Evidence for existence of thyroid hormone inducible semicarbazide-sensitive amine oxidase (SSAO) in rat heart cytosol

Abstract

Background

Semicarbazide-sensitive amine oxidase (SSAO; EC; 1.4.3.6.) has widespread tissue distribution, and the physiological role of SSAO is quite well known through its involvement in several pathological states.

Aims

The present study examined modulators of SSAO which might be present in the rat heart cytosol and looked for changes in SSAO modulatory activity.

Methods

An endogenous inhibitor of SSAO was separated by gel filtration from 105,000 g supernate of T4-treated rat heart cytosol. SSAO inhibition fraction was referred to as “endogenous SSAO inhibitor”.

Results

The inhibition by this inhibitor was concentration-dependent. Inhibition of SSAO was not enhanced by varying the time of preincubation of the enzyme, indicating reversible inhibition of SSAO. The molecular weight of this inhibitor was estimated to be 1000–1100 by gel filtration. The isoelectric point (pI) value was determined to be 4.8 isoelectric focusing. This inhibitor was found to be heat-stable and resistant to protease treatment. SSAO inhibition activity was much lower in the cytosol of thyroidectomized, non-T4-treated rats than T4-treated rats, suggesting that this inhibitor was induced by thyroid hormone T4. SSAO activity in rat heart might be regulated by the level of this inhibitor.

Conclusion

These results suggest the presence of SSAO inhibitor in T4-treated rat cytosol and that the level of this inhibitor is regulated by thyroid hormone.

1. Introduction

Semicarbazide-sensitive amine oxidase (SSAO)1, 2 differs from monoamine oxidase (MAO; EC 1.4.3.4) and deaminates various monoamines.³ Although they are all classified as the amine oxidase (copper-containing) (EC 1.4.3.6), they comprise a large group of enzymes with different substrate specificities and tissue distributions. SSAO is the name used for the benzylamine oxidizing activity that remains after pretreatment with acetylenic MAO inhibitor, clorgyline or deprenyl.⁴ SSAO activity is suspected to cause damage, such as diabetes in humans.⁵ SSAO has two primary functions, (a) production of metabolites with cytotoxic effects or reactive oxygen species and (b) as an adhesion molecule, in leukocyte trafficking, in regulating glucose uptake, and in adipocyte homeostasis.⁶ The role of SSAO is quite well known through its involvement in several pathological states, where its increased serum activity has been found: diabetes mellitus, congestive heart failure, multiple types of cerebral infarction, uremia, and liver cirrhosis. It plays a detrimental role in vascular diseases, particularly atherosclerosis, and its role in the pathophysiology of these conditions has been extensively investigated, from which its role may be deduced.

One of the important actions of thyroid hormone is thought to be the regulation of protein synthesis and enzyme activities.⁷ On the contrary, MAO activity in various organs of was shown to be diminished by thyroid hormone⁸ or by a pituitary factor,⁹ which is known to increase the secretion of thyroid hormones. In the latter case, it is not clear, whether thyroid hormones affect the protein synthesis of MAO or act by another mechanism (e.g. induction of specific modulator). There are many reports on the possible existence of the multiple modulators of MAO being present in the cytosol fractions of various organs of animals.10, 11, 12 These studies imply that endogenous MAO modulators might be important in the physiological regulation of MAO activity. None of these investigators, however, provide any information about existence of endogenous SSAO inhibitor (ESI).

In the present study, we found an ESI in rat heart cytosol, and this inhibitor could be induced by thyroid hormone T4.

2. Methods

2.1. Chemicals

Semicarbazide, clorgyline hydrochloride, deprenyl hydrochloride, benzylamine hydrochloride, T4 sodium salt, subtilisin (protease Type III), and protease E (protease Type XIV) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). The radioactive substrate [7-¹⁴C]-benzylamine hydrochloride (1.85–2.29 Gbq/mmol) was obtained from Amersham International (Amersham, England). Servalyte (pH 2–11) was purchased from Serva Fine Biochemical Co. (St. Louis, MO, USA).

