Phosphorylation and dephosphorylation of purified phospholamban and associated phosphatidylinositides

Phospholamban, the putative regulator for the calcium pump, was purified to apparent homogeneity and in high yields from canine cardiac sarcoplasmic reticulum membranes. Purified phospholamban migrated with an apparent Mr of 27,000 in alkaline sodium dodecyl sulfate-polyacrylamide gels, and upon boiling in 7.5% sodium dodecyl sulfate, it dissociated into a lower molecular weight component of 5500-6000. Purified phospholamban contained 0.62 +/- 0.09 mumol of lipid Pi/mg of protein, and the major phospholipids were phosphatidylserine (34%), phosphatidylcholine (22%), sphingomyelin (17%), phosphatidylinositol (13%), and phosphatidylethanolamine (9%). Phospholamban was phosphorylated by cAMP-dependent protein kinase to a level of 207 nmol of Pi/mg, and this would indicate an incorporation of 1 mol of phosphate/mol of protein, assuming a molecular weight of 5500 for phospholamban. Phosphorylation of phospholamban could be reversed by a "phospholamban phosphatase" isolated from canine cardiac cytosol. Phospholipids associated with the purified phospholamban were also phosphorylated in the presence of the catalytic subunit of cAMP-dependent protein kinase, and the maximal phosphate incorporation was 4 nmol/mg of protein. The main phospholipids phosphorylated were phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate. Phosphorylation of phospholipids was inhibited by the heat-stable inhibitor protein of the cAMP-dependent protein kinase, and it could be also reversed by the phospholamban phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid methylation and taurine content of synaptosomes from cerebral cortex of developing rat.

Changes in taurine concentration and rate of methylation of phosphatidylethanolamine have been examined in rat brain synaptosomes over the course of development. At 7, 14, 21, 28 and 56 days of age, rats were injected i.p. with 300 microCi/kg [3H-methyl]methionine. Synaptosomes (P2B fraction) were isolated from the cerebral cortex 9 h later and incorporation of the methionine methyl group into phospholipid and protein was investigated. Synaptosomal taurine and methionine concentrations were determined at the same ages, as were the concentrations of the major classes of phospholipids (phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine and phosphatidylserine). Methionine concentration increased between day 7 and 14 and fell thereafter. Phospholipid methylation rates calculated from the specific activity of synaptosomal methionine were high from days 7 and 14 and then fell, whereas protein methionylation increased between day 7 and 28 and then decreased. A strong correlation was found between the taurine concentration of the synaptosome and phospholipid methylation rates during brain development. Protein methionylation rates, however, showed no correlation with taurine concentration.     

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.     

Phosphorylation of phospholipids in isolated guinea pig hearts stimulated with isoprenaline.

Phosphorylation of phospholipids was studied in Langendorff perfused guinea pig hearts subjected to beta-adrenergic stimulation. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi and freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline. 32P incorporation into total phospholipids, individual phospholipids and polyphosphoinositides was analysed in whole tissue homogenates and membranes, enriched in sarcoplasmic reticulum, prepared from the same hearts. Isoprenaline stimulation of the hearts did not result in any significant changes in the levels of phosphate incorporation in the total phospholipid present in cardiac homogenates (11.6 +/- 0.4 nmol of 32P/g for control hearts and 12.4 +/- 0.5 nmol of 32P/g for isoprenaline-treated hearts; n = 6), although there was a significant increase in the degree of phospholipid phosphorylation in sarcoplasmic reticulum (3.5 +/- 0.3 nmol of 32P/mg for control hearts and 6.7 +/- 0.2 nmol of 32P/mg for isoprenaline-treated hearts; n = 6). Analysis of 32P incorporation into individual phospholipids and polyphosphoinositides revealed that isoprenaline stimulation of the hearts was associated with a 2-3-fold increase in the degree of phosphorylation of phosphatidylinositol monophosphate and bisphosphate as well as phosphatidic acid in both cardiac homogenates and sarcoplasmic reticulum membranes. In addition, there was increased phosphate incorporation into phosphatidylinositol in sarcoplasmic reticulum membranes. Thus, perfusion of guinea pig hearts with isoprenaline is associated with increased formation of polyphosphoinositides and these phospholipids may be involved, at least in part, in mediating the effects of beta-adrenergic agents in the mammalian heart.     

