Synaptosomal Release Research Articles

P-368 - Effects of tetrahydroaminoacridine on acetylcholine release, membrane potential and intracellular pH in cerebrocortical synaptosomes of the rats

P-368 - Effects of tetrahydroaminoacridine on acetylcholine release, membrane potential and intracellular pH in cerebrocortical synaptosomes of the rats

Convulsions and wet-dog shakes produced by systemic or intrahippocampal administration of ruthenium red in the rat

In this work we have studied in the rat the behavioral effects of the intraperitoneal (i.p.) and intrahippocampal (i.h.) administration of ruthenium red (RuR), an inorganic dye which has been shown to inhibit neurotransmitter release in synaptosomes. The i.p. injection induced initially flaccid paralysis and subsequently generalized tonic-clonic convulsions. It contrast, unilateral RuR microinjection into the CA1 area of the hippocampus produced complex seizure behavior and wet-dog shakes (WDS). The i.p. administration of the serotonin receptor antagonist ketanserin markedly inhibited the WDS induced by i.h. RuR. In contrast, the i.h. injection of ketanserin and of the gamma-aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol(THIP) and baclofen together with RuR did not affect the frequency of WDS nor the seizure behavior. However, the i.h. injection of the GABA uptake blocker nipecotic acid, simultaneously with RuR, increased the frequency of WDS. The release of [3H]GABA, measured in synaptosomes of different cerebral structures of the rats injected i.p. with RuR, and in slices of the CA1 area after i.h. injection of the dye, was not affected. Histological observations of the injected area showed a specific and intense staining of the somas of the CA1 pyramidal neurons. It is concluded that the convulsant action induced by i.h. RuR microinjection is probably the result of an increased excitability of these CA1 neurons, which is independent of any action on GABA release.

Regulation of histamine release in rat hypothalamus and hippocampus by presynaptic galanin receptors

The effect of galanin, a peptide present in a subpopulation of histaminergic neurons emanating from the rat posterior hypothalamus, was investigated on K +-evoked [ 3H]histamine release in slices and synaptosomes from rat cerebral cortex, striatum, hippocampus and hypothalamus. Porcine galanin (0.3 μM) significantly inhibited histamine release induced by 25 mM K + in slices from hypothalamus and hippocampus, but not from cerebral cortex and striatum, i.e., only in regions in which a colocalization of histamine and galanin has been described. The inhibitory effect of galanin was concentration dependent, with an EC 50 value of 5.8±1.9 nM. The maximal inhibition was of 30–40% in hypothalamic and hippocampal slices depolarized with 25 mM K +. The galanin-induced inhibition observed in hypothalamic slices was not prevented in the presence of 0.6 μM tetrodotoxin and also occurred in hippocampal and hypothalamic synaptosomes, strongly suggesting the activation by galanin of presynaptic receptors located upon histaminergic nerve endings. The maximal inhibitory effect of galanin in slices or synaptosomes was lower than that previously reported for histamine acting at H 3-autoreceptors, possibly suggesting that not all histaminergic axon terminals, even in the hypothalamus and hippocampus, are endowed with galanin receptors. It increased progressively in hypothalamic and hippocampal synaptosomes as the strength of the depolarizing stimulus was reduced. It is concluded that galanin modulates histamine release via presynaptic receptors, presumably autoreceptors located upon nerve terminals of a subpopulation of cerebral histaminergic neurons.

Antagonism by antidepressant drugs of the inhibitory effect of trifluoromethylphenylpiperazine (TFMPP) on [ 3H]acetylcholine release in rat or guinea-pig hippocampal synaptosomes

The effects of antidepressant drugs on the m-trifluoromethylphenylpiperazine (TFMPP)-induced inhibition of K +-evoked [ 3H]acetylcholine ( 3H-ACh) release were studied in rat or guinea-pig hippocampal synaptosomes. The serotonergic agonist TFMPP dose-dependently inhibited the K +-evoked release of 3H-ACh in rat hippocampus (IC 50=53 μM). Chlorimipramine (5–500 nM), a typical tricyclic antidepressant, and minaprine (1–100 nM), an atypical antidepressant drug, partially antagonized the effect of TFMPP on 3H-ACh release in a dose-dependent manner. Other antidepressants (imipramine, citalopram, indalpine, fluoxetine, fluvoxamine, oxaprotiline, mianserine, nomifensine), at concentrations ranging from 10 to 500 nM, produced similar effects. Drugs with no antidepressant effect, such as chlorpromazine, clobazam, and cocaine (50, 100 and 500 nM), were without significant influence on the TFMPP effect. In guinea-pig hippocampal synaptosomes, minaprine (50 nM) also reduced the TFMPP-induced inhibition of 3H-ACh release, whilst clobazam (50 nM) was inactive. These results suggest that antidepressant drugs interact in vitro with heterologous serotonergic presynaptic receptors on cholinergic nerve terminals in rat and guinea-pig hippocampus.

