Effects of potassium, veratridine, and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro

Abstract

1. 45-Ca uptake by pinched-off nerve terminals (synaptosomes) of rat brain incubated in standard physiological saline (including 132 mM-Na + 5mM-K + 1-2 mM-Ca) at 30 degrees C averages about 0-5 mumole Ca per g protein per minute. This may be equivalent to a Ca influx of about 0-03 p-mole/cm-2 sec. 2. The rate of 45-Ca uptake is increased when the concentration of K in the medium is increased above 15-20 mM, K replacing Na isosmotically. Maximum stimulation, a three- to six-fold increase in the rate of Ca uptake, occurs when [K]o is about 60 mM. The effect of increased [K]o is reversible. 3. The K-stimulated Ca uptake is associated primarily with the nerve terminal fraction of brain homogenates. The entering Ca is not accompanied by extracellular markers such as mannitol or inulin. Replacement of external chloride by methylsulphate or sulphate does not prevent the stimulation by K. 4. The effects of external K are quantitatively mimicked by Rb. Caesium also stimulates Ca uptake, but is only about one fifth as effective as K or Rb; Li is ineffective. 5. Two other depolarizing agents also stimulate Ca uptake by synaptosomes: veratridine (7-5 times 10- minus 6 to 7-5 times 10- minus 5 M) and scorpion (Leirus quinquestriatus) venom (6-7 times 10- minus 7 to 6-7 times 10- minus g/ml.). The stimulatory effects of veratridine and scorpion venom, but not of increased [K] are blocked by 2 times 10- minus 7 M tetrodotoxin. 6. Internal K also influences the rate of 45-Ca uptake by synaptosomes: lowering [K]i reduces the stimulatory effect of external K and veratridine. 7. Replacement of external Na by choline markedly inhibits the response to veratridine, but has a much smaller effect on the response to increased [K]o. 8. The Ca uptake mechanism has an apparent dissociation constant for Ca (KCa) of about 0-8 mM. Increasing [K]o increases the maximal rate of Ca uptake, but has no effect on KCa. The K-induced 45-Ca uptake is competitively inhibited by Mg-2+, Mn-2+ and La-3+. 9. The release of acetylcholine and noradrenaline was also studied. Increasing [K]o stimulates external Ca-dependent acetylcholine release. Scorpion venom stimulates noradrenaline release from synaptosomes; this effect could be prevented by adding tetrodotoxin or removing external Ca. 10. These results indicate that synaptosomes may increase their permeability to Ca, accumulate Ca and release neural transmitter substances, when stimulated by depolarizing agents under appropriate physiological conditions.