The vasoactive peptide urotensin II stimulates spontaneous release from frog motor nerve terminals.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca(2+) three-fold had no effect on the MEPP frequency increase to 25 microM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca(2+) caused a 61% reduction in the MEPP frequency increase to 25 microM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 microM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP(3)) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 microM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP(3) and cyclic AMP/protein kinase A.

Nicotinic acid adenine dinucleotide phosphate enhances quantal neurosecretion at the frog neuromuscular junction: possible action on synaptic vesicles in the releasable pool.

Inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine diphosphate-ribose (cADPR) are second messengers that enhance neurosecretion by inducing Ca(2+) release from smooth endoplasmic reticulum (SER). The putative intracellular messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), releases Ca(2+) from stores that are distinct from SER. Evidence is presented here that NAADP causes a concentration-dependent increase in quantal output that is associated with an increase in probability of transmitter release at the frog neuromuscular junction. This effect is mimicked by A23187, a Ca ionophore that promotes Ca(2+) entry at the plasmalemma. The response to NAADP is potentiated by IP(3) but antagonized by cADPR. Thapsigargin completely blocks IP(3) and cADPR responses and decreases but does not prevent the response to NAADP. We conclude that NAADP, whose receptors are widely distributed in the brain, enhances neurosecretion by releasing Ca(2+) from an internal store near the plasmalemma, possibly from synaptic vesicles in the releasable pool. These data also support the hypothesis of a two-pool model for Ca(2+) oscillations at the presynaptic site.

Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum.

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+. The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid or release of Ca2+ from intracellular stores such as mitochondria. Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose, so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10(-4)M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10(-4)M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.

Effect of hypertonicity on augmentation and potentiation and on corresponding quantal parameters of transmitter release.

Augmentation and (posttetanic) potentiation are two of the four components comprising the enhanced release of transmitter following repetitive nerve stimulation. To examine the quantal basis of these components under isotonic and hypertonic conditions, we recorded miniature endplate potentials (MEPPs) from isolated frog (Rana pipiens) cutaneous pectoris muscles, before and after repetitive nerve stimulation (40 s at 80 Hz). Continuous recordings were made in low Ca2+ high Mg2+ isotonic Ringer solution, in Ringer that was made hypertonic with 100 mM sucrose, and in wash solution. Estimates were obtained of m (no. of quanta released), n (no. of functional release sites), p (mean probability of release), and vars p (spatial variance in p), using a method that employed MEPP counts. Hypertonicity abolished augmentation without affecting potentiation. There were prolonged poststimulation increases in m, n, and p and a marked but transient increase in vars p in the hypertonic solution. All effects were completely reversed with wash. The time constants of decay for potentiation and for vars p were virtually identical. The results are consistent with the notion that augmentation is caused by Ca2+ influx through voltage-gated calcium channels and that potentiation is due to Na+-induced Ca2+ release from mitochondria. The results also demonstrate the utility of this approach for analyzing the dynamics of quantal transmitter release.

Angiotensin II and related peptides alter liposomal membrane fluidity.

We investigated the effects of angiotensinogen (Ang), angiotensin I (Ang I), and angiotensin II (Ang II) on the fluidity of phosphatidylcholine vesicles. Changes in fluidity were assessed by changes in anisotropy values calculated from fluorescence polarization measurements. All three compounds produced an increase in membrane fluidity when localized inside the phosphatidylcholine vesicles. When placed outside the vesicles, Ang II increased bilayer rigidity (decreased fluidity), whereas Ang and Ang I produced no effect. These results suggest the possibility that these peptides may alter the fluidity of cell membranes by a direct action on the phospholipid bilayer, which may in turn interfere with receptor-mediated effects.

Real-time detection of mitochondrial inhibition at frog motor nerve terminals using increases in the spatial variance in probability of transmitter release.

The effects of Hg2+, methyl mercury, and flufenamic acid, all of which inhibit mitochondria, were examined at frog motor nerve terminals. Unbiased estimates of m (no. of transmitter quanta released), n (no. of functional release sites), p (probability of release), and vars p (spatial variance in p) were obtained using K(+)-induced asynchronous neurosecretion (m, n and p not having the same definitions as with nerve-evoked release). Transient but significant increases in m, n, p and vars p were found with all three agents. These findings indicate that mitochondrial inhibition and release of sequestered Ca2+ can be detected as a real-time increase in vars p. The results also suggest that changes in vars p might be used to differentiate between cellular (membrane) and subcellular (organellar) actions of drugs at the nerve terminal.

