Bioassayable cholecystokinin in the brain of the goldfish, Carassius auratus.

Cholecystokinin (CCK)-gastrin peptides are shown to be present in the brain of all the species ranging from coelenterates to mammals. Differentiation between CCK and gastrin, presumed to be evolved from a common ancestral cerulein-like peptide, has been suggested to occur at the level of the amphibians. We examined the presence of bioassayable CCK, as determined by its potency to stimulate enzyme secretion from isolated rat pancreatic acini, in the brain of the goldfish Carassius auratus, a more primitive vertebrate than the amphibian. Among the various regions tested, the brain stem, telencephalon and spinal cord possess the highest levels of bioassayable CCK followed in decreasing order by gustatory lobes, optic tectum and duodenum. No detectable levels of CCK were found in the cerebellum. The observed bioactivity was not due to gastrin because: radioimmunoassay of the brain homogenates for gastrin revealed very low or nondetectable levels of gastrin; amylase release dose-response curves for standard CCK8 and the brain homogenate were identical; and proglumide, a competitive antagonist of CCK8, inhibited homogenate CCK-induced enzyme release with a parallel rightward shift in the dose-response curve. These observations provide evidence for the distinct presence of CCK in the brain of the goldfish suggesting that the differentiation of CCK as a distinct neuropeptide, from that of gastrin, occurs at the level of Osteichthyes (bony fish).

Interaction of enkephalins and des-tyrosyl-enkephalins with synaptosomal plasma membrane vesicles: enkephalin binding and inhibition of proline transport.

Leucine- and methionine-enkephalins inhibit the Na+-dependent transport of proline into plasma membrane vesicles derived from synaptosomes. Glycine transport is weakly inhibited by enkephalins whereas there is no inhibition of transport of glutamic acid, aspartic acid, or gamma-aminobutyric acid. The inhibition of proline uptake is observed with des-tyrosyl-enkephalins but not with morphine, dynorphin(1-13), or beta-endorphins. Furthermore, enkephalin-induced inhibition of proline transport is not antagonized by naloxone. [Leu]enkephalinamide and modified [Leu]enkephalins with greater selectivity for the delta-subclass of enkephalin binding sites are less effective than [Leu]enkephalin in the inhibition of proline transport. Specific binding of [3H]Leu-enkephalin to the plasma membrane vesicles is demonstrated, and des-Tyr-[Leu]enkephalin competes with Leu-enkephalin for [Leu]enkephalin binding sites. The similarity in the concentrations of des-Tyr-[Leu]enkephalin required to compete for specific [Leu]enkephalin binding and to inhibit proline transport suggests that a specific subclass of enkephalin binding sites, distinguished by their recognition of both the enkephalins and their des-tyrosyl derivatives, may be associated with the synaptic proline transport system.

Partial purification of molecular weight 12 000 fatty acid binding proteins from rat brain and their effect on synaptosomal Na+-dependent amino acid uptake.

High-affinity, Na+-dependent synaptosomal amino acid uptake systems are strongly stimulated by proteins which are known to bind fatty acids, including the Mr 12 000 fatty acid binding protein (FABP) from liver. To explore the possibility that such a function might be served by fatty acid binding proteins intrinsic to brain, we examined the 105000g supernatant of brain for fatty acid binding. Observed binding was accounted for mainly by components excluded by Sephadex G-50, and to a small degree by the Mr 12 000 protein fraction (brain FABP fraction). The partially purified brain FABP fraction contained a protein immunologically identical with liver FABP as well as a FABP electrophoretically distinct from liver FABP. Brain FABP fraction markedly stimulated synaptosomal Na+-dependent, but not Na+-independent, amino acid uptake, and also completely reversed the inhibition of synaptosomal Na+-dependent amino acid uptake induced by oleic acid. Palmitic, stearic, and oleic acids were endogenously associated with the brain FABP fraction. These data are consistent with the hypothesis that Mr 12 000 soluble FABPs intrinsic to brain may act as regulators of synaptosomal Na+-dependent amino acid uptake by sequestering free fatty acids which inhibit this process.

Stimulation of synaptosomal uptake of neurotransmitter amino acids by insulin: possible role of insulin as a neuromodulator.

