Intracellular metabolism of biogenic amines in paraneurons

Arch. Histol. Cytol., Vol. 52, Suppl. (1989) p. 69-74

Intracellular

Metabolism

Hiroyuki

HASEGAWA,

Takaaki

Department

of Biochemistry, Hamamatsu

of Biogenic

KOBAYASHI,

Fumio

INOUE

Amines

and

Arata

University School of Medicine, Hamamatsu,

in Paraneurons

ICHIYAMA Japan

from tryptophan are representative biogenic amines, but the scope of this article will be confined to the biosynthesis of serotonin (an indoleamine) in paraneuron cells of the mouse intestinal mucosa with references to that in mouse mastocytoma and rat basophilic leukemia cells.

Summary. Serotonin is one of the representative biogenic amines produced from tryptophan in the gastrointestinal tract as well as in the brain. The pineal gland also synthesizes serotonin, but as an intermediate in the biosynthesis of melatonin. The first enzymic step in the biosynthesis of serotonin is the hydroxylation of L-tryptophan catalyzed by tryptophan 5-monooxygenase. The 5-hydroxy-L-tryptophan (L-5HTP) formed is then decarboxylated to serotonin. Since the second step is catalyzed by non-specific aromatic L-amino acid decarboxylase, whether or not a given cell has the capacity to synthesize serotonin depends on the existence of tryptophan monooxygenase, and regulation of the serotonin biosynthesis is achieved mainly by modulating the activity of the first step enzyme. The activity of tryptophan monooxygenase was detected in extracts of the mouse stomach and intestines, although this activity was as low as approximately 1/20 of that in similar extract of the mouse brainstem. The upper small intestine and colon had a higher level of activity than other parts, and in the upper small intestine the enzyme was found to reside primarily in enterochromaffin cells of the mucosa. The intestinal tryptophan monooxygenase shared a common antigenic character with the enzyme from murine mastocytoma and was immunologically different from the brain enzyme. The enzymic properties were also similar to those of mastocytoma tryptophan monooxygenase which requires loosely-bound functional iron (Fe2+) for the activity. It seemed likely that the activity of tryptophan monooxygenase in paraneurons such as mastocytoma and enterochromaffin cells depends on available ferrous iron, and is therefore regulated, at least in part, by the cytosolic level of chelatable iron.

MATERIALS

AND

METHODS

Sources of nude (BALB/c-nu/nu) and mast celldeficient (WBB6F1-W/W°) mice, their control mice (BALB/c and WBB6F1- + / +) and other strains of mice used have been described previously (INoUE et al., 1985, 1987 ; HASEGAWAet al., 1987). The animals were usually sacrificed by cervical dislocation and phlebotomized by cutting the carotid artery. Tissues were then quickly isolated. The small intestine was divided into proximal (upper small intestine), middle and distal thirds. Mouse mastocytoma P-815 cells were grown and harvested as described previously (YANAGISAWAet al., 1982). A rat basophilic leukemia cell-line (RBL2H3) was supplied from the Cell Bank, Division of Mutagenesis, Institute of Hygienic Sciences, Tokyo. Mastocytoma tryptophan monooxygenase was purified from the P-815 cells according to the method by NAKATA and FUJISAWA (1982). Anti(mastocytoma tryptophan monooxygenase)-Igs were obtained by immunizing guinea-pigs and rabbits with the purified enzyme (HASEGAWA et al., 1987). The extraction of serotonin from tissues and its measurement by high performance liquid chromatography (HPLC) were performed as described previously (INoUE et al., 1985). For the assay of tryptophan monooxygenase in the gastrointestinal tissues and mastocytoma cells, homogenates were prepared in a medium containing 0.3 mM Fe(NH4)2(S04)2i followed by centrifugation at 105,000 x g. The supernatant was then subjected to gel-filtration on a Sephadex G-25 column to remove endogenous small molecules and added iron (YANAGISAWAet al., 1984). The enzyme

Biogenic amines are produced in neurons and paraneurons from respective precursor amino acids by decarboxylation, and play crucial roles in the signal transmission between cells and the regulation of cellular processes as a neurotransmitter, hormone or local chemical mediator, y-Aminobutyric acid produced from glutamic acid, histamine from histidine, catecholamines from tyrosine, and indoleamines 69

70

H.

