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Compensation of the natural magnetic field does not alter N-acetyltransferase activity and melatonin content of rat pineal gland
Neuroscience Letters, 76 (1987) 215 220 Elsevier Scientific Publishers Ireland Ltd.
215
NSL I)4561
Compensation of the natural magnetic field does not alter N-acetyltransferase activity and melatonin content of rat pineal gland Roderich Khoory Centre q["Human Genetics Counselling, University q/" Bremen, Bremen ( F. R.G. i (Received 29 December 1986: Revised version received and accepted 19 January !987)
Key word~'." Magnetic field: N-Acetyllransferase: Melatonin: Pineal gland: Rat The amr of this study was to investigate if compensation of the natural magnetic tield (NM F) inltuences pincal melatonin production. Rats were exposed to an artiticial magnetic lield / A M F ) that compcnsalcd the N M F during the night for 30 rain. In another experiment rals were treated with the [t-adrenergic agonist isoprolerenol prior to treatment with the same A M F d u r m g the day. Pineal N-acetyltransfcrase activity was determined by a radioenzymatic assay and melatonin content was measured by radioinrmunoassa}. No statistically significant differences in these parameters were found in rats treated with this AM F \allen compared to control animals.
The pineal hormone melatonin is synthesized rhythmically. Its formation from the essential amino acid tryptophan requires 4 enzymatic steps. The most important of these is the acetylation of the neurotransmitter serotonin by the enzyme indoleamine N-acetyltransferase (E.C. 2.3.1.5: NAT) or, as has been postulated recently, by arylalkylamine N-acetyltransferase (E.C. 2.3.1.87 [7]). This enzyme activity changed during the day, being high during the night and low during the day [8]. Exposing animals to light at night reveals low levels of pineal NAT (P-NAT) activity usually seen during the day after as little as one minute of exposure [4, 9]. A similar response is obtained when rats are transferred during the night into an artificial magnetic field (AMF) that inverts the horizontal component of the natural magnetic field ( N M F [17]). Although the location of the receptor of magnetic fields has not been found, the visual system seems to be crucial [12, 14, 15]. Red light, unable to diminish P-NAT activity, is necessary for the effects of AMF upon P-NAT [12]. However, it is unknown whether the melatonin synthesizing mechanism is disturbed by the absence of the NMF. The effects of unusual magnetic conditions upon human physiology became important alter men were sent to extreme environments such as Antarctica or even space. Astronauts stayed in an orbit for up to several Correspondence: R. Khoory, Centre of H u m a n Genetics Counselling, University of Bremen, D-28I)0 Bremen 33, F.R,G. 0 3 0 4 - 3 9 4 0 8 7 5 03.50 @ 1987 Elsevier Scientific Publishers Ireland Ltd.
216 months under a magnetic field of very low intensity. Thus, the aim of this study was to investigate possible physiologic consequences o f ' n e a r zero" magnetic fields. Male Wistar rats were purchased t~rom Zentralinstitut for Versuchsticre, Hannover, F.R.G. They were kept tk~r at least tWO weeks in our facilities. Details of housing conditions have been described [6]. Pipe-shaped coils (i.d. 10 cm), that were oriented parallel to the inclination of the N M F, generated the A M F. A direct current was applied to the coils, which generated an A M F that compensated the N M F . Usually the field strength of the N M F in the building where the experiments took place was 0.5 G with very little variation. The magnetic field within the coils might not have been exactly "zero' during all the experiments, but it never exceeded 0,025 G (5% of the NMF). Strength and orientation of A M F and N M F were monitored with a Gaussmeter Type 640 (Bell