Biomedical subjects
L L Iversen
Publications and source records attributed to L L Iversen.
Molecular cloning of a serotonin receptor from human brain (5HT1E): a fifth 5HT1-like subtype.
Degenerate primers, suitable for polymerase chain reaction studies and based on the conserved structure of G protein-coupled receptors, were used to isolate cDNA clones encoding putative G protein-coupled receptors from a human hippocampal cDNA library. One clone isolated by this approach (AC1) encoded a putative receptor with 39% amino acid sequence identity to the serotonin 5HT1A receptor and 47% identity to the 5HT1D receptor. When expressed transiently in the human embryonic kidney cell line 293, AC1 cDNA-encoded receptor displayed high affinity (Kd = 15 nM) and saturability for [3H]serotonin, suggesting that AC1 encodes a 5HT1-like receptor. However, 5-carboxamidotryptamine demonstrated low affinity (pKi = 5.15) compared with serotonin (pKi = 8.14), consistent with the observed binding of the putative 5HT1E receptor. The excellent correlation observed between the pharmacology of the expressed receptor encoded by AC1 and the human brain 5HT1E binding site confirms that AC1 encodes a 5HT1E receptor and establishes a fifth 5HT1-like receptor subtype.
L-687,306: a functionally selective and potent muscarinic M1 receptor agonist.
The oxadiazole L-687,306 is a high affinity muscarinic agonist with a N-methylscopolamine/oxotremorine-M binding profile predictive of a partial agonist. L-687,306 showed marked selectivity in functional pharmacological assays. L-687,306 was a partial agonist at muscarinic M1 receptors in the rat ganglion but a high affinity competitive antagonist at guinea-pig cardiac M2 and ileal M3 muscarinic receptors. This compound gives an opportunity to study receptor reserve involved in muscarinic receptors in vitro and in vivo.
Autoradiographic assessment of the effects of N-methyl-D-aspartate (NMDA) receptor antagonists in vivo.
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Pharmacological approaches to the treatment of ischaemic neuronal damage.
Retina is particularly susceptible to ischaemic damage following exposure to anoxia or hypoglycaemia. In animal models the neuronal damage following ischaemia resembles that caused by exposure to glutamate or other excitotoxic agents which act on excitatory amino acid receptors. There are a number of pharmacological approaches designed to limit neuronal damage following ischaemia. These include free radical scavenging agents, calcium channel blockers, kappa opiate agonists and excitatory amino acid antagonists. Recent studies with antagonists acting at both NMDA and non-NMDA receptors for glutamate show that such compounds can protect against ischaemic damage, and are effective even when administered several hours after the ischaemic insult.
Cholecystokinin receptors: synthetic antagonists with selectivity for receptor subtypes and possible clinical applications.
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Pharmacological specificity of novel, synthetic, cyclic peptides as antagonists at tachykinin receptors.
1. The interaction at tachykinin receptors of a series of novel cyclic hexapeptides has been examined by use of radioligand binding assays (NK1 and NK3 sites in rat cortex, NK2 sites in hamster urinary bladder) and functional pharmacological assays (guinea-pig ileum, rat vas deferens and rat portal vein for NK1, NK2 and NK3 receptors, respectively). 2. The compounds cyclo(GlnTrpPhe(R)Gly[ANC-2]LeuMet) (L-659,837) and cyclo(GlnTrpPheGly-LeuMet) (L-659,877) were powerful and selective displacers of NK2 binding (pIC50 6.9 and 8.0, respectively), and were competitive antagonists of responses to stimulation of NK2 receptors in rat vas deferens (pKB for antagonism of responses to eledoisin 6.7 and 8.1, respectively). Responses in the NK1 and NK3 pharmacological assays were blocked only weakly, if at all. 3. In the longitudinal muscle of the small intestine of the rat, responses to stimulation of the putative NK2 receptor by eledoisin, neurokinin A or neurokinin B were antagonized by both cyclo(GlnTrpPhe(R)-Gly[ANC-2]LeuMet) and cyclo (GlnTrpPheGlyLeuMet) in a manner consistent with the presence in this tissue of a uniform population of receptors, indistinguishable from the NK2 receptor of the rat vas deferens. 4. The compounds cyclo(GlnTrpPheGlyLeuMet) and the lactam-containing analogue are among the most selective antagonists for the NK2 receptor that have been described; their availability should be of value in the characterization of the receptors mediating responses to tachykinins, and in elucidating the physiological functions of the tachykinin receptors.
