of action of 5-HT and the specific 5-HT receptor subtype mediating synovial 5-HT-induced plasma extravasation. 5-HT (1 microM) stimulates synovial plasma extravasation 7-fold above base-line levels. Surgical lumbar sympathectomy, but not C-fiber depletion by neonatal capsaicin, dramatically reduces 5-HT-induced synovial plasma extravasation (P < .001), indicating that sympathetic efferents mediate this effect. Polymerase chain reaction amplification of 5-HT receptor cDNA demonstrates that 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A and 5-HT3, but not the 5-HT2C, receptor subtypes are present in lumbar sympathetic ganglia. With selective ligands for these receptor subtypes, we demonstrate that 5-HT-induced synovial plasma extravasation is mediated via the 5-HT2A receptor. These findings suggest a role for 5-HT2A antagonists in various synovial inflammatory pain states.

(R)-(-)-[77Br]4-bromo-2,5-dimethoxyamphetamine labels a novel 5-hydroxytryptamine binding site in brain membranes.

(R)-(-)-[77Br]4-Bromo-2,5-dimethoxyamphetamine [(R)-(-)-[77Br] DOB] was synthesized to a high specific activity (1875 +/- 50 Ci/mmol) and used to label membrane-associated recognition sites in rat brain. (R)-(-)-[77Br]DOB displayed high affinity (KD = 0.60 +/- 0.08 nM) for a relatively low density of binding sites (Bmax = 1.2 +/- 0.08 pmol/g of tissue) in rat cortical membranes as compared with [3H]ketanserin (KD = 0.65 +/- 0.1 nM; Bmax = 6.2 +/- 0.6 pmol/g of tissue). Guanine, but not adenine, nucleotides were found to inhibit specific (R)-(-)-[77Br]DOB binding. GTP (10(-4) M) did not eliminate specific (R)-(-)-[77Br]DOB binding but caused a competitive inhibition of the radioligand. Drug competition studies of 5-hydroxytryptamine (5-HT) and related agents indicate that both putative agonists and antagonists display nanomolar potency for these sites. A significant correlation (p less than 0.01) exists between drug potencies for (R)-(-)-[77Br]DOB-labeled sites and both 5-HT2 (r = 0.64) and 5-HT1C (r = 0.68) binding sites. However, the sites do not appear to be identical. Moreover, a significant correlation exists between drug potencies for (R)-(-)-[77Br]DOB-labeled sites and human hallucinogenic drug potencies (r = 0.89; p less than 0.01). We conclude that (R)-(-)-[77Br]DOB labels a unique 5-HT recognition site in rat brain that does not coincide with previously described 5-HT binding site subtypes. The (R)-(-)-[77Br]DOB site does not appear to be a high affinity "state" of the 5-HT2 receptor but may label a subset of heterogeneous 5-HT2 recognition sites.