The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: electron microscopy and cytotoxicity studies.

Neurosecretory PC12 cells were exposed in a variety of experimental conditions to nanomolar concentrations of alpha-latrotoxin purified from the venom of the black widow spider. When applied in a modified Ringer medium containing millimolar Ca2+ the toxin rapidly elicited a marked stimulation of exocytosis, as indicated by the appearance of typical images of granule-plasmalemma interaction and by the decreased density (number/unit area) of secretion granules in the cytoplasm. Without Ca2+ in the medium this early toxin effect was delayed and evolved less rapidly, but was still clearly appreciable. These morphological results appear in good quantitative agreement with the biochemical data on dopamine release reported in the preceding article. The stimulation of exocytosis was followed after a short delay by a stimulation of endocytosis, as revealed by an increased accumulation of the extracellular tracer, [14C]sucrose, within the toxin-treated cells. At later times after the application of alpha-latrotoxin other effects appeared, but only in the presence of Ca2+: these included changes in cell shape; focal alterations of the mitochondrial matrix (clear discrete areas and dense precipitates) and frank signs of cytotoxicity (rupture of the plasmalemma, clearing of the cytoplasmatic matrix). The toxin-induced cell death was studied quantitatively by using trypan blue exclusion as well as the 51Cr test, and was found to be dependent on alpha-latrotoxin concentration, temperature of incubation and Ca2+ concentration in the medium. Ionic substitutions concerning anions as well as cations other than Ca2+ had minor or no consequences. Thus, the early effect of alpha-latrotoxin in PC12 cells (stimulation of exocytosis, at least partially Ca2+-independent) can be dissociated from the late 'toxic' effect (strictly Ca2+-dependent).

The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: studies on toxin binding and stimulation of transmitter release.

alpha-Latrotoxin of black widow spider venom was found to bind with high affinity (KA = 1.8 X 10(9)M-1) to specific sites present in discrete number (approximately 6300/cell, approximately 12/micron2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (approximately 2-fold) in the presence of mM concentrations of various divalent cations (Ca2+, Mn2+ and Co2+) while Ba2+ and Sr2+ had a smaller effect and Mg2+ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The alpha-latrotoxin-induced stimulation of 3H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca2+-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca2+-free, Mg2+-containing Ringer. Introduction of Ca2+ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na+ and Ca2+ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca2+ and Mg2+ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and 1.5 mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca2+ and Mg2+, the relative efficacy being Sr2+ greater than Ca2+ greater than Ba2+ much greater than Mn2+ greater than Mg2+ greater than Co2+. Moreover, the response occurring at suboptimal concentration of Ca2+ (0.4 mM) was potentiated by the concomitant addition of either Mg2+, Mn2+ or Co2+. The effect(s) of divalent cations in supporting the alpha-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because (a) the stimulatory effects of the various cations on release were not matched by parallel effects on binding, and (b) Ca2+ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca2+-free medium. Delays in the release responses were observed when cells were exposed to alpha LTx in Na+-free, glucosamine or methylamine-based media, or depolarized with high K+ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg2+ to support the alpha LTx response was considerably decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity of sympathetic nerves in heart during cardiopulmonary bypass in patients.

The sympathetic nerve activity in heart during cardiopulmonary bypass was studied in 20 patients. The rate of release of dopamine-beta-hydroxylase from cardiac nerves was used as an indicator. It was found that the thoracotomy alone does not represent such a stimulus which would lead unambiguously to sympathetic overactivity in heart. Cardiopulmonary bypass represents, on the other hand, an intensive sympathomimetic stimulus, as it was demonstrated by significnat increase of coronary A-V difference negativity of dopamine-beta-hydroxylase as early in 15th minute of its duration. The washout of enzyme to the end of 30 min. duration of bypass still increased. After finishing of bypass the coronary A-V diff. of dopamine-beta-hydroxylase again decreased.