The effects of thyroxine or a GnRH analogue on thyroid hormone deiodination in the olfactory epithelium and retina of rainbow trout, Oncorhynchus mykiss, and sockeye salmon, Oncorhynchus nerka.

Using low (0.5nM) substrate levels we determined the activities of thyroxine (T4) outer-ring deiodination (ORD), T4 inner-ring deiodination (T4IRD) and 3,5,3(')-triiodothyronine (T3) IRD activities in the olfactory epithelium (OLF) and retina (RET) of laboratory-held immature 1-year-old rainbow trout and immature 2.5-year-old sockeye salmon. In both species all three deiodination activities were detected in OLF and RET. For OLF, no particular pathway predominated and activities were similar to those of brain. For RET, T3IRD activity was greater than T4ORD activity and in sockeye RET T3IRD activity exceeded that of liver. Trout immersion for 6 weeks in 100ppm T4 increased plasma T4 levels 3-fold and plasma T3 levels by 50% and caused the anticipated autoregulatory responses in brain and liver deiodination ( downward arrow T4ORD, upward arrow T4IRD, and upward arrow T3IRD); OLF deiodination and RET T4ORD activity were unaltered but RET T4IRD and T3IRD activities increased dramatically. Two injections of a GnRH analogue (20 microgkg(-1)) into sockeye increased plasma T3 levels but not T4 levels and decreased RET T4IRD and T3IRD activities without changing liver, brain, or OLF deiodination. We conclude that in salmonids the main TH deiodination pathways occur in OLF but show no regulation by T4 or GnRH. In contrast, T3IRD activity predominates in RET and can be regulated by T4 and GnRH, suggesting that for RET plasma may be the major T3 source. These findings have implications for thyroidal regulation of sensory functions during salmonid diadromous migrations.

Characterization of hepatic low-K(m) outer-ring deiodination in red drum (Sciaenops ocellatus).

The more biologically active thyroid hormone 3,5,3'-triiodothyronine (T(3)), is primarily derived from peripheral deiodination of thyroxine (T(4)). We characterized hepatic deiodination for a commercially important, warm water teleost fish, the red drum (Sciaenops ocellatus). Low K(m) outer-ring deiodination (ORD) activity was determined by production of free iodide ((125)I) upon incubation of hepatic microsomes with radiolabeled T(4). HPLC analysis demonstrated that (125)I, and T(3) were produced in equal amounts, thereby validating 125I as a measure of T(3) production. A small amount of 3,3',5'-triiodothyronine (reverse T(3)) was also produced by inner-ring deiodination. Production of (125)I was linear over a range of 0--100 microg protein/ml and for incubations of 30 min--4 h. Maximal ORD activity was measured at pH 6.6, 50 mM dithiothreitol (DTT) and an incubation temperature of 20 degrees C. Double reciprocal plots demonstrated that the average apparent K(m) was 5.1 nM and the average V(max) was 3.7 pmol T(4) converted/h per mg protein. ORD was not inhibited by propylthiouracil but was 50% inhibited by 90 microM of iodoacetic acid and 7 microM of gold thioglucose. The substrate analog preference was T(4) = tetraiodoacetic acid = reverse T(3) > triiodoacetic acid >> T(3). In relation to other tissues, ORD for liver>gill>intestine>kidney. Similar hepatic deiodination activity was present in adult wild, aquacultured and laboratory-reared red drum, but in adult wild red drum the optimum temperature was higher. Red drum hepatic low-K(m) deiodination activity appears to most closely resemble rainbow trout hepatic and mammalian Type II deiodination. Evidence of inner-ring T(4) deiodination suggests a more active hepatic iodothyronine catabolic pathway than in other teleost species.

Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa.

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.

Changes in intestinal and hepatic thyroid hormone deiodination during spontaneous metamorphosis of the sea lamprey, Petromyzon marinus.

