Regulation of the Muscarinic Acetylcholine Receptor: Effects of Phosphorylating Conditions on Agonist and Antagonist Binding

Incubation of rat brain synaptic membranes under phosphorylating conditions (i.e., in the presence of Mg2+, ATP, and cyclic AMP) leads to a loss in muscarinic acetylcholine receptors, detectable as specific binding of the muscarinic antagonist L-[3H]quinuclidinyl benzilate. A role for protein phosphorylation in this receptor loss is indicated by the finding that 5'-adenylyl imidodiphosphate, a nonhydrolysable analogue of ATP, does not support receptor loss. Furthermore, receptor loss is inhibited by adenosine and 2-deoxyadenosine, both of which inhibit protein kinase activity. The loss of muscarinic receptors is calmodulin dependent, and it has been demonstrated here that this requirement is probably at the level of calmodulin-dependent phosphorylation. An investigation of the effects of phosphorylation on the binding of the agonist carbachol to synaptic membranes from the cortex and cerebellum demonstrated that phosphorylation altered the relative proportions of the super-high-, high-, and low-affinity binding sites. The results were consistent with an apparent conversion of high- into super-high-affinity sites. In the presence of 5'-guanylyl imidodiphosphate, agonist binding demonstrated the properties expected of a population of largely low-affinity sites. This conversion of super-high- and high-affinity sites into low-affinity sites by 5'-guanylyl imidodiphosphate was partially inhibited by phosphorylation.     

Calcium- and Calmodulin-Dependent Phosphorylation of Diphosphoinositide in Acetylcholine Receptor-Rich Membranes from Electroplax of Narke japonica

The phosphorylation of phosphoinositides in the acetylcholine receptor (AChR)-rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR-rich membranes were incubated with [gamma-32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri- and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR-rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P-incorporation into these lipids was realized by the membrane-bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane-bound DPI kinase was activated by Ca2+ (greater than 10(-6) M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+-dependent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the phosphorylation of DPI in the AChR-rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+- and calmodulin-dependent DPI kinase responding to the change in the intracellular Ca2+ level.     

Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

Continuous Determination by a Chemiluminescent Method of Acetylcholine Release and Compartmentation in Torpedo Electric Organ Synaptosomes

Abstract: The detection of acetylcholine (ACh) with a chemiluminescent procedure enables one to follow continuously the release of transmitter from stimulated synaptosomes and to study the compartmentation of ACh in resting and active nerve terminals. A compartment of ACh liberated almost entirely by a single freezing and thawing could be directly measured and compared with a compartment of ACh resistant to several cycles of freezing and thawing but liberated by a detergent (60–70% of the total). It is the compartment liberated by freezing and thawing that is reduced when synaptosomes are stimulated. Up to half the total synaptosomal ACh content is readily releasable provided the calcium entry is maintained, or if a strong releasing agent such as the venom of Glycera convoluta is used. In addition, it is shown that synaptosomes contain only negligible amounts of choline, and that the proportion of the two ACh compartments is not influenced by changing extracellular calcium just before their determination.     

Gastrocnemius Muscle Lipids in Relation to Diet in Two Mouse Mutants, 129Re-dy and A2G-adr, with Abnormal Muscle Function

The lipids of gastrocnemius muscle from normal and dystrophic (dy) mice of the Bar Harbor, 129Re strain were studied. Animals were fed diets containing either 3.1% or 1.1% of total calories as linoleic acid. Lipid analyses were also done on muscle from a new mouse mutant, A2G-adr, which has abnormal muscle function, characterised by an arrested development of the righting response. These animals were fed the "high" linoleic acid diet only. Total lipid, triacylglycerol, and cholesterol were elevated in the 129Re-dy irrespective of the diet, whereas A2G-adr possessed significantly higher levels of cholesterol. Total phosphorus (micrograms P/g muscle) and cholesterol/phospholipid ratios were elevated in the dy strains only. Cardiolipin was raised in the dy ("low" linoleic diet) and adr muscle, whereas phosphatidylcholine was lower in the adr strain only. Linoleic acid esterified to phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine was elevated whereas arachidonic acid in phosphatidylserine was decreased in both mutants. Docosahexanoic acid (22:6) in all three dy phospholipids was decreased, independent of dietary treatment. The adr strain possessed normal levels of this fatty acid. The results specifically point to an abnormality in long-chain polyunsaturated fatty acid metabolism in gastrocnemius muscle in the 129Re-dy mutant; in the adr mutant they could reflect an abnormal increase in the number of muscle mitochondria.     

