Adenylyl Cyclases: mRNA and Characteristics of Enzyme Activity in Three Areas of Brain

Abstract: Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca²⁺-dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca²⁺ ionophore ionomycin. The Ca²⁺ dependence of this effect was established, as removal of Ca²⁺ eliminated evoked release and the lipid-dependent potentiation. Also, Ca²⁺ channel blockers attenuated ionomycin- and KCI-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca²⁺, it may not involve an enhancement of evoked Ca²⁺ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca²⁺ accumulation. Also, the facilitation may not depend on inhibition of K⁺ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCI- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca²⁺ and involves multiple presynaptic effects, some of which depend on protein kinase C.     

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

Ethylenediamine as a Specific Releasing Agent of γ-Aminobutyric Acid in Rat Striatal Slices

Abstract: Following incubation with [¹⁴C]y-aminobutyric acid (GABA) or [³H]dopamine, slices of rat striatum were superfused with media containing 36 mM K⁺ or ethylenediamine (EDA), 1 or 5 mM. Both K⁺ and EDA induced a release of [¹⁴C]GABA, the K⁺-induced release being largely Ca²⁺-dependent, while the EDA-induced release was not. Whereas K⁺ also evoked a Ca²⁺-dependent release of [³H]dopamine, EDA evoked no release of dopamine. EDA may therefore have potential as a specific GABA releasing agent.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.     

Presynaptic Modulation of Cholecystokinin Release by Protein Kinase C in the Rat Hippocampus

Abstract: The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4β-phorbol 12,13-dibutyrate (β-PDBu) dose dependently (5–5,000 n M ) increased CCK-8 release in a strictly Ca²⁺-dependent way. This effect was observed only when synaptosomes were stimulated with the K⁺_A channel blocker 4-aminopyridine (4-AP; 1 m M ) but not with KCI (10–30 m M ). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by α-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. β-PDBu (50–100 n M ) also stimulated the 4-AP-evoked Ca²⁺-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of colocalizing neurotransmitters via modulation of presynaptic K⁺ channels in rat hippocampus.     

Barium-Evoked Glutamate Release from Guinea-Pig Cerebrocortical Synaptosomes

Abstract: Ba²⁺ has multiple effects on presynaptic terminals. The ion inhibits the K⁺ channels responsible for stabilizing the plasma membrane potential in the same way as previously reported for dendrotoxin and 4-aminopyridine. Secondly, the ion can substitute fully for Ca²⁺ in supporting KCl-evoked release of glutamate from guinea-pig cerebrocortical synaptosomes. In the latter case, the kinetics of glutamate release in the presence of saturating Ca²⁺ or Ba²⁺ are essentially identical. Substantially lower external concentrations of Ba²⁺ are required to achieve the same release kinetics as with Ca²⁺. The average internal free Ba²⁺ concentration attained during KCl depolarization is some 10-fold higher than that for Ca²⁺. However, because the fura-2 signal reflects predominantly the overflow of divalent cation after dissociation from the release trigger, it is not the valid parameter to compare effectiveness of the cations in triggering glutamate exocytosis. In view of the established inability of Ba²⁺ to interact with calmodulin, these results are discussed in relation to theories in which Ca²⁺/calmodulin-dependent protein kinase-mediated phosphorylation is a prerequisite for synaptic vesicle exocytosis.     

Oxidative Stress, Hypoxia, and Ischemia-Like Conditions Increase the Release of Endogenous Amino Acids by Distinct Mechanisms in Cultured Retinal Cells

Abstract: The aim of this study was to elucidate the mechanisms by which retinal cells release endogenous amino acids in response to ascorbate/Fe²⁺-induced oxidative stress, as compared with chemical hypoxia or ischemia. In the absence of stimulation, oxidative stress increased the release of aspartate, glutamate, taurine, and GABA only when Ca²⁺ was present. Under hypoxia or ischemia, the release of aspartate, glutamate, glycine, alanine, taurine, and GABA increased mainly by a Ca²⁺-independent mechanism. The increased release observed in N -methyl- d -glucamine⁺ medium suggested the reversal of the Na⁺-dependent amino acid transporters. Upon oxidative stress, the release of aspartate, glutamate, and GABA, occurring through the reversal of the Na⁺-dependent transporters, was reduced by about 30%, although the release of taurine was enhanced. An increased release of [³H]arachidonic acid and free radicals seems to affect the Na⁺-dependent transporters for glutamate and GABA in oxidized cells. All cell treatments increased [Ca²⁺]_i (1.5 to twofold), although no differences were observed in membrane depolarization. The energy charge of cells submitted to hypoxia or oxidative stress was not changed. However, ischemia highly potentiated the reduction of the energy charge, as compared with hypoglycemia or hypoxia alone. The present work is important for understanding the mechanisms of amino acid release that occur in vivo upon oxidative stress, hypoxia, or ischemia, frequently associated with the impairment of energy metabolism.     

