Depolarization-induced Release Research Articles

Alterations in Ca 2+-dependent and Ca 2+-independent release of catecholamines in preparations of rat brain produced by ethanol treatment in vivo

Compared to preparations from control animals, superfused striatal slice preparations from brains of rats treated chronically with ethanol released a significantly greater fraction of stored [ 3H] dopamine on depolarisation in 40 mM K +. Similarly, the electrically-evoked release of [ 3H]-norepinephrine from cortical slices and of [ 3H]-dopamine from striatal slices is also increased, although with this mechanism of depolarisation the change is significant only in the case of [ 3H] norepinephrine release. In contrast to this tendency to enhancement of Ca 2+-dependent depolarisation-induced release, a reduced fraction of stored [ 3H]-catecholamines was released from these preparations by the indirect sympathomimetics tyramine and (+)-amphetamine. The catecholamine release induced by these indirect sympathomimetics is largely independent of external Ca 2+ and the results are interpreted as suggesting that chronic alcohol treatment changes the distribution of catecholamine neurotransmitters between storage pools in the nerve terminal which do or do not require Ca 2+ entry for release.

ENHANCED [ <sup>3</sup> H]-NORADRENALINE RELEASE IN SYNAPTOSOMES FROM ETHANOL-TOLERANT ANIMALS: THE ROLE OF NERVE TERMINAL CALCIUM ION CONCENTRATIONS

Cortical slices from brains of ethanol-tolerant rats have previously been shown to release a greater fraction of uptake [3H]-noradrenaline ([3H]-NA) on K+-depolarisation than slices from control animals. In the present experiments, when stimulated with the Ca2+ ionophore A23187 (30 microM), untreated cortical synaptosomes release only a very small fraction of uptaken [3H]-NA. Under these conditions synaptosomal preparations from ethanol-tolerant animals released a greater fraction of uptaken [3H]-NA but the difference was not significant. However, after incubation with ouabain (100 microM, to increase intrasynaptosomal [Ca2+]) A23187 now produced a much greater enhancement of [3H]-NA release in preparations from ethanol-tolerant rats. Further, after superfusion of the synaptosomal preparation with 1mM EGTA and A23187 for 30 min (a procedure which should reduce intrasynaptosomal [Ca2+]) the reintroduction of Ca2+ to the superfusing fluid caused a marked release of [3H]-NA which was also significantly greater in preparations from ethanol-tolerant animals. Ca2+/Mg2+-ATPase activity was also higher in these synaptosomes but no difference could be detected in the release of [3H]-NA from a combined synaptic vesicle: synaptic plasma membrane preparation. The results suggest that the development of ethanol tolerance is associated with a fundamental change in the dynamic control of Ca2+ concentrations within the nerve terminal which potentiates the depolarisation-induced release of neurotransmitters.

Presynaptic muscarinic receptor activation enhances striatal dopamine release evoked by depolarization but not that induced by non-depolarizing stimuli

The release of [ 3H]dopamine stimulated by depolarization with 15 mM KCl of superfused rat striatal synaptosomes was potentiated by acetylcholine through the activation of presynaptic muscarinic receptors. In contrast, acetylcholine did not potentiate the release of [ 3H]dopamine elicited by d-amphetamine nor that caused by the calcium ionophore A23187. The dopamine carrier blocker nomifensine prevented the releasing action of amphetamine but not that of acetylcholine. The results suggest that the activation of muscarinic receptors on dopamine terminals in the rat corpus striatum selectively affects the calcium-dependent depolarization-induced release of the [ 3H]catecholamine. Moreover, the [ 3H]dopamine release caused by acetylcholine seems to occur independently of the membrane dopamine carrier.

