22 1
Indole Metabolism in the Pineal Gland of the Rat; Some Regulatory Aspects M.G.M. BALEMANS
Zoological Laboratory, State University of Utrecht, Utrecht ( m e Netherlands)
About twenty years ago, Lerner et al. (1958) isolated an indolic compound from the pineal, which at that moment was important for its blanching reaction in amphibian tadpoles. Lerner et al. (1959) and Lerner and Case (1960) identified this compound, which was termed melatonin, as 5-methoxy-N-acetyltryptamine.During the following years, the influence of melatonin on various organs was investigated, especially on those which are related to reproduction (Reiter and Fraschini, 1969). The results described motivate the intensive research that has been done on indole metabolism. In the indole metabolism the aminoacid tryptophan (Fig. 1) is converted to 5-hydroxytryptophan by hydroxylation (Lovenberg et al., 1967; Jequier et al., 1969). Decarboxylation of this indole derivative (Lovenberg et al., 1962; Snijder and Axelrod, 1964) leads to the formation of 5-hydroxytryptamine (serotonin). Serotonin can be acetylated to N-acetyl serotonin (Weissbach et al., 1960), oxidized to 5-hydroxyindole-3-acetic acid (Lerner and Case, 1960) or metabolized to 5-hydroxytryptophol (McIsaac and Page, 1959). The 5-hydroxyindoles 5-hydroxytryptophan, 5-hydroxytryptamine, 5 -hydroxyindole-3-acetic acid, 5-hydroxytryptophol and N-acetyl serotonin can be methylated by hydroxyindole-0-methyl transferase (HIOMT) (Axelrod and Weissbach, 1960, 1961; Cardinali and Wurtman, 1972; Axelrod and Lauber, 1968). From the methylated products, melatonin (Lerner et al., 1959; Lerner and Case, 1960) and 5-methoxytryptophol (McIsaac et al., 1965) seem to be physiologically the most important substances. Studies, especially directed to the enzyme HIOMT (Wurtman et al., 1964; Moore et id., 1967) on pineal serotonin content (Fiske, 1964; Illnerova, 197 1) and on N-acetyltransferase activity (Klein and Weller, 1970) showed that changes of the light/dark periods have a marked influence on indole metabolism. Measuring HIOMT (Moore et al., 1967) and N-acetyltransferase (Moore and Klein, 1974) activity in the pineal after blinding, lesions in the optic tract and on several places in the central nervous system led to the description of the visual pathway from the retina via the optic nerve, suprachiasmatic nucleus, median forebrain bundle, intermedio-lateral cell column, superior cervical ganglion and nervi conarii (Kappers, 1960) to the pineal. The sympathetic nerve endings in the pineal contain norepinephrine (Pellegrino de Iraldi and Zieher, 1966; Zieher and Pellegrino de Iraldi, 1966). If this catecholamine (Fig. 2) is released, it may be methylated to normetanephrine, taken up by the nerve fibre and probably also by the pineal cell or may stimulate a &receptor located in the pinealocyte cell membrane (Deguchi and Axelrod, 1972a, b; Backstrom and Wetterberg, 1973). The o-receptor activates adenylylcyclase activity (Weiss and Costa, 1967, 1968a,b; Weiss, 1969, 1971;Weiss
222
(Try)
(5-HTP)
Tryptopbn hydror ylare
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Hydms yindole-Omethyl transfernre
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(N-Ac-5-HT)
Hydrox yindok-O-
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N-Acetyl5-Hydrox ytryptamine
methyl tmnrlerose (HIOMT)
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5-Hydroxytryptophd (5-HTL)
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Hydroxyindok-0meihyl Ironsferase
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5-Methoxyindole acetic acid (5-MIAA)
5-Methoxytryptophol (5-MTL)
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Fig. 1. The indole metabolism in the pineal gland.
and Strada, 1972), the enzyme responsible for the conversion of ATP to CAMP. From the investigations of Shein and Wurtman (1969, 1971) it is known that norepinephrine and CAMPstimulate tryptophan hydroxylase activity. Furthermore, Klein et al. (1 970a,b) have demonstrated a stimulatory effect of both substances on Nacetyltransferase activity (Fig. 3). Experiments of Zatz and O’Dea (1976) proved that the effect of CAMPon N-acetyltransferase activity is mediated by a protein kinase. This probably means that N-acetyltransferase activity depends on phosphorylation by a protein kinase.
223 NMN
/
/
Fig. 2. Norepinephrine (NE)released from the sympathetic nerve ending may be methylated to normetanephrine (NMN) by means of catechola-methyltransferase (COMT) or taken up by the nerve fibre and probably by the pinealocyte, or may stimulate a p-receptor.
The central position of serotonin in indole metabolism is probably proved by its inhibiting effect on norepinephrine-induced activity of adenylylcyclase in the pineal (Weiss and Costa, 1968a). In this way a feedback regulation mechanism is established for its own synthesis and conversion to N-acetyl serotonin and melatonin (Fig. 3). The amount of norepinephrine (Wurtman and Axelrod, 1966; Wurtman et al., 1967) increases during the night up to the onset of light (Fig. 4), then it decreases and is minimal during daytime. The stimulatory effect of this catecholamine on tryptophan hydroxylase (Shein and Wurtman, 1971) and on N-acetyltransferase (Klein et al., 1970a,b) occurs also during the night (Shibuya et al., 1978; Klein and Weller, 1970, respectively). In contrast to the maximal amount of norepinephrine present at the end of the night, the activity of both enzymes is decreasing as the end of the night approaches. This may probably be explained in the following way; the sensitivity of the &receptor increases during daytime (Romero and Axelrod, 1974, 1975). Since norepinephrine rapidly decreases during daytime, cAMP is not produced in a sufficient quantity. After the onset of darkness, however, the increasing amount of norepinephrine produces a rapid increase in quantity of CAMP, although the
1
Met
Fig.3. The influence of norepinephrine and cAMP on indole metabolism and the feed back regulation of serotonin (for abbreviations see Fig. 1).
224
I
5-HTP
Me1
Try-hydroxylase
5 HT
Me1
Fig. 4. Scheme of day/night rhythms of norepinephrine, tryptophan hydroxylase, serotonin, Nacetyltransferase, HIOMT and melatonin, and the influence of norepinephrine on tryptophan-hydroxylase and Nacetyltransferase.
sensitivity of .the &receptor is declining during the night (Weiss, 1971). The increasing amount of CAMPis sufficient for the stimulation of both enzymes. A further decrease in receptor sensitivity results in a decreased synthesis of CAMP at the end of the night (Weiss, 9171). This may explain the decrease of both enzymes at the end of the night. The
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decarboxyla'se
Fig. 5. Scheme of the synthesis of melatonin from tryptophan, which is probably realized within 36 hr. Above: Try + 5-HTP during one single night: below: N-Ac-5-HT + Me1 during the following night.
