All procedures and behavioral experiments involving vertebrate animals were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.
Full-length expression constructs for mouse TM-PAP (nt 64- 1314 from GenBank accession # NM_207668) and human TM-PAP (nt 51-1304 from GenBank accession # BC007460) were generated by RT-PCR amplification using C57BL/6 mouse trigeminal cDNA or human placental cDNA (Clontech) as template and Phusion polymerase. The red fluorescent protein mCherry was then fused in-frame to the C-terminus of all TM-PAP constructs. Mouse TM-PAP(H12A) was generated by PCR-based mutagenesis using mouse TM-PAP as template (His12 corresponds to His43 of the mPAP preprotein). This active site mutant was previously described and lacks catalytic activity (Schneider et al., 1993
; Ostanin et al., 1994
). All constructs have a Kosak consensus sequence, were cloned into pcDNA3.1 and were sequence verified. We obtained additional constructs from others (see Acknowledgments). We confirmed that adenosine receptors were expressed in Rat1 fibroblasts by RT-PCR (A1
R primers: 5’ CATTGGGCCACAGACCTACT and 5’ GGCAGAAGAGGGTGATACA).
Cell lines were grown on glass bottom culture dishes (MatTek Corp, P35G-0-10-C) in DMEM containing 10% Fetal Bovine Serum, 100 U/mL penicillin and 100 µg/mL streptomycin and transfected with Lipofectamine Plus (Invitrogen) according to manufacturer’s protocol. The total amount of DNA per transfection was adjusted to 1 µg by adding pcDNA 3.1. Following transfection (18–24 hours), cells were loaded for one hour at room temperature with 2 µM Fura-2 AM (Invitrogen, F-14185) in Hank’s Buffered Salt Solution (HBSS + Calcium and Magnesium) assay buffer (HBSS + 9 mM HEPES + 11 mM D-Glucose + 0.1% fatty-acid free BSA, pH 7.4). Cells were then washed 3 times with HBSS assay buffer and sat for at least 30 min prior to imaging. A Nikon TE2000U microscope and Sutter DG4 light source were used to image calcium responses (excitation 340 nm / 380 nm; emission 510 nm). We manually pipetted and aspirated solutions for all calcium imaging experiments. Cells were stimulated with 1 µM capsaicin, 100 nM LPA, 1 U/mL thrombin, 10 µM ATP or 1 µM bradykinin for 1–5 min, washed in HBSS assay buffer for 1 min, then stimulated with 0.006% SDS to evoke maximal calcium responses for normalization. We did not use ionomycin to normalize responses because this calcium ionophore activates calcium-dependent PLC enzymes. As a result, the magnitude of the ionomycin-induced calcium response is also proportional to PIP2 levels in cells.
Calcium responses were normalized by calculating the area under the curve (AUC) during ligand stimulation for each cell, and then dividing by the maximum SDS-evoked calcium response in each cell. These values were averaged over all cells for a given condition and then normalized relative to untransfected cells in the same field of view or relative to control cells (with the untransfected or control cell response set to 100%).
For experiments with capsaicin, Rat1 fibroblasts were transfected with TRPV1-GFP alone or TRPV1-GFP plus various constructs. The same amount of TRPV1-GFP was used for each transfection and the total amount of DNA per transfection was adjusted to 1 µg by adding pcDNA 3.1. Cells were stimulated with 1 µM capsaicin (from 100 mM stock in 100% DMSO, dissolved to final concentration in HBSS assay buffer) for 1 min, followed by a 5 min wash in HBSS assay buffer, then stimulation with 0.006% SDS.
For experiments with the PIP2 shuttle, Rat1 fibroblasts were stimulated with 100 nM LPA for 1 min, followed by a 15 min wash with HBSS assay buffer + 3 nM PIP2 + 3 nM Carrier 2 (PIP2 Shuttle Kit, Echelon, P-9045) or HBSS assay buffer + 3 nM Carrier 2 alone. Cells were then stimulated with 100 nM LPA again, washed for 1 min with HBSS assay buffer, and stimulated with 0.006% SDS.
For thapsigargin experiments, HBSS assay buffer lacking calcium and containing 1 mM EGTA was used to eliminate extracellular calcium. 10 µM thapsigargin was added for 5 min, the cells were then washed for 2 min with HBSS assay buffer, and stimulated with 0.006% SDS. For PTX experiments, Rat1 fibroblasts were incubated for 18 hours with 500 ng/mL PTX prior to loading with Fura-2 AM and stimulation with 100 nM LPA. For experiments with adenosine receptor antagonists, PLC inhibitor (U73122), PKC inhibitor (staurosporine), or PKA inhibitor (KT5720), cells were incubated with antagonists for 3–4 hours, loaded in the presence of antagonists/inhibitors with Fura-2 AM for one hour, and then stimulated with pronociceptive ligands.
