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Central monoamine oxidase and phenolsulfotransferase activities in spontaneously hypertensive rats
Life Sciences, Vol. 48, pp. 1985-1990 Printed in the U.S.A.
Pergamon Press
CENTRAL MONOAMINE OXIDASE AND PHENOLSULFOTRANSFERASE ACTIVITIES IN SPONTANEOUSLY HYPERTENSIVE RATS M.K. Sire
Department of Pharmacology, Faculty of Medicine National University of Singapore, Singapore 0511 (Received in final form March 14, 1991)
Summary
The activities of monoamine oxidase and phenolsulfotransferase in the hypothalamus and anterior pituitary gland of spontaneously hypertensive rats and the normotensive control (Wistar Kyoto rat) rats were investigated. The monoamine oxidase activity (determined using dopamine as substrate) in both these tissues was not significantly different between the normo- and hypertensive animals. Hypothalamic phenolsulfotransferase does not sulfate-conjugate dopamine at pH of 6.5 and pituitary phenolsulfotransferase does not sulfate-conjugate dopamine or 3,4-dihydroxyphenylacetic acid at the same pH. Hypothalamic phenolsulfotransferase activity determined using 3,4-dihydroxyphenylacetic acid as substrate was significantly higher in the spontaneously hypertensive than the Wistar Kyoto rats, while pituitary enzyme (determined using phenol as substrate) was the same in both strains of animals. We proposed that in the spontaneously hypertensive rats the higher level of hypothalamic phenolsulfotransferase could (by removing 3,4-dihydroxyphenylacetic acid as sulfated acid) increase the deamination of dopamine by monoamine oxidase. This could in turn result in the presence of high amount of sulfated 3,4-dihydroxyphenylacetic acid in the anterior pituitary gland reported in our earlier study, and be partly responsible for the reduced central dopaminergic activity found in the hypertensive rats.
We recently showed that direct injection of dopamine into the arcuate nucleus of spontaneously hypertensive rats (SHR) produced a significantly greater drop in the blood pressure of these animals than in the control normotenslve Wistar Kyoto rats (WK¥) ( I ) . Since the dopaminergic neurons of the arcuate nucleus together with their axons in the median eminence are part of the tuberoinfundlbular tract that produces and transports dopamine to the anterior pituitary gland via the hypophysaal portal circulation (2), our results indicate that the tract is a possible site for the alleged reduced central dopaminergic activity in the SHR (3-5). In a later study examining the contents of dopamine 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
1986
MAO and Sulfotransferase in SHR
Vol. 48, No. 20, 1991
and its metabolites in the anterior pituitary gland, we found that the glandular levels of free and sulfate-conjugated dopamine in the SHR were not significantly different from those in the WKY, but that the levels of free and sulfateconjugated 3,4-dihydroxyphenylacetic acid (DOPAC) were respectively 6- and 15-fold higher in the former animal (6). Since DOPAC is the immediate product of dopamine oxidation by the enzyme monoamine oxidase (MAO), and sulfate-conjugated DOPAC by the enzyme phenolsulfotransferase (PST), the findings indicate that the metabolism of dopamine in the hypothalamo-pituitary region of the SHR is abnormal. In an attempt to locate the site (pituitary or extra-pituitary) and identify the enzymes involved in the abnormality, we studied the activities of MAO and PST in the anterior pituitary gland and hypothalamus of the SHR and WIRY. Methods
Animals: The animals used were 3-4 months old male SHR and WKY. They were purchased from the Animal Resource Centre, Murdoch, Western Australia. The mean blood pressure (± SEM) of the SHR and WKY, measured by the tail cuff method, was 172 ± 9 and 114 ± 12 mm Hg, respectively. Dru~s and Chemicals: [7- 14C] Dopami n e (specific acti vl"ty of 56 m C ~ mmole) was obtained from Amarsham Inter national. 3'-Phosphoa denosine-5'-phospho [~ S ]sulphate ([ S]PAPS) of 1.6 Ci/mmole specific activity was obtained from New England Nuclide. Dopamine and DOPAC were purchased from Sigma and phenol from Merck. Pentachlorophenol and 2,6-dichloro-4-nitrophenol were obtained from Fluka.
