Coupling of metabotropic glutamate receptors to phosphoinositide mobilisation and inhibition of cyclic AMP generation in the guinea-pig cerebellum.

1. The effects of metabotropic glutamate receptor (mGluR) agonists on cyclic nucleotide and phosphoinositide turnover were investigated in adult guinea-pig cerebellar slices by use of radioactive precursors. 2.L-Glutamate, 1-aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD) and RS-3,5-dihydroxyphenylglycine (DHPG) evoked concentration-dependent increases in the accumulation of [3H]-inositol phosphates with pEC50 values of 2.98 +/- 0.02, 4.45 +/- 0.06 and 4.47 +/- 0.07, respectively. Maximal responses to these agents were 43 +/- 8, 52 +/- 12 and 84 +/- 11% of the response to 1 mM histamine, respectively. 3. The phosphoinositide response to 1S,3R-ACPD was antagonized in the presence of (+)-alpha-methyl-4-carboxyphenylglycine, with a calculated pKi value of 3.55 +/- 0.03. 4. Forskolin-stimulated accumulation of [3H]-cyclic AMP was not significantly altered in the presence of 10 microM DCG-IV or 300 microM 1S,3R-ACPD. Similarly, 300 microM 1S,3R-ACPD failed to alter isoprenaline-(1 microM) or 2-chloroadenosine (2-CA, 30 microM)-stimulated accumulation of [3H]-cyclic AMP. 5. Forskolin-stimulated accumulation of [3H]-cyclic AMP was concentration-dependently inhibited in the presence of L-glutamate and L-serine-O-phosphate (L-SOP) with pIC50 values of 2.91 +/- 0.17 and 2.86 +/- 0.04 with maximal inhibitions of 47 +/- 2 and 92 +/- 3%, respectively. L-2-Amino-4-phosphonobuty-rate (L-AP4) inhibited the forskolin response without saturating, evoking an inhibition of 71 +/- 7% at 3 mM. 6. 2-CA-evoked accumulation of [3H]-cyclic AMP was also inhibited by L-glutamate and L-SOP with pIC50 values of 2.71 +/- 0.03 and 2.72 +/- 0.08 and maximal inhibitions of 51 +/- 5 and 99 +/- 0%, respectively. L-AP4 inhibited the 2-CA response without saturating, evoking an inhibition of 68 +/- 1% at 3 mM. 7. Isoprenaline-evoked accumulation of [3H]-cyclic AMP was inhibited by L-glutamate and L-SOP with pIC50 values of 3.21 +/- 0.01 and 2.96 +/- 0.08 and maximal inhibitions of 88 +/- 2 and 93 +/- 3%, respectively. 8. These results suggest that the guinea-pig cerebellum expresses Group I and Group III mGluRs coupled to phosphoinositide turnover and inhibition of cyclic AMP generation, respectively.     

Pharmacological characterization of the metabotropic glutamate receptor inhibiting D-[3H]-aspartate output in rat striatum.

1. The effects of several agonists of the metabotropic glutamate receptor (mGluR) were studied in adult rat striatal slices by measuring (i) KCl (30 mM)-induced output of previously taken up D-[3H]-aspartate (Asp), (ii) forskolin (30 microM)-induced adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation and (iii) phophoinositide (PI) hydrolysis. 2. K(+)-induced efflux of D-[3H]-Asp was inhibited by the following mGluR agonists: (1S,3S,4S)-(carboxycyclopropyl)glycine (L-CCG-I), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and quisqualic acid (Quis). 2-Amino-4-phosphonobutyrate (L-AP4) was inactive up to 300 microM. The maximal inhibition of D-[3H]-Asp output was 60 +/- 8%. The EC50s of mGluR agonists were: 0.5 microM for L-CCG-I, 100 microM for 1S,3R-ACPD and 100 microM for Quis. 3. Forskolin-induced cyclic AMP accumulation was also inhibited by mGluR agonists. The maximal inhibition was 50 +/- 4% and was obtained at a concentration of 10 microM for L-CCG-I and 100 microM for 1S,3R-ACPD. The EC50s for this inhibition were: 0.9 microM for L-CCG-I and 20 microM for 1S,3R-ACPD. Quis (300 microM) inhibited cyclic AMP accumulation by approximately 20%. L-AP4 slightly potentiated cyclic AMP accumulation. 4. PI hydrolysis was stimulated by mGluR agonists. The most potent compound was Quis (100 microM), which increased inositol phosphate formation up to 2.2 fold over control values. Its EC50 was 15 microM. L-CCG-I and 1S,3R-ACPD increased inositol phosphate formation by approximately 1.8 fold and their EC50 values were 30 and 25 microM, respectively. L-AP4 did not affect PI hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-activity relationships for a series of phenylglycine derivatives acting at metabotropic glutamate receptors (mGluRs).

