Article Figures & Data
Figures
- Figure 1.
Validation of cDNA microarray data. Selected genes from the Affymetrix experiment were further analyzed by real-time RT-PCR. Validated primers were used for each gene, and expression was normalized to the housekeeping gene 18S. Data are presented as mean ± SEM (n = 5 per group; **p < 0.01 and ***p < 0.001 vs respective vehicle control, two-tailed unpaired t test). N. Rec, Nuclear receptor subfamily 1, group D, member 1; 12-LOX, 12-lipoxygenase; IL 1-α, interleukin-1α; n.s., not significant.
- Figure 2.
Estrogen regulates PRL expression in TG neurons. A, Real-time RT-PCR experiments were performed with TG RNA samples from proestrous female rats and OVX female rats pretreated with either vehicle or estradiol (80 μg · kg−1 · d−1 for 10 d). Reactions were performed using primers specific for PRL gene and the internal control (18S). Data were normalized to the relative amount of OVX (vehicle) PRL mRNA/18S. Data are presented as mean ± SEM (n = 5 per group; **p < 0.01 vs PRL mRNA of the OVX/vehicle group, one-way ANOVA with Bonferroni’s post hoc test). B, Representative immunoblot of TG protein extracts. PRL was detected as a single band migrating at ∼25 kDa. Proteins were separated by 12.5% SDS-PAGE and probed with an antibody against PRL. Incubation of the antibody with the control peptide (20×) significantly blocked the immunoreactivity seen in TG protein samples from proestrous rats. C, Quantification of immunoblots of TG protein extracts from OVX rats injected with estradiol (2–80 μg/kg). Proteins were separated in 12.5% SDS-PAGE and probed with an antibody against PRL. Data are presented as mean ± SEM (n = 4 per group; *p < 0.05). D, Release of immunoreactive PRL from cultured TG neurons harvested from intact female rats. Data are presented as mean ± SEM percentage of basal release (n = 6 per group; *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni’s post hoc test). E, Immunohistochemistry was performed to demonstrate colocalization of PRL with TRPV1 (horizontal arrows) in the proestrous rat TG. Vertical arrow shows a PRL-positive neuron that is not positive for TRPV1. F, The cell size distribution of the immunoreactive PRL-containing neurons. Approximately, of all PRL-positive cells, 50% were of small diameter (0–20 μm), 46.12% were of medium diameter (20–40 μm), and 3.7% were of large diameter (40–60 μm). The mean cell size was 21.4 μm (n = 1160). n.s., Not significant; Veh, vehicle.
- Figure 3.
The expression of PRLR long form is regulated by estradiol. A, Representative immunoblot. Proteins were separated in a 12.5% SDS-PAGE and probed with an antibody against PRLR. The antibody recognized two bands migrating at ∼100 and 40 kDa, corresponding to the long and short PRLR isoforms, respectively. Blots were stripped and probed with an antibody against β-actin to normalize for loading differences. B, C, Quantification of immunoblots. TG proteins from OVX treated with E2 (0, 2, 20, or 80 μg/kg) were separated in a 12.5% SDS-PAGE and probed with an antibody against PRLR. Data are presented as mean ± SEM (n = 4 per group; *p < 0.05 and **p < 0.01, one-way ANOVA with Bonferroni’s post hoc test). U.OD., Uncalibrated OD; n.s., not significant; Veh, vehicle.
- Figure 4.
