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night differences in pineal indoles in the adult pigeon (Columba livia)
Camp. Biochem.
f’hrsiol.
Vol. 78C, No. 1,pp. 141.-143, 1984
03064492184$3.00fO.00 c‘ 1984Pergamon PressLtd
Printed in Great B&in
DAY/NIGHT DIFFERENCES IN PINEAL INDOLES IN THE ADULT PIGEON (COLUMBA LIVIA) RICHARD K. GRADY, JR, ALBERT CALICURI and IVAN N. MEFFORD Boston College Department of Chemistry, Chestnut Hill. MA 02167, USA. Telephone: (617) 552 3615 (Receioed 3 I August 1983) Abstract-l. Day/night differences in concentrations of 5-hydroxy and 5-methoxy indole metabolites in the pineal gland of the pigeon are described. 2. A simultaneous determination of 5-hydroxytryptamine (serotonin), 5-hydroxyindol~cetic acid, 5-hydroxytryptophol~ ~~-a~tyl-5-hydroxytryp~dmine (N-acetyl serotonin), 5-methoxyindolea~etic acid. S-methoxytryptophol, tryptophan, indoleacetic acid and melatonin was accomplished using a recently developed procedure employing high-performance liquid chromatography with electrochemical detection. 3. As in mammalian species, an inverse relationship was observed between N-acetylated indoles and serotonin and its acid metabolites. 4. Melatonin and N-acetyl serotonin were increased approximately three-fold at night to concentrations of 0.730 and 1.79 ng/pineal respectively. 5. Daytime serotonin values were 44.9 + 13.0 ngjpineal and decreased to 12.3 + 6.5 ng/pineal during the dark phase.
INTRODUCTION
the results obtained.
Extensive literature describes the rhythm in indole metabolism in several mammalian species (Reiter, 1980; Young and Anderson, 1982; Illnerova, 1971; Rollag et al., 1980). Production of the active pineal indole hormone, melatonin, is regulated by the light/dark cycle, via the induction of serotonin Nacetyl transferase (SNAT) (Klein and Moore, 1979). The avian pineai has been less widely studied although it is apparent that it may possess both photoreceptive and endocrine functions (Doi et al., 1983). Several authors have shown that avian pineal SNAT shows a marked diurnal rhythm synchronized with the light/dark cycle (Brinkley et al., 1974, 1978). Few of the various indoles associated with the synthesis of me~atonin have been measured in the avian pineal. It has been established that melatonin shows a distinct rhythm in the chick (Brinkley et al., 1974; Pelham, 1975; Brinkley, 1983) and quail (Ralph et al., 1967), and that serotonin and 5-hydroxyindoleacetic acid show a diurnal rhythm in the pigeon (Quay, 1966). We have examined the concentrations of the entire spectrum of hydroxyindoles, methoxyindoles and N-acetylated indoles in the pigeon pineal during the light and dark phase to ascertain the similarities between indole metabolism in the avian and mammalian pineal. MATERIALS AND METHODS Adult domestic homing pigeons (Co~ffmbu hia) bred and raised at a local rookery were used. Animals were adapted to natural external lighting patterns and were sacrificed in mid-June during a daily photoperiod of 15 hr light; 9 hr darkness (sunrise at about 0530 hr; darkness at about 2030 hr). Animals varied in age from 1to 2 years. Both sexes were used as no apparent sex differences were observed in
Sacrifice during the light cycle was accomplish~ between 1200 and 1400 hr and during the dark cycle between 2400 and 0200 hr. Pineals were removed and immediately frozen on dry ice and stored at -80 C until analyzed. Sample preparaiion
To each frozen pineal was added 100 p I of 0.2 M HCIO,. The tissue was sonicated (Branson Sonitier cell disruptor 200) for approximately 5 set to thoroughly disrupt cell membranes. Tissue debris was removed by centrifugation at 12,800g for 5min (Fisher Microcentrifuge Model No. 235A). The clear supernatant was then applied directly to the chromatography system. Separation of the various indoles was accomplished using reversed-phase high-performance liquid chromatography on a C18 anslytical col;mn (IBM Inst;uments, inc..-Da&&y, CT, USA) 15 cm length x 4.5 mm i.d., 5 urn oarticle size. Isocratic elution was &complished using a solvent of 0. I M ammonium acetate, 0.1 M acetic acid and 10% acetone (v/v),
a modification
of our previously
reported
method
(Mefford and Barchas, 1980). A flowrate of 2.0 mljmin was maintained using a Milton Roy minipump with additional pulse dampening. Sample was applied to the column with a 20~1 fixed loop injection valve (Rheodyne Model 7125). Detection of the separated indoles was a~omplished amperometricaily at a glassy carbon electrode mainldined at 4-0.9 V vs a Ag/AgCl reference. A model LC-2A amperometric controller (BioAnalytical Systems, Inc., West Lafayette, Indiana, U.S.A.) was used. Chromatograms were recorded on a strip chart recorder. Peak identities were established with authentic standards (Sigma Chemical Co., St Louis, Missouri, U.S.A.). Calibration curves established linearity for all compounds from 0.01 to 100 ng injected.