1. The actions of a series of twelve phenylglycine derivatives at metabotropic glutamate receptors (mGluRs) linked to both phosphoinositide hydrolysis (PI) and cyclic AMP were investigated. 2. PI hydrolysis was determined by the accumulation of [3H]-inositol-monophosphate ([3H]-IP1) in neonatal ral cortical slices prelabelled with [3H]-myo-inositol. The non-selective mGluR agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid ((1S,3R)-ACPD) produced a concentration-dependent increase in [3H]-IP1 (EC50 approximately 20 microM). This agonist was subsequently used to investigate potential antagonist activity of the phenylglycine derivatives. Of the compounds tested (+)-alpha-methyl-4-carboxyphenylglycine (M4CPG) and (RS)-alpha-ethyl-4-carboxyphenylglycine (E4CPG) were the most active with KP values of 0.184 +/- 0.04 mM and 0.367 +/- 0.2 mM respectively. 3. Activity at adenylyl cylase-coupled mGluRs was investigated by determining the accumulation of [3H]-cyclic AMP in adult rat cortical slices. [3H]-cyclic AMP accumulation, elicited by 30 microM forskolin, was inhibited by (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-1) and L-2-amino-4-phosphonobutanoate (L-AP4) with respective EC50 values of 0.3 microM and 10 microM. Neither agonist was able to inhibit completely forskolin stimulated cyclic AMP accumulation; this is evidence that not all adenylyl cyclase is susceptible to modulation by mGluRs. Phenylglycine derivatives were examined for their ability to antagonize the inhibition of [3H]-cyclic AMP accumulation by L-CCG-1 or L-AP4 at their EC50 concentrations. 4. A rank order of potency of the phenylglycine derivatives as antagonists of L-AP4 and L-CCG-1 was obtained. The most effective compound. (RS)-alpha-methyl-3-carboxymethylphenylglycine (M3CMPG) had IC50 values in the order of 1 microM against L-AP4 and 0.4 microM against L-CCG-1. 5. The results from this study indicate that phenylglycine-derived compounds can discriminate between groups of metabotropic glutamate receptors and may also display some selective activity between subtypes within groups. Future work based on these findings may lead to the development of more selective and potent compounds as important pharmacological tools.     

Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor-mediated cyclic AMP responses to NECA and 2-chloroadenosine.     

Natriuretic peptide-induced cyclic GMP accumulation in adult guinea-pig cerebellar slices.

1. Second messenger responses to natriuretic peptides were studied in guinea-pig cerebellar slices by use of radioactive precursors. 2. The rank order of potency of the different natriuretic peptides in generating [3H]-guanosine 3':5'-cyclic monophosphate (cyclic GMP) was atrial natriuretic peptide (ANP) > brain natriuretic peptide (BNP) >> C-type natriuretic peptide (CNP) with EC50 values of 19.5 +/- 8.8 nM for ANP and 169 +/- 41 nM for BNP. CNP induced [3H]-cyclic GMP accumulation only at concentrations greater than 1 microM. 3. An additive response to ANP (1 microM) was observed in the presence of the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA, 10 microM) or the soluble guanylyl cyclase activator, sodium nitroprusside (SNP, 100 microM) for [3H]-cyclic GMP accumulation. 4. ANP, BNP and CNP (all at 1 microM) failed to alter significantly either basal-, forskolin- (10 microM), isoprenaline- (100 microM), or NECA- (10 microM) induced [3H]-cyclic AMP generation. Natriuretic peptides also did not change the [3H]-cyclic AMP steady-state reached after 10 min of treatment with 10 microM forskolin. 5. Natriuretic peptides failed to elicit significant accumulation of [3H]-inositol phosphates at concentrations up to 10 microM. 6. These data are consistent with the presence of ANPA, rather than ANPB or clearance receptors (C-receptors), linked to second messenger cascades in guinea-pig cerebellar slices.     

