Cholinergic Mechanisms and Psychopharmacology, 1978
Numerous mechanisms have already been proposed for the regulation of ACh synthesis by CAT (AcCoA:... more Numerous mechanisms have already been proposed for the regulation of ACh synthesis by CAT (AcCoA: choline-O-acetyltransferase, EC:2.3. 1.6). They are: regulation by mass action (14, 35); inhibition by ACh (16) and regulation by salts (27). None of these proposed mechanisms seems sufficient to explain the perfect regulation of ACh synthesis in cholinergic nerve terminals (2, 9, 10).
Tremendous excitement has been generated by the isolation, purification, and subsequent synthesis... more Tremendous excitement has been generated by the isolation, purification, and subsequent synthesis of the opioid peptides. Our collaborative efforts have been directed at the questions of where β-endorphin is stored in brain and pituitary, how such structures are related to those storing the enkephalins and possibly other biogenic substances, and the functions of these peptides expressed through their electrophysiological properties. Although none of these questions is yet completely answered, sufficient data have been accumulated (Bloom et al., 1976, 1977a, b; Guillemin et al., 1977a, b, c; Rossier et al., 1977a, b, c; Nicoll et al., 1977; Henriksen et al., 1977; French et al., 1977) to enable us to give this overview and progress report.
Ausgehend von dem Ester (I) werden über das Hydrazid (II) das Azid (III) sowie die Aminoverbindun... more Ausgehend von dem Ester (I) werden über das Hydrazid (II) das Azid (III) sowie die Aminoverbindung (IVa) dargestellt und anschließend zu den N‐substituierten Amino‐β‐carbolinen (IVb)‐(VIII) umgesetzt.
The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) ... more The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) was iodinated and purified by high performance liquid chromatography. Monoiodo-azido-DTLET displayed a high affinity (KD = 15 nM) and is selective (Kl mu/Kl delta = 9.8) for rat brain delta opioid receptors (for comparison, the corresponding values for tritiated azido-DTLET are KD = 1.66 nM and Kl mu/Kl delta = 27). On rat brain sections, the anatomical distribution of [125I]azido-DTLET binding sites revealed by autoradiography corresponds to that of delta receptors. On rat brain membrane homogenates and NG108-15 hybrid cells, UV irradiation of the receptor-ligand complex results in the irreversible binding to membrane proteins of 14% of the bound radioactivity Gel electrophoresis of [125I]azido-DTLET-labeled proteins followed by autoradiography shows a different pattern in rat brain and NG108-15 cells. In rat brain, labeling of two of these proteins, with molecular weights of 44,000 and...
Publisher Summary This chapter discusses the biosynthesis of enkephalins by adrenal chromaffin ce... more Publisher Summary This chapter discusses the biosynthesis of enkephalins by adrenal chromaffin cells. Immunohistochemical studies have shown that bovine adrenal chromaffin cells contain immunoreactive enkephalin. Leu- and Met-enkephalin represent only a part of this immune-reactivity. These cells contain a large amount of a newly characterized enkephalin: met-enkephalin-Arg-Phe. The concentration of this new compound is higher than that of met-enkephalin. The chromaffin granules contain several large Mr peptides containing the aminoacid sequences of the enkephalins. The steady state levels of the enkephalins and their precursors in primary monolayer bovine adrenal chromaffin cells may indicate that they actively synthesize these molecules de-novo.
A number of recent studies have established the fact that enkephalins are released, together with... more A number of recent studies have established the fact that enkephalins are released, together with catecholamines, from the adrenal medulla, both in vitro and in vivo, in response to various stimuli (Kilpatrick et aI., 1980; Stine et aI., 1980; Livett et aI., 1981; Hexum et aI., 1980; Chaminade et aI., 1984; Edwards et aI., 1986). It further appears that these pep tides are present in adrenergic, but not noradrenergic, chromaffin cells in the adrenal medulla (Livett et aI., 1982; Pelto-Huikko et aI., 1982; Roisin et aI., 1983), but the functional significance of the phenomenon remains obscure. Chaminade et aI., (1984) showed that, in the isolated perfused adrenal gland of the cat, the release of free [Met]enkephalin-Iike immunoreactivity was invariably associated with that of much larger precursor molecules. The extent to which these latter were released depended upon the intensity of the stimulus and it appeared that the smaller molecules, presumably obtained by the more complete processing of precursors, were secreted preferentially during more moderate forms of stimulation. In the present study the "adrenal clamp" technique (Edwards et aI., 1974) has been employed to examine the release of these peptides under the most physiological conditions that have yet been achieved i.e. in response to splanchnic nerve stimulation in conscious animals. The results show that large molecular weight precursor forms of en kephalin are indeed released under these conditions and in amounts that far exceed those of free [Met]enkephalin. However, the amount of free peptide in adrenal effluent plasma during nerve stimulation was invariably much higher than that in extracts of the gland, indicating that release is associated with a final processing step or steps. Alternatively, these results could be taken to indicate preferential release of granules containing more highly processed enkephalin-like material. The finding that the ratio of adrenaline to total enkephalin-like material that was released was almost three times higher in adrenal effluent plasma during stimulation of the splanchnic nerve at 15Hz than at 4Hz is consistent with the hypothesis that the population of releasable granules is heterogeneous. METHODS Animals: Pedigree Jersey calves were obtained from local farms shortly after birth and used at ages ranging between 25 and 52 days (26.4-31.2 kg body weight). Thereafter, they were kept in individual pens in the laboratory animal house and maintained on a diet of either cow's milk or artificial milk (Easy-Mix Volac; Volac Ltd.) at a rate of 2-4 I/day. Food was withheld for at least 14 h before surgery and each experiment.
