Biogenic amine binding sites in rabbit spermatozoa

The binding of the neuroleptic, [3H] spiperone, to rabbit spermatozoa was investigated. Binding was partially inhibited by haloperidol, mianserin or epinephrine. Mixtures of two of the three inhibitors reduced binding further, but binding of the radioligand to spermatozoa was lowest in the presence of all three inhibitors. Unlabeled spiperone eliminated binding. Cholinergic ligands, GABA, and beta-adrenergic ligands, had little effect on binding. Spiperone binding occurred to both heads and tails suggesting that alpha 1-adrenergic, D2-dopamine and S2-serotonin binding sites are present in the head and/or tail of rabbit spermatozoa.     

Energetics of reserpine binding and occlusion by the chromaffin granule biogenic amine transporter

The energetics of reserpine binding to the bovine adrenal biogenic amine transporter suggest that H+ ion translocation converts the transporter to a form which binds reserpine essentially irreversibly. Reserpine binding to bovine adrenal chromaffin granule membrane vesicles is accelerated by generation of a transmembrane pH difference (delta pH) (interior acid) or electrical potential (delta psi) (interior positive). Both components of the electrochemical H+ potential (delta mu H+) must be dissipated to block reserpine binding, and generation of either one stimulates the binding rate. Reserpine binding is less dependent than amine transport on the delta pH, suggesting that translocation of fewer H+ ions is required to expose the high-affinity site than are required for net transport. Bound reserpine dissociates very slowly, if at all, from the transporter. Binding is stable to 1% cholate, 1.5% Triton X-100, 1 M SCN-, and 8 M urea, but sodium dodecyl sulfate (0.035%) and high temperatures (100 degrees C) released bound reserpine, indicating that binding is noncovalent. The results raise the possibility that the transporter, by translocating one H+ ion outward down its concentration gradient, is converted to a form that can either transport a neutral substrate molecule inward or occlude reserpine in a dead-end complex.     

Preferential adsorption of dopamine antagonist binding sites by fluphenazine-agarose

Separation of dopamine (DA) agonist and antagonist receptors was attempted by means of a covalently-bound fluphenazine-agarose (Flu-agarose). Incubation of striatal membranes with Flu-agarose resulted in loss of 3H-spiroperidol (3H-SPI) binding sites, while incubation with non-coupled agarose did not cause any change. The loss of 3H-SPI binding to the Flu-agarose treated membranes was not attributed to the release of fluphenazine from Flu-agarose as justified by several criteria. Flu-agarose adsorbed more effectively striatal membranes with 3H-SPI binding sites than those with 3H-DA binding sites. Following incubation of the membranes with Flu-agarose (2.5 ml beads/100 mg membrane protein), the density of 3H-SPI binding sites in the resulting membranes was reduced to 29%, whereas the density of 3H-DA binding sites to the same membranes was not changed. In addition, the potencies of DA antagonists to inhibit 3H-N-propylnorapomorphine binding to the membranes were decreased more than a hundred times, while the potencies of DA agonists were little affected. These results suggest that in the striatal membranes exist at least two populations of DA receptors.     

Simulation of biogenic amine metabolism in the brain

The metabolism of a number of biogenic amines has been simulated by using data obtained from studies of the individual enzymes from pig brain. It is shown that beta-hydroxylated amines such as noradrenaline and octopamine are metabolized primarily to the alcoholic metabolite whereas amines lacking this group [e.g. dopamine (3,4-dihydroxyphenethylamine) and 5-hydroxytryptamine] are metabolized at low concentrations to give the corresponding acid. Increase in the amine concentration results in an increase in the proportion of the alcoholic metabolite formed and this may in part account for the effects of the drug reserpine on amine metabolism. The effects of disulfiram (Antabuse) and ethanol (acting through its metabolite acetaldehyde) on amine metabolism may be understood in terms of this simulated model. It is shown that drugs that affect this system also cause alterations in the steady-state concentrations of the intermediate aldehydes and the possible implications of this are discussed.     

Advances in sodium-ion coupled biogenic amine transporters.

The sodium-ion coupled transporters for 5-hydroxytryptamine (5HT), noradrenaline and dopamine function to reduce extracellular levels of biogenic amines. Over the past fifteen years selective inhibitors of these transport systems have been developed including fluoxetine, citalopram, paroxetine, litoxetine (for 5HT), nisoxetine, desipramine, maprotiline (for noradrenaline) and GBR-12935 (for dopamine). Some of these inhibitors, including drugs selective for noradrenaline transport and particularly those selective for the 5HT transport system are currently widely used in the clinical management of affective disorders. Selective biogenic amine uptake inhibitors have, in addition, provided tools to undertake molecular pharmacological and biochemical studies of their respective transporters. By this means, the rat brain 5HT and dopamine transporters have been identified as polypeptides with relative molecular masses of 73,000 and 80,000, respectively, using affinity-chromatographic purification and photoaffinity-labelling techniques. Recently, the biogenic amine transporters have been cloned and a comparison of their predicted amino acid sequences reveals that these proteins share a considerable degree of similarity with notably 12-13 transmembrane spanning domains. Perspectives for future fundamental and clinical research on biogenic amine transport systems using molecular biological techniques are discussed.     

