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Query: UNIPROT:P06889 (Mol)
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The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT) binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high affinity [3H]5-HT binding is relatively insensitive to NEM. This study examines the NEM sensitivity of the various high affinity [3H]5-HT binding subtypes, using selective ligands, tissues, and pharmacological masks to study each subtype. Reconstitution of NEM-inactivated binding by addition of GTP-binding proteins (G proteins, Gi and Go) is also described. Agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites in rat brain and to 5-HT1A and 5-HT1D sites in bovine brain is sensitive to NEM. Binding of [3H]dihydroergotamine and [125I]iodocyanopindolol, both of which are weak partial agonists to 5-HT1B sites is relatively insensitive to NEM. Binding of [3H]5-HT to 5-HT1C sites in rat and bovine brain and choroid plexus is relatively insensitive to NEM. In the presence of spiperone to mask binding of 5-HT2 sites, binding of antagonist [( 3H]mesulergine) to 5-HT1C sites is also insensitive to NEM. Likewise, binding of the agonist [3H]4-bromo-2,5-dimethoxyphenylisopropylamine and of the antagonist [3H]ketanserin to 5-HT2 sites is not inhibited by NEM treatment of membranes. These findings suggest that agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites is sensitive to NEM alkylation. Binding of neither agonist nor antagonist to 5-HT1C and 5-HT2 sites is sensitive to NEM. Inability of high concentrations of a variety of ligands to protect the sensitive binding sites against NEM inactivation indicates that the critical sulfhydryl group(s) are not located in the ligand binding domain of the NEM-sensitive binding sites. When membranes are treated with NEM, displacement of [125I]iodocyanopindolol by 5-HT is no longer sensitive to 5'-guanylyl imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity of agonist displacement of 5-HT1B binding to NEM-treated membranes is restored by addition of purified guanine nucleotide binding proteins (Gi plus Go). In addition, NEM-inactivated binding to 5-HT1A and 5-HT1D sites can be restored by addition of Gi plus Go. These data suggest that NEM exerts its effects on 5-HT1A, 5-HT1B, and 5-HT1D binding sites by inactivating the G protein(s) associated with the 5-HT receptor subtypes.
Mol Pharmacol 1988 Oct
PMID:Differential inactivation and G protein reconstitution of subtypes of [3H]5-hydroxytryptamine binding sites in brain. 313 89

[3H]ICS 205-930 recognition sites were analyzed in membranes prepared from murine neuroblastoma N1E-115 cells. [3H]ICS 205-930 bound rapidly, reversibly, and stereoselectively to a homogeneous population of high affinity recognition sites: Bmax = 40 +/- 5 fmol/mg of protein, pKD = 9.20 +/- 0.05 (n = 11). Nonlinear regression and Scatchard analysis of saturation data suggested the existence of a single class of [3H]ICS 205-930 recognition sites on N1E-115 cells. The affinity of [3H]ICS 205-930 determined in kinetic studies was in agreement with that obtained under equilibrium conditions. Competition studies carried out with a large variety of agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. [3H]ICS 205-930-binding sites displayed the pharmacological profile of a 5-HT3 receptor. Potent 5-HT3 receptor antagonists showed nM affinities for [3H]ICS 205-930-binding sites with the following rank order of potency: SDZ 206-830 greater than SDZ 206-792 greater than ICS 205-930 greater than BRL 43694 greater than quipazine greater than BRL 24924 greater than MDL 72222 greater than GR 38032F. Methiothepine, mCPP, and metoclopramide showed sub-microM affinity. The rank order of potency of agonists was: 5-HT greater than phenylbiguanide = 2-methyl-5-HT much greater than 5-methoxytryptamine = 5-carboxamidotryptamine. All antagonist competition curves were steep (pseudo-Hill coefficients not lower than 1), monophasic, and best fit for a one-site model; 5-HT and 2-methyl-5-HT produced pseudo-Hill coefficients of 1.2-1.4. Drugs acting at 5-HT1, 5-HT2, alpha- and beta-adrenergic, dopaminergic, and histaminergic receptors (methysergide, ketanserin, propranolol, phentolamine, sulpiride, SCH 23390, cimetidine) were essentially inactive at 10 mumol/liter. The binding of [3H]ICS 205-930 was not affected by guanine and adenine nucleotides (GTP, GppNHp, and ATP) at 1 mmol/liter. These nucleotides did not affect the binding of agonists, suggesting that 5-HT3 recognition sites are not coupled to G-proteins. The interactions of agonists and antagonists with [3H]ICS 205-930 recognition sites were competitive in nature, as demonstrated by saturation experiments carried out with [3H]ICS 205-930 in the presence and the absence of unlabeled compounds: apparent Bmax values were not reduced, whereas apparent KD values were increased in the presence of competing ligands.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1988 Mar
PMID:Identification of serotonin 5-HT3 recognition sites in membranes of N1E-115 neuroblastoma cells by radioligand binding. 335 95