Effect of Soman and Sarin on Phosphatidylcholine Asymmetry in the Electroplax from the Electric Eel

Some effects of organophosphorus anticholinesterase compounds that are unrelated to cholinesterase inhibition and that are sometimes long lasting may be due to alterations at the cellular membrane level. Phosphatidylcholine exchange protein was used to assess the effects of sarin and soman on phosphatidylcholine asymmetry in the inner and outer leaflets of the plasma membrane bilayer of the electroplax. Exposure of electroplax (30 min in vitro) to soman (10(-4), 10(-6) M) or sarin (10(-4), 10(-6), 5 x 10(-9) M) increased the percentage of phosphatidylcholine in the outer monolayer of the innervated plasma membrane bilayer and decreased the percentage in the inner monolayer. These changes by sarin were observed at concentrations that produced 100% cholinesterase inhibition (10(-4), 10(-6) M) and at a concentration (5 x 10(-9) M) where no inhibition occurred, suggesting that these effects are not directly due to cholinesterase inhibition. A 1-week exposure of live eels to soman (10(-8) M) in vivo caused an increase in phosphatidylcholine labeling in the outer monolayer of the innervated and noninnervated surfaces of the electroplax. Two weeks after stopping exposure to soman, increased labeling was still observed, suggesting that this may be a long-term effect. Because the organophosphates did not increase the permeability of the electroplax, all of these changes in labeling appear to be due to a redistribution of phosphatidylcholine from the inner to the outer monolayer of the plasma membrane bilayer.     

Comparative Thermodynamics of Benzodiazepine Receptor Ligand Interactions in Rat Neuronal Membranes

The effects of temperature on the interaction of various ligands with the benzodiazepine receptor were studied in rat brain membrane preparations. The affinities of all ligands studied were reduced on raising the temperature from 4 to 37 degrees C. The variation of affinity constant with temperature deviated from the classical relationship for both the anticonvulsant ligand [3H]flunitrazepam and the proconvulsant ligand [3H]ethyl beta-carboline-3-carboxylate. This implies a variation of observed enthalpy change of binding with temperature. Possible reasons for this are discussed. Gamma-Aminobutyric acid and sodium chloride both enhance the binding of [3H]flunitrazepam--the former by an increase in the entropic component of the binding energy, and the latter by an increase in the enthalpic component. In a series of ligands of different biological activities, no simple correlation was observed between biological activity and temperature dependence of binding.     

Calcium-Independent Release of Acetylcholine from Electric Organ Synaptosomes and Its Changes by Depolarization and Cholinergic Drugs

Chemiluminescent detection was applied to measure the continuous spontaneous Ca2+-independent liberation of acetylcholine (ACh) from Torpedo electric organ synaptosomes. Differentiation between the release of ACh and choline was achieved by inhibiting cholinesterases with phospholine, and a way to quantify the continuous release was devised. The method permitted measurements during short time intervals from minute amounts of tissue and without an accumulation of ACh in the medium. Synaptosomes continuously liberated small amounts of ACh during incubations in the presence of 3 mM K+ and in the absence of Ca2+. The spontaneous liberation of ACh was similar both quantitatively and qualitatively at pH values of 8.6 and 7.8. It was unaltered by MgCl2 (10.4 mM), 2-(4-phenylpiperidino)cyclohexanol (10 microM), ouabain (104 microM), atropine (10 microM), and valinomycin (102 nM). Carbamoylcholine brought about a decrease, which could be partially reversed by atropine. The Ca2+-independent output of ACh was increased considerably when the concentration of K+ ions was raised (eightfold at 103 and 35-fold at 203 mM K+). Carbamoylcholine (104 microM) blocked the increase in ACh release produced by high K+; this effect of carbamoylcholine was not reversed by atropine (10 microM). When Ca2+ was added to synaptosomes depolarized by a high concentration of K+, the amount of ACh released during the first 1-3 min after the addition of Ca2+ was at least 20 times higher than in the absence of Ca2+, but the release returned rapidly to predepolarization values. Similarly high values of ACh release could be achieved by adding Ca2+ plus the ionophore A23187 and even higher values by adding Ca2+ plus gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of Acetate into Acetylcholine, Acetylcarnitine, and Amino Acids in the Torpedo Electric Organ

