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Endocrinology Vol. 148, No. 4 1921-1927
Copyright © 2007 by The Endocrine Society

Thyronamines Are Substrates for Human Liver Sulfotransferases

C. A. Pietsch, T. S. Scanlan and R. J. Anderson

Veterans Affairs Medical Center (C.A.P., R.J.A.), Omaha, Nebraska 68105; University of California, San Francisco (T.S.S.), San Francisco, California 94143; and Creighton University Medical Center (R.J.A.), Omaha, Nebraska 68131

Address all correspondence and requests for reprints to: Robert J. Anderson, M.D., Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, Nebraska 68105. E-mail: Robert.Anderson4{at}va.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sulfotransferases (SULTs) catalyze the sulfation of many endogenous compounds that include monoamine neurotransmitters, such as dopamine (DA), and thyroid hormones (iodothyronines). Decarboxylation of iodothyronines results in formation of thyronamines. In the mouse, thyronamines act rapidly in a nongenomic fashion to initiate hypothermia and decrease cardiac output and heart rate. These effects are attenuated after 1–4 h, and metabolism of thyronamines via sulfation may be a mechanism for termination of thyronamine action. We carried out this study to test thyronamine (T0AM), 3-iodothyronamine (T1AM), 3,5-diiodothyronamine (T2AM), and 3,5,3'-triiodothyronamine (T3AM) as substrates for human liver and cDNA-expressed SULT activities. We characterized several biochemical properties of SULTs using the thyronamines that acted as substrates for SULT activities in a human liver high-speed supernatant pool (n = 3). T1AM led to the highest SULT activity. Activities with T0AM and T3AM were 10-fold lower, and there was no detectable activity with T2AM. Thyronamines were then tested as substrates with eight cDNA-expressed SULTs (1A1, 1A2, 1A3, 1C2, 1E1, 2A1, 2B1a, and 2B1b). Expressed SULT1A3 had the greatest activity with T0AM, T1AM, and T3AM, whereas SULT1A1 showed similar activity only with T3AM. Expressed SULT1E1 had low activity with each substrate. T1AM, the most active thyronamine pharmacologically, was associated with the greatest SULT activity of the thyronamines tested in the liver pool and in both the expressed SULT1A3 and SULT1E1 preparations. Our results support the conclusion that sulfation contributes to the metabolism of thyronamines in human liver and that SULT activities may regulate the physiological effects of endogenous thyronamines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECENTLY, THERE HAS been considerable interest in alternative pathways of thyroid hormone metabolism (1). One metabolic avenue involves decarboxylation of T4 and its deiodinated derivatives, probably by an aromatic amino acid decarboxylase (2). This proposed path results in the production of a family of thyronamines (TxAMs, where x represents the number of iodine atoms in the molecule) (Fig. 1Go). Thyronamines have been noted for their structural similarity to biogenic amines, such as DA, serotonin, tyramine, and octopamine. Tyramine and octopamine, which are trace amines in vertebrates, act as neurotransmitters in invertebrates (3). There is emerging interest in the possible role of trace amines as neurotransmitters or neuromodulators in the vertebrate system (3, 4, 5). It has been postulated that thyroid hormones also might act as neurotransmitters (6, 7).


Figure 1
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  		FIG. 1. Structures of thyronamines that are substrates for human liver SULT activities.

 
Very few studies have been performed using thyronamines. The previous work involved only four of these compounds. Thyronamine (T0AM) and 3,5,3'-triiodothyronamine (T3AM; also called triam) have been the subjects of the most publications (8, 9, 10, 11, 12, 13, 14), whereas 3,5-diiodothyronamine (T2AM) and 3,5,3',5'-tetraiodothyronamine (T4AM, also called thyroxamine) were studied less frequently in the past (11, 14, 15). Most studies were completed before 1985. The recent discovery and classification of a special group of mammalian orphan G protein-coupled receptors (4, 5), now called the trace amine-associated receptors (TAARs) (16), have renewed interest in thyronamines. In humans and other mammals, TAARs are closely related to receptors for monoamine neurotransmitters, including serotonin and DA. Currently, nine TAAR genes have been identified in humans (17, 18). Recent initial studies involving 3-iodothyronamine (T1AM) indicated that this compound was the most potent agonist of human embryonic kidney cell-expressed mouse and rat TAAR1 of nine thyronamines tested (2). T0AM was approximately an order of magnitude less potent. T0AM and T1AM were found to induce hypothermia in mice and to cause a decrease in cardiac output and heart rate in a dose-dependent manner. Their actions were rapid, indicating that these compounds operated in a nongenomic fashion. The effects lasted up to 4 h (2).

