Tunicamycin partially delays release of newly synthesized hyaluronate from Swarm rat chondrosarcoma chondrocytes

Tunicamycin (5-100 micrograms/ml) inhibits total [3H]hyaluronate synthesis in cultures of Swarm rat chondrosarcoma chondrocytes by approx. 15%. In agreement with previous results (Lohmander, L.S., Fellini, S.K., Kimura, J.H., Stevens, R.L. and Hascall, V.C. (1983) J. Biol. Chem. 258, 12280-12286) the relative decrease in [3H]hyaluronate radioactivity in the culture medium was greater than in the cell layer. Treated cultures show a concentration-related decrease in the proportion of medium 35S-labelled proteoglycans forming 'natural aggregates'. Pulse-chase experiments in cultures pretreated with tunicamycin (100 micrograms/ml, 13 h) showed that 30-40% of the total [3H]hyaluronate synthesized is released more slowly from these chondrocytes than from control culture chondrocytes. Release of some hyaluronate molecules may be delayed by 6 h or more. After a 24 h chase period almost all the [3H]hyaluronate is released from the cells. The proportion of 35S-labelled proteoglycans present as aggregates in the 24 h chase medium (57%) remained depressed compared to controls (81%), although the monomers could form aggregates if exogenous hyaluronate was added. Hyaluronate synthesized in the presence of tunicamycin has the same hydrodynamic size as control culture hyaluronate, as assessed by its sedimentation profile in CsSO4 gradients and its chromatographic profile on a dissociative Sephacryl S-1000 column.     

Tolerance and cross tolerance with morphine resulting from physiological release of endogenous opiates

Mice which had been exposed to a chronic schedule of warm water swimming showed the development of a significant tolerance to the antinociceptive response (tail-flick latency) and a significant, two-fold increase in the ED50 of morphine (tail-flick latency and abdominal constriction response). These results suggest the involvement of endogenous opiates during swim stress in mice and are consistent with the hypothesis that during chronic stress the opiate receptors are activated in a manner analogous to the repeated application of exogenous opiates producing tolerance, morphine cross tolerance and (as previously reported) withdrawal-like behaviour.     

Plasma-mediated enhancement of enzyme secretion in Aspergillus oryzae

Technical bottlenecks in protein production and secretion often limit the efficient and robust industrial use of microbial enzymes. The potential of non-thermal atmospheric pressure plasma to overcome these technical barriers was examined. Spores of the fermenting fungus Aspergillus oryzae (A. oryzae) were submerged in potato dextrose broth (PDB) (5 × 10⁶ per ml) and treated with micro dielectric barrier discharge plasma at an input voltage of 1.2 kV and current of 50 to 63 mA using nitrogen as the feed gas. The specific activity of α-amylase in the broth was increased by 7.4 to 9.3% after 24 and 48 h of plasma treatment. Long-lived species, such as NO₂⁻ and NO₃⁻, generated in PDB after plasma treatment may have contributed to the elevated secretion of α-amylase. Observations after 24 h of plasma treatment also included increased accumulation of vesicles at the hyphal tip, hyphal membrane depolarization and higher intracellular Ca²⁺ levels. These results suggest that long-lived nitrogen species generated in PDB after plasma treatment can enhance the secretion of α-amylase from fungal hyphae by depolarizing the cell membrane and activating Ca²⁺ influx into hyphal cells, eventually leading to the accumulation of secretory vesicles near the hyphal tips.     

One-electron reduction characteristics of N(3)-substituted 5-fluorodeoxyuridines synthesized as radiation-activated prodrugs

We designed and synthesized N(3)-substituted 5-fluorodeoxyuridines as radiation-activated prodrugs of the antitumor agent, 5-fluorodeoxyuridine (5-FdUrd). A series of 5-FdUrd derivatives possessing a 2-oxoalkyl group at the N(3)-position released 5-FdUrd in good yield via one-electron reduction initiated by hypoxic irradiation. Cytotoxicity of the 5-FdUrd derivative possessing the 2-oxocyclopentyl group (3d) was low, but was enhanced by hypoxic irradiation resulting in 5-FdUrd release.     

