Neomycin inhibits K+- and veratridine-stimulated noradrenaline release in rat brain slices and rat brain synaptosomes

The possible involvement of phosphoinositides' turnover in the process of neurotransmitter release in the central nervous system (CNS) was studied using rat brain slices and synaptosomes. A depolarizing concentration of potassium chloride (25 mM) induces an 8.6 +/- 0.4% increase of [3H]noradrenaline [( 3H]NA) fractional release in cerebral cortical slices above spontaneous release, and 15 mM KCl induces a 3-fold increase of [3H]NA release in rat brain synaptosomes. Neomycin, an aminoglycoside which binds phosphoinositides, inhibits the potassium-induced release in cortical slices with an IC50 = 0.5 +/- 0.07 mM and with IC50 = 0.2 +/- 0.03 mM in synaptosomes. Veratridine, a veratrum alkaloid which increases membrane permeability to sodium ions and causes depolarization of neuronal cells, induces a net 13.4 +/- 0.3% increase of [3H]NA fractional release above spontaneous release in cortical slices. In analogy to K+ stimulation, neomycin inhibits the veratridine-stimulated release in cortical slices with an IC50 = 0.65 +/- 0.1 mM. It appears that the recycling of phosphoinositides, which is necessary for Ca2+ mobilization, participates in the Ca2+-dependent induced neurotransmitter release in the central nervous system.     

Effects of cocaine on sodium dependent dopamine uptake in rat striatal synaptosomes

Initial velocity of uptake of dopamine (DA) has been measured in the presence of 1M cocaine as a function of both [DA] and [Na]. Although DA uptake is overwhelmingly dependent on sodium, it appears that a small amount of DA uptake takes place in the absence of sodium. This contrasts with a previous study of the sodium dependence of uptake without cocaine (referred to below as control), in which uptake was found to be 100% sodium dependent. The data were fitted to several rapid equilibrium models and the minimal best fit model identified. The interaction of transporter (C), DA (S), and Na⁺ (Na) in this present model is identical to the reaction scheme found previously to fit control data (no cocaine). Whereas the control model required translocation only as CNa₂S, in the presence of cocaine (I), two additional translocated species are required to fit the data (CS and CNaS). Another previous study of the interaction of carrier and cocaine at a constant [Na]₀ predicted that cocaine interacts with a transporter site other than the DA binding site and that uptake takes place as CS and CSI. The present results are consistent with the assumption that the CS and CNaS forms of the present model are actually CSI and CNaSI, since they are required to fit a model of the sodium dependence in the presence of cocaine, but are not required in the absence of cocaine.     

Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle

This report demonstrates that the commonly used anesthetic agent, pentobarbital sodium, in concentrations of 1 · 10^?4 to 2 · 10^?3 M inhibits calcium (Ca²⁺) uptake in both rat aortic and portal venous smooth muscle. The data indicate that total exchangeable Ca²⁺ in portal vein is reduced by about 15% in 1 · 10^?4 M pentobarbital sodium, while the intracellular exchangeable Ca²⁺ is reduced by 24%. On the other hand, in aortic smooth muscle, while 5–20 · 10^?4 M pentobarbital sodium reduces total exchangeable Ca²⁺ by about 15%, intracellular Ca²⁺ is reduced by 22% in 5 · 10^?4 M pentobarbital sodium and by 38% in 2 · 10^?3 M pentobarbital sodium. The present studies thus reveal that concentrations of pentobarbital sodium known to be present during induction of surgical anesthesia can exert significant inhibitory effects on exchangeability and transmembrane movement of Ca²⁺ in at least two different types of blood vessels.     

Preincubation of synaptosomes in the presence of sodium affects Ca2+ uptake

Preincubation of synaptosomes in standard physiological medium stimulates 2-fold Ca²⁺ uptake as compared to non-preincubated synaptosomes. When the sodium concentration in the preincubation medium has been halved, Ca²⁺ uptake was reduced by approximately 50 percent. The addition of ouabain to the preincubation medium decreases depolarization-stimulated Ca²⁺ uptake by about 40 percent. A steady-state level of Ca²⁺ uptake is achieved by synaptosomes preincubated for 0,5 or 10 min. These findings suggest that Ca²⁺ uptake might depend on the Na-gradient formed during the preincubation of synaptosomes under control conditions.     