One possible mechanism for termination of thyronamine action other than glucuronidation or monoamine oxidation is through sulfation by cytosolic sulfotransferases (SULTs) (Fig. 2Go). SULTs play key roles in the regulation of monoamine neurotransmitters and hormones (including thyroid hormone), in defense against potentially damaging phenolic chemicals as phase II metabolizing enzymes and in the regulation of hormone availability during early development (19, 20, 21). SULTs function by transferring the sulfuryl group from the donor molecule 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to hydroxylated substrates. Substrates for SULTs include many steroids, phenolic drugs, and xenobiotics. Sulfation inactivates most of these compounds, but it may activate substrates such as minoxidil (22). DA and other biogenic amines (including trace amines) also are readily metabolized by SULTs. A potential role for SULTs in thyronamine metabolism includes inactivation to regulate the potency and duration of their effects. Three thyronamine analogs lacking the hydroxyl group at the 4' position had similarly potent effects in inducing hypothermia in mice at a dosage of 50 mg/kg, but they exhibited obvious toxicity within a week in treated mice (23). T1AM and 3-methyl-T0AM, another hydroxylated compound, were not toxic at the same dosage. Because sulfation of the 4'-hydroxyl group of thyroid hormones is important in their metabolism, we hypothesized that sulfation via SULTs is also a key metabolic route for termination of the effects of thyronamines. We carried out these studies to determine whether thyronamines served as substrates for human liver SULTs and to characterize the biochemical properties of these SULT activities with several thyronamines as substrates.


Figure 2
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  		FIG. 2. SULT reaction with T1AM as the substrate. S*, Radiolabeled 35S; PAP, 3'-phosphoadenosine-5'-phosphate.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
[35S] PAPS (specific activity from 1.0–3.0 Ci/mmol; lot nos. 1105, 0206, and 0306) was purchased from PerkinElmer LAS (Shelton, CT). Dithiothreitol was purchased from CalBiochem (La Jolla, CA). BSA and NaCl were obtained from Sigma Chemical Company (St. Louis, MO). Protein assay reagent was obtained from Bio-Rad Laboratories (Richmond, CA), and Bio-Safe II scintillation fluor was purchased from Research Products International Corp. (Mount Prospect, IL). COS-1 cells were obtained from the American Type Culture Collection (Rockville, MD). The human liver SULT1A1, SULT1A2, SULT1A3, SULT1E1, SULT1C2, SULT2A1, SULT2B1a, and SULT2B1b cDNAs were gifts from Dr. R. M. Weinshilboum (24, 25, 26, 27, 28). The thyronamines were synthesized as previously described (23).

Frozen human liver samples obtained at surgery were acquired from the University of Nebraska Medical Center tissue bank. A pool of livers (n = 3) was prepared by our previous methods (29) to screen thyronamines for potential activities with human SULTs. Liver was chosen because it is known to contain high levels of multiple SULT activities. Human brain and heart tissue samples were obtained from the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. The brain pool consisted of three male and one female frontal cortex samples obtained at autopsy. Cardiac left atrial tissue was a surgical sample from one male. All samples were stored at –80 C until preparation. These studies were approved by the Veterans Affairs Nebraska-Western Iowa Health Care System Institutional Review Board and Research and Development committees.

Tissue samples were homogenized for 15–30 sec in 4 volumes of 2.5 mM dithiothreitol and 1.25 mM disodium EDTA in 5 mM potassium phosphate buffer (pH 7.5) with a Polytron Tissue Homogenizer (Kinematica, Lucerne, Switzerland). Homogenates were centrifuged at 12,900 x g for 10 min at 4 C. The supernatant was removed and centrifuged at 100,000 x g for 1 h at 4 C. Aliquots of the high-speed supernatant (HSS) were stored at –80 C until assay. Equal volumes of individual liver or brain HSS were combined to create a pool. COS-1 cells were transfected with SULT cDNA and processed by our previously published procedures (22, 30, 31, 32).

Thyronamines were dissolved in 60% (vol/vol) dimethylsulfoxide (DMSO) and double distilled water in the same percentage of DMSO that was used with procedures for the thyronamine preparation in studies with mice (2). One millimolar pargyline, a monoamine oxidase inhibitor, was used with all thyronamine assays. SULT activities were measured by the method of Foldes and Meek (33), as modified by Anderson and Liebentritt (34). Optimal assay conditions with each thyronamine as the substrate were established with regard to pH, incubation time, HSS protein, and substrate concentrations.

