Timothy J. Searl

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.

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.

Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endings

Phorbol esters and adenosine have been proposed to interact at common sites downstream of calcium entry at amphibian motor nerve endings. We thus studied the actions and interactions of phorbol esters and adenosine using electrophysiological recording techniques in conjunction with both binomial statistical analysis and high‐frequency stimulation at the amphibian neuromuscular junction. To begin this study, we confirmed previous observations that synchronous evoked acetylcholine (ACh) release (reflected as endplate potentials, EPPs) is well described by a simple binomial distribution. We then used binomial analysis to study the effects of the phorbol ester phorbol dibutyrate (PDBu, 100 nm) and adenosine (50 µm) on the binomial parameters n (the number of calcium charged ACh quanta available for release) and p (the average probability of release), where the mean level of evoked ACh release (m) =np. We found that PDBu increased m by increasing the parameter n whilst adenosine reduced m by reducing n; neither agent affected the parameter p. PDBu had no effect on either the potency or efficacy of the inhibition produced by adenosine. Subtle differences between these two agents were revealed by the patterns of EPPs evoked by high‐frequency trains of stimuli. Phorbol esters increased ACh release during the early phase of stimulation but not during the subsequent plateau phase. The inhibitory effect of adenosine was maximal at the beginning of the train and was still present with reduced efficacy during the plateau phase. When taken together with previous findings, these present results suggest that phorbol esters increase the immediately available store of synaptic vesicles by increasing the number of primed vesicles whilst adenosine acts at a later stage of the secretory process to decrease the number of calcium‐charged primed vesicles.

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.

Modulation of Ca2+-dependent and Ca2+-independent miniature endplate potentials by phorbol ester and adenosine in frog

1 Phorbol esters and adenosine modulate transmitter release from frog motor nerves through actions at separate sites downstream of calcium entry. However, it is not known whether these agents have calcium‐independent sites of action. We therefore characterised calcium independent miniature endplate potentials (mepps) generated in response to 4‐aminoquinaldine (4‐AQA) and then compared the modulation of these mepps by phorbol esters and adenosine with that of normal calcium dependent mepps. 2 Application of 30 μM 4‐AQA resulted in the appearance of a population of mepps with amplitudes greater than twice the total population mode (mepp>2M). In the presence of 4‐AQA, K+ depolarisation or hypertonicity increased the numbers of normal amplitude mepps (meppN) but had no effect on the frequency of mepp>2M events, suggesting that mepp>2M are not dependent on calcium. 3 Treatment with the botulinum toxin (Botx) fractions C, D, or E (which selectively cleave syntaxin, synaptobrevin and SNAP‐25, respectively) produced equivalent reductions in both normal and 4‐AQA induced mepps, suggesting that both mepp populations have equal dependence on the intact SNARE proteins. 4 Phorbol dibutyrate (PDBu, 100 nM) increased the frequencies of both populations of mepps recorded in the presence of 4‐AQA. Adenosine (25 μM) selectively reduced the numbers of meppN with no effect on the frequency of mepp>2M events. 5 These results suggest that mepp>2M events released in response to 4‐AQA are dependent on intact forms of syntaxin, synaptobrevin and SNAP‐25, but unlike meppN are independent of a functional calcium sensor. The selective action of adenosine, to reduce the numbers of normal amplitude mepps without effecting the frequency of mepp>2M events, suggests that adenosine normally inhibits transmitter release through a mechanism that is dependent on the presence of a functional calcium sensor.

Evidence for two distinct processes in the final stages of neurotransmitter release as detected by binomial analysis in calcium and strontium solutions

The statistical parameters underlying acetylcholine (ACh) release were studied using Ca2+ and Sr2+ ions to promote ACh secretion. Experiments were performed at frog neuromuscular junctions using electrophysiological recording techniques. Increases in asynchronous ACh release, reflected as the frequency of occurrence of miniature end‐plate potentials (MEPPf), were evoked by high potassium depolarization in either Ca2+ or Sr2+ solutions. Increases in MEPPf mediated by Ca2+ were of very low probability and well‐described by a Poisson distribution whilst similar MEPPf increases mediated by Sr2+ were best described as a simple binomial distribution. From the binomial distribution in Sr2+ solutions, values for the average probability of release (p) and the number of releasable ACh quanta (n) may be determined (whereby mean MEPPf=np). In Sr2+ solutions, values of p were independent of both bin width and of the value of n, suggesting that both n and p were stationary. Calculations of p using the simple binomial distribution in Sr2+ solutions gave theoretical values for the third moment of the mean which were indistinguishable from the experimental distribution. These results, in conjunction with Monte Carlo simulations of the data, suggest that spatial and temporal variance do not measurably affect the analysis. Synchronous ACh release evoked by nerve impulses (end‐plate potentials, EPPs) follow a simple binomial distribution in both Ca2+ and Sr2+ solutions. Similar mean levels of synchronous ACh release (m, where m=np) were produced by lower values of p and higher values of n in Ca2+ as compared to Sr2+. The statistical analyses suggest the presence of two different Ca2+‐dependent steps in the final stages of neurotransmitter release. The results are discussed in accordance with (i) statistical models for quantal neurotransmitter release, (ii) the role of Sr2+ as a partial agonist for evoked ACh release, and (iii) the specific loci that may represent the sites of Ca2+ and Sr2+ sensitivity.