Preparation of Oraan Extracts and Assay of Enzymes: The preparation of organ extracts and assay of MAO and PST were carried out as described previously (7). Briefly, rats were killed by cervical dislocation and the brain and anterior pituitary gland were removed. The hypothalamus was dissected out according to the method described by Glowiski et al. (8). The pituitary gland (5-7 rag) and hypothalamus (17-20 rag) were sonicated in 100 and 200 ul respectively of 0.5 M phosphate buffer, pH 7.4 (for assay of MAO) and pH 6.5 (for assay of PST), for 10 rain in a Vibra ultra sound sonicator (Vibra Cell, Sonic & Materials Inc., Connecticut) to obtain the respective organ homogenates. Total protein in the various homogenates was determined by the method of Lowry et al. (9). The a s s a y f o r MAO was s t a r t e d by a d d i n g 30 u l of homogenate t o an i n c u b a t i o n m i x t u r e c o n t a i n i n g 50 u M r a d i o l a b e l e d [ 7 - ' ~ C ] dopamine ( s p e c i f i c a c t i v i t y of 0.93 mCi/mmole) i n a t o t a l volume of 100 u l 0 . 5 M phosphate b u f f e r pH 7 . 4 ; and t h e a s s a y f o r PST s t a r t e d ~ y a d d i n g 30 u l of homogenate t o an i n c u b a t i o n m i x t u r e c o n t a i n i n g 1.7 ,M of [ ~ S ] PAPS ( s p e c i f i c a c t i v i t y of 1.6 Ci/mmole) and 50 uM of one of t h e f o l l o w i n g s u b s t r a t e s : dopamine, dopac or phenol i n a f i n a l volume of 100 u l 10 mM phosphate b u f f e r pH 6 . 4 . Assay of PST c a r r i e d i n t h e p r e s e n c e of 2 , 6 - d i c h l o r o - 4 - n i t r o p h e n o l or p e n t a c h l o r o p h e n o l c o n t a i n e d v a r i o u s c o n c e n t r a t i o n s ( 0 . 0 5 t o 10 ,M) of e i t h e r i n h i b i t o r i n t h e same i n c u b a t i o n m i x t u r e . The enzyme a c t i v i t y of both a s s a y s was s t o p p e d by a d d i n g 30 ul 2N HC1 a t v a r i o u s i n c u b a t i o n p e r i o d s and t h e c o n t r o l a s s a y s o b t a i n e d by a d d i n g t h e a c i d t o t h e i n c u b a t i o n m i x t u r e s p r i o r to t h e a d d i t i o n of homogenete or s u p e r n a t a n t . The a c i d i f i e d i n c u b a t i o n m i x t u r e s were t h e n p r e p a r e d f o r chromatography by e i t h e r c e n t r i f u g a t i o n a t 100,000 x g f o r 3 h o u r s or f i l t r a t i o n through u l t r a f i l t e r s (Ultrafree-HC, Hillipore). The s u p e r n a t a n t s or f i l t r a t e s were t h e n t r a n s f e r r e d t o and s t o r e d i n capped t u b e s t i l l r e q u i r e d . The r a d i o l a b e l l e d p r o d u c t s were s e p a r a t e d from t h e s u b s t r a t e s by high performance l i q u i d chromatography and t h e r a d i o a c t i v i t y
Vol. 48, No. 20, 1991
MAO and Sulfotransferase in SHR
1987
determined by a coupled radioisotope detector as described previously (7). With this method, the MAO and PST in small brain samples can be assayed individually and accurately i.e. without having to pool samples; the acid sulfate (DOPAC-sulfate) formed is not lost during barium sulfate precipitation to remove unreacted PAPS (I0); and the product as well as the substrate of the enzyme reaction can be simultaneously determined which in turn facilitates accurate monitoring of enzyme kinetics and product identification.