Effects of morphine on cold-induced TRH release from the median eminence of unanesthetized rats

The effect of morphine perfusion into the median eminence on cold-induced TRH secretion was studied in unanesthetized rats by push-pull cannulation. Perfusion with 10(-6)M morphine blocked the cold-induced TRH peak occurring about 40 min after the transfer of rats from 24 degrees C to 4 degrees C. This inhibition by morphine was blunted by concomitant administration of naloxone (10(-6)M or 10(-5)M), but naloxone alone had no effect on either basal or cold-induced TRH release. We conclude that specific opiate receptors may be located on TRH nerve endings in the ME, and that endogenous opiates may not have any physiological role in the cold-induced TRH response, at least during the two hours that follow cold exposure.     

Controlled drug release: new water-soluble prodrugs of an HIV protease inhibitor

We designed and synthesized a series of highly water-soluble prodrugs of an HIV protease inhibitor, KNI-727 (1), containing tandem-linked two auxiliary units, a solubilizing moiety and a self-cleavable spacer. Prodrugs with an ionized amino group at the solubilizing moiety exhibited a remarkable increase of water-solubility (>10(4) fold) compared to the parent drug 1. These prodrugs released I not enzymatically, but chemically via an intramolecular cyclization-elimination reaction through an imide formation in physiological conditions. Diversified rates of parent drug release were observed when the chemical structure of both the solubilizing and the spacer moieties were modified. This new approach for water-soluble prodrugs will enable to control chemically the release of parent drug as well as to maintain high water-solubility.     

Botulinum neurotoxin inhibits the release of newly synthesized acetylcholine from torpedo electric organ synaptosomes

In previous reports, we have shown that botulinum neurotoxin inhibits acetylcholine release from Torpedo marmorata electric organ and from its synaptosomal fraction. Here, we have focussed our attention on the study of the effect of botulinum neurotoxin on the metabolism of acetylcholine, namely, the precursors supply, the synthesis activity and the storage of the neurotransmitter into nerve endings isolated from Torpedo electric organ. Radiolabelled acetylcholine precursors (acetate and choline) uptake, choline O-acetyltransferase activity, and the compartmentalization of the transmitter into the synaptosomes were not modified by botullinum neurotoxin. When labelled nerve ending were depolarized by K⁺, the specific radioactivity of acetylcholine in the free pool fell markedly, but the specific radioactivity in the bound pool remained constant. Botulinum neurotoxin prevented this K⁺-induced decrease of specific radioactivity in the free pool.     

Hypoxia-driven elimination of thiopurines from their nitrobenzyl prodrugs

A novel bioreductive prodrug of 6-thioguanine, 2-amino-6-[2-(4-nitrophenyl)prop-2-ylsulfanyl]-9H-purine, containing a gem-dimethyl thioether linkage, was synthesised and compared with its unsubstituted analogue. In A549 whole cell experiments hypoxia selective release of 6-thioguanine was observed with the substituted prodrug only.     

Acute effects of heroin and morphine on newly synthesized serotonin in rat brain.

Heroin and morphine, in acute intraperitoneal doses of 2 and 10 mg/kg respectively, produced significant increments in the formation of newly formed brain serotonin from tritiated (³H)-L-tryptophan to ³H-serotonin. Opiate analgesia, Straub tail sign and catatonia, were observed during the increase in the synthesis of serotonin. The transport of radio-labelled tryptophan into the rat brain was not increased by the acute injection of the opiates, but brain levels of ³H-serotonin and of its main metabolite, 5-hydroxyindoleacetic acid, were significantly elevated. These opiates do not interfere with the accumulation of serotonin or with the transport of its metabolite in serotonergic neurons after inhibition of monoamine oxidases with Pargyline. An increase in the activity of tryptophan hydroxylases was more pronounced in the forebrain than in the brain stem. Stimulation of newly synthesized serotonin is probably mediated by an increase in tryptophan hydroxylase activity and not by an increase in the transport of tryptophan into the brain.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Development of enzymatically cleavable prodrugs derived from dendritic polyglycerol

In this Letter we report the synthesis and in vitro studies of cleavable polymer–drug conjugates derived from dendritic polyglycerol and maleimide-bearing prodrugs of doxorubicin and methotrexate that are cleaved by cathepsin B. Cleavage properties and cytotoxicity of the new conjugates are presented.     