2.2. Isolation of SSAO inhibitor

Thyroidectomized rats (Male Wistar), weighing 100–150 g, were used for experiments. In the case of T4 administered rats, T4 (dissolved in saline) was injected subcutaneously to the rats after 10 days from operation at a dose of 200 μg/kg per day for 2 weeks. The rats were killed by decapitation and heart quickly removed and homogenized in 10 vol. of 10 mM phosphate buffer, pH 7.4 containing 0.25 M sucrose. The homogenates was centrifuged at 105,000 g for 60 min, and the supernate (cytosol fraction) was applied on a Sephadex G-25 column (1.0 × 60 cm), previously equilibrated with 20 mM phosphate buffer (pH 7.4). The column was eluted with the same buffer at a rate of 10 ml/hr and the fractions were collected in 2.5 ml each. An aliquot of each fraction was assayed for SSAO inhibition activity, and active fractions were combined and used for further characterization. This fraction is referred to as “ESI”.

2.3. Assay of SSAO activity

Rat heart homogenate fraction was used as a source of SSAO activity. The 10% homogenate of this was prepared in 0.25 M sucrose with 10 mM phosphate buffer, pH 7.4. SSAO activity was assayed radiochemically.¹³ Assay mixture contained 20 μl of homogenate fraction (100 μg/ml protein), [¹⁴C]-benzylamine (1 μM), in 20 mM phosphate buffer, pH 7.4 (20 μl) in the presence of ESI (0–140 μl). After a 10-min preincubation at 37 °C, the mixture was diluted with the solution of respective unlabeled amines, and the reaction was stopped by adding 2 N HCl (200 μl). The products of the reaction were extracted with 2 ml of benzene-ethyl acetate (1:1, v/v) saturated with water. Triton X-100 toluene scintillation liquid (10 ml) was added to 1.0 ml samples of the extract, and the radioactivity was measured in Beckmann LS-9000 scintillation spectrometer. In inhibition studies, enzyme preparation was preincubated with various concentration of semicarbazude, clorgyline and deprenyl for 30 min at 37 °C before adding [¹⁴C]-benzylamine for assay of remaining SSAO activity. The reaction products were extracted with ethyl acetate-benzene (1:l, v/v) and the radioactivity was counted. Protein concentrations of the preparations were measured by the method of Lowry et al. with bovine serum albumin as the standard.¹⁴

2.4. Isoelectric focusing (IEF)

Gel IEF was performed by the method of Fawcett.¹⁵ The final composition of the gel was 5% acrylamide, 0.2% methylene bisacrylamide, 0.75% Triton X-100, 2% servalyte (pH 2–11), 0.0002% ribofiavin, 0.01% ammonium persulfate, 0.05% TEMED (N,N,N′,N′-tetramethylenediamine). The gel was mounted on a vertical apparatus containing 0.01 M H₃PO₄ in the upper tank (anode) and 0.02 M NaOH in the lower tank (cathode). The current was at 100 V for the first 1 h, 200 V for the next 2 h and then 300 V for 2 h. After electrophoresis, the gel was cut into 4 mm thick slices, and each sliced gel was placed in a test tube and incubated for 1 h at room temperature by adding 1 ml of distilled water and bubbled with N₂ gas. After the measurement of slice pH, a minimum amount of 0.5 M H₃PO₄ was added to adjust the pH to 7.4. An aliquot of each slice suspension was then assayed for SSAO inhibition activity with benzylamine as a substrate.

2.5. Statistical analysis

All values are presented as means ± S.E.M. The significance of difference was determined by using ANOVA with Fisher's post hoc test. A p value of less than 0.05 was regarded as being statistically significant.

3. Results

3.1. Inhibition of SSAO activity in rat

As shown in Fig. 1, in vitro inhibition of SSAO activity in rat heart homogenate by clorgyline, deprenyl, and semicarbazide was determined with 1 μM benzylamine as substrate. When the enzyme was preincubated at 37 °C for 30 min with various concentrations of clorgyline and deprenyl, the deamination of 1 μM benzylamine was not inhibited at high concentration of these inhibitors, while it was highly sensitive for semicarbazide. The complete inhibition at 10⁻⁴ M semicarbazyde was obtained.

Fig. 1.

Inhibition of benzylamine deamination in rat heart by clorgyline, deprenyl and semicarbazide. The enzyme preparation was preincubated with clorgyline (open circle), deprenyl (triangle), and semicarbazide (square) at 37 °C for 30 min. Enzyme activity was assayed radiochemically by addition of 1 μM benzylamine as substrate at 37 °C for 10 min. Each point represents the mean percentages (±S.E.) on the control SSAO activity in triplicate experiments.