225
maximal activity of N-acetyltransferase during the night results in a high synthesis of N-acetyl serotonin (Klein and Weller, 1970). Also during the night there is a maximal activity of HIOMT (Axelrod et al., 1965) and, consequently, a maximal production of melatonin (Lynch, 1971). The synthesis of melatonin from tryptophan occurs for the greater part in darkness. Only the synthesis of serotonin (Figs. 4 and 5), catalyzed by 5-hydroxytryptophan decarboxylase, is high during daytime and low during darkness (Snijder et al., 1965; Pellegrino de Iraldi and Rodriquez de Lores Arnaiz, 1964). This probably means that the synthesis from tryptophan to 5-hydroxytryptophan occurs during one single night; during the day following this night serotonin will be synthesized and the next night serotonin will be converted to N-acetyl serotonin and melatonin. Thus the synthesis of melatonin from tryptophan is probably realized within 36 hours (Fig. 5). Consequently, the serotonin concentration during daytime (Quay, 1963) is crucial for the amount of N-acetyl serotonin to be synthesized. During a long photoperiod, the 0-receptors become more sensitive to norepinephrine which results in a still higher activity of N-acetyltransferase (Deguchi and Axelrod, 1972a,b, 1973a,b) and of tryptophan hydroxylase (Shein and Wurtman, 1971). A high activity of tryptophan hydroxylase means that a great amount of 5-hydroxytryptophan is synthesized and, consequently, also of serotonin during daytime. The high amount of serotonin synthesized during a long photoperiod probably has a dual function. It serves as a substrate for the synthesis of a high amount of N-acetyl serotonin and melatonin, while, by its inhibitory effect on the norepinephrine induced activation of adenylylcyclase, serotonin regulates both the activity of N-acetyltransferase (Fig. 3) and its own synthesis. The regulation of HIOMT activity has been investigated by several authors. Weiss (1968) showed that norepinephrine inhibits HIOMT activity. This observation is in agreement with the data of Brownstein and Heller (1968), who found a decrease in HIOMT activity after nerve stimulation. According to Wartman et al. (1969), acetylcholine is involved in the increase in HIOMT activity. The influence of a parasympathetic agent is observed only during darkness. This suggests that acetylcholine cancels the inhibition of norepinephrine which shows a maximal concentration in darkness. Other substances are probably also involved in the regulation of HIOMT activity in the pineal. Investigations of Cremer-Bartels et al. (1975) and Cremer-Bartels and Hollwich (1 978) on the influence of 2,4,7-triamino-6-phenylpteridineon retinal and pineal HIOMT and the demonstration of the presence of pteridines in the pineal by Ebels et al. (1979) suggest that pteridines may regulate HIOMT activity in the pineal. Therefore, 3 pterins: pterin-6-aldehyde, reduced neopterin and isoxanthopterin were tested in vitro, with the method of Balemans et al. (1978), for the influence on HIOMT activity in the pineal of 4 2 d a y d d , 180 k 10 g, male Wistar rats (Balemans et al., submitted for publication). Every 4 hours animals were decapitated. Three of the extirpated pineals were preincubated separately with pterind-aldehyde, 3 separately with reduced neopterin and 6 separately with solvent system only. In the method used no substrate is added. The 5-hydroxy component present in the pineal can all be methylated to their 5-methoxyproducts after addition of the methyl donor S-adenosylmethionine 3H. In these experiments the pteridine was added to the incubation medium half an hour before addition of the methyl donor. As an indication of HIOMT activity, in the month of October complete day/ night rhythms of the following methylated products were tested: 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-aceticacid and melatonin/5-methoxytryptophol together .
226 DPM/pneal
-1
t
I \
I
I
I
f
\
I
I/
Time (clock hwrsl-
Fig. 6. The day/night rhythm of HIOMT in synthesizing melatonin/5-methoxytyptophol(MEL/S-MTL) (-- - - - -) and the influence of pterindaldehyde (. . . . * .) and reduced neopterin (-) on this enzyme activity during the day/night period. Each point represents the mean i SEM.
Addition of isoxanthopterin to the incubation medium has no influence on the HIOMT activity in the formation of the above mentioned 5-methoxyindoles.Pterin-6-aldehyde, however, causes a shift to the left, towards daytime, on the methylation of 5-HTP, 5-HIAA and SHTL/N-ac-S-HT, and a stimulation of the methylation of 5-HT during daytime. Reduced neopterin causes a shift to the end of darkness on the methylation of 5-HTL/N-ac-5-HTand a stimulation of the methylation of 5-HTP, 5 H T and 5-HIAA during the night period. This means that the synthesis of the 5-methoxyindoles is projected to the light or to the dark period, respectively. The melatonin/5-methoxytryptopholcurve is presented in Fig. 6. It is of interest that Balemans et al. (1979) observed comparable shifts of HIOMT activity in the formation of melatonin/5-methoxytryptopholduring the year. Whether there is any correlation between the duration of the light period and the amount of pteridines or pterins present in the pineal has not been investigated further. These experiments also lead to the question of where and how the pteridines or pterins act in the indole metabolism. REFERENCES Axelrod, J. and Lauber, J.K. (1968) HydroxyindoleO-methyltransferase in several avian species. Bioc h e n Pylarmawl., 11: 828-830. Axelrod, J. and Weissbach, H. (1960) Enzymatic 0-methylation of N-acetylserotonin to melatonin. Science, 131: 1312. Axelrod, J. and Weissbach, H. (1961) Purification and properties of hydroxyindo1e-O-methyl transferase. J. bwl. Chem., 236:211-213. Axelrod, J., Wurtman, R.J. and Snijder, S. (1965) Control of hydroxyindole0-methyltransferaseactivity in the rat pineal gland by environmental lighting. J. bwl. Chem.. 240: 949-955. Backstrtim, M. and Wetterberg, L. (1973) Melatonin formation in rat pineal gland organ culture: induction by an adrenergic p-stimulating drug. Yale J. biol. Med., 46: 630.