Labs., Cleveland, OH, U.S.A.). [i4C]Acetyl coenzyme A was obtained from Amersham Buchler, Braunschweig, F.R,G. Serotonin oxalate complex ( > 98%) was from Sigma. Munich. F.R.G. Pineal melatonin was quantitated by a commercially available iodinated radioimmunoassay (RIA; Tecova, Wohlen, Switzerland). All other chemicals were from commercial sources. Activity of P - N A T was determined as described previously [6]. In brief, pineal homogenate is incubated with 4.15 nmol of AcCoA and 10 mmol/l of serotonin in 45/tl of sodium phosphate buffer (0.1 mol/l; pH 6.5). Enzymatically formed N-acetylserotonin (aHT)* is extracted with 20% of isoamyl alcohol in toluene (v:v). In our hands recovery for a H T under these conditions is 72.6% [5]. For extraction of pineal melatonin (P-aMT) 400 ~1 of buffer are added to 100 ltl of homogenate. After adding 2.5 ml diethylether samples are thoroughly shaken for 10 rain in the cold ( 4 C ) . The organic phase is removed and dried under a gentle stream of nitrogen. Resuspended residues are subjected to RIA. Experiment l. Five and a half hours alter "lights off' rats were lightly anesthetized with ether and immobilized physically. After 30 min of exposure to the A M F rats were killed under a dim red light and the pineal glands removed within one minute. Control animals were treated similarily and killed at the same time of day, but were not exposed to an AMF. Glands were frozen in liquid nitrogen and transferred to a deep freezer on the next morning. Biochemical assays were run within two weeks. This experiment was done 4 times independently between May and July. E.rperiment 2. Three hours after "lights on' rats were lightly anesthetized with ether, immobilized physically, and injected with isoproterenol (10 mg/kg body wt.). This treatment stimulates P-NAT activity to levels comparable to those during the night. Two and a half hours later, the animals were subjected to the same A M F described above. This experiment was also done 4 times independently between October and November. Different dilutions of pineal homogenate paralleled the standard curve of the melatonin RIA (Fig. 1). Injection of isoproterenol, a/]-receptor agonist, during the day, *Abbreviations used in this paper for indolic compounds follow a "one-letter-code'proposed by Dr. 1. Smith and published in Psychoneuroendocrinology. 8 (1983) 41 60.
217
go
5O
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~1~
--
~,--
~
~
2'0
&~o
2'o
,~o ~o
~0 .~ ~6o
26o~o
6601~%-/r~tl
Fig. I. Dilfcrenl dilutions of pineal homogenate measured by RIA I'or mehttonin. Pineal homogenalc "~Mis diluted 1:62.5, 1:25, 1:10 and 1:5. Samples wcrc then subjected to RIA. F'or comparison, the standard curve is also shown. Note thai data are presented as a logit,'log plot.
caused enhanced P-aMT (Fig. 2A), whereas giving light to the animals at night caused decreased P-aMT levels (Fig. 2B). These data show that the RIA used in this study is valid for d e t e r m i n a t i o n o f P - a M T content. K e e p i n g rats at night in an enviro n m e n t a l m o s t free o f a m a g n e t i c field does not influence N A T activity or a M T conLight:Dork
12h:12h
P - oMT P-aMT
bmo'l
h~m ~.0"
1,0 2/,h: Oh
0.5
B
0
Fig. 2. Pineal melatonin after different treatments of the rats, A: rats were killed in the middle oF the light phase (untreated). or after they had been rejected with 10 mg/kg body wt. of ascorbic acid in physiologic saline (vehicle), or after they had been injected with 10 mg/kg body wt. ofisoprotcrenol m vehicle (stimulated). Pineal glands were removed, melatonm vvas extracted and determined as described. B: rats were killed in the middle of the dark phase after they had been exposed to either 12 h of light per day or constant light. Pineal glands were removed, melatonin was extracted and determined as described above. ~ 4_-S. E. M. of each group ( n - 4 ) are shown.
