Comparative study of acute in vitro and short-term in vivo triiodothyronine treatments on the contractile activity of isolated rat thoracic aortas

Abstract

We aimed to characterize the participation of rapid non-genomic and delayed non-genomic/genomic or genomic mechanisms in vasoactive effects to triiodothyronine (T3), emphasizing functional analysis of the involvement of these mechanisms in the genesis of nitric oxide (NO) of endothelial or muscular origin. Influences of in vitro and in vivo T3 treatments on contractile and relaxant responsiveness of isolated rat aortas were studied. in vivo T3-treatment was 500 μg·kg^-1·d^-1, subcutaneous injection, for 1 (T3_1d) and 3 (T3_3d) days. In experiments with endothelium- intact aortic rings contracted with phenylephrine, increasing concentrations of T3 did not alter contractility. Likewise, in vitro T3 did not modify relaxant responses induced by acetylcholine or sodium nitroprusside (SNP) nor contractile responses elicited by phenylephrine or angiotensin II in endothelium-intact aortas. Concentration- response curves (CRCs) to acetylcholine and SNP in endothelium-intact aortic rings from T3_1d and T3_3d rats were unmodified. T3_3d, but not T3_1d, treatment diminished CRCs to phenylephrine in endothelium-intact aortic rings. CRCs to phenylephrine remained significantly depressed in both endothelium-denuded and endothelium- intact, nitric oxide synthase inhibitor-treated, aortas of T3_3d rats. In endotheliumdenuded aortas of T3_3d rats, CRCs to angiotensin II, and high K⁺ contractures, were decreased. Thus, in vitro T3 neither modified phenylephrine-induced active tonus nor CRCs to relaxant and contractile agonists in endothelium-intact aortas, discarding rapid non-genomic actions of this hormone in smooth muscle and endothelial cells. Otherwise, T3_3d-treatment inhibited aortic smooth muscle capacity to contract, but not to relax, in an endothelium- and NO-independent manner. This effect may be mediated by delayed non-genomic/genomic or genomic mechanisms.

Keywords: Genomic effect; Rapid non-genomic effect; Rat aorta; Triiodothyronine.

Fig. 1. Triiodothyronine (T3) applied in vitro did not influence previously established aortic active tonus.

Cumulative administration of increasing concentrations of T3, every 30 min, in endothelium intact (Endo +) aortic rings contracted with phenylephrine (1 μM). Temporal controls received the corresponding concentration of vehicle (V). Values are presented as means ± standard error of the means (n = 16). p = not significant (two-way ANOVA).

Fig. 2. Lack of effect of 3,5,3’ triiodo-L-thyronine (T3) in vitro treatments on the relaxing responses of agonists in aortic tissues.

(A, B) Relaxing responses to acetylcholine and (C, D) sodium nitroprusside (SNP) in endothelium-intact aortic rings (Endo +) contracted with phenylephrine (1 μM), treated previously with T3 (0.01, 0.1, and 1 μM) or vehicle (V) during 30 min or 2 h. Vehicle concentration corresponds to that required to dissolve T3 1 μM. Relaxing responses were calculated as decreases of phenylephrine-induced tension (grams). Values are presented as means ± standard error of the means (n = 15–16). p = not significant (two-way ANOVA).

Fig. 3. Lack of effect of in vitro 3,5,3’ triiodo-L-thyronine (T3) treatments on the contractile responses to agonists in aortic segments.

(A, B) Cumulative concentration-response curves to Phenylephrine and (C, D) Angiotensin II in annular segments of aortas with endothelium (Endo +) treated previously with T3 (0.01, 0.1, and 1 μM) or vehicle (V) for 30 min and 2 h. Vehicle concentration corresponded to that required to dissolve T3 1 μM. The contractile responses represent grams of developed tension. Values are presented as means ± standard error of the means (n = 16). p = not significant (two-way ANOVA).

Fig. 4. Short-term in vivo 3,5,3’ triiodo-L-thyronine (T3) treatment did not affect relaxant responses of agonists in aortic tissues.

