Effect of Atrial Natriuretic Peptide on the Release of 19-Endorphin from Rat Hypothalamo- Neurohypophysial Complex

Endocrinol. Jappon. 1989,36 (5),647-653

YUKIHIRO IKEDA1*, ISSEI TANAKA1, YUTAKA OKI1, YOSHIO IKEDA2 AND TERUYA YOSHIMII

1Second Department of Internal Medcine, Hamamatsu Uniyersity School of Medicine, Hamamatsu 431-31 and 2Department of Neuro-Psychiatry, Fujita Gakuen Health University School of Medicine, Toyoake 470-11, Japan

Abstract

The aim of this study was to determine whether atrial natriuretic peptide (ANP) alters 13-endorphin (ƒÀ-END) secretion from rat intermediate pituitary and whether this effect is a direct action on the intermediate pituitary or an indirect one mediated by hypothalamic factor(s). We studied the release of

β-END from rat neuro-intermediate lobes of the pituitary(NIL)and from the hypothalamo-neurohypophysial complex (HNC), which consists of the hypothalamus, pituitary stalk, intermediate and posterior lobes of the pituitary, by means of an in vitro perifusion system. NIL and HNC were prepared from male Wistar rats and individually perifused for 30 min with perifusion medium followed by 20 min perifusion with medium containing a-rat ANP and/or dopamine (DA). Samples of perifusion medium were collected every 5 min and subjected to RIA for n-END. The basal release of ƒÀ-END from NIL was 180% of that from HNC (p<0.01), which provides further support for the presence of hypothalamic factors that inhibit (ƒÀ-END release from the intermediate pituitary. The perifusion of HNC with ANP at 10-7 and 10-6M increased the ƒÀ-END concentration by 25 and 50%, respectively (p<0.01). In contrast, ANP (10-8 to 10-6M) had no effect on ƒÀ-END release from NIL. The inhibitory eect of DA (10-6M) on ƒÀ-END release from NIL and HNC (51% and 50% of the basal release, respectively, p<0.01) was confirmed. However, this inhibitory effect was not reversed by ANP: Therefore, it seems unlikely that the stimulatory effect of ANP on ƒÀ-END release results from antagonizing DA action on the intermediate pituitary. Since ANP had no direct effect on ƒÀ-END release from NIL, the stimulatory effect of ANP on the release of ƒÀ-END is thought to be an indirect one, mediated by hypothalamic factor(s).

Received Febrary 28, 1989 Society. This work was supported by Grants-in-Aid * To whom all correspondence should be ad- for Research on Intractable Disease from the dressed: YUKIHIRO IKEDA Second Depart- Ministry of Health and Welfare of Japan. ment of Internal Medicine, Hamamatsu Uni- A part of this report was presented at the versity School of Medicine, 3600 Handa-cho, 60th Annual Meeting of the Japan Endocrine Hamamatsu 431-31, Japan Endpcrinol. Japon. 648 IKEDA et al. October 1989

Atrial natriuretic peptide (ANP), which tary lobes, has been also used to study the is synthesized in and released from the release of vasopressin in vitro (Januszewictz cardiac atria, is a potent natriuretic and et al., 1986; Sladek et al., 1977). vasodilating hormone (Currie et al., 1983; In this study, we used rat NIL and de Bold et al., 1981) and has direct inhi- HNC to examine the effect of ANP on the bitory effects on aldosterone and renin release of ƒÀ-endorphin (ƒÀ-END) from the secretion (Atarashi et al., 1984; Obana et intermediate lobe of the pituitary. al., 1985). Effects of ANP on pituitary hormone secretion have also been studied

by several investigators, since the existence Materials and Methods of ANP and its specific binding sites in the hypothalamus has been reported (Jaco- Chemicals

bowitz et al., 1985; Quirion et al., 1986; Synthetic a-rat ANP was purchased from the Tanaka et al., 1984). ANP has been re- Protein Research Foundation, Minoh, Japan. Synthetic camel ƒÀ-END was obtained from ported to inhibit vesopressin secretion, in Peninsula Laboratories, Belmont, CA, USA. vivo and in vitro (Januszewicz et al., 1986; Dopamine (DA) was obtained from Sigma Obana et at., 1985; Samson, 1985). How- Chemical Company, St. Louis, MO, USA. ever, the effects of ANP on anterior and intermediate pituitary hormone release are Tissue preparation controversial. Shibasaki et al. (1986) found Male Wistar rats weighing 200 to 250g were decapitated, and the HNC was prepared according that ANP inhibited the release of pro- to the method described by Sladek and Knigge opiomelanocortin (POMC) derived peptides (1977). Briefly, the brain was removed from from cultured rat anterior and intermediate the skull by a caudal approach, and the dura pituitary cells. On the other hand, Horvath was carefully cut to allow removal of the intact et al. (1986) observed that ANP increased pituitary, pituitary stalk and hypothalamus. ACTH release from superfused rat anterior The ventral hypothalamus was taken as a trian-

