Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4789-4792, June 1989 Physiological Sciences Role of in the control of the release of thyrotropin in the rat (third ventricular injection/plasma thyrotropin/neuromedin B antiserum/in vitro anterior pituitary incubation/thyrotropin releasing hormone) VALERIA RETTORI, LJILJANA MILENKOVIC*, ASSEM M. FAHIMt, JULIA POLAKO, STEPHAN R. BLOOM§, AND SAMUEL M. MCCANN¶ Department of Physiology, Division, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9040 Contributed by Samuel M. McCann, March 13, 1989

ABSTRACT Neuromedin B (NB), a -like pep- that NB is present in the hypothalamus and in various regions tide, was first isolated from porcine spinal cord and subse- of the brain and also in the thyrotropes of the rat anterior quently found in the central nervous systems of rat, cat, and pituitary (5). To the best of our knowledge, it has not been human. Immunocytochemical studies have shown that NB is detected in other pituitary cell types. Although a number of present in hypothalamus and various other regions of the brain fibers of presumed NB-containing neurons were localized to and in thyrotrophs of the anterior pituitary of the rat. The the hypothalamus, no cell bodies of NB-containing neurons possible physiological role ofNB in the hypothalamic-pituitary were detected in the hypothalamus of rats, even after col- axis is not known. Therefore, we studied the in vivo effect ofthis chicine treatment (5). on the plasma levels of thyrotropin (TSH) after ad- Polak and coworkers (5) studied the effects of hyper- and ministering NB to conscious freely moving adult male rats. hypothyroidism on the levels of thyrotropin (TSH) ,8 and NB When injected i.v., only the highest dose of NB (50 jpg, 44.2 in the pituitary, as determined by radioimmunoassay, and nmol) significantly lowered plasma TSH levels relative to levels also on the content of the peptide, as estimated from im- in saline controls. When injected intraventricularly, NB doses munocytochemical studies. The content of NB in the pitu- of 0.5 and 5 ,ug (0.44 and 4.42 nmol, respectively) lowered itary and the density of its staining of thyrotropes paralleled plasma TSH levels with a 15-min latency period. Responsive- the content and density of staining of TSH. Therefore, the ness of the pituitary to TSH-releasing hormone (TRH) was results suggest that NB and TSH are cosecreted from thy- tested after the effective dose of NB was administered i.v. The rotropes. The secretion ofboth molecules would be enhanced increase in plasma TSH levels after the i.v. injection of 1 /g of in TRH was not altered by NB. NB at 10-9 to 10-7 M also reduced after reduced negative feedback by thyroid hormone TSH release from hemipituitaries incubated in vitro without hypothyroidism and diminished with elevated levels of thy- decreasing the response to TRH. Consequently, NB appears to roid hormones in hyperthyroidism. These suppositions are have a direct inhibitory effect on TSH release from thyro- based only on content changes. Consequently, it is not clear trophs. Since the response to TRH was not depressed,, NB without measurement of the content of NB in either pituitary appears to act on a separate NB thyrotroph receptor that sinusoidal blood or peripheral blood whether or not NB is suppresses TSH release by a TRH-independent mechanism. actually released with TSH. Alternatively, it might play a After antiserum directed against NB was inJected into the third modulatory role at the thyrotrope level to alter TSH release. ventricle, there was a highly significant elevation of the plasma Stimulated by these interesting findings, we have begun to level of TSH, beginning at 4 hr and increasing at 6 hr. relative evaluate the role of NB in modulating the activity of the to initial levels and levels in normal rabbit serum-injected hypothalamic-pituitary unit. We have evaluated the effects controls. These results indicate that NB has a tonic suppressive of NB after its in vivo injection i.v. or intraventricularly into effect on TSH release and is a physiologically significant the third cerebral ventricle of conscious