Inhibitors of metalloendoprotease activity prevent K+-stimulated neurotransmitter release from the retina of Xenopus laevis

Metalloendoprotease activity was identified in retinal homogenates using a synthetic fluorogenic metalloendoprotease substrate and specific metalloendoprotease inhibitors. The requirement of metalloendoprotease activity in neurotransmitter release was examined during the depolarization-induced release of [3H]glycine from the retina of Xenopus laevis. Neurons with high affinity uptake and calcium-dependent, K+-stimulated release of glycine have been described previously in this retina. When isolated retinas preloaded with [3H]glycine are depolarized by 22 mM K+, the usual efflux of [3H]glycine is completely abolished by the metalloendoprotease inhibitor, 1,10-phenanthroline (100 micrograms/ml). The inhibition of [3H]glycine release by 1,10-phenanthroline is dose dependent; furthermore, 1,10-phenanthroline blocks release by chelating metal and not Ca2+, since addition of equimolar calcium does not alter the inhibition. The metalloendoprotease inhibitor, carbobenzoxy (CBZ)-L-phenylalanine, also prevents release, whereas the amino acid, L-phenylalanine, has no effect. Synthetic carbobenzoxy dipeptide amides which are metalloendoprotease substrates (e.g., CBZ-Gly-Leu-amide, CBZ-Ser-Leu-amide, and CBZ-Gly-Phe-amide) also prevent [3H]glycine release in a dose-dependent and reversible manner. The synthetic dipeptide CBZ-Gly-Gly-amide, however, is not a metalloendoprotease substrate and has no effect on release. The ability of synthetic dipeptides to inhibit neurotransmitter release is amino acid specific, dose dependent, reversible, and matches their ability to interact with characterized metalloendoproteases. Depolarization-stimulated transmitter release may therefore require the activity of a metalloendoprotease.

Effects of antidepressants on the uptake and release of norepinephrine from rat cerebral cortex.

The effects of several antidepressants on the release of (3H)-norepinephrine (NE) from homogenates of rat cerebral cortex were studied. A continuous superfusion collection system was used in order to differentiate these effects from effects on reuptake. Amitriptyline, maprotiline, mianserin, and trazodone produced a statistically significant decrease in spontaneous tritium efflux when present in the superfusion medium at a concentration of 1.0 microM. The other antidepressants studied had no effect. We used a buffer with the K+ concentration raised to 56 mM as a model of depolarization-induced release. Desipramine, fluoxetine, and iprindole (again at 1.0 microM) caused a significant decrease in this measure. These results indicate that some of both the tricyclic and atypical antidepressants may alter spontaneous or depolarization-induced release of NE.

The neurobiology of vascular head pain.

Nervous connections between the trigeminal ganglia and cerebral blood vessels have recently been identified in experimental animals and have been termed the trigeminovascular system. Existence of this system in humans is inferential. Trigeminovascular neurons and their peripheral unmyelinated nerve fibers contain the neurotransmitter peptide substance P. Most newly synthesized substance P is transported from ganglion cell bodies to afferent nerve fibers, where depolarization-induced release of neurotransmitter into the wall of the cerebral blood vessel occurs. Substance P dilates pial arteries, increases vascular permeability, and activates cells that participate in the inflammatory response. The relationship of trigeminovascular fibers to the pathogenesis of vascular head pain sheds light on possible mechanisms of migraine and other central nervous system conditions associated with headache and inflammation.

Effect of a sulfonium analog of dopamine on the depolarization-induced release of [ 3H]acetylcholine from mouse striatal slices

