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Influence of δ-sleep inducing peptide on melatonin synthesis in the rat pineal gland
Ncur~;sciem'e Letters, 70 (1986) 127 131 Elsevier
127
NSL 04154
Influence of,4-sleep inducing peptide on melatonin synthesis in the rat pineal gland Sol O a k n i n l, M a u r e e n E. Troiani l, Susan M. W e b b 2 and Russel J. Reiter t il)cl ~artutcHl O/('e/IMar and Structural Bioloau. 4uloHomou.s' { uivcr.~Jl.l', Barc{'lo#la f .~'p(Ihl ) ( Received 22 April 1986: Acceptcd 12 .Iune 1986)
Key wor~£" (5-Sleepinducing peptide
Pineal gland
N-Acetyltransferase
Melatonin
Rat
The influence of
('orre.v;omlence. R.J. Reitcr, Department of Cellular and Structural Biology, The University of Texas. [ [ealth Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. 0304-3940 8 6 S 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
12~
The observations of G r a f et al. [7] in reference to the suppressive effects of DSIP on pineal NAT were of particular interest to us since one of the goals of this laboratory is to define factors controlling the nocturnal rise in pineal melatonin production. NAT, the enzyme which converts serotonin to N-acetylserotonin (the immediate precursor of melatonin), seems to be especially important in controlling the quantity of melatonin formed [2]. Typically, substances or conditions that interfere with the nocturnal rise in the activity of the acetylating enzyme also depress the nighttime increase in melatonim an important hormone of the pineal gland [13, 14]. Thus, the purpose of the present study was to examine the effect of DSIP on the activity of the serotonin acetylating enzyme and the content of melatonin in the pineal gland of rats treated with DSIP. It was of particular importance to investigate the levels of these constituents at night when the values are normally the highest. Young adult male rats were purchased from Charles River (Cambridge, MA). The animals were maintained in a light- and temperature (22_+ 2°C)-controlled room; the light d a r k (LD) cycle was maintained at 14:10 (in hours) during the course of the study. Lights were automatically turned on at 06.00 h and off at 20.00 h daily. The rats were provided with standard laboratory chow and tap water ad libitum. Rats received an i.p. injection of either diluent alone or 15, 30 or 60 nmot/kg DSIP at 20.00 h, immediately before the onset of darkness. Groups of animals from each of the groups were subsequently killed at 3 points during the dark phase (23.00, 01.00 and 03.00 h) and 08.00 h during the early light phase of the LD cycle. To aid in the collection of the pineal glands, animals were decapitated under a dim red light; red light has been shown not to influence the nocturnal rise in either pineal N A T or melatonin. After removing the pineal glands they were individually frozen on solid CO2 and stored frozen until the time of assay of NAT and melatonin. The procedure for _ • Saline
0 Low Dose (15 rnmole/kg) (-
Med Dose (30 mmole/kg) E] High Dose (60 mmole/kg)
O_
>.. -~-> ° - -
2-
° - -
L)
1/
Z O-
20.00
24.00
04.00
08.00
Figi 1. N A T activity in the pineal gland of rats treated with either saline or DSIP before lights out. The black bar represents the period of darkness, An indication of variation within the individual groups is not included in the figure because they would have greatly confounded the presentation. * P < 0 . 0 2 vs saline-treated controls.
