RESUMEN
Parasympathetic control of murine urinary bladder consists of contractile components mediated by both muscarinic and purinergic receptors. Using intracellular recording techniques, the purinergic component of transmission was measured as both evoked excitatory junctional potentials (EJPs) in response to electrical field stimulation and spontaneous events [spontaneous EJPs (sEJPs)]. EJPs, but not sEJPs, were abolished by the application of the Na(+) channel blocker tetrodotoxin and the Ca(2+) channel blocker Cd(2+). Both EJPs and sEJPs were abolished by the application of the P2X(1) antagonist 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF279). Application of phorbol dibutyrate (PDBu) increased electrically evoked EJP amplitudes with no effect on mean sEJP amplitudes. Similar increases in EJP amplitudes were produced by PDBu in the presence of either the nonselective protein kinase inhibitor staurosporine or the specific protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X). These results suggest that PDBu increases the purinergic component of detrusor transmission through increasing neurogenic ATP release via a PKC-independent mechanism.
Asunto(s)
Unión Neuromuscular/efectos de los fármacos , Neurotransmisores/metabolismo , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/metabolismo , Receptores Purinérgicos/metabolismo , Vejiga Urinaria/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Estimulación Eléctrica/métodos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Unión Neuromuscular/metabolismo , Ésteres del Forbol/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Purinérgicos/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Suramina/análogos & derivados , Suramina/farmacología , Transmisión Sináptica/efectos de los fármacos , Vejiga Urinaria/enzimología , Vejiga Urinaria/metabolismoRESUMEN
Ethanol has been shown to have both presynaptic and postsynaptic effects on synaptic transmission. However, the mechanisms by which ethanol affects evoked neurotransmitter release have not been studied at the mouse neuromuscular junction, a synapse at which binomial analysis of neurotransmitter release and measurements of prejunctional ionic currents can be made. Ethanol (400 mM) increased neurotransmitter release independently of both the cAMP and phorbol ester/Munc13 signaling pathways. Binomial analysis of neurotransmitter release revealed that ethanol increases the average probability of secretion without an effect on the immediately available store of the neurotransmitter. Application of ethanol also resulted in an inhibition of potassium currents in the motor nerve endings. These results suggest that the potentiating effects of ethanol on neurotransmitter release at the skeletal neuromuscular junction are mediated by an inhibition of the delayed rectifier potassium current, thus increasing both calcium entry into the nerve ending and the probability of neurotransmitter release. Identifying the mechanism through which ethanol enhances neurotransmitter release at the neuromuscular junction may be useful in determining the processes underlying the enhancement of neurotransmitter release at other synapses.
Asunto(s)
Etanol/farmacología , Unión Neuromuscular/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Acetilcolina/metabolismo , Animales , Canales de Calcio/metabolismo , Cloruro de Calcio/farmacología , AMP Cíclico/metabolismo , Técnicas In Vitro , Ratones , Ratones Endogámicos , Modelos Estadísticos , Unión Neuromuscular/metabolismo , Técnicas de Placa-Clamp , Ésteres del Forbol/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Cloruro de Potasio/farmacología , Potenciales Sinápticos/efectos de los fármacosRESUMEN
The actions of the active L-isomer of vesamicol, an inhibitor of the vesicular storage of acetylcholine, has been studied on spontaneous and evoked acetylcholine release at the snake neuromuscular junction. Miniature endplate currents and endplate currents were recorded from cut muscle fibres of the garter snake, Thamnophis sirtalis. In controls, prolonged periods of high frequency nerve stimulation produced a bimodal distribution of miniature endplate current amplitudes. The stimulation induced "small-mode" miniature endplate currents had a mean amplitude of around 40-55% of the pre-stimulation miniature endplate current. Relative to the normal-sized post-stimulation miniature endplate current, the proportion and, to a lesser extent, amplitude of the small-mode miniature endplate currents was related to both the frequency and duration of nerve stimulation and to the extracellular calcium ion concentration. In unstimulated preparations, L-vesamicol (2-5 microM) did not affect either endplate current quantal content or miniature endplate current amplitude or frequency. However, at these doses, the mean amplitude of the stimulation-induced, small-mode miniature endplate current was reduced by L-vesamicol in a concentration-dependent manner such that they were not visible at the highest dose. L-Vesamicol had no affect on the mean or coefficient of variance of amplitude of the larger, normal-sized miniature endplate current. Additionally, the stimulation-induced increase in overall miniature endplate current frequency seen in controls was abolished by 5 microM L-vesamicol. After prolonged 10 Hz nerve stimulation endplate current amplitude was markedly reduced in both controls (by 94%) and in the presence of 5 microM L-vesamicol (by 98%). Analysis of endplate current amplitude variance showed that in control the decrease was due to reductions in both quantal content and quantal size while in L-vesamicol the decrease was due entirely to a change in quantal content with no change in quantal size. Thus, we have observed that L-vesamicol selectively reduces the amplitude of a population of stimulation-induced small-mode quanta both as miniature endplate currents and as constituents of endplate currents. We suggest that these quanta are derived from a highly active, readily releasable pool. An action of L-vesamicol on this labile pool is consistent with previous observations on its ability to inhibit the vesicular storage of acetylcholine.