We have synthesized a chemical analog or dopamine in which the amino group has been replaced by a charged dimethylsulfonium group. The dopaminergic activity of this drug was evaluated by determining its ability to inhibit the depolarization-evoked release of [ 3H]acetylcholine from mouse striatal slices. The slices were preincubated with [ 3H]choline (0.1 μM) and then superfused in physiological medium. [ 3H]Acetylcholine release was induced by exposure of the slices to a high potassium medium (12.5 mM) for 5 min. The sulfonium analog of dopamine, dopamine, and apormorphine inhibited the evoked [ 3H]acetylcholine release with IC 50 values of approximately 10, 2.0, and 0.3 μM respectively. The inhibition by the sulfonium analog was reversed by fluphenazine (1 μM), suggesting that the inhibition of [ 3H]acetylcholine release was due to the activation of dopaminergic receptors. The sulfonium analog also inhibited the uptake of [ 3H]dopamine into striatal slices and caused the release of exogenously taken up [ 3H]dopamine from these slices. The release of [ 3H]dopamine by the sulfonium analog was inhibited by cocaine (3 μM), suggesting that the drug-induced release of [ 3H]dopamine was dependent on the carrier-mediated uptake of the sulfonium analog into dopaminergic neurons. The inhibition of the evoked [ 3H]acetylcholine release by high concentrations (30 and 60 μM) of the sulfonium analog did not appear to be mediated by endogenous dopamine release, since the analog still inhibited [ 3H]acetylcholine release from slices after reserpine-α- methyl-p- tyrosine treatment, However, the inhibitory effect of the sulfonium analog at 10 μM was reduced by reserpine-α- methyl-p- tyrosine treatment, suggesting that the inhibition at lower concentrations was mediated through endogenous DA release. These results suggest that a charged compound can act as a substrate for the dopamine carrier and can activate the dopamine receptor regulating acetylcholine release. They also indicate that the nitrogen on the dopamine molecule is not essential for dopamine agonist activity.

Comparison of two superfusion systems for study of neurotransmitter release from rat cerebral cortex slices.

Depolarization-induced release of [3H]gamma-aminobutyric acid (GABA) and [3H]noradrenaline (NA) from rat cerebral cortex slices was studied in two superfusion systems: one with stationary and the other one with continuously shaken slice compartments. Calcium-dependent depolarization-induced release of GABA and NA could be demonstrated only with shaken slices. GABA, but not NA, could also be released by high K+ media and veratridine from stationary slices. Synaptic transmitter releasing mechanisms are apparently damaged in stationary slices, possibly due to impaired energy metabolism.

Stimulation-induced uptake and release of zinc in hippocampal slices.

The mossy-fibre axons of the hippocampus form a dense plexus, uniquely rich in chelatable zinc. Because the metal is apparently concentrated within the terminal bags of the axons, it has been hypothesized that the zinc is involved in mossy-fibre synaptic transmission. Although some electrophysiological findings have favoured the hypothesis, neither preferential uptake of zinc into the hippocampus nor depolarization-induced release of zinc from hippocampal tissue has previously been found. Using the hippocampal slice preparation, we now report that the mossy-fibre neuropil and cells of origin (dentate granule cells) take up zinc preferentially, and that electrical stimulation selectively facilitates both uptake of exogenous zinc into mossy-fibre neuropil and release of previously incorporated 65Zn from the tissue. The results suggest that the role of zinc in mossy-fibre axons is dynamically linked to neural signalling processes.

Amino acid transport by synaptosomes isolated from post mortem human brain.

Synaptosomes isolated from post mortem human brain release the transmitter amino acids upon depolarisation. Active preparations can be isolated for up to 24 hours post mortem. The amount of depolarisation-induced release of the transmitter amino acids is correlated with the rate of oxygen uptake. Only when this correlation is taken into consideration would it be possible to detect disease-related changes in presynaptic amino-acid transport systems.

K +-induced thyrotropin-releasing hormone release from superfused mediobasal hypothalami in rats. Inhibition by somatostatin

Somatostatin (SRIF), in concentration of 10 −6 M, significantly inhibited the depolarization-induced release of immunoreactive thyrotropin-releasing hormone (IR-TRH) from superfused mediobasal hypothalami (MBH) containing mainly the median eminence (ME), without affecting the basal release of TRH. The total amount of K +-induced TRH release was 0.24 ± 0.02 and 0.61 ± 0.08 pg/MBH/min, respectively, in the presence and absence of SRIF in the medium. The data are consistent with a role of SRIF as a neuromodulator on TRH release from the ME. In contrast, superfusion with Locke medium containing triiodothyronine (10 −6 M) had no effect on basal and K +-induced IR-TRH release in our system.