Effect of the putative cognitive enhancer, linopirdine (DuP 996), on quantal parameters of acetylcholine release at the frog neuromuscular junction.

1. The subcellular mechanism and site of action of linopirdine or DuP 996 (3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one) was investigated at the frog neuromuscular junction, using miniature endplate potential (m.e.p.p.) counts and a new method for obtaining unbiased estimates of n (number of functional release sites), p (probability of release), and varsp (spatial variance in p). 2. DuP 996 produced an increase in m (no. of quanta released), which was due to an increase in n and p. The increase in m was concentration-dependent over a range of 0.1-100 microM and completely reversible with 15 min of wash. There was a saturation in the increase in p, but not in the increase in m and n, for [DuP 996] > 10 microM. By contrast, there was no major change in varsp. 3. Block of presynaptic Na(+)- and Ca(2+)-channels with 3 microM tetrodoxin and 1.8 mM Co2+ prevented the m.e.p.p. frequency increase to DuP996, and this effect was completely reversed by washing. 4. Application of the neuronal Ca(2+)-channel blocker, omega-conotoxin GVIA (1 microM) brought about a rapid and profound decrease in the m.e.p.p. frequency increased produced by DuP996. The effect of the toxin was not reversed by prolonged washing. 5. Block of voltage-gated K(+)-channels with 100 microM 4-aminopyridine (4-AP) resulted in only a small (28%) increase in m. The combination of 4-AP (100 microM) and DuP996 (10 microM) produced an increase in m (189%) which was much greater than the sum of the responses to each agent alone. This increase in m was due solely to an increase in n, as p and varsp were unchanged.6. For [DuP 996] up to 100 gM, there was no apparent change in the mean size, amplitude distribution,or time course of m.e.p.ps, signifying that it had no anticholinesterase activity.7. It is concluded that DuP 996 increases the release of quantal transmitter but not the postsynaptic response to the quanta. This appears to involve an effect at the nerve terminal membrane, most likely an increase in Ca2+-conductance, and not an action to block K+-conductance or to release Ca2+ from intraterminal organelles.

Unbiased estimates of quantal release parameters and spatial variation in the probability of neurosecretion.

A procedure was developed for dealing with two problems that have impeded the use of quantal parameters in studies of transmitter release. The first, involving temporal and spatial biasing in the estimates for the number of functional release sites (n) and probability of release (p), was addressed by reducing temporal variance experimentally and calculating the bias produced by spatial variance in p (var(s)p). The second, involving inaccuracies in the use of nerve-evoked endplate potentials (EPPs), was circumvented by using only miniature EPPs (MEPPs). Intracellular recordings were made from isolated frog cutaneous pectoris, after decapitation and pithing of the animals, and the concentration of K+ ([K+]) was raised to 10 mM to increase the level of transmitter release. The number of quanta released (m) by the EPP was replaced by the number of MEPPs in a fixed time interval (bin), and 500 sequential bins used for each quantal estimate. With the use of 50-ms bins, estimates for var(s)p were consistently negative. This was due to too large a bin (and introduction of undetected temporal variance) because the use of smaller bins (5 ms) produced positive estimates of var(s)p. Increases in m, n, and p but not var(s)p were found in response to increases in [K+] or [Ca2+]/[Co2+]. La3+ (20 microM) produced increases in m and n, which peaked after 20 min and declined toward zero. There were also large increases in p and var(s)p, which peaked and declined only to initial control values. The increase in var(s)p was presumed to reflect La(3+)-induced release of Ca2+ from intracellular organelles. The results suggest that this approach may be used to obtain unbiased estimates of n and p and that the estimates of var(s)p may be useful for studying Ca2+ release from intraterminal organelles.

Subcellular mechanism and site of action of ionic lanthanum at the motor nerve terminal.

The mechanism by which ionic lanthanum (La3+) increases and subsequently decreases spontaneous transmitter release was investigated by recording miniature endplate potentials (MEPPs) at frog neuromuscular junctions. Addition of tetrodotoxin and Co2+ delayed the onset of MEPP frequency increase but did not otherwise prevent the response. Dinitrophenol substantially reduced but did not eliminate the increase, whereas 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) completely abolished it. Thus, La3+ does not act by depolarizing the terminal or by substituting for Ca2+ at transmitter release sites. Instead, it appears to enter the terminal through Na+ channels and promote Ca2+ release from intracellular organelles. The profound depletion of transmitter with time may be due to the high turnover of transmitter coupled with the inhibition of metabolic processes by La3+.