227 Balemans, M.G.M., Legerstee, W.C. and Van Benthem, J. (1979) Day and night rhythms in the methylation of N-acetylserotonin/S-hydroxytryptopholin the pineal gland of male rates of different ages, J. neural Transm., 45: In press. Balemans, M.G.M., Noordegraaf, E.M., Bary, F.A.M. and Van Berlo, M.F. (1978) Estimation of the methylathg capacity of the pineal gland. With special reference to indole metabolism. Experientia (Basel), 34: 887-888. Balemans, M.G.M., Van Benthem, J., Legerstee, W.C., De MorBe, A., Noteborn, H.J.P.M. and Ebels, I., The influence of some synthetic pterins on the circadian rhythmicity of HIOMT in synthesizing several 5-methoxyindoles in the pineal gland of 42 days old male Wistar rats. Submitted for publication. Brownstein, M.J. and Heller, A. (1968) Hydroxyindole-O-methyltransferaseactivity: Effect of sympathetic nerve stimulation. Science 162: 367-368. Cardinali, D.P. and Wurtman, R.J. (1972) Hydroxyindole-0-methyltransferasein rat pineal, retina and harderian gland. Endocrinology. 91: 247-252. Cremer-Bartels, G. and Hollwich, F. (1978) Effect of triaminophenylpteridine on hydroxyindole-0methyltransferase of rat pineal gland and bovine retina. J. neural Transm., Suppl. 13: 360. Cremer-Bartels, G., Hollwich, F. and Kotulla, W. (1975) Melatoninbiosynthese in der Sugetierretina in Abhangigkeit von der Adaptation an Belichtung. Klin. Mbl. Augenheilk., 165 : 88-93. Deguchi, T. and Axelrod, J. (1972a) Control of circadian change of serotonin N-acetyltransferaseactivity in the pineal organ by the p-adrenergic receptor. Proc. Nut. Acad. Sci. U.S.A., 69: 2547-2550. Deguchi, T. and Axelrod, J. (1972b) Induction and superinduction of serotonin Nacetyltransferase by adrenergic drugs and denervation in rat pineal organ. Proc. Nut. Acad. Sci. U.S.A.,69: 2208-2211. Deguchi, T. and Axelrod, J. (1973a) Supersensitivity and subsensitivity of the 8-adrenergic receptor in pineal gland regulated by catecholamine transmitter. Proc. Nut. Acad. Sci. U.S.A., 70: 24112414. Deguchi, T. and Axelrod, J. (1973b) Superinduction of serotonin N-acetyltransferase and supersensitivity of adenyl cyclase to catecholamines in denervated pineal gland.Mo1. Pharmacol., 9: 612-618. Ebels, I., De MorBe, A., HusCitharel, A. and Moszkowska, A. (1979) A survey of some active sheep pineal fractions and a discussion on the possible significance of pteridines in those fractions in in vitro and in vivo assays. J. neural Transm., 44: 97-116. Fiske, V.M. (1964) Serotonin rhythm in the pineal organ: Control by the sympathetic nervous system. Sience 146: 253-254. Illnerova, H. (1971) Effect of light on the serotonin content of the pineal gland. Life Sci., 10: 955-960. Jequier, E., Robinson, D.S., Lovenberg, W. and Sjoerdsma, A. (1969) Further studies on tryptophan hydroxylase in rat brainstem and beef pineal. Biochem. Pharmacol., 18: 1071-1081. Kappers, J.A. (1960) The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z. Zellforsch., 52: 163-215. Klein, D.C. and Weller, J.L. (1970) Indole metabolism in the pineal gland: A circadian rhythm in N-acetyltransferase. Science, 169: 1093-1095. Klein, D.C., Berg, G.R. and Weller, J.L. (1970a) Melatonin synthesis; adenosine 3’,5’-monophosphate and norepinephrine stimulate N-acetyltransferase. Science, 168: 979-980. Klein, D.C., Berg, G.R., Weller, J.L. and Glinsmann, W. (1970b) Pineal gland: Dibutyryl cyclic adenosine monophosphate stimulation of labeled melatonin production. Science, 167: 1738-1740. Lerner, A.B. and Case, J.D. (1960) Melatonin. Fed. Proc., 19: 590-593. Lerner, A.B., Case, J.D., Takahashi, Y.,Lee, T.H. and Mori, W. (1958) Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Amer. chem. Soc., 80: 2587. Lemer, A.B., Case, J.D., Biemann, K., Anthony, R.V. and Krivis, A. (1959). Isolation of S-methoxyindole-3-acetic acid from bovine pineal glands. J. Amer. chem. Soc., 81 : 5 264. Lovenberg, W., Weissbach, H. and Udenfriend, A. (1962) Aromatic lamino acid decarboxylase. J. Biol. Chem., 237: 89-93. Lovenberg, W., Jequier, E. and Sjoerdsma, A. (1967) Tryptophan hydroxylation. Measurement in pineal gland, brainstem and carcinoid tumor. Science, 155: 217-219. Lynch, H.J. (1971) Diurnal oscillations in pineal melatonin content,Life Science, 10: 791-795. McIsaac, W.M. and Page, I. (1959) The metabolism of serotonin (S-hydroxytryptamine). J. bwl. Chem., 234: 858-864. McIsaac, W.M., Farrell, G., Taborsky, R.G. and Taylor, A.N. (1965) Indole compounds: Isolation from pineal tissue. Science, 148: 102-103.
228 Moore, R.Y. and Klein, D.C. (1974) Visual pathways and the central neural control of a circadian rhythm in pineal serotonin N-acetyltransferase activity. Brain Res., 71 : 17-33. Moore, R.Y., Heller, A., Wurtman, R.J. and Axelrod, J. (1967) Visual pathway mediating pineal response to environmental light. Science, 155: 220-223. Pellegrino de Iraldi, A. and Rodriquez de Lores Amah, G. (1964) 5-Hydroxytryptophandecarboxylase activity in normal and denervated pineal gland of rats. Life Sci., 3: 589-593. Pellegrino de lraldi, A. and Zieher, L.M. (1966) Noradrenaline and dopamine content of normal, decentralized and denervated pineal gland of the rat. Life Sci., 5: 149-154. Quay, W.B. (1963) Circadian rhythm in rat pineal serotonin and its modifications by estrous cycle and photoperiod. Gen. comp. Endocrinol., 3: 473-479. Reiter, R.J. and Fraschini, F. (1969) Endocrine aspects of the mammalian pineal gland: A review. Neurendocrinol., 5 : 2 19 -25 5. Romero,. J.A. and Axehod, J. (1974) Pineal Padrenergic receptor: Diurnal variation in sensitivity. Science, 184: 1091-1092. Romero, J .A. and Axelrod, J. (1975) Regulation of sensitivity to beta-adrenergic stimulation in induction of pineal N-xetyltransferase. Proc. Nut. Acad. Sci. U.S.A., 72: 1661-1665. Shein, H.M. and Wurtman, R.J. (1969) Cyclic adenosine monophosphate: Stimulation of melatonin and serotonin sylrthesis in cultured rat pineals. Science, 166: 5 19-520. Shein, HcM. and Wurtman, R.J. (1971) Stimulation of 4C tryptophan 5-hydroxylation by norepinephrine and dibutyryl adenosine 3’,5‘monophosphate in rat pineal organ cultures. Life Sci., 10: 935940. Shibuya, H., Toru, M. and Watanabe, S. (1978) A circadian rhythm of tryptophan hydroxylase in rat pineals. Brain Res.. 138: 364-368. Snijder, S.H. and Axelrod, J. (1964) A sensitive assay for Shydroxytryptophan decarboxylase, Biochem. Pharmacol., 13: 805-806. Snijder, S.H., Axelrod, J., Wurtman, R.J. and Fischer, J.E. (1965) Control of 5-hydroxytryptophan decarboxylase activity in the rat pineal by sympathetic nerves.J. Pharmucol. exp. Ther., 147: 371-375. Wartman, S.A., Branch, B.J., George, R. and Newman Taylor, A. (1969) Evidence