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Biomedical subjects

E Heilbronn

Publications and source records attributed to E Heilbronn.

At least 37 records · Page 2Linked to original sources
PubMed1987

Forskolin blocks carbachol-mediated ion-permeability of chick myotube nicotinic receptors and inhibits binding of 3H-phencyclidine to Torpedo microsac nicotinic receptors.

Forskolin, a commonly used adenylate cyclase activator, was found to inhibit reversibly the carbachol-induced ion-translocating capacity of the nicotinic acetylcholine receptor (nAChR) on chick myotubes in a dose- (IC50 = 20 microM) and time-dependent manner. This effect was not correlated to increases in cellular cAMP. Forskolin, at a concentration (50 microM) that totally blocked the carbachol-induced 86Rb influx, caused no change in carbachol or alpha-bungarotoxin binding to chick myotube nAChR in situ. In contrast, in the presence of carbachol, forskolin inhibited (IC50 = 10 microM) the binding of 3H-phencyclidine, a putative nAChR ion-channel ligand, to Torpedo microsac nAChR. Inhibition of 3H-phencyclidine binding in the absence of carbachol was not complete. Membrane leakage studies on myotubes, measuring 3H-efflux from 2-deoxy-D(1-3H)-glucose loaded cells and electrophysiological measurements of membrane properties supported the interpretation that forskolin induced decreases in plasma membrane permeability. In conclusion, forskolin blocks the carbachol-mediated increase in permeability of the nAChR channel by (1) binding to the ion-channel (open state) and (2) generally perturbing the plasma membrane function possibly by interfering with the protein-lipid interface.

Animals↗
PubMed1987

Effect of long-term administration of manganese on biogenic amine levels in discrete striatal regions of rat brain.

The effect of long-term manganese exposure of rats on biogenic amine levels in striatal brain regions is described. Four groups of male Sprague-Dawley rats received manganese as MnCl2 continuously in the drinking water for 60, 100, 165 and 265 days, respectively. Discrete regions within the caudate-putamen were punched out. Dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5-hydroxyindoleacetic acid were determined by high performance liquid chromatography with electrochemical detection. Rats exposed for 60 and 165 days showed significantly increased levels of dopamine and 3,4-dihydroxyphenylacetic acid in discrete regions of the dorsal caudate-putamen. The affected regions were possibly not identical in the two age groups but they were adjacently situated. These alterations were not found in rats exposed for 100 or 265 days.

Animals↗
PubMed1987

Effects of manganese oxide on monkeys as revealed by a combined neurochemical, histological and neurophysiological evaluation.

Four monkeys were exposed to a total of 8 g each of manganese as oxide by repetitive subcutaneous injections during 5 months, after which they were left for 1 week to 6 months before they were sacrificed. All animals developed hyperactive behaviour after about 2 months. About 5 months after the start of the exposure the animals became hypoactive with an unsteady gait, and subsequently an action tremor appeared in some of the animals. The animals lost power in both upper and lower limbs and the movements of the hands and feet were very clumsy. The serum content of manganese rose 10-40 times during the exposure time and the content in brain was generally increased more than 10 times, with the highest content found in globus pallidus and putamen. The observed neurochemical effects were also largest in globus pallidus and putamen. In these regions there was a considerable depletion of dopamine and 3,4-dihydroxyphenylacetic acid, while the homovanillic acid content remained almost unchanged. A severe neuronal cell loss was observed in globus pallidus but not in other regions. This is in accordance with results from the most recent neuropathological study of a human suffering from chronic manganese poisoning [Yamada et al. (1986) Acta Neuropathol 70: 273-278] where globus pallidus was devoid of neuronal cells while the content of pigmented cells in substantia nigra was normal. Our data suggest a reduction in number of dopaminergic nerve terminals, as the activity of the dopamine synthesizing enzyme DOPA-decarboxylase was also lowered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Some biochemical characteristics and cell membrane actions of a toxic phospholipase A2 isolated from the venom of the pit viper Agkistrodon halys (Pallas).