A model of the sodium dependence of dopamine uptake in rat striatal synaptosomes

Initial velocity of uptake of dopamine (DA) has been measured in rat striatal synaptosomes as a function of both [DA] and [Na]. Carrier mediated uptake is totally dependent on external sodium. The data were fitted to a rapid equilibrium model which has been found in previous studies to fit, with appropriate simplification, uptake data for glutamate, GABA, and choline in several brain regions under varying conditions. This model also gives a good fit to the dopamine data. The minimal best fit simplification of this model allows for DA uptake along with two sodium ions and predicts that apparent maximal velocity of uptake should increase with [Na], while the Michaelis-Menten constant should decrease. The minimal best fit model for DA, and a number of kinetic parameters which quantitate the model, are compared to those for the GABA, glutamate, and choline transporters. The results are consistent with a symmetrical, rapid equilibrium model, which has been presented previously for other neurotransmitters and precursors (18). This model offers a unifying basis for understanding the sodium and membrane potential dependence of neurotransmitter transport and the possible participation of transporters in depolarization induced release throughout the CNS.     

Vinpocetine blockade of sodium channels inhibits the rise in sodium and calcium induced by 4-aminopyridine in synaptosomes

The objective of this study was to get a more understandable picture of the mechanism underlying the anticonvulsant action of vinpocetine. The question of how the cerebral excitability is affected was investigated by determining the effect of vinpocetine on the changes on the internal concentrations of Na(+) (Na(i)) and Ca(2+) (Ca(i)) induced by different concentrations of the convulsing agent 4-aminopyridine (4-AP) in striatal isolated nerve endings. The cytosolic concentrations of Na(i) and Ca(i) were detected fluorimetrically with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. Vinpocetine, like the Na(+) channel blocker, tetrodotoxin, abolished the increase in Na(i) induced by 0.1 mM 4-AP and only inhibited in 30% the rise in Na(i) induced by 1mM 4-AP. In contrast with the different sensitivity of the rise in Na(i) induced by 0.1 and 1mM 4-AP to vinpocetine and tetrodotoxin, the rise in Ca(i) induced by the two concentrations of 4-AP was markedly inhibited by vinpocetine (and tetrodotoxin), indicating that only the voltage-sensitive sodium channels (VSSC)-mediated fraction of the rise in Na(i) induced by 4-AP is linked with the activation of pre-synaptic Ca(2+) channels. The elevation of Ca(2+) induced by high K(+) (30 mM) does not require a Na(+) gradient and is vinpocetine and tetrodotoxin insensitive. In contrast, the elevation of Ca(i) induced by 4-AP, requires a physiological (out/in) Na(+) gradient and is vinpocetine and tetrodotoxin-sensitive. It is concluded that by blocking the tetrodotoxin-sensitive fraction of the rise in Na(i) induced by 4-AP, vinpocetine inhibits the concomitant rise in Ca(i) induced by 4-AP. The inhibitory effect of vinpocetine on pre-synaptic voltage-sensitive sodium channels may underlie the in vivo anticonvulsant action of vinpocetine.     

Ethanol inhibits L-arginine uptake and enhances NO formation in human placenta.

The acute effects of ethanol (20-60 mM) on L-arginine uptake and nitric oxide (NO) formation was investigated in human placental cotyledons perfused at constant flow. Ethanol (40 mM) decreased L-[3H]arginine uptake from 27.6 +/- 2.3 to 15.8 +/- 1.3 per cent (P < 0.05) of the injected dose and significantly enhanced NO levels in the perfusate from 0.88 +/- 0.11 to 2.80 +/- 0.39 microM. Ethanol also elicited the constriction of placental vessels. The effects of ethanol (20-60 mM) on L-arginine uptake and endothelial NO synthase (eNOS) activity were also investigated in cultured human umbilical vein endothelial cells (HUVEC). After 60 min of ethanol (40 mM) exposure, basal L-[3H]arginine uptake (4.7 +/- 0.3 pmol/microg protein/min) was inhibited by 60 per cent (P < 0.05). Basal eNOS activity in HUVEC determined under "no flow" (static) conditions was significantly increased (approximately 1.8 fold) by 60 mM ethanol. These data are consistent with a stimulatory effect of ethanol on eNOS activity in both basal and flow-stimulated conditions, which may serve a protective role against its vasoconstrictive acute effect. While acute ethanol administration inhibits L-arginine uptake, the present results do not allow us to speculate on the effects of chronic ethanol exposure on NO formation in the fetoplacental unity.     

