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Biomedical subjects

M E Eldefrawi

Publications and source records attributed to M E Eldefrawi.

At least 73 records · Page 4Linked to original sources
PubMed1985

Biochemical identification of a putative glutamate receptor in housefly thoracic membranes.

Specific stereoselective binding of [3H]L-glutamate was detected to membranes prepared from housefly thorax to which were added several antiproteases. A single high affinity binding site was detected (KD 0.5 +/- 0.04 microM), but total binding varied from preparation to preparation (5-60 pmoles/mg protein). Specific binding was inhibited by preincubation of the membranes with trypsin, chymotrypsin or protease, or by exposure to 70 degrees C for 5 min. It was also inhibited by several compounds, the most potent being L-glutamate and L-aspartate, followed by L-glutamate diethylester, then D-glutamate, N-methyl-D-aspartate and ibotenate. Quisqualate had little effect, while kainate, proctolin and D-aspartate had none. d-Tubocurarine stimulated [3H]L-glutamate binding. The data suggest that [3H]L-glutamate is binding to an L-glutamate receptor in housefly thoracic muscle membranes.

Animals↗
PubMed1985

The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist.

The secondary amine, mecamylamine, interacts with the nicotinic receptor ionic channel complex as a noncompetitive antagonist. Mecamylamine (1-10 microM) blocked indirect muscle twitches with no discernible effect on the membrane potential, overshoot, or amplitude of the action potential. It also produced a voltage- and concentration-dependent depression of the peak amplitude of the endplate currents (EPC) and induced nonlinearity in the current-voltage relationship. The decay time constant of the EPC (TEPC) was significantly shortened. The linear relationship between the reciprocal of TEPC and the drug concentration suggested an open channel blockade. Patch-clamp studies, in agreement with the noise analysis results, revealed that mecamylamine (1-8 microM) shortened the lifetime of the open channels. Further, the single channel studies showed that at high concentrations mecamylamine reduced the double exponential nature of the distribution of open times characteristic of channels recorded from myoballs. Closed times had a complex distribution that could not be fitted to a single exponential function because of the presence of short closures or "flickers" during the open state. Although the frequency of channel openings progressively decreased with increasing drug concentration, the single channel conductance remained unchanged at all the concentrations tested. Biochemical studies showed that mecamylamine (up to 100 microM) did not block [3H]acetylcholine binding to the nicotinic receptor of the Torpedo electroplax, but inhibited the binding of [3H]perhydrohistionicotoxin to its channel site, both in the resting and the activated state. These results suggested that, at the nicotinic receptors of the neuromuscular junction, mecamylamine acted as a noncompetitive blocker, binding primarily to the receptor's open channel conformation. Most of the alterations of EPCs were consistent with the predictions of a sequential model for open channel blockade. Biochemical and patch-clamp results, however, could not be fully explained by this model and provided some evidence of the existence of additional blocked states most likely through pathways into desensitized species. In contrast to a competitive antagonism of acetylcholine receptors reported at autonomic ganglia, there was no such action of the drug at the neuromuscular junction; thus, mecamylamine is a useful tool to characterize the nicotinic receptors from different synapses.

Amphibian Venoms↗
PubMed1985

Comparison of the actions of carbamate anticholinesterases on the nicotinic acetylcholine receptor.

Neostigmine (Neo), pyridostigmine (Pyr), and physostigmine (Phy) at low concentrations inhibited acetylcholine (ACh) esterase, thereby indirectly potentiating ACh enhancement of [3H]perhydrohistrionicotoxin (H12-HTX) binding to the channel sites of the nicotinic ACh receptor of Torpedo membranes. However, at higher concentrations, they inhibited ACh action due to their direct binding to the ACh receptor. They displaced binding of [3H]ACh and 125I-alpha-bungarotoxin (alpha-BGT) to the receptor sites with the following order of decreasing potency: Neo greater than Phy greater than Pyr. Furthermore, Neo and Pyr potentiated [3H] H12-HTX binding to the receptor's channel sites. Preincubation of ACh receptors with any of the three carbamates reduced the rate of binding of 125I-alpha-BGT and increased the potency of carbamylcholine in inhibiting 125I-alpha-BGT binding, suggesting that the three carbamates act as partial agonists and potentiate receptor desensitization. Although none of the three carbamates inhibited [3H]H12-HTX binding to the receptor's closed channel conformation, only Phy was a potent inhibitor of [3H]H12-HTX binding to the carbamylcholine-activated conformation. The potency of Phy was not due to the absence of positive charge since Phy methiodide acted similarly. The data suggest that the major action of the three carbamates at nicotinic cholinergic synapses is inhibition of ACh-esterase. Their interactions with the nicotinic ACh receptor are with its "receptor" as well as allosteric "channel" sites, but they differ in their effects. Neo and Pyr act mainly as partial agonists, while Phy is mostly an inhibitor of the channel in the activated receptor conformation.