3,4-Methylenedioxyamphetamine (MDA) analogues exhibit differential effects on synaptosomal release of 3H-dopamine and 3H-5-hydroxytryptamine

The effect of various analogues of the neurotoxic amphetamine derivative, MDA (3,4-methylenedioxyamphetamine) on carrier-mediated. calcium-independent release of 3H-5-HT and 3H-DA from rat brain synaptosomes was investigated. Both enantiomers of the neurotoxic analogues MDA and MDMA (3,4-methylenedioxymethamphetamine) induce synaptosonal release of 3H-5-HT and 3H-DA in itro. The release of 3H-5-HT induced by MDMA is partially blocked by 10 −6 M fluoxetime. The (+) enantiomers of both MDA and MDMA are more potent than the (−) enantiomers as releasers of both 3H-5-HT and 3H-DA. Eleven analogues, differing from MDA with respect to the nature and number of ring and/or side chain substituents, also show some activity in the release experiments, and are more potent as releasers of 3H-5-HT than of 3H-DA. The amphetamine derivatives (±)fenfluramine, (±)norfenfluramine, (±)MDE, (±)PCA, and d-methamphetamine are all potent releasers of 3H-5-HT and show varying degrees of activity as 3H-DA releasers. The hallucinogen DOM does not cause significant release of either 3H-monoamine. Possible long-term serotonergic neurotoxicity was assessed by quantifying the density of 5-HT uptake sites in rats treated with multiple doese of selected analogues using 3H-paroxetine to label 5-HT uptake sites. In the neurotoxicity study of the compounds investigated, only (+)MDA caused a significant loss of 5-HT uptake sites in comparison to saline-treated controls. These results are discussed in terms of the apparent structure-activity properties affecting 3H-monoamine release and their possible relevance to neurotoxicity in this series of MDA congeners.

The effects of (−)‐daurisoline on Ca2+ influx in presynaptic nerve terminals

1. The effects of (-)-daurisoline on 45Ca2+ uptake and [3H]-gamma-aminobutyric acid ([3H]-GABA) release from synaptosomes of rat cerebral cortex and on contractile activity of rat aorta were examined. 2. Application of (-)-daurisoline (1-100 microM) produced concentration-related inhibition of high K(+)-stimulated 45Ca2+ uptake and [3H]-GABA release (IC50 = 7.7 +/- 0.9 microM and 10.0 +/- 1.5 microM, respectively) in synaptosomes but verapamil was only weakly active. 3. Neither (-)-daurisoline (100 microM) nor verapamil (100 microM) modified 45Ca2+ uptake in control medium (5 mM K+, resting uptake) and [3H]-GABA release in Ca-free medium (45 mM K+ basal release). 4. High K+ and noradrenaline-evoked contractions of rat aorta were inhibited by both (-)-daurisoline and verapamil. 5. In conclusion, (-)-daurisoline, which differed from verapamil in its mode of blocking Ca2+ influx may be a potent Ca2+ antagonist of Ca2+ channels in neurones.

Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.

Chronic ethanol treatment alters ω-conotoxin and Bay K 8644 sensitive calcium channels in rat striatal synaptosomes

Two groups of adult Sprague-Dawley rats were maintained on a nutritionally complete liquid diet. In one group, 37% of the calories normally provided by dextrin were replaced with ethanol. Animals were maintained on this diet for eight weeks. The addition of ethanol (200 mM) in vitro significantly inhibited both calcium influx and dopamine release from control synaptosomes but did not alter calcium influx or dopamine release from synaptosomes isolated from ethanol-treated rats. The dihydropyridine calcium channel agonist Bay K 8644 (1 nM) significantly increased both calcium entry and dopamine release from control synaptosomes depolarized with 15 mM KCl. Bay K 8644 (1 nM) had no significant effect on either calcium entry or dopamine release in synaptosomes isolated from ethanol-treated animals. This loss of functional effect was accompanied by a slight (15%) but statistically insignificant increase in the binding of 3H-nitrendipine to striatal membranes from ethanol-treated rats as compared to control. The calcium-channel blocker, ω-conotoxin (500 nM) had no effect on voltage-dependent calcium uptake into synaptosomes prepared from control or ethanol-treated rats. Conotoxin (500 nM) inhibited the voltage-dependent release of endogenous dopamine from synaptosomes isolated from both groups by 36–44%. Ethanol (200 mM) added in vitro to control synaptosomes did not alter conotoxin's inhibition of dopamine release but completely abolished the ω-conotoxin-induced inhibition of dopamine release in synaptosomes isolated from ethanol-treated animals. These results suggest that DHP-sensitive and ω-conotoxin-sensitive calcium channels in rat brain respond differentially to chronic exposure to ethanol.