The effect of sodium on depolarization-induced calcium uptake and acetylcholine release by synaptosomes

The influence of external sodium concentration on potassium (depolarizing agent)-stimulated calcium uptake and Ca⁺-dependent acetylcholine release by rat cerebral cortex synaptosomes has been studied. It was found that increased sodium concentration decreases both the Ca²⁺ uptake and the acetylcholine release, whereas a low external sodium concentration is stimulatory.     

Indomethacin inhibits the uptake of sodium by ovine trophoblastic tissue in vitro

Blastocysts from several species synthesize prostaglandins in vitro, but the exact functions of the prostaglandins are unknown. The purpose of this study was to determine if indomethacin, an inhibitor of prostaglandin synthesis, would inhibit the uptake of ²²sodium ([²²Na]) by ovine trophoblastic tissue. To determine the concentration of indomethacin that would inhibit the synthesis of PGF_2α and 13,14-dihydro-15-keto-PGF_2α (PGFM) by blastocysts, blastocysts were collected from ewes 16 days after mating, sliced into pieces approximately 2 mm in length and incubated for 48 h at 37°C in 2 ml of medium containing either 0, 0.2, 0.4, 0.8 or 1.6 mM of indomethacin. Concentrations of indomethacin mM reduced (P<.01) trophoblastic release (ng/μg DNA) of PGF_2α from

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, reduced PGFM from

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, and inhibited formation of trophoblastic vesicles. In a second experiment, blastocysts were recovered from ewes 16 days after mating and pieces of trophoblast were incubated with [²²Na] and either 0 or 0.4 mM of indomethacin. Indomethacin reduced the uptake of [²²Na], which is an indirect measure of the transport of water across epithelia, from 3680 ± 1118 to 934 ± 248 cpm/μg DNA (P<.03) and prevented formation of trophoblastic vesicles. Prostaglandins produced by ovine blastocysts might be involved in controlling uptake of water, which is essential for expansion of blastocysts.     

d-glucosamine uptake by rat brain synaptosomes

Uptake of d-glucosamine by rat brain synaptosomes occurs via a saturable transport process (K_m 2.1 mM, V 3.0 nmol/mg per min) which was clearly distinguishable from simple diffusion. This transport process is highly sensitive to cytochalasin (K_i = 7 · 10^?5 mM. d-Glucose competitively inhibits d-glucosamine uptake with a K_i value of 8 · 10^?1 mM.     

Indomethacin inhibits the uptake of 22sodium by ovine trophoblastic tissue in vitro

Blastocysts from several species synthesize prostaglandins in vitro, but the exact functions of the prostaglandins are unknown. The purpose of this study was to determine if indomethacin, an inhibitor of prostaglandin synthesis, would inhibit the uptake of 22sodium ([22Na]) by ovine trophoblastic tissue. To determine the concentration of indomethacin that would inhibit the synthesis of PGF2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM) by blastocysts, blastocysts were collected from ewes 16 days after mating, sliced into pieces approximately 2 mm in length and incubated for 48 h at 37 degrees C in 2 ml of medium containing either 0, 0.2, 0.4, 0.8 or 1.6 mM of indomethacin. Concentrations of indomethacin greater than or equal to 0.2 mM reduced (P less than .01) trophoblastic release (ng/micrograms DNA) of PGF2 alpha from 205 +/- 71.2 to less than or equal to 3.3 +/- 0.2, reduced PGFM from 0.7 +/- 0.1 to less than or equal to 0.17 +/- 0.01, and inhibited formation of trophoblastic vesicles. In a second experiment, blastocysts were recovered from ewes 16 days after mating and pieces of trophoblast were incubated with [22Na] and either 0 or 0.4 mM of indomethacin. Indomethacin reduced the uptake of [22Na], which is an indirect measure of the transport of water across epithelia, from 3680 +/- 1118 to 934 +/- 248 cpm/micrograms DNA (P less than .03) and prevented formation of trophoblastic vesicles. Prostaglandins produced by ovine blastocysts might be involved in controlling uptake of water, which is essential for expansion of blastocysts.     

Opposite effects of extracellular sodium removal on the uptake of tryptophan into rat cortical slices and synaptosomes

Abstract: The removal of extracellular sodium decreased L-tryptophan (L-TRP) uptake into brain slices but in-creased uptake into synaptosomes. The uptake of 5-hy-droxytryptamine (5-HT) was inhibited in both preparations under these conditions. It is suggested that the dif-ferent effects on L-TRP uptake may be due to differencesin cellular composition between the two preparationsrather than to any differences in sample handling.     

