Depolarization reverses age-related decrease of spontaneous transmitter release.

The effect of increasing extracellular Ca concentration on spontaneous transmitter release was studied at soleus nerve terminals of young (10 mo) and old (24 mo) C57BL/6J mice depolarized by high extracellular K concentration ([K]o). By using intracellular recording, miniature end-plate potentials (MEPPs) were first recorded in a normal [K]o Krebs solution. Subsequently, MEPPs were recorded in high [K]o Krebs solutions with four different Ca concentrations: Ca-free/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 0.5, 1.5, and 2.5 mM Ca. In both the normal [K]o Krebs and the Ca-free-high [K]o Krebs solutions, MEPP frequency was lower at old than at young nerve terminals. In the three high [K]o Krebs solutions with Ca, MEPP frequency was progressively higher at old than at young nerve terminals with higher Ca concentrations. Periodic oscillations were observed in MEPP frequency of depolarized nerve terminals. The period of oscillation was inversely proportional to spontaneous transmitter release. These results demonstrate that when the nerve terminal is depolarized, permeability of the terminal membrane to Ca increases because of opening of voltage-dependent Ca channels. In the present study resting MEPP frequency was lower at old than at young terminals. On depolarization, MEPP frequency became higher at old than at young terminals. The study demonstrates that voltage-dependent Ca entry increases during aging at the soleus nerve terminal.     

Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.     

Dual action of intracellularly released calcium on the quantal mediator secretion

The mice diaphragm muscle and microelectrode technique were used to check the influence of ryanodine (0.5 mcM) on spontaneous and evoked mediator release under conditions of potassium depolarization (8-16 mM [K+]ex or rhythmic (4-100 Hz) stimulation of motor nerve terminals. Weak tonic calcium loading (by muscle exposition to 8 mM [K+]ex) caused a two-fold frequency increase if miniature and plate potentials (MEPPs), which was returned to the basal level by subsequent application of ryanodine. This inhibitory effect of ryanodine was blocked by apamin (500 nM) a blocker of K+(Ca)-channels. A greater calcium load of terminals (in solution with 16 mM [K+]ex) caused a 15-fold increase of MEPPs frequency. Subsequent ryanodine application caused an additional 2-3-fold increase of MEPPs frequency. During rhythmic activity of motor synapses, ryanodine was able to decrease the amplitude of EPP by 60% at plateau phase at short low frequency (4 Hz) of discharges and to increase the amplitude of EPP by 60-150% at high frequency (70-100 Hz) of discharges. It is concluded that rynodine induced calcium release from intraterminal Ca2+-stores can influence dual: excitatory or inhibitory, action on spontaneous and evoked mediator release, due to different intraterminal calcium loads and regimen of synaptic activity.     

The influence of hindlimb unloading on the effectiveness of modulation of the mediator secretion via the autoreceptor system

In experiments on neuromuscular synapses of rat fast (m. Extensor digitorum longus, EDL) and slow (m. soleus) skeletal muscles, changes in the intensity of spontaneous quantal mediator secretion in response to the activation of presynaptic cholinoreceptors by the nonhydrolyzable acetylcholine analogue carbachol and to an increase in K+ concentration in the control group of animals and in animals subjected to different terms of unloading of hindlimbs have been compared. The intensity of spontaneous secretion of mediator quanta was evaluated from the mean frequency of miniature endplate potentials. In the control group of animals, the frequency of miniature endplate potentials by the action of carbachol increased by 363% in m. EDL and by 62% in m. soleus. The frequency of miniature endplate potentials in the synapses of m. EDL was more sensitive to K(+)-induced depolarization too. The bearing unloading of hindlimbs abolished the sensitivity of spontaneous secretion to carbachol in the synapses of m. EDL, whereas in m. soleus it was unchanged. However, the preservation of sensitivity of nerve endings of fast muscle to K(+)-induced depolarization allows one to assume that the hindlimb unloading leads to a decrease in the number of functioning presynaptic receptors.     