218
@
P- NAT
[i
[m 1
l 2000
P-aMT gland]
AMF
NMF
®
P- NAT
I pmol 11 5:glandJ 2000-
P--aM T glandj
AMF
NMF
5 1000
1000
19 19
16 16
ji
17 18
18 19
Fig. 3. Pineal NAT activity and melatonin content after exposure to an AMF during the night. Rats experienced either an AMF or the NMF for 30 min during the night. They were killed in the middle of the dark phase. Pineal glands were removed, melatonin was extracted and determined as described above. Open bars represent P-NAT activity, hatched bars P-aMT content. Numbers at the bottom of the bars indicate number of investigated animals. ~ + S.E.M. are shown. Fig. 4. Stimulated pineal NAT activity and melatonin content alter exposure to an AMF during the day. Animals were injected with isoproterenol and then subjected either to an AMF or to the NMF for 30 min during the day. They were killed in the middle of the light phase (for details see text). Pineal glands were removed, melatonin was extracted and determined as described above. Open bars represent P-NAT activity, hatched bars P-aMT content. Numbers at the bottom of the bars indicate number of investigated animals. ~_+ S.E.M. are shown.
tent in the pineal g l a n d (Fig. 3). A c c o r d i n g l y , e x p o s u r e d u r i n g the d a y is also ineffective in altering s t i m u l a t e d P - N A T o r P - a M T (Fig. 4). Previous w o r k has shown (i) that a l t e r a t i o n o f the N M F by a p p l i c a t i o n o f an A M F for 15 rain o r m o r e d r a m a t i c a l l y reduces P - a M T biosynthesis in the rat d u r i n g the night, but not d u r i n g the d a y [17], (ii) that an intact visual p a t h w a y is necessary for this response [12], a n d (iii) t h a t m a g n e t i c fields s t r o n g e r than the N M F are ineffective [13]. M o r e o v e r , this kind o f A M F is also c a p a b l e to alter m e l a t o n i n biosynthesis in the pineal gland a n d retina o f quails, as well as the night vision acuity in h u m a n s [3]. T o g e t h e r with e l e c t r o p h y s i o l o g i c a l studies [14, 15] these results s t r o n g l y suggest that the m a g n e t i c r e c e p t o r is tightly c o u p l e d to the process o f vision. In s p a r r o w s "near zero' m a g n e t i c fields have been shown to influence c i r c a d i a n r h y t h m s o f activity [2], which in this species are linked to m e l a t o n i n ' s synthetic p a t h w a y in the pineal g l a n d [1]. To elucidate w h e t h e r c o m p e n s a t i o n o f the N M F w o u l d influence c i r c a d i a n r h y t h m i c i t y in m a m m a l s , rats were e x p o s e d to a ' n e a r z e r o ' m a g n e t i c field. Figs. 3 a n d 4 clearly show t h a t u n d e r e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d in this study, m e l a t o n i n f o r m a t i o n in the pineal g l a n d o f the r a t is n o t changed. Either the magnetic stimulus, i.e. the a b s e n c e o f a m a g n e t i c field, was i n a p p r o p r i a t e , o r s o m e o t h e r factor(s) p r e v e n t e d the r e c e p t i o n o f the stimulus. F u r t h e r e x p e r i m e n t s are required to clarify this question, because very recent d a t a indicate that an inverted