for a cholinergic influence on pineal hydroxyindo1e-O-methyl transferase activity with changes in environmental lighting. Life Sci., 8: 1263-1270. Weiss, B. (1968) Discussion of the formation, metabolism and physiologic effects of melatonin. Aduanc. Pharmacol., 6A: 152-155. Weiss, B. (1969) Effects of environmental lighting and chronic denervation on the activation of adenyl cyclase of rat pineal gland by norepinephrine and sodium fluoride. J. Pharmucol. exp. Ther., 168: 146 -1 53. Weiss. B. (1971) On the regulation of adenyl cyclase activity in the rat pineal gland. Ann. N.Y. Acad. Soc., 185: 505-519. Weiss, B. and Costa, E. (1967) Adenyl cyclase activity in rat pineal gland: Effect of chronic denervation and norepinephrine. Science, 156: 1750-1752. Weiss, B. and Costa, E. (1968a) Selective stimulation of adenyl cyclase of rat pineal gland by pharmacologically active catecholamines. J. Pharmacol. exp. Ther., 161: 310-319. Weiss, B. and Costa, E. (1968b) Regional and subcellular distribution of adenylcyclase and 3’,5’cyclic nucleotide phosphodiesterase in brain and pineal gland. Biochem. Pharmucol., 17: 2107-2116. Weiss, B. and Strada, S.J. (1972) Neuroendocrine control of the cyclic AMP system of brain and pineal gland. Adu. cycl. nucl. Res., 1: 357-375. Weissbach, H.,Redfield, B.G. and Axelrod, J. (1960) Biosynthesis of melatonin. Enzymic conversion of serotonin to N-acetylserotonin. Biochim. biophys. Acta, 43: 352-353. Wurtman, R.J. and Axelrod, J. (1966) A 24hours rhythm in the content of norepinephrine in the pineal and salivary glands of the rat. Life Sci., 5: 665-669. Wurtman, R.J., Axelrod, J. and Fischer, J.E. (1964) Melatonin synthesis in the pineal gland: Effect of light mediated by the sympathetic nervous system. Science, 143: 1328-1330. Wurtman, R.J., Axelrod, J., Sedvall, G. and Moore, R.Y. (1967) Photic and neural control of the 24-hour norepinephrine rhythm in the rat pineal gland. J. Pharmucol. exp. Ther., 157: 487-493. Zatz, M. and O’Dea, F. (1976) Regulation of protein kinase in rat pineal: Increased V max. in supersensitive glands. J. cycl. nucl. Res., 2: 427-439. Zieher, L.M. and Pellegrino de Iraldi, A. (1966). Central control of noradrenaline content in rat pineal and submaxillary glands, Life Sci., 5 : 155-161.
229
DISCUSSION I. NIR: (1) The late peak of noradrenaline towards the end of the night contrary to midnight melatonin and the enzymes involved in its synthesis would fit also into the 36-hr rhythm suggested by you. - (2) We found an inversion in NAT to occur following a reversion in the light/dark cycle. This is a rather slow process taking 48 up to 72 hr. It would be interesting to examine whether any pteridines are formed in the pineal during this process and, if so, whether such an inversion could be performed using pteridines. - (3) Regarding HIOMT determination we have a publication in press on a simplified procedure for its determination using TLC instead of extractions. It gives reproducible and exact results. We encountered difficulties in Fnding a diurnal HIOMT rhythm. M.G.M. BALEMANS: As far as your second question is concerned I can answer that we started only a short time ago with Dr. Ebels with an investigation on the influence of pteridines o n indole mctabolism. So, this matter is something for future research. My answer to your third question is that the HIOMT rhythms mentioned were all obtained with the method developed in our laboratory and not with Axelrod’s method. H. ILLNEROVA: (1) What was the concentration of adenosylmethionine in your in vitro estimation of HIOMT activity? You found a profound rhythm which we have not been able to obtain. We observed a rhythm in HIOMT activity only if we used lower concentrations of adenosylmethionine in the in vitro assay, such as 2-4 X lo6 M. However, when using higher concentrations, such as 4 X lo* M , we could not observe any rhythm. - We have found at least two types of HIOMT in the rat pineal gland according to the kinetics of the reaction: one enzyme with a lower and another with a higher affinity towards adenosylmethionine. Our preliminary investigations show that the enzyme with the higher affinity prevails during night time, while the enzyme with the lower affinity prevails during daytime. - (2) Would not it be more exact to call your rhythms “rhythms in the production of methylated hydroxyindoles” rather than rhythms in the activity of HIOMT as you added no external substrate, thus measuring the activity of the enzyme as well as the concentration of the substrate proper at the same time? - (3) What is the physiological significance of the effect of pteridines on HIOMT activity? Are there any pteridines in the pineal gland? M.G.M. Balemans: (1) The concentration of S-aden~syl-L-[methyl-~H]methionine we used was 0.7 pCi and 1.5 pCi ( a specific activity of 12.2 Ci/mmol; 31 mCi/mg). With these two concentrations we obtained identical results. Using our method it is possible to measure the synthesis of several 5-methoxy components. The experiments carried out with this method suggest the presence of more than one HIOMT enzyme the activity of which is present during the night and during daytime. - (2) I termed the rhythms “rhythms in HIOMT activity in the production of melatonin (or other 5-methoxyindoles)”. I think this agrees with your suggestion. - (3) Dr. Ebels isolated pteridines from pineal tissue, but about their physiological significance related to HIOMT activity we do not know anything, so far. I. SMITH: As a comment on the remarks of Dr. NU on noradrenaline rhythms I should like to remark that the concentrations of pineal noradrenaline and 5 N T are some lo6 times greater than that of melatonin. Hence, rhythms in which concentrations of 5-HT + NA drop, say to 10’ times greater than that of melatonin, seem unlikely to have any effect on HIOMT and melatonin synthesis. M.G.M. BALEMANS: I do not know in which concentrations NA and 5HT exert physiological effects on the indole metabolism in vivo. On the other hand these two compounds also may have other functions in the pineal gland. L. VOLLRATH: I was very much impressed by your last slide showing an inverse HIOMT rhythm in May and September. How many times did you repeat this experiment? Karyometric studies carried out in my laboratory show that even within one week an inversion of the circadian rhythm may be demonstrable. M.G.M. BALEMANS: The HIOMT rhythms were investigated in 1977 and 1978. The results obtained in both years were completely identical. In addition to your studies I need to mention that all experiments have been done from Tuesday 08.00 to Wednesday 08.00.