Stimulatory effects of the putative metabotropic glutamate receptor antagonist L-AP3 on phosphoinositide turnover in neonatal rat cerebral cortex.

1. The effects of the metabotropic glutamate receptor (mGluR) antagonist, L-2-amino-3-phosphonopropionate (L-AP3) on phosphoinositide turnover in neonatal rat cerebral cortex slices has been investigated. 2. At concentrations of < or = 300 microM, L-AP3 inhibited total [3H]-inositol phosphate ([3H]-InsPx) and Ins(1,4,5)P3 mass responses stimulated by the selective mGluR agonist, 1-amino-cyclopentane-1S, 3R-dicarboxylic acid (1S, 3R-ACPD). Comparison with the competitive mGluR antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG) clearly demonstrated that L-AP3 caused inhibition by a mechanism that was not competitive, as L-AP3 decreased the maximal response to 1S, 3R-ACPD (by approximately 40% at 300 microM L-AP3) without significantly affecting the concentration of 1S, 3R-ACPD required to cause half-maximal stimulation of the [3H]-InsPx response. 3. In contrast, at a higher concentration L-AP3 (1 mM) caused a large increase in [3H]-InsPx accumulation which was similar in magnitude in both the absence and presence of 1S, 3R-ACPD (300 microM). D-AP3 (1 mM) had no stimulatory effect alone and did not affect the response evoked by 1S, 3R-ACPD. L-AP3 (1 mM) also caused a large increase in Ins(1,4,5)P3 accumulation. The magnitude of the response (4-5 fold increase over basal) approached that evoked by a maximally effective concentration of 1S, 3R-ACPD, but differed substantially in the time-course of the response. The stimulatory effects of 1S, 3R-ACPD and L-AP3 on Ins(1,4,5)P3 accumulation were also similarly affected by decreases in extracellular calcium concentration. 4. Detailed analysis of the inositol phospholipid labelling pattern and the inositol (poly)phosphate isomeric species generated following addition of L-AP3 was also performed. In the continued presence of myo-[3H]-inositol, L-AP3 (1 mM) stimulated a significant increase in phosphatidylinositol labelling, but not that of the polyphosphoinositides, and the inositol (poly)phosphate profile suggested that substantial Ins(1,4,5)P3 metabolism occurs via both 5-phosphatase and 3-kinase routes. 5. A significant stimulatory effect of L-AP3 (1 mM) on [3H]-InsPx accumulation was also observed in neonatal rat hippocampus, and cerebral cortex and hippocampus slices prepared from adult rat brain. 6. These data demonstrate that whilst L-AP3 antagonizes mGluR-mediated phosphoinositide responses at concentrations of < or = 300 microM, higher concentrations substantially stimulate this response. The ability of (+/-)-MCPG (1 mM) to attenuate significantly L-AP3-stimulated [3H]-InsPx accumulation, suggests that both the inhibitory and stimulatory effects of L-AP3 may be mediated by mGluRs.     

Excitatory amino acid receptor-stimulated phosphoinositide turnover in primary cerebrocortical cultures.