219 horizontal c o m p o n e n t of the N M E is effective in c h a n g i n g P - N A T activity u n d e r the c o n d i t i o n s prevailing in the a u t h o r ' s laboratory. A l t h o u g h in this study a rat strain was used other than in previous studies [11 13, 17], the lack of effect of the A M F reported here is not a t t r i b u t e d to strain specific differences, since a l b i n o rats were used in either case (Sprague Dawley a n d Wistar) and even rats with pigmented eyes ( L o n g Evans) respond to an A M F [1 1]. Red light is necessary if A M F s should influence P - N A T [I 2]. Hence, the perception of light, even of a wavelength u n a b l e to influence P - N A T alone, is necessary for thc perception of an A M F . D u r i n g this experiment rats were exposed to only a few lux of red light ( M a v o l u x electronic: Gossen. Erlangen, F . R . G . ) , which might have been below the threshold necessary for the action of A M F u p o n the pineal gland. The existence of a threshold in light effects u p o n the pineal gland is not u n c o m m o n . White light reduces n o c t u r n a l P - N A T activity in the rat only when a certain intensity is exceeded [10, 16]. T h u s it is postulated that the i n h i b i t o r y effect of A M F upon PN A T a n d P - a M T requires synergistic effects of red light of sufficient intensity and a suitable magnetic stimulus. It is interesting to note that a l t h o u g h P - a M T c o n c e n t r a t i o n s are similar in isoproterenol-stimulated a n d n o c t u r n a l animals, P - N A T is more active in a n i m a l s treated with isoproterenol (Figs. 3 and 4). This indicates that besides P - N A T activity other factor(s) also regulate(s) n o c t u r n a l pineal rnelatonin content. This study was supported by the Deutsche F o r s c h u n g s g e m e i n s c h a f t (Kh 8'1-1). The a u t h o r is grateful to Prof. W. Schloot for his support, to Prof. A. MayerHeinricci for help in designing the A M F generating coils and to Laboserv G m b H , Giessen, lk~r placing a ),-counter at his disposal. T h a n k s also to Mrs. E. Klenke for technical assistance, to Mr. M. Bach for i m p r o v i n g the English and to Mrs. G. Taddigs for d o i n g the artwork. I Binkley.S., Circadian organization in mammals and birds, Photochem. Pholobiol., 35 (19821 XS7 890. 2 Bliss, V.L. and Hcppner. F.H., Circadian aclivily rhythm mlluenccd by near zero magnetic lield, Nalure (London). 261 (1976) 411 412. 3 ('rcmer-Bartels. G.. Krause, K. and Kfichle, It.J., Influence of low magnetic tield strength variations on Hie retina and pineal gland of quail and humans. Graefe's Arch. Clin. Exp. Ophthalmol., 220 (I 9S 1) 248 252. 4 lllnerowt, H. and Vanecek, J., Response of rat pineal serotonin N-acetyltransferase to one minute light pulse at different night times, Brain Res., 167 (1979) 431 434. 5 Khoory. R., Dubbels, R. and Schloot, W.. A moditied assay for rat pineal N-acetyltransferase with enhanced sensitivity and indicating ;in inhibitory principle of the homogenale. In (}.M. Brown and S.D. Wainwright (Eds.), The Pineal Gland Endocrine Aspects, Pergamon, Oxford. 1985. pp, 53 58. 6 Khoory, R. and Schloot, W., Inhibition of da3, lime, but not isoprolerenol-stimulatcd pineal N-acet\,ltransferasc activity by an unidentified pineal compound, J. Neural Transm., 66 (1986) 281 289. 7 Klein,D.C., Voisin. P. and Namboodiri, M.A.A., The pineal fumily of aromatic amine N-acet>ltransferascs. Bioessays, 3 (1985) 217 22(I. S Klein. D.('. and Weller, J.L., lndole metabolism in the pineal gland: a circadian rhythm in N-acetyltransferase, Science, 169 (1970)1093 1095. 9 Klein. I).C. and Weller, J.L., Rapid light-induced decrease in pineal scrotonin N-acetyllransfcrasc activity, Science, 17711972) 532 533.
