Eugene M. Silinsky

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

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.

ATP mediates excitatory synaptic transmission in mammalian neurones

Adenosine 5′‐triphosphate (ATP, 0.1–100 μm), produced inward currents in patch‐clamped coeliac neurones from guinea‐pig when studied in either the whole cell configuration or in excised (outside‐out) patches. The P2‐purinoceptor antagonists suramin (80–230 μm) or reactive blue 2 (2–20 μm) depressed the ATP‐induced currents but not those produced by acetylcholine. Excitatory post‐synaptic currents (e.p.s.cs) were observed in cultured neurones. E.p.s.cs had similar current‐voltage relationships to currents evoked by ATP in excised patches and were reduced by suramin or reactive blue 2 to a similar extent as ATP currents. The results suggest that ATP is the excitatory neurotransmitter in cultures of these neurones.

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.

On the excitatory effects of ATP and its role as a neurotransmitter in coeliac neurons of the guinea‐pig.

1. The effects of ATP on neurons from guinea‐pig coeliac ganglia were studied to evaluate the possibility that this nucleotide acts as an excitatory neurotransmitter substance. 2. In experiments with intracellular microelectrodes, ATP (> or = 10 nM) depolarized coeliac neurons from the resting potential and produced an increase in the membrane conductance. These excitatory effects of ATP were observed in isolated coeliac ganglia, in acutely dissociated neurons or in cultured neurons. ATP also produced membrane conductance increases in neurons clamped at the resting potential using a single electrode voltage clamp. 3. When studied in the whole‐cell configuration of the patch clamp (intracellular Cs+ to block K+ currents; ‐50 mV holding potential), ATP evoked inward currents in a manner more potent and efficacious than acetylcholine (ACh). 4. Whole‐cell currents induced by ATP were inwardly rectifying and reversed at ‐13 mV in normal Na+ solutions. Changes in extracellular Na+ concentration altered the reversal potential in a manner predicted by the Goldman‐Hodgkin‐Katz bi‐ionic equation with a ratio of Na+ to Cs+ permeability (PNa/PCs) = 0.6. 5. Single channel currents were evoked by ATP in excised (outside‐out) patches. Current‐voltage relationships for single channel currents exhibited inward rectification. The mean single channel conductance was 22 pS at ‐50 mV. 6. Antagonists of ATP‐gated channels (suramin, Reactive Blue 2) reduced the effects of ATP but not ACh. 7. Antagonists at nicotinic receptors/ion channels (hexamethonium or tubocurarine) reduced the effects of ACh but not ATP. 8. Excitatory synaptic currents were observed in cultures of coeliac neurons. Synaptic currents possessed similar current‐voltage relationships to currents produced by ATP, were increased in frequency by K+ depolarization in a Ca(2+)‐dependent manner, and were selectively antagonized by ATP antagonists. 9. Local K+ depolarization of the ends of neurites evoked single channel currents characteristic of ATP in outside‐out patches when patches were positioned near the region of apparent synaptic contact but not when patches were positioned at remote regions. 10. The results suggest that ATP receptors are linked to ion channels and mediate excitatory synaptic transmission between coeliac neurons.

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.

Phorbol esters and neurotransmitter release: more than just protein kinase C?

This review focuses on the effects of phorbol esters and the role of phorbol ester receptors in the secretion of neurotransmitter substances. We begin with a brief background on the historical use of phorbol esters as tools to decipher the role of the enzyme protein kinase C in signal transduction cascades. Next, we illustrate the structural differences between active and inactive phorbol esters and the mechanism by which the binding of phorbol to its recognition sites (C1 domains) on a particular protein acts to translocate that protein to the membrane. We then discuss the evidence that the most important nerve terminal receptor for phorbol esters (and their endogenous counterpart diacylglycerol) is likely to be Munc13. Indeed, Munc13 and its invertebrate homologues are the main players in priming the secretory apparatus for its critical function in the exocytosis process.

