Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function.

Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ-/- mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ-/- muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ-/- neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.

[A reminder of the structure and function of the skeletal neuromuscular junction]. / Rappels structuraux et fonctionnels de la jonction neuromusculaire squelettique.

The skeletal neuromuscular junction has been considered as a model of chemical synapses due to its relatively simple organization. It is made up of three cellular partners including the motoneuron nerve terminals, the peri-synaptic Schwann cells and a specialized region of skeletal muscle fibers. It has been extensively studied revealing its ultrastructural complexity involving many molecular actors. The neuromuscular junction is a highly specialized structure, optimized for the rapid transmission of information from the presynaptic nerve terminal to the post-synaptic muscle fiber. This rapid transmission requires a very close apposition of plasmic membranes of pre- and post-synaptic partners, and a strict structural and molecular arrangement on both sides of the narrow synaptic cleft separating nerve terminal and muscle membranes. In this short review, we summarize the knowledge regarding pre- and post-synaptic ultrastructural specializations and give an overview of some functional aspects of neuromuscular transmission, including the quantal acetylcholine release process, which will help to better understand the pharmacological actions of botulinum toxins in esthetic and corrective dermatology.

[Mechanisms of action of botulinum toxins and neurotoxins]. / Mécanismes d'action des toxines et neurotoxines botuliques.

Several bacteria of the Clostridium genus (C. botulinum) produce 150 kDa di-chainal protein toxins referred as botulinum neurotoxins or BoNTs. They associate with non-toxic companion proteins and form a complex termed botulinum toxin. BoNTs specifically inhibit vesicular neurotransmitter release. The cellular action of BoNTs can be depicted according to a multi-step model : The toxin's heavy chain mediates binding to specific receptors comprised of a ganglioside moiety and a vesicular protein (SV2 for BoNT type A, synaptotagmin for BoNT type B), followed by endocytotic internalisation of the BoNT/receptor complex. Vesicle recycling induces BoNT internalisation. Upon acidification of vesicles, the light chain of the neurotoxin is translocated into the cytosol. Here, this zinc-endopeptidase cleaves one or two among three synaptic proteins (VAMP-synapto-brevin, SNAP25, and syntaxin). As the three protein targets of BoNT play major role in fusion of synaptic vesicles at the release sites, their cleavage is followed by blockade of neurotransmitter exocytosis. Importantly, as the BoNT receptors and intracellular targets are present in all nerve terminals, the BoNTs are not specific for cholinergic transmission. Duration of their inhibitory action is mainly determined by the the life-time of the toxin's light chain in the cytosol. Sprouting of new nerve-endings, which are retracted when the poisoned nerve terminals have recovered full functionality, may lead to anticipated recovery of the poisoned nerve terminals.

Regulation of autophagy by the inositol trisphosphate receptor.

The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.

Outcome of acetylcholinesterase deficiency for neuromuscular functioning.

Acetylcholinesterase (AChE) plays an essential role in neuromuscular transmission, therefore it is surprising that AChE knockout (KO) mice could live to the adulthood. Neuromuscular functioning in KO and normal (wild type, WT) mice were studied, at different age (1.5-, 4- and 9-month-old). Hindlimb muscle force productions in response to nerve or muscle electric stimulation were recorded in situ and in vitro. Our results show that contrary to WT mice, 1.5-, 4- and 9-month-old KO mice exhibited a decreased in tetanic force during short periods (500 ms) of repetitive nerve stimulations (tetanic fade). Nevertheless submaximal muscle forces in response to single or repetitive nerve stimulation were increased (potentiation) in 1.5-, 4- and 9-month-old KO mice as compared to WT mice (p<0.05). Tetanic fade and potentiation were absent when muscles were directly stimulated, indicating neuromuscular transmission alterations in KO mice. Contrary to younger mice, muscle weight and maximal tetanic force in response to repetitive nerve stimulation were not reduced in 4- and 9-month-old KO mice as compared to WT mice (p>0.05). In conclusion AChE deficit leads to marked neuromuscular alterations in hind limb muscle functioning and a prominent symptom is the lack of resistance to fatigue.