Assays of expressed SULTs 1A1, 1A3, 1E1, and 2A1 activities were performed with their respective model substrates [e.g. p-nitrophenol (p-NP), DA, estrone, and dehydroepiandrosterone] as previously published (29, 30). Each thyronamine was assayed at optimal substrate concentrations with COS-1-expressed SULTs 1A1, 1A2, 1A3, 1C2, 1E1, 2A1, 2B1a, and 2B1b using standard human liver conditions. Each expressed SULT had documented enzyme activity with standard substrates before testing with thyronamines.

Reactions were incubated at 37 C for 25 min, and blank samples containing the DMSO vehicle and no sulfate acceptor substrate were used as controls. Radioactivity was measured using a Packard model 1600 TR liquid scintillation counter. One unit of enzyme activity represented the formation of 1 nmol sulfated product/h incubation at 37 C. Assays were performed in triplicate, and values reported were the means ± SEM of three experiments unless otherwise specified. Protein concentrations were measured by the dye-binding method of Bradford (35) with BSA as the standard.

Effects of varying NaCl concentrations on liver SULT activities toward T1AM were examined by our published methods (29). Thermal stability of enzyme activities with T3AM was tested by the methods of Reiter et al. (36) as modified by Anderson et al. (34, 37). Aliquots of HSS were preincubated for 15 min at various temperatures before dilution and assay. Aliquots of the same preparation were kept on ice as a control. Each aliquot was then diluted to contain 3.14 µg liver protein/tube and assayed for SULT activity.

Apparent Km and Vmax values were determined by the direct linear plot method of Eisenthal and Cornish-Bowden (38) using the Enzpak 3 program by Williams (Elsevier-Biosoft, Cambridge, UK) and were presented as the mean ± SEM of three assays. The 50% inactivation temperature (T50) and IC50 with NaCl were determined using a curve-fitting program (Prism3, GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human liver SULT assay conditions with thyronamines
The human liver pool was assayed with T0AM, T1AM, T2AM, and T3AM. Four final thyronamine concentrations, from 10–100 µM, led to negligible activity with T2AM and moderate activity with T0AM and T3AM. Elevated SULT activity that was more than 10-fold higher than the activity with T0AM was detected with T1AM (Fig. 3Go). Human liver SULT activities with thyronamines increased in a linear fashion with HSS protein (1.57–15.7 µg protein/assay tube). All assays were within this linear range. A 50 mM potassium phosphate buffer (pH 8.0) was used in all liver pool assays with T0AM and T1AM to provide an optimum tissue pH of 7.6 (Table 1Go). With T3AM, 100 mM glycine-sodium hydroxide buffer (pH 9.0) was used to give an optimum tissue pH of 8.1. The HSS liver pool SULT activities with thyronamines represented a composite effect of several known hepatic SULT activities. The possibility of endogenous thyroid hormone interference with SULT activity was considered. When human liver HSS preparation was tested with T1AM as the substrate and assayed in the presence of either 10 nM T3 or T4, there was no detectible inhibition of the SULT activity measured with T1AM. The same experiment with T3AM as the substrate showed no inhibition of the SULT activity in the presence of T3 or T4 (data not shown).


Figure 3
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  		FIG. 3. Human liver pool SULT activities measured with varying concentrations of T0AM, T1AM, T2AM, and T3AM as the substrates. One unit of activity represents 1 nmol sulfated product formed per hour of incubation.

 

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  		TABLE 1. Biochemical properties of human liver SULTs

 
Effects of varying cosubstrate concentrations
The effects of a range of concentrations of each thyronamine on liver SULT activities were determined (Fig. 4Go). T0AM was assayed at concentrations from 0.5–300 µM with activity first observed at 10 µM. With T1AM, final assay concentrations were evaluated from 5 nM to 100 µM. Activity was first noted at 0.5 µM. With T3AM, a range of 1 nM to 300 µM was assayed, and first activity occurred at approximately 1 µM. Substrate inhibition of the liver SULT activities occurred at T3AM concentrations greater than 30 µM. T2AM was not evaluated further because of the minimal activity found with the liver pool.


Figure 4
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  		FIG. 4. Human liver pool SULT activities with varying concentrations of T0AM (A), T1AM (C), and T3AM (E). Double reciprocal plots derived from the corresponding substrate data are shown in B, D, and F, respectively. Each point represents the mean ± SEM of three separate experiments with three replicates per experiment. Liver protein concentrations per assay tube for each substrate were as follows: T0AM, 15.7 µg; T1AM, 1.57 µg; and T3AM, 3.14 µg. One unit of activity represents 1 nmol sulfated product formed/h incubation.