Presence of Base-Exchange Activity in Rat Brain Nerve Endings: Dependence on Soluble Substrate Concentrations and Effect of Cations

The calcium-dependent, energy-independent incorporations of 14C-labeled bases, choline, ethanolamine, and serine, into their corresponding membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, were compared in microsomes and in subcellular fractions prepared from a lysed crude mitochondrial (P2) pellet of whole rat brain. When activities were measured in the presence of an extracellular (1.25 mM) concentration of Ca2+, recovered activities were highest in the microsomal fraction, although substantial activity remained associated with the P2 homogenate even after repeated washing of the pellet. When this washed P2 homogenate was subfractionated, enrichment of all three exchange activities was obtained only in a fraction that was fivefold enriched over the homogenate and sevenfold enriched over the microsomal fraction in Na+, K+-ATPase, a plasma membrane marker. This strongly suggests that the base-exchange enzymes are normal constituents of synaptosomal plasma membranes. The three exchange activities were measured in synaptosomes prepared from whole rat brain in the presence of various substrate (base) concentrations, and kinetic constants were calculated. The Vmax values for choline, ethanolamine, and serine exchange were, respectively, 1.27 +/- 0.09, 1.60 +/- 0.17, and 0.56 +/- 0.06 nmol/mg of protein/h; the respective Km (apparent) values were 241 +/- 29, 65 +/- 18, and 77 +/- 22 microM. Endogenous levels of the three bases, choline, ethanolamine, and serine, in whole (microwaved) rat brains were 20 +/- 8, 78 +/- 28, and 639 +/- 106 nmol, respectively. That ethanolamine and serine incorporations had lower Km values than choline incorporation suggests that these bases are preferentially incorporated into their respective phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of the skeletal sarcoplasmic reticulum Ca2(+)-pump: influence of negatively charged phospholipids.

The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.     

Proteolipids of brain myelin and synaptosomes in the frog and hen

Proteolipid complex of Folch-Lees has been obtained and purified from the myelin and synaptosomes of the brain of the frog Rana temporaria and hen Gallus domesticus. Relative content of this proteolipid and glycolipids in the myelin is almost twice higher, whereas that of phospholipids--1 1/2 times lower than in the synaptosomal membranes of the same animal. Protein content of this complex is higher for myelin than for synaptosomal membranes; opposite relation was found with respect to phospholipid content. Within this complex, lipids are presented mainly by phospholipids, especially by acid ones which amount to 30-60%. Proteolipid complexes fro the myelin and synaptosomes differ from each other by their lipid component. Myelin proteolipid complex contains mainly phosphatidylserine and phosphatid acid, whereas synaptosomal one--phosphatidylserine and diphosphatediglycerol. No significant differences were found in fatty acid composition of phospholipids from proteolipid complex from myelin and synaptosomes as compared to this composition in the initial membranes.     

Studies on the effects of acetylcholine and antiepileptic drugs on32Pi incorporation into phospholipids of rat brain synaptosomes

Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated³²P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated³²P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the³²P labeling of phospholipids nor on the specific radioactivity of [³²P]ATP. Omission of Na⁺ drastically reduced both the³²P labeling of synaptosomal phospholipids and the specific radioactivity of [³²P]ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate Na⁺ and Ca²⁺ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca²⁺ and Na⁺ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.Abbreviations used ACh Acetylcholine - PA phosphatidic acid - PI phosphatidylinositol - poly PI polyphosphoinositides (diphosphoinositide and triphosphoinositide) - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - S.A. specific radioactivity     

Mature and immature synaptosomal membranes have a different lipid composition

Subfractionation of the optic tectum in chick embryos results in the isolation of two fractions enriched in synaptosomes (fraction A and fraction B). In chicks after hatching, this fractionation results in the isolation of a single synaptosomal fraction (fraction B) and of a fraction enriched in myelin membranes devoid of synaptosomes (fraction A). The lipid composition of synaptosomal fractions (A and B) and corresponding synaptosomal plasma membranes has been analyzed and compared to the lipid composition of similar fractions isolated from 2–3 day-old chicks. The phospholipid composition of fraction A in embryos was mainly represented by phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PE content was significantly lower than that of PC, which accounted for by approximately 50%. Sphingomyelin (SP) and phosphatidylinositol (PI) accounted for by only 6% of the total membrane phopsholipids. Fraction A isolated from the young chicks showed many significant changes. PC accounted for by approximately 40% and PE made up 35%. The amount of phosphatidylserine (PS) and SP increased. These data parallel our previous morphological observations, which showed that fraction A contains immature synaptosomes in embryos but myelin membranes and no synaptosomes in the young chicks. Fraction B has been shown to contain synaptosomes at all stages considered. It possessed in embryos a lipid composition similar to fraction A, except that PC content was higher in young embryos. The analyses on membrane fractions confirmed these results. On the contrary, this fraction showed many significant changes after hatching. The content of PC was significantly reduced, PE/PC ratio was significantly increased as well as ethanolamine plasmalogen (PLE) content. The percentage of PS, PI and SP were increased. The composition of fatty acids of the total fraction of phospholipids was also examined. The results parallel the observations on phospholipid classes.     

