- Email: [email protected]
Saline ingestion stimulates the in situ molar activity of tyrosine hydroxylase in the median eminence and superior cervical ganglion
Brain Research, 446 (1988) 363-368 Elsevier
363
BRE 13458 ,--$~
Saline ingestion stimulates the in situ molar activity of tyrosine hydroxylase in the median eminence and superior cervical ganglion Paulus S. Wang*, Hector A. Gonzalez and John C. Porter Cecil H. and Ida Green Center for Reproduciive Biology Sciences, University of Texas Health Science Center at Dallas, Departments of Obstetrics and Gynecology and Physiology, Dallas, TX 75235 (U.S.A. ) (Accepted 6 October 1987)
Key words: Tyrosine hydroxylase mass; Tyrosine hydroxylase activity; Sodium chloride ingestion; Median eminence; Superior cervical ganglion
The effects of drinking saline for 7 days on the mass and in situ activity of tyrosine hydroxylase (TH) in the ~.aedianeminence (ME) and superior cervical ganglion (SCG) of rats were investigated. TH mass was quantified by immunoblot assay. In situ TH activity was calculated from the rate of intracellular accumulation of L-dihydroxyphenylalanine(DOPA). In rats that drank 10 mM, 30 mM, and 100 mM NaCl for 7 days, TH activity in the ME was 34 + 4, 36 + 5, and 45 + 3 (mean and S.E.M.) tool of DOPA-h-l.mol ofTH -I. respectively, compared to 30 + 2 for rats that drank water. The activity of TH in the SCG of animals that drank 10 mM, 30 raM, and 100 mM NaCi was 143 + 24, 167 + 12, and 272 + 13 mol DOPA.h-l.mol TH -l, respectively, compared to 119 +_ 10 for animals that drank water. The mass of TH in the ME and SCG decreased as a function of the concentration of NaCI in the drinking water. In animals that drank water, 10 mM, 30 mM, and 100 mM NaCl, the amounts (pmol) of TH were, respectively, 0.28 + 0.03, 0.31 +_0.04, 0.23 + 0.02. and 0.21 _+0.01 per ME and 0.67 + 0.06, 0.72 + 0.11, 0.37 + 0.01, and 0.34 _+0.02 per SCG. TH activity in the ME or SCG was unaffected by treatment for 7 days with arginine vasopressin. The mean concentration of dopamine and that of norepinephrine in hypophysial portal plasma of the animals that drank 100 mM NaC! were more than twice that of animals that drank water. It is concluded that the in situ molar activity of TH in the ME and SCG as well as the secretion of dopamine into portal blood are stimulated by ingestion of saline.
INTRODUCTION t e r / n e u r o h o r m o n e s is a function of their rates of bioThe synthesis of d o p a m i n e in d o p a m i n e r g i c neurons and n o r e p i n e p h r i n e in n o r a d r e n e r g i c neurons involves two reactions that are c o m m o n to both types of cells 2°. These reactions are (a) the oxidative conversion of L-tyrosine to L-dihydroxyphenylalanine ( D O P A ) and (b) the decarboxylation of D O P A to form dopamine. In both types of cells, tyrosine hydroxylase (TH) [tyrosine 3 - m o n o o x y g e n a s e , L-tyrosine, hydropteridine: oxygen oxidoreductase (3-hydroxylating, E.C.1.14.16.2)] is believed to be the regulatory enzyme 16. Thus, any mechanism(s) that modulate(s) the intracellular activity of T H is important since the secretion of these neurotransmit-
synthesis. It was shown by Cahill and Perlman 5 that veratridine stimulated under in vitro conditions the in situ activity of T H of the superior cervical ganglion (SCG). Later, Porter 24 r e p o r t e d that incubation of hypothalamic tissue in the presence of veratridine resulted in a dose-dependent increase in the activity of T H of the median eminence ( M E ) , a region of the brain that contains neurites of tuberoinfundibular dopaminergic neurons. Inasmuch as veratridine stimulates the intraceilular flux of sodium ions, these findings raise the question of w h e t h e r ingestion of sodium chloride would lead to an alteration in the in
* Present address: Department of Physiology, National Yang-Ming Medical College, Taipei, Taiwan. Correspondence: J.C. Porter, Cecil H. & I. Green Center for Reproductive Biology Sciences, University of Texas Health Science Center at Dallas, Department of Obstetrics and Gynecology, 5323 Harry Hines Blvd., Dallas TX 75235, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
364 situ molar activity of TH in dopaminergic and noradrenergic neurons. MATEP,I.kLS AND METHODS Animals Female rats (3-4 months of age) of the LongEvans strain were maintained under conditions of controlled temperature (20-22 °C) and lighting (14 h of light; 10 h of darkness). Food was available at all times. To obviate the variable of ovarian function in these studies, the animals were ovariectomized 3 weeks before the beginning of each treatment. For 7 days prior to their use, rats were given 10, 30, or 100 mM NaCI to drink or were treated with arginine vasopressin (AVP). AVP was dissolved in a slow-release zinc phosphate solution l° and injected daily (2Ftg AVP per rat, s.c.). Control animals for the saline-treated rats were allowed to drink distilled water; control animals for the AVP-treated rats were injected with zinc phosphate solution. All animals were used at the end of the 7-day treatment period. In siul activity of TH The in situ activity of TH in the ME or SCG was assayed between 12.30 h and 14.30 h by determining the rate of accumulation of DOPA following the administration of 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015), an inhibitor of DOPA decarboxylase activity in the brain 6-9. NSD 1015 (50 mg/ml) was dissolved immediately before use in a buffered solution containing the following (mM): NaCl 116, KCI 5.5, CaCl 2 1.8, glucose 5, MgSO4 0.8, Hepes buffer 25 and NaH2PO4 10/zM; the solution was adjusted to pH 7.4 with 10 M NaOH. Each rat was injected with NSD 1015 (100 mg/kg b. wt., i.p.) at 0 t;me. Thirty min later, the animal was decapitated, and the ME and right SCG were excised with the aid of a dissecting microscope. The ME and SCG were homogenized in 50 and 200/d of ice-cold water, respectively, and centrifuged for 1 min at 10,000 g. An aliquot (20/A) of each supernate was mixed with an equal volume of 2x Laemmli buffer 15, heated for 3 min in a boiling water bath, and analyzed for TH. Another aliquot (25/~1) of each supernate was diluted with 100#1 of 0.133 M perchloric acid (PCA), and the mixture was centrifuged for 1 min at 10,000 g. The supernate was analyzed for DOPA.
