Thyroid hormones profoundly alter cardiac function and systemic vascular resistance . Although the cardiovascular manifestations of thyroid disease states have been recognized for more than a century, the therapeutic utility of thyroid hormones as cardioactive and/or vasomotor drugs has only recently been addressed [2,3]. The administration of triiodothyronine (T3) to brain-dead organ donors was among the first clinical applications of T3 therapy in which the drug was targeted specifically at improving hemodynamic performance and enhancing organ retrieval [4,5].
In animal models, use of exogenous T3 after cardiopulmonary bypass improves ventricular performance without oxygen wasting [6,7]. Clinical trials of T3 in cardiac surgery have yielded mixed results [8-11], and the routine use of T3 as an inotropic drug in cardiac surgery is not recommended [12,13]. However, administration of T3 has been proposed to increase cardiac output and to decrease vascular resistance in certain clinical settings when conventional inotropic drugs prove insufficient . Patients with poor ventricular function may benefit the most from use of T3 through its reduction of the need for conventional inotropic drugs . Additionally, T3 enhances the recovery of ventricular function after ischemia in experimental animals . Furthermore, unlike conventional inotropic drugs, which improve cardiac function at the cost of increased oxygen consumption and, thus, decreased efficiency, T3 may improve cardiac function without additional cost in the myocardial oxygen consumption by decreasing afterload, increasing coronary blood flow, and improving diastolic function as well as systolic function [2,3,7,15]. Finally, because the mechanism of action of T3 may be different from that of conventional inotropic drugs, T3 may be used to achieve synergism [16,17]. As a result, routine use of T3 in fast-track myocardial revascularization in the elderly or in patients with significant left ventricular dysfunction has been advocated by some authors [18,19].
The purported cardiovascular effects of T3 include effects on both the myocardium and the peripheral vasculature : thyroid hormones improve myocardial performance and decrease vascular resistance. Reduction in vascular resistance seems greater than can be accounted for by a thyroid hormone-induced increase in tissue metabolism and the consequent release of local vasodilators because oxygen extraction actually decreases despite increased oxygen consumption . Therefore, thyroid hormones may either have a direct vasodilatory effect or increase shunting in vascular beds.
Our previous study using smooth muscle cells isolated from rat aorta and cultured on a deformable matrix demonstrated that exposure to T3 causes these cells to relax rapidly, as evidenced by a decrease in tension generated within the matrix . In the present study, we examined the hypothesis that thyroid hormones, T3, and levothyroxine (T4) have direct vasodilatory effects on rat skeletal muscle resistance arteries. We also examined the role of the endothelium in this effect.
In accordance with institutional animal care committee standards, Wistar rats of either sex, weighing 100-150 g, were anesthetized by injecting ketamine 40 mg/kg and xylazine 5 mg/kg intraperitoneally. The proximal hind leg skeletal muscles were harvested and placed in cold (4[degree sign]C) modified Krebs buffer (NaCl 120 mM, KCl 5.9 mM, dextrose 11.1 mM, NaHCO3 25 mM, NaH2 PO4 1.2 mM, MgSO (4) 1.2 mM, CaCl2 2.5 mM). Resistance arteries of approximately 100 micro m were dissected carefully from the surrounding tissue. Each vessel was placed in a vessel chamber, cannulated with dual micropipettes measuring 50-75 micro m in diameter, and secured with a 10-0 Ethilon suture. The vessel was continuously bathed with modified Krebs buffer, gassed with 95% O2/5% CO2 mixture, and maintained at 36.5-37.5[degree sign]C and a pH of 7.35-7.45. PO2 in the vessel chamber exceeded 400 mm Hg. Because the vessel was studied in a no-flow state, the pressure in the micropipettes was maintained at 40 mm Hg to provide distention. The vessel was visualized, and its internal lumen diameter was measured and recorded as previously described . The stability of similarly prepared vessel preparations over 2.5 h has been demonstrated previously .
T3 and T4 were obtained from Sigma Chemical Company (St. Louis, MO). For each, a stock solution of 10-3 M was made up in 0.1 N NaOH and stored at -20[degree sign]C. The stock solution was diluted as needed in modified Krebs buffer, and the pH was adjusted to 7.4 with phosphoric acid.
