R.S. Redman

Synchronous release of ATP and neurotransmitter within milliseconds of a motor nerve impulse in the frog.

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 microM) 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 microns of the motor nerve ending. This multichannel phasic response was followed by trail of discrete channel openings characteristic of ATP‐gated channels. 4. The selective ATP antagonist suramin (50 microM) 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.

Mutual occlusion of P2X ATP receptors and nicotinic receptors on sympathetic neurons of the guinea-pig

1 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. 2 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. 3 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). 4 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. 5 The results suggest that nicotinic receptors and P2X ATP receptors do not act independently in these sympathetic neurons.

Opposing effects of phorbol esters on transmitter release and calcium currents at frog motor nerve endings

1 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. 2 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. 3 Blockade of Ca2+ channels by Cd2+ prevented the action of PKC stimulators on perineural waveforms. 4 The inactive compound 4‐α‐phorbol 12‐myristate 13‐acetate (150 nm) did not affect EPP amplitudes or perineural currents. 5 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. 6 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.

Quantal ATP release from motor nerve endings and its role in neurally mediated depression

Publisher Summary This chapter reviews the mechanisms of presynaptic neurotransmitter modulation within the framework of a physiologically relevant behavior—namely, the process of neurally mediated prejunctional depression at the skeletal neuromuscular junction. At the frog and mouse neuromuscular junctions, depression appears to be because of the release of endogenous ATP that after degradation to adenosine acts back on the nerve ending to inhibit the subsequent release of the neurotransmitter acetylcholine. The chapter also presents the evidence that ATP is released synchronously from motor nerve endings within milliseconds of a solitary nerve impulse and in the appropriate concentration range to mediate neuromuscular depression. The data suggest that, in the frog, adenosine mediates the inhibition of both spontaneous and evoked ACh release and by an action at the secretory apparatus and the downstream of calcium entry.

Decrease in calcium currents induced by aminoglycoside antibiotics in frog motor nerve endings

1 The effects of the aminoglycoside antibiotics, streptomycin, neomycin and gentamicin were examined on perineural currents and evoked acetylcholine (ACh) release at frog motor nerve endings. 2 In the standard solutions used previously to measure Ca2+ currents, streptomycin reduced the peak amplitude of the Ca2+ component of the perineural current. 3 In a solution in which changes in both Ca2+ currents and evoked ACh release can be recorded simultaneously, both Ca2+ currents and evoked ACh release were reduced by aminoglycosides in the potency order neomycin > streptomycin > gentamicin. This potency sequence is similar to that reported previously for these agents as inhibitors of neurally‐evoked contractions of mammalian skeletal muscle. 4 These data suggest that the presynaptic inhibitory effects of aminoglycoside antibiotics at the neuromuscular junction occur as a consequence of a reduction in Ca2+ currents in the motor nerve terminal.

Neurotransmitter release evoked by nerve impulses without Ca2+ entry through Ca2+ channels in frog motor nerve endings.

1. The requirement for extracellular Ca2+ in the process of evoked acetylcholine (ACh) release by nerve impulses was tested at endplates in frog skeletal muscle. Ca(2+)‐containing lipid vesicles (Ca2+ liposomes) were used to elevate cytoplasmic Ca2+ concentrations under conditions in which Ca2+ entry from the extracellular fluid was prevented. 2. In an extracellular solution containing no added Ca2+ and 1 mM Mg2+ (‘Ca(2+)‐free’ solution), Ca2+ liposomes promoted the synchronous release of ACh quanta, reflected electrophysiologically as endplate potentials (EPPs), in response to temporally isolated nerve impulses. 3. Motor nerve stimulation generated EPPs during superfusion with Ca2+ liposomes in Ca(2+)‐free solutions containing the Ca2+ channel blocker Co2+ (1 mM), and the Ca2+ chelator EGTA (2 mM). As a physiological control for Ca2+ leakage from the liposomes to the extracellular fluid, the effect of Ca2+ liposomes on asynchronous evoked ACh release mediated by Ba2+ was examined. In contrast to the effects of 0.2‐0.3 mM extracellular Ca2+, which generated EPPs but antagonized Ba(2+)‐mediated asynchronous ACh release, Ca2+ liposomes generated EPPs but did not reduce asynchronous release mediated by Ba2+. The effects of Ca2+ liposomes were thus not due to leakage of Ca2+ from the liposome to the extracellular fluid. 4. Morphological studies using fluorescently labelled liposomes in conjunction with a confocal microscope demonstrate that lipid is transferred from the liposomes to nerve endings and liposomal contents are delivered to the nerve terminal cytoplasm. 5. The results suggest that when intracellular Ca2+ is elevated using liposomes as a vehicle, evoked ACh release can occur in the absence of Ca2+ entry via Ca2+ channels.