In vitro studies To investigate the effect of NaC! on the activity of TH in the ME under in vitro conditions, the hypothalamus was excised according t~ the procedure of Arita and Kimura ! and incubated as described earlier 24. The concentration of NaC! in the medium varied from 0 to 116 raM. To maintain isotonicity, NaC! omitted from the medium was replaced with an appropriate quantity of sucrose. At the end of a 60min incubation under oxygen in the presence of l0 mM NSD 1015, the ME was excised and analyzed for DOPA and TH. Secretion of catecholamines into hypophysial blood Ovariectomized rats were allowed to drink distilled water, 10 mM NaCI, or 100 mM NaC! for 7 days. At. the ead of the treatment, each animal was anesthetized with urethane (1.4 g/kg b. wt., i.p. ), and hypophysiai portal blood was collected for 2 h from a single portal vessel, using the vessel draining the medial region of the ME as described previously 25"27. Throughout this 2-h period, arterial blood was also obtained from the animal at the rate of 10/zl/min. At the end of the collection, the plasma was harvested and mixed with an equal volume of 0.6 M PCA containing 2.7 mM EGTA. The mixture was centrifuged at 16000 g for 1 min, and the supernate was analyzed for catecholamines. A n alytical p rocedu res DOPA in the PCA extract of the ME or SCG was assayed by means of HPLC with electrochemical detection using the procedure of Felice et al.l-' with minor adaptations 24. TH in the aqueous extract of the ME or SCG was quantified by an immunobiot assay23, using purified rat TH as the standard. Catecholamines in hypophysial portal plasma and arterial plasma were measured by a radioenzymatic assay 3. In situ TH activity is expressed as mol of DOPA. h-~.mol of TH -l , and is called molar activity. In some cases, the treatment means were tested for homogeneity using a one-way analysis of variance, and the difference between specific means was tested for significance using Duncan's multiple range test ~t. In other cases, Student's t test was employed 13. A difference between two means was considered statistically significant when P < 0.05.
365
TABLE I Effect of saline ingestion on the mass of TH in the ME and SCG Treatment
Number TH mass in ME TH mass in SCG (pmol TH.ME -I) (pmol TH.SCG -t) animals
of H20 10 mM NaCI 30 mM NaC! 100mMNaCi
10 10 10 10
0.28 + 0.03* 0.31 + 0.04 0.23 _+0.02 0.21 _+0.01"*
0.67 _+0.06 0.72 + 0.11 0.37 + 0.01"** 0.34+0.02***
~litMean + S.E.M. Significantly (**P < 0.05; ***P < 0.01) ferent from animals that drank water.
RESULTS The mass of T H in the M E of rats that d r a n k 100 mM NaCI was significantly less than that of animals that drank water (Table I). In animals that d r a n k 10 mM NaC1, the mass of T H in the M E was similar to that of the rats that d r a n k water; whereas the mass of T H in the ME of rats that d r a n k 30 mM NaCI was between that of rats that d r a n k 10 mM NaCI and those that drank 100 mM NaCI. The quantity of T H in SCG of rats that d r a n k 30 m M or 100 mM NaC! was significantly less than that of animals that d r a n k water (Table I).
The ingestion of 10, 30, and 100 m M NaCI resulted in a c o n c e n t r a t i o n - d e p e n d e n t increase in the in situ molar activity of T H in the M E as well as the SCG (Table II). T h e molar activities of T H in the ME and SCG of rats that drank 100 m M NaCI were increased significantly c o m p a r e d to rats that d r a n k water. T h e release of d o p a m i n e and n o r e p i n e p h r i n e into hypophysial portal blood was stimulated by the drinking of 100 m M NaCI. As shown in Table III, the concentrations of d o p a m i n e and n o r e p i n e p h r i n e of rats that d r a n k 100 mM NaCI for 7 days were significantly greater than those of animals that drank water. C o m p a r e d to water, 10 m M NaCi had no effect on the c o n c e n t r a t i o n of d o p a m i n e or n o r e p i n e p h r i n e in portal plasma. The c o a c e n t r a t i o n in a~erial plasma of d o p a m i n e or n o r e p i n e p h r i n e was unaffected by 100 m M NaCl. The concentration of epinephrine in portal plasma was similar in all t r e a t m e n t groups. In contrast to d o p a m i n e and n o r e p i n e p h r i n e , the concentration of epinephrine in portal plasma was appreciably less than that in arterial plasma, indicating extractio~ of epinephrine from arterial blood during its transit through the ME. I n a s m u c h as saline ingestion could be expected to stimulate the release of vasopressin, the effect of t r e a t m e n t of rats with AVP on the mass and activity
TABLE II Effect of saline ingestion on the in situ activity of TH in the ME and SCG Treatment
Number of animals
TH activity in ME (tool DOPA.h-t.mol TH -t )
TH activity in SCG (tool DOPA.h-t.mol TH -t)
H20
10 10 10 l0
30 + 2* 34 _+4 36 + 5 45 + 3**
119 + 10
10 mM NaCI 30 mM NaCI 100mM NaCI
143 + 24 167 + 12"* 272 _+ 13"*
* Mean + S.E.M. ** Significantly (P < 0.01) different from animals that drank water. TABLE III Concentrations of catecholamines in hypophysial portal plasma and arterial plasma of ovariectomized rats after drinking saline for 7 days Source of plasma
Drinking solution
Number of animals
Dopamine (ng/mi )
Norepinephrine (ng/ml)
Epinephrine (ng/ml)
Hypophysial portal vessel
H20 10 mM NaCI 100 mM NaCI H,O 10 mM NaCI 100 mM NaCI
11 10 9 11 10 9
0.36 _+0.08* 0.32 :_~0.09 0.84 _+0.23** <0.1 <0.1 <0.1
0.32 + 0.05 0.30 _+0.05 0.67 + 0.17"* 0.33 __.0.06 0.48 + 0.09 0.36 +_0.13
0.92 +_0.14 0.85 +_0.18 0.86 +_0.26 1.61 _.+0.18 1.77 +_0.27 1.81 +_0.44
Femoral artery
* Mean + S.E.M. ** Significantly (P < 0.05) different from animals that drank water.
