Adenosine Transport into Guinea-pig Synaptosomes

Abstract: Kinetics for transport of adenosine into guinea-pig neocortex synaptosomes were studied by incubating them with [¹⁴C]adenosine for up to 30 s. The apparent K _m value of the high-affinity transport system for adenosine was 21.1 μM and the V _max value was 257.3 pmol/min/mg protein. The transport system was inhibited by both compounds structurally related (compounds 554 and 555) and unrelated (dipyridamole) to adenosine. Because electrically stimulated synaptosomes release up to 1.5% of the adenosine derivative content per min, the physiological significance of adenosine uptake is discussed as a possible mechanism to compensate for the loss of adenine nucleotides from synaptosomes preparations.     

Some Biochemical Properties of the Rapid Adenosine Uptake System in Rat Brain Synaptosomes

Abstract: The rapid uptake of adenosine into rat brain cortical synaptosomes is mediated by a facilitated diffusion process. The carrier mediated uptake is sensitive to pH and temperature. The average Q₁₀ value for the system is approximately 1.77 and the necessary activation energy ( E _a) is estimated to be 8870 cal/mol. These values are essentially in agreement with values reported for adenosine uptake carriers of other tissues. Substrate specificity of the uptake system in the CNS demonstrates that nucleotides do not interact with the carrier until they have been hydrolyzed to nucleosides. Structural modification of the purine moiety at the "2" position did not have a profound effect on the ability of the molecule to serve as a substrate for the uptake system. Competitive inhibition by sulfhydryl reagents, p -chloromercuribenzoate, and N- ethylmaleimide on adenosine uptake suggests a direct involvement of sulfhydryl group(s) in the uptake mechanism. Other purines and pyrimidines also inhibited adenosine uptake, suggesting that a variety of nucleosides can interact with a common carrier system.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

Kinetic and inhibitor specificity of adenosine transport in guinea pig cerebral cortical synaptosomes: evidence for two nucleoside transporters

The transport of [³H]adenosine at 22°C was investigated in guinea pig cerebral cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions adenosine was not significantly metabolized during the incubation period used to determine the initial rates of adenosine transport. The dose response curves for the inhibition of adenosine transport by nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole were biphasic—approx. 50–60% of the transport activity was inhibited with IC₅₀ values of 0.7, 1 and 9 nM respectively, but the remaining activity was insensitive to concentrations as high as 1 μ M. Adenosine influx by both components was saturable (K_m values of 17 ± 3 and 68 ± 8 μ M; V_max values of 2.8 ± 0.3 and 6.1 ± 0.4 pmol/mg protein per s for NBMPR-sensitive and -insensitive components, respectively), and inhibited by other nucleosides and benzodiazepines. The two transport components also differed in their sensitivity to inhibition by other nucleosides and benzodiazepines indicating that the NBMPR-sensitive component of nucleoside transport in guinea pig synaptosomes exhibits a higher affinity than the NBMPR-insensitive component. However, both components have a broad specificity. Inhibition of adenosine transport by NBMPR was associated with high affinity binding of NBMPR to the synaptosomes (K_d 88 ± 6 pM). Binding of NBMPR to these sites was blocked by dilazep and dipyridamole with K₁ values similar to those measured for inhibiting NBMPR-sensitive adenosine influx. These results, together with previous findings using NBMPR and dipyridamole as ligand probes, suggest that there are two components of nucleoside transport in mammalian cerebral cortical synaptosomes that differ in their sensitivity to inhibition by NBMPR and other transport inhibitors.     

Do benzodiazepines bind at adenosine uptake sites in CNS?

Benzodiazepines inhibit adenosine uptake into rat cerebral cortical synaptosomes and their potency as inhibitors of adenosine uptake is closely correlated with therapeutic efficacy. Agents which possess “benzodiazepine like” activities such as CL218,872, zopiclone and fominoben and which displace benzodiazepine binding to brain cell membranes, are also inhibitors of adenosine uptake into brain synaptosomes. The IC₅₀ values of all these compounds as inhibitors of adenosine uptake are in close agreement with the IC₅₀ values obtained for the displacement of benzodiazepine binding to the brain receptors. Adenosine uptake inhibitors (dipyridamole, hexobendine, papaverine, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine) which competitively inhibit adenosine uptake, presumably by blocking adenosine binding to its carrier-protein, are competitive inhibitors of diazepam binding to the brain membrane receptors. The finding of a pronounced correlation between inhibition of benzodiazepine binding and inhibition of adenosine uptake further supports the proposal that benzodiazepines may exert part of their pharmacological action through the inhibition of adenosine uptake.     

