Rat Cerebrocortical Synaptosomes Research Articles

A late phase of exocytosis from synaptosomes induced by elevated [Ca2+]i is not blocked by Clostridial neurotoxins.

Treatment of rat cerebrocortical synaptosomes with botulinum toxin types E and C1 or tetanus toxin removed the majority of intact SNAP-25, syntaxin 1A/1B, and synaptobrevin and diminished Ca(2+)-dependent K+ depolarization-induced noradrenaline secretion. With botulinum toxin type E, <10% of intact SNAP-25 remained and K(+)-evoked release of glutamate and GABA was inhibited. The large component of noradrenaline release evoked within 120 s by inclusion of the Ca2+ ionophore A23187 with the K+ stimulus was also attenuated by these toxins; additionally, botulinium neurotoxin type E blocked the first 60 s of ionophore-induced GABA and glutamate exocytosis. However, exposure to A23187 for longer periods induced a phase of secretion nonsusceptible to any of these toxins (>120 s for noradrenaline; >60 s for glutamate or GABA). Most of this late phase of release represented exocytosis because of its Ca2+ dependency, ATP requirement, and sensitivity to a phosphatidylinositol 4-kinase inhibitor. Based on these collective findings, we suggest that the ionophore-induced elevation of [Ca2+]i culminates in the disassembly of complexes containing nonproteolyzed SNAP receptors protected from the toxins that can then contribute to neuroexocytosis.

Modulation of glutamate exocytosis by redox changes of superficial thiol groups in rat cerebrocortical synaptosomes

The treatment of cerebral cortex synaptosomes with the membrane impermeable thiol reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) induces a long-lasting partial inhibition (about 40%) of the KCl-stimulated Ca2+-dependent exocytosis of glutamate. Synaptosomes are not damaged by the treatment. The increase of cytoplasmic free Ca2+ concentration ([Ca2+]i) upon depolarization is not affected by DTNB. The inhibition is observed also if exocytosis is induced with the Ca2+-ionophore ionomycin. In all cases the inhibition is reversed by the impermeable reductant glutathione (GSH). Similarly the inhibition of exocytosis by H2O2 (Zoccarato, F., Valente, M. and Alexandre, A., Hydrogen peroxide induces a long-lasting inhibition of the Ca2+-dependent glutamate release in cerebrocortical synaptosomes without interfering with cytosolic Ca2+. J. Neurochem., 64 (1995) 2552–2558.) is reversed by GSH. It is concluded that redox changes (possibly thiol-disulfide transitions) of superficial groups modulate the exocytotic apparatus directly. In an attempt to identify the protein(s) involved in this novel type of control, we evidenced DTNB (H2O2) reactive bands at 35 and at 85–150 kDa which can be labeled with a monobromotrimethylammoniobimane bromide (qBBr) derivatization.

PhTx4, a new class of toxins from Phoneutria nigriventer spider venom, inhibits the glutamate uptake in rat brain synaptosomes

We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [ 3 H ]- l-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC 50 value obtained was 2.35±0.9 μg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3±0.69%, n=3).

Group I mGlu receptors potentiate synaptosomal [ 3H]glutamate release independently of exogenously applied arachidonic acid

