Specific Receptor-Mediated Inhibition of Cyclic AMP Synthesis by Dopamine in a Neuroblastoma × Brain Hybrid Cell Line NCB-20

Abstract: Dopamine and dopamine receptor agonists were found to inhibit adenylate cyclase activity dose-de-β ndently in a neuroblastoma × Chinese hamster brain explant hybrid cell line NCB-20. Apomorphine (with an IC₅₀ value of 10 n M ) was the most effective inhibitor, followed by 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydro-naphthaline (ADTN), dopamine, and N -dipropyldopa-mine. The inhibition was potently reversed by sulpiride, butaclamol, and flupenthixol in a stereospecific manner, but was unaffected by yohimbine, except at high concentrations. Clonidine also inhibited adenylate cyclase activity in these cells and this was reversed by the α₂-adrenoreceptor antagonist yohimbine, but not by sulpiride. [ d -Ala², d -Leu⁵]Enkephalin inhibited adenylate cyclase activity in NCB-20 cells at nanomolar concentrations; this was reversed by naloxone. All three inhibitory neurotransmitters were able to reverse the stimulation of cyclic AMP synthesis by serotonin or prostaglandin E₁The dopamine receptor that modulates cyclic AMP synthesis in NCB-20 cells is pharmacologically quite distinct from a high-affinity spiperone binding site identified in these cells, but shows the pharmacologic specificity of the D₂ receptor previously described in mammalian brain.     

Secretin Receptors on Neuroblastoma Cell Membranes: Characterization of 125I-Labeled Secretin Binding and Association with Adenylate Cyclase

Secretin, a gut-brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin greater than vasoactive intestinal peptide greater than peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I-labeled secretin to the neuroblastoma plasma membranes was saturable, time-dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5'-(beta, gamma-imino)-triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.     

Possible Role of Gangliosides in Regulating an Adenylate Cyclase-Linked 5-Hydroxytryptamine (5-HT1) Receptor

Cultured NCB-20 hybrid cells express adenylate cyclase-coupled receptors for 5-hydroxytryptamine (5-HT) that correspond biochemically and pharmacologically to 5-HT1 receptors in rodent brain membrane preparations, apart from a much-reduced affinity for 5-HT (160 nM compared to less than 5 nM in brain). Since NCB-20 cells also differ from rodent brain both qualitatively and quantitatively in their ganglioside composition, the effects of exogenously added gangliosides on the affinity of the 5-HT1 receptor for 5-HT were tested. Both GM1 ganglioside (the cholera toxin receptor) and tetrasialoganglioside GQ1b produced a 10-fold increase in receptor affinity for [3H]5-HT, measured by binding studies. All gangliosides, at submicromolar concentrations, resulted in significantly reduced EC50 values for 5-HT-mediated elevation of intracellular cyclic AMP levels. GQ1b had the capacity to most dramatically enhance the potency of 5-HT in mediating increases in cyclic AMP levels. Gangliosides had no effect on the potency of DADLE or 3,4-dihydroxyphenylethylamine (dopamine)-mediated depression of cyclic AMP levels, suggesting some specificity for 5-HT. Our data are interpreted as implying a specific role for polysialogangliosides in modulating the affinity of the 5-HT1 receptor and the coupling of the 5-HT1 receptor-guanine nucleotide binding protein adenylate cyclase complex.     

Cell-Free Desensitization of Opioid Inhibition of Adenylate Cyclase in Neuroblastoma × Glioma NG108–15 Hybrid Cell Membranes

When membranes from neuroblastoma X glioma NG108-15 hybrid cells were incubated in a cell-free system with opioid agonists, a time-, temperature-, and dose-dependent desensitization to opioid inhibition of adenylate cyclase activity was observed. The composition of the system during the incubation was manipulated to elucidate the biochemical mechanisms of desensitization. Receptor coupling appeared to be a prerequisite for desensitization, because both magnesium and sodium, which are necessary for coupling, were required for desensitization. Removal of ATP and addition of cyclic AMP or cyclic GMP had no effect on desensitization.     