Indole Metabolism in the Pineal Gland of the Rat; Some Regulatory Aspects M.G.M. BALEMANS
Zoological Laboratory, State University of Utrecht, Utrecht ( m e Netherlands)
About twenty years ago, Lerner et al. (1958) isolated an indolic compound from the pineal, which at that moment was important for its blanching reaction in amphibian tadpoles. Lerner et al. (1959) and Lerner and Case (1960) identified this compound, which was termed melatonin, as 5-methoxy-N-acetyltryptamine.During the following years, the influence of melatonin on various organs was investigated, especially on those which are related to reproduction (Reiter and Fraschini, 1969). The results described motivate the intensive research that has been done on indole metabolism. In the indole metabolism the aminoacid tryptophan (Fig. 1) is converted to 5-hydroxytryptophan by hydroxylation (Lovenberg et al., 1967; Jequier et al., 1969). Decarboxylation of this indole derivative (Lovenberg et al., 1962; Snijder and Axelrod, 1964) leads to the formation of 5-hydroxytryptamine (serotonin). Serotonin can be acetylated to N-acetyl serotonin (Weissbach et al., 1960), oxidized to 5-hydroxyindole-3-acetic acid (Lerner and Case, 1960) or metabolized to 5-hydroxytryptophol (McIsaac and Page, 1959). The 5-hydroxyindoles 5-hydroxytryptophan, 5-hydroxytryptamine, 5 -hydroxyindole-3-acetic acid, 5-hydroxytryptophol and N-acetyl serotonin can be methylated by hydroxyindole-0-methyl transferase (HIOMT) (Axelrod and Weissbach, 1960, 1961; Cardinali and Wurtman, 1972; Axelrod and Lauber, 1968). From the methylated products, melatonin (Lerner et al., 1959; Lerner and Case, 1960) and 5-methoxytryptophol (McIsaac et al., 1965) seem to be physiologically the most important substances. Studies, especially directed to the enzyme HIOMT (Wurtman et al., 1964; Moore et id., 1967) on pineal serotonin content (Fiske, 1964; Illnerova, 197 1) and on N-acetyltransferase activity (Klein and Weller, 1970) showed that changes of the light/dark periods have a marked influence on indole metabolism. Measuring HIOMT (Moore et al., 1967) and N-acetyltransferase (Moore and Klein, 1974) activity in the pineal after blinding, lesions in the optic tract and on several places in the central nervous system led to the description of the visual pathway from the retina via the optic nerve, suprachiasmatic nucleus, median forebrain bundle, intermedio-lateral cell column, superior cervical ganglion and nervi conarii (Kappers, 1960) to the pineal. The sympathetic nerve endings in the pineal contain norepinephrine (Pellegrino de Iraldi and Zieher, 1966; Zieher and Pellegrino de Iraldi, 1966). If this catecholamine (Fig. 2) is released, it may be methylated to normetanephrine, taken up by the nerve fibre and probably also by the pineal cell or may stimulate a &receptor located in the pinealocyte cell membrane (Deguchi and Axelrod, 1972a, b; Backstrom and Wetterberg, 1973). The o-receptor activates adenylylcyclase activity (Weiss and Costa, 1967, 1968a,b; Weiss, 1969, 1971;Weiss
222
(Try)
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(N-Ac-5-HT)
Hydrox yindok-O-
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N-Acetyl5-Hydrox ytryptamine
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Hydroxyindok-0meihyl Ironsferase
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5-Methoxyindole acetic acid (5-MIAA)
5-Methoxytryptophol (5-MTL)
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Fig. 1. The indole metabolism in the pineal gland.
and Strada, 1972), the enzyme responsible for the conversion of ATP to CAMP. From the investigations of Shein and Wurtman (1969, 1971) it is known that norepinephrine and CAMPstimulate tryptophan hydroxylase activity. Furthermore, Klein et al. (1 970a,b) have demonstrated a stimulatory effect of both substances on Nacetyltransferase activity (Fig. 3). Experiments of Zatz and O’Dea (1976) proved that the effect of CAMPon N-acetyltransferase activity is mediated by a protein kinase. This probably means that N-acetyltransferase activity depends on phosphorylation by a protein kinase.
223 NMN
/
/
Fig. 2. Norepinephrine (NE)released from the sympathetic nerve ending may be methylated to normetanephrine (NMN) by means of catechola-methyltransferase (COMT) or taken up by the nerve fibre and probably by the pinealocyte, or may stimulate a p-receptor.
The central position of serotonin in indole metabolism is probably proved by its inhibiting effect on norepinephrine-induced activity of adenylylcyclase in the pineal (Weiss and Costa, 1968a). In this way a feedback regulation mechanism is established for its own synthesis and conversion to N-acetyl serotonin and melatonin (Fig. 3). The amount of norepinephrine (Wurtman and Axelrod, 1966; Wurtman et al., 1967) increases during the night up to the onset of light (Fig. 4), then it decreases and is minimal during daytime. The stimulatory effect of this catecholamine on tryptophan hydroxylase (Shein and Wurtman, 1971) and on N-acetyltransferase (Klein et al., 1970a,b) occurs also during the night (Shibuya et al., 1978; Klein and Weller, 1970, respectively). In contrast to the maximal amount of norepinephrine present at the end of the night, the activity of both enzymes is decreasing as the end of the night approaches. This may probably be explained in the following way; the sensitivity of the &receptor increases during daytime (Romero and Axelrod, 1974, 1975). Since norepinephrine rapidly decreases during daytime, cAMP is not produced in a sufficient quantity. After the onset of darkness, however, the increasing amount of norepinephrine produces a rapid increase in quantity of CAMP, although the
1
Met
Fig.3. The influence of norepinephrine and cAMP on indole metabolism and the feed back regulation of serotonin (for abbreviations see Fig. 1).
224
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5-HTP
Me1
Try-hydroxylase
5 HT
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Fig. 4. Scheme of day/night rhythms of norepinephrine, tryptophan hydroxylase, serotonin, Nacetyltransferase, HIOMT and melatonin, and the influence of norepinephrine on tryptophan-hydroxylase and Nacetyltransferase.
sensitivity of .the &receptor is declining during the night (Weiss, 1971). The increasing amount of CAMPis sufficient for the stimulation of both enzymes. A further decrease in receptor sensitivity results in a decreased synthesis of CAMP at the end of the night (Weiss, 9171). This may explain the decrease of both enzymes at the end of the night. The
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Fig. 5. Scheme of the synthesis of melatonin from tryptophan, which is probably realized within 36 hr. Above: Try + 5-HTP during one single night: below: N-Ac-5-HT + Me1 during the following night.
