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Non-coordinate localization of corticotroph-neurophysin and β-endorphin in the anterior pituitary gland of the rat
Neuroscience Letters, 69 (1986) 89 93
89
Elsevier Scientific Publishers Ireland Ltd. NSL 04085 NON-COORDINATE LOCALIZATION OF C O R T I C O T R O P H - N E U R O P H Y S I N A N D ]3-ENDORPHIN IN THE ANTERIOR PITUITARY G L A N D OF THE RAT
WAYNE B. WATKINS Po.w~tra~hmte School ~/' Obstetrics and Gynaecology. National Women's Hospital. Claude Road. Epsotn. ..flu'kland (New Zealand)
(Received December 2rid, 1985; Revised version received April l lth, 1986: Accepted May 5th, 1986)
Kel" wm'~t~': rat
corticotroph adrenocorticotrophin hormone histochemistry biosynthesis
[~-endorphin neurophysin immuno-
Anti-neurophysin serum was applied in the immunohistochemical technique to anterior pituitary tissues obtained t¥om normal and chronically dehydrated rats and also from rats with chronic diabetes insipidus (Brattleboro strain). In all cases there was a positive staining in the corticotrophs, which also stained for either [~-endorphin ([3-END) or adrenocorticotrophin hormone (ACTH). It was concluded that corticotroph neurophysin may be synthesized independently of either ACTH or [3-END.
The anterior pituitary gland has received much attention with regard to the biosynthetic relationships between the h o r m o n e s o f the corticotrophs and the neuro-intermediate lobe viz, a d r e n o c o r t i c o t r o p h i n h o r m o n e ( A C T H ) , [3-1ipotrophin (!3-LPH), ctmelanocyte-stimulating h o r m o n e (ct-MSH) and !3-endorphin ([3-END). That these protein h o r m o n e s are the products o f cleavage from a larger 31 k D a precursor (proopiocortin) has been supported by the studies of Nakanishi et al. [9]. He showed that the primary amino acid sequence o f the h o r m o n e s resides within the precursor molecule. Further evidence for a c o m m o n biosynthetic p a t h w a y comes from immunohistochemical experiments in which colocalization o f the peptides occurs within the corticotrophs o f the anterior pituitary gland and cells o f the intermediate lobe [2, 3, 8, 14, 15]. More recently, however, it has been reported from this laboratory that the colocalization o f ]3-END and A C T H does not necessarily occur within the same corticotroph [13]. Using immunohistochemical techniques, neurophysin-like immunoreactivity (denoted c o r t i c o t r o p h - n e u r o p h y s i n ) , has been identified in the corticotrophs o f rat pituitary gland [10] and also in the corticotrophs o f dogs, sheep, man, pigs and cattle (W.B. Watkins, unpublished results). The aim o f this present study was to establish whether there is coordinate localization o f c o r t i c o t r o p h - n e u r o p h y s i n , [3-END and A C T H . O f particular interest was the examination o f those tissues which previously demonstrated n o n - c o o r d i n a t e localization o f [3-END and A C T H [1 1].