Asunto(s)
Acetilcolina/farmacocinética , Unión Neuromuscular/fisiología , Inhibidores de la Captación de Neurotransmisores/farmacología , Fenciclidina/análogos & derivados , Piperidinas , Serpientes/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Estimulación Eléctrica , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Fenciclidina/farmacologíaRESUMEN
The effects of the phosphatidylinositol 4-kinase inhibitor, phenylarsine oxide (PAO), on acetylcholine (ACh) release and on prejunctional Ca(2+) currents were studied at the frog neuromuscular junction using electrophysiological recording techniques. Application of PAO (30 microM) 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 microM) also inhibited the perineural voltage change associated with Ca(2+) currents through N-type Ca(2+) channels (prejunctional Ca(2+) 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 Ca(2+) current. The effects of PAO on N-type Ca(2+) channels were investigated more directly using the whole cell patch clamp technique on acutely dissociated sympathetic neurons. Application of PAO (30 - 40 microM) to these neurons decreased the voltage-activated calcium currents through N-type Ca(2+) 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 Ca(2+) entry via N-type calcium channels. The relationship between the effects of PAO on N-type Ca(2+) channels in motor nerve endings and in neuronal soma is discussed.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Arsenicales/farmacología , Canales de Calcio Tipo N/metabolismo , Calcio/metabolismo , Neurotransmisores/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Quelantes/farmacología , Dimercaprol/farmacología , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Ganglios Simpáticos/efectos de los fármacos , Ganglios Simpáticos/fisiología , Técnicas In Vitro , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Rana pipiensRESUMEN
BACKGROUND AND PURPOSE: Ethanol is known to have both pre-synaptic and post-synaptic effects at a range of loci in the mammalian nervous system, including the neuromuscular junction. However, the effects of ethanol on evoked synaptic transmission have not been previously studied at the mouse neuromuscular junction. Here, we report on the effects of ethanol on evoked neuromuscular transmission and the interaction of ethanol with non-depolarizing blocking drugs. EXPERIMENTAL APPROACH: Electrophysiological techniques to measure synaptic potentials and synaptic currents were employed in this study. KEY RESULTS: Ethanol (≥100 mM) produced increases in the amplitudes of both spontaneous and evoked synaptic events. Under conditions in which neuromuscular transmission was blocked by (+)-tubocurarine, ethanol (12-100 mM) produced greater increases in evoked response amplitude than in spontaneous response amplitude recorded in the absence of (+)-tubocurarine. Ethanol (100 mM) did not affect evoked neurotransmitter release in low-calcium/high-magnesium solutions. With respect to the clinically used neuromuscular blocking drugs, ethanol (100 mM) interfered with the blocking action of vecuronium, but not cisatracurium. CONCLUSIONS AND IMPLICATIONS: Under these conditions, the stimulant effect of ethanol on neuromuscular transmission is exclusively on the post-junctional elements, both to enhance transmission through nicotinic receptors and also via interactions with neuromuscular blocking agents. These actions of ethanol on neuromuscular transmission may affect the dosage of neuromuscular blockers required in patients who have imbibed significant amounts of alcohol.
