Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits.

The possibility that Spodoptera frugiperda (Sf9) cells can provide an intact cell setting for reconstitution of the human 5-hydroxytryptamine1A (5-HT1A) receptor with mammalian G protein subunits was explored. The 5-HT1A receptor was found to assume an uncoupled phenotype when expressed alone in Sf9 cells at relatively high levels (5-34 pmol of receptor/mg of membrane protein), i.e. agonist-binding to the receptor was characterized by a relatively high Kd and an insensitivity to GTP. Co-expression of the receptor with members of the alpha i "family" together with various combinations of beta 1 and gamma subunits increased the affinity for agonists to that observed for the coupled form of receptor in mammalian cells, concomitant with conferrance of guanosine 5'-(beta,gamma-imino)triphosphate sensitivity. The agonists employed were [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin ([3H]8-OH-DPAT) and [125I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl) amino]tetralin ([125I]8-OH-PIPAT). The binding of an antagonist, [125I]4-(2'-methoxyphenyl)-1-[2'-[N-(2"- pyridinyl)-p-iodobenzamido]ethyl]piperazine ([125I]p-MPPI), was unaffected by co-expression of G protein subunits. Both alpha and beta gamma subunits were required for optimal coupling. No differences were evident among alpha i1, alpha i2, alpha i3, alpha o, and alpha z when expressed with beta 1 gamma 2 in this regard, nor among most permutations of beta 1 gamma subunits when expressed with alpha i1 (beta 1 gamma 2 approximately beta 1 gamma 3 approximately beta 1 gamma 5 > beta 1 gamma 1). Alpha s and alpha q expressed with beta 1 gamma 2 did not participate in coupling. These data support the conclusion that normal interactions between a mammalian receptor and a select array of G proteins can be established in intact Sf9 cells, and extend previous observations of 5-HT1A receptor coupling to G(o) and the pertussis toxin-insensitive G protein Gz.

Lack of discrimination by agonists for D2 and D3 dopamine receptors.

The affinities of D3 dopamine receptors for antagonists are similar to those of D2 receptors. D3 receptors have been reported, however, to have affinities nearly 100-fold higher than those of D2 receptors for some agonists, including (+/-)-7-hydroxy-n,n-dipropyl-aminotetralin (7-OH-DPAT) and quinpirole. This has led to the use of these agonists to try to identify functional responses mediated by D3 receptors in vivo. However, D2 receptors exist in multiple states having high and low affinities for agonists. The G protein-coupled state of D2 receptors is believed to be the functional state of these receptors. When receptors were labeled with the D2 receptor antagonist [125I]-(S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethoxysalicylamide ([125I]-NCQ-298) under conditions that promote uncoupling of receptors from G proteins, the affinities of D3 receptors were approximately 130-fold higher than those of D2 receptors for 7-OH-DPAT and quinpirole. When receptors were labeled with the D2 receptor agonist [125I]-(R)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'- propenyl)-amino]tetralin ([125I]-7-OH-PIPAT) under conditions that favor interactions of receptors with G proteins, the affinities of D3 receptors were less than sevenfold higher than the affinities of D2 receptors for the same drugs. Similarly, small differences in the affinities of D2 and D3 receptors for other agonists were seen when receptors were labeled with [125I]-7-OH-PIPAT. These data demonstrate that putative D3 receptor-selective agonists also interact with a high-affinity, G protein-coupled state of D2 receptors. The similarities in affinities of the agonist-preferring state of D2 and D3 receptors means that currently available agonists cannot be used to discriminate between behavioral effects mediated by D2 and D3 receptors.

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain.

Binding characteristics of a novel radioiodinated ligand, [125I]R(+)trans-7-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl) aminotetralin ([125I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [125I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (Kd = 0.42 nM). A majority (70%) of the sites labeled by [125I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [125I]R(+)trans-7-OH-PIPAT to 5-HT1A receptors, with a Kd value of 1.4 nM and a Bmax value of 210 fmol/mg protein, was observed. Binding of [125I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D2 and 5-HT1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [125I]R(+)trans-7-OH-PIPAT. [125I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D3 receptors.

Characterization of [125I](R)-trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin binding to dopamine D3 receptors in rat olfactory tubercle.