3.2. Separation of ESI

The gel-filtration of T4-treated rat heart cytosol with Sephacryl S-200 column showed that the fractions to inhibit SSAO activity were eluted in a low molecular weight area (<2000) (data not shown). Therefore, we used Sephadex G-25 column to separate the inhibitor and determine the molecular size (Fig. 2A). The fractions 15–17 were found to inhibit SSAO activity with 1 μM benzylamine being used as a substrate. The molecular weight of the inhibitor was estimated to be 1000–1100 (Fig. 2B). Some endogenous SSAO activity was observed in fractions 6–9. When thyroidectomized, non-T4-treated rats were used (ESI-control), the inhibition activities for 1 μM benzylamine were very low. T4-treatment remarkably enhanced SSAO inhibition activities for substrate. These results suggest that the inhibitor could be induced by T4 treatment. The effect of concentration of the inhibitor on SSAO activity was examined using 1 μM benzylamine as a substrate. The inhibition curves of SSAO activity by ESI were concentration dependent, and IC₅₀ values for 1 μM benzylamine oxidation were 105 μl (Fig. 3).

Fig. 2.

Sephadex G-25 chromatography of cytosolic fraction from T4-treated rat heart. Cytosolic fraction (10 mg protein) was applied to a Sephadex G-25 column (1.0 × 60 cm). (A) ESI-control (closed circle) or ESI-T4 (open circle) was obtained from thyroidectomized rat or thyroidectomized, T4-treated rat, respectively, as described in the text. The activity of SSAO in homogenate was assayed with 1 μM benzylamine as a substrate. Arrows indicate the position of marker compounds. The molecular for markers used were as follows: (a) cyanocobalamine (MW, 1355); (b) FAD (MW, 786); (c) DNP-alanine (MW, 255). Vo; dextran (MW, 2,000,000). The broken line shows the absorbance at 280 nm. (B) Estimation of molecular weight of ESI-T4 by gel filtration with Sephadex G-25. The arrows show the mobility of the peak of inhibition activity.

Fig. 3.

Concentration-dependent effect of ESI (open circle) without preincubation (closed circle). In the presence of 150 μl of ESI T4, SSAO activity in rat heart homogenate was determined 1 μM benzylamine as substrate at 37 °C for 10 min. The control activities for 1 μM benzylamine were 0.23 ± 0.02 nmol/min/mg protein. Values are means ± S.E.M. for six animals. *p < 0.05 versus control (buffer) (ANOVA and Fisher test). ^§p < 0.001 versus significant difference between the data connected by each bracket (ANOVA and Fisher's test).

3.3. Isoelectric point (pI) of ESI

The pI value of ESI was determined by gel isoelectric-focusing (Fig. 4). SSAO inhibition activity was found as a single peak at a position 2.8 cm from the top of the gel. The pH measurement after IEF revealed that ESI has a pI value of about 4.8.

Fig. 4.

Effect of preincubation time with ESI on SSAO in the rat heart. Rat heart homogenates were preincubated with ESI at 37 °C for the incubation times, and then remaining SSAO activity was assayed, as described in the text. Values were expressed as percent inhibiting SSAO activity in preparations preincubated with distilled water for the same periods. Each point represents the mean percentage activity (±S.E.) assayed in triplicate experiments with respect to homogenates preincubated for the same length of the time but with distilled water instead of ESI.

3.4. Some properties of ESI

Study of the time-course inhibition of SSAO by ESI inhibited that extent of the inhibition did not change even for preincubation time up to 90 min at 37 °C (Fig. 5). When ESI were treated at 100 °C for 10 min, the inhibition activity was not changed. Also, treatment of ESI with subtilisin or pronase at 37 °C for 12 h did not affect the inhibition activity of ESI. These results suggest that ESI is a non-peptide inhibitor (Fig. 6).

Fig. 5.

Estimation of pI value of ESI-T4 in rat heart cytosol by gel isoelectric focusing. ESI-T4 fraction was collected and was solubilized with 0.75% Triton X-100. Isoelectric focusing was performed as described in Materials and Methods. After IEF-gel electrophoresis, the gel was cut into 4-mm slices, and the pH (open circle) of each gel slice was determined. After adjusting pH to 7.4, the SSAO activity for 1 μM benzylamine was determined in the presence of each gel slice (closed circle) without preincubation. The control activity for benzylamine was 0.23 ± 0.02 nmol/min/mg protein. Each point represents the mean percentages (±S.E.) on the control SSAO activity in triplicate experiments.

Fig. 6.

