ASPECTS OF ACETYLCHOLINE METABOLISM IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA

Abstract— —The synthesis of acetylcholine and its compartmentation were studied in the electric organ of Torpedo marmorata. When electric organ was homogenized in iso-osmotic NaCl-sucrose some 55 per cent of its acetylcholine content was lost unless very potent cholinesterase inhibitors were present. Slices of electric organ incubated in a suitable medium were found to synthesize radioactive-labelled acetylcholine from [ N-Me-³ H] choline. The specific activity of the labelled acetylcholine was higher in the trichloracetic acid extract of the organ slices than in an NaCl-sucrose homogenate. Acetylcholine-containing vesicles isolated from the NaCl-sucrose homogenate contained labelled acetylcholine with about the same specific activity as the parent homogenate. There was thus a fraction of acetylcholine in the incubated tissue of higher specific radioactivity that was lost when the tissue was homogenized. The acetylcholine-containing vesicles lose their acetylcholine when submitted to gel filtration under hypo-osmotic conditions. On standing at 5°C there were only small losses of acetylcholine from the vesicles but at 20°C the losses were substantial. Vesicles containing labelled acetylcholine were studied. On gel filtration under iso-osmotic conditions there was a considerable loss of labelled acetylcholine without a concomitant loss of bio-assayable acetylcholine. The pools of radioactive and bio-assayable acetylcholine are therefore not homogeneous in the vesicles as isolated.     

THE FINE STRUCTURE OF THE ELECTRIC ORGAN OF TORPEDO MARMORATA

The fine structure of the electric organ of the fish Torpedo marmorata has been examined after osmium tetroxide or potassium permanganate fixation, acetone dehydration, and Araldite embedment. This organ consists of stacks of electroplaques which possess a dorsal noninnervated and a ventral richly innervated surface. Both surfaces are covered with a thin basement membrane. A tubular membranous network whose lumen is continuous with the extracellular space occupies the dorsal third of the electroplaque. Nerve endings, separated from the ventral surface of the electroplaque by a thin basement membrane, contain synaptic vesicles (diameter 300 to 1200 A), mitochondria, and electron-opaque granules (diameter 300 A). Projections from the nerve endings occupy the lumina of the finger-like invaginations of the ventral surface. The cytoplasm of the electroplaques contains the usual organelles. A "cellular cuff" surrounds most of the nerve fibers in the intercellular space, and is separated from the nerve fibre and its Schwann cell by a space containing connective tissue fibrils. The connective tissue fibrils and fibroblasts in the intercellular space are primarily associated with the dorsal surface of the electroplaque.     

ADENOSINE UPTAKE BY CHOLINERGIC SYNAPTOSOMES FROM TORPEDO ELECTRIC ORGAN

Pure cholinergic synaptosomes prepared from the electric organ of Torpedo are able to take adenosine up efficiently and convert it to ATP. The apparent K_m of the adenosine uptake is 2.4 μM and the V_m: 518 pmol/30′/mg prot. The uptake system exhibits a high specificity towards adenosine, as shown by the lack of competition with analogues. Tubercidin blocks the uptake competitively and dipyridamole is a very potent non competitive inhibitor (K_i= 4 × 10^-8 M). Considering that during nerve activity ATP is released extracellularly and can modulate transmitter release, the physiological significance of adenosine uptake is discussed as a possible mechanism to terminate the ATP action.     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.     

EFFECT OF CHOLINE ON THE RATES OF SYNTHESIS and OF RELEASE OF ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

Abstract— Slices of electric organ of Torpedo marmorata were chopped and incubated in a saline-urea-sucrose medium. This preparation of minced tissue exhibited a relative enrichment in ACh and nerve endings, which was attributed to a loss of electroplaque cytoplasm. Electron microscopic controls showed nerve endings of normal morphology, some of them forming 'chaplets' separated from electro-plaques. Miniature endplate potentials were recorded on sealed fragments also present in this preparation. ACh levels remained unchanged during incubation periods as long as 19 h. The time course of the incorporation of [1-¹⁴C]acetate of [2-¹⁴C]pyruvate into ACh pools was studied. These incorporations were similarly affected by the choline added to the medium. In the presence of increasing choline concentrations (up to 10^-4 m ), the incorporation of [¹⁴C]acetate or [¹⁴C]pyruvate into ACh increased. They both diminished when choline was added above 10^-4M. The ACh content of the tissue was not affected by added choline. From the constancy of ACh levels in the presence of various choline concentrations and from the steady state of our preparation, we can conclude that the release of transmitter varied in parallel to the incorporation rate of the precursor of the acetyl moiety of ACh. This fact was also found using the efflux of [¹⁴C]acetate as an evaluation of ACh release. The values of release calculated by this method were in good agreement with those determined from the incorporations of acetate and pyruvate into ACh. It is suggested that the primary action of choline is on its high affinity carrier system. This triggers a secondary action on the ACh release mechanisms.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

