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Pineal effects upon pituitary protein and nucleic acid synthesis in vitro
Neuroscience Letters, 2 (1976) 207--210
207
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
PINEAL EFFECTS UPON PITUITARY PROTEIN AND NUCLEIC ACID SYNTHESIS IN VITRO NANCY S. PERESS, GOLLAPUDI G. MURTHY and ROBERT J. BALCOM Veterans Administration Hospital, Northport, N. Y. and Department of Pathology, State University of New York, Stony Brook, N. Y. (U.S.A.)
(Received May 6th, 1976) (Accepted May llth, 1976)
SUMMARY The effects of various dilutions of pineal extract upon leucine and thymidine incorporation by pituitary cells were studied in vitro, using a miniature cell culture system. A pineal extract dose-dependent increase in thymidine and leucine incorporation by these pituitary cells was observed. These effects may be related to either increased synthesis and/or decreased secretion of newly synthesized proteins.
Pineal lesions have been associated with both precocious and delayed puberty. This has been attributed to either decreased or augmented melatonin synthesis [3,6]. In view of the anatomical proximity of the pineal and the hypothalamic-pituitary axis, these effects of melatonin are thought to be mediated through the pituitary gland [4,7,13 ]. The effects of other pineal secretory products upon pituitary cellular function are uncertain [ 1,2,9--11 ]. We have employed a miniature pituitary cell culture system to study the effects of pineal cell-free extracts on pituitary protein synthesis. Pituitary cell suspensions were prepared from young male Wistar rat pituitaries b y the method of Vale et al. [12] with the following modifications: (1) 0.15% hyaluronidase {Sigma Co~ was used, (23 't2"% fetal calf serum was included and (3) the final cell suspension was in tissue culture medium 199. [ 14C] Leucine (0.03 ~Ci/ml of medium; specific activity 324 mCi/mmole) and [3H]thymidine (0.6 ~Ci/ml of medium; specific activity 18.4 Ci/mmole, New England Nuclear) were added to the final cell suspension which was uniformly distributed in the microwells of Limbro Multi Dish Disposo Trays. Pineal cell-free extracts were prepared from pineals obtained immediately following decapitation of the same animals used as pituitary sources. The pineals were placed in tissue culture media 199 and then sonicated in a Branson Model W-350 sonicator equipped with a microtip. Following this step they were centrifuged at 15,000 rpm for 5 rain. The supernatant was serially diluted and 0.1 ml aliquots of the various dilutions were added to the cell suspensions
208
in triplicate. Pituitary cell cultures not exposed to pineal extract served as the controls. After incubation for 20 h in a CO2 incubator at 37°C, the cells were harvested on a glass fiber filter paper (Reeve Angel -- Grade 934AH), using a cell harvester originally devised for l y m p h o c y t e culture [5]. The cells were washed five times with either cold saline or cold saline and leucine. The filter paper discs were removed and counted in toluene POPOP using liquid scintillation counter Teledyne Intertechnique, model SL30. The experiments were repeated on three different mornings using 20--30 rats per experiment. In order to compare the data from these experiments the control values were arbitrarily set at 100% and the experimental results were plotted as percentages of the control. The data were analyzed using a Student's t-test. The cell suspension system was able to reproducibly incorporate both leucine and thymidine over the relatively short period of the experiment. When pineal cell-free extracts were added to the pituitary cell suspension there was an increase in both leucine and thymidine incorporation (Figs. 1 and 2). The observed increases were statistically significant at all the dose levels employed when compared to the control. Maximal leucine uptake was observed at a 1:4 dilution while highest thymidine uptake was noted at a 1:2 dilution of the pineal cell-free extract. $50
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PINEAL EXTRACT DILUTIONS Fig. 1. [ 14C] Leucine uptake by the pituitary cells (vertical axis) at various dilutions of pineal cell-free e x t r a c t ( h o r i z o n t a l axis). The various values are represented in r e l a t i o n t o the control w h i c h was a r b i t r a r i l y fixed at 100%. E a c h bar r e p r e s e n t s a m e a n -+ S.E.M. o f 9 observations. Statistically significant values are r e p r e s e n t e d b y ~ (P --- < 0 . 0 5 ) a n d b y **
(P= < 0.01).