366 TABLE
TABLE VI
IV
Effect of arginine vasopressin on the mass of TH in the ME and SCG Treatment
Solvent vehicle AVP
Number TH mass in ME TH mass in SCG of (pmol TH.ME -t) (pmol TH.SCG -t) animals
10 10
0.21 +_0.01" 0.22 +_0.02
0.51 + 0.02 0.60 +_0.02**
* Mean + S.E.M. ** Significantly (P < 0.01) different from solvent-treated animals. of TH was evaluated. As shown in Table IV, AVP had no effect on the mass of TH in the ME. However, the mass of TH in the SCG of AVP-treated animals was slightly but significantly greater than that of the controls. Treatment with AVP had no effect on the in situ molar activity of TH in the ME or SCG (Table V). When hypothalamic tissue was incubated in medium containing 0-116 mM NaCI, it was found that NaC! had no demonstrable effect on the in situ activity of TH in the ME (Table VI). DISCUSSION The biosynthesis of dopamine in dopaminergic neurons and norepinephrine in noradrenergic neurons is believed to be dependent on the enzymic activity of TH, the putative rate-limiting enzyme that catalyzes the conversion of L-tyrosine to D O P A 16. Although not fully documented, the activity of TH in dopaminergic and noradrenergic neurons can be expected to be modulated by the mass of the enzyme, phosphorylation of the enzyme, availability of L-tyrosine, the precursor of D O P A , intraceiluiar level of the co-substrate, tetrahydrobiopterin, and end-product inhibition2°. It has been shown under in vitro conditions that
Effect of the concentration of NaC! in medium on the in situ activity q( TH in the ME under in vitro conditions Concentration of NaCI (raM)
Number of animals
TH activity (mol DOPA. mol TH -t. h- t)
116
3
14 + 2*
87 58 29 0
3 3 3 3
17 + 3 11+ 1 15 + 3 16+4
* Mean + S.E.M.
some circumstances that lead to increased phosphorylation of TH in cells of the SCG 5 and in neurites of the ME 24 also increase the in situ activity of TH. However, the converse does not always appear to be true. We have recently observed that prolactin causes an increase in the in situ activity of TH of the ME without affecting phosphorylation of the enzyme (unpublished observations). As with olactin, the increased in situ molar activity of TH in the ME was associated with an increase in the secretion of dopamine and norepinephrine into hypophysiai portal blood, a result we attribute to increased D O P A biosynthesis. Given the possibility of multiple mechanisms of controlling intraneuronai activity of TIt, it is of interest that the drinking of 100 mM NaC! for 7 days causes a significant reduction in the mass of TH in the ME as well as SCG. In this respect, the effect of ingesting 100 mM NaC! is similar to that seen in rats treated with progesterone, i.e. a reduction in the intracellular mass of TH and an increase in the molar activity of the enzyme 31. It is unclear whether t!;e reduction in the mass of TH is due to increased ra:~ of metabolism of the enzyme or decreased rate of synthesis. It is noteworthy that a marked reduction in the mass of TH in the ME
TABLE V Effect of arginine vasopressin on the in situ activity of TH in the ME and SCG Treatment
Number of animals
TH activity in ME (mol DOPA.h-I.mol TH -I)
TH activity in SCG (mol DOPA. It- I mol TH- i )
Solvent vehicle AVP
10 10
37 _+3.3* 37 + 4.1
168 + 11.6 157 + 10.5
* Mean + S.E.M.
367 occurs in the rat between the days of proestrus and estrus 23, which may be due to secretion of progesterone that increases at this time of the ovulatory cycle 4. The results obtained under in vivo conditions raise the question of whether or not NaCI acts directly on TH-containing neurons. When hypothalamic tissue was incubated in m e d i u m containing various concentrations of NaCI, it was found that t~',e absence of NaCi had no greater effect on TH activity than did the highest concentration of NaCI studied, i.e. 116 raM. This finding suggests that the effect of drinking saline on TH mass and activity may not be due to a direct action of NaCI on catecholaminergic neurons of the hypothalamus. Moreover, it seems that the influence of NaCI on the quantity and molar activity of TH is not due to AVP since the quantity and in situ molar activity of TH in ME and SCG were unaffected by treatment with this hormone. Intravenous infusion of isotonic saline has been shown to produce a 2- to 3-fold increase in the circulating concentration of the atrial natriuretic factor (ANF), a hormone secreted by the heart cauring natriuresis 2:9"22'2s, in rats 14 and in human 2s. A dose-dependent increase in the A N F in plasma has been observed in the human following the ingestion of NaC! for 4 days 29. Since A N F is present in the hypothalamus and other regions of the brain 17"3°as well as the heart and circulation, we speculate that the modulatory effect of NaCi intake on the mass and in situ molar activity of TH in ME and SCG of rats may be a
REFERENCES l Arita, J. and Kimura, F., In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: involvemeat of voltage-dependent Ca-'+ channels, Brain Research. 347 ( 19851299-3(15. 2 Atlas, S.A., Atrial natriuretic factor: a new hormone of cardiac origin, Recent Prog. Horm. Res., 42 (1986) 207-249. 3 Ben-Jonathan, N. and Porter, J.C., A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue, Endocrinology, 98 (1976) 1497-1507. 4 Butcher, R.L., Col!ins, W.E. and Fugo, N.W., Plasma concentration of LH, FSg, Frolaetin, progesterone and estradio!-17fl throughout the 4-day estrous cycle of the rat, Endocrinology, 94 (1974) 1704-1708. 5 Cahill, A.L. and Perlman, R.L., Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion, Biochim. Biophys, Acta, 805 (1984) 217-226.