Tetrahydroaminoacridine and physostigmine have opposing effects on probability of transmitter release at the frog neuromuscular junction.

The effect of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on quantal transmitter release was examined at the frog neuromuscular junction. THA (3 microM) caused an increase in m (no. of quanta released) as measured by K(+)-evoked miniature endplate potential (MEPP) frequency. This was due to an increase in p (probability of release), as n (no. of functional release sites) was unchanged. The increase in p was dose-dependent over a range of 0.3-10 microM. By contrast, physostigmine (3 microM) caused a decrease in p, and neostigmine, which does not cross the nerve membrane, had no consistent effect on p. At the postsynaptic site, neostigmine produced the largest increase in MEPP size (79.2%), and THA produced the smallest (17.5%). The divergent effects of THA and physostigmine on p indicate a fundamental difference in their actions at the nerve terminal.

Effect of sodium and calcium channel blockade on the increase in spontaneous transmitter release produced by the mitochondrial inhibitor, dinitrophenol.

The effect of 2,4-dinitrophenol (DNP) was investigated at the frog neuromuscular junction to elucidate the mechanism by which mitochondrial inhibitors increase transmitter release. After a prolonged lag time (30 min), DNP (10(-4) M) caused an increase in miniature endplate potential frequency (MEPP F), which peaked within 10 min and then declined toward, but not reaching, control values. The time course and peak effect of the response were not altered by Na and Ca channel blockade [produced by 3 microM tetrodotoxin (TTX) plus 1.8 mM Co++], but the lag time was sufficient to prevent influex of ions at the nerve terminal, as the same concentration caused complete block of a 50-fold MEPP F increase to 15 mM [K+]. The results suggest that mitochondrial inhibitors produce their effect primarily by reversing the Ca++ uniporter and not by a Na+-mediated stimulation of the Na+-Ca++ antiporter. Their mechanism in this regard differs from that underlying the MEPP F increase produced by cardiac glycosides. The long lag time with DNP may be due to Na pump inhibition and nerve terminal swelling, and the shortening of lag time by TTX plus Co++ to an inhibition of Na+ influx and swelling. Finally, the block of MEPP F with TTX plus Co++ indicates that the MEPP F increase with Co++ alone is probably due to entry of Co++ through unblocked Na channels.

Probability of quantal transmitter release from nerve terminals: theoretical considerations in the determination of spatial variation.

The release of transmitter occurs in discrete quantal units, such that the number released (m) is equal to the number available (n) times the average probability of release (p). Although a common method of estimating these parameters is to use simple binomial statistics, results may be biased if there is spatial or temporal variation in n and p (vars p, vart n, vart p). The problem arises in the simultaneous analysis of five variables, which is impractical due to the complexity and margin of error involved. The proposed solution is to eliminate two variables (vart n, vart p) by assuming stationarity and to obtain the required information from the first three moments of m. The resulting quadratic equation gives two solutions, p1 and p2. Computer simulation of quantal output as a function of vars p indicates that p1 is the better estimator of p when vars p is small, but that p2 is better when vars p is large. This changeover or "inflection" occurs at points which correspond to the maximum vars p obtainable by unimodal distributions of p (larger vars p being obtained by bimodal distributions). Comparison of the simulated histogram of m with those predicted by p1 and p2 shows that p1 provides the better fit, whether vars p is large or small. This discrepancy indicates that histogram analysis is unable to distinguish the appropriate estimate. The major limitations in the procedure can be met by assuming (1) stationarity (which can be attained and tested experimentally), and (2) normal distribution of p (since vars p is then less than "inflection" point, p1 will always be the correct estimate). The overall findings demonstrate that vars p and unbiased estimates of n and p may be calculated, provided reasonable assumptions are made. This in turn should allow the continued use of quantal parameters for describing transmitter release.

Hg2+ causes neurotoxicity at an intracellular site following entry through Na and Ca channels.