209
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150
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100
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Z F, >I
50
-r eo
0
I CONTROL
I 1.4
1,2
I 1.1
PINEAL EXTRACT DILUTIONS
Fig. 2. [ 3H ] ThYmidine uptake by the pituitary cells (vertical axis) at various dilutions of pineal cell-free extract (horizontal axis). The various values are represented in relation to the control which was arbitrarily fixed at 100%. Each bar represents a mean ± S.E.M. o f 3 observations. Statistically significant values are represented by * (P = < 0.05) and by **
(P = < 0.01).
We have thus demonstrated an effect of pineal extract on the pituitary. This effect may be related to either a stimulation of protein and nucleic acid synthesis and/or an inhibition of secretion of newly synthesized proteins. In the only study to date which has dealt with the effects of melatonin directly on pituitary protein synthesis, melatonin was administered systemically over a five day period, and in vitro pituitary leucine incorporation into protein was studied [ 8 ] . The data showed an inhibitory effect upon pituitary protein synthesis by melatonin. It is difficult to compare these findings with ours because we dealt with the direct in vitro effects of whole pineal extracts upon pituitary nucleic acid and protein synthesis within a 24 h period.
210 REFERENCES 1 Benson, B., Matthews, M.J. and Rodin, A.E., A melatonin-free extract of bovine pineal with antigonadotropic activity, Life Sci., 10 (1971 ) 607--612. 2 Benson, B., Matthews, M.J. and Rodin, A.E., Studies on a non-melatonin pineal antogonadotrophin, Acta Endocr. (Kbh.), 69 (1972) 257--266. 3 Cardinali, D.P., Melatonin and the endocrine role of the pineal organ, Curr. Topics exp. Endocr., 2 (1974) 107--128. 4 Kamberi, I.A., Mical, R.S. and Porter, J.C., Effects of melatonin and gerotonin on the release of FSH and prolactin, Endocrinology, 88 (1971) 1288--1293. 5 Kaplan, J.H. and Razzano, A.F., A miniature phytohemagglutinin assay using disposable microtiter plates, Immunol. Commun., 2 (1973) 507--519. 6 Kitay, J.I. and Altschule, M.D., The pineal glands Cambridge, Mass., 1954. 7 0 n d o , J.G., Eskay, R.L., Mical, R.S. and Porter, J.C., Release of LH by LRF injected into the CSF: a transport role for the median eminence, Endocrinology, 93 (1973) 231--237. 8 0 r s i , L., Denari, J.H., Nagle, C.A., Cardinali, D.P. and Rosner, J.M., Effects of melatonin on the synthesis of proteins by the rat hypothalamus, hypophysis and pineal organ, J. Endocr., 58 (1973) 131--132. 9 0 r t s , R.J. and Benson, B., Inhibitory effects on serum and pituitary LH by a melatoninfree extract of bovine pineal glands, Life Sci., 12 (1973) 513--519. 10 Pavel, S., Dumitru, I., Klepsh, I. and Dorcescu, M., A gonadotropin inhibiting principle in the pineal of human fetuses. Evidence for its identity to arginine vasotocin, Neuroendocrinology, 13 (1973/74) 41--46. 11 Reiter, R.J., Vaughan, M.K., Blask, D.E. and Johnson, L.Y., Melatonin: its inhibition of pineal antigonadotrophic activity in male hamsters, Science, 185 ( 1974) 1169--1171. 12 Vale, W., Grant, G., Amoss, M.A., Blackwell, R. and Guillemin, R., Culture of enzymatically dispersed pituitary cells: functional validation of a method, Endocrinology, 91 (1972) 562--572. 13 Wurtman, R.J. and Anton-Tay, F., The mammalian pineal as a neuroendocrine transducer, Hormone Res., 25 (1969) 493--522.
