Biomedical subjects
Publications and source records attributed to C J Duncan.
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The thiol-oxidizing agent diamide markedly increases m.e.p.p. frequency at the frog neuromuscular junction, even at low [Ca2+]0 and also when the mitochondria are uncoupled with DNP. The effect is reversed by dithioerythritol and is very temperature-sensitive, with a marked transition at 16 degrees C; m.e.p.p. frequency is raised 2- to 5-fold at 13-15 degrees C and 55- to 60-fold at 17-20 degrees C. Diamide increases the frequency of large amplitude m.e.p.p.s, the effect being explicable as the fusion of two or more vesicles. It is concluded that (a) diamide does not act at the Ca2+ channels of the plasma membrane, nor at the mitochondria. It affects the release system directly via an alteration of membrane protein --SH groups; (b) the eventual decline in m.e.p.p. frequency after DNP treatment is because of the exhaustion of mitochondrial Ca2+ rather than a depletion of quanta; (c) the major effect of temperature is on the release mechanism, perhaps via a phase-change in the phospholipoproteins of the plasmalemma or vesicles, rather than an elevation of [Ca2+]i; (d) either diamide or temperatures above 16 degrees C make Ca2+ more effective in promoting vesicle-plasmalemma fusion.
The characteristic effect of temperature on m.e.p.p. frequency at the amphibian neuromuscular junction is unaltered by the presence of Dantrolene (an agent that is believed to reduce the efflux of Ca2+ from intracellular stores) or by changes in [Ca2+)o. It is concluded that temperature affects the release system directly, with a transition temperature at about 16 degrees C.
Experimentally-induced rises in intracellular calcium ([Ca2+]i) promote rapid myofilament degradation in amphibian and mammalian cardiac muscle strips. The relevance of these studies to the subcellular injury produced by ischaemia, the possible involvement of lysosomal enzymes and similarities with skeletal muscle are discussed.
Exposure of the frog neuromuscular junction at 17 degrees C or 23 degrees C to salines with low [Ca2+], buffered with EGTA, cause mepp frequency to fall after 4--6 min to about 20% of the control rate. Results obtained in the presence of verapamil suggest that this fall is a consequence of a lower Ca2+-influx coupled with the action of the extracellular EGTA in promoting Ca2+-efflux from the terminals. These findings confirm the suggestion that [Ca2+]i has a major role in determining mepp frequency. At 13 degrees C, the fall in mepp frequency after addition of EGTA is preceded by a transient (1--2 min) rise in mepp rate which is not present at 17 degrees C or in the presence of verapamil. This transient acceleration in spontaneous release is believed to be because EGTA promotes the emptying of a Ca2+-reservoir on or beneath the inner face of the membrane, thus causing a rapid Ca2+-efflux via the Ca2+-sensitive sites that trigger exocytosis of transmitter. The significance of the sensitivity of the response to temperature is discussed. The suppressive effect of higher temperatures can be reversed to some extent by hyperosmotic salines, an effect that may reflect the action of hypertonicity on the plasmalemma. It is concluded that the characteristics of the release system may change markedly at about 16 degrees C. Ca2+-EGTA buffers are widely used; it is suggested that extracellular EGTA can also modify [Ca2+]i in cellular systems.
1. The possibility that rapid Ca2+-uptake by skeletal muscle mitochondria may cause local reductions in pHi (by H+/Ca2+ exchange) and so promote lysosomal breakdown has been explored using amphibian and mammalian preparations. Recent studies suggested that such a sequence of events is possible in cardiac muscle. 2. However, extensive muscle damage can still be initiated in skeletal muscle when the mitochondria are uncoupled so that Ca2+-uptake is prevented. 3. DNP alone induces extensive myofilament degradation which is similar to that produced by A23187 and caffeine and described previously. 4. It is suggested that (a) the known action of DNP in promoting lysosomal labilization in living cells is produced by mitochondrial uncoupling and the release of stored Ca2+, (b) raised [Ca2+]i promotes lysosomal breakdown in skeletal muscle, so that the hydrolases released effect myofilament dissolution rapidly. 5. DNP also rapidly causes septation and division of the mitochondria in mammalian skeletal muscle.
The rapid formation (in less than 45 min) of internal septa and the apparent subdivision in situ of mitochondria for cardiac and skeletal muscle are described following a variety of experimental treatments. For example, the ionophore A23187, caffeine, DNP, ruthenium red, and the insecticide lindane have been applied to intact, glycerinated, and chemically skinned skeletal muscle fibers and to cardiac muscle strips from both amphibians and mammals. In some mitochondria, the two compartments are in the same configurations; in others they are different. The significance of these mitochondrial septa is discussed, and it is suggested that the findings are consistent with the hypothesis that a variety of experimental procedures can promote rapid mitochondrial division.
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The actions on amphibian embryos of UV-irradiation, exposure to Li+ or exposure to ouabain show interesting parallels with their effects on spontaneous release at the presynaptic terminals of the neuromuscular junction. It is suggested that these treatments serve to raise intracellular Ca2+ ([Ca2+]i) in these examples, and that UV-promoted abnormalities in embryogenesis are a consequence of changes in [Ca2+]i at critical stages in development.
