Paradoxical nifedipine facilitation of 45Ca uptake into rat hippocampal synaptosomes

Nifedipine has a high incidence of neurologic adverse reactions as compared with other dihydropyridine Cav1 (L-type) channel blockers used for treating cardiovascular diseases. The mechanism mediating neuronal excitation by nifedipine is still in debate. Nifedipine caused a dual role on veratridine-induced 45Ca uptake by rat hippocampal synaptosomes. In the nanomolar range (0.001-0.3 microM), nifedipine decreased 45Ca uptake in a cadmium-sensitive manner. In contrast with nitrendipine (0.001-10 microM), nifedipine consistently facilitated 45Ca accumulation when used in low micromolar concentrations (0.3-10 microM). The cadmium-insensitive nifedipine facilitation became less evident upon increasing veratridine concentration from 5 to 20 microM and was not detected when the synaptosomes where depolarised with 30 mM KCl. Na+ substitution by N-methyl-D-glucamine (132 mM) or blockade of Na+ currents with tetrodotoxin (1 microM) both prevented nifedipine excitation. The Na+/Ca2+-exchanger inhibitor, KB-R7943 (3-50 microM), did not reproduce nifedipine actions. Data suggest that tetrodotoxin-sensitive Na+ channels may operate paradoxical nifedipine facilitation of 45Ca uptake by rat hippocampal synaptosomes.     

Ca-dependence of accumulation of monoamines into synaptosomes and its inhibition by copper

Effects of various concentrations of extracellular Ca on the high affinity uptake of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) as well as the effects of Ca on Cu-induced uptake inhibition were studied by using striatal, cortical and hypothalamic synaptosomes, respectively. The spontaneous release of all three amines from preloaded synaptosomes by Cu increased slightly with increasing external Ca up to 2.5 mM. The sensitivity to Ca of the accumulation of the amines into synaptosomes decreased in the order of DA greater than NA greater than 5-HT. Below 0.3 mM Ca, DA uptake increased due to concurrent decreased release of DA from synaptosomes. The pattern was similar but less evident for NA while 5-HT uptake and its spontaneous release did not change upon buffer Ca at all. The uptake inhibition of none of the amines depended on extracellular Ca. Zn reversed the Cu-induced inhibition of 5-HT uptake but increased additively the inhibition of DA uptake and that of NA uptake in vitro. The potency of Cu to inhibit uptake decreased when the protein concentration in the incubation medium was increased. Cysteine, added concurrently with Cu, did not protect uptake from the toxic influence of Cu. The results so far suggest that the primary mechanism in the Cu-induced inhibition of uptake is blocking the membrane uptake carrier. The release of an accumulated amine from synaptosomes may rather be secondary and result from the inhibition of the reuptake.     

Cannabinoid receptor-mediated inhibition of acetylcholine release from hippocampal and cortical synaptosomes

In previous studies cannabinoid agonists have been found to inhibit and cannabinoid antagonists to enhance electrically-evoked [(3)H]-acetycholine (ACh) release in hippocampal slices. The present study was undertaken to determine if similar cannabinoid effects could be observed in synaptosomes. [(3)H]-ACh release was evoked by two methods, both sensitive to presynaptic receptor effects. The first involved the addition of 1.3 mM calcium following perfusion with calcium-free Krebs and the second the addition of 11 mM potassium following perfusion with normal Krebs. In hippocampal synaptosomes the 1.3 mM calcium-evoked release and the high potassium-evoked [(3)H]-ACh release were inhibited by the cannabinoid agonist, WIN 55212-2, by 59 and 39%, respectively, and with an EC(50) of approximately 1 nM. WIN 55212-2 produced a similar, although less potent, inhibition of [(3)H]-ACh release in cortical synaptosomes. No inhibitory effect of WIN 55212-2 on [(3)H]-ACh release in striatal synaptosomes was observed, supporting previous data collected in this area with brain slices. The cannabinoid antagonist, SR 141716A, produced a robust enhancement of 1.3 mM calcium-evoked [(3)H]-ACh release in hippocampal synaptosomes (EC(50)<0.3 nM) but had no effect on potassium-evoked release or on [(3)H]-ACh release in the cortex or striatum. In conclusion our data demonstrates the inhibitory effects of WIN 55212-2 observed on ACh release in brain slices can be observed in hippocampal and cortex synaptosomes, suggesting a direct action of these compounds on the synaptic terminals. The SR 141716A-induced enhancement of ACh release can similarly be observed in hippocampal synaptosomes and is probably due to an inverse agonist action at constitutively active receptors.     

