Inhibition of Voltage-Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells

Abstract: The role of the A_2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ could be inhibited with CGS21680, an A_2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A_2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp -adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A_2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca²⁺ concentration induced by 70 m M K⁺, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca²⁺ influx without VSCC activity after cobalt or 70 m M K⁺ pretreatment. These data suggest that the A_2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A_2A receptors induce inhibition of VSCCs as well as ATP-induced Ca²⁺ influx, the two inhibitory effects are clearly distinct from each other.     

Adenosine Receptors Modulate [Ca2+]i in Hippocampal Astrocytes In Situ

Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca²⁺]_i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca²⁺]_i that were blocked by the adenosine receptor antagonist 8-( p -sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P_2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca²⁺]_i, whereas the P₂ agonist adenosine 5'- O -(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P₁ purinergic receptors coupled to increases in [Ca²⁺]_i, whereas a small minority appear to contain P₂ purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca²⁺]_i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.     

5'-(N-Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Abstract: We investigated the effect of the adenosine receptor agonist 5'-( N -ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A_2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30–40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca²⁺]_i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca²⁺ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca²⁺]_i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca²⁺/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca²⁺ influx pathway (e.g., L-type Ca²⁺ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

Adenosine Receptor Agonists Inhibit K+-Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes

Abstract: The uptake of Ca²⁺ by a K⁺-depolarized rat brain cerebral cortical crude synaptosomal preparation (P₂ fraction) was investigated. The characteristics of the Ca²⁺ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca²⁺ uptake and neurotransmitter release, as it is generally accepted that K⁺-evoked Ca²⁺ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K⁺-evoked Ca²⁺ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A₂-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K⁺- evoked uptake of Ca²⁺ by the synaptosomal fraction.     

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A_2A receptor on the A₁ receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A_2A receptor agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low-affinity binding site for the A₁ receptor agonist 2-chloro- N ⁶-cyclopentyladenosine (CCPA). This effect was blocked by the A_2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C-mediated heterologous desensitisation of the A₁ receptor by the A_2A receptor.     

Inhibition of Striatal GABA Release by the Adenosine A2a Receptor Is Not Mediated by Increases in Cyclic AMP

Abstract: The adenosine A_2a receptor inhibition of potassium (15 m M )-evoked GABA release from striatal nerve terminals has been examined. High extracellular calcium concentrations (4 m M ) reduced the effect of the A_2a receptor agonist CGS-21680 (1 n M ). CGS-21680 inhibited GABA release in the presence of the L-type calcium channel blocker nifedipine, which itself inhibited evoked GABA release (by 16 ± 4%). ω-Conotoxin inhibited the evoked release by 45 ± 4% and prevented the action of CGS-21680. Forskolin and 8-bromo cyclic AMP both stimulated evoked GABA release at low concentrations, but at higher concentrations they abolished the inhibition by CGS-21680 without affecting the evoked release. The nonselective protein kinase inhibitor H-7 inhibited both the evoked release and the inhibition by CGS-21680, whereas the selective protein kinase A and G inhibitor HA-1004 had no effect on either evoked release or the action of CGS-21680. Pretreatment with pertussis toxin did not affect the A_2a receptor-mediated inhibition. Therefore, the effect of A_2a receptor stimulation was not mediated by protein kinases A or G but was inhibited by elevated cyclic AMP levels and mimicked by inhibitors of the N-type calcium channel and protein kinase C.     

Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP-Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells

Abstract: To study how growth factors affect stimulus-secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP-induced [Ca²⁺]_i rise and dopamine secretion in PC12 cells. After a 4-day incubation of cells, all three factors increased ATP-induced dopamine secretion significantly. We then examined which step of ATP-induced secretion was affected by the growth factors. Cellular levels of dopamine-β-hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP-induced [Ca²⁺]_i rise was also enhanced after growth factor treatment. The EC₅₀ of ATP for inducing [Ca²⁺]_i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca²⁺]_i of dopamine secretion was increased significantly only in NGF-treated cells. Our results suggest that for EGF- and insulin-treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca²⁺]_i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca²⁺ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca²⁺]_i.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Activation of Phosphodiesterase IV During Desensitization of the A2A Adenosine Receptor-Mediated Cyclic AMP Response in Rat Pheochromocytoma (PC12) Cells