90 Antisera were raised in rabbits against porcine neurophysin-II, ACTH 17 39 and ovine 13-END using methods previously described [10]. Anterior pituitary glands were collected from physiologically normal male and female Sprague-Dawley rats, tixed in formalin (4%, v/v) and embedded in paraffin at 55°C. Tissue sections (4 lain) were cut from these tissues as well as from the anterior pituitaries of homozygous Brattleboro rats and rats given 2% saline to drink for 7 days as reported [13]. Immunofluorescence and immunoperoxidase histochemical techniques employed were the same as those published previously [15]. In the normal rat there is coordinate localization of neurophysin and 13-END consistent with our previous observations [13] (compare Fig. l a with l b). However, in the Brattleboro rat, the distribution of corticotroph-neurophysin is similar to that of ACTH (compare Fig. 2a with 2el. In these animals there is little staining of the corticotrophs with J3-END antiserum (Fig. 2c). When tissues from chronically dehydrated rats are subjected to immunohistochemistry, the distribution of cells in the anterior pituitary, staining with anti-neurophysin serum, is similar to those cells that stain positively for [3-END (compare Fig. 2b with 2d). As previously reported, there was an absence of staining in these tissues with ACTH antiserum (Fig. 20 [13]. It has been reported earlier from this laboratory that the rat corticotroph contains a substance(s) which cross-reacts immunologically with a selection of antisera raised against porcine neurophysin-lI. This corticotroph-neurophysin has been purified from sheep anterior pituitary glands and has been shown to possess an N-terminal amino acid sequence identical to that of ovine neurophysin-III (the vasopressin-associated neurophysin) up to residue number 35 [12]. Further immunohistochemical evidence tor the presence of vasopressin in the anterior pituitary has been provided by Lolait et al. [7], who demonstrated that, in the rat, there were a few scattered vasopressin-containing cells. This immunoreactive vasopressin extracted from SpragueDawley and Long-Evans rat coelutes on high-performance liquid chromatography
Fig. 1. a and b: immunofluorescencelocalizationof rat corticotrophs using antisera raised against neurophysin (a) and [3-END (b). a and b are serial sections, x 204. The position of the asterisk represents a common landmark on the pituitary tissue.
91
!
¢
~'11.
Fig. 2. a-f: immunoperoxidase histochemical localization of neurophysin (a and b), [3-END (c and d) and ACTH (e and f) in serial sections of the anterior pituitary gland of Brattleboro (a, c and e) and Sprague Dawley (b, d and f) rats dehydrated with 2% saline for 7 days. x 475. The position of the asterisk represents a common capillary landmark.
92 with authentic vasopressin. Other workers have also partially purified vasopressin from rat and pig anterior pituitary [4]. In contrast to the differential localization o f A C T H and [3-END in both the dehydrated and Brattleboro rats, there was a consistent appearance o f neurophysin in the corticotrophs. We earlier proposed that the physiological stress on the two groups o f experimental rats m a y cause c o n f o r m a t i o n a l changes in the structure o f A C T H and ]3-END, which, after formalin fixation, renders the molecules inaccessible to interaction with the corresponding antisera [ll]. Such interaction with a fixative does nol appear to be o f importance in the case o f the immunolocalization o f corticotroph-neurophysin. The nucleotide sequence o f cloned c D N A encoding bovine neurophysin-II precursor does not contain the p r o o p i o c o r t i n sequence [5], nor is the neurophysin sequence present in the c D N A encoding ovine proopiocortin [9]. This would suggest that the c o r t i c o t r o p h - n e u r o p h y s i n is synthesized independently o f either 13-END or A C T H . Such a proposal is supported by the present immunohistochemical observations. L i p i d - n e u r o p h y s i n complexes obtained from bovine posterior pituitary glands also contain biologically active A C T H - l i k e material [1]. Similarly, high-moleculaweight (80 k D a ) species containing neurophysin and vasopressin also possess the amino acid sequences o f A C T H and [3-END [6]. N e u r o p h y s i n - A C T H @ - E N D complexes are present within the h y p o t h a l a m o - n e u r o h y p o p h y s i a l system [6]. Conversely, it m a y not be unexpected that regions which are p r e d o m i n a n t l y responsible for the synthesis o f ]3-END and A C T H m a y also possess genetic material capable o f synthesizing significant quantities o f neurophysin and their associated neurohypophysial hormones. The significance o f these latter peptides in the anterior pituitary gland is not known. This work was financed by the Medical Research Council o f New Zealand. I Audhya, T.K., Seil, S.M., Robinson, A.G., Krieger, D.T., Arruda, J.A.L., Kurtzman, N.A. and Walter, R., Biological and immunological studies of lipid-containing neurophysin of rat and reaggregated lipid complex. Biochem. Biophys. Acta, 572 (1979)483-491. 2 Begoet, M., Dubois, M.P. and Dubois, P.M., Immunologic localization of a- and [3-endorphins and !3-1ipotrophin in corticotrophic cells of the normal and anencephalic fetal pituitaries, Cell Tissue Res., 191 (1978) 413 422. 3 Bloom, F.E., Rossier, J., Battenbery, E.L.F., Bayon, A., French, E., Henriksen, S.J., Siggins, G.R., Segal, D., Browne, R., Ling, N. and Guillemin, R., 13-Endorphin:cellular localization, electrophysiological and behavioural effects. In E. Costa and M. Trabucchi (Eds.), Advances in Biochemical Psychopharmacology, Vol. 18, Raven Press, New York, 1978, pp. 89-109. 4 Chateau, M., Marchetti, J., Burlet, A. and Boulange, M., Evidence of vasopressin in adenohypophysis: research into its role in corticotrope activity, Neuroendoerinology, 28 (1979) 25-35. 5 Land, H., Schutz, G., Schmale, H. and Richter, D., Nucleotide sequence of cloned eDNA encoding bovine arginine vasopressin-neurophysin II precursor, Nature (London), 295 (1982) 299-303. 6 Lauber, M., Nicolas, P., Boussetla, tt.. Fahy, C., B6guin, P., Camier, M., Vaudry, H and Cohen, P., The Mr 80,000 common forms of neurophysin and vasopressin from bovine neurohypophysis have corticotroph- and [3-endorphin-likesequences and liberate by proteolysis biologically active corticotropin, Proc. Natl. Acad. Sci. USA, 78 (1981) 6086-6090. 7 Lolait, S.J., Markwick, A.J., McNally, M., Smilh. A.I. and Funder, J.W, Anterior pituitary cells l¥om
93
8 9
10
11 12
13
14
15
Brattleboro (DI/DI), Long-Evans (LE) and Sprague-Dawley (SD) rats contain immunoreactive argininc vasopressin. Ncuroendocrinology, submitted. Martin, R., Weber, E. and Voigt, K.H., Localization of corticotropin- and endorphin-related peptides in the intermediate lobe of the rat pituitary, Cell Tissue Res., 196 (1979) 307-319. Nakanishi, S., lnone, A., Kita, T., Numa, S., Chang, A.C.Y., Cohen, S.N., Nunberg, J. and Schimke, R.J., Construction of bacterial plasmids that contain nucleotide sequence for bovine corticotropin [~lipotropin precursor. Proc. Natl. Acad. Sci. USA, 75 (1978)6021-6025. Watkins. W.B., Presence of neurophysin-like material in the pituitary corticotrophs and melanotrophs and cells of the arcuate nucleus of the rat as revealed by immunocytochemistry, Neuropeptides, 3 (1983) 477 492. Watkins, W.B., Differential immunostaining of adenohypophysial cells with antisera to ACTH and [3-cndorphin, Reg. Peptides, 1, ( 1981 ) 375-385. Watkins, W.B., Moore, L.G., Rivier, J.E. and Spiess, J., Characterization of neurophysin from sheep anterior pituitary glands. Abstracts of the 7th International Congress of Endocrinology, 1984, No. 2667. Watkins, W.B., Yen, S.S.C. and Moore, R.Y., Presence of [3-endorphin-like immunoreactivity in thc anterior pituitary gland of rat and man and evidence for the differential localization with ACTH, Cell Tissue Res., 215 (1981) 577 589. Weber, F., Voigt, K.H. and Martin, R., Concomitant storage of ACTH and endorphin-like immunorcactivity in the secretory granules of anterior pituitary corticotrophs, Brain Res., 157 (1978) 385 39(/. Wilkes, M.M., Watkins, W.B., Stewart, R.D. and Yen, S.S.C., Localization and quantitation of 13endorphin in human brain and pituitary, Neuroendocrinology, 30 (1980) 113-121.
