Korean J Physiol Pharmacol.  2020 Jul;24(4):339-348. 10.4196/kjpp.2020.24.4.339.

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

• López RM
• López JS
• Lozano J
• Flores H
• Carranza RA
• Franco A
• Castillo EF

• KMID: 2503327
• DOI: http://doi.org/10.4196/kjpp.2020.24.4.339

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.

Keyword

Genomic effect; Rapid non-genomic effect; Rat aorta; Triiodothyronine

Figure

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  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 (T31d, T33d and V1d, V3d, 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 (T31d, T33d and V1d, V3d, respectively). Contractile responses are expressed as grams (g) of developed force. Values are presented as means ± SEM (n = 16). *p < 0.05 T33d vs. V3d (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 NG-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 (T33d and V3d, 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 T33d (Endo +) vs. V3d (Endo +); #p < 0.05 T33d (Endo –) vs. V3d (Endo –); ψp < 0.05 T33d (Endo +) vs. T33d (Endo –); &p < 0.05 V3d (Endo +) vs. V3d (Endo –). Two-way ANOVA with Sidak’s post-hoc test. (B) (n = 14–16) *p < 0.05 T33d vs. V3d; #p < 0.05 T33d L-NAME vs. V3d L-NAME; ψp < 0.05 T33d vs. T33d L-NAME; &p < 0.05 V3d vs. V3d L-NAME. Two-way ANOVA with Sidak’s post-hoc test.

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  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 (T33d and V3d, 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. V3d (two-way ANOVA with Sidak’s post-hoc test). (B) (n = 13–15) *p < 0.05 vs. V3d (K+ 40 mM); #p < 0.05 vs. V3d (K+ 80 mM) (unpaired Student t-test).

Reference

1. Brożek JL, Bousquet J, Agache I, Agarwal A, Bachert C, Bosnic-Anticevich S, Brignardello-Petersen R, Canonica GW, Casale T, Chavannes NH, Correia de Sousa J, Cruz AA, Cuello-Garcia CA, Demoly P, Dykewicz M, Etxeandia-Ikobaltzeta I, Florez ID, Fokkens W, Fonseca J, Hellings PW, et al. 2017; Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines-2016 revision. J Allergy Clin Immunol. 140:950–958. DOI: 10.1016/j.jaci.2017.03.050. PMID: 28602936.

2. D'Amato G, Pawankar R, Vitale C, Lanza M, Molino A, Stanziola A, Sanduzzi A, Vatrella A, D'Amato M. 2016; Climate change and air pollution: effects on respiratory allergy. Allergy Asthma Immunol Res. 8:391–395. DOI: 10.4168/aair.2016.8.5.391. PMID: 27334776. PMCID: PMC4921692.

3. Ricketti PA, Alandijani S, Lin CH, Casale TB. 2017; Investigational new drugs for allergic rhinitis. Expert Opin Investig Drugs. 26:279–292. DOI: 10.1080/13543784.2017.1290079. PMID: 28141955.

4. Kakli HA, Riley TD. 2016; Allergic rhinitis. Prim Care. 43:465–475. DOI: 10.1016/j.pop.2016.04.009. PMID: 27545735.

5. Greiner AN, Meltzer EO. 2006; Pharmacologic rationale for treating allergic and nonallergic rhinitis. J Allergy Clin Immunol. 118:985–998. DOI: 10.1016/j.jaci.2006.06.029. PMID: 17088121.

6. Wheatley LM, Togias A. 2015; Clinical practice. Allergic rhinitis. N Engl J Med. 372:456–463. DOI: 10.1056/NEJMcp1412282. PMID: 25629743. PMCID: PMC4324099.

7. Dantzer JA, Wood RA. 2018; The use of omalizumab in allergen immunotherapy. Clin Exp Allergy. 48:232–240. DOI: 10.1111/cea.13084. PMID: 29315922.

8. Long R, Zhou Y, Huang J, Peng L, Meng L, Zhu S, Li J. 2015; Bencycloquidium bromide inhibits nasal hypersecretion in a rat model of allergic rhinitis. Inflamm Res. 64:213–223. DOI: 10.1007/s00011-015-0800-6. PMID: 25690567.

9. Widdicombe JH, Wine JJ. 2015; Airway gland structure and function. Physiol Rev. 95:1241–1319. DOI: 10.1152/physrev.00039.2014. PMID: 26336032.