Adenosine A1 Receptor Inhibition of Glutamate Exocytosis and Protein Kinase C-Mediated Decoupling

Abstract: The adenosine modulation of glutamate exoeytosis from guinea pig cerebrocortical synaptosomes is investigated. Endogenously leaked adenosine is sufficient to cause a partial tonic inhibition of 4-aminopyridine-evoked glutamate release, which can be relieved by adenosine deaminase. The adenosine A₁ receptor is equally effective in mediating inhibition of glutamate exocytosis evoked by 4-aminopyridine (where K⁺-channel activation would inhibit release) and by elevated KC1 (where K⁺-channel activation would have no effect), arguing for a central role of Ca²⁺-channel modulation. In support of this, the plateau phase of depolarization-evoked free Ca²⁺ elevation is decreased by adenosine with both depolarization protocols. No effect of adenosine agonists is seen on membrane potential in polarized or KC1- or 4-aminopyridine-stimulated synaptosomes. The interaction of protein kinase C with the A₁ receptormediated inhibition is examined. Activation of protein kinase C by 4β-phorbol dibutyrate has been shown previously by this laboratory to modulate glutamate release via K⁺-channel inhibition, and is shown here to have an additional action of decoupling the adenosine inhibition of glutamate exocytosis.     

Release of Glutamate and Aspartate from CA1 Synaptosomes: Selective Modulation of Aspartate Release by Ionotropic Glutamate Receptor Ligands

Abstract: Synaptosomes prepared from area CA1 of the rat hippocampus were used to determine (a) whether Schaffer collateral-commissural-ipsilateral associational terminals release both aspartate and glutamate in a Ca²⁺-dependent manner when reuptake of released glutamate is minimal and (b) whether autoreceptor mechanisms described in CA1 or hippocampal slices could reflect direct actions of glutamate receptor ligands on the synaptic terminal. When challenged for 1 min with either 25 m M K⁺ or 300 µ M 4-aminopyridine, CA1 synaptosomes released both glutamate and aspartate in a Ca²⁺-dependent manner. The glutamate/aspartate ratio was ∼5:1 in each case. K⁺-evoked glutamate release was unaffected by ligands active at NMDA or ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Unlike glutamate release, the release of aspartate was enhanced by NMDA, and this effect was blocked by d -2-amino-5-phosphonovalerate ( d -AP5). Kainate selectively depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) selectively increased the K⁺-evoked release of aspartate. AMPA enhanced aspartate release, like the antagonist CNQX. When applied in the presence of diazoxide, which blocks the desensitization of AMPA receptors, AMPA and kainate both depressed aspartate release. These findings support the view that Schaffer collateral-commissural-ipsilateral associational terminals release aspartate as well as glutamate and that these two release processes are regulated by different autoreceptor mechanisms.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium

The SV channel encoded by the TPC1 gene represents a Ca²⁺- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca²⁺ signals and cytosolic Ca²⁺ is required for SV channel function, we here studied the Ca²⁺-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca²⁺ sensitivity and Ca²⁺ impermeability. K⁺ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca²⁺ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca²⁺ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca²⁺. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca²⁺ before SV channel activity vanishes and K⁺ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca²⁺ signals resulting in overproduction of jasmonate.     

Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.     