function of the ANF. It is of interest that Kurihara et al.lS have demonstrated the presence of ANF receptors in several regions of the rat hypothalamus, suggesting a role for this peptide in the control of neuronal function. Nakao et al. 21 found that ANF, administered intracerebroventricularly to rats, induced a preferential reduction in the concentration o f dopamine in the septurn and hypothalamus, compared to other regions c~f the brain. They interpreted this action of A N F as inhibitory to the dopaminergic system of the brain. However, it is possible that this effect of A N F was the result of dopamine release that exceeded synthesis. Racz et al. 26 found that dopamine synthesis in pheochromocytoma cells was increased in the presence of ANF. Thus, the results of the present study are consistent with other findings that are suggestive of a role for A N F in the control of catecholaminergic neurons. ACKNOWLEDGEMENTS The autl',ors thank Kay Stanley for editorial assistance and Robert Lipsey, Sharyn Monroe, Nhu-Y Dong, and Renon Mical for excellent technical assistance. This research was supported by grants AM01237, AG-04344, and AG-00306 from the National Institutes of Health. P.S.W. is the recipient of a Fogarty International Fellowship. H.A.G. is a fellow supported by the Chilton Foundation.
6 Carlsson, A., Davis, J.N., Kehr, W., Lindqvist, M. and Atack, C.V., Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in viva using an inhibitor of the aromatic amino acid decarboxylase, Naunyn Schmiedebergs' Arch. Pharmacal., 275 (19721 153-168. 7 Carlsson, A. and Lindqvist, M., In-vivo measurements of tryptophan and tyrosine hydroxylase activities in mouse brain, J. Neural Transm., 34 (1973) 79-91. 8 Demarest, K.T., AIper, R.H. and Moore, K.E., Dopa accumulation is a measure of dopamine synthesis in the median eminence of posterior pituitary, J. Neural T~ansm., 46 (1979) 183-193. 9 Demarest, K.T. and Moore, K.E., Accumulation of LDOPA in the median eminence: an index of tuberoinfundibular dopaminergic nerve activity, Endocrinology, 106 (1980) 463-468. 10 De Wied, D., Inhibitory effect of ACTH and related peptides on extinction of conditioned avoidance behavior in rats, Proc. Soc. Exp. Biol. Mad., 122(1966128-32.
368 11 Duncan. D.B., Multiple range and multiple F tests, Biometrics, 11 (1955) 1-42. 12 Felice, L.J., Felice, J.D. and Kissinger, P.T., Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography, J. Neurochem., 31 (1978) 1461-1465. 13 Fisher, R.A., Statistical Methods for Research Workers, 12th edn., Hafner, New York, 1954, pp. 114-173. 14 Lang, R.E., Tholken, H., Ganten, D., Luft, F.C., ,~uskoaho, H. and Ungcr, T., Atrial natriuretic factor-- a circulating hormone stimulated by volume loading, Nature (London), 314 (1985) 264-266. 15 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London), 227 (1970) 680-685. 16 Levitt, L., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. Exp. T~er., 148 (1965) 1-8. 17 Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. and Sano, Y., Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry, Neuroscience, 16 (1985) 521-546. 18 Kurihara, M., Saavedra, J.M. and Shigematsu, K., Localization and characterization of atrial natriuretic peptide binding sites in discrete areas of rat brain and pituitary gland by quantitative autoradiography, Brain Research, 408 (1987) 31-39. 19 Maack, T., Atlas, S.A., Camargo, M.J.F.and Cogan, M.G., Renal hemodynamic and natriuretic effects of atrial natriuretic factor, Fed. Proc., 45 (1986) 2128-2132. 20 Nagatsu, T., Biochemistry of Catecholamines: The Biochemical Method, University Park Press, Baltimore, 1973, p. 162. 21 Nakao, K., Katsuura, G., Morii, N., Itoh, H., Shiono, S., Yamada, T., Sugawara, A., Sakamoto, M., Saito, Y., Eigyo, M., Matsushita, A. and Imura, H., Inhibitory effect of centrally administered atrial natriuretic polypeptide on the brain dopaminergic system in rats, Eur. J. Pharmacol., 131
(1986) 171-177. 22 Needleman, P., Adams, S.P., Cole, B.R., Currie, M.G., GeUer, D.M., Michener, M.L., Saper, C.B., Schwartz, D. and Sandaert, D.G., Atropeptins as cardiac hormones, Hypertension, 7 (1985) 469-482. 23 Porter, J.C., Relationship of age, sex and reproductive status to the quantity of tyrosine hydroxylase in the median eminence and superior cervical ganglion of the rat, Endocrinology, 118 (1985) 1426-1432. 24 Porter, J.C., In situ activity and phosphorylation of tyrosine ilydroxylase in the median eminence, Mol. Cell. Endocrinol., 46 (1986) 21-27. 25 Porter, J.C., Mical, R.S., Kamberi, I.A. and Grazia, Y.R., A procedure for the cannulation of a pituitary stalk portal vessel and perfusion of the pars distalis in the rat, Endocrinology, 87 (1970) 197-201. 26 Racz, K., Kuchel, O., Buu, N.T., Debinski, W., Cantin, M. and Genest, J., Atrial natriuretic factor, catecholamines, and natriuresis, New Engl. J. Med., 314 (1986) 321-322. 27 Re:',,n,ond, M.J., Speciale, S,G. and Porter, J.C., Dopamine in plasma of lateral and medial hypophysial portal vessels: evidence for regional variation in the release of hypothalamic dopamine into hypophysial portal blood, Endocrinology, 112 (1983) 1958-1963. 28 Sagnella, G.A. and MacGregor, G.A., Structural and functional features of atrial natriuretic peptides, T.I.B.S., 11 (1986) 299-302. 29 SagneUa, G.A., Markand, N.D., Shore, A.C. and MacGregor, G.A., Effects of changes in dietary sodium intake and saline infusion on immunoreactive atrial natriuretic peptide in human plasma, Lancet, 2 (1985) 1208-1211. 30 Skofitsch, G., Jacobowitz, D.M., Eskay, R.L. and Zamir, N., Distribution of atrial natriuretic factor-like immunoreactive neurons in the rat brain, Neuroscience, 16 (1985) 917-948. 31 Wang, P.S. and Porter, J.C., Hormonal modulation of the quantity and in situ activity of tyrosine hydroxylase in neurites of the median eminence, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9804-9806.