225
maximal activity of N-acetyltransferase during the night results in a high synthesis of N-acetyl serotonin (Klein and Weller, 1970). Also during the night there is a maximal activity of HIOMT (Axelrod et al., 1965) and, consequently, a maximal production of melatonin (Lynch, 1971). The synthesis of melatonin from tryptophan occurs for the greater part in darkness. Only the synthesis of serotonin (Figs. 4 and 5), catalyzed by 5-hydroxytryptophan decarboxylase, is high during daytime and low during darkness (Snijder et al., 1965; Pellegrino de Iraldi and Rodriquez de Lores Arnaiz, 1964). This probably means that the synthesis from tryptophan to 5-hydroxytryptophan occurs during one single night; during the day following this night serotonin will be synthesized and the next night serotonin will be converted to N-acetyl serotonin and melatonin. Thus the synthesis of melatonin from tryptophan is probably realized within 36 hours (Fig. 5). Consequently, the serotonin concentration during daytime (Quay, 1963) is crucial for the amount of N-acetyl serotonin to be synthesized. During a long photoperiod, the 0-receptors become more sensitive to norepinephrine which results in a still higher activity of N-acetyltransferase (Deguchi and Axelrod, 1972a,b, 1973a,b) and of tryptophan hydroxylase (Shein and Wurtman, 1971). A high activity of tryptophan hydroxylase means that a great amount of 5-hydroxytryptophan is synthesized and, consequently, also of serotonin during daytime. The high amount of serotonin synthesized during a long photoperiod probably has a dual function. It serves as a substrate for the synthesis of a high amount of N-acetyl serotonin and melatonin, while, by its inhibitory effect on the norepinephrine induced activation of adenylylcyclase, serotonin regulates both the activity of N-acetyltransferase (Fig. 3) and its own synthesis. The regulation of HIOMT activity has been investigated by several authors. Weiss (1968) showed that norepinephrine inhibits HIOMT activity. This observation is in agreement with the data of Brownstein and Heller (1968), who found a decrease in HIOMT activity after nerve stimulation. According to Wartman et al. (1969), acetylcholine is involved in the increase in HIOMT activity. The influence of a parasympathetic agent is observed only during darkness. This suggests that acetylcholine cancels the inhibition of norepinephrine which shows a maximal concentration in darkness. Other substances are probably also involved in the regulation of HIOMT activity in the pineal. Investigations of Cremer-Bartels et al. (1975) and Cremer-Bartels and Hollwich (1 978) on the influence of 2,4,7-triamino-6-phenylpteridineon retinal and pineal HIOMT and the demonstration of the presence of pteridines in the pineal by Ebels et al. (1979) suggest that pteridines may regulate HIOMT activity in the pineal. Therefore, 3 pterins: pterin-6-aldehyde, reduced neopterin and isoxanthopterin were tested in vitro, with the method of Balemans et al. (1978), for the influence on HIOMT activity in the pineal of 4 2 d a y d d , 180 k 10 g, male Wistar rats (Balemans et al., submitted for publication). Every 4 hours animals were decapitated. Three of the extirpated pineals were preincubated separately with pterind-aldehyde, 3 separately with reduced neopterin and 6 separately with solvent system only. In the method used no substrate is added. The 5-hydroxy component present in the pineal can all be methylated to their 5-methoxyproducts after addition of the methyl donor S-adenosylmethionine 3H. In these experiments the pteridine was added to the incubation medium half an hour before addition of the methyl donor. As an indication of HIOMT activity, in the month of October complete day/ night rhythms of the following methylated products were tested: 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-aceticacid and melatonin/5-methoxytryptophol together .
226 DPM/pneal
-1
t
I \
I
I
I
f
\
I
I/
Time (clock hwrsl-
Fig. 6. The day/night rhythm of HIOMT in synthesizing melatonin/5-methoxytyptophol(MEL/S-MTL) (-- - - - -) and the influence of pterindaldehyde (. . . . * .) and reduced neopterin (-) on this enzyme activity during the day/night period. Each point represents the mean i SEM.
Addition of isoxanthopterin to the incubation medium has no influence on the HIOMT activity in the formation of the above mentioned 5-methoxyindoles.Pterin-6-aldehyde, however, causes a shift to the left, towards daytime, on the methylation of 5-HTP, 5-HIAA and SHTL/N-ac-S-HT, and a stimulation of the methylation of 5-HT during daytime. Reduced neopterin causes a shift to the end of darkness on the methylation of 5-HTL/N-ac-5-HTand a stimulation of the methylation of 5-HTP, 5 H T and 5-HIAA during the night period. This means that the synthesis of the 5-methoxyindoles is projected to the light or to the dark period, respectively. The melatonin/5-methoxytryptopholcurve is presented in Fig. 6. It is of interest that Balemans et al. (1979) observed comparable shifts of HIOMT activity in the formation of melatonin/5-methoxytryptopholduring the year. Whether there is any correlation between the duration of the light period and the amount of pteridines or pterins present in the pineal has not been investigated further. These experiments also lead to the question of where and how the pteridines or pterins act in the indole metabolism. REFERENCES Axelrod, J. and Lauber, J.K. (1968) HydroxyindoleO-methyltransferase in several avian species. Bioc h e n Pylarmawl., 11: 828-830. Axelrod, J. and Weissbach, H. (1960) Enzymatic 0-methylation of N-acetylserotonin to melatonin. Science, 131: 1312. Axelrod, J. and Weissbach, H. (1961) Purification and properties of hydroxyindo1e-O-methyl transferase. J. bwl. Chem., 236:211-213. Axelrod, J., Wurtman, R.J. and Snijder, S. (1965) Control of hydroxyindole0-methyltransferaseactivity in the rat pineal gland by environmental lighting. J. bwl. Chem.. 240: 949-955. Backstrtim, M. and Wetterberg, L. (1973) Melatonin formation in rat pineal gland organ culture: induction by an adrenergic p-stimulating drug. Yale J. biol. Med., 46: 630.