Exocytotic and Nonexocytotic Modes of Glutamate Release from Cultured Cerebellar Granule Cells During Chemical Ischaemia

Abstract: The mechanism of glutamate release from cultured cerebellar granule neurones in response to a chemical model of ischaemia (10 m M 2-deoxyglucose plus 1 m M sodium cyanide) was investigated. In the first 2 min of ischaemia, release of preloaded d -[³H]aspartate could be extensively attenuated by tetanus toxin and bafilomycin A₁ and was dependent on the activation of Ca²⁺ channels sensitive to the "Q" type Ca²⁺ channel antagonist, ω-conotoxin-MVIIC. During this period, ATP/ADP ratios fell rapidly. The extent of release in the first 2 min was comparable to that evoked by 2-min depolarization by 50 m M KCl. Free Ca²⁺ concentrations, determined in neurites and somata, did not increase until after 2 min. The neurite increase in cellular Ca²⁺ precedes that of the cell somata. Release of d -[³H]aspartate was partially inhibited by the NMDA receptor antagonist MK-801, which also delayed the increase in free Ca²⁺ concentration. Prolonging the period of ischaemia to 6 and 10 min produced no further increase in the apparently exocytotic component of release, but initiated an extensive nonexocytotic release of the amino acid. Studies with the synaptic vesicle membrane probe FM1-43 in which released amino acid was removed by superfusion indicated that Ca²⁺-dependent exocytosis was delayed in this system. It is concluded that chemical ischaemia initiates an initial exocytotic followed by nonexocytotic release and that the former is facilitated by NMDA receptor activation. These events occur in cells that are still able to exclude propidium iodide, indicating that cell death has not yet occurred.     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Extracellular Ionic Composition Alters Kinetics of Vesicular Release of Catecholamines and Quantal Size During Exocytosis at Adrenal Medullary Cells

Abstract: The temporal resolution of carbon-fiber microelectrodes has been exploited to examine the plasticity of quantal secretory events at individual adrenal medullary cells. The size of individual quantal events, monitored by amperometric oxidation of released catecholamines, was found to be dependent on the extracellular ionic composition, the secretagogue, and the order of depolarization delivery. Release was observed with either exposure to 60 m M K⁺ in the presence of Ca²⁺ or exposure to 3 m M Ba²⁺ in solutions of different pH, with and without external Ca²⁺. Ba²⁺ was demonstrated to induce Ca²⁺-independent exocytotic release for an extended period of time (>4 min) relative to release induced by K⁺ (∼30 s), which is Ca²⁺ dependent. In all cases, simultaneous changes of intracellular divalent cations, monitored by fura-2 fluorescence, accompanied quantal release and had a similar time course. Exocytosis caused by Ba²⁺ in Ca²⁺-free medium had a larger mean spike area at pH 8.2 than at pH 7.4. When Ba²⁺-induced spikes measured at pH 7.4 were compared, the spikes in Ca²⁺-free medium were found to be broader and shorter but had the same area. Release induced by K⁺ after exposure to Ba²⁺ was comprised of larger quantal events when compared with preceding K⁺ stimulations. Finally, spikes obtained with Ba²⁺ exposure at an extracellular pH of 5.5 had a different shape than those obtained in more basic solutions. These changes in spike size and shape are consistent with the interactions between catecholamines and other intravesicular components.     

Control of Noradrenaline Release from Hippocampal Synaptosomes

Abstract Potassium-evoked tritiated noradrenaline (NA) release from hippocampal synaptosomes was measured with a superfusion method. A single 2-min high-K⁺ pulse released 39% of the vesicular NA by a Ca²⁺-dependent mechanism; the Ca²⁺-independent release was negligible. After changing the vesicular NA store size by pretreating rats with either α-methyl-para-tyrosine, 500 mg/kg, or tranylcypromine, 10 mg/kg, a single K⁺ pulse released a constant percentage of the vesicular NA. With two K⁺ pulses, however, there was a reduction in the percentage of vesicular N A released in response to the second pulse.     

Extracellular Calcium Has Distinct Effects on Fast and Slow Components of the Depolarization-Induced Secretory Response from Chromaffin Cells

Abstract: An increase in extracellular Ca²⁺ concentration from 0.25 to 10 m M enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K⁺ concentration (75 m M ). The increment in extracellular Ca²⁺ concentration also increased the observed peak inward Ca²⁺ current in response to long (10-s) depolarizing pulses from a holding potential of −55 mV to +5 mV, from about −26 to −400 pA. However, the total amount of Ca²⁺ influx into the cell only increased when the extracellular Ca²⁺ concentration was raised from 0.25 to 1 m M and then remained constant up to 10 m M extracellular Ca²⁺. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 m M extracellular Ca²⁺, of ∼4 and 41 s, being the fast component of secretion produced by the exocytosis of ∼220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca²⁺ concentration.