Each vessel was equilibrated in the vessel chamber for a minimum of 30 min. The baseline diameter (Dbaseline) was measured at the end of initial equilibration. Each vessel was then preconstricted with the thromboxane analog U46619 1 micro m, and the constricted diameter (Dconst) was measured. Each vessel was then subjected to increasing concentrations of T3 or T4 (10-10 to 10-7 M) for 20 min at each concentration. At each concentration, the internal diameter was measured (D (relax)), and the percentage of relaxation (% relaxation) from U46619-induced preconstriction was calculated: Equation 1
At the end of each experiment, the vessel chamber was flushed with fresh Krebs buffer, and the vessel was reequilibrated at 37[degree sign]C. KCl was then added to a final concentration of 100 mM, and the internal lumen diameter was measured. Preservation of endothelial function was confirmed by response to the endothelium-dependent dilator adenosine diphosphate (ADP) 10 micro M. ADP produced 73% +/- 5% relaxation in endothelium-intact vessels. Only those vessels that constricted by at least 15% to KCl at the end of each experiment were considered still viable and included for data analysis. For comparison, the dilatory responses of U46619-preconstricted skeletal muscle resistance arteries to isoproterenol (10-7 M) were also measured.
Because the vasodilatory effect of T4 was quite modest, we assessed the endothelial dependence of the vasodilatory effect of T3 only. Concentration response curves for T3 were obtained, as described above, in the presence of the NO synthase inhibitor NG-nitro-L-arginine (L-NNA) 10 micro M , the cyclooxygenase inhibitor indomethacin 10 micro M , the adenosine triphosphate (ATP)-sensitive K+ (KATP) channel blocker glibenclamide 1 micro M , and the beta-adrenergic blocker propranolol 1 micro M , or after endothelial denudation. Endothelial denudation was achieved by gently rubbing the inside of the vessel lumen with a piece of human hair, as previously described , and was confirmed by lack of dilation to the endothelium-dependent dilator ADP 10 micro M. Viability of the vessels was tested as above.
No animal contributed more than one vessel to any one experimental group. Therefore, the number for each group represents the number of animals as well as the number of vessels. All data are presented as mean +/- SD.
Whether there is a concentration-dependent relaxation to T3 or T4 was tested by one-way analysis of variance (ANOVA) (linear contrast). The effects of a blocker or endothelial denudation on the concentration response curves to T3 were analyzed by two-way ANOVA with a repeated-measures factor and post hoc multiple pairwise comparison (Neuman-Keuls) and stratified z-tests to identify the concentrations in which the differences in response were significant. Our null hypothesis was that the blocker or intervention had no effect on the response of the vessels to T3. Similarly, the concentration response curves to T3 and T4 were compared by using a two-way ANOVA with a repeated-measures factor. Significance was considered as P < 0.05. All statistics were calculated using True Epistat[trademark symbol] software (Epistat Services, Richardson, TX).
Both T3 and T4 demonstrated concentration-dependent dilatory effects on U46619-preconstricted skeletal muscle resistance arteries (Figure 1) (P < 0.001 for each) (T3: n = 9, baseline vessel size 100 +/- 9 micro M; T4: n = 7, vessel size 98 +/- 9 micro M). The vasodilatory effect of T3 was greater than that of T4 (P < 0.05). For comparison, isoproterenol 10-7 M produced a 56% +/- 6% relaxation.
T3-mediated dilation of U46619-preconstricted skeletal muscle resistance arteries was attenuated but not abolished by endothelial denudation (P < 0.01) (denuded vessels: n= 8, size 95 +/- 10 micro M), pretreatment with the NO synthase inhibitor L-NNA (P < 0.01) (L-NNA-pretreated vessels: n = 6, size 100 +/- 10 micro M), the cyclooxygenase inhibitor indomethacin (P < 0.05) (indomethacin-pretreated vessels: n = 8, size 92 +/- 15 micro M), or the KATP channel blocker glibenclamide (P < 0.01) (glibenclamide-pretreated vessels: n = 8, size 94 +/- 10 micro M) (Table 1). The effect of endothelial denudation reached statistical significance only in the supraphysiologic concentrations of >or=to10-8 M. However, pretreatment with the beta-adrenergic receptor blocker propranolol did not influence the response of the vessels to T3 (P = 0.99) (propranolol-pretreated vessels: n = 6, size 97 +/- 8 micro M) (Table 1).
In the present study, we examined the direct vasomotor effect of thyroid hormones on skeletal muscle resistance arteries in vitro. Because skeletal muscle vessels constitute a large proportion of the systemic circulation, changes in skeletal muscle vascular resistance have a great impact on systemic vascular resistance (SVR). Our main findings are (a) that both T3 and T4 can have direct vasodilatory effects on skeletal muscle resistance arteries, with the former more potent than the latter (36% +/- 9% relaxation at 10-7 M T3 vs 24% +/- 6% relaxation at 10-7 M T4), but they are modest in comparison to isoproterenol (56% +/- 6% relaxation at 10-7 M); (b) that the T3-mediated effect has both an endothelium-independent and an endothelium-dependent component; (c) that the endothelium-independent effect predominates in physiological concentrations; and (d) that the endothelium-dependent effect is most obvious in supraphysiologic concentrations.
An increase in serum thyroid hormones, whether caused by hyperthyroidism or exogenous administration, is associated with a decrease in SVR [2,3]. Part of the decrease in SVR may be due to the thyroid hormone-associated increase in cellular respiration, which leads to the release of local vasodilators such as adenosine . However, although total body oxygen consumption increases markedly in hyperthyroidism, the arteriovenous difference in oxygen content, i.e., oxygen extraction, is less than that in euthyroidism . If the vasodilatory effect of thyroid hormones is entirely indirect via the release of local vasodilators, then the increase in flow should be proportional to the increase in metabolism, and oxygen extraction should not decrease. Therefore, thyroid hormones may either have a direct vasodilatory effect or increase arteriovenous shunting in vascular beds.
Previous studies examining the direct vasomotor effect of thyroid hormones used conductance artery preparations. Ishikawa et al.  used vessel strips prepared from rabbit superior mesenteric artery to demonstrate that supraphysiologic concentrations of T4 and T3 (>10-6 M in their study) relaxed KCl-precontracted muscles. Ojamaa et al.  measured distortion of the matrix on which rat aortic smooth muscle cells were cultured and demonstrated relaxation of the cells with physiologic concentration (10-10 m) of T3 but not of T4. The present study is the first in vitro demonstration of the direct vasodilatory effect of thyroid hormones on resistance arteries at both physiologic (10-10 to 10-9 M)  and supraphysiologic (10-8 to 10-7 M) concentrations. The greater vasodilatory effect of T3 compared with T4 reached statistical significance only at high concentrations.
We have further demonstrated that the net vasodilatory effect of T3 is composed of both endothelium-independent and endothelium-dependent components. Endothelial denudation attenuated but did not abolish the T3-mediated effect. The endothelium-independent effect of T3 is consistent with our previous demonstration of the T3 effect in cultured smooth muscle cells, in which endothelial cells are absent . Whether the effect could be strengthened by a coculture with endothelial cells was not examined.
Vascular endothelium can release at least three different mediators of vasodilation-namely, nitric oxide, prostacyclin, and endothelium-dependent hyperpolarizing factor [27,28]. Endothelium-dependent hyperpolarizing factor opens KATP channels in the vascular smooth muscle, which leads to vasodilation . In our study, the T3-mediated effect was attenuated by L-NNA, indomethacin, and glibenclamide. This finding indicates that the endothelium-dependent component of the T3 effect is mediated by multiple factors, involving all three known endothelium-derived relaxing factors. In contrast, the beta-adrenergic antagonist propranolol had no effect on T3-mediated vasodilation. This finding is consistent with that of Ishikawa et al. , who reported that neithier alpha- nor beta-adrenergic blockade inhibited the vasorelaxant effect of T3 and T4 on vessel strips.
A limitation of our study is that it is an in vitro study in an experimental animal preparation. Although our findings provide suggestive evidence for a direct vasodilatory effect of T3, accounting for part of its observed cardiovascular effects, further studies are needed for in vivo validation and to establish the presence of the action in higher species.
In summary, both T3 and T4 have direct vasodilatory effects in vitro on rat skeletal muscle resistance arteries. The vasodilatory effect of T3 seems to be multifactorial, with both endothelium-independent and endothelium-dependent components. This finding suggests that direct vasodilation by T3 contributes to the observed decrease in SVR noted with either the exogenous administration or the endogenous increase of the hormone.
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