Regulated expression of the proteoglycan SPOCK in the neuromuscular system.

SPOCK is prevalent in developing synaptic fields of the central nervous system (Charbonnier et al., 2000. Mech. Dev. 90, 317-321). The expression of SPOCK during neuromuscular junction (NMJ) formation was compared to agrin and acetylcholine receptor (AChR) distribution. SPOCK is detected within the myogenic masses during the early steps of embryonic development, and distributed in the cytoplasm of myotubes before coclustering with AChRs. In the adult, SPOCK is present in axons and is highly expressed by Schwann cells. SPOCK altered expression pattern after nerve lesioning, or cholinergic transmission blockade, strongly indicate that its cellular distribution at the NMJ depends on innervation.

Nerve terminal sprouting in botulinum type-A treated mouse levator auris longus muscle.

The marked outgrowth of the motor nerve terminal arborization triggered by an in vivo local injection of Clostridium botulinum type-A toxin in the mouse levator auris longus muscle was studied with morphological and immunochemical approaches. The increase in total nerve terminal length depended on the time elapsed after toxin administration and was due to both increased number of terminal branches and branch length as revealed by a quantitative morphological analysis of whole mounts using the combined cholinesterase-silver stain. Nerve terminal sprouts increased in number, length and complexity even after the functional recovery of neuromuscular transmission had occurred as revealed by electrophysiological examination. Although we cannot exclude that transmitter release sites from the original nerve terminal arborization may still be functional after botulinum type-A toxin (BoTx-A) treatment, it is likely that newly formed functional release sites on the sprouts play a major role in the functional recovery of neuromuscular transmission. The presence of an immunoreactivity to synaptophysin and synaptotagmin-II, integral proteins of synaptic vesicles, gives support to our previous findings suggesting that nerve terminal sprouts have the molecular machinery for acetylcholine release.

A new type of transmitter release at the neuromuscular junction.

Examination of spontaneous miniature endplate potentials (MEPPs) in murine skeletal muscle has revealed that in conditions such as botulinum poisoning, during nerve terminal regeneration or in the presence of the drug 4-aminoquinoline, two types of acetylcholine release are responsible for the MEPPs. In addition to the MEPPs which correspond to the quantal component of a nerve impulse-evoked endplate potential a second type of acetylcholine release occurs. The latter type of transmitter release gives rise to MEPPs with a more prolonged time-to-peak and frequently a larger than normal amplitude. It is unaffected by nerve terminal depolarization and transmembrane Ca2+ fluxes. The relationship between MEPP frequency and temperature has a Q10 of about 12 compared to 2-3 for normal MEPPs. In botulinum-poisoned muscles this secretory type of transmitter release dominates, being exclusively present in muscles where nerve stimulation fails to release transmitter. In normal muscle such a release is induced by 4-aminoquinoline which may cause up to 45% of all the spontaneous MEPPs to be of that kind. It is suggested that the described spontaneous secretion of acetylcholine serves in inductory and neurotrophic function.

Effects of carbonyl cyanide m-chlorophenylhydrazone on quantal transmitter release and ultrastructure of frog motor nerve terminals.