We measured microsomal low-K(m) outer-ring deiodination (ORD) and inner-ring deiodination (IRD) activities for thyroxine (T(4)) and 3, 5,3'-triiodothyronine (T(3)) in intestine and liver in nonmetamorphosing (undersized) larvae, immediately premetamorphic larvae, animals in stages 1-7 of metamorphosis, and immediately postmetamorphic sea lampreys (Petromyzon marinus). For intestine: T(4)ORD activity was relatively low in nonmetamorphosing larvae, increased in premetamorphic individuals, was highest in stages 1 and 2 and was very low during stages 3-7; T(4)IRD activity was negligible until stage 3 but increased 4.7-fold through stages 3 to 7 such that T(4)IRD activity was 14 times T(4)ORD activity at stage 6; T(3)ORD activity was undetectable; T(3)IRD activity was not measured through stages 3-7 but correlated with T(4)IRD activity at other stages. For liver: deiodination was only measured up to stage 2 and in postmetamorphic animals; in contrast to intestine, T(4)ORD activity fell to low levels at stage 2 and was low during postmetamorphosis; T(4)IRD and T(3)IRD activities were very low and uninfluenced by developmental stage; T(3)ORD activity was undetectable. We conclude that (1) deiodination activity is usually much higher in intestine than in liver, (2) intestinal ORD and IRD activities change reciprocally so that ORD predominates in early metamorphosis but IRD predominates in mid and late metamorphosis, and (3) changes in intestinal deiodination may contribute to the characteristic depression of plasma T(4) and T(3) levels during spontaneous metamorphosis. J. Exp. Zool. 286:305-312, 2000.

Thyroid hormone deiodination in tissues of American plaice, Hippoglossoides platessoides: characterization and short-term responses to polychlorinated biphenyls (PCBs) 77 and 126.

We have described the tissue distribution and properties of thyroid hormone (TH) deiodination activities of the marine American plaice, Hippoglossoides platessoides. We then studied the 1- or 4-week responses of the plaice liver and brain deiodination activities and the plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels to an intraperitoneal injection (5-500 ng/g) of the polychlorinated biphenyl (PCB) congeners 77 (3,3'-4,4'-tetrachlorobiphenyl) or 126 (3,3',4,4',5-pentachlorobiphenyl). T4 and 3,3'5'-triiodothyronine (rT3) outer-ring deiodination (ORD) activities were greater in liver than in kidney, gill, heart, brain, intestine or muscle; inner-ring deiodination (IRD) activity occurred in all tissues but was consistently higher in brain. Deiodination characteristics (optimal pH, optimal dithiothreitol concentration, responses to inhibitors and apparent Km values of 0.6-4 nM) fell in the same rage as those of low-Km deiodinases in other teleosts. Deiodination activities were maximal when assayed at 25 degrees C but uniformly low over the natural range of 0-9 degrees C. Neither PCB 77 nor PCB 126 altered brain T4ORD activity or plasma T4 levels (P < 0.05). However, at 1 week post injection hepatic T4ORD activity was increased and plasma T3 levels lowered by PCB 77 (5 and 25 ng/g), while hepatic IRD activity was increased by PCB 126 (50 and 500 ng/g). Neither PCB 77, PCB 126 nor selected hydroxylated. PCBs given in vitro compared with T4 for binding sites on plasma proteins or altered hepatic deiodination activity, indicating no direct action on plasma proteins or deiodinases We conclude that plaice TH deiodination tissue distribution and characteristics resemble those of other teleosts. Deiodination activities are low at natural assay temperatures but at 1 week show some responses to PCBs 77 and 126.

Effect of T(3) treatment and food ration on hepatic deiodination and conjugation of thyroid hormones in rainbow trout, Oncorhynchus mykiss.

We studied the 7-day effects of 3,5,3'-triiodothyronine (T(3)) hyperthyroidism (induced by 12 ppm T(3) in food) and food ration (0, 0.5, or 2% body weight/day) on in vitro hepatic glucuronidation, sulfation, and deiodination of thyroxine (T(4)), T(3), and 3,3', 5'-triiodothyronine (rT(3)). T(3) treatment doubled plasma T(3) with no change in plasma T(4), depressed hepatic low-K(m) (1 nM) outer-ring deiodination (ORD) of T(4), induced low-K(m) (1 nM) inner-ring deiodination (IRD) of both T(4) and T(3) but did not alter high-K(m) (1 microM) rT(3)ORD, glucuronidation, or sulfation of T(4), T(3), or rT(3). Plasma T(4) levels were greater for 0 and 2% rations than for a 0.5% ration. Fasting decreased low-K(m) T(4)ORD activity and increased high-K(m) rT(3)ORD activity but did not alter T(4)IRD or T(3)IRD activities. T(4), T(3), and rT(3) glucuronidation were greater for 0 and 0.5% rations than for a 2% ration. T(3) glucuronidation was greater for a 0.5% ration than for a 0% ration. T(3) and rT(3) sulfation were greater for a 2% ration than for a 0 or a 0.5% ration; ration did not change T(4) sulfation. We conclude that (i) modest experimental T(3) hyperthyroidism induces T(3) autoregulation by adjusting hepatic low-K(m) ORD and IRD activities but not high-K(m) rT(3)ORD or conjugation activities; (ii) in contrast, ration level changes both deiodination and conjugation pathways, suggesting that the response to ration does not solely reflect altered T(3) production; (iii) deiodination and conjugation appear complementary in regulating thyroidal status in response to ration; and (iv) high-K(m) rT(3)ORD in trout differs from rat type I deiodination in that it does not respond to T(3) hyperthyroidism and it increases, rather than decreases, its activity during fasting.

