Amino acid incorporation into proteins of the supraoptic nucleus of the rat after osmotic stress

BRAIN RESEARCH

95

A M I N O A C I D I N C O R P O R A T I O N I N T O P R O T E I N S OF T H E S U P R A O P T I C N U C L E U S OF T H E R A T A F T E R O S M O T I C STRESS

ANDERS NORSTROM, SVERKER ENESTROM AND ANDERS HAMBERGER Institute of Neurobiology, University of GSteborg, GSteborg, and Department of Pathology, The Region Hospital, Link6ping (Sweden)

(Accepted August 15th, 1970)

1NTRODUCTION The neurohypophyseal hormones, vasopressin and oxytocin, are synthesized in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) and then transported in the axons to the nerve terminals in the posterior pituitary where they are stored and released 21. The production of vasopressin occurs largely in the neurones of the SON 13,93,z6. A depletion of stored vasopressin has been observed both in the SON s and in the posterior pituitary after osmotic stress lz,22. Striking morphological changes in the SON neurones 27 as well as changes in enzymic activity levels and RNA content were induced after thirst or salt water administrationa, 3,5,9,19. An increased incorporation of R N A precursors into the SON and posterior pituitary was observed upon functional stimulation17, go. Under similar conditions Takabatake and Sachs 24 observed an increased incorporation of labelled amino acids into vasopressin. In the present study the protein biosynthesis of the SON in vivo and in vitro has been examined by measuring the extent of incorporation of radioactive labelled leucine under various experimental conditions. The SON is a unique and well-defined region of the CNS which can be specifically stimulated by relatively physiological means and thus represents a useful unit for studies correlating metabolism of macromolecules with cell function. MATERIALSAND METHODS Animals

Male Sprague-Dawley rats weighing 200-250 g were kept at room temperature with standard food ad lib#urn. Osmotic stress was produced by deprivation of drinking water for 4, 7 or 11 days, or by giving 1.5 or 2 ~ NaCI solutions. The controls had fresh water ad lib#urn. Brain Research, 26 (1971) 95-103

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A. NORSTROMet at.

Incorporation of leucine into brain proteins in vivo In the first series of experiments a 20-gauge cannula was stereotactically implanted into the subarachnoid space at a point proved to be localized at the level of the SON. The animals were allowed to recover completely for at least 7 days. Fifteen #Ci L-[4,5-ZH]leucine (specific activity 29 Ci/mmole, The Radiochemical Centre, Amersham, Great Britain) diluted in 30 #1 of sterile 0.9~, NaCt were then slowly injected through the cannula by a chromatographic needle coupled to a calibrated teflon tube. The injection could be carried out without anaesthesia. No neurological signs were observed during or after the injection, T h e rats were killed by decapitation 2 h after the injection. The brains were removed within 2 rain. A frontal section at the level of the optic chiasma was made with a razor blade through the centre of the SON. The nucleus was identified, isolated and dissected free as precisely as possible. The neurohypophysis was carefully isolated from the anterior lobe of the pituitary gland. The cortex sample was taken from the upper surface of the slice from which the SON was dissected. In a second series of experiments the rats were injected intraperitoneally (1 /~Ci [3H]leucine/g body weight, in 1.5 ml of 0.9 ~o NaCI). The animals were killed by decapitation after 30, 90 or 180 min. Incorporation of leucine into brain proteins in vitro All animals were killed by decapitation without anaesthesia and the brain areas were isolated as described. The tissue samples were sliced with a razor blade into pieces around 0.5 mm thick. In a preliminary series of experiments the region of the brain containing the SON was chopped in a mechanical tissue chopper (Mickle Eng. Inc., Great Britain) set at 0.3 mm and the SON was dissected from the slices: Material from the cerebral cortal was treated similarly. Incubation of the slices was carried out in 1 ml medium per SON tissue in a shaking water-bath at 37°C in closed 15 ml erlenmeyer flasks gassed with pure oxygen for 1 min. The incubation medium had the following composition (final concentrations): Tris-chloride, 35 mM; sodium phosphate buffer, pH 7.6, 5 mM; NaCI, 120 mM; KC1, 5 mM; MgCI~, 2.5 mM; glucose, 5 mM: adenosine diphosphate, 2.5 m M and [3H]leucine (spec. act. 100 mCi/mM, 5 or 10 /~Ci/ml); The incubation was terminated after 60 rain by transfer of the flasks to ice. The pieces of tissue were rapidly removed and quickly rinsed in cold incubation medium without isotope. Radioactivity measurements All tissue samples were homogenized in 0.32 mM sucrose, 10 mM Tris-HC1 (pH 7.4). Aliquots of the homogenate (0.1 ml) were applied to discs of Whatman No. 3 filter paper, 2.5 cm in diameter. The papers were dried at room temperature and then washed twice in cold 5 % TCA (trichloroacetic acid), heated at 90°C for 30 min in 5 % TCA, and finally washed twice in 5 % TCA. Lipid extraction was done Brain Research, 26 (1971) 95-t03