227 Balemans, M.G.M., Legerstee, W.C. and Van Benthem, J. (1979) Day and night rhythms in the methylation of N-acetylserotonin/S-hydroxytryptopholin the pineal gland of male rates of different ages, J. neural Transm., 45: In press. Balemans, M.G.M., Noordegraaf, E.M., Bary, F.A.M. and Van Berlo, M.F. (1978) Estimation of the methylathg capacity of the pineal gland. With special reference to indole metabolism. Experientia (Basel), 34: 887-888. Balemans, M.G.M., Van Benthem, J., Legerstee, W.C., De MorBe, A., Noteborn, H.J.P.M. and Ebels, I., The influence of some synthetic pterins on the circadian rhythmicity of HIOMT in synthesizing several 5-methoxyindoles in the pineal gland of 42 days old male Wistar rats. Submitted for publication. Brownstein, M.J. and Heller, A. (1968) Hydroxyindole-O-methyltransferaseactivity: Effect of sympathetic nerve stimulation. Science 162: 367-368. Cardinali, D.P. and Wurtman, R.J. (1972) Hydroxyindole-0-methyltransferasein rat pineal, retina and harderian gland. Endocrinology. 91: 247-252. Cremer-Bartels, G. and Hollwich, F. (1978) Effect of triaminophenylpteridine on hydroxyindole-0methyltransferase of rat pineal gland and bovine retina. J. neural Transm., Suppl. 13: 360. Cremer-Bartels, G., Hollwich, F. and Kotulla, W. (1975) Melatoninbiosynthese in der Sugetierretina in Abhangigkeit von der Adaptation an Belichtung. Klin. Mbl. Augenheilk., 165 : 88-93. Deguchi, T. and Axelrod, J. (1972a) Control of circadian change of serotonin N-acetyltransferaseactivity in the pineal organ by the p-adrenergic receptor. Proc. Nut. Acad. Sci. U.S.A., 69: 2547-2550. Deguchi, T. and Axelrod, J. (1972b) Induction and superinduction of serotonin Nacetyltransferase by adrenergic drugs and denervation in rat pineal organ. Proc. Nut. Acad. Sci. U.S.A.,69: 2208-2211. Deguchi, T. and Axelrod, J. (1973a) Supersensitivity and subsensitivity of the 8-adrenergic receptor in pineal gland regulated by catecholamine transmitter. Proc. Nut. Acad. Sci. U.S.A., 70: 24112414. Deguchi, T. and Axelrod, J. (1973b) Superinduction of serotonin N-acetyltransferase and supersensitivity of adenyl cyclase to catecholamines in denervated pineal gland.Mo1. Pharmacol., 9: 612-618. Ebels, I., De MorBe, A., HusCitharel, A. and Moszkowska, A. (1979) A survey of some active sheep pineal fractions and a discussion on the possible significance of pteridines in those fractions in in vitro and in vivo assays. J. neural Transm., 44: 97-116. Fiske, V.M. (1964) Serotonin rhythm in the pineal organ: Control by the sympathetic nervous system. Sience 146: 253-254. Illnerova, H. (1971) Effect of light on the serotonin content of the pineal gland. Life Sci., 10: 955-960. Jequier, E., Robinson, D.S., Lovenberg, W. and Sjoerdsma, A. (1969) Further studies on tryptophan hydroxylase in rat brainstem and beef pineal. Biochem. Pharmacol., 18: 1071-1081. Kappers, J.A. (1960) The development, topographical relations and innervation of the epiphysis cerebri in the albino rat. Z. Zellforsch., 52: 163-215. Klein, D.C. and Weller, J.L. (1970) Indole metabolism in the pineal gland: A circadian rhythm in N-acetyltransferase. Science, 169: 1093-1095. Klein, D.C., Berg, G.R. and Weller, J.L. (1970a) Melatonin synthesis; adenosine 3’,5’-monophosphate and norepinephrine stimulate N-acetyltransferase. Science, 168: 979-980. Klein, D.C., Berg, G.R., Weller, J.L. and Glinsmann, W. (1970b) Pineal gland: Dibutyryl cyclic adenosine monophosphate stimulation of labeled melatonin production. Science, 167: 1738-1740. Lerner, A.B. and Case, J.D. (1960) Melatonin. Fed. Proc., 19: 590-593. Lerner, A.B., Case, J.D., Takahashi, Y.,Lee, T.H. and Mori, W. (1958) Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Amer. chem. Soc., 80: 2587. Lemer, A.B., Case, J.D., Biemann, K., Anthony, R.V. and Krivis, A. (1959). Isolation of S-methoxyindole-3-acetic acid from bovine pineal glands. J. Amer. chem. Soc., 81 : 5 264. Lovenberg, W., Weissbach, H. and Udenfriend, A. (1962) Aromatic lamino acid decarboxylase. J. Biol. Chem., 237: 89-93. Lovenberg, W., Jequier, E. and Sjoerdsma, A. (1967) Tryptophan hydroxylation. Measurement in pineal gland, brainstem and carcinoid tumor. Science, 155: 217-219. Lynch, H.J. (1971) Diurnal oscillations in pineal melatonin content,Life Science, 10: 791-795. McIsaac, W.M. and Page, I. (1959) The metabolism of serotonin (S-hydroxytryptamine). J. bwl. Chem., 234: 858-864. McIsaac, W.M., Farrell, G., Taborsky, R.G. and Taylor, A.N. (1965) Indole compounds: Isolation from pineal tissue. Science, 148: 102-103.
228 Moore, R.Y. and Klein, D.C. (1974) Visual pathways and the central neural control of a circadian rhythm in pineal serotonin N-acetyltransferase activity. Brain Res., 71 : 17-33. Moore, R.Y., Heller, A., Wurtman, R.J. and Axelrod, J. (1967) Visual pathway mediating pineal response to environmental light. Science, 155: 220-223. Pellegrino de Iraldi, A. and Rodriquez de Lores Amah, G. (1964) 5-Hydroxytryptophandecarboxylase activity in normal and denervated pineal gland of rats. Life Sci., 3: 589-593. Pellegrino de lraldi, A. and Zieher, L.M. (1966) Noradrenaline and dopamine content of normal, decentralized and denervated pineal gland of the rat. Life Sci., 5: 149-154. Quay, W.B. (1963) Circadian rhythm in rat pineal serotonin and its modifications by estrous cycle and photoperiod. Gen. comp. Endocrinol., 3: 473-479. Reiter, R.J. and Fraschini, F. (1969) Endocrine aspects of the mammalian pineal gland: A review. Neurendocrinol., 5 : 2 19 -25 5. Romero,. J.A. and Axehod, J. (1974) Pineal Padrenergic receptor: Diurnal variation in sensitivity. Science, 184: 1091-1092. Romero, J .A. and Axelrod, J. (1975) Regulation of sensitivity to beta-adrenergic stimulation in induction of pineal N-xetyltransferase. Proc. Nut. Acad. Sci. U.S.A., 72: 1661-1665. Shein, H.M. and Wurtman, R.J. (1969) Cyclic adenosine monophosphate: Stimulation of melatonin and serotonin sylrthesis in cultured rat pineals. Science, 166: 5 19-520. Shein, HcM. and Wurtman, R.J. (1971) Stimulation of 4C tryptophan 5-hydroxylation by norepinephrine and dibutyryl adenosine 3’,5‘monophosphate in rat pineal organ cultures. Life Sci., 10: 935940. Shibuya, H., Toru, M. and Watanabe, S. (1978) A circadian rhythm of tryptophan hydroxylase in rat pineals. Brain Res.. 138: 364-368. Snijder, S.H. and Axelrod, J. (1964) A sensitive assay for Shydroxytryptophan decarboxylase, Biochem. Pharmacol., 13: 805-806. Snijder, S.H., Axelrod, J., Wurtman, R.J. and Fischer, J.E. (1965) Control of 5-hydroxytryptophan decarboxylase activity in the rat pineal by sympathetic nerves.J. Pharmucol. exp. Ther., 147: 371-375. Wartman, S.A., Branch, B.J., George, R. and