Deiodination and deconjugation of thyroid hormone conjugates and type I deiodination in liver of rainbow trout, Oncorhynchus mykiss.

We studied the hepatic in vitro deconjugation and deiodination of glucuronide (G) and sulfate (S) conjugates of the thyroid hormones (TH) thyroxine (T(4)), 3,5,3'-triiodothyronine (T(3)), and 3,3', 5'-triiodothyronine (rT(3)) in trout. These conversions have not been studied in nonmammals. Deconjugation of T(4)G, T(3)G, rT(3)G, or rT(3)S was negligible in all subcellular fractions. Some T(4)S desulfation occurred but T(3)S was desulfated to the greatest extent by freshly isolated hepatocytes and by the mitochondrial/lysosomal and microsomal fractions. Deiodination of T(4)G, T(3)G, rT(3)G, T(4)S, T(3)S, and rT(3)S (1 or 1000 nM) was negligible in control trout and in trout treated with T(3) to induce inner-ring deiodination (IRD) but simultaneously tested rat microsomes rapidly deiodinated T(4)S, T(3)S, and rT(3)S. Furthermore, T(4)S, T(3)S, and rT(3)S (1-100 nM) were less effective than their unsulfated forms in competitively inhibiting trout hepatic outer-ring deiodination (ORD) of T(4) (0.8 nM), and rT(3)ORD (100 nM) was not competitively inhibited by T(4)S, T(3)S, or rT(3)S (100 nM) or by T(4) or T(3) (1 microM). Thus, there is no evidence in trout liver for THS deiodination, which is a key property of rat type I deiodination. We therefore studied other properties of trout hepatic high-K(m) deiodination, which has been considered homologous to rat type I deiodination. We found that it resembled rat type I deiodination in its rT(3)ORD ability, its optimum pH (7.0), and its requirement for dithiothreitol (DTT). However, it differed from rat type I deiodination not only in its negligible deiodination of T(4) and THS but also in its low DTT optimum (2.5 mM), its low apparent K(m) for rT(3) (200 nM), its lack of IRD ability, its extremely weak propylthiouracil inhibition (IC(50), 1 mM), its weaker inhibition by iodoacetate (IC(50), 10 microM) and aurothioglucose (IC(50), <3 microM), its activation by fasting, and its unresponsiveness to T(3) hyperthyroidism. We conclude that most conjugated TH are neither deconjugated nor deiodinated by trout liver and are therefore eliminated in bile. However, T(3)S can be desulfated. Substrate preference and other properties suggest that trout hepatic high-K(m) ORD shares some properties with rat type I deiodination but differs functionally in several other respects and may contribute negligibly to hepatic T(3) production in trout.

Sulfation of thyroid hormones by liver of rainbow trout, Oncorhynchus mykiss.

We studied hepatic sulfation of thyroid hormones (TH) in rainbow trout, Oncorhynchus mykiss. Sulfation of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) was detected in the cytosolic (63-67%), microsomal (12-16%), nuclear (12-14%) and mitochondrial/lysosomal (7-8%) fractions. Using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor, sulfation of T4 and T3 by the cytosolic fraction depended on protein concentration and time. The pH profiles for T4- and T3-sulfation were broad and overlapping with optimal pH values of about 6.5 and 7.0 U respectively. At pH 7.0, apparent K(m) (microM), Vmax (pmol/mg cytosolic protein per hour) and catalytic efficiency (Vmax/K(m)) values were 3,5',3'-triiodothyronine (reverse T3, rT3) = 0.7, 583 and 832; T4 = 1.7, 46 and 27; T3 = 11.5, 840 and 73. Inhibitor profiles for both T4- and T3-sulfation were not significantly different with a common inhibitor preference of rT3 > pentachlorophenol > triiodothyroacetic acid > tetraiodothyroacetic acid T4 = T3 = 3,5-diiodothyronine. T4-, T3- and rT3-sulfation activity decreased with increasing pre-incubation temperature (12, 24, 36 degrees C); however, there were no significant differences in T4-, T3- and rT3-sulfation activity at each pre-incubation temperature. We conclude that: (i) in trout, hepatic sulfation of TH is enzymatic and obeys Michaelis-Menten kinetics; (ii) like mammalian hepatic sulfotransferases (STs), trout hepatic STs are heat-sensitive cytosolic proteins using PAPS as a sulfate donor; (iii) unlike mammalian sulfation of TH, trout hepatic sulfation of T4, T3 and rT3 may be catalyzed by a single form of ST preferring rT3 as substrate and with a catalytic efficiency of rT3 >>> T3 > T4.