AMINO ACID INCORPORATION IN RA~[ SUPRAOPTIC NUCLEUS

97

at room temperature for 30 rain in ethanol-ether (1:4, v/v) 15. The discs were dried in counting vials, and 0.5 ml Soluene (Packard) was added to each vial. Scintillation fluid containing PPO (2,5-diphenyloxazole, Packard) in toluene (6 g/1000 ml) was added. The radioactivity was measured in a Packard Tri-Carb spectrometer at 25-30 !Yo efficiency. The TCA-soluble radioactivity was measured in order to estimate the amount of leucine in the tissue samples. Aliquots of the homogenates were precipitated with an equal volume of cold l0 ~ TCA, and the radioactivity of the supernatant was determined after centrifugation. Protein was determined according to Lowry eta/. 1~ with serum albumin as standard, RESULTS

Incorporation of leucine in vivo Rats with an implanted cannula were observed for up to 6 weeks and showed no neurological disturbances. Weight gain, fertility and diurnal behaviour did not differ from control animals. However, a zone of localized hyperaemia and leucocyte infiltration was found where the cannula penetrated the cortical tissue. The food intake and weight curves for the experimental animals have been reported previously~, ,~. The results after subarachnoidal injection of tritiated leucine are shown in Table I, Salt loading induced an increased incorporation of leucine into the TCAprecipitated residue by 4 0 0 ~ in the SON and by 150~ in the posterior pituitary. Thirst was somewhat less effective, but induced a stimulation of 5 0 ~ in the SON and 70% in the posterior pituitary. The effect of thirst and salt loading on the cerebral cortex was slight and, with one exception, not statistically significant. The much lower concentration of radioactive precursor around the upper cortical surface was reflected by the low radiolabelling of cortical proteins. Table II shows the protein-bound radioactivity in the SON, neurohypophysis and cerebral cortex as a function of time after intraperitoneal injection of tritiated leucine. This experiment was performed in order to investigate whether differences in protein turnover rates between control and experimental animals would influence comparisons. The results did not indicate any appreciable differences. The extent of incorporation was increased upon osmotic stimulation in the SON and posterior pituitary (40 and 8 0 ~ , respectively), but slightly decreased in the cerebral cortex after 7 days of thirst. Incorporation of leucine in vitro A series of experiments were performed to study the effect of tissue slice thickness on leucine incorporation. Cerebral cortex slices 0.3 mm thick consistently incorporated to a higher extent than slices 0.5 mm thick. Slices from the SON region, however, showed twice as high an incorporation at 0.5 mm as at 0.3 mm thickness. Brain Research, 26 (1971) 95-103

xD ..4

to o~

S O N , NEUROHYPOPHYSIS AND CEREBRAL CORTEX 2 h AFTER A SUBARACHNO1DAL INJECTION OF [3H]LEUCINE

SON Posterior pituitary Cerebral cortex SON Posterior pituitary Cerebralcortex

2 T h i r s t f o r 7 days or 1 . 5 ~ o N a C l f o r 1 2 d a y s

19500 +- 1170 1305 +- 80 705 +- 110

12900 +- 800 1450_~ ~ 400 450 ± 40 4 4 4

7 7 7 32800 +- 1940 2020 ± 230 940 i 170

25500 +- 2930 2 6 5 0 ~ - 280 595 +- 70

Means

n

Region

Means

Thirst

Control

1 Thirst for 4 days or2~NaClfor4days

E x p e r i m e n t no.

n.s. ~ not significant.

4 4 4

7 7 7

n

n.s.

~z 0.01 ~0.05

-< 0.001 <0.01 n.s.

P

44450 +- 1940 2190 +- 130 710 ~ 150

55890 -~ 8260 3680+_ 330 745 _:: 120

Means

S a l t loading

4 4 4

7 7 7

n

n.s.

