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Immunohistochemical localization of neurotensin and β-endorphin in the rat anterior pituitary gland
386
Brain Research, 331 (1985) 386-388
Elsevier BRE 20727
Immunohistochemical localization of neurotensin and fl-endorphin in the rat anterior pituitary gland RIE MIYOSHI, SHOZO KITO, TAKENOBU KISHIDA, EIKO ITOGA and NORIO OGAWA Third Department of lnternal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734 and Department of Neurochemistry, Institute for Neurobiology, Okayama University Medical School, 2-5-1 Schikata-cho, Okayama 700 (Japan)
(Accepted November 20th, 1984) Key words: anterior pituitary gland-- immunohistochemistry- - fl-endorphin - - neurotensin--
enzyme-labeled antibody technique - - immunoreactivity
Nakane's enzyme-labeled antibody technique revealed that cells containing neurotensin-like immunoreactivitywere widely distributed in the anterior lobe of the pituitary body. Immunohistochemicalstudies on serial sections showed that a part of neurotensin positive anterior lobe cells contained fl-endorphin-like peptide simultaneously. The results show that fl-endorphin and neurotensin occur together in certain pituitary cells and this is an evidence of coexistence of more than one peptide within one anterior pituitary cell. Existence of A C T H and fl-endorphin in pituitary anterior lobe cells has been well-documented. On the other hand, neurotensin-like immunoreactivity in the pituitary body has been confirmed through various experimental techniques including radioimmunoassayl, fluorescent antibody technique 9 and combination of radioimmunoassay (RIA) and high per-" formance liquid chromatography (HPLC) 2. In this paper the authors studied on mutual relationship between neurotensin- and fl-endorphin-containing cells in the anterior lobe with use of Nakane's enzyme-labeled antibody technique. The pituitary body of the Wistar strain male rat (150 g b.wt.) was fixed by Zamboni solution for 4 h after decapitation. After fixation, the organ was washed out by 5% ice-cold sucrose phosphate buffer (pH 7.4) for 17.5 h and embedded in paraffin. Serial sections of 4 ~m thickness of the organ were prepared and these sections were incubated with either fl-endorphin- or neurotensin-specific antibody alternatively at 4 °C for 30 h. As the next step, the sections were incubated with peroxidase-labeled antirabbit IgG goat IgG at 4 °C for 24 h. For staining, 4-Cl-l-naphthol for fl-endorphin-like immunoreac-
tivity and DAB for neurotensin-like immunoreactivity were used. At DAB staining pre-incubation with 20 mg DAB containing 100 ml Tris buffer at room temperature for 30 rain was done. There was no similarity between amino acid sequences of fl-LPH (a precursor peptide of fl-endorphin) and neurotensin. Antibody against fl-endorphin was prepared by injection of camel fl-endorphin into rabbit 6. This antibody showed no cross-reactivity with a-, y-endorphin, enkephalin analogs, a-MSH, ACTH, vasopressin, insulin, glucagon, TRH, LH-RH, bombesin, myelin basic protein, growth hormone, prolactin, morphine and naloxone. To obtain antibody against neurotensin, synthetic neurotensin was injected into a rabbit. Thus obtained antibody was specific to the C-terminal peptide chain of neurotensin. Cross-reactivity between the fl-endorphin and neurotensin antibodies was examined by Ouchterlony method with negative results. As the results, authors observed numerous fl-endorphin positive anterior lobe cells (Fig. 1A), while neurotensin-like immunoreactivity was also noticed in considerable number of anterior lobe cells (Fig. 1B). Fig. 2 shows photographs of higher mag-
Correspondence: R. Miyoshi, Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
387 In 1976, Carraway pointed out that there was positive neurotensin-like immunoreactivity within the pituitary body using R I A 1. This finding was confirmed morphologically by Uhl through fluorescent antibody technique in 19779 . These two works were further verified by Goedert when he proved that neurotensin itself existed in anterior lobe cells by combination of H P L C and R I A 2. The present authors showed that there were neurotensin-positive anterior lobe cells in the pituitary body. Moreover, we discovered that a part of the neurotensin-positive anterior lobe cells also contained substances with positive fl-endorphin-like immunoreactivity. This fact was noteworthy because it meant one anterior lobe cell excreted at least two hormones which were not related in their intracellular processing each
Fig. 1. fl-Endorphin-like immunoreactivity (upper figure) and neurotensin-like immunoreactivity (lower figure) in the rat anterior pituitary gland were obtained by use of Nakane's enzyme-labeled antibody technique, fl-Endorphin- and neurotensin-like immunoreactivity were found in many anterior lobe cells. The cells in the central part of both pictures were proved to be the same cell by serial sections.