Blocking of KC1O4-induced metamorphosis in premetamorphic sea lampreys by exogenous thyroid hormones (TH); effects of KC1O4 and TH on serum TH concentrations and intestinal thyroxine outer-ring deiodination.

Immediately premetamorphic larval sea lampreys (Petromyzon marinus) (>/=120 mm in length) were treated for 4, 8, or 16 weeks with one of two concentrations of either exogenous thyroxine (T4; 1 or 0.5 mg/L) or 3,5,3'-triiodothyronine (T3; 1 or 0.25 mg/L) in the presence or absence of the goitrogen potassium perchlorate (KC1O4; 0.05%) as well as with KC1O4 alone. Larvae from all treatments were examined for signs of metamorphosis, changes in serum T4 and T3 concentrations (serum T4 and serum T3), and changes in intestinal T4 outer-ring (5') deiodination to T3 (T4ORD). KC1O4 depressed both serum T4 and T3 and induced metamorphosis in 80% of larvae treated for 8 weeks or longer. However, neither effect was observed in larvae exposed to KC1O4 combined with either thyroid hormone (TH). These data confirm previous suggestions that exogenous TH blocks KC1O4-induced metamorphosis by elevating serum TH concentrations, and provide evidence that declines in serum TH concentrations are mandatory for precocious metamorphosis. Serum T4, but not serum T3, was elevated following exogenous T4 treatment in the presence or absence of KC1O4. This maintenance of serum T3 at control levels, in the presence of a T4 challenge, was not due to decreases in intestinal T4ORD activity, since T4ORD activity was not affected by any treatments in the study. Exogenous T3 elevated both serum T4 and T3. However, serum T3 in T3-treated larvae decreased with time, suggesting a stringent T3 regulation. Elevation of serum T4 following T3 treatment may have been a result of either inhibition of T4 metabolism, or stimulation of T4 secretion by the endostyle. Based on these results, we conclude that (i) exogenous TH blocks KClO4-induced metamorphosis in sea lampreys and (ii) serum T3 is maintained at control levels despite elevations in serum T4 (its immediate precursor), but this does not involve any changes in intestinal T4ORD activity.

Factors influencing the steady-state distribution and exchange of thyroid hormones between red blood cells and plasma of rainbow trout, Oncorhynchus mykiss.

We studied effects of in vitro conditions on the steady-state distribution and exchange of thyroid hormones (TH) between red blood cells (RBC) and plasma of rainbow trout. At steady state at 12 degrees C the RBC contained 5-11% of L-thyroxine (T4), 14-23% of 3,5,3'-triiodo-L-thyronine (T3), and 23-24% of 3,3',5'-triiodo-L-thyronine (rT3) present in whole blood. The steady-state distribution was (i) higher in immature than in mature trout for T3, (ii) increased by incubation temperature from 0 to 22 degrees C for both T4 and T3, (iii) unaltered by blood T4 or T3 concentration (0-40 ng/ml), and (iv) increased by O2 gassing and decreased by N2 gassing for T4 and rT3, but negligibly for T3. The exchange between RBC and plasma was more rapid for T3 (50% of maximal influx or efflux at 30-40 s) than rT3 (14 min) than T4 (30 min). Efflux of T4 and T3 was greatly reduced in the absence of plasma protein. Incubation with 10% bovine serum albumin extracted > 98% of labeled T4 and T3 from RBC. We conclude that for trout (i) steady-state distribution and exchange kinetics between RBC and plasma differ greatly for T4, T3, and rT3 and vary with the in vitro conditions, (ii) almost all TH in RBC are reversibly bound to intracellular sites, (iii) efflux is strongly influenced by plasma binding sites, (iv) T3 exchange is rapid and may allow T3 access to RBC TH receptors, buffer plasma T3 levels, or aid T3 delivery to tissues, (v) T4 exchange is slow and this may prevent oxygenation state from altering T4 uptake into RBC, and (vi) rT3 uptake into RBC may contribute to low rT3 levels in trout plasma.

Effects of long-term temperature acclimation on thyroid hormone deiodinase function, plasma thyroid hormone levels, growth, and reproductive status of male Atlantic cod, Gadus morhua.