10. Rogers DF. 2003; Airway hypersecretion in allergic rhinitis and asthma: new pharmacotherapy. Curr Allergy Asthma Rep. 3:238–248. DOI: 10.1007/s11882-003-0046-1. PMID: 12662474.

11. Kremer B, den Hartog HM, Jolles J. 2002; Relationship between allergic rhinitis, disturbed cognitive functions and psychological well-being. Clin Exp Allergy. 32:1310–1315. DOI: 10.1046/j.1365-2745.2002.01483.x. PMID: 12220469.

12. Galietta LJ, Pagesy P, Folli C, Caci E, Romio L, Costes B, Nicolis E, Cabrini G, Goossens M, Ravazzolo R, Zegarra-Moran O. 2002; IL-4 is a potent modulator of ion transport in the human bronchial epithelium in vitro. J Immunol. 168:839–845. DOI: 10.4049/jimmunol.168.2.839. PMID: 11777980.

13. Zhou Y, Shapiro M, Dong Q, Louahed J, Weiss C, Wan S, Chen Q, Dragwa C, Savio D, Huang M, Fuller C, Tomer Y, Nicolaides NC, McLane M, Levitt RC. 2002; A calcium-activated chloride channel blocker inhibits goblet cell metaplasia and mucus overproduction. Novartis Found Symp. 248:150–165. discussion 165–170. 277–282. DOI: 10.1002/0470860790.ch10. PMID: 12568493.

14. Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra-Moran O, Galietta LJ. 2008; TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science. 322:590–594. DOI: 10.1126/science.1163518. PMID: 18772398.

15. Cho HJ, Choi JY, Yang YM, Hong JH, Kim CH, Gee HY, Lee HJ, Shin DM, Yoon JH. 2010; House dust mite extract activates apical Cl^- channels through protease-activated receptor 2 in human airway epithelia. J Cell Biochem. 109:1254–1263. DOI: 10.1002/jcb.22511. PMID: 20186875.

16. Rievaj J, Davidson C, Nadeem A, Hollenberg M, Duszyk M, Vliagoftis H. 2012; Allergic sensitization enhances anion current responsiveness of murine trachea to PAR-2 activation. Pflugers Arch. 463:497–509. DOI: 10.1007/s00424-011-1064-9. PMID: 22170096.

17. Kang JW, Lee YH, Kang MJ, Lee HJ, Oh R, Min HJ, Namkung W, Choi JY, Lee SN, Kim CH, Yoon JH, Cho HJ. 2017; Synergistic mucus secretion by histamine and IL-4 through TMEM16A in airway epithelium. Am J Physiol Lung Cell Mol Physiol. 313:L466–L476. DOI: 10.1152/ajplung.00103.2017. PMID: 28546154.

18. Huang F, Zhang H, Wu M, Yang H, Kudo M, Peters CJ, Woodruff PG, Solberg OD, Donne ML, Huang X, Sheppard D, Fahy JV, Wolters PJ, Hogan BL, Finkbeiner WE, Li M, Jan YN, Jan LY, Rock JR. 2012; Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction. Proc Natl Acad Sci U S A. 109:16354–16359. DOI: 10.1073/pnas.1214596109. PMID: 22988107. PMCID: PMC3479591.

19. Scudieri P, Caci E, Bruno S, Ferrera L, Schiavon M, Sondo E, Tomati V, Gianotti A, Zegarra-Moran O, Pedemonte N, Rea F, Ravazzolo R, Galietta LJ. 2012; Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia. J Physiol. 590:6141–6455. DOI: 10.1113/jphysiol.2012.240838. PMID: 22988141. PMCID: PMC3530122.

20. Qiao X, He WN, Xiang C, Han J, Wu LJ, Guo DA, Ye M. 2011; Qualitative and quantitative analyses of flavonoids in Spirodela polyrrhiza by high-performance liquid chromatography coupled with mass spectrometry. Phytochem Anal. 22:475–483. DOI: 10.1002/pca.1303. PMID: 21465598.

21. Lee HJ, Kim MH, Choi YY, Kim EH, Hong J, Kim K, Yang WM. 2016; Improvement of atopic dermatitis with topical application of Spirodela polyrhiza. J Ethnopharmacol. 180:12–17. DOI: 10.1016/j.jep.2016.01.010. PMID: 26778605.