Some newly synthesized leukotriene B4 analogs inhibit LTB4-induced lysozyme release from rat polymorphonuclear leukocytes

Antagonistic activities of newly synthesized LTB4 analogs, which possess the same olefinic geometry and the same hydroxyl group stereochemistry as natural LTB4, were investigated by studying lysozyme release from rat polymorphonuclear leukocytes (PMNLs). 14,15-Dihydro LTB4(LTB3) induced lysozyme release from PMNLs as well as LTB4, while 14,15-dehydro LTB4 did not cause lysozyme release but instead clearly inhibit LTB4-induced lysozyme release. Compounds containing aminocarbonyl groups partially retained lysozyme releasing activity. A displacement of the hydrocarbon chain at C13-20 by cyclohexenyl and beta-cyclohexylethyl groups had little effect on lysozyme release, but did strongly inhibit release induced by LTB4. These results may be useful in developing LTB4 antagonists.     

Effects of atropine on the release of newly synthesized acetylcholine from rat striatal slices at various concentrations of calcium ions

The release of acetylcholine (ACh) from brain tissue is known to be inhibited by muscarinic autoreceptors on cholinergic nerve terminals but the mechanism of the inhibition is not understood. Atropine brings about an increase of ACh release by removing the inhibitory action of autoreceptors. We investigated whether the effect of atropine on the release of [¹⁴C]ACh newly synthesized during incubations from [U-¹⁴C] glucose depends on the concentration of Ca²⁺ in the medium. In rat striatal slices incubated in the presence of an inhibitor of cholinesterases and of 30 mmol/l K⁺, significant increases in the release of [¹⁴C]ACh elicited by atropine were only observed during incubations with very low concentrations of Ca²⁺. This finding supports the view that the activation of presynaptic muscarinic autoreceptors in the brain affects the release of ACh by reducing the availability of Ca²⁺ that is required for transmitter liberation.     

Counteraction by morphine of stress-induced inhibition of growth hormone release in the rat

The purpose of this study was to determine the effect of morphine (MOR) administration on pituitary growth hormone (GH) release during stress in the male rat. Circulating GH levels were significantly decreased following a brief (2 min) exposure to either, during repetitive etherization coupled with blood withdrawal, and during continuous immobilization. Under all three stress conditions, systemic administration of MOR resulted in a significant increase in plasma GH levels compared to the vehicle-treated group. These results indicate that the pathway for opiate-induced stimulation of GH release is functional during stress, and suggest that the suppressive effect of stress does not involve a blockade of opiate receptor stimulation of GH. Thus, the present findings, taken together with reports that the overall activity of central opioid neurons is enhanced during stress, support the view that the decline in GH is due to the overriding inhibitory influence of an independent nonopioid mechanism. However, MOR can apparently increase opiate receptor stimulation sufficiently to counteract this inhibitory signal, implying that stress and the opiates may influence GH release via separate mechanisms.     

Differential inhibition of the release of endogenous and newly synthesized acetylcholine from Torpedo synaptosomes by presynaptic muscarinic receptors

Activation of Torpedo presynaptic muscarinic acetylcholine (ACh) receptors with the agonist oxotremorine (20 μM) results in the inhibition of Ca²⁺-dependent release of endogenous ACh from Torpedo synaptosomes. This effect is reversed by the muscarinic antagonist atropine (1 μM) which, by itself, has no effect. In contrast, under the same conditions the amount of newly synthesized radiolabeled [³H]ACh released is not affected by muscarinic ligands. These findings suggest that presynaptic muscarinic inhibition in the Torpedo is due to interference with the mobilization of ACh from a storage pool.