Arachidonic Acid Increases Inositol Phospholipid Metabolism and Glutamate Release in Synaptosomes Prepared from Hippocampal Tissue

We have been interested in the possibility that arachidonic acid or one of its 12-lipoxygenase metabolites may function as a retrograde messenger in long-term potentiation (LTP) in the dentate gyrus of the hippocampus. One criterion required of a retrograde messenger is that it stimulates presynaptic changes. Here, two possible presynaptic actions of arachidonic acid and its 12-lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 12-hydroperoxyeicosatetraenoic acid (HPETE), are examined. We report that arachidonic acid, HETE, and HPETE significantly increase both K(+)-stimulated release of [3H]glutamate and [3H]inositol labelling of inositol phosphates in synaptosomes, whereas other biologically important fatty acids (oleic, palmitic, and stearic) failed to induce a similar response. The findings of these experiments are consistent with the hypothesis that arachidonic acid, HETE, or HPETE may play the role of a retrograde messenger in LTP.

Biochemical and behavioral evidence for muscarinic autoreceptors in the CNS

Muscarinic autoreceptors of the M 2 subclass were examined in rat forebrain using a number of different methodologies, including receptor autoradiography and image analysis, regulation of acetylcholine release, phosphoinositide turnover, low- K m GTP hydrolysis, and behavioral analysis. The relatively minor population of M 2 receptors in coronal sections was visualized by autoradiography and image analysis using [ 3H]quinuclidinyl benzilate in the presence of a concentration of pirenzepine that blocked most of M 1 (and M 4) receptors. The highest densities of M 2 receptors in forebrain regions were found in the outer layers of the cortex, CA1 region of the hippocampus and striatum. The M 2-, but not M 1-selective antagonists were able to block the oxotremorine-induced attenuation of acetylcholine release in forebrain synaptosomes. Low concentrations of the M 2-selective antagonist gallamine increased phosphoinositide turnover, which is thought to be an M 1 postsynaptic response in the forebrain, in brain slices by a Ca 2+-dependent mechanism. The M 2-selective agonist oxotremorine produced a substantial stimulation of low- K m GTPase in cortical membranes, suggesting that M 2 forebrain receptors are efficiently coupled to G-proteins in the cortex. Behavioral signs of cholinergic stimulation were observed after intracerebroventricular injections of M 2-, but not M 1-selective antagonists. It is suggested that a minor population of foregrain M 2 receptors regulates acetylcholine release by a mechanism that includes coupling through G-proteins presynaptically at synapses for which the postsynaptic response involves phosphoinositide turnover. Selective blockade of these receptors produces both biochemical and behavioral signs of acetylcholine release.

Verapamil corrects abnormalities in norepinephrine metabolism of brain synaptosomes in CRF

Abnormalities in norepinephrine (NE) metabolism of brain synaptosomes occur in chronic renal failure (CRF), and this has been attributed to the parathyroid hormone (PTH)-induced accumulation of calcium in synaptosomes. The present study examined the effect of treatment with the calcium-channel blocker verapamil on NE content, release, and uptake, on Na(+)-K(+)-ATPase activity, and on calcium content of brain synaptosomes from rats with 21 days of CRF. Verapamil treatment of normal rats for 21 days did not affect synaptosomal NE content, release, or uptake, Na(+)-K(+)-ATPase activity, or calcium content. Rats with 21 days of CRF displayed a significant (P less than 0.01) reduction in their synaptosomal NE content, release, and uptake, an increase in Na(+)-K(+)-ATPase activity, and a significant (P less than 0.01) increase in calcium content of synaptosomes. The treatment of CRF rats with verapamil normalized synaptosomal NE content and release and Na(+)-K(+)-ATPase activity, produced a significant (P less than 0.01) improvement in NE uptake, and prevented the accumulation of calcium in synaptosomes. The data of the present study are consistent with the notion that the abnormalities in synaptosomal NE metabolism and Na(+)-K(+)-ATPase in CRF are mainly the result of PTH-induced accumulation of calcium in synaptosomes and could be prevented by a calcium-channel blocker.