The recent collapse of the Northwestern Atlantic cod fisheries has coincided with a cooling of water temperatures. During this time the condition factor of cod has been poor. The objective of the present study was to determine the effects of long-term temperature acclimation on growth reproduction and thyroid function in laboratory held Atlantic cod (Gadus morhua). One of the key parameters used to assess thyroid function is the peripheral metabolism of L-thyroxine (T4) by microsomal deiodinase enzymes. Deiodinase function has not been described for gadid fish. T4 outer-ring deiodinating activity (apparent K(m) 1-2 nM) was confined primarily to liver. Its properties resembled those for hepatic T4ORD activity of other teleosts and the mammalian type II deiodinase. The T4ORD activity of cod liver exceeded that of salmonids and could explain the high plasma T3 levels (10-18 ng/ml), which were 2-5 times greater than T4 levels. T4 and T3 inner-ring deiodination was confined mainly to brain. In order to determine the effects of long-term temperature acclimation on cod, somatic growth, reproduction, and thyroidal status were assessed monthly in 400-900-g satiation-fed male Atlantic cod captured in June from the St. Lawrence Estuary and then acclimated from August to the following June under a natural photoperiod at 2-4 degrees C (LT) or 6-10 degrees C (HT). Reproductive status was determined from the gonadosomatic index (GSI), plasma testosterone (T) and 11-ketotestosterone (11-KT) levels, and the appearance of milt; thyroidal status was determined from plasma T4 and 3,5,3'-triiodo-L-thyronine (T3) levels and hepatic T4ORD activity to produce biologically active T3. Testis maturation (high levels of 1 and 11-KT, and milt release) occurred in April and May and was uninfluenced by acclimation temperature. LT cod grew more slowly than HT cod. Differences in body weight were particularly evident from December to February. In conclusion, (i) cod possess outer- and inner-ring deiodinase activities, predominating respectively in liver and brain, and with properties resembling those of other teleosts, (ii) T4ORD activity of liver is unusually high and may account for the high plasma T3 levels in this species, (iii) T4ORD activity tends to increase during periods of increased somatic growth, and (iv) chronic acclimation of male cod to 2-4 degrees C, as opposed to 6-10 degrees C, decreases somatic growth but does alter circulating levels of thyroid hormones and androgens and it does not change the time of sexual maturation.

Glucuronidation of thyroxine and 3,5,3'-triiodothyronine by hepatic microsomes in rainbow trout, Oncorhynchus mykiss.

We studied the glucuronidation of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) in the hepatic microsomal fraction of rainbow trout, Oncorhynchus mykiss. Uridine diphosphoglucuronosyl transferase (UDPGT) activity toward T4 depended on both protein concentration (linear up to about 0.75 mg/ml) and time (linear up to 60 min). The optimal pH for glucuronidation was 6.8-7.8 units for T4 and > or = 8.5 units for T3. At a common pH of 7.8, the apparent Km and Vmax values were, respectively, 6.0 muM and 42 nmol/mg protein/hr for T4, and 1.1 muM and 4.3 nmol/mg protein/hr for T3. At concentrations of 100 muM, T4 inhibited T3-glucuronidation, but T3 did not inhibit T4-glucuronidation. T4-glucuronidation was inhibited by 3,3', 5'-triiodothyronine (rT3) and tetraiodothyroacetic acid (Tetrac); T3-glucuronidation was inhibited by rT3, T4, Tetrac, and triiodothyroacetic acid. Therefore, analogues with two outer-ring iodines were the most effective inhibitors, showing that outer-ring iodine substitution influences UDPGT substrate specificity. Following a 15-min pre-incubation at 24 degrees C, UDPGT thermal stability was higher for T4 than T3. Polyoxyethylene 10 cetyl ether (Brij 56) maximally stimulated UDPGT activity for both T4 and T3 about two-fold at 0.0125% (w/v) detergent, suggesting that the UDPGTs are transmembrane proteins with the active site facing the lumen of the microsomal vesicles. Clofibrate did not affect either T4- or T3-UDPGT activity. On a per mg microsomal protein basis, T4-glucuronidation in the rat liver exceeded that in the trout 3-fold. We conclude that (a) trout liver microsomes have more than one form of UDPGT for the glucuronidation of T4 and T3 and (b) these trout UDPGTs share several properties of with those of the rat.

Thyroid hormone deiodination in various tissues of larval and upstream-migrant sea lampreys, Petromyzon marinus.