pituitary cells, and Lamacz et al. (1987) gular block, the anterior margin of which was demonstrated a stimulatory effect of ANP just rostral to the optic chiasm. Lateral cuts tapering to the anterior margin of the mamillary on ƒ¿-MSH release from perifused frog bodies were made. The width of the rostral neurointermediate lobes of pituitary (NIL). portion of the block was approximately 6mm, These discrepancies may be explained by and the depth was 2-3 mm. The stalk and the differences in the experimental apparatus, pituitary gland remained attached to the block. species or tissues. Another question in The anterior lobe was carefully removed by regard to the effects of ANP on the pitui- dissection. NIL was prepared by removing the tary hormone secretion is whether ANP hypothalamus from the HNC. has a direct action on pituitary hormone Perifusion procedure secretion or an indirect action mediated by One NIL or HNC was placed in a 0.5ml changes in hypothalamic factor(s). chamber and perifused at 37•Ž with the peri- The isolated hypothalamic-pituitary com- fusion medium (Krebs Ringer bicarbonate buffer,

plex has been used by several investigators pH 7.4, containing 0.1% glucose, 0.1% ascorbic (Gill et al., 1983; Karanth et al., 1987; acid, 0.003% Bacitracin and 0.1% bovine serum Miyachi et al., 1973) to study the influence albumin), which was saturated with a 95% 02, 5% CO2 mixture. The medium was delivered of the hypothalamus on anterior pituitary to the chamber by means of a peristaltic pump hormone secretion in vitro. The hypothala- (Pharmacia Fine Chemicals, Uppsala, Sweden) mo-neurohypophysial complex (HNC), which at a flow rate of 0.2ml/min. The perifused consists of the hypothalamus, pituitary medium was collected every 5 min and subjected stalk, and intermediate and posterior pitui- to radioimmunoassay (RIA) for ƒÀ-END. a-Rat Vol.36, No.5 EFFECT OF ANP ON ƒÀ-ENDORPHIN RELEASE 649

ANP and DA were dissolved in the freshly were analyzed by Duncan's multiple range test. gassed perifusion medium. The high potassium medium, which contained 50 mM KCl, was prepared by reducing the NaCl concentration by Results 50mM to maintain the constant ionic strength and osmotic pressure. After 150 min of preperifusion, tissue was perifused for 30 min to es- Basal release of n-END tablish the rate of basal ƒÀ-END release, then The experiment was started after 150 for 20 min with one of the test substances and min of preperifusion when the spontaneous subsequently for 10 min with high potassium release of ƒÀ-END from NIL and HNC was medium. rather stable. The mean basal concentra-

RIA tion of ƒÀ-END in the effluent medium The antibody to ƒÀ-END was raised in a New from chambers containing NIL was signi- Zealand White rabbit immunized with human ficantly greater than that from chambers β-END conjugated to bovine thyroglobulin using containing HNC (773•}61pg/fraction and carbodiimide. RIA for rat ƒÀ-END was perform- 424•}50pg/fraction, respectively,p<0.01) ed with synthetic camel n-END as the standard (Fig. 1). and labeled tracer. ƒÀ-END was radioiodinated The gel chromatographic pattern of the with Na 125I (Amersham Japan, Tokyo) by the effluent from a chamber containing the chloramine T method. The labeled ƒÀ-END was HNC is depicted in Fig. 2. More than purified by adsorbing to silicic acid (Mallincrodt Chemical Works, St. Louis, MO, USA) and had 92% of the total immunoreactive ƒÀ-END a specific activity of 150ƒÊCi/ƒÊg. For RIA, 100

μl of the standard or media sample was incubated for 2 days at 4•Ž with 200ƒÊl of assay buffer β- END (0.05M phosphate buffer, pH 7.4, containing 500 kallikrein inactivator units/ml of aprotinin, 1mM EDTA, 0.02% sodium azide, 0.4% 2- mercaptoethanol and 0.1% bovine serum albumin), 100 pl of antiserum (final dilution 1: 100,000) and 100ƒÊl of labeled n-END. Antibody bound and free peptide were separated by adding 1% bovine r-globulin and 25% polyethylene- glycol. The minimal detectable quantity of camel ƒÀ-END was 3pg/tube. As expected, this antiserum crossreacts 100% with ƒÀ-lipotropin, but does not recognize ACTH, Met-enkephalin, Leu-enkephalin, arginine vasopressin or a-rat ANP.