nification of Fig. 1. The cells in the central part of the pictures of the upper and lower plates represent those of the actually same cell as observed by serial sections. This means that this cell contains both fl-endorphinlike-immunoreactivity and neurotensin-like-immunoreactivity in its c~¢toplasm. Fig. 3 shows anterior lobe cells which also contain both fl-endorphin- and neurotensin-like immunoreactivity. We have to mention that such coexistence of fl-endorphin and neurotensin within one anterior lobe cells was not c o m m o n but in many occasions, pituitary cells contained only one of these two peptides whenever our histochemical reaction was positive.
Fig. 2. Photographs with higher magnification of Fig. 1. fl-Endorphin-like immunoreactivity (upper figure) and neurotensinlike immunoreactivity (lower figure) in the rat anterior pituitary gland.
388
Fig. 3. fl-Endorphin-like immunoreactivity (left figure) and neurotensin-like immunoreactivity (right figure) in the rat anterior pituitary gland. An anterior lobe cell contains both fl-endorphin- and neurotensin-like immunoreactivity (arrow).
other. For the past several years it has been known that A P U D cells in the gastrointestinal tract contain both bioactive amines and neuropeptides simultaneously. Moreover, it has been taken for granted for central nerve cells3,10 and adrenal medulla cells 4 to contain two kinds of biologically active substances. Both neurotensin7 and fl-endorphin8 increase prolactin secretion, whereas neurotensin is considered to promote A C T H secretion 5. From this viewpoint,
1 Carraway, R. and Leeman, S. E., Characterization of radioimmunoassayable neurotensin in the rat, J. Biol. Chem., 251 (1976) 7041-7052. 2 Goedert, M., Lightman, S. L., Nagy, J. I., Marley, P. D. and Emson, P. C., Neurotensin in the rat anterior pituitary gland, Nature (Lond.), 298 (1982) 163-165. 3 Hfkfelt, T., Johansson, O., Ljungdahl,/~ke, Lundberg, J. M. and Schultzberg, M., Peptidergic neurons, Nature (Lond.), 284 (1980) 515-521. 4 H6kfelt, T., Lundberg, J. M., Schultzberg, M., Johansson, O., Ljungdahl, A. and Rehfetd, J., Neural Peptides and Neuronal Communication, Raven Press, New York, 1980, pp. 1-23. 5 Leeman, S. E., Morg, E. A. and Carraway, R. E., Substance P and neurotensin. In H. Gainer (Ed.), Peptides in Neurology, Plenum, New York, 1977, pp. 99. 6 Ogawa, N., Panerai, A. E., Lee, S., Forsbach, G., Havli-
functional relationship between neurotensin and flendorphin within one anterior lobe cell is considered to be a target of future research. The authors express their sincere gratitude to Professor Leeman and Dr. Carraway of Massachusetts Medical School who generously provided neurotensin antibody.
7
8 9 10
cek, V. and Friesen, H. G., fl-Endorphin concentration in the brain of intact and hypophysectomized rats, Life Sci., 25 (1979) 317-326. Rivier, C., Brown, M. and Vale, W., Effect of neurotensin, substance P and morphine sulfate on th¢ secretion of prolactin and growth hormone in the rat, Endocrinology, 100 (1977) 751-754. Rossier, J., Functions offl.Endorphin and Enkephalins in the Pituitary, Raven Press, New York, 1982, pp. 191-209. Uhl, G. R., Kuhar, M. J. and Snyder, S. H., Neurotensin; immunohistochemical localization in rat central nervous system, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 4059-4063. Vincent, S. R., Johansson, O., H6kfelt, T., Meyerson, B., Sachs, C., Elde, R. P., Terenius, O. and Kimmel, J., Neuropeptide coexistence in human cortical neurons, Nature (Lond.), 298 (1982) 65-67.