Phospholipid modulation of low-Km cyclic AMP phosphodiesterase activity on rat adipocyte microsomes

The ability of nine phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase was examined in microsomal fractions of rat adipocytes. The enzyme was activated by phosphatidylserine (21% at 300 microM) and phosphatidylglycerol (36% at 300 microM). The activation was concentration dependent over the range 1-1000 microM. Six other phospholipids were without effect. Phosphatidylinositol 4-phosphate inhibited the activity of the enzyme over the same range of concentrations (26% at 300 microM). Phosphatidylserine also activated a partially purified preparation of the enzyme, whereas phosphatidylinositol 4-phosphate was ineffective. The mechanism of the activation of the enzyme by phosphatidylserine and phosphatidylglycerol involved an increase in the apparent Vmax of the enzyme, while the inhibition by phosphatidylinositol 4-phosphate was associated with an increase in the Km of the enzyme for substrate. The phospholipid modulators of low-Km cyclic AMP phosphodiesterase activity did not alter the activity of high-Km cyclic AMP phosphodiesterase. The ability of phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase in native membranes suggests a possible role for phospholipids in metabolic regulation.     

Low affinity binding of taurine to phospholiposomes and cardiac sarcolemma

A sarcolemma-enriched membrane fraction was prepared from the hearts of Sprague-Dawley rats and its ability to bind taurine (0.5-150 mM) was measured. In the absence of cations, the sarcolemma bound a maximum of 661 nmol taurine/mg protein, with a dissociation constant of 19.2 mM and a Hill coefficient of 1.9, indicating positive cooperativity. Scatchard analysis of taurine binding to sarcolemma gave a bell-shaped curve. Neither beta-alanine nor guanidinoethane sulfonate, inhibitors of taurine transport, affected the degree of taurine binding to sarcolemma. However, hypotaurine was an effective antagonist. Equimolar concentrations of Ca2+, Na+ or K+ also reduced taurine binding. Heterogeneous phospholipid vesicles of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine (18:19:2:1) also bound taurine with positive cooperativity, yielding a bell-shaped Scatchard curve. The affinity of taurine for these mixed phospholipid vesicles was enhanced by the inclusion of cholesterol (50%). Taurine associated in a maximum ratio of 1:1 with homogeneous vesicles of phosphatidylcholine or phosphatidylserine. Vesicles of phosphatidylethanolamine bound taurine in a maximum ratio of 2:1, whereas those of phosphatidylinositol bound insignificant amounts of taurine. These studies demonstrate a low affinity binding to sarcolemma of taurine at concentrations normally present in rat heart. Similar levels of binding were observed in phospholipid vesicles, suggesting that the interaction of taurine with biological membranes involves phospholipids.     

Rapid decreases in phosphatidylinositol in isolated luteal plasma membranes after stimulation by luteinizing hormone

Phospholipid concentrations were determined in plasma membrane preparations from porcine corpora lutea after incubation for 15 to 120 s without or with 0.5 microgram/ml luteinizing hormone (LH) or 2 microM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Treatment with LH caused a dramatic loss of 9 nmol in plasma membrane phosphatidylinositol (PI)/mg protein after 15 s of incubation, but no significant changes in other measurable phospholipids. Also, phospholipid concentrations were unchanged in untreated and dbcAMP-treated plasma membranes. The nature of the LH-induced decrease in PI was studied by incubating plasma membrane preparations for 15 s with [gamma 32P] adenosine 3',5'-triphosphate (ATP). 32P was incorporated only into three phospholipids: phosphatidic acid, phosphatidylinositol 4'-phosphate (PIP), and phosphatidylinositol 4',5'-bisphosphate (PIP2). Although LH generated small but significant increases in labeling of PIP and PIP2, less than 0.5 nmol of total phospholipids/mg protein were radiolabeled in 15 s. Phosphatidylinositol kinase activity, the enzyme that converts PI into PIP, was not affected by LH or dbcAMP treatment. However, incubation of luteal plasma membranes for 15 s with LH resulted in an increase of approximately 2 nmol 1,2-diacylglycerol/mg protein more than that observed in untreated or dbcAMP-treated plasma membranes. In summary, these experiments suggest that LH may stimulate hydrolysis of PI (and possibly PIP and PIP2) in isolated luteal plasma membranes.     

