The Journal of Neuroscience, October 26, 2005, ():
Postsynaptically Synthesized Prostaglandin E2 (PGE2) Modulates Hippocampal Synaptic Transmission via a Presynaptic PGE2 EP2 Receptor
J. Neurosci. Sang et al.
25: 9858
Supplemental data
Files in this Data Supplement:
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Supplementary figure 2. Thansfection with shRNA-encoding plasmid targeting EP2 reduces mRNA and protein of the EP2 receptor in RAW264.7 macrophages. A. Real-time RT PCR analysis of mRNA of EP2 in RAW264.7 macrophages transfected with EP2-shRNA plasmid. RAW264.7 macrophages (ATCC, TIB-71) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL, Gaithersburg, MD), supplemented with 10% heat inactivated fetal bovine serum. Transfection with shRNA-encoding plasmid (EP2-shRNA or luciferase-shRNA) into RAW264.7 cells was conducted using Lipofectamine 2000 (Invtrogen). 1X106 cells were plated onto 6 well culture chambers one day before transfection. DNA (4 mg) and lipofectamine reagent (8 ml) were mixed and added into the cultures. Total RNA was isolated 48 hours after transfection and used for real-time RT PCR analysis as described in the Experimental procedures section. Results are obtained from 3 independent cultures with duplicate wells. B. Western blot analysis of EP2 expression in RAW264.7 macrophages transfected with EP2-shRNA plasmid. C. Graphic quantitation of EP2 protein protein expression (normalized to the control) Proteins were harvested 72 hours following transfection for Immunoblot analysis. Cells were lysed by incubation on ice for 30 min in lysis buffer containing 50 mM Tris–HCl (pH 8), 1% triton X-100, 150 mM NaCl, 0.1% SDS, 0.5%DOC and 1Xprotease inhibitors cocktail (BOEHRINGER MANNHEIM GmbH,Germany). The extract was pelleted by centrifugation at 14,000g for 15 min at 4 oC, and the supernatants were then collected. Samples were loaded on 4-20% SDS gel, transferred to nitrocellulose membrane. The membrane was incubated with a rabbit anti-EP2 polyclonal antibody (dilution of 1:1000, Cayman, Ann Arbor, MI) at 4oC over night. The blot was washed, and incubated with a secondary antibody (goat anti rabbit 1:10000, Vector Laboratories, Burlingame, CA) at room temperature for 1 hour. Bound antibody was visualized using chemiluminescent substrate (ECL, Habersham, Arlington Heights, IL), and exposed to X-ray film (Hyperfilm-ECL Amersham). To check the equal loading, the membrane was incubated with re-probed solution (Geno Technology,St Lonis MO) at room temperature for 3 hours, then membrane was incubated with a monoclonal primary antibody (mouse anti β-actin 1: 4000 Sigma) at 4oC overnight. The blot was washed, and incubated with a secondary antibody (goat anti mouse 1: 20000, PIERCE Rockford, IL) at room temperature for 1 hour. The Bound antibody was visualized using chemiluminescent substrate (ECL, Habersham, Arlington Heights, IL).and exposed to X-ray film (Hyperfilm-ECL Amersham). Autoradiograph films were scanned using Digital imaging and analysis systems (alpha Innotech) to obtain integrated densitometric values.
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Supplemental table.
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Figure 1- Forskolin increases synaptic transmission in primary hippocampal neurons in culture.
a. Representative sweeps of mEPSCs in the absence or presence of Forskolin (20 μM) and washout.
b. Cumulative probability of mEPSC frequency in the absence of presence of forskolin and washout.
c. Mean percentage changes in the frequency of mEPSCs at 10 min after the application of forskolin.
d. Cumulative probability of mEPSC amplitude in the absence or presence of forskolin and washout.
e. Mean percentage changes in the frequency of mEPSCs at 10 min after the application of forskolin.
f. Time courses of forskolin-induced changes in frequency and amplitude of mEPSCs.
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Figure 3- Supplemental figure 3. Cartoon illustrating the COX-2-mPGES-1/2-synthesized PGE2 as a retrograde messenger in hippocampal synaptic signaling. Presynaptic depolarization-induced glutamate release increases an influx of Ca2+ through the postsynaptic NMDA receptor. The elevated Ca2+ modulates several signaling pathways in the postsynaptic dendritic space, including the activation of phospholipase A2 (PLA2) that releases arachidonic acid (AA). AA is converted by COX-2 to PGH2. PGH2 is the substrate of microsomal prostaglandin E synthase-1 (mPGES-1) and mPGES-2. PGE2 is released from the postsynaptic sites, and functions as a retrograde messenger through the presynaptic EP2/4 receptors by increasing the probability of the release of glutamate. PGE2 may also be a paracrine messenger on EP (EP1?) receptors in neighboring astrocytes and release glutamate through a Ca2+-dependent mechanism (Bezzi et al., 1998), and possibly an autocrine signal on the EP1/3 receptors at postsynaptic sites. Activation of the presynaptic EP2/4 receptors increases the levels of cAMP and PKA in presynaptic terminals, which leads to the enhanced probability of the release of the neurotransmitter. In addition, PGE2 is also released from astrocytes where COX-2 is expressed. Under normal physiologic conditions, PGE2 derived from the constitutive COX-2 dynamically regulates basal synaptic transmission. However, PGE2 derived from the inducible COX-2 may contribute to synaptic plasticity resulting from neural activity-induced enhancement of COX-2 expression, and may lead to neurotoxicity resulting from abnormally excessive expression of COX-2.