Facilitation of spontaneous acetylcholine release induced by activation of cAMP in rat neuromuscular junctions

Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. Activation of the cAMP-protein kinase A (PKA) pathway has been shown to facilitate synaptic transmission in mammalian neuromuscular synapses, although the relevant phosphorylation targets are mostly unknown. We found that the inhibitor of the phosphodiesterase aminophylline (1 mM AMIN), the membrane-permeable analog of cAMP, 8-Br-cAMP (5 mM) and, the direct adenylate cyclase activator, forskolin (20 microM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltage-dependent calcium channels (VDCC), since these proteins are known to be phosphorylated by PKA. But this possibility was ruled out, since the increase in MEPPs frequency was not attenuated by the VDCC blocker Cd2+ (100 microM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2+]o and of Ca2+ influx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca2+]i was diminished by exposing the preparations to zero Ca2+-EGTA solution (isotonic condition) or when nerve terminals were loaded with a permeant Ca2+ chelator (BAPTA-AM) (hypertonic condition), indicate that cAMP-mediated presynaptic facilitation is a function of nerve terminal Ca2+ concentration. We also found that AMIN exerted a comparable increase in MEPPs frequency in control and high K+ (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.     

Protons resolve dual effects of calcium on miniature end-plate potential frequency at frog neuromuscular junctions

Inhibition of transmitter release by protons (H+) was studied at the frog neuromuscular junction at various extracellular concentrations of calcium ([Ca++]o) and potassium ([K+]o) by recording miniature end-plate potential (MEPP) frequency with the intracellular microelectrode. H+ decreased K+ -stimulated MEPP frequency. A double logarithmic graph of MEPP frequency at 7.5 mM K+ vs. [H+]o yielded a straight line with negative slope. At 10 mM K+, there was a parallel shift to the right of the graph. According to the surface charge model, K+ acts solely to depolarize the prejunctional membrane in accordance with the Nernst equation. By decreasing the prejunctional negative surface charge, H+ decreases K+ -stimulated MEPP frequency by decreasing [Ca++]o at the Ca++ channel. An estimated pKa of 4.20 may represent an acidic site at the Ca++ channel associated with Ca++ influx. As [Ca++]o increased above 1 mM for pH 7.40 and 10 mM K+, MEPP frequency decreased, i.e., the inhibitory component of dual effects of Ca++ occurred. At pH 6.40, the inhibitory component was abolished, unmasking the stimulatory effect of Ca++ on MEPP frequency. Reversal of Ca++ action by H+ could not be explained by surface charge theory alone. A double logarithmic graph of MEPP frequency vs. [K+]o at 8.5-10.5 mM was linear with a slope of 4. There were parallel shifts to the right of this graph for changes in pH from 7.40 to 6.90 and in [Ca++]o from 1 to 2.5 mM. These results are explained on the hypothesis that K+ also acts at an acidic prejunctional site to increase Ca++ -dependent quantal transmitter release. This action of K+ was inhibited by H+ and raised Ca++. Based on kinetic theory, the estimated pKa of the acidic prejunctional K+ site was 6.31. Based on free energy calculations, its cation preference was H+ greater than K+ greater than Ca++.     

[3H]Noradrenaline Release from Brain Slices Induced by an Increase in the Intracellular Sodium Concentration: Role of Intracellular Calcium Stores

Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10-120 mM K+) or to veratrine (1-25 microM). In the absence of extracellular Ca2+ veratrine, in contrast to K+-depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 microM). The Ca2+ antagonist Cd2+ (50 microM), which strongly reduced K+-induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 microM), known to inhibit Ca2+-entry into mitochondria, enhanced veratrine-induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine-induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

Effects of black widow spider venom and Ca2+ on quantal secretion at the frog neuromuscular junction

A modification of the classical procedure of fluctuation analysis is used to measure the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of miniature endplate potentials (MEPPs) at frog cutaneous pectoris neuromuscular junctions treated with black widow spider venom (BWSV). MEPP parameters are determined from the power spectrum of the fluctuating potential and the second (variance), third (skew), and fourth semi-invariants (cumulants) of high-pass-filtered records of the potential. The method gives valid results even when the mean potential undergoes slow changes unrelated to MEPPs and when the MEPP rate is not stationary; it detects changes in the distribution of MEPP amplitudes and corrects for the nonlinear summation of MEPPs. The effects of Ca2+ on BWSV-induced secretion are studied in detail. When Ca2+ is absent, the power spectrum of the fluctuations is shaped like the spectrum of w(t) and secretion is quasi-stationary; (r) rises smoothly to peak values of approximately 1,500/s and then quickly subsides to levels near 10/s. Many relatively small and some "giant" MEPPs occur at the ends of the experiments, and the distribution of MEPP amplitudes broadens. When the effects of this broadening are corrected for, we find that approximately 0.7 X 10(6) MEPPs occurred during the 30 min of intense secretion. Since BWSV depletes nerve terminals of their quanta of transmitter and their synaptic vesicles, this figure is an upper limit for the quantal store in a resting terminal. When Ca2+ is present, the noise spectrum deviates from the spectrum of w(t) and secretion is nonstationary; (r) rises to similar peak values but is sustained at levels near 400/s for up to an hour and at least 1.5 X 10(6) quanta are secreted within this period. Thus, the quantal store must have turned over at least twice under this condition. Data previously obtained at junctions treated with La3+ are corrected for nonlinear summation and for the distribution of MEPP amplitudes. The two corrections roughly compensate each other, and the corrected results confirm the previous conclusion that the number of quanta secreted from La3+-treated terminals during 1 h is not strongly dependent upon the extracellular concentration of Ca2+; approximately 2 X 10(6) quanta are released even when Ca2+ is absent.     