89
Elsevier Scientific Publishers Ireland Ltd. NSL 04085 NON-COORDINATE LOCALIZATION OF C O R T I C O T R O P H - N E U R O P H Y S I N A N D ]3-ENDORPHIN IN THE ANTERIOR PITUITARY G L A N D OF THE RAT
WAYNE B. WATKINS Po.w~tra~hmte School ~/' Obstetrics and Gynaecology. National Women's Hospital. Claude Road. Epsotn. ..flu'kland (New Zealand)
(Received December 2rid, 1985; Revised version received April l lth, 1986: Accepted May 5th, 1986)
Kel" wm'~t~': rat
corticotroph adrenocorticotrophin hormone histochemistry biosynthesis
[~-endorphin neurophysin immuno-
Anti-neurophysin serum was applied in the immunohistochemical technique to anterior pituitary tissues obtained t¥om normal and chronically dehydrated rats and also from rats with chronic diabetes insipidus (Brattleboro strain). In all cases there was a positive staining in the corticotrophs, which also stained for either [~-endorphin ([3-END) or adrenocorticotrophin hormone (ACTH). It was concluded that corticotroph neurophysin may be synthesized independently of either ACTH or [3-END.
The anterior pituitary gland has received much attention with regard to the biosynthetic relationships between the h o r m o n e s o f the corticotrophs and the neuro-intermediate lobe viz, a d r e n o c o r t i c o t r o p h i n h o r m o n e ( A C T H ) , [3-1ipotrophin (!3-LPH), ctmelanocyte-stimulating h o r m o n e (ct-MSH) and !3-endorphin ([3-END). That these protein h o r m o n e s are the products o f cleavage from a larger 31 k D a precursor (proopiocortin) has been supported by the studies of Nakanishi et al. [9]. He showed that the primary amino acid sequence o f the h o r m o n e s resides within the precursor molecule. Further evidence for a c o m m o n biosynthetic p a t h w a y comes from immunohistochemical experiments in which colocalization o f the peptides occurs within the corticotrophs o f the anterior pituitary gland and cells o f the intermediate lobe [2, 3, 8, 14, 15]. More recently, however, it has been reported from this laboratory that the colocalization o f ]3-END and A C T H does not necessarily occur within the same corticotroph [13]. Using immunohistochemical techniques, neurophysin-like immunoreactivity (denoted c o r t i c o t r o p h - n e u r o p h y s i n ) , has been identified in the corticotrophs o f rat pituitary gland [10] and also in the corticotrophs o f dogs, sheep, man, pigs and cattle (W.B. Watkins, unpublished results). The aim o f this present study was to establish whether there is coordinate localization o f c o r t i c o t r o p h - n e u r o p h y s i n , [3-END and A C T H . O f particular interest was the examination o f those tissues which previously demonstrated n o n - c o o r d i n a t e localization o f [3-END and A C T H [1 1].