RESULTS The concentrations in the adult pigeon Significant day/night 141
of the various indoles present pineal are shown in Table I. differences were observed for
142
R. K.
GRADY. JR et
al.
most but not all of the compounds measured. Serotonin was decreased at night from 45 ng/pineal to 12.3 ng/pineal. Considerable inter-animal variability was observed. Both melatonin and N-acetyl serotonin were found to be present during the light period. Only a three-fold increase was observed for these products of N-acetylation during the dark phase. Products of monoamine oxidase (MAO), 5-HIAA, 5-MIAA and 5hydroxytryptophol were decreased at night as has been previously observed in the rat (Mefford et al., 1983). However fi-methoxytryprophol, which was detected in seven of the ten animals, did not show the nighttime decrease observed with other indole metabolites originating from MAO degradation. Neither tryptophan nor indole-3acetic acid showed a diurnal variation. DISCUSSION
Our results indicate that diurnal rhythms in indole metabolism in the adult pigeon pineal are similar to those observed in mammals (Reiter, 1980; Young and Anderson, 1982; Illnerova, 1971; Rollag et al., 1980; Mefford et al., 1983). Numerically, our results are comparable to those observed in the rat, but are somewhat at variance with other published data on avian pineal. Quay (1966) reported midday levels of serotonin in pigeon pineal of approximately 150 ng/ pineal and nighttime levels of approximately 50 ng/ pineal. In this same work, daytime 5-HIAA concentrations ranged from 7 to 12 ng/pineal. Nighttime concentrations decreased to 4 ng/gland at the middle of the dark cycle. The present results show essentially the same trends although the absolute values observed in this work are somewhat lower. The observed nighttime rise in N-acetyl serotonin and melatonin concentrations are also somewhat less than anticipated, although no literature values were available for the pigeon. In domestic chickens, melatonin has been reported to vary by a factor of ten (Brinkley et al., 1974). N-acetyltransferase and HIOMT activities have also been reported to be considerably higher in the chicken than the rat, although melatonin concentrations are reported to be similar (Brinkley et al., 1974). One factor which may contribute to the lower than expected observed concentrations of melatonin has been recently described (Rieter et al., 1983). Thirteen-lined ground squirrels acclimated to natural external lighting showed lower nighttime pineal melatonin concentrations than squirrels under a fixed-cycle laboratory lighting schedule, presumably because of the intensity of the natural sunlight exposure. The pigeons used in the present study were raised under natural lighting conditions; therefore, a similar mechanism could be in play. Acid and neutral metabolites and serotonin were decreased at night in the pigeon. This phenomenon has been reported in mammals, and presumably occurs via mass action as the increase in N-acetylase activity shunts the metabolism of serotonin to the N-acetylated products instead of monoamine oxidase products (Mefford et al., 1983). Although no enzymatic measurements were made in this work, these data support the position that serotonin metabolism in the pigeon is similar to that of other avians and to
Pineal indoles in pigeons mammals. The failure to observe a rhythm in tryptophan concentrations suggests that no marked changes in tryptophan hydroxylase activity or tryptophan uptake occur through the LD cycle consistent with observations in mammals. We have applied a sensitive and specific analytical technique, high-performance liquid chromatography with electrochemical detection, to simultaneous measurement of the entire spectrum of indole metabolites in the pineal of the adult pigeon (Columba /ivia). Our results indicate that indole metabolism in this species is similar to that observed in other birds, as well as in mammals. REFERENCES
Brinkley S. (1983) Rhythms in ocular and pineal Nacetyltransferase: a portrait of an enzyme clock. Camp. Biochem. Phvsiol. 75A. 123-129.
