Leu27 insulin-like growth factor-II, an insulin-like growth factor-II analog, attenuates depolarization-evoked GABA release from adult rat hippocampal and cortical slices.

Accumulated evidence suggests that the single transmembrane domain insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6P or IGF-II receptor) plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of insulin like growth factor (IGF-II). However, the role of this receptor in signal transduction following IGF-II binding remains controversial. In the present study, we revealed that Leu(27)IGF-II, an analog which binds preferentially to the IGF-II receptor, can attenuate K(+)-as well as veratridine-evoked GABA release from the adult rat hippocampal formation. Tetrodotoxin failed to alter the effects of Leu(27)IGF-II on GABA release, thus suggesting the lack of involvement of voltage-dependent Na(+) channels. Interestingly, the effect is found to be sensitive to pertussis toxin (PTX), indicating the possible involvement of a Gi/o protein-dependent pathway in mediating the release of GABA from the hippocampal slices. Additionally, Leu(27)IGF-II was found to attenuate GABA release from frontal cortex but not from striatum. These results, together with the evidence that IGF-II receptors are localized on GABAergic neurons, raised the possibility that this receptor, apart from mediating intracellular trafficking, may also be involved in the regulation of endogenous GABA release by acting directly on GABAergic terminals.

Mitochondrial Na+/Ca2+-exchanger blocker CGP37157 protects against chromaffin cell death elicited by veratridine.

Mitochondrial calcium (Ca(2+)) dyshomeostasis constitutes a critical step in the metabolic crossroads leading to cell death. Therefore, we have studied here whether 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; CGP), a blocker of the mitochondrial Na(+)/Ca(2+)-exchanger (mNCX), protects against veratridine-elicited chromaffin cell death, a model suitable to study cell death associated with Ca(2+) overload. Veratridine produced a concentration-dependent cell death, measured as lactate dehydrogenase released into the medium after a 24-h incubation period. CGP rescued cells from veratridine-elicited death in a concentration-dependent manner; its EC(50) was approximately 10 microM, and 20 to 30 microM caused near 100% cytoprotection. If preincubated for 30 min and washed out for 3 min before adding veratridine, CGP still afforded significant cytoprotection. At 30 microM, CGP blocked the veratridine-elicited free radical production, mitochondrial depolarization, and cytochrome c release. At this concentration, CGP also inhibited the Na(+) and Ca(2+) currents by 50 to 60% and the veratridine-elicited oscillations of cytosolic Ca(2+). This drastic cytoprotective effect of CGP could be explained in part through its regulatory actions on the mNCX.

Organic and inorganic calcium antagonists inhibit veratridine-induced epileptiform activity in CA3 neurons of the guinea pig.

Veratridine is believed to cause epileptiform discharges via its effects on sodium channels. We addressed the question whether calcium currents, known to contribute to the generation of paroxysmal depolarization shifts (PDS) in most models of epilepsies, also contribute to veratridine-induced epileptiform activity. Therefore, we recorded from CA3 neurons (n=50) of veratridine-treated hippocampal slices and analyzed the effects of two calcium antagonists. Veratridine (0.5-1.0 microM) elicited spontaneous epileptiform bursts, paroxysmal depolarization shifts (PDS) lasting 100-300 ms, and depolarizations (LD) lasting up to several minutes. Most often PDS directly preceded LD which resulted in typical composite depolarizations termed veratridine-induced complexes (VC). VC persisted even in the presence of CNQX and APV (25 micromol/l, both), or in nominally calcium-free saline, revealing the non-synaptic nature of these potentials. Cobalt (1-2mM) abolished VC within minutes, but allowed LD type-like potentials to be elicited by depolarizing current pulses. Verapamil (50 microM) also diminished or abolished amplitudes of VC. All inhibitory effects of cobalt and verapamil were at least partly reversible. Due to the effects of both calcium antagonists we conclude that veratridine-induced epileptiform activity depends not only on sodium, but also on calcium currents.

Depolarization preconditioning produces cytoprotection against veratridine-induced chromaffin cell death.

The hypothesis that K(+) channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K(+) solutions or with tetraethylammonium (TEA), a K(+) channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K(+) (20-80 mM) or TEA (10-30 mM) for 24 h caused 20-60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca(2+) ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K(+) or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca(2+) signal. High K(+) shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K(+) channels that depolarize the cell and cause a cytosolic Ca(2+) signal, leading to enhanced expression of BDNF and Bcl-2.