Acetylcholine↗
PubMed1984

Distinction between high-affinity [3H]phencyclidine binding sites and muscarinic receptors in guinea-pig ileum muscle.

[3H]Phencyclidine ([3H]PCP) binding was studied in guinea-pig ileum muscle membranes. Specific binding of [3H]PCP was time dependent, reversible and saturable, with an equilibrium dissociation constant of 154 nM and maximum binding of 12.9 pmol/mg of protein at pH 9. Its pH dependency suggests that the unionized PCP is the pharmacologically active form. The binding site was on a protein which was sensitive to heat, proteolytic enzymes and the carboxylic group reagent dicyclohexylcarbodiimide, but insensitive to phospholipase A and C, concanavalin A, dithiothreitol and N-ethylmaleimide. Specific [3H]PCP binding was displaced effectively by several PCP analogs and Ca++ channel antagonists including verapamil, to which these sites had a high affinity. These high-affinity PCP-binding sites were found at a much higher concentration in the same membrane preparation than muscarinic receptor sites identified by their specific binding of [3H]quinuclidinyl benzilate. PCP bound to both sites, but with a lower affinity to the muscarinic receptor sites. The PCP and muscarinic receptor sites differed in their sensitivities to pH and drug specifities .

Animals↗
PubMed1984

Interactions of phencyclidine with crayfish muscle membranes. Sensitivity to calcium channel antagonists and other drugs.

[3H]Phencyclidine ( [3H]PCP) bound to crayfish abdominal muscle membranes at pH 7.4 with two affinities (Kd of 0.96 nM for 0.38 pmole/mg of protein, and 18.9 nM for 7.6 pmoles/mg of protein). Binding affinities increased at higher pH, suggesting that binding may be due mostly to the un-ionized form of [3H]PCP. This high-affinity [3H]PCP binding was sensitive to the actions of trypsin, protease, and dicyclohexylcarbodiimide, but insensitive to phospholipase A, concanavalin A,N-ethylmaleimide, and dithiothreitol. Calcium channel antagonists were most potent in inhibiting the high-affinity [3H]PCP binding with the following descending order of potencies: bepridil greater than nicardipine = diltiazem = verapamil greater than cinnarizine greater than (+)-D-600 greater than (-)-D-600 greater than 4-NO2-nifedipine greater than 2-NO2-nifedipine. The binding was also highly sensitive to several PCP analogues, antipsychotics, piperocaine , and tilorone, and moderately sensitive to d-tubocurarine, atropine, imipramine, nortryptyline , and tetracaine. Although verapamil and nifedipine inhibited the action potential of crayfish muscle, PCP did not and actually prolonged slightly the falling phase of the action potential. Although it is unlikely that the [3H]PCP binding protein in crayfish muscles is a Ca2+ channel, it is possible that it may be a K+ channel.

Action Potentials↗
PubMed1983

Neurotransmitter receptors as targets for pesticides.

