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Biomedical subjects

J M Ritchie

Publications and source records attributed to J M Ritchie.

At least 109 records · Page 6Linked to original sources
PubMed1975

Mechanism of action of local anaesthetic agents and biotoxins.

Four current theories for the mechanism of action of local anaesthetics are that these drugs: (1) interfere with some chemical, such as acetylcholine, that is involved in nervous conduction; (2) alter the density of fixed charges on the surface of the membrane; (3) cause an expansion of some volume of membrane that is critical for conduction; and (4) react with some specific receptor in the nerve membrane. The first theory fails for lack of evidence that acetylcholine is involved physiologically in conduction. The other three theories, however, all seem to apply.

Acetylcholine↗
PubMed1974

Evidence that tetrodotoxin and saxitoxin act at a metal cation binding site in the sodium channels of nerve membrane.

The effects of monovalent, divalent, and trivalent cations on the binding of tetrodotoxin and saxitoxin to intact nerves and to a preparation of solubilized nerve membranes have been examined. All eight divalent and trivalent cations tested, and the monovalent ions Li(+), Tl(+), and H(+) appear to compete reversibly with the toxins for their binding site. The ability of lithium to reduce toxin binding is paralleled by its ability to reduce tetrodotoxin-sensitive ion fluxes through the nerve membrane. We conclude that the toxins act at a metal cation binding site in the sodium channel and suggest that this site is the principal coordination site for cations (normally Na(+) ions) as they pass through the membrane during an action potential. The dissociation constant for Li(+) is 0.1-0.2 M and for Na(+) > 0.6 M, reflecting the weak binding necessary for rapid passage of sodium ions through the channel.

Animals↗
PubMed1974

The binding of tetrodotoxin and alpha-bungarotoxin to normal and denervated mammalian muscle.

1. The binding of [(3)H]tetrodotoxin and [(125)I]iodo-alpha-bungarotoxin to innervated and denervated rat diaphragm muscle has been measured.2. A saturable component of tetrodotoxin binding, which was inhibited by saxitoxin, was detected in addition to considerable non-saturable binding. The saturable component had an equilibrium constant of K = 6.1 nM (limits 4.7-7.8 nM) and binding capacity M = 2.5 f-mole/mg wet wt. (limits 2.1-2.8 f-mole/mg).3. If the saturable component consisted of one-to-one binding of tetrodotoxin to sodium channels, the density of sodium channels would be about 21/mum(2) of surface membrane, a figure similar to that found in other excitable membranes.4. After denervation the specific tetrodotoxin binding, as measured by the ratio M/K, fell by a factor of 2.8. This change appeared to be due to a fall in binding capacity rather than a decrease in affinity.5. After denervation the maximum rate of rise of the action potential fell by 27% and became partially resistant to tetrodotoxin. The maximum rate of rise was at first reduced by tetrodotoxin in similar concentrations to those affecting normal muscle, but even large concentrations which completely blocked normal muscle only reduced the maximum rate of rise by a factor of about 2.6. Detubulation with glycerol did not appreciably affect the tetrodotoxin sensitivity of normal or denervated muscle.7. Tetrodotoxin resistance was not observed after denervation of the frog sartorius muscle.8. [(125)I]iodo-alpha-bungarotoxin binding amounted to 3.8 +/- 0.7 f-mole/mg in innervated muscle and 44.5 +/- 2.1 f-mole/mg in denervated muscle. Most of the uptake was inhibitable by (+)-tubocurarine.9. The increase in the labelled bungarotoxin binding is much larger than the specific tetrodotoxin binding of innervated muscle, which renders implausible the possibility that the acetylcholine receptors which appear after denervation are related to the tetrodotoxin resistant-sodium channels.

Action Potentials↗
PubMed1973

9 -Tetrahydrocannabinol: effects on mammalian nonmyelinated nerve fibers.

Delta(9)-Tetrahydrocannabinol can be applied to tissue in vitro by dissolving it in Pluronic F68 and ethanol. It causes a decrease in size of the compound action potential of the nonmnyelinated fibers of the vagus nerve of the rabbit. This effect appears to be dose-related and chloride-dependent. Effects on other measurable parameters of nerve function seem to be minimal. Although the amounts required seem to be higher than those required to produce hallucinogenic effects in man, this effect is consistent with other work on Delta(9)-tetrahydrocannabinol and may ultimately account for a significant portion of the pharmacological activity of this drug.