A HEK293-TRPV1 stable cell line (Kim et al., 2008
) was transfected with TM-PAP-mCherry or TM-PAP(H12A)-mCherry. Patch clamp recordings were made from mCherry-expressing cells using a Multiclamp 700B amplifier and pClamp 9.2 software as described (Campagnola et al., 2008
). Heat ramps were generated by exchanging bath solution with a pre-heated solution via a 2-to-1 port. Solution was preheated with an in-line heater controlled by a TC-324B temperature controller modified for high temperature (Warner Instruments). Only one current recording was made per cover slip. The bath solution consisted of (in mM) NaCl 140, KCl 4, CaCl2
2, NaHEPES 10, Glucose 5, (pH 7.4, mOsm 295–310) and was perfused at a rate of 2–3 mL/min by gravity flow. Electrodes were pulled from borosilicate glass on a Sutter Instruments P-2000 and filled with intracellular solution that contained (in mM), KCl 135, MgATP 3, HEPES 10, Na2
ATP 0.5, CaCl2
1.1, EGTA 2, Glucose 5, with pH adjusted to 7.5 with HCl and osmolarity adjusted to 300 mOsm with sucrose. Tip resistances ranged from 2.5 to 5 MΩ. Series resistance was not compensated; however, recordings with series resistances greater than 15 MΩ were discarded.
For quantification of PIP2 in vitro, HEK293 cells or Rat1 fibroblasts were plated onto glass coverslips and transfected with the construct PLCδ-PH-GFP along with indicated constructs using Lipofectamine Plus (Invitrogen), according to the manufacturer’s protocol. 18–24 hours later, the cells were fixed with 4% paraformaldehyde (PFA)-PBS. Cells were imaged on a confocal microscope. GFP fluorescence on the plasma membrane of cells compared to the cytoplasm was quantified using ImageJ by taking cross-sectional averages of pixel intensity at the plasma membrane and dividing by the average of pixel intensity in the cytoplasm.
For quantification of PIP2 in DRG, age-matched, adult male mice were injected i.t. with 5 µL of 15% lidocaine + 50 U/mL hPAP (250 mU total), 15% lidocaine + 3 nM Carrier 2 (Echelon, P-9C2), 15% lidocaine + 3 nM PIP2 (Echelon, P-9045) + 3 nM Carrier 2 or 15% lidocaine alone. Prior to injection, an equimolar mixture of PIP2 + Carrier 2 was incubated for 15 min. at room temperature. Lidocaine causes transient (5–20 min.) paralysis of both hindlimbs, permitting us to visually determine if each mouse received a successful i.t. injection (we only quantified PIP2 levels in mice that showed transient bilateral paralysis). One day later, mice were sacrificed and L3-L6 DRGs were dissected bilaterally (n=8 ganglia / sample) and placed in PBS on ice. For each sample, DRG wet weight was determined then lipids were extracted and quantified using the PI(4,5)P2 Mass ELISA Kit from Echelon (K-4500) following the manufacturer’s protocol. PIP2 levels were normalized by dividing by the wet weight of DRG tissue.
/J) mice were backcrossed to C57BL/6 mice for at least 10 generations. For all other experiments male, C57BL/6 mice were purchased from Jackson Laboratories. Male, 2–4 month-old mice were used for all behavioral studies. All mice were acclimated to testing room, equipment, and experimenter for 1–3 days before behavioral testing. The experimenter was blind to genotype and drug treatment during behavioral testing. Thermal and mechanical sensitivity were measured as described previously (Zylka et al., 2008
). For intrathecal drug delivery, 5 µL was injected into unanesthetized mice using the direct lumbar puncture method (Fairbanks, 2003
). The CFA model of inflammatory pain and the SNI model of neuropathic pain were performed as described previously (Shields et al., 2003
; Zylka et al., 2008
S-hPAP (Sigma, P1774) and heat-inactivated S-hPAP were prepared as described previously (Zylka et al., 2008
). 18:1 Lysophosphatidic acid (Avanti Polar Lipids, 857130) was dissolved in 0.9% ethanol and then diluted to final concentrations in either HBSS assay buffer (calcium imaging) or 0.9% saline (injections). Adenosine 5’-triphosphate (ATP, Sigma, A26209) was dissolved in either HBSS assay buffer (calcium imaging) or 0.9% saline (injections). Capsaicin (Sigma, 2028 – 1 mg) was dissolved in 0.9% saline/10% ethanol/0.5% Tween 80 and 5 µL was injected for intrathecal delivery, while 20 µL was injected for intraplantar delivery. U73122 (Tocris, 1268) was first dissolved into DMSO, then further diluted in 0.9% saline for i.t. injection. The PI(4,5)P2
Shuttle PIP Kit (Echelon, P-9045) was used to increase PIP2
levels in vivo
was first dissolved into 10% DMSO in 0.9% saline. Carrier 2 (Histone H1) was dissolved into 0.9% saline. Prior to injection, PIP2
and Carrier 2 were mixed in a 1:1 molar ratio and incubated at room temperature for 15 min. Thrombin (Sigma, T4648) was first dissolved to 100 Units/mL in 0.1% BSA and further diluted in HBSS assay buffer to final concentrations. BK was dissolved to 1 mM in DMSO and further diluted in HBSS assay buffer to final concentrations. PTX (Sigma, P7208) and caffeine (Sigma, C0750) were dissolved in water. 8-Cyclopentyl-1,3-dimethylxanthine (CPT) (Sigma, C102), 8-Cyclopentyl-1,3-dipropylxanthine (CPX) (Sigma, C101), SCH58261 (Sigma, S4568), MRS1754 (Sigma, M6316), MRS 1523 (Sigma, M1809), staurosporine (Sigma, S4400), KT5720 (Tocris, 1288), and U73122 (Tocris, 1268) were dissolved in DMSO and further diluted in HBSS assay buffer to final concentrations.