DIFFERENT RECOVERY RATES OF THE ELECTROPHYSIOLOGICAL, BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS IN THE CHOLINERGIC SYNAPSES OF THE TORPEDO ELECTRIC ORGAN AFTER STIMULATION

—During stimulation there occurred a decay in electrical response, vesicular acetylcholine, ATP and nucleotide as well as a loss of vesicle number and a decrease in vesicle diameter in the electric organ of Torpedo. These alterations were re-established during a subsequent recovery period. The different parameters recovered at different rates. Firstly, electrical response to single pulses recovered to prestimulation values within about 5 h. Vesicle number and diameter as well as bouton size were found to be re-established fully after 24 h. The newly formed vesicles appeared to be empty as vesicular acetylcholine, ATP and total nucleotide recovered much more slowly and were back to control values after about three days. Acetylcholine reappeared more quickly in the vesicles than ATP. Only after recovery of the vesicular pool of transmitter and ATP did the electric organ regain full stability of the electric discharge pattern on restimulation.     

亲和层析法分离腺苷结合蛋白质

本文报告一种新的腺苷亲和层析凝胶的合成方法。利用这种凝胶可从大鼠心脏、肝脏及小牛主动脉平滑肌的水溶部份分离出几种腺苷结合蛋白质,其亚基分子量(据SDS-PAGE)分别为35,000、37,000、46,000、43,000及15,300Dal。现已证明,35,000Dal蛋白质是乳酸脱氢酶及苹果酸脱氢酶,43,000Dal蛋白质是腺苷激酶,46,000Dal蛋白质可能是S-腺苷同型半胱氨酸水解酶。15,000Dal蛋白质前人未有报道。它对腺苷具有高度特导性和亲和力,推测是腺苷的细胞内受体和/或载体。测定了这种低分子量腺苷结合蛋白质的氨基酸组成及某些物理常数:pI=6.5;沉降系数2.42S,微分比容0.727cm~3/g,与腺苷复合物的解离常数K_D=2.3μM。     

BREAKDOWN OF CREATINE PHOSPHATE AND ATP IN NERVE TERMINALS AND ELECTROPLAQUES OF THE TORPEDO ELECTRIC ORGAN: COMPARISON WITH THE ELECTRICAL ENERGY DISSIPATED

Abstract— The electrical work performed by the electric organ of Torpedo was compared with the energy provided by the net breakdown of ATP and creatine phosphate (CrP). The electrical work was calculated for single impulses and for repetitive stimulations. The content in CrP and ATP was measured at different times in the course of stimulation and during the period of recovery. The chemical expenditure due to activity of the nerve terminals was distinguished from the total expenditure by the use of curare which interrupts synaptic transmission but does not interfere to any great extent with the release of acetylcholine. In the presence of curare the breakdown of phosphagen started only after more than 1 min of stimulation; it represented the loss of about 20-25% of the initial store. In untreated tissue the breakdown of CrP and ATP occurred in two phases and continued within the first minute after the end of the stimulation; as much as 77% of the phosphagen content was utilized under these conditions. The recovery of ATP and CrP was completed only 3-5 h after stimulation, a long time after the restoration of the physical capabilities of the tissue. The electrical energy dissipated during activity was smaller than the chemical energy provided by the net breakdown of phosphagens. This suggests that only a fraction of the chemical energy is utilized directly to compensate for the physical work accomplished, i.e. for the restoration of the ionic electromotive force. The electric organ also requires chemical energy for other purposes, particularly in the nerve endings where the presynaptic machinery seems to utilize an important fraction of the high energy phosphates stored in the tissue.     

CHARACTERIZATION OF MEMBRANES OBTAINED FROM ELECTRIC ORGAN OF THE ELECTRIC EEL BY SUCROSE GRADIENT FRACTIONATION AND BY MICRODISSECTION

