(1S,3R)-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Induced Increases in Cyclic AMP Formation in the Neonatal Rat Hippocampus Are Mediated by a Synergistic Interaction Between Phosphoinositide- and Inhibitory Cyclic AMP-Coupled mGluRs

Abstract: A pharmacological approach was used to investigate the cellular mechanism and metabotropic glutamate receptor (mGluR) subtypes that mediate stimulation of basal cyclic AMP (cAMP) formation in slices of the neonatal rat hippocampus. (1 S ,3 R )-1-Aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD), which is an agonist for phosphoinositide-coupled and inhibitory-coupled cAMP-linked mGluRs in cloned and in situ preparations, produced prominent stimulations of basal cAMP levels (five- to 10-fold). However, the agonists 3,5-dihydroxyphenylglycine (DHPG) and (2 R ,4 R )-4-aminopyrrolidine-2,4-dicarboxylate (2 R ,4 R -APDC), which selectively act on phosphoinositide-coupled and inhibitory cAMP-coupled mGluRs, respectively, only weakly increased cAMP levels. When these two mGluR subtype-selective agonists were added in combination, robust increases in cAMP levels, similar to those observed for 1 S ,3 R -ACPD, were found. Stimulations of cAMP content evoked by 1 S ,3 R -ACPD and combined additions of DHPG plus 2 R ,4 R -APDC occurred at concentrations of these agents that directly couple to other mGluR second messenger responses. However, these stimulatory cAMP responses were prevented by the presence of adenosine deaminase and 8- p -sulfophenyltheophylline (an adenosine receptor antagonist), as well as (+)-α-methyl-4-carboxyphenylglycine (an mGluR receptor antagonist). Thus, 1 S ,3 R -ACPD-induced increases in cAMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between mGluRs coupled to phosphoinositide (group 1) and inhibitory cAMP (group 2), which are indirectly expressed by potentiation of cAMP responses to other agonists (in this case, endogenous adenosine).     

4-Bromohomoibotenic Acid Selectively Activates a 1-Aminocyclopentane-1S,3R-Dicarboxylic Acid-Insensitive Metabotropic Glutamate Receptor Coupled to Phosphoinositide Hydrolysis in Rat Cortical Slices

Abstract: Glutamate activates a family of receptors, known as metabotropic glutamate receptors (mGluRs), that are coupled to various second messenger systems through G proteins. All mGluR subtypes characterized to date in rat brain slices are activated by the glutamate analogue 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (1 S ,3 R -ACPD). However, few agonists are available that selectively activate specific mGluR subtypes. We report that the glutamate analogue ( R,S )-4-bromohomoibotenate (BrHI) stimulates phosphoinositide hydrolysis in rat cerebral cortical slices in a concentration-dependent manner (EC₅₀ = 190 µ M ). The response to BrHI is stereoselective and is not blocked by ionotropic glutamate receptor antagonists. It is interesting that the responses to BrHI and 1 S ,3 R -ACPD are completely additive, suggesting that these responses are mediated by different receptor subtypes. Consistent with this, the response to BrHI is insensitive to l -2-amino-3-phosphonopropionic acid ( l -AP3), whereas the response to 1 S ,3 R -ACPD is partially blocked by l -AP3. BrHI does not activate metabotropic receptors coupled to changes in cyclic AMP accumulation or activation of phospholipase D. Thus, BrHI seems to activate specifically a phosphoinositide hydrolysis-linked mGluR that is insensitive to 1 S ,3 R -ACPD. This compound may prove useful as a tool for elucidating the roles of different mGluR subtypes in mammalian brain.     

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Abstract: Phospholipase D (PLD) is activated by many neuro-transmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) time- and concentrationdependently enhanced the formation of [³H]phosphatidylpropanol ([³H]PP) and of [³H]phosphatidic acid in the presence of 2% propanol. N -MethylD-aspartate and kainate (both 1 m M ) caused small, but significant increases (∼50%). whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 μ M ) was ineffective. Maximally effective concentrations of glutamate (1 m M ) and of 1 S ,3 R -ACPD (300 μ M ) increased the PLD activity to almost 300% of basal activity; the EC₅₀ values were 199 and 47 μ M , respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3). DL-2-aminc-5-phosphonovalenic acid, and kynurenate (all 1 m M ) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1 S ,3 R -ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-β-hydroxarnate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.     

Forskolin Stimulation of Cyclic AMP Accumulation in Rat Brain Cortex Slices Is Markedly Enhanced by Endogenous Adenosine

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.     