Asunto(s)
Etanol/farmacología , Bloqueantes Neuromusculares/farmacología , Unión Neuromuscular/efectos de los fármacos , Potenciales Sinápticos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ratones , Ratones Endogámicos , Tubocurarina/farmacologíaAsunto(s)
Neuronas Motoras/metabolismo , Terminaciones Nerviosas/metabolismo , Fármacos Neuromusculares Despolarizantes/farmacología , Neurotransmisores/metabolismo , Fenciclidina/análogos & derivados , Piperidinas , Animales , Estimulación Eléctrica , Técnicas In Vitro , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/fisiología , Fenciclidina/farmacología , RatasRESUMEN
Adenosine reduces both evoked and spontaneous calcium-dependent acetylcholine (ACh) release through a mechanism downstream of calcium entry at amphibian motor nerve endings (Silinsky EM. J Physiol 1984; 346: 243-56). LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), an inhibitor of both phosphoinositide-3 kinase (PI-3 kinase) and casein kinase II, has been reported to increase spontaneous ACh release reflected in miniature endplate potential (MEPP) frequencies independently of intraterminal calcium at the frog neuromuscular junction (Rizzoli SO, Betz WJ. J Neurosci 2002; 22: 10680-9). It has been suggested that the increase in MEPP frequency caused by LY 294002, is mediated through an action on synaptotagmins, vesicle associated calcium sensors believed to trigger synaptic vesicle exocytosis. We thus examined the effects of adenosine on MEPP frequencies and evoked ACh release reflected as endplate potentials (EPPs) in order to determine if the presumed calcium-independent ACh release is affected by adenosine. We also wanted to determine if PI-3 kinase or casein kinase II is involved in mediating or modulating the inhibitory effects of adenosine. To these ends, we examined the effects of adenosine in the presence of LY 294002, wortmannin (a highly selective the PI-3 kinase inhibitor), or DRB (5,6-dichlorobenzimidazole riboside, an inhibitor of casein kinase II). LY 294002 reduced the sensitivity of both MEPP frequencies and the nerve-evoked calcium dependent EPPs to adenosine. The occlusive effects of LY 294002 on the actions of adenosine on MEPPs and EPPs were overcome by increasing adenosine concentration. Neither wortmannin nor DRB had any effect on the sensitivity of the EPPs to adenosine indicating that neither PI-3 kinase nor casein kinase II inhibition mediates the reduction in motor-nerve terminal sensitivity to adenosine produced by LY 294002. The results indicate a competitive relationship between LY 294002 and adenosine at A(1) receptors at the frog neuromuscular junction. This effect is independent of the previously described effects of LY 294002 on the exocytotic process, and is also independent of PI-3 kinase or casein kinase II.
RESUMEN
Recent work from our laboratory has demonstrated that phorbol esters known to stimulate protein kinase C (PKC) also stimulate acetylcholine (ACh) secretion by an action at a strategic component of the secretory apparatus [ J Physiol (Lond) 501:41-48]. In an attempt to determine whether the stimulatory effects of phorbols are mediated by PKC, we examined the effects of several PKC antagonists on ACh release promoted by phorbol 12,13-dibutyrate (PDBu) at the frog neuromuscular junction. PKC antagonists that act at the ATP binding site (C3 domain) were examined for their ability to antagonize the stimulatory action of PDBu. Neither the nonselective PKC inhibitor, staurosporine (at concentrations as high as 1 microM), nor its more selective derivative, GF109203X (at concentrations as high as 10 microM), attenuated the stimulatory effects of PDBu. PKC antagonists that act at the phorbol ester binding site (C1 domain) were examined for their ability to antagonize the stimulatory action of PDBu. Neither sphingosine (500 microM) nor calphostin C (25 microM) reduced the stimulatory actions of PDBu on ACh release. These results suggest that a presynaptic protein possessing a phorbol ester receptor and not the enzyme PKC is the target site for the stimulatory effects of phorbol esters at motor nerve endings.
Asunto(s)
Acetilcolina/metabolismo , Carcinógenos/farmacología , Unión Neuromuscular/efectos de los fármacos , Ésteres del Forbol/farmacología , Proteína Quinasa C/efectos de los fármacos , Estaurosporina/farmacología , Animales , Inhibidores Enzimáticos/farmacología , Naftalenos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Rana pipiens , Esfingosina/farmacologíaRESUMEN
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 microM) 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.
