Modulation of cyclic AMP formation by putative metabotropic receptor agonists.

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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)     

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.     

Inhibition of histamine-stimulated inositol phospholipid hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle.

1. The effect on histamine-stimulated [3H]-inositol phosphate accumulation of a range of agents which increase the accumulation, or mimic the actions, of cyclic AMP has been investigated in bovine tracheal smooth muscle. 2. Salbutamol (1 microM), forskolin (1 microM) and vasoactive intestinal peptide (VIP, 1 microM) inhibited the inositol phosphate response to 0.1 mM histamine and increased the accumulation of [3H]-cyclic AMP in [3H]-adenine-labelled slices of bovine tracheal smooth muscle. The effect on inositol phospholipid hydrolysis was mimicked by the membrane permeant analogues of cyclic AMP, dibutrylcyclic AMP (1 mM) and 8-bromo-cyclic AMP (1 mM). 3. In contrast to salbutamol, which was equally effective at producing the two effects, forskolin produced large increases in [3H]-cyclic AMP accumulation (EC50 = 1.2 microM) at much higher concentrations than those required for inhibition of histamine-stimulated [3H]-inositol phosphate accumulation (EC50 = 0.09 microM). However, significant increases in [3H]-cyclic AMP accumulation, of similar magnitude to those obtained with salbutamol and VIP, were observed over the concentration range appropriate for inhibition of the inositol phosphate response to histamine. 4. In the presence of histamine (0.1 mM), isobutylmethylxanthine (IBMX, 1 mM) and rolipram (0.1 mM) both significantly (P less than 0.05) elevated tissue [3H]-cyclic AMP levels. IBMX, rolipram and (to a lesser extent) SKF 94120 significantly (P less than 0.05) reduced histamine-stimulated [3H]-inositol phosphate accumulation by 81%, 68% and 20%, respectively. M&B 22948 was without a significant effect on either [3H]-cyclic AMP or histamine-induced [3H]-inositol phosphate accumulation. 5. Both rolipram and forskolin reduced the increase in incorporation of [3H]-inositol into membrane phospholipids which followed stimulation with histamine. However, a significant inhibition of [3H]-inositol phosphate accumulation could be demonstrated under conditions in which there was no change in the level of [3H]-inositol incorporation.     

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.     

Metabotropic glutamate receptors, transmitter output and fatty acids: studies in rat brain slices.

1. The effects of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), a non-selective agonist of the metabotropic glutamate receptors (mGluRs), have been studied in rat cortical and striatal slices by measuring the depolarization-induced output of D-[3H]-aspartate (D-[3H]-Asp) and of [3H]-glutamate ([3H]-Glu), neosynthesized from [3H]-glutamine. 2. In cortical slices, 1S,3R-ACPD potentiated the depolarization-induced (KCl, 30 mM) output of both D-[3H]-Asp and [3H]-Glu. The potentiation, obtained at 300 microM 1S,3R-ACPD was 65 +/- 6% for D-[3H]-Asp and 56 +/- 10% for [3H]-Glu. Conversely, in striatal slices, 1S,3R-ACPD reduced the depolarization-induced transmitter output. The reduction, obtained at 300 microM of the agonist, was 60 +/- 8% for D-[3H]-Asp and 50 +/- 5% for neosynthesized [3H]-Glu. 3. Bovine serum albumin (BSA, 15 microM), which is able to bind locally produced fatty acids, completely eliminated the potentiating effect 1S,3R-ACPD had on D-[3H]-Asp output from cortical slices. Low concentrations of arachidonic acid (1-10 microM) or of oleic acid (1-10 microM) added to BSA-containing perfusion medium, restored this potentiating effect. BSA, however, had no effect on the inhibitory action of 1S,3R-ACPD in striatal slices. 4. Bromophenacyl bromide (100 microM), an inhibitor of phospholipase A2, and RG80267 (100 microM), an inhibitor of diacylglycerol lipase, have been shown to inhibit fatty acid production. These compounds prevented the potentiating effect of 1S,3R-ACPD on D-[3H]-Asp-output in cortical slices. 5. Indomethacin (100 microM), an inhibitor of cyclo-oxygenases, plus nordihydroguaiaretic acid (100 microM), an inhibitor of lipoxygenases, increased D-[3H]-Asp output in cortical slices perfused with BSA-containing medium. 6. These experiments suggest that the mGluR-mediated potentiation of transmitter output requires the availability of unsaturated fatty acids, such as arachidonic or oleic acids, in cortical slices. In contrast, the mGluR-induced inhibition of transmitter output is not dependent upon fatty acid availability in striatal slices. The requirement of both unsaturated fatty acids and 1S,3R-ACPD in the facilitation of transmitter exocytosis may play an important role in the regulation of synaptic plasticity.     

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.     