Depolarization promotes caffeine induced [3H]-noradrenaline release in calcium-free solution from peripheral sympathetic nerves

The transmitter releasing action of caffeine was studied in the absence of extracellular Ca2+ from the peripheral sympathetic nerves of the rabbit main pulmonary artery. Caffeine (10 mM) increased the release of [3H]-noradrenaline moderately, but not significantly in Ca2(+)-free (+1 mM EGTA) Krebs solution. When peripheral nerve endings/varicosities were depolarized by elevating extracellular K+ to 47.2 mM and 70.8 mM in Ca2(+)-free solution, the transmitter releasing effect of 10 mM caffeine became significant. Ca2+ removal itself transiently increased the [3H]-noradrenaline outflow. In the individual experiments the amount of the caffeine evoked transmitter release at 47.2 mM and 70.8 mM K(+)-depolarization was inversely correlated to the release evoked by Ca2(+)-removal. Our results suggest that caffeine-sensitive calcium stores are present in peripheral nerve terminals of rabbit pulmonary artery, and part of the caffeine sensitive calcium stores may discharge during Ca2(+)-removal from the extracellular solution.     

Effect of presynaptic neurotoxin notechis II-5 from tiger snake venom on the motor nerve endings of mice]

The neurotoxin notechis II-5 (N-II-5) from tiger snake venom (Notechis scutatus) induces three-phasic changes in miniature end-plate potential (MEPP) frequency recorded in the mouse diaphragm muscle: an initial fall of frequency followed by increase and decrease in MEPP frequency up to complete blockade. The effect of N-II-5 was enhanced with rising of the solution temperature from 20 to 30 and 35 degrees C. Removal of Ca2+ from the solution prevented the presynaptic effect of N-II-5. After washing out of the muscle from N-II-5 with Ca-free solution, addition of Ca2+ to the solution provoked the development of the effect typical of the effect of typical of the toxin. In the presence of N-II-5 an increase in K+ concentration in the solution up to 20 mM did not result in a sharp rise of MEPP frequency characteristic of depolarized nerve terminals. The agents that raise Ca2+ axoplasmic concentration not on account of depolarization of nerve terminals (hypertonic solution, ionofor A23187) preserved the capacity for increasing MEPP frequecy. It is suggested that the presynaptic effect of N-II-5 is related to its phospholipase activity and can be explained by disturbance of the activity of release sites rather than by depletion of transmitter stores.     

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.     

Influence of hindlimb unloading on the modulation of neurotransmitter secretion through the autoreceptor system

In experiments on neuromuscular junctions of fast (m. extensor digitorum longus, EDL) and slow (m. soleus) muscles of rats under hindlimb unloading of varied duration, we compared the intensity of spontaneous quantal secretion of neurotransmitter in response to potassium depolarization and activation of presynaptic cholinoreceptors with a nonhydrolyzable acetylcholine analog. Secretion was assessed by the mean frequency of miniature endplate potentials. In the controls, carbachol raised this index by 363% in EDL and by 62% in soleus. Secretion in the fast muscle was also more sensitive to [K⁺]. Hindlimb unloading abolished the sensitivity to carbachol in EDL while in soleus it did not change. Preservation of the sensitivity of the fast muscle to potassium depolarization suggested that unloading reduced the number of functional presynaptic receptors.     

Effect of low extracellular calcium and ryanodine on muscle contraction of the mouse during postnatal development.