Cannabinoid CB1 receptor-mediated inhibition of glutamate release from rat hippocampal synaptosomes

Cannabinoid receptors are widely expressed in the brain and have been shown to regulate synaptic transmission through a presynaptic mechanism. Using synaptosomal preparation, I show here that 2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)-pyrrolo-1,4-benzoxazin-6-yl-1-naphthalenylmethanone (WIN 55212-2) strongly depressed 4-aminopyridine-evoked glutamate release in a concentration-dependent manner, and this effect was reversed by the selective cannabinoid CB(1) receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM 281). The inhibitory modulation by WIN 55212-2 was not due to a decrease in synaptosomal excitability or a direct effect on the release machinery because WIN 55212-2 did not alter 4-aminopyridine-mediated depolarization and ionomycin-induced glutamate release. In addition, the WIN 55212-2-mediated inhibition of glutamate release was blocked by the G(i)/G(o) protein inhibitor pertussis toxin, but not by the protein kinase A inhibitor 2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo-benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720). Furthermore, this inhibitory effect was associated with a decrease in 4-aminopyridine-evoked Ca(2+) influx, which could be completely prevented in synaptosomes pretreated with the N- and P/Q-type Ca(2+) channel blockers. Together, these observations indicate that activation of cannabinoid CB(1) receptors inhibit 4-aminopyridie-evoked glutamate release from hippocampal synaptosomes through a inhibitory G protein to suppress N- and P/Q-type Ca(2+) channel activity.     

Opioid inhibition of nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells.

The ability of a number of opioid agonists and antagonists to affect nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells has been investigated. High (10 microM) concentrations of the opioid agonist bremazocine produced a significant inhibition of nicotine-induced 45Ca2(+)-uptake throughout the 15 min time course examined. The opioid subtype-selectivity of this inhibition was investigated; mu and delta selective agonists produced only minor effects whereas the kappa selective agonist U50-488H and the endogenous opioid peptides dynorphin(1-13) and metorphamide almost abolished nicotine-induced 45Ca2(+)-uptake. The U50-488H inhibition was significant at 10 nM concentrations with an IC50 of approximately 1 microM. U50-488H inhibition could not be reversed or reduced by the opioid antagonists naxolone, diprenophine or Mr2266. Furthermore, Mr2266 and its optical isomer Mr2267 also produced marked inhibition of 45Ca2(+)-uptake. The inhibition was specific to nicotine-induced 45Ca2(+)-uptake in that a similar level of uptake evoked by potassium depolarization was unaffected by high concentrations of U50-488H. These data indicate that opioid inhibition of nicotine-induced 45Ca2(+)-uptake does not involve classical, stereospecific opioid receptors and suggests the involvement of a pharmacologically distinct opioid recognition site. It is speculated that this may be associated with the nicotine receptor-ionophore complex.     

Adenosine and adenosine triphosphate decrease 45Ca uptake by synaptosomes stimulated by potassium.