rats and determined TSH-release-inhibitory factor. its effect on the responsiveness of the thyrotropes to TSH- releasing hormone (TRH). We have evaluated the effects of Neuromedin B (NB) was isolated from porcine spinal cord NB on TSH release in vitro. The results indicate that NB has using a bioassay based on uterine contractility (1). Since it is a suppressive effect on TSH release that is mediated at the related chemically to bombesin, it was given the name pituitary level by direct suppression of the release of TSH neuromedin B. A comparison of the structures of bombesin without suppressing the response of the thyrotropes to TRH. (the amphibian skin peptide), -releasing peptide (GRP, The physiologic significance of NB in control of TSH release the mammalian counterpart of bombesin also found in the is indicated by elevation of plasma levels of TSH after brain), and NB is given in Fig. 1. NB is a decapeptide differing from the C-terminal decapeptide sequence of bombesin in Abbreviations: NB, neuromedin B; TSH, thyrotropin; TRH, TSH- only three loci. In position 3 of NB, is replaced by releasing hormone; NRS, normal rabbit serum; GRP, gastrin- glutamine in bombesin. Threonine in position 6 of NB is releasing peptide. replaced by in bombesin and in position 8 of NB *Present address: Institute of Molecular Biology and Endocrinology -09, Boris Kidric Institute, P.O. Box 522, 11001 Belgrade, Yugo- is replaced by leucine in bombesin. Both slavia. are C-terminally amidated, as is the case with many tPresent address: Zoology Department, Faculty of Science, Cairo biologically active peptides (2). University, Giza, Egypt. NB has subsequently been found in cat, rat, and human tPresent address: Department of Histochemistry, University of spinal cord (3, 4). Immunocytochemical studies have shown London, Royal Postgraduate Medical School, Hammersmith Hos- pital, Ducane Road, London W12 OHS, England. §Present address: Department of Medicine, Royal Postgraduate The publication costs of this article were defrayed in part by page charge Medical School, Hammersmith Hospital, Ducane Road, London payment. This article must therefore be hereby marked "advertisement" W12 OHS, England. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom correspondence should be addressed.

4789 Downloaded by guest on September 23, 2021 4790 Physiological Sciences: Rettori et al. Proc. Natl. Acad. Sci. USA 86 (1989) pGlu-GIn-Arg-Leu-Gly-Asn-Gin-Trp-Ala-Val-Gly-His-Leu-Met-NH2: BOMBES IN Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys- Met-Tyr-Pro-Arg-GIy-Asn-His-Trp-Ala-Val-GIy-His-Leu-Met-NH 2: GRP Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH 2: NEUROMEDIN B

FIG. 1. Structures of bombesin, GRP, and NB. pGlu, pyroglutamic acid. injection of highly specific antiserum against NB into the a Dubnoff metabolic shaker. After removal of preincubation third ventricle. medium, the hemipituitaries were resuspended in 1 ml of medium alone or medium containing NB at a final concen- tration of 10-11 to 10-7 M in the presence or absence of 200 MATERIALS AND METHODS ng of TRH and incubated for 2 hr. After incubation, the Male Sprague-Dawley rats ranging in weight from 220 to 250 medium was removed and stored frozen at -20°C until g were purchased from Holtzmann (Madison, WI). The measurement of hormones. animals were housed in individual cages in a room with Radioimmunoassay. TSH levels in plasma samples and controlled lighting (on from 0500 to 1900) and temperature incubation medium were determined using kits supplied by (22-240C). Animals had free access to rat chow and water. the National Institute of Diabetes and Digestive and Kidney In Vivo Studies. A stainless steel cannula (23 gauge) was Diseases and expressed in terms of the preparation (RP2) implanted into the third ventricle by using a David-Kopf reference provided. stereotaxic instrument (6). The rats were housed in single Statistics. Analysis of variance with repeated measures cages thereafter. Five to eight days after implantation of the followed by the Student-Newman-Keul multiple comparison cannula into the third ventricle, an indwelling catheter