22. Nam JH, Jung HW, Chin YW, Yang WM, Bae HS, Kim WK. 2017; Spirodela polyrhiza extract modulates the activation of atopic dermatitis-related ion channels, Orai1 and TRPV3, and inhibits mast cell degranulation. Pharm Biol. 55:1324–1329. DOI: 10.1080/13880209.2017.1300819. PMID: 28290212. PMCID: PMC6130684.

23. Di Capite J, Parekh AB. 2009; CRAC channels and Ca²⁺ signaling in mast cells. Immunol Rev. 231:45–58. DOI: 10.1111/j.1600-065X.2009.00808.x. PMID: 19754889.

24. Kircher S, Merino-Wong M, Niemeyer BA, Alansary D. 2018; Profiling calcium signals of in vitro polarized human effector CD4+ T cells. Biochim Biophys Acta Mol Cell Res. 1865:932–943. DOI: 10.1016/j.bbamcr.2018.04.001. PMID: 29626493.

25. Park BK, Park YC, Jung IC, Kim SH, Choi JJ, Do M, Kim SY, Jin M. 2015; Gamisasangja-tang suppresses pruritus and atopic skin inflammation in the NC/Nga murine model of atopic dermatitis. J Ethnopharmacol. 165:54–60. DOI: 10.1016/j.jep.2015.02.040. PMID: 25721805.

26. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, Park SP, Lee J, Lee B, Kim BM, Raouf R, Shin YK, Oh U. 2008; TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature. 455:1210–1215. DOI: 10.1038/nature07313. PMID: 18724360.

27. Namkung W, Phuan PW, Verkman AS. 2011; TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells. J Biol Chem. 286:2365–2374. DOI: 10.1074/jbc.M110.175109. PMID: 21084298. PMCID: PMC3023530.

28. Kunzelmann K, Kongsuphol P, Aldehni F, Tian Y, Ousingsawat J, Warth R, Schreiber R. 2009; Bestrophin and TMEM16-Ca²⁺ activated Cl^- channels with different functions. Cell Calcium. 46:233–241. DOI: 10.1016/j.ceca.2009.09.003. PMID: 19783045.

29. Fischer H, Illek B, Sachs L, Finkbeiner WE, Widdicombe JH. 2010; CFTR and calcium-activated chloride channels in primary cultures of human airway gland cells of serous or mucous phenotype. Am J Physiol Lung Cell Mol Physiol. 299:L585–L594. DOI: 10.1152/ajplung.00421.2009. PMID: 20675434. PMCID: PMC2957417.

30. Banga A, Flaig S, Lewis S, Winfree S, Blazer-Yost BL. 2014; Epinephrine stimulation of anion secretion in the Calu-3 serous cell model. Am J Physiol Lung Cell Mol Physiol. 306:L937–L946. DOI: 10.1152/ajplung.00190.2013. PMID: 24705724. PMCID: PMC4025061.

31. Seo Y, Lee HK, Park J, Jeon DK, Jo S, Jo M, Namkung W. 2016; Ani9, a novel potent small-molecule ANO1 inhibitor with negligible effect on ANO2. PLoS One. 11:e0155771. DOI: 10.1371/journal.pone.0155771. PMID: 27219012. PMCID: PMC4878759.

32. Seo Y, Ryu K, Park J, Jeon DK, Jo S, Lee HK, Namkung W. 2017; Inhibition of ANO1 by luteolin and its cytotoxicity in human prostate cancer PC-3 cells. PLoS One. 12:e0174935. DOI: 10.1371/journal.pone.0174935. PMID: 28362855. PMCID: PMC5376326.

33. Zhuo RG, Peng P, Zheng JQ, Zhang YL, Wen L, Wei XL, Ma XY. 2017; The glycine hinge of transmembrane segment 2 modulates the subcellular localization and gating properties in TREK channels. Biochem Biophys Res Commun. 490:1125–1131. DOI: 10.1016/j.bbrc.2017.06.200. PMID: 28676394.

34. Devor DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, Bridges RJ. 1999; Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol. 113:743–760. DOI: 10.1085/jgp.113.5.743. PMID: 10228185. PMCID: PMC2222914.