Insulin stimulates in a dose-dependent manner (concentration range of 0.1 - 10 microM) the synaptosomal uptake of amino acids characterized by high-affinity, Na+-dependent, veratridine-sensitive transport systems. This stimulation is observed in synaptosomes prepared from each of several regions of the adult rat brain. Both the initial rate of amino acid uptake and the overall capacity for amino acid accumulation are increased. Since these transport systems have been associated with the neurotransmitter role of the amino acids, we postulate that insulin can modulate neurotransmission in the rat central nervous system by increasing the efficiency of neuroactive amino acid reuptake.

Iminoglycine transport system in synaptosomes and its interaction with enkephalins.

Evidence is presented which suggests that proline, pipecolic acid, and glycine are accumulated by a common transport system in rat brain cortical synaptosomes and synaptosomal plasma membrane vesicles. This system is Na+ dependent and appears to be similar to the iminoglycine transport system present in renal tubules and in renal brush border membranes. The opioid pentapeptides Leu- and Met-enkephalin specifically inhibit the uptake of these three imino/amino acids, presumably by interaction with a nonopioid receptor, since the inhibition is not affected by the opiate antagonist naloxone and occurs with des-tyrosyl enkephalins as well as with the intact pentapeptides.

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.

Release of neurotransmitter amino acids from synaptosomes: enhancement of calcium-independent efflux by oleic and arachidonic acids.

The release of preloaded [14C]neuroactive amino acids (glutamic acid, proline, gamma-aminobutyric acid) from rat brain synaptosomes can occur via a time-dependent, Ca2+-independent process. This Ca2+-independent efflux is increased by compounds that activate Na+ channels (veratridine, scorpion venoms), by the ionophore gramicidin D, and by low concentrations of unsaturated fatty acids (oleic acid and arachidonic acid). Saturated fatty acids have no effect on the efflux process. Neither saturated nor unsaturated fatty acids have an effect on the release of [14C]leucine, an amino acid not known to possess neurotransmitter properties. The increase in the efflux of neuroactive amino acids by oleic and arachidonic acids can also be demonstrated using synaptosomal membrane vesicles. Under conditions in which unsaturated free fatty acids enhance amino acid efflux, no effect on 22Na+ permeability is observed. Since Na+ permeability is not altered by fatty acids, the synaptosomes are not depolarized in their presence and, thus, the Na+ gradient can be assumed to be undisturbed. We conclude that unsaturated fatty acids represent a potentially important class of endogenous modulators of neuroactive amino acid transport in nerve endings and further postulate that their action is the result of an uncoupling of amino acid transport from the synaptosomal Na+ gradient.

Presynaptic tyrosine availability in the phenylketonuric brain: a hypothetical evaluation.

From measured effects of amino acids on synaptosomal tyrosine uptake and from published data on human CNS levels of amino acids hypothetical calculations were made to compare CNS tyrosine availability for catecholamine synthesis in the hyperphenylalaninemic and non-hyperphenylalaninemic condition. These calculations indicate an approximately two-fold reduction in the availability of tyrosine in phenylketonuria that is due solely to a reduction in CNS tyrosine alone.

Modulation of membrane transport by free fatty acids: inhibition of synaptosomal sodium-dependent amino acid uptake.

High-affinity, Na+-dependent synaptosomal amino acid uptake systems are strongly stimulated by proteins which are known to bind free fatty acids. The rate of uptake as well as the overall level of accumulation is increased by such proteins as bovine serum albumin, hepatic fatty acid binding protein, beta-lactoglobulin, and fetuin. Such a stimulation is not observed with proteins which do not bind fatty acids. The transport activity of synaptosomal preparations can be directly correlated with the free fatty acid content of the preparation. Thus, incubation with albumin reduces the free fatty acid content of synaptosomal preparations, suggesting that the stimulatory effects of the proteins are related to their removal of inhibitory fatty acids formed by hydrolysis of membrane lipids during incubation. Inhibition of amino acid uptake is seen with most cis-unsaturated long chain fatty acids while saturated and trans-unsaturated fatty acids have relatively little or no effect. Under conditions in which the ionophore gramicidin D causes an increase of 22Na flux into synaptosomes, oleic acid (50 microM) has no effect on the influx. These data are consistent with the hypothesis proposed earlier by us [Rhoads, D. E., Peterson, N. A., & Raghupathy, E. (1982) Biochemistry 21, 4782] that Na+-dependent amino acid transport carrier proteins reside in a relatively fluid lipid domain in the synaptosomal membrane and that the effects of cis-unsaturated fatty acids are mediated by interactions with such domains.