[125I](R,S)-trans-7-Hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)- amino]tetralin ([125](R,S)-trans-7-OH-PIPAT) has been shown to bind with high affinity to dopamine D3 receptors expressed in Spodoptera frugiperda cells. No specific binding was seen in Spodoptera frugiperda cells expressing a high density of D2 receptors. It was therefore, suggested that [125I] (R,S)-trans-7-OH-PIPAT selectively labels D3 receptors. In the present study, saturation binding of [125I](R)-trans-7-OH-PIPAT to membranes from rat olfactory tubercle resulted in markedly curvilinear Scatchard plots, suggesting that the radioligand was binding to more than one receptor class or affinity state. [125I] (R)-trans-7-OH-PIPAT bound with high affinity to membranes from human embryonic kidney-293 cells expressing transfected D2 or D3 receptors and to membranes from Chinese hamster ovary cells expressing serotonin1A receptors. Binding of [125I](R)-trans-7-OH-PIPAT to serotonin1A and D2 receptors was decreased or eliminated in the presence of NaCl and/or guanylyl-imidodiphosphate [Gpp(NH)p]. In the presence of Gpp(NH)p and NaCl, a linear Scatchard plot with a Kd value of 0.4 nM and a density of 100 fmol/mg of protein was obtained in membranes from rat olfactory tubercle. Agonists and antagonists inhibited binding of [125I](R)-trans-7-OH-PIPAT with a rank order of potency consistent with an interaction at D3 receptors. These results suggest that, in the presence of Gpp(NH)p and NaCl, [125I](R)-trans-7-OH-PIPAT specifically labels D3 receptors.

Unsurmountable antagonism of brain 5-hydroxytryptamine2 receptors by (+)-lysergic acid diethylamide and bromo-lysergic acid diethylamide.

Lysergic acid diethylamide (LSD) and its structural analogue 2-bromo-lysergic acid diethylamide (BOL) act as unsurmountable antagonists of serotonin-elicited contractions in smooth muscle preparations. Two different models, allosteric and kinetic, have been invoked to explain these findings. The present studies investigate the mechanism of antagonism of brain 5-hydroxytryptamine (5HT)2 receptors, utilizing cells transfected with 5HT2 receptor cDNA cloned from rat brain. A proximal cellular response, phosphoinositide hydrolysis, was examined in order to minimize possible postreceptor effects. Even though LSD behaved as a partial agonist and BOL as a pure antagonist, both drugs blocked the effect of serotonin in an unsurmountable manner, i.e., increasing concentrations of serotonin could not overcome the blocking effect of LSD or BOL. Radioligand binding studies showed that preincubation of membranes with either LSD or BOL reduced the density of [3H]ketanserin binding sites, suggesting that the drugs bind tightly to the 5HT2 receptor and are not displaced during the binding assay. Two additional experiments supported this hypothesis. First, the off-rate of [3H] LSD was slow (20 min), relative to that of [3H]ketanserin (approximately 4 min). Second, when the length of incubation with [3H]ketanserin was increased to 60 min, the LSD-induced decrease in Bmax was essentially eliminated. The possibility that LSD and BOL decrease [3H]ketanserin binding by interacting with an allosteric site was rejected, because neither drug altered the rate of dissociation of [3H]ketanserin. The most parsimonious interpretation of these results is that unsurmountable antagonism reflects prolonged occupancy of the receptor by slowly reversible antagonists.

(+)Lysergic acid diethylamide, but not its nonhallucinogenic congeners, is a potent serotonin 5HT1C receptor agonist.