Detection and Characterization of the Serotonin 5-HT1D Receptor in Rat and Human Brain

In the presence of 1 microM ( +/- )-pindolol [to block 5-hydroxytryptamine (5-HT, serotonin) 5-HT 1A and 5-HT 1B receptors] and 100 nM mesulergine (to block 5-HT 1C receptors), 2.0 nM [3H]5-HT binding to rat cortical homogenates is specific, saturable, and reversible. Scatchard analysis of [3H]5-HT binding, in the presence of 1 microM ( +/- )-pindolol and 100 nM mesulergine, produced a KD of 3.2 nM and Bmax of 43 fmol/mg protein. Distribution studies show this site to be present in most rat brain regions. This site is also detectable in human caudate. The pharmacological profile of this site is distinct from the previously identified 5-HT receptor subtypes. Compounds with high affinity for 5-HT 1A (8-hydroxydipropylaminotetralin), 5-HT 1B (trifluoromethylphenylpiperazine), 5-HT 1C (mesulergine), 5-HT 2 (4-bromo-2,5-dimethoxyphenylisopropylamine), and 5-HT3 (ICS 205-930) receptors have low affinity for this site. These data suggest the presence of an additional, previously unidentified, 5-HT binding site in rat and human brain tissue. This putative novel 5-HT receptor has a similar pharmacology to the "5-HT 1D" site detected in bovine brain by Heuring and Peroutka.     

Chronic Activation of Inhibitory δ-Opioid Receptors Cross-Regulates the Stimulatory Adenylate Cyclase-Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108-15) Hybrid Cells

Abstract: The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E₁ (PGE₁) receptor system in neuroblastoma × glioma (NG108-15) hybrid cells. Persistent activation of δ-opioid receptors by morphine (10 µmol/L; 3 days) substantially down-regulates the number of PGE₁ binding sites by ～30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTPγS (100 µmol/L) further revealed that the remaining PGE₁ binding sites are still capable of interacting functionally with their associated stimulatory G proteins, G_s. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the α subunit of G_s (G_sα) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc^? reconstitution experiments, respectively. Evaluation of the functional interaction between PGE₁ receptors and G_s by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of G_sα revealed a significant increase in the ability of PGE₁ receptors to activate G_sα (3.3-fold increase in EC₅₀; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 µmol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 µmol/L) had no further effect on sensitization in PGE₁ receptor/G_s coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE₁ receptor system represents a potential target of chronic δ-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.     

Native Serotonin 5-HT3 Receptors Expressed in Xenopus Oocytes Differ from Homopentameric 5-HT3 Receptors

Abstract: Efficacies of the 5-hydroxytryptamine (serotonin) 5-HT₃ receptor (5-HT₃R) agonists 2-methyl-5-HT, dopamine, and m -chlorophenylbiguanide on 5-HT₃R native to N1E-115 cells and on homopentameric 5-HT₃R expressed in Xenopus oocytes were determined relative to that of 5-HT. Efficacies of 2-methyl-5-HT and dopamine on 5-HT₃R native to differentiated N1E-115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5-HT₃R-A_L and 5-HT₃R-A_s receptors expressed in oocytes (4–8%). m -Chlorophenylbiguanide does not distinguish between 5-HT₃R in N1E-115 cells and in oocytes. The distinct pharmacological profile of 5-HT₃R native to differentiated N1E-115 cells is conserved when poly(A)⁺ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5-HT₃R subunits, N1E-115 cells express additional proteins involved in 5-HT₃R function.     