Results
Table I shows that the activity of hypothalamic and anterior pituitary MAO in the SHR, though lower, was not significantly different from that of the WKY. TABLE
I
Activity of monoamine oxidase in the anterior pituitary and hypothalamus of the WKY and SHR IMonoamine Oxidase Activity (umole/mg protein) Anterior Pituitary Hypothalamus Mean SEM Mean SEM ~Y SHR
9.5 8.1
Values a r e the mean dopamine as substrate
of
6-8
1.5 1.3 separate
TABLE Activity
of
17.9 16.2
2.1 1.9
determinations.
Iusing
II
sulphotransferase in the anterior pituitary gland and hypothalamus of the WKY and SHR Sulphotransferase A~tivity (cpm/mg protein) Anterior Pituitary" Hypothalamus ~ Mean SEM Mean SEM
WKY
2302
240
45069
4235
SHR
2497
255
~108379
10105
V a l u e s a r e t h e R a n of 6 - 8 s e p a r a t e d e t e r m i n a t i o n s . 1Using phenol as substrate. - - U s i n g DOPAC a s s n b s t r a t e . See t e x t f o r d e t a i l s . *Significantly different (p < 0 . 0 5 , S t u d e n t ' s t _ - t e s t ) from t h e v a l u e of WKY.
1988
MAO and Sulfotransferase in SHR
Vol. 48, No. 20, 1991
Hypothalamic PST activity (assayed using DOPAC as substrate) was significantly higher in the SHR than WK¥, while anterior pituitary PST (assayed using phenol) was not significantly different between the normo- and hypertensive animals (see Table 2 for details). Table III shows that both pituitary and hypothalamic PST were inhibited concentration-dependently by 2,6-dichloro-4-nitrophenol and pentachorophenol, respectively. The inhibition of the pituitary enzyme at each concentration of the inhibitors was not significantly different from that of the hypothalamic enzyme. TABLE III
Inhibition of pituitary and hypothalamic PST by 2,6-dichloro-4nitrophenol and pentachlorophenol
Concentration
(.~)
0.05 0.10 1.0 10
Inhibition of PST (% of Control) 2,6-Dichl~ro-4-nitrophenol^ Fentac~lorophenol Pituitary* Hypothalamic z Pituitary* Hypothalamic ~ Mean
SEM
Mean
SEM
Mean
SEM
Mean
SEM
18 23 51 100
2 2.7 4.9
16 20 48 100
1.7 2.1 4
6 11 48 100
0.7 1.2 3.9
6 12 51 100
0.6 1 4
IAssayed using phenol as substrate. 2Aasayed using DOPAC as substrate. The values are the mean of three separate determinations.
