Higher environmental temperature-induced change in synaptosomal acetylcholinesterase activity of brain regions

Expcsure of adult male albino rats to higher environmental temperature (HET) at 35° for 2–12 hr or at 45° for 1–2 hr increases hypothalamic synaptosomal acetylcholinesterase (AChE) activity. Synaptosomal AChE activity in cerebral cortex of rats exposed to 35° for 12 hr and in cerebral cortex and pons-medulla of rats exposed to 45° for 1–2 hr are also activated. AChE activity of synaptosomes prepared from normal rat brain regions incubated in-vitro at 39° or 41° for 0.5 hr increases significantly in cerebral cortex and hypothalamus. The activation of AChE in ponsmedulla is also observed when this brain region is incubated at 41° for 0.5 hr. Increase of (a) the duration of incubation at 41° and (b) the incubation temperature to 43° under in-vitro condition decreases the synaptosomal AChE activity. Lioneweaver-Burk plots indicate that (a) in-vivo and invitro HET-induced increases of brain regional synaptosomal AChE activity are coupled with an increase ofV _max without any change inK _m (b) very high temperature (43° under in-vitro condition) causes a decrease inV _max with an increase inK _m of AChE activity irrespective of brain regions. Arrhenius plots show that there is a decrease in transition temperature in hypothalamus of rats exposed to either 35° or 45°; whereas such a decrease in transition temperature of the pons-medulla and cerebral cortex regions are observed only after exposure to 45°. These results suggests that heat exposure increases the lipid fluidity of synaptosomal membrane depending on the brain region which may expose the catalytic site of the enzyme (AChE) and hence activate the synaptosomal membrane bound AChE activity in brain regions. Further the in-vitro higher temperature (43°C)-induced inhibition of synaptosomal AChE activity irrespective of brain regions may be the cause iof partial proteolysis/disaggregation of AChE oligomers and/or solubilization of this membrane-bound enzyme.To whom to address reprint requests:     

Uptake and metabolism of GABA in astrocytes cultured from dissociated mouse brain hemispheres

Uptake kinetics and contents of GABA in cultured, normal (i.e. nontransformed) glia cells obtained from the brain hemispheres of newborn mice were measured together with the activity of the GABA transaminase. During three weeks of culturing the activity of the transaminase rose from a low neonatal value toward the level in the adult brain. The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics. Both theK _m (40 M) and theV _max (0.350 nmol×min^–1×mg^–1 cell protein) were reasonably comparable to the corresponding values in brain slices, and theV _max was much higher than that reported for other glial preparations. The GABA content was low (<5 nmol/mg cell protein), which is in agreement with the high activity of the GABA transaminase.     

Effect of external high potassium and pH on the uptake of choline in glial and neuronal cells in culture

TheV _max of the uptake of choline was increased in nerve cell cultures by lowering (from 7.4 to 6.5) or increasing (from 7.4 to 8.1) the pH. In neurons no effect was observed on the value of theK _m's of the uptake of either the apparent high or low affinity components. In glial cells only a low affinity component was measured at pH 6.5 and diffusion was observed at pH 8.1. An excess of K⁺ ions in the incubation medium reproduced the increase inV _max observed with changes in pH suggesting a possible dependence of the uptake of choline upon the H⁺ and OH^– gradients. Taking into account the characteristics already known of the transport of choline into nerve cells, such a dependence adds new insight in the mechanisms underlying the transport and indicates another possible regulation of choline entry, eventually directed towards the synthesis of acetylcholine.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Action of barbiturates on activity of acetylcholinesterase from synaptosomal membranes

The acetylcholinesterase from synaptosomal membranes is inhibited by anesthetics: Nembutal, brietal, and thiopental. Nembutal and brietal decrease theK _m for acetylthiocholine, without changes inV _max. A noncompetitive type of inhibition is produced by thiopental. This anesthetic decreases Arrhenius plot discontinuity by about 4°C and increases activation energies. Nembutal and brietal do not change Arrhenius plot discontinuities, but they increase activation energies. These results suggest that barbiturates change lipid-protein interactions in synaptosomal membranes.     

Some properties of choline acetyltransferase isolated from the electric organ ofTorpedo marmorata: Specificity for choline analogues and inhibition by certain amines and amino acids

(1) Choline acetyltransferase ofTorpedo marmorata electric organ was studied by using soluble tissue extracts partially purified by (NH₄)₂SO₄ fractionation. (2) Linear enzymatic rates were observed at 30°C, in the presence of 350 M acetyl-CoA and 50 mM choline, over a 30–40 min incubation period. (3) A number of analogues of choline, including mono-, di-, and triethylcholine and pyrrolcholine were synthesized and theK _m (apparent) andV (maximum velocity) values determined. TheK _m (apparent) for choline (11.5 mM), with theTorpedo enzyme, was high in comparison to values reported for mammalian or invertebrate nervous tissue. TheTorpedo enzyme was also not so specific for choline in comparison with the other choline analogues (based onK _m values) as were other sources of the enzyme. TheV values for choline and mono-, di-, and triethylcholine with theTorpedo enzyme indicated a direct relationship between enzyme activity andN-alkyl substitution. (4) Several amines and amino acids inhibited choline acetyltransferase fromTorpedo. Histamine (15 mM) brought about a 60% inhibition and was found to be a noncompetitive inhibitor with respect to choline.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