At motor nerve terminals, Hg2+ causes (a) irreversible depolarization, (b) increase in transmitter release, and (c) subsequent irreversible block of transmitter release. All effects are antagonized when a Na channel blocker (tetrodotoxin, TTX) and a Ca channel blocker (Co2+) are present, but not when either blocker is used alone. The effects are not antagonized by TTX plus Co2+ when the mercurial is lipid-soluble (methylmercury). This indicates that the neurotoxic action of Hg2+ is at an intracellular site and that entry is gained through both Na and Ca channels. The results suggest that metals may inhibit transmitter release at either the Ca channel or at the release site, but that irreversible toxicity is due to an intracellular action, possibly involving SH groups.

Effects of physiologic alterations on binomial transmitter release at magnesium-depressed neuromuscular junctions.

1. Transmitter release from Mg2+-treated frog neuromuscular junctions can be described by binomial statistics. Good agreement between the observed amplitude-frequency distribution of e.p.p.s. and that predicted by binomial statistics is observed with relatively low concentrations of Mg2+. Conversely, good agreement is found with Poisson predictions when higher concentrations of Mg2+ are used to depress transmission. 2. Binomial analysis at these junctions shows that Mg2+ reduces quantal content (m), the probability of release (p) and to a lesser extent the available stores of transmitter (n). Raising Ca2+ causes an increase in n and p and a small but significant increase in n. K+ increases m and p but not n. 3. During 'frequency-facilitation' (1-6 Hz), e.p.p.s., m and n are increased but p is unaffected. 4. It is concluded that binomial statistics can be used to estimate the quantal parameters of transmitter release and that these parameters can be identified as discrete entities.

Binomial analysis of quantal transmitter release at glycerol treated frog neuromuscular junctions.

1. Transmitter release was analysed at frog neuromuscular junctions pre-treated with 400 mM glycerol Ringer. In the absence of added drugs, end-plate potentials (e.p.p.s) and miniature e.p.p.s (m.e.p.p.s) could be recorded at selected junctions. 2. E.p.p.s were unusually large and calculations of quantal content indicated a high level of release. Also recorded were anomalous action potentials resembling e.p.p.s but these could be distinguished using a summation test. 3. Plots of coefficient of variation of e.p.p.s (0-5 Hz stimulation) versus direct quantal content (M1) showed a marked deviation from Poisson expectations with high M1. Analysis of these results with amplitude-frequency histograms showed a progressively better fit to binomial predictions with increasing M1. 4. The use of binomial statistics allowed direct calculations of the mean probability of release (p) and the readily available store (n). Increasing Ca/Mg caused increases in both n and p. 5. Plots of M1 vs. Ca/Mg showed a power relationship of 3.58. Maximum m.e.p.p. amplitude occurred at control Ca/Mg. Both results were consistent with studies in muscles not treated with glycerol and indicated that glycerol treatment caused no major alterations pre- or post-junctionally. 6. Estimates of quantum size using Poisson assumptions showed an over-all increase when stimulus frequency was raised, indicating a shift from binomial to a Poisson distribution. 7. The combined findings demonstrate that the glycerol treated preparation can be used to examine the release process during high output. Such release conforms to binomial statistics and allows direct determinations of the parameters n and p.

Quantal release parameters during fade of endplate potentials.

An analog of hemicholinium-3 caused the fade of endplate potentials during repetitive stimulation at low rates. The quantal parameters of release were estimated during steady-state conditions before and in the presence of the drug. The fade of EPP's was associated with a decrease in quantal content (m). The decrease in m was, in turn, associated with a decrease in the binomial parameter n and an increase in the binomial parameter p. The changes in n and p are discussed in relation to available stores of transmitter and the probability of transmitter release.

Enhancement by carbachol of transmitter release from motor nerve terminals.

In the endplates of rat phrenic nerve-diaphragm, application of the acetylcholine-like compound, carbachol, causes a marked increase in transmitter release, as measured electrophysiologically using miniature endplate potential frequency. Washing out of carbachol reverses the increase in frequency. The ability of carbachol to increase transmitter release is greatly enhanced by perfusion of the preparation with Ringer solution containing elevated K(+). At concentrations of carbachol greater than 30 muM, the onset of the postjunctional blocking action of carbachol is too rapid and obscures the increase in miniature potential frequency. The rate of increase in transmitter release is dependent on the concentration of carbachol applied and can be antagonized by d-tubocurarine (10-60 nM) and other blocking compounds. These findings, in contrast to previous reports, indicate that cholinergic nerve endings, like adrenergic nerve endings, respond to applied acetylcholine-like drugs with measurable increases in transmitter output.