[3H]Ketanserin binding studies were performed on purified chromaffin granule membranes. Binding was found to occur on one class of sites and was temperature dependent. At 30 degrees the equilibrium dissociation constant KD was 45 nM. At 0 degrees, a KD value of 6 nM and a half-life of dissociation of 40 sec were measured. Methysergide, an antagonist of 5-hydroxytryptamine2 (5-HT2) receptors structurally unrelated to ketanserin, did not displace ketanserin binding. Tetrabenazine, an inhibitor of the monoamine transporter of chromaffin granules, displaced [3H]ketanserin binding. Conversely, ketanserin inhibited the binding of [3H] dihydrotetrabenazine, a ligand that specifically binds to the monoamine transporter. The inhibition was of the competitive type, indicating that both drugs bind to the same site. Ketanserin binding did not depend upon ATP-induced energization of chromaffin granules. ATP-dependent 5-HT uptake by chromaffin granule ghosts was inhibited by ketanserin with an IC50 value of 70 nM. A series of ketanserin derivatives were tested for their ability to displace [3H]dihydrotetrabenazine; EC50 values differed by more than 2 orders of magnitude and were not correlated to affinities on 5-HT2 receptors. In mouse brain, [3H]ketanserin was found to bind to methysergide-sensitive and to tetrabenazine-sensitive sites. In the striatum, tetrabenazine-sensitive sites represented a larger fraction than the methysergide-sensitive ones, whereas the reverse was true in the frontal cortex. It is concluded that nonspecific displaceable binding sites of [3H]ketanserin previously described in the striatum are tetrabenazine binding sites associated with the synaptic vesicle monoamine transporter.
Mol Pharmacol 1988 Jun
PMID:Ketanserin binds to the monoamine transporter of chromaffin granules and of synaptic vesicles. 338 81

The putative serotonin (5-HT) agonist RU 24969 [5-methoxy-3-1,2,3,6-tetrahydropyridin-4-yl)indole; 5-MeO-THPI] has been extensively used in the study and classification of 5-HT receptors. In order to study molecular determinants for recognition of THPIs at central 5-HT recognition sites, about 25 additional THPI derivatives were synthesized, incorporating, among others, 16 different indole-5-substituents and three different pyridine-N substituents in various combinations. Two saturated derivatives (piperidin-4-ylindoles) and two 2-methyl analogs were also included. Binding affinities at 5-HT1A, 5-HT2, and total 5-HT1 sites were obtained and the data were incorporated in quantitative structure-activity relationships (QSARs) using a combined linear free energy/molecular modeling approach. The QSAR analyses suggest distinct differences in the structural features that determine optimal potency at 5-HT1A sites versus those directing optimal potency for 5-HT2 sites. The parameter of the indole-5 substituent that almost exclusively determines potency for 5-HT1A sites is volume, the optimal size being about 24 cubic angstroms (calculated by fitting the activity versus volume data to a bilinear function). This is approximately the size of a carboxamide group. In contrast, at the 5-HT2 site both volume and hydrophobicity play major but opposing roles for the 5-substituent. A balance between the smallest possible volume and the greatest possible hydrophobicity is required for maximal 5-HT2 potency. Benzyl groups on the indole-1 or pyridyl-1 positions also favor potency at the 5-HT2 site (probably largely due to increased hydrophobic binding) while decreasing potency at the 5-HT1A site. A minor electronic contribution to the QSARs involving the charge on the indole 5-carbon is of opposite sign for 5-HT1A versus 5-HT2 sites and thus may also be useful for selective drug design. The data are consistent with the possibility that the indole and pyridyl rings are in a coplanar configuration when binding at both 5-HT1A and 5-HT2 sites, because the indole-2-methyl substituent, which provides a large energy barrier to the coplanar configuration, greatly reduces the potency of THPIs at both binding sites. Similarities in analog selectivity patterns suggest that the indolic portion of these compounds binds similarly to that of other indole derivatives such as tryptamines; thus, it is possible that optimally selective substituents predicted by these QSARs may be extrapolated to tryptamines and other indoles.
Mol Pharmacol 1988 Jul
PMID:Molecular determinants for recognition of RU 24969 analogs at central 5-hydroxytryptamine recognition sites: use of a bilinear function and substituent volumes to describe steric fit. 339 40

