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. 1994 Dec 1;481(Pt 2):251–271. doi: 10.1113/jphysiol.1994.sp020436

Regulation of intracellular calcium and calcium buffering properties of rat isolated neurohypophysial nerve endings.

E L Stuenkel 1
PMCID: PMC1155926  PMID: 7738824

Abstract

1. Electrophysiological measurements of Ca2+ influx using patch clamp methodology were combined with fluorescent monitoring of the free intracellular calcium concentration ([Ca2+]i) to determine mechanisms of Ca2+ regulation in isolated nerve endings from the rat neurohypophysis. 2. Application of step depolarizations under voltage clamp resulted in voltage-dependent calcium influx (ICa) and increase in the [Ca2+]i. The increase in [Ca2+]i was proportional to the time-integrated ICa for low calcium loads but approached an asymptote of [Ca2+]i at large Ca2+ loads. These data indicate the presence of two distinct rapid Ca2+ 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+ transients. More than 99% of Ca2+ influx at low Ca2+ loads is immediately buffered 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 s of dialysis of the nerve endings. These properties indicated that this Ca2+ buffer component was either immobile or of high molecular weight and slowly diffusible. 5. In the presence of large Ca2+ loads a second distinct Ca2+ buffer mechanism was resolved which limited increases in [Ca2+]i to approximately 600 nM. This Ca2+ buffer exhibited high capacity but low affinity for Ca2+ and its presence resulted in a loss of proportionality between the integrated ICa and the increase in [Ca2+]i. This buffering mechanism was sensitive to the mitochondrial Ca2+ uptake inhibitor Ruthenium Red. 6. Basal [Ca2+]i, depolarization-induced changes in [Ca2+]i and recovery of [Ca2+]i to resting levels following an induced increase in [Ca2+]i were unaffected by thapsigargin and cyclopiazonic acid, specific inhibitors of intracellular Ca(2+)-ATPases. Caffeine and ryanodine were also without effect on Ca2+ regulation. 7. Evoked increases in [Ca2+]i, as well as rates of recovery from a Ca2+ load, were unaffected by the extracellular [Na+], suggesting a minimal role for Na(+)-Ca2+ exchange in Ca2+ regulation in these nerve endings. 8. Application of repetitive step depolarizations for a constant period of stimulation resulted in a proportional frequency (up to 40 Hz)-dependent increase in [Ca2+]i. On the other hand, for a constant number of stimuli a reduction in the [Ca2+]i. On the other hand, for a constant number of stimuli a reduction in the [Ca2+]i increase per impulse was observed at higher frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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