Stimulation of spontaneous transmitter release at the frog neuromuscular junction by 12-O-tetradecanoylphorbol-13-acetate occurs in the absence of extracellular Ca2+ and is enhanced by depolarization

Summary The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release of transmitter at the frog neuromuscular junction has been investigated electrophysiologically. TPA (100 nmol/l) caused a gradual rise in miniature end-plate potential (MEPP) frequency. After 20–30 min MEPP frequency had risen by approximately 40%. This action of the drug was not inhibited by bathing preparations in either Ca²⁺-free medium (0 Ca²⁺-1 mmol/l EGTA) or high Mg²⁺ medium, or by pretreatment with verapamil (5 mol/l). The inactive TPA analogue 4--TPA had no effect on release rate. There was no indication of any positive correlation between resting MEPP frequency and the size of the subsequent response to TPA treatment. Any synergism between [Ca²⁺]_i and TPA treatment is therefore likely to occur at a site other than that which determines spontaneous release rate.The stimulatory effect of TPA was enhanced 2-fold by carrying out the experiments in a partially depolarising saline (10 mmol/l K⁺). When TPA was applied to preparations bathed in Ca²⁺-free depolarising saline, the response to the drug was still significantly greater than that in non-depolarised preparations. It is concluded that responsiveness to TPA is enhanced by depolarisation, but that little, if any, of this enhancement can be attributed to the consequent influx of Ca²⁺.Send offprint requests to S. J. Publicover at the above addressPEL was in receipt of an S.E.R.C. postgraduate awardZYS was in receipt of financial support from Umm Al Qura University, Saudi Arabia     

The depressing effect of tetracaine and ryanodine on the slow outward current correlated with that of contraction in voltage-clamped frog muscle fibres

The effects of tetracaine (10–50 M) and ryanodine (0.1–10 M) were tested on the slow outward K⁺ current (I _so) and the mechanical tension of isolated frog muscle fibres in a voltage-clamp device (double mannitol-gap) connected to a mechanoelectric transducer. In the concentration range tested, both drugs induced a simultaneous inhibition of tension and current. In all cases the effect on tension was twice that on current. The tetracaine-induced current and tension blocks were fully reversible and dose-dependent. In contrast the ryanodine effects on current and tension were not reversible and did not exhibit a dose dependence except for the delay before the onset of the response, which was shortened when the concentration was raised. Linear regression analysis of the time-dependent and dose-dependent effects of both drugs indicated a strong correlation between the decreases in tension and current. It is concluded that the slow outward current is partly under the control of the Ca²⁺ release from sarcoplasmic reticulum during contraction.     

Binding of botulinum neurotoxin to pure cholinergic nerve terminals isolated from the electric organ of Torpedo

Summary Torpedo electric organ has been used to study the binding of botulinum neurotoxin type A to pure cholinergic synaptosomes and presynaptic plasma membrane.¹²⁵I-labeled botulinum neurotoxin type A exhibits specific binding to cholinergic fractions. Two binding sites have been determined according to data analysis: a high affinity binding site (synaptosomes: K_d=0.11±0.03 nM, B_max=50±10 fmol · mg prot^–1; presynaptic plasma membrane: K_d=0.2±0.05 nM, B_max=150±15 fmol · mg prot^–1) and a low affinity binding site (synaptosomes: K_d 26 nM, B_max 7.5 pmol · mg prot^–1; presynaptic plasma membrane: K_d 30 nM, B_max 52 pmol · mg prot^–1). The binding of¹²⁵I-botulinum neurotoxin type A is decreased by previous treatment of synaptosomes by neuraminidase and trypsin, and by a preincubation with bovine brain gangliosides or antiserum raised against Torpedo presynaptic plasma membrane. When presynaptic plasma membranes are blotted to nitrocellulose sheet, either¹²⁵I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a M_r 140,000 protein. Botulinum toxin-gold complexes have also been used to study the toxin internalization process into Torpedo synaptosomes. The images fit the three step sequence model in the pathway of botulinum neurotoxin poisoning.     

Further evidence for the dynamic formation of transmitter quanta at the neuromuscular junction.