Brinkley S., M&Bride S. E., Klein D. C. and Ralph C. L. (1974) Pineal enzymes: regulation of avian melatonin synthesis. Science 181, 273-275. Brinkley S., Riebman J. B. and Reilly K. B. (1977) Timekeeping by the pineal gland. Science 197, 1181-l 183. Doi 0, Koyama E., Nakamura T. and Tanabe Y. (1983) Photoperiodic regulation of serotonin N-acetyltransferase activity in the pineal gland of the chicken. Comp. Biochem. Physiol. 74, 195-I 98. Illnerova H. (1971) The effect of light on the serotonin content of the pineal gland. Life Sci. 10, 955-960. Klein D. C. and Moore R. Y. (1979) Pineal N-acetyltransferase and hydroxyindole-O-methyltransferasecontrol by retino-hypothalamic tract and the suprachiasmatic nucleus, Brain Res. 174, 245-262.
143
Mefford I. N. and Barchas J. D. (1980) Determination of tryptophan and metabolites in rat brain and pineal tissue by reverse-phase high-performance liquid chromatography with electrochemical detection. J. Chromat. 181, 187-193. Mefford I. N., Chang P., Klein D. C., Namboodiri M. A. A., Sugden D. and Barchas J. D. (1983) Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal. Endocrinology 113, 1582-1586. Pelham R. W. (1975) A serum melatonin rhythm in chickens and its abolition by pinealectomy. Endocrinology 96, 543-546. Quay W. B. (1966) Rhythmic and light-induced changes in levels of pineal 5-hydroxyindole in the pigeon (Columha livia). Gen. camp. Endocrin. 6, 371-371.
Ralph C. L., Hedlund L. and Murphy W. A. (1967) Diurnal cycles of melatonin and bird pineal bodies, Camp. Biothem. Physiol. 22, 591-599.
Reiter R. J. (1980) The pineal and its hormones in the control of the reproduction in mammals. Endocrine Rev. 1, 109-131. Reiter R. J., Steinlechner S., Richardson B. A. and King T. S. (1983) Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 134ined ground squirrels (Spermophilus tridecemlineatus). Life Sci. 32, 2625-2629. Rollag M. D., Panke E. S., Trakuhungsi N. K., Trakulrungsi C. and Reiter R. J. (1980) Quantitation of daily melatonin synthesis in the hamster pineal gland. Endocrinology 106, 23 l-236. Young S. N. and Anderson G. M. (1982) Factors influencing melatonin, 5-hydroxytryptophol, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine and tryptophan in rat pineal glands. Neuroendocrinology 35, 464-468.
f’hrsiol.
Vol. 78C, No. 1,pp. 141.-143, 1984
03064492184$3.00fO.00 c‘ 1984Pergamon PressLtd
Printed in Great B&in
DAY/NIGHT DIFFERENCES IN PINEAL INDOLES IN THE ADULT PIGEON (COLUMBA LIVIA) RICHARD K. GRADY, JR, ALBERT CALICURI and IVAN N. MEFFORD Boston College Department of Chemistry, Chestnut Hill. MA 02167, USA. Telephone: (617) 552 3615 (Receioed 3 I August 1983) Abstract-l. Day/night differences in concentrations of 5-hydroxy and 5-methoxy indole metabolites in the pineal gland of the pigeon are described. 2. A simultaneous determination of 5-hydroxytryptamine (serotonin), 5-hydroxyindol~cetic acid, 5-hydroxytryptophol~ ~~-a~tyl-5-hydroxytryp~dmine (N-acetyl serotonin), 5-methoxyindolea~etic acid. S-methoxytryptophol, tryptophan, indoleacetic acid and melatonin was accomplished using a recently developed procedure employing high-performance liquid chromatography with electrochemical detection. 3. As in mammalian species, an inverse relationship was observed between N-acetylated indoles and serotonin and its acid metabolites. 4. Melatonin and N-acetyl serotonin were increased approximately three-fold at night to concentrations of 0.730 and 1.79 ng/pineal respectively. 5. Daytime serotonin values were 44.9 + 13.0 ngjpineal and decreased to 12.3 + 6.5 ng/pineal during the dark phase.
INTRODUCTION
the results obtained.
