Jody K. Hirsh

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.

Palytoxin-induced single-channel currents from the sodium pump synthesized by in vitro expression.

Palytoxin, the most potent animal toxin, is proposed to convert Na+/K(+)-ATPase into a cation-selective ion channel. Because of the ubiquity of pumps and channels in the living tissues used to study its mechanism of action, it is difficult to rule out that another site may be involved. In order to show that palytoxin selectively acts on Na+/K(+)-ATPase, two entirely in vitro methods were employed: (1) a cell-free expression system to synthesize the rat alpha 3 and beta 1 subunit proteins, and (2) single-channel recording of the synthetic Na+/K(+)-ATPase reconstituted in a planar lipid bilayer. Upon addition of palytoxin, single-channel currents were induced which had a conductance of 10 pS, in agreement with previous studies. In control experiments, when the cDNAs for Na+/K(+)-ATPase subunits were omitted, no single-channel currents were induced with palytoxin. Thus, the results show unambiguously that the Na+/ K(+)-ATPase is the site of action for palytoxin. Because palytoxin turns the Na+/K(+)-ATPase into a channel which can be detected by the exquisitely sensitive single-channel recording technique, the present results are the first to demonstrate the activity of in vitro synthesized Na+/K(+)-ATPase.

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.

Pertussis toxin prevents the inhibitory effect of adenosine and unmasks adenosine‐induced excitation of mammalian motor nerve endings

Pertussis toxin (PTX), which blocks certain classes of guanine nucleotide binding proteins (G proteins), consistently blocked the inhibitory effects of adenosine (100 μm‐250 μm) on quantal acetylcholine (ACh) secretion in rat phrenic nerve hemidiaphragm preparations. PTX pretreatment also highlighted long‐lasting increases in evoked ACh release elicited by adenosine. The results suggest that specific G proteins are involved in mediating the inhibitory effects of adenosine at motor nerve endings.

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.

Regulation by Rab3A of an endogenous modulator of neurotransmitter release at mouse motor nerve endings

Rab3A, a small GTP‐binding protein attached to synaptic vesicles, has been implicated in several stages in the process of neurosecretion, including a late stage occurring just prior to the actual release of neurotransmitter. The inhibitory neuromodulator adenosine also targets a late step in the neurosecretory pathway. We thus compared neuromuscular junctions from adult Rab3A−/‐ mutant mice with those from wild‐type mice with respect to: (a) the basic electrophysiological correlates of neurotransmitter release at different stimulation frequencies, and (b) the actions of exogenous and endogenous adenosine on neurotransmitter release in normal calcium solutions. Neither the spontaneous quantal release of acetylcholine (ACh) nor basal evoked ACh release (0.05 Hz) differed between the mutant and wild‐type mice. At 50‐100 Hz stimulation (10‐19 stimuli), facilitation of release was observed in the mutant mice but not in wild‐type, followed by a depression of ACh release in both strains. ACh release at the end of the stimulus train in the mutant mouse was approximately double that of the wild‐type mouse. The threshold concentration for inhibition of ACh release by exogenous adenosine was over 20‐fold lower in the mutant mouse than in the wild‐type mouse. The adenosine A1 receptor antagonist 8‐cyclopentyltheophylline (CPT) increased ACh release (0.05‐1 Hz stimulation) in the mutant mouse under conditions in which it had no effect in the wild‐type mouse. CPT had no effect on the pattern of responses recorded during repetitive stimulation in either strain. The results suggest that Rab3A reduces the potency of adenosine as an endogenous mediator of neuromuscular depression.

The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings.

1 The importance of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP‐dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2 Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.p.f) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3 The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8‐(4‐Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4 All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5 The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (30–100 μm) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel. 6 H7 prevented the increases in spontaneous ACh release produced by CPT cyclic AMP (250 μm). Thus H7 is likely to inhibit PK A in frog motor nerve endings. 7 H7 did not alter the inhibitory effect of adenosine on evoked and spontaneous ACh release. 8 The results suggest: (i) that the adenylyl cyclase‐cyclic AMP‐PK A system is compartmentalized within the motor nerve terminal, (ii) that phosphorylation does not play a major role in ACh release and (iii) the cyclic AMP‐PK A system modulates rather than mediates the inhibitory effects of adenosine.

The effects of TMB-8 on acetylcholine release from frog motor nerve: interactions with adenosine

The putative intracellular calcium (Ca) antagonist TMB-8 was shown to reduce postjunctional sensitivity and quantal acetylcholine (ACh) release at low micromolar concentrations. At 10-fold higher concentrations, TMB-8 also blocked caffeine-induced Ca release (as monitored electrophysiologically by changes in ACh release) but did not impair the ability of adenosine to inhibit quantal ACh release. This last result implies that TMB-8 and adenosine exert their inhibitory actions at different steps in the depolarization-secretion coupling sequence.

Inhibition of spontaneous acetylcholine secretion by 2‐chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction

Previous studies have reported discrepancies in the potencies of A1 adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) in mediating the inhibitory effects of A1 receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2‐chloroadenosine (CADO). CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 μM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 μM) produced highly statistically‐significant reductions in spontaneous ACh release. In the presence of the non‐selective protein kinase inhibitor, H7 (50 μM), the potency of CADO was increased such that 1 μM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 μM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory effect of 1 μM CADO. A number of other non‐selective PKA inhibitors also failed to increase the potency of CADO. The results suggest that an endogenous H7‐sensitive process modulates the sensitivity of the mouse A1 adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition.

The effect of reduced temperature on the inhibitory action of adenosine and magnesium ion at frog motor nerve terminals

1 A study was made to exclude the notion that adenosine receptor agonists exert a direct physical blockade of the depolarization‐secretion process. Reduced temperature was employed as a tool for distinguishing between physico‐chemical processes (such as those which mediate evoked transmitter release) and biochemical mechanisms (such as those which involve second messenger substances) in the action of adenosine. Adenosine and 2‐chloroadenosine were used as agonists in this electrophysiological study of the release of acetylcholine (ACh) from frog motor nerve terminals. 2 The ability of these two adenosine receptor activators to reduce neurally‐evoked ACh release was prevented or greatly attenuated by maintaining the preparation at temperatures between 5 and 10°C. Such low temperatures inhibit the activation of receptors coupled to second messengers via guanine nucleotide binding proteins (e.g. adenylate cyclase). Low temperature alone did not substantially alter evoked ACh secretion under the conditions of these experiments. 3 Inhibition of evoked ACh release by the extracellular Ca antagonist Mg, which acts directly to block Ca channels, was not affected by low temperature. 4 The results are consistent with the hypothesis that a temperature‐sensitive second messenger system controls the intracellular events linked to extracellular adenosine receptor activation.