HASEGAWA

et a!.:

activity was determined both with and without the activation by anaerobic preincubation with dithiothreitol (ICHIYAMAet al., 1974) by an HPLC procedure (HASEGAWA et al., 1984, 1987). A gastrin-releasing peptide (GRP)-like immunoreactive substance was kindly measured by Dr. N. YANAIHARA, Laboratory of Bioorganic Chemistry, University of Shizuoka Pharmaceutical School, using an antiserum raised with a synthetic GRP.

RESULTS

AND

Organ distribution

DISCUSSION

of serotonin

in the mouse

It has been known that the biosynthesis of serotonin from tryptophan occurs independently in the brain and in the periphery, especially the gastrointestinal tract (UDENFRIEND and WEISSBACH, 1958; BERTACCINI, 1960). Figure 1 shows the organ distribution of serotonin in a strain of mouse (WBB6F1). In the case of rodents, mast cells also contain serotonin, and therefore the organ distribution in a mast cell-deficient mutant mouse (WBB6F1-W/WV) was compared with that of a normal mouse of the same strain (WBB6F1-+/+). Mice were sacrificed by cervical dislocation. The serotonin content of the brain was relatively small in spite of the high activity for the synthesis, reflecting its rapid turnover : the turnover times of serotonin in the brain and gastrointestinal tract have been estimated at about 1 and 10-17 h, respectively (NEEF and TOZER,1968; UDENFRIENDand WEISSBACH, 1958). In the periphery, large amounts of serotonin were detected in the blood and lung as well as the gastrointestinal tract, in agreement with many previous workers (BERTACCINI,1960, etc.), but blood platelets are known to acquire serotonin by uptake when they come into the immediate vicinity of the intestine (HARDISTY and STACEY, 1955, etc.). Recently, we found that cervical dislocation very rapidly induces the entrapping of platelets in the pulmonary capillaries; hence, the high content of serotonin in the excised lung tissue is due to these entrapped platelets (YAMAMOTOet al., 1986, 1988). The spleen also contained serotonin, but this organ was shown to owe its high serotonin content to the local destruction of a large number of platelets (MELLINKOFFet al., 1962). These studies thus confirmed the prevailing view that the major site of serotonin biosynthesis in the periphery is the gastrointestinal tract. In addition, the pineal gland synthesizes serotonin as an intermediate

Fig.

Fig.

1.

2.

Organ

distribution

Biosynthesis

of serotonin

of catecholamines

in the biosynthesis of melatonin, regulating reproductive functions pothalamo-hypophyseo-gonadal

in WBB6F1

mice.

and indoleamines.

a neurohormone through the hy-

axis (REITER, 1973).

Intracellular

Biosynthesis

of catecholamines

Metabolism

of Biogenic

71

Amines

and indoleamines

Figure 2 shows that indoleamines and catecholamines are synthesized by a similar mechanism from tryptophan and tyrosine, respectively. The closed arrow represents reactions occurring in both the brain and peripheral paraneurons, and the hatched arrow those occurring only or predominantly in the paraneuron cells. Hydroxylase is another name for monooxygenase. In the synthesis of serotonin and catecholamines, the first enzymic step is the hydroxylation of the precursor amino acids to form 5-hydroxy-L-tryptophan (L-5HTP) and 3, 4-dihydroxy-L-phenylalanine (L-dopa), respectively, catalyzed by very similar but different pterin-dependent monooxygenases, tryptophan 5-monooxygenase and tyrosine 3-monooxygenase. The L-5HTP and L-dopa thus formed are then decarboxylated to serotonin and dopamine, respectively, by aromatic L-amino acid decarboxylase. Since the second step of the serotonin and catecholamine biosyntheses is catalyzed by the same decaboxylase (CHRISTENSONet al., 1972), whether or not a given cell has the capacity to synthesize serotonin or catecholamines depends on the existence of the monooxygenases, and the individual regulation of serotonin and catecholamine biosyntheses is achieved by modulating the activity of the first step enzymes. Hydroxylation