Depolarization-induced release of l-glutamic acid from isolated-resealed synaptic membrane vesicles

l-Glutamic acid actively loaded into resealed brain synaptic membrane vesicles was rapidly released into the incubation medium following the introduction of KCl and CaCl 2, or nigericin, or veratridine into the external medium. The KCl-induced release was enhanced by the presence of low (0.1 mM), extravesicular [Ca 2+]. Neither the KCl-induced nor the veratridine-stimulated l-glutamate efflux were carrier-mediated processes. Finally, the KCl-stimulated l-glutamate efflux was dependent on the ratio of intra- to extravesicular [K +]. The observations described in this study were indicative of depolarization-induced l-glutamate release from isolated synaptic plasma membrane vesicles.

Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures

Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum, for 2,5,8 or 12 days in vitro (DIV), in conditions giving a purity > 90%. The results obtained can be summarized as follows: (1) Light microscopic autoradiography showed that cultured granule cells and their processes can accumulate the glutamate analog [ 3H] d-aspartate once they have reached an advanced degree of morphological differentiation (8 and 12 DIV), but, even then, only a limited number of cells was heavily labeled. In contrast, astrocytes were heavily labeled at all stages. (2) Calcium-dependent, high [K +]-induced release, or tetrodotoxin-sensitive, veratridine-induced release of [ 3H] d-aspartate from granule cell-enriched cultures was detectable only in cultures of 8 or 12 DIV. (3) When subject to 3 consecutive depolarizations, cultured granule cells maintained their ability to release [ 3H] d-aspartate and endogenous glutamate almost unchanged. (4)_Newly synthesized [ 3H]glutamate was autoradiographically localized in both neurons and astrocytes (the latter, however, were not preferentially labeled as with [ 3H d-aspartate), but was specifically release from neuronal structures (perikarya and processes) by depolarizing stimuli.

γ-Aminobutyrate as an autonomic neurotransmitter: Release and uptake of [ 3H]γ-Aminobutyrate in guinea pig large intestine and cultured enteric neurons using physiological methods and electron microscopic autoradiography

To provide further evidence that some enteric neurons use γ-aminobutyrate (GABA) as a neurotransmitter, we have demonstrated a depolarization-induced release of [ 3H]GABA from isolated myenteric ganglia in culture, and from segments of large intestine containing the myenteric plexus. In addition, light and electron microscopic autoradiography has been employed to visualize the putative GABAergic neurons and their projections, both in cultured ganglia and in sections from the gut wall. Explant cultures of the guinea-pig myenteric plexus, containing only neurons and glia intrinsic to the gut, were incubated with 0.14μ M [ 3H]GABA, washed and then repeatedly depolarized by 62 mM K +. The depolarizations always evoked release of [ 3H]GABA. The evoked release was reversibly blocked by 5mM Co 2+, suggesting a dependence on Ca 2+ influx, a characteristic of neuronal transmitter release. Strips of the guinea-pig taenia coli, containing the myenteric plexus, were incubated with 0.14 or 0.7μM [ 3H]GABA, washed and subjected to electrical field stimulation. This caused release of [ 3H]GABA, which could be evoked successively on repeated stimulation. The release was of neuronal origin and Ca 2+ dependent, since it was abolished by 3μ M tetrodotoxin and reversibly blocked by 10 mM Co 2+. By combined electrophoresis and chromatography the released tritium was identified as being attached to GABA. Autoradiography, following incubation with low concentrations of [ 3H]GABA, was used to identify specifically putative GABAergic neurons. Light microscopic autoradiography of cultured ganglia, and electron microscopic autoradiography of sections from the taenia coli including the myenteric plexus, were in good agreement, showing a selective and heavy labelling over a sub-population of neurons, and light labelling over glial cells. The majority of neurons and the non-neural cells were unlabelled. The electron microscopic autoradiographs also showed heavy labelling over some, but not all, axons in the fine axon bundles that innervate the longitudinal muscle of the taenia. These results strongly support our previous suggestion that a population of myenteric neurons are GABAergic, although more work is needed before this is fully established. Our data also suggest that some of the putative GABAergic neurons project out to the gut musculature, where their axons, together with axons from other neurons, run in bundles to form the intramuscular nerves. We therefore propose that one of the roles of these neurons in the gut is the presynaptic modulation of transmitter released from neighbouring axons in these nerves.

