The novel 5-Hydroxytryptamine(1A) antagonist LY426965: effects on nicotine withdrawal and interactions with fluoxetine.

LY426965 [(2S)-(+)-1-cyclohexyl-4-[4-(2-methoxyphenyl)-1-piperazinyl]2-methyl- 2-phenyl-1-butanone monohydrochloride] is a novel compound with high affinity for the cloned human 5-hydroxytryptamine (HT)(1A) receptor (K(i) = 4.66 nM) and 20-fold or greater selectivity over other serotonin and nonserotonin receptor subtypes. Both in vitro and in vivo studies indicate that LY426965 is a full antagonist and has no partial agonist properties. LY426965 did not stimulate [(35)S]guanosine-5'-O-(3-thio) triphosphate (GTPgammaS) binding to homogenates of cells expressing the cloned human 5-HT(1A) receptor in vitro but did inhibit 300 nM 5-HT-stimulated [(35)S]GTPgammaS binding with a K(i) value of 3.07 nM. After both p.o. and s.c. administration, LY426965 blocked the lower lip retraction, flat body posture, hypothermia, and increase in rat serum corticosterone induced by the 5-HT(1A) agonist 8-OH-DPAT (8-hydroxy-2-dipropylaminotetralin). In pigeons, LY426965 dose-dependently blocked the stimulus cue induced by 8-OH-DPAT but had no 8-OH-DPAT-like discriminative properties. LY426965 completely reversed the effects of nicotine withdrawal on the auditory startle reflex in rats. In microdialysis experiments, LY426965 administered together with fluoxetine significantly increased extracellular levels of serotonin above those achievable with fluoxetine alone. In electrophysiological studies, the administration of LY426965 produced a slight elevation of the firing rate of 5-HT neurons in the dorsal raphe nucleus of anesthetized rats and both blocked and reversed the effects of fluoxetine on 5-HT neuronal activity. These preclinical results indicate that LY426965 is a selective, full 5-HT(1A) antagonist that may have clinical use as pharmacotherapy for smoking cessation and depression and related disorders.

Native 5-HT1B receptors expressed in OK cells display dual coupling to elevation of intracellular calcium concentrations and inhibition of adenylate cyclase.

The opossum kidney (OK) cell line has been shown previously to express endogenous 5-HT1B receptors which negatively couple to adenylate cyclase. Since other Gi-linked receptors have been shown to inhibit adenylate cyclase and to elevate intracellular calcium concentrations ([Ca2+]i), studies were initiated to determine whether native opossum 5-HT1B receptors could also display dual coupling to these signal transduction mechanisms. Saturation studies using [125I](-)-iodocyanopindolol ([125I]CYP) to radiolabel the 5-HT1B receptor in OK cell membranes (in the presence of 3 microM (-)-isoproterenol to mask beta-adrenergic receptors) yielded an equilibrium dissociation constant (pKd) of 10.04 and binding site density (Bmax) of 55 fmol/mg protein. Exposure of intact OK cells to 5-HT, CP 93,129, a selective rodent 5-HT1B receptor agonist, and (+/-)-cyanopindolol, a mixed 5-HT1A/1B receptor agonist/antagonist, produced concentration-dependent inhibitions of forskolin (3 MM)-stimulated cAMP accumulation (FSCA; Emax=90-95%) and elevations of [Ca2+]i (Emax approximately 200 nM increase above basal levels). Agonist potencies (pEC50) ranged from 9.7 to 8.1 and were comparable between the two second messenger assays, although slightly higher agonist potencies (approximately three-fold) were observed in the cAMP assay. GR 127,935, a selective 5-HT1B/1D receptor antagonist, behaved as a strong partial agonist in both the cAMP and calcium assays, with an intrinsic activity of 0.7 relative to 5-HT. Methiothepin, a nonselective 5-HT receptor antagonist, competitively antagonized the inhibitory cAMP response elicited by CP 93,129, yielding an apparent pKb value of 7.3. Methiothepin (10 microM) completely antagonized the stimulatory calcium response evoked by a saturating concentration of CP 93,129 (100 nM). Pertussis toxin pretreatment blocked the CP 93,129-induced inhibition of FSCA and elevation of [Ca2+]i in OK cells, indicating the involvement of Gi/o proteins in transducing these second messenger responses. The agonist properties of (+/-)-cyanopindolol and GR 127,935 observed in both second messenger assays suggests that a large degree of receptor reserve may be present, even though 5-HT1B receptor expression is low in OK cells. The OK cell line continues to serve as a model system to investigate 5-HT1B receptor-mediated signaling events.