The quantal acetylcholine release and the ultrastructural effects of the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone have been examined at frog neuromuscular junctions. Carbonyl cyanide m-chlorophenylhydrazone (2 microM) caused a temperature-dependent block of evoked quantal transmitter release accompanied by an increase in the rate of spontaneous quantal release. The carbonyl cyanide m-chlorophenylhydrazone-induced increase in miniature endplate potential frequency was neither antagonized nor prevented by tetrodotoxin. It also occurred in a Ca2+-free medium and after replacement of Ca2+ by Sr2+, indicating that it does not depend upon a Na+ or Ca2+ influx from the external medium but may act by releasing Ca2+ from intraterminal stores. Spontaneous quantal transmitter release was exhausted irreversibly within 4 h of carbonyl cyanide m-chlorophenylhydrazone (2 microM) action, during which time an average of 4.7 x 10(5) acetylcholine quanta were released per junction. The morphologic analysis revealed a significant temperature and time-dependent reduction in the number of synaptic vesicles with swelling and dispersion of mitochondria within the motor nerve terminals. Changes in synaptic vesicle number appear to be directly related to the intensity of transmitter release. The good correlation observed between the number of quanta secreted and the number of vesicles lost by nerve terminals in the absence of vesicle recycling provides an estimate of the initial store of transmitter quanta.

Sodium-dependent increase in quantal secretion induced by brevetoxin-3 in Ca2+-free medium is associated with depletion of synaptic vesicles and swelling of motor nerve terminals in situ.

Brevetoxin-3 at nanomolar concentrations markedly enhanced spontaneous quantal transmitter release from neuromuscular junctions equilibrated in a Ca2+-free EGTA medium. After about 3 h, the sustained increase in miniature endplate potential frequency led to an exhaustion of transmitter release. This increase still occurred after loading the nerve terminals with the Ca2+ chelator bis-(aminophenoxy)ethanetetra-acetate or after pretreatment with various pharmacological agents known to prevent Ca2+ release from intracellular pools, but was completely prevented by the Na+ channel blocker tetrodotoxin. Brevetoxin-3 also increased miniature endplate potential frequency from junctions treated with botulinum type-A toxin, but to a smaller extent than at normal junctions. At normal junctions, brevetoxin-3 exposure for 2 h increased the three-dimensional projected area of living motor nerve terminals in situ by about 74% while at botulinum type-A poisoned junctions a similar toxin exposure caused only a 29% increase. Tetrodotoxin prevented such effects, indicating that they are related to both Na+ entry into the terminals and increased quantal transmitter release. Ultrastructural examination of nerve terminals from junctions exposed for 3 h to brevetoxin-3 revealed profound depletions of clear and large dense core synaptic vesicles and an increase in coated vesicles and axolemma infoldings. These results indicate that brevetoxin-3 impairs the recycling of clear synaptic vesicles and are consistent with our immunofluorescent observations showing that synaptophysin epitopes can be revealed without nerve terminal permeabilization. In contrast, no such changes were detected in nerve terminals poisoned with botulinum type-A toxin which, after 3 h exposure to brevetoxin-3, retained their synaptic vesicles and had a normal appearance. We conclude that tetrodotoxin-sensitive Na+ entry into motor nerve terminals induced by brevetoxin-3 triggers external Ca2+-independent asynchronous quantal transmitter release, blocks synaptic vesicle recycling and induces swelling of the terminals. We suggest that an excess of cytoplasmic Na+ per se can activate the asynchronous neurotransmitter release process.

Nodal swelling produced by ciguatoxin-induced selective activation of sodium channels in myelinated nerve fibers.

Ciguatoxin-1b, the major toxin involved in ciguatera fish poisoning, and D-mannitol were examined on frog nodes of Ranvier using confocal laser scanning microscopy and conventional current- and voltage-clamp techniques. During the action of 10 nM ciguatoxin-1b, an increase in nodal volume was observed as determined by digital image processing and three-dimensional reconstruction of axons. The increase was prevented by blocking Na+ channels with tetrodotoxin. Ciguatoxin-1b (10 nM) induced high frequency action potential discharges up to 70-100 Hz. Analysis of Na+ current revealed that the toxin modified a current fraction which was activated at resting membrane potential and failed to inactivate. Increasing the osmolality of the external solution by about 50% with D-mannitol restored the nodal volume to its control value and suppressed spontaneous action potentials. In addition, D-mannitol affected unmodified and ciguatoxin-1b-treated Na+ currents in a similar manner causing a reduction of maximum conductance, negative shifts of current reversal potential and modification of the voltage-dependence of current activation and inactivation. In conclusion, ciguatoxin-1b induced a tetrodotoxin-sensitive swelling of nodes of Ranvier and selectively affected the Na+ current of myelinated axons. It is proposed that ciguatoxin-1b, by modifying Na+ current, increased intracellular Na+ concentration which caused water influx and nodal swelling. This may explain some of the reported symptoms of ciguatera fish poisoning. D-mannitol, an agent used for ciguatera treatment, was found to reverse the effects of ciguatoxin-1b by reducing Na+ entry and increasing the efflux of water through its osmotic action. It is the first time that osmotic changes produced by the selective activation of ionic channels, i.e. Na+ channels, are reported.