220 10 Minneman. K . P , Lynch, H. and Wurtman, R.J,, Relationship between environmental light intensit~ and retina-mediated suppression of rat pineal serotonin N-acetyltransferase, Life Sci., 15 I1975) 179 I 1796, I I Olcese, J. and Reuss. S., Magnetic field effects on pineal gland melatonin synthesis: comparative studic~, on albino and pigmented rodents, Brain Res., 369 (1986) 365 368. 12 Olcese, J., Reuss, S. and Vollrath, L., Evidence for the involvement of the visual system in mediating magnetic field effects on pineal melatonin synthesis in the rat, Brain Res., 333 (1985) 382 384. 13 Reuss, S., Olcesc, J., Vollrath, L., Skalej, M. and Meves, M., Lack of effect of NMR-strength magnetic fields on rat pineal metatonin synthesis, 1RCS Med. Sci., 13 (1985) 471. 14 Serum, P. and Demaine, C.. NeurophysiologicaI properties of magnetic cells in the pigeon's visual sys~ tern, J. Comp. Physiol. A, 159 (1986) 619 625. 15 Serum, P.. Nohr, D., Demainc, ('. and Wiltschko. W., Neural basis of the magnetic compass: interactions of visual, magnetic and vestibular inputs in the pigeon's brain, J. Comp. Physiol. A, 155 (1984) 283 288. 16 Thiele, G., Holtort, A., Steinlcchner, S. and Reiter, R.J., The influence of different light irradiances on pineal N-acetyltransferase activity and melatonin levels in the cotton rat, Life Sci., 33 (1983) 1543 1547. 17 Welker, H.A., Serum, P., Willig, R.P., Commentz, J.C., Wiltschko, W. and Vollrath, L.. Effects of an artificial magnetic field on serotonin N-acetyltransferase activity and melatonin content of the rat pineal gland, Exp. Brain Res., 50 (1983) 426 432.
215
NSL I)4561
Compensation of the natural magnetic field does not alter N-acetyltransferase activity and melatonin content of rat pineal gland Roderich Khoory Centre q["Human Genetics Counselling, University q/" Bremen, Bremen ( F. R.G. i (Received 29 December 1986: Revised version received and accepted 19 January !987)
Key word~'." Magnetic field: N-Acetyllransferase: Melatonin: Pineal gland: Rat The amr of this study was to investigate if compensation of the natural magnetic tield (NM F) inltuences pincal melatonin production. Rats were exposed to an artiticial magnetic lield / A M F ) that compcnsalcd the N M F during the night for 30 rain. In another experiment rals were treated with the [t-adrenergic agonist isoprolerenol prior to treatment with the same A M F d u r m g the day. Pineal N-acetyltransfcrase activity was determined by a radioenzymatic assay and melatonin content was measured by radioinrmunoassa}. No statistically significant differences in these parameters were found in rats treated with this AM F \allen compared to control animals.
The pineal hormone melatonin is synthesized rhythmically. Its formation from the essential amino acid tryptophan requires 4 enzymatic steps. The most important of these is the acetylation of the neurotransmitter serotonin by the enzyme indoleamine N-acetyltransferase (E.C. 2.3.1.5: NAT) or, as has been postulated recently, by arylalkylamine N-acetyltransferase (E.C. 2.3.1.87 [7]). This enzyme activity changed during the day, being high during the night and low during the day [8]. Exposing animals to light at night reveals low levels of pineal NAT (P-NAT) activity usually seen during the day after as little as one minute of exposure [4, 9]. A similar response is obtained when rats are transferred during the night into an artificial magnetic field (AMF) that inverts the horizontal component of the natural magnetic field ( N M F [17]). Although the location of the receptor of magnetic fields has not been found, the visual system seems to be crucial [12, 14, 15]. Red light, unable to diminish P-NAT activity, is necessary for the effects of AMF upon P-NAT [12]. However, it is unknown whether the melatonin synthesizing mechanism is disturbed by the absence of the NMF. The effects of unusual magnetic conditions upon human physiology became important alter men were sent to extreme environments such as Antarctica or even space. Astronauts stayed in an orbit for up to several Correspondence: R. Khoory, Centre of H u m a n Genetics Counselling, University of Bremen, D-28I)0 Bremen 33, F.R,G. 0 3 0 4 - 3 9 4 0 8 7 5 03.50 @ 1987 Elsevier Scientific Publishers Ireland Ltd.