of tryptophan

Figure 3 illustrates

the reaction

catalyzed

by trypto-

phan monooxygenase. This enzyme is a monooxygenase, and hence incorporates one oxygen atom from molecular oxygen into L-tryptophan to yield L-5HTP, and concomitantly the other oxygen atom is reduced to water by a cofactor, tetrahydrobiopterin. The quinonoid dihydrobiopterin formed is then reduced back to the tetrahydro form by another enzyme, dihydropteridine reductase. Thus the enzymic conversion of tryptophan to 5HTP requires the cofactor tetrahydrobiopterin and its generating system (dihydropteridine reductase and NADH or NADPH), in addition to tryptophan monooxygenase and molecular oxygen. Tryptophan tract

monooxygenase

in the gastrointestinal

In our study of tryptophan monooxygenase from the bovine pineal gland and mouse mastocytomas, we have found that the properties of the non-neural

Fig.

3.

Hydroxylation

of tryptophan.

tryptophan monooxygenase are distinctly different from those of the brain enzyme (ICHIYAMAet al., 1974; NUKIWA et al., 1974; YANAGISAWAet al., 1982 etc.). This was in contrast to the case of catecholamine biosynthesis in which the tyrosine hydroxylation in the brain and adrenal medulla appears to be catalyzed by the same tyrosine 3-monooxygenase. The HPLC method developed during the course of the study for measuring the tryptophan hydroxylation (HASEGAWA et al., 1984), together with accumulated knowledge of the non-neural tryptophan monooxygenase, has allowed us to measure reasonably the low activity of this enzyme in the digestive tract. Although the activity determined in extracts of the mouse stomach and intestines was only approximately 1/20 of that in similar extract of the brainstem, the strict dependency of the activity on both molecular oxygen and tetrahydrobiopterin was observed, indicating that the determined activity is decidedly of tryptophan monooxygenase (HASEGAWA et al., 1987). One of the remarkable features of tryptophan monooxygenase from the bovine pineal gland and mouse mastocytomas was the activation by anaerobic preincubation with dithiothreitol (ICHIYAMAet al., 1974 ; HASEGAWA et al., 1982) ; apparently the same activation has been observed with the enzyme in the intestinal extract (HASEGAWA et al., 1987). Localization gastrointestinal

of tryptophan

monooxygenase

in the

tract

The tryptophan monooxygenase activity was detected throughout the mouse gastrointestinal tract, but the upper small intestine and colon showed a higher

72

Table

H.

1.

HASEGAWA

et al.:

Number of mucosal oxygenase activity

mast cells, serotonin in mouse stomach

content

level of activity than other parts. It was unlikely that mast cells contributed to some of the tryptophan monooxygenase activity of the upper small intestine, because the extract from mast cell-deficient mutant mouse (WBB6F1-W/W") contained approximately the same level of activity (HASEGAWAet al., 1987). In the stomach, however, the tryptophan monooxygenase activity as well as the serotonin content of the mast cell-deficient mutant mouse was less than half that of the control, as shown in Table 1. In addition, both the enzyme activity and serotonin content in the stomach of an athymic nude mouse (BALB/c-nu/nu) were significantly above the control level, and in accord with this numerous cells showing metachromasia were observed in the stomach mucosa of the nude mouse by toluidine blue staining. These cells were small in size, variable in shape and sparsely granulated; these morphological features resembled those of mucosal mast cells (INouE et al., 1987). These results thus suggested, although not proved, that mucosal