129 600 i
Sabine
o Law Dose (15 mmole/kg) -~ c-
50O
A Med Dose (30 mmole/kg) //,' ///
400
tO
-4---
300
,
X), '
D High Dose (60 mmale/kg)
/7 o ,
i
0.._
/
"
\
2004i
0 100
---7
20.00
24.00
04.00
08.00
Fig. 2. Mehtlonin levels in the pineal gland of rats treated with either saline or DSIP before lights out. "File black bar represents the period of darkness. An indication of variation within the individual groups is not inchided in the figure because they would have greatly conflmndcd the presentation. *P<0.02 vs saline-treated controls. enzyme analysis [1] and the r a d i o i m m u n o a s s a y for melatonin [16] have been previously described. Data were statistically analyzed using an A N O V A followed by a Student N e w m a n Keuls test. The procedures used in this experiment have been previously employed in this laboratory to investigate the metabolism o f serotonin to melatonin within the pineal gland [I]. In all animals darkness was associated with an increase in the activity o f the enzyme which ,N-acetylates serotonin as well as in melatonin. As reported by G r a f et al. [7], one dose o f D S I P (15 rnmol/kg), injected in the evening before lights out, caused a significant inhibition ( P < 0 . 0 2 ) o f pineal N A T activity at 3 h into the dark phase (Fig. I). The inhibition, however, was not apparent at the subsequent time points. Melatonin exhibited a similar response. Again, the lowest dose o f D S I P (15 nmol/kg) significantly ( P < 0 . 0 2 ) retarded the nighttime rise in pineal melatonin at 11.00 h but the effect was not apparent at the other two nighttime points or at 08.00 h alter light onset (Fig. 2). As did G r a f et al. [7], we report here that the administration o f D S I P just before darkness onset delays the subsequent rise in pineal N A T activity; furthermore, we found that melatonin levels are likewise inhibited. It is also clear from the present data, as well as the earlier findings, that the inhibitory effect is highly dose specific. Hence, at only one dosage (15 nmol/kg) was D S I P able to significantly alter the nocturnal rise in enzyme activity and, therefore, melatonin production. This dosage dit L fers from the dose reported by G r a f et al. to inhibit pineal N A T activity [7] (15 nmol/ kg in the present study vs 30 n m o l / k g in the experiment o f G r a f et al.). This could be a result o f the use of different strains o f rats which m a y metabolize D S I P at slightly different rates or due to some u n k n o w n factor. A rapid metabolism o f D S I P could also explain the short-lived nature o f the suppressive effect o f the peptide on
130
pineal melatonin production. G r a f et al. [7] noted the inhibition at 4 h into the dark phase whereas in the present study a significant reduction in N A T activity and melatonin level was observed only 3 h after D S I P was administered. On the whole, the differences that were seen in the DSIP-injected animals were not great. Whether the changes were sufficiently great to alter the physiology o f the pineal gland relative to its endocrine actions c a n n o t be determined at this time. The fact that a similar effect was obtained in two separate studies conducted in different laboratories suggests the alteration is real; however, the physiological consequences o f the change remain the subject o f subsequent investigations. The changes induced by D S I P are similar to those which follow endomethacin administration [15]; endomethacin is a prostaglandin synthesis inhibitor. Thus, there appear to be at least some endogenously produced c o m p o u n d s that can, under certain circumstances, at least slightly modify pineal indoleamine metabolism. It is unlikely that the change observed was merely due to a phase shift o f the rise in melatonin production. Typically, the onset o f darkness is associated with a rise in both N A T activity and melatonin content o f the rat pineal gland [il]; these values usually return to daytime levels before lights on. After D S I P injection this pattern was also noted. The rise occurred, although it was blunted, and likewise the drop anticipated lights on with basal daytime levels being apparent the subsequent morning. This is only one o f a n u m b e r o f effects of D S l P in rats. Usually, its administration is associated with changes in circadian l o c o m o t o r activity [5]. The rhythms in the pineal gland as well as the 24-h l o c o m o t o r rhythm both originate from electrical activity in the suprachiasmatic nuclei (SCN) o f the hypothalamus. Thus, the c o m m o n site o f action for D S I P for both parameters could be the SCN. The S C N are k n o w n to be important relay nuclei in the transfer o f information from tire retina to the pineal gland [14]. Interestingly, the S C N have been proposed as a possible site o f action o f m e l a t o n i n as well [13]. In summary, these data suggest a m i n o r role for D S I P in controlling the circadian rhythmicity o f the pineal gland. The effect seems to be highly dose dependent and rather short lived. Besides an effect on N A T and melatonin, D S I P could have other actions in the pineal gland. Research supported by N S F G r a n t 8410592, G r a n t CCA-8411106 from the Spanish American Committee and Caja A h o r r o s del Tenerife, La L a g u n a University. 1 Champney, T.H., Holtorf, A.P., Steger, R.W. and Reiter, R.J., Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland, J. Neurosci. Res., 11 (1984) 59-66. 2 Ebadi, M., Regulation of the synthesis of melatonin and its significance to neuroendocrinology. In R.J. Reiter (Ed.), The Pineal Gland, Raven Press, New York, 1984,pp. l 37. 3 Feldman, S.C. and Kastin, A.J., Localization of neurons containing immunoreactive delta sleep inducing peptide in the rat brain: an immunocytochemical study, Neuroscience, 11 (1984) 303 318. 4 Gral, M., Christen, H., Tobler, H.J., Maier, P.F. and Schoenenberger, G.A., Effects of repeated DSIP and DSIP-P administration on the circadian locomotor activity of rats, Pharmacol. Biochem. Behav., 15(1981)717 721.