DISCUSSION
Our results show that the anterior pituitary PST of the SHR and WKY sulfateconjugates phenol but not dopamine or DOPAC. The presence of a PST that sulfateconjugates phenol in the rat anterior pituitary has also been reported by Foldes and Meek (11). Together with the inhibition profile of the enzyme by 2,6dichloro-4-nitrophenol (see Table III), it appears that the pituitary PST is probably the P form of the enzyme as defined by Rein et al. (12). From this it can be concluded that the sulfate-conjugated derivatives of dopamine and DOPAC found in the anterior pituitary gland in an earlier study of ours (6) are probably from extrapituitary source. The fact that phenol is a substrate of pituitary PST may indicate that the enzyme is specific for the metabolism of steroids that regulate pituitary functions. On the other hand hypothalamic PST of the SHR and WKY sulfate-conjugates phenol and DOPAC but not dopamine at pH 6.5. Together with our similar finding for the PST in the Sprague Dawley rat (7), the present results may indicate that dopamine is not a substrate of this particular form of hypothalamic PST. Rat hypothalamic PST that sulfate-conjugates phenol (11) and rat brain PST that
Vol. 48, No. 20, 1991
MAO and Sulfotransferase in SHR
1989
sulfate-conjugates phenol and DOPAC (I0) at pH 6.5 have also been reported. It thus appears that hypothalamic PST of the rat could be a variant of the P form of PST in that it acts on DOPAC in addition to phenol. Furthermore, hypothalamic PST cannot be differentiated from pituitary PST by specific inhibitors, 2,6-dichloro4-nitrophenol or pentachlorophenol (see Table III). The presence of high level of hypothalamic PST that sulfate-conjugates DOPAC in the SHR (see Table 2 for details) could, thus, be responsible for the high amount of sulfate-conjugated DOPAC found in the anterior pituitary gland of these animals (6). Another form of rat hypothalamic PST which has pHoptimum of 9.0 has been described by Foldes and Meek (10), and Wong (13) to sulfate-conjugate dopamine. However, the activity of this form of PST in the hypothalamus and other brain regions of the SHR was found not to be significantly different from its corresponding activity in normotensive WKY (14). The MAOactivity in both the hypothalamusand anterior pituitary gland of the SHR, although lower than that of the WKY, was not significantly different. In spite of this, the significantly higher level of PST in the hypothalamus of the SHR could, by rapidly removing DOPAC as sulfated DOPAC, increase the rate of deamination of dopamine by MAO. It has been known that SHR exhibit increased plasma prolactin level ( 1 5 , 16), and that prolactin stimulates the tuberoinfundibular neurons to increase the production of dopamine (17, 18). In normal animals, the dopamine produced by an excess prolactin would have acted on the D 2 dopaminargic receptors in the anterior pituitary gland to reduce the production of prolactin as a negative feedback for maintaining the correct level of the hormone in the blood (2). The fact that the level of anterior pituitary dopamine in the SHR was not elevated (6), despite the high plasma prolactin level, may indicate that these animals could not increase their hypothalamlc dopamine due to the presence of high level of PST as explained above in this paragraph.
The aim of the present study, namely to locate the possible site and enzymes involved in the production of high amount of sulfate-conjugated DOPAC found in the anterior pituitary gland of the SHR, has thus been achieved. In addition, the data on PST support the statements that "sulfotransferases are a family of separate enzymes and isozymes with differing but often overlapping substrate specificity" (19) and that the "classification is temporary and will probably have to be modified in the future as additional substrates emerge" (20).
Acknowledsements This study was supported by a Grant RP880351 from the National University of Singapore. The author would like to thank Ms T.P. Hsu and L. Tan for their technical assistance.
References I. 2. 3. 4.
J.S.L. MOK, J.S. HUTCHINSON and M.K. SIM, Clin. Exp. Hypertens. AI2 15-36 (1990). J. TOUMISTO and P. MANNISTO, Pharmacol. Rev. 37 249-332 (1985). H. KAWABE, K. KONDO and T. SARUTA, Clin. Exp. Hypertens. A5 1703-1716 (1983). J.S. HUTCHINSON and J.S.L. MOK, Clin. Exp. Hypertens. A6 2055-2058 (1984).
1990
MAO and Sulfotransferase in SHR
Vol. 48, No. 20, 1991
5, 6. 7. 8. 9.