We have examined the effects of low Ca2+ solutions, Co2+, and ryanodine on the isometric tension and contraction speed of isolated, developing mouse EDL muscles. Twitch responses of young muscles (7-14 days postnatal) were more sensitive to lowered [Ca2+]o than those of more fully developed muscles (22-35 days postnatal). Responses of EDL muscles from a middle-aged group (15-21 days postnatal) were intermediate between the two other groups. Overall, the time course of contraction in a single twitch was accelerated by low [Ca2+]o. Ca(2+)-free solution induced a 7.95 and 9.25 mV depolarization in young and "old" muscle fibres, respectively. The presence of cobalt ions (5 mM) in the Krebs solution had a similar effect as Ca(2+)-free Krebs in terms of reduction of the isometric twitch and tetanic tensions of EDL muscles from the various age groups. In contrast, the shortening of the contraction time seen with Ca(2+)-free solution did not take place following exposure to Co(2+)-containing solutions. Finally, young (7-14 days postnatal) muscles were less sensitive to the inhibitory action of ryanodine on the twitch compared with more fully developed muscles (22-35 days postnatal). Taken together, our results indicate that from birth to maturity, there is a gradual change in the spectrum of calcium utilization for the contractile process.     

The effect of sodium ions of MEPP frequency at the frog neuromuscular junction.

MEPP frequency at the frog neuromuscular junction maintained in saline with normal [K]_o was insensitive to reductions in [Na]_o at 130°C. However, at 23°C, decreasing [Na]_o causes a progressive rise in MEPP frequency; there is an approximately linear relationship between log [Na]_o and the rate of spontaneous release. The effect of reducing [Na]_o is dependent on [Ca]_o; thus MEPP frequency is unaffected, even at 23°C, by changes in [Na]_o when [Ca]_o is reduced to the low level of 5 × 10^?7M. It is suggested that: (i) MEPP frequency is determined by [Ca]_i at the presynaptic terminals (ii) Reductions in [Na]_o cause an increase in Ca-influx. At 13°C the presynaptic terminals are able to maintain [Ca]_i constant when challenged whereas, at 23°C, there is a mobilization of Ca from intracellular storage sites and under these conditions [Ca]_i is not maintained constant in the face of a rise in Ca-influx (associated with a reduction in [Na]_o) and MEPP frequency consequently rises. The ways in which both extracellular and intracellular Na affect [Ca]_i and MEPP frequency are discussed.     

The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP_8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.     

Effects of resting membrane potential and intactness of the T-tubules on caffeine contractures in rat skeletal muscle

We have studied the effects of changes in the resting membrane potential (Vm) and T-tubules on caffeine contracture (25 mM) elicited in rat soleus muscle in vitro at 34 degrees C. In high [K]o (30-140 mM, [K]o X [Cl]o constant) caffeine contractures were reduced by about 40-50% and had a faster time course than in normal Krebs ([K]o = 5 mM). Detubulation of the muscles by an osmotic treatment produces a reduction of about 30% in the caffeine contracture tension. Our results with high K solutions suggest a reduced sensitivity of the myofibrils to calcium released by caffeine. The effects of detubulation on caffeine contracture suggest that caffeine may have a direct effect on sarcolemma in addition to its well known action on the sarcoplasmic reticulum (SR). However, a depletion of the calcium content in the SR of depolarized muscle fibres as well as an anatomical damage produced by the osmotic treatment can not be ruled out as an explanation for the reduced caffeine contracture.     

Postsynaptic end-plate ionic currents in fast and slow chick muscle fibers. Effect of cholinesterase inhibition

Miniature end-plate potentials (MEPPs) were recorded in fast and slow chick muscle fibers extracellularly (focally) and under voltage clamp conditions. The duration of the MEPPs in synapses of slow fibers was on average 2.5 times longer than their duration in synapses of fast fibers. Inhibition of acetylcholinesterase (AChE) lengthened MEPPs by a varied degree: by 1.5 times in synapses of slow fibers and by 3.5 times in those of fast fibers. As a result the difference in the decay time of MEPPs in synapses of these types disappeared almost completely, and only a small difference remained in the rise time of the MEPPs. The MEPP decay time during hyperpolarization in the slow fiber synapse was rather less dependent on potential than in synapses of fast fibers; after inhibition of AChE it became even less dependent. Similar changes in potential dependence were found after lengthening of the MEPP by the action of ethanol. The functional significance of differences in AChE activity and in the activating effect of the mediator for the kinetics of MEPPs in synapses of fast and slow fibers is discussed.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 390–397, July–August, 1981.     

Depolarization, intracellular calcium and exocytosis in single vertebrate nerve endings.

