1. It has been suggested that ATP is released together with the neurotransmitter acetylcholine (ACh) and, after hydrolysis to adenosine, is the primary physiological mediator of prejunctional neuromuscular depression. To evaluate whether ATP is released with sufficient rapidity to mediate prejunctional depression, outside-out patches containing both ATP-gated and ACh-gated ion channels were made from acutely dissociated guinea-pig sympathetic neurons and used to detect the co-release of nucleotide and neurotransmitter in frog cutaneous pectoris nerve-muscle preparations. 2. In a normal bathing solution in which muscle nicotinic receptors were blocked, a single stimulus to the motor nerve produced channel openings in the detector patch characteristic of both ATP and ACh. 3. In the remaining experiments, preparations were treated with sufficient hexamethonium (200 #M) to block nicotinic responses in the detector patch. In these experiments, a single temporally isolated nerve impulse caused the synchronous opening of ATP-gated channels in the detector patch with a latency of <5 ms when patches were placed within 10 ,um of the motor nerve ending. This multichannel phasic response was followed by a trail of discrete channel openings characteristic of ATP-gated channels. 4. The selective ATP antagonist suramin (50 uM) reversibly eliminated the response to nerve stimulation. 5. The results suggest that ATP is released synchronously together with the neurotransmitter ACh in response to an individual nerve impulse and with a brief latency characteristic of quantal release from synaptic vesicles.
Phorbol esters activate protein kinase C (PKC) and also increase the secretion of neuro‐transmitter substances by an unknown mechanism. To evaluate whether the stimulatory effects of such agents on acetylcholine (ACh) secretion occur as a consequence of stimulation of Ca2+ entry, we made electrophysiological measurements of ACh secretion (i.e. endplate potentials, EPPs) and the component of the prejunctional perineural voltage change associated with nerve terminal calcium currents (perineural calcium current) at frog neuro‐muscular junctions.
In the first series of experiments, modest concentrations of K+ channel blockers were employed so that simultaneous measurements of EPP amplitudes and perineural calcium currents could be made. In these experiments, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA; 162 nm) and phorbol 12,13‐dibutyrate (PDBu; 100‐200 nm) each increased ACh release but simultaneously decreased the calcium component of the prejunctional perineural current. TPA and PDBu also inhibited perineural calcium currents in the presence of higher concentrations of K+ channel blockers.
Blockade of Ca2+ channels by Cd2+ prevented the action of PKC stimulators on perineural waveforms.
The inactive compound 4‐α‐phorbol 12‐myristate 13‐acetate (150 nm) did not affect EPP amplitudes or perineural currents.
The extracellular [Ca2+]‐ACh release relationship was increased in maximum by PDBu without any change in the potency of Ca2+ to support evoked ACh release.
The results demonstrate that phorbol esters increase neurotransmitter secretion whilst simultaneously decreasing the nerve ending calcium currents that promote evoked release. The results, which suggest that the optimal control point for secretion might not be the calcium channel but rather a component of the secretory apparatus, are discussed in conjunction with the possible target sites for phorbol esters in the nerve ending.
The interaction of ion channels activated by nicotinic receptor agonists with ion channels gated by extracellular ATP (i.e. P2X receptors) was studied on sympathetic neurons acutely dissociated from coeliac ganglia of the guinea‐pig. Patch clamp methods were used to measure the inward current generated through these non‐selective cationic channels under voltage clamp.
At the whole cell level, the specific nicotinic receptor agonists nicotine (5‐100 μM) or cytisine (50‐75 μM) and the P2X receptor agonists ATP (0.1‐7 μM) or α,β‐methylene ATP (6 μM) were examined separately and in the presence of the other receptor activator. When a nicotinic and P2X receptor agonist were applied together, mutually occlusive effects were generally observed. This occurred even with concentrations of agonists that in themselves generated little to no inward current.
The occlusive effects of nicotinic agonists on ATP‐gated currents were blocked by the nicotinic receptor/ion channel blocker hexamethonium (150 μM). The occlusive effects of ATP analogues on inward currents generated by nicotinic agonists were blocked by the P2X receptor antagonist suramin (100 μM).
Mutual occlusion of the effects of nicotinic agonists and ATP analogues were also observed when currents through single channels were studied in excised (outside‐out) patches.
The results suggest that nicotinic receptors and P2X ATP receptors do not act independently in these sympathetic neurons.
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