The action of adenosine and adenosine triphosphate on the uptake of ⁴⁵Ca has been studied on the synaptosome-rich preparation from the rat cerebral cortex. The preparation was depolarized by high potassium. Adenosine (0.1–2mM) and adenosine triphosphate (0.2 mM) decreased the uptake of ⁴⁵Ca. Adenosine triphosphate (1 mM) did not change significantly the uptake of ⁴⁵Ca and adenosine triphosphate (2 mM) even increased it. These results indicate that the inhibitory effects of these substances on neurotransmission result from its inhibitory effects on the entry of calcium into the nerve endings.     

Depolarization-dependent 45Ca uptake by synaptosomes of rat cerebral cortex is enhanced by L-triiodothyronine

Depolarization-induced release of neurotransmitters and other secretions from nerve endings is triggered by the rapid entry of Ca++ through voltage-sensitive channels. Calcium entry is thought to occur in two distinct phases or processes: a fast-phase response to an action potential, which initiates release; and a slow phase associated with extended stimulation of the neuron. Thyroid hormones are sequestered by nerve terminals and can produce changes in behaviour and mood. They may therefore be involved in modulating central synaptic transmission. We studied the effects of L-triiodothyronine (T3), L-thyroxine (T4), reverse T3 (rT3) and D-T3 on depolarization-induced uptake of 45Ca by synaptosomes from euthyroid and hypothyroid rats. T3, but not T4, rT3, or D-T3 significantly enhanced depolarization-induced 45Ca uptake at physiologically relevant (1 to 10 nmol/L) concentrations. The stimulatory effect of 10 nmol/L T3 on depolarization-induced uptake after 2 seconds (21%) was greater than after 5 (10%) or 30 (8%) seconds, indicating that T3 enhanced primarily the fast-phase process. There was no effect of T3 or other hormones tested on nondepolarization-induced 45Ca uptake. Preincubation of synaptosomes with T3 prior to depolarization did not enhance the effect of T3; in fact, preincubations of 30 seconds or more resulted in diminished T3 effects. Preincubation of synaptosomes for 15 seconds with D-T3 or the addition of D-T3 and T3 together reduced the effect of T3. We found no difference in the effect of T3 on 45Ca uptake by synaptosomes from euthyroid and hypothyroid rats. These results suggest a novel mechanism of action of thyroid hormones in the brain.     

Somatostatin inhibition of VIP-induced somatostatin release, cyclic AMP accumulation and 45Ca2+ uptake in diencephalic cells

The effect of somatostatin (SRIF) on VIP induction of SRIF secretion, cyclic AMP accumulation and 45Ca2+ influx was investigated in cultured diencephalic cells. [D-Trp8]SRIF suppressed VIP-stimulated SRIF release and decreased VIP-stimulated cyclic AMP accumulation in a dose-dependent manner. SRIF-14 blocked basal and VIP-stimulated 45Ca2+ entry into cells. The data suggest that the inhibitory effect of SRIF on VIP-induced SRIF release is partly due to a decrease in Ca2+ entry into cells.     

Adenosine receptor-mediated modulation of acetylcholine release from rat striatal synaptosomes.

1. The effects of A1 and A2a adenosine receptor agonists on the veratridine-evoked release of [3H]-acetylcholine ([3H]-ACh) from rat striatal synaptosomes was investigated by use of the A1-selective agonist, R-PIA and the 185 fold selective A2a agonist, CGS 21680. The effects of NECA, which is equipotent at both receptor subtypes, were also studied. 2. The evoked release of [3H]-ACh was significantly enhanced by the A2a agonist CGS 21680 but decreased by the A1 agonist, R-PIA. The effects of NECA were dependent on the concentration used, with high concentrations inhibiting and low concentrations enhancing the evoked release of [3H]-ACh. In the absence of any antagonists, the rank order of potency for these three drugs on increasing [3H]-ACh release was CGS 21680 > NECA > R-PIA. 3. The stimulatory effects of CGS 21680 and low NECA concentrations on evoked [3H]-ACh release were antagonized by the A2a receptor antagonists, CP66,713 (300 nM) and CGS 15943A (50 nM) whilst the inhibitory effects of R-PIA were reversed by the selective A1 antagonist, DPCPX (4 nM). In the presence of DPCPX, NECA greatly enhanced the evoked release of [3H]-ACh at all concentrations studied when, during such A1 receptor blockade, the rank order of potency was NECA >> CGS 21680 > R-PIA. 4. These results demonstrate that both A1 and A2a adenosine receptors modulate the veratridine-evoked release of [3H]-ACh from rat striatal synaptosomes.     