Effect of cerebral ischemia on synaptosomal uptake and release of3H-5-hydroxytryptamine in adult and young mongolian gerbils

Cerebral ischemia induced by bilateral common carotid artery occlusion (15 min) with and without release (1 hr) served as a model for comparative regional studies of synaptosomal 3H-5-hydroxytryptamine (3H-5-HT) uptake and release in adult and young gerbils. A decreased uptake and an increased release of 5-HT was observed in the adult after ischemia alone and/or ischemia with reflow. At the same time, 5-HT uptake was not affected except in the cortex and the release was reduced in the young. These findings indicate that the same ischemic insults affect differently the synaptosomal uptake and/or release of 5-HT in adult and young brain.

Influence of tetrahydrobiopterin on serotonin synthesis, metabolism and release in synaptosomes

The effects of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH(4)) on the uptake of tryptophan, its conversion to serotonin (5-HT) and 5-hydroxyindoleacetic acid and on the basal release of 5-HT was studied in rat brain synaptosomes. When BH(4), the essential cofactor for tryptophan hydroxylase, was incubated with synaptosomes in concentrations varying from 10 to 200 ?M, there was no effect on 5-HT formation, metabolism or release. Concentrations of 1-2 mM BH(4) had strong inhibitory effects on 5-HT synthesis. The incubation of synaptosomes with tryptophan increased the synthesis of 5-HT, but BH(4) did not further increase this effect. BH(4) was taken up into synaptosomes in a concentration-dependent manner under all incubation conditions and was stable in the chemically reduced (tetrahydro-) form. These results indicate that increases in the synaptosomal concentration of BH(4) do not increase the synthesis and release of 5-HT. It is concluded from the present results that tryptophan hydroxylase is saturated with BH(4) in synaptosomes.

The action of notexin from tiger snake venom ( Notechis scutatus scutatus) on acetylcholine release and compartmentation in synaptosomes from electric organ of Torpedo marmorata

At rest, in the presence of calcium, notexin induced a rapid and concentration-dependent leakage of acetylcholine from nerve endings. In the presence of 20 nM notexin (5 min), synaptosomes were well-preserved structurally and they responded to addition of A23187 ionophore by a normal calcium-dependent acetylcholine release. When stimulated by high-K + depolarization, evoked acetylcholine release was increased when notexin was present. These findings demonstrate that notexin (up to 100 nM) does not inhibit the acetylcholine release process itself. Further studies on intracellular acetylcholine compartmentation showed that, in the presence of calcium, nm concentrations of notexin were able to mobilize vesicular acetylcholine, the amount of which strongly decreased and fed the cytoplasmic compartment leading to an important redistribution of the neurotransmitter. Other metabolic studies under notexin confirmed the inhibition of the synaptosomal membrane choline transport, but failed to elicit changes in the choline acetyltransferase activity. In order to distinguish between the phospholipase A 2 activity of notexin and its neurotoxic effects, we compared effects of notexin to those obtained with a non-neurotoxic pancreatic phospholipase A 2. The latter exhibits similar effects but at a higher range of concentration than notexin.

3H]acetylcholine release and the change in cytosolic free calcium level induced by high K + and ouabain in rat brain synaptosomes

High K + (50 mM) increased both [ 3H]acetylcholine ([ 3H]ACh) release and cytosolic free calcium level ([Ca 2+] i) in rat brain synaptosomes in the presence of extracellular Ca 2+. Ouabain (5 × 10 −8 to 5 × 10 −4 M) also caused a dose-dependent increase in [ 3H]ACh release, but not in [Ca 2+] i, in the absence of Ca 2+. The effects of high K + and ouabain on [ 3H]ACh and/or [Ca 2+] i were inhibited by the intracellular Ca 2+ antagonist TMB-8 (10 −4 M). These results suggest that unlike high K +, ouabain increases transmitter release from nerve endings through a mechanism which is independent of [Ca 2+] i, but sensitive to TMB-8.

Reduced inhibitory effects of clonidine and neuropeptide Y on 3H-noradrenaline release from synaptosomes of the medulla oblongata of the spontaneously hypertensive rat.