216 months under a magnetic field of very low intensity. Thus, the aim of this study was to investigate possible physiologic consequences o f ' n e a r zero" magnetic fields. Male Wistar rats were purchased t~rom Zentralinstitut for Versuchsticre, Hannover, F.R.G. They were kept tk~r at least tWO weeks in our facilities. Details of housing conditions have been described [6]. Pipe-shaped coils (i.d. 10 cm), that were oriented parallel to the inclination of the N M F, generated the A M F. A direct current was applied to the coils, which generated an A M F that compensated the N M F . Usually the field strength of the N M F in the building where the experiments took place was 0.5 G with very little variation. The magnetic field within the coils might not have been exactly "zero' during all the experiments, but it never exceeded 0,025 G (5% of the NMF). Strength and orientation of A M F and N M F were monitored with a Gaussmeter Type 640 (Bell Labs., Cleveland, OH, U.S.A.). [i4C]Acetyl coenzyme A was obtained from Amersham Buchler, Braunschweig, F.R,G. Serotonin oxalate complex ( > 98%) was from Sigma. Munich. F.R.G. Pineal melatonin was quantitated by a commercially available iodinated radioimmunoassay (RIA; Tecova, Wohlen, Switzerland). All other chemicals were from commercial sources. Activity of P - N A T was determined as described previously [6]. In brief, pineal homogenate is incubated with 4.15 nmol of AcCoA and 10 mmol/l of serotonin in 45/tl of sodium phosphate buffer (0.1 mol/l; pH 6.5). Enzymatically formed N-acetylserotonin (aHT)* is extracted with 20% of isoamyl alcohol in toluene (v:v). In our hands recovery for a H T under these conditions is 72.6% [5]. For extraction of pineal melatonin (P-aMT) 400 ~1 of buffer are added to 100 ltl of homogenate. After adding 2.5 ml diethylether samples are thoroughly shaken for 10 rain in the cold ( 4 C ) . The organic phase is removed and dried under a gentle stream of nitrogen. Resuspended residues are subjected to RIA. Experiment l. Five and a half hours alter "lights off' rats were lightly anesthetized with ether and immobilized physically. After 30 min of exposure to the A M F rats were killed under a dim red light and the pineal glands removed within one minute. Control animals were treated similarily and killed at the same time of day, but were not exposed to an AMF. Glands were frozen in liquid nitrogen and transferred to a deep freezer on the next morning. Biochemical assays were run within two weeks. This experiment was done 4 times independently between May and July. E.rperiment 2. Three hours after "lights on' rats were lightly anesthetized with ether, immobilized physically, and injected with isoproterenol (10 mg/kg body wt.). This treatment stimulates P-NAT activity to levels comparable to those during the night. Two and a half hours later, the animals were subjected to the same A M F described above. This experiment was also done 4 times independently between October and November. Different dilutions of pineal homogenate paralleled the standard curve of the melatonin RIA (Fig. 1). Injection of isoproterenol, a/]-receptor agonist, during the day, *Abbreviations used in this paper for indolic compounds follow a "one-letter-code'proposed by Dr. 1. Smith and published in Psychoneuroendocrinology. 8 (1983) 41 60.
217
go
5O
° o x
~1~
--
~,--
~
~
2'0
&~o
2'o
,~o ~o
~0 .~ ~6o
26o~o
6601~%-/r~tl
Fig. I. Dilfcrenl dilutions of pineal homogenate measured by RIA I'or mehttonin. Pineal homogenalc "~Mis diluted 1:62.5, 1:25, 1:10 and 1:5. Samples wcrc then subjected to RIA. F'or comparison, the standard curve is also shown. Note thai data are presented as a logit,'log plot.
caused enhanced P-aMT (Fig. 2A), whereas giving light to the animals at night caused decreased P-aMT levels (Fig. 2B). These data show that the RIA used in this study is valid for d e t e r m i n a t i o n o f P - a M T content. K e e p i n g rats at night in an enviro n m e n t a l m o s t free o f a m a g n e t i c field does not influence N A T activity or a M T conLight:Dork
12h:12h
P - oMT P-aMT
bmo'l
h~m ~.0"
1,0 2/,h: Oh
0.5
B
0
Fig. 2. Pineal melatonin after different treatments of the rats, A: rats were killed in the middle oF the light phase (untreated). or after they had been rejected with 10 mg/kg body wt. of ascorbic acid in physiologic saline (vehicle), or after they had been injected with 10 mg/kg body wt. ofisoprotcrenol m vehicle (stimulated). Pineal glands were removed, melatonm vvas extracted and determined as described. B: rats were killed in the middle of the dark phase after they had been exposed to either 12 h of light per day or constant light. Pineal glands were removed, melatonin was extracted and determined as described above. ~ 4_-S. E. M. of each group ( n - 4 ) are shown.