Neuronal electrical high frequency stimulation enhances GABA outflow from human neocortical slices.

Electrical high frequency stimulation of the globus pallidus internus or the subthalamic nucleus has beneficial motor effects in advanced Parkinson's disease. The mechanisms underlying these clinical results remain, however, unclear. From previous studies it is proposed that the gamma-aminobutyric acid (GABA) system is involved in the effectiveness of electrical high frequency stimulation. In these experiments, human neocortical slices were stimulated electrically (130 Hz) in vitro, and GABA outflow was measured after o-phthaldialdehyde sulphite derivatization using HPLC with electrochemical detection. Our results could demonstrate that high frequency stimulation (HFS) significantly increased basal GABA outflow in the presence of submaximal concentrations of the voltage-gated sodium channel opener veratridine. This effect could be abolished by the GABA antagonists bicuculline or picrotoxin. These results suggest that HFS has an activating effect on GABAergic neuronal terminals in human neocortical slices, depending on sodium and chloride influx. Since GABA plays a role in CNS disorders of basal ganglia, anxiety and epilepsy, its neocortical modulation by HFS may be (patho)physiologically relevant.

Ability of some plant extracts, traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators.

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.

PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, elicits its function in primary neuronal cultures by up-regulating Bcl-2 expression.

Neurotoxicity in primary neurons was induced using hypoxia/hypoglycemia (H/H), veratridine (10microM), staurosporine (1microM) or glutamate (100microM), which resulted in 72%, 67%, 75% and 66% neuronal injury, respectively. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811; 10microM; Panacea Pharmaceuticals, Gaithersburg, MD) pretreatment for 24 h provided maximal neuroprotection of 89%, 42%, 47% and 89% against these toxicities, respectively. Glutamate or H/H treatment of cells increased cytosolic cytochrome c levels, which was blocked by pretreatment of cells with PAN-811. Pretreatment of neurons with PAN-811 produced a time-dependent increase in the protein level of Bcl-2, which was evident even after glutamate or H/H treatments. An up-regulation in the expression of the p53 and Bax genes was also observed following exposure to these neurotoxic insults; however, this increase was not suppressed by PAN-811 pretreatment. Functional inhibition of Bcl-2 by HA14-1 reduced the neuroprotective efficacy of PAN-811. PAN-811 treatment also abolished glutamate or H/H-mediated internucleosomal DNA fragmentation.

Antagonism by local anesthetics of sodium channel activators in the presence of scorpion toxins: two mechanisms for competitive inhibition.

1. The interaction of veratridine (VTD), a Na+ channel activator, scorpion alpha-toxin (LQ), an open state Na+ channel stabilizer, and the local anesthetic, lidocaine (LID), a channel inhibitor, at the neuronal sodium channel was assessed by measuring VTD-dependent slow depolarizations of frog sciatic nerve using the sucrose-gap method. 2. The slow depolarizing action of veratridine was potentiated more than 10-fold by the peptide LQ toxin, whereas its competitive inhibition by lidocaine was unchanged by LQ. 3. We conclude that the antagonism between VTD and a LID molecule during slow depolarization is allosteric, involving a trapping of the Na+ channel by LID in the inactivated state that has a very low affinity for VTD. 4. The binding of VTD to the open state of the channel, which is stabilized by LQ, may be inhibited by orthosteric competition at overlapping sites since both LID and VTD bind avidly and rapidly to open channels.

A novel toxicity-based assay for the identification of modulators of voltage-gated Na+ channels.

Voltage-gated Na+ channels are promising drug targets. Screening of large numbers of putative modulators, however, can be demanding and expensive. In this study, a simple, cheap, and robust assay to test the pharmacological modulation of Na+ channel function is presented. The assay makes use of the fact that the intracellular accumulation of Na+ ions can be cytotoxic. The toxicity of the Na+ channel activator veratridine in the presence of an inhibitor of the Na+/K+ ATPase (ouabain) in a Nav1.2a (rat brain IIA alpha) expressing cell line is assessed. Na+ channel blockers should reduce toxicity in this model. CHO cells which recombinantly expressed rat Nav1.2a subunits were seeded in 96-well plates, and cell survival was tested after 24 h incubation in medium containing veratridine and ouabain in the presence or absence of Na+ channel blockers. Propidium iodide fluorescence was used as toxicity readout. Veratridine (100 microM) or ouabain alone (500 microM) were not toxic to the cells. In the presence of 500 microM ouabain, however, veratridine induced halfmaximal cell death with an EC50 value of 15.1 +/- 2.3 microM. Ouabain's EC50 was 215.3 +/- 16.7 microM (with 30 microM veratridine). The effects of a number of Na+ channel blockers were tested and compared with their Na+ channel blocking activity measured in voltage-clamp experiments. Blockers from various chemical classes reduced toxicity half maximally with IC50 values ranging from 11.7 +/- 1.4 nM (tetrodotoxin) to 280.5 +/- 48.0 microM (lamotrigine). There was a linear relationship between the log IC50 values obtained by the two methods (slope: 1.1 +/- 0.08; correlation coefficient: 0.93). In summary, these data show that this novel toxicity assay is well suited to test Na+ channel blockers.