131
5 (h'af. M., Uhi-isten, H. and Schoenenberger, G.A., DSIP/DSIP-P and circadian motor activity of rats under continuous light, Peptides, 3 (1982) 623 626. 6 Graf, M. and Kastin. A.J., Delta-sleep-inducing peptide: a review, Neurosci. Biobehav. Rex'.. g (1984) S3 9:;. 7 ( h'af, M., Kastin. A.J. and Schoenenberger, G.A., Delta sleep reducing peptide and t ~ o of its analogs rcducc nocturnal increase of N-acetyllransfizrase activity in rat pineal gland, J. Ncurochem., 44 (198'~) 629 632. ,v Kastm, A.J., ('astellanos, P.F., Banks, W.A. and Coy, D.H., Radioimmunoassay of DSIP like material in h u m a n hlood: possible protein binding, Pharmacol. Biochem. Behav., 15 (1981)969 974. 9 Kaslin, A.J., Dickson, J.C. and Fischman, A.J., DSIP like material in rats running a c~mlplex maze, Ph>si,',l. Bchav.. 33 (1984) 427 431. l0 Kastin. A.J.. Nissen. C. and Coy. D.H.. Permeability of the bl,.',od bram barrier to DSIP pcplides. Pharmac,.~l. Biochem. Behav.. 15 ( 19gl ) 955 959. I I Kastin, A.J., Nissen, ('.. Schally. A.V. and Coy, D H . , Additional evidence that small a m o u n t s of peptides can cross t h e h l o o d h r a i n h a r r i e r , Pharmacol. Biochem. Behav., II (1979) 717 719. [ 2 Monnicr, M. and 1loesli, L., Dialysis of sleep and waking factors in hlood of rahhit, Science, 146 (1964) 796 798. 13 Reitcr. RJ., The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev., 1 {1980) 109 131. [4 Rcitcr, R.,I., Ncurocndocrine effects of the pineal gland and of melatonin. In W.F. ( i a n o n g and 1.. Martini (Eds.I. Frontiers in Neuroendocrinology, Vol. I, Raven Press, New York. 1982, pp. 287 316. 15 Relier, R.J.. Steinlechner, S. and Richardson. B.A., Daytime and nighttime pincal N-acetyltransferase activity and melatonm content in male rats treated with indomethacin, a prostaglandin synthesis inhihitor, Neuroendocr. Lett.,2(1986) 281 287. 16 Rollag. M.I). and Niswender, G.D., Radioimmunoassay of serum concentrations of melatonin m shccp exposed to different lighting regimens. Endocrinology, 98 (I 976) 482 489. 17 Schoenenherg, (}.A. and Monnier, M., ('haracterization of a delta-electroencephalogram (-sleepl inducingpeptidc, Proc. Nall. Acad. Sci. USA, 74(1977) 1281 1286.
127
NSL 04154
Influence of,4-sleep inducing peptide on melatonin synthesis in the rat pineal gland Sol O a k n i n l, M a u r e e n E. Troiani l, Susan M. W e b b 2 and Russel J. Reiter t il)cl ~artutcHl O/('e/IMar and Structural Biolo
Key wor~£" (5-Sleepinducing peptide
Pineal gland
N-Acetyltransferase
Melatonin
Rat
The influence of
('orre.v;omlence. R.J. Reitcr, Department of Cellular and Structural Biology, The University of Texas. [ [ealth Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. 0304-3940 8 6 S 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
12~
The observations of G r a f et al. [7] in reference to the suppressive effects of DSIP on pineal NAT were of particular interest to us since one of the goals of this laboratory is to define factors controlling the nocturnal rise in pineal melatonin production. NAT, the enzyme which converts serotonin to N-acetylserotonin (the immediate precursor of melatonin), seems to be especially important in controlling the quantity of melatonin formed [2]. Typically, substances or conditions that interfere with the nocturnal rise in the activity of the acetylating enzyme also depress the nighttime increase in melatonim an important hormone of the pineal gland [13, 14]. Thus, the purpose of the present study was to examine the effect of DSIP on the activity of the serotonin acetylating enzyme and the content of melatonin in the pineal gland of rats treated with DSIP. It was of particular importance to investigate the levels of these constituents at night when the values are normally the highest. Young adult male rats were purchased from Charles River (Cambridge, MA). The animals were maintained in a light- and temperature (22_+ 2°C)-controlled room; the light d a r k (LD) cycle was maintained at 14:10 (in hours) during the course of the study. Lights were automatically turned on at 06.00 h and off at 20.00 h daily. The rats were provided with standard laboratory chow and tap water ad libitum. Rats received an i.p. injection of either diluent alone or 15, 30 or 60 nmot/kg DSIP at 20.00 h, immediately before the onset of darkness. Groups of animals from each of the groups were subsequently killed at 3 points during the dark phase (23.00, 01.00 and 03.00 h) and 08.00 h during the early light phase of the LD cycle. To aid in the collection of the pineal glands, animals were decapitated under a dim red light; red light has been shown not to influence the nocturnal rise in either pineal N A T or melatonin. After removing the pineal glands they were individually frozen on solid CO2 and stored frozen until the time of assay of NAT and melatonin. The procedure for _ • Saline
0 Low Dose (15 rnmole/kg) (-
Med Dose (30 mmole/kg) E] High Dose (60 mmole/kg)
O_
>.. -~-> ° - -
2-
° - -
L)
1/
Z O-
20.00
24.00
04.00
08.00
Figi 1. N A T activity in the pineal gland of rats treated with either saline or DSIP before lights out. The black bar represents the period of darkness, An indication of variation within the individual groups is not included in the figure because they would have greatly confounded the presentation. * P < 0 . 0 2 vs saline-treated controls.