1. Characterization of excitatory amino acid-induced accumulation of [3H]-phosphoinositides was carried out in primary cerebrocortical cultures isolated from foetal rats. 2. All of the excitatory amino acid receptor agonists examined caused concentration-dependent enhancement of phosphoinositide (PI) formation. The most potent excitatory amino acid receptor agonists were quisqualate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD), ibotenate and glutamate with mean EC50 values of 0.9 +/- 0.4 microM, 15 +/- 5 microM, 15 +/- 3 microM and 41 +/- 8 microM respectively. 3. The selective ionotropic receptor antagonists kynurenic acid (1 mM), 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX, 10 microM) and (+/-)-4-(3-phosphonopropyl)-2 piperazinecarboxylic acid (CPP, 100 microM), failed to block responses to quisqualate, (1S,3R)-ACPD or glutamate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) did not block 1S,3R-ACPD or quisqualate-induced PI turnover, but had an additive effect with quisqualate or (1S,3R)-ACPD. 4. Exposure of cultures to agonists in the absence of added extracellular calcium reduced the maximal quisqualate response by approximately 45%, revealing a two-component concentration-response curve. Concentration-response curves to ibotenate and glutamate became flattened by omission of extracellular calcium, whereas (1S,3R)-ACPD-stimulated PI turnover was unaffected. 5. Pretreatment of cultures with pertussis toxin markedly inhibited PI responses evoked by (1S,3R)-ACPD. 6. These results suggest that excitatory amino acid-stimulated PI turnover in cerebrocortical cultures is independent of ionotropic receptor activation and is mediated via specific G-protein-linked metabotropic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R-ACPD in neonatal rat cerebral cortex.

1. The effect of NMDA-receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) has been examined in neonatal rat cerebral cortex slices. 2. Total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 5 mM LiCl, in [3H]-inositol pre-labelled slices was concentration-dependently increased by 1S,3R-ACPD (EC50 16.6 microM) and, at a maximally effective concentration, 1S,3R-ACPD (300 microM) increased [3H]-InsPx accumulation by 12.8 fold over basal values. 3. [3H]-InsPx accumulation stimulated by 1S,1R-ACPD was enhanced by low concentrations of NMDA (3-30 microM), but not by higher concentrations (> 30 microM). [3H]-InsPx accumulations stimulated by 1S,3R-ACPD in the absence or presence of 10 microM NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 microM NMDA the initial enhancement of 1S,3R-ACPD-stimulated phosphoinositide hydrolysis progressively decreased with time. 4. In the presence of a maximal enhancing concentration of NMDA (10 microM), the response to 1S,3R-ACPD (300 microM) was increased 1.9 fold and the EC50 for agonist-stimulated [3H]-InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration-dependently inhibited by competitive and uncompetitive antagonists of NMDA-receptor activation. 5. 1S,3R-ACPD also stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5-6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1-10 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells.

1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 microM) and WAY 100635 (5-HT1A; 1 microM). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pKb = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pKb = 10.44 +/- 0.06). 5. Pretreatment with PTX (200 ng ml-1 for 4 h) completely attenuated the Ca2+ response to 100 microM 5-HT. 6. In untransfected CHO-K1 cells, 5-HT (1 microM), RU 24969 (1 microM), and 5-CT (1 microM) elicited increases in [Ca2+]i similar to those observed in CHO-A1 cells. 7. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s).     

Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells.

1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of A2 adenosine receptor-induced cyclic AMP generation in cerebral cortex and relaxation of pre-contracted aorta.