Fig. 4. Localization of tryptophan in the intestinal mucosa.

monooxygenase

and

tryptophan

mono-

mast cells or mucosal mast cell-like cells contribute significantly to the tryptophan monooxygenase activity in the stomach mucosa of mouse. Therefore, all subsequent studies were performed with the upper small intestine. Tryptophan monooxygenase in the upper small intestine was then found to reside between the upper villus region and the bottom of the crypt (HASEGAWA et al., 1987). In this experiment, a luminal layer of the upper small intestine was scraped as evenly as possible with the edge of a glass slide. Following this, the residual sheet of the intestinal tissue was throughly scraped. These scrapings were referred to as the upper and lower layers, respectively (Fig. 4). The lower layer mass comprised about one-third that of the upper layer in wet weight. As shown in Figure 4, thymidine kinase, a marker enzyme of mitotically active cells of the crypt, was distributed evenly between the two layers, indicating that the mucosa was separated into the two layers at the level of the bottom zone of the crypt. Although not shown in the figure, the larger part of the GRPlike immunoreactivity was detected in the lower layer, indicating that most enteric nerve plexuses were included in the lower layer mass. Villi were largely scraped off in the upper layer as visualized by the distribution of alkaline phosphatase (Fig. 4). With these tissue preparations, we found 86% of the tryptophan monooxygenase activity to be localized in the upper layer (Fig. 4) ; the distribution of this enzyme in the upper layer was 10% less than that of alkaline phosphatase and 35% more than that of thymidine kinase. These results suggested that the tryptophan monooxygenase activity detected in extracts of the upper small intestine is mainly of mucosal paraneurons and not of submucosal nerve plexus origin. In fact, Dr. T. IWANAGAof the Department of Anatomy, Niigata University School of

Intracellu

Medicine, recently demonstrated the localization of tryptophan monooxygenase in enterochromafl'in cells, immunocytochemically using an antibody raised in rabbits against the mastocytoma enzyme.

Immunological monooxygenase

properties

of intestinal

tryptophan

Tryptophan monooxygenase in extracts of the upper small intestine was precipitated by a guinea-pig antibody against mastocytoma tryptophan monooxygenase as effectively as the mastocytoma enzyme, while the enzyme in extracts of the mouse brain was not (HASEGAWA et al., 1987). It was evident that mouse intestinal tryptophan monooxygenase shares a common antigenic character with the mastocytoma enzyme and is immunologically different from the brain enzyme. Enzymic properties of tryptophan monooxygenase of mouse intestine and mastocytoma The apparent Km values of intestinal tryptophan monooxygenase for L-tryptophan and tetrahydrobiopterin were determined to be approximately 75 and 45 pM, respectively (HASEGAWA et al., 1987). These values were within the physiological range and were similar to those of the mastocytoma enzyme (YANAGISAWAet al., 1984). Other enzymic properties examined so far were also similar to those of the mastocytoma enzyme. Most of the characteristic properties observed with mastocytoma tryptophan monooxygenase are due to the fact that the enzyme absolutely requires looselybound ferrous iron for the activity. When the purified mastocytoma enzyme was mixed with 10 mM ethylenediaminetetraacetate (EDTA) and then freed of EDTA and other small molecules by passing through a Sephadex G-25 column, no activity was detectable unless ferrous iron was added to the reaction mixture. The easy removal of iron indicated that the monooxygenase is not a metalloenzyme which binds functional iron tightly as a prosthetic atom, but is an iron-activated enzyme which binds iron less tightly and whose activity depends on available free iron. In fact, the dissociation constant of the binding of ferrous iron to the monooxygenase was estimated to be around 10-8 to 10-6 M. The enzyme also bound ferric iron more tightly, but the enzyme bound with ferric iron was totally devoid of the enzyme activity, making the competition by ferric iron very serious. In addition, ferrous

lar Metabolism

of Biogenic

Amines

73

iron in neutral aqueous solutions, either free or loosely bound to the enzyme, was susceptible to the rapid oxidation to the ferric form under aerobic conditions. Therefore, manifestation of the full activity of this enzyme required special conditions in which ferrous iron was supplied while ferric iron was being removed from the enzyme. Dithiothreitol used to activate the enzyme to its fully active state is an ideal reagent for that purpose in that it possesses both the reducing and metal-chelating activities.