J . S . L . MOK and M.K. SIM, Clin. Exp. Hypertens. A9 1615-1635 (1987). M.K. SIM and T.P. HSU, Clin. Exp. Hypertens. AI2 343-353 (1990). M.K. SIM and T.P. HSU, J. Pharmacol. Methods 24 157-163 (1990). J. GLOWINSKI and L.L. IVERSEN, J. Neurochem. 13 655-669 (1966). O.H. LOWRY, N.J. ROSEBRDUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 10. A. FOLDES and J.L. MEEK, Biochem. Biophys. Acta. 327 365-373 (1973). 11. A. FOLDES and J.L. MEEK, J. Neurochem. 23 303-307 (1974). 12. G. REIN, V. GLOVER and M. SANDLER, In Phenolsulfotransferase in Mental Health Research 98-126 M. SANDLER and E. USDIN Ed. Macmillan Publishers Ltd., London
(1981). 13. K.P. WONG, Biogenic Amines ~ 189-200 (1987). 14. J.S.L. MOK, J.S. HUTCHINSON and K.P. WONG,Biogenic Amines ! 475-481 (1990). 15. J.R. SOWERS, G. RESCH, G. TEMPEL, J. HERZOGand M. CALANTINO, Acta Endocrinol. 90 1-7 (1979). 16. F.P. McMURTY, N. KAZAMAand B.C. WEXLER, Proc. Soc. Exp. Biol. Med. 161 186188 (1979). 17. N.A. PERKINS, T.C. WESTFALL, C.V. PAUL, R.M. MecLEOD and A.D. ROGOL, Brain Res. 160 431-444 (1979). 18. K.T. DEMARE, G.D. RIS2LE and K.E. MOORE, Neuroendocrinology 38 467-475 (1984). 19. G.J. MULDERand W.B. JAKOBY, In Conjugation Reactions in Drug Metabolism: An I n t e g r a t e d Approach 107-162 G.J. Mulder Ed. Taylor & Francis, London (1990). 20. M. MESNIL, B. TESTA and P. JENNER, Adv. Drug Res. 13 95-207 (1984).
Pergamon Press
CENTRAL MONOAMINE OXIDASE AND PHENOLSULFOTRANSFERASE ACTIVITIES IN SPONTANEOUSLY HYPERTENSIVE RATS M.K. Sire
Department of Pharmacology, Faculty of Medicine National University of Singapore, Singapore 0511 (Received in final form March 14, 1991)
Summary
The activities of monoamine oxidase and phenolsulfotransferase in the hypothalamus and anterior pituitary gland of spontaneously hypertensive rats and the normotensive control (Wistar Kyoto rat) rats were investigated. The monoamine oxidase activity (determined using dopamine as substrate) in both these tissues was not significantly different between the normo- and hypertensive animals. Hypothalamic phenolsulfotransferase does not sulfate-conjugate dopamine at pH of 6.5 and pituitary phenolsulfotransferase does not sulfate-conjugate dopamine or 3,4-dihydroxyphenylacetic acid at the same pH. Hypothalamic phenolsulfotransferase activity determined using 3,4-dihydroxyphenylacetic acid as substrate was significantly higher in the spontaneously hypertensive than the Wistar Kyoto rats, while pituitary enzyme (determined using phenol as substrate) was the same in both strains of animals. We proposed that in the spontaneously hypertensive rats the higher level of hypothalamic phenolsulfotransferase could (by removing 3,4-dihydroxyphenylacetic acid as sulfated acid) increase the deamination of dopamine by monoamine oxidase. This could in turn result in the presence of high amount of sulfated 3,4-dihydroxyphenylacetic acid in the anterior pituitary gland reported in our earlier study, and be partly responsible for the reduced central dopaminergic activity found in the hypertensive rats.