Methyl-β-carboline 3 carboxylate (β-CCM) is a benzodiazepine receptor ligand that has been shown ... more Methyl-β-carboline 3 carboxylate (β-CCM) is a benzodiazepine receptor ligand that has been shown to have pharmacological properties that are opposite to those of benzodiazepines in all situations tested. Thus this molecule is a convulsant whereas benzodiazepines are known anticonvulsants; β-CCM is anxiogenic whereas benzodiazepines are known anxiolytics. β-CCM can therefore be considered as an inverse agonist of benzodiazepines. Since benzodiazepines have been shown to be amnesic drugs, we have hypothetized that β-CCM could have memory enhancing effects. This hypothesis was tested in two different behavioral situations. 1 — Imprinting in chicks. According to the technique used in our laboratory, chicks placed in front of a moving decoy during an acquisition session learn to follow it. This behavior of following the decoy is tested in the same situation 24 h later (test session). When chicks were injected with β-CCM (2.5 mg/kg i.p.) before the acquisition session, following of the decoy was enhanced in the test session (without drug). On the other hand, the classical benzodiazepine diazepam (DZ) (0.25 mg/kg i.p.), administered before the acquisition session, reduced following of the decoy during the test session. 2 — Latent learning in mice.
Cholinergic Mechanisms and Psychopharmacology, 1978
Numerous mechanisms have already been proposed for the regulation of ACh synthesis by CAT (AcCoA:... more Numerous mechanisms have already been proposed for the regulation of ACh synthesis by CAT (AcCoA: choline-O-acetyltransferase, EC:2.3. 1.6). They are: regulation by mass action (14, 35); inhibition by ACh (16) and regulation by salts (27). None of these proposed mechanisms seems sufficient to explain the perfect regulation of ACh synthesis in cholinergic nerve terminals (2, 9, 10).
Tremendous excitement has been generated by the isolation, purification, and subsequent synthesis... more Tremendous excitement has been generated by the isolation, purification, and subsequent synthesis of the opioid peptides. Our collaborative efforts have been directed at the questions of where β-endorphin is stored in brain and pituitary, how such structures are related to those storing the enkephalins and possibly other biogenic substances, and the functions of these peptides expressed through their electrophysiological properties. Although none of these questions is yet completely answered, sufficient data have been accumulated (Bloom et al., 1976, 1977a, b; Guillemin et al., 1977a, b, c; Rossier et al., 1977a, b, c; Nicoll et al., 1977; Henriksen et al., 1977; French et al., 1977) to enable us to give this overview and progress report.
Ausgehend von dem Ester (I) werden über das Hydrazid (II) das Azid (III) sowie die Aminoverbindun... more Ausgehend von dem Ester (I) werden über das Hydrazid (II) das Azid (III) sowie die Aminoverbindung (IVa) dargestellt und anschließend zu den N‐substituierten Amino‐β‐carbolinen (IVb)‐(VIII) umgesetzt.
The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) ... more The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) was iodinated and purified by high performance liquid chromatography. Monoiodo-azido-DTLET displayed a high affinity (KD = 15 nM) and is selective (Kl mu/Kl delta = 9.8) for rat brain delta opioid receptors (for comparison, the corresponding values for tritiated azido-DTLET are KD = 1.66 nM and Kl mu/Kl delta = 27). On rat brain sections, the anatomical distribution of [125I]azido-DTLET binding sites revealed by autoradiography corresponds to that of delta receptors. On rat brain membrane homogenates and NG108-15 hybrid cells, UV irradiation of the receptor-ligand complex results in the irreversible binding to membrane proteins of 14% of the bound radioactivity Gel electrophoresis of [125I]azido-DTLET-labeled proteins followed by autoradiography shows a different pattern in rat brain and NG108-15 cells. In rat brain, labeling of two of these proteins, with molecular weights of 44,000 and...