In Vitro Evidence for Increased Facilitation of Striatal Acetylcholine Release via Pre- and Postsynaptic NMDA Receptotors in Hemiparkinsonian Rats

Abstract : The NMDA-evoked acetylcholine release from striatal slices and synaptosomes was investigated in rats subjected to unilateral injection of 6-hydroxydopamine into the substantia nigra. In slices prepared from the striatum contralateral to the lesion, the NMDA-evoked endogenous acetylcholine release was not significant at 10 μ M NMDA and maximal at 100 μ M NMDA (124 ± 19%). Conversely, in slices taken from the dopamine-depleted striatum, NMDA was effective even at 10 μ M (41 ± 4%), and at 100 μ M (196 ± 24%) efficacy was nearly doubled. In synaptosomes prepared from the contralateral striatum, NMDA maximally stimulated 20 m M KCl-induced endogenous acetylcholine release at 1 μ M (66 ± 5.1%), with lower concentrations (0.01-0.1 μ M ) being ineffective. Conversely, in synaptosomes prepared from the dopamine-depleted striatum, NMDA maximally enhanced the K⁺-evoked acetylcholine release at 0.1 μ M (118 ± 12.4%). Concentration-response curves of NMDA-evoked acetylcholine release in sham-operated rats could be superimposed on those observed in the contralateral striatum of the 6-hydroxydopamine-lesioned animals. The present data support the view of an increased glutamatergic regulation of striatal acetylcholine release via pre- and postsynaptic NMDA receptors during Parkinson's disease.     

Adenosine and Cyclic AMP in Rat Cerebral Cortical Slices: Effects of Adenosine Uptake Inhibitors and Adenosine Deaminase Inhibitors

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.     

Interactions between neuropeptide Y and alpha 2-adrenoceptors in selective rat brain regions.

Neuropeptide Y significantly reduced the potassium-stimulated release of [3H]norepinephrine [( 3H]NE) from slices of rat hippocampus, hypothalamus and frontal cortex but not from slices of parieto-occipital cortex. The NPY-induced inhibition of [3H]NE release from frontal cortical slices was concentration dependent, reaching statistical significance at 10 nM. The alpha 2-adrenoceptor partial agonist, clonidine, also reduced the potassium-stimulated release of [3H]NE. The combination of NPY and clonidine in hippocampal slices produced a greater reduction of stimulated [3H]NE release than either of the two compounds alone, suggesting a potentiation of their activity, whereas in frontal cortical slices, the effect was additive. When NPY and clonidine were added to frontal cortical slices, they independently produced a significant concentration-dependent reduction in forskolin-stimulated cAMP accumulation. However, NPY and clonidine combined did not produce a further reduction in forskolin-induced cAMP accumulation than either compound when used alone. These results suggest that the ability of NPY to potentiate alpha 2-adrenoceptor-induced inhibition of [3H]NE release in discrete brain regions does not depend on the reductions in cAMP.     

3,5-Dihydroxyphenylglycine Is a Highly Selective Agonist for Phosphoinositide-Linked Metabotropic Glutamate Receptors in the Rat Hippocampus

Abstract: Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein-coupled glutamate receptors that are linked to multiple second messenger systems in the CNS. In this study the selectivity of mGluR agonists for different mGluR second messenger effects was characterized in slices of the rat hippocampus. The mGluR agonists (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid and (2 S ,3 S ,4 S )α-(carboxycyclopropyl)glycine produced multiple effects on second messengers that included enhanced phosphoinositide hydrolysis in both adult and neonatal rat hippocampus, inhibition of forskolin-stimulated cyclic AMP (cAMP) formation in adult tissue, and increases in basal cAMP formation in the neonatal hippocampus. In contrast, 3,5-dihydroxyphenylglycine was potent and effective in increasing phosphoinositide hydrolysis in both adult and neonatal hippocampus but unlike the other mGluR agonists did not inhibit forskolin-stimulated cAMP formation (in the adult) or substantially enhance basal cAMP formation (in the neonate). Thus, in the rat hippocampus mGluR agonist-mediated increases or decreases in cAMP formation are not secondary to mGluR-mediated changes in phosphoinositide hydrolysis. Furthermore, 3,5-dihydroxyphenylglycine can be used to activate subpopulations of mGluRs coupled to phosphoinositide hydrolysis with minimal effects on cAMP-mGluR second messenger systems.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

M3 Muscarinic Receptor-Mediated Enhancement of NMDA-Evoked Adenosine Release in Rat Cortical Slices In Vitro

Abstract: Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep-promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA-evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 µ M ) alone did not affect the basal release of adenosine. However, carbachol (100 µ M ) induced a 253% increase in NMDA (20 µ M )-evoked adenosine release in the presence of Mg²⁺. In the absence of Mg²⁺, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 µ M ) blocked the facilitatory effect of carbachol on NMDA-evoked adenosine release, and this was mimicked by the M3-selective antagonist 4-diphenylacetoxy- N -methylpiperidine (1 µ M ). Neither an M1-selective dose of pirenzepine (50 n M ) nor the M2-selective antagonist methoctramine (1 µ M ) affected carbachol's action on NMDA-evoked adenosine release. Carbachol had no effect on adenosine release evoked by α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor-mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long-term basis.     