Phencyclidine, dizocilpine, and cerebrocortical neurons.
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Novel quinuclidine-based ligands for the muscarinic cholinergic receptor.
Recent clinical studies on Alzheimer's patients have implied that only agents displaying high efficacy at the cortical muscarinic receptor have yielded encouraging results. This paper describes the design, synthesis, and biochemical characterization of novel quinuclidine-based muscarinic agonists which can readily penetrate into the central nervous system and which are capable of displaying high efficacy at cortical sites. With use of a biochemical assay capable of measuring receptor affinity and predicting cortical efficacy, it has been discovered that an oxadiazole ring and related heterocycles can function as bioisosteric replacements for the ester moiety found in several known muscarinic ligands. Within this series there exist compounds which span the efficacy range from high-efficacy agonist through partial agonists to antagonists with affinity comparable or superior to that of classical quaternary ammonium ligands. Consistent with recent molecular biology studies, structure-activity trends are interpreted in terms of separate binding sites for agonists and antagonists with H-bonding interactions characterizing agonist behavior and lipophilic binding characterizing antagonist behavior. Thus the aminooxadiazole moiety has structural features which are optimized for an agonist profile.
Enantiomers of HA-966 (3-amino-1-hydroxypyrrolid-2-one) exhibit distinct central nervous system effects: (+)-HA-966 is a selective glycine/N-methyl-D-aspartate receptor antagonist, but (-)-HA-966 is a potent gamma-butyrolactone-like sedative.
The antagonist effect of (+/-)-3-amino-1-hydroxypyrrolid-2-one (HA-966) at the N-methyl-D-aspartate (NMDA) receptor occurs through a selective interaction with the glycine modulatory site within the receptor complex. When the enantiomers of (+/-)-HA-966 were resolved, the (R)-(+)-enantiomer was found to be a selective glycine/NMDA receptor antagonist, a property that accounts for its anticonvulsant activity in vivo. In contrast, the (S)-(-)-enantiomer was only weakly active as an NMDA-receptor antagonist, but nevertheless it possessed a marked sedative and muscle relaxant action in vivo. In radioligand binding experiments, (+)-HA-966 inhibited strychnine-insensitive [3H]glycine binding to rat cerebral cortex synaptic membranes with an IC50 of 12.5 microM, whereas (-)-HA-966 had an IC50 value of 339 microM. In electrophysiological experiments, (+)-HA-966 selectively antagonized NMDA receptor responses in rat cortical slices, whereas the (-)-enantiomer was much weaker. On cultured cortical neurones (+)-HA-966 inhibited glycine-potentiated NMDA responses with an IC50 = 13 microM compared with (-)-HA-966, which has an IC50 = 708 microM. In agreement with findings with racemic HA-966, even high concentrations of (+)-HA-966 did not completely inhibit NMDA responses, suggesting that (+)-HA-966 is a low-efficacy partial agonist. (+)-HA-966 produced parallel shifts to the right of the glycine concentration curve for potentiation of NMDA responses, resulting in an estimated pKb = 5.6. In mice, (+)-HA-966 antagonized sound and N-methyl-DL-aspartic acid (NMDLA)-induced seizures with ED50 values of 52.6 mg/kg of body weight (i.p.) and 900 mg/kg (i.v.), respectively. The coadministration of D-serine dose-dependently (10-100 micrograms into the cerebral ventricles per mouse) antagonized the anticonvulsant effect of a submaximal dose of (+)-HA-966 (100 micrograms administered directly into the cerebral ventricles) against NMDLA-induced seizures. The sedative/ataxic effect of racemic HA-966 was mainly attributable to the (-)-enantiomer, which was greater than 25-fold more potent than the (+)-enantiomer. It is suggested that, as in the case of the sedative gamma-butyrolactone, disruption of striatal dopaminergic mechanisms may be responsible for this action.
Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site.
Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes. Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing. Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions. Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain. Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon. Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site. Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated. Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site. This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes.
Therapeutic potential of NMDA receptor antagonists as neuroprotective agents.
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In vitro selectivity of agonists and antagonists for beta 1- and beta 2-adrenoceptor subtypes in rat brain.