ACCELERATION OF CHOLINE UPTAKE AFTER DEPOLARIZATION-INDUCED ACETYLCHOLINE RELEASE IN RAT CORTICAL SYNAPTOSOMES

Abstract— Activation of nerve elements in vivo and in vitro is associated with an increased rate of choline uptake by a Na⁺-dependent high affinity transport system. Following the methodology of B arker (1976), rat cortical synaptosomes were depolarized (37°C, 10min) by 25mM-KCl in the presence of CaCl₂ (1 mM) or other divalent cations. After reisolation by centrifugation, the rate of ³H-choline uptake (1.25μM) was measured by Millipore filtration. KCl treatment alone failed to accelerate the rate of uptake in the reisolated synaptosomes. CaCl₂, BaC1₂ or SrCl₂ (but not MgCl₂ or MnCl₂) were necessary (1 mM) to observe the KCl induced acceleration. Moreover, RbCl, but not LiCl or CsCl, also produced the calcium-dependent rate enhancement in the reisolated synaptosomes. The conditions mediating the enhanced rate of choline uptake correlated strongly with those associated with neurotransmitter release. To test this possibility, synaptosomal acetylcholine content was measured in response to the various salt treatments. Treatment with KCI (25 mM) and CaCl₂ (1 mM), but not KCl alone, reduced the synaptosomal acetylcholine content from 154 to 113pmol/mg protein. The respective rates of choline uptake increased about 60%. The increased rate was reversed by incubation with 50 μM-choline followed by synaptosome reisolation. This procedure also normalized the acetylcholine content. In summary, the rate of choline uptake by the high affinity choline uptake system is inversely related to the synaptosomal acetylcholine content.     

COMPARATIVE STUDIES ON SYNAPTOSOMES: UPTAKE OF [N-Me-3H]CHOLINE BY SYNAPTOSOMES FROM SQUID OPTIC LOBES

Abstract— The uptake of [ N -Me-³H]choline into synaptosomes from squid optic lobes was studied using a Millipore filtration technique. When incubated in an artificial sea water medium at 26°C, but not at 0°C, the synaptosomes rapidly accumulated choline, most of which could be recovered as unchanged free choline. The accumulated choline was readily released by treatment of the synaptosomes with Triton X-100 or exposing them to hypo-osmotic conditions. The influx of choline increased with increasing concentrations of choline and could be resolved into saturable and non-saturable components. Kinetic analysis revealed the presence of two saturable components one of high affinity ( K _m about 2 μ m ) and one of lower affinity ( K _m 25 μ m ). The rate of choline uptake by these synaptosomes was considerably greater than by mammalian brain synaptosomes. Both high and low affinity systems were Na⁺-requiring and inhibited by hemicholinium no. 3, levorphanol and dextrorphan. NaCN, 2,4-dinitrophenol and ouabain also inhibited choline uptake, the high affinity system being particularly sensitive to these agents. It is suggested that the high affinity system is specific for cholinergic terminals.     

Nitrobenzylthioinosine binding in brain: an interspecies study

The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ( [3H]NBI) to cerebral cortical membrane preparations from human, dog, guinea pig, rat, and mouse was investigated. Reversible, specific, saturable, high affinity binding was found in all five species with similar kinetic parameters. (Kd = 0.16-0.44 nM; Bmax = 128-196 fmol/mg prot.). Dilazep, hexobendine, and dipyridamole were all high potency inhibitors of [3H]NBI binding in human, dog, guinea pig and mouse preparations but not in rat. These compounds showed a competitive inhibition of [3H]NBI binding indicating that they are acting at the same site. Discrepancies seen in the rat appear to be a unique, species related anomaly. The dihydropyridine calcium antagonists also inhibited binding with lower potency than the adenosine uptake blockers. This inhibition was most potent in dog and human and suggests a relationship between the calcium channel and the adenosine uptake site.     