129 600 i
Sabine
o Law Dose (15 mmole/kg) -~ c-
50O
A Med Dose (30 mmole/kg) //,' ///
400
tO
-4---
300
,
X), '
D High Dose (60 mmale/kg)
/7 o ,
i
0.._
/
"
\
2004i
0 100
---7
20.00
24.00
04.00
08.00
Fig. 2. Mehtlonin levels in the pineal gland of rats treated with either saline or DSIP before lights out. "File black bar represents the period of darkness. An indication of variation within the individual groups is not inchided in the figure because they would have greatly conflmndcd the presentation. *P<0.02 vs saline-treated controls. enzyme analysis [1] and the r a d i o i m m u n o a s s a y for melatonin [16] have been previously described. Data were statistically analyzed using an A N O V A followed by a Student N e w m a n Keuls test. The procedures used in this experiment have been previously employed in this laboratory to investigate the metabolism o f serotonin to melatonin within the pineal gland [I]. In all animals darkness was associated with an increase in the activity o f the enzyme which ,N-acetylates serotonin as well as in melatonin. As reported by G r a f et al. [7], one dose o f D S I P (15 rnmol/kg), injected in the evening before lights out, caused a significant inhibition ( P < 0 . 0 2 ) o f pineal N A T activity at 3 h into the dark phase (Fig. I). The inhibition, however, was not apparent at the subsequent time points. Melatonin exhibited a similar response. Again, the lowest dose o f D S I P (15 nmol/kg) significantly ( P < 0 . 0 2 ) retarded the nighttime rise in pineal melatonin at 11.00 h but the effect was not apparent at the other two nighttime points or at 08.00 h alter light onset (Fig. 2). As did G r a f et al. [7], we report here that the administration o f D S I P just before darkness onset delays the subsequent rise in pineal N A T activity; furthermore, we found that melatonin levels are likewise inhibited. It is also clear from the present data, as well as the earlier findings, that the inhibitory effect is highly dose specific. Hence, at only one dosage (15 nmol/kg) was D S I P able to significantly alter the nocturnal rise in enzyme activity and, therefore, melatonin production. This dosage dit L fers from the dose reported by G r a f et al. to inhibit pineal N A T activity [7] (15 nmol/ kg in the present study vs 30 n m o l / k g in the experiment o f G r a f et al.). This could be a result o f the use of different strains o f rats which m a y metabolize D S I P at slightly different rates or due to some u n k n o w n factor. A rapid metabolism o f D S I P could also explain the short-lived nature o f the suppressive effect o f the peptide on
130
pineal melatonin production. G r a f et al. [7] noted the inhibition at 4 h into the dark phase whereas in the present study a significant reduction in N A T activity and melatonin level was observed only 3 h after D S I P was administered. On the whole, the differences that were seen in the DSIP-injected animals were not great. Whether the changes were sufficiently great to alter the physiology o f the pineal gland relative to its endocrine actions c a n n o t be determined at this time. The fact that a similar effect was obtained in two separate studies conducted in different laboratories suggests the alteration is real; however, the physiological consequences o f the change remain the subject o f subsequent investigations. The changes induced by D S I P are similar to those which follow endomethacin administration [15]; endomethacin is a prostaglandin synthesis inhibitor. Thus, there appear to be at least some endogenously produced c o m p o u n d s that can, under certain circumstances, at least slightly modify pineal indoleamine metabolism. It is unlikely that the change observed was merely due to a phase shift o f the rise in melatonin production. Typically, the onset o f darkness is associated with a rise in both N A T activity and melatonin content o f the rat pineal gland [il]; these values usually return to daytime levels before lights on. After D S I P injection this pattern was also noted. The rise occurred, although it was blunted, and likewise the drop anticipated lights on