Some properties of the ESI treatment. ESI-T4 was tested in various ways, such as nontreatment (N), heat treatment (H; 100 °C for 10 min), trypsin (T; 37 °C for 30 min), subtilisin (S; 37 °C for 12 h), and pronase (P; 37 °C for 12 h). Control (C) values for SSAO activity were 0.23 ± 0.02 nmol/min/mg protein. Values of percentage SSAO activity are means ± S.E.M. for six animals, *p < 0.05 versus control (ANOVA and Fisher test).

4. Discussion

It is well established that one of the actions of thyroid hormones is thought to be the regulation of protein synthesis and enzyme activities.7, 16, 17 Thyroid hormones were also found to influence of MAO activity in the rat heart.¹⁸ Ichikawa et al.¹⁹ reported that endogenous MAO modulators isolated from the rat heart cytosol. SSAO differs from MAO and deaminates various monoamines.²⁰ The cardiac MAO activity was increased in hyperthyroid rats or by administration of thyromimetic compounds.²¹ Thyroid hormone is shown to be protective against cardiac injury. The cardiovascular system is an important target for thyroid hormones. However, there is no information about SSAO activity. It is possible that thyroid hormone regulates the SSAO activity in rat heart. It is not known, whether a decrease in SSAO activity in hyperthyroid state is a direct effect of thyroid hormone or is mediated through specific mechanism. In the present study, we found the presence of thyroid hormone inducible endogenous SSAO modulator in rat cardiac cytosol fraction, which could inhibit SSAO activity. To our knowledge, this is first report on SSAO modulator in rat heart.

Although its physiological role is not yet clear, SSAO in certain rat cardiovascular tissues deaminate various monoamine such as benzylamine, β-phenylethylamine, kynuramine and dopamine.²² This enzyme in other rat tissues deaminates other monoamines. Benzylamine is the best substrate so far examined for this enzyme. It is well known that low concentration of benzylamine (1 μM) was deaminated solely by SSAO.²³ Metabolism of benzylamine by SSAO has a Km of around 5 μM, which is much lower than for benzylamine metabolism by MAO activities.²⁴ Therefore, the complete inhibition at 10⁻⁴ M semicarbazide was obtained (Fig. 1). When corresponding experiments were performed with 100 μM benzylamine, the opposite results were obtained (not illustrated). Although SSAO is present in plasma, SSAO activity in blood was very low (data not shown).

There have been many reports on the effects of thyromimetic compounds on MAO activity, whereas the others report that the cardiac MAO activity was increased in hyperthyroid rats or by administration of thyromimetic compound.²⁵ We found an endogenous SSAO modulator of a low molecular weight (1000–1100) in heart cytosol of T4-treated rats (Fig. 2). The inhibitory effects of this inhibitor were non-linear concentration-dependent (Fig. 3). This molecular weight is much smaller than that reported for rat MAO modulator¹⁹ (8500–35,000). When thyroidectomized, nontreated rats were used, inhibition activities for benzylamine were very low. However, T4-treatment remarkably enhanced SSAO inhibition activities for benzylamine (Fig. 3). The fact that the inhibitory activity in the hyperthyroid state suggests the possibility that thyroid hormone may regulate the SSAO activity by induction of this inhibitory modulator. We previously reported that an endogenous MAO inhibitor of a low molecular weight (600–700) in liver cytosol of T4-treated rats.²⁶ This molecular weight is similar to ESI (1000–1100) in cardiac cytosol of T4-treated rats. It can be concluded that the rat heart contains low molecular weight materials which act like SSAO inhibitor drugs. The nature of the inhibition of SSAO by ESI was studied. Time-course experiments indicated that this enzyme was inhibited equally effectively in the absence of preincubation as after 90 min of preincubation (Fig. 5). Moreover, dilution experiments showed that the dilution of the reaction mixture restored the activity to the level at final concentration of the inhibitor (data not shown), suggesting that the inhibition is reversible. These may indicate that ESI was a reversible inhibitor of SSAO.

The present study showed that this compound is heat-stable and resist to protease and subtilisin treatment (Fig. 6). Our findings suggested that thyroid hormone-inducible SSAO inhibitor may have an important role in SSAO activity. We also discussed the physiological role of involvement in the thyroid hormone-mediated regulation of vascular function.

5. Conclusion

In the present study, we found a new SSAO inhibitory modulator in T4-treated rat cardiac cytosol, and that the level of this modulator is regulated by thyroid hormone. Although the physiological role of this inhibitor still remains unclear, we consider that this modulator may play some role in regulating the SSAO activity in rat heart. While the role of these modulators is no doubt important, their mechanisms remain to be elucidated. Further study will be necessary to clarify this point.

Conflicts of interest

The authors have none to declare.