Action of the venom of the scorpion Tityus trinitatis on pancreatic insulin secretion.

In vivo, canine pancreas was stimulated to secrete insulin by the venom of the scorpion Tityus trinitatis; the venom also caused a rise in plasma glucose level as well. The venom-induced insulin secretion was also observed under in vitro conditions in rat pancreatic slices, and this stimulation was dose-related. Maximal effect was observed at 20 micrograms/ml. Atropine (3 x 10(-6) M) completely abolished both venom (20 micrograms/ml)- and acetylcholine (3 x 10(-7) M)-induced insulin secretion. It is suggested that the venom-induced insulin secretion is mediated through muscarinic cholinergic mechanisms.

Proline transport by synaptosomal membrane vesicles isolated from rat brain: energetics and inhibition by free fatty acids.

Synaptosomal membrane vesicles have been employed to study the energetics of proline transport and the inhibition of proline transport by unsaturated free fatty acids. Active uptake of proline into synaptosomal membrane vesicles requires extravesicular Na+ and is primarily driven by a Na+ gradient created by diluting K+-loaded vesicles into Na+-containing buffers. Uptake of proline under these conditions is enhanced up to 2-fold by a valinomycin-induced diffusion potential (interior negative). Proline transport is reduced in the absence of external C1- or internal K+. Strong (40-90%) inhibition of proline uptake occurs upon collapse of the Na+ gradient by ionophores such as gramicidin D or activation of the action potential Na+ channel by veratridine or Tityus serrulatus venom. Less (15-25%) inhibition is obtained with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, which also prevents the stimulation of proline uptake by the valinomycin-induced diffusion potential. Unsaturated free fatty acids inhibit proline uptake. The inhibition is greatest for arachidonic acid and was somewhat less for oleic acid. The saturated fatty acids palmitic and stearic have little or no inhibitory capacity. Endogenous unsaturated free fatty acids may exert similar inhibitory effects on the reuptake systems for neuroactive amino acids and thus modulate their action in the central nervous system.

Inhibitory effects of scorpion venom on the uptake of amino acids by synaptosomes and synaptosomal membrane vesicles.

Scorpion (Tityus serrulatus) venom strongly inhibited the Na+-dependent uptake of (( 14C ))proline by rat brain synaptosomal preparations. In addition, the efflux of proline was enhanced markedly by scorpion venom. The inhibitory effects of the venom were also demonstrated in synaptosomal vesicle preparations where proline uptake was energized by an artificially imposed Na+ gradient. In both preparations, the effect of scorpion venom was additive with the inhibitory effect of veratridine on Na+-dependent amino acid uptake. The inhibitory effects of both compounds were abolished by tetrodotoxin. The Na+-dependent uptakes of amino acids (e.g. proline, glutamic acid, and gamma-aminobutyric acid) were much more sensitive to inhibition by the toxin than the Na+-independent uptakes (e.g. leucine and phenylalanine). The results of the present study indicate that the scorpion venom may exert its inhibitory effect on Na+-dependent transport by decreasing the transmembrane Na+ gradient. Efflux of accumulated proline, which is presumably controlled by maintenance of this Na+ gradient, was stimulated 3- to 4-fold by the scorpion venom.

Effects of free fatty acids on synaptosomal amino acid uptake systems.

The Na+-dependent synaptosomal uptakes of proline, aspartic acid, glutamic acid, and gamma-aminobutyric acid were strong inhibited by monounsaturated fatty acids. With oleic acid, half-maximal inhibition was observed at about 15 microM. 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.

Stimulation of Na+-dependent, veratridine-sensitive synaptosomal amino acid uptakes by serum albumin.

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 gamma-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.