Selective inhibition of synaptosomal proline uptake by leucine and methionine enkephalins

The high affinity, sodium-dependent uptake of proline by rat brain synaptosomes was inhibited by the opioid pentapeptides, Leu-enkephalin and Met-enkephalin. The synaptosomal uptake of other putative neurotransmitter amino acids including glutamic acid, aspartic acid, gamma-aminobutyric acid, and taurine was not altered in the presence of enkephalins. The uptake of a neuroinactive amino acid, leucine, was also unaffected by enkephalins. The extent of proline uptake was half-maximal at a Leu-enkephalin concentration of 1 microM. Both the initial rate of transport and the overall capacity for proline accumulation were reduced. The effect of the enkephalins was vectorial since carrier-mediated efflux of proline was not altered in the presence of enkephalins. Morphine and the opioid peptides, dynorphin and beta-endorphin, were without effect on proline uptake. The inhibition of proline uptake by enkephalins was not diminished by prior incubation of the synaptosomal preparation with naloxone; however, the inhibition was attenuated by 1-butanol. The des-tyrosyl fragments of the enkephalins were as inhibitory as the intact pentapeptides. A modified enkephalin ([D-Ser2]Leu-enkephalin-Thr) with selective affinity for the delta subclass of enkephalin receptor was effective in inhibiting proline uptake. On the basis of the selectivity of these effects, we propose that there is a specific population of nerve endings in the cerebral cortex that contains both a proline-transport system and binding sites for Leu- and Met-enkephalin and furthermore, that these binding sites may be related to the putative delta receptor.     

Stimulation of Na+-Dependent, Veratridine-Sensitive Synaptosomal Amino Acid Uptakes by Serum Albumin

Abstract: Bovine serum albumin (BSA) is shown to stimulate selectively the synaptosomal uptakes of those amino acids that are dependent on external Na⁺ and that are inhibited by veratridine. Thus, the stimulation can be seen in the case of aspartic acid, glutamic acid, glycine, proline, and γ-aminobutyric acid, but not with serine and threonine. Further, studies on the interaction of veratridine, valinomycin, and BSA on the uptake of proline suggest that the primary action of the albumin is to increase the influx of proline. Such an action could result as a consequence of stabilization of the Na⁺ gradient by increased endogenous levels of ATP. Intrasynaptosomal ATP was increased in the presence of BSA but significantly decreased by veratridine.     

The effects of benzyladenine, cycloheximide, and cordycepin on wilting-induced abscisic Acid and proline accumulations and abscisic Acid- and salt-induced proline accumulation in barley leaves

Benzyladenine inhibits proline accumulation in wilted, abscisic acid (ABA)-treated, and salt-shocked barley leaves. It does not affect ABA accumulation or disappearance in wilted leaves. Inhibition of proline accumulation in salt-shocked leaves was observed both when benzyladenine was added at the beginning of or after salt treatment. Cycloheximide (CHX) and cordycepin inhibited both ABA and proline accumulations in wilted barley leaves and proline accumulation in ABA-treated leaves. In salt-shocked leaves, cordycepin inhibited proline accumulation when added after salt treatment but before proline began to accumulate but not when added after the onset of proline accumulation. CHX delayed the accumulation of proline in salt-shocked leaves but, after a period of time, proline accumulated in the CHX-treated leaves at rates comparable to the salt-treated control. This delay and subsequent accumulation was observed when CHX was added before, during, and after salt treatment. However, the earlier in the salt treatment period that CHX was given, the longer was the observed delay. These results are interpreted to indicate that gene activation is involved in proline accumulation in response to wilting, to ABA, and to salt in barley leaves. This gene activation is in addition to the gene activation that is required for ABA accumulation in wilted leaves. If ABA accumulation is required for proline accumulation in wilted barley leaves, then two sets of gene activation are involved in wilting-induced proline accumulation. All of our results are consistent with this possibility but do not prove it. The inhibition of proline accumulation by benzyladenine is probably neither due to an effect on gene activation nor to an effect on the ABA level.     