-,-~0.001 < 0.01

-~0.001 ,::0.05

,::i 0.001

P

Radioactivity expressed as c o u n t s / r a i n / r a g protein; M e a n s -# S.E.M. n ~= n u m b e r of a n i m a l s ; P :~: probability o f a significant difference f r o m c o n t r o l s ;

SPECIFIC ACTIVITY OF PROTEIN IN THE

TABLE I

©:

L¢3 ,-'d

z G

Oo

99

AMINO ACID INCORPORATION IN RAT SUPRAOPTIC NUCLEUS T,X.BLE 11 SPECIFIC ACTIVITY OF PROTEIN IN S O N ,

NEUROHYPOPHYSIS AND CEREBRAL CORTEX AFTER INTRAPERI-

TONEAL INJECTION OF [3H]LEUCINE

Radioactivity expressed as counts/rain/rag protein.

SON

Posterior pituitary

Cerebral cortex

Time after injection (rain)

Control

Thirst 7 days

30 90 180 30 90 180 30 90 180

246 242 297 500 448 515 236 225 340

219 289 416 813 787 914 184 237 184

TABLE llI EFFECTS OF THIRST AND SALT LOADING ON THE INCORPORATION

in vitro

OF [3H]LEUCINE INTO T C A -

PRECIPITABLE RESIDUE BY THE S O N , NEUROHYPOPHYSIS AND CEREBRAL CORTEX

The chopped tissue was incubated for 60 rain at 37~C. Activity expressed as counts/rain/rag protein. Each value represents the mean for 4-5 animals.

Experirnent

Tissue

Control

tlO.

1 2 3 4

5 6

SON Cortex SON Cortex SON Cortex SON Posterior pituitary Cortex SON Cortex SON Cortex

902 1059 2256 1250 1718 1205 1250

Duration o f thirst (days)

Duration o f 2 % NaCl treatment days

4 1598 954 2107 1414 1859 703

2789 417 1132 960 2218 1330

Counts/rain/rag in the SON as per cent of counts/ min/mg in cortex (means of experiments 1 to 6 5- S.E.M.) 166 ~ 31

7

11

14

2617 1281 2098 752 1346

2728 998 1728 764

2094 897

3060 397 1315 563 1786 1046

194~36

274~39

244:~15

202(233,171)

Brain Research, 26 ( 197 I) 95-103

100

A. NORSTROMet at.

Therefore, 0.5 mm slices were used in the main experimental series for both cortex and SON, since optimal conditions for incorporation by the SON were desired; The influence of variations in osmolarity of the incubation medium was studiedl Both SON and cerebral cortex incorporated leucine at optimal rates in the isotonic medium used in the main experimental series. Table III shows the TCA-precipitable radioactivity from the experiments in vitro. In the control material the SON incorporated leucine per unit weight of protein 50~0 more than cerebral cortex. Thirst for 4, 7 or I1 days caused a moderate increase in the incorporation of leucine per unit weight of protein into the SON, approximately 20°/ in all groups. A decreased radiolabelling was found after salt loading. The results are summarized at the bottom of Table II1, where the changes in the SON are related to the effects of a cortical area, which is not involved in the specific osmotic response. A relative stimulation of the SON was thus seen for all the experimental groups, with a maximum after 7 days of thirst (P in the range of 0.05). The series performed on the posterior pituitary hi vitro (8 animals) showed approximately a 15°/o increase in leucine incorporation into proteins after thirst. The TCA-soluble radioactivity was, however, over 50~o higher than in the controls. The TCA-soluble radioactivity in the SON and cortical slices was not affected by osmotic stress. In all experiments, however, the TCA-soluble radioactivity per unit weight of protein was about 40°/,, higher in the cerebral cortex than m the SON. DISCUSSION Microanalyses on isolated cells of the SON performed in our laboratory have shown a significant increase in total cell volume, RNA content and respiration in osmotically stimulated rats3, 5, A number of studies of isolated cells in other CNS units have demonstrated that this kind of response is common for specifically stimulated neurones2,9,11 . Since the SON is well delineated and the rauo neuronal.non-neuronal matter is unusually high, it is well suited for studies on protein synthesis on a macro scale which can be correlated with the previous findings on the single cell level. We measured the incorporation of leucine into TCA-insoluble proteins in order to estimate changes in the capacity for protein synthesis without special reference to the formation of the specific hormone. The radiolabelling will include both sedentary proteins and exportable proteins, such as neurophysins, but not vasopressin, since leucine is not present in its polypeptide chain~. The results demonstrate that the incorporation of leucine into the SON and posterior pituitary proteins was enhanced both in vivo and in vitro after osmotic stress. The experiments carried out in vivo showed a greater response to osmouc stress compared with those involving slice incubation. The parallel use of in vivo and in vitro conditions for protein biosynthesis may give a better documentation of a physiological response. The correlation was relatively good in this study, although the qualitative pattern of protein synthesis differs between in vivo and in vitro conditions TM. The differences between conditions in vivo-in vitro can be accounted for Brain Research, 26 (1971) 95-103

