Calcium-Wirkungen auf die Noradrenalinspeicherung isolierter Milznervengranula

Abstract

1. Granules containing 0.7–1.5 μg noradrenaline/mg protein were prepared from an homogenate of postganglionic bovine splenic nerves by differential centrifugation in isotonic phosphate buffer followed by centrifugation over 1.1 M sucrose. 2. Splenic nerve granules were depleted to one third of their noradrenaline content by preincubation. A 10 min incubation at 37°C with 1.5×10−5 M noradrenaline, 4 mM Mg++ and 5 mM ATP restored their original noradrenaline content. As shown by experiments with C14-noradrenaline, there is a 90% exchange of exogenous for endogenous noradrenaline after 20 min, and complete exchange after 40 min. 3. Granules were equilibrated with 1.5×10−5 M C14-noradrenaline, During a second incubation at 20° C with 2.5×10−10 M unlabelled noradrenaline and ATP, there was no net loss of noradrenaline and no statistically significant loss of C14-activity within 30 min. At 20° C, after dilution of the noradrenaline in the incubation medium and after the addition of 2.5 mM Ca++ noradrenaline is lost from the granules at 1.5±0.6% and C14-activity at 1.8±0.6% of the granular content per minute. Ca++ accelerates the loss of granular catecholamines at 37° C but has no effect at 0°C. 4. Ca++ releases noradrenaline from granules only after dilution of exogenous noradrenaline. 0.5–4.0 mM Ca++ does not inhibit the net uptake of noradrenaline into granules during incubation with 1.5×10−5 M noradrenaline, 4 mM Mg++ and 5 mM ATP. 5. The Ca++-induced in vitro release of noradrenaline occurs after a disturbance of the dynamic equilibrium between bound and free noradrenaline in the presence of ATP and Mg++; it is suggessted that it may be important for the transmitter release in response to nerve stimulation in vivo.