In the current study, we have characterized group I metabotropic glutamate (mGlu) receptor enhancement of 4-aminopyridine (4AP)-evoked [ 3H]glutamate release from rat cerebrocortical synaptosomes. The broad spectrum mGlu receptor agonist (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1 S,3 R)-ACPD, 10 μM) increased 4AP-evoked [ 3H]glutamate release (143.32±2.73% control) only in the presence of exogenously applied arachidonic acid; an effect reversed by the inclusion of bovine serum albumin (BSA, fatty acid free). In contrast, the selective group I mGlu receptor agonist ( S)-3,5-dihydroxyphenylglycine (DHPG) potentiated (EC 50=1.60±0.25 μM; E max=147.61±10.96% control) 4AP-evoked [ 3H]glutamate release, in the absence of arachidonic acid. This potentiation could be abolished by either the selective mGlu 1 receptor antagonist ( R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA, 1 mM) or the selective PKC inhibitor (Ro 31-8220, 10 μM) and was BSA-insensitive. The selective mGlu 5 receptor agonist ( R, S)-2-chloro-5-hydroxyphenylglycine (CHPG, 300μM) was without effect. DHPG (100 μM) also potentiated both 30 mM and 50 mM K +-evoked [ 3H]glutamate release (121.60±12.77% and 121.50±4.45% control, respectively). DHPG (100 μM) failed to influence both 4AP-stimulated 45Ca 2+ influx and 50 mM K +-induced changes in synaptosomal membrane potential. Possible group I mGlu receptor suppression of tonic adenosine A 1 receptor, group II/III mGlu receptors or GABA B receptor activity is unlikely since 4AP-evoked [ 3H]glutamate release was insensitive to the selective inhibitory receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine, ( R, S)-α-cyclopropyl-4-phosphonophenylglycine or CGP55845A, respectively. These data suggest an ‘mGlu 1 receptor-like’ receptor potentiates [ 3H]glutamate release from cerebrocortical synaptosomes in the absence of exogenously applied arachidonic acid. This PKC dependent effect is unlikely to be via modulation of synaptosomal membrane potential or voltage-activated Ca 2+ channels and not via a suppression of tonically active inhibitory adenosine A 1 receptor, group II/III mGlu receptors or GABA B receptors.

Regulation of calcium-dependent [ 3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca 2+-dependent exocytosis of [ 3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca 2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca 2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [ 3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca 2+ ionophore calcimycin (10 μM) induced more extensive [ 3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 μM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [ 3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca 2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca 2+-dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.

P–8 - Maitotoxin-induced elevation of cytosolic free calcium level in rat cerebrocortical synaptosomes in the absence of extracellular calcium

P–8 - Maitotoxin-induced elevation of cytosolic free calcium level in rat cerebrocortical synaptosomes in the absence of extracellular calcium

Bilirubin inhibits Ca2+-dependent release of norepinephrine from permeabilized nerve terminals.

Although the well-known neurotoxic agent bilirubin can induce alterations in neuronal signaling, direct effects on neurotransmitter release have been difficult to demonstrate. In the present study we have used permeabilized nerve terminals (synaptosomes) from rat brain prelabeled with [3H]norepinephrine to examine the effects of bilirubin on transmitter release. Rat cerebrocortical synaptosomes were permeabilized with streptolysin-O (2 U/ml) in the absence or presence of bilirubin (10 microM-320 microM) and Ca2+ (100 microM), and the amount of radiolabeled transmitter released during 5 min to the medium was analysed. Low levels of bilirubin decreased Ca2+-evoked release in a dose-dependent manner, with half-maximal effect at approx 25 microM bilirubin. Higher levels of bilirubin (100-320 microM) increased [3H]norepinephrine efflux in the absence of Ca2+, suggesting that high bilirubin levels induced leakage of transmitter from vesicles. The nontoxic precursor biliverdin had no effect on Ca2+-dependent exocytosis. Our data indicate that bilirubin directly inhibits both exocytotic release and vesicular storage of brain catecholamines.

Effects of intravenous general anesthetics on [3H]GABA release from rat cortical synaptosomes.