Effect of incubation of guinea-pig taenia coli in potassium-free media on arachidonate release and lipid metabolism

We studied the effects of immersion of guinea-pig taenia coli strips in potassium-free media on arachidonate stores and other lipid fractions. Control studies obtained with the strips in Krebs solution showed that greater than 97% of arachidonate was found esterified in phospholipid with the following distribution: phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol. 30 min incubation of the strips with [3H]arachidonate complexed to albumin resulted in incorporation of this isotope into phospholipid and neutral lipid fractions, phosphatidylcholine greater than neutral lipid greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. 30 min incubations with 32PO4(2-)-resulted in an isotope incorporation into phospholipids, phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. After 'loading' with [3H]arachidonate and 32P, placing the strips in potassium-free media caused the following: there was an increased release of [3H]arachidonate from the tissue into the bathing solution. [3H]Arachidonate and 32P radioactivity in phosphatidylinositol fell without a change in phosphatidylinositol content. [3H]Arachidonate and 32P radioactivity in other phospholipid fractions was unchanged. Arachidonate specific activity fell and arachidonate content increased in the phosphatidylserine plus phosphatidylinositol fraction. [3]Arachidonate in neutral lipid did not change significantly. We conclude that exposure of taenia coli to potassium-free media activates turnover of phosphatidylinositol, which results in release of arachidonate.     

Inhibition of hormone-stimulated adenylate cyclase activity after altering turkey erythrocyte phospholipid composition with a nonspecific lipid transfer protein. Phosphatidylinositol uncouples catecholamine binding from adenylate cyclase activation

The nonspecific lipid transfer protein from beef liver was used to modify the phospholipid composition of intact turkey erythrocytes in order to study the dependence of isoproterenol-stimulated adenylate cyclase activity on membrane phospholipid composition. Incorporation of phosphatidylinositol into turkey erythrocytes inhibited isoproterenol-stimulated cyclic AMP accumulation in a linear, concentration-dependent manner. Inhibition was relatively specific for phosphatidylinositol; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid were from 3 to 7 times less effective as inhibitors of hormone-stimulated cyclase activity. Inhibition by phosphatidylinositol was not reversible when up to 90% of the incorporated phosphatidylinositol was removed, either by incubation with phosphatidylinositol-specific phospholipase C or a second incubation with transfer protein; possibly adenylate cyclase activity depends on a small pool of phosphatidylinositol that is inaccessible to either phospholipase C hydrolysis or removal by lipid transfer protein. Phosphatidylinositol incorporation inhibits adenylate cyclase activity by uncoupling beta-adrenergic receptors from the remainder of the cyclase complex. Phosphatidylinositol incorporation had no effect on stimulation of cAMP accumulation by either cholera toxin or forskolin, indicating that inhibition occurs only at the level of receptor. Phosphodiesterase activity was not altered in phosphatidylinositol-modified cells. Inhibition of cAMP accumulation was not the result of changes in either membrane fluidity or in cAMP transport out of modified turkey erythrocytes. Phosphatidylinositol inhibition of isoproterenol-stimulated cyclase activity may serve as a useful model system for hormone-induced desensitization.     

Fatty acid release in incubations of serum with synaptosome and myelin subfractions of brain

To study lipid breakdown in brain membranes following hemorrhage, synaptosome and myelin fractions isolated from rat brain were incubated with rat serum. After 3 h in vitro at 37 degrees C, 0.43 and 0.26 mumol of fatty acid were released in incubations containing synaptosomes (1.37 mumols phospholipid) or myelin (1.23 mumols phospholipid), respectively, in the presence of 0.25 mL serum. Less than 0.05 mumol of fatty acid was liberated in incubations containing only serum, synaptosomes, or myelin. For synaptosomes and serum, docosahexaenoate was the principal fatty acid released (28 mol% of total) after 3 h of incubation. This fatty acid and arachidonate made up 43 mol% of the liberated fatty acid. The presence of free docosahexaenoate was of interest, as this fatty acid is particularly enriched in phosphatidylserine and phosphatidylethanolamine, phospholipids found in the cytoplasmic half of the synaptosomal plasma membrane and in interior synaptosomal membranes. In incubations of serum and myelin, oleate was the major free fatty acid produced in 30 min to 3 h of incubation (29-35 mol% of total). After 3 h, docosahexaenoate contributed 20 mol% to the total. The release of fatty acids from the membranes may be mediated by serum phospholipase(s) or possibly by activated endogenous lipolytic activities.     