Properties and activities of four potential thyroid hormone (TH) monodeiodinating pathways (T4ORD, L-thyroxine (T4) outer-ring (5') deiodination to 3,5,3'-triiodo-L-thyronine (T3); T4IRD, T4 inner-ring (5) deiodination to 3,3',5'-triiodo-L-thyronine (reverse T3); T3ORD, T3 outer-ring deiodination to 3,5-diiodo-L-thyronine; T3IRD, T3 inner-ring deiodination to 3,3'-diiodo-L-thyronine) were studied in microsomes of liver, kidney, muscle, and intestine of unmetamorphosed larvae and nontrophic upstream-migrant (spawning-phase) sea lampreys, Petromyzon marinus. T4ORD properties (pH optimum, dithiothreitrol cofactor requirement, apparent K(m), substrate preference and potency of potential inhibitors) were similar in most respects to those described previously for teleosts. T4ORD activity was detected in all larval tissues examined and was highest in intestine. In upstream migrants, T4ORD was also greatest in intestine, but low in muscle and kidney and undetectable in liver. T3ORD activity was not found in any tissue of either developmental stage. T4IRD and T3IRD activities were negligible in larval tissues, but present in kidney and particularly intestine of upstream migrants. We conclude that depending on developmental/physiological state, sea lampreys possess low-K(m) outer-ring and inner-ring monodeiodinases, which in most respects correspond functionally with those of teleosts. However, in contrast to teleosts, deiodination is particularly active in larval intestine, perhaps reflecting the release from the endostyle of TH into the lumen of the alimentary canal.

Iodine metabolism and thyroid-related functions in organisms lacking thyroid follicles: are thyroid hormones also vitamins?

Thyroid-related functions in organisms devoid of follicular thyroid tissue have been reviewed. In the lamprey, a primitive vertebrate, the larva concentrates iodide and synthesizes thyroid hormones (TH) by iodoperoxidase (IP)-mediated iodination of a thyroglobulin (TG)-like molecule in a subpharyngeal afollicular endostyle. The endostyle is the thyroid homolog, and it reorganizes into a follicular thyroid at metamorphosis to the adult. Ascidians and amphloxus, invertebrate protochordate relatives of vertebrates, also concentrate iodide and synthesize TH in a subpharyngeal afollicular endostyle, but the endostyle never transforms to follicles. Ascidian plasma contains L-thyroxine and its more biologically active derivative 3,5,3'-triiodo-L-thyronine, and TH receptors exist, but TH effects are poorly understood. No other invertebrates possess an endostyle. Several invertebrates concentrate iodide at other sites and form protein-incorporated iodohistidines and iodotyrosines; however, de novo iodothyronine biosynthesis through IP-mediated TG iodination has not been established. Nevertheless, TH occur in invertebrates, and exogenous iodothyrosines or iodothyronines have effects on jellyfish, insects, and sea urchins. Furthermore, gut bacteria metabolize TH, and plants may synthesize TH by nonenzymatic oxidative iodination. Thus, TH occur in many organisms and, after ingestion and enteric absorption, can enter the food chain. Indeed, sea urchin larvae obtain TH required to induce metamorphosis from plant diatoms. Thyroid hormones can therefore have vitamin-like effects and, in conjunction with vitamin D, and possibly with other steroids, may be more aptly termed vitamones. Availability of exogenous TH has implications for models of invertebrate and vertebrate TH metabolism and iodine salvaging, and it may explain the prominent and probable ancestral role of peripheral mechanisms in regulating thyroidal status.

Effects of L-thyroxine on brain monoamines during parr-smolt transformation of Atlantic salmon (Salmo salar L.).

During spring, seaward migrating juvenile Atlantic salmon (Salmo salar) undergo parr-smolt transformation (PST) which involves changes in physiology, including one or two peaks in plasma thyroxine (T4). To investigate if changes in plasma T4 influence neural function, we measured levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and also measured serotonin (5-hydroxytryptamine, 5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in brain regions of two groups of Atlantic salmon parr on an 8:16 h light/dark photoperiod. One group was treated with ambient T4 to simulate the natural smolt peak in plasma T4. T4 treatment depressed DOPAC levels as well as DOPAC/DA and 5-HIAA/5-HT ratios in the olfactory system but with no changes in the optic tectum. We conclude that during PST monoaminergic functions in specific brain regions of juvenile Atlantic salmon are affected by T4 treatment.

Characteristics of the uptake of 3,5,3'-triiodo-L-thyronine and L-thyroxine into red blood cells of rainbow trout (Oncorhynchus mykiss).