(A) Cumulative concentration-relaxing response curves to acetylcholine and (B) sodium nitroprusside (SNP) in endothelium-intact (Endo +) thoracic aortic rings isolated from rats treated subcutaneously with T3 (500 μg·kg^–1·d^–1) or vehicle (V; 1 ml·kg^–1·d^–1) for 1 and 3 days (T3_1d, T3_3d and V_1d, V_3d, respectively). Relaxing responses were calculated as decreases of phenylephrine-induced tension. Values are presented as means ± standard error of the means (n = 14–16). p = not significant (two-way ANOVA).

Fig. 5. Delayed depression of Phenylephrine-induced contractions in aortic tissues from short-term thyroid hormone-treated rats.

Cumulative concentration-contractile response curves to phenylephrine, in endothelium-intact (Endo +) thoracic aortic rings isolated from rats treated subcutaneously with 3,5,3’ triiodo-L-thyronine (T3; 500 μg·kg^–1·d^–1) or vehicle (V; 1 ml·kg^–1·d^–1) for 1 and 3 days (T3_1d, T3_3d and V_1d, V_3d, respectively). Contractile responses are expressed as grams (g) of developed force. Values are presented as means ± SEM (n = 16). *p < 0.05 T3_3d vs. V_3d (two-way ANOVA with Sidak’s post-hoc test).

Fig. 6. Endothelium-independent inhibition of contractile responses to Phenylephrine in aortic tissues from 3,5,3’ triiodo-L-thyronine (T3) in vivo treated rats.

(A) Cumulative concentration-response curves (CRCs) to phenylephrine in annular segments of aortas with (Endo +) and without (Endo –) endothelium. (B) CRCs to phenylephrine in endothelium-intact (Endo +) thoracic aortic rings in the presence or absence of N^G-Nitro-L-arginine methyl ester (L-NAME, 100 μM). Aortic tissues were obtained from rats treated with subcutaneous injections of T3 (500 μg·kg^–1·d^–1) or vehicle (1 ml·kg^–1·d^–1) for 3 days (T3_3d and V_3d, respectively). All data are expressed as grams (g) of developed force. Values are presented as means ± standard error of the means. (A) (n = 16) *p < 0.05 T3_3d (Endo +) vs. V_3d (Endo +); ^#p < 0.05 T3_3d (Endo –) vs. V_3d (Endo –); ^ψp < 0.05 T3_3d (Endo +) vs. T3_3d (Endo –); ^&p < 0.05 V_3d (Endo +) vs. V_3d (Endo –). Two-way ANOVA with Sidak’s post-hoc test. (B) (n = 14–16) *p < 0.05 T3_3d vs. V_3d; ^#p < 0.05 T3_3d L-NAME vs. V_3d L-NAME; ^ψp < 0.05 T3_3d vs. T3_3d L-NAME; ^&p < 0.05 V_3d vs. V_3d L-NAME. Two-way ANOVA with Sidak’s post-hoc test.

Fig. 7. Endothelium-independent inhibition of contractile responses to angiotensin II and high K⁺ in aortic tissues from thyroid hormone-treated rats.

(A) Cumulative concentration-response curves to angiotensin II in endothelium-denuded (Endo –) thoracic aortic rings. (B) Contractile responses to high K⁺ (40 and 80 mM) in endothelium-denuded (Endo –) thoracic aortic rings. Aortic tissues were obtained from rats treated with subcutaneous injections of 3,5,3’ triiodo-L-thyronine (500 μg·kg^–1·d^–1) or vehicle (1 ml·kg^–1·d^–1) for 3 days (T3_3d and V_3d, respectively). All data are expressed as grams (g) of developed force. Values are presented as means ± standard error of the means. (A) (n = 15) *p < 0.05 vs. V_3d (two-way ANOVA with Sidak’s post-hoc test). (B) (n = 13–15) *p < 0.05 vs. V_3d (K⁺ 40 mM); ^#p < 0.05 vs. V_3d (K⁺ 80 mM) (unpaired Student t-test).