The trematode Schistosoma mansoni possesses an adenylate cyclase that is stimulated by serotonin. During development from the newly transformed schistosomulum to the adult stage, the serotonin-stimulated activity of adenylate cyclase increases. Our results show that the apparent affinity of the receptor for serotonin is the same at all stages tested. Serotonin receptors were characterized by testing the ability of agonists and antagonists to influence cyclase activity. The order of potency of antagonists is similar to that seen in Fasciola hepatica and is different from that characteristic of mammalian serotonin receptors. The nature of serotonin receptors in S. mansoni appears to be unchanged during development from cercaria to adult. Forskolin, which activates adenylate cyclase at the catalytic subunit, increases cyclase activity at all stages of development with no change in affinity. Forskolin and serotonin act synergistically to activate cyclase, and the degree of potentiation is the same (four-fold) at all stages of development, indicating that the coupling between the serotonin receptor and the catalytic component of cyclase is fully developed in the schistosomulum. The synergism between serotonin and forskolin is characterized by an increase in Vmax with no effect on the affinity for either serotonin or forskolin. The Ka for potentiation of serotonin action by forskolin is lower than the Ka for direct activation of cyclase by forskolin. The change in adenylate cyclase activity during development from schistosomulum to adult does not appear to be attributable to a change in the nature of the serotonin receptor, the catalytic component, or the coupling mechanism between these two components. Instead, there appears to be an increase in the total receptor-coupled enzyme system.
Mol Biochem Parasitol 1987 Nov
PMID:Nature of serotonin-activated adenylate cyclase during development of Schistosoma mansoni. 343 67

Neurons from rat superior cervical ganglia were grown in coculture with pineal cells. Action potentials of neurons in cocultures were 25% longer and were 25% greater in amplitude than those recorded from neurons grown in the presence of ganglionic nonneuronal cells alone. Neurons showed an increase in action potential duration with increasing time in culture. This may have been related to the recovery of nonneuronal cell populations after an initial exposure to the antimitotic agent Ara-C. In cultures not initially exposed to Ara-C, a subsequent exposure after 7 days in culture resulted in a shortening of the action-potential duration. Neuronal cultures were exposed to gel slabs containing the pineal indolamines, serotonin, N-acetylserotonin, and melatonin. Serotonin and N-acetylserotonin showed no effect on the neuronal action potentials at the concentrations tested. Melatonin caused an increase in action-potential duration that was associated not with an increase in action-potential amplitude, but with a decrease in action-potential rise rates. The effects of long-term exposure in melatonin appeared to be reversible in some cells but not in all. Short-term effects of melatonin were observed in older cultures and in younger cultures after the cells were stimulated repeatedly. Older cultures also had higher levels of spontaneous activity. The dependence of the short-term effects of melatonin on electrical activity may suggest a role for melatonin as a neuromodulator.
Cell Mol Neurobiol 1986 Dec
PMID:Effects of pineal factors on the action potentials of sympathetic neurons. 354 91

Histamine-N-methyltransferase, a major histamine-degrading enzyme in the skin, was purified from guinea pig skin about 150-fold. The enzymological characteristics including pH optimum, Km values for substrates, and molecular weight were almost consistent with those reported in the brain. Regulatory mechanism of the enzyme activity by biogenic amines was investigated using the purified specimen. Serotonin, tryptamine, and 5-methoxytryptamine intensely inhibited the activity while tryptophan, melatonin, N-acetylserotonin, tryptophol, and 5-hydroxyindole acetic acid had no significant effects. Dopamine, tyramine, 3-methyltyramine, and phenylethylamine also inhibited the activity while no particular effects were obtained by adrenaline, noradrenaline, tyrosine, and DOPA. Spermidine and cadaverine caused significant but weaker inhibition. These amines acted competitively with respect to histamine, although varying manners were observed with respect to S-adenosyl-L-methionine. From these results, it was concluded that the enzyme activity was inhibited by such compounds in which a certain chemical structure, CH2-CH2-NH2 group neighboring the hydrophobic group, was contained. A possible mechanism of inhibition by the amines is postulated, and possible roles of such compounds in the inflammation by impairing the histamine metabolism is discussed.
Exp Mol Pathol 1986 Dec
PMID:Regulation of the activity of histamine-N-methyltransferase from guinea pig skin by biogenic amines. 379 10