Evidence for a Ca2+-independent association between calpain II and phospholipid vesicles

Possible interactions between calpain II and phospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylcholine were studied using fluorescence and gel filtration techniques. Changes in fluorescence intensity of purified calpain II show that the enzyme strongly interacts with phosphatidylinositol and phosphatidylserine and to a lesser extent with phosphatidylcholine. These results are corroborated by the gel filtration technique which permits the isolation of the enzyme phospholipid complex. Association between calpain II and various phospholipid vesicles can occur in the absence of calcium. Such binding occurs without any observable change of the molecular mass of the two subunits on SDS-polyacrylamide gel electrophoresis.     

Phospholipid composition of myelin and synaptosomal proteolipid from vertebrate brain

1. The phospholipid composition of the main proteolipid complexes of the nervous system was studied in myelin and synaptosomal membranes from brains of representatives of various vertebrate classes. 2. The relative content of acid phospholipids was much higher in proteolipid complexes from myelin and synaptosomal membranes of all vertebrates studied as compared to their content in the initial lipid extract (28-80% and 11-20% of total phospholipid content, respectively). 3. The relative content of acid phospholipids in proteolipid complexes of myelin membranes was much lower in brain of fishes and amphibia as compared to higher vertebrates. 4. The main acid phospholipids of proteolipid complexes was phosphatidylserine, phosphatidic acid being characteristic for myelin proteolipids and diphosphatidyl glycerol for synaptosomal proteolipids of all vertebrates studied.     

Artificial gravity and functional plasticity of nerve system. L-[14C]-glutamate uptake by nerve terminals from rat cerebellum and cerebral hemispheres under hypergravity stress

We have investigated the effects of altered gravity on the kinetic parameters of glutamate transport activity. We observed no differences in Km values for cerebellum and cerebral hemisphere nerve terminals (synaptosomes) between control rats- 18,2 +/- 7,6 micromoles (cerebellum), 10,7 +/- 2,5 micromoles (cerebral hemispheres) and animals exposed to hypergravity- 23,3 +/- 6,9 micromoles (cerebellum), 6,7 +/- 1,5 micromoles (cerebral hemispheres). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate in cerebellum and cerebral hemispheres was not sensitive to hypergravity stress. The maximal velocity of L-[14C]-glutamate uptake (Vmax) reduced for cerebellum synaptosomes from 9,6 +/- 3,9 nmol/min/mg of protein in control group to 7,4 +/- 2,0 nmol/min/mg of protein in animals, exposed to hypergravity stress. For cerebral hemisphere synaptosomes the maximal velocity significantly decreased from 12,5 +/- 3,2 nmol/min/mg of protein to 5,6 +/- 0,9 nmol/min/mg of protein, respectively.     

Acute starvation affects rat adrenal steroidogenesis

To determine how starvation affects adrenal steroidogenesis we measured the activities of 3 adrenal enzymes involved in corticosterone biosynthesis in a group of adult female rats. The animals were either starved for 7 days or fed ad libitum for the same period. Relative adrenal weight and plasma corticosterone levels were increased in the experimental group of animals compared to the control group (40 +/- 2 vs 27 +/- 1 mg/100 g body weight, P less than 0.001, and 45 +/- 4 vs 30 +/- 5 ng/dl, P less than 0.05 respectively). There were no differences in plasma ACTH levels between the groups (34 +/- 5 vs 26 +/- 4 pg/ml). 11-Hydroxylase activity was increased in the starved group of animals (18 +/- 3 vs 8 +/- 2 nmol/mg protein/min, P less than 0.01). 3 beta-Hydroxysteroid dehydrogenase and 21-hydroxylase activities were not different between the groups (19 +/- 2 vs 16 +/- 1 nmol/mg protein/min, and 100 +/- 10 vs 110 +/- 10 pmol/mg protein/min respectively). These results suggest that acute starvation in rats produces an increase in adrenal 11-hydroxylase activity.     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.