A radioreceptor binding assay was developed to determine the selectivity of beta-adrenoceptor agents in rat brain. This was achieved by using the highly selective unlabelled antagonists CGP 20712A and ICI 118-551 to block beta 1- or beta 2-sub-populations respectively in rat cerebral cortex membranes. This permitted the selective labelling of beta-adrenoceptors with the antagonist (-)-[125I]pindolol. Using this method, compounds could be routinely screened and selectivity profiles for binding in the CNS determined with a high degree of sensitivity and resolution.
Test models and new directions in dementia research.
Psychopharmacology research in the dementia field is particularly difficult because there are no effective treatments on which to base models. Much effort is devoted to attempts to replace defective brain chemical transmitters or neuropeptides, with particular emphasis on the cholinergic system. There is also interest in noradrenaline, serotonin and somatostatin deficits. New directions include the study of glutamate-related excitotoxins such as quinolinic acid as possible causative agents for cerebral degeneration in dementia. Glutamate receptor antagonists, e.g. MK-801, may have the potential to limit or slow down neurodegenerative processes. Neurotrophic factors, e.g. nerve growth factor, may also possess the ability to protect neurons from irreversible loss.
7-Chlorokynurenic acid is a selective antagonist at the glycine modulatory site of the N-methyl-D-aspartate receptor complex.
Glycine markedly potentiates N-methyl-D-aspartate (N-Me-D-Asp) responses in mammalian neurons by an action at a modulatory site on the N-Me-D-Asp receptor-ionophore complex. Here we present evidence that 7-chlorokynurenic acid (7-Cl KYNA) inhibits N-Me-D-Asp responses by a selective antagonism of glycine at this modulatory site. In rat cortical slices 7-Cl KYNA (10-100 microM) noncompetitively inhibited N-Me-D-Asp responses, and this effect could be reversed by the addition of glycine (100 microM) or D-serine (100 microM). Radioligand binding experiments showed that 7-Cl KYNA had a much higher affinity for the strychnine-insensitive [3H]glycine binding site (IC50 = 0.56 microM) than for the N-Me-D-Asp (IC50 169 microM), quisqualate (IC50 = 153 microM), or kainate (IC50 greater than 1000 microM) recognition sites. In whole-cell patch-clamp recordings from rat cortical neurones in culture, the inhibitory effects of 7-Cl KYNA on N-Me-D-Asp-induced currents could not be overcome by increasing the N-Me-D-Asp concentration but could be reversed by increasing the glycine concentration. 7-Cl KYNA could completely abolish N-Me-D-Asp responses, including basal responses in the absence of added glycine, suggesting that it may possess negative modulatory effects at the glycine site. These findings indicate that the glycine modulatory site is functional in intact adult tissue and that 7-Cl KYNA should prove to be a selective tool for elucidating the involvement of this site in physiological and pathological events mediated by N-Me-D-Asp receptors.
Differences between high-affinity forskolin binding sites in dopamine-rich and other regions of rat brain.
[3H]Forskolin bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into one rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebellum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.
Relative affinities of drugs acting at cholinoceptors in displacing agonist and antagonist radioligands: the NMS/Oxo-M ratio as an index of efficacy at cortical muscarinic receptors.
1. Radioligand binding assays using [3H]-N-methylscopolamine (NMS) and [3H]-oxotremorine M (Oxo-M) have been devised to predict the efficacy of test compounds at muscarinic receptors in rat cerebral cortex. 2. Muscarinic antagonists, including non-selective and both M1- and M2-selective compounds, displayed similar affinity for both binding assays. 3. Full agonists such as carbachol and muscarine possessed a ratio of potencies against the antagonist versus the agonist ligand (NMS/Oxo-M ratio) of greater than 4000. 4. Compounds which have been shown previously to display partial agonist activity in functional assays e.g. pilocarpine and RS86 had intermediate NMS/Oxo-M ratios of 100-150. A second group of compounds which included oxotremorine had somewhat higher ratios (500-1400). 5. The ratio of affinity constants for the two assays predicted the ability of agonists to stimulate cortical phosphatidyl-inositol turnover. 6. These results suggest that the NMS/Oxo-M ratio may be a useful prediction of efficacy for novel compounds acting at cortical muscarinic receptors.
Biochemical measurement of muscarinic receptor efficacy and its role in receptor regulation.
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