A novel type of serotonergic binding site, termed the 5-HT1c site, was recently identified on choroid plexus epithelial cells. In the present study, we describe the solubilization of pig choroid plexus 5-HT1c sites by the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS). High affinity labeling of both membrane-bound and solubilized 5-HT1c sites was obtained by use of the serotonergic radioligand N1-methyl-2-[125I]lysergic acid diethylamide (125I-MIL). In solubilized preparations, 125I-MIL exhibited a dissociation constant (Kd) of 2.0 nM and a 60-75% ratio of specific to total binding. Approximately 45% of the membrane binding sites were solubilized by CHAPS as measured by a postlabeling, polyethylene glycol precipitation method. The CHAPS-solubilized 5-HT1c site fulfilled the accepted criteria for receptor solubilization. The affinities of a series of serotonergic antagonists for the 5-HT1c site showed little or no change upon solubilization of the site. Serotonin, however, showed a 20-fold increase in affinity for the 5-HT1c site after solubilization, which may indicate the loss of a modulatory component during detergent treatment. Both gel filtration and equilibrium sedimentation experiments indicate that the CHAPS-solubilized site is a large molecular weight complex. These studies demonstrate that the pig choroid plexus 5-HT1c site can be solubilized with retention of its binding activity in a form suitable for further purification and characterization.
Mol Pharmacol 1986 Feb
PMID:Solubilization and characterization of the serotonin 5-HT1c site from pig choroid plexus. 395 27

A potential photoaffinity probe for the substrate-binding polypeptide of the neuronal serotonin uptake system has been synthesized. Under dark conditions, 3-(beta-(4-azidobenzamidino)ethyl-5-hydroxyindole (serotonin azidobenzamidine (SABA) was found to inhibit competitively [3H]5-hydroxytryptamine uptake by rat cortical synaptosomes with a K1 of 130 nM. The selectivity of this action was indicated by SABA's much lower potency as an inhibitor of synaptosomal [3H]norepinephrine uptake (K1 = 7 microM). When synaptosomes were irradiated in the presence of SABA, serotonin uptake was irreversibly inhibited in a concentration-dependent fashion with the maximum effect occurring at 1 microM SABA. At this concentration, approximately 40% of the serotonin uptake activity could not be recovered upon repeated washing of the synaptosomes. This inhibition was determined not to result from the production of a potent inhibitory photolysis product of SABA. The photoinactivation of serotonin transport by SABA was found to depend on the time of irradiation and could be prevented by the presence of agents that interact with the uptake system. Serotonin, p-chloroamphetamine, fenfluramine, and alaproclate protected the serotonin carrier against SABA's irreversible effects in a concentration-dependent manner. The presence of high concentrations of Tris or p-aminobenzoic acid, two nitrene-scavenging agents, did not reduce the level of photoinactivation of serotonin uptake by SABA, indicating that the irreversible inhibition is a result of true photoaffinity labeling of the carrier.
Mol Pharmacol 1985 Aug
PMID:Photoinactivation of serotonin uptake by an arylazido derivative of 5-hydroxytryptamine. 402 1

The effects of serotonin (5-HT) on membrane potential, membrane resistance, and select ionic currents were examined in large pedal neurons (LP1, LP3) of the mollusk Hermissenda. Calcium (Ca) action potentials were evoked in sodium-free artificial seawater containing tetramethylammonium, tetraethylammonium, and 4-aminopyridine (0-Na, 4-AP, TEA ASW). They failed at stimulation rates greater than 0.5/sec and were blocked by cadmium (Cd). Under voltage clamp the calcium current (ICa) responsible for them also failed with repeated stimulation. Thus, ICa inactivation accounts for refractoriness of the Ca action potential. The addition of 10 microM 5-HT to 0-Na, 4-AP, TEA ASW produced a slight depolarization and increased excitability and input resistance. Under voltage clamp the background current decreased. The voltage-dependent inward, late outward, and outward tail currents, sensitive to Cd, increased. ICa inactivation persisted. Under voltage clamp with Ca influx blocked by Cd, the addition of 10 microM 5-HT decreased the remaining current uniformly over membrane potentials of -10 to -100 mV. Thus, 5-HT reduces a background current that is active within the physiological range of the membrane potential, voltage insensitive, independent of Ca influx, noninactivating, and not blocked by 4-AP or TEA.
Cell Mol Neurobiol 1985 Dec
PMID:Serotonin decreases a background current and increases calcium and calcium-activated current in pedal neurons of Hermissenda. 408 49


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