Nicotinic and muscarinic acetylcholine (ACh) receptors have been identified biochemically by means of their specific binding of [3H] alpha-bungarotoxin ([3H]alpha-BGT) and [3H]quinuclidinyl benzilate, respectively. There are some differences in the drug specificities, and sensitivities to active group reagents, of these receptors in insects when compared to those in vertebrates. Also, insect brain contains more nicotinic than muscarinic receptors, while the reverse is found in mammalian brain. Insect brain contains a third kind of putative ACh-receptor that is relatively soluble and is both nicotinic and muscarinic in its pharmacology but does not bind alpha-BGT. Toxic nicotine and analogs bind to it with high affinities. Several organophosphorus and carbamate insecticides and nereistoxin bind with high affinities to the nicotinic ACh-receptor of the electric organ of Torpedo. A few chlorinated hydrocarbon insecticides and derivatives interact with Torpedo nicotinic ACh-receptors, not at their 'receptor' sites but at their allosteric or 'channel' sites (which are identified by their specific binding of [3H]perhydrohistrionicotoxin). A few also bind to mammalian brain muscarinic receptors. The most potent on both receptors is the acaricide chlorobenzilate. Pyrethrins and synthetic pyrethroids also bind with high affinities to the channel sites of the Torpedo nicotinic ACh-receptor, though not to its receptor sites. Another group that binds to ACh-receptors is the organic and inorganic mercury compounds, which interact with both the Torpedo nicotinic and rat brain muscarinic receptors. Thus, neurotransmitter receptors act as molecular targets, primary or secondary for different pesticides.

Acetylcholinesterase↗
PubMed1982

Allethrin interactions with the nicotinic acetylcholine receptor channel.

Interactions of the synthetic pyrethroid allethrin with the nicotinic acetylcholine (ACh) receptor/channel were studied in membranes from Torpedo electric organ. Allethrin did not inhibit binding of [3H]ACh to the receptor sites, but inhibited noncompetitively binding of [3H]perhydrohistrionicotoxin ([3H]H12-HTX) to the ionic channel sites in a dose-dependent manner. The inhibition constant (Ki) of [3H]H12-HTX binding in absence of receptor agonist was 30 micro M, while in presence of 100 micro M carbamylcholine it was 4 micro M. This inhibitory effect of allethrin had a negative temperature coefficient. The high affinity binding of allethrin to the channel sites of the nicotinic ACh-receptor may be indicative of a postsynaptic site of action for pyrethroids, in addition to their known action on the sodium channel.

Acetylcholine↗
PubMed1982

Labelled-replica techniques: post-shadow labelling of intramembrane particles in freeze-fracture replicas.

Three methods are described for direct post-fracture, post-shadow labelling of individual classes of intramembrane particles (IMPs) in freeze-fracture replicas of biological membranes. The P-face IMPs corresponding to the acetylcholine receptor complexes (AChRs) of vertebrate neuroeffector junctions are identified by post-replication labelling with ferritin-antibody complexes and with neurotoxin-biotin-avidin-colloidal gold affinity ligands. (The freeze-etch nomenclature of Branton et al., 1975, is used in this report.) These post-shadow labelling techniques resemble conventional en bloc labelling techniques except that the labelling reagents must penetrate a thin but discontinuous layer of platinum superimposed on the molecules of interest. In the 'sectioned labelled-replica technique', the replicated and labelled tissues are stained, embedded in plastic and sectioned parallel to the replica-tissue interfaces. In the direct 'labelled-replica techniques', the replicated and labelled samples are freeze-dried or critical point dried, the labelled surfaces are stabilized by carbon coating, and the underlying tissues are dissolved, allowing the labelled-replicas to be examined as conventional freeze-fracture replicas. The unshadowed side of each AChR IMP is shown to retain sufficient biochemical information to permit both immunospecific and neurotoxin specific labelling despite formaldehyde fixation, freezing, fracturing, platinum shadowing, and thawing in aqueous media. A new mixed ferricyanide-osmium staining method reveals electron opaque structures spanning the membrane bilayer in the same size, number and distribution as the labelled IMPs. These experiments demonstrate the feasibility of identifying individual IMPs in freeze-fracture replicas and may allow the identification of specific membrane lesions in human disease.

Animals↗
PubMed1982

Pumiliotoxin-C and synthetic analogues. A new class of nicotinic antagonists.