We recently showed that direct injection of dopamine into the arcuate nucleus of spontaneously hypertensive rats (SHR) produced a significantly greater drop in the blood pressure of these animals than in the control normotenslve Wistar Kyoto rats (WK¥) ( I ) . Since the dopaminergic neurons of the arcuate nucleus together with their axons in the median eminence are part of the tuberoinfundlbular tract that produces and transports dopamine to the anterior pituitary gland via the hypophysaal portal circulation (2), our results indicate that the tract is a possible site for the alleged reduced central dopaminergic activity in the SHR (3-5). In a later study examining the contents of dopamine 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
1986
MAO and Sulfotransferase in SHR
Vol. 48, No. 20, 1991
and its metabolites in the anterior pituitary gland, we found that the glandular levels of free and sulfate-conjugated dopamine in the SHR were not significantly different from those in the WKY, but that the levels of free and sulfateconjugated 3,4-dihydroxyphenylacetic acid (DOPAC) were respectively 6- and 15-fold higher in the former animal (6). Since DOPAC is the immediate product of dopamine oxidation by the enzyme monoamine oxidase (MAO), and sulfate-conjugated DOPAC by the enzyme phenolsulfotransferase (PST), the findings indicate that the metabolism of dopamine in the hypothalamo-pituitary region of the SHR is abnormal. In an attempt to locate the site (pituitary or extra-pituitary) and identify the enzymes involved in the abnormality, we studied the activities of MAO and PST in the anterior pituitary gland and hypothalamus of the SHR and WIRY. Methods
Animals: The animals used were 3-4 months old male SHR and WKY. They were purchased from the Animal Resource Centre, Murdoch, Western Australia. The mean blood pressure (± SEM) of the SHR and WKY, measured by the tail cuff method, was 172 ± 9 and 114 ± 12 mm Hg, respectively. Dru~s and Chemicals: [7- 14C] Dopami n e (specific acti vl"ty of 56 m C ~ mmole) was obtained from Amarsham Inter national. 3'-Phosphoa denosine-5'-phospho [~ S ]sulphate ([ S]PAPS) of 1.6 Ci/mmole specific activity was obtained from New England Nuclide. Dopamine and DOPAC were purchased from Sigma and phenol from Merck. Pentachlorophenol and 2,6-dichloro-4-nitrophenol were obtained from Fluka.
Preparation of Oraan Extracts and Assay of Enzymes: The preparation of organ extracts and assay of MAO and PST were carried out as described previously (7). Briefly, rats were killed by cervical dislocation and the brain and anterior pituitary gland were removed. The hypothalamus was dissected out according to the method described by Glowiski et al. (8). The pituitary gland (5-7 rag) and hypothalamus (17-20 rag) were sonicated in 100 and 200 ul respectively of 0.5 M phosphate buffer, pH 7.4 (for assay of MAO) and pH 6.5 (for assay of PST), for 10 rain in a Vibra ultra sound sonicator (Vibra Cell, Sonic & Materials Inc., Connecticut) to obtain the respective organ homogenates. Total protein in the various homogenates was determined by the method of Lowry et al. (9). The a s s a y f o r MAO was s t a r t e d by a d d i n g 30 u l of homogenate t o an i n c u b a t i o n m i x t u r e c o n t a i n i n g 50 u M r a d i o l a b e l e d [ 7 - ' ~ C ] dopamine ( s p e c i f i c a c t i v i t y of 0.93 mCi/mmole) i n a t o t a l volume of 100 u l 0 . 5 M phosphate b u f f e r pH 7 . 4 ; and t h e a s s a y f o r PST s t a r t e d ~ y a d d i n g 30 u l of homogenate t o an i n c u b a t i o n m i x t u r e c o n t a i n i n g 1.7 ,M of [ ~ S ] PAPS ( s p e c i f i c a c t i v i t y of 1.6 Ci/mmole) and 50 uM of one of t h e f o l l o w i n g s u b s t r a t e s : dopamine, dopac or phenol i n a f i n a l volume of 100 u l 10 mM phosphate b u f f e r pH 6 . 