The metabolism of acetate was investigated in the nerve-electroplaque system of Torpedo marmorata. In intact fragments of electric organ, radiolabeled acetate was incorporated into acetylcholine (ACh), acetylcarnitine (ACar), and three amino acids: aspartate, glutamate, and glutamine. These compounds were identified by TLC, high-voltage electrophoresis, column chromatography, and enzymic tests. The system responsible for acetate transport and incorporation into ACh displayed a higher affinity but a lower Vmax than that involved in the synthesis of ACar and amino acids. Choline, when added to the medium, increased the rate of acetate incorporation into ACh but decreased (at concentrations greater than 10(-5) M) that into ACar and amino acids. Monofluoroacetate slightly depressed ACh and ACar synthesis from external acetate but inhibited much more the synthesis of amino acids. During repetitive nerve stimulation, the level of the newly synthetized [14C]ACh was found to oscillate together with that of endogenous ACh, but the level of neither [14C]ACar nor the 14C-labeled amino acids exhibited any significant change as a function of time. This means that there is probably no periodic transfer of acetyl groups between ACh and the investigated metabolites in the course of activity. Acetate metabolism was also tested in the electric lobe (which contains the cell bodies of the neurons innervating the electric organ) and in Torpedo synaptosomes (which are nerve terminals isolated from the same neurons). Radioactive pyruvate and glutamine were also assayed in some experiments for comparison with acetate. These observations are discussed in connection with ACh metabolism under resting and active conditions in tissues where acetate is the preferred precursor of the neurotransmitter.     

Effects of Steroid Exposure on Ligand Binding and Functional Activities of Diverse Nicotinic Acetylcholine Receptor Subtypes

Abstract: Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. The present study tests whether nAChR are potential targets for steroids. Acute or short-term (5 min) preexposure to steroids such as progesterone (which acts most potently), estradiol, corticosterone, or dexamethasone inhibits function of human muscle-type (α1β1γδ) or ganglionic (α3β4) nAChR measured using ⁸⁶Rb⁺ efflux assays in TE671/RD clonal or SH-SY5Y neuroblastoma cells. Absolute (high nanomolar to intermediate micromolar range) and rank-order potencies for steroid-mediated functional inhibition are similar across nAChR subtypes but differ for some steroid derivatives. At concentrations that produce blockade of nAChR function, steroids do not affect binding of radioligands such as ¹²⁵I-labeled α-bungarotoxin or [³H]acetylcholine to muscle-type or ganglionic nAChR or to neuronal toxin-binding nAChR that contain α7 subunits (α7-nAChR). Steroid-mediated blockade of nAChR function is insurmountable by increasing agonist concentrations, and cell-impermeant progesterone:bovine serum albumin conjugates have full potency as inhibitors of ganglionic or muscle-type nAChR function. Chronic (48 h) exposure to progesterone or estradiol, but not the other steroids, also produces blockade of nAChR function, without significant effects on numbers of nAChR radioligand-binding sites. Collectively, these results suggest that steroids act noncompetitively at extracellular sites to inhibit nAChR function with unique potencies for different steroid-nAChR subtype combinations. Thus, nAChR could be among the targets mediating physiologically relevant effects of steroid action in the nervous system.     

发菜膜脂及其脂肪酸组成

对野生发菜（Ｎｏｓｔｏｃ ｆｌａｇｅｌｌｉｆｏｒｍｅ Ｂｏｒｎ．ｅｔ Ｆｌａｈ）的膜脂（主要成分为类囊体膜脂）及其脂肪酸组成进行了测定分析。发菜的膜脂由单半乳糖甘油二酯（ＭＧＤＧ）、双半乳糖甘油三酯（ＤＧＤＧ）、磷酯酰甘油（ＰＧ）和硫代异鼠李糖甘油二酯（ＳＱＤＧ）组成,其酯酰基连接有棕榈酸（１６：０）、十六碳烯酸（１６：１）、硬脂酸（１８：０）、油酸（１８：１）、亚油酸（１８：２）和亚麻酸（１８：     

Alteration in Fatty Acid Composition of Adult Rat Brain Capillaries and Choroid Plexus Induced by a Diet Deficient in n-3 Fatty Acids: Slow Recovery After Substitution with a Nondeficient Diet