A toxic component (AgTx) from the venom of Agkistrodon halys (Pallas) was isolated using DEAE-cellulose DE11 and CM-Sephadex C50 column chromatography and finally purified to homogeneity by FPLC on a MonoQ column. The toxin is a neutral (pI 6.9) single chain polypeptide with a mol. wt of 14,000 and an amino acid composition (123 residues) roughly similar to that of notexin. AgTx was found to have phospholipase A2 activity which was dependent on calcium and stimulated by sodium deoxycholate. The toxin caused efflux of 2-deoxy-(1-3H)-glucose-6-phosphate (a cell membrane integrity probe) as well as of [3H]acetylcholine from rat brain synaptosomes. No cell membrane damage was induced by AgTx on cultured N1E 115 neuroblastoma cells and chick myotube cultures. The LD50 ws 150 micrograms/kg (i.p.) in mice. The main symptom observed was respiratory paralysis. The results obtained show that AgTx can be classified as a toxic phospholipase A2 with a presynaptic site of action.

Acetylcholine↗
PubMed1987

Isolation and pharmacological characterization of a new alpha-neurotoxin (alpha-AgTx) from venom of the viper Agkistrodon halys (Pallas).

A hitherto unknown alpha-neurotoxin, alpha-agkistrodotoxin, was isolated from the venom of the pit viper Agkistrodon halys (Pallas). It's molecular weight was approx. 8000 +/- 80 (SDS-polyacrylamide electrophoresis). The toxin crossreacted with antiserum directed against alpha-bungarotoxin and inhibited binding of 125I-alpha-bungarotoxin to the nicotinic acetylcholine receptor of cultured myotubes (IC50 = 2 X 10(-9) M). The association and dissociation rates were 4.85 X 10(5) per mole per min and 3.55 X 10(-4) per min, respectively, giving a Kd of 7.3 X 10(-10) M. The toxin also inhibited carbachol-induced influx of cations through the nAChR (IC50 = 6 X 10(-8) M).

Animals↗
PubMed1987

Effects of a toxic phospholipase A2 (AgTx) from the venom of the Pit viper (Agkistrodon halys (Pallas)) on the crayfish stretch receptor neuron.

Neurotoxicity of an isolated fraction (V) of the venom from the snake Agkistrodon halys (Pallas) was studied on the crayfish stretch receptor using a two-electrode voltage-clamp technique. The toxin was previously shown to have less phospholipase A2 activity but to be more toxic in mice compared with bee venom phospholipase A2. At 5 micrograms ml-1 (0.35 microM) the AgTx had very little effects on the electrophysiological properties of the receptor neuron whereas at 50 micrograms ml-1 (3.5 microM) the leak conductance was increased about three times and the net outward (K) current was reduced to 70% of control. The net inward (Na) current was not affected except for a small (+ 10 mV) shift in the I-V relationship. The resting membrane potential and the membrane capacitance were not changed by AgTx. These effects of AgTx were similar to those seen after exposure to bee venom phospholipase A2 at concentrations 10-20 times lower. At concentrations of AgTx which presynaptically affect the frog neuromuscular transmission (5-10 micrograms ml-1) the effects on ion channels in the stretch receptor neuron are very small and probably reflect the low phospholipase A2 activity compared with that of bee venom phospholipase A2.

Animals↗
PubMed1986

Interaction with chick myotube cholinergic receptors of an alpha-neurotoxin isolated from venom of the banded krait (Bungarus fasciatus).

A postsynaptic acting short chain alpha-toxin, B.f. III, was isolated from venom of the banded krait (Bungarus fasciatus) using ion-exchange chromatography. The toxin, a basic protein (pI = 10) has an apparent molecular weight of 6,500 as determined by SDS-polyacrylamide gel electrophoresis. It shares immunological determinants with alpha-bungarotoxin, as it cross-reacted with antibodies raised in rabbits against alpha-bungarotoxin. B.f. III inhibits binding of 125I-alpha-bungarotoxin to cultured chick myotubes with an IC50 of 3 X 10(-10) M. The rate of association with chick myotube nAChR was 3 times faster than that of alpha-bungarotoxin, and binding was slowly reversible. The toxin is a less potent antagonist than alpha-bungarotoxin; in ion flux experiments, measuring influx of 86Rb in chick myotubes, B.f. III inhibited carbachol-induced influx of 86Rb (IC50 = 5 X 10(-9) M) at concentrations higher than those needed for alpha-bungarotoxin (IC50 = 6 X 10(-10) M).

