Ca(2+) dynamics in synaptosomes isolated from the squid optic lobe.

Synaptosomes from the optic lobes of squid (Loligo forbesi) were prepared by homogenization and allowed to settle onto glass coverslips. Synaptosomes were loaded with Ca(2+) sensitive dyes (Fura-2 AM, Calcium Green-1 AM and Calcium Green-5N AM), visualized by light microscopy and Ca(2+) sensitive fluorescence signals recorded and analyzed. With Fura-2, resting Ca(2+) was found to be 80 nM (n = 10, SEM 5.7). Addition of K(+) (30 mM), caffeine (3 mM) and thapsigargin (10 microM) evoked transient increases in cytoplasmic Ca(2+). Addition of BAPTA-AM (20 microM) decreased intrasynaptosomal free Ca(2+). Similar results were obtained with Calcium Green-1 AM but not with Calcium Green-5N AM. We conclude that synaptosomes from the squid optic lobe posses intact membranes and mechanisms to regulate intrasynaptosomal free [Ca(2+)], as well as caffeine sensitive Ca(2+) stores. The results of this study are discussed with respect to the role of Ca(2+) in presynaptic protein synthesis.

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve.

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.

Calcium efflux from platelet vesicles of the dense tubular system. Analysis of the possible contribution of the Ca2+ pump.

The ATP dependent Ca2+ uptake of platelet vesicles was inhibited by the two hydrophobic drugs trifluoperazine (TFP) and propranolol (PROP). Inhibition was significantly lowered when Pi was used instead of oxalate as a precipitant agent. When the ATPase ligands substrate (Mg2+ and Pi) were absent of the efflux medium, a slow release of Ca2+ which did not couple with ATP synthesis (passive Ca2+ efflux) was observed. Both, TFP and PROP enhanced the passive Ca2+ efflux. This enhanced efflux was partially inhibited only when Mg2+ and Pi were added together to the efflux reaction media, but it was not affected by spermidine, ruthenium red or thapsigargin (TG). The Ca2+ ionophores A23187 and ionomycin, also enhanced passive Ca2+ efflux. However, in this case, Ca2+ efflux was inhibited just by inclusion of Mg2+ to the medium. Ca2+ efflux promoted by Triton X-100 was not affected by either Mg2+ or Pi, included together or separately into the efflux medium. The ATP <==> Pi measured in the presence of Triton X-100 and millimolar Ca2+ concentrations was inhibited by both TFP and PROP, but not by Ca2+ ionophores up to 4 microM. The data suggest that the observed enhancement of passive Ca2+ efflux promoted by TFP and PROP could be attributed to a direct effect of these drugs over the platelet Ca2+ pump isoforms (Sarco Endoplasmic Reticulum Calcium ATPase, SERCA2b and SERCA3) themselves, as it was reported for the sarcoplasmic reticulum Ca2+ ATPase (SERCA1).

Ribosomes and polyribosomes are present in the squid giant axon: an immunocytochemical study.

Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.

Protein synthesis in presynaptic endings from squid brain: modulation by calcium ions.

Previous biochemical, autoradiographic, and ultrastructural data have shown that, in the synaptosomal fraction of the squid optic lobe, protein synthesis is largely due to the presynaptic terminals of the retinal photoreceptor neurons (Crispino et al. [1993a] Mol. Cell. Neurosci. 4:366-374; Crispino et al. [1993b] J. Neurochem. 61:1144-1146; Crispino et al. [1997] J. Neurosci. 17:7694-7702). We now report that this process is close to its maximum at the basal concentration of cytosolic Ca++, and is markedly inhibited when the concentration of this ion is either decreased or increased. This conclusion is supported by the results of experiments with: 1) compounds known to increase the level of cytosolic Ca++, such as A23187, ionomycin, thapsigargin, and caffeine; 2) compounds sequestering cytosolic calcium ions such as BAPTA-AM; and 3) agents that block the role of Ca++ as second messenger, such as TFP and W7, which inhibit calmodulin, and calphostin, which inhibits protein kinase C. We conclude that variations in the level of cytosolic Ca++ induced in presynaptic terminals by neuronal activity may contribute to the modulation of the local synthesis of protein.

Active polysomes are present in the large presynaptic endings of the synaptosomal fraction from squid brain.

