LYSOPHOSPHATIDIC ACID STIMULATES CALCIUM TRANSIENTS IN ENTERIC GLIA B. J. SEGURA, W. ZHANG, R. A. COWLES, L. XIAO, T. R. LIN, C. LOGSDON AND M. W. MULHOLLAND* Department of Surgery, University of Michigan, 2101 Taubman Cen- ter, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346, USA Abstract—The enteric nervous system plays an integral role in the gastrointestinal tract. Within this intricate network, enteric glia are crucial in the maintenance of normal bowel function, yet their signaling mechanisms are poorly under- stood. Enteric glia, and not enteric neurons, selectively re- sponded to lysophosphatidic acid (LPA), a product of phos- phatidylcholine metabolism, with dose-dependent calcium (Ca 2 ) signaling over a range from 100 pM to 10 M. The elicited calcium transients involved both the mobilization of intracellular Ca 2 stores and the influx of extracellular Ca 2 as LPA signals were obliterated following the depletion of intracellular Ca 2 and attenuated by the removal of Ca 2 from the perfusion buffer. Pretreatment with pertussis toxin (100 ng/ml) reduced the magnitude of LPA Ca 2 transients (9520 nM vs 16817 nM for controls). Repetitive exposure yielded diminished responsiveness, with a 25% reduction in [Ca 2 ] i between first and second exposures. Inhibition of the inositol 1,4,5-trisphosphate (IP 3 ) receptor with 200 M 2-ami- noethoxydiphenylborate (2APB) abolished LPA signals. RT- PCR analysis demonstrated the presence of two LPA-cou- pled endothelial differentiation gene (EDG) receptor mRNAs (EDG-2 and EDG-7) in myenteric plexus primary cultures. EDG-2 expression in glial cells of the ENS was confirmed immunocytochemically. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: enteric nervous system, sphingolipid, EDG re- ceptors. A family of signaling molecules and growth factors, collec- tively known as “bioactive lipids,” includes sphingosine-1- phosphate (S1P) and lysophosphatidic acid (LPA). Both are products of membrane lipid catabolism. S1P is enzy- matically generated from sphingomyelin whereas LPA is derived from phosphatidylcholine. Each lysophospholipid has been implicated in a diverse array of cellular events including cell growth and differentiation, cytoskeletal changes and cell migration, and calcium (Ca 2+ ) signaling (An et al., 1999; Edsall et al., 1997; Postma et al., 1996; Sadahira et al., 1992; Wang et al., 1999). The Ca 2+ ion is a critical second messenger which serves to transduce extracellular signals into biological responses. In neural systems, cytosolic levels of Ca 2+ are an integral factor in gene expression, apoptosis, intercellular communication, synaptic neurotransmission, and long-term potentiation— events in which glia participate (Araque et al., 1999). LPA and S1P are distinctive in that, although lipids, they act as extracellular signaling molecules in a receptor- mediated fashion (Moolenaar, 1999). A family of G protein- coupled receptors with affinity for S1P (endothelial differ- entiation gene receptors: EDG-1, EDG-3, EDG-5, EDG-6, and EDG-8) and LPA (EDG-2, EDG-4, and EDG-7) has been described (Bandoh et al., 1999; Kon et al., 1999; Zondag et al., 1998). Although the evidence in support of S1P and LPA functioning via extracellular receptors is convincing, the mechanisms responsible for Ca 2+ mobili- zation are not completely defined. A role for these bioac- tive lipids in the enteric nervous system has not been recognized. We hypothesized that LPA could serve as a signaling molecule within the myenteric plexus and that it would function through putative LPA receptors, EDG-2, EDG-4, or EDG-7. The current studies of the mechanisms by which LPA affects intracellular Ca 2+ concentration ([Ca 2+ ] i ) in enteric glia demonstrate that: 1) LPA causes dose-depen- dent Ca 2+ signaling; 2) LPA transients involve intracellular and extracellular Ca 2+ ; 3) LPA-induced Ca 2+ responses desensitize with repetitive exposure; 4) LPA effects are attenuated by pertussis toxin (PTX) exposure; 5) LPA mo- bilizes thapsigargin- and inositol-1,4,5-trisphosphate-sen- sitive intracellular Ca 2+ stores; and 6) myenteric plexus cultures express EDG-2, and EDG-7 receptor mRNA, with EDG-2 demonstrated immunocytochemically in enteric glia. EXPERIMENTAL PROCEDURES LPA, ATP, collagenase type V, thapsigargin, U73122, U73343, trypsin-EDTA, soybean trypsin inhibitor (type I-S), penicillin-strep- tomycin solution, HEPES, and saponin were from Sigma Chemi- cal (St. Louis, MO, USA). Pertussis toxin (PTX) was from Calbio- chem (San Diego, CA, USA). Hanks’ balanced salt solution, me- dium 199, and L-glutamine were from GIBCO BRL (Grand Island, NY, USA). Rat tail collagen was from Boehringer Mannheim (Mannheim, Germany). New Serum I (NSI) was from Collabora- tive Research (Bedford, MA, USA). 2-Aminoethoxydiphenyl bo- rate was from Tocris (Ballwin, MO, USA). Fura-2-AM, fluorescein- conjugated streptavidin (S-869), DAPI cytological nuclear coun- terstain kit (C-7590) and prolong antifade kit (p-7481) were from Molecular Probes, Inc. (Eugene, OR, USA). TRIzol reagent, DNAse I, SuperScript II RNase H - Reverse Transcriptase (RT), and RT buffer were from Life Technologies, Inc. (Grand Island, *Corresponding author. Tel: +1-734-936-3236; fax: +1-734-763- 5615. E-mail address: micham@umich.edu (M. W. Mulholland). Abbreviations: [Ca 2+ ] i , intracellular Ca 2+ concentration; EDG, endo- thelial differentiation gene; HEPES, N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid; IP 3 , inositol 1,4,5-trisphosphate; LPA, lysophos- phatidic acid; NSI, New Serum I; PCR, polymerase chain reaction; PTX, pertussis toxin; RT, reverse transcriptase/transcription; SPC, sphingosylphosphorylcholine; S1P, sphingosine-1-phosphate; 2APB, 2-aminoethoxydiphenylborate. Neuroscience 123 (2004) 687– 693 0306-4522/04$30.00+0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.10.003 687