Animals↗
PubMed1986

Pitfalls in the assay of cyclic AMP-dependent protein kinase activity in microsacs from Torpedo marmorata.

Endogenous phosphorylation of the nicotinic acetylcholine receptor (nAChR) in microsacs from Torpedo marmorata was found to be affected by several reagents commonly used in the preparation of cyclic AMP (cAMP)-dependent protein kinases and in its activity determination. The presence of a Na+,K+-ATPase inhibitor is essential to avoid a rapid depletion of ATP, even when a membrane fraction highly enriched in the nAChR is used. The presence of the thiol reducing agent dithiothreitol was found to abolish the cAMP dependence of nAChR phosphorylation, whereas the less potent reagent 2-mercaptoethanol did not affect the assay. Concentrations in the millimolar range of the chelators EDTA and EGTA were found to inhibit nAChR phosphorylation effectively. This inhibition was not due to a withdrawal of Ca2+ by the chelators, but rather to a reversible inhibition by the Mg2+ complexes. These observations may explain some of the discrepancies found in the literature concerning endogenous and exogenous nAChR phosphorylation.

Animals↗
PubMed1986

Effects of manganese chloride on the rat developing nervous system.

Sprague-Dawley rats were exposed to sublethal doses of manganese chloride during their postnatal development period up to 44 days of age. They showed transient clinical signs of disease and a decreased homovanillic acid (HVA) content in the striatum and hypothalamus between 15 and 22 days of age. The manganese content in the brain was increased 20-40 times during this period as determined with neutron activation analyses. In spite of this no structural damage or signs of maturation disturbances in the nervous system were found. Interruption of manganese exposure reversed the changes in HVA content.

Animals↗
PubMed1985

Oxotremorine acts as a partial nicotinic agonist on cultured chick myotubes.

Oxotremorine, a compound widely used as a muscarinic agonist, was found to inhibit binding of 125I-alpha-bungarotoxin to chick myotube nicotinic acetylcholine receptors, IC50 = 79 +/- 5 microM. Oxotremorine also induced a d-tubocurarine sensitive influx of 86Rb in the myotubes (EC50 = 50 +/- 15 microM). Comparative ion-flux studies with carbachol suggested a partial agonist mode of action of oxotremorine.

Animals↗
PubMed1985

ATP-induced cation influx in myotubes is additive to cholinergic agonist action.

Using biochemical methods, ATP was shown to induce an inward flux of 86Rb into cultured chick myotubes. A biphasic dose-response curve was observed, the first part of which was saturable and had an EC50 value of approximately 10 microM. beta, gamma-imido ATP inhibited the ATP-induced uptake. ADP, AMP and adenosine were less potent than ATP in producing influx. ATP-induced influx of 86Rb was found to be additive to carbachol-induced influx and, in contrast to the latter, not blocked by the nicotinic acetylcholine receptor antagonist alpha-bungarotoxin. The results suggest the presence of an ATP recognition-site on myotubes, triggering ion-permeation.

Adenosine Triphosphate↗
PubMed1984

Effects of manganese on the nervous system.

The acute effect of manganese on the synaptosomal sodium-dependent choline uptake was studied. Manganese (Mn) revealed a mixed competitive-noncompetitive inhibition of the choline uptake, at Mn concentrations in the mM range. We do not believe that lack of choline for transmitter synthesis is of importance in Mn poisoning. Manganese chloride intubation of neonatal rats resulted in reduced homovanillic acid content in the striatum and hypothalamus. No other alteration in the dopamine or serotonin metabolism was revealed. A mechanism of cytotoxic action of manganese is discussed in terms of its transition metal properties and interaction with reactive oxygen compounds which result in the production of substances that alkylate or oxidize the cellular thiols.