1. A comparative study was carried out between the adenosine receptor mediating a stimulation of cyclic AMP formation in guinea-pig cerebral cortical slices with the adenosine receptor mediating relaxation of phenylephrine precontracted guinea-pig aortic rings. 2. [3H]-cyclic AMP accumulation in [3H]-adenine-prelabelled guinea-pig cerebral cortical slices was stimulated by adenosine and its analogues with the following EC50 values (microM): 5'-N-ethylcarboxamidoadenosine (3.1 +/- 0.3) > 2-chloroadenosine (10 +/- 2) > adenosine (109 +/- 15). 3. 2-Chloroadenosine and adenosine elicited maximal responses for [3H]-cyclic AMP accumulation that were 100 +/- 7 and 71 +/- 6% of the maximal response to 5'-N-ethylcarboxamidoadenosine, respectively. CGS 21680 (100 microM) and DPMA (100 microM) elicited -2 +/- 2 and 12 +/- 3% of the response to 100 microM 5'-N-ethylcarboxamidoadenosine. 4. Estimation of antagonist potencies at the A2 adenosine receptor of cerebral cortex showed a rank order of potency (K1, nM): xanthine amino congener (35 +/- 3) > 8-cyclopentyl-1,3-dipropylxanthine (130 +/- 22) > PD 115,199 (407 +/- 82) > 3,7-dimethyl-1-propargylxanthine (13 +/- 2 microM). 5. Adenosine analogues produced long-lasting relaxation of phenylephrine-precontracted aortic rings with the following rank order of potency (EC50 values, microM): 5'-N-ethylcarboxamidoadenosine (0.68 +/- 0.06) > 2-chloroadenosine (4.3 +/- 0.6) > adenosine (104 +/- 13). Maximal relaxations elicited by these agents were 71 +/- 3, 98 +/- 1, and 100 +/- 1%, respectively. CGS 21680 and DPMA at 100 microM elicited smaller relaxations of the precontracted tissues (12 +/- 2 and 43 +/- 15%, respectively). 6. Antagonism by xanthine derivatives of the 5'-N-ethylcarboxamidoadenosine-induced relaxation of aortic rings showed the following rank order of potency (Ki, nM): xanthine amino congener (17 +/- 4) > 8-cyclopentyl-1,3-dipropylxanthine (171 +/- 36) > PD 115,199 (341 +/- 64) > 3,7-dimethyl-1-propargylxanthine (5520 +/- 820).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor-stimulated phosphoinositide turnover.

1. The abilities of the four diastereoisomers of 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methylcarboxyphenylglycine (MCPG) to inhibit, phosphoinositide turnover in neonatal rat cerebral cortex have been studied. Two indices of phosphoinositide cycle activity were assessed; inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation, and total inositol phosphate [3H]-InsPx accumulation (in the presence of Li+) in myo-[3H]-inositol prelabelled slices. 2. The diastereoisomers of ACPD stimulated each response with a rank order of potency of 1S, 3R > 1R, 3R > 1S, 3S >> 1R, 3S. The response to 1R, 3R-ACPD was largely prevented by pre-addition of the NMDA-receptor antagonist, MK-801, or omission of extracellular Ca2+, suggesting that this isomer acts indirectly on phosphoinositide responses through activation of NMDA-type ionotropic glutamate receptors. In contrast, the responses to 1S, 3R- and 1S, 3S-ACPD were unaffected by prior addition of MK-801, but were blocked by MCPG. 3. The concentration of 1S, 3R-ACPD required to half-maximally stimulate the Ins(1,4,5)P3 response (-log EC50 (M), -4.09 +/- 0.10) was significantly higher than that required to exert a similar effect on [3H]-InsPx accumulation (-log EC50 (M), -4.87 +/- 0.07; P < 0.01; n = 4). A similar marked 8-9 fold discrepancy between these two values was observed for the 1S, 3S isomer, which elicited similar maximal responses to those caused by 1S, 3R-ACPD. 4. Significant differences were also observed with respect to the ability of (+/-)-MCPG (1 mM) to cause a rightward shift in the concentration-response relationships for 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 (5.59 +/- 0.24 fold shift) and [3H]-InsPx (3.04 +/- 0.34 fold shift; P < 0.01; n = 4) responses, giving rise to Kd values of 218 and 490 microM for (+/-)-MCPG antagonism of the respective responses. 5. The potency difference between the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 and [3H]-InsPx responses was reduced when experiments were performed in nominally calcium-free medium ([Ca2+]e = 2 - 5 microM) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+]e < or = 100 nM). Similarly, the Kd value for (+/-)-MCPG antagonism of the 1S, 3R-ACPD-stimulated [3H]-InsPx response decreased under [Ca2+]e-free conditions, approaching those obtained for the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 response in the presence of normal [Ca2+]e. 6. These data suggest that estimates of the activities of mGluR agonists and antagonists, derived by measuring phosphoinositide turnover, can differ significantly depending on whether Ins(1,4,5)P3 mass or [3H]-InsPx responses are measured. In particular, the possibility that the mGluR-mediated [3H]-InsPx response may not simply reflect direct receptor/G protein/phosphoinositidase C (PIC) activation, but may also be the consequence of stimulation of a facilitatory Ca2+-influx pathway is discussed.     

Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP.

1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells.     

Adenosine induces cyclic-AMP formation and inhibits endothelin-1 production/secretion in guinea-pig tracheal epithelial cells through A(2B) adenosine receptors

1. The adenosine receptor subtype mediating adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin-1 (ET-1) secretion were studied in primary cultures of tracheal epithelial cells. 2. Adenosine analogues showed the following rank order of potency (pD(2) value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5'-N-ethylcarboxyamidoadenosine (NECA, A(1)/A(2A)/A(2B), pD(2): 5.44+/-0.16)>adenosine (ADO, non selective, pD(2): 4.99+/-0. 09; 71+/-9% of NECA response) >/=2-Cl-adenosine (2CADO, non selective, pD(2): 4.72+/-0.14; 65+/-9% of NECA response)>CGS21680 (A(2A); inactive at up to 100 microM). 3. Cyclic AMP formation stimulated by NECA in guinea-pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA(2) value): Xanthine amine congeners (XAC, A(2A)/A(2B), 7.89+/-0.22)>CGS15943 (A(2A)/A(2B), 7.24+/-0. 26)>ZM241385 (A(2A), 6.69+/-0.14)>DPCPX (A(1), 6.51+/-0. 14)>3n-propylxanthine (weak A(2B), 4.30+/-0.10). This rank order of potency is typical for A(2B)-adenosine receptor. 4. Adenosine decreased basal and LPS-stimulated irET production in a concentration-dependent manner. Moreover, NECA but not CGS21680 inhibited LPS-induced irET production. 5. The inhibitory effect of NECA on LPS-induced irET production was reversed by XAC (pA(2)=8.84+/-0. 12) and DPCPX (pA(2)=8.10+/-0.22). 6. These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea-pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A(2B) subtype. This adenosine receptor may be involved in the regulation of the level of ET-1 production/secretion by guinea-pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.     

Characterization of the interaction between muscarinic M2 receptors and beta-adrenoceptor subtypes in guinea-pig isolated ileum.

1. Contraction of guinea-pig ileum to muscarinic agonists is mediated by M3 receptors, even though they account for only 30% of the total muscarinic receptor population. The aim of this study was to characterize the biochemical and functional effects of stimulation of the predominant M2 muscarinic receptor (70%) and to investigate the hypothesis that M2 receptors specifically oppose beta-adrenoceptor-mediated effects in the ileum. 2. In guinea-pig ileal longitudinal smooth muscle slices, isoprenaline, a non-selective beta-adrenoceptor agonist, and BRL 37344 (sodium-4-[2-[2-hydroxy-2-(3- chlorophenyl)ethylamino]propyl]-phenoxyacetate sesquihydrate), a beta 3-adrenoceptor selective agonist, increased cyclic AMP accumulation with -log EC50 values of 6.6 +/- 0.1 and 5.8 +/- 0.1 respectively. Maximal stimulation by BRL 37344 (10 microM) was 26.4 +/- 5.2% of that observed with isoprenaline (10 microM). Isoprenaline (10 microM)-stimulated cyclic AMP accumulation was significantly, but not completely, inhibited by propranolol (5 microM), with a propranolol-resistant component of 28.2 +/- 6.8% of the maximal stimulation to isoprenaline. In contrast, basal and BRL 37344 responses were resistant to this antagonist. These data provide evidence that both beta 1- and beta 3-adrenoceptors activate adenylyl cyclase in guinea-pig ileum. 3. Isoprenaline (10 microM)-stimulated cyclic AMP accumulation was inhibited (67.4 +/- 0.9%) by the muscarinic agonist (+)-cis-dioxolane (-log EC50 = 7.3 +/- 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)     

(S)-homoquisqualate: a potent agonist at the glutamate metabotropic receptor.