218
@
P- NAT
[i
[m 1
l 2000
P-aMT gland]
AMF
NMF
®
P- NAT
I pmol 11 5:glandJ 2000-
P--aM T glandj
AMF
NMF
5 1000
1000
19 19
16 16
ji
17 18
18 19
Fig. 3. Pineal NAT activity and melatonin content after exposure to an AMF during the night. Rats experienced either an AMF or the NMF for 30 min during the night. They were killed in the middle of the dark phase. Pineal glands were removed, melatonin was extracted and determined as described above. Open bars represent P-NAT activity, hatched bars P-aMT content. Numbers at the bottom of the bars indicate number of investigated animals. ~ + S.E.M. are shown. Fig. 4. Stimulated pineal NAT activity and melatonin content alter exposure to an AMF during the day. Animals were injected with isoproterenol and then subjected either to an AMF or to the NMF for 30 min during the day. They were killed in the middle of the light phase (for details see text). Pineal glands were removed, melatonin was extracted and determined as described above. Open bars represent P-NAT activity, hatched bars P-aMT content. Numbers at the bottom of the bars indicate number of investigated animals. ~_+ S.E.M. are shown.
tent in the pineal g l a n d (Fig. 3). A c c o r d i n g l y , e x p o s u r e d u r i n g the d a y is also ineffective in altering s t i m u l a t e d P - N A T o r P - a M T (Fig. 4). Previous w o r k has shown (i) that a l t e r a t i o n o f the N M F by a p p l i c a t i o n o f an A M F for 15 rain o r m o r e d r a m a t i c a l l y reduces P - a M T biosynthesis in the rat d u r i n g the night, but not d u r i n g the d a y [17], (ii) that an intact visual p a t h w a y is necessary for this response [12], a n d (iii) t h a t m a g n e t i c fields s t r o n g e r than the N M F are ineffective [13]. M o r e o v e r , this kind o f A M F is also c a p a b l e to alter m e l a t o n i n biosynthesis in the pineal gland a n d retina o f quails, as well as the night vision acuity in h u m a n s [3]. T o g e t h e r with e l e c t r o p h y s i o l o g i c a l studies [14, 15] these results s t r o n g l y suggest that the m a g n e t i c r e c e p t o r is tightly c o u p l e d to the process o f vision. In s p a r r o w s "near zero' m a g n e t i c fields have been shown to influence c i r c a d i a n r h y t h m s o f activity [2], which in this species are linked to m e l a t o n i n ' s synthetic p a t h w a y in the pineal g l a n d [1]. To elucidate w h e t h e r c o m p e n s a t i o n o f the N M F w o u l d influence c i r c a d i a n r h y t h m i c i t y in m a m m a l s , rats were e x p o s e d to a ' n e a r z e r o ' m a g n e t i c field. Figs. 3 a n d 4 clearly show t h a t u n d e r e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d in this study, m e l a t o n i n f o r m a t i o n in the pineal g l a n d o f the r a t is n o t changed. Either the magnetic stimulus, i.e. the a b s e n c e o f a m a g n e t i c field, was i n a p p r o p r i a t e , o r s o m e o t h e r factor(s) p r e v e n t e d the r e c e p t i o n o f the stimulus. F u r t h e r e x p e r i m e n t s are required to clarify this question, because very recent d a t a indicate that an inverted