Fatt and Katz (Nature 166:597-598, 1950; J Physiol 117:109-128, 1952) attributed miniature endplate potentials (MEPPs) to the action of a standard quantity of transmitter, the quantum (Del Castillo and Katz, J Physiol 124:560-573, 1954). Quantal packets of transmitter were proposed to be preformed (Del Castillo and Katz, In CNRS Paris (Ed): "Microphysiologie comparée des éléments excitables" 67:245-258, 1957) and stored in large numbers in the motor nerve terminal. Statistical analyses of intervals between MEPPs and numbers of quanta composing small endplate potentials indicated that quantal release was a random process and that release sites functioned independently of each other. With the discovery of synaptic vesicles it was proposed that each contained one quantum of transmitter. The quantal-vesicular hypothesis (Del Castillo and Katz, as cited above) fails, however, to explain amplitude distributions of MEPPs that are skewed and/or that show multiple peaks (Kriebel et al., Brain Res Review 15:167-178, 1990). The drop formation process (Shaw, "The Dripping Faucet as a Model Chaotic System," Santa Cruz, CA: Aerial Press, Inc., 1984) was shown to generate amplitude classes of drops that were similar to classes of MEPPs which suggested that rapid changes in quantal size and ratios of skew- to bell-MEPPs could be explained with a simple dynamic process which determines quantal size at the moment of release (Kriebel et al., as cited above, 1990). Further similarities between miniature endplate currents (MEPCs) and the formation of drops are reported here. We found that rapid changes in MEPC amplitudes and time courses, which accompany an increase in frequency, mimic changes in drop sizes that accompany increases in flow rate. MEPC intervals have a minimum and their distributions are comparable to those of drop intervals. During an increased rate of transmitter release, MEPP amplitudes and intervals were positively correlated. The results suggest that spontaneously released transmitter "packets" are formed at the moment of release and that transmitter supply to the process that forms packets is continuous.     

Reactivity and toxicity of atracurium and its metabolitesin vitro

Cytotoxicity of atracurium and of its metabolites was tested in vitro.Exposure of isolated rat hepatocytes to atracurium produced cellular damage evidenced by extrusion of an intracellular enzyme, lactate dehydrogenase (LDH), into the incubation medium. Leakage of LDH was directly related to the concentration of atracurium in the medium (250 to 800 μM). If the spontaneous degradation of atracurium (presumably via Hofmann elimination) was first carried out in vitroand the degradation products subsequently added to the isolated hepatocytes, the leakage of LDH was also dose-dependent but larger than that observed after the addition of the parent drug. When l-cysteine was admixed to the products of the spontaneous degradation of atracurium prior to their addition to the liver cells, no leakage of LDH was observed. The results are compatible with the working hypothesis that atracurium itself and, even more so, acrylates formed in Hofmann elimination of atracurium, are reactive toward nucleophiles and damage the cells by alkylating nucleophiles present in cellular membranes. Antecedent covalent binding of acrylates to the nucleophile cysteine, i.e., the formation of acrylatecysteine adducts, saturated the reactive capacity of acrylates for nucleophiles and thus prevented the reactive metabolites from alkylating the endogenous nucleophiles. Possible clinical consequences resulting from in vivogeneration of reactive metabolites are not clear at the present time but are projected to be related to (a) the dose of atracurium administered, (b) the amount of acrylates generated, (c) the functional importance of the endogenous nucleophiles alkylated, and (d) the pathway and the speed of detoxification of atracurium and its metabolites.     

Effect of vasoactive intestinal polypeptide on the release of serotonin from the in vitro vascularly perfused small intestine of guinea pig

