Functional characterisation of the human cloned 5-HT7 receptor (long form); antagonist profile of SB-258719

1. The functional profile of the long form of the human cloned 5-HT7 receptor (designated h5-HT7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT7(a) receptor. 2. The receptor binding profile, determined by competition with [3H]-5-CT, was consistent with that previously reported for the h5-HT7(a) receptor. The selective 5-HT7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]ben zene sulfonamide) displayed high affinity (pKi 7.5) for the receptor. 3. In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT > 5-HT > 8-OH-DPAT) was the same in functional and binding studies. 4. Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA2 of 7.2+/-0.2 and slope not significantly different from 1, consistent with competitive antagonism. 5. The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT7 receptor-mediated and to reflect inverse agonism. 6. It is concluded that in this expression system, the h5-HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.     

Stimulation of type 1 and type 8 Ca2+/calmodulin-sensitive adenylyl cyclases by the Gs-coupled 5-hydroxytryptamine subtype 5-HT7A receptor

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT6 and 5-HT7 couple to stimulation of adenylyl cyclases through Gs and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT6 or 5-HT7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT6 receptor functioned as a typical Gs-coupled receptor in that it stimulated AC5, a Gs-sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by Gs-coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. 5-HT also increased intracellular Ca2+ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin.     

Cross-modulation of synaptic plasticity by beta-adrenergic and 5-HT1A receptors in the rat basolateral amygdala

Neurotransmitter receptors are often colocalized in a neuron with other receptors, and activation of one receptor can either amplify or antagonize the response to a colocalized receptor. The aim of this study was to investigate the cross-regulation of synaptic transmission by beta-adrenergic and serotonin 1A (5-HT1A) receptors and to elucidate their underlying mechanisms. Stimulation of presynaptic beta-adrenergic receptors with isoproterenol (Iso) in the basolateral amygdala resulted in a long-lasting increase in synaptic transmission. This effect was mimicked by forskolin, an activator for adenylyl cyclase and a cAMP analog. In addition, the effect of forskolin was blocked by catalytic and regulatory site antagonists for cAMP-dependent protein kinase (PKA), indicating a PKA-mediated mechanism. Application of 5-HT depressed the synaptic transmission and blocked Iso- and forskolin-induced potentiation. The effect of 5-HT was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was blocked by the selective 5-HT1A antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine, indicating its mediation by 5-HT1A receptors. To determine the locus of interaction, Sp-cAMPS, a membrane-permeable activator of PKA, was applied, and the potentiation produced by Sp-cAMPS was completely blocked in slices pretreated with 5-HT. These results suggest that the interaction between the intracellular signaling pathways activated by 5-HT1A and beta-adrenergic receptors occurs at a step downstream from cAMP production.     

The human and dog 5-HT1D receptors can both activate and inhibit adenylate cyclase in transfected cells

The cloned human serotonin 1D (5-HT1D) receptor has been shown to inhibit adenylate cyclase while the corresponding cloned dog receptor has been characterized by its enhancement of cAMP accumulation. To resolve this apparent discrepancy, the human 5-HT1D receptor has been cloned and expressed in Chinese hamster ovary (CHO) cells and the corresponding dog receptor expressed in mutant Y1 adrenal (Y1 Kin-8) cells. It is shown that both receptors when activated by sumatriptan depress forskolin induced adenosine 3'5'-cyclic monophosphate (cAMP) accumulation by a pertussis toxin sensitive mechanism, presumably involving Gi (the adenylate cyclase inhibitory GTP transducing protein). In the absence of forskolin, the dog receptor enhances cAMP accumulation, thus activating Gs (the adenylate cyclase stimulatory GTP transducing protein). When its overriding action on Gi is blocked by pertussis toxin pretreatment, the human receptor also enhances cAMP accumulation. Thus both 5-HT1D receptors activate markedly Gi and to a lesser extent Gs and can exert opposite effects on the same effector system, adenylate cyclase.     