Publisher Summary This chapter discusses the biosynthesis of enkephalins by adrenal chromaffin ce... more Publisher Summary This chapter discusses the biosynthesis of enkephalins by adrenal chromaffin cells. Immunohistochemical studies have shown that bovine adrenal chromaffin cells contain immunoreactive enkephalin. Leu- and Met-enkephalin represent only a part of this immune-reactivity. These cells contain a large amount of a newly characterized enkephalin: met-enkephalin-Arg-Phe. The concentration of this new compound is higher than that of met-enkephalin. The chromaffin granules contain several large Mr peptides containing the aminoacid sequences of the enkephalins. The steady state levels of the enkephalins and their precursors in primary monolayer bovine adrenal chromaffin cells may indicate that they actively synthesize these molecules de-novo.
A number of recent studies have established the fact that enkephalins are released, together with... more A number of recent studies have established the fact that enkephalins are released, together with catecholamines, from the adrenal medulla, both in vitro and in vivo, in response to various stimuli (Kilpatrick et aI., 1980; Stine et aI., 1980; Livett et aI., 1981; Hexum et aI., 1980; Chaminade et aI., 1984; Edwards et aI., 1986). It further appears that these pep tides are present in adrenergic, but not noradrenergic, chromaffin cells in the adrenal medulla (Livett et aI., 1982; Pelto-Huikko et aI., 1982; Roisin et aI., 1983), but the functional significance of the phenomenon remains obscure. Chaminade et aI., (1984) showed that, in the isolated perfused adrenal gland of the cat, the release of free [Met]enkephalin-Iike immunoreactivity was invariably associated with that of much larger precursor molecules. The extent to which these latter were released depended upon the intensity of the stimulus and it appeared that the smaller molecules, presumably obtained by the more complete processing of precursors, were secreted preferentially during more moderate forms of stimulation. In the present study the "adrenal clamp" technique (Edwards et aI., 1974) has been employed to examine the release of these peptides under the most physiological conditions that have yet been achieved i.e. in response to splanchnic nerve stimulation in conscious animals. The results show that large molecular weight precursor forms of en kephalin are indeed released under these conditions and in amounts that far exceed those of free [Met]enkephalin. However, the amount of free peptide in adrenal effluent plasma during nerve stimulation was invariably much higher than that in extracts of the gland, indicating that release is associated with a final processing step or steps. Alternatively, these results could be taken to indicate preferential release of granules containing more highly processed enkephalin-like material. The finding that the ratio of adrenaline to total enkephalin-like material that was released was almost three times higher in adrenal effluent plasma during stimulation of the splanchnic nerve at 15Hz than at 4Hz is consistent with the hypothesis that the population of releasable granules is heterogeneous. METHODS Animals: Pedigree Jersey calves were obtained from local farms shortly after birth and used at ages ranging between 25 and 52 days (26.4-31.2 kg body weight). Thereafter, they were kept in individual pens in the laboratory animal house and maintained on a diet of either cow's milk or artificial milk (Easy-Mix Volac; Volac Ltd.) at a rate of 2-4 I/day. Food was withheld for at least 14 h before surgery and each experiment.
Methyl-β-carboline 3 carboxylate (β-CCM) is a benzodiazepine receptor ligand that has been shown ... more Methyl-β-carboline 3 carboxylate (β-CCM) is a benzodiazepine receptor ligand that has been shown to have pharmacological properties that are opposite to those of benzodiazepines in all situations tested. Thus this molecule is a convulsant whereas benzodiazepines are known anticonvulsants; β-CCM is anxiogenic whereas benzodiazepines are known anxiolytics. β-CCM can therefore be considered as an inverse agonist of benzodiazepines. Since benzodiazepines have been shown to be amnesic drugs, we have hypothetized that β-CCM could have memory enhancing effects. This hypothesis was tested in two different behavioral situations. 1 — Imprinting in chicks. According to the technique used in our laboratory, chicks placed in front of a moving decoy during an acquisition session learn to follow it. This behavior of following the decoy is tested in the same situation 24 h later (test session). When chicks were injected with β-CCM (2.5 mg/kg i.p.) before the acquisition session, following of the decoy was enhanced in the test session (without drug). On the other hand, the classical benzodiazepine diazepam (DZ) (0.25 mg/kg i.p.), administered before the acquisition session, reduced following of the decoy during the test session. 2 — Latent learning in mice.
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