Pumiliotoxin-C (PTX-CI), a cis-decahydroquinoline alkaloid, and synthetic analogues inhibited directly and indirectly elicited twitches in rat phrenic nerve diaphragm preparations. At 40 microM the most potent analogue, 2,5-di-n-propyl-cis-decahydroquinoline (PTX-CII), reversibly blocked the indirectly elicited twitch and markedly depressed the amplitude of end-plate potentials and miniature end-plate potentials without affecting muscle membrane potential. PTX-CII did not depress the directly elicited twitch in diaphragm muscle treated with alpha-bungarotoxin. At 40 microM, the compound blocked extrajunctional acetylcholine (ACh) sensitivity in chronically denervated soleus muscle of the rat. The peak amplitude of the end-plate current in frog sartorius muscle was depressed by PTX-CII at both positive and negative membrane potentials. Nonlinearity in the current-voltage relationship appeared at membrane potentials greater than -100 mV with concentrations of PTX-CII from 2.5 to 7.5 microM. The decay phase of the end-plate current was shifted to lower values such that it decayed faster at all membrane potentials but retained voltage sensitivity. PTX-CII, the most potent of the analogues, inhibited binding of [3H]perhydrohistrionicotoxin to Torpedo electroplax membranes with an IC50 value of 1.5 microM in the absence of carbamylcholine (Carb) and 0.1 microM in the presence of Carb. The other analogues were less potent, but all showed higher affinities in the presence of Carb. PTX-CI, PTX-CII, and PTX-CIII had no effect on the binding of [125I]alpha-bungarotoxin to ACh receptors in Torpedo membranes while slightly enhancing the binding of [3H]ACh, whereas PTX-CIV gave slight inhibition of both receptor ligands at concentrations greater than 10 microM. The pumiliotoxin-C class of alkaloids therefore appears to block neuromuscular transmission primarily via interactions with sites associated with the ion channel controlled by the ACh receptor.

Acetylcholinesterase↗
PubMed1982

Interactions of d-tubocurarine with the nicotinic acetylcholine receptor/channel molecule.

The interactions of d-tubocurarine (d-TC) with the ionic channel of the nicotinic acetylcholine receptor were studied by biochemical methods in Torpedo electric organ membranes and by electrophysiological methods on frog sciatic nerve-sartorius muscle preparation. Torpedo membranes were treated with alpha-bungarotoxin to inhibit the acetylcholine receptor sites, then binding of [3H]perhydrohistrionicotoxin to the ionic channel sites was studied and found to be inhibited by d-TC. At 37 degrees C, the Ki of d-TC was 10 microM, and at 22 degrees C it was 100 microM. The affinity of d-TC for the ionic channel sites relative to that of perhydrohistrionicotoxin was constant at temperatures from 2-20 degrees C, but increased at higher temperatures up to 37 degrees C. The peak endplate current amplitude was depressed with 1 to 2 microM d-TC in a voltage-dependent manner, with considerable departure from linearity at 10 and 30 degrees C. The effect of d-TC on spontaneous miniature endplate currents was similar and slightly more potent. The time constant of endplate current decay was decreased by d-TC (1 and 2 microM) at temperatures of 10, 15 and 30 degrees C. The channel lifetime was reduced by d-TC, but channel conductance was unaffected. It is suggested that d-TC interacts with both the acetylcholine receptor sites as well as its ionic channel sites in closed and open conformations.

Amphibian Venoms↗
PubMed1982

Nicotinic acetylcholine receptor desensitization studied by [3H]perhydrohistrionicotoxin binding.

Desensitization of the nicotinic acetylcholine (ACh) receptor of Torpedo electric organs was studied by monitoring activated [3H]perhydrohistrionicotoxin binding to the channel sites of the receptor. Receptor desensitization was produced by incubating Torpedo membranes that had been pretreated with diisopropylfluorophosphate with ACh and was quenched by the addition of AChesterase. Desensitization caused concentration- and time-dependent decrease in the binding of [3H]perhydrohistrionicotoxin to carbamylcholine-activated receptors and an increase in the apparent affinity of carbamylcholine to the receptor. d-Tubocurarine and concanavalin A antagonized the desensitizing effects of ACh. The kinetics of this desensitization showed an extremely rapid temperature- and concentration-dependent component which was complete by the earliest measurement (i.e., 6 sec) and a much slower component (T1/2 = 55 sec) which reached a plateau after 120 sec. The rate of this slow component was temperature- and concentration-independent. Recovery from desensitization was temperature dependent, but independent of the ACh concentration used for desensitization. It also showed biphasic kinetics: half the receptors recovered fast (T1/2 = 36 sec), whereas the other was slow (T1/2 = 180 sec).

Acetylcholinesterase↗