Receptor subtype and density determine the coupling repertoire of the 5-HT2 receptor subfamily

The 5-Hydroxytryptamine (5-HT)2C receptor (originally known as the 5-HT1C receptor) is a member of the 5-HT2 subfamily of G protein coupled receptors, which is known to couple to phospholipase C. Within the 5-HT2 subfamily, only the 5-HT2C receptor also coupled to inhibition of forskolin-stimulated cAMP production when expressed at high density (12 pmol/mg membrane protein) in stably transformed AV12 cells. The 5-HT2C receptor coupled with high efficacy to both phospholipase C as measured by IP3 (inositol 1,4,5-trisphosphate) production and to inhibition of forskolin-stimulated cAMP production (EC50 = 2.98 nM +/- 0.9 and IC50 = 47.99 nM +/- 10.25 respectively). The 5-HT2A and 5-HT2B receptors, while coupling to phospholipase C with high affinity (EC50s of 19.24 nM +/- 6.44 and 1.24 nM +/- 0.136 respectively), did not decrease adenylyl cyclase activity. The 5-HT2C receptor actions in both systems showed the expected pharmacology for the 5-HT2C receptor, e.g., mesulergine antagonized the effects of 5-HT and spiperone did not. Preincubation of cells with PTX showed that the G protein coupling of the 5-HT2C receptor to phospholipase C is PTX insensitive, while the G protein coupling to inhibition of adenylyl cyclase is PTX sensitive, even to concentrations as low as 20 ng/ml of PTX. PTX pretreatment of the 5-HT2C bearing cells also unmasked a small stimulatory effect on adenylyl cyclase. When expressed at low density the 5-HT2C receptor potentiated forskolin-stimulated cAMP production by 2 fold while still maintaining its ability to enhance PI hydrolysis. A more modest potentiation of cAMP production was noted with low density expression of the 5-HT2B receptor. Thus the ability of the 5-HT2C receptor to interact with several effectors through at least two different G proteins is, in part, receptor subtype specific but also influenced by receptor density.     

5-Hydroxytryptamine-induced contraction of the marmoset aorta is mediated by a 5-HT1-like receptor

1. 5-Hydroxytryptamine (5-HT) exerts both contractile and relaxant effects in the marmoset isolated aorta, actions that are unaffected by the 5-HT2 antagonist ketanserin. The aim of the present study was to define the receptors mediating the contractile activity of 5-HT in the marmoset aorta. 2. Contractile responses were elicited in aortic rings that were either: (i) precontracted submaximally with the thromboxane A2 agonist U44069 in order to amplify the responses; or (ii) exposed to N(omega)-nitro-L-arginine (100 micromol/L) plus LY 53857 (0.1 micromol/L; a 5-HT2 receptor antagonist shown previously to inhibit relaxation). The effect of 5-HT on adenosine 3',5'-cyclic monophosphate (cAMP) formation was also investigated. 3. The effects of agonists and antagonists comprised: (i) agonist potencies in the order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin; (ii) inhibition of contractile action of 5-HT by the 5-HT1D antagonist GR 127935; (iii) a contractile response to methysergide; (iv) a lack of effect of tropisetron, an antagonist of 5-HT3 and 5-HT4 receptors; and (v) inhibition of forskolin-stimulated cAMP formation by 5-HT (in the presence of LY 53857), indicative of negative coupling to adenylate cyclase. 4. The above effects fulfill the criteria for a 5-HT1-like receptor. In view of the previous finding that this contractile response is insensitive to ketanserin, it is concluded that the contractile effects of 5-HT in the marmoset aorta are mediated exclusively by a 5-HT1-like receptor.     

Changes in agonist-antagonist EMG, muscle CSA, and force during strength training in middle-aged and older people

Serotonin (5-HT) is one of the major transmitters involved in supraspinal control of somatic sensation and nociception. The aim of the present study was to investigate if the 5-HT-induced modulation of sensory transmission in the dorsal horn could be due to regulation of neuronal responses to excitatory amino acids. Experiments were performed in an in vitro preparation of the young rat spinal cord. Responses to dorsal root stimulation (DR-EPSP) and to droplet application of N-methyl-D-aspartic acid (NMDA) and alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) were obtained by means of intracellular recordings of dorsal horn neurons. Bath applications of 5-HT (50 microM) generally caused reductions in amplitude and integrated area of DR-EPSPs and of responses to NMDA but the responses to AMPA were unaltered. A linear correlation was found between the effects of 5-HT on the DR-EPSP and on the NMDA response measured as percentage change in amplitude (r2 = 0.45; P < or = 0.01) and integrated area (r2 = 0.77; P < or = 0.001). The NMDA receptor antagonist d-AP5 (50 microM) completely abolished NMDA responses and caused a depression of the DR-EPSP similar to that of 5-HT. The 5-HT1 receptor agonist 5-carboxamidotryptamine (5-CT; 1 microM) mimicked the depressant effects of 5-HT but had a stronger depressant action on the DR-EPSP than 5-HT. The depression of NMDA responses induced by 5-HT and 5-CT was tetrodotoxin (1 microM) resistant. It is concluded that 5-HT-induced depression of NMDA responses explains partially the depressant action of 5-HT on dorsal horn synaptic transmission activating a postsynaptic site sensitive to 5-CT. The possible activation of coadjuvant mechanisms is discussed.     

