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A latent ATP-dependent Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. The data indicate that a fraction of the Ca uptake (measured with 45Ca) is associated with the intraterminal mitochondria; it is blocked by ruthenium red, by FCCP, and by azide + dinitrophenol + oligomycin. There is, however, a residual ATP-dependent Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondrial) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. The proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the ATP-dependent Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca2+ concentration (buffered with EGTA) is less than 0.3 micrometer; brain mitochondria take up little Ca when the ionized Ca2+ level is this low. Preliminary subfractionation studies indicate that the nonmitochondrial Ca storage system does not sediment with synaptic vesicles. We propose that this Ca storage system, which has many properties comparable to those of skeletal muscle sarcoplasmic reticulum, may be associated with intraterminal smooth endoplasmic reticulum. This Ca-sequestering organelle may help to buffer intracellular Ca.

The kinetic properties of the nonmitochondrial ATP-dependent Ca sequestering mechanism in disrupted nerve terminal (synaptosome) preparations have been investigated with radioactive tracer techniques; all solutions contained DNP, NaN3, and oligomycin, to block mitochondrial Ca uptake. The apparent half-saturation constant, KCa, for the nonmitochondrial Ca uptake is approximately 0.4 micrometer Ca; the Hill coefficient is approximately 1.6. Mg is also required for the Ca uptake, and the apparent KMg is approximately 80 micrometer. ATP and deoxy-ATP, but not CTP, GTP, ITP, UTP, ADP, or cyclic AMP, promote Ca uptake; the KATP, is approximately 10 micrometer. ATP analogs with blocked gamma-phosphate groups are unable to replace ATP. Particulate fractions from the disrupted synaptosomes possess Ca-dependent ATPase activity in the presence of Mg; the apparent KCa for this activity is 0.4--0.8 micrometer Ca, and the Hill coefficient is approximately 1.6. The Ca uptake and ATPase kinetic data suggest that the hydrolysis of 1 ATP may energize the transport of two Ca2+ ions into the storage vesicles. The second part of the article concerns the intraterminal distribution of Ca in "intact" terminals. When the terminals are disrupted after 45Ca loading, about one-half of the 45Ca is retained in the particulate material; some of this Ca, presumably stored in mitochondria, is released by the uncoupler, FCCP. Some of the 45Ca is released by A-23187, but not by FCCP; this fraction may be Ca stored in the nonmitochondrial sites described above. The proportion of 45Ca stored in the nonmitochondrial sites is increased when the Ca load is reduced or when the mitochondria are blocked with ruthenium red. These data indicate that the nonmitochondrial Ca storage sites are involved in intraterminal Ca buffering; they may play an important role in synaptic facilitation and post-tetanic potentiation, which result from Ca retention after neural activity.

Treatment of synaptosomes with sera containing antibodies (Ab) directed against synaptosomal membranes and complement (C) alters the plasma membrane so that it becomes selectively permeable to small molecules and ions but not to proteins. When synaptosomes are incubated with Ab and C, a rapid release of intracellular K occurs. This release does not occur after treatment with antiserum alone, or with normal serum + C. Ab + C treatment releases approximately the same amount of K as does detergent treatment or hypotonic lysis, two procedures that extensively disrupt the plasma membrane. The selectivity of the complement-induced lesion is consistent with the equivalent pore radius determined in other systems (Michaels and Mayer 1978; Sears et al. 1964). The lesions are large enough to allow the rapid permeation of small ions, but too small to permit the escape of the soluble cytoplasmic enzyme, lactate dehydrogenase. In addition, electron microscopic studies indicate that Ab + C treatment does not lead to gross morphological disruption of the synaptosomes. Ab + C treated synaptosomes are also permeable to calcium and ATP, as demonstrated by the stimulation of Ca sequestration into endoplasmic reticulum when 45Ca and ATP are added to the incubation medium.

Isolated cholinergic synaptosomes from elasmobranch electric organ release both ACh and ATP when depolarized in the presence of calcium. The conditions that trigger ATP release are the same as those known to stimulate neurosecretion. The ratio of ACh to ATP released is the same as that found in purified elasmobranch synaptic vesicles. Both ACh and ATP release are inhibited either by the removal of extracellular calcium or the addition of the "calmodulin antagonist" trifluoperazine (TFP). Taken together, these data suggest that both ACh and ATP are released by exocytosis from synaptic vesicles within nerve terminals. A number of drugs, collectively known as calmodulin antagonists, were examined for their effect on neurosecretion. TFP completely inhibited secretion in a rapid and reversible manner. Other related drugs had similar effects, though with relative efficacies different from those reported for inhibiting calmodulin. It therefore seems likely that a calmodulin-like component of the nerve terminal, distinct from calmodulin itself, is essential for the process of neurosecretion.

The particulate fraction from osmotically shocked synaptosomes ('synaptosomal membrances') sequesters Ca when incubated with ATP]containing solutions. This net accumulation of Ca can reduce the free [Ca2+] of the bathing medium to sub-micromolar levels (measured with arsenazo III). Two distinct types of Ca sequestration site are responsible for the Ca2+ buffering. One site, presumed to be smooth endoplasmic reticulum, operates at low [Ca2+] (less than 1 microM), and has a relatively small capacity. Ca sequestration at this site is prevented by the Ca2+ ionophore, A-23187, but not by mitochondrial poisons. The secone (mitochondrial) site, in contrast, is blocked by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and oligomycin. Since the intraterminal organelles can buffer [Ca2+] to about 0.3-0.5 microM, this may be an upper limit to the normal resting level of [Ca2+]i in nerve terminals. In the steady state, total cell Ca and [Ca2+]i will be governed principally be Ca transport mechanisms in the plasmalemma; the intracellular organelle transport systems then operate in equilibrium with this [Ca2+]. During activity, however, Ca rapidly enters the terminals and [Ca2+]i rises. The intracellular buffering mechanisms then come into play and help to return [Ca2+]i toward the resting level; the non-mitochondrial Ca sequestration mechanism probably plays the major role in this Ca buffering.