 
Apparent Km values for human liver SULTs with each thyronamine were calculated based on the means of three experiments (Table 1Go). Of the three thyronamines tested, the lowest apparent Km value of 11.1 µM was found with T3AM (Table 1Go). The optimal concentrations used in subsequent assays of liver and COS-1-expressed SULTs were 200 µM T0AM, 50 µM T1AM, and 25 µM T3AM. The effects of varying [35S]-PAPS concentrations up to 4 µM on SULT activities were determined at the optimal concentration of each thyronamine. The apparent Km value with PAPS in the liver pool was 1.79 µM with T1AM and 1.37 µM with T3AM. A value for T0AM could not be calculated.

Expressed SULT activities with thyronamines
Each substrate was screened with eight COS-1-expressed liver SULTs to elucidate which enzymes contributed to the composite liver pool SULT activities (Fig. 5Go). SULT1A3 showed the most activity with each thyronamine. The prototypical substrate for SULT1A3 is DA, a monoamine neurotransmitter. Individual thyronamine profiles varied for the remaining SULTs. With T1AM, SULTs 1A2 and 1E1 showed high activities, and SULTs 1A1, 1C2, 2A1, and 2B1b showed limited activities. These SULTs had much lower activity with T0AM. With T3AM, SULTs 1A1, 1A2, and 1A3 had relatively similar levels of activity, and SULT1E1 had lower but readily detectable activity (Fig. 5Go).


Figure 5
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  		FIG. 5. COS-1-expressed human liver SULT activities with 200 µM T0AM, 50 µM T1AM, and 25 µM T3AM as substrates. Each bar represents the mean ± SEM of two to three experiments with three replicates per experiment. One unit of activity represents 1 nmol sulfated product formed/h incubation. Each expressed SULT had confirmed enzyme activity with its corresponding prototypical substrate.

 
Based on these observations, the liver pool SULT activities were assayed with T1AM using varying concentrations of NaCl to determine whether SULT1A3 activity or SULT1E1 activity was primarily responsible for liver T1AM sulfation. NaCl is helpful for analysis of HSS tissue SULT preparations because it enhances SULT1E1 and SULT2A1 activities (25, 30), whereas it inhibits SULT1A1 activity more readily than SULT1A3 activity (29). The results showed definite NaCl inhibition of T1AM sulfation in the liver at an IC50 similar to the value reported for SULT1A3 (29) (Fig. 6Go). The IC50 value with NaCl of 146 mM for the liver pool SULT activity corresponded to the value of 178.7 mM with SULT1A3 using DA as the substrate in the thyroid published previously by our group (29). In comparison, the IC50 value for SULT1A1 activity using p-NP as the substrate in the thyroid was 62.5 mM (29). This supported the conclusion that sulfation of T1AM was primarily due to SULT1A3 activity in the liver pool. Although there was demonstrable activity for expressed SULT1A2 with T1AM and T3AM, actual expression of SULT1A2 in human tissue is minimal to undetectable (29, 39).


Figure 6
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  		FIG. 6. NaCl inhibition of liver pool SULT activity with 50 µM T1AM as the substrate. Results are expressed as the percentage of initial activity remaining at the given concentration of NaCl. Each point represents the mean ± SEM of three experiments with three replicates per experiment. The pattern is consistent with SULT1A3 activity as the major liver pool activity with T1AM.

 
Because T3AM was associated with high activity of both the thermostable SULT1A1 and thermolabile SULT1A3, the thermal stabilities of the liver pool SULT activities were tested to determine which of these SULTs contributed more to T3AM sulfation. The patterns of the liver SULT thermal stability when assayed with T3AM would indicate which isozyme was more involved (36). The thermal stability of human liver SULT activities was determined after preincubation of tissue aliquots for 15 min at temperatures from 35–47 C (Fig. 7Go). DA (SULT1A3) and p-NP (SULT1A1) activities were tested for comparison. The calculated T50 value of 42.7 ± 0.4 C (mean ± SEM) demonstrated that T3AM sulfation was catalyzed more by thermostable SULT1A1 than by more thermolabile SULT1A3 or SULT1E1 activities (25). The T50 found previously by our group for SULT1A1 with p-NP was 44.3 C in human platelets and 43.8 ± 0.6 C in thyroid tissue (29, 34), whereas the T50 for SULT1A3 with DA was 38.9 ± 0.3 C in thyroid tissue (29).


Figure 7
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  		FIG. 7. Thermal stability of human liver pool SULT activities with 25 µM T3AM as the substrate. Results are expressed as the percentage of initial activity remaining after preincubation of liver pool enzymes at the given temperatures. Each point for T3AM represents the mean ± SEM of three experiments with three replicates per experiment. Liver pool SULT activities with p-NP for SULT1A1 activity and with DA for SULT1A3 activity are shown as representative experiments for comparison. The pattern is consistent with SULT1A1 as the major liver pool activity with T3AM.