Potentiation by general anesthetics of gamma-aminobutyric acid (GABA)-mediated inhibitory transmission in the central nervous system is attributed to GABA(A) receptor-mediated postsynaptic effects. However, the role of presynaptic mechanisms in general anesthetic action is not well characterized, and evidence for anesthetic effects on GABA release is controversial. The effects of several intravenous general anesthetics on [3H]GABA release from rat cerebrocortical synaptosomes (isolated nerve terminals) were investigated. Purified synaptosomes were preloaded with [3H]GABA and superfused with buffer containing aminooxyacetic acid and nipecotic acid to inhibit GABA metabolism and reuptake, respectively. Spontaneous and elevated potassium chloride depolarization-evoked [3H]GABA release were evaluated in the superfusate in the absence or presence of various anesthetics, extracellular Ca2+, GABA receptor agonists and antagonists, and 2,4-diaminobutyric acid. Propofol, etomidate, pentobarbital, and alphaxalone, but not ketamine, potentiated potassium chloride-evoked [3H]GABA release (by 1.3 to 2.9 times) in a concentration-dependent manner, with median effective concentration values of 5.4 +/- 2.8 microM (mean +/- SEM), 10.1 +/- 2.1 microM, 18.8 +/- 5.8 microM, and 4.4 +/- 2.0 microM. Propofol also increased spontaneous [3H]GABA release by 1.7 times (median effective concentration = 7.1 +/- 3.4 microM). Propofol facilitation of [3H]GABA release was Ca2+ dependent and inhibited by bicuculline and picrotoxin, but was insensitive to pretreatment with 2,4-diaminobutyric acid, which depletes cytoplasmic GABA pools. Low concentrations of propofol, etomidate, pentobarbital, and alphaxalone facilitated [3H]GABA release from cortical nerve terminals. General anesthetics may facilitate inhibitory GABA-ergic synaptic transmission by a presynaptic mechanism in addition to their well-known postsynaptic actions.

The alkaloid 6-benzoylheteratisine inhibits voltage-gated Na + channels in rat brain synaptosomes

The effects of the Aconitum alkaloid 6-benzoylheteratisine on the aconitine-, veratridine-, oubain- and KCl-induced alterations in free synaptosomal Na + ([Na +] i) and Ca 2+ ([Ca 2+] i) and the release of endogenous glutamate from rat cerebrocortical synaptosomes were investigated. [Na +] i and [Ca 2+] i were fluorometrically determined employing SBFI and Fura-2 as the Na + and Ca 2+ sensitive dyes, respectively. Glutamate was detected by a continuous enzyme-linked fluorometric assay. The study revealed a concentration-dependent inhibitory effect of 6-benzoylheteratisine on aconitine-induced increases in [Na +] i, [Ca 2+] i and the release of glutamate. The IC 50 values were 4.1 μM (Na +), 4.8 μM (Ca 2+) and 4.8 μM (glutamate release). Application of 100 μM 6-benzoylheteratisine after stimulation with 5 μM veratridine also reduced the induced [Na +] i and [Ca 2+] i with half-lives of 72.1 and 44.7 s, respectively. Furthermore, 100 μM 6-benzoylheteratisine reduced the ouabain-induced Na + influx to the same extent as the Na + channel inhibitor tetrodotoxin, which points to an inhibition of non-activated Na + channels by 6-benzoylheteratisine. Additionally, 100 μM 6-benzoylheteratisine failed to affect the release of glutamate and the increase in [Ca 2+] i induced by 30 mM KCl, indicating that voltage-gated Ca 2+ channels were not affected by 6-benzoylheteratisine. The data suggest an inhibitory effect of 6-benzoylheteratisine on voltage-gated Na + channels as the only target, whereas mechanisms of Na + and Ca 2+ homoeostasis and pathways of glutamate release seem not to be affected by the drug.