Summary Isolated segments of the guinea pig small intestine were vascularly perfused and the release of endogenous serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) into the portal vein was measured. All test substances were intraarterially perfused. Vasoactive intestinal polypeptide (VIP, 1 pmol/l — 100 nmol/1) inhibited the spontaneous release of 5-HT and 5-HIAA. The maximal inhibitory effect (about 60%) was seen at 100 pmol/1. The effect of VIP on the spontaneous release of 5-HT and 5-HIAA was not changed in the presence of 1 ol/l tetrodotoxin (TTX).Raising intraluminal pressure by 500 Pa for 5 min increased the release of 5-HT and 5-HIAA by about 25%. Raising the intraluminal pressure in the presence of VIP reduced the release of 5-HT and 5-HIAA by about 75%. In the presence of TTX (1 gmol/l), raising intraluminal pressure also caused a decrease of the release of 5-HT and 5-HIAA which was unaffected by the additional presence of VIP. The fluid volume expelled during peristaltic activity was not affected by VIP, but reduced by about 90% in the presence of TTX.In conclusion the results demonstrate a direct inhibitory effect of VIP on the release of 5-HT from the enterochromaffin cells. In addition, VIP appears to interfere with the neuronally mediated stimulation of 5-HT release during peristaltic activity. Send offprint request to H. Schwörer at the above address     

Meta-analysis of gross insertions causing human genetic disease: novel mutational mechanisms and the role of replication slippage

Although gross insertions (>20 bp) comprise <1% of disease-causing mutations, they nevertheless represent an important category of pathological lesion. In an attempt to study these insertions in a systematic way, 158 gross insertions ranging in size between 21 bp and approximately 10 kb were identified using the Human Gene Mutation Database (www.hgmd.org). A careful meta-analytical study revealed extensive diversity in terms of the nature of the inserted DNA sequence and has provided new insights into the underlying mutational mechanisms. Some 70% of gross insertions were found to represent sequence duplications of different types (tandem, partial tandem, or complex). Although most of the tandem duplications were explicable by simple replication slippage, the three complex duplications appear to result from multiple slippage events. Some 11% of gross insertions were attributable to nonpolyglutamine repeat expansions (including octapeptide repeat expansions in the prion protein gene [PRNP] and polyalanine tract expansions) and evidence is presented to support the contention that these mutations are also caused by replication slippage rather than by unequal crossing over. Some 17% of gross insertions, all >or=276 bp in length, were found to be due to LINE-1 (L1) retrotransposition involving different types of element (L1 trans-driven Alu, L1 direct, and L1 trans-driven SVA). A second example of pathological mitochondrial-nuclear sequence transfer was identified in the USH1C gene but appears to arise via a novel mechanism, trans-replication slippage. Finally, evidence for another novel mechanism of human genetic disease, involving the possible capture of DNA oligonucleotides, is presented in the context of a 26-bp insertion into the ERCC6 gene.     

Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1

Before completion of polarization, Madin-Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca²⁺ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV-I in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. © Wiley-Liss, Inc.     

Vasopressin release and firing of supraoptic neurosecretory neurones during drinking in the dehydrated monkey

A close relationship exists between drinking and the release of vasopressin, the two main factors responsible for the maintenance of body water content. Whereas the participation of peripheral factors, such as oropharyngeal stimulation, seems obvious in the metering of fluid intake and in thirst satiation, very little is known about their influence on vasopressin release. In the present experiments, the influence of drinking on vasopressin release was studied using both biochemical and electrophysiological approaches.In one group of monkeys made thirsty by water deprivation, the subsequent drinking of water during a 5–8 min induced: i) a short-term response, consisting of an abrupt fall in plasma vasopressin concentration which was independent of osmolality, occurred at the time of drinking and was partly reversed after the cessation of drinking, and ii) a longer lasting response, consisting of a slow diminution of plasma vasopressin concentration as the intestinal absorption of water progressed. In another group of thirsty monkeys, extracellular recordings were made during drinking from cells which were identified as neurosecretory neurones of the supraoptic nucleus, a number of them being considered vasopressin secreting on the basis of their phasic pattern of firing. Their firing decreased considerably during the periods of water intake and recovered to control levels immediately after-wards.The decrease in vasopressin release at the onset of water intake, the diminution in the firing rate of the neurones, the short latency and the reversibility of these events after cessation of drinking, suggest that a reflex inhibition of vasopressin-secreting neurones occurs which is probably induced by peripheral stimuli and most likely via oropharyngeal or other visceral receptors. It is postulated that this reflex inhibition of vasopressin release may participate in some active manner in the anticipatory mechanisms of thirst satiation.