 
Human brain and left atrial tissue SULT activities with T1AM as the substrate
Human brain and cardiac samples were tested with T1AM as the substrate to evaluate these tissues that were known targets of thyronamine action in the mouse and rat (2). T1AM (50 µM, the optimal concentration with liver) was screened with a human frontal cortex pool (6.57 µg protein/assay tube) and a left atrial sample (0.85 µg protein/assay tube). The standard assay conditions for the liver pool were used due to limited tissue availability. A test of T1AM concentrations from 1–100 µM showed brain pool SULT activity at 10 µM and higher concentrations. T3AM was also assayed from 0.5–25 µM but failed to show evaluable activity. The brain pool had previously been shown by RT-PCR to strongly express SULTs 1A1, 1A3, 1C2, and 4A1 with lesser expression of SULTs 1E1 and 2B1a (39). Frontal cortex SULT activity with T1AM as the substrate was 0.170 ± 0.018 U/mg protein. This value was comparable with SULT1A3 activity measured previously in this frontal cortex pool (39). The left atrial sample was also tested for sulfation of T1AM. Using 50 µM T1AM and the conditions developed with the liver, an activity of 7.59 ± 1.75 U/mg protein was calculated. Both the frontal cortex and left atrial samples showed substantial SULT1A1 and SULT1A3 activities with p-NP and DA as substrates, respectively. The findings supported the conclusion that both human brain and cardiac tissue are capable of T1AM sulfation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that SULTs, known as phase II drug-metabolizing enzymes, use several proposed endogenous thyronamines as substrates. The potential importance of this SULT action is the attenuation, and, thus, regulation of the thyronamine-induced physiological effects of hypothermia and decreased cardiac output shown in mice (2). The effects in humans are not known. Our results with human liver enzyme preparations represented a composite of SULT activities toward thyronamines as a result of multiple SULTs documented in liver tissue (40, 41). T1AM led to the highest levels of activities with the liver SULT preparations. The relative order of liver pool SULT activities detected with thyronamines were, from highest to lowest: T1AM > T3AM >> T0AM (Table 1Go). Based on Vmax/Km values, the specificities of the substrates with liver pool SULTs were T3AM = T1AM >> T0AM. No determinations were possible with T2AM.

Screening of T0AM, T1AM, and T3AM with eight available cDNA-expressed SULT activities showed that SULT1A3 (DA SULT) had the greatest activity with each thyronamine (Fig. 5Go). Because the major activities with T1AM were noted with expressed SULT1A3 and SULT1E1, a targeted experiment testing the liver pool preparation responses to sodium chloride was done with T1AM as the substrate. The result supported SULT1A3 as the primary liver SULT involved with T1AM because the tissue SULT activity was inhibited rather than activated in the presence of NaCl, a typical response for SULT1A3 (29). Because both SULT1A1 and SULT1A3 yielded high activity with T3AM as the substrate, a separate targeted thermal stability experiment with the liver pool SULT preparation was done with T3AM as the substrate to help differentiate the effects of SULT1A1, a thermostable SULT, from SULT1A3, a thermolabile SULT (29, 31, 36). More thermostable SULT activity supported SULT1A1 as the main SULT affecting T3AM in the human liver preparations. Although expressed SULT1A3 showed higher enzyme activity with two of the three thyronamines tested, endogenous liver SULT1A1 appeared to be the major enzyme involved in the sulfation of T3AM. This corresponds with previous findings of hepatic SULT1A1 as the major SULT responsible for sulfation of T3 (42, 43). This may also be due, in part, to the higher expression of SULT1A1 than SULT1A3 in the liver. Thyroid hormones are sulfated by a number of SULTs that include 1A1, 1A3, 1B1, 1E1, and 2A1 (1, 29, 30, 32). We also would expect the involvement of multiple SULTs in the sulfation of thyronamines as we have seen with the expressed SULT activities. The relative contribution to the regulation of each thyronamine in human tissues remains an important area for investigation.

The livers used in the pool for this study have been tested for endogenous T0AM and T1AM using liquid chromatography coupled to tandem mass spectrometry (2). Trace amounts of both compounds were found in all samples, with larger amounts of T0AM than T1AM (data not shown). T1AM has also been found in extracts of rat brain, guinea pig brain, and mouse brain, heart, liver, and blood using the same techniques (2).