The nootropic drug vinpocetine inhibits veratridine-induced [Ca2+]i increase in rat hippocampal CA1 pyramidal cells.

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3alpha,16alpha-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca2+]i, this study investigated the effect of vinpocetine on [Ca2+]i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na+ influx, causing depolarization and increasing [Ca2+]i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca2+]i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 microM) significantly inhibited the elevation in [Ca2+]i induced by veratridine (10 microM). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca2+]i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca2+]i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.

Tetrapentylammonium block of chloramine-T and veratridine modified rat brain type IIA sodium channels.

Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel alpha subunit was studied using whole cell patch clamp. Results indicate that TPeA blocks the inactivating brain sodium channel in a potential and use-dependent manner similar to that of the cardiac sodium channel. Removal of inactivation using chloramine-T (CT) unmasks a time-dependent block by TPeA consistent with slow blocking kinetics. On the other hand, no time dependence is observed when inactivation is abolished by modification with veratridine. TPeA does not bind in a potential-dependent fashion to veratridine-modified channels and does not significantly affect gating of veratridine-modified channels suggesting that high affinity binding of TPeA to the brain sodium channel is lost after veratridine modification.

NMDA receptor dependent and independent components of veratridine toxicity in cultured cerebellar neurons are prevented by nanomolar concentrations of terfenadine.

Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate. Terfenadine reduced tetrodotoxin-sensitive inward currents, and reduced intracellular cGMP formation following veratridine exposure. Our data suggest that nanomolar concentrations of TEF may reduce excitatory aminoacid release following neuronal depolarization via a presynaptic mechanism involving voltage sensitive sodium channels, and therefore may be considered as a prototype for therapeutic drugs in the treatment of diseases that involve excitatory aminoacid neurotransmission.

Veratridine-evoked release of dopamine from guinea pig isolated cochlea.

Dopamine released from the lateral olivocochlear efferent system is thought to inhibit the toxic effect of the extreme glutamate outflow from the inner hair cells during ischemia or acoustic trauma. Using in vitro microvolume superfusion, we have studied the release of [(3)H]dopamine from the lateral olivocochlear efferent bundle of guinea pig in response to accumulation of [Na(+)](i), under condition characteristics of ischemia. Veratridine, that acts only on excitable membranes as a specific activator of voltage-sensitive sodium channels, significantly increased the electrically evoked release of [(3)H]dopamine, which was completely inhibited by tetrodotoxin. Dizocilpine (MK-801), a non-competitive NMDA-receptor antagonist, and GYKI-52466, a selective non-NMDA-receptor antagonist, had no effect on veratridine-induced [(3)H]dopamine release. Our data provide further evidence that the cochlear release of dopamine is of neural origin and possibly independent on a local effect of glutamate. The veratridine-induced transmitter release in the cochlea will be a very useful method in studying the effect of drugs on ischemic injury.

Veratridine-stimulated amylase secretion from rabbit pancreatic lobules: role of cholinergic and noncholinergic receptors.

