adenosine-5--(n-ethylcarboxamide) and 1-amino-1-3-dicarboxycyclopentane

Binding properties of the subtypes of adenosine A2 receptors in membrane preparations and the effects of adenosine receptor ligands on cAMP accumulation in slices from the optic tectum of neonatal chicks have been investigated. [3H]2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxaminoadenosin e (CGS 21680), a selective ligand for adenosine A2a receptors, did not bind to optic tectal membranes, as observed with rat striatal membranes. CGS 21680 also did not induce cyclic AMP accumulation in optic tectum slices. However, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloro-adenosine or adenosine induced a 2.5- to 3-fold increase on cyclic AMP accumulation in this preparation. [3H]NECA binds to fresh non-washed-membranes obtained from optic tectum of chicks, displaying one population of binding sites, which can be displaced by NECA, 8-phenyltheophylline, 2-chloro-adenosine, but is not affected by CGS 21680. The estimated K(D) value was 400.90 +/- 80.50 nM and the Bmax was estimated to be 2.51 +/- 0.54 pmol/mg protein. Guanine nucleotides, which modulate G-proteins activity intracellularly, are also involved in the inhibition of glutamate responses by acting extracellularly. Moreover, we have previously reported that guanine nucleotides potentiate, while glutamate inhibits, adenosine-induced cyclic AMP accumulation in slices from optic tectum of chicks. However, the guanine nucleotides, GMP or GppNHp and the metabotropic glutamate receptors agonist, 1S,3R-ACPD did not alter the [3H]NECA binding observed in fresh non-washed-membranes. Therefore, the adenosine A2 receptor found in the optic tectum must be the adenosine A2b receptor which is available only in fresh membrane preparations, and its not modulated by guanine nucleotides or glutamate analogs.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cell Membrane; Chickens; Cyclic AMP; Cycloleucine; In Vitro Techniques; Phenethylamines; Radioligand Assay; Rats; Receptors, Purinergic P1; Superior Colliculi

The effects of group 2- versus group 3-selective metabotropic glutamate (mGlu) receptor agonists were examined against forskolin (10 microM)-, vasoactive intestinal peptide (VIP; 1 microM)- and 5'-N-ethylcarboxamidoadenosine (NECA; 10 microM)-stimulated cAMP accumulations in adult rat hippocampal slices (in the presence of adenosine deaminase). Group 2 mGlu receptor-selective ((1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD) and (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG I)) and group 3 mGlu receptor-selective (L-2-amino-4-phosphonobutyric acid (L-AP4) and L-serine-O-phosphate) agonists greatly inhibited forskolin-stimulated cAMP formation ( > 80% at maximally effective concentrations). In contrast, stimulation of cAMP by VIP or NECA was inhibited by group 3, but not by group 2, mGlu receptor agonists. In fact, group 2 mGlu receptor agonists greatly potentiated cAMP accumulation evoked by NECA. Both the inhibitory effects of 1S,3R-ACPD on forskolin-stimulated cAMP and the potentiating effects on NECA-stimulated cAMP accumulation were reversed by the competitive group 1/2 mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG). However, (+)-MCPG had no effects on L-AP4 inhibition of cAMP. Thus, the effects of group 2 versus group 3 mGlu receptor agonists on cAMP coupling can be pharmacologically as well as functionally differentiated in the rat hippocampus.

Topics: Adenosine; Adenosine Deaminase; Adenosine-5'-(N-ethylcarboxamide); Amino Acids, Dicarboxylic; Aminobutyrates; Animals; Colforsin; Cyclic AMP; Cycloleucine; Excitatory Amino Acid Agonists; Hippocampus; In Vitro Techniques; Male; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Vasoactive Intestinal Peptide