We tested the brain for sulfation of T1AM because of the potent thermoregulatory effect this compound produced in mice (2). The presence of TAAR receptors in many areas of the brain may indicate an important function for trace amines such as thyronamines in neurotransmission or neuromodulation (4, 5). Recent reviews have speculated about the role that trace amines may play in psychiatric disorders (depression, schizophrenia), neurological disorders (attention deficit hyperactivity disorder, Parkinson’s disease), drug addiction, and headaches (44, 45). Trace amines such as ß-phenylethylamine and tyramine have shown an inhibitory effect on dopaminergic neurons (46). Action of trace amines on these neurons are through interference with GABAB receptor-mediated processes (47, 48). Thyronamines have not been tested for these effects. SULT1A3 has been shown previously to sulfate monoamine neurotransmitters and trace amines and, now, thyronamines. Our results show SULT enzymatic activity toward T1AM in the frontal lobe. Activity in other areas of the brain have yet to be elucidated, particularly those where TAAR1 receptors have been detected using RT-PCR (4).

In the heart, T1AM was the most potent inducer of cardiovascular effects of thyronamines tested (2). The rapid action of thyronamines on the heart are opposite of the genomic effects observed with thyroid hormones. In hyperthyroid patients, T3 increases cardiac output and heart rate (49). T1AM directly decreased cardiac output and heart rate in ex vivo rat heart preparations (2). We speculate that metabolism of thyroid hormone to thyronamines may serve as a protective mechanism in a nongenomic fashion in hyperthyroid patients. Our demonstration of sulfation of T1AM in the left atrium may account in part for the attenuation of thyronamine effects that were seen in the cardiovascular system in rodents (2). Finally, thyronamines could be used as potential agents to safely induce hypothermia for medical purposes.

This report is the first systematic investigation of human tissue sulfation of thyronamines. It has established the radiochemical enzymatic assay for human liver SULT activities and has identified SULT1A3 as the major contributor to sulfation of T0AM and T1AM, whereas SULT1A1 is the major contributor to sulfation of T3AM. Ether bond cleavage and glucuronidation are other potential metabolic pathways, but these were not the focus of this investigation. Our assay method is not affected by these enzyme reactions. Studies of thyronamine metabolism through routes such as sulfation will increase our understanding of these thyroid hormone derivatives and the termination of their effects.


   Acknowledgments
 
We thank Dr. Richard M. Weinshilboum (Mayo Clinic, Rochester, MN) for providing the SULT cDNAs used in this study.


   Footnotes
 
This work was supported by the VA Research service (to R.J.A.) and by the National Institutes of Health (Grant DK-52798 to T.S.S.).

The authors have nothing to disclose.

First Published Online January 4, 2007

Abbreviations: DA, Dopamine; DMSO, dimethylsulfoxide; HSS, high-speed supernatant; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; p-NP, p-nitrophenol; SULT, sulfotransferase; T50, 50% inactivation temperature; TAAR, trace amine-associated receptor; T0AM, thyronamine; T1AM, 3-iodothyronamine; T2AM, 3,5-diiodothyronamine; T3AM, 3,5,3'-triiodothyronamine.

Received August 28, 2006.

