Influence of Hyperthyroidism on the Activity of Liver Nitric Oxide Synthase in the Rat

NITRIC OXIDE: Biology and ChemistryVol. 1, No. 6, pp. 463–468 (1997)Article No. NO970149

Influence of Hyperthyroidism on the Activityof Liver Nitric Oxide Synthase in the Rat

Virginia Fernandez,1 Pamela Cornejo, Gladys Tapia, and Luis A. Videla

Programa de FarmacologıB a Molecular y Clinica, Instituto de Ciencias Biomedicas, Facultad de Medicina,Universidad de Chile, Casilla 70086, Santiago 7, Chile

Received July 22, 1997, and in revised form September 2, 1997

Thyroid hormones play a significant role in cellularHyperthyroidism enhances the prooxidant activ- processes related to energy production in the adult

ity of the liver by elevating superoxide radical and/ vertebrate (1). The signal transduction pathway in-or hydrogen peroxide generation in microsomal, mi- volves thyroid hormone binding to specific nuclear re-tochondrial, and peroxisomal fractions, with an in- ceptors in target cells, leading to the expression ofcreased respiratory burst of Kupffer cells. In this enzymatic activities related to several redox processesstudy, the influence of daily doses of 0.1 mg 3,3*,5- (2). The development of a hyperthyroid state in mam-triiodothyronine (T3)/kg for three consecutive days

mals increments their basal metabolic rate, an effecton liver nitric oxide (NO) synthase (NOS) was as-known as thyroid calorigenesis, which is characterizedsessed, as a possible contributory mechanism to T3-by an enhancement in the total rate of O2 consumptioninduced liver prooxidant activity. Thyroid calori-of target tissues coupled to higher rates of cellulargenesis was paralleled by a progressive increment inelectron transfer systems (1, 2). In the liver, this condi-the rate of NO generation, with significant increasestion leads to an elevated generation of reactive O2after 2 (47%) and 3 days (70%) of T3 treatment, and aspecies, namely, superoxide radical (O•0

2 ) and/or hy-net 45% (P õ 0.05) enhancement in the NG-methyl-L-drogen peroxide (H2O2) in microsomal, mitochondrial,arginine-sensitive NO production, compared to con-

trol values. These enhancement effects were re- and peroxisomal fractions (3, 4). Recently, hyperthy-versed to control levels after 3 days of hormone with- roidism was found to enhance Kupffer cell function,drawal, concomitantly with the normalization of which may represent an alternate source of free radi-hepatic respiration. Enhancement of liver NOS ac- cal species to that induced in parenchymal cells (5).tivity in hyperthyroid animals was diminished by In addition, thyroid hormone elicits a depression of27% (P õ 0.05) by the selective in vivo inactivation key antioxidant mechanisms of the liver (6, 7), leadingof Kupffer cells by gadolinium chloride (GdCl3), to a significant increase in the oxidative stress statuswithout direct actions of GdCl3 on the enzyme. These of the tissue and the consequent lipid peroxidationdata demonstrate that hyperthyroidism leads to a response (3, 8–11). Thyroid hormone-induced alter-significant and reversible enhancement in rat liver ations in oxidative stress-related parameters haveNOS activity, an effect that is exerted at hepatocyte

also been observed in different extrahepatic tissues,and Kupffer cell levels, thus representing an addi-such as skeletal muscle (12), heart (12), mesenterictional source of prooxidants to those of reactive oxy-lymph nodes (13), and the brain of newborn rats (14).gen species. q 1997 Academic Press

Nitric oxide is an inorganic free radical speciesKey Words: Hyperthyroidism; liver oxidativesynthesized from L-arginine by NO synthasesstress; hepatic nitric oxide synthase; Kupffer cells.(NOS)2 in an NADPH-requiring reaction, a process

2 Abbreviations used: NOS, nitric oxide synthase; T3, L-3,3*,5-1 To whom correspondence should be addressed. Fax: 56-2- triiodothyronine; L-NMA, NG-methyl-L-arginine; ONOO0, perox-

ynitrite anion.7355580. E-mail: [email protected].