The release of 3H-noradrenaline (3H-NA) evoked by high-K+ (15 mM) was studied in synaptosomes isolated from the medulla oblongata of the normotensive Wistar-Kyoto and spontaneously hypertensive male rat (14 weeks old) using a superfusion apparatus. Based on concentration-response curves clonidine was shown to have a reduced ability to inhibit 3H-NA release in synaptosomes isolated from the spontaneously hypertensive rat (SHR) versus the normotensive rat. Furthermore, only a high concentration of NPY (100 nM) had the ability to enhance the inhibitory effects of clonidine on 3H-NA release in synaptosomes isolated from the medulla oblongata of the SH male rat, while 1 nM of NPY was effective in synaptosomes isolated from the medulla oblongata of the normotensive Wistar-Kyoto rat. These results may indicate a reduced presynaptic alpha 2-adrenoceptor and NPY receptor function to inhibit 3H-NA release from NA and/or adrenaline (A) nerve terminals in the medulla oblongata of the adult 14 weeks old SHR.

Abnormal norepinephrine uptake and release in brain synaptosomes in chronic renal failure

Abnormalities in the function of the central nervous system exist in chronic renal failure (CRF) and some of these derangements may be related to excess parathyroid hormone (PTH) which causes a rise in brain calcium. The latter may affect metabolism of neurotransmitters such as norepinephrine (NE) in brain synaptosomes. We measured NE content, uptake and release in brain synaptosomes of CRF rats and studied whether excess PTH affects these parameters. Synaptosomes from rats with 21 days of CRF compared to those from normal animals have higher calcium content (11.4 +/- 0.92 vs. 7.1 +/- 0.50 nmol/mg protein, P less than 0.01) and lower Na-K ATPase activity (6.5 +/- 0.81 vs. 11.4 +/- 0.76 mumol Pi/mg protein/hr, P less than 0.01). NE content (11.0 +/- 0.60 vs. 13.6 +/- 0.55 pmol/mg protein/hr, P less than 0.01), uptake (46 +/- 4.5 vs. 110 +/- 5.9 pmol/mg protein times 50 min, P less than 0.01) and release (2.0 +/- 0.2 vs. 5.1 +/- 0.47 pmol/mg protein times 10 min, P less than 0.01). Parathyroidectomy (PTX) in CRF rats kept normocalcemic reversed these abnormalities in brain synaptosomes; indeed calcium content, Na-K ATPase activity and NE content, uptake and release in synaptosomes from PTX-CRF rats were not different from those seen in normal rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Reduced inhibitory effects of clonidine and neuropeptide Y on 3H-noradrenaline release in synaptosomes of spontaneously hypertensive rats

Reduced inhibitory effects of clonidine and neuropeptide Y on 3H-noradrenaline release in synaptosomes of spontaneously hypertensive rats

Compared Effects of Two Vesicular Acetylcholine Uptake Blockers, AH5183 and Cetiedil, on Cholinergic Functions in Torpedo Synaptosomes: Acetylcholine Synthesis, Choline Transport, Vesicular Uptake, and Evoked Acetylcholine Release

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.

Neuropeptide Y increases the inhibitory effects of clonidine on potassium evoked 3H-noradrenaline but not 3H-5-hydroxytryptamine release from synaptosomes of the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat.

The release of 3H-noradrenaline (3H-NA) and of 3H-5-hydroxytryptamine (3H-5-HT) evoked by high-K+ (15 mM) was studied in synaptosomes isolated from the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat using a superfusion apparatus. Based on concentration-response curves obtained by analyzing the full-time course of the inhibitory effects of clonidine on 3H-NA and on 3H-5-HT release neuropeptide Y (NPY) (1 nM) was shown to significantly increase the ability of clonidine to inhibit 3H-NA release in synaptosomes isolated from the hypothalamus and from the frontoparietal cortex. NPY (1 nM) alone had no effect on K+-evoked 3H-NA release from these regions. In contrast, NPY (1 nM) did not modulate the inhibitory effects of clonidine on 3H-5-HT release in the above mentioned regions. These results indicate that NPY can increase the sensitivity of the alpha 2-autoreceptors belonging to hypothalamic NA and/or to adrenaline nerve terminals and to cortical NA nerve terminals, while the alpha 2-heteroreceptors inhibiting 3H-HT release in the same brain regions appear not to be regulated by high affinity NPY receptors. Thus, alpha 2-autoreceptors and alpha 2-heteroreceptors appear to be differentially controlled by high affinity NPY receptors at least with regard to regulation of 3H-NA and 3H-5-HT release, respectively.