Acute effects of thyroid hormone analogs on sodium currents in neonatal rat myocytes.

We previously reported that T3(3,3',5-triiodo-L-thyronine) acutely increases sodium currents (INa) in neonatal rat myocytes. Here we compare the effects of several thyroid hormone analogs, including T4(3,3',5,5'-tetraiodo-L-thyronine), rT3(3,3',5'-triiodo-L-thyronine), D-T3(3,3',5-triiodo-D-thyronine), 3,5-T2(3,5-diiodo-L-thyronine), DIT (3,5-diiodo-L-tyrosine), MIT (3-monoiodo-L-tyrosine), tetrac (3,3',5,5'-tetraiodo-thyroacetic acid), triac (3, 3',5-triiodo-thyroacetic acid), and tyrosine, on INa in cultured neonatal rat myocytes (n ranged from 9 to 28 for each comparison). T4, T3, 3,5-T2, and DIT (10 n m) all increased current density relative to control to a similar degree: to 1.22+/-0.2, 1.21+/-0.03, 1.16+/-0.02 and 1.16+/-0.03, respectively, P<0.05. In contrast, thyroid hormone analogs with an altered side group of the inner iodophenyl ring, including tetrac, triac, and D-T3, had no effect on INa nor did rT3, MIT or tyrosine. Pretreatment with rT3 inhibited the effects of T4, T3, 3,5-T2, and DIT. Conversely, the dose-dependent inhibitory effect of amiodarone, an iodinated benzofuran derivative that antagonizes thyroid hormone actions, on INa was blocked when myocytes were pretreated with T3(100 n m, n=3), suggesting an interaction of T3 with amiodarone. The enhancement of INa by T3 and 3, 5-T2 could not be blocked by propranolol, suggesting that the effects are not mediated through beta -adrenergic signaling pathways. In conclusion, the present results suggest that the acute effects of thyroid hormone and analogs on cardiac INa are mediated by a non-genomic thyroid hormone receptor with a unique structure-activity relationship.

Identification of thyroxine-sulfate (T4S) in human serum and amniotic fluid by a novel T4S radioimmunoassay.

Recently, we identified significant amounts of thyroxine sulfate (T4S) in fetal sheep serum, meconium, bile, and amniotic and allantoic fluids. Little is known, however, about sulfate conjugation of thyroxine in humans. In this study, we employed a novel, sensitive T4S RIA to address this question. The rabbit antiserum was quite specific; T4, T3, rT3, and 3,3'-T2 showed less than 0.002% cross-reactivity. Other analogs cross-reacted less than 0.0001%. Only rT3S and T3S cross-reacted significantly (9.9% and 2.0%, respectively). The mean serum T4S concentration (ng/dL) was 8.6 in euthyroid subjects, 14.4 in hyperthyroid subjects, 5.0 in hypothyroid subjects, 5.9 in pregnancy, and 4.5 in patients with nonthyroid illnesses. T4S concentration in amniotic fluid from women at 18-19 weeks of gestation (25.5 ng/dL) was higher than that at 14-15 weeks of gestation (14.3 ng/dL). A significant rise in serum T4S was detected in hyperthyroid patients 1 day after ingestion of 1 g of ipodate. These data suggest that T4S is a normal component of human serum and amniotic fluid, and it is mostly derived from T4 peripherally and accumulates when type I 5'-monodeiodinating activity is low in fetuses or inhibited by drugs, such as ipodate.

Acute thyroid hormone promotes slow inactivation of sodium current in neonatal cardiac myocytes.

Sodium current (INa) inactivation kinetics in neonatal cardiac myocytes were analyzed using whole cell voltage clamp before and after acute treatments with thyroid hormone (3,5,3'-triiodo-L-thyronine, T3). In untreated neonatal myocytes, INa inactivation was predominantly mono-exponential, with 93 +/- 3% (S.D.; n = 9) of the peak amplitude decaying with a time constant, tau h1, of 1.8 +/- 0.5 ms at -30 mV. The remaining 7% of control INa decayed more slowly, with a time constant, tau h2, of 9.3 +/- 3.0 ms at -30 mV. The contribution of slowly-inactivating channels to peak current was increased from 7% to 43 +/- 27% within 5 min of exposure to 5-20 nM T3 (nine cells; P less than 0.005). The time constants for both the fast- and slow-inactivating components of peak current (tau h1 and tau h2) were not significantly changed by acute T3 treatment, nor was steady-state INa inactivation (h infinity) affected. Thyroid hormone action on sodium inactivation was partially reversible by lidocaine. These findings indicate that T3 acts at the neonatal cardiac cell membrane to promote slow inactivation kinetics in sodium channels.