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) receptor isoform coupled to adenylate cyclase stimulation

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) (h5-HT7(a)) receptor isoform was performed using stably transfected LM(tk-) cells. Expression levels of the h5-HT7(a) receptor determined from saturation studies using either a labeled agonist ([3H]5-HT) or antagonist ([3H]LSD) were very similar (Bmax = 160-190 fmol/mg protein), suggesting that all receptors may exist in the high affinity (G protein-coupled) state. In intact cells, 5-HT produced a concentration-dependent elevation of intracellular cAMP levels ([cAMP]i) with an EC50 value of 80 nM and a maximal response of 5-fold increase above basal levels. The rank order of agonist potencies in the second messenger assay paralleled their rank order of binding affinities: 5-carboxamidotryptamine > 5-hydroxytryptamine >/= 5-methoxytryptamine > 8-hydroxy N,N-dipropyl aminotetralin > sumatriptan. Agonist potencies (EC50 values) to stimulate [cAMP]i were more than 25-fold lower relative to their respective binding affinities (Ki values) obtained in [3H]5-HT competition assays. In contrast, antagonist potencies (Kb values) to block 5-HT-stimulated [cAMP]i were in close agreement with their corresponding Ki values. These data may indicate low efficiency of receptor-effector coupling to adenylate cyclase stimulation. Pretreatment of stably transfected cells with cholera toxin abolished the 5-HT-mediated elevation of [cAMP]i, indicating that the 5-HT7(a) subtype directly interacts with Galphas protein(s) to activate adenylate cyclase(s). Clonal cell lines stably expressing h5-HT7 receptor isoforms will serve as valuable cellular models to study their function and regulation, as well as assist in the development of selective 5-HT7 receptor agents to uncover the biological roles and potential therapeutic applications of this novel receptor subtype.     

7-OH-DPAT antagonizes dopamine D2 receptor-inhibited adenylyl cyclase activity

(+/-)7-OH-DPAT (7-hydroxy-2-(di-n-propylamino) tetralin) binds to both dopamine D2 and D3 receptor subtypes. In 7315c pituitary tumor cell membranes, which express only the D2 type of dopamine receptor, dopamine inhibited, and 7-OH-DPAT had no effect on adenylyl cyclase activity. When combined, 7-OH-DPAT antagonized the inhibition of adenylyl cyclase produced by dopamine. Thus, it appears that 7-OH-DPAT acts as an antagonist at dopamine D2 receptors.     

Dopamine-dependent cyclic AMP generating system in chick retina and its relation to melatonin biosynthesis

Stimulation of a D4-like dopamine (DA) receptors inhibits a cAMP-dependent increase in serotonin N-acetyltransferase activity and melatonin biosynthesis in the chick retina. In order to gain more insight into the molecular mechanisms underlying this suppressive action of DA, the effects of selective stimulation of the D2-family of DA receptors (including the D4-subtype) on cAMP formation were examined in chick retina using two experimental approaches: measurements of adenylyl cyclase activity in retinal homogenates, and cAMP accumulation in eye cup preparation prelabeled with [3H]adenine. The DA-sensitive adenylyl cyclase system is well expressed in chick retina. DA increased both basal and forskolin-stimulated adenylyl cyclase activity. This effect of DA was antagonized by SCH 23390 (a blocker of D1-family of DA receptors) and not affected by sulpiride (a D20-family blocker). Incubation of retinal homogenates with quinpirole (a predominant agonist of D3/D4 DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D4-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retina maintained in "eye-cup" preparations. It is suggested that D4-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.     