Kinetics of tryptophan accumulation into synaptosomes of various regions of rat brain

The kinetics of synaptosomal tryptophan accumulation has been determined in five regions of the rat brain. For tryptophan concentrations ranging from 2.5–20 M, we found an active uptake in all the structures studied, i.e.: Corpus striatum, midbrain, brainstem, hypothalamus and cerebral cortex + hippocampus. The Vm of tryptophan uptake was highest in the cortex, followed in descending order by corpus striatum, hypothalamus, midbrain and brainstem, while the Km was highest in the cortex, then in descending order corpus striatum, brainstem, midbrain and hypothalamus. In spite of the possible nonspecific high affinity tryptophan uptake into serotoninergic neurons, we found a correlation between the Vm of tryptophan uptake and the different results in the literature concerning uptake and release of serotonin. These observations might indicate a correlation between the Vm of tryptophan uptake and the functional activity of serotonergic neurons.This research was supported by INSERM(FRA 5) and CNRS(ERA 560)     

Effect of some stereoisomeric tricyclic antidepressants on45Ca uptake in synaptosomes from rat hippocampus

The present study has examined the inhibition of synaptosomal⁴⁵calcium uptake by trimipramine, oxaprotiline and doxepin, and their stereoisomers, in synaptosomes from the rat hippocampus. No significant difference in potency could be established for inhibition of net depolarization-induced⁴⁵calcium uptake for any pair of antipodes, and the IC₅₀ values for calcium channel blockade were in the vicinity of 30 µM for this group of compounds. Trimipramine, doxepin and oxaprotiline also inhibited the⁴⁵calcium uptake mediated by Na⁺-Ca²⁺ exchange, with IC₅₀ values of 71 µM, 110 µM, and 100 µM, respectively. The similar potency of doxepin isomers for inhibition of voltage-dependent calcium channels is in harmony with their reported similar potency in the clinic. A slight difference in potency is reported between the isomers of oxaprotiline in the behavioral despair test in rats, and the dextrorotatory isomer of trimipramine is reported to be a much more potent antidepressant than the levorotatory isomer: these order of potencies do not correspond perfectly with the similar potency of the antipodes against voltage-dependent calcium channels. The present study of stereoisomeric tricyclic antidepressants therefore fails to provide unequivocal support for the hypothesis that calcium channel blockade by tricyclic antidepressants is involved in their therapeutic effect.     

Kinetics of serotonin accumulation into synaptosomes of rat brain--effects of amphetamine and chloroamphetamines

Synaptosomes from whole brain of rats accumulated serotonin by means of a high affinity process and a low affinity process, distinguishable kinetically. The high affinity process was stronly inhibited by 4-chloroamphetamine. 3-Chloroamphetamine, 2-chloroamphetamine and amphetamine were progressively weaker inhibitors of that process. 4-Chloroamphetamine was a less potent inhibitor of the low affinity process (about equal to amphetamine). When 4-chloroamphetamine was administered to rats and synaptosomes were isolated from whole brain, the high affinity uptake of serotonin was inhibited for as long as 22·5 hr after drug administration. In vitro, 4-chloroamphetamine but not chloroimipramine at 5 μM caused a release of serotonin from synaptosomes preincubated with radioactive serotonin. The ability of 4-chloroamphetamine to inhibit the high affinity uptake of serotonin by synaptosomes and to release serotonin from synaptosomes is probably related to the prolonged lowering of brain serotonin levels in rats treated with 4-chloroamphetamine.     