Effects of Free Fatty Acids on Synaptosomal Amino Acid Uptake Systems

Abstract: The Na⁺-dependent synaptosomal uptakes of proline, aspartic acid, glutamic acid, and γ-aminobutyric acid were strongly inhibited by monounsaturated fatty acids. With oleic acid, half-maximal inhibition was observed at about 15 μM. The Na⁺-independent uptakes of leucine, phenylalanine, histidine, and valine were less sensitive to inhibition by the unsaturated fatty acids. In contrast, the uptakes of all of these amino acids were unaffected by saturated fatty acids. The inhibition of proline uptake (and that of the other Na⁺-dependent amino acids) by oleic acid was overcome by the addition of serum albumin and the data presented further indicate that the previously reported stimulation of proline uptake by albumin could be related to its fatty acid binding properties.     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

Inactivation by detergents of the proline transport system in membrane vesicles from Escherichia coli and its reactivation by bovine serum albumin

The proline transport system of membrane vesicles from Escherichia coli was inactivated by a low concentration of detergents such as deoxycholate, dodecyl sulfate and Triton X-100. The addition of a large amount of bovine serum albumin to membrane vesicles which had been treated with one of these detergents resulted in the restoration of the proline transport activity. The restoration of the transport activity by bovine serum albumin was most remarkable with the deoxycholate-inactivated membrane vesicle. 80% inactivation of the transport system with 0.005% deoxycholate was completely overcome by the addition of albumin. The degree of restoration was dependent on the concentration of albumin. Although albumin stimulated the proline transport activity itself, the stimulatory effect could not account for the restoration transport activity. The binding of deoxy[¹⁴C]cholate to the membrane vesicle was roughly proportional to the amount of detergent added. Deoxycholate once bound to the membrane vesicle was removed almost completely by the incubation with albumin. It is concluded that the removal of detergent from the membrane vesicle by bovine serum albumin results in the restoration of the proline transportactivity.     

Effect of verapamil on CRF-induced abnormalities in phospholipid contents of brain synaptosomes

Chronic renal failure is associated with significant reductions in total phospholipids, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine of brain synaptosomes. These derangements in synaptosomal phospholipid metabolism were attributed to the state of secondary hyperparathyroidism of chronic renal failure (CRF) and the parathyroid hormone-induced accumulation of calcium in synaptosomes. This study examined whether a calcium channel blocker, verapamil, would prevent this synaptosomal calcium accumulation and correct the abnormalities in synaptosomal phospholipids in CRF. Verapamil treatment of normal rats for 21 days did not affect synaptosomal content of calcium or phospholipids. CRF of 21 days' duration was associated with a significant (P less than 0.01) increase in synaptosomal calcium (10.2 +/- 0.5 vs 7.4 +/- 0.6 nmol/mg protein) and a significant reduction (P less than 0.01) in total phospholipids (397 +/- 12 vs 529 +/- 19 nmol phospholipid P/mg protein), phosphatidylinositol (2.7 +/- 0.22 vs 4.6 +/- 0.27 nmol phospholipid P/mg protein), and phosphatidylserine (37 +/- 1.9 vs 83 +/- 5.2 nmol phospholipid P/mg protein). Simultaneous treatment of CRF rats with verapamil for 21 days reversed the synaptosomal abnormalities in calcium and phospholipid contents. Our data support the notion that the effect of excess parathyroid hormone of CRF on synaptosomal phospholipids is mainly due to the parathyroid hormone-induced calcium accumulation.     

Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.     

Senescence of Rice Leaves : VII. PROLINE ACCUMULATION IN SENESCING EXCISED LEAVES

Proline content increased greatly in detached rice (Oryza sativa cv. Taichung Native 1) leaves during senescence. There was a slight but significant increase in proline level after one day of incubation, and, subsequently, proline accumulated relatively rapidly. By 4 days after excision, the level of proline had increased 30- to 50-fold, which is similar to the level seen in the water-stressed detached rice leaves. It is unlikely that the proline accumulation in detached leaves is to be derived solely from protein hydrolysis, since the addition of l-glutamic acid increased the proline level during senescence. The proline analog, 3,4-dehydroproline, did not affect the level of proline during senescence. It seems that accumulation of proline may, at least in part, result from an increased rate of synthesis, possibly due to a disruption of the normal feedback inhibition of proline synthesis. Potassium cyanide and 2,4-dinitrophenol strongly inhibited proline accumulation, indicating that some energy compound(s) may participate in proline accumulation during senescence of excised rice leaves.     

Removal by bovine serum albumin of fatty acids from membrane vesicles and its effect on proline transport activity in Escherichia coli.

Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.     