Evidence for specific adenosine receptors at cholinergic nerve endings.

1 An electrophysiological study was made to determine if adenosine and adenine nucleotides affect cholinergic nerve endings to frog skeletal muscle through relatively non‐specific nucleotide receptors or through specific adenosine receptors. 2 Non‐hydrolysable derivatives of adenosine triphosphate failed to alter the mean number of acetylcholine (ACh) quanta released by a nerve impulse (m̄) or the miniature endplate potential frequency (m.e.p.p.f) but N6‐methyladenosine and 2‐chloroadenosine, two adenosine analogues with an unsubstituted ribose moiety (R‐site agonists), produced marked reductions in m̄ and m.e.p.p.f. 3 In contrast, 2′‐deoxyadenosine, a derivative with an unsubstituted purine ring (P‐site agonist), generally produced increases in m̄ and m.e.p.p.f, which further increased after removing the drug. Other P‐site agonists such as 5′‐deoxyadenosine (in the presence of theophylline) and 9‐β‐d‐arabinofuranosyl adenine also increased m̄ and m.e.p.p.f. 4 The results suggest that two types of adenosine receptors may be present at cholinergic nerve endings, one type (R‐site) mediating depression and the other type (P‐site) producing enhancement of ACh release.

Calcium currents at motor nerve endings: absence of effects of adenosine receptor agonists in the frog.

1. The effects of adenosine (50 microM) and 2‐chloroadenosine (1‐25 microM) were studied on Ca2+ currents in frog motor nerve endings. 2. Ca2+ currents associated with the synchronous, neurally evoked release of acetylcholine (ACh) were measured using either perineural or patch recording methods. Tetraethylammonium and/or 3,4‐diaminopyridine were employed to block K+ currents. 3. Ca2+ currents were depressed by omega‐conotoxin (1.5‐2.5 microM), Cd2+ (100 microM‐2 mM), Co2+ (500 microM‐5 mM) or by a reduction of the extracellular calcium concentration. Such currents were also observed when Sr2+ was substituted for Ca2+. Both ACh release and Ca2+ currents at motor nerve endings have been reported to be insensitive to 1,4‐dihydropyridine antagonists in this species. 4. Adenosine receptor agonists did not affect Ca2+ currents at concentrations that produced maximal inhibition of ACh release. 5. The effects of adenosine receptor agonists were examined on asynchronous K(+)‐dependent ACh release under conditions in which the Ca2+ concentration gradient is likely to be reversed (Ca(2+)‐free Ringer solution containing 1 mM EGTA). ACh release was measured by monitoring the frequency of occurrence of miniature endplate potentials (MEPPs). In Ca(2+)‐free solutions containing 1 mM EGTA, high K+ depolarization caused a decrease in MEPP frequency, presumably because it elicits the efflux of Ca2+ from the nerve ending via membrane Ca2+ channels in a reverse Ca2+ gradient. 6. The Ca2+ channel blocker Co2+, which blocks the exit of Ca2+ from the nerve ending, increased the frequency of MEPPs in a concentration‐dependent manner in a reverse Ca2+ gradient. 7. Adenosine or 2‐chloroadenosine inhibited ACh release in a reverse Ca2+ gradient. 8. The results suggest that blockade of Ca2+ entry is not responsible for the inhibitory effects of adenosine at frog motor nerve endings.

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.

Inhibition of transmitter release by adenosine: are Ca2+ currents depressed or are the intracellular effects of Ca2+ impaired?

Abstract It has been suggested that blockade of nerve terminal Ca 2+ channels by extracellular adenosine is responsible for the inhibitory effects of adenosine and its congeners on the secretion of neurotransmitter substances. Several lines of evidence, however, are not in accord with this suggestion. Eugene M. Silinsky presents evidence to indicate that the impairment of Ca 2+ -dependent processes at an intracellular site associated with the secretory apparatus provides the best single explanation for the inhibitory effects of adenosine derivatives on transmitter release.