207
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
PINEAL EFFECTS UPON PITUITARY PROTEIN AND NUCLEIC ACID SYNTHESIS IN VITRO NANCY S. PERESS, GOLLAPUDI G. MURTHY and ROBERT J. BALCOM Veterans Administration Hospital, Northport, N. Y. and Department of Pathology, State University of New York, Stony Brook, N. Y. (U.S.A.)
(Received May 6th, 1976) (Accepted May llth, 1976)
SUMMARY The effects of various dilutions of pineal extract upon leucine and thymidine incorporation by pituitary cells were studied in vitro, using a miniature cell culture system. A pineal extract dose-dependent increase in thymidine and leucine incorporation by these pituitary cells was observed. These effects may be related to either increased synthesis and/or decreased secretion of newly synthesized proteins.
Pineal lesions have been associated with both precocious and delayed puberty. This has been attributed to either decreased or augmented melatonin synthesis [3,6]. In view of the anatomical proximity of the pineal and the hypothalamic-pituitary axis, these effects of melatonin are thought to be mediated through the pituitary gland [4,7,13 ]. The effects of other pineal secretory products upon pituitary cellular function are uncertain [ 1,2,9--11 ]. We have employed a miniature pituitary cell culture system to study the effects of pineal cell-free extracts on pituitary protein synthesis. Pituitary cell suspensions were prepared from young male Wistar rat pituitaries b y the method of Vale et al. [12] with the following modifications: (1) 0.15% hyaluronidase {Sigma Co~ was used, (23 't2"% fetal calf serum was included and (3) the final cell suspension was in tissue culture medium 199. [ 14C] Leucine (0.03 ~Ci/ml of medium; specific activity 324 mCi/mmole) and [3H]thymidine (0.6 ~Ci/ml of medium; specific activity 18.4 Ci/mmole, New England Nuclear) were added to the final cell suspension which was uniformly distributed in the microwells of Limbro Multi Dish Disposo Trays. Pineal cell-free extracts were prepared from pineals obtained immediately following decapitation of the same animals used as pituitary sources. The pineals were placed in tissue culture media 199 and then sonicated in a Branson Model W-350 sonicator equipped with a microtip. Following this step they were centrifuged at 15,000 rpm for 5 rain. The supernatant was serially diluted and 0.1 ml aliquots of the various dilutions were added to the cell suspensions
208
in triplicate. Pituitary cell cultures not exposed to pineal extract served as the controls. After incubation for 20 h in a CO2 incubator at 37°C, the cells were harvested on a glass fiber filter paper (Reeve Angel -- Grade 934AH), using a cell harvester originally devised for l y m p h o c y t e culture [5]. The cells were washed five times with either cold saline or cold saline and leucine. The filter paper discs were removed and counted in toluene POPOP using liquid scintillation counter Teledyne Intertechnique, model SL30. The experiments were repeated on three different mornings using 20--30 rats per experiment. In order to compare the data from these experiments the control values were arbitrarily set at 100% and the experimental results were plotted as percentages of the control. The data were analyzed using a Student's t-test. The cell suspension system was able to reproducibly incorporate both leucine and thymidine over the relatively short period of the experiment. When pineal cell-free extracts were added to the pituitary cell suspension there was an increase in both leucine and thymidine incorporation (Figs. 1 and 2). The observed increases were statistically significant at all the dose levels employed when compared to the control. Maximal leucine uptake was observed at a 1:4 dilution while highest thymidine uptake was noted at a 1:2 dilution of the pineal cell-free extract. $50
I
100
~-
u LIJ
z u uJ
LJ
50
0
I CONTROL
I 18
I
I
~4
12
I1
PINEAL EXTRACT DILUTIONS Fig. 1. [ 14C] Leucine uptake by the pituitary cells (vertical axis) at various dilutions of pineal cell-free e x t r a c t ( h o r i z o n t a l axis). The various values are represented in r e l a t i o n t o the control w h i c h was a r b i t r a r i l y fixed at 100%. E a c h bar r e p r e s e n t s a m e a n -+ S.E.M. o f 9 observations. Statistically significant values are r e p r e s e n t e d b y ~ (P --- < 0 . 0 5 ) a n d b y **
(P= < 0.01).