Asunto(s)
Adenosina/fisiología , Terminaciones Nerviosas/fisiología , Unión Neuromuscular/fisiología , Neurotransmisores/metabolismo , Ésteres del Forbol/farmacología , Acetilcolina/metabolismo , Adenosina/farmacología , Anfibios/fisiología , Animales , Distribución Binomial , Calcio/farmacología , Relación Dosis-Respuesta a Droga , Electrofisiología , Inhibidores Enzimáticos/farmacología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Indoles/farmacología , Maleimidas/farmacología , Método de Montecarlo , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Rana pipiens , Estaurosporina/farmacología , Vesículas Sinápticas/fisiologíaRESUMEN
The statistical parameters underlying acetylcholine (ACh) release were studied using Ca(2+) and Sr(2+) 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 (MEPP(f)), were evoked by high potassium depolarization in either Ca(2+) or Sr(2+) solutions. Increases in MEPP(f) mediated by Ca(2+) were of very low probability and well-described by a Poisson distribution whilst similar MEPP(f) increases mediated by Sr(2+) were best described as a simple binomial distribution. From the binomial distribution in Sr(2+) solutions, values for the average probability of release (p) and the number of releasable ACh quanta (n) may be determined (whereby mean MEPP(f) = np). In Sr(2+) 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 Sr(2+) 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 Ca(2+) and Sr(2+) 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 Ca(2+) as compared to Sr(2+). The statistical analyses suggest the presence of two different Ca(2+)-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 Sr(2+) as a partial agonist for evoked ACh release, and (iii) the specific loci that may represent the sites of Ca(2+) and Sr(2+) sensitivity.
Asunto(s)
Acetilcolina/metabolismo , Calcio/farmacología , Estroncio/farmacología , Potenciales de Acción/fisiología , Animales , Distribución Binomial , Simulación por Computador , Electrofisiología , Método de Montecarlo , Placa Motora/fisiología , Unión Neuromuscular/fisiología , Distribución de Poisson , Rana pipiens , SolucionesRESUMEN
1. The presynaptic mechanisms governing the release and recycling of synaptic vesicles have been studied by examining the effects of nerve stimulation, (-)-vesamicol (an inhibitor of acetylcholine transport into synaptic vesicles) and troxypyrrolium (an inhibitor of the high-affinity, sodium-dependent, choline uptake system) on endplate currents (EPCs) and miniature endplate currents (MECPs) recorded from motor endplates in cut rat hemidiaphragm preparations. 2. In control experiments, 5 min of 10 Hz nerve stimulation had no effect on either the mean or the distribution of MEPC amplitudes. 3. Nerve stimulation in the presence of (-)-vesamicol (25 nM-10 microM) revealed a population of MEPCs that was unaffected by the compound and a population of MEPCs whose mean amplitude was selectively reduced by the compound. 4. Nerve stimulation in the presence of troxypyrrolium (20 microM) produced a uniform reduction in the amplitude of all MEPCs with no change in the coefficient of variance of MEPC amplitudes. 5. The concentration-dependent effects of (-)-vesamicol on the amplitude of the evoked EPCs paralleled the concentration-dependent effects of the compound on MEPC amplitudes. 6. The results are consistent with the hypothesis that both recycled and performed synaptic vesicles are heterogeneously released from rat motor nerve terminals and that (-)-vesamicol acts selectively on recycling vesicles. In addition, a model of vascular loading that accounts for the different effects of nerve stimulation on MEPC amplitudes in the presence of (-)-vesamicol and troxypyrrolium is described.
Asunto(s)
Acetilcolina/metabolismo , Placa Motora/efectos de los fármacos , Piperidinas/farmacología , Pirrolidinas/farmacología , Acetilcolina/fisiología , Animales , Técnicas In Vitro , Placa Motora/metabolismo , Placa Motora/fisiología , Fármacos Neuromusculares Despolarizantes/farmacología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/fisiología , Ratas , Ratas Endogámicas , Transmisión Sináptica , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/fisiologíaRESUMEN
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 microM) or cytisine (50-75 microM) and the P2X receptor agonists ATP (0.1-7 microM) or alpha,beta-methylene ATP (6 microM) 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 microM). The occlusive effects of ATP analogues on inward currents generated by nicotinic agonists were blocked by the P2X receptor antagonist suramin (100 microM). 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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Neuronas/metabolismo , Receptores Nicotínicos/metabolismo , Receptores Purinérgicos P2/metabolismo , Sistema Nervioso Simpático/metabolismo , Animales , Estimulación Eléctrica , Electrofisiología , Cobayas , Técnicas In Vitro , Masculino , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2X , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
1. Phorbol esters activate protein kinase C (PKC) and also increase the secretion of neurotransmitter 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 neuromuscular 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-alpha-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.