Modulation of fluoroaluminate-induced inositol phosphate formation by increases in tissue cyclic AMP content in bovine tracheal smooth muscle.

1. The effect of fluoroaluminate complexes (AlCl3 plus NaF) upon smooth muscle tone, [3H]-inositol phosphate accumulation and [3H]-cyclic AMP accumulation has been investigated in slices of bovine tracheal smooth muscle. 2. Fluoroaluminate (10 microM AlCl3 + various concentrations of NaF) elicited concentration-dependent contractions of bovine tracheal smooth muscle strips at concentrations of NaF in the range 1-10 mM. The resultant contractile response was reversed by isoprenaline (50 nM) and was preserved in calcium-free medium. 3. Fluoroaluminate stimulated [3H]-inositol phosphate formation at concentrations of NaF over 1 mM. The response to 20 mM NaF + 10 microM AlCl3 was 164 +/- 29% of the response to 1 mM histamine. Fluoroaluminate also increased the incorporation of [3H]-myo-inositol into membrane phospholipids. 4. Fluoroaluminate produced a small rise in [3H]-cyclic AMP levels (2.1 fold increase over basal with 20 mM NaF). The response to forskolin (1 microM, 8.6 fold over basal) was reduced by fluoroaluminate in a concentration-dependent manner, but still remained significantly (P less than 0.05) elevated over the response to fluoroaluminate alone. 5. The [3H]-inositol phosphate response to fluoroaluminate was inhibited by salbutamol (maximum inhibition 60%, IC50 = 0.08 microM), forskolin (1 microM, 46% inhibition) and isobutylmethylxanthine (1 mM, 73% inhibition). 6. These data suggest that inhibition of agonist-induced inositol phospholipid turnover by cyclic AMP in this tissue can occur at the post-receptor level.     

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)     

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).     

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.     

The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells.

1 Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the mu-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+]i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors. 2 E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHO micro and SH-SY5Y membranes with pKi values of 8.02+/-0.09 and 8.54+/-0.13 respectively. E-2 displaced [3H]-DPN binding in CHOmu and SH-SY5Y cells with pKi values of 7.82+/-0.11 and 8.43+/-0.13 respectively. E-1/E-2 bound weakly to CHOdelta and CHOkappa membranes, with IC50 values of greater than 10 microM. 3 In CHOmu cells, E-1/E-2 inhibited forskolin (1 microM) stimulated cyclic AMP formation with pIC50 values of 8.03+/-0.16 (Imax = 53.0+/-9. 3%) and 8.15+/-0.24 (Imax = 56.3+/-3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72+/-0.13 (Imax=46.9+/-5.6%) and 8.11+/-0.31 (Imax = 40.2+/-2.8%) respectively. 4 E-1/E-2 (1 microM) increased [Ca2+]i in fura-2 loaded CHOmu cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106+/-28 and 69+/-6.7 nM respectively. In single adherent cells E-1/E-2 (1 microM) increased [Ca2+]i with a mean 340/380 ratio change of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOdelta, CHOkappa and SH-SY5Y cells. 5 These data show that E-1/E-2 bind with high affinity and selectivity to mu-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the mu-opioid receptor.     

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)     

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)     

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)     

Characterisation of α1B-adrenoceptors linked to inositol phosphate formation and calcium mobilisation in human astrocytoma U373 MG cells

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.     

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 dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus.

1. We studied the release of [3H]-dopamine and [3H]-noradrenaline (NA) from hippocampal synaptosomes induced by glutamate receptors and the associated Ca2+ influx through Ca2+ channels. The release of tritiated neurotransmitters was studied by use of superfusion system and the intracellular free Ca2+ concentration ([Ca2+]i) was determined by a fluorimetric assay with Indo-1 as a probe for Ca2+. 2. Presynaptic glutamate receptor activation induced Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA from rat hippocampal synaptosomes. Thus, L-glutamate induced the release of both neurotransmitters in a dose-dependent manner (EC50 = 5.62 microM), and the effect of 100 microM L-glutamate was inhibited by 83.8% in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX), but was not affected by 1 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801). 3. Other glutamate receptor agonists also stimulated the Ca(2+)-dependent release of [3H]-dopamine and [3H]-NA as follows: N-methyl-D-aspartate (NMDA), at 200 microM, released 3.65 +/- 0.23% of the total 3H catecholamines, and this effect was inhibited by 81.2% in the presence of 1 microM MK-801; quisqualate (50 microM), S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA) (100 microM) or kainate (100 microM) released 1.57 +/- 0.26%, 1.93 +/- 0.17% and 2.09 +/- 0.22%, of the total 3H catecholamines, respectively. 4. The ionotropic glutamate receptor agonist, AMPA, induced an increase in the [Ca2+]i which was inhibited by 58.6% in the presence of 10 microM CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)