Animals↗
PubMed1983

Extraction of peripheral proteins from nicotinic acetylcholine receptor-enriched membranes.

The solubilisation of membrane proteins from nicotinic acetylcholine receptor-enriched membranes from the electric organ of Torpedo marmorata was studied. Chaotropic ions were shown to be ineffective in extracting peripheral proteins from these membranes. Two different anhydrides, 2, 3-dimethylmaleic and 3,4,5,6-tetrahydrophthalic anhydride, released certain peripheral membrane proteins but not the integral receptor protein. Treatment of membranes containing greater than 3 nmol alpha-bungarotoxin binding sites per mg protein with anhydride resulted in a 43 kDa polypeptide as the major constituent of the solubilised material. The nature of the 43 kDa polypeptide is discussed. Gentle anhydride treatment did not change the alpha-bungarotoxin and carbamoylcholine binding properties of the receptor.

Animals↗
PubMed1983

Changes in the redox state of neuroblastoma cells after manganese exposure.

The toxicological effects of manganese chloride on the redox state of thiols and on the lipid peroxidation in cultures of the neuroblastoma clone N1E 115 were studied. The cell cultures were exposed, after a stationary growth phase was attained, to manganese chloride (25-100 microM) for up to 9 days. The non-protein thiols decreased at the most 27% as compared to the controls. Significant effects were obtained at all manganese concentrations tested. The total thiol content was maximally reduced by 40%. This reduced thiol content was also reflected in a lowered activity of the thiolenzyme, glyceraldehyde-3-phosphate dehydrogenase in manganese exposed cells. In addition the lipid peroxide level in the cells was decreased during the manganese treatment.

Cells, Cultured↗
PubMed1983

Morphological changes observed in rats immunized with the Torpedo acetylcholine receptor alpha-chain.

Lewis rats were immunized with nicotinic acetylcholine receptor (nAChR) polypeptides purified from the electric tissue of Torpedo marmorata. Animals immunized with the acetylcholine binding, 41.500 daltons, polypeptide showed comparable fragmentation of the postsynaptic membrane and indications of rearrangement of the adjacent muscle fiber as earlier found in rabbits and rats immunized with the nAChR complex. Thus, these results demonstrate that the alpha-polypeptide chain carries all determinants necessary for onset of Experimental Autoimmune Myasthenia Gravis (EAMG).

Animals↗
PubMed1983

Microanalysis of endogenous acetylcholine released from the hemidiaphragm of the rat.

A chemiluminescence method for the determination of endogenous acetylcholine was adapted to transmitter released from the hemidiaphragm of the rat. Released choline and acetylcholine were isolated and concentrated using KI3 precipitation. Between 1 and 20 pmol of acetylcholine may be measured without sample dilution. The detection limit for acetylcholine luminescence is 1.0 pmol; thus, with an observed precipitation recovery of approximately 30%, the lowest detectable released amount is 3.5 pmol. The limitations of the method are discussed.

Acetylcholine↗
PubMed1983

Release of acetylcholine at the motor endplate of the rat - evidence against a muscarinic acetylcholine autoreceptor.

The effect of some drugs on the release of endogenous acetylcholine from the phrenic nerve-hemidiaphragm preparation of the rat was measured. Muscarinic ligands had no effect. 8-Br-cyclic GMP, a penetrating analogue of cyclic guanosine 3',5'-monophosphate (cyclic GMP) was also without effect. 8-Br-cyclic AMP somewhat enhanced the basal release while the potassium-induced release remained unaltered. In supersensitivity experiments, no specific binding of ligand [3H]-quinuclidinylbenzilate [( 3H]-QNB) was found in homogenates of the diaphragm, either before or after atropine treatment, while concomitant binding studies in the CNS demonstrated the expected increase in muscarinic binding sites after atropine. Our conclusion is that muscarinic acetylcholine receptors are probably absent from the presynaptic motor endplate area of the rat. Certain preliminary results suggest that a presynaptic nicotinic mechanism might be involved in the release of acetylcholine.

Acetylcholine↗