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The human 5-hydroxytryptamine-2C (5-HT2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be l-threo-alpha-d-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 microM) markedly enhanced [3H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([3H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT-activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[35S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [3H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT2A, 5-HT2B, 5-HT6, 5-HT7, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the alpha-d-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [3H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.

Overexpression of the D2 dopamine receptor has been proposed to be part of the pathology of schizophrenia. The isolation of a D2 dopamine receptor clone has assisted the molecular characterization of D2 receptor. We have now isolated an identical rat clone along with two other clones--a second related rat clone (RD-2in) and a homologous bovine clone (BD-2in), both of which contain an insert encoding an additional 29 amino acids relative to the original rat clone (RD-2o). All three clones encode D2 receptor binding sites when expressed in COS-7 cells. The amino-acid insert encoded by D-2in lies in the domain of the receptor believed to interact with the GTP-binding proteins (G proteins) of various signal transduction pathways. By using oligonucleotide probes specific for either D-2o or D-2in RNA transcripts, we have found that the level of expression of the D-2in-encoded form of the receptor is seven times that of the D-2o form in the caudate nucleus, the richest brain source of D2 receptors.

Human 5-HT7A receptors positively modulated adenylyl cyclases via Gs subtypes of G proteins in human embryonic kidney 293 cells, and bound 5-hydroxytryptamine (HT) with high and low affinity (K(I) values of 1.5 +/- 0.3 and 93 +/- 4 nM). More than 60% of 5-HT7A receptors, however, displayed the high-affinity 5-HT binding with no sensitivity to 5'-guanylylimidodiphosphate. In this study, we found that select amphipathic agents affected the high-affinity 5-HT binding to 5-HT7A. Oleic acid at low concentrations (<15 microM), but not palmitic, stearic, and arachidonic acids, increased maximal [3H]5-HT binding without affecting its K(D) value and [3H]mesulergine (antagonist) binding. Fatty acid-free bovine serum albumin (FF-BSA), a scavenger of fatty acids and lipid metabolites, substantially reduced maximal [3H]5-HT binding (no change in K(D) value and antagonist binding) but lost its action upon treatment with inactive stearic acid. FF-BSA and oleic acid produced no appreciable effects on [3H]5-HT binding to analogous 5-HT receptors 5-HT1D and 5-HT2C. Among various lysophospholipids, lysophosphatidyl choline (50 microM) decreased maximal [3H]5-HT binding, and a similar zwitterion, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; 0.1%), increased it (no change in K(D)). Functionally, 5-HT-induced guanosine-5'-O-(3-[35S]thio)triphosphate (GTPgamma35S) binding was enhanced by oleic acid and CHAPS, but reduced by FF-BSA and lysophosphatidyl choline; the amphipathic agents and FF-BSA did not affect dopamine-induced GTPgamma35S binding at D1, a prototypic Gs-coupled receptor. At 5-HT7A, oleic acid, FF-BSA, CHAPS, and lysophosphatidyl choline also brought about corresponding changes in the half-maximal 5-HT concentration for cAMP production, without affecting the maximal and basal levels. We propose that endogenous, amphipathic lipid metabolites may modulate 5-HT7A receptors allosterically to promote high-affinity 5-HT binding and to enable receptors to couple more efficiently to Gs subtypes of G proteins.

The present study describes the preclinical pharmacology of a highly selective 5-HT1D receptor agonist PNU-142633. PNU-142633 binds with a Ki of 6 nm at the human 5-HT1D receptor and a Ki of> 18 000 nm at the human 5-HT1B receptor. The intrinsic activity of PNU-142633 at the human 5-HT1D receptor was determined to be 70% that of 5-HT in a cytosensor cell-based assay compared with 84% for that of sumatriptan. PNU-142633 was equally effective as sumatriptan and a half-log more potent than sumatriptan in preventing plasma protein extravasation induced by electrical stimulation of the trigeminal ganglion. Like sumatriptan, PNU-142633 reduced the increase in cat nucleus trigeminal caudalis blood flow elicited by electrical stimulation of the trigeminal ganglion compared with the vehicle control. The direct vasoconstrictor potential of PNU-142633 was evaluated in vascular beds. Sumatriptan increased vascular resistance in carotid, meningeal and coronary arteries while PNU-142633 failed to alter resistance in these vascular beds. These data are discussed in relation to the clinical findings of PNU-142633 in a phase II acute migraine study.

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The type 2 serotonin (5-HT(2)) receptor subfamily is known to couple to phosphoinositide hydrolysis (PI) and the subsequent mobilization of intracellular Ca(2+), as well as the release of arachidonic acid (AA). Less is known of 5-HT(2)-mediated activation of the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK1/2) signaling. The present study measured the relative efficacies and potencies of 5-HT agonists to activate ERK2 in non-neuronal cells expressing recombinant human 5-HT(2A), 5-HT(2B), and 5-HT(2C(ISV)) receptors. 5-HT agonists stimulated ERK2 activity via all three 5-HT(2) subtypes. There were no meaningful differences in the potencies or relative efficacies of these agonists to affect ERK2 activity vs. PI accumulation or Ca(2+) mobilization, suggesting that these pathways may be sequentially linked. Indeed, ERK2 activity was very sensitive to PKC inhibition and calcium chelation and insensitive to tyrosine kinase and PI-3-kinase inhibition. 5-HT(2) receptors efficiently couple to MAPK activation via sequential PI hydrolysis, and Ca(2+) mobilization. This profile differs from reports of "agonist-directed trafficking of receptor stimulus" between PI/Ca(2+) and AA pathways activated by 5-HT(2) receptors.

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