363
BRE 13458 ,--$~
Saline ingestion stimulates the in situ molar activity of tyrosine hydroxylase in the median eminence and superior cervical ganglion Paulus S. Wang*, Hector A. Gonzalez and John C. Porter Cecil H. and Ida Green Center for Reproduciive Biology Sciences, University of Texas Health Science Center at Dallas, Departments of Obstetrics and Gynecology and Physiology, Dallas, TX 75235 (U.S.A. ) (Accepted 6 October 1987)
Key words: Tyrosine hydroxylase mass; Tyrosine hydroxylase activity; Sodium chloride ingestion; Median eminence; Superior cervical ganglion
The effects of drinking saline for 7 days on the mass and in situ activity of tyrosine hydroxylase (TH) in the ~.aedianeminence (ME) and superior cervical ganglion (SCG) of rats were investigated. TH mass was quantified by immunoblot assay. In situ TH activity was calculated from the rate of intracellular accumulation of L-dihydroxyphenylalanine(DOPA). In rats that drank 10 mM, 30 mM, and 100 mM NaCl for 7 days, TH activity in the ME was 34 + 4, 36 + 5, and 45 + 3 (mean and S.E.M.) tool of DOPA-h-l.mol ofTH -I. respectively, compared to 30 + 2 for rats that drank water. The activity of TH in the SCG of animals that drank 10 mM, 30 raM, and 100 mM NaCi was 143 + 24, 167 + 12, and 272 + 13 mol DOPA.h-l.mol TH -l, respectively, compared to 119 +_ 10 for animals that drank water. The mass of TH in the ME and SCG decreased as a function of the concentration of NaCI in the drinking water. In animals that drank water, 10 mM, 30 mM, and 100 mM NaCl, the amounts (pmol) of TH were, respectively, 0.28 + 0.03, 0.31 +_0.04, 0.23 + 0.02. and 0.21 _+0.01 per ME and 0.67 + 0.06, 0.72 + 0.11, 0.37 + 0.01, and 0.34 _+0.02 per SCG. TH activity in the ME or SCG was unaffected by treatment for 7 days with arginine vasopressin. The mean concentration of dopamine and that of norepinephrine in hypophysial portal plasma of the animals that drank 100 mM NaC! were more than twice that of animals that drank water. It is concluded that the in situ molar activity of TH in the ME and SCG as well as the secretion of dopamine into portal blood are stimulated by ingestion of saline.
INTRODUCTION t e r / n e u r o h o r m o n e s is a function of their rates of bioThe synthesis of d o p a m i n e in d o p a m i n e r g i c neurons and n o r e p i n e p h r i n e in n o r a d r e n e r g i c neurons involves two reactions that are c o m m o n to both types of cells 2°. These reactions are (a) the oxidative conversion of L-tyrosine to L-dihydroxyphenylalanine ( D O P A ) and (b) the decarboxylation of D O P A to form dopamine. In both types of cells, tyrosine hydroxylase (TH) [tyrosine 3 - m o n o o x y g e n a s e , L-tyrosine, hydropteridine: oxygen oxidoreductase (3-hydroxylating, E.C.1.14.16.2)] is believed to be the regulatory enzyme 16. Thus, any mechanism(s) that modulate(s) the intracellular activity of T H is important since the secretion of these neurotransmit-
synthesis. It was shown by Cahill and Perlman 5 that veratridine stimulated under in vitro conditions the in situ activity of T H of the superior cervical ganglion (SCG). Later, Porter 24 r e p o r t e d that incubation of hypothalamic tissue in the presence of veratridine resulted in a dose-dependent increase in the activity of T H of the median eminence ( M E ) , a region of the brain that contains neurites of tuberoinfundibular dopaminergic neurons. Inasmuch as veratridine stimulates the intraceilular flux of sodium ions, these findings raise the question of w h e t h e r ingestion of sodium chloride would lead to an alteration in the in
* Present address: Department of Physiology, National Yang-Ming Medical College, Taipei, Taiwan. Correspondence: J.C. Porter, Cecil H. & I. Green Center for Reproductive Biology Sciences, University of Texas Health Science Center at Dallas, Department of Obstetrics and Gynecology, 5323 Harry Hines Blvd., Dallas TX 75235, U.S.A.
0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
364 situ molar activity of TH in dopaminergic and noradrenergic neurons. MATEP,I.kLS AND METHODS Animals Female rats (3-4 months of age) of the LongEvans strain were maintained under conditions of controlled temperature (20-22 °C) and lighting (14 h of light; 10 h of darkness). Food was available at all times. To obviate the variable of ovarian function in these studies, the animals were ovariectomized 3 weeks before the beginning of each treatment. For 7 days prior to their use, rats were given 10, 30, or 100 mM NaCI to drink or were treated with arginine vasopressin (AVP). AVP was dissolved in a slow-release zinc phosphate solution l° and injected daily (2Ftg AVP per rat, s.c.). Control animals for the saline-treated rats were allowed to drink distilled water; control animals for the AVP-treated rats were injected with zinc phosphate solution. All animals were used at the end of the 7-day treatment period. In siul activity of TH The in situ activity of TH in the ME or SCG was assayed between 12.30 h and 14.30 h by determining the rate of accumulation of DOPA following the administration of 3-hydroxybenzylhydrazine dihydrochloride (NSD 1015), an inhibitor of DOPA decarboxylase activity in the brain 6-9. NSD 1015 (50 mg/ml) was dissolved immediately before use in a buffered solution containing the following (mM): NaCl 116, KCI 5.5, CaCl 2 1.8, glucose 5, MgSO4 0.8, Hepes buffer 25 and NaH2PO4 10/zM; the solution was adjusted to pH 7.4 with 10 M NaOH. Each rat was injected with NSD 1015 (100 mg/kg b. wt., i.p.) at 0 t;me. Thirty min later, the animal was decapitated, and the ME and right SCG were excised with the aid of a dissecting microscope. The ME and SCG were homogenized in 50 and 200/d of ice-cold water, respectively, and centrifuged for 1 min at 10,000 g. An aliquot (20/A) of each supernate was mixed with an equal volume of 2x Laemmli buffer 15, heated for 3 min in a boiling water bath, and analyzed for TH. Another aliquot (25/~1) of each supernate was diluted with 100#1 of 0.133 M perchloric acid (PCA), and the mixture was centrifuged for 1 min at 10,000 g. The supernate was analyzed for DOPA.