Prolactin augments capsaicin-evoked calcium influx in acutely cultured trigeminal ganglia neurons from intact female rats but not from OVX rats. A, Results of Ca2+ imaging experiments performed in acutely dissociated TG neurons. Data are presented as mean ± SEM percentage of vehicle (n = 30 per group; ***p < 0.001, two-way ANOVA). B, Representative traces of Ca2+ imaging experiments performed in neurons from proestrous rats. Neurons were treated with 40 nm PRL or vehicle for 10 min, followed by a 40 s capsaicin (30 nm) application. PRL did not evoke Ca2+ influx on its own but significantly potentiated capsaicin-evoked Ca2+ influx. Data are presented as mean ± SEM F340/F380 (n = 8 per group; error bars are SEM). C, Ca2+ imaging experiments performed in neurons from proestrous rats. Neurons were treated as described in the previous experiment (B). In addition, neurons were cotreated with PRL and the PRL receptor antagonist Δ1-9-G129R-hPRL (800 nm). The PRLR antagonist significantly blocked PRL potentiation of capsaicin-evoked Ca2+ influx, whereas it had no effect on its own. Data are presented as mean ± SEM percentage of vehicle (n = 19–40; *p < 0.05, two-way ANOVA). D, iCGRP release experiments were performed with TG neurons from intact female and OVX rats cultured for 5 d in the presence of E2 (50 nm) or vehicle. iCGRP was measured by radioimmunoassay. Data are presented as mean ± SEM percentage of vehicle (n = 4 per group; *p < 0.05 and **p < 0.01 vs iCGRP levels of each control group, two-tailed unpaired t test). Veh, Vehicle.
- Figure 5.
Prolactin sensitizes ICAP in TG neurons from naive, but not OVX, female rats. A, PRL concentration dependently sensitized ICAP with maximal potentiation observed at 4 nm (i.e., 0.1 μg/ml) with EC50 of 0.27 nm in cultured trigeminal neurons from naive female rats. Data are presented as mean ± SEM of PRL-treated ΔICAP (third ICAP − second ICAP) normalized to vehicle-treated ΔICAP (n = 8–12 per group; *p < 0.05 and ***p < 0.001, two-way ANOVA). B, PRL pretreatment increased capsaicin potency. Acutely dissociated TG neurons from female rats were pretreated with vehicle or PRL (40 nm) for 60 s, followed by a single capsaicin pulse (40 s, 0.1 nm to 5 μm). Data are shown as mean ± SEM (n = 12–20 per group; *p < 0.05 and ***p < 0.001 vehicle vs PRL, two-way ANOVA). C, D, Representative traces of ICAP recordings from intact (C) and OVX (D) female rat TG neurons. Concentration of applied CAP and PRL are indicated. CAP and PRL application durations were 30 and 60 s, respectively. EST, Estrogen treated; n.s., not significant.
- Figure 6.
Prolactin lowers the activation threshold temperature and potentiates heat-evoked inward currents in TG neurons from female rats. A, Pretreatment with PRL significantly decreased the threshold temperature for Iheat activation in cultured trigeminal neurons from naive female rats. Data are presented as mean ± SEM (n = 8–12 per group; ***p < 0.001 vs vehicle control, two-tailed unpaired t test). B, C, Pretreatment with PRL significantly potentiated inward currents evoked by 43°C (B) and 47°C (C). Data are presented as mean ± SEM (n = 8–12 per group; ***p < 0.001 vs vehicle control, two-tailed unpaired t test). D, E, Representative traces of Iheat recordings from TG neurons pretreated with PRL or vehicle. PRL lowered the activation temperature threshold and potentiated the magnitude of Iheat. Veh, Vehicle.
- Figure 7.
Prolactin induces the phosphorylation of TRPV1. A, Representative autoradiograph showing the incorporation of 32P in TRPV1 in acutely dissociated neurons from OVX and proestrous rats after treatment with PRL or vehicle. Proteins were separated in a 15% SDS-PAGE, and the immunoprecipitated TRPV1 was probed with a specific antibody. B, Quantification of 32P incorporation in TRPV1 from acutely cultured TG neurons from OVX and proestrous rats. Autoradiograph uncalibrated OD (U. OD.) was normalized by the TRPV1 U. OD. of each treatment group. PRL significantly induced TRPV1 phosphorylation in neurons from proestrous but, as observed in previous experiments, had no effect in neurons from OVX rats. Data are presented as mean ± SEM (n = 3 per group; *p < 0.05, two-tailed unpaired t test). Autorad, Autoradiography; WB, Western blot; n.s., not significant; Veh, vehicle; FEM, female.
- Figure 8.