The mechanisms of uptake of thyroid hormones (TH) 3,5,3'-triiodo-L-thyronine (T3) and L-thyroxine (T4) by trout red blood cells (RBC) were characterized by coincubating washed RBC with [125I]T3 or [125I]T4 and a variety of inhibitors and potentially competing amino acids and TH analogues. Nonsaturable uptake of both TH was unaffected by almost all agents tested, consistent with a diffusion process. Saturable T3 uptake was unaffected by a major (80%) cyanide- and iodoacetate-induced reduction in RBC nucleotide triphosphate content or by inhibitors of Na+ transport, but was depressed by (i) inhibitors of protein binding (bromosulfophthalein, 8-anilino-1-naphthalene sulfonic acid, phloretin, and 5,5'-diphenylhydantoin, (ii) sulfhydryl blockers (p-hydroxy-mercuribenzoate and N-ethylmaleimide), (iii) endocytotic inhibitors (chloroquine, colchicine, and monodansylcadaverine), (iv) tryptophan and phenylalanine, and (v) certain TH analogues, particularly 3,3',5'-triiodo-L-thyronine (reverse T3). In contrast, saturable T4 uptake was depressed only by inhibitors or protein binding. Cross-competition between T3 and T4 for transport occurred only at highly pharmacologic TH levels. We conclude that T3 and T4 are transported into trout RBC by two separate systems. The relatively slow T4 uptake depends on binding to certain proteins. The much more rapid T3 uptake depends on protein binding, but is Na(+)-independent, and involves a system comparable to the T-system of aromatic amino acid transport. However, participation od receptor-mediated endocytosis cannot be excluded.

3,5,3'-Triiodo-L-thyronine and L-thyroxine uptake into red blood cells of rainbow trout, Oncorhynchus mykiss.

Uptake of the thyroid hormones (TH) 3,5,3'-triiodo-L-thyronine (T3) and L-thyroxine (T4) by trout red blood cells (RBC) was studied by incubating washed RBC in a balanced salts medium containing glucose and [125I]TH at the fish acclimation temperature of 12 degrees. RBC were separated from the medium by centrifugation through silicone oil and glycine buffer (pH 10.5). Maximal [125I]T3 uptake occurred by 10-15 min, but not by 60 min for [125I]T4. First-order uptake was measured at 30 sec for T3 and at 90 sec for T4. Total T4 uptake was enhanced 15-fold from pH 8 to 6 and was affected most below pH 7.2; total T3 uptake was maximal between pH 6.4 and 7.0, but was relatively insensitive to pH. At 0.2 nM, nonsaturable uptake of T3 exceeded that of T4 3- to 6-fold, accounting for 3% (T3) and 50% (T4) of total uptake. Saturable TH uptake was described by Michaelis-Menten kinetics. The saturable transport system for T3 had an apparent K(t) (carrier affinity) of 70-119 nM and J(max) (maximal uptake velocity) of 540-1116 pmol . 10(6) cells(-1) . min(-1). A saturable system was also found for T4, with an apparent K(t) of 99 pM-1.1 nM and J(max) of 8-77 fmol . 10(6) cells(-1) . min(-1). Saturable uptake of both TH depended on temperature. Activation energies for the nonsaturable component were 48 (T4) and 64 (T3) KJ . mol(-1) over the range 0-21 degrees. Activation energies for the saturable components were 52 KJ . mol(-1) (T4, 0-21 degrees), 52 KJ . mol(-1) (T3, 0-10 degrees), and 3 KJ . mol(-1) (T3, 10-21 degrees). During a 16-month study saturable and nonsaturable uptake of both TH increased, probably due to fish age. We conclude that in trout RBC, rapid T3 uptake by a pH- and temperature-sensitive saturable carrier greatly exceeds T4 uptake. The rate of T3 uptake exceeds by 100- to 1000-fold that of mammals and amphibia, and in contrast to those taxa some saturable T4 uptake also occurs.

Thyroid hormone deiodination pathways in brain and liver of rainbow trout, Oncorhynchus mykiss.