In vitro studies To investigate the effect of NaC! on the activity of TH in the ME under in vitro conditions, the hypothalamus was excised according t~ the procedure of Arita and Kimura ! and incubated as described earlier 24. The concentration of NaC! in the medium varied from 0 to 116 raM. To maintain isotonicity, NaC! omitted from the medium was replaced with an appropriate quantity of sucrose. At the end of a 60min incubation under oxygen in the presence of l0 mM NSD 1015, the ME was excised and analyzed for DOPA and TH. Secretion of catecholamines into hypophysial blood Ovariectomized rats were allowed to drink distilled water, 10 mM NaCI, or 100 mM NaC! for 7 days. At. the ead of the treatment, each animal was anesthetized with urethane (1.4 g/kg b. wt., i.p. ), and hypophysiai portal blood was collected for 2 h from a single portal vessel, using the vessel draining the medial region of the ME as described previously 25"27. Throughout this 2-h period, arterial blood was also obtained from the animal at the rate of 10/zl/min. At the end of the collection, the plasma was harvested and mixed with an equal volume of 0.6 M PCA containing 2.7 mM EGTA. The mixture was centrifuged at 16000 g for 1 min, and the supernate was analyzed for catecholamines. A n alytical p rocedu res DOPA in the PCA extract of the ME or SCG was assayed by means of HPLC with electrochemical detection using the procedure of Felice et al.l-' with minor adaptations 24. TH in the aqueous extract of the ME or SCG was quantified by an immunobiot assay23, using purified rat TH as the standard. Catecholamines in hypophysial portal plasma and arterial plasma were measured by a radioenzymatic assay 3. In situ TH activity is expressed as mol of DOPA. h-~.mol of TH -l , and is called molar activity. In some cases, the treatment means were tested for homogeneity using a one-way analysis of variance, and the difference between specific means was tested for significance using Duncan's multiple range test ~t. In other cases, Student's t test was employed 13. A difference between two means was considered statistically significant when P < 0.05.
365
TABLE I Effect of saline ingestion on the mass of TH in the ME and SCG Treatment
Number TH mass in ME TH mass in SCG (pmol TH.ME -I) (pmol TH.SCG -t) animals
of H20 10 mM NaCI 30 mM NaC! 100mMNaCi
10 10 10 10
0.28 + 0.03* 0.31 + 0.04 0.23 _+0.02 0.21 _+0.01"*
0.67 _+0.06 0.72 + 0.11 0.37 + 0.01"** 0.34+0.02***
~litMean + S.E.M. Significantly (**P < 0.05; ***P < 0.01) ferent from animals that drank water.
RESULTS The mass of T H in the M E of rats that d r a n k 100 mM NaCI was significantly less than that of animals that drank water (Table I). In animals that d r a n k 10 mM NaC1, the mass of T H in the M E was similar to that of the rats that d r a n k water; whereas the mass of T H in the ME of rats that d r a n k 30 mM NaCI was between that of rats that d r a n k 10 mM NaCI and those that drank 100 mM NaCI. The quantity of T H in SCG of rats that d r a n k 30 m M or 100 mM NaC! was significantly less than that of animals that d r a n k water (Table I).
The ingestion of 10, 30, and 100 m M NaCI resulted in a c o n c e n t r a t i o n - d e p e n d e n t increase in the in situ molar activity of T H in the M E as well as the SCG (Table II). T h e molar activities of T H in the ME and SCG of rats that drank 100 m M NaCI were increased significantly c o m p a r e d to rats that d r a n k water. T h e release of d o p a m i n e and n o r e p i n e p h r i n e into hypophysial portal blood was stimulated by the drinking of 100 m M NaCI. As shown in Table III, the concentrations of d o p a m i n e and n o r e p i n e p h r i n e of rats that d r a n k 100 mM NaCI for 7 days were significantly greater than those of animals that drank water. C o m p a r e d to water, 10 m M NaCi had no effect on the c o n c e n t r a t i o n of d o p a m i n e or n o r e p i n e p h r i n e in portal plasma. The c o a c e n t r a t i o n in a~erial plasma of d o p a m i n e or n o r e p i n e p h r i n e was unaffected by 100 m M NaCl. The concentration of epinephrine in portal plasma was similar in all t r e a t m e n t groups. In contrast to d o p a m i n e and n o r e p i n e p h r i n e , the concentration of epinephrine in portal plasma was appreciably less than that in arterial plasma, indicating extractio~ of epinephrine from arterial blood during its transit through the ME. I n a s m u c h as saline ingestion could be expected to stimulate the release of vasopressin, the effect of t r e a t m e n t of rats with AVP on the mass and activity
TABLE II Effect of saline ingestion on the in situ activity of TH in the ME and SCG Treatment
Number of animals
TH activity in ME (tool DOPA.h-t.mol TH -t )
TH activity in SCG (tool DOPA.h-t.mol TH -t)
H20
10 10 10 l0
30 + 2* 34 _+4 36 + 5 45 + 3**
119 + 10
10 mM NaCI 30 mM NaCI 100mM NaCI
143 + 24 167 + 12"* 272 _+ 13"*
* Mean + S.E.M. ** Significantly (P < 0.01) different from animals that drank water. TABLE III Concentrations of catecholamines in hypophysial portal plasma and arterial plasma of ovariectomized rats after drinking saline for 7 days Source of plasma
Drinking solution
Number of animals
Dopamine (ng/mi )
Norepinephrine (ng/ml)
Epinephrine (ng/ml)
Hypophysial portal vessel
H20 10 mM NaCI 100 mM NaCI H,O 10 mM NaCI 100 mM NaCI
11 10 9 11 10 9
0.36 _+0.08* 0.32 :_~0.09 0.84 _+0.23** <0.1 <0.1 <0.1
0.32 + 0.05 0.30 _+0.05 0.67 + 0.17"* 0.33 __.0.06 0.48 + 0.09 0.36 +_0.13
0.92 +_0.14 0.85 +_0.18 0.86 +_0.26 1.61 _.+0.18 1.77 +_0.27 1.81 +_0.44
Femoral artery
* Mean + S.E.M. ** Significantly (P < 0.05) different from animals that drank water.