Anticonvulsant and glutamate release-inhibiting properties of the highly potent metabotropic glutamate receptor agonist (2 S,2′ R,3′ R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV)

The anticonvulsant effects of intracerebral administration of the highly potent group II metabotropic glutamate receptor agonist, DCG-IV, were tested in fully kindled rats following daily electrical stimulation of the basolateral amygdala. The agonist caused a dose-dependent increase in the generalized seizure threshold (GST) of these seizure susceptible animals within the dose range tested (0.01–1.0 nmol). The estimated GST 100 value (dose causing a 100% increase in GST) for this effect was 0.22 nmol. The anti-seizure activity of DCG-IV was fully inhibited in the presence of the group II metabotropic glutamate receptor antagonist (2 S,1′ S,2′ S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG; 40 nmol), while MCCG alone showed no significant inhibitory effect on seizure activity. DCG-IV also powerfully inhibited depolarization-induced release of [ 3 H ] d-aspartate from rat cerebrocortical synaptosomes, with an IC 50 value of 0.39 μM. In this respect, DCG-IV was approximately 70-fold more potent than the clinically effective anticonvulsant drug lamotrigine (IC 50=27.7 μM), a proposed neurotransmitter release inhibitor known to inhibit glutamate release, also tested in this assay. These findings demonstrate the high potency of DCG-IV as an anticonvulsant agent and confirm a key role for group II metabotropic glutamate receptors in the control of seizure activity via their modulatory action on neuronal glutamate release.

Halothane and Isoflurane Alter Calcium Dynamics in Rat Cerebrocortical Synaptosomes

An increase in synaptosomal Ca2+ triggers neurotransmitter release and volatile anesthetics have been shown to inhibit neurotransmitter release by inhibition of Ca2+ entry.We have examined the effect of isoflurane and halothane on the kinetics of increase and decrease of Ca2+ in rat cerebrocortical synaptosomes ([Ca2+]in). We have also used specific Ca2+ antagonists to examine the role of L-, N-, and P-type Ca2+ channels. Synaptosomal [Ca2+]in was measured spectrofluorometrically using fura-2 as a Ca2+ reporter; Ca2+ transients were initiated by depolarization with 40 mM KCl. We found that <or=to1 minimum alveolar anesthetic concentration halothane and isoflurane decreased peak [Ca2+]in by approximately 40%, that both anesthetics decreased the rate of [Ca2+]in increase and decrease, that specific voltage-dependent calcium channel antagonists had little effect on peak or plateau [Ca2+]in, and that the volatile anesthetics increased the permeability of synaptosomal membranes to Ca2+. These results suggest that the volatile anesthetics, at clinically relevant concentrations, can alter Ca2+ homeostasis in the synapse. Implications: Clinically relevant concentrations of halothane and isoflurane markedly depress K+ -evoked increases in rat cerebrocortical synaptosomal calcium (Ca2+) unrelated to L-, N-, and P-type voltage-dependent calcium channels and increase the Ca2+ permeability of the synaptosomal membrane. These changes in Ca2+ dynamics could have profound effects on Ca2+ signaling in the synapse. (Anesth Analg 1998;87:701-10)

Halothane and isoflurane alter calcium dynamics in rat cerebrocortical synaptosomes.

An increase in synaptosomal Ca2+ triggers neurotransmitter release and volatile anesthetics have been shown to inhibit neurotransmitter release by inhibition of Ca2+ entry. We have examined the effect of isoflurane and halothane on the kinetics of increase and decrease of Ca2+ in rat cerebrocortical synaptosomes ([Ca2+]in). We have also used specific Ca2+ antagonists to examine the role of L-, N-, and P-type Ca2+ channels. Synaptosomal [Ca2+]in was measured spectrofluorometrically using fura-2 as a Ca2+ reporter; Ca2+ transients were initiated by depolarization with 40 mM KCl. We found that < or = 1 minimum alveolar anesthetic concentration halothane and isoflurane decreased peak [Ca2+]in by approximately 40%, that both anesthetics decreased the rate of [Ca2+]in increase and decrease, that specific voltage-dependent calcium channel antagonists had little effect on peak or plateau [Ca2+]in, and that the volatile anesthetics increased the permeability of synaptosomal membranes to Ca2+. These results suggest that the volatile anesthetics, at clinically relevant concentrations, can alter Ca2+ homeostasis in the synapse. Clinically relevant concentrations of halothane and isoflurane markedly depress K+-evoked increases in rat cerebrocortical synaptosomal calcium (Ca2+) unrelated to L-, N-, and P-type voltage-dependent calcium channels and increase the Ca2+ permeability of the synaptosomal membrane. These changes in Ca2+ dynamics could have profound effects on Ca2+ signaling in the synapse.