Mobilization of intracellular calcium and stimulation of calcium fluxes by endothelin-2 in cultured mouse swiss 3T3 fibroblasts

Specific receptor-induced signal transduction mechanisms for the endothelin-2 isoform (ET-2), a potent vasoconstrictor of vascular smooth muscle, were examined in Swiss 3T3 cells. Half-maximal binding (EC₅₀) and maximal, saturable binding (B_max) were estimated from Scatchard analyses and were found to be 24.2 ± 3.3 pM and 56500 ± 1700 sites/cells, respectively. A saturating concentration of ET-2 (100 nM) increased intracellular free calcium (measured by Fura-2 fluorescence) from a resting level of 100 nM to a peak level of 600–800 nM. The initial increase in intracellular free calcium was transitory and was followed by a smaller maintained elevation (250 nM). In the absence of extracellular calcium, ET-2 induced a transitory response equal in size to the peak in the presence of extracellular calcium, but the maintained response was absent. ET-2 increased intracellular free calcium in a concentration-dependent manner with an EC₅₀ of 1 nM. In calcium free solution (2 mM EGTA), ET-2 increased the efflux of ⁴⁵Ca from cells loaded to isotopic equilibrium (3 h) with ⁴⁵Ca. The intracellular second messenger, IP₃, also increased the calcium efflux from saponin permeabilized 3T3 cells loaded with ⁴⁵Ca (pCa 6) in the presence of MgATP. In the presence of extracellular calcium, ET-2 significantly increased calcium uptake into 3T3 cells by 92 ± 36.6 pmoles/million cells/2 min (n = 8). It is suggested that ET-2 binds to specific, high affinity receptors in 3T3 cells and that this receptor interaction increases the intracellular free calcium by IP₃-induced mobilization of calcium from cellular stores and by increasing influx of extracellular calcium.     

Effects of Forskolin, Dibutyryl Cyclic AMP, and 5''N-Ethylcarboxamide Adenosine on 22Na Uptake by Rat Brain Synaptosomes Stimulated by Veratridine

The effects of forskolin, dibutyryl cyclic AMP, and 5'-N-ethylcarboxamide adenosine on specific 22Na uptake by synaptosomes stimulated by veratridine were investigated. All substances inhibited 22Na uptake, with forskolin more potent than 5'-N-ethylcarboxamide and this latter one more potent than dibutyryl cyclic AMP. In the absence of preincubation with forskolin, this substance caused little or no effect on 22Na uptake by synaptosomes. In the presence of the adenosine antagonist dipropylsulfophenylxanthine, the inhibitory effect of 5'-N-ethylcarboxamide adenosine on 22Na uptake was consistently antagonized. The results were interpreted as forskolin and 5'-N-ethylcarboxamide adenosine increasing cyclic AMP accumulation, and dibutyryl cyclic AMP mimicking it, and by these mechanisms decreasing sodium uptake through the sodium channels.     

Biphasic effect of local anesthetics on the adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes

Previous studies have shown that an adenosine triphosphate-dependent calcium uptake activity in lysed brain synaptosomes is attributable to the neuronal endoplasmic reticulum elements. The present study has examined the effects of tetracaine, lidocaine, and dibucaine on this calcium uptake process. The adenosine triphosphate-dependent uptake of 45Ca2+ was measured (in the absence and in the presence of drug) by Millipore filtration and liquid scintillation spectrometry. The local anesthetics studied exhibited a biphasic effect on 45Ca2+ uptake by lysed synaptosomes from rat brain cortex. High concentrations (5 mM tetracaine, 50 mM lidocaine, 0.6 mM dibucaine) inhibited the uptake of 45Ca2+; the order of potency for this effect was dibucaine greater than tetracaine greater than lidocaine. Lower concentrations of these local anesthetics produced either no effect on 45Ca2+ uptake (2 mM tetracaine or 30 mM lidocaine) or a stimulation of 45Ca2+ uptake (1 mM tetracaine, 10 mM lidocaine, and 0.3 mM or 0.1 mM dibucaine); the order of potency for stimulation of 45Ca2+ uptake was dibucaine greater than tetracaine greater than lidocaine.     

Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles

Abstract— Microsomes and synaptosomes from rat brain accumulated ^4,5Ca against a concentration gradient by an ATP-dependent process. Calcium accumulation occurred to the same extent in microsomes prepared from white matter and from grey matter, an observation suggesting that calcium uptake may be in part an activity of the axonal membrane. Microsomes and synaptosomes accumulated calcium to a similar extent but less actively than mitochondria. By contrast, synaptic vesicles showed relatively little calcium accumulation. Isotonic concentrations of sucrose, NaCl, KCl and choline chloride inhibited calcium accumulation, with NaCl and KCl the least effective of these inhibitory agents. No consistent effects on calcium uptake were obtained with adenosine 3′,5′-monophosphate, dibutyryl cyclic AMP or the methyl xanthines. Incubation of prelabelled microsomes resulted in a release of ⁴⁵Ca, and ATP inhibited this release process. In the absence of added ATP, isotonic NaCl promoted calcium release to a significantly greater extent than KCl choline chloride or sucrose. In the presence of ATP, these agents all promoted a similar degree of release. Adenosine 3′,5′-monophosphate or agents that affect its metabolism did not significantly affect calcium release. Magnesium ions reduced calcium release under all conditions tested.     

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature-dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent K_m value of 0.9μM and a V_max value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na⁺ with choline chloride in the medium. Ca²⁺ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co²- or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15-min incubation period was released under the same conditions. [³H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl-evoked release mechanisms were found to be at least partially Ca²⁺-dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca²⁺-independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.     

The Characterization of [3H] Adenosine Uptake into Rat Cerebral Cortical Synaptosomes

: Uptake of adenosine, a putative inhibitory transmitter or modulator, was investigated in rat cerebral cortical synaptosomes. The accumulation of [³H]adenosine into synaptosomes, using an adenosine concentration of 10 μ.m , was linear for 30 min at 37°C. The uptake appeared to be mediated by kinetically saturable processes with apparent K_m's of 1 μam (“high-affinity A”) and 5 μm (“high-affinity B”), both of which were partially sensitive to the presence of external sodium and calcium ions. Both uptake processes were partially inhibited by 2,4-dinitrophenol, implying the presence of active uptake and diffusional components. A study of the metabolites of adenosine taken up by the two uptake systems indicates that the major metabolites were adenosine and nucleotides. However, adenosine incorporated by the high-affinity A uptake system is more likely to form deaminated metabolites, such as hypoxanthine and inosine, indicating a possible functional difference between the two uptake processes. A detailed comparison of the inhibitory properties of certain adenosine analogues and other pharmacological agents has revealed differences between the two adenosine uptake systems. Since the glial contamination in synaptosomal preparations is well established, one of the uptake systems we observed in the present study might be of glial origin. This notion is supported by the findings that the K_m values and kinetic properties of papaverine action in the synaptosomal high-affinity A uptake system are similar to those of astrocytes reported in the literature. In conclusion, the uptake processes of synaptosomal preparations show that accumulation of adenosine into neuronal (and possibly glial) elements may play a major role in regulating the extracellular adenosine concentration. Uptake inhibitors, such as diazepam, may exert, at least in part, their pharmacological actions by interfering with the regulation of extracellular adenosine concentrations.     

The Effect of Spider Toxin PhTx3-4, ω-Conotoxins MVIIA and MVIIC on Glutamate Uptake and on Capsaicin-Induced Glutamate Release and [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Spinal cord Synaptosomes

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and ω-conotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or ω-conotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca²⁺-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca²⁺]_i in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.     

The effect of oxidative stress on levels of cytosolic calcium within and uptake of calcium by synaptosomes

The ability of synaptosomes subjected to oxidative stress, to maintain homeostasis has been evaluated using various indices of cellular integrity. These include levels of cytosolic calcium and leakiness of the plasma membrane. The status of a neural characteristic; depolarization-induced calcium entry into the cytoplasm, has also been studied. The presence of 5 μM FeSO₄ and 0.1 mM ascorbic acid increased peroxidative activity as judged by the rate of thiobarbituric acid reactive material production, and depressed levels of free ionic calcium [Ca²⁺]_i as determined using the calcium-sensitive flouorescent indicator dye fura-2. Depolarization-induced influx of ⁴⁵Ca²⁺ was greatly depressed under these conditions, while basal calcium uptake was inhibited to a much lesser degree. The efflux of fura-2 from synaptosomes was enhanced in the oxidizing environment, suggesting increased permeability of the synaptosomal outer limiting membrane.