with basal daytime levels being apparent the subsequent morning. This is only one o f a n u m b e r o f effects of D S l P in rats. Usually, its administration is associated with changes in circadian l o c o m o t o r activity [5]. The rhythms in the pineal gland as well as the 24-h l o c o m o t o r rhythm both originate from electrical activity in the suprachiasmatic nuclei (SCN) o f the hypothalamus. Thus, the c o m m o n site o f action for D S I P for both parameters could be the SCN. The S C N are k n o w n to be important relay nuclei in the transfer o f information from tire retina to the pineal gland [14]. Interestingly, the S C N have been proposed as a possible site o f action o f m e l a t o n i n as well [13]. In summary, these data suggest a m i n o r role for D S I P in controlling the circadian rhythmicity o f the pineal gland. The effect seems to be highly dose dependent and rather short lived. Besides an effect on N A T and melatonin, D S I P could have other actions in the pineal gland. Research supported by N S F G r a n t 8410592, G r a n t CCA-8411106 from the Spanish American Committee and Caja A h o r r o s del Tenerife, La L a g u n a University. 1 Champney, T.H., Holtorf, A.P., Steger, R.W. and Reiter, R.J., Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland, J. Neurosci. Res., 11 (1984) 59-66. 2 Ebadi, M., Regulation of the synthesis of melatonin and its significance to neuroendocrinology. In R.J. Reiter (Ed.), The Pineal Gland, Raven Press, New York, 1984,pp. l 37. 3 Feldman, S.C. and Kastin, A.J., Localization of neurons containing immunoreactive delta sleep inducing peptide in the rat brain: an immunocytochemical study, Neuroscience, 11 (1984) 303 318. 4 Gral, M., Christen, H., Tobler, H.J., Maier, P.F. and Schoenenberger, G.A., Effects of repeated DSIP and DSIP-P administration on the circadian locomotor activity of rats, Pharmacol. Biochem. Behav., 15(1981)717 721.
131
5 (h'af. M., Uhi-isten, H. and Schoenenberger, G.A., DSIP/DSIP-P and circadian motor activity of rats under continuous light, Peptides, 3 (1982) 623 626. 6 Graf, M. and Kastin. A.J., Delta-sleep-inducing peptide: a review, Neurosci. Biobehav. Rex'.. g (1984) S3 9:;. 7 ( h'af, M., Kastin. A.J. and Schoenenberger, G.A., Delta sleep reducing peptide and t ~ o of its analogs rcducc nocturnal increase of N-acetyllransfizrase activity in rat pineal gland, J. Ncurochem., 44 (198'~) 629 632. ,v Kastm, A.J., ('astellanos, P.F., Banks, W.A. and Coy, D.H., Radioimmunoassay of DSIP like material in h u m a n hlood: possible protein binding, Pharmacol. Biochem. Behav., 15 (1981)969 974. 9 Kaslin, A.J., Dickson, J.C. and Fischman, A.J., DSIP like material in rats running a c~mlplex maze, Ph>si,',l. Bchav.. 33 (1984) 427 431. l0 Kastin. A.J.. Nissen. C. and Coy. D.H.. Permeability of the bl,.',od bram barrier to DSIP pcplides. Pharmac,.~l. Biochem. Behav.. 15 ( 19gl ) 955 959. I I Kastin, A.J., Nissen, ('.. Schally. A.V. and Coy, D H . , Additional evidence that small a m o u n t s of peptides can cross t h e h l o o d h r a i n h a r r i e r , Pharmacol. Biochem. Behav., II (1979) 717 719. [ 2 Monnicr, M. and 1loesli, L., Dialysis of sleep and waking factors in hlood of rahhit, Science, 146 (1964) 796 798. 13 Reitcr. RJ., The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev., 1 {1980) 109 131. [4 Rcitcr, R.,I., Ncurocndocrine effects of the pineal gland and of melatonin. In W.F. ( i a n o n g and 1.. Martini (Eds.I. Frontiers in Neuroendocrinology, Vol. I, Raven Press, New York. 1982, pp. 287 316. 15 Relier, R.J.. Steinlechner, S. and Richardson. B.A., Daytime and nighttime pincal N-acetyltransferase activity and melatonm content in male rats treated with indomethacin, a prostaglandin synthesis inhihitor, Neuroendocr. Lett.,2(1986) 281 287. 16 Rollag. M.I). and Niswender, G.D., Radioimmunoassay of serum concentrations of melatonin m shccp exposed to different lighting regimens. Endocrinology, 98 (I 976) 482 489. 17 Schoenenherg, (}.A. and Monnier, M., ('haracterization of a delta-electroencephalogram (-sleepl inducingpeptidc, Proc. Nall. Acad. Sci. USA, 74(1977) 1281 1286.