Gel exclusion chromatography Gel chromatography was performed on a Sephadex G-75 column (1 •~ 55 cm) equilibrated with the assay buffer at 4•Ž. The descending flow rate was 2ml/hr, and the fractionvolume was 1.0ml. Each fraction was subjected to RIA for (ƒÀ-END. The column was calibrated with blue dextran for the void volume, 125I for the salt peak and synthetic ƒÀ-END. Recovery of n-END applied to the column was 95%. Fig. 1. Basal release of ƒÀ-END from rat HNC Statistical analysis (N=27) and NIL (N=25). mean•}S.E. The data are expressed as mean•}S.E. and *:p<0 .01 Endocrinol. Japon. 650 IKEDA et al. October 1989 eluted at the elution position of synthetic Effect of ANP on the release of ƒÀ- END from NIL and from HNC β-END. There was no detectable peak of immunoreactive ƒÀ-END at the expected When the HNC were perifused with ƒ¿- elution position of ƒÀ-lipotropin. rat ANP (10-8 to 10-6M) for 20 min, the ƒÀ-END concentration in the effluent fractions did not change with 10-8 M ANP, but increased 25 and 50% (p<0.01) with 10-7 and 10-6M ANP, respectively, (Fig. 3). The high potassium medium did not increase ƒÀ-END release. In contrast, a-rat ANP (10-8 to 106-M) did not affect the β-END concentration in the effluent medium

from NIL. The high potassium medium also did not increase the ƒÀ-END concentration in the effluent medium.

Effect of DA on the release of ƒÀ-END The ƒÀ-END responses to 10-6M DA alone or a mixture of 10-6M DA and 10-8

or 10-6M ANP are shown in Fig. 4. DA

perifusion significantly decreased ƒÀ-END Fig. 2. Gel chromatographic profile of immu- release from NIL and HNC (51% and 50% noreactive ƒÀ-END released from rat HNC of the basal level, respectively, p<0.01). obtained by a Sephadex G-75 column (1•~55 α-Rat ANP at the concentrations of 10-8

cm). M to 10-6M did not reverse the inhibitory effect of DA on ƒÀ-END release from NIL or HNC.

Fig. 3. Left panel: Effects of 10-8, 10-7 and 10-6M ANP on β-END release ffom rat HNC (N=5, 5 and 7, respectively).

Right panel: Effects of 10-8, 10-7 and 10-6

M ANP on ƒÀ-END release from rat NIL

(N=5, 5 and 6, res-

pectively). mean•}S.E.

Fraction number Vol.36, No.5 EFFECT OF ANP ON ƒÀ-ENDORPHIN RELEASE 651

Vermes et al., 1980). Our observation that 10-6M DA equally inhibited ƒÀ-END release from NIL and from HNC confirms and extends previous studies. It is unlikely that hypothalamic factors might reach intermediate pituitary via the hypothalamo-

pituitary portal system because of the absence of blood flow and removal of the anterior pituitary. However, it is likely that hypothalamic neural factors can reach the intermediate pituitary, because the

neural pathways are preserved in the HNC

(Sladek et al., 1977). Therefore, our result showing a lower immunoreactive ƒÀ-END concentration in the effluent medium from the HNC than in that from the intermediate

pituitary suggests the presence of hypothalamic neural factors which inhibit ƒÀ- END release from the intermediate pituitary. Gel chromatographic study revealed that immunoreactive ƒÀ-END in the effluent from a chamber containing the HNC consisted primarily of a single peak eluting at the elution position of synthetic ƒÀ-END. Because ƒÀ-lipotropin is a major component of B-END immunoreactivity released from Fig. 4. Upper panel: Effects of 10-6M DA and 10-8M or 10-6M ANP (each N=5) on the anterior pituitary, its absence from the effluent from the HNC rules out possible β-END release from rat HNC. Lower panel: Effects of 10-6 M DA and contamination of the HNC by anterior

10-8M (N=4) or 10-6 M ANP (N=5) on ƒÀ- pituitary tissue. The possibility that ƒÀ- END release from rat NIL. mean•}S.E. END released from the hypothalamus may contribute to the release of ƒÀ-END from the HNC cannot be ruled out, although Discussion the amount of ƒÀ-END in the hypothalamus is less than 1% of that in the intermediate

This study demonstrates that the basal pituitary (Rossier et al., 1977). In addition, release of immunoreactive f-END from the the absence of a measurable ƒÀ-END increase HNC is significantly lower than that from from the HNC in response to treatment the NIL. The intermediate lobe of the with a high potassium concentration, which pituitary is innervated by dopaminergic others have shown causes ƒÀ-END release nerve fibers arising in the arcuate nucleus from the hypothalamus but not the inter- (Annunziato et al., 1980) and its secretion mediate pituitary (Osborne et al., 1979; of POMC derived peptides is under inhibi- Przewlocki et al., 1978), is further evidence tory dopaminergic regulation involving the that the hypothalamic contribution to ƒÀ- D-2 dopamine receptor (Beaulieu et al., END released from HNC is minimal. 1984; Cote et al., 1982; Cote et al., 1986; This study also demonstrates a stimu- Endocrinol. Japon. 652 IKEDA et al. October 1989

