Leslie L. Iversen

Naloxone as a GABA antagonist: Evidence from iontophoretic, receptor binding and convulsant studies

From the following three lines of evidence, it is proposed that at least part of the convulsant activity of naloxone is a result of GABA receptor blockade. Firstly, iontophoretic naloxone reversibly antagonized GABA-evoked depression of firing rate in 21 of 27 neurons tested in the rat olfactory tubercle-nucleus accumbens region, without blocking inhibition evoked in the same cells by glycine (15 cells) or morphine (6 cells). Secondly, i.p. naloxone in high doses caused convulsions in mice, and potentiated the convulsant activity of bicuculline, but not that of strychnine. Diazepam, which protected mice against convulsions elicited by bicuculline, but not by strychnine, also protected mice against naloxone. Thirdly, naloxone, morphine, levorphanol and its non-analgesic enantiomer dextrorphan displaced 3H-GABA from GABA receptor sites in homogenates of human cerebellum, all with comparable low potencies (IC50 = 250--400 micron). There was no correlation with affinities at the stereospecific receptor sites that mediate opiate-induced analgesia, since the potent opiates etorphine and diprenorphine were relatively inactive (IC50 greater than 3 mM). In addition naloxone displaced 3H-GABA from receptor sites in rate forebrain and cerebellum, with similar low potency.

Multiple tachykinin binding sites in peripheral tissues and in brain

Tachykinin binding sites in guinea pig urinary bladder (GPUB), rat salivary gland (RSG), hamster urinary bladder (HUB), rat vas deferens (RVD) and rat cerebral cortex (RCC) were compared using 125I-Bolton Hunter conjugates of substance P (125I-BHSP), eledoisin (125I-BHE) and neurokinin A (125I-BHNKA). In typical SP-P tissues (GPUB, RSG) and in RCC, SP was the most potent displacer of 125I-BHSP and [Glp6,D-Pro9]-SP(6-11) was 90 times less active than [Glp6,L-Pro9]-SP(6-11) while SP methyl ester (SPOMe) was 5-10 times more active than the Bolton Hunter conjugate of SPOMe (I-BHSPOMe). On the other hand, in typical SP-E tissues (HUB, RVD), neurokinin A was most potent in displacing 125I-BHE and [Glp6,D-Pro9]-SP(6-11) was over 300 times more active than [Glp6,L-Pro9]-SP(6-11) while SPOMe was 160 times less active than I-BHSPOMe. In rat cerebral cortex, the rank order of potency of tachykinins and related analogues in displacing 125I-BHE was distinct from that of peripheral SP-E sites, with neurokinin B being the most potent displacer, and SPOMe was over 1,000 times more active than I-BHSPOMe; 125I-BHE binding sites in CNS may represent a third category of tachykinin receptor, designated SP-N.

Calcitonin gene-related peptide: Novel neuropeptide

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide encoded in the calcitonin gene. Its expression is dependent on tissue-specific alternative RNA processing: mRNA for CGRP predominates in the brain, whilst calcitonin (CT) mRNA predominates in thyroid C cells. The existence of this hitherto unsuspected peptide was predicted by mRNA analysis and demonstrated using antibodies raised against a synthetic peptide corresponding to the predicted C-terminal sequence of CGRP. The distribution of CGRP in the central and peripheral nervous system and its co-localization in some neurons with substance P (SP) or acetylcholine suggests several possible roles in autonomic, sensory and motor functions. Its actions appear to depend on the existence of specific CGRP receptors in target tissues, distinct from the receptors for CT but bearing some resemblance to them.

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 > 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.

Competitive as well as uncompetitive N-methyl-D-aspartate receptor antagonists affect cortical neuronal morphology and cerebral glucose metabolism

The studies examined the effects of three antagonists (CPP, CGS 19755, and CGP 37849) that act competitively at the glutamate recognition site of the NMDA receptor complex on cortical neuronal morphology and cerebral limbic glucose metabolism. Responses were compared to the effects of dizocilpine, an uncompetitive NMDA receptor ion channel antagonist as a positive control. CGS 19755 and CGP 37849 (100 mg kg−1i.p.) caused vacuolation in cortical pyramidal neurons in the posterior cingulate cortex four hours after dosing and this dose of CGP 37849 caused a pattern of limbic glucose metabolism activation similar to that seen after dizocilpine. CPP was without effect at 100 mg/kg i.p. probably due to poor brain penetration. The data indicates that the functional consequences (structural and metabolic) of NMDA receptor blockade with NMDA antagonists acting competitively at the glutamate recognition site and uncompetitively in the receptor ion channel are ultimately the same. Comparisons of the potential therapeutic window for CGS 19755 and CGP 37849 with dizocilpine (neuroprotection versus vacuolation) suggests that the window for the competitive antagonists is greater. This indicates that the potential therapeutic window for the different classes of NMDA antagonists may vary with the site in the receptor complex at which they interact.