was test was employed for assessment of significance. advanced into the right atrium through the right jugular vein after ether anesthesia (7). Twenty-four hours later, cannu- RESULTS lated animals were moved to a quiet laboratory and extension tubing was attached to the jugular cannula at its exit in the Effect of Intraventricular Injection of NB on TSH Release. dorsum of the neck. Animals were then left undisturbed for The injection of the saline diluent into the third ventricle 1 hr. Then a basal heparinized blood sample (0.7 ml) was produced no changes in plasma TSH during the 2 hr of the collected, and a solution of 0.5 ,&g or 5 pug of NB in 1.5 p.l of experiments (Fig. 2). In contrast intraventricular injection of 0.9% NaCl (saline) or an equal volume of saline for control 0.5 jig (0.44 nmol) of NB induced a significant decline below animals was injected into the third ventricle. NB was pur- initial values in levels ofplasma TSH, noticeable at 15 and 30 chased from Peninsula Laboratories. Blood samples (0.7 ml) min. Values continued to decline gradually over the 2 hr of were obtained 5, 15, 30, 60, and 120 min after the injection. In this and subsequent experiments, after removal of each blood sample, an equal volume of a suspension of 40% of Saline or NB washed rat erythrocytes in saline was injected to maintain 80 blood volume. For i.v. testing, atrial cannulae were implanted as de- scribed above. After removal of the initial blood sample (0.7 ml), 0.5 ml of 0.9% NaCl (saline) alone or saline containing 0.5 pug, 5 ttg, or 50 pug of NB was injected through the atrial cannula. Blood samples (0.7 ml) were obtained 5, 15, 30, 60, 601 120, 150, and 180 min after the injection. After the removal E of the blood sample at 120 min, a challenge dose of 1 pug of TRH was injected in 0.2 ml of saline in a bolus, and the 0 responsiveness of the pituitary was studied by removing cn blood samples at 15, 30, and 60 min after the injection ofTRH 0)Q and measuring plasma TSH levels. In additional experiments highly specific antiserum against 401 NB diluted 1:10 with saline was injected into the third cC, ventricle of conscious rats. The NB antiserum was raised in rabbits. The specificity ofthe antiserum was tested by adding p

30

20k Basal 10-11 10-9 10-7 TRH 10-11 10-9 10-7 NB, M NB, M 10 p

120 150 bated in vitro. (Left) Control hemipituitaries. (Right) Hemipituitaries 05 15 30 60 120135150 180 incubated in 0.5 x 10-7 M TRH. Glands were incubated with various Time, min doses of NB in the presence (o) or absence (e) of TRH. Values are mean ± SEM of three experiments, with six to eight tubes per FIG. 3. Effect of i.v. injection of NB on p1 lasma TSH. At 120 min experiment and each tube containing one hemipituitary selected at after injection of saline or NB, an injection (of TRH was given i.v. random. *, P < 0.01, for TSH values in medium from pituitaries There were six to eight rats per group. incubated with NB vs. medium from control pituitaries. Downloaded by guest on September 23, 2021 4792 Physiological Sciences: Rettori et al. Proc. Natl. Acad. Sci. USA 86 (1989) DISCUSSION Passive neutralization of NB by a highly specific anti-NB antiserum indicates that the NB suppression of TSH release In line with its localization in thyrotropes, NB had a pro- has physiological significance, since plasma concentrations nounced effect on the release of TSH, whereas NB had little of TSH rose (after a delay) after intraventricular injection of effect on the release of or growth hormone either in the antiserum. The delay is probably the time required for the vivo or in vitro (unpublished data), suggesting that the effect antiserum to penetrate to the hypothalamic tissue and to of NB on TSH release is specific. After its intraventricular diffuse to the site of its interaction with NB, as observed with injection, NB acts to lower the plasma level of TSH. It is antiserum against GRP (8). We cannot say with certainty puzzling that the response to the lower dose was more whether this indicates that the peptide has a physiologically prolonged and greater than the response to the higher NB significant effect