The effects of L-2,4-diaminobutyric acid on the uptake of gamma-aminobutyric acid by a synaptosomal fraction from rat brain

l-2,4-diaminobutyric acid was studied as an inhibitor of gamma-aminobutyric acid uptake by a synaptosomal fraction isolated from rat brain. Competitive inhibition was observed during short-term exposure of the synaptosomal fraction to the inhibitor but noncompetitive inhibition was observed following prolonged exposure. Studies on the mode of action of l-2,4-diaminobutyric acid showed that the synaptosomal fraction was capable of accumulating this compound and that both the uptake and the effectiveness of the inhibitor were sodium-dependent and temperature-sensitive. In addition, the degree of inhibition of gamma-aminobutyric acid uptake was related to the amount of l-2,4-diaminobutyric acid accumulated. It is suggested that the observed noncompetitive inhibition of gamma-aminobutyric acid uptake by l-2,4-diaminobutyric acid is a result of the accumulation of the inhibitor which exerts its effect from within the synaptosomes. Raising the external concentration of gamma-aminobutyric acid to saturating levels did not completely inhibit the accumulation of l-2,4-diaminobutyric acid. Thus, the transport of l-2,4-diaminobutyric acid appears to be mediated, at least in part, by a carrier which is not involved in the transport of gamma-amiuobutyric acid.     

The Requirement for Low-Intensity Light in the Accumulation of Proline as a Response to Water Deficit

The effect of light on proline accumulation has been examinedin excised segments from the first leaf of barley (cv. Prior)that were subjected to water deficit by exposure to polyethyleneglycol solutions. Illumination either of the intact plant priorto leaf excision or of the leaf segments after excision promotedproline accumulation. This promotion was not due to a changein tissue water potential and the response was saturated ata low irradiance (15–75 µE m ²s ¹ depending uponprevious illumination of the intact plant). Saturation was notdue to a finite capacity of the leaf segments to accumulateproline, but was apparently associated with a finite rate ofaccumulation. The stimulation of proline accumulation by lightwas apparent at all levels of external water potential imposedbut the magnitude of accumulation increased as water potentialwas decreased both in light and darkness. Proline. or its precursor,was readily translocated within the leaf, since leaves thatwere partially shaded whilst subjected to water deficit containedthe same proline concentration throughout. It is concluded thatproline accumulation is not evoked by light through a triggeringmechanism but rather is stimulated by the continuous presenceof light. Key words: Light intensity, Barley, Water deficit, Proline accumulation     

Effect of Bilirubin on the Membrane Potential of Rat Brain Synaptosomes

The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin-induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2-14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.     

STRUCTURAL REQUIREMENTS FOR AMINO ACID INHIBITION OF Na+-DEPENDENT PROLINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The structural requirements for amino acid inhibition of Na⁺-dependent proline uptake by rat brain synaptosomal fractions were investigated. It is shown that the amino group has to be in the α-position to strongly inhibit proline uptake. Hydroxyamino acids are less potent inhibitors than the parent amino acids. Amino acids with net positive or negative charges on their molecules exert no effect, whereas elimination of the net charge results in compounds with profound inhibitory effects. Blocking of the carboxyl group reduces the inhibition, but does not abolish it. Since acetylation of the α-amino group results in elimination of the inhibitory effect whereas N -methylation does not, it is concluded that in the interaction of an amino acid with the proline transport site the positive charge on the amino group plays the most critical role.     

Proline accumulation induced by excess nickel in detached rice leaves

The regulation of proline accumulation in detached rice leaves exposed to excess NiSO₄ was investigated. NiSO₄ treatment increased proline and Ni contents but had no effect on relative water content, indicating that proline accumulation in Ni-exposed detached rice leaves was due to Ni uptake per se, rather than to water stress. Proline accumulation caused by NiSO₄ was related to protein hydrolysis, a decrease in proline dehydrogenase activity, and a decrease in proline utilization. It seems that an increase in the content of ammonia and an increase in the activities of Δ¹-pyrroline-5-carboxylate reductase and ornithine-δ-aminotransferase play minor if any role in Ni-induced proline accumulation in detached rice leaves.     

Osmotic stress and water stress have opposite effects on putrescine and proline production in excised rice leaves

The effects of water stress and osmotic stress (sorbitol treatment) on the production of putrescine and proline in excised rice leaves were compared. Osmotic stress and water stress were found to affect differentially the levels of putrescine and proline in excised rice leaves. Putrescine accumulation is induced by osmotic stress, whereas proline accumulation is induced by water stress. The effects of ABA on the levels of proline and putrescine are similar to those of water stress, whereas the effects of jasmonic acid methyl ester (JA-Me) are similar to those of osmotic stress. Water stress results in an increase of endogenous ABA is excised rice leaves. However, neither osmotic stress nor JA-Me has effect on endogenous ABA levels in excised rice leaves. Of particular interest is the finding that proline levels increase when putrescine levels induced by osmotic stress or JA-Me are reduced by D-arginine and -methylornithine. L-arginine and L-ornithine applied exogenously also cause an increase in proline levels. It seems that L-arginine and L-ornithine are preferentially utilized as precursors for putrescine accumulation in excised rice leaves treated with osmotic stress and JA-Me, and for proline accumulation in excised rice leaves exposed to water stress and ABA.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - HPLC high performance chromatography - JA-Me jasmonic acid methyl ester - PVP poly-vinylpyrrolidone     