Terminal sprouting in mouse neuromuscular junctions poisoned with botulinum type A toxin: morphological and electrophysiological features.

Functional properties of terminal sprouts elicited by an in vivo injection of Clostridium botulinum type A toxin were studied in endplates of the Levator auris longus muscle of the mouse poisoned from a few days to 28 days beforehand. For this purpose, morphological observations of the extent of terminal sprouts and localization of acetylcholine receptors was performed in whole mount preparations. Sprouts appeared as thin unmyelinated filaments that run usually parallel to the longitudinal axis of the muscle fibres; labelling acetylcholine receptors revealed their line-shaped accumulation co-localized with the sprouts. In addition, presynaptic membrane currents elicited by nerve stimulation were recorded by external electrodes applied under visual control onto the membrane of pre-existing motor endings and newly formed sprouts. These recordings showed the presence of widespread triphasic waveforms which indicated active impulse propagation of the action potential over most of the length of the poisoned endings. Ca2+ influx and Ca2(+)-dependent K+ currents in the sprout membrane were found to be similar to those described in unpoisoned endings. The presence of normal Ca2+ influx, upon active depolarization, in the terminal sprout membranes together with the localization of acetylcholine receptors in front of these membranes, indicates that the terminal sprouts may play a role in the recovery of neuromuscular transmission after Clostridium botulinum poisoning.

Ciguatoxin enhances quantal transmitter release from frog motor nerve terminals.

1. Ciguatoxin (CTX), a marine toxin produced by the benthic dinoflagellate Gambierdiscus toxicus, is responsible for a complex endemic disease in man known as ciguatera fish poisoning. In the present study we have investigated the effects of purified CTX extracted for Gymnothorax javanicus moray-eel liver on frog isolated neuromuscular preparations with conventional electrophysiological techniques. 2. CTX (1-2.5 nM) applied to cutaneous pectoris nerve-muscle preparations induced, after a short delay, spontaneous fibrillations of the muscle fibres that could be suppressed with 1 microM tetrodotoxin (TTX) or by formamide to uncouple excitation-contraction. 3. In preparations treated with formamide, CTX (1-2.5 nM) caused either spontaneous or repetitive muscle action potentials (up to frequencies of 60-100 Hz) in response to a single nerve stimulus. Recordings performed at extrajunctional regions of the muscle membrane revealed that during the repetitive firing a prolongation of the repolarizing phase of the action potential occurred. At junctional sites the repetitive action potentials were triggered by repetitive endplate potentials (e.p.ps). 4. CTX (2.5 nM) caused a TTX-sensitive depolarization of the muscle membrane. 5. In junctions equilibrated in solutions containing high Mg2+ + low Ca2+, addition of CTX (1.5 nM) first induced an average increase of 239 +/- 36% in the mean quantal content of e.p.ps. Subsequently CTX reduced and finally blocked nerve-evoked transmitter release irreversibly. 6. CTX (1.5-2.5 nM) increased the frequency of miniature endplate potentials (m.e.p.ps) in junctions bathed either in normal Ringer, low Ca2(+)-high Mg2+ medium or in a nominally Ca2(+)-free solution containing EGTA.2+ Extensive washing with toxin-free solutions did not reverse the effect. Furthermore, Cd2 + (0.1 mM), a potent calcium channel blocker, neither antagonized nor abolished the increase in transmitter release caused by CTX. 7. TTX (1 microM) completely prevented the effect of CTX (2.5nM) on m.e.p.p. frequency. This effect was independent of the presence of extracellular Ca2 +. TTX, when added after CTX (2.5 nM) exposure, antagonized the increase in m.e.p.p. frequency. The antagonism was complete in Ca2 +-free medium. These results strongly suggest that increased permeability of the nerve terminal to Na+ is responsible for the increase in m.e.p.p. frequency caused by CTX. It is likely that CTX may trigger calcium release from internal stores due to an increase of intraterminal Na+ concentration. 8. It is concluded that CTX exerts, in the nanomolar concentration range, a selective action on sodium channels of the neuromuscular junction causing both pre- and postsynaptic effects.