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K_M. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition ( N -[2-( p -bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4-β-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.     

N-Acetylaspartylglutamate Inhibits Forskolin-Stimulated Cyclic AMP Levels via a Metabotropic Glutamate Receptor in Cultured Cerebellar Granule Cells

Abstract: The neuronal dipeptide N -acetylaspartylglutamate (NAAG) fulfills several of the criteria for classification as a neurotransmitter including localization in synaptic vesicles, calcium-dependent release after neuronal depolarization, and low potency activation of N -methyl- d -aspartate receptors. In the present study, the influence of NAAG on metabotropic receptor activation in cerebellar granule cells was examined in cell culture. Stimulation of granule cell adenylate cyclase with forskolin increased cyclic AMP (cAMP) several hundredfold above basal levels within 10 min in a concentration-dependent manner. Although gluta-mate, NAAG, and the metabotropic receptor agonist frans-1-amino-1, 3-cyclopentanedicarboxylic acid did not alter the low basal cAMP levels, the application of 300 μ M glutamate or NAAG or trans-1-amino-1, 3-cyclopentanedicarboxylic acid reduced forskolin-stimulated cAMP in granule cells by 30–50% in the absence or presence of inhibitors of ionotropic acidic amino acid receptors, as well as 2-amino-4-phosphonobutyrate. No additivity in the inhibition of cAMP was found when 300 μ M NAAG and trans -1-amino-1, 3-cyclopentanedicarboxylic acid were coapplied. The β-analogue of NAAG failed to reduce cAMP levels. Similar effects of NAAG and glutamate were obtained under conditions of inhibition of phosphodiesterase activity and were prevented by pretreatment of the cells with pertussis toxin. These data are consistent with the activation by NAAG of a metabotropic acidic amino acid receptor coupled to an inhibitory G protein. In contrast, the metabotropic acidic amino acid receptor coupled to phosphoinositol turnover in these cells was not activated by NAAG. Granule cells in culture expressed very low levels of extracellular peptidase activity against NAAG, converting to glutamate <0.1% of the 10 μ M through 1 m M NAAG applied to these cells during 15-min in vitro assays.     

Protein Kinase C-Mediated Suppression of the Presynaptic Adenosine A1 Receptor by a Facilitatory Metabotropic Glutamate Receptor

Abstract: KCl-evoked glutamate exocytosis from cerebrocortical synaptosomes can be inhibited by the adenosine A1 receptor agonist cyclohexyladenosine (CHA). Inhibition is associated with a decreased KCl-evoked Ca²⁺ level elevation, and the effect of the agonist is occluded by prior incubation with the Agelenopsis aperta neurotoxin ω-agatoxin-IVA at 250 n M . The inhibition is suppressed in the presence of 3 n M phorbol dibutyrate (PDBu) or by activation of the protein kinase C (PKC)-coupled metabotropic glutamate receptor by 100 µ M (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )ACPD]. A tonic inhibition of release by leaked exogenous adenosine can be reversed by adenosine deaminase or by PDBu addition. The CHA-induced inhibition can be enhanced by the PKC inhibitor Ro 31-8220. The mechanism for the suppression of the adenosine A1 receptor-mediated inhibition is distinct from that previously described for the (1 S ,3 R )ACPD-evoked, PKC-mediated, facilitatory pathway, which enhances phosphorylation of the MARCKS protein, 4-aminopyridine-induced action potentials, and release of glutamate because the latter requires at least 100 n M PDBu [or the combination of (1 S ,3 R )ACPD and arachidonic acid] and is not seen following KCl depolarization. Both PKC-mediated pathways may be involved in the presynaptic events associated with the establishment of synaptic plasticity.     

Developmental Changes in the Modulation of Cyclic AMP Formation by the Metabotropic Glutamate Receptor Agonist 1S,3R-Aminocyclopentane-1,3-Dicarboxylic Acid in Brain Slices

Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide-binding regulatory protein-coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,1R-ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin-stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R-ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N-methyl-D-aspartate, kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and was antagonised by L-2-amino-3-phosphonopropionate (L-AP-3). L-AP-3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R-ACPD on forskolin-stimulated cAMP formation. In hippocampal slices from 1-, 8-, or 15-day-old rats, 1S,3R-ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar development pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R-ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin-stimulated cAMP formation by 1S,3R-ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.     