Extensive literature describes the rhythm in indole metabolism in several mammalian species (Reiter, 1980; Young and Anderson, 1982; Illnerova, 1971; Rollag et al., 1980). Production of the active pineal indole hormone, melatonin, is regulated by the light/dark cycle, via the induction of serotonin Nacetyl transferase (SNAT) (Klein and Moore, 1979). The avian pineai has been less widely studied although it is apparent that it may possess both photoreceptive and endocrine functions (Doi et al., 1983). Several authors have shown that avian pineal SNAT shows a marked diurnal rhythm synchronized with the light/dark cycle (Brinkley et al., 1974, 1978). Few of the various indoles associated with the synthesis of me~atonin have been measured in the avian pineal. It has been established that melatonin shows a distinct rhythm in the chick (Brinkley et al., 1974; Pelham, 1975; Brinkley, 1983) and quail (Ralph et al., 1967), and that serotonin and 5-hydroxyindoleacetic acid show a diurnal rhythm in the pigeon (Quay, 1966). We have examined the concentrations of the entire spectrum of hydroxyindoles, methoxyindoles and N-acetylated indoles in the pigeon pineal during the light and dark phase to ascertain the similarities between indole metabolism in the avian and mammalian pineal. MATERIALS AND METHODS Adult domestic homing pigeons (Co~ffmbu hia) bred and raised at a local rookery were used. Animals were adapted to natural external lighting patterns and were sacrificed in mid-June during a daily photoperiod of 15 hr light; 9 hr darkness (sunrise at about 0530 hr; darkness at about 2030 hr). Animals varied in age from 1to 2 years. Both sexes were used as no apparent sex differences were observed in
Sacrifice during the light cycle was accomplish~ between 1200 and 1400 hr and during the dark cycle between 2400 and 0200 hr. Pineals were removed and immediately frozen on dry ice and stored at -80 C until analyzed. Sample preparaiion
To each frozen pineal was added 100 p I of 0.2 M HCIO,. The tissue was sonicated (Branson Sonitier cell disruptor 200) for approximately 5 set to thoroughly disrupt cell membranes. Tissue debris was removed by centrifugation at 12,800g for 5min (Fisher Microcentrifuge Model No. 235A). The clear supernatant was then applied directly to the chromatography system. Separation of the various indoles was accomplished using reversed-phase high-performance liquid chromatography on a C18 anslytical col;mn (IBM Inst;uments, inc..-Da&&y, CT, USA) 15 cm length x 4.5 mm i.d., 5 urn oarticle size. Isocratic elution was &complished using a solvent of 0. I M ammonium acetate, 0.1 M acetic acid and 10% acetone (v/v),
a modification
of our previously
reported
method
(Mefford and Barchas, 1980). A flowrate of 2.0 mljmin was maintained using a Milton Roy minipump with additional pulse dampening. Sample was applied to the column with a 20~1 fixed loop injection valve (Rheodyne Model 7125). Detection of the separated indoles was a~omplished amperometricaily at a glassy carbon electrode mainldined at 4-0.9 V vs a Ag/AgCl reference. A model LC-2A amperometric controller (BioAnalytical Systems, Inc., West Lafayette, Indiana, U.S.A.) was used. Chromatograms were recorded on a strip chart recorder. Peak identities were established with authentic standards (Sigma Chemical Co., St Louis, Missouri, U.S.A.). Calibration curves established linearity for all compounds from 0.01 to 100 ng injected.
RESULTS The concentrations in the adult pigeon Significant day/night 141
of the various indoles present pineal are shown in Table I. differences were observed for
142
R. K.
GRADY. JR et
al.
most but not all of the compounds measured. Serotonin was decreased at night from 45 ng/pineal to 12.3 ng/pineal. Considerable inter-animal variability was observed. Both melatonin and N-acetyl serotonin were found to be present during the light period. Only a three-fold increase was observed for these products of N-acetylation during the dark phase. Products of monoamine oxidase (MAO), 5-HIAA, 5-MIAA and 5hydroxytryptophol were decreased at night as has been previously observed in the rat (Mefford et al., 1983). However fi-methoxytryprophol, which was detected in seven of the ten animals, did not show the nighttime decrease observed with other indole metabolites originating from MAO degradation. Neither tryptophan nor indole-3acetic acid showed a diurnal variation. DISCUSSION