Tryptophan

hydroxylation

philic leukemia

in cultured

rat

baso-

cells

From the enzymic properties of non-neural tryptophan monooxygenase described above, it seemed likely that the cellular activity of this enzyme is regulated, at least in part, by the cytosolic level of chelatable iron. Some preliminary experiments were carried out at the cellular level to test this possibility. In the experiment shown in Figure 5, a serotonin producing rat basophilic leukemia cell (RBL2H3) was cultured for 5 h in Dalbecco's modified Eagle's minimal essential medium containing 10% fetal calf serum in the presence or absence of 50 ji M hemin. Hemin had been believed to increase the intracellular chelatable iron pool by delivering iron to it (ROUAULT et al., 1985). The tryptophan monooxygenase activity in the cell was then determined by supplying the cells with the substrate, L-tryptophan, and a decarbox-

Fig. 5. Effect of hemin on tryptophan monooxygenase activity in cultured RBL2H3 cells. Hemin (H, 50 pM) was added to either the preculture (PreLoad) or the assay (Load in assay) or both. + BPH4 and - BPH4 show whether or not exogenous tetrahydrobiopterin was supplied to the cell before initiation

of the assay.

74

H. HASEGAWA

et al.

ylase inhibitor, NSD-1055, followed by incubation at 24°C for 0-45 min. As shown in Figure 5, hemin did not affect the tryptophan hydroxylation when added just before the initiation of the assay, but significantly increased the intracellular tryptophan monooxygenase activity when loaded beforehand. At present, detailed direct studies on the regulation of intestinal tryptophan monooxygenase are limited by the low activity of this enzyme and unavailability of isolated culture-adapted enterochromaffin cells. We hope that studies on tryptophan monooxygenase of similar properties in other paraneuronal cells are not only valuable in themselves but also useful as a model or guide for study of the serotonin biosynthesis in the intestinal paraneurons.

REFERENCES BERTACCINI, G.: Tissue 5-hydroxytryptamine and urinary 5-hydroxyindoleacetic acid after partial or total removal of the gastrointestinal tract in the rat. J. Physiol. (Lond.) 153: 239-249 (1960). CHRISTENSON, J. G., W. DAIRMAN and S. UDENFRIEND: On the identity of DOPA decarboxylase and 5-hydroxytryptophan decarboxylase. Proc. Nat. Acad. Sci. USA 69: 343-347 (1972). HARDISTY, R. M. and R. S. STACEY: 5-Hydroxytryptamine in normal human platelets. J. Physiol. (Lond.) 130: 711-720 (1955). HASEGAWA, H., M. YANAGISAWA and A. _ICHIYAMA: Three discrete activity states of mastocytoma tryptophan monooxygenase. In: (ed. by) M. NOzAKI, S. YAMAMOTO,Y. ISHIMURA,M. J. COON, L. ERNSTER and R. W. ESTABROOK: Oxygenases and oxygen metabolism. Academic Press, Inc., Tokyo, 1982 (p. 293-304). HASEGAWA, H., M. YANAGISAWA and A. ICHIYAMA: Assay of tryptophan hydroxylase and aromatic L-amino acid decarboxylase based on rapid separation of the reaction product by high performance liquid chromatography. J. Biochem. 95: 1751-1758 (1984). HASEGAWA, H., M. YANAGISAWA, F. INOUE, N. YANAIHARA and A. ICHIYAMA: Demonstration of non-neural tryptophan 5-monooxygenase in mouse intestinal mucosa. Biochem. J. 248: 501-509 (1987). ICHIYAMA,A., S. HORI, Y. MASHIMO, T. NUKIWA and H. MAKUUCHI: The activation of bovine pineal tryptophan 5-monooxygenase. FEBS Lett. 40: 88-91 (1974). INOUE, F., H. HASEGAWA, M. NISHIMURA, M. YANAGISAWAand A. ICHIYAMA: Distribution of 5-hydroxytryptamine (5HT) in tissues of a mutant mouse deficient in mast cells (W/W"). Demonstration of the contribution of mast cells to the 5HT content in various organs. Agents Actions 16: 295-301 (1985). INOUE, F., H. HASEGAWA,M. YAMADA and A. ICHIYAMA: The serotonin content and tryptophan 5-monooxy-