Acute effects of thyroid hormone on sodium currents in neonatal myocytes.

Sodium currents and action potentials were recorded from myocytes of neonatal rats during acute exposure to thyroid hormone (5-20 nM). One to 5 minutes after addition of thyroid hormone to the bath, decay from peak Na current was slowed, with the fractional current flowing 20 ms after onset (relative to peak current) increasing from 6 +/- 5% to 17 +/- 13% (p less than 0.01, n = 12). Action potential durations were increased from 55 +/- 14 to 86 +/- 36 msec (p less than 0.05, n = 6). The effects of thyroid hormone were partially reversed by lidocaine (60 microM, n = 5), a specific blocker of a slow sub-population of Na channels. Thus thyroid hormone interacts directly with myocyte membrane, probably by slowing of inactivation of Na channels.

Effects of cholecystographic agents and sulfobromophthalein on binding of thyroid hormones to serum proteins.

A number of interactions between thyroid hormones and cholecystographic agents have previously been demonstrated. In the present study we show that cholecystographic agents also interfere with the binding of thyroid hormones to serum proteins. A commercial kit (Tri-Tab) was used in which the uptake of labeled hormone from serum by a silicate adsorbent tablet is measured. In the presence of cholecystographic agents or sulfobromophthalein (BSP), the amount of labeled hormone bound to adsorbent increased in a dose-dependent fashion, reflecting displacement from protein-binding sites. The order of potency was BSP greater than ipodate greater than iopanoate greater than tyropanoate. Displacement of hormone was confirmed by a second methodology in which graded amounts of unlabeled T4 were added to the system. This allowed a Scatchard analysis to be performed for binding sites on T4-binding globulin. The cholecystographic agents and BSP caused displacement of the Scatchard slopes, again demonstrating interference with binding to serum protein sites. A method is described in which the change in Scatchard slope produced by an inhibitor is employed to compute the association constant between T4-binding sites on T4-binding globulin and the inhibitors. The values were: BSP, 14.6 X 10(3) M-1; ipodate, 4.7 X 10(3) M-1, iopanoate, 2.2 X 10(3) M-1; and tyropanoate, 0.1 X 10(3) M-1. Because of these relatively low values and the rapidity with which these agents are normally cleared from serum, it seems likely that effects on free hormone levels would be transient and of small magnitude during routine cholecystography. Also, ipodate, in the 1 g/day dose that has been employed experimentally to treat hyperthyroidism, should have a negligible effect on protein binding. On the other hand, when high levels of these compounds are used in experimental settings to study other aspects of thyroid hormone metabolism, changes in protein binding can occur and confound interpretation of results.

Sudden infant death syndrome and hypertriiodothyroninemia: comparison of neonatal and postmortem measurements.

Serum triiodothyronine concentrations in victims of sudden infant death syndrome, when compared with those of both living and dead controls, were found to be elevated to a degree comparable to those found in a previous study. Thyroxine, thyroid-stimulating hormone, and thyroglobulin values were not elevated. Neonatal triiodothyronine and thyroxine values, determined on specimens collected within a few days of birth for most of the sudden infant death syndrome victims, did not differ significantly from those of comparable peers in their birth cohort. These results indicate that hypertriiodothyroninemia may serve as a useful postmortem diagnostic marker for the syndrome but not as a premortem predictor. Parenthetically, thyroid hormones appear to remain stable in either a frozen or dried state for up to two years.

Interactions of iron deficiency, anemia, and thyroid hormone levels in response of rats to cold exposure.

