1. Conventional electrophysiological techniques were used to record from isolated rat phrenic nerve-hemidiaphragm preparations. After periods of rest (20 min) or nerve stimulation (7/sec for 20 min) the bathing medium of the preparation was removed and assayed for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) using a sensitive modification of the firefly luciferase method (Silinsky, 1974). 2. In the presence of tubocurarine and normal (2 mM) calcium, fourteen periods of nerve stimulation (eight preparations) caused the appearance of ATP and/or ADP in amounts ranging from 28 to 641 p-mole. Experiments using carbachol (30 muM or 1 mM) suggested that this nucleotide efflux was not produced by a secondary action of released acetylcholine (ACh). 3. Stimulation of isolate phrenic nerve trunks at 7/sec for 20 min did not cause the efflux of ATP or ADP. 4. In solutions of normal osmotic pressure and reduced calcium concentrations (0-1 mM or 'calcium-free'), stimulation failed to release adenine nucleotide from non-contracting preparations. 5. Diaphragms were bathed in normal calcium and indirectly stimulated at 11/sec for 80-90 min in the presence of 5 times 10-minus 5 M hemicholinium-3. After all detectable signs of ACh release were eliminated, nerve stimulation failed to release ATP or ADP. 6. These results in conjunction with experiments on the hydrolysis of exogenous ATP suggest that ATP is released from the motor nerve ending and is subsequently degraded by enzymatic activity. It is also suggested that the released nucleotide may be derived from the cholinergic vesicle.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1975.sp010925

On the association between transmitter secretion and the release of adenine nucleotides from mammalian motor nerve terminals.

Organization and synaptic physiology of crustacean neuromuscular systems

INVESTIGATION of the adrenal medulla1,2 and the Torpedo electroplax3 suggested that ATP plays some part in the storage and release of chemical transmitters. The fluid bathing contracting amphibian muscle contains ATP, but this ATP was thought to be released from the muscle4. We present here evidence that ATP is released from motor nerve terminals on indirect stimulation of a mammalian nerve-muscle preparation.

Biological Sciences: Release of ATP from Rat Motor Nerve Terminals

Mechanism of transmitter release.

The sections in this article are:


1
I. Motoneurons
1.1
A. Alpha Motoneurons

1.2
Gamma Motoneurons




2
II. Interneurons
2.1
A. General Considerations

2.2
B. Functionally Identified Interneuron Systems




3
III. Tract Cells
3.1
General Considerations

3.2
B. Specific Systems




4
IV. Intraspinal Primary Afferents and Presynaptic Modulation
4.1
A. General Considerations

4.2
B. Intrinsic Properties of Intraspinal Afferents

4.3
C. Evidence for the Existence of Primary Afferent Depolarization (PAD)

4.4
D. Afferent Terminals Are Subjected Only to Depolarizing Input

4.5
E. Interneurons Are Involved in the Mechanism of PAD Generation

4.6
F. Possible Mechanisms of PAD Production by Interneurons

4.7
G. Changes in Synaptic Transmission Related to PAD

4.8
H. Possible Mechanisms for Presynaptic Inhibition

4.9
I. Consequences of Presynaptic Modulation of Synaptic Transmission

4.10
J. Organization of PAD




5
V. Concluding Comment

Spinal Neurons and Synapses

1. Motor nerve terminals in magnesium-poisoned rat hemidiaphragm-phrenic nerve preparations in vitro were stimulated with short depolarizing pulses of approximately threshold strength and the evoked antidromic responses recorded from the phrenic nerve. The percentage of these 1/sec or 0·5/sec stimuli to which there was no antidromic response was used as a quantitative measure of the terminal excitability. After standard tetanic stimulation (1000 impulses at 100/sec) the excitability of the terminals was depressed for an average duration of 60-70 sec, during most of which time no antidromic responses to stimuli of pretetanic intensity were recorded. There was no significant interaction between stimuli to the terminals at rates of 1 or 0·5/sec.

2. Potassium-free solutions at first increased, then decreased, the post-tetanic depression of excitability. Raising [K]o threefold (15 m M) abolished the post-tetanic depression and often converted it to an exaltation of excitability.

3. Polarizing currents were applied to the terminals with a second electrode. Depolarizing currents increased, while hyperpolarizing currents decreased, the post-tetanic depression of excitability.

4. In solutions with 70% of the normal NaCl content replaced by sucrose, the post-tetanic depression of excitability was reversibly prolonged.

5. In the presence of 7·7 × 10−6 M digoxin or 0·42 m M ouabain there was a small reversible reduction of post-tetanic excitability.

6. After exposure to solutions containing no glucose or to solutions containing 3-5 m M sodium azide the excitability of the terminals was not altered by the tetanus. After washing with the control solution, post-tetanic depression of excitability returned. Antimycin-A (1·8 × 10−6 M) had little or no effect upon post-tetanic excitability.

7. It was concluded that the post-tetanic depression of excitability reflected hyperpolarization of the terminals and that this hyperpolarization was caused by a shift of the membrane potential towards the potassium equilibrium potential because of an increase in potassium permeability.

J. I. Hubbard

Papers

The origin of the post-tetanic hyperpolarization of mammalian motor nerve terminals