Effects of fenfluramine on accumulation of 5-hydroxytryptamine and other neurotransmitters into synaptosomes of rat brain.

Earlier studies have demonstrated a marked long-lasting reduction in the brain level of 5-hydroxytryptamine after injection of fenfluramine. The decrease in the brain levels of dopamine and noradrenaline were less marked and disappeared one day after treatment. In this study, the effects of fenfluramine upon the uptake of 5-hydroxytryptamine and other neurotransmitters (dopamine, γ-aminobutyric acid) or their precursors (tryptophan, tyrosine and glutamic acid) into synaptosomes of rat brain were investigated. In vivo, fenfluramine is a competitive inhibitor of the high affinity synaptosomal uptake of 5-hydroxytryptamine. Even though fenfluramine produced a small reduction in the uptake of dopamine, γ-aminobutyric and glutamic acids 4 hr after injection, this effect disappeared 1 day later. In vitro, addition of fenfluramine into a synaptosomal fraction reduced uptake of 5-hydroxytryptamine, dopamine, γ-aminobutyric acid, glutamic acid, tryptophan and tjrosine. [¹⁴C] Fenfluramine itself was bound to synaptosomes. Ten days after 5–6 dihydroxytryptamine treatment, both the accumulation of [¹⁴C] fenfluramine in the brain in vivo, and the binding of [¹⁴C] fenfluramine to synaptosomes in vitro, were reduced. These results indicate that 5-hydroxytryptamine synaptosomal sites are involved in some actions of fenfluramine.     

The kappa-opiate agonist U50488H decreases the entry of 45Ca into rat cortical synaptosomes by inhibiting N- but not L-type calcium channels

The selective kappa-opiate agonist U50488H (1-100 microM) significantly reduced the uptake of 45Ca into cortical synaptosomes from the brain of the rat, in a time- and dose-dependent manner. In physiological medium, the maximum inhibition occurred after 2 min; this was approximately 55% (at 100 microM) and the IC50 was 80 nM. Nifedipine (1 microM) had no significant effect on the influx of Ca2+ in physiological medium (containing 5 mM K+), though, in fact, there was an approximately 20% decrease in the presence of 100 microM of drug. Nifedipine, however, did cause a significant blockade of the entry of 45Ca in medium containing 10 or 15 mM K+, demonstrating that L-type channels on synaptosomes were operational under depolarising conditions. Under these depolarising conditions, there was an additive inhibitory effect on entry of 45Ca into synaptosomes when U50488H (1 microM) and nifedipine (1 microM) were incubated together. Treatment of synaptosomes with omega-conotoxin (omega-CgTx, 0.5 microM) resulted in a 35% reduction in the uptake of 45Ca. omega-Conotoxin (0.5 microM) or naloxone (20 microM) abolished the inhibitory effect of U50488H on the uptake of 45Ca, but naloxone did not alter the blockade of L-type Ca2+ channels, caused by nifedipine. In conclusion, the data demonstrate that under depolarising conditions, there are functional L-type calcium channels on nerve endings in the CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flunarizine and R 56865 suppress veratridine-induced increase in oxygen consumption and uptake of 45Ca2+ in rat cortical synaptosomes.