The phosphatidylinositol 4-kinase inhibitor phenylarsine oxide blocks evoked neurotransmitter release by reducing calcium entry through N-type calcium channels

The effects of the phosphatidylinositol 4‐kinase inhibitor, phenylarsine oxide (PAO), on acetylcholine (ACh) release and on prejunctional Ca2+ currents were studied at the frog neuromuscular junction using electrophysiological recording techniques. Application of PAO (30 μM) increased both spontaneous ACh release reflected as miniature end‐plate potential (mepp) frequencies and evoked ACh release reflected as end‐plate potential (epp) amplitudes with a similar time course. Following the initial increase in epp amplitudes produced by PAO, epps slowly declined and were eventually abolished after approximately 20 min. However, mepp frequencies remained elevated over this time period. PAO (30 μM) also inhibited the perineural voltage change associated with Ca2+ currents through N‐type Ca2+ channels (prejunctional Ca2+ currents) at motor nerve endings. Addition of British anti‐lewisite (BAL, 1 mM), an inactivator of PAO, partially reversed both the inhibition of epps and the inhibition of the prejunctional Ca2+ current. The effects of PAO on N‐type Ca2+ channels were investigated more directly using the whole cell patch clamp technique on acutely dissociated sympathetic neurons. Application of PAO (30–40 μM) to these neurons decreased the voltage‐activated calcium currents through N‐type Ca2+ channels, an effect that was partially reversible by BAL. In combination, these results suggest that inhibition of neurotransmitter release by PAO occurs as a consequence of the inhibition of Ca2+ entry via N‐type calcium channels. The relationship between the effects of PAO on N‐type Ca2+ channels in motor nerve endings and in neuronal soma is discussed.

Cross-talk between apparently independent receptors

Nicotinic receptors and P2X ATP receptors both incorporate transmembrane cation channels as part of the receptor moiety. However, the nicotinic and P2X ATP receptors are regarded as entirely independent molecular entities and the receptor subunits have distinctly different transmembrane topologies. Given this difference, it is of interest that recent papers (Searl et al. 1998; and two papers appearing in this issue of The Journal of Physiology: Zhou & Galligan, 1998; Barajas-Lopez et al. 1998) have confirmed earlier findings (Nakazawa et al. 1991; Silinsky & Gerzanich, 1993) that co-application of nicotinic agonists and P2X ATP receptor agonists produce less than the additive responses predicted by independent receptor activation. These papers, when taken together, suggest that such interdependent, mutually occlusive interactions are: (i) receptor mediated (as antagonists reveal full responses of the unaffected agonist); (ii) not mediated by soluble second messengers (as non-additivity is also seen in excised patches); and (iii) not due to pH changes produced by high concentrations of agonist (Wildman et al. 1997). Figure 1 depicts three mechanisms that could account for non-additive responses to nicotinic and ATP receptor agonists. In A, both receptors are intimately linked, such that the binding of agonist to one type of receptor inhibits the opening of the other. In B, both receptors are closely associated and inward cationic current through one receptor inhibits the opening of the other. In C, the receptors are independent but each receptor contains an inhibitory binding site for the other receptor agonist. Figure 1 Three mechanisms by which non-additivity between nicotinic and P2X ATP receptors could take place Several experiments should distinguish between these models. The work of Barajas-Lopez et al. (1998), performed using saturating concentrations of agonists, suggests that inward current through one class of receptor acts locally to inhibit the activation of the other receptor class, whilst outward currents are merely additive (Fig. 1B). If this model is true, then selective nicotinic channel blockers, such as procaine, known not to bind to the agonist recognition site, should reverse the occlusion of the ATP-receptor channel. Possibly, mere occupation of a strategic site in the channel by cations entering from the outside occludes the gating of the other channel. Intriguingly, the P2X ATP receptor in guinea-pig ganglia exhibits profound inward rectification (see Silinsky & Gerzanich, 1993), as may the nicotinic receptor in some instances. Models using ion channels constructed from artificial peptides suggest that inward rectification can be attributed to a dipole potential produced by parallel peptide helices in the pore-forming region of the channel (see e.g. Kienker et al. 1994). Perhaps a permeant ion entering from the outside can interact with such a dipole potential, maybe in conjunction with fixed charges, and influence the gating properties and the conductance of its own channel and that of a closely associated receptor. In this regard, Zhou & Galligan (1998), also using saturating concentrations of agonists, found that when agonists at GABAA and 5-HT3 receptors (receptors that do not exhibit inward rectification of the single channel conductance in guinea-pig autonomic neurons) were combined with ACh or ATP, additivity of responses occurred. In may also be that these other receptors are not as intimately positioned as the nicotinic and P2X ATP receptors in these preparations. In contrast, Nakazawa et al. (1995) and Searl et al. (1998) have found that inhibition can occur using concentrations of the occluding agonist that produce by themselves little to no inward currents. Nakazawa et al. (1995) reported that ATP (100 fM) occluded responses of recombinant ganglionic nicotinic receptors in Xenopus oocytes. This latter finding suggests that the model in Fig. 1C could be correct in some instances. Outside-out patches containing only one receptor class could be examined to test this. At present our understanding of the mechanisms by which nicotinic and P2X ATP receptor agonists could interact is hampered by our lack of knowledge about P2X receptors, e.g. whilst nicotinic receptors contain nicotinic and ATP binding sites, the ATP binding site on P2X receptors is still equivocal. It will be interesting to see whether interactions occur using cloned neuronal nicotinic receptors expressed in HEK cells (which appear to closely resemble the intact ganglion; Sivilotti et al. 1997) and cloned human P2X3 ATP receptors (whose agonist selectivity sequence resembles those of some ganglionic P2X receptors; see Silinsky & Gerzanich, 1993).