Vipoxin. A protein from Russell's viper venom with high affinity for biogenic amine receptors

We have purified a small, basic protein with high affinity and selectivity for biogenic amine receptors to apparent homogeneity from the venom of Russell's viper (Vipera russelli). This protein, which we designate "vipoxin," has Mr = 13,000, and appears to exist in solution as a single polypeptide chain. It may contain 2 atypical amino acids. Vipoxin inhibits in a dose-dependent manner the binding of 3H-ligands to biogenic amine receptors, with apparent Ki values of 3 nM at alpha 1-adrenergic receptors, 5 nM at alpha 2-adrenergic receptors, 15 nM at dopamine receptors, and 32 nM at serotonin receptors. At concentrations up to 1 microM, vipoxin is inactive at beta-adrenergic, histamine, nicotinic cholinergic, muscarinic cholinergic, adenosine, gamma-aminobutyric acid, benzodiazepine, or opiate receptor binding sites. The effect of vipoxin is essentially irreversible over 20 h at alpha 1- and alpha 2-adrenergic receptors and serotonin receptors and is only slightly reversible at dopamine receptors. Norepinephrine protects alpha-adrenergic receptors from inhibition by vipoxin, while dopamine does not. Vipoxin has no protease activity but does have phospholipase A2 activity, which cannot account for its action on receptors, since receptor binding is assayed in the presence of 1 mM CoSO4 which completely and selectively inhibits the phospholipase activity. Other phospholipases A2 in the same venom lack vipoxin's action on receptors. In physiologic experiments, vipoxin behaves as an agonist at alpha 2-adrenergic receptors in the rat vas deferens and is over an order of magnitude more potent than norepinephrine itself. At alpha 1-adrenergic receptors, it is neither a simple agonist nor an antagonist, but selectively potentiates norepinephrine. Vipoxin may be a useful tool for biogenic amine receptor characterization.     

Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine.

Antagonism of glutamate-receptor responses activated by N-methyl-D-aspartic acid (NMDA) was studied using whole cell voltage clamp recording from mouse dissociated hippocampal neurons cultured for 10-15 d. The kinetics of onset of and recovery from NMDA receptor block during continuous application of NMDA together with either glycine, or L-alanine, were recorded in response to concentration jump application of NMDA- and glycine-binding site directed competitive antagonists, applied with a multibarrel flow pipe under conditions which allowed rapid solution changes around the cell less than 10 ms. Mathematical solutions for both one- and two-equivalent site models for competitive antagonism were determined according to the differential equations outlined by Colquhoun and Hawkes (1977. Proc. R. Soc. Lond. B. 199:231-262). The kinetics of action of D-CPP and D-AP5, NMDA binding site antagonists, and 7Cl-kynurenic acid, a glycine binding site antagonist, were examined for each model. For all these antagonists, the kinetics for the onset of and recovery from antagonism were better fit by the two-equivalent site model, which yielded antagonist microscopic kBoff/kBon values which closely approximated Ki values determined from analysis of equilibrium dose response curves. These results suggest that two molecules of NMDA and two molecules of glycine must bind to the NMDA receptor for activation of ion channel gating.     

LIM homeobox gene-dependent expression of biogenic amine receptors in restricted regions of the C. elegans nervous system

Biogenic amines regulate a variety of behaviors. Their functions are predominantly mediated through G-protein-coupled 7-transmembrane domain receptors (GPCR), 16 of which are predicted to exist in the genome sequence of the nematode Caenorhabditis elegans. We describe here the expression pattern of several of these aminergic receptors, including two serotonin receptors (ser-1 and ser-4), one tyramine receptor (ser-2), and two dopamine receptors (dop-1 and dop-2). Moreover, we describe distinct but partially overlapping expression patterns of different splice forms of the ser-2 tyramine receptor locus. We find that each of the aminergic receptor genes is expressed in restricted regions of the nervous system and that many of them reveal significant overlap with the expression of regulatory factors of the LIM homeobox (Lhx) gene family. We demonstrate that the expression of several of the biogenic amine receptors is abrogated in specific cell types in Lhx gene mutants, thus establishing a role for these Lhx genes in regulating aspects of neurotransmission. We extend these findings with other cell fate markers and show that the lim-4 Lhx gene is required for several but not all aspects of RID motor neuron differentiation and that the lim-6 Lhx gene is required for specific aspects of RIS interneuron differentiation. We also use aminergic receptor gfp reporter fusions as tools to visualize the anatomy of specific neurons in Lhx mutant backgrounds and find that the development of the elaborate dendritic branching pattern of the PVD harsh touch sensory neuron requires the mec-3 Lhx gene. Lastly, we analyze a mutant allele of the ser-2 tyramine receptor, a target of the ttx-3 Lhx gene in the AIY interneuron class. ser-2 mutants display none of the defects previously shown to be associated with loss of AIY function.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Molecular and pharmacological properties of insect biogenic amine receptors: lessons from Drosophila melanogaster and Apis mellifera