Newman Taylor, A. (1969) Evidence for a cholinergic influence on pineal hydroxyindo1e-O-methyl transferase activity with changes in environmental lighting. Life Sci., 8: 1263-1270. Weiss, B. (1968) Discussion of the formation, metabolism and physiologic effects of melatonin. Aduanc. Pharmacol., 6A: 152-155. Weiss, B. (1969) Effects of environmental lighting and chronic denervation on the activation of adenyl cyclase of rat pineal gland by norepinephrine and sodium fluoride. J. Pharmucol. exp. Ther., 168: 146 -1 53. Weiss. B. (1971) On the regulation of adenyl cyclase activity in the rat pineal gland. Ann. N.Y. Acad. Soc., 185: 505-519. Weiss, B. and Costa, E. (1967) Adenyl cyclase activity in rat pineal gland: Effect of chronic denervation and norepinephrine. Science, 156: 1750-1752. Weiss, B. and Costa, E. (1968a) Selective stimulation of adenyl cyclase of rat pineal gland by pharmacologically active catecholamines. J. Pharmacol. exp. Ther., 161: 310-319. Weiss, B. and Costa, E. (1968b) Regional and subcellular distribution of adenylcyclase and 3’,5’cyclic nucleotide phosphodiesterase in brain and pineal gland. Biochem. Pharmucol., 17: 2107-2116. Weiss, B. and Strada, S.J. (1972) Neuroendocrine control of the cyclic AMP system of brain and pineal gland. Adu. cycl. nucl. Res., 1: 357-375. Weissbach, H.,Redfield, B.G. and Axelrod, J. (1960) Biosynthesis of melatonin. Enzymic conversion of serotonin to N-acetylserotonin. Biochim. biophys. Acta, 43: 352-353. Wurtman, R.J. and Axelrod, J. (1966) A 24hours rhythm in the content of norepinephrine in the pineal and salivary glands of the rat. Life Sci., 5: 665-669. Wurtman, R.J., Axelrod, J. and Fischer, J.E. (1964) Melatonin synthesis in the pineal gland: Effect of light mediated by the sympathetic nervous system. Science, 143: 1328-1330. Wurtman, R.J., Axelrod, J., Sedvall, G. and Moore, R.Y. (1967) Photic and neural control of the 24-hour norepinephrine rhythm in the rat pineal gland. J. Pharmucol. exp. Ther., 157: 487-493. Zatz, M. and O’Dea, F. (1976) Regulation of protein kinase in rat pineal: Increased V max. in supersensitive glands. J. cycl. nucl. Res., 2: 427-439. Zieher, L.M. and Pellegrino de Iraldi, A. (1966). Central control of noradrenaline content in rat pineal and submaxillary glands, Life Sci., 5 : 155-161.
229
DISCUSSION I. NIR: (1) The late peak of noradrenaline towards the end of the night contrary to midnight melatonin and the enzymes involved in its synthesis would fit also into the 36-hr rhythm suggested by you. - (2) We found an inversion in NAT to occur following a reversion in the light/dark cycle. This is a rather slow process taking 48 up to 72 hr. It would be interesting to examine whether any pteridines are formed in the pineal during this process and, if so, whether such an inversion could be performed using pteridines. - (3) Regarding HIOMT determination we have a publication in press on a simplified procedure for its determination using TLC instead of extractions. It gives reproducible and exact results. We encountered difficulties in Fnding a diurnal HIOMT rhythm. M.G.M. BALEMANS: As far as your second question is concerned I can answer that we started only a short time ago with Dr. Ebels with an investigation on the influence of pteridines o n indole mctabolism. So, this matter is something for future research. My answer to your third question is that the HIOMT rhythms mentioned were all obtained with the method developed in our laboratory and not with Axelrod’s method. H. ILLNEROVA: (1) What was the concentration of adenosylmethionine in your in vitro estimation of HIOMT activity? You found a profound rhythm which we have not been able to obtain. We observed a rhythm in HIOMT activity only if we used lower concentrations of adenosylmethionine in the in vitro assay, such as 2-4 X lo6 M. However, when using higher concentrations, such as 4 X lo* M , we could not observe any rhythm. - We have found at least two types of HIOMT in the rat pineal gland according to the kinetics of the reaction: one enzyme with a lower and another with a higher affinity towards adenosylmethionine. Our preliminary investigations show that the enzyme with the higher affinity prevails during night time, while the enzyme with the lower affinity prevails during daytime. - (2) Would not it be more exact to call your rhythms “rhythms in the production of methylated hydroxyindoles” rather than rhythms in the activity of HIOMT as you added no external substrate, thus measuring the activity of the enzyme as well as the concentration of the substrate proper at the same time? - (3) What is the physiological significance of the effect of pteridines on HIOMT activity? Are there any pteridines in the pineal gland? M.G.M. Balemans: (1) The concentration of S-aden~syl-L-[methyl-~H]methionine we used was 0.7 pCi and 1.5 pCi ( a specific activity of 12.2 Ci/mmol; 31 mCi/mg). With these two concentrations we obtained identical results. Using our method it is possible to measure the synthesis of several 5-methoxy components. The experiments carried out with this method suggest the presence of more than one HIOMT enzyme the activity of which is present during the night and during daytime. - (2) I termed the rhythms “rhythms in HIOMT activity in the production of melatonin (or other 5-methoxyindoles)”. I think this agrees with your suggestion. - (3) Dr. Ebels isolated pteridines from pineal tissue, but about their physiological significance related to HIOMT activity we do not know anything, so far. I. SMITH: As a comment on the remarks of Dr. NU on noradrenaline rhythms I should like to remark that the concentrations of pineal noradrenaline and 5 N T are some lo6 times greater than that of melatonin. Hence, rhythms in which concentrations of 5-HT + NA drop, say to 10’ times greater than that of melatonin, seem unlikely to have any effect on HIOMT and melatonin synthesis. M.G.M. BALEMANS: I do not know in which concentrations NA and 5HT exert physiological effects on the indole metabolism in vivo. On the other hand these two compounds also may have other functions in the pineal gland. L. VOLLRATH: I was very much impressed by your last slide showing an inverse HIOMT rhythm in May and September. How many times did you repeat this experiment? Karyometric studies carried out in my laboratory show that even within one week an inversion of the circadian rhythm may be demonstrable. M.G.M. BALEMANS: The HIOMT rhythms were investigated in 1977 and 1978. The results obtained in both years were completely identical. In addition to your studies I need to mention that all experiments have been done from Tuesday 08.00 to Wednesday 08.00.