We have investigated the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve terminals. The change of [Ca2+]i and vasopressin release were measured with a time resolution of less than 1 s in response to K(+)-induced depolarization. Exocytosis was also monitored in the whole-terminal patch-clamp configuration by time resolved capacitance measurements while [Ca2+]i was simultaneously followed by fura-2 fluorescence measurements. In intact as well as patch-clamped nerve terminals sustained depolarization leads to a sustained rise of [Ca2+]i. The rate of vasopressin release from intact nerve terminals rises in parallel with [Ca2+]i but then declines rapidly towards basal (t1/2 approximately 15 s) despite the maintained high [Ca2+]i indicating that only a limited number of exocytotic vesicles can be released. We demonstrate that in nerve terminals exocytosis can be followed during step depolarization by capacitance measurements. The capacitance increase starts instantaneously whereas [Ca2+]i rises with a half time of several hundred milliseconds. An instantaneous steep capacitance increase is followed by a slow increase with a slope of 25-50 fF/s indicating the sequential fusion of predocked and cytoplasmic vesicles. During depolarization the capacitance slope declines to zero with a similar time course as the vasopressin release indicating a decrease in exocytotic activity. Depolarization per se in the absence of a sufficient rise of [Ca2+]i does not induce exocytosis but elevation of [Ca2+]i in the absence of depolarization is as effective as in its presence. The experiments suggest that a rapid rise of [Ca2+]i in a narrow region beneath the plasma membrane induces a burst of exocytotic activity preceding the elevation of bulk [Ca2+]i in the whole nerve terminal.     

The effects of pentachlorophenol (PCP) at the toad neuromuscular junction

1. Effects of PCP at the frog neuromuscular junction were studied in vitro in sciatic nerve sartorius muscle of the toad Pleurodema-thaul. 2. Within the concentration 0.003-0.1 mM, PCP caused a dose-time-dependent block of evoked transmitter release acompanied by an increase in the rate of spontaneous quantal release. 3. PCP induced an increase in miniature endplate potential (MEPP) frequency and it was not antagonized in a Ca2(+)-free medium, indicating that it does not depend upon Ca2+ influx from the external medium, but may act by releasing Ca2+ from intraterminal stores. 4. The present data, together with previous results concerning PCP at eighth sympathetic ganglia indicate that 3,4-diaminopyridine (3,4-DAP) counteracts the effects of PCP on synaptic transmission. This result suggests that PCP interfering Ca2+ influx occurs during depolarization of motor nerve terminals.     

Calcium permeability changes and neurotransmitter release in cultured brain neurons. II. Temporal analysis of neurotransmitter release

The coupling between depolarization-induced calcium entry and neurotransmitter release was studied in rat brain neurons in culture. The endogenous dopamine content of the cells was determined by high performance liquid chromatography utilizing electrochemical detection. The amount of dopamine in unstimulated cells was found to be about 16 ng/mg of protein. Depolarization of the neurons by elevated K+ caused a Ca2+-dependent release of dopamine from the cells. Following 1 min of depolarization, the cellular dopamine content and the amount of [3H]dopamine in cells preloaded with the radioactive transmitter were reduced by 35%. The release of [3H]dopamine by the neurons was measured at 1.5-6-s intervals by a novel rapid dipping technique. Depolarization in the presence of Ca2+ (1.8 mM) enhanced the rate of neurotransmitter release by 90-fold (0.072 +/- 0.003 s-1) over the basal release in the presence of Ca2+. The evoked release consisted of a major rapidly terminating phase (t1/2 = 9.6 s) which comprised about 40% of the neurotransmitter content of the cells and a subsequent slower efflux (t1/2 = 575 s) which was observed during following prolonged depolarization. Predepolarization of the cells in the absence of extracellular Ca2+ did not affect the kinetics of the evoked release. The fast evoked release could be re-elicited in the cells after 20 min "rest" in reference low K+ buffer. The effects of varying the extracellular Ca2+ concentrations on the kinetic parameters of the evoked release were measured. The amount of neurotransmitter released during the fast kinetic phase was very sensitive to the external Ca2+ (from 0% in the absence of Ca2+ to 40% of the neurotransmitter content at Ca2+ 0.3 mM). The rate constant of the fast release did not depend on the extracellular Ca2+, whereas the rate constant of the slow release increased from 0.0004 +/- 0.0001 s-1 at 0.4 mM Ca2+ to 0.0012 +/- 0.0002 s-1 at 0.8 mM Ca2+. The fast evoked release was inhibited by verapamil in a concentration-dependent manner. By contrast, verapamil enhanced the basal and the slow release independent of the presence of Ca2+. Both fast and slow phases of the evoked release were blocked by Co2+. Addition of Co2+ within the first 6 s after the onset of depolarization inhibited the fast release but failed to do so when added later on.(ABSTRACT TRUNCATED AT 400 WORDS)