209
D
150
T
u
100
.< a. u,J
Z F, >I
50
-r eo
0
I CONTROL
I 1.4
1,2
I 1.1
PINEAL EXTRACT DILUTIONS
Fig. 2. [ 3H ] ThYmidine uptake by the pituitary cells (vertical axis) at various dilutions of pineal cell-free extract (horizontal axis). The various values are represented in relation to the control which was arbitrarily fixed at 100%. Each bar represents a mean ± S.E.M. o f 3 observations. Statistically significant values are represented by * (P = < 0.05) and by **
(P = < 0.01).
We have thus demonstrated an effect of pineal extract on the pituitary. This effect may be related to either a stimulation of protein and nucleic acid synthesis and/or an inhibition of secretion of newly synthesized proteins. In the only study to date which has dealt with the effects of melatonin directly on pituitary protein synthesis, melatonin was administered systemically over a five day period, and in vitro pituitary leucine incorporation into protein was studied [ 8 ] . The data showed an inhibitory effect upon pituitary protein synthesis by melatonin. It is difficult to compare these findings with ours because we dealt with the direct in vitro effects of whole pineal extracts upon pituitary nucleic acid and protein synthesis within a 24 h period.
210 REFERENCES 1 Benson, B., Matthews, M.J. and Rodin, A.E., A melatonin-free extract of bovine pineal with antigonadotropic activity, Life Sci., 10 (1971 ) 607--612. 2 Benson, B., Matthews, M.J. and Rodin, A.E., Studies on a non-melatonin pineal antogonadotrophin, Acta Endocr. (Kbh.), 69 (1972) 257--266. 3 Cardinali, D.P., Melatonin and the endocrine role of the pineal organ, Curr. Topics exp. Endocr., 2 (1974) 107--128. 4 Kamberi, I.A., Mical, R.S. and Porter, J.C., Effects of melatonin and gerotonin on the release of FSH and prolactin, Endocrinology, 88 (1971) 1288--1293. 5 Kaplan, J.H. and Razzano, A.F., A miniature phytohemagglutinin assay using disposable microtiter plates, Immunol. Commun., 2 (1973) 507--519. 6 Kitay, J.I. and Altschule, M.D., The pineal glands Cambridge, Mass., 1954. 7 0 n d o , J.G., Eskay, R.L., Mical, R.S. and Porter, J.C., Release of LH by LRF injected into the CSF: a transport role for the median eminence, Endocrinology, 93 (1973) 231--237. 8 0 r s i , L., Denari, J.H., Nagle, C.A., Cardinali, D.P. and Rosner, J.M., Effects of melatonin on the synthesis of proteins by the rat hypothalamus, hypophysis and pineal organ, J. Endocr., 58 (1973) 131--132. 9 0 r t s , R.J. and Benson, B., Inhibitory effects on serum and pituitary LH by a melatoninfree extract of bovine pineal glands, Life Sci., 12 (1973) 513--519. 10 Pavel, S., Dumitru, I., Klepsh, I. and Dorcescu, M., A gonadotropin inhibiting principle in the pineal of human fetuses. Evidence for its identity to arginine vasotocin, Neuroendocrinology, 13 (1973/74) 41--46. 11 Reiter, R.J., Vaughan, M.K., Blask, D.E. and Johnson, L.Y., Melatonin: its inhibition of pineal antigonadotrophic activity in male hamsters, Science, 185 ( 1974) 1169--1171. 12 Vale, W., Grant, G., Amoss, M.A., Blackwell, R. and Guillemin, R., Culture of enzymatically dispersed pituitary cells: functional validation of a method, Endocrinology, 91 (1972) 562--572. 13 Wurtman, R.J. and Anton-Tay, F., The mammalian pineal as a neuroendocrine transducer, Hormone Res., 25 (1969) 493--522.