1 The psychoactive cannabinoids (-)-A9-tetrahydrocannabinol ((-)-A9-THC) and the 1,l-dimethylheptyl homologue of (-)-11-hydroxy-A8-tetrahydrocannabinol ((-)-DMH) both inhibited electricallyevoked contractions of the mouse isolated vas deferens and the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine. 2 Concentrations of (-)-A9-THC and (-)-DMH that decreased twitch heights by 50% were 6.3 and 0.15nM respectively in the mouse vas deferens and 60nM and 1.4nM respectively in the myenteric plexus preparation. (-)-DMH was about 40 times more potent than (-)-A9-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 (-)-A9-THC were not attenuated by 300 nM naloxone. 5 The findings that (-)-A9-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.

α-Latrotoxin (α-LTX) is a neurotoxin that accelerates spontaneous exocytosis independently of extracellular Ca2+. Although α-LTX increases spontaneous transmitter release at synapses, the mechanism is unknown. We tested the hypothesis that α-LTX causes transmitter release by mobilizing intracellular Ca2+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 α-LTX increases transmitter release after release of a physiologically relevant concentration of intracellular Ca2+. Neither the blockade of Ca2+release nor the depletion of Ca2+from endoplasmic reticulum affected Ca2+signals produced by α-LTX. The Ca2+source is likely to be mitochondria, because the effects on Ca2+mobilization of CCCP (which depletes mitochondrial Ca2+) and of α-LTX are mutually occlusive. The release of mitochondrial Ca2+is partially attributable to an increase in intracellular Na+, suggesting that the mitochondrial Na+/Ca2+exchanger is activated. Effects of α-LTX were not blocked when Ca2+increases were reduced greatly in saline lacking both Na+and Ca2+and by application of intracellular Ca2+chelators. Therefore, although increases in intracellular Ca2+may facilitate the effects of α-LTX on transmitter release, these increases do not appear to be necessary. The results show that investigations of Ca2+-independent α-LTX mechanisms or uses of α-LTX to probe exocytosis mechanisms would be complicated by the release of intracellular Ca2+, which itself can trigger exocytosis.

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.

In response to concerns about interactions of academic and public health investigators with industry, the Canadian Association for Immunization Research and Evaluation (CAIRE), in collaboration with six major vaccine manufacturers, developed guidelines for participation in industry-sponsored clinical trial and epidemiology contract research within Canada. Topics addressed include definition of investigators, data ownership, protocol development, data management, data analysis, producing a study report and publication of the results of the study.