366 TABLE
TABLE VI
IV
Effect of arginine vasopressin on the mass of TH in the ME and SCG Treatment
Solvent vehicle AVP
Number TH mass in ME TH mass in SCG of (pmol TH.ME -t) (pmol TH.SCG -t) animals
10 10
0.21 +_0.01" 0.22 +_0.02
0.51 + 0.02 0.60 +_0.02**
* Mean + S.E.M. ** Significantly (P < 0.01) different from solvent-treated animals. of TH was evaluated. As shown in Table IV, AVP had no effect on the mass of TH in the ME. However, the mass of TH in the SCG of AVP-treated animals was slightly but significantly greater than that of the controls. Treatment with AVP had no effect on the in situ molar activity of TH in the ME or SCG (Table V). When hypothalamic tissue was incubated in medium containing 0-116 mM NaCI, it was found that NaC! had no demonstrable effect on the in situ activity of TH in the ME (Table VI). DISCUSSION The biosynthesis of dopamine in dopaminergic neurons and norepinephrine in noradrenergic neurons is believed to be dependent on the enzymic activity of TH, the putative rate-limiting enzyme that catalyzes the conversion of L-tyrosine to D O P A 16. Although not fully documented, the activity of TH in dopaminergic and noradrenergic neurons can be expected to be modulated by the mass of the enzyme, phosphorylation of the enzyme, availability of L-tyrosine, the precursor of D O P A , intraceiluiar level of the co-substrate, tetrahydrobiopterin, and end-product inhibition2°. It has been shown under in vitro conditions that
Effect of the concentration of NaC! in medium on the in situ activity q( TH in the ME under in vitro conditions Concentration of NaCI (raM)
Number of animals
TH activity (mol DOPA. mol TH -t. h- t)
116
3
14 + 2*
87 58 29 0
3 3 3 3
17 + 3 11+ 1 15 + 3 16+4
* Mean + S.E.M.
some circumstances that lead to increased phosphorylation of TH in cells of the SCG 5 and in neurites of the ME 24 also increase the in situ activity of TH. However, the converse does not always appear to be true. We have recently observed that prolactin causes an increase in the in situ activity of TH of the ME without affecting phosphorylation of the enzyme (unpublished observations). As with olactin, the increased in situ molar activity of TH in the ME was associated with an increase in the secretion of dopamine and norepinephrine into hypophysiai portal blood, a result we attribute to increased D O P A biosynthesis. Given the possibility of multiple mechanisms of controlling intraneuronai activity of TIt, it is of interest that the drinking of 100 mM NaC! for 7 days causes a significant reduction in the mass of TH in the ME as well as SCG. In this respect, the effect of ingesting 100 mM NaC! is similar to that seen in rats treated with progesterone, i.e. a reduction in the intracellular mass of TH and an increase in the molar activity of the enzyme 31. It is unclear whether t!;e reduction in the mass of TH is due to increased ra:~ of metabolism of the enzyme or decreased rate of synthesis. It is noteworthy that a marked reduction in the mass of TH in the ME
TABLE V Effect of arginine vasopressin on the in situ activity of TH in the ME and SCG Treatment
Number of animals
TH activity in ME (mol DOPA.h-I.mol TH -I)
TH activity in SCG (mol DOPA. It- I mol TH- i )
Solvent vehicle AVP
10 10
37 _+3.3* 37 + 4.1
168 + 11.6 157 + 10.5
* Mean + S.E.M.
367 occurs in the rat between the days of proestrus and estrus 23, which may be due to secretion of progesterone that increases at this time of the ovulatory cycle 4. The results obtained under in vivo conditions raise the question of whether or not NaCI acts directly on TH-containing neurons. When hypothalamic tissue was incubated in m e d i u m containing various concentrations of NaCI, it was found that t~',e absence of NaCi had no greater effect on TH activity than did the highest concentration of NaCI studied, i.e. 116 raM. This finding suggests that the effect of drinking saline on TH mass and activity may not be due to a direct action of NaCI on catecholaminergic neurons of the hypothalamus. Moreover, it seems that the influence of NaCI on the quantity and molar activity of TH is not due to AVP since the quantity and in situ molar activity of TH in ME and SCG were unaffected by treatment with this hormone. Intravenous infusion of isotonic saline has been shown to produce a 2- to 3-fold increase in the circulating concentration of the atrial natriuretic factor (ANF), a hormone secreted by the heart cauring natriuresis 2:9"22'2s, in rats 14 and in human 2s. A dose-dependent increase in the A N F in plasma has been observed in the human following the ingestion of NaC! for 4 days 29. Since A N F is present in the hypothalamus and other regions of the brain 17"3°as well as the heart and circulation, we speculate that the modulatory effect of NaCi intake on the mass and in situ molar activity of TH in ME and SCG of rats may be a
REFERENCES l Arita, J. and Kimura, F., In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: involvemeat of voltage-dependent Ca-'+ channels, Brain Research. 347 ( 19851299-3(15. 2 Atlas, S.A., Atrial natriuretic factor: a new hormone of cardiac origin, Recent Prog. Horm. Res., 42 (1986) 207-249. 3 Ben-Jonathan, N. and Porter, J.C., A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue, Endocrinology, 98 (1976) 1497-1507. 4 Butcher, R.L., Col!ins, W.E. and Fugo, N.W., Plasma concentration of LH, FSg, Frolaetin, progesterone and estradio!-17fl throughout the 4-day estrous cycle of the rat, Endocrinology, 94 (1974) 1704-1708. 5 Cahill, A.L. and Perlman, R.L., Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion, Biochim. Biophys, Acta, 805 (1984) 217-226.
function of the ANF. It is of interest that Kurihara et al.lS have demonstrated the presence of ANF receptors in several regions of the rat hypothalamus, suggesting a role for this peptide in the control of neuronal function. Nakao et al. 21 found that ANF, administered intracerebroventricularly to rats, induced a preferential reduction in the concentration o f dopamine in the septurn and hypothalamus, compared to other regions c~f the brain. They interpreted this action of A N F as inhibitory to the dopaminergic system of the brain. However, it is possible that this effect of A N F was the result of dopamine release that exceeded synthesis. Racz et al. 26 found that dopamine synthesis in pheochromocytoma cells was increased in the presence of ANF. Thus, the results of the present study are consistent with other findings that are suggestive of a role for A N F in the control of catecholaminergic neurons. ACKNOWLEDGEMENTS The autl',ors thank Kay Stanley for editorial assistance and Robert Lipsey, Sharyn Monroe, Nhu-Y Dong, and Renon Mical for excellent technical assistance. This research was supported by grants AM01237, AG-04344, and AG-00306 from the National Institutes of Health. P.S.W. is the recipient of a Fogarty International Fellowship. H.A.G. is a fellow supported by the Chilton Foundation.