Changes of quantal transmitter release caused by gadolinium ions at the frog neuromuscular junction.

1. The actions of the trivalent cation, gadolinium (Gd3+), were studied on frog isolated neuromuscular preparations by conventional electrophysiological techniques. 2. Gd3+ (450 microM) applied to normal or formamide-treated cutaneous pectoris nerve-muscle preparations induced, after a short delay, a complete block of neuromuscular transmission. The reversibility of the effect was dependent on the time of exposure. 3. Gd3+ (5-450 microM) had no consistent effect on the resting membrane potential of the muscle fibres. 4. Gd3+ (5-40 microM) applied to preparations equilibrated in solutions containing high Mg2+ and low Ca2+ reduced the mean quantal content of endplate potentials (e.p.ps) in a dose-dependent manner. Under those conditions, 3,4-diaminopyridine (10 microM) consistently reversed the depression of evoked quantal release. 5. The calcium current entering motor nerve terminals, revealed after blocking presynaptic potassium currents with tetraethylammonium (10 mM) in the presence of elevated extracellular Ca2+ (8 mM), was markedly reduced by Gd3+ (0.2-0.5 mM). 6. Gd3+ (40-200 microM) increased the frequency of spontaneous miniature endplate potentials (m.e.p.ps) in junctions bathed either in normal Ringer solution or in a nominally Ca(2+)-free medium supplemented with 0.7 microM tetrodotoxin. This effect may be due to Gd3+ entry into the nerve endings since it is not reversed upon removal of extracellular Gd3+ with chelators (1 mM EGTA or EDTA). Gd3+ also enhanced the frequency of me.p.ps appearing after each nerve stimulus in junctions bathed in a medium containing high Mg2+ and low Ca2+. 7. Gd3+, in concentrations higher than 100 microM, decreased reversibly the amplitude of m.e.p.ps suggesting a postsynaptic action. 8. It is concluded that the block of nerve-impulse evoked quantal release caused by Gd3 + is related to its ability to block the calcium current entering the nerve endings, supporting the view that Gd3 + blocks N-type Ca2+ channels; while the enhancement of spontaneous quantal release is probably the result of Gd3 + entry into motor nerve endings. Besides its dual prejunctional effects on quantal release it is suggested that Gd3 + exerts a postsynaptic action on the endplate acetylcholine receptor-channel complex.

Selective depolarization of the muscle membrane in frog nerve-muscle preparations by a chromatographically purified extract of the dinoflagellate Ostreopsis lenticularis.