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.

Pharmacological characterizations of recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes.

Recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D alpha cell line) and Y-1 adrenocortical tumor cells (1D beta cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated cAMP accumulation (FSCA). [3H]LSD saturation studies indicated that 5-HT1D receptor expression levels were slightly higher in the 1D beta cell line (Bmax = 1334 +/- 134 fmol/mg protein) than in the 1D alpha cell line (Bmax = 900 +/- 218 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC50 approximately 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine > or = 5-carboxamidotryptamine (5-CT) > LSD > or = 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), with Ki/EC50 ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT1D alpha and 5-HT1D beta receptors with apparent dissociation constants (Kb values) of 12 +/- 1 nM and 3 +/- 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D alpha cell line, these compounds behaved as partial agonists in the 1D beta cell line. To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D beta cell line whereas nearly linear relationships were observed for these two compounds in the 1D alpha cell line. Moreover, KA/EC50 ratios of these compounds were significantly larger in the 1D beta (10-32) as compared to the 1D alpha (0.9-2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger-receptor reserve in the 1D beta cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists. Although the potencies (EC50 values) of compounds matched their respective affinity constants (Ki values) for the closely-related 5-HT1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT1D alpha receptor relative to the human 5-HT1D beta subtype may be responsible for the higher intrinsic activities observed in the 1D alpha cell line, even though receptor reserve is apparently lower in this system.

Dual coupling of cloned human 5-hydroxytryptamine1D alpha and 5-hydroxytryptamine1D beta receptors stably expressed in murine fibroblasts: inhibition of adenylate cyclase and elevation of intracellular calcium concentrations via pertussis toxin-sensitive G protein(s).

The second messenger coupling of cloned human 5-hydroxytryptamine (5-HT)1D alpha and 5-HT1D beta receptors stably expressed in murine fibroblasts (LM (tk-)) was investigated. Clonal cell lines expressing similar receptor densities (Bmax = 750-950 fmol/mg) were used in this study. 5-HT (EC50 = 1.5-2.0 nM) and sumatriptan (EC50 = 6-14 nM), a selective 5-HT1D agonist, produced dose-dependent inhibition of forskolin-stimulated cAMP accumulation in intact cells transfected with the 5-HT1D alpha or 5-HT1D beta receptor gene. The maximal inhibitory responses elicited by these agonists were slightly greater with the 5-HT1D alpha receptor (approximately 90%) than the 5-HT1D beta receptor (approximately 80%). 5-HT (EC50 = 1.7-2.4 nM) and sumatriptan (EC50 = 8-18 nM) also evoked dose-dependent elevations in intracellular calcium concentrations ([Ca2+]i), with EC50 values that were indistinguishable from those for inhibition of forskolin-stimulated cAMP accumulation. Cells expressing 5-HT1D beta receptors displayed significantly larger 5-HT-induced increases in [Ca2+]i than did cells expressing 5-HT1D alpha receptors (206 nM versus 114 nm increase; p < 0.01). Dose-dependent elevations in inositol phosphates (IP) were also observed after application of 5-HT (EC50 = 29-54 nM) or sumatriptan (EC50 = 73-481 nM); the maximal increases in IP accumulation were modest (51-69%) for both 5-HT1D subtypes. In contrast to the cAMP and calcium responses, the concentration-response curves for IP accumulation were shifted to the right at least 10-fold. Methiothepin, a nonselective 5-HT1 antagonist, competitively antagonized the cAMP response, yielding an apparent dissociation constant (Kb) of 3-4 nM for the 5-HT1D receptors. Methiothepin (10 microM) significantly reduced the elevations in [Ca2+]i (> 90%) and IP (> 75%) evoked by saturating concentrations (1 microM) of agonists. All three functional responses were significantly attenuated (> 90%) by pretreatment with 100 ng/ml pertussis toxin. The sumatriptan-induced elevation of [Ca2+]i via activation of the 5-HT1D subtypes may provide a molecular mechanism of action by which sumatriptan could directly constrict cerebral blood vessels and alleviate migraine symptoms.