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by a number of factors: difficulties in control of permeability and pool factors in vivo and partial inactivation of the biosynthetic mechanisms in vitro. The moderate effect of salt loading in the experiments in vitro which contrasted with the striking changes in vivo may, however, be due to differences in the length of the salt loading periods. Cerebral cortex tissue was processed in parallel with the SON in all experiments as an indicator of possible general effects on brain. Surprisingly, osmotic stress induced a tendency towards increased incorporation of leucine in vivo, whereas a slightly decreased incorporation was observed for cortical slices in vitro. This discrepancy might be due to local cortical reactions to the implanted cannula. In spite of the rich vascularization in the SON which would suggest a high sensitivity to hypoxia, we find a higher degree of radioactive labelling in vitro with 0.5 mm thick pieces compared with 0.3 mm pieces which was the opposite of the findings for cortical slices. Consistent with our findings are the reports by Grenell and Kabat ~ and the high content of enzymes involved in anaerobic glycolysisa, 6. It is possible that the integrity of the tissue is more important than optimal oxygen tension for amino acid incorporation into SON tissue, it is improbable that the effects on labelling in vitro would be due to changed transport rates, sinse the TCA-soluble radioactivity in the SON or the cortex were at a similar level in control and experimental animals. The labelling of the posterior pituitary in vivo may represent both locally synthesized proteins and transported neurosecretory material is, whereas the material labelled in vitro is solely of pituitary origin. Takabatake and Sachs 2~ reported up to a five-fold increase in vasopressin labelling after osmotic stress, when whole hypotha[amic-median eminence slices were incubated with [3'~S]cysteine. However, the authors found only a small increase in incorporation into TCA-precipitable proteins. The more significant enhancement of incorporation in the present study could well be explained since only the supraoptic region was used. A striking increase in labelling of supraoptic neurones was also found by Murray ~6, who monitored the effect of osmotic stress by quantitative autoradiography. The combined results of amino acid incorporation into proteins of the SON in vivo and i , vitro are consistent with the findings on single SON neurones which showed a striking increase in cell volume, RNA, and respiratory enzymes as a result of osmotic stress on the animal 3,4. It is apparent that the higher content of proteins in the cells after specific stimulation is due at least to a large extent to an increased rate of protein synthesis, since the rate of decay of SON proteins is not significantly changed during osmotic stimulation 16. SUMMARY

Incorporation of tritiated leucine into the supraoptic nucleus and posterior pituitary was measured in vivo after subarachnoidal administration of the precursor and in vitro by incubation of slices from the same regions. Osmotic stimulation induced by thirst or salt loading produced a striking increase of the amino acid incorporation into the proteins of the supraoptic nucleus and posterior pituitary in vivo, Brain Research, 26 (1971) 95--103

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A. NORSTROM et aL

w h e r e a s o n l y a m o d e r a t e s t i m u l a t i o n was o b t a i n e d in vitro. T h e effect o f slice t h i c k n e s s o n a m i n o a c i d i n c o r p o r a t i o n was i n v e s t i g a t e d . T h e results a r e discussed in c o n n e c t i o n w i t h results o b t a i n e d e a r l i e r o n single cells o f t h e s u p r a o p t i c region. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d by g r a n t s f r o m t h e S w e d i s h M e d i c a l R e s e a r c h C o u n c i l (B70-12X-164-06A) and from the Medical Faculty, University of G6teb0rg. W e are i n d e b t e d to D r . A l a n W a l d m a n f o r his h e l p f u l c o m m e n t s a n d c r i t i c i s m .

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Brain Research, 26 (1971) 95-103