Verapamil is known to reduce Ca2+ entry in a variety of cells. At 10(-5) M it produces a small reduction in MEPP frequency at the frog neuromuscular junction, whereas the rate of spontaneous release rises following treatment at a concentration of 10(-4) M. This latter effect is augmented by raising [Ca2+]0 to 9 mM or, more especially, by raising the temperature from 17 to 23 degrees C. It is argued that both these opposing effects are related to the action of verapamil in modifying [Ca2+]i at the presynaptic terminals and it is suggested that the drug can affect both inward Ca2+ flux (so reducing the steady-state position of [Ca2+]i) and also, at higher concentration, either inhibit the membrane Ca2+ pump or cause the release of Ca2+ from intracellular Ca2+ stores (so raising [Ca2+]i).
Diamide (10-(4)M), a thiol oxidizing agent, rapidly promotes septation in isolated frog liver mitochondria an also in situ in liver slices. The effect is partially inhibited by dithioerythritol. DNP does not have this effect, and it is concluded that diamide does not promote septation via an uncoupling action. The septate mitochondria have a different appearance from typical dividing mitochondria previously described; it is suggested that (1) diamide may act by favouring the fusion of the internal membranes, and (2) -SH oxidation is of importance in mitochondria in ageing and in various pathological conditions. The ways in which septa may develop in mitochondria in the orthodox and condensed configuration are discussed.
Lindane (5 x 10(-5) M) causes a progressive and marked rise in MEPP frequency at the frog neuromuscular junction. Concentrations over a range of 5 x 10(-6) M to 5 x 10(-4) M were tested. The results suggest that it has two actions in promoting this effect. Its major effect is probably to cause an increase in Ca2+-permeability and a rise in Ca2+ entry. Its second, smaller effect, which persists in the absence of extracellular Ca2+, is probably also because of a rise in [Ca2+]i. The ways in which these effects might be produced and the significance of these findings for explaining the known pharmacological actions of lindane are discussed.
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Treatment of stage 5 Xenopus embryos with the ionophore A23187 for only 10 min, in the absence of extracellular Mg2+ and Ca2+, causes cortical contractions and a high incidence of abnormal embryos during subsequent development. Cation analysis shows that divalent ions are not lost from the embryos, but that Ca2+ is redistributed within the subcellular fractions. Ca2+ is probably released from yolk platelets and/or pigment granules by the action of A23187, [Ca2+] rises in the cytosol, and the mitochondria attempt to take up this free Ca2+. The mitochondria concomitantly undergo characteristic ultrastructural transformations, changing towards energized-twisted and energized-zigzag conformations. A23187 allows these changes to be demonstrated in situ. Extracellular divalent cations (10(-4) M) interfere with this intracellular action of A23187. Intracellular accumulation of Na+ (by treatment with ouabain) or Li+ also causes abnormal development, probably by promoting a release of Ca2+ from the mitochondria. It is suggested (a) that all these treatments cause a rise in [Ca2+]i which interferes with normal, integrated cell division, so causing, in turn, abnormal embryogenesis, (b) that levels of [Ca2+]i are of importance in regulating cleavage, (c) that the mitochondria could well have a function in regulating [Ca2+]i during embryogenesis in Xenopus, and (d) that vegetalizing agents may well act by promoting a rise in [Ca2+]i in specific cells in the amphibian embryo.
1. The cholinesterase inhibitors neostigmine, edrophonium and eserine (7 x 10(-7) M) reduced m.e.p.p. frequency by some 50% at the frog neuromuscular junction. Neostigmine also produced a small reduction in quantal content. 2. Tetraisopropylpyrophosphoramide, with a high specificity for non-specific cholinesterase, has a similar effect on m.e.p.p. frequency but ambenonium, with a high specificity for acetylcholinesterase, was markedly less effective in this respect. 3. Carbachol (10(-5) M) and the muscarinic agonists muscarine and metacholine (7 x 10(-7) M) also reduced the rate of spontaneous release. 4. The action of neostigmine was antagonized by atropine, but not by D-tubocurarine. Muscarine did not have any further effect when m.e.p.p. frequency was reduced with neostigmine. 5. Experiments with reduced extracellular Ca2+ concentration suggest that the cholinergic agents reduce Ca2+ permeability directly at the presynaptic terminals and that they do not act via a change in PNa or PK. It is suggested that the consequent reduction in Ca2+ entry causes a fall in both evoked release and in intracellular Ca2+ concentration, thereby reducing m.e.p.p. frequency. 6. It is concluded that non-specific cholinesterases present on the presynaptic terminals can act as inhibitory muscarinic cholinergic receptors. This form of presynaptic inhibition at the amphibian neuromuscular junction contrasts with that described in the mammalian preparation in which the sites are blocked by D-tubocurarine and are excitatory.