alpha-Latrotoxin releases calcium in frog motor nerve terminals.

alpha-Latrotoxin (alpha-LTX) is a neurotoxin that accelerates spontaneous exocytosis independently of extracellular Ca(2+). Although alpha-LTX increases spontaneous transmitter release at synapses, the mechanism is unknown. We tested the hypothesis that alpha-LTX causes transmitter release by mobilizing intracellular Ca(2+) in frog motor nerve terminals. Transmitter release was measured electrophysiologically and with the vesicle marker FM1-43; presynaptic ion concentration dynamics were measured with fluorescent ion-imaging techniques. We report that alpha-LTX increases transmitter release after release of a physiologically relevant concentration of intracellular Ca(2+). Neither the blockade of Ca(2+) release nor the depletion of Ca(2+) from endoplasmic reticulum affected Ca(2+) signals produced by alpha-LTX. The Ca(2+) source is likely to be mitochondria, because the effects on Ca(2+) mobilization of CCCP (which depletes mitochondrial Ca(2+)) and of alpha-LTX are mutually occlusive. The release of mitochondrial Ca(2+) is partially attributable to an increase in intracellular Na(+), suggesting that the mitochondrial Na(+)/Ca(2+) exchanger is activated. Effects of alpha-LTX were not blocked when Ca(2+) increases were reduced greatly in saline lacking both Na(+) and Ca(2+) and by application of intracellular Ca(2+) chelators. Therefore, although increases in intracellular Ca(2+) may facilitate the effects of alpha-LTX on transmitter release, these increases do not appear to be necessary. The results show that investigations of Ca(2+)-independent alpha-LTX mechanisms or uses of alpha-LTX to probe exocytosis mechanisms would be complicated by the release of intracellular Ca(2+), which itself can trigger exocytosis.

alpha-latrocrustatoxin increases neurotransmitter release by activating a calcium influx pathway at crayfish neuromuscular junction.

alpha-latrocrustatoxin (alpha-LCTX), a component of black widow spider venom (BWSV), produced a 50-fold increase in the frequency of spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their amplitude distribution. During toxin action, periods of high-frequency mEPSP discharge were punctuated by periods in which mEPSP frequency returned toward control levels. EPSPs were increased in amplitude during periods of enhanced mEPSP discharge. alpha-LCTX had no effect when applied in Ca(2+)-free saline, but subsequent addition of Ca(2+) caused an immediate enhancement of mEPSP frequency even when alpha-LCTX was previously washed out of the bath with Ca(2+)-free saline. Furthermore removal of Ca(2+) from the saline after alpha-LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus alpha-LCTX binding is insensitive to Ca(2+), but toxin action requires extracellular Ca(2+) ions. Preincubation with wheat germ agglutinin prevented the effect of alpha-LCTX but not its binding. These binding characteristics suggest that the toxin may bind to a crustacean homologue of latrophilin/calcium-independent receptor for latrotoxin, a G-protein-coupled receptor for alpha-latrotoxin (alpha-LTX) found in vertebrates. alpha-LCTX caused "prefacilitation" of EPSP amplitudes, i.e., the first EPSP in a train was enhanced in amplitude to a greater degree than subsequent EPSPs. A similar alteration in the pattern of facilitation was observed after application of the Ca(2+) ionophore, A23187, indicating that influx of Ca(2+) may mediate the action of alpha-LCTX. In nerve terminals filled with the Ca(2+) indicator, calcium green 1, alpha-LCTX caused increases in the fluorescence of the indicator that lasted for several minutes before returning to rest. Neither fluorescence changes nor toxin action on mEPSP frequency were affected by the Ca(2+) channel blockers omega-agatoxin IVA or Cd(2+), demonstrating that Ca(2+) influx does not occur via Ca(2+) channels normally coupled to transmitter release in this preparation. The actions of alpha-LCTX could be reduced dramatically by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid. We conclude that induction of extracellular Ca(2+) influx into nerve terminals is sufficient to explain the action of alpha-LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions.

The fade of the purinergic neurogenic contraction of the guinea-pig vas deferens: analysis of possible mechanisms.

The purinergic response of the guinea-pig vas deferens to long trains of pulses at high frequency consists of an initial twitch followed by a much lower plateau. Mechanical, neurochemical and electrophysiological techniques were used to examine the reason for the fade. Mechanical measurements. In tissues stimulated by trains of 180 pulses/10 Hz and treated with prazosin to suppress the noradrenergic contraction component, the response to alpha, beta-methylene ATP and to exogenous ATP was as high during the secondary plateau of the purinergic neurogenic contraction as it was outside electrical stimulation periods; the response to 50 pulses/100 Hz was also unchanged during the low plateau. The plateau was not increased by reactive blue 2,8-(p-sulphophenyl)theophylline, propranolol or capsaicin. Neurochemical measurements. In tissues preincubated with [3H]-noradrenaline, electrical stimulation elicited an overflow of tritium and of ATP. In the absence of drugs as well as in the presence of prazosin and suramin to suppress contractions, the overflow of tritium per pulse decreased slightly in the course of trains of 90 pulses/10 Hz; the overflow of ATP per pulse decreased to a greater extent on average, but the decrease was not statistically significant. In the presence of prazosin and nifedipine, also to suppress contractions, the overflow of tritium per pulse again decreased slightly in the course of trains of 105 pulses/10 Hz, but the overflow of ATP per pulse if anything tended to increase. Electrophysiological measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of certain enantiomeric cannabinoids in the mouse vas deferens and the myenteric plexus preparation of guinea-pig small intestine.

1. The psychoactive cannabinoids (-)-delta 9-tetrahydrocannabinol ((-)-delta 9-THC) and the 1,1-dimethyl-heptyl homologue of (-)-11-hydroxy-delta 8-tetrahydrocannabinol ((-)-DMH) both inhibited electrically-evoked contractions of the mouse isolated vas deferens and the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine. 2. Concentrations of (-)-delta 9-THC and (-)-DMH that decreased twitch heights by 50% were 6.3 and 0.15 nM respectively in the mouse vas deferens and 60 nM and 1.4 nM respectively in the myenteric plexus preparation. (-)-DMH was about 40 times more potent than (-)-delta 9-THC in both preparations, supporting the notion that their mode of action in each tissue is the same. 3. The psychically inactive cannabinoid, (+)-DMH, had no inhibitory effect in the mouse vas deferens at a concentration of 30 nM, showing it to be at least 1000 times less potent than (-)-DMH. In the myenteric plexus preparation, (+)-DMH was about 500 times less potent than its (-)-enantiomer. 4. The inhibitory effects of sub-maximal concentrations of (-)-delta 9-THC were not attenuated by 300 nM naloxone. 5. The findings that (-)-delta 9-THC and (-)-DMH are highly potent as inhibitors of the twitch response of the mouse vas deferens and guinea-pig myenteric plexus preparation and that DMH shows considerable stereoselectivity suggest that the inhibitory effects of cannabinoids in these preparations are mediated by cannabinoid receptors.