The effect of NaCl on proline accumulation in rice leaves

The regulation of proline accumulation in detached leaves of rice(Oryza sativa cv. Taichung Native 1) was investigated.Increasing concentrations of NaCl from 50 to 200 mM progressivelyincreased proline content in detached rice leaves. NaCl induced prolineaccumulation was mainly due to the effect of both Na⁺ andCl^– ions. Proline accumulation caused by NaCl was related toprotein proteolysis, an increase in ornithine--aminotransferaseactivity,a decrease in proline dehydrogenase activity, a decrease in prolineutilisation,and an increase in the content of the precursors of proline biosynthesis,ornithine and arginine. Results also show that proline accumulation caused byNaCl was associated with ammonium ion accumulation.     

Serum Albumin Washing Specifically Enhances Arachidonate Incorporation into Synaptosomal Phosphatidylinositols

Arachidonate incorporation into synaptosomal phospholipids was shown to be affected by factors including the procedure for preparation of the membrane fractions and preincubation of synaptosomes prior to assay of incorporation of arachidonate into both phosphatidylcholine (PC) and phosphatidylinositol (PI). However, the inhibition toward incorporation into PIs, but not PCs, was fully reversed when the membranes were washed with bovine serum albumin. A twofold increase in arachidonate incorporation into PIs was also observed when freshly prepared synaptosomes were washed with serum albumin immediately before assay of incorporation activity. The inhibitory action is thought to be due to an increase in polyunsaturated fatty acids and/or their oxidation products which may then elicit a special effect on the acyltransferase responsible for transferring arachidonate into phosphatidylinositols. The differences in fatty acid uptake and response to serum albumin also suggest the presence of different acyltransferase for acyl transfer to PIs and PCs.     

Characteristics of the Induction of the Accumulation of Proline by Abscisic Acid and Isobutyric Acid in Detached Rice Leaves

The effects of abscisic acid and isobutyric acid on levels ofproline in detached rice leaves were compared. The lowest concentrationof abscisic acid that induces accumulation of proline in detachedrice leaves was much lower than that of isobutyric acid. Theaccumulation of porline induced by isobutyric acid was associatedwith acidification of the cell sap, whereas abscisic acid increasedlevels of proline without decreasing the pH of the cell sap.Potassium chloride enhanced the accumulation of proline thatwas induced by abscisic acid, but it did not stimulate thatinduced by isobutyric acid. Of particular interest is the findingthat detached rice leaves treated with fusicoccin showed anincrease in levels of proline and a decrease in the pH of thecell sap. A synergistic increase in levels of proline was observedwhen fusicoccin and abscisic acid applied simultaneously. However,fusicoccin had no effect on the induction of the accumulationof pro-line by isobutyric acid. Benzyladenine inhibited theinduction by abscisic acid of the accumulation of proline, butit did not inhibit the induction by isobutyric acid. It is concludedthat the mode of action of abscisic acid in inducing the accumulationof proline differs from that of isobutyric acid. Our resultsalso indicate that factors other than acidification of the cellsap are involved in regulation of the accumulation of proline. (Received September 25, 1990; Accepted December 20, 1990)     

Triphasic effects of short chain n-alcohols on synaptic membrane transport of choline and of gamma-aminobutyric acid

n-Alcohols, when added in increasing concentrations, had an unusual triphasic effect on the uptake of choline and of gamma-aminobutyric acid by isolated synaptosomes. There was slight inhibition of these uptakes at low n-alcohol concentrations, followed by a sharp peak of uptake enhancement, and then greater inhibition. The n-alcohol concentrations required for these effects were proportional to published n-alcohol membrane/buffer partition coefficients, with the peaks of uptake enhancement occurring at 60 mM n-propanol, 20 mM n-butanol and 7.5 mM n-pentanol. Synaptosomal membrane potential, as estimated from synaptosomal accumulation of the permeant cation [3H]tetraphenylphosphonium, was not affected by n-alcohols in the concentrations used in this study, suggesting that neither the inhibitory or enhancing effects of these n-alcohols were attributable to changes in trans-synaptosomal membrane ion gradients. The inhibiting and enhancing effects of n-alcohols could be reproduced in determinations of gamma-aminobutyric acid uptake by isolated synaptic plasma membranes, suggesting that the observed effects are due to a direct action of the n-alcohols on the synaptosomal plasma membrane. These effects may be attributable to a change in membrane binding of these alcohols from the membrane core to the membrane surface as alcohol concentration is increased.