Stimulation of pancreatic nerves results in marked increases in exocrine secretion. However, the neurotransmitters and pre- and postsynaptic receptors, which determine synaptic transmission between nerves and acinar cells, are poorly defined. We used rabbit pancreatic lobules, which contain nerve terminals and secrete independently of the influences of vascular perfusion or gastrointestinal hormones, to study the role of cholinergic and noncholinergic nerves in regulating amylase secretion. Pancreatic nerves were stimulated by veratridine (Ver; 50-200 microM), an activator of voltage-dependent sodium channels, in a concentration-dependent and tetrodotoxin-sensitive manner, resulting in an increase of 138+/-15% in amylase secretion above basal at 100 microM. This stimulation was unaffected by either hexamethonium (100 microM) or the combination of phentolamine and propranolol (10 microM). Atropine (5 microM) inhibited Ver-stimulated secretion by approximately 65-70%. Bethanechol (Bch; 0.01-100 microM) increased amylase secretion in a concentration-dependent manner (EC50, 6.2 microM), with a maximal stimulation of 177+/-15% above basal. Antagonism of Bch-stimulated secretion with 4-diphenylacetoxy-N-methyl-piperidine, pirenzepine (Pzp), or methoctramine (Met) resulted in IC50 values of 7.9 nM, 282 nM, and 79.8 microM, respectively. Ver-stimulated secretion was unaffected by Pzp (0.1 and 1 nM) or Met (1 and 100 nM) at concentrations that had no significant effect on Bch-stimulated secretion. Thus cholinergic nerves, activating postsynaptic M3 receptors, provided the predominant stimulatory innervation of rabbit pancreatic acini. Nonadrenergic, noncholinergic nerves also made a significant contribution to secretion. Adrenergic nerves did not appear to innervate acini or the excitatory cholinergic nerves directly.

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer.

Glutamate concentration increases significantly in the extracellular compartment during brain ischaemia and anoxia. This increase has an important Ca(2+)-independent component, which is due in part to the reversal of glutamate transporters of the plasma membrane of neurons and glia. The toxin phoneutriatoxin 3-4 (Tx3-4) from the spider Phoneutria nigriventer has been reported to decrease the evoked glutamate release from synaptosomes by inhibiting Ca(2+) entry via voltage-dependent Ca(2+) channels. However, we report here that Tx3-4 is also able to inhibit the uptake of glutamate by synaptosomes in a time-dependent manner and that this inhibition in turn leads to a decrease in the Ca(2+)-independent release of glutamate. No other polypeptide toxin so far described has this effect. Our results suggest that Tx3-4 can be a valuable tool in the investigation of function and dysfunction of glutamatergic neurotransmission in diseases such as ischaemia.

Neuroprotective role of Na+/myo-inositol cotransporter against veratridine cytotoxicity.

Na+/myo-inositol cotransporter has been shown to protect cells from the perturbing effects of hypertonic stress by the accumulation of myo-inositol. Here we report a regulatory mechanism for the cotransporter. Induction of myo-inositol cotransporter mRNA was observed after exposure to veratridine, a voltage-gated sodium channel opener. The veratridine-elicited induction was inhibited when Na+ was eliminated from the bath, although calcium chelation failed to modify the gene expression. Veratridine evoked an accumulation of Na+ in the cells, which paralleled the abundance of the mRNA. These results strongly suggested that an increase in Na+ influx due to sodium channel opening affected transcription of the cotransporter gene. Activity of the myo-inositol cotransporter was also up-regulated after veratridine exposure. To clarify the possible roles of myoinositol accumulation under veratridine exposure, we next examined the neurotoxic effects of veratridine when myo-inositol uptake was blocked. Neither 30 microM veratridine nor 500 microM 2-O,C-methylene myo-inositol, a competitive inhibitor of myo-inositol, elicited apparent cytotoxicity. However, a combination of these agents markedly increased cytotoxicity in culture, suggesting that an adequate amount of myo-inositol was necessary when the cells were stimulated with veratridine.

Inhibitory effects of tacrine and physostigmine on catecholamine secretion and membrane currents in guinea-pig adrenal chromaffin cells.

The effects of tacrine and physostigmine on catecholamine secretion induced by veratridine and high K+, and on voltage-dependent Na+ and Ca2+ currents, were investigated in guinea-pig adrenal chromaffin cells. In perfused adrenal glands, tacrine (100 microM) caused an inhibition of veratridine-induced catecholamine secretion, but physostigmine (100 microM) did not. In dispersed cells, both tacrine (1 microM-1 mM) and physostigmine (1 microM-1 mM) decreased catecholamine secretion induced by veratridine in a dose-dependent manner. The inhibitory effect of tacrine was much greater than that of physostigmine. Tacrine alone at a high concentration (such as 1 mM) caused a substantial increase in catecholamine secretion by itself and completely abolished the veratridine-induced secretory response in dispersed cells. High-concentration physostigmine showed a similar effect, but to a much lesser extent. The high K+ (46.2 mM)-evoked catecholamine secretion from dispersed cells was not affected by tacrine (1-100 microM) or physostigmine (1 microM-1 mM). In fura-2 loaded cells, tacrine (100 microM) almost abolished [Ca2+]i rise induced by veratridine, but only slightly reduced that evoked by high K+. In voltage-clamped cells, tacrine (300 microM) depressed the voltage-dependent Na+ and Ca2+ current by about 93% and 69%, and physostigmine (300 microM) depressed them by about 30% and 17%, respectively. These results suggest that tacrine decreases the veratridine-induced catecholamine secretion primarily by inhibiting the voltage-dependent Na+ channels rather than the Ca2+ channels. Physostigmine acts in a manner similar to tacrine, but its potency is much lower than that of tacrine.

