beta(3)-adrenoceptor regulation and relaxation responses in mouse ileum

Abstract

1. This study examines the relationship between beta(3a)- and beta(3b)-adrenoceptor (AR) mRNA levels, beta(3)-AR binding and changes in ileum responses in mice treated with the beta(3)-AR agonist (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1, 3-benzodioxole-2,2-dicarboxylate (CL316243), or the beta(3)-AR antagonist 3-(2-ethylphenoxy)-1-[(1S)-1,2,3, 4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A), or dexamethasone or forskolin. 2. Levels of beta(3a)- and beta(3b)-AR mRNA and the maximum number of binding sites (B(max)) in ileum were unaffected following CL316243 treatment, although responses to CL316243 were reduced by 50% following 4 and 24 h treatment, indicating another desensitization mechanism not involving changes in receptor expression or number. beta(3a)-AR mRNA levels were reduced in both brown (BAT) and white adipose tissue (WAT) but beta(3b)-AR mRNA levels were significantly reduced only in WAT. Levels of beta(3a)- and beta(3b)-mRNA returned towards normal with continued treatment. 3. SR59230A treatment markedly increased beta(3)-AR mRN levels in ileum and BAT but not in WAT. The increase in beta(3)-AR mRNA levels in ileum was associated with increased B(max) levels in binding analysis and increased responses to CL316243, suggesting these as the cause of sensitization. 4. Treatment with forskolin (4 h) or dexamethasone (4 h) significantly reduced beta(3a)-AR mRNA levels in BAT and WAT but did not alter levels in ileum. Responses to CL316243 in ileum were unaffected by either treatment. 5. In summary, the beta(3)-AR is differently regulated in adipose tissue and ileum: Treatment with SR59230A increased beta(3)-AR number, mRNA and responsiveness in ileum, whereas treatment with CL316243 reduced responses without affecting beta(3)-AR number or mRNA levels.

Figure 1

Effect of in vivo CL316243 (1 h) treatment (1 mg kg⁻¹) on β_3a-AR and β_3b-AR mRNA levels in WAT, BAT and ileum and on responses to CL316243 in ileum. (a,b) β_3a-AR mRNA levels in WAT and ileum were not altered but levels in BAT (^**P<0.01) were significantly reduced following 1 h CL316243 treatment. β_3b-AR mRNA levels were unchanged in WAT, BAT and ileum. Bars show mean±s.e.mean (n=6). (c) β₃-AR mediated responses in ileum were unaffected following 1 h CL316243 treatment (2-way ANOVA ns). Points show mean and vertical lines indicate s.e.mean (n=8).

Figure 2

Effect of in vivo CL316243 (4 h) treatment (1 mg kg⁻¹) on β_3a-AR and β_3b-AR mRNA levels in WAT, BAT and ileum and on responses to CL316243 in ileum. (a,b) β_3a-AR mRNA levels were significantly reduced in WAT (^***P<0.001) and BAT (^***P<0.001) but not ileum following treatment. β_3b-AR mRNA levels were significantly reduced in WAT (^***P<0.001) following treatment. Bars show mean±s.e.mean (n=5–6). (c) 4 h CL316243 treatment desensitized β₃-AR mediated responses in mouse ileum with a significant reduction in the R_max response of the tissue with no change in pEC₅₀ (2-way ANOVA ^***P<0.0001). Points show mean and vertical lines indicate s.e.mean (n=12–13).

Figure 3

Effect of in vivo CL316243 (24 h) treatment (1 mg kg⁻¹) on β_3a-AR and β_3b-AR mRNA levels in WAT, BAT and ileum and on responses to CL316243 in ileum. (a,b) β_3a-AR and β_3b-AR mRNA levels in WAT were significantly reduced (^*P<0.05) following 24 h CL316243 treatment but levels were unaffected in BAT or ileum. Bars show mean±s.e.mean (n=5–6). (c) β₃-AR mediated responses in ileum were desensitized following treatment, with the R_max responses to CL316243 significantly reduced with no change in pEC₅₀ values (2-way ANOVA ^***P<0.0001). Points show mean and vertical lines indicate s.e.mean (n=6).

Figure 4

Saturation binding curve of (−)-[¹²⁵I]-CYP to mouse ileal membrane preparations. Graphs show specific binding; (a) shows the significant increase in (−)-[¹²⁵I]-CYP binding following SR59230A (4 h) (2-way ANOVA ^***P<0.0001) and CL316243 (4 h) treatment (2-way ANOVA ^*P<0.05); (b) shows that 24 h SR59230A or CL316243 treatment had no significant effect on (−)-[¹²⁵I]-CYP binding (2-way ANOVA ns). Points show mean±s.e.mean (n=5–6).

Figure 5

Effect of in vivo SR59230A (4 h) treatment (1 mg kg⁻¹) on β_3a-AR and β_3b-AR mRNA levels in WAT, BAT and ileum and on responses to CL316243 in ileum. (a,b) SR59230A treatment significantly increased β_3a-AR mRNA levels in BAT (^*P<0.05) and ileum (^*P<0.05) but not in WAT. β_3b-AR mRNA levels were significantly increased in BAT (^*P<0.05) and ileum (^**P<0.01) but not in WAT following treatment. Bars show mean±s.e.mean (n=3–6). (c) SR59230A treatment increased β₃-AR mediated responses in mouse ileum, significantly increasing the R_max response of the tissue while pEC₅₀ values were significantly different following treatment (2-way ANOVA ^***P<0.0001). Points show mean and vertical lines indicate s.e.mean (n=9–11).

Figure 6

Effect of in vivo 4 h forskolin treatment (3 mg kg⁻¹) on β_3a-AR and β_3b-AR mRNA levels in WAT, BAT and ileum and on responses to CL316243 in ileum. (a,b) β_3a-AR, but not β_3b-AR, mRNA levels in WAT and BAT are significantly reduced (^*P<0.05) following treatment. β_3a- and β_3b-AR mRNA levels were unaffected following treatment. Bars show mean±s.e.mean (n=4–6). (c) 4 h forskolin treatment did not alter β₃-AR mediated responses in ileum (2-way ANOVA ns). Points show mean and vertical lines indicate s.e.mean (n=13).

Figure 7

Effect of in vivo dexamethasone treatment (4 h, 1 mg kg⁻¹) on β_3a-AR and β_3b-AR mRNA levels in WAT, BAT and ileum and on responses to CL316243 in ileum. (a,b) β_3a-AR mRNA levels were significantly reduced in WAT (^*P<0.05) and BAT (^*P<0.05) but not ileum following dexamethasone treatment. β_3b-AR mRNA levels were significantly reduced in BAT (^*P<0.05) but not in WAT or ileum following treatment. Bars show mean±s.e.mean (n=3–6). (c) Dexamethasone treatment did not affect β₃-AR mediated responses (2-way ANOVA ns). Points show mean and vertical lines indicate s.e.mean (n=5–8).