The treatment of synaptosomes with 25 μM -tocopherol succinate before and during exposure to the Fe²⁺/ascorbate mixture prevented many of the changes otherwise induced by the oxidizing system. Similar pretreatment with β-carotene or superoxide dismutase did not have any protective effect. Ganglioside GM₁ pre-exposure did not alter the Fe²⁺/ascorbate-induced changes in calcium-related parameters, but mitigated synaptosomal plasma membrane damage as judged by fura-2 leakage. Thus exogenous agents may be capable of reducing the severity of oxidative stress in nervous tissue.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

Transport of Histidine into Synaptosomes of the Rat Central Nervous System

Abstract: Histidine transport into synaptosomes was studied in order to characterize this aspect of histamine synthesis in neurons. Histidine transport was found to be independent of sodium, calcium, and magnesium ions and dependent upon potassium and chloride ions. Histidine transport was also found to be energy dependent, and subcellular fractionation studies suggested it was highly localized to nerve terminals. Kinetic analysis of histidine transport in several brain regions indicated the presence of two uptake sites, a high-affinity site with a K _m of approximately 35 μ M and a low-affinity site with a K _m in the millimolar range. Density of the high-affinity site, as reflected by V_max, correlates well with density of proposed histaminergic innervation. Rate of histidine transport was not altered by prior depolarization of the synaptosomes, indicating that histidine transport probably does not play a regulatory role in histamine synthesis.     

Regulation of L-Type Calcium Channels in GH4 Cells via A1 Adenosine Receptors

Abstract: Identification of A₁ adenosine receptors (A₁Rs) in a tumor cell line derived from rat pituitary (GH₄ cells) was performed by ligand binding and immunological experiments. Subsequently, the involvement of A₁Rs in the regulation of calcium conductance was studied in these cells. The agonist N ⁶-( R )-(2-phenylisopropyl)adenosine ( R -PIA) did not modify the intracellular calcium basal levels, whereas it inhibited the increase produced by 15 m M KCl depolarization. The antagonist 1,3-dipropyl-8-cyclopentylxanthine led to the opening of voltage-dependent cell surface calcium channels in the absence of exogenous KCl. The channels were of the L type because the effect was abolished by calciseptine and by verapamil. These results suggest that endogenous adenosine exerts a tonic inhibitory effect on calcium transport. This was confirmed by the high adenosine concentration found in cell supernatants (up to 1 µ M ) and by the calcium mobilization produced by exogenously added adenosine deaminase. In depolarizing conditions, the calcium peak in the presence of adenosine deaminase was reduced when cells were preincubated with R -PIA, thus suggesting that A₁R activation regulates the intensity of depolarization. These results demonstrate that adenosine is an important regulator of the physiological state of pituitary tumor cells by modulating, in an autocrine manner, the activity of L-type voltage-dependent calcium channels.     

Interactions between the presynaptically active neurotoxins alpha-latrotoxin and omega-conotoxin GVIA: studies on calcium fluxes and binding parameters in rat and chicken synaptosomes

Possible interactions between alpha-latrotoxin, an activator of synaptosomal calcium uptake, and omega-conotoxin GVIA, an inhibitor of voltage-sensitive calcium channels of the N-type, were investigated in rat and chicken synaptosomal preparations. While omega-conotoxin GVIA potently and effectively inhibited calcium uptake induced by elevated potassium in chick synaptosomes, little or no effect of omega-conotoxin GVIA was observed either in potassium-treated rat synaptosomes or in alpha-latrotoxin-exposed synaptosomes of either spaces. In contrast to the lack of effect of omega-conotoxin on stimulated calcium uptake in rat synaptosomes, cadmium effectively inhibited calcium uptake induced by either potassium or alpha-latrotoxin. Synaptosomal calcium transport induced by alpha-latrotoxin can be bidirectional, since alpha-latrotoxin also induced efflux of preaccumulated calcium. Competition experiments revealed that binding of 125I-labelled omega-conotoxin and 125I-labelled alpha-latrotoxin was similar in either chicken or rat synaptosomes. Neither alpha-latrotoxin nor omega-conotoxin competed with the binding of the other ligand in either species. The results reported here show that (1) elevated potassium evokes calcium uptake principally through N-channels in avian but not in rat synaptosomes; (2) alpha-latrotoxin-activated calcium fluxes are omega-conotoxin insensitive but cadmium sensitive; (3) the molecular acceptors for the two ligands are likely to be unrelated synaptic membrane constituents.