Serotonin 5-HT2C Receptor RNA Editing Alters Receptor Basal Activity

Rat and human serotonin 5-HT2C receptor isoforms were evaluated for agonist-independent activation of inositol phosphate production in COS-7 cells. The nonedited isoform (5-HT(2C-INI)) displayed the greatest basal activity, stimulating inositol phosphate production fourfold over the fully edited isoform (5-HT(2C--VGV)). All of the other isoforms tested displayed intermediate levels of basal activity. Decreasing receptor expression levels by 50% produced a parallel decrease in basal activity. 5-HT stimulated inositol phosphate production twofold over basal levels through the 5-HT(2C-INI) receptor and eightfold over basal levels through the 5-HT(2C-VGV) receptor but produced similar maximal levels of inositol phosphate. 5-HT competition for [3H]mesulergine binding to 5-HT(2C-INI) best fit a two-site analysis with K(H) = 7.6 nM and K(L) = 160 nM, whereas 5-HT(2C-VGV) best fit a one-site model with Ki = 163 nM. [3H]5-HT labeled 36% of the total population of 5-HT(2C-INI) receptors labeled by [3H]mesulergine but only 12% of 5-HT(2C-VGV) receptors. [H]5-HT K(D) values increased from 5.1 nM for 5-HT(2C-INI) to 20 nM for 5-HT(2C-VGV). [3H]Mesulergine K(D) values were the same for both isoforms. 5-HT EC50 values for inositol phosphate production increased from 6.1 nM for 5-HT(2C-INI) to 30 nM for 5-HT(2C-VGV). These results demonstrate that RNA editing decreases 5-HT2C receptor basal activity, agonist affinity, and potency, indicating that RNA editing may play a role in regulating serotonergic signal transduction and response to drug therapy.     

Characterization of a Mouse Serotonin 5-HT3 Receptor Purified from Mammalian Cells

Abstract: A serotonin 5-HT₃ receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding ∼50% of the histidine-tagged 5-HT₃ receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of ∼5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT₃ receptor is a pentameric homooligomer. The secondary structure of the 5-HT₃ receptor as determined by circular dichroism appeared to consist of mainly α-helices (50%) and β-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.     

Cloning, Characterization, and Chromosomal Localization of a Human 5-HT6 Serotonin Receptor

Abstract: We describe the cloning and characterization of a human 5-HT₆ serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5-HT₆ cDNA encodes a 440-amino-acid polypeptide whose sequence diverges significantly from that published for the rat 5-HT₆ receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5-HT₆ amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5-HT₆ receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5-HT_1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5-HT₆ receptor also reveals an Rsa I restriction fragment length polymorphism within the coding region.     

Differential Membrane Targeting and Pharmacological Characterization of Chimeras of Rat Serotonin 5-HT1A and 5-HT1B Receptors Expressed in Epithelial LLC-PK1 Cells

Abstract: The serotonin 5-HT_1A and 5-HT_1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT_1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT_1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT_1A and 5-HT_1B receptors still able to bind specifically [³H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT_1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT_1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT_1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT_1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).     

Cloning and Expression of a Human Serotonin 5-HT4 Receptor cDNA

Abstract: Using a combination of library screening and nested PCR based on a partial human serotonin 5-HT₄ receptor sequence, we have cloned the complete coding region for a human 5-HT₄ receptor. The sequence shows extensive similarity to the published porcine 5-HT_4A and rat 5-HT_4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5-HT₄ sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5-HT₄ receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5-HT₄ receptor, transiently expressed in COS-7 cells, showed radioligand binding properties similar to 5-HT₄ receptors in guinea pig striatal tissue. [³H]GR 113808 revealed K _D values of 0.15 ± 0.01 n M for the human receptor and 0.3 ± 0.1 n M in the guinea pig tissue. Binding constants were determined for four investigated 5-HT₄ antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT₄-specific agonists caused an increase in cyclic AMP levels.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Lasting Increase in Serotonin 5-HT1A but Not 5-HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5-HT) release and uptake in hippocampal synaptosomes and 5-HT_1A and 5-HT₄ receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8-[³H]hydroxy-2-(di- n -propylamino)tetralin ([³H]8-OH-DPAT) to 5-HT_1A receptors was selectively increased by 20% on average ( p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average ( p < 0.05) 1 week after stage 5 (tonic-clonic seizures) compared with sham-stimulated rats. A 20% increase ( p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5-HT₄ receptors, which colocalize with 5-HT_1A receptors on hippocampal neurons, were not modified in kindled tissue. [³H]5-HT uptake and its release as well as the 5-HT_1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg⁻¹ 8-OH-DPAT or 1,000 µg kg⁻¹ WAY-100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5-HT_1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Detection of a Novel Serotonin Receptor Subtype (5-HT1E) in Human Brain: Interaction with a GTP-Binding Protein