In vivo microdialysis study of (+/-)-kavain on veratridine-induced glutamate release.

This is the first microdialysis study to address the effects of (+/-)-kavain on veratridine-induced glutamate release in freely moving rats. (+/-)-Kavain (100 mg/kg, p.o.) significantly reduced veratridine-induced glutamate release compared with that of vehicle-treated controls. Maximum extracellular glutamate levels were obtained 20-40 min after veratridine stimulation (500 microM, added to the perfusate). In the control group the increase was 301% and in the (+/-)-kavain group the increase was significantly reduced to 219% (the basal value was 100%). These results demonstrate that (+/-)-kavain reduces veratridine-induced glutamate release in vivo, which confirms previous in vitro data.

Effects of drugs interfering with sodium channels and calcium channels on the release of endogenous dopamine from superfused substantia nigra slices.

The importance of voltage-dependent sodium channels and different types of voltage-sensitive calcium channels for depolarisation-induced release of endogenous dopamine from dendrites and cell bodies in superfused guinea pig substantia nigra slices was investigated. The stimulatory effect of veratridine (10 microM) on dopamine release was only marginally attenuated in Ca(2+)-free medium but was completely blocked by tetrodotoxin (1 microM) and by the dopamine reuptake inhibitor GBR 12909 (10 microM). Low extracellular concentration of Na+ stimulated the dopamine release. Potassium-evoked dopamine release was completely Ca(2+)-dependent, not blocked by GBR 12909 and partially blocked by tetrodotoxin. Nifedipine (20 microM), omega-conotoxin GVIA (0.5 microM), penfluridol (5 microM), and Ni2+ (20 microM) had no effect, amiloride (1 mM) attenuated and neomycin (350 microM), and omega-agatoxin IVA (1 microM) almost totally blocked the potassium-induced dopamine release. The results suggest that veratridine released dopamine mostly by reversing the dopamine transporter. High concentrations of potassium induced release of nigral dopamine by opening of voltage-sensitive calcium channels of P/Q type but not L-type, N-type and probably not T-type. The depolarisation evoked by high concentrations of potassium seems to open voltage-sensitive calcium channels both by the depolarisation induced by potassium per se and by the secondary depolarisation induced by opening of voltage-dependent sodium channels.

Adenosine A2A receptors facilitate 45Ca2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes.

In the hippocampus, the neuromodulatory role of adenosine depends on a balance between inhibitory A1 responses and facilitatory A2A responses. Since the presynaptic effects of hippocampal inhibitory A1 adenosine receptors are mostly mediated by inhibition of Ca2+ channels, we now investigated whether presynaptic facilitatory A2A adenosine receptors would modulate calcium influx in the hippocampus. The mixed A1/A2 agonist, 2-chloroadenosine (CADO; 1 microM) inhibited veratridine (20 microM)-evoked 45Ca2+ influx into hippocampal synaptosomes of the CA1 or CA3 areas by 24.2 +/- 4.5% and 17.2 +/- 5.8%, respectively. In the presence of the A, antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), the inhibitory effect of CADO (1 microM) on 45Ca2+ influx was prevented in CA1 synaptosomes, but was converted into a facilitatory effect (14.2 +/- 6.7%) in CA3 synaptosomes. The A2A agonist, CGS 21680 (3-30 nM) facilitated 45Ca2+ influx in CA3 synaptosomes, with a maximum increase of 22.9 +/- 3.9% at 10 nM, and was virtually devoid of effect in CA1 synaptosomes. This facilitatory effect of CGS 21680 (10 nM) in CA3 synaptosomes was prevented by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC; 200 nM), but not by the A1 antagonist, DPCPX (20 or 100 nM). The facilitatory effect of CGS 21680 on 45Ca2+ uptake by CA3 synaptosomes was prevented by the class A calcium channel blocker, omega-agatoxin-IVA (200 nM). These results indicate that presynaptic adenosine A2A receptors facilitate calcium influx in the CA3 but not the CA1 area of the rat hippocampus through activation of class A calcium channels.