Accepted for publication December 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wu S-Y, Green WL, Huang W-S, Hays MT, Chopra IJ 2005 Alternate pathways of thyroid hormone metabolism. Thyroid 15:943–958[CrossRef][Medline]
  2. Scanlan TS, Suchland KL, Hart ME, Chiellini G, Huang Y, Kruzich PJ, Frascarelli S, Crossley DA, Bunzow JR, Ronca-Testoni S, Lin ET, Hatton D, Zucchi R, Grandy DK 2004 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nat Med 10:638–642[CrossRef][Medline]
  3. Berry MD 2004 Mammalian central nervous system trace amines. Pharmacologic amphetamines, physiologic neuromodulators. J Neurochem 90:257–271[CrossRef][Medline]
  4. Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, Durkin MM, Lakhlani PP, Bonini JA, Pathirana S, Boyle N, Pu X, Kouranova E, Lichtblau H, Ochoa FY, Branchek TA, Gerald C 2001 Trace amines: Identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci USA 98:8966–8971[Abstract/Free Full Text]
  5. Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, Darland T, Suchland KL, Pasumamula S, Kennedy JL, Olson SB, Magenis RE, Amara SG, Grandy DK 2001 Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol 60:1181–1188[Abstract/Free Full Text]
  6. Dratman MB, Gordon JT 1996 Thyroid hormones as neurotransmitters. Thyroid 6:639–647[Medline]
  7. Mason GA, Walker CH, Prange AJ 1993 L-Triiodothyronine: is this peripheral hormone a central neurotransmitter? Neuropsychopharmacology 8:253–258[Medline]
  8. Buu-Hoi NP, Pham-Huu-Chanh, Petit L 1969 Thyronamine, a new substance with long-acting positive inotropic effect. Pharmacology 2:281–287[Medline]
  9. Côté P, Polumbo RA, Harrison DC 1974 Thyronamine, a new inotropic agent: its cardiovascular effects and mechanism of action. Cardiovas Res 8:721–730[Medline]
  10. Buu-Hoi NP, Pham-Huu-Chanh, Petit L 1966 Some biological effects of thyronamine. Med Pharmacol Exp 15:17–23
  11. Cody V, Meyer T, Dohler KD, Hesch RD, Rokos H, Marko M 1984 Molecular structure and biochemical activity of 3,5,3'-triiodothyronamine. Endocr Res 10:91–99[Medline]
  12. Meyer T, Hesch R-D 1983 Triiodothyronamine: a ß-adrenergic metabolite of triiodothyronine? Horm Metab Res 15:602–606[Medline]
  13. Boissier JR, Giudicelli JF, Larno S, Advenier C 1973 Differential inotropic-chronotropic action of thyronamine. Eur J Pharmacol 22:141–149[Medline]
  14. Tomita K, Lardy HA 1956 Synthesis and biological activity of some triiodinated analogues of thyroxine. J Biol Chem 219:595–604[Free Full Text]
  15. Felt V, Ploc I 1982 Effect of theophylline on binding of triiodothyronine, thyroxine, thyroxamine, tetraiodothyroacetic acid and cortisol in the cytosol of human leukocytes. Endokrinologie 79:315–317[Medline]
  16. Lindemann L, Ebeling M, Kratochwil NA, Bunzow JR, Grandy DK, Hoener MC 2005 Trace amine-associated receptors form structurally and functionally distinct subfamilies of novel G protein-coupled receptors. Genomics 85:372–385[CrossRef][Medline]
  17. Gloriam DEI, Bjarnadóttir TK, Schiöth HB, Fredriksson R 2005 High species variation within the repertoire of trace amine receptors. Ann NY Acad Sci 1040:323–327[CrossRef][Medline]
  18. Lewin AH 2006 Receptors of mammalian trace amines. AAPS J 8:E138–E145
  19. Glatt HR, Meinl W 2004 Pharmacogenetics of soluble sulfotransferases (SULTs). Naunyn Schmiedebergs Arch Pharmacol 369:55–68[CrossRef][Medline]
  20. Coughtrie MWH 2002 Sulfation through the looking glass: recent advances in sulfotransferase research for the curious. Pharmacogenomics J 2:297–308[CrossRef][Medline]
  21. Blanchard RL, Freimuth RR, Buck J, Weinshilboum RM, Coughtrie MWH 2004 A proposed nomenclature system for the cytosolic sulfotransferase (SULT) superfamily. Pharmacogenetics 14:199–211[CrossRef][Medline]
  22. Kudlacek PE, Anderson RJ, Liebentritt DK, Johnson GA, Huerter CJ 1995 Human skin and platelet minoxidil sulfotransferase activities: biochemical properties, correlations and contribution of thermolabile phenol sulfotransferase. J Pharmacol Exp Ther 273:582–590[Abstract/Free Full Text]
  23. Hart ME, Suchland KL, Miyakawa M, Bunzow JR, Grandy DK, Scanlan TS 2006 Trace amine-associated receptor agonists: synthesis and evaluation of thyronamines and related analogues. J Med Chem 49:1101–1112[CrossRef][Medline]
  24. Otterness DM, Weiben ED, Wood TC, Watson RWG, Madden BJ, McCormick DJ, Weinshilboum RM 1992 Human liver dehydroepiandrosterone sulfotransferase: molecular cloning and expression of cDNA. Mol Pharmacol 41:865–872[Abstract]
  25. Aksoy IA, Wood TC, Weinshilboum RM 1994 Human liver estrogen sulfotransferase: identification by cDNA cloning and expression. Biochem Biophys Res Commun 200:1621–1629[CrossRef][Medline]