Outer-ring (5') deiodination (ORD) and inner-ring (5) deiodination (IRD) of L-thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3) were studied in whole-brain microsomes of rainbow trout and compared with liver deiodination. Brain T4ORD activity (apparent Km = 1.2-2.5 nM; V(max) = 0.10-0.14 pmol/hr/mg microsomal protein) was less than T4IRD activity (apparent Km = 4.9; V(max) = 0.32) and T3IRD activity (apparent Km = 5.2-5.4; V(max) = 1.1-2.0); T3ORD activity was negligible. All three brain deiodinase pathways shared the following properties: pH optima between 7.0 and 7.3, activity enhanced by dithiothreitol (10 mM), weak inhibition by 6-n-propyl-2-thiouracil and iodoacetate, but stronger inhibition by aurothioglucose. Based on competitive inhibition, the substrate preference for brain T4ORD was T4 = tetraiodothyroacetic acid (TETRAC) > 3,3',5'-triiodo-L-thyronine (rT3) > 3,5,3'-triiodothyroacetic acid (TRIAC) >> T3 > 3,5-diiodo-L-thyronine (3,5-T2). A comparable substrate preference profile was obtained for liver T4ORD (Km 1 nM). Both T4IRD and T3IRD in brain had similar substrate preference profiles (rT3 > 3,5-T2 > T4 > T3) which differed from that of T4ORD. Negligible T4IRD and T3IRD activities existed in liver. We conclude that for rainbow trout (i) T4ORD systems in brain and liver are similar, and consistent with a common enzyme that does not match exactly either mammalian type I or II deiodinases, (ii) brain T4IRD and T3IRD enzymes share several common properties, and correspond functionally to the mammalian type III deiodinase, and (iii) under normal physiological conditions the predominant deiodinase pathways in brain (T4IRD and T3IRD) are poised toward T4 and T3 degradation, while that in liver (T4ORD) is poised toward T3 generation.

Kinetic analysis of thyroid hormone secretion and interconversion in the 5-day-fasted rainbow trout, Oncorhynchus mykiss.

Estimating the 3,5,3'-triiodothyronine (T3) thyroidal secretion rate and the rate of extrathyroidal conversion of thyroxine (T4) to T3 are two difficult and important quantitative endocrine system problems in vertebrates. To address these questions in fish, two thyroid hormone tracer studies were modeled and analyzed, based on data from two groups of rainbow trout maintained at 12 degrees and fasted for 5 days. The data consisted of three time series: plasma concentrations of radioactive 125I-labeled T3 (T3*) following T3* injection, and both labeled T4 (T4*) and T3* following T4* injection. To facilitate model parameter estimation, plasma volumes were determined independently by injection of labeled bovine serum albumin. The T4* injectate was contaminated by an unknown amount of T3*, and this was considered an additional unknown. A six-compartment model was formulated in terms of 13 uniquely identifiable (quantifiable) parameters, which were estimated simultaneously from the three data sets using a sophisticated optimization algorithm built into a new model-fitting software package called FITMOD. The rates of interest, plus other kinetic indices, were estimated successfully using additional analysis. We found that the thyroid gland secreted 0.835 +/- 0.707 (mean +/- SD) pmol/hr of T3 and 2.44 +/- 2.09 pmol/hr of T4 per 100 g body weight (BW). Also, 8.19 to 11.2% of secreted T4 was monodeiodinated to T3, forming 0.200 to 0.274 pmol/hr of T3 per 100 g BW. This means that 75 to 81% of all T3 produced was secreted by the thyroid in these starved fish--a rather surprising result--while the remaining 19 to 25% resulted from T4 to T3 conversion.

Identification of thyroid hormone conjugates produced by isolated hepatocytes and excreted in bile of rainbow trout, Oncorhynchus mykiss.

We studied thyroid hormone (TH) conjugation in fasted trout by incubating isolated hepatocytes with either [125I]T4 or [125I]T3, and by analyzing bile from trout injected with either [125I]T4 or [125I]T3. Glucuronide conjugates were identified by hydrolysis with beta-glucuronidase and sulfate conjugates by acid solvolysis with ethyl acetate/trifluoroacetic acid (1%). We used Sephadex LH-20 chromatography to concentrate the conjugate fractions from hepatocyte incubates prior to HPLC analysis. Glucuronide conjugates of T4 and T3 were produced in vitro and glucuronides of T4, T3, and 3,3'-T2 were found in vivo. Sulfation of T4 occurred in vitro and in vivo. T3 sulfation was not established in vitro, but sulfate conjugates of T3 and T2 were found in bile. Significant proportions of unconjugated T4 and T3 also occurred in the bile. We conclude that (i) as in other vertebrates, iodothyronines undergo hepatic glucuronidation and are excreted as such in the bile, (ii) T4 and T3 undergo sulfation, and in contrast to mammals, are excreted in significant amounts in the bile, (iii) 3,3'-T2, a prominent deiodination product of T3, is excreted as both glucuronide and sulfate conjugates, and (iv) the isolated hepatocyte system is appropriate for studying aspects of TH metabolism in trout.