at the hypothalamic or pituitary level. We dose. are in the same situation with respect to interpretation of the Only the highest dose of NB used suppressed TSH release results from the intraventricular or i.v. injection ofNB. Since after i.v. injection and the responsiveness to i.v. injection of the direction of change is the same after the injection of NB TRH was not suppressed. This result in vivo is in agreement by either path, it is probable that when NB is injected with the in vitro results in which the release of TSH by intraventricularly it is taken up by portal vessels and deliv- hemipituitaries was suppressed by NB at 10-9 and 1i-O M, ered to its site of action in the pituitary. Similarly, the without suppression, in fact with enhancement, of the re- antiserum may act to block the inhibitory effect of endoge- nous NB on TSH release only after uptake by portal vessels sponse to TRH at the highest NB dose. Since the effect of and delivery to the pituitary. We can consider the pituitary intraventricular injection of NB was similar-namely a sup- site of action established. Further experiments are necessary pression of TSH release-to that seen after exposure of to determine whether NB has a parallel action, to alter the pituitary cells to NB, it is possible that the response to release of TRH, in the hypothalamus. In contrast to the intraventricular NB injection may be caused by uptake ofNB effects of the NB antiserum on TSH release, there was little by portal vessels and delivery to the pituitary where it exerts effect on the plasma levels of either growth hormone or its suppressive effect. To establish an additional hypotha- prolactin after intraventricular injection of the antiserum lamic site of action of NB, it will be necessary to measure its (unpublished data), which lends credence to the concept that effect on the release of TRH either in vivo or in vitro. NB may not be physiologically significant in the control of On the other hand, the suppressive effect of NB on TSH these two pituitary hormones. release from the thyrotropes appears to be established from these studies. Since the response to TRH was not suppressed We thank Ms. Judy Scott for typing the manuscript. This work was and, if anything, was enhanced by coincubation of the supported by Grant HD09988 from the National Institutes of Health. pituitary cells with both NB and TRH, the results indicate that the suppressive effect of NB is not mediated by the same 1. Minamino, N., Kangawa, K. & Matsuo, H. (1983) Biochem. receptor that mediates the stimulatory effect of TRH. Since Biophys. Res. Commun. 114, 541-548. both NB and TSH are localized to the thyrotropes, one can 2. Mutt, V. (1989) Introductory Remarks in Nobel Symposium on , eds. Mutt, V., Fuxe, K. & H6kfelt, T. (Nobel postulate that NB has a modulatory effect on TSH release Found., Stockholm), in press. from thyrotropes, which at least in an animal with normal 3. Namba, M., Ghatei, M. A., Gibson, S. J., Polak, J. M. & thyroid status is suppressive. NB may act on its receptors in Bloom, S. R. (1985) Neuroscience 15, 1217-1226. the thyrotropes to suppress the release of TSH by a mech- 4. Namba, M., Ghatei, M. A., Anand, P. & Bloom, S. R. (1985) anism independent of TRH. In other words, NB is a TSH- Brain Res. 342, 183-186. release-inhibiting peptide. In view of the parallel changes in 5. Steel, J. H., Van Noorden, S., Ballesta, J., Gibson, S. J., TSH and NB content in thyrotropes under different thyroid Ghatei, M. A., Burrin, J., Leonhardt, U., Domin, J., Bloom, S. R. & Polak, J. M. (1988) Endocrinology 122, 270-282. states (4), it is possible that this inhibitory modulatory role of 6. Antunes-Rodrigues, J. & McCann, S. M. (1970) Proc. Soc. Exp. NB could change under different conditions of thyroid ac- Biol. Med. 133, 1464-1470. tivity. Consequently, it will be necessary to evaluate the 7. Harms, P. & Ojeda, S. R. (1974) J. Appl. Physiol. 36, 391-392. effects of NB on TSH release in conditions of hypo- and 8. Kentroti, S., Dees, W. L. & McCann, S. M. (1988) Proc. Natl. hyperthyroidism. Acad. Sci. USA 85, 953-957. Downloaded by guest on September 23, 2021