(B) Basal cAMP levels were 0

(B) Basal cAMP levels were 0.80 0.16 pmol/well versus 0.91 0.19 pmol/well, Veh and NTI, respectively, and PGE2-stimulated cAMP levels were 186% 33% above basal and 130% 26% above basal, Veh and NTI, respectively (mean S.E.M., = 5). et al., 2010). Recently, we provided evidence for functional DOR-KOR heteromers in peripheral pain-sensing neurons (nociceptors) in main cultures and in vivo (Berg et al., 2012). We found that KOR coimmunoprecipitated with DOR from cultures of adult rat sensory neurons from your trigeminal ganglia and that a DOR-KOR heteromer-selective antibody augmented the antinociceptive efficacy of the DOR agonist [D-Pen2,5]-enkephalin (DPDPE). Moreover, we found that antagonists selective for KOR altered the potency and/or efficacy of selective DOR agonists in a ligand-dependent manner both in sensory neuron cultures and in leniolisib (CDZ 173) behavioral antinociception assessments in vivo. Such ligand-dependent effects are a hallmark of allosterism (Smith and Milligan, 2010; Christopoulos et al., 2014). These results suggested a functional role leniolisib (CDZ 173) for DOR-KOR heteromers in rat sensory neurons and that orthosteric KOR ligands regulated the function of orthosteric DOR ligands via intraheteromer allosteric interactions. In addition to ligand dependence, another hallmark of allosterism is usually reciprocity (Smith and Milligan, 2010). Thus, it is expected that if the KOR antagonist regulation of DOR agonist function is due to intraheteromer allosteric regulation, then leniolisib (CDZ 173) DOR ligands should regulate the function of KOR agonists. In this study, we examined the effects of DOR antagonists on KOR agonist-mediated inhibition of rat peripheral sensory neurons in culture and in vivo. We found that DOR antagonists altered the function of KOR agonists in a ligand-dependent manner. Moreover, we discovered that the putative DOR-KOR heteromer-selective activity of 6?-guanidinonaltrindole (6?-GNTI) in peripheral sensory neurons was due to allosteric enhancement of 6?-GNTI efficacy at KOR via its occupancy of DOR and that this effect was blocked by DOR transmembrane peptides both in culture and in vivo. Taken together, these results provide strong evidence for a functional role of DOR-KOR heteromers in regulating the activity of Rabbit Polyclonal to MEN1 rat peripheral pain-sensing neurons. Materials and Methods Materials. We purchased 6?-GNTI, 2-(3,4-dichlorophenyl)-is usually the measured response at a given agonist concentration (test. When only a single drug concentration was used, statistical analysis was done with one-way analysis of variance (ANOVA) followed by Dunnetts post test. < 0.05 was considered statistically significant. For behavior experiments, monotonic doseCresponse curve data were evaluated using nonlinear regression analysis (eq. 1). The significance of treatment effects around the mean fit values for ED50 and <0.05 was considered statistically significant. Results Allosteric Regulation of KOR Agonist-Mediated Responses by DOR Antagonists in Peripheral Nociceptors Ex lover Vivo and In Vivo Ligand-Dependent Effects of DOR Antagonists on KOR Agonist Responses. As shown in Fig. 1A, the concentrationCresponse curve (CRC) for the KOR agonist ICI-199441 for inhibition of PGE2-stimulated cAMP accumulation in cultures of sensory neurons was shifted to the left approximately 20-fold with no switch in the test, < 0.05). The mean maximal inhibition of PGE2-stimulated cAMP levels for ICI-199441 was 40% 3% versus 37% 2%, mean S.E.M. for vehicle and NTI, respectively, = 5 (paired test, = 0.96). As we found previously (Berg et al., 2011; Jamshidi et al., 2015), neither basal nor PGE2-stimulated cAMP levels were altered by NTI alone (see story of Fig. 1). Open in a separate windows Fig. 1. Ligand-dependent effects of the DOR antagonist naltrindole (NTI) on concentrationCresponse curves for the KOR agonists (A) ICI-199441 and (B) "type":"entrez-nucleotide","attrs":"text":"U50488","term_id":"1277101"U50488 for inhibition of PGE2-stimulated cAMP accumulation. Main cultures of peripheral sensory neurons were pretreated with NTI (20 nM) or vehicle (Veh) for 15 minutes. After pretreatment, cells were treated with numerous concentrations of ICI-199441.