Variations in hypothalamic luteinizing hormone releasing hormone content and release in vitro and plasma concentrations of luteinizing hormone and testosterone in developing cockerels.

An in-vitro superfusion system was used to study age-dependent changes in the functional activity of LH releasing hormone (LHRH) neurones terminating in the mediobasal hypothalamus (MBH) of the cockerel. Fragments of MBH tissue were obtained from cockerels killed at 8, 12, 16 and 20 weeks of age and both the rate of release of LHRH in vitro and the residual content of LHRH were determined by radioimmunoassay. Blood was collected from a similar group of cockerels of the same age for determination of plasma LH and testosterone concentrations. Superfused cockerel MBH showed both basal and depolarization-induced release of LHRH and the calcium-dependency of the release process was demonstrated. The viability of MBH in vitro was indicated by the observation that the rate of CO2 production by the tissue remained constant during a 3-h period of superfusion. Both the basal rate of release of LHRH (7.24 +/- 0.63 pg/2MBH per h; mean +/- S.E.M., n = 4) and the residual content of LHRH (1.10 +/- 0.24 ng/2MBH) were lowest in the 8-week-old cockerel and increased progressively to reach levels three (P less than 0.001) and 13 (P less than 0.001) times greater, respectively, by 20 weeks of age. Concentrations of LH and testosterone in plasma did not increase significantly until 16 weeks of age when the respective values were 5.86 +/- 0.37 micrograms/l and 1.88 +/- 0.31 nmol/l (n = 11). Whereas plasma testosterone increased further to 5.76 +/- 0.42 nmol/l (n equal 11) at 20 weeks of age, plasma LH fell significantly (P less than 0.02) to a concentration of 4.08 +/- 0.41 micrograms/l (n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)

Muscimol Potentiation of Acidic Amino Acid Release from Cerebellar Synaptosomes Is Chloride Dependent

In previous studies we have shown that the depolarization-induced release of preaccumulated acidic amino acids and newly synthesized glutamate from cerebellar synaptosomal preparations is potentiated by gamma-aminobutyric acid (GABA) agonists through a GABAergic presynaptic mechanism. Here we report a systematic analysis of the ionic requirements of the potentiating effect of muscimol on the high K+-evoked release of D-[3H]aspartate. Our studies show that: Ca2+, Na+, and Mg2+ are not required for muscimol to exert its effect; a depolarizing concentration of K+ is a necessary, but not sufficient, condition to observe the presynaptic effect in question; and a minimal Cl- concentration (50-70 mM) is also required. A possible model based on these findings is proposed.

L-Glutamic acid, a neuromodulator of dopaminergic transmission in the rat corpus striatum

A superfusion system was used to study the effects of neuroexcitatory amino acids upon spontaneous and depolarization-evoked release of exogenously taken up and newly synthesized [ 3H]dopamine by rat striatal slices. Neither l-glutamate nor other aminoacids such as l-aspartate and d-glutamate (5 × 10 −5 M) modified the spontaneous release of exogenous [ 3H]dopamine from rat striatal slices. In contrast, these neuroexcitatory aminoacids did potentiate spontaneous release of striatal [ 3H]dopamine newly synthesized from [ 3H]tyrosine. A different pattern of effects emerged when depolarization-evoked release of dopamine was studied. Only l-glutamate (5 × 10 −6-1 × 10 −4 M) potentiated dopamine release under these experimental conditions in a rather specific and stereoselective manner. In addition, similar results were obtained regardless of whether depolarization-induced release of exogenous or newly synthesized [ 3H]dopamine was studied. The effect of l-glutamate on depolarization-induced release depended both upon the degree of neuronal depolarization and upon the presence of external Ca 2+ in the superfusion medium and it was blocked by l-glutamate diethylester. Furthermore, this effect of l-glutamate seemed quite specific with regard to regional localization within the brain as it was only demonstrated in slices from striatum and not in slices from olfactory tubercle or hippocampus. It is suggested that during depolarization a Ca 2+-dependent event occurs at the striatal membrane level which changes the sensitivity of the dopamine release process to neuroexcitatory aminoacids in such a way as to render it relatively more specific and stereoselective towards l-glutamate stimulation. The findings reported have led us to propose that l-glutamic acid could play a role as a neuromodulator of dopaminergic transmission in the rat corpus striatum.