Prolactin sensitizes female rats to capsaicin-evoked nocifensive behavior in an estrogen-dependent manner. A, Immunohistochemistry was performed in cornea cryosections from an intact female rat. The fixed cryosection was probed with antibodies against TRPV1 and PRLR. PRL receptors (green) are colocalized (white arrows) with TRPV1-containing fibers (red) innervating the cornea. B, Effect of pretreatment with PRL on capsaicin-induced eye wiping in proestrous rats. PRL (1 μg/μl in saline) or vehicle (40 μl) was applied in the eye, immediately followed by application of 0.01% capsaicin or vehicle (40 μl), and the total time spent grooming the injected eye per 5 min bins was measured by observers blinded to treatment allocation. Data are shown as mean ± SEM (n = 6 per group; *p < 0.05 and ***p < 0.001, two-way ANOVA). C, Effect of pretreatment with PRL on capsaicin-induced eye wiping in OVX rats treated with vehicle. The behavioral assay was conducted as described in B. Data are shown as mean ± SEM (n = 6 per group; two-way ANOVA). D, Effect of pretreatment with PRL on capsaicin-induced eye wiping in OVX rats treated with E2 (20 μg · kg−1 · d−1 for 10 d). The behavioral assay was conducted as described in B. Data are shown as mean ± SEM (n = 6 per group; **p < 0.01, two-way ANOVA). Veh, Vehicle.
Tables
- Table 1.
Effect of estradiol administration (80 μg/kg for 10 d) versus vehicle on gene expression in trigeminal ganglia of ovariectomized rats as assessed by Affymetrix microarray
Gene name Probe set ID number NCBI accession number SAMa GSa Concordant increasers Prolactin E03166cds_s_at NM_012629 6.9 ≥1.5 Prolactin V01244_at NM_012629 12.8 ≥1.5 Prolactin V01250cds_s_at NM_012629 3 ≥1.5 TrkA 3′ mRNA sequence AA958274_at AA958274 1.5 ≥1.5 Histidine ammonia lyase AB002393_at NM_017159 1.5 ≥1.5 Ania-10 early gene mRNA, 3′ UTR AF050662UTR#1_at NM_031593 1.6 ≥1.5 Ania-12 early gene mRNA, 3′ UTR AF050664UTR#1_at XM_220080 1.5 ≥1.5 Insulin receptor substrate 2 (IRS-2) mRNA AF087674_at XM_573948 1.4 ≥1.5 Interleukin 1α D00403_g_at NM_017019 1.3 ≥1.5 Aldosterone synthase, exon 9 D14097cds_s_at NM_012537 1.5 ≥1.5 Cytochrome P450, 3a18 D38381_s_at NM_145782 1.3 ≥1.5 Prepro bone inducing protein D49494cds_s_at NM_024375 1.3 ≥1.5 Nuclear receptor subfamily 1, group D, member 1 M25804_g_at NM_145775 1.5 ≥1.5 Cytochrome P450, 1a2 M26127_s_at NM_012541 1.3 ≥1.5 Interleukin 5 (colony-stimulating factor, eosinophil) X54419cds_at NM_021834 1.3 ≥1.5 Cytosolic epoxide hydrolase X60328_at NM_022936 1.7 ≥1.5 Similar to Mus musculus pre 45S pre rRNA gene rc_AA859966_i_at AA859966 4.1 ≥1.5 Cytochrome P450, subfamily IIC6 rc_AA945571_s_at NM_001013904 1.7 ≥1.5 Concordant decreasers Arachidonate 12-lipoxygenase S69383_at NM_031010 0.5 ≤1.5 Protein phosphatase 3, catalytic subunit, β isoform M31809_at NM_017042 0.6 ≤1.5 Electron transf. flavoprotein (Etfa), mRNA rc_AA894174_at NM_001009668 0.7 ≤1.5 -
↵aAffymetrix microarray experiment was performed with TG RNA isolated from vehicle-treated or E2-treated (80 μg/kg for 10 d) rats (n = 5 per group). Results were individually analyzed by two independent methods: statistical analysis of microarrays (SAM) and GeneSpring 5.1 (GS). There were 18 genes found to be upregulated in both analyses (concordant increasers), and three genes were found to be downregulated in both analyses (concordant decreasers). Data are presented as means with false discovery rate of 10% for SAM and change cutoff for the GS. UTR, Untranslated region.
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