4631089-8603/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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464 FERNANDEZ ET AL.

known to be widely distributed among different cell (36–377C), without recirculation of the perfusate,and O2 consumption was determined in the effluenttypes (15). Hepatic synthesis of NO has been found

to occur in both hepatocytes (16, 17) and Kupffer perfusate collected via a cannula placed in the venacava and allowed to flow past a Clarke-type O2 elec-cells (18, 19), catalyzed by a Ca2/-independent NOS

inducible by endotoxin (17, 19). In view of these trode (5, 7). Separate groups of control animals andrats receiving T3 for 3 days were simultaneouslyconsiderations, the current work was undertaken to

assess the influence of hyperthyroidism on hepatic given either saline or gadolinium chloride (GdCl3)(10 mg/kg by tail vein injection) on the first andNO generation, as a possible additional mechanism

contributing to thyroid hormone-induced liver pro- third day of treatment.oxidant activity. For this purpose, NOS activity wasdetermined in the liver of control rats and animalsmade hyperthyroid by L-3,3*-5-triiodothyronine (T3) Assay of NOS Activitytreatment, and the contribution of Kupffer cells toliver NO production was assessed in separate Animals were anesthetized with sodium pentobar-

bital (50 mg/kg, ip) and the liver was perfused ingroups of animals subjected to liver macrophage in-activation by gadolinium chloride (GdCl3) adminis- situ with 150 ml of a cold solution containing 150

mM KCl and 5 mM Tris, pH 7.4, in order to removetration (20).blood. Livers were kept on ice before homogeniza-tion. One gram of tissue was homogenized on ice

MATERIALS AND METHODS in a Tri-R Stir-R homogenizer (Model S63C, Tri-RInstruments Inc., Rockville Centre, NY), at maximalAnimals and T3 Treatmentspeed, with 5 ml of a buffer containing 10 mM Hepes,0.32 M sucrose, 0.1 mM EDTA, 1 mM dithiothreitol,Female Sprague–Dawley rats (Instituto de Salud

Publica, Santiago) weighing 220–270 g were fed ad 10 mg soybean trypsin inhibitor/ml, 10 mg leupeptin/ml, 2 mg aprotinin/ml, and 1 mg phenylmethanesul-libitum and received daily ip injections of either T3

(0.1 mg/kg) or equivalent volumes of T3 diluent (0.1 fonyl fluoride/ml, adjusted to pH 7.4 with NaOH.The homogenates were centrifuged at 100,000g forN NaOH) (controls) for one to three consecutive

days, and studies were performed 24 h after the last 1 h and the liver supernatant was removed and kepton ice until NOS activity assay. Determinationstreatment. T3-treated animals for three consecutive

days were studied at 1, 2, and 3 days after treatment were carried out within 3 h of preparation.NO synthesis was measured as described byto assess the reversibility of the changes in the stud-

ied parameters. Under these conditions, the param- Knowles et al. (17), by a method in which the oxida-tion of oxyhemoglobin to methemoglobin by NO iseters of thyroid calorigenesis were determined as

follows: serum T3 levels were measured by a Gamma monitored spectrophotometrically. The change in ab-sorbance at 401 nm versus 411 nm was registeredCoat [125I]T3 radioimmunoassay kit (Baxter Health-

care Corp., Cambridge, MA), the rectal temperature with a Lambda 2 spectrometer (Perkin Elmer Corp.,Ueberlingen, Germany). The reaction medium, con-of the animals was measured with a thermocouple

(Model 8112-20, Cole-Parmer Instrument Co., Chi- taining 1.6 mM oxyhemoglobin, 1 mM MgCl2, 40 mMpotassium phosphate, pH 7.2, and 100 ml of livercago, IL), and the rate of O2 consumption of the liver

was assessed polarographically in perfusion experi- supernatant, was preincubated for 5 min at 377C.The reaction was initiated by the addition of L-argi-ments (5, 7). For this purpose, rats were anesthe-

tized with sodium pentobarbital (50 mg/kg ip) and nine (1 mM) and NADPH (1 mM) and NO productionwas measured at 377C for 2 min, the time at whichperfusate (118 mM NaCl, 4.8 mM KCl, 1.2 mM

KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM the rate remained linear. Separate determinationswere performed after the addition of 1 mM NG-NaHCO3, and 10 mM glucose, equilibrated with an

O2/CO2 mixture (19/1, v/v) to give pH 7.4) was methyl-L-arginine (L-NMA) to the reaction medium.The rate was calculated by using the absorption coef-pumped into the liver via a cannula placed in the

portal vein. Perfusions were carried out at constant ficient of methemoglobin, 38,600 M01 cm01, for thewavelength pair 401 minus 411 (17). The resultsflow rates (3.5–4.0 ml/g liver/min) and temperature

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465LIVER NITRIC OXIDE SYNTHASE IN HYPERTHYROIDISM

are expressed as nmol NO/mg protein/min or nmolNO/g liver/min.