Action Potentials↗
PubMed1973

The binding of labelled saxitoxin to the sodium channels in nerve membranes.

1. Tritium labelled saxitoxin has been prepared and purified, and its binding both to intact rabbit vagus nerves and to a solubilized preparation of garfish olfactory nerve membranes has been examined.2. In intact and solubilized nerves there is a saturable binding component of magnitude equal to that previously obtained for labelled tetrodotoxin.3. This component of bound saxitoxin is displaced competitively by tetrodotoxin, and it is concluded that the two toxins bind to the same site.4. The saturable saxitoxin (STX) interaction with the nerve membrane is reversible and can be described by the equation STX + R right harpoon over left harpoon STX.R where R is the binding site or receptor. With the solubilized preparation of garfish nerve membranes the saxitoxin-receptor reaction rates are almost four times faster than those of tetrodotoxin. The half-life of the saxitoxin-receptor complexes is 13 sec compared with 44 sec for the tetrodotoxin-receptor complex.5. A number of agents were tested for their ability to displace the labelled saxitoxin. Calcium and thallous ions each produced significant reversible reduction in binding, with apparent equilibrium dissociation constants of about 20-30 mM. Toxin binding is also inhibited reversibly in acidic solutions by protons competing with toxin for a binding site with a pK(a) of 5.6-5.9. All three ions are known to block sodium currents in myelinated nerve at similar concentrations. Our experiments indicate that they do so at the site of toxin binding.6. Lidocaine and veratrine do not affect the binding of saxitoxin.

Animals↗
PubMed1972

The interaction at equilibrium between tetrodotoxin and mammalian non-myelinated nerve fibres.

1. As a preliminary to chemical studies an estimate has been made of the equilibrium dissociation constant (K) for the interaction of tetrodotoxin (TTX) with the non-myelinated fibres of the rabbit desheathed vagus nerve.2. TTX causes a parallel shift to the right of the curves obtained when either the height or the conduction velocity of the compound action potential are plotted against the logarithm of the external sodium concentration.3. A model has been formulated based on the independence principle and the Hodgkin-Huxley theory, and the experimental results shown to be consistent with it. On this basis, and on the assumptions that one TTX molecule blocks one sodium channel, and that binding is Langmuir, K was estimated to be about 3-5 nM at about 20 degrees C. Other simple non-Langmuir models gave essentially similar low values for K.4. An alternative method of computing K that makes rather different assumptions gives a similar low value.5. Despite the low value for K, a TTX concentration of at least 100 nM is needed to block conduction completely and this seems to be related to the fact that conduction is not completely blocked until the external sodium concentration falls below 7% of its value in normal Locke.6. The minimum sodium concentration needed to support conduction increased with temperature.

Action Potentials↗
PubMed1972

The binding of labelled tetrodotoxin to non-myelinated nerve fibres.

1. Tritiated tetrodotoxin has been prepared and purified, and its binding to rabbit, lobster, and garfish non-myelinated nerve fibres examined.2. In each case a component of the binding curve was found that saturated at concentrations of a few nanomolar.3. In addition, non-specific binding, indicated by a linear dependence of the amount bound on concentration, occurred.4. The kinetics of wash-in and wash-out of the radioactive toxin were consistent with a model in which all binding was rapid and reversible and in which diffusion into and out of the nerve bundle was rate-limiting. This was shown by numerical solution of the appropriate non-linear diffusion equation. An extension of the limited biophase model that allows for non-specific binding was shown to give good semiquantitative approximations to the proper diffusion equation; and (unlike the latter) the extension was shown to have a relatively simple solution.5. A number of pharmacological agents were tested for competition with, or perturbation of, tetrodotoxin binding: sodium, calcium and hydrogen ions, lidocaine, batrachotoxin and saxitoxin. Apart from a small calcium effect, only saxitoxin, whose effect on sodium current is similar to that of tetrodotoxin, was found to interfere with binding. Increasing saxitoxin concentrations resulted in reduced amounts of tetrodotoxin binding in a manner consistent with a competition between the two toxins for the same site.