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genase activity in the stomach of an athymic nude mouse and mast cell-deficient mouse. Biomed. Res. 8: 5359 (1987). MELLINKOFF, S., C. CRADDOCK,M. FRANKLAND, F. KEN. DRICKSand M. GREIPEL: Serotonin concentration in the spleen. Amer. J. Dig. Dis. 7: 347-355 (1962). NAKATA, H. and H. FUJISAWA: Tryptophan 5-monooxygenase from mouse mastocytoma P815. A simple purification and general properties. Eur. J. Biochem. 124: 595-601 (1982). NEEF, N. H. and T. N. TozER : In vivo measurement of brain serotonin turnover. In: (ed. by) S. GARATTINI and P. A. SHORE: Advances in pharmacology, Vol. 6, Part A. Academic Press, Inc., New York-London, 1968 (p. 97-109). NUKIWA, T., C. TOHYAMA, C. OKITA, T. KATAOKAand A. ICHIYAMA: Purification and some properties of bovine pineal tryptophan 5-monooxygenase. Biochem. Biophys. Res. Commun. 60: 1029-1035 (1974). REITER, R. J.: Comparative physiology: Pineal gland. Ann. Rev. Physiol. 35: 305-328 (1973). ROUAULT, T., K. RAO, J. HARFORD, E. MATTIA and R. D. KLAUSNER: Hemin, chelatable iron, and the regulation of transferrin receptor biosynthesis. J. Biol. Chem. 260: 14862-14866 (1985). UDENFRIEND, S. and H. WEISSBACH: Turnover of 5hydroxytryptamine (serotonin) in tissues. Proc. Soc. Exp. Biol. 97: 748-751 (1958). YAMAMOTO,Y., H. HASEGAMWA, F. INOUE, K. IKEDA and A. ICHIYAMA: Serotonin in the lung. Demonstration of a close correlation to blood platelets. Agents Actions 18: 352-358 (1986). YAMAMOTO,Y., H. HASEGAWA, K. IKEDA and A. ICHIYAMA: Cervical dislocation of mice induces rapid accumulation of platelets serotonin in the lung. Agents Actions 25: 48-56 (1988). YANAGISAWA, M., H. HASEGAWA and A. ICHIYAMA: Tryptophan hydroxylase from mouse mastocytoma P-815. Reversible activation by ethylenediaminetetraacetate. J. Biochem. 92: 449-456 (1982). YANAGISAWA, M., H. HASEGAWA, A. ICHIYAMA, S. HOSODA and W. NAKAMURA: Comparison of serotoninproducing murine mastocytomas, P-815 and FMA3: Determination of tryptophan hydroxylase, aromatic z-amino acid decarboxylase, and cellular concentration of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. Biomed. Res. 5: 19-28 (1984).

Prof. Arata ICHIYAMA Department of Biochemistry Hamamatsu University School of Medicine 3600 Handa-cho, Hamamatsu 431-31 Japan