The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft.

Abstract

SUMMARY1. The kinetics of tubocurarine inhibition were studied at the post-synaptic membrane of frog skeletal muscle fibres. Acetylcholine (ACh) and (+ )-tubocurarine were ionophoresed from twin-barrel micropipettes, and the membrane potential of the muscle fibre was recorded intracellularly. Tubocurarine-receptor binding was measured by decreases in the response to identical pulses of ACh. 2. The responses to both ACh and tubocurarine had brief latencies and reached their maxima rapidly. It is suggested that under these conditions the kinetics of tubocurarine action are not slowed by diffusion in the space outside the synaptic cleft.3. After a pulse of tubocurarine, recovery from inhibition proceeds along a roughly exponential time course with a rate constant, l/T0! -0. 5 see -1. This rate constant does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25).4. After a sudden maintained increase in tubocurarine release, the ACh responses decrease and eventually reach a new steady-state level. Inhibition develops exponentially with time and the apparent rate constant, 1/Ton, is greater than 1/Tof!. When the steady-state inhibition reduces the ACh response to 1/n of its original level, the data are summarized by the relation, 1/Ton = n(1/Toff).5. When the ACh sensitivity is reduced with cobra toxin, both I/Ton and 1/TO!! increase. Thus, the kinetics of tubocurarine inhibition depend on the density of ACh receptors in the synaptic cleft. 6. After treatment with collagenase, part of the nerve terminal is displaced and the post-synaptic membrane is exposed directly to the external solution. Under these circumstances, 1/Tofr increases more than tenfold.7. Bath-applied tubocurarine competitively inhibits the responses to brief ionophoretic ACh pulses with an apparent equilibrium dissociation constant, K = 0 5 /IM. 8. In denervated frog muscle fibres, extrasynaptic receptors have a lower apparent affinity for tubocurarine. After a pulse of tubocurarine, inhibition decays tenfold more rapidly at these extrasynaptic sites than at the synapse.9. It is suggested that each tubocurarine molecule binds repeatedly to several

The Effect of Tubocurarine Competition on the Kinetics of Agonist Action on the Nicotinic Receptor

“…One method is to measure the rate of recovery of the response to an agonist following a brief ionophoretic application of tubocurarine (Castillo & Katz, 1957;Waud, 1967;Armstrong & Lester, 1979). However, it has been shown that rates measured in this way are likely to be limited by diffusion, even when the nerve terminal is lifted away from the endplate (Waud, 1967;Armstrong & Lester, 1979 Ginsborg & Stephenson (1974).…”

“…However, it has been shown that rates measured in this way are likely to be limited by diffusion, even when the nerve terminal is lifted away from the endplate (Waud, 1967;Armstrong & Lester, 1979 Ginsborg & Stephenson (1974). They inferred that the halftime for dissociation of tubocurarine is around a millisecond in rat diaphragm endplates, and even briefer in amphibian endplates; that is, the dissociation rate constant is about 700 s-' in rat, and faster in amphibia.…”

“…In the same cell, a high carbachol concentration (600 pM) caused a faster rise to a peak, followed by rapid desensitization as shown in Figure lc. This rapid rise may have been partly a result of the more rapid diffusion of the agonist that would be expected as-a result of both the a-bungarotoxin treatment (100 nm for 21 min) that preceded the response and the high agonist concentrations used (Colquhoun, Henderson & Ritchie, 1972;Katz & Miledi, 1973;Armstrong & Lester, 1979). But the main reason for the early peak in Figure lc is, no doubt, that desensitization cuts short the rapid rise long before a plateau can be reached.…”

Preliminary investigations of the clinical pharmacology of three short-acting non-depolarizing neuromuscular blocking agents, Org 9453, Org 9489 and Org 9487

“…Others explained this relationship using the concept of buffered diffusion. 19 The relationship, however, may also be explained by differences in pharmacokinetic behaviour between compounds. A more rapid initial decay of the plasma concentration coincides with a more rapid equilibration between plasma and biophase, with subsequent shortening of the onset time.…”

Communications

“…By comparing pulse responses of the model system with results obtained in real synapses (Armstrong & Lester, 1979), it is calculated that a steady terminal leakage could account for the extracellular curare if less than fifty curare releasing endings are formed by the injected neurone. However, if their number exceeds fifty then the 60P PROCEEDINGS OF THE PHYSIOLOGICAL SOCIETY, JUNE 1981 extracellular curare can only be explained by an (additional or exclusive) action potential-dependent release.…”