latory effect of ANP on ƒÀ-END release Grewe, M. Munemura, J. C. Stoof, K. Tsuruta from the HNC, but no direct effect of and J. W. Kebabian (1982). Biochemical and ANP on ƒÀ-END release from NIL. Intr- physiological studies of the beta-adrenoceptor and the D-2 dopamine receptor in the inter- estingly, Nakao et al. (1986) reported an mediate lobe of the rat pituitary gland: A inhibitory effect of ANP on the brain review. Neuroendocrinology 35, 217-224. dopaminergic system. Therefore, the stimu- Cote, T. E., R. Felder, J. W. Kebabian, R. D. latory effect of ANP on ƒÀ-END release Sekura, T. Reisine and H. U. Affolter (1986). from the HNC might result from the inhi- D-2 dopamine receptor-mediated inhibition of bitory effect of ANP on hypothalamic DA. pro-opiomelanocortin synthesis in rat intermediate lobe. J. Biol. Chem. 261, 4555-4561. It seems unlike that ANP directly at- Currie, M. G., D. M. Geller, B. R. Cole, T. tenuates the inhibitory effect of DA on ƒÀ- G. Boylan, W. Yuseng, S. W. Holmberg and END release at the pituitary level, because P. Needleman (1983). Bioactive cardiac sub- ANP did not reduce the effect of DA on stances: potent vasoreluxant activity in mam- β-END release from NIL It may also be malian atria. Science 221, 71-73. possible that ANP stimulates a hypothalamic de Bold, A. J., H. B. Borenstein, A. T. Veress factor which increases ƒÀ-END release from and H. A. Sonnenberg (1981). A rapid and the HNC. Further studies are necessary potent natriuretic response to intravenous injection of atrial myocardial extracts in rat. to elucidate the mechanism by which ANP Life Sci. 28, 89-94. causes ƒÀ-END release from the HNC. Gill, M. K., A. Dutt and H. S. Juneja (1985). Effect of castration and steroid treatment on the release of gonadotropins by the rat Acknowledgements pituitary-hypothalamus complex in vitro (1985). Horm. metabol. Res. 17, 141-146. We are greatly indebted to Dr. C. Rowan Horvath, J., T. Ertl and A. V. Schally (1986). DeBold, Vanderbilt University, Nashville, TN, Effect of atrial natriuretic peptide on gonado- USA, for his kind review and suggestions in tropin release in superfused rat pituitary preparing this manuscript, and to Dr. Masataka cells. Proc. Natl. Acad. Sci. USA 83, 3444- Nanno, Hamamatsu University School of Medi- 3446. cine, for his invaluable advise and assistance. Jacobowitz, D. M., G. Scofitch, H. R. Keiser, R. L. Eskay and V. Zamir (1985). Evidence of the existence of atrial natriuretic factor- References containing neurons in the rat brain. Neuro- endocrinology 40, 92-94. Annunziato, L. and R. I. Weiner (1980). Januszewicz, P., G. Thibault, R. Garcia, J. Characteristics of dopamine uptake and 3, 4- Gutkowska, J. Genest and M. Cartin (1986). dehydroxyphenylacetic acid (DOPAC) form- Effect of synthetic atrial natriuretic factor on ation in the dopaminergic terminals of the arginine vasopressin release by the rat hypo- neurointermediate lobe of the pituitary gland. thalamo nuerohypophysical complex in organ Neuroendocrinology 31, 8-12. culture. Biochem. Biophys. Res. Commun. 134, Atarashi, K., P. J. Murlow, R. Franco-Saenz, 652-658. R. Snajdar and J. Rapp (1984). Inhibition of Karanth, S., A. Dutt and H. S. Juneja (1987). aldosterone production by an atrial extract. Age-related release of prolactin by the pituitary Science 224, 992-994. and the pituitary-hypothalamic complex in Beaulieu, M., M. E. Goldman, K. Miyazaki, vitro: an attempt to describe the development E. A. Frey, R. L. Eskay, J. W. Kebabian of the hypothalamic prolactin-inhibiting and and T. E. Cote (1984). Bromocriptine-induced -releasing activities in male rats. J. Endocr. changes in the biochemistry, physiology, and 115, 405-409. histology of the intermediate lobe of the rat Lamacz, M., P. Netchitailo, M. C. Tonon, M. pituitary gland. Endocrinology 114, 1871-1884. Feuilloley, N. Ling, G. Pelletier and H. Cote, T. E., R. L. Eskay, E. A. Frey, C. W. Vaudry (1987). Atrial natriuretic factor (ANF) Vol.36, No.5 EFFECT OF ANP ON ƒÀ-ENDORPHIN RELEASE 653

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