Abstract— Two membrane fractions were obtained from electric organ tissue of the electric eel by sucrose gradient centrifugation of tissue homogenates. Electron microscopic examination showed that both fractions contained mainly vesicular structures (microsacs). Both the light and heavy fractions had a-bungarotoxin-binding capacity and Na⁺-K⁺ ATPase activity, while only the light fraction had AChE activity. The polypeptide patterns of vesicles derived from both the light and heavy fractions were examined by SDS-polyacrylamide gel electrophoresis and found to be very similar. The ratio of protein to phospholipid in the light vesicles was much lower than in the heavy vesicles, but the relative amounts of individual phospholipids in the two fractions were similar. A marked difference in the permeability of the light and heavy vesicles was observed by measuring efflux of both [¹⁴C]sucrose and ²²Na⁺, and also by monitoring volume changes induced by changing the osmotic strength of the medium. All three methods showed the heavy vesicles to be much more permeable than the light ones. Only the light vesicles displayed increased sodium efflux in the presence of carbamylcholine. The AChE in the light fraction does not appear to be membrane-bound, but is rather a soluble enzyme, detached from the membrane during homogenization, which migrates on the gradient similarly to that of the light vesicles. This is supported by the fact that the bulk of the AChE is readily removed by washing the vesicles. Moreover, under the conditions employed in our sucrose gradient separations,‘native’14 S + 18 S AChE exists in the form of aggregates which migrate very similarly to the major peak of AChE activity of tissue homogenates. Separated innervated and non-innervated surfaces of isolated electroplax were obtained by microdissection. α-Bungarotoxin-binding capacity was observed only in the innervated membrane. About 80% of the AChE was in the innervated membrane, and about 70% of the Na⁺-K⁺ ATPase in the non-innervated membrane. The data presented indicate that the light and heavy vesicle fractions separated by sucrose gradient centrifugation are not derived exclusively from the innervated and non-innervated membranes respectively, as previously suggested by others, but contain membrane fragments from both sides of the electroplax. The separation of two populations on sucrose gradients may be explained both by the differences in permeability and in protein to phospholipid ratios.     

CHARACTERIZATION OF AN α-BUNGAROTOXIN BINDING COMPONENT FROM DROSOPHILA MELANOGASTER

Abstract— We describe an α-bungarotoxin binding component from Dromphila melanoyaster that has the properties expected of an acetylcholine receptor. Toxin binding to a paniculate form of this component has been shown to be proportional to amount of extract, to be saturable and to be destroyed by heat. Localization studies using ¹²⁵I-α-bungarotoxin binding to frozen sections has shown toxin binding to be restricted to synaptic areas of the Drosophila CNS. We have also shown that this toxinbinding component can be treated with Triton X-100 without significantly altering its toxin-binding and pharmacological specificity. The ability of preincubation with cholinergic ligands to block labeled α-bungarotoxin binding to both particulate and detergent treated extracts has been studied. The nicotinic agents nicotine, d-tubocurarine, and acetylcholine are the most effective blocking agents. All of the muscarinic agents tested and the nicotinic agent decamethonium were less effective than acetylcholine in preventing α-bungarotoxin binding.     

THE MECHANISM OF α-ADRENERGIC AGONIST ACTION IN LIVER

1. Although the mechanism of action of a-adrenergic agonists in liver tissue is somewhat complex, a number of experimental approaches can be usefully employed to identify the molecular details of the events that occur. 2. Receptors specific for α₁-adrenergic agonists located on the plasma membranes of rat liver cells have been partially characterized using pharmacological agents, affinity labels and monoclonal antibodies. Much of this work has employed isolated plasma membrane fractions and does not take account of tissue-related factors which may now be studied in the intact perfused rat liver, following the development of an appropriate assay system. 3. Because a redistribution of cellular Ca²⁺ is central to the mechanism of action of a-adrenergic agonists in liver, it is important to first gain an understanding of basic cellular Ca²⁺ regulation. Knowledge about the compartmentation of cellular calcium and about Ca²⁺-translocation systems located in the mitochondria, plasma membrane and endoplasmic reticulum is now quite extensive. However, the role of mitochondria in the regulation of intracellular Ca²⁺ is still unclear; it now appears that the mitochondrial calcium content is much less than considered previously. This may have important implications for such a regulatory role. 4. The sequence of Ca²⁺ movements that may occur when a-adrenergic agonists interact with liver have been identified and are as follows: (a) Ca²⁺ is mobilized from an intracellular pool(s) (mitochondria plus endoplasmic reticulum and/or plasma membranes). (b) This elevates the cytoplasmic free Ca²⁺ concentration and leads to an efflux of the ion from the cell. (c) At this time, Ca²⁺-sensitive metabolic events in the cytoplasm are activated and an increase in Ca²⁺-cycling occurs across the plasma membrane. (d) Immediately after the hormone is withdrawn, there is a net influx of Ca²⁺ into the cell, and the intracellular Ca²⁺ pools and transmembrane fluxes are restored to the pre-induced states. In this model, Ca²⁺ movements across the plasma membrane play a key role in regulating the cytoplasmic Ca²⁺ concentration. 5. In the perfused rat liver it has been possible to define in quite precise terms the amounts and rates of Ca²⁺ mobilized in each of these stages. 6. Although several proposals for ‘second messengers’ to link the hormone-receptor interaction with initial Ca²⁺ mobilization have been made, at this time only polyphosphoinositide turnover appears to be a suitable candidate.