5-HT1B receptor antagonist properties of novel arylpiperazide derivatives of 1-naphthylpiperazine

A new series of arylpiperazide derivatives of 1-naphthylpiperazine of general formula 4 has been prepared and evaluated as 5-HT1B antagonists. Binding experiments at cloned human 5-HT1A, 5-HT1B, and 5-HT1D receptors show that these derivatives are potent and selective ligands for 5-HT1B/1D subtypes with increased binding selectivity versus the 5-HT1A receptor when compared to 1-naphthylpiperazine (1-NP). Studies of inhibition of the forskolin-stimulated cAMP formation mediated by the human 5-HT1B receptor demonstrate that the nature of the arylpiperazide substituent modulates the intrinsic activity of these 1-NP derivatives. Among them, 2-[[8-(4-methylpiperazin-1-yl)naphthalen-2-yl]oxy] -1-(4-o-tolylpiperazin-1-yl)ethanone (4a) was identified as a potent neutral 5-HT1B antagonist able to antagonize the inhibition of 5-HT release induced by 5-CT (5-carbamoyltryptamine) in guinea pig hypothalamus slices. Moreover, 4a was found to potently antagonize the hypothermia induced by a selective 5-HT1B/1D agonist in vivo in the guinea pig following oral administration (ED50 = 0.13 mg/kg).     

Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene

A cyclic AMP-responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose-dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein-coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5-HT) receptor agonist ligands caused an inhibition of forskolin-stimulated luciferase expression in the rank order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin. The response to 5-HT was reversed by the 5-HT1 receptor antagonists cyanopindolol and pindolol, but not the 5-HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene-related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8-37). These results were consistent with the presence of 5-HT(1B-like) and calcitonin (C1a-like) receptors in CHO cells, with the responses to 5-HT and CGRP being pertussis and cholera toxin-sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein-coupled receptors linked to adenylyl cyclase.     

Selective effects of ethanol on the generation of cAMP by particular members of the adenylyl cyclase family

A selective action of ethanol on major signal transduction proteins, such as adenylyl cyclase, has been considered to be important for certain actions of ethanol, and alcoholics have been demonstrated to differ from controls in measures of platelet adenylyl cyclase activity. Recent advances in identification and characterization of isoforms of adenylyl cyclase have demonstrated that there exists at least eight different forms of this enzyme. To examine whether the effect of ethanol on generation of cAMP is modified by the presence of particular isoforms of adenylyl cyclase within a cell, we transiently expressed each of six adenylyl cyclases in human embryonic kidney (HEK293) cells and measured cAMP accumulation in whole cells in the presence and absence of ethanol. The treatment of cells expressing the various adenylyl cyclases with ethanol alone did not enhance cAMP generation. In the presence of prostaglandin E1, cAMP generation by type I and type III adenylyl cyclases was insensitive to ethanol. cAMP accumulation generated by the other adenylyl cyclases was, however, increased by incubation of cells with ethanol in the presence of stimulatory agonists (e.g., prostaglandin E1). Stimulation by ethanol of cAMP generation by type VII adenylyl cyclase was 2- to 3-fold greater than that seen with the other tested adenylyl cyclases. The noted stimulation of cAMP generation by ethanol was dose-dependent and required concurrent activation of adenylyl cyclase through the stimulatory G protein. The effects of ethanol were reversible and mimicked by butanol but not by chloroform.     

Vascular 5-HT1-like receptors mediating vasoconstriction and vasodilatation: their characterization and distribution in the intact canine cardiovascular system