Endogenous Adenosine Modulation of 22Na Uptake by Rat Brain Synaptosomes

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence ²²Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1–5U/ml) caused an excitatory effect on ²²Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25M). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1–10M) and the adenosine deaminase inhibitor EHNA (10–25M) inhibited ²²Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.     

Metabotropic Glutamate Receptor (mGluR)-Mediated Potentiation of Cyclic AMP Responses Does Not Require Phosphoinositide Hydrolysis: Mediation by a Group II-Like mGluR

Abstract: Metabotropic glutamate receptors (mGluRs) in the CNS are coupled to a variety of second messenger systems, the best characterized of which is activation of phosphoinositide hydrolysis. Recently, we found that activation of mGluRs in rat brain slices by the selective mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (1 S ,3 R -ACPD) potentiates cyclic AMP (cAMP) responses elicited by activation of other receptors coupled to G_s. It has been suggested that mGluR-mediated potentiation of cAMP responses is secondary to activation of phosphoinositide hydrolysis. However, preliminary evidence suggests that this is not the case. Therefore, we designed a series of experiments to test more fully the hypothesis that mGluR-mediated potentiation of cAMP responses is secondary to phosphoinositide hydrolysis. Inhibitors of both protein kinase C and intracellular calcium mobilization failed to antagonize 1 S ,3 R -ACPD-stimulated potentiation of cAMP responses. Further, coapplication of phorbol esters and 1 S ,3 R -ACPD induced a cAMP response that was greater than additive. Finally, ( RS )-3,5-dihydroxyphenylglycine, a selective agonist of mGluRs coupled to phosphoinositide hydrolysis, failed to potentiate cAMP responses, whereas (2 S ,1' R ,2' R ,3' R )-2-(2,3-dicarboxycyclopropyl)glycine, an mGluR agonist that does not activate mGluRs coupled to phosphoinositide hydrolysis, elicited a robust potentiation of cAMP responses. In total, these data strongly suggest that mGluR-mediated potentiation of cAMP responses is not secondary to activation of phosphoinositide hydrolysis and is likely mediated by a group II mGluR.     

Kainate-Evoked Release of Adenosine from the Hippocampus of the Anaesthetised Rat: Possible Involvement of Free Radicals

Abstract: Using microdialysis in the hippocampus of anaesthetised rats, the concentration of extracellular adenosine was estimated to be 0.8 µ M . Kainic acid (0.1–25 m M ) in the perfusate evoked a concentration-dependent release of adenosine with an EC₅₀ of 940 µ M . Two 5-min pulses of 1 m M kainic acid in the perfusate increased the dialysate levels with an S2/S1 ratio of 0.52 ± 0.03. Kainate-evoked release of adenosine was reduced significantly by 10 µ M tetrodotoxin and by a κ-receptor agonist, U50,488H (100 µ M ). The S2/S1 ratio was reduced by 4.5 µ M 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA receptor antagonist, but not by the NMDA receptor blockers (+)-MK-801 (dizocilpine; 100 µ M ) or (±)-2-amino-5-phosphonopentanoic acid (1 m M ), indicating a non-NMDA receptor-mediated process. The S2/S1 ratio was also reduced significantly by 10 m M ascorbic acid, 10 m M glutathione (a scavenger of hydroperoxides), and 1 m M oxypurinol (a xanthine oxidase inhibitor), indicating the possible involvement of free radicals. Neither the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (100 µ M ) nor the A1 adenosine receptor agonist R (−)- N ⁶-(2-phenylisopropyl)adenosine (100 µ M ) affected release. Adenosine release evoked by kainic acid is therefore mediated by activation of non-NMDA receptors and may involve the propagation of action potentials and the production of free radicals.     

Influence of cyclic AMP on DNA synthesis in slices of regenerating rat liver.

DNA synthesis in slices of regenerating rat liver is inhibited by adenosine cyclic 3',5'-monophosphate [cAMP]. The number of cells synthesizing DNA as assayed by 2-14C-thymidine incorporation is reduced by 65% in the presence of 10(-3) M cAMP. The inhibition of cAMP is not specific; other adenosine compounds, N6,O2,-dibutyryl adenosine 3',5'-monophosphate, 5'AMP and adenosine have the same effect. Moreover, the concentration of cAMP in the cell required for this inhibition is much higher than the normal levels of cAMP in liver cells.     

AMP is an adenosine A1 receptor agonist

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.