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 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.

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.

Down-regulation of Na channel expression by A23187 in N1E-115 neuroblastoma cells.

Growth of cultured N1E-115 neuroblastoma cells in 1 microM A23187 for 2 days to elevate internal Ca reduced both membrane Na current and the transient, but not steady state, component of outward K current. Na channel mRNA abundance was reduced by an average value of 45% without effect on Kv3.1. Increases in internal Ca may therefore control excitability by independent regulation of Na and K channel mRNA abundance in neurons.

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.

Aminopyridine block of potassium channels in mouse neuroblastoma cells.

Although 4-aminopyridine (4-AP) is known to block a variety of voltage-dependent K channels, details as to the site of action and the mechanism of block are known for relatively few. Single channel analysis has not been extensively used to answer these questions. The actions of 4-AP on whole cell K currents and single voltage-dependent K channels that exhibit fast activation and inactivation were therefore examined in N1E-115 neuroblastoma cells. The concentration for half block (K0.5) of the whole cell K current for externally applied compounds was found to be 56 microns for 4-AP and 0.3 mM for 3,4-diaminopyridine. 4-AP slowed the rate of development of outward K current, and the rate of decay after repolarization. These effects were consistent with the idea that 4-AP preferentially blocked a type of K channel generating a transient current. Block of this component of current was time- and use-dependent. 4-AP blocked the channel responsible for the transient outward current by decreasing the probability of an open channel in inside-out patches. 4-AP reduced the open time, indicating that 4-AP can interact with the open channel. The first latency to opening was also increased. 4-Aminopyridine methiodide (4-APMI), a permanently charged derivative, blocked the whole cell current with a K0.5 = 0.19 mM. Block by 4-APMI was found to be by a different mechanism at a different site compared to 4-AP.(ABSTRACT TRUNCATED AT 250 WORDS)

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 microM) 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 microM). 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.

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 microM-250 microM) 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.

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 degrees 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.