1. In anaesthetized dogs, intra-left atrial administration of 5-hydroxytryptamine (5-HT) and selected tryptamine analogues (5-carboxamidotryptamine, 5-CT; 5-methyl tryptamine, 5-MT; alpha-methyl 5-hydroxytryptamine, alpha-HT; sumatriptan, Sum) in the presence of ketanserin and MDL72222 (5-HT2 and 5-HT3 receptor antagonists, respectively), produced dose-related changes in carotid, coronary and renal vascular conductance mediated by vascular 5-HT1-like receptors. 2. In the carotid vascular bed, 5-HT, 5-MT, alpha-HT and Sum were vasoconstrictors with a rank order of potency (comparing ED50 values) of 5-HT = Sum > 5-MT > alpha-HT. By contrast in this vascular bed, 5-CT was a potent vasodilator. 3. In the coronary vascular bed, 5-HT, 5-CT, 5-MT and alpha-HT were vasodilators with a rank order of potency (comparing ED50 values) of 5-CT > 5-HT > 5-MT > alpha-HT. In this vascular bed, Sum was without effect. 4. In the renal vascular bed, 5-HT, 5-CT, 5-MT, alpha-HT and Sum were vasoconstrictors with a rank order of potency (comparing ED50 values) of 5-CT > 5-HT > Sum > 5-MT > alpha-HT. 5. The coronary (and carotid) vasodilator responses to 5-CT were antagonized by the 5-HT1-like receptor antagonists, spiperone (1 mg kg-1) and methiothepin (0.1 mg kg-1), whereas the renal vasoconstrictor responses to this tryptamine analogue were antagonized only by methiothepin. 6. It is concluded from these studies that agonist finger-printing in vivo, using tryptamine analogues,identifies and confirms the functional presence of at least two pharmacologically distinct subtypes of the 5-HT1-like receptor in the intact canine cardiovascular system. These two subtypes are located on the vascular smooth muscle and mediate direct vasoconstriction and vasodilatation responses in vivo.7. In addition, these studies confirm that the distribution of these subtypes within the major vascular beds, shows a marked heterogeneity. The carotid vascular responses to the tryptamine analogue sindicate the presence of both the vasodilator and the vasoconstrictor subtypes. The coronary vascular responses to these analogues are, however, consistent with presence of the vasodilator subtype, only. By contrast, the renal vascular responses to these analogues indicates only the presence of the vasoconstrictor subtype.     

Dopamine modulates inwardly rectifying hyperpolarization-activated current (Ih) in cultured rat olfactory receptor neurons

The presence of dopamine receptors in olfactory receptor neurons (ORNs) suggests that odor sensitivity may be modulated by neurotransmitters at the level of primary sensory neurons. Using standard patch-clamp techniques on rat ORNs, we found that 1 microM dopamine, 500 microM SQ 22536 (SQ, an adenylyl cyclase inhibitor), 20 and 50 microM quinpirole (a selective dopamine D2 receptor agonist), and 1 mM adenosine 3', 5'-cyclic monophosphate (cAMP) modulate the hyperpolarization-activated current Ih. On hyperpolarizing from a holding potential of -58 mV, a small Cs+-sensitive inwardly rectifying current (Ih) was observed. Increases in extracellular K+ increased Ih amplitude without shifting its voltage dependence of activation, whereas increases in temperature produced an increase in Ih amplitude and a hyperpolarizing shift in the activation curve. Application of 1 microM dopamine reversibly shifted Ih activation to more negative potentials and decreased Ih current amplitudes. These effects were blocked by concomitant application of dopamine with sulpiride, a selective dopamine D2 receptor antagonist. The effects of dopamine were mimicked by quinpirole. Quinpirole (20 microM) decreased Ih current amplitude, but was without effect on Ih voltage dependence of activation. However, 50 microM quinpirole produced both a reduction of Ih peak currents and a hyperpolarizing shift in the activation curve for Ih. External application of the adenylyl cyclase inhibitor SQ 22536 produced a reversible decrease in peak currents but had no effect on Ih voltage dependence of activation, whereas internal application of cAMP shifted Ih activation to more depolarized potentials. Because Ih modulates cell excitability and spike frequency adaptation, our findings support a role for dopamine in modulating the sensitivity and output of rat ORNs to odorants.     

Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol

Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs.     

The selective 5-HT1B receptor inverse agonist 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro- spiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289) potently blocks terminal 5-HT autoreceptor function both in vitro and in vivo

5-HT1 receptors are members of the G-protein-coupled receptor superfamily and are negatively linked to adenylyl cyclase activity. The human 5-HT1B and 5-HT1D receptors (previously known as 5-HT1Dbeta and 5-HT1Dalpha, respectively), although encoded by two distinct genes, are structurally very similar. Pharmacologically, these two receptors have been differentiated using nonselective chemical tools such as ketanserin and ritanserin, but the absence of truly selective agents has meant that the precise function of the 5-HT1B and 5-HT1D receptors has not been defined. In this paper we describe how, using computational chemistry models as a guide, the nonselective 5-HT1B/5-HT1D receptor antagonist 4 was structurally modified to produce the selective 5-HT1B receptor inverse agonist 5, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6, 7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289). This compound is a potent antagonist of terminal 5-HT autoreceptor function both in vitro and in vivo.