Activation of central serotonin 5HT2 receptors is believed to be the primary mechanism whereby lysergic acid diethylamide (LSD) and other hallucinogens induce psychoactive effects. This hypothesis is based on extensive radioligand binding and electrophysiological and behavioral studies in laboratory animals. However, the pharmacological profiles of 5HT2 and 5HT1C receptors are similar, making it difficult to distinguish between effects due to activation of one or the other receptor. For this reason, it was of interest to investigate the interaction of LSD with 5HT1C receptors. Agonist-stimulated phosphoinositide hydrolysis in rat choroid plexus was used as a direct measure of 5HT1C receptor activation. (+)LSD potently stimulated phosphoinositide hydrolysis in intact choroid plexus and in cultures of choroid plexus epithelial cells, with EC50 values of 9 and 26 nM, respectively. The effect of (+)LSD in both systems was blocked by 5HT receptor antagonists with an order of activity consistent with interaction at 5HT1C receptors. Neither (+)-2-bromo-LSD nor lisuride, two nonhallucinogenic congeners of LSD, were able to stimulate 5HT1C receptors in cultured cells or intact choroid plexus. In contrast, lisuride, like (+)LSD, is a partial agonist at 5HT2 receptors in cerebral cortex slices and in NIH 3T3 cells transfected with 5HT2 receptor cDNA. The present finding that (+)LSD, but not its nonhallucinogenic congeners, is a 5HT1C receptor agonist suggests a possible role for these receptors in mediating the psychoactive effects of LSD.

Phosphoinositide hydrolysis linked 5-HT2 receptors in fibroblasts from choroid plexus.

A serotonin (5-HT)-mediated phosphoinositide hydrolysis response was characterized in fibroblasts cultured from rabbit choroid plexus. 5-HT elicited a maximum 8-fold increase in [3H]inositol-phosphate ([3H]IP) formation, while the partial agonists, (+)-lysergic acid diethylamide and (-)-1-(4-bromo-2,5-dimethyoxyphenyl)-2-aminopropane caused 2- and 5-fold increases, respectively. Mianserin, ketanserin, and spiperone were equipotent at blocking the 5-HT-mediated response. Thus, agonist and antagonist profiles indicate interactions with 5-HT2 receptors.

Lysergic acid diethylamide and 2,5-dimethoxy-4-methylamphetamine are partial agonists at serotonin receptors linked to phosphoinositide hydrolysis.

Based on electrophysiological, radioligand binding, and behavioral studies in laboratory animals, it is generally believed that the psychotomimetic effects of the phenethylamine and indolealkylamine hallucinogens are mediated by central serotonin (5-HT) receptors, in particular the 5-HT-2 subtype. Agonist-stimulated phosphoinositide hydrolysis was utilized to determine the potency and efficacy of racemic 1-(2,5)-dimethoxy-4-methyl-phenyl)-2-aminopropane (DOM), and d-lysergic acid diethylamide (LSD) at the 5-HT-2 receptor in rat cerebral cortex and the 5-HT-1c receptor in rat choroid plexus. Both DOM and LSD stimulated phosphoinositide hydrolysis in cerebral cortex. These effects were blocked by the 5-HT-2 antagonists, ketanserin and spiperone, but not by antagonists of muscarinic, alpha-1 adrenergic or histaminergic receptors. The maximum responses of DOM and LSD, respectively, were 76% and 25% of the maximum response to 5-HT. However, LSD was 500 times more potent than was racemic DOM. Consistent with a partial agonist effect, LSD partially blocked the effect of 5-HT, with a maximal inhibition equivalent to the intrinsic activity of LSD alone. In choroid plexus, DOM and LSD stimulated phosphoinositide hydrolysis and both responses were blocked by mianserin and less effectively by spiperone. The maximum effect of DOM was 67% of that of 5-HT, whereas the maximum effect of LSD was only 34% of the maximum response of 5-HT. LSD was 50 times more potent than was racemic DOM. LSD partially antagonized the effect of 5-HT in the choroid plexus, consistent with a partial agonist effect at the 5-HT-1c receptor in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidation of brain lipids following cyanide intoxication in mice.

Lipid peroxidation of brain lipids as determined by the conjugated diene method was observed in mice following administration of sublethal doses of potassium cyanide (KCN). Conjugated diene production was dose- and time-dependent; 10 mg/kg KCN produced detectable levels of conjugated dienes at 30 min post cyanide, whereas, 15 mg/kg produced marked levels of conjugated dienes over a 10-60-min period after KCN. Pretreatment of mice with either diltiazem (600 micrograms/kg, i.v.) or allopurinol (25 mg/kg, i.v.) blocked the generation of conjugated dienes. These results suggest lipid peroxidation of neuronal membranes play a role in cyanide intoxication and this action is related to altered regulation of neuronal calcium homeostasis and activation of xanthine oxidase.