Ba 2+ does not support synaptic vesicle retrieval in rat cerebrocortical synaptosomes

To investigate whether any specific requirement for extracellular Ca 2+ exists in the control synaptic vesicle retrieval, we examined the ability of the divalent cation Ba 2+ to substitute for Ca 2+ in both vesicle exocytosis and endocytosis. Ba 2+ stimulated glutamate release from rat cerebrocortical synaptosomes. Ba 2+-evoked release was inhibited by bafilomycin A1, indicating release was via exocytosis of synaptic vesicles. However, Ba 2+ did not stimulate vesicle retrieval, monitored by a FM2-10-based retrieval assay. Therefore synaptic vesicle retrieval in central nerve terminals has a specific requirement for extracellular Ca 2+ and the Ca 2+ receptor for retrieval has a different cation specificity to the Ca 2+ receptor for exocytosis.

Investigation of the effect of PhTx2, from the venom of the spider Phoneutria nigriventer, on the release of [ 3H]-acetylcholine from rat cerebrocortical synaptosomes

Venoms from spiders are an important source of toxins that can help on the dissection of mechanisms involved in neurotransmission. Among them is the venom of the spider Phoneutria nigriventer that contains several toxic fractions with different targets in mammals and/or insects. We here report that one of these fractions (PhTx2) is able to evoke acetylcholine release from rat cortical synaptosomes and that this effect is dependent on extracellular calcium and is inhibited by the sodium channel blocker tetrodotoxin.

Presynaptic GABA(B) receptor modulation of glutamate exocytosis from rat cerebrocortical nerve terminals: receptor decoupling by protein kinase C.

GABA and the GABA(B) receptor agonist (-)-baclofen inhibited 4-aminopyridine (4AP)- and KCl-evoked, Ca2+-dependent glutamate release from rat cerebrocortical synaptosomes. The GABA(B) receptor antagonist CGP 35348, prevented this inhibition of glutamate release, but phaclofen had no effect. (-)-Baclofen-mediated inhibition of glutamate release was insensitive to 2 microg/ml pertussis toxin. As determined by examining the mechanism of GABA(B) receptor modulation of glutamate release, (-)-baclofen caused a significant reduction in 4AP-evoked Ca2+ influx into synaptosomes. The agonist did not alter the resting synaptosomal membrane potential or 4AP-mediated depolarization; thus, the inhibition of Ca2+ influx could not be attributed to GABA(B) receptor activation causing a decrease in synaptosomal excitability. Ionomycin-mediated glutamate release was not affected by (-)-baclofen, indicating that GABA(B) receptors in this preparation are not coupled directly to the exocytotic machinery. Instead, the data invoke a direct coupling of GABA(B) receptors to voltage-dependent Ca2+ channels linked to glutamate release. This coupling was subject to regulation by protein kinase C (PKC), because (-)-baclofen-mediated inhibition of 4AP-evoked glutamate release was reversed when PKC was stimulated with phorbol ester. This may therefore represent a mechanism by which inhibitory and facilitatory presynaptic receptor inputs interplay to fine-tune transmitter release.

Specific binding of L-triiodothyronine modulates Na(+)-K(+)-ATPase activity in adult rat cerebrocortical synaptosomes.