1. The effects of various metabotropic glutamate receptor agonists on [3H]-cyclic AMP accumulation and phosphoinositide hydrolysis were investigated in guinea-pig cerebral cortical slices prelabelled with [3H]-adenine or [3H]-inositol. 2. 1-Aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD), L-2-amino-4-phosphonobutanoate (L-AP4) and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited concentration-dependent inhibitions of forskolin-stimulated [3H]-cyclic AMP accumulation, with IC50 values of 2.1 +/- 0.3, 71 +/- 17 and 0.2 +/- 0.1 microM respectively. 3. 1S,3R-ACPD and L-CCG-I increased the cyclic AMP responses to histamine H2 receptor stimulation with EC50 values of 7 +/- 2 microM and 19 +/- 2 microM respectively. 1S,3R-ACPD (EC50 values 17 +/- 2 microM) and L-CCG-I (EC50 value 15 +/- 3 microM) potentiated the cyclic AMP responses to the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 microM). This potentiating effect of L-CCG-I was reduced in the presence of a protein kinase C inhibitor, and also in the absence of extracellular calcium. In contrast, L-AP4 inhibited the NECA response in a concentration-dependent manner, with an IC50 value of 120 +/- 20 microM. 4. L-AP4 (at concentrations up to 1 mM) failed to stimulate phosphoinositide hydrolysis in guinea-pig cerebral cortical slices, but both 1S,3R-ACPD (EC50 value 35 +/- 6 microM) and L-CCG-I (approximately 160 microM) elicited concentration-dependent stimulations of phosphoinositide turnover. 5. These results confirm the existence of at least two distinct subtypes of metabotropic receptor in guinea-pig cortex. The data also substantiate previous studies indicating the importance of the agonist L-CCG-I as a pharmacological tool for distinguishing metabotropic receptor subtypes.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Amino Acids, Dicarboxylic; Aminobutyrates; Animals; Cerebral Cortex; Colforsin; Cyclic AMP; Cycloleucine; Guinea Pigs; Hydrolysis; In Vitro Techniques; Phosphatidylinositols; Receptors, Metabotropic Glutamate

Inhibition of forskolin-stimulated cAMP formation by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) in rat hippocampal slices was partially obliterated by the adenosine-depleting enzyme, adenosine deaminase, or by the adenosine receptor agonist, 5'-(N-ethylcarboxamido)-adenosine, suggesting that activation of metabotropic glutamate receptors (mGluRs) modulates the release of endogenous adenosine. Consistent with this hypothesis, forskolin stimulated the release of purines from rat hippocampal slices, and this effect was reduced by 1S,3R-ACPD. To establish which transduction pathway is involved in the modulation of forskolin-stimulated purine release, we have tested the novel mGluR2 agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), which reduced forskolin-stimulated cAMP formation but, as opposed to 1S,3R-ACPD, did not stimulate polyphosphoinositide hydrolysis. DCG-IV was highly potent and more efficacious than 1S,3R-ACPD in inhibiting forskolin-stimulated purine release. Neither DCG-IV nor 1S,3R-ACPD reduced the release of purines stimulated by depolarizing concentrations of K+, suggesting that their effect was stimulus-specific. These results indicate that, in rat hippocampal slices, activation of mGluR2 receptors attenuates the release of purines induced by forskolin, a process that amplifies the final effect of forskolin on cAMP formation as a result of A2 purinergic receptor activation. Thus, the final effect of mGluR agonists on forskolin-stimulated cAMP formation in hippocampal slices depends on both a direct inhibition of adenylyl cyclase and the inhibition of adenosine release.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Colforsin; Cycloleucine; Cyclopropanes; Glycine; Hippocampus; In Vitro Techniques; Male; Neurotoxins; Purines; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Synaptic Transmission