Previous data have suggested that the large nerve terminals present in the synaptosomal fraction from squid optic lobe are capable of protein synthesis (Crispino et al., 1993a,b). We have further examined this issue by comparing the translation products of synaptosomal and microsomal polysomes. Both preparations programmed an active process of translation, which was completely abolished by their previous treatment with EDTA. After immunoabsorption of the newly synthesized neurofilament (NF) proteins, the labeling ratio of the 60 and 70 kDa NF proteins was found to differ, in agreement with comparable differences obtained with intact synaptosomes. These observations indicate that the set of mRNAs translated by synaptosomes differs from that translated by nerve cell bodies. Hence, because NF proteins are neuron-specific, they support the view that the active synaptosomal polysomes are mostly localized in the large nerve terminals that represent the most abundant neuronal component of the fraction. This hypothesis was confirmed (1) by electron spectroscopic data demonstrating the presence of ribosomes and polysomes within the large nerve endings of the synaptosomal fraction, as well as in the carrot-like nerve endings of the retinal photoreceptors that constitute the only large terminals in the optic lobe, and (2) by light and high resolution autoradiography of synaptosomal samples incubated with [3H]leucine, showing that most labeled proteins are associated with the large nerve endings. This response was abolished by cycloheximide. Taken together, the data provide the first unequivocal demonstration that presynaptic nerve terminals are capable of protein synthesis.

Reversal of the Ca2+ pump of blood platelets.

In this study, the endoplasmic Ca2+ transport ATPase of blood platelets was compared with the Ca2+ ATPase of sarcoplasmic reticulum skeletal muscle. Similar to the muscle enzyme, the Ca2+ ATPase from platelets was found to catalyse an ATP<-->P(i) exchange both in the presence and in the absence of a transmembrane Ca2+ gradient. When platelet vesicles are loaded with Ca2+ and diluted in medium containing ADP, P(i) and EGTA, the ATPase catalyses Ca2+ efflux coupled to synthesis of ATP. The stoichiometry between Ca2+ ion released and ATP synthesized by platelet Ca2+ ATPase is 1, while that of skeletal muscle is 2. Thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases, inhibited both the Ca(2+)-dependent ATPase activity and the reversal of the platelet Ca2+ pump. The possibility is discussed that the differences observed between the two transport systems is related to the distinct amino acid sequences of the enzymes.

Correlation between Ca2+ uptake, Ca2+ efflux and phosphoenzyme level in sarcoplasmic-reticulum vesicles.

Previously [Inesi & de Meis (1989) J. Biol. Chem. 264, 5929-5936] it was shown that dimethyl sulphoxide (Me2SO) increases the amount of Ca2+ accumulated by sarcoplasmic-reticulum vesicles. This effect was related to a decrease in the enzyme affinity for ADP from less than 20 microM to 1 mM. In the present work the apparent affinity of the ADP-sensitive phosphoenzyme for ADP was determined by measuring the rate of ATP synthesis in vesicles previously loaded with Ca2+, at different pH values and in the presence and absence of Me2SO (20%, v/v) and KCl. In all conditions tested, addition of Me2SO never promoted an increase of the apparent Km for ADP to a value higher than 25 microM. ADP inhibits the phosphorylation of the enzyme by Pi. Two components, with Ki values of 80 microM and 8 mM, were detected when vesicles previously loaded with Ca2+ were used. The high-affinity component (Ki 80 microM) was abolished after addition of Me2SO to the medium. Empty vesicles, on the other hand, exhibited only the low-affinity component (Ki 8 mM). During ATP synthesis in a totally aqueous medium, there was a decrease in the phosphoenzyme formed by Pi, after addition of 80-100 microM-ADP to the medium. In the presence of Me2SO this decrease was smaller. The sum of the fractions of phosphoenzyme formed by ATP and by Pi during Ca2+ uptake was higher in the presence of Me2SO than in a totally aqueous medium. Me2SO decreased the passive efflux of Ca2+ measured in the presence of 0.1 mM-Pi and 0.1 mM-MgCl2. In a totally aqueous medium the same decrease was observed when Pi and MgCl2 concentrations were raised to 4 mM. These data suggest that ADP binds to two different enzyme conformations. The binding to one of these conformations (*E) inhibits the phosphorylation of the enzyme by Pi, increases the efflux of Ca2+ and decreases the amount of Ca2+ accumulated by the vesicles.