Brain Research, 331 (1985) 386-388
Elsevier BRE 20727
Immunohistochemical localization of neurotensin and fl-endorphin in the rat anterior pituitary gland RIE MIYOSHI, SHOZO KITO, TAKENOBU KISHIDA, EIKO ITOGA and NORIO OGAWA Third Department of lnternal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734 and Department of Neurochemistry, Institute for Neurobiology, Okayama University Medical School, 2-5-1 Schikata-cho, Okayama 700 (Japan)
(Accepted November 20th, 1984) Key words: anterior pituitary gland-- immunohistochemistry- - fl-endorphin - - neurotensin--
enzyme-labeled antibody technique - - immunoreactivity
Nakane's enzyme-labeled antibody technique revealed that cells containing neurotensin-like immunoreactivitywere widely distributed in the anterior lobe of the pituitary body. Immunohistochemicalstudies on serial sections showed that a part of neurotensin positive anterior lobe cells contained fl-endorphin-like peptide simultaneously. The results show that fl-endorphin and neurotensin occur together in certain pituitary cells and this is an evidence of coexistence of more than one peptide within one anterior pituitary cell. Existence of A C T H and fl-endorphin in pituitary anterior lobe cells has been well-documented. On the other hand, neurotensin-like immunoreactivity in the pituitary body has been confirmed through various experimental techniques including radioimmunoassayl, fluorescent antibody technique 9 and combination of radioimmunoassay (RIA) and high per-" formance liquid chromatography (HPLC) 2. In this paper the authors studied on mutual relationship between neurotensin- and fl-endorphin-containing cells in the anterior lobe with use of Nakane's enzyme-labeled antibody technique. The pituitary body of the Wistar strain male rat (150 g b.wt.) was fixed by Zamboni solution for 4 h after decapitation. After fixation, the organ was washed out by 5% ice-cold sucrose phosphate buffer (pH 7.4) for 17.5 h and embedded in paraffin. Serial sections of 4 ~m thickness of the organ were prepared and these sections were incubated with either fl-endorphin- or neurotensin-specific antibody alternatively at 4 °C for 30 h. As the next step, the sections were incubated with peroxidase-labeled antirabbit IgG goat IgG at 4 °C for 24 h. For staining, 4-Cl-l-naphthol for fl-endorphin-like immunoreac-
tivity and DAB for neurotensin-like immunoreactivity were used. At DAB staining pre-incubation with 20 mg DAB containing 100 ml Tris buffer at room temperature for 30 rain was done. There was no similarity between amino acid sequences of fl-LPH (a precursor peptide of fl-endorphin) and neurotensin. Antibody against fl-endorphin was prepared by injection of camel fl-endorphin into rabbit 6. This antibody showed no cross-reactivity with a-, y-endorphin, enkephalin analogs, a-MSH, ACTH, vasopressin, insulin, glucagon, TRH, LH-RH, bombesin, myelin basic protein, growth hormone, prolactin, morphine and naloxone. To obtain antibody against neurotensin, synthetic neurotensin was injected into a rabbit. Thus obtained antibody was specific to the C-terminal peptide chain of neurotensin. Cross-reactivity between the fl-endorphin and neurotensin antibodies was examined by Ouchterlony method with negative results. As the results, authors observed numerous fl-endorphin positive anterior lobe cells (Fig. 1A), while neurotensin-like immunoreactivity was also noticed in considerable number of anterior lobe cells (Fig. 1B). Fig. 2 shows photographs of higher mag-
Correspondence: R. Miyoshi, Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
387 In 1976, Carraway pointed out that there was positive neurotensin-like immunoreactivity within the pituitary body using R I A 1. This finding was confirmed morphologically by Uhl through fluorescent antibody technique in 19779 . These two works were further verified by Goedert when he proved that neurotensin itself existed in anterior lobe cells by combination of H P L C and R I A 2. The present authors showed that there were neurotensin-positive anterior lobe cells in the pituitary body. Moreover, we discovered that a part of the neurotensin-positive anterior lobe cells also contained substances with positive fl-endorphin-like immunoreactivity. This fact was noteworthy because it meant one anterior lobe cell excreted at least two hormones which were not related in their intracellular processing each
Fig. 1. fl-Endorphin-like immunoreactivity (upper figure) and neurotensin-like immunoreactivity (lower figure) in the rat anterior pituitary gland were obtained by use of Nakane's enzyme-labeled antibody technique, fl-Endorphin- and neurotensin-like immunoreactivity were found in many anterior lobe cells. The cells in the central part of both pictures were proved to be the same cell by serial sections.