The synthetic quisqualate analogue, (S)-homoquisqualate was examined for activity at the glutamate metabotropic receptor, in relation to its ability to stimulate phosphoinositide hydrolysis in rat pup cerebro-cortical slices. The compound produced a robust increase in hydrolysis (EC50 = 50.2 +/- 1.6 microM), which, in common with responses to quisqualate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), was antagonized uncompetitively by L-2-amino-3-phosphonopropionate (L-AP3). In contrast to quisqualate which exhibits low efficacy, (S)-homoquisqualate behaves as a full agonist at the metabotropic receptor.     

Pharmacological characterization of metabotropic glutamate receptors potentiating NMDA responses in mouse cortical wedge preparations.

1. Mouse cortical wedge preparations were used in order to study the effects of metabotropic glutamate receptor (mGluR) agonists and antagonists on the depolarization induced by N-methyl-D-aspartate (NMDA) or by (S)-alpha-amino-4-bromo-3-hydroxy-5-isoxazolepropionic acid (AMPA). 2. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (30-300 microM) significantly potentiated the depolarizations induced by NMDA, leaving unchanged those mediated by AMPA. This potentiation developed slowly and lasted for up to 60 min provided that the slices were continuously perfused with the mGluR agonist. 3. Concentration-response curves to NMDA in the absence and in the presence of 1S,3R-ACPD (100 microM) indicated that the potentiation was due to increased affinity of the NMDA receptor complex for its agonist. The maximal responses to NMDA were not potentiated. 4. Selective agonists of group 1 mGluR such as quisqualate (Quis) (30 microM) or (RS)-3,5-dihydroxyphenylglycine (DHPG) (300 microM) did not potentiate NMDA responses. Similarly, selective agonists of group 2 mGluRs, such as (2S,3S,4S)-alpha-carboxycyclopropyl-glycine (L-CCG-I) (3-30 microM), and of group 3, such as L-2-amino-4-phosphonobutyric acid (L-AP4) (100 microM) were inactive in our test. A number of other putative mGluR agents having partial agonist activity on mGluRs in brain slices and in expression systems, such as 1R,3S-ACPD (500 microM), DL-2-amino-3-phosphonopropionic acid (DL-AP3) (300 microM) and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG; 500 microM), when placed in the experimental protocol we used, did not change NMDA responses. 5. Available mGluR antagonists, such as DL-AP3 (1 mM), (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) (500 microM), S-4-carboxyphenylglycine (4CPG; 500 microM) and S-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG; 500 microM), did not reduce 1S,3R-ACPD potentiation of NMDA responses. 6. It is concluded that the potentiation of NMDA currents induced by the mGluR agonist 1S,3R-ACPD, in mouse cortical wedges, has a pharmacological profile which is different from that of the three mGluR groups so far described in expression systems.     

Signal transduction pathways involved in the acute potentiation of NMDA responses by 1S,3R-ACPD in rat hippocampal slices.

1. A grease-gap recording technique has been used to investigate the mechanisms underlying the acute potentiation of N-methyl-D-aspartate (NMDA) responses by aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) in area CA1 of rat hippocampal slices. 2. 1S,3R-ACPD (10 microM), but not 1R,3S- ACPD (10 microM), potentiated submaximal responses to NMDA (dose-ratio of 0.81 +/- 0.02 (mean +/- s.e.mean); n = 55), but not to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), in a readily reversible manner. Potentiation also occurred in slices treated with 0.2 microM tetrodotoxin, and in slices perfused with Mg(2+)-free medium. 3. 1S,3R-ACPD-induced potentiation was unaffected by the protein kinase inhibitors K-252b (0.1 microM) and staurosporine (1 microM) and the intracellular Ca2+ store depletor, thapsigargin (10 microM). Coapplication of staurosporine and thapsigargin was also without effect. 4. 1S,3R-ACPD-induced potentiation was unaffected by inhibitors of arachidonic acid formation, bromophenacyl bromide (50 microM) and RG80267 (100 microM). Arachidonic acid (10-50 microM) depressed reversibly NMDA-induced responses. The potentiation was unaffected by 8-bromo cyclic AMP (500 microM) or the PKA inhibitor Rp-adenosine 3,5-cyclic monophosphothioate triethylamine (Rp-cAMPS; 50 microM). 5. 1S,3R-ACPD-induced potentiation was abolished in slices perfused with Ca(2+)-free medium. The potentiation was also blocked by phorbol-12,13-diacetate (1 microM), in a staurosporine-sensitive manner. 6. It is concluded that the potentiation of NMDA responses by 1S,3R-ACPD is not mediated by protein kinase A or C, by release of Ca2+ from intracellular stores or by arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of NMDA effects on agonist-stimulated phosphoinositide turnover by memantine in neonatal rat cerebral cortex.