4 . Assay of PST c a r r i e d i n t h e p r e s e n c e of 2 , 6 - d i c h l o r o - 4 - n i t r o p h e n o l or p e n t a c h l o r o p h e n o l c o n t a i n e d v a r i o u s c o n c e n t r a t i o n s ( 0 . 0 5 t o 10 ,M) of e i t h e r i n h i b i t o r i n t h e same i n c u b a t i o n m i x t u r e . The enzyme a c t i v i t y of both a s s a y s was s t o p p e d by a d d i n g 30 ul 2N HC1 a t v a r i o u s i n c u b a t i o n p e r i o d s and t h e c o n t r o l a s s a y s o b t a i n e d by a d d i n g t h e a c i d t o t h e i n c u b a t i o n m i x t u r e s p r i o r to t h e a d d i t i o n of homogenete or s u p e r n a t a n t . The a c i d i f i e d i n c u b a t i o n m i x t u r e s were t h e n p r e p a r e d f o r chromatography by e i t h e r c e n t r i f u g a t i o n a t 100,000 x g f o r 3 h o u r s or f i l t r a t i o n through u l t r a f i l t e r s (Ultrafree-HC, Hillipore). The s u p e r n a t a n t s or f i l t r a t e s were t h e n t r a n s f e r r e d t o and s t o r e d i n capped t u b e s t i l l r e q u i r e d . The r a d i o l a b e l l e d p r o d u c t s were s e p a r a t e d from t h e s u b s t r a t e s by high performance l i q u i d chromatography and t h e r a d i o a c t i v i t y
Vol. 48, No. 20, 1991
MAO and Sulfotransferase in SHR
1987
determined by a coupled radioisotope detector as described previously (7). With this method, the MAO and PST in small brain samples can be assayed individually and accurately i.e. without having to pool samples; the acid sulfate (DOPAC-sulfate) formed is not lost during barium sulfate precipitation to remove unreacted PAPS (I0); and the product as well as the substrate of the enzyme reaction can be simultaneously determined which in turn facilitates accurate monitoring of enzyme kinetics and product identification.
Results
Table I shows that the activity of hypothalamic and anterior pituitary MAO in the SHR, though lower, was not significantly different from that of the WKY. TABLE
I
Activity of monoamine oxidase in the anterior pituitary and hypothalamus of the WKY and SHR IMonoamine Oxidase Activity (umole/mg protein) Anterior Pituitary Hypothalamus Mean SEM Mean SEM ~Y SHR
9.5 8.1
Values a r e the mean dopamine as substrate
of
6-8
1.5 1.3 separate
TABLE Activity
of
17.9 16.2
2.1 1.9
determinations.
Iusing
II
sulphotransferase in the anterior pituitary gland and hypothalamus of the WKY and SHR Sulphotransferase A~tivity (cpm/mg protein) Anterior Pituitary" Hypothalamus ~ Mean SEM Mean SEM
WKY
2302
240
45069
4235
SHR
2497
255
~108379
10105
V a l u e s a r e t h e R a n of 6 - 8 s e p a r a t e d e t e r m i n a t i o n s . 1Using phenol as substrate. - - U s i n g DOPAC a s s n b s t r a t e . See t e x t f o r d e t a i l s . *Significantly different (p < 0 . 0 5 , S t u d e n t ' s t _ - t e s t ) from t h e v a l u e of WKY.
1988
MAO and Sulfotransferase in SHR
Vol. 48, No. 20, 1991
Hypothalamic PST activity (assayed using DOPAC as substrate) was significantly higher in the SHR than WK¥, while anterior pituitary PST (assayed using phenol) was not significantly different between the normo- and hypertensive animals (see Table 2 for details). Table III shows that both pituitary and hypothalamic PST were inhibited concentration-dependently by 2,6-dichloro-4-nitrophenol and pentachorophenol, respectively. The inhibition of the pituitary enzyme at each concentration of the inhibitors was not significantly different from that of the hypothalamic enzyme. TABLE III
Inhibition of pituitary and hypothalamic PST by 2,6-dichloro-4nitrophenol and pentachlorophenol
Concentration
(.~)
0.05 0.10 1.0 10
Inhibition of PST (% of Control) 2,6-Dichl~ro-4-nitrophenol^ Fentac~lorophenol Pituitary* Hypothalamic z Pituitary* Hypothalamic ~ Mean
SEM
Mean
SEM
Mean
SEM
Mean
SEM
18 23 51 100
2 2.7 4.9
16 20 48 100
1.7 2.1 4
6 11 48 100
0.7 1.2 3.9
6 12 51 100
0.6 1 4
IAssayed using phenol as substrate. 2Aasayed using DOPAC as substrate. The values are the mean of three separate determinations.