The Ferrier Lecture, 1983. Amino acids and peptides: fast and slow chemical signals in the nervous system?

The classical monoamine neurotransmitters, acetylcholine and the catecholamines, are used by only a small proportion of synapses in mammalian c.n.s. The amino acids GABA (y-aminobutyric acid) and L-glutamate may be the principal inhibitory and excitatory neurotransmitters used for fast point-to-point transmission in the c.n.s. The monoamines and the large number of neuropeptides (over 30) now known to exist in c.n.s. may be chemical signals used for a different type of chemically addressed form of information transmission between neurons in c.n.s. characterized by less precise spatial connections, a slower time course and a far richer diversity of chemical signals than used in classical synaptic neurotransmission. In this context the brain can be viewed as a neuroendocrine secretory organ of great complexity.

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 cyclc(GlnTrpPhe(R)Gly[ANC‐2]LeuMet) (L‐659,837) and cyclo(GlnTrpPheGlyLeuMet) (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.

Autoradiographic assessment of the effects of N-methyl-d-aspartate (NMDA) receptor antagonists in vivo

Twenty years ago, autoradiography occupied only a minor role in neurochemistry, although autoradiographic techniques had a secure place in neuroanatomy to localise the sites of anterogradely transported tritiated amino acids after their intracerebral injection (1). The utility of autoradiographic assessment of the in vivo uptake of freely diffusible tracers had long been recognised in the investigation of local cerebral haemodynamics in animals (2,3), but autoradiographic methods were not in widespread use because of the absence of a suitable tracer (4). Autoradiography had been used to define regional heterogeneity in a number of neurochemical processes, e.g., the incorporation, after systemic tracer administration, of tritiated thymidine in DNA or tritiated amino acid into protein (5). As a direct and indirect consequence of the innovations of Sokoloff and his research group, quantitative autoradiography is now widely used in neurobiology to explore a diverse array of CNS processes. The pivotal development was the advent of the [14C]2-deoxyglucose technique which allows the assessment of local functional events simultaneously in all the macroscopic subunits of the CNS (6,7). Sokoloff and his associates were responsible for the development of similar autoradiographic methods to measure local cerebral blood flow

The design of novel muscarinic partial agonists that have functional selectivity in pharmacological preparations in vitro and reduced side-effect profile in vivo

Antagonist/agonist binding ratios (NMS/Oxo-M ratio) were used as an index of the efficacy of novel compounds acting at muscarinic receptors. These binding ratios have been used with a range of functional pharmacological assays to investigate the effects of varying the efficacy of muscarinic agonists. This strategy has been used as a means of obtaining functional receptor selectivity by exploiting differences in effective receptor reserves. The oxadiazole and pyrazine muscarinic agonists L-670,548 (NMS/Oxo-M ratio 1100) and L-680,648 (NMS/Oxo-M ratio 690) are amongst some of the most potent and efficacious agonists known. Decreasing the efficacy of compounds from these series, resulted in compounds with functional selectivity. The chloropyrazine L-689,660 (NMS/Oxo-M ratio 28) was an agonist on the rat superior cervical ganglion (M1), a partial agonist on the guinea-pig ileum (M3), but was an antagonist in the guinea-pig atria (M2). Synthesis of compounds with even lower predicted efficacy, such as the cyclopropyloxadiazole L-687,306 (NMS/Oxo-M ratio 15), maintained agonist activity in the ganglion, but showed antagonist activity in the M3 ileal, as well as the M2 atrial preparations. When tested in vivo these compounds did not produce many of the side effects associated with more efficacious agonists, particularly those associated with the cardiovascular system. However, they were active in reversing scopolamine-induced deficits in a variety of behavioural paradigms. This approach shows how functional selectivity for muscarinic receptor subtypes can be achieved in vitro, that in vivo reduces the dose-limiting side effects normally associated with muscarinic agonists.

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

Abstract: GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23°C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]‐γ‐aminobutyric acid ([3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA > isoguvacine > 3‐aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) > 4,5,6,7‐tetrahydroisoxazolo (4,5,6)‐pyridin‐3‐ol (THIP) = taurine > piperidine 4‐sulphonic acid (P4S). At 37°C, the potencies of agonists remained unchanged, but isoguvacine, 3APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist‐induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor‐receptor in teraction under near‐physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABAA receptor ligands.