6 Carlsson, A., Davis, J.N., Kehr, W., Lindqvist, M. and Atack, C.V., Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in viva using an inhibitor of the aromatic amino acid decarboxylase, Naunyn Schmiedebergs' Arch. Pharmacal., 275 (19721 153-168. 7 Carlsson, A. and Lindqvist, M., In-vivo measurements of tryptophan and tyrosine hydroxylase activities in mouse brain, J. Neural Transm., 34 (1973) 79-91. 8 Demarest, K.T., AIper, R.H. and Moore, K.E., Dopa accumulation is a measure of dopamine synthesis in the median eminence of posterior pituitary, J. Neural T~ansm., 46 (1979) 183-193. 9 Demarest, K.T. and Moore, K.E., Accumulation of LDOPA in the median eminence: an index of tuberoinfundibular dopaminergic nerve activity, Endocrinology, 106 (1980) 463-468. 10 De Wied, D., Inhibitory effect of ACTH and related peptides on extinction of conditioned avoidance behavior in rats, Proc. Soc. Exp. Biol. Mad., 122(1966128-32.
368 11 Duncan. D.B., Multiple range and multiple F tests, Biometrics, 11 (1955) 1-42. 12 Felice, L.J., Felice, J.D. and Kissinger, P.T., Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography, J. Neurochem., 31 (1978) 1461-1465. 13 Fisher, R.A., Statistical Methods for Research Workers, 12th edn., Hafner, New York, 1954, pp. 114-173. 14 Lang, R.E., Tholken, H., Ganten, D., Luft, F.C., ,~uskoaho, H. and Ungcr, T., Atrial natriuretic factor-- a circulating hormone stimulated by volume loading, Nature (London), 314 (1985) 264-266. 15 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London), 227 (1970) 680-685. 16 Levitt, L., Spector, S., Sjoerdsma, A. and Udenfriend, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea-pig heart, J. Pharmacol. Exp. T~er., 148 (1965) 1-8. 17 Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. and Sano, Y., Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry, Neuroscience, 16 (1985) 521-546. 18 Kurihara, M., Saavedra, J.M. and Shigematsu, K., Localization and characterization of atrial natriuretic peptide binding sites in discrete areas of rat brain and pituitary gland by quantitative autoradiography, Brain Research, 408 (1987) 31-39. 19 Maack, T., Atlas, S.A., Camargo, M.J.F.and Cogan, M.G., Renal hemodynamic and natriuretic effects of atrial natriuretic factor, Fed. Proc., 45 (1986) 2128-2132. 20 Nagatsu, T., Biochemistry of Catecholamines: The Biochemical Method, University Park Press, Baltimore, 1973, p. 162. 21 Nakao, K., Katsuura, G., Morii, N., Itoh, H., Shiono, S., Yamada, T., Sugawara, A., Sakamoto, M., Saito, Y., Eigyo, M., Matsushita, A. and Imura, H., Inhibitory effect of centrally administered atrial natriuretic polypeptide on the brain dopaminergic system in rats, Eur. J. Pharmacol., 131
(1986) 171-177. 22 Needleman, P., Adams, S.P., Cole, B.R., Currie, M.G., GeUer, D.M., Michener, M.L., Saper, C.B., Schwartz, D. and Sandaert, D.G., Atropeptins as cardiac hormones, Hypertension, 7 (1985) 469-482. 23 Porter, J.C., Relationship of age, sex and reproductive status to the quantity of tyrosine hydroxylase in the median eminence and superior cervical ganglion of the rat, Endocrinology, 118 (1985) 1426-1432. 24 Porter, J.C., In situ activity and phosphorylation of tyrosine ilydroxylase in the median eminence, Mol. Cell. Endocrinol., 46 (1986) 21-27. 25 Porter, J.C., Mical, R.S., Kamberi, I.A. and Grazia, Y.R., A procedure for the cannulation of a pituitary stalk portal vessel and perfusion of the pars distalis in the rat, Endocrinology, 87 (1970) 197-201. 26 Racz, K., Kuchel, O., Buu, N.T., Debinski, W., Cantin, M. and Genest, J., Atrial natriuretic factor, catecholamines, and natriuresis, New Engl. J. Med., 314 (1986) 321-322. 27 Re:',,n,ond, M.J., Speciale, S,G. and Porter, J.C., Dopamine in plasma of lateral and medial hypophysial portal vessels: evidence for regional variation in the release of hypothalamic dopamine into hypophysial portal blood, Endocrinology, 112 (1983) 1958-1963. 28 Sagnella, G.A. and MacGregor, G.A., Structural and functional features of atrial natriuretic peptides, T.I.B.S., 11 (1986) 299-302. 29 SagneUa, G.A., Markand, N.D., Shore, A.C. and MacGregor, G.A., Effects of changes in dietary sodium intake and saline infusion on immunoreactive atrial natriuretic peptide in human plasma, Lancet, 2 (1985) 1208-1211. 30 Skofitsch, G., Jacobowitz, D.M., Eskay, R.L. and Zamir, N., Distribution of atrial natriuretic factor-like immunoreactive neurons in the rat brain, Neuroscience, 16 (1985) 917-948. 31 Wang, P.S. and Porter, J.C., Hormonal modulation of the quantity and in situ activity of tyrosine hydroxylase in neurites of the median eminence, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9804-9806.