Wistar rats were fed for three generations with a semisynthetic diet containing either 1.5% sunflower oil (940 mg% of C18:2n-6, 6 mg% of C18:3n-3) or 1.9% soya oil (940 mg% of C18:2n-6, 130 mg% of C18:3n-3). At 60 days of age, the male offspring of the third generation were killed. The fatty acyl composition of isolated capillaries and choroid plexus was determined. The major changes noted in the fatty acid profile of isolated capillaries were a reduction (threefold) in the level of docosahexaenoic acid and, consequently, a fourfold increase in docosapentaenoic acid in sunflower oil-fed animals. The total percentage of polyunsaturated fatty acids was close to that in the soya oil-fed rats, but the ratio of n-3/n-6 fatty acids was reduced by threefold. In the choroid plexus, the C22:6n-3 content was also reduced, but by 2.6-fold, whereas the C22:5n-6 content was increased by 2.3-fold and the ratio of n-3/n-6 fatty acids was reduced by 2.4-fold. When the diet of sunflower oil-fed rats was replaced with a diet containing soya oil at 60 days of age, the recovery in content of n-6 and n-3 fatty acids started immediately after diet substitution; it progressed slowly to reach normal values after 2 months for C22:6n-5 and 2.5 months for C22:6n-3. The recovery in altered fatty acids of choroid plexus was also immediate and very fast. Recovery in content of C22:5n-6 and C22:6n-3 was complete by 46 days after diet substitution.     

Effect of Inoculation with Various Rhizobia Strains on the Fatty Acid Composition of Peribacteroid and Bacteroid Membranes of the Nodule Symbiosomes

The fatty acid (FA) composition of bacteroid and peribacteroid membranes was studied in the symbiotic pairs differing in their nitrogen-fixing efficiency; the results are compared with the FA composition of plasmalemma and free-living rhizobia. The experiments involved lupine plants inoculated with strains of Bradyrhizobium lupini359a (Nod⁺Fix⁺) and 400 (Nod⁺Fix L) manifesting high and low nitrogen-fixing efficiency, respectively, and broad bean plants inoculated with strains of Rhizobium leguminosarum97 (Nod⁺Fix⁺) and 87 (Nod⁺Fix L) of high and low nitrogen-fixing efficiency, respectively. We showed that the rhizobia of the strains 359a and 97 were able to form nodules with peribacteroid membranes containing FA mainly or exclusively of plant origin. These strains were able to develop effective symbiotic pairs with legume plants. The use of strains 400 and 87 resulted in the formation of nodules with peribacteroid membranes containing typical bacterial (branched-chain) FAs; these strains were characterized by an ineffective symbiosis.     

Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor

Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right-side-out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low-affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed.     

Novel Pharmacological Sensitivity of the Presynaptic Calcium Channels Controlling Acetylcholine Release in Skate Electric Organ

Abstract: The presynaptic terminals of skate ( Raja montagui ) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca²⁺ concentrations triggered by K⁺ depolarisation were studied. ACh release was measured as ³H efflux from slices of organ prelabelled with [³H]choline. Depolarisation caused a marked, Ca²⁺-dependent increase in ³H efflux that was completely blocked by 100 µ M Cd²⁺ and by 300 n M ω-conotoxin-MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC₅₀ of ∼20 n M ) and reversible. No inhibition was seen with nifedipine (5 µ M ) or the two other peptide antagonists studied: ω-conotoxin-GVIA (GVIA) at 5 µ M and ω-agatoxin-IVA (Aga-IVA) at 1 µ M . In a "nerve plate" preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca²⁺ concentrations were measured by microfluorimetry using fluo-3. An increase in fluorescence, indicating a rise in the free [Ca²⁺], rapidly followed K⁺ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 µ M ), GVIA (5 µ M ), and Aga-IVA (300 n M ) but almost completely abolished by MVIIC (1 µ M ). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.     

Unsaturated Free Fatty Acids Increase Benzodiazepine Receptor Agonist Binding Depending on the Subunit Composition of the GABAA Receptor Complex

Abstract: It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABA_A receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABA_A/benzodiazepine receptor complexes formed by different subunit compositions (α_xβ_yγ₂, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10^?4M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (>200% of control values) the binding of [³H]flunitrazepam ([³H]FNM) to the α₃β₂γ₂ receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [³H]FNM binding to α₁β_xγ₂ and α₂β_xγ₂ receptor combinations were observed, and weak effects (130% of control values) were detected using the α₅β₂γ₂ receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [³H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [³H]FNM only if an α₁β₂γ₂ receptor combination was used. Given the heterogeneity of the GABA_A/benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.     