In rat cerebral cortical slices, the 1-aminocyclopentyl-1S,3R-dicarboxylate (1S,3R-ACPD) isomer of the selective metabotropic excitatory amino acid agonist ACPD inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in a concentration-dependent manner with a maximal inhibition of 51 +/- 3% and a half-maximally effective concentration of 8.8 +/- 3.4 microM. Similarly, 1R,3S-ACPD inhibited the forskolin response in a concentration-dependent manner, but with an inhibition of 80 +/- 5% at 3 mM. In addition to inhibiting forskolin-stimulated cAMP levels, 1S,3R-ACPD, but not 1R,3S-ACPD, enhanced the cAMP response to A2b adenosine receptor activation. In the presence of 1.2 U/ml of adenosine deaminase (included to reduce the contribution of endogenous adenosine), the efficacy of 1S,3R-ACPD was increased (88 +/- 3% inhibition), but the potency was unchanged. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine also increased the inhibitory effect of 100 microM 1S,3R-ACPD, from 57 +/- 1 to 78 +/- 5%. These results indicate that endogenous adenosine plays an important role in regulating the apparent efficacy of 1S,3R-ACPD inhibition of forskolin-stimulated cAMP accumulation in rat cerebral cortical slices and that previous studies in rat hippocampus and hypothalamus in the absence of added adenosine deaminase may have underestimated the efficacy of this compound.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cerebral Cortex; Colforsin; Cyclic AMP; Cycloleucine; Female; In Vitro Techniques; Kinetics; Male; Neurotoxins; Rats; Rats, Inbred Strains; Xanthines

The 1R,3R- and 1R,3S-isomers of 1-aminocyclopentane-1,3-dicarboxylate (ACPD) failed to stimulate phosphoinositide turnover or modify A2b adenosine receptor-stimulated cyclic AMP accumulation in guinea-pig cerebral cortical slices. In contrast, both 1S,3R- and 1S,3S-ACPD elicited concentration-dependent stimulations of phosphoinositide turnover (EC50 values 35 and 97 microM, respectively) and potentiated A2b-stimulated cAMP formation (17 and 58 microM, respectively). When forskolin was used to elevate cyclic AMP levels, however, all four isomers elicited concentration-dependent inhibitions of cyclic AMP formation to the same extent (approximately 90% inhibition). For this response the rank order of potencies were (IC50 values): 1S,3S-(0.9 microM) > 1S,3R-(2.1 microM) > 1R,3R-(237 microM) > 1R,3S-ACPD (approximately 1 mM). These data suggest the presence in guinea-pig cerebral cortex of two distinct subtypes of ACPD receptor coupled to phosphoinositide hydrolysis (and the potentiation of A2b receptor-stimulated cAMP formation) and the inhibition of forskolin-stimulated cAMP accumulation. Furthermore, our results indicate the usefulness of 1S,3S-ACPD as a tool to selectively activate one of these subtypes.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cerebral Cortex; Colforsin; Cyclic AMP; Cycloleucine; Glutamates; Guinea Pigs; In Vitro Techniques; Phosphatidylinositols; Receptors, Amino Acid; Receptors, Purinergic; Stereoisomerism

Incubation of guinea-pig cerebral cortical slices with the excitatory amino acid agonists DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N-methyl-D-aspartate (NMDA) failed to significantly alter either basal or 5'-N-ethylcarboxamidoadenosine- (NECA-)stimulated cAMP levels. Quisqualate, L-glutamate and DL-1-aminocyclopentane-trans-1,3- dicarboxylate (t-ACPD) were also without effect on basal levels of cAMP but increased NECA-stimulated levels of cAMP to approximately 190, 220 and 290% of control levels, respectively. Analysis of t-ACPD concentration-response data for the potentiation of NECA-stimulated cAMP elevations and the stimulation of phosphoinositide turnover gave EC50 values of 51 and 107 microM, respectively. The enhancing effect of t-ACPD on cAMP levels was maintained for up to 40 minutes as was the stimulation of phosphoinositide turnover. We conclude that the cAMP response to A2b adenosine receptor stimulation is augmented by the rigid glutamate analogue t-ACPD, possibly through the action of products of phosphoinositide turnover.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Biotransformation; Cerebral Cortex; Cyclic AMP; Cycloleucine; Female; Guinea Pigs; Ibotenic Acid; In Vitro Techniques; Inositol Phosphates; Male; N-Methylaspartate; Receptors, Amino Acid; Receptors, Purinergic; Vasodilator Agents