DISCUSSION
Our results show that the anterior pituitary PST of the SHR and WKY sulfateconjugates phenol but not dopamine or DOPAC. The presence of a PST that sulfateconjugates phenol in the rat anterior pituitary has also been reported by Foldes and Meek (11). Together with the inhibition profile of the enzyme by 2,6dichloro-4-nitrophenol (see Table III), it appears that the pituitary PST is probably the P form of the enzyme as defined by Rein et al. (12). From this it can be concluded that the sulfate-conjugated derivatives of dopamine and DOPAC found in the anterior pituitary gland in an earlier study of ours (6) are probably from extrapituitary source. The fact that phenol is a substrate of pituitary PST may indicate that the enzyme is specific for the metabolism of steroids that regulate pituitary functions. On the other hand hypothalamic PST of the SHR and WKY sulfate-conjugates phenol and DOPAC but not dopamine at pH 6.5. Together with our similar finding for the PST in the Sprague Dawley rat (7), the present results may indicate that dopamine is not a substrate of this particular form of hypothalamic PST. Rat hypothalamic PST that sulfate-conjugates phenol (11) and rat brain PST that
Vol. 48, No. 20, 1991
MAO and Sulfotransferase in SHR
1989
sulfate-conjugates phenol and DOPAC (I0) at pH 6.5 have also been reported. It thus appears that hypothalamic PST of the rat could be a variant of the P form of PST in that it acts on DOPAC in addition to phenol. Furthermore, hypothalamic PST cannot be differentiated from pituitary PST by specific inhibitors, 2,6-dichloro4-nitrophenol or pentachlorophenol (see Table III). The presence of high level of hypothalamic PST that sulfate-conjugates DOPAC in the SHR (see Table 2 for details) could, thus, be responsible for the high amount of sulfate-conjugated DOPAC found in the anterior pituitary gland of these animals (6). Another form of rat hypothalamic PST which has pHoptimum of 9.0 has been described by Foldes and Meek (10), and Wong (13) to sulfate-conjugate dopamine. However, the activity of this form of PST in the hypothalamus and other brain regions of the SHR was found not to be significantly different from its corresponding activity in normotensive WKY (14). The MAOactivity in both the hypothalamusand anterior pituitary gland of the SHR, although lower than that of the WKY, was not significantly different. In spite of this, the significantly higher level of PST in the hypothalamus of the SHR could, by rapidly removing DOPAC as sulfated DOPAC, increase the rate of deamination of dopamine by MAO. It has been known that SHR exhibit increased plasma prolactin level ( 1 5 , 16), and that prolactin stimulates the tuberoinfundibular neurons to increase the production of dopamine (17, 18). In normal animals, the dopamine produced by an excess prolactin would have acted on the D 2 dopaminargic receptors in the anterior pituitary gland to reduce the production of prolactin as a negative feedback for maintaining the correct level of the hormone in the blood (2). The fact that the level of anterior pituitary dopamine in the SHR was not elevated (6), despite the high plasma prolactin level, may indicate that these animals could not increase their hypothalamlc dopamine due to the presence of high level of PST as explained above in this paragraph.
The aim of the present study, namely to locate the possible site and enzymes involved in the production of high amount of sulfate-conjugated DOPAC found in the anterior pituitary gland of the SHR, has thus been achieved. In addition, the data on PST support the statements that "sulfotransferases are a family of separate enzymes and isozymes with differing but often overlapping substrate specificity" (19) and that the "classification is temporary and will probably have to be modified in the future as additional substrates emerge" (20).
Acknowledsements This study was supported by a Grant RP880351 from the National University of Singapore. The author would like to thank Ms T.P. Hsu and L. Tan for their technical assistance.
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