Changes in the alpha-adrenoceptors in the medulla oblongata including nucleus tractus solitarii of spontaneously hypertensive rats

The nucleus tractus solitarii (NTS) is a brain stem center mediating depression of blood pressure. In order to elucidate a possible mechanism for the central regulation of blood pressure, we studied noradrenergic indices in the medulla oblongata, a region including the NTS, in spontaneously hypertensive rats (SHR) as compared with normotensive controls of the Wistar Kyoto strain (WKY) at 12 weeks of age. The medulla oblongata was the only brain region showing a significantly low noradrenaline level in the SHR as compared with WKY rats; the level is also significantly decreased at 8 weeks of age. The alpha 1-adrenergic binding sites, as measured with 2-(2, 6-dimethoxy) phenoxyethylamine-methylbenzodioxan [³H]WB4101 showed significant increases inK _D andB _max values in medulla oblongata homogenates from rats of both strains from 4–12 weeks after birth, with no significant interstrain difference. On the other hand, theK _D andB _max of the alpha 2-sites, measured by [³H]yohimbine binding, were reduced in SHR as compared to WKY animals, even at 4 weeks after birth when hypertension was not yet apparent. As expected, the relatively selective alpha 2-antagonist, clonidine, was a potent inhibitor of [³H]yohimbine binding but not of [³H]WB4101 binding in these homogenates. The results suggest that some genetic disorder in the alpha 2-adrenergic transmission system in the NTS region may be involved in the development of hypertension in the SHR rats.Dedicated to Professor Yasuzo Tsukada.     

Effects of systematically administered lithium on tryptophan transport and exchange in plasma-membrane vesicles isolated from rat brain

The effect of lithium on the sodium-dependent high-affinity system for tryptophan uptake was examined in plasma membrane vesicles derived from rat brain. We demonstrated that Na⁺ could be replaced by lithium in the external medium and the presence of lithium produced an increase in theV _max of the tryptophan transport whereas it had no significant effect on theK _m for the substrate. Plasma membrane vesicles derived from synaptosomes obtained from long-term lithium-treated rats are able to accumulate tryptophan to a greater extent than normal rats and maintain a more negative membrane potential than controls. Our data support the idea that the stimulation by lithium of the high-affinity uptake system for tryptophan by maintaining adequate membrane potentials across the membrane, could lead to the stabilization of serotonin production, as has been demonstrated in long termlithium treatment.     

Nystatin affects zinc uptake in human fibroblasts

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 μg nystatin/mL or 0.1% dimethyl-sulfoxide for 10 min at 37°C. The cells were then incubated with 1 to 30 μM ⁶⁵zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced theK _m 56% and theV _max 69%. In the AE fibroblasts, nystatin treatment significantly reduced theV _max 59%, but did not significantly affect theK _m. The AE mutation alone affected theV _max for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction inV _max compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also effects zinc transport by reducing zinc transport.     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

Receptors for phorbol esters are primarily localized in neurons: Comparison of neuronal and glial cultures

Binding of [³H]PDB has been measured in the present study to determine the levels of protein kinase C in the neuronal and astrocytic glial cells in culture from rat brain. Binding of [³H]PDB to homogenates of cultured neuronal cells from the brains of normotensive and hypertensive rats was time-dependent and specific. The relative potency for competition by various phorbol esters to [³H]PDB binding was TPA > -PDD > POE > -PDD 4phorbol. Scatchard analysis showed that neuronal cultures from normotensive rat brains contained 2–3 fold more phorbol ester receptors compared with the glial cultures from the same brains. No differences in theK _d andB _max were observed between neuronal cultures from normotensive and spontaneously hypertensive rat brains. These studies suggest that the phorbol ester receptors are primarily localized in neuronal cells.     

Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in theputP gene affect the kinetics of proline transport

Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of threeputP substrate specificity mutants. One class of mutation increases theK _m over 120-fold but only decreases theV _max fourfold. SuchK _m mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV _max 80-fold without changing theK _m .V _max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both theK _m andV _max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na⁺/proline symport.     