1. The ability of memantine (1-amino-3,5-dimethyladamantane) to antagonize the modulatory effects of N-methyl-D-aspartate (NMDA) on phosphoinositide turnover stimulated by muscarinic cholinoceptor- and metabotropic glutamate receptor-agonists has been examined in neonatal rat cerebral cortex slices. 2. Memantine antagonized the inhibitory effect of NMDA (100 microM) on both total [3H]-inositol phosphate ([3H]-InsPx) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulations stimulated by carbachol (1 mM) with EC50 values of 21 and 16 microM respectively. 3. Memantine concentration-dependently antagonized (IC50 24 microM) the ability of NMDA (10 microM) to potentiate [3H]-InsPx accumulation in response to a sub-maximal concentration of the metabotropic glutamate receptor agonist, 1S,3R-ACPD (10 microM). 4. The small (approx. 3 fold), concentration-dependent increase in [3H]-InsPx accumulation stimulated by NMDA was completely antagonized by the prototypic NDMA receptor-channel blocker, MK-801 (1 microM) at all concentrations of NDMA studied (1-1000 microM). In contrast, antagonism by memantine (100 microM) was observed only at low concentrations of NMDA (1-10 microM), whilst [3H]-InsPx accumulation stimulated by high concentrations of NMDA (300-1000 microM) was markedly enhanced by memantine. 5. Assessment of the incorporation of [3H]-inositol into inositol phospholipids revealed that memantine (100 microM) caused an approximate 2 fold increase in the labelling of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine receptor-induced second messenger production in adult guinea-pig cerebellum.

1. The effects of adenosine receptor agonists on cyclic nucleotides accumulation were investigated in adult guinea-pig cerebellar slices by use of radioactive precursors. 2. Adenosine elicited a rapid and maintained increase in cyclic AMP, that was fully reversed upon addition of adenosine deaminase. Adenosine analogues stimulated cyclic AMP generation up to 40 fold with the rank order of potency: 5'-N-ethylcarboxamidoadenosine (0.6 microM) > 2-chloroadenosine (6 microM) > adenosine (13 microM). CGS 21680 (10 microM) elicited only a small stimulation (1.2 fold). 3. The cyclic AMP response to NECA was reversed by the 1,3-dipropylxanthine-based adenosine receptor antagonists 8-[4-[[[[(2-aminoethyl)amino]amino]carbonyl]methyl]oxy]- phenyl]-1,3-dipropylxanthine (XAC), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(dimethylamino)ethyl]N-methyl-4-(1,3-dipropylxanthine)benzene sulphonamide (PD 115,199) with estimated apparent inhibition constants of 15, 81 and 117 nM, respectively. 4. Pretreatment with adenosine also potentiated the cyclic GMP response to sodium nitroprusside, abolishing the decline in [3H]-cyclic GMP observed with sodium nitroprusside alone, and allowing [3H]-cyclic GMP levels to be maintained for at least an additional 10 min. This potentiation was fully reversed by adenosine deaminase. 5. Adenosine analogues potentiated the sodium nitroprusside-elicited cyclic GMP generation with the rank order of potency: 5'-N-ethylcarboxamidoadenosine (0.7 microM) > 2-chloroadenosine (6 microM) > adenosine (42 microM). 6. NECA potentiation of cyclic GMP formation was reversed by the antagonists XAC, DPCPX and PD 115,199 with apparent inhibition constants of 17, 102 and 242 nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)