Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Effect of stobadine on indomethacin- and ethanol-induced stomach lesions and gastric secretion.

Stobadine was found to inhibit the ulcerogenic activity of indomethacin in relation to the dose but was ineffective against the direct necrotizing action of ethanol. It also inhibited gastric acid secretion when administered intraduodenally. Although stobadine is considered to be a scavenger of free radicals, our results indicate that, under the given experimental conditions, it is rather the inhibition of gastric acid secretion that is responsible for its antiulcerogenic effect. The preliminary results do not allow the exclusion of other mechanisms for explaining its antiulcerogenic effect.     

The Roberts syndrome

Summary Four siblings with the autosomal recessive Roberts syndrome are reported, and we discuss the phenotypic overlap of this syndrome with other similar radial, aplasia syndromes.     

Retrograde Inhibition of Transmitter Release by ATP

Abstract: After labelling ACh tissue stores in Torpedo electric organ prisms with radioactive acetate, the release of ACh and ATP triggered by electrical stimulation or KCI depolarization was measured in the same perfusate samples. The luciferin-luciferase reaction for ATP was first counted, then the radioactive content of the sample determined. Further evidence showing that ATP release resulted from postsynaptic transmitter action was that carbachol could induce the release of ATP. A dose-response curve was obtained. Curare or α-bungarotoxin block the release of ATP elicited by carbachol. When triggered by KCI depolarization the increased efflux of ACh and ATP returned to low levels in spite of the maintained depolarization. After two successive KCI depolarizations, it was possible to dissociate the release of both substances. The efflux of ATP was exhausted while ACh release was maintained. If the second KCI depolarization was delayed ATP release recovered, but the release kinetics of ACh and ATP were sustained. The exhaustion of endogenous ATP release or the action of exogenous ATP had little or no effect on the release of ACh triggered by KCI depolarization. On the contrary, the release of ACh induced by electrical stimulation was sensitive to the action of adenine nucleotides, and a quantitative estimation of the inhibition of ACh release by ATP and adenosine could be made. At the onset of stimulation ATP release predominated, being gradually replaced by adenosine, which can be reuptaken. This would terminate the inhibitory action of the nucleotide. Carbachol inhibits evoked ACh release, while the effect of α-bungarotoxin was to increase spontaneous ACh release. These effects could be respectively mediated by an increased or a reduced release of ATP resulting from the postsynaptic action of ACh agonists or antagonists. However, a direct presynaptic effect of these substances is not excluded. It seems possible that the action of ATP on ACh release can be explained through its inhibition of the depolarization-evoked Ca²⁺ entry.     

Severe mental deficiency,proportionate dwarfism,and delayed sexual maturation

Summary Two 46,XY brothers were found to have a previously undescribed syndrome characterized by severe mental deficiency, proportionate dwarfism, and delayed sexual development. A recessive mode of inheritance, either autosomal or X-linked, is assumed.     

Synaptosomal uptake

Using purified synaptosomal preparations from rat brain, the uptake ofl-tryptophan and norepinephrine was studied. We were unable to replicate some of the results of the experiments obtained with crude mitochondrial, fractions (P₂). Thus we examined the validity of the results of uptake studies obtained with the crude synaptosomes and established conditions which would simulate the biochemical milieu in which the nerve terminals functionin vivo, such as active substrate-dependent respiration, respiratory coupling on addition of ADP, low impurity of noncharacteristic markers, exogenous added proteins (e.g. bovine serum albumin), and verification by electron microscopy. All uptake studies withl-TRP and NE were completed in a system designed for simultaneous recording of respiration and the effect of added ADP. This system was also employed in comparative studies with mitochondria purified by multiple density gradients derived either from the perikaryon or from synaptosomes. Synaptosomal or mitochondrial preparations which did not conform to the above criteria invariably showed significantly lowered ability of uptake ofl-TRP or NE. This was found to be related to impairment in their respiratory and coupling ability. When the experimental conditions of others were employed, the time course of uptake of TRP for crude synaptosomes (P₂) was 100 nmol/g/min and was linear for 2.5 min, while for the purified synaptosomes it was 20 nmol/g/min with a l-min linearity. The mitochondria purified from P₂ displayed 30 nmol/g/min uptake withl-TRP with a linearity of 2.5 min. Reconstituted system of purified synaptosomes and mitochondria gave 60 nmol/g/min ofl-TRP transport with 2.5 min linearity. Also examined was the effect of eight different media. It was found that Krebs-Ringer solution containing glucose (40 mM), pyruvate and malate (10 mM), and ADP (250 nmol) gave optimal uptake of TRP both for synaptosomes and for mitochondria, increasing it to 60 and 86 nmol/g/min. The above conditions also enhanced the uptake of NE by synaptosomes and mitochondria. Uptake of NE was not proportional to protein concentration when the protein content exceeded 0.4 mg. Purified synaptosomal mitochondria accumulated NE more actively than the purified nonsynaptic free mitochondria, albeit at the same rate. Synaptic and free mitochondria had an impaired uptake of NE in presence of DNP, antimycin A, and rotenone, and unlike withl-TRP, pyruvate and malate also reduced uptake of NE. Significant differences were noted for the cytochrome oxidase activity between the synaptosomal and free michondria when compared to that of the homogenate.