  26. Her C, Kaur GP, Athwal RS, Weinshilboum RM 1997 Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization. Genomics 41:467–470[CrossRef][Medline]
  27. Raftogianis RB, Wood TC, Weinshilboum RM 1999 Human phenol sulfotransferases SULT1A2 and SULT1A1. Genetic polymorphisms, allozyme properties, and human liver genotype-phenotype correlations. Biochem Pharmacol 58:605–616[CrossRef][Medline]
  28. Her C, Wood TC, Eichler EE, Mohrenweiser HW, Ramagli LS, Siciliano MJ, Weinshilboum RM 1998 Human hydroxysteroid sulfotransferase SULT2B1: Two enzymes encoded by a single chromosome 19 gene. Genomics 53:284–295[CrossRef][Medline]
  29. Ebmeier CC, Anderson RJ 2004 Human thyroid phenol sulfotransferase enzymes 1A1 and 1A3: activities in normal and diseased thyroid glands, and inhibition by thyroid hormones and phytoestrogens. J Clin Endocrinol Metab 89:5597–5605[Abstract/Free Full Text]
  30. Li X, Anderson RJ 1999 Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases. Biochem Biophys Res Commun 263:632–639[CrossRef][Medline]
  31. Dubin RL, Hall CM, Pileri CL, Kudlacek PE, Li X-Y, Yee JA, Johnson ML, Anderson RJ 2001 Thermostable (SULT1A1) and thermolabile (SULT1A3) phenol sulfotransferases in human osteosarcoma and osteoblast cells. Bone 28:617–624[Medline]
  32. Li X-Y, Clemens DL, Cole JR, Anderson RJ 2001 Characterization of human liver thermostable phenol sulfotransferase (SULT1A1) allozymes with 3,3',5-triiodothyronine as the substrate. J Endocrinol 171:525–532[Abstract]
  33. Foldes A, Meek JL 1973 Rat brain phenolsulfotransferase: partial purification and some properties. Biochim Biophys Acta 327:365–374[Medline]
  34. Anderson RJ, Liebentritt D 1990 Human platelet thermostable phenol sulfotransferase: assay of frozen samples and correlation between frozen and fresh activities. Clin Chim Acta 189:221–230[CrossRef][Medline]
  35. Bradford MM 1976 A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  36. Reiter C, Mwaluko G, Dunnette J, Van Loon JA, Weinshilboum RM 1983 Thermolabile and thermostable human platelet phenol sulfotransferase. Naunyn Schmiedebergs Arch Pharmacol 324:140–147[CrossRef][Medline]
  37. Anderson RJ, Jackson BL, Liebentritt D 1988 Human platelet thermostable phenol sulfotransferase from blacks and whites: biochemical properties and variations in thermal stability. J Lab Clin Med 112:773–783[Medline]
  38. Eisenthal R, Cornish-Bowden A 1974 The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J 139:715–720[Medline]
  39. Ebmeier CC, Pietsch CA, Anderson RJ 2006 Sulfotransferase (SULT) mRNA expression in the human anterior pituitary and brain. Program of the 88th Annual Meeting of The Endocrine Society, Boston, MA, 2006, p 400 (Abstract P2-15)
  40. Chen G, Zhang D, Jing N, Yin S, Falany CN, Radominska-Pandya A 2003 Human gastrointestinal sulfotransferases: identification and distribution. Toxicol Appl Pharmacol 187:186–197[CrossRef][Medline]
  41. Aksoy IA, Sochorová V, Weinshilboum RM 1993 Human liver dehydroepiandrosterone sulfotransferase: nature and extent of individual variation. Clin Pharmacol Ther 54:498–506[Medline]
  42. Young WF, Gorman CA, Weinshilboum RM 1988 Triiodothyronine: a substrate for the thermostable and thermolabile forms of human phenol sulfotransferase. Endocrinology 122:1816–1824[Abstract]
  43. Anderson RJ, Babbitt LL, Liebentritt DK 1995 Human liver triiodothyronine sulfotransferase: copurification with phenol sulfotransferases. Thyroid 5:61–66[Medline]
  44. Branchek TA, Blackburn TP 2003 Trace amine receptors as targets for novel therapeutics: legend, myth and fact. Curr Opin Pharmacol 9:90–97
  45. D’Andrea G, Terrazzino S, Fortin D, Cocco P, Balbi T, Leon A 2003 Elusive amines and primary headaches: historical background and prospectives. Neurol Sci 24:S65–S67
  46. Geracitano R, Federici M, Prisco S, Bernardi G, Mercuri NB 2004 Inhibitory effects of trace amines on rat midbrain dopaminergic neurons. Neuropharmacology 46:807–814[CrossRef][Medline]
  47. Federici M, Geracitano R, Tozzi A, Longone P, Di Angelantonio S, Bengtson CP, Bernardi G, Mercuri NB 2005 Trace amines depress GABAB response in dopaminergic neurons by inhibiting G-ß{gamma}-gated inwardly rectifying potassium channels. Mol Pharmacol 67:1283–1290[Abstract/Free Full Text]
  48. Berretta N, Giustizieri M, Bernardi G, Mercuri NB 2005 Trace amines reduce GABAB receptor-mediated presynaptic inhibition at GABAergic synapses of the rat substantia nigra pars compacta. Brain Res 1062:175–178[CrossRef][Medline]
  49. Klein I, Ojamaa K 2001 Mechanisms of Disease: Thyroid hormone and the cardiovascular system. N Engl J Med 344:501–509[Free Full Text]



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