1. The actions of a chromatographically identified extract of the marine dinoflagellate Ostreopsis lenticularis, named ostreotoxin-3 (OTX-3), were studied on frog isolated neuromuscular preparations. 2. OTX-3 (1-10 microg ml(-1)) applied to cutaneous pectoris nerve-muscle preparations depolarized skeletal muscle fibres and caused spontaneous contractions. The depolarization was neither reversed by prolonged washing nor by (+)-tubocurarine. 3. OTX-3 decreased the amplitude of miniature end plate potentials (m.e.p.ps) but did not affect their frequency. 4. Extracellular recording of compound action potentials revealed that OTX-3 affected neither excitability nor conduction along intramuscular nerve branches. 5. End-plate potentials (e.p.ps) elicited by nerve stimulation were reduced in amplitude by OTX-3 and even showed reversed polarity in junctions deeply depolarized by the toxin. 6. Membrane depolarization induced by OTX-3 was decreased about 70% in muscles pretreated for 30 min with 10 microM tetrodotoxin. In contrast, muscles pretreated with 5 microM mu-conotoxin GIIIA were completely insensitive to OTX-3-induced depolarization. 7. OTX-3 did not affect e.p.p. amplitude and the quantal content of e.p.ps in junctions in which muscle depolarization was abolished by mu-conotoxin GIIIA. 8. OTX-3 is a novel type of sodium-channel activating toxin that discriminates between nerve and skeletal muscle membranes.

Ca(2+)-dependent changes of acetylcholine release and IP3 mass in Torpedo cholinergic synaptosomes.

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP3) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP3 mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP3 mass levels under resting conditions. The IP3 mass was neither modified by changes in external Ca2+ nor by a Ca(2+)-free medium containing EGTA. IP3 mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K+ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca2+ emphasizing that Ca2+ entry, independently of the influx mechanism involved, leads to an IP3 increase. The phospholipase C beta inhibitors U-73122 and U-73343 reduced K(+)-stimulated IP3 levels while K(+)-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP3 by agents that stimulate ACh release may suggest a possible link between IP3 metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C.

Ca(2+)-dependent changes of acetylcholine release and IP3 mass in Torpedo cholinergic synaptosomes.

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP3) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP3 mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP3 mass levels under resting conditions. The IP3 mass was neither modified by changes in external Ca2+ nor by a Ca(2+)-free medium containing EGTA. IP3 mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K+ and by the ionophores A-23/87 and gramicidin-D in a manner dependent on external Ca2+ emphasizing that Ca2+ entry, independently of the influx mechanism involved, leads to an IP3 increase. The phospholipase C beta inhibitors U-73122 and U-73343 reduced K(+)-stimulated IP3 levels while K(+)-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP3 by agents that stimulate ACh release may suggest a possible link between IP3 metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C.

The relationship between quantal content and delayed quantal release.

Following an endplate potential there is a period of elevated spontaneous quantal release, known as delayed release. We have estimated the relationship between evoked quantal release and delayed release in two ways. First, in a solution in which the Na+ was replaced with methylamine, the number of delayed release evoked by electronic depolarization of the nerve terminal changed little over a 30-fold range of output. Second, we varied [Ca2+]out stepwise in Na solution with added Mg2+ and measured delayed release and EPP amplitude at single junctions. Over much of the range, elevating the EPP amplitude many-fold increased delayed release only slightly, if at all. Our interpretation is that the effects of residual Ca2+ are not proportional to the amount of Ca2+ entering to trigger quantal release.

The wheat proteins puroindoline-a and alpha1-purothionin induce nodal swelling in myelinated axons.

The effects of two basic cysteine-rich lipid-binding proteins isolated from wheat seedlings, puroindoline-a and alpha1-purothionin, were studied on single frog myelinated axons stained with the fluorescent dye FM1-43 using confocal laser scanning microscopy. During exposure to either puroindoline-a or alpha1-purothionin (10 and 100 microM) a marked swelling of nodes of Ranvier was observed, provided NaCl was present in the external solution. It is suggested that these proteins increase the internal osmolality by forming pores in the axonal membrane and induce water influx to compensate for such an increase. Moreover, in the presence of alpha1-purothionin (100 microM), the intensity of the axonal staining with FM1-43 was increased. It is the first time, to our knowledge, that basic proteins containing domains of a cysteine-rich repeated motif are reported to produce swelling and water movements across neuronal cell membranes.