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

Abstract

1 The abilities of the four diastereoisomers of 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mGluR) antagonist (±)-α-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]-InsP(x) 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]-InsP(x), 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]-InsP(x), (3.04 ± 0.34 fold shift; P < 0.01; n=4) responses, giving rise to K(d) values of 218 and 490 μM for (+)-MCPG antagonism of the respective responses. 5 The potency difference between the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 and [3H]-InsP(x), responses was reduced when experiments were performed in nominally calcium-free medium ([Ca2+](e) = 2-5 μM) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+](e) ≤ 6100 nM). Similarly, the K(d) value for (±)-MCPG antagonism of the 1S, 3R-ACPD-stimulated [3H]-InsP(x), 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]-InsP(x), responses are measured. In particular, the possibility that the mGluR-mediated [3H]-InsP(x), 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.