The occurrence of analgesia in an animal model of hypertension

The relationship between blood pressure and pain sensitivity in a spontaneously hypertensive strain of rats (SH) was studied. Both analgesia and systolic blood pressure (SBP) were determined in SH and Wistar-Kyoto (WK) rats between 30 and 70 days of age. The SBP of the 30 day old SH rat was not significantly different from that of the WK rat (SH = 102±10 mmHg; WK = 93±12 mm Hg). Howeverm, the yooung SH rat exhibited considerably more analgesia than corresponding, age matched, WK rats. The SH rat jump latency was 45±10 seconds whereas the WK rat latency was 21±6 seconds. Both naloxone and atropine attenuated the analgesia of the SH rat but did not significantly alter the response of the WK rat. Methyl-alropine did not affect either the SH or WK rat response. These data suggest the presence of higher endorphin-like activity in the SH rat and a possible relationship between endogenous opioid pathways and the development of hypertension.     

Isotopic labeling of synaptosomes that accumulate choline and the effect of narcotic drugs

Subcellular studies of choline uptake of rat striatum indicated a correspondence between the Na⁺-dependent uptake and choline acetyltransferase (ChAc), whereas there was a lack of correspondence between the Na⁺-independent uptake and ChAc. Subcellular studies also showed a correspondence between the Na⁺-dependent uptake and hemicholinium-3 inhibition, and more important, particles that accumulate choline were shown to consist of at least two subcellular populations. A comparison was made of kinetic data from three areas of the rat brain: corpus striatum, cerebral cortex, and hypothalamus. Taken together, our data on choline uptake give added support to the idea that the Na⁺-dependent choline transport is concentrated in the striatum and specifically related to cholinergic nerve endings. Morphine and methadone in vitro inhibited the Na⁺-dependent choline uptake. In vivo morphine induced a significant lowering of theV _max in the rat cerebral cortex, but not in the striatum. This finding is consistent with the known action of morphine on acetylcholine turnover.Preliminary reports of this work were presented at the Fifth Meeting of the American Society for Neurochemistry in New Orleans, March 1974, and the Fall ASPET Meeting in Montreal, August 1974 (1,2).     

Choline and ethanolamine phosphotransferase activities in glomerular particles isolated from bovine cerebellar cortex

Isolated cerebellar glomeruli provide a relatively homogenous subcellular fraction, which can be used to study the biochemical events related to chemical transmission within a well-characterized central synapse. Choline and ethanolamine phosphotransferase activities were identified and partially characterized in this nerve ending preparation. Choline phosphotransferase associated with the glomerular particles required Mg²⁺, while ethanolamine phosphotransferase required Mn²⁺ for optimal activities. Both enzymes were inhibited by exogenous Ca²⁺. The apparent V_max values were 35.9 and 10.0 nmol/hr per mg protein for the choline and ethanolamine phosphotransferases, respectively. The apparentK _m value for the CDPcholine substrate was 28.6 M, and theK _m for CDPethanolamine was 8.3 M. Neither enzyme responded to the various adenine nucleotides, neurotransmitters or neurotransmitter agonists tested. However, exposure of the glomerular particles to cytidine nucleotides inhibited ethanolamine phosphotransferase activity and stimulated choline phosphotransferase activity.     

Effects of hemicholinium-3 and sodium ions on choline uptake system in excised superior cervical sympathetic ganglia of rats

Active choline uptake by rat superior cervical sympathetic ganglia (SCG), which contain abundant cholinergic nerve terminals, was studied with respect to sensitivity to inhibition by hemicholinium-3 (HC-3) and dependence on extracellular Na⁺ under standard conditions of assay. Choline was taken up by a single saturable process with apparentK _m=3.07×10^–5 M and Vmax=286 pmoles/min/mg protein. Neither denervation followed by degeneration of cholinergic nerve terminals nor axotomy with successive neuronal degeneration significantly decreased in choline uptake by the ganglia in vitro. HC-3 dose-dependently inhibited ganglionic choline uptake more effectively at lower than at higher choline concentrations. HC-3 sensitive inhibition of ganglionic choline uptake was not seen in young rats one week after birth but appeared with maturity, attaining approximately 50% maximal inhibition in adult SCG. Extent of inhibition by HC-3 and Na⁺ dependence of ganglionic choline uptake was not altered by denervation or axotomy.Abbreviations used (HC-3) hemicholinium-3 - (HAChU) high affinity choline uptake - (LAChU) low affinity choline uptake - (SCG) superior cervical ganglia - (Ch) choline - (ACh) acetylcholine     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

Amino acid transport: alterations due to synaptosomal depolarization

The effect of a preliminary high potassium depolarization on the synaptosomal transport of a number of compounds was tested. It was found that a preliminary depolarization increased the transport of glutamic acid, aspartic acid, glycine and choline, all of which are known or suspected neurotransmitters or neurotransmitter precursors. Depolarization had no effect on the transport of a number of other compounds including the neurotransmitter precursor, tyrosine. The increased transports of glutamate, aspartate, glycine and choline were found to be into osmotically sensitive compartments and were not due to decreases in leakiness of synaptosomes. Kinetically, the increase in choline transport was characterized by an increase in V_max and the increases in glutamate and glycine transport were characterized by decreases in K_T. Thus, selected transport systems in synaptosomal preparations can be altered by a previous depolarization.