nification of Fig. 1. The cells in the central part of the pictures of the upper and lower plates represent those of the actually same cell as observed by serial sections. This means that this cell contains both fl-endorphinlike-immunoreactivity and neurotensin-like-immunoreactivity in its c~¢toplasm. Fig. 3 shows anterior lobe cells which also contain both fl-endorphin- and neurotensin-like immunoreactivity. We have to mention that such coexistence of fl-endorphin and neurotensin within one anterior lobe cells was not c o m m o n but in many occasions, pituitary cells contained only one of these two peptides whenever our histochemical reaction was positive.
Fig. 2. Photographs with higher magnification of Fig. 1. fl-Endorphin-like immunoreactivity (upper figure) and neurotensinlike immunoreactivity (lower figure) in the rat anterior pituitary gland.
388
Fig. 3. fl-Endorphin-like immunoreactivity (left figure) and neurotensin-like immunoreactivity (right figure) in the rat anterior pituitary gland. An anterior lobe cell contains both fl-endorphin- and neurotensin-like immunoreactivity (arrow).
other. For the past several years it has been known that A P U D cells in the gastrointestinal tract contain both bioactive amines and neuropeptides simultaneously. Moreover, it has been taken for granted for central nerve cells3,10 and adrenal medulla cells 4 to contain two kinds of biologically active substances. Both neurotensin7 and fl-endorphin8 increase prolactin secretion, whereas neurotensin is considered to promote A C T H secretion 5. From this viewpoint,
1 Carraway, R. and Leeman, S. E., Characterization of radioimmunoassayable neurotensin in the rat, J. Biol. Chem., 251 (1976) 7041-7052. 2 Goedert, M., Lightman, S. L., Nagy, J. I., Marley, P. D. and Emson, P. C., Neurotensin in the rat anterior pituitary gland, Nature (Lond.), 298 (1982) 163-165. 3 Hfkfelt, T., Johansson, O., Ljungdahl,/~ke, Lundberg, J. M. and Schultzberg, M., Peptidergic neurons, Nature (Lond.), 284 (1980) 515-521. 4 H6kfelt, T., Lundberg, J. M., Schultzberg, M., Johansson, O., Ljungdahl, A. and Rehfetd, J., Neural Peptides and Neuronal Communication, Raven Press, New York, 1980, pp. 1-23. 5 Leeman, S. E., Morg, E. A. and Carraway, R. E., Substance P and neurotensin. In H. Gainer (Ed.), Peptides in Neurology, Plenum, New York, 1977, pp. 99. 6 Ogawa, N., Panerai, A. E., Lee, S., Forsbach, G., Havli-
functional relationship between neurotensin and flendorphin within one anterior lobe cell is considered to be a target of future research. The authors express their sincere gratitude to Professor Leeman and Dr. Carraway of Massachusetts Medical School who generously provided neurotensin antibody.
7
8 9 10
cek, V. and Friesen, H. G., fl-Endorphin concentration in the brain of intact and hypophysectomized rats, Life Sci., 25 (1979) 317-326. Rivier, C., Brown, M. and Vale, W., Effect of neurotensin, substance P and morphine sulfate on th¢ secretion of prolactin and growth hormone in the rat, Endocrinology, 100 (1977) 751-754. Rossier, J., Functions offl.Endorphin and Enkephalins in the Pituitary, Raven Press, New York, 1982, pp. 191-209. Uhl, G. R., Kuhar, M. J. and Snyder, S. H., Neurotensin; immunohistochemical localization in rat central nervous system, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 4059-4063. Vincent, S. R., Johansson, O., H6kfelt, T., Meyerson, B., Sachs, C., Elde, R. P., Terenius, O. and Kimmel, J., Neuropeptide coexistence in human cortical neurons, Nature (Lond.), 298 (1982) 65-67.