90 Antisera were raised in rabbits against porcine neurophysin-II, ACTH 17 39 and ovine 13-END using methods previously described [10]. Anterior pituitary glands were collected from physiologically normal male and female Sprague-Dawley rats, tixed in formalin (4%, v/v) and embedded in paraffin at 55°C. Tissue sections (4 lain) were cut from these tissues as well as from the anterior pituitaries of homozygous Brattleboro rats and rats given 2% saline to drink for 7 days as reported [13]. Immunofluorescence and immunoperoxidase histochemical techniques employed were the same as those published previously [15]. In the normal rat there is coordinate localization of neurophysin and 13-END consistent with our previous observations [13] (compare Fig. l a with l b). However, in the Brattleboro rat, the distribution of corticotroph-neurophysin is similar to that of ACTH (compare Fig. 2a with 2el. In these animals there is little staining of the corticotrophs with J3-END antiserum (Fig. 2c). When tissues from chronically dehydrated rats are subjected to immunohistochemistry, the distribution of cells in the anterior pituitary, staining with anti-neurophysin serum, is similar to those cells that stain positively for [3-END (compare Fig. 2b with 2d). As previously reported, there was an absence of staining in these tissues with ACTH antiserum (Fig. 20 [13]. It has been reported earlier from this laboratory that the rat corticotroph contains a substance(s) which cross-reacts immunologically with a selection of antisera raised against porcine neurophysin-lI. This corticotroph-neurophysin has been purified from sheep anterior pituitary glands and has been shown to possess an N-terminal amino acid sequence identical to that of ovine neurophysin-III (the vasopressin-associated neurophysin) up to residue number 35 [12]. Further immunohistochemical evidence tor the presence of vasopressin in the anterior pituitary has been provided by Lolait et al. [7], who demonstrated that, in the rat, there were a few scattered vasopressin-containing cells. This immunoreactive vasopressin extracted from SpragueDawley and Long-Evans rat coelutes on high-performance liquid chromatography
Fig. 1. a and b: immunofluorescencelocalizationof rat corticotrophs using antisera raised against neurophysin (a) and [3-END (b). a and b are serial sections, x 204. The position of the asterisk represents a common landmark on the pituitary tissue.
91
!
¢
~'11.
Fig. 2. a-f: immunoperoxidase histochemical localization of neurophysin (a and b), [3-END (c and d) and ACTH (e and f) in serial sections of the anterior pituitary gland of Brattleboro (a, c and e) and Sprague Dawley (b, d and f) rats dehydrated with 2% saline for 7 days. x 475. The position of the asterisk represents a common capillary landmark.
92 with authentic vasopressin. Other workers have also partially purified vasopressin from rat and pig anterior pituitary [4]. In contrast to the differential localization o f A C T H and [3-END in both the dehydrated and Brattleboro rats, there was a consistent appearance o f neurophysin in the corticotrophs. We earlier proposed that the physiological stress on the two groups o f experimental rats m a y cause c o n f o r m a t i o n a l changes in the structure o f A C T H and ]3-END, which, after formalin fixation, renders the molecules inaccessible to interaction with the corresponding antisera [ll]. Such interaction with a fixative does nol appear to be o f importance in the case o f the immunolocalization o f corticotroph-neurophysin. The nucleotide sequence o f cloned c D N A encoding bovine neurophysin-II precursor does not contain the p r o o p i o c o r t i n sequence [5], nor is the neurophysin sequence present in the c D N A encoding ovine proopiocortin [9]. This would suggest that the c o r t i c o t r o p h - n e u r o p h y s i n is synthesized independently o f either 13-END or A C T H . Such a proposal is supported by the present immunohistochemical observations. L i p i d - n e u r o p h y s i n complexes obtained from bovine posterior pituitary glands also contain biologically active A C T H - l i k e material [1]. Similarly, high-moleculaweight (80 k D a ) species containing neurophysin and vasopressin also possess the amino acid sequences o f A C T H and [3-END [6]. N e u r o p h y s i n - A C T H @ - E N D complexes are present within the h y p o t h a l a m o - n e u r o h y p o p h y s i a l system [6]. Conversely, it m a y not be unexpected that regions which are p r e d o m i n a n t l y responsible for the synthesis o f ]3-END and A C T H m a y also possess genetic material capable o f synthesizing significant quantities o f neurophysin and their associated neurohypophysial hormones. The significance o f these latter peptides in the anterior pituitary gland is not known. This work was financed by the Medical Research Council o f New Zealand. I Audhya, T.K., Seil, S.M., Robinson, A.G., Krieger, D.T., Arruda, J.A.L., Kurtzman, N.A. and Walter, R., Biological and immunological studies of lipid-containing neurophysin of rat and reaggregated lipid complex. Biochem. Biophys. Acta, 572 (1979)483-491. 2 Begoet, M., Dubois, M.P. and Dubois, P.M., Immunologic localization of a- and [3-endorphins and !3-1ipotrophin in corticotrophic cells of the normal and anencephalic fetal pituitaries, Cell Tissue Res., 191 (1978) 413 422. 3 Bloom, F.E., Rossier, J., Battenbery, E.L.F., Bayon, A., French, E., Henriksen, S.J., Siggins, G.R., Segal, D., Browne, R., Ling, N. and Guillemin, R., 13-Endorphin:cellular localization, electrophysiological and behavioural effects. In E. Costa and M. Trabucchi (Eds.), Advances in Biochemical Psychopharmacology, Vol. 18, Raven Press, New York, 1978, pp. 89-109. 4 Chateau, M., Marchetti, J., Burlet, A. and Boulange, M., Evidence of vasopressin in adenohypophysis: research into its role in corticotrope activity, Neuroendoerinology, 28 (1979) 25-35. 5 Land, H., Schutz, G., Schmale, H. and Richter, D., Nucleotide sequence of cloned eDNA encoding bovine arginine vasopressin-neurophysin II precursor, Nature (London), 295 (1982) 299-303. 6 Lauber, M., Nicolas, P., Boussetla, tt.. Fahy, C., B6guin, P., Camier, M., Vaudry, H and Cohen, P., The Mr 80,000 common forms of neurophysin and vasopressin from bovine neurohypophysis have corticotroph- and [3-endorphin-likesequences and liberate by proteolysis biologically active corticotropin, Proc. Natl. Acad. Sci. USA, 78 (1981) 6086-6090. 7 Lolait, S.J., Markwick, A.J., McNally, M., Smilh. A.I. and Funder, J.W, Anterior pituitary cells l¥om
93
8 9
10
11 12
13
14
15
Brattleboro (DI/DI), Long-Evans (LE) and Sprague-Dawley (SD) rats contain immunoreactive argininc vasopressin. Ncuroendocrinology, submitted. Martin, R., Weber, E. and Voigt, K.H., Localization of corticotropin- and endorphin-related peptides in the intermediate lobe of the rat pituitary, Cell Tissue Res., 196 (1979) 307-319. Nakanishi, S., lnone, A., Kita, T., Numa, S., Chang, A.C.Y., Cohen, S.N., Nunberg, J. and Schimke, R.J., Construction of bacterial plasmids that contain nucleotide sequence for bovine corticotropin [~lipotropin precursor. Proc. Natl. Acad. Sci. USA, 75 (1978)6021-6025. Watkins. W.B., Presence of neurophysin-like material in the pituitary corticotrophs and melanotrophs and cells of the arcuate nucleus of the rat as revealed by immunocytochemistry, Neuropeptides, 3 (1983) 477 492. Watkins, W.B., Differential immunostaining of adenohypophysial cells with antisera to ACTH and [3-cndorphin, Reg. Peptides, 1, ( 1981 ) 375-385. Watkins, W.B., Moore, L.G., Rivier, J.E. and Spiess, J., Characterization of neurophysin from sheep anterior pituitary glands. Abstracts of the 7th International Congress of Endocrinology, 1984, No. 2667. Watkins, W.B., Yen, S.S.C. and Moore, R.Y., Presence of [3-endorphin-like immunoreactivity in thc anterior pituitary gland of rat and man and evidence for the differential localization with ACTH, Cell Tissue Res., 215 (1981) 577 589. Weber, F., Voigt, K.H. and Martin, R., Concomitant storage of ACTH and endorphin-like immunorcactivity in the secretory granules of anterior pituitary corticotrophs, Brain Res., 157 (1978) 385 39(/. Wilkes, M.M., Watkins, W.B., Stewart, R.D. and Yen, S.S.C., Localization and quantitation of 13endorphin in human brain and pituitary, Neuroendocrinology, 30 (1980) 113-121.