Reduction by intracellular calcium chelation of acetylcholine secretion without occluding the effects of adenosine at frog motor nerve endings.

1 The calcium chelators bis‐(aminophenoxy)ethane‐tetraacetic acid (BAPTA) or dimethyl‐BAPTA (DMBAPTA) were introduced into the cytoplasm of frog motor nerve endings by use of the AM loading technique. The effects of intracellular Ca2+ chelation were studied on quantal acetylcholine (ACh) release and on the action of adenosine. 2 Intracellular BAPTA or DMBAPTA prevented the increases in quantal ACh secretion normally evoked by caffeine. 3 Intracellular DMBAPTA decreased the number of ACh quanta released by individual nerve impulses and virtually eliminated the fast phase of facilitation in response to paired nerve impulses. 4 Adenosine reduced both spontaneous and evoked secretion of ACh quanta with its usual potency and efficacy in the presence of intracellular DMBAPTA. Adenosine had no significant effect on facilitation. 5 The results, which suggest that adenosine and intracellular DMBAPTA reduce ACh secretion by different mechanisms, are consistent with the hypothesis that adenosine inhibits ACh release by reducing the ability of Ca2+ to promote ACh secretion from frog motor nerve endings.

Adenosine Derived from Neurally Released ATP is the Physiologic Cause of Skeletal Neuromuscular Depression

The efficiency of skeletal neuromuscular transmission is limited by the neuromuscular depression that ensues with even brief repetitive nerve stimulation [1–4]. Indeed, in patients with neuromuscular disorders such as myasthenia gravis, neuromuscular depression can be severely debilitating [5]. Neuromuscular depression is prejunctional in origin, occurring as a consequence of a reduction in the number of acetylcholine (ACh) quanta released by a nerve impulse [1–4,6]. Most attempts to quantify neuromuscular depression have been based on the assumption that the phenomenon is due to a reduction in the available vesicular store of ACh [2]. While depletion of vesicular stores of ACh contributes to depression at enormous, unphysiologic ACh outputs in the presence of very high concentrations of K+ channel blockers [3], there is no evidence to support the depletion hypothesis at normal levels of ACh output.

α-Bag cell peptide inhibits bag cell adenylate cyclase via a GTP-dependent mechanism

alpha-Bag cell peptide (alpha-BCP), one of several secreted peptides encoded in the precursor to the egg-laying hormone (proELH) of the neurosecretory bag cells of Aplysia, has been variously reported to have autoexcitatory or autoinhibitory effects on the cells which secrete it. Since we had found previously that alpha-BCP reduces stimulated cAMP levels in intact bag cells, an effect that would be consistent with electrophysiological inhibition, we investigated the direct effect of the peptide on adenylate cyclase in bag cell membrane preparations. alpha-Bag cell peptide did not affect basal adenylate cyclase activity, but reduced forskolin-stimulated activity by about 30%. The potency of the peptide in this assay was within the range reported for observable physiological effects: half-maximal inhibition was seen at approximately 100 nM peptide. Both basal and forskolin-stimulated enzyme activity were dependent on GTP, and the inhibitory effect of alpha-BCP was inversely dependent on the nucleotide. The non-hydrolyzable analogue, GTP-gamma-S, stimulated both basal and forskolin-stimulated enzyme activity and enhanced alpha-BCPs effect to the extent that the peptide completely inhibited forskolins stimulation of the enzyme. The peptides effect could be blocked by pretreatment with pertussis toxin. We conclude that alpha-BCP inhibits bag cell adenylate cyclase, an effect which is consistent with an autoinhibitory role in bag cell function. Moreover, this inhibition appears to be mediated by a GTP-binding protein.

Determinants of potency and temperature-dependent function in the Aplysia bag cell peptides

Structure-activity relationships were determined for the natural bag cell peptides (BCPs) and for a series of synthetic analogues in terms of their ability to stimulate (at 30 degrees C) and to inhibit (at 15 degrees C) bag cell adenyl cyclase. We found that the core RLRF motif shared by all these peptides is active in this assay, and is stimulatory. The histidine residue C-terminal to this motif in beta-BCP is superfluous in this respect. An electronegative residue C-terminal to RLRF is sufficient to induce temperature-dependent function. The Ala-Pro pair that is N-terminal to this motif in alpha-BCP increases potency, but does not alter function.