Inactivation of depolarization-induced calcium uptake in rat brain synaptosomes

The inactivation of depolarization-induced Ca uptake into rat brain synaptosomes was demonstrated biochemically by comparing⁴⁵Ca fluxes after various intervals of predepolarization achieved by abruptly increasing {K⁺}₀. The chemical composition of the medium was maintained throughout the predepolarization and Ca uptake steps. Under these conditions, inactivation was dependent on depolarization, i.e., basal unstimulated Ca uptake in the presence of 5 mM {K⁺}₀ did not inactivate. Inactivation of stimulated Ca uptake was dependent on the predepolarization interval, moderately dependent on {Ca}₀ and relatively independent of membrane potential, i.e., {K⁺}₀ and ions such as Ni²⁺ and Co²⁺ that blocked Ca uptake. Both cinnarizine and lidoflazine blocked stimulated Ca uptake in a concentration-dependent manner without affecting the % inactivation. Although the amount of stimulated uptake increased greatly between 10 and 30°C, the % inactivation was unaffected by temperature. These findings suggest that inactivation of the presynaptic Ca uptake is an intrinsic property of the channel independent of calcium uptake.     

Preparation of synaptosomes and vesicles with sodium diatrizoate

Abstract— Synaptosomes were prepared from rat brain on a continuous gradient of sodium diatrizoate and compared with synaptosomes obtained on a discontinuous sucrose gradient. The yield of synaptosomal protein using diatrizoate was double that obtained with sucrose. No significant differences in quality between the two preparations were found based on measurement of: β-glucuronidase, RNA polymerase, 2′,3′-cyclic nucleotide 3′-phosphohydrolase, total and Na⁺, K⁺ -ATPase, acetylcholinesterase, lactic acid dehydrogenase, glucose utilization, and serotonin uptake. Electron microscopy showed the vesicles in the diatrizoate synaptosomes to be better preserved. Vesicles prepared on diatrizoate segregated into two distinct bands which differed in electron microscopic appearance and enzyme activity. The less dense vesicles were smaller, had much higher Mg²⁺ -ATPase activity, and a lower content of acetylcholinesterase than the more dense vesicles. The less dense vesicle preparation was very homogeneous morphologically, free of myelin and mitochondria, and contained occasional organelle fragments and double membrane structures.     

Kainate-induced uptake of calcium by synaptosomes from rat brain

Kainic acid induces a rapid increase in 45Ca2+ uptake by crude synaptosomal fractions isolated from rat brain. This enhanced Ca2+ permeability occurs with a half-time of approx. 1 s, similar to the fast phase of depolarization-induced calcium uptake. The depolarization-induced uptake of calcium is inhibited 85% by 3 mM CoCl2, 80% by 100 microM quinacrine and 50% by 15 microM trifluoperazine while these agents had little effect on the kainate-induced uptake. It is proposed that kainate induces receptor-mediated opening of a class of calcium channels with properties different from those of the voltage-dependent channels.     

Effect of lithium on dopamine uptake by brain synaptosomes

Lithium chloride exerts two opposite effects on dopamine uptake by synaptosomes isolated from rat caudate nucleus. Added in vitro, it inhibits dopamine uptake; whereas administered chronically in vivo, it enhances dopamine uptake in vitro. Thus, in vitro, 1, 2.5, 5 and 10 meqiv.l-1 of lithium chloride decrease [3H]dopamine uptake by 13, 17, 25 and 31%, respectively. Synaptosomes isolated from rats treated with lithium chloride for 20 days, show a 23% increase in [3H]dopamine uptake with respect to synaptosomes isolated from control rats. It is suggested that chronic lithium treatment stimulates a compensatory mechanism which overcomes its direct inhibitory effect on [3H]dopamine uptake.     

Inhibition of 3-H-noradrenalin uptake in synaptosomes by substance P]

The uptake kinetics of DL-[3H]-norepinephrine into synaptosomes from rat brain hypothalamus can be described as a two stage process with uptake constants of Km = 0.9 micron and Km = 0.06 micron, respectively. Substance P exerts an inhibiting influence on both uptakes. The possible importance of this inhibition for the regulation of noradrenergic transmission is discussed.