Abstract: Prolonged activation of an A_2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A_2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A_2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)-selective inhibitor (Ro 20-1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A_2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A_2A-selective agonist (CGS21680) leads to increased PDE4 activity in a dose-dependent manner, which can be blocked by an A_2A-selective antagonist [8-(3-chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4-immunoreactive bands (72 and 79 kDa) were increased significantly during A_2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A_2A desensitization could be blocked by a protein kinase A (PKA)-selective inhibitor (H89) and was not observed in a PKA-deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA-dependent pathway plays a critical role in dampening the signal of the A_2A receptor.     

Nitric Oxide Disrupts Ca2+ Homeostasis in Hippocampal Neurons

Abstract: Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In [Ca²⁺]_i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S -nitrosocysteine produced a delayed rise in [Ca²⁺]_i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca²⁺]_i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca²⁺]_i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca²⁺]_i fluorimetry experiments, S -nitrosocysteine had no effect on the resting [Ca²⁺]_i or on the recovery kinetics of [Ca²⁺]_i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca²⁺]_i homeostasis in hippocampal neurons by impairing Ca²⁺ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca²⁺]_i may contribute to the delayed neurotoxicity of nitric oxide.     

GABA Alters GABAA Receptor mRNAs and Increases Ligand Binding

Abstract: Adenosine A_2a receptors have been localized to GABAergic striatopallidal neurons, but their functional role is unknown. To address this question, the modulation of endogenous GABA release by adenosine A_2a receptors was examined in slices of rat globus pallidus. The selective adenosine A_2a receptor agonist CGS-21680 (3.0–10 n M ) significantly increased electrically stimulated release (overflow) of GABA, with 10 n M CGS-21680 resulting in a 44% increase compared with the control. Both the nonselective adenosine receptor antagonist 8-phenyltheophylline (10 μ M ) and the selective A_2a receptor antagonist KF-17837 (100 n M ) abolished the CGS-21680-induced increase in GABA overflow. Higher concentrations of CGS-21680 (0.10–1.0 μ M ) decreased GABA overflow by ˜25%.8-Phenyltheophylline (10 μ M ) antagonized these effects, whereas KF-17837 (100 n M ) did not, suggesting actions of CGS-21680 on other adenosine receptors at these concentrations. These results demonstrate that activation of adenosine A_2a receptors augments electrically stimulated release of GABA from globus pallidus slices and suggest a mechanism by which adenosine may modulate GABAergic output from the striatopallidal efferent system.     

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.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.     

Photoaffinity Labeling with 2-[2-(4-Azido-3-[125I]-Iodophenyl)ethylamino]adenosine and Autoradiography with 2-[2-(4-Amino-3-[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain

Abstract: The A_2a adenosine receptor agonist 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [¹²⁵I]APE, discriminates between high- and low-affinity conformations of A_2a adenosine receptors. In this study, [¹²⁵I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [¹²⁵I]APE binding is consistent with that expected of an A_2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [¹²⁵I]APE binding to these brain regions is abolished by 1 µ M 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 n M 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [¹²⁵I]APE to the corresponding arylazide results in [¹²⁵I]AzPE. The rank-order potency of compounds to compete for [¹²⁵I]AzPE binding in the dark is CGS-21680 > d -( R )- N ⁶-phenylisopropyladenosine > N ⁶-cyclopentyladenosine, indicating that it also is an A_2a-selective ligand. Specific photoaffinity labeling by [¹²⁵I]AzPE of a single polypeptide (42 kDa) corresponding to A_2a adenosine receptors is reduced 55 ± 4% by 100 µ M guanosine 5'- O -(3-thiotriphosphate) and 91 ± 1.3% by 100 n M CGS-21680. [¹²⁵I]APE and [¹²⁵I]AzPE are valuable new tools for characterizing A_2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.