We have earlier shown that iron-deficient rats have increased urinary norepinephrine (NE) excretion. They also have an exaggerated rise in urinary NE when placed in the cold, a stimulus known to cause increased NE excretion in normal rats. Nonetheless, they fall to maintain body temperature. We have now examined the thyroidal response to cold in iron-deficient rats. As others have shown, control rats had a rise in plasma levels of thyroxine (T4) and triiodothyronine (T3) soon after entering the cold environment (4 degree C); they also maintained a rectal temperature above 36 degree C. In the iron-deficient rats, basal levels of T3 and T4 were normal, but there was little or no increase after 6 hr in the cold, and, as before, body temperatures fell. Injections of T3, 10 microgram/kg, 15 min before cold exposure improved the ability of iron-deficient rats to maintain body temperature, but they still did not do as well as the controls. We conclude that the inability of iron-deficient rats to increase T3 levels after cold exposure is one factor in their poor resistance to cold. The defect could involve inability to augment thyroid secretion, imparied ability to convert T4 to T3 in peripheral tissues, or both. Preliminary data suggest that anemia is an important and perhaps critical factor in the cold sensitivity of iron deficiency. Transfusing iron-deficient rats from their usual hematocrit of 15-20 to one of 30 restores cold resistance to normal. Transfusion also allows a more normal thyroid response, with a rise in T3 and T4 levels, so thyroid hormones may be a factor in the improvement produced by transfusion.

Inhibition of hepatic binding of thyroxine by cholecystographic agents.

Subsequent to studies indicating that cholecystographic agents and sulfobromophthalein (BSP) inhibit uptake of thyroxine (T(4)) by rat liver slices, the effect of such compounds on hepatic storage of T(4) in man has been examined. After intravenous administration of [(125)I]T(4) to five normal subjects, hepatic radioactivity, estimated by external gamma counting, rose to a peak in approximately 4 h and then declined in parallel with serum radioactivity. When a 6-g dose of the cholecystographic agent, tyropanoate (Bilopaque), was administered orally 3 d later, estimated hepatic extravascular radioactivity fell 50-60% within 4 h and then rose toward the pretyropanoate value. Concomitant with the fall in hepatic radioactivity, serum radioactivity rose 57-70%, as did stable T(4) levels in serum, suggesting that hormone discharged from the liver entered the serum. Both uptake of T(4) and discharge by tyropanoate were much less in two patients with liver disease. Ipodate (Oragrafin), 12 g orally in two subjects, caused much smaller changes in hepatic and serum radioactivity. However, ipodate also caused a doubling of the percent free T(4) in the serum as judged by equilibrium dialysis, and the ratio of hepatic radioactivity to free [(125)I]T(4) in serum declined markedly after ipodate, similar to the fall in hepatic:serum (125)I ratios after tyropanoate. BSP, 20 mg/kg i.v. in three subjects, caused a smaller change; the decline in hepatic T4 content averaged 19%. We conclude that these organic anions, tyropanoate, ipodate, and BSP, all can displace T(4) from the liver. The results imply a link between T(4) transport and organic anion transport, and indicate a mechanism for altering hepatic T(4) content in man that could be the site of physiologic regulation or of disease. A method is described whereby analysis of the change in hepatic and plasma radioactivity after tyropanoate could be employed to estimate hepatic T(4) content in diverse clinical circumstances.

Postpartum thyrotoxicosis in a patient with Graves' disease. Association with low radioactive iodine uptake.

A patient with previously diagnosed Graves' hyperthyroidism had a transient episode of thyrotoxicosis three months postpartum. This was associated with a diffusely enlarged thyroid gland, a rise in antithyroid microsomal antibody titer, a 24-hour radioactive iodine uptake (RAIU) of 1%, and an aspiration biopsy specimen suggestive of chronic lymphocytic thyroiditis. An alternative cause for the depressed RAIU was not discovered. This case would suggest that patients with previously diagnosed Graves' disease can have thyrotoxicosis without an enhanced RAIU and that the postpartum thyrotoxic syndrome may involve an immunologic injury causing a release of performed thyroid hormone.

Decreased adrenal responsiveness in hypothermic patients.

We have performed ACTH simulation tests in a total of 14 subjects who were hypothermic at the time of initial presentation. Plasma cortisol values were measured before and 1 h after an iv dose of 25 U synthetic ACTH. The cortisol response was depressed in these subjects, with a mean rise of 32% and an absolute mean rise of 5.0 microgra/dl. There appeared to be a temperature threshold effect, with only minimal responses observed below 32 C. A subgroup of 5 patients with sluggish responses to ACTH while hypothermic (mean cortisol rise, 12.5%) were retested after warming and responded normally (mean rise, 166%). Thus, ACTH stimulation tests may be misleading in the hypothermic patient and should be performed only after body temperature has returned to normal.

Metabolism of thyroid hormones by rat thyroid tissue in vitro.

