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Demonstration of the vasopressin associated glycopeptide in the brain and peripheral tissues of the Brattleboro rat
Neuropeptides7: 79-85, 1986
DEMONSTRATIONOF THE VASOPBESSINASSOCIATEDGLYCOPEPTIDEIN THE BRAIN AND PERIPHERALTISSUESOF THE BRATTLEBORORAT iva Mezey+, N.G. Seidah*,M. Chretien*,and M.J. Brownstein +Laboratoryof Cell Biology,NIMH, Bethesda,MD 20892, U.S.A. and *ClinicalResearch Instituteof Montreal,Montreal,Canada H2W 1RT (reprintrequeststo EM) ABSTRACT An antibodythat recognizes precursor
was used to visualize
the C-terminal portion of