Interactions of L-triiodothyronine (T3) in adult rat cerebrocortical synaptosomes were studied in vitro. Scatchard plot analysis revealed two sets of T3 binding sites. The degree of saturation of T3 binding sites (putative receptor) correlated well with the dose-dependent inhibition of Na(+)-K(+)-ATPase activity in synaptosomes. The relative binding affinities and relative inhibition of enzyme activities for different TH analogues were L-T3 > T3-amine > TRIAC = L-T4 > r-T3 > T2 and L-T3 > T3-amine > TRIAC > L-T4 > r-T3 > T2, respectively. The present study demonstrates the nature of inhibition of synaptosomal Na(+)-K(+)-ATPase activity may be as a function of T3 occupancy of synaptosomal receptor sites in adult mammalian brain.

Arachidonic acid potentiates the duration of the metabotropic, protein kinase C-mediated, suppression of the inhibitory adenosine A1 receptor pathway in glutamatergic nerve terminals from rat cerebral cortex

The KCl-evoked exocytotic release of glutamate from rat cerebrocortical synaptosomes is inhibited by a presynaptic adenosine A1 receptor decreasing voltage-activated Ca 2+ entry. This inhibition was transiently suppressed by (1 S,3 R)-1-aminocyclopenthyl-1,3-dicarboxylate (ACPD) but was restored within 1 min in the continued presence of the metabotropic agonist. In the presence of 2 μM arachidonic acid ACPD initiated a prolonged suppression of the adenosine-mediated inhibition persisting for at least 10 min. Arachidonic acid (20–40 pmol) was bound per mg synaptosomal protein. Prolonged ACPD-mediated phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C-kinase substrate (MARCKS) was detected in the presence but not the absence of arachidonic acid, but arachidonic acid added 2 min after ACPD was ineffective. It is concluded that arachidonic acid synergistically prolongs the metabotropic glutamate receptor-mediated activation of presynaptic PKC, suppressing inhibitory receptor pathways.

P-352 - Mechanism of maitotoxin-induced elevation of cytosolic free calcium level in rat cerebrocortical synaptosomes

P-352 - Mechanism of maitotoxin-induced elevation of cytosolic free calcium level in rat cerebrocortical synaptosomes

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Effect of halothane on conventional protein kinase C translocation and down-regulation in rat cerebrocortical synaptosomes

Protein kinase C (PKC) is a key regulatory enzyme that has been implicated as a molecular target for the action of general anaesthetics. We have determined the effects of halothane on the translocation and down-regulation of conventional PKC (cPKC) by analysing the subcellular distribution of PKC activity, [3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding and PKC immunoreactivity in intact rat cerebrocortical synaptosomes, a subcellular fraction that contains functional nerve terminals. Halothane alone (2.4 vol%) reduced membrane-associated (P < 0.05) and increased cytosol (P < 0.01) PKC activity, while phorbol-12-myristate, 13-acetate (PMA) 0.1 mumol litre-1, a metabolically stable activator of PKC, reduced membrane (P < 0.01) without altering cytosol PKC activity. Halothane and PMA in combination reduced membrane PKC activity to undetectable levels and reduced cytosol PKC activity (P < 0.01). Halothane alone had no significant effects on the distribution of [3H]PDBu binding, while PMA alone significantly reduced both membrane and cytosol [3H]PDBu binding (P < 0.01). Halothane and PMA in combination reduced membrane and cytosol [3H]PDBu binding further, but this effect was not significantly different from the effect of PMA alone. Experiments using isoform-selective antibodies to PKC alpha, PKC beta or PKC gamma demonstrated synergistic interactions between halothane and PMA in promoting translocation of the three conventional PKC isoforms from the cytosol to the membrane fraction of synaptosomes and down-regulation of their immunoreactivity. Halothane and PMA together reduced cytosol PKC alpha/beta/gamma immunoreactivity significantly more (P < 0.05) than PMA alone. Halothane thus has two distinct actions on PKC in synaptosomes: activation of endogenous PKC activity and potentiation of activation-induced cPKC translocation and down-regulation. These potentially competing effects may underlie some of the conflicting results obtained with halothane on PKC-mediated processes in intact cells.