In the central nervous system (CNS) of both vertebrates and invertebrates, biogenic amines are important neuroactive molecules. Physiologically, they can act as neurotransmitters, neuromodulators, or neurohormones. Biogenic amines control and regulate various vital functions including circadian rhythms, endocrine secretion, cardiovascular control, emotions, as well as learning and memory. In insects, amines like dopamine, tyramine, octopamine, serotonin, and histamine exert their effects by binding to specific membrane proteins that primarily belong to the superfamily of G protein-coupled receptors. Especially in Drosophila melanogaster and Apis mellifera considerable progress has been achieved during the last few years towards the understanding of the functional role of these receptors and their intracellular signaling systems. In this review, the present knowledge on the biochemical, molecular, and pharmacological properties of biogenic amine receptors from Drosophila and Apis will be summarized. Arch.     

Agonist and antagonist binding to tachykinin peptide NK-2 receptors

The binding of tachykinin peptides and fragments to NK-2 receptor sites in hamster urinary bladder membranes was examined and compared to binding to NK-1 receptor sites in rat submandibular gland. Neurokinin A (NKA) and its C-terminal fragments bound with highest NK-2 affinity and selectivity. N-terminal fragments of NKA did not bind to either type of receptor. Kassinin and eledoisin were NK-2 selective while physalaemin, phyllomedusin, and uperolein were NK-1 selective. Of fifteen tachykinin antagonists examined, none exhibited appreciable affinity or selectivity (relative to agonists) for NK-1, NK-2, or rat cerebral cortical NK-3 receptor sites. NKA binding to NK-2 sites was stimulated by Mn++ greater than Mg++ greater than Ca++. At the optimal concentration, the Mn++ stimulation was due to both an increased Bmax and increased affinity. The nonhydrolyzable guanine nucleotide, GppNHp, reduced agonist binding but not antagonist binding to NK-2 receptor sites. The nucleotide effect was due to a reduction in both Bmax and affinity and was potentiated by Mn++. The results indicate that tachykinin NK-2 receptor sites possess distinct structural requirements for agonists and are linked to a G-protein coupling system.     

Differentiation by ascorbic acid of dopamine agonist and antagonist binding sites in striatum

Ascorbic acid, which is usually included in the incubation mixture when studying dopamine (DA) receptor binding sites, inhibits the specific binding of the agonists ADTN and apomorphine but not the antagonists haloperidol and spiroperidol. Other reducing agents have a similar action. This observation is consistent with the notion that DA agonists and antagonists sites are separate and distinct. Moreover, they suggest the possibility that the number of agonist sites $\text{in vivo}$ may be modulated, in part, by the state of oxidation of the binding sites.     

Mapping of the leptin binding sites and design of a leptin antagonist

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.     

Current status of the biogenic amine theory of depression

An attempt has been made to assess critically the clinical and experimental evidence that implicates a malfunctioning of amine neurotransmitter systems in the aetiology of depression. The evidence available does provide indirect evidence in favour of the biogenic amine theory of depression. However, emphasis is now being placed more on the abnormal responsiveness of the amine receptors, particularly the noradrenergic receptors, to the presynaptic release of the neurotransmitter rather than on the mechanisms governing the synthesis per se.     

Orthosteric and allosteric binding sites of P2X receptors

P2X receptors for ATP comprise a distinct family of ligand gated ion channels with a range of properties. They have been shown to be involved in a variety of physiological processes including blood clotting, sensory perception, pain sensation, bone formation as well as inflammation and may provide a number of novel drug targets. In addition to the orthosteric site for ATP binding it has been suggested that there may be additional allosteric sites that regulate agonist action at the receptor. There is currently no crystal structure available for P2X receptors and the lack of sequence similarity to other ATP binding proteins has meant that a mutagenesis-based approach has been used primarily to investigate receptor structure-function. This review aims to provide an overview of recent work that gives an insight into residues involved in ATP action and allosteric regulation.