GPR detection by reverse transcription
GPR35 detection by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA of cultured DRG neurons was extracted using the RNAqueous kit (Ambion). Complementary DNA (cDNA) was synthesized from 1μg of isolated total RNA with SuperScript III reverse transcriptase (Invitrogen). An aliquot of cDNA was used as a template for subsequent PCR. cDNA of adult rat DRG was a kind gift from Dr. Y. Taniguchi. To amplify rat GPR35, PCR was performed with the following two primers : 5′-AAATTGTAGCATCCTCCCGTGGCC-3′ and 5′-TATCTTGGCTCTTGTGGGGTGTGC-3′. The following conditions were used for amplification: 35 cycles of denaturation at 94°C for 30s, annealing at 58°C for 30s, and extension at 72°C for 1min. A final extension period of 7min at 72°C completed the PCR. The ‘housekeeping’ gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control using the following two primers: 5′-GTCTTCACCACCATGGAGAAGGCT-3′ and 5′-GTGATGGCATGGACTGTGGTCATGA-3′. PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining.
Immunohistochemistry for the expression of GPR35. DRGs were isolated from lumber level L5 of adult rats and fixed in 4% formaldehyde. After cryoprotection in 20% sucrose in PBS, DRGs were mounted with OCT compound and sectioned at 8μm with a cryostat. Sections were incubated in CAS-BLOCK (Invitrogen) containing 0.1% Triton X-100, rabbit anti-GPR35 antibody (1:200 dilution; LifeSpan Biosciences), and goat rat TRPV1 antibody (1:100 dilution; Santa Cruz Biotechnology) for 16h at room temperature. After washing, sections were incubated with secondary Annexin V-Cy3 Apoptosis Kit Plus mg conjugated to AlexaFluor-488 or -633 (1:500 dilution, respectively; Invitrogen) for 2h at room temperature. Cultured DRG neurons were fixed in 4% formaldehyde and incubated in PBS containing 0.1% Triton X-100, 2% BSA, and anti-GPR35 antibody (1:200 dilution) for 16h at 4°C, followed by incubation with a secondary antibody conjugated to AlexaFluor-488 for 1h at room temperature. Confocal fluorescence images were acquired using the LSM5 Pascal confocal microscope with a 20X objective (Carl Zeiss).
Cell transfection and cAMP accumulation. An expression plasmid for rat GPR35 in pcDNA3.1 (a kind gift from Dr. Y. Taniguchi) was transfected into Chinese hamster ovary (CHO) cells with Lipofectamine2000 reagent (Invitrogen). Stable clones of transfected cells were selected with 1mg/ml G418. A single stable clone was expanded in a flask to 80% confluence, harvested with 0.53mM EDTA–PBS, and then resuspended in Hanks’ buffered salt solution (HBSS) containing 20mM Hepes and 100μM 3-isobutyl-1-methylxanthine (IBMX; Sigma). The cells were plated on 96-well plates at a density of 10,000 cells per well and then stimulated with 10μM forskolin (Sigma) in addition to the indicated compounds for 20min at 37°C. Cyclic AMP (cAMP) levels were determined with the cAMP-screen system (Applied Biosystems). In some experiments, cells were incubated with 300ng/ml pertussis toxin (Sigma) for 16h prior to the cAMP assay.
Cyclic AMP accumulation in DRG sensory neurons. To examine the effect of agonists on adenylate cyclase, cultured DRG neurons were rinsed two times in HBSS containing 20mM Hepes and 100μM IBMX, then stimulated with 10μM forskolin along with the indicated compounds for 30min at 37°C. As indicated, 300ng/ml pertussis toxin was incubated with the cells for 16h prior to the cAMP assay.
Data analysis. Data obtained in cAMP accumulation assays were analyzed using Prism 4.0 (GraphPad Software). Statistical significance was assessed by two-tailed Student’s t test. Values with p<0.05 were considered to be statistically significant.
Discussion The present study demonstrates that GPR35 is expressed by nociceptive neurons in the DRG. Two agonists for GPR35, kynurenic acid and zaprinast, inhibited forskolin-stimulated cAMP production by cultured DRG neurons. The effects of both agonists were Gi/o protein-dependent and were abolished by pretreatment with the Gi/o protein inhibitor pertussis toxin that catalyzes the ADP-ribosylation of α subunits of the Gi/o protein family. The effects of agonist treatment on DRG neurons were similar to the effects of agonist treatment of heterologous cells expressing rat GPR35. From these experiments we conclude that GPR35 is negatively coupled to adenylate cyclase-cAMP signaling in DRG neurons and propose that GPR35 modulates nociceptive signaling.