Lysophosphatidic acid increases intracellular free calcium ion levels in neuro 2A and CHO.A2A cells

Anongnard Kasorn, David A. Kendall and Stephen P.H. Alexander, Institute of Neuroscience and School of Biomedical Sciences, University of Nottingham Medical School, Queens Medical Centre, Nottingham, NG7 2UH, ENGLAND

LPA (lysophosphatidic acid) receptors are thought to couple to multiple G proteins to elicit cellular responses. One particular cellular response described for LPA receptors is the elevation of intracellular free calcium ion levels (Ca2+i, Ishii et al., 2000). In this report, we have investigated the expression and functional coupling of LPA receptors to elevation of Ca2+ i in Neuro 2a neuroblastoma and CHO.A2A fibroblast cells.

LPA-induced increases of Ca2+ i in Neuro 2a (albino mouse neuroblastoma) and CHO.A2A (Chinese hamster ovary cells transfected with human A2A adenosine receptors) cells were assessed using Fura 2 on a 96-well FlexStation (Molecular Devices). Confluent Neuro 2A and CHO.A2A cells were grown in 96 well plates coated with laminin and poly-L-lysine for a week before assessing changes in Ca 2+ i. Ionomycin (4 μM) was used as a normalization stimulus, examining responses in triplicate wells over 3-6 different experiments, with statistical analysis by one way ANOVA with Newman-Keuls post-hoc test. Using parallel cultures, the expression of LPA receptors (LPA1-4) in these cells was also investigated using reverse transcriptase-polymerase chain reaction (RT-PCR).

RT-PCR analysis (27-35 cycles) suggested expression of LPA2 receptors in both cells, while LPA1 and LPA 4 receptor expression was only detected in CHO.A2A cells. In the absence of added bovine serum albumin (BSA), LPA (10 μM) was without significant effect on Ca2+i (4 ± 2 % ionomycin response) in Neuro 2a cells. However, in the presence of 0.1 % BSA, 10 μM LPA induced a significant increase in Ca2+i in Neuro 2a cells, using either fatty acid-containing or fatty acid-free BSA, with responses of 68 ± 12 and 74 ± 14 % relative to ionomycin, respectively (P < 0.01). Ca2+ i responses to LPA in Neuro 2a cells were concentration-dependent with pEC50 values of 6.6 ± 0.1 and 6.5 ± 0.04, and maximal responses of 84 ± 5 and 78 ± 6 % ionomycin response, in the presence of 0.1 % fatty acid-containing and fatty acid-free BSA, respectively (n = 6). The putative antagonist dioctanoylglycerol pyrophosphate (DGPP, 150 μM, Fischer et al., 2001) evoked an elevation of Ca2+ i in both Neuro 2a and CHO.A2A cells (35 ± 3, P<0.01 and 9 ± 3 % ionomycin response, P<0.05), respectively. In CHO.A2A cells, DGPP failed to alter LPA-evoked Ca2+ i responses (control 48 ± 7; 20 µM 43 ± 14; 150μM 43 ± 5 % ionomycin response). In Neuro 2a cells, however, DGPP caused a concentration-dependent reduction in LPA-evoked Ca2+i (control 99 ± 3; 20 μM 57 ± 13, P<0.05; 150 μM 38 ± 4 % ionomycin response, P<0.01).

In conclusion, LPA receptors appear differentially expressed in Neuro 2a and CHO.A2A cells, with a distinct sensitivity to the putative antagonist DGPP.

Fischer, D.J., Nusser, N., Virag, T., et al. (2001). Mol. Pharmacol., 60, 776-784.
Ishii, I., Contos, J.J.A., Fukushima, N., et al. (2000). Mol. Pharmacol., 58, 895-902.