Fig 1: Cav3.2 protein expression is enhanced in ipsilateral L5 DRG tissues during the sub-acute phase (Day 2) following carrageenan injection.Cav3.2 protein expression was analyzed and normalized relative to the expression of the housekeeping protein ß-actin. The inset of a representative, original immunoblot shows the up-regulation of Cav3.2 protein (n = 6 mice for each group). *p < 0.05 compared with the saline control (unpaired Student's t-test).
Fig 2: Cav3.2 mRNA is expressed in a subset of DRG neurons in normal mice.(A and B) Macroautoradiographic images of an [35S]-labeled probe from an in situ hybridization showing the specificity of the Cav3.2 probe (A; sense probe) and Cav3.2 mRNA expression (B; anti-sense probe) in mouse DRG tissues. Hybridization signals were obtained only when the anti-sense probe was used. The medium DRG neurons (arrows) were densely labeled, whereas smaller neurons (arrow head) were only weakly labeled. Scale bar = 50 µm (C) Microscopic images of a DIG-labeled probe for in situ hybridization (n = 3 mice for each probe). Scale bar = 50 µm. (D) The proportion of Cav3.2 mRNA-positive neurons relative to the total number of DRG neurons was determined. (E) Histogram showing the proportions of Cav3.2 mRNA-labeled cells based on a cross-sectional area (left). The proportion of cells labeled by an anti-NF-H antibody is shown as a reference on the right side. Only labeled cells that included nuclei were subjected to area measurements.
Fig 3: Cav3.2 mRNA is gradually upregulated in ipsilateral L5 DRG tissues during the sub-acute phase (Days 1–2) following carrageenan injection.(A and B) Representative gel images of semi-quantitative RT-PCR analyses showing an upregulation of Cav3.2 mRNA (A) and data (proportion relative to ß-actin) quantified by densitometry (B). (C) Representative images of semi-quantitative RT-PCR analyses of Cav3.1 and Cav3.3 mRNA on Day 2 showing no upregulation (n = 3 mice for Day 1 and n = 4 mice for Day 2). (D) Summary of data from the quantitative RT-PCR (qRT-PCR) analyses. The results were similar to the results of the semi-quantitative RT-PCR: Cav3.2 mRNA was increasingly upregulated during the sub-acute phase of hyperalgesia (n = 4 mice for each group). *p < 0.05 compared with saline-treated controls (one-way ANOVA with the Bonferroni post hoc test). n.s., not significant; SA, saline; CA, carrageenan; ipsi, ipsilateral; contra, contralateral.
Fig 4: Intracellular calcium concentration is reduced during activation of Cav3.2-/- and Ni2+-treated platelets. (A) Change in global calcium content in WT and Cav3.2-/- platelets (p = .015, wild-type [WT] vs. Cav3.2-/-). (B) Calcium mobilization within the first 2 min with no calcium between Cav3.2-/- and WT platelets. Calcium influx initiated after the addition of calcium (2 mM) in Cav3.2-/- platelets versus WT controls (*p = .01, WT vs. Cav3.2-/-). (C) Calcium influx mediated by thrombin 10 mU/ml in the presence of apyrase 5 U/ml (p = .002, WT vs. Cav3.2-/-). (D) Change in global calcium content and (E) calcium influx in Ni2+-treated platelets (p = .002, for global calcium concentration; *p = .01, for calcium influx, vehicle vs. Ni2+). (F) Calcium influx mediated by thrombin 10 mU/ml in the presence of apyrase 5 U/ml (*p = .04, Veh vs. Ni2+-treated platelets). Data are mean ± SEM (N = 3–5) and were analyzed by unpaired t-test with Mann-Whitney U test and ANOVA with Tukey's multiple comparison test. ANOVA, analysis of variance; veh, vehicle.
Fig 5: Expression of Cav3.2 T‐type calcium channel in mouse platelets. (A) Detection of Cav3.2 mRNA expression by reverse transcriptase‐polymerase chain reaction. BM, bone marrow; MK, megakaryocytes; WT, wild‐type. (B) Detection of human and mouse clones of Cav3.2 expressed in HEK 293 cells. (C) Detection of Cav3.2 in mouse platelets and testes. (D) Transmission electron microscopy of platelets. The yellow arrows show the dense granules and the green arrows the alpha granules. Scale bar = 0.5 μm.
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