Dietary ratio of n-6 to n-3 polyunsaturated fatty acids and periodontal disease in community-based older Japanese: a 3-year follow-up study

The longitudinal relationship between dietary n-6 to n-3 PUFAs ratio and periodontal disease in 235 Japanese subjects for whom data were available for the years 2003-2006 was investigated. PUFAs intake was assessed at baseline with a brief-type self-administered diet history questionnaire. Full-mouth periodontal status, measured as the clinical attachment level (CAL), was recorded at baseline and once a year for 3 years. The number of teeth with a change in the loss of CAL ≥3 mm at any site over a year was calculated as ‘periodontal disease events’. Poisson regression analysis was conducted, with dietary n-6 to n-3 PUFAs ratio as the main predictor, to estimate its influence on periodontal disease events.A high dietary n-6 to n-3 PUFAs ratio was significantly associated with greater number of periodontal disease events. The findings suggest the dietary n-6 to n-3 PUFAs ratio is associated with periodontal disease among older Japanese.     

A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies

Several studies have shown that the fatty acid composition of circulating lipids reflects dietary fat intake, in turn being related to health status. The fatty acid composition of plasma lipids is therefore an important parameter in studies on dietary interventions. The aim of our study was to develop a rapid and inexpensive method for the analysis of circulating fatty acids applicable to large population groups. Drops of blood collected from fingertips have been directly subjected to transmethylation for gas chromatography analysis. This new method, validated for reproducibility, has been compared with the conventional method, based on withdrawal of blood from the antecubital vein followed by lipid extraction, and identical data have been obtained with the two techniques. Observed and predicted differences between blood and plasma fatty acids are related to the contribution of circulating cell membranes in blood. Finally the application of the methods to samples from 100 healthy subjects and the assessed correlation between dietary habits and blood fatty acid profiles demonstrate the validity of the new method and its applicability to nutritional and epidemiological studies.     

Calcium-Dependent and -Independent Acetylcholine Release from Electric Organ Synaptosomes by Pardaxin: Evidence of a Biphasic Action of an Excitatory Neurotoxin

Abstract: The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 × 10⁻⁷ M ), the release was calcium-dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 × 10⁻⁷ M to 10⁻⁵ M , the pardaxin-induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10⁻⁶ M pardaxin-treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations ( 10⁻⁵ M ), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium-dependent mechanisms of neurotransmitter release in the nervous system.     

Evidence for an Involvement of Membrane Lipids in the Control of Neuronal Nicotinic Receptor Function Using Bungarotoxin II-S1

Previous work has shown that a toxin fraction, bungarotoxin (BGT) II-S1, isolated from Bungarus multicinctus venom could inhibit nicotinic receptor-mediated function. Experimental evidence suggested that this effect of the toxin might be due to a direct interaction of the toxin at the acetylcholine binding site and/or to its phospholipase activity. The toxin's enzymic activity has been further characterized; it has phospholipase activity of the A2 type with a Vmax of 12 pmol/min/ng protein and a Km of 300 microM. Phospholipases can produce their effects on a tissue through a variety of mechanisms including the disruption of important lipid protein bonds or the production of free fatty acids which interact with the tissue. To test for this latter possibility, various concentrations of fatty acid-free bovine serum albumin were added to the incubation medium. Fatty acid-free bovine serum albumin partially reversed the inhibition of carbachol-stimulated 1-[1,2-3H(N)]amino-4-guanidobutane ([3H]agmatine) uptake (used as a measure of ion flux) into the ganglion produced by BGT II-S1 (1.0 microM). In an attempt to determine which fatty acids might be responsible for this effect, various fatty acids were added to the incubation medium and their effect on nicotinic receptor-mediated [3H]agmatine uptake determined. Arachidonic acid decreased amine uptake by approximately 50% over the control carbachol-stimulated uptake; linoleic and oleic acid, on the other hand, did not significantly affect the response. This observation could imply that arachidonic acid is the fatty acid produced by the action of BGT II-S1 on the tissue to mediate the toxin's inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)