Rat thyroid lobes or hemilobes have been incubated in Krebs-Ringer phosphate buffer containing labeled T4 and/or T3, and the products were separated by paper chromatography. Labeled T4 was actively degraded; about half of the T4 metabolized was recovered as T3. Labeled T3 was also metabolized, but less rapidly than T4. Other than T3 produced from T4, the major products from both hormones were inorganic iodide and iodoprotein; the latter was presumably a secondary product of iodide organification because its formation was inhibited by hypoxia and methimazole. Feeding the animals a low iodine diet increased their hormone-metabolizing activity. Incubation under nitrogen did not affect the rate of T4 degradation, but partially inhibited T3 degradation. Degradation of both hormones was unchanged in the presence of methimazole and ascorbate, was markedly inhibited by 1 mM propylthiouracil (PTU), and was partially inhibited by azide and cyanide. Thyroid tissues concentrated both hormones, tissue to medium gradients averaging 5.4 for T4 and 20.7 for T3; none of the conditions affecting hormone degradation (incubation under nitrogen or with azide, cyanide, or PTU) significantly altered these gradients. It is concluded that the thyroid can metabolize both of its major hormones by a system distinct from thyroidal peroxidase. Hormone metabolism, therefore, is a potentially important factor in net hormone secretion. In its resistance to hypoxia, methimazole, and ascorbate and its sensitivity to PTU, the thyroid's system for generating T3 from T4 resembles T3-forming systems of liver and kidney. The thyroid, because T3 formation is its dominant pathway for T4 metabolism, may provide a useful model for study of this reaction.

Triiodothyronine (T3)-binding immunoglobulins in a euthyroid woman: effects on measurement of T3 (RIA) and on T3 turnover.

A 36-year-old woman with nodular goiter, nervousness, and tachycardia was evaluated for T3 toxicosis. Her serum thyroxine level, resin T3 uptake, and thyroidal radioiodine uptake were normal. Her T3 (RIA), by a technique employing charcoal to separate bound and free T3, was reported as indeterminate due to an interfering substance; by a double-antibody method, her T3 (RIA) was 325 ng/dl. Further studies of the patient's serum revealed an abnormal T3-binding protein which misgrated in the beta-gamma globulin zone on paper electrophoresis and gel filtration chromatography (Sephadex G-200), and was precipitated from serum by rabbit anti-human Fab antibody. The gamma globulin fraction of the patient's serum, separated by a standard technique, showed strong binding activity toward [125I]T3, with an association constant of 4.1 X 10(8) 1/mole (Scatchard plot). In a similar system, labeled T4 was not bound. To avoid artefacts which this T3-binding protein might produce in assaying unextracted serum, T3 (RIA) was performed on an ethanol extract of serum and found to be 191 ng/dl, a slight elevation. However, the metabolic clearance rate of injected [125I]T3, estimated by non-compartmental analysis of the serum decay curve or by the specific activity or urinary T3, was about 16 1/day, a low value, so that the T3 production rate, 31 mug/day, was normal. The patient's symptoms disappeared with the resolution of domestic problems, and she appeared clinically euthyroid. Serum TSH was 5.0 uU/ml and antithyroglobulin titer, 1:16. A test for antibodies to thyroid microsomes was negative. We postulate that this subject was euthyroid, but had a concentration of T3-binding immunoglobulin which was sufficient to produce modest slowing of T3 turnover, borderline elevation of extractable T3 (RIA), and a major artefact in the T3 (RIA) measurement of unextracted serum. A similar abnormality may account for other instances of high T3:T4 ratios in serum.

Thyrotropin levels in hyperlipidemic survivors of myocardial infarction.

In an earlier investigations, the prevalence of hyperlipidemia in a group of patients who survived myocardial infarction was determined, and family studies were performed to allow genetic classification of patients with hyperlipidemia. Radioimmunoassay for thyrotropin (TSH) has now been performed on plasmas from most of these hyperlipidemic survivors. Elevated TSH values were found in five of the 18 hyperlipidemic women over age 60, and in seven of the remaining 104 hyperlipidemic subjects. Among the various genetically defined types of hyperlipidemia, the highest prevalence of TSH elevations was seen in women with sporadic (nonfamilial) hypertriglyceridemia; four of the ten had an abnormal TSH level, and two of the remainder were receiving thyroid medication. Hypercholesterolemia was not strongly correlated with TSH abnormalities. These data support the hypothesis that clinically inapparent thyroid damage may be associated with coronary artery disease.