219 horizontal c o m p o n e n t of the N M E is effective in c h a n g i n g P - N A T activity u n d e r the c o n d i t i o n s prevailing in the a u t h o r ' s laboratory. A l t h o u g h in this study a rat strain was used other than in previous studies [11 13, 17], the lack of effect of the A M F reported here is not a t t r i b u t e d to strain specific differences, since a l b i n o rats were used in either case (Sprague Dawley a n d Wistar) and even rats with pigmented eyes ( L o n g Evans) respond to an A M F [1 1]. Red light is necessary if A M F s should influence P - N A T [I 2]. Hence, the perception of light, even of a wavelength u n a b l e to influence P - N A T alone, is necessary for thc perception of an A M F . D u r i n g this experiment rats were exposed to only a few lux of red light ( M a v o l u x electronic: Gossen. Erlangen, F . R . G . ) , which might have been below the threshold necessary for the action of A M F u p o n the pineal gland. The existence of a threshold in light effects u p o n the pineal gland is not u n c o m m o n . White light reduces n o c t u r n a l P - N A T activity in the rat only when a certain intensity is exceeded [10, 16]. T h u s it is postulated that the i n h i b i t o r y effect of A M F upon PN A T a n d P - a M T requires synergistic effects of red light of sufficient intensity and a suitable magnetic stimulus. It is interesting to note that a l t h o u g h P - a M T c o n c e n t r a t i o n s are similar in isoproterenol-stimulated a n d n o c t u r n a l animals, P - N A T is more active in a n i m a l s treated with isoproterenol (Figs. 3 and 4). This indicates that besides P - N A T activity other factor(s) also regulate(s) n o c t u r n a l pineal rnelatonin content. This study was supported by the Deutsche F o r s c h u n g s g e m e i n s c h a f t (Kh 8'1-1). The a u t h o r is grateful to Prof. W. Schloot for his support, to Prof. A. MayerHeinricci for help in designing the A M F generating coils and to Laboserv G m b H , Giessen, lk~r placing a ),-counter at his disposal. T h a n k s also to Mrs. E. Klenke for technical assistance, to Mr. M. Bach for i m p r o v i n g the English and to Mrs. G. Taddigs for d o i n g the artwork. I Binkley.S., Circadian organization in mammals and birds, Photochem. Pholobiol., 35 (19821 XS7 890. 2 Bliss, V.L. and Hcppner. F.H., Circadian aclivily rhythm mlluenccd by near zero magnetic lield, Nalure (London). 261 (1976) 411 412. 3 ('rcmer-Bartels. G.. Krause, K. and Kfichle, It.J., Influence of low magnetic tield strength variations on Hie retina and pineal gland of quail and humans. Graefe's Arch. Clin. Exp. Ophthalmol., 220 (I 9S 1) 248 252. 4 lllnerowt, H. and Vanecek, J., Response of rat pineal serotonin N-acetyltransferase to one minute light pulse at different night times, Brain Res., 167 (1979) 431 434. 5 Khoory. R., Dubbels, R. and Schloot, W.. A moditied assay for rat pineal N-acetyltransferase with enhanced sensitivity and indicating ;in inhibitory principle of the homogenale. In (}.M. Brown and S.D. Wainwright (Eds.), The Pineal Gland Endocrine Aspects, Pergamon, Oxford. 1985. pp, 53 58. 6 Khoory, R. and Schloot, W., Inhibition of da3, lime, but not isoprolerenol-stimulatcd pineal N-acet\,ltransferasc activity by an unidentified pineal compound, J. Neural Transm., 66 (1986) 281 289. 7 Klein,D.C., Voisin. P. and Namboodiri, M.A.A., The pineal fumily of aromatic amine N-acet>ltransferascs. Bioessays, 3 (1985) 217 22(I. S Klein. D.('. and Weller, J.L., lndole metabolism in the pineal gland: a circadian rhythm in N-acetyltransferase, Science, 169 (1970)1093 1095. 9 Klein. I).C. and Weller, J.L., Rapid light-induced decrease in pineal scrotonin N-acetyllransfcrasc activity, Science, 17711972) 532 533.
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