[3H]Serotonin (5-hydroxytryptamine, [3H]5-HT) was used as a radioligand probe of brain 5-HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 microM pindolol (to block 5-HT1A and 5-HT1B receptors), and 100 nM mesulergine (to block 5-HT1C and 5-HT2 receptors). One of these sites demonstrated high affinity for 5-carboxyamidotryptamine (5-CT) and ergotamine, consistent with the known pharmacology of the 5-HT1D receptor; the second site demonstrated low affinity for 5-CT and ergotamine. Computer-assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5-CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5-CT and ergotamine, respectively) for 45% of the sites. To investigate the non-5-HT1D component of the binding, 100 nM 5-CT (to block 5-HT1A, 5-HT1B, and 5-HT1D receptors) was coincubated with [3H]5-HT, membranes, and mesulergine. The remaining [3H]5-HT binding (hereafter referred to as "5-HT1E") displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5-CT and ergotamine displayed the highest selectivity for the 5-HT1D site versus the 5-HT1E site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine Brain Coated Vesicles Contain Adenosine A1 Receptors. Presence of Adenylate Cyclase Coupled to the Receptor

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.     

Identification of Residues in Transmembrane Regions III and VI that Contribute to the Ligand Binding Site of the Serotonin 5-HT6 Receptor

Abstract: We have examined the ligand binding site of the serotonin 5-HT₆ receptor using site-directed mutagenesis. Replacing the highly conserved Asp¹⁰⁶ in transmembrane region III by asparagine eliminated d -[³H]lysergic acid diethylamide ([³H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5-HT and LSD to stimulate adenylyl cyclase was reduced by 3,600- and 500-fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5-HT and D106 is essential for high-affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp¹⁰²) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy-terminal half of transmembrane region VI (Ala²⁸⁷ and Asn²⁸⁸) for leucine and serine resulted in a mutant receptor with increased affinities (seven- to 30-fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5-HT₆ receptor ligand binding site.     

Cross-Talk of the Receptors for Bradykinin, Serotonin, and ATP Shown by Single Cell Ca2+ Responses Indicating Different Modes of Ca2+ Activation in a Neuroblastoma × Glioma Hybrid Cell Line

Abstract: Modes of Ca²⁺ activation by bradykinin, serotonin, and ATP and the possible receptor cross-talk were investigated in mouse neuroblastoma × rat glioma hybrid cells (108CC15) by monitoring fura-2 fluorescence in single cells. A transient rise of cytosolic Ca²⁺ activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca²⁺ stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca²⁺-free medium abolished the Ca²⁺ response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, Induces the release of Ca²⁺ from intracellular stores. Serotonin, in contrast, activates Ca²⁺ entry from the extracellular space. To investigate whether ATP releases Ca²⁺ from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca²⁺. The application of ATP before that of bradykinin reduced the size of a following bradykinin-induced Ca²⁺ response in Ca²⁺-free medium, but not in Ca²⁺-containing medium. This suggests that bradykinin may interact with the ATP-activated mechanism by cross-desensitization. Possibly, bradykinin receptors are coupled to additional Ca²⁺ stores not accessible to ATP that are refilled by extracellular Ca²⁺. Cyclic AMP and cyclic GMP apparently do not affect the Ca²⁺ responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.