Protein content was assayed according to Lowryet al. (21)

All reagents used were obtained from SigmaChemical Co. (St Louis, MO). Values shown corre-spond to the means { SE for the indicated numberof separate observations. The significance betweenmean values was assessed by either Student’s t testfor unpaired data or one-way ANOVA and the New-man–Keuls’ test as indicated.

RESULTS

T3 administration to fed rats elicited a significantenhancement in the circulating levels of the hor-mone, reaching maximal values at 1 day after treat-ment, with a significant decrease after the third doseof T3 (Fig. 1A), concomitantly with enhancement inthe rectal temperature of the animals (Fig. 1A) andin the rate of O2 uptake by the liver (Fig. 1B). Thesechanges were normalized 3 days after the last T3

administration (Figs. 1A and B). Under these condi-tions, hyperthyroid rats exhibited a progressive in-crement in the rate of liver NO generation (Fig. 1B)compared to controls, with significant effects beingobserved after 2 days (47%) and 3 days (70%) of T3

treatment that returned to control values after 3days of hormone withdrawal (Fig. 1B).

Liver NO generation in euthyroid and hyperthy-roid rats was drastically decreased by the addition

FIG. 1. Serum T3 levels, rectal temperature of the animals (A),of the NOS inhibitor L-NMA to the reaction medium,liver O2 uptake, and liver nitric oxide (NO) production (B) in

leading to a significant 45% enhancement in the he- control rats and T3-treated animals. Rats were given daily dosespatic L-NMA-sensitive NO generation in hyperthy- of 0.1 mg T3/kg ip for three consecutive days (arrows) or T3 vehicle

(controls, time zero). Values shown correspond to means { SEroid rats, compared to control values (Table I).for 5–11 animals per experimental group. Significance studiesThe administration of GdCl3 to euthyroid animalswere carried out by one-way ANOVA and the Newman–Keuls’did not modify the activity of liver NOS, whereastest: (a) P õ 0.05 compared to control values at time zero; (b) P

that in rats treated with T3 for three consecutive õ 0.05 compared to values at 1 day of T3 treatment. Rates of NOdays was significantly decreased (27%) by the Kupf- production expressed as nmol/g liver/min are: euthyroid rats, 3.99fer cell inactivator (Fig. 2). These effects were ob- { 0.74 (n Å 11); T3-treated 1 day, 4.68 { 0.34 (n Å 7); T3-treated

2 days, 6.17 { 0.58 (n Å 6) (a); T3-treated 3 days, 7.29 { 0.99served in the absence of significant changes in liver(n Å 11) (a); T3-treated 3 days/2 days after hormone withdrawal,NOS activity by GdCl3 added in vitro [euthyroid rats:7.59 { 0.56 (n Å 6) (a); T3-treated 3 days/3 days after hormoneno additions, 0.083 { 0.003 (n Å 3) nmol/mg protein/withdrawal, 5.61 { 0.72 (n Å 8).

min; 50 mM GdCl3, 0.082 { 0.005 (n Å 3). T3-treatedrats: no additions, 0.166 { 0.010 (n Å 3); 50 mMGdCl3, 0.177 { 0.015 (n Å 3)].

DISCUSSIONSimilar results were observed when liver NOS ac-

tivity was expressed as nmol/g liver/min (Figs. 1 and Liver NOS is primarily present in hepatocyteswhereas the nonparenchymal-cell fraction con-2, Table I).

