REGULATION OF INTRACELLULAR CALCIUM AND CALCIUM BUFFERING PROPERTIES OF RAT ISOLATED NEUROHYPOPHYSEAL NERVE-ENDINGS

1. Electrophysiological measurements of Ca2+ influx using patch clamp methodology were combined with fluorescent monitoring of the free intr acellular calcium concentration ([Ca2+]i) to determine mechanisms of C a2+ regulation in isolated nerve endings from the rat neurohypophysis. 2. Application of step depolarizations under voltage clamp resulted i n voltage-dependent calcium influx (I-ca) and increase in the [Ca2+]i. The increase in [Ca2+]i was proportional to the time-integrated I-ca for low calcium loads but approached an asymptote of [Ca2+]i, at large Ca2+ loads. These data indicate the presence of two distinct rapid Ca 2+ buffering mechanisms. 3. Dialysis of fura-2, which competes for Ca2 + binding with the endogenous Ca2+ buffers, reduced the amplitude and increased the duration of the step depolarization-evoked Ca2+ transien ts. More than 99 % of Ca2+ influx at low Ca2+ loads is immediately buf fered by this endogenous buffer component, which probably consists of intracellular Ca2+ binding proteins. 4. The capacity of the endogenous buffer for binding Ca2+ remained stable during 300 a of dialysis of t he nerve endings. These properties indicated that this Ca2+ buffer com ponent was either immobile or of high molecular weight and slowly diff usible. 5. In the presence of large Ca2+ loads a second distinct Ca2buffer mechanism was resolved which limited increases in [Ca2+]i to ap proximately 600 nar. This Ca2+ buffer exhibited high capacity but low affinity for Ca2+ and its presence resulted in a loss of proportionali ty between the integrated I-ca and the increase in [Ca2+]i. This buffe ring mechanism was sensitive to the mitochondrial Ca2+ uptake inhibito r Ruthenium Reel. 6. Basal [Ca2+]i, depolarization-induced changes in [Ca2+]i and recovery of [Ca2+]i to resting levels following an induced increase in [Ca2+], were unaffected by thapsigargin and cyclopiazonic acid, specific inhibitors of intracellular Ca2+-ATPases. Caffeine and ryanodine were also without effect on Ca2+ regulation. 7. Evoked incr eases in [Ca2+]i, as well as rates of recovery from a Ca2+ load, were unaffected by the extracellular [Na+], suggesting a minimal role for N a+-Ca2+ exchange in Ca2+ regulation in these nerve endings. 8. Applica tion of repetitive step depolarizations for a constant period of stimu lation resulted in a proportional frequency (up to 40 Hz)-dependent in crease in [Ca2+]i. On the other hand, for a constant number of stimuli a reduction in the [Ca2+]i increase per impulse was observed at highe r frequencies. Application of step depolarizations, mimicking in durat ion and frequency those occurring: during impulse bursting of a vasopr essinergic neuron, raised [Ca2+]i to values where, in addition to buff ering- by the endogenous cytoplasmic component, there occurred bufferi ng by a Ruthenium Red-sensitive mechanism and by plasma membrane Ca2+- ATPase activity. 9. It is suggested that the Ca2+ regulatory propertie s of the neurohypophysial nerve endings may, together with existing: e lectrophysiological and secretory data on these nerve endings, explain the importance of phasic impulse bursting to potentiation of neuropep tide release from this system.