Animals↗
PubMed1971

Optical studies on the kinetics of the sodium pump in mammalian non-myelinated nerve fibres.

1. A study has been made of the changes in the fluorescence of desheathed rabbit cervical vagus nerves that occur during and after electrical stimulation of its non-myelinated fibres.2. Stimulation for 5 sec at 30 shocks/sec produces a maximal decrease, of about 1% of the resting fluorescence. Stimulation for less than 0.5 sec fails to produce responses visible above the inherent noise in the recording system.3. A pharmacological dissection (with ouabain, metabolic inhibitors, and calcium) has revealed four phases of fluorescence change:(a) under conditions where the sodium pump is functioning, there is a prolonged decrease in the fluorescence following electrical activity;(b) even in the absence of pumping the mere entry of sodium into the nerve causes an initial decrease in fluorescence;(c) the entry of calcium ions with electrical activity also causes an initial rapid decrease in fluorescence;(d) following these phases of decreased fluorescence there is a phase of increased fluorescence.4. These changes in fluorescence are related to changes in the NADH concentration in the nerve resulting from:(a) the splitting of ATP during sodium extrusion;(b) the initial binding of sodium to the sodium- and potassium-dependent ATPase, which is the sodium pump;(c) the stimulation of mitochondrial respiration by calcium that has entered during the spike; and(d) an increased glycogenolysis as a result of the calcium entry during activity.

Action Potentials↗
PubMed1971

On the control of glycogenolysis in mammalian nervous tissue by calcium.

1. A study has been made of the increase in fluorescence of the desheathed cervical vagus nerve that occurs after electrical stimulation (usually 5 sec at 30/sec) of its non-myelinated fibres.2. At room temperature this increase in fluorescence is normally masked by the decrease in fluorescence caused by mitochondrial oxidative phosphorylation. However, at higher temperatures (30-35 degrees C) the increasing fluorescence phase predominates and the net change on stimulation is an increase.3. At room temperature the increase in fluorescence is seen clearly only when ATP splitting has been prevented by ouabain, by bathing the nerve in lithium-Locke solution, or when oxidative phosphorylation has been prevented by metabolic inhibitors.4. The increasing fluorescence response is absent when calcium is removed from the external medium; it increases with increasing calcium concentration.5. It is argued that the increasing fluorescence response is due to an increase in glycogenolysis (leading to an increase in the reduced pyridine nucleotide concentration) brought about by the increased calcium entry during the action potential. This calcium presumably increases the activity of phosphorylates a or phosphofructokinase.6. Calcium entry also speeds mitochondrial oxidative phosphorylation.

Action Potentials↗
PubMed1971

The binding of tetrodotoxin to nerve membranes.

1. The% reduction in size of the externally recorded action potential produced by concentrations of tetrodotoxin (TTX) in the range 6-300 nM was determined for the small non-myelinated fibres of the rabbit cervical vagus nerve and of the walking leg nerves of crab and lobster. The concentration of TTX for 50% reduction was around 80 nM for rabbit vagus and 14 nM for crab nerve.2. Bio-assay procedures were devised to measure the amount of TTX taken up by a nerve when it was exposed to a very small volume of a solution whose TTX content was just great enough to produce 100% block of conduction. The extracellular space of each nerve was determined with [(14)C]sugar so that an allowance could be made for extracellular dilution.3. The TTX binding by rabbit, crab and lobster nerve was respectively 0.064, 0.053 and 0.036 p-mole/mg wet weight of nerve.4. The binding of saxitoxin was measured in rabbit vagus nerve, and found to be much the same as that of TTX.5. Control experiments on rabbit sciatic nerve, where the area of excitable membrane was much smaller, showed that there was relatively little unspecific binding of TTX.6. In view of the evidence presented here and elsewhere that the blocking of sodium conductance by TTX involves the attachment of only one TTX molecule at each sodium site, and that unspecific binding of TTX does not cause serious errors, these results suggest that in 1 mum(2) of nerve membrane the number of sodium sites is 75 for rabbit, 49 for crab, and 36 for lobster nerve.

Action Potentials↗