Substrate specificity and developmental aspects of a presynaptic GABA receptor regulating glutamate release in the rat cerebellum.

In order to better characterize the presynaptic GABA receptors regulating glutamate release in the cerebellum [Levi and Gallo, 1981], a number of GABA agonists and GABA transport inhibitors were tested for their ability to potentiate the depolarization-induced release of the glutamate analog D-[3H]aspartate from superfused cerebellar synaptosomes. Of all the compounds tested, only those which are known to interact specifically with GABA receptors were effective when tested on synaptosomal preparations. The order of effectiveness found was the following: muscimol congruent to 3-APS greater than or equal to P4S greater than isoguvacine greater than THIP. GABA uptake inhibitors were unable to enhance D-[3H]aspartate evoked release from synaptosomes, but were effective when tested in cerebellar slices; in the latter case, the activation of the GABA receptors may be achieved indirectly, through an increase of the extracellular GABA concentrations. The substrate specificity of the presynaptic GABA receptors regulating cerebellar acidic amino acid release appears to be similar to that reported for GABA receptors in radioligand binding studies and for GABA autoreceptors. Studies on synaptosomes from immature cerebella suggested that the presence of the potentiating effect on the acidic amino acid release by GABA agonists is correlated with the development of the parallel fiber terminals, which are believed to be the main site from which glutamate is released in the adult cerebellum.

Depolarization-induced release of [ 3H]histamine by high potassium concentrations, electrical stimulation and veratrine from rat brain slices after incubation with the radiolabelled amine

Brain slices obtained from neocortex, hypothalamus or hippocampus were incubated with [ 3H]histamine and subsequently superfused and exposed to different depolarizing stimuli, viz. high K +-concentrations, electrical field stimulation and veratrine. K +-induced release of tritium was completely calcium-dependent and its magnitude depended on the K +-concentration, with maximal release being reached at 56 mM K +. Electrically-evoked release of tritium increased with increasing frequencies and reached its maximum at about 20 Hz. The electrically-evoked release appeared to be totally calcium-dependent and it was strongly inhibited by tetrodotoxin. Veratrine (5–100 μM) also induced a release of tritium; maximal release was obtained at 100 μM veratrine. Veratrine-induced release was partially calcium-dependent and was strongly reduced by tetrodotoxin. Taken together the data indicate that the depolarization-induced release of tritium from brain slices pre-labelled with [ 3H]histamine, represents [ 3H]histamine release from neurons and not from either mast cells or glial cells. It remains to be established whether these neurons are specifically histaminergic.

Enhancement of depolarization-induced release of gamma-aminobutyric acid from brain slices by antibodies to ganglioside.

The effect of antibodies to GM1 ganglioside on release of neurotransmitters from rat brain slices was studied. Depolarization-induced (40 mM-KCl or veratrine) release of gamma-aminobutyric acid was markedly enhanced. Depolarization-induced release of norepinephrine was only slightly enhanced, whereas that of serotonin was unaffected. No effect on spontaneous release was observed for any of these three neurotransmitters. These results show that antibodies that can bind to synaptic membrane antigens may alter neurotransmitter release and that antibodies directed against GM1 ganglioside exhibit a measure of specificity in producing such an effect.