The effect of the anti-ischemic compounds flunarizine and R 56865 on the veratridine-induced uptake of Ca2+ and Na+ was observed in cortical synaptosomes in the rat. The veratridine-induced uptake of Na+ and Ca2+ was determined by means of a measurement of synaptosomal oxygen consumption and a method for the uptake of 45Ca2+, respectively. Veratridine (10(-5) M) was found to induce a 3-fold increase in synaptosomal oxygen consumption (uptake of Na+) and uptake of 45Ca2+, both of which were inhibited by tetrodotoxin (10(-5) M). Nitrendipine (10(-5) M) and omega-conotoxin (5 x 10(-7) M) were ineffective on the veratridine-induced response. Nimodipine (10(-5) M) suppressed the veratridine-induced uptake of 45Ca2+ but also diminished the unstimulated uptake of 45Ca2+. The veratridine-induced uptake of Na+ was not influenced by nimodipine. Flunarizine (3 x 10(-6)-10(-5) M), as well as R 56865 (10(-6)-10(-5) M), attenuated the veratridine-induced uptake of both Na+ and 45Ca2+. In conclusion, the veratridine-induced uptake of Na+ and 45Ca2+ was shown to be closely correlated to the activity of Na+ channels but not to voltage-operated Ca2+ channels. Secondly, flunarizine and R 56865 seemed to evoke their effects by interfering with the permeability of Na+ channels. Since veratridine-induced uptake of Na+ and Ca2+ shares some similarities with ischaemia-induced uptake of Na+ and Ca2+, it is proposed, that flunarizine and R 56865 exert their anti-ischaemic effects by reducing ischaemia-induced Na+ and Ca2+ load, probably by inhibiting a TTX-sensitive Na+ channel.     

D2-dopamine receptor-mediated inhibition of intracellular Ca2+ mobilization and release of acetylcholine from guinea-pig neostriatal slices.

The effect of dopamine receptor activation on electrically- or high K+ (30 mM)-evoked neurotransmitter release and rise in intracellular Ca2+ concentration was investigated using slices of guinea-pig neostriatum. A specific D2-dopamine receptor agonist, LY-171555 (a laevorotatory enantiomer of LY-141865: N-propyl tricyclic pyrazole) at 10(-6) M inhibited electrical stimulation- and high K+-evoked release of [3H]-acetylcholine ([3H]-ACh) to 47.7 +/- 6.0% and 54.1 +/- 5.0% of control, respectively. The maximal inhibition by LY-171555 at 10(-5) M was 54.8 +/- 5.1% reduction of the control. The half-maximal effective concentration (EC50) of LY-171555 for the inhibition of [3H]-ACh release was 2.3 X 10(-7) M. A specific D2-dopamine receptor antagonist, (-)-sulpiride (10(-7) M) reversed the inhibition of [3H]-ACh release induced by LY-171555. A specific D1-dopamine receptor agonist, SK&F 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-benzazepine) (10(-5) M) had no effect on the release of [3H]-ACh. LY-171555 (10(-6) M) also inhibited the high K+-evoked endogenous glutamate release, by 47% of control. This inhibitory effect was reversed by (-)-sulpiride (10(-7) M). We used a fluorescent, highly selective Ca2+ indicator, 'quin 2' to measure intracellular free Ca2+ concentrations ([Ca2+]i). Electrical stimulation of slices preloaded with quin 2 led to an elevation of relative fluorescence intensity and this response was reduced by the removal of Ca2+ from the bathing medium. These results indicate that the enhanced elevation in fluorescence intensity in the quin 2-loaded slices reflects the increase of intracellular free Ca2+ concentration, [Ca2+]i. The mixed D1- and D2-receptor agonist, apomorphine and LY-171555 inhibited the increase of [Ca2+]i induced by electrical stimulation or high K+ medium, in a concentration-dependent manner, while SK&F 38393 did not affect the increase of [Ca2+]i. The maximal inhibitory effect of LY-171555 at 3 X 10(-5) M was 35 +/- 3% reduction in control values. The inhibitory effect of LY-171555 was antagonized by (-)-sulpiride (10(-7) M). There was a high correlation (r = 0.997, P less than 0.05) between the D 2-receptor-mediated inhibition of the stimulated rise of [Ca2+]i and [3H]-ACh release. When the slices were superfused with the Ca2+-free medium containing EGTA (10(-4) M) for 5 min, the rise in [Ca2+]i was markedly suppressed to 18.0% of control by LY-171555 (10(-6) M). These data indicate that activation of the D2-dopamine receptor suppresses the elevation of [Ca2+]i induced by depolarizing stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)