Pertussis toxin-sensitive guanine nucleotide-binding protein(S) couple adenosine A1 and 5-hydroxytryptamine1A receptors to the same effector systems in rat hippocampus: biochemical and electrophysiological studies.

Distinct membrane receptors that elicit similar cellular responses may share elements of signal transduction. In the present study, rat hippocampal adenosine (AD) and 5-hydroxytryptamine (5-HT) receptors were chosen to test this possibility using biochemical and electrophysiological techniques. Responses elicited by the AD receptor that mediates the inhibition of forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes and hyperpolarization of resting membrane potential (RMP) in rat hippocampal pyramidal cells were characterized and compared, in the same preparation, with those analogous responses elicited by the 5-HT1A receptor. A series of AD agonists including the selective AD A1 agonist (R)-phenylisopropyladenosine [(R)-PIA] inhibited forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes in a concentration-dependent manner. Cyclopentyltheophylline (CPT), a selective AD A1 antagonist, was a potent, competitive antagonist of this response with a dissociation constant (Kb) of 6 nM (Schild analysis). The rank order of agonist EC50 values and antagonist Kb values, as well as stereoselectivity, are consistent with the classification of this receptor as the AD A1 receptor. Spiperone, a potent 5-HT1A antagonist, competitively antagonized 5-HT-mediated inhibition of forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes with a Kb value of 14 nM. Intracellular recording techniques revealed that AD, (R)-PIA, 5-HT, and 5-carboxyamidotryptamine (5-CT) elicited concentration-dependent hyperpolarization of RMP within the same hippocampal pyramidal cell. The maximal hyperpolarization obtained for the AD or 5-HT analogs was the same for individual pyramidal cells. CPT and spiperone antagonized the hyperpolarization by (R)-PIA and 5-CT, respectively. Saturating concentrations of spiperone failed to antagonize (R)-PIA-mediated responses and CPT did not block responses elicited by 5-HT in either the biochemical or electrophysiological preparations. The combination of saturating concentrations of 5-HT and (R)-PIA evoked nonadditive biochemical responses relative to those observed with (R)-PIA alone. Similarly, electrophysiological experiments conducted under voltage-clamp conditions demonstrated that maximally effective concentrations of AD and 5-CT exhibited nonadditive behavior. Because the amount of outward current elicited when these agonists were coperfused was significantly less than the algebraic sum of the currents evoked individually by these agents, we infer that a population of AD A1 and 5-HT1A receptors activates a common pool of guanine nucleotide-binding proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of nicotine on beta-endorphin, alpha MSH, and ACTH secretion by isolated perfused mouse brains and pituitary glands, in vitro.

The effects of nicotine on secretion of the pituitary peptides beta-endorphin, alpha MSH, and ACTH were studied using the isolated perfused mouse brain (IPMB) and isolated superfused pituitaries of C3H mice. Nicotine (6.1 microM) stimulated secretion of beta-endorphin immunoreactivity from C3H IPMB approximately twofold. Secretion of alpha MSH immunoreactivity was stimulated approximately two- and sixfold by 6.1 microM and 12.2 microM nicotine, respectively. However, nicotine (6.1 microM) had no direct effect on the secretion of beta-endorphin, alpha MSH, or ACTH immunoreactivities from the isolated superfused pituitaries. The data suggest nicotine acts in the brain to stimulate pituitary secretion of alpha MSH and beta-endorphin. Electrocorticographic (ECoG) activity of the IPMB was monitored. Nicotine induced characteristic ECoG changes including a reduction of input voltage, a biphasic response of rapid desynchronization followed by prolonged synchronization, and seizure at high doses (12.2 microM).