Catabolism of Ap4A and Ap5A by rat brain synaptosomes

Adenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5',5'''-P1,P5-pentaphosphate (Ap5A) are stored in and released from rat brain synaptic terminals. In the present study we investigated the hydrolysis of dinucleotides (Ap4A and Ap5A) in synaptosomes from the cerebral cortex of adult rats. Ap4A and Ap5A, but not Ap3A, were hydrolyzed at pH 7.5 in the presence of 20 mM Tris/HCl, 2.0 mM MgCl2, 10 mM glucose and 225 mM sucrose at 37oC. The disappearance of the substrates measured by FPLC on a mono-Q HR column was both time and protein dependent. Since synaptosome integrity was at least 90 percent at the end of the assay, hydrolysis probably occurred by the action of an ecto-enzyme. Extracellular actions of adenine dinucleotides at central nervous system terminate due to the existence of ecto-nucleotidases which specifically cleave these dinucleotides. These enzymes in association with an ATP diphosphohydrolase and a 5'-nucleotidase are able to promote the complete hydrolysis of dinucleotides to adenosine in the synaptic cleft

ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats subjected to chemically induced phenylketonuria

ATP diphosphohydrolase (apyrase)(EC3.6.1.5) activity was measured in synaptosomes from cerebral cortex of Wistar rats of both sexes subjected to experimental phenylketonuria, i.e., chemical hyperphenylaninemia induced by subcutaneous administration of 5.2 µmol phenylalanine/g body weight (twice a day) plus 0.9 µmol p-chlorophenylalanine/g body weight (once a day). ATP diphosphohydrolase specific activity (nmol Pi min-1 mg protein-1) of synaptosomes was significantly decreased compared to controls for both ATp (from 147.6 to 129.9) and ADP (from 70.2 to 63.1) hydrolysis one hour after single administration of the drugs to 35-day old rats. Chronic treatment was performed from the 6th to the 28th postpartum day. The enzyme specific activity of synaptosomes was measured one week after the last administration of the drugs and was significantly reduced compared to controls for both ATP (from 164.1 to 150.2) and ADP (from 76.3 to 62.1) hydrolysis. The in vitro effects of the drugs on the synaptosome enzyme specific activity were also investigated. Phenylalnine alone or associated with p-chlorophenylalanine significantly reduced enzyme specific activity for both ATP (from 150.2 to 136.0) and ADP (from 70.5 to 59.3) nucleotides as substrates. Since ATP diphosphohrolase seems to play an important role in neurotransmission, these findings may be related to the neurological dysfunction characteristic of human phenylketonuria

Lithium-induced inhibition of Na-K ATPase and Ca ATPase activities in rat brain synaptosome

To explore the action mechanism of lithium in the brain, the author investigated the effects of lithium on Na-K ATPase and Ca ATPase in rat brain synaptosomes prepared from forebrains by the method of Booth and Clark. The activities of Na-K ATPase and Ca ATPase were assayed by the level of inorganic phosphate liberated from the hydrolysis of ATP. Lithium at the optimum therapeutic concentration of 1 mM decreased the activity of Na-K ATPase from the control value of 19.08 +/- 0.29 to 18.27 +/- 0.10 micromoles Pi/mg protein/h and also reduced the activity of Ca ATPase from 6.38 +/- 0.12 to 5.64 +/- 0.12 micromoles Pi/mg protein/h. The decreased activity of Na-K ATPase will decrease the rate of Ca2+ efflux, probably via an Na-Ca exchange mechanism and will increase the rate of Ca2+ entry by the depolarization of nerve terminals. The reduced activity of Ca ATPase will result in the decreased efflux of Ca2+. As a Conclusion, it can be speculated that lithium elevates the intrasynaptosomal Ca2+ concentration via inhibition of the activities of Na-K ATPase and Ca ATPase, and this increased [Ca2+]i will cause the release of neurotransmitters and neurological effects of lithium.

A brain soluble fraction inhibits synaptosomal membrane but not astrocyte ATPase activity

Since a brain soluble fraction (peak II) is known to be able to inhibit synaptosomal membrane Na+, K+-ATPase activity, here we attempted to compare its effect on cellular and subcellular brain components such as synaptosomal and astrocytic membranes, as well as mitochondrial preparations. Peak II highly inhibited total ATPase in synaptosomal membranes but failed to modify enzyme activity in astrocytic and mitochondrial preparations. Findings suggest cellular and subcellular specificity of peak II on brain ATPase activity.

Effects of undernutrition during suckling on ATP and ADP hydrolysis by synaptosomes from the cerebral cortex of adult rats

Early undernutrition can cause permanent functional changes in the nervous system. Alterations in enzymes involved in neurotransmiter metabolism have been reported to result from early undernutrition. In a previous study, we demonstrated that undernutrition during suckling decreaseATP and ADP hydrolysis by synaptosomes from cerebral cortex by abouth 20% of the value found in 20-day-old well-nourished rats (j.B.T. Rocha, C.F. Melo, J.J.F.Sarkis and R.D. Dias, British Journal of Nutrition, 63:273-283, 1990). In the present study, we investigated whether this deficit persists in synaptosomes from cerebral cortex of nutritionally rehabilitated adult rats. rats were undernourished from birth to 25 days of life by feeding their dams a 7% casein (w/w) diet, while well-nourished offspring were fed by mothers maintained on a 28% casein diet. In contrast to the results previously obtained in young rats, the synaptosomes obtained from the cerebral cortex of early undernourished adult rats hydrolyzed ATP and ADP more efficiently than did those obtained from well-nourished rats. Specific activity (nmol min-1 mg protein-1, mean ñ SD) was 114.9ñ9.5 for undernourished rats (N=8) for ATP, and 50.4ñ6.1 (N=8) vs 38.8ñ4.5 (N=8) for ADP. These results suggest that the deficits found in young rats disappear in rehabilitation adult rats

Synaptosomal apyrase in the hypiothalamus of adult rats

1. The synaptosomal fraction isolated from hypothalamus of adult rats on sucrose density gradient hydrolyzes the labile phosphatase from ATP and ADP, thereby satisfying the general definition of apyrase activity. 2. The parallel behavior of ATPase and ADPase activities under different reaction conditions suggests the presence of a "true" apyrase enzyme. The optimum conditions for the are the same for both nucleotides: pH 8.0, 0.6 mM nucleotide and 1.5 mM cation. At temperatures between 10 and 40-C, both activities increase with no change in the ATP/ADP hydrolysis ratio. Thermal inactivation or inhibition of the enzyme activity by iodoacetamide, p-hydroxynercuribenzoate or 2- mercaptoethanol affected the hydrolysis of both substrates in a similar manner. 3- Adenylate Kinase and phyrophosphatase activities were not detected in the preparation. 4. The enzyme is located on the outer surface of the synaptosomal membrane: intact and lysed synaptosomes have similar activity and the supernatant obtained by centrifugation of intact synaptosomal preparations does not hydrolyze ATP or ADP