Our results indicate that diurnal rhythms in indole metabolism in the adult pigeon pineal are similar to those observed in mammals (Reiter, 1980; Young and Anderson, 1982; Illnerova, 1971; Rollag et al., 1980; Mefford et al., 1983). Numerically, our results are comparable to those observed in the rat, but are somewhat at variance with other published data on avian pineal. Quay (1966) reported midday levels of serotonin in pigeon pineal of approximately 150 ng/ pineal and nighttime levels of approximately 50 ng/ pineal. In this same work, daytime 5-HIAA concentrations ranged from 7 to 12 ng/pineal. Nighttime concentrations decreased to 4 ng/gland at the middle of the dark cycle. The present results show essentially the same trends although the absolute values observed in this work are somewhat lower. The observed nighttime rise in N-acetyl serotonin and melatonin concentrations are also somewhat less than anticipated, although no literature values were available for the pigeon. In domestic chickens, melatonin has been reported to vary by a factor of ten (Brinkley et al., 1974). N-acetyltransferase and HIOMT activities have also been reported to be considerably higher in the chicken than the rat, although melatonin concentrations are reported to be similar (Brinkley et al., 1974). One factor which may contribute to the lower than expected observed concentrations of melatonin has been recently described (Rieter et al., 1983). Thirteen-lined ground squirrels acclimated to natural external lighting showed lower nighttime pineal melatonin concentrations than squirrels under a fixed-cycle laboratory lighting schedule, presumably because of the intensity of the natural sunlight exposure. The pigeons used in the present study were raised under natural lighting conditions; therefore, a similar mechanism could be in play. Acid and neutral metabolites and serotonin were decreased at night in the pigeon. This phenomenon has been reported in mammals, and presumably occurs via mass action as the increase in N-acetylase activity shunts the metabolism of serotonin to the N-acetylated products instead of monoamine oxidase products (Mefford et al., 1983). Although no enzymatic measurements were made in this work, these data support the position that serotonin metabolism in the pigeon is similar to that of other avians and to
Pineal indoles in pigeons mammals. The failure to observe a rhythm in tryptophan concentrations suggests that no marked changes in tryptophan hydroxylase activity or tryptophan uptake occur through the LD cycle consistent with observations in mammals. We have applied a sensitive and specific analytical technique, high-performance liquid chromatography with electrochemical detection, to simultaneous measurement of the entire spectrum of indole metabolites in the pineal of the adult pigeon (Columba /ivia). Our results indicate that indole metabolism in this species is similar to that observed in other birds, as well as in mammals. REFERENCES
Brinkley S. (1983) Rhythms in ocular and pineal Nacetyltransferase: a portrait of an enzyme clock. Camp. Biochem. Phvsiol. 75A. 123-129.
Brinkley S., M&Bride S. E., Klein D. C. and Ralph C. L. (1974) Pineal enzymes: regulation of avian melatonin synthesis. Science 181, 273-275. Brinkley S., Riebman J. B. and Reilly K. B. (1977) Timekeeping by the pineal gland. Science 197, 1181-l 183. Doi 0, Koyama E., Nakamura T. and Tanabe Y. (1983) Photoperiodic regulation of serotonin N-acetyltransferase activity in the pineal gland of the chicken. Comp. Biochem. Physiol. 74, 195-I 98. Illnerova H. (1971) The effect of light on the serotonin content of the pineal gland. Life Sci. 10, 955-960. Klein D. C. and Moore R. Y. (1979) Pineal N-acetyltransferase and hydroxyindole-O-methyltransferasecontrol by retino-hypothalamic tract and the suprachiasmatic nucleus, Brain Res. 174, 245-262.
143
Mefford I. N. and Barchas J. D. (1980) Determination of tryptophan and metabolites in rat brain and pineal tissue by reverse-phase high-performance liquid chromatography with electrochemical detection. J. Chromat. 181, 187-193. Mefford I. N., Chang P., Klein D. C., Namboodiri M. A. A., Sugden D. and Barchas J. D. (1983) Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal. Endocrinology 113, 1582-1586. Pelham R. W. (1975) A serum melatonin rhythm in chickens and its abolition by pinealectomy. Endocrinology 96, 543-546. Quay W. B. (1966) Rhythmic and light-induced changes in levels of pineal 5-hydroxyindole in the pigeon (Columha livia). Gen. camp. Endocrin. 6, 371-371.
Ralph C. L., Hedlund L. and Murphy W. A. (1967) Diurnal cycles of melatonin and bird pineal bodies, Camp. Biothem. Physiol. 22, 591-599.
Reiter R. J. (1980) The pineal and its hormones in the control of the reproduction in mammals. Endocrine Rev. 1, 109-131. Reiter R. J., Steinlechner S., Richardson B. A. and King T. S. (1983) Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 134ined ground squirrels (Spermophilus tridecemlineatus). Life Sci. 32, 2625-2629. Rollag M. D., Panke E. S., Trakuhungsi N. K., Trakulrungsi C. and Reiter R. J. (1980) Quantitation of daily melatonin synthesis in the hamster pineal gland. Endocrinology 106, 23 l-236. Young S. N. and Anderson G. M. (1982) Factors influencing melatonin, 5-hydroxytryptophol, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine and tryptophan in rat pineal glands. Neuroendocrinology 35, 464-468.