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TABLE I

Liver L-Arginine (L-ARG)-Dependent Nitric Oxide Production in the Absence and Presenceof NG-Methyl-L-arginine (L-NMA) in Control Rats and T3-Treated Animalsa

NO production

Conditions Control rats (n Å 4) T3-treated rats (n Å 4) P

(nmol/mg protein/min)

L-ARG (a) 0.091 { 0.018 0.156 { 0.020 0.05L-ARG / L-NMA (b) 0.008 { 0.002 0.017 { 0.002 0.05L-NMA-sensitive production (a–b) 0.083 { 0.016 0.120 { 0.002 0.05

(nmol/g liver/min)

L-ARG (a) 5.27 { 0.88 10.39 { 1.63 0.05L-ARG / L-NMA (b) 0.43 { 0.12 1.03 { 0.23 0.05L-NMA-sensitive production (a–b) 4.84 { 0.79 9.36 { 1.02 0.05

a Animals were given daily doses of either 0.1 mg/kg of T3 for 3 consecutive days or equivalent volumes of T3 diluent (0.1 N NaOH)(controls). NO production was measured in liver postmicrosomal fractions in a reaction medium containing 1.6 mM oxyhemoglobin, 1mM MgCl2, 40 mM potassium phosphate buffer, pH 7.2, 1 mM L-ARG, and 1 mM NADPH, in either the absence or presence of 1 mML-NMA, as described under Materials and Methods. Values shown correspond to means { SE for the number of separate experimentsindicated in parentheses (n). Significance studies were carried out by Student’s t test for unpaired data.

sisting of endothelial cells and Kupffer cells has alower activity (17). The enzyme corresponds to aCa2/-independent and inducible isoform, whose ex-pression is under complex control (22). In fact, endo-toxin and cytokines such as tumor necrosis factor-a,interleukin-1, and interferon-g act synergistically toup-regulate gene expression for hepatocyte NOS,whereas glucocorticoids are down-regulators (22).However, Kupffer cells (17, 19) and mouse peritonealmacrophages (23) or murine macrophages (24) ex-hibit induction of NOS by only two stimuli, namely,endotoxin and interferon-g. Data presented in thiswork indicate that, in addition to the above-men-tioned factors, thyroid hormone also modulates NOSin rat liver. In fact, thyroid hormone-induced calori-genesis, involving enhanced liver O2 consumptionFIG. 2. Effect of gadolinium chloride (GdCl3) administration onrates, leads to progressive increments in the activityliver nitric oxide (NO) production in control rats and T3-treated

animals. Control rats treated with T3 vehicle and animals receiv- of hepatic NOS, with significantly higher rates of L-ing 0.1 mg T3/kg ip for three consecutive days were simultane- NMA-sensitive NO generation being elicited in ratsously given either saline or GdCl3 (10 mg/kg by tail vein injection) treated with T3 for three consecutive days. This ef-on the first and third day of treatment, and measurements were

fect of hyperthyroidism on liver NOS activity is re-carried out 24 h after the last injection. Values shown correspondversed toward control values after 3 days of hormoneto means { SE for 4–11 animals per experimental group. The

significance of differences between mean values (P õ 0.05) is withdrawal, concomitantly with normalization of he-shown by the letters over the histograms, assessed by one-way patic respiration. The enhancement in liver NOSANOVA and the Newman–Keuls’ test. Rates of NO production achieved by T3 treatment seems to be exerted at leastexpressed as nmol/g liver/min are: control rats (a), 3.99 { 0.46

in both hepatocytes and Kupffer cells. This view is(n Å 4); GdCl3-treated (b), 3.51 { 0.20 (n Å 4); T3-treated (c), 7.30supported by the finding that the in vivo inactivation{ 0.99 (n Å 11) (a, b, d); GdCl3/T3-treated (d), 4.39 { 0.31

(n Å 4) (a, c). of Kupffer cells in hyperthyroid animals by GdCl3

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467LIVER NITRIC OXIDE SYNTHASE IN HYPERTHYROIDISM

administration lowered the increased NOS activity the adaptive increase of the enzyme in this hormonedysfunction.by 27%, an effect that is independent of direct effects

of GdCl3 on the enzyme. In addition to hepatocytesand Kupffer cells, synthesis of NO has been found ACKNOWLEDGMENTSto occur in endothelial cells (25), a process that could

This work was supported by Grant 1970300 from FONDECYT.also be increased by thyroid hormone. EnhancementThe technical assistance of C. Almeyda and M. Suarez is grate-of NO generation may be conditioned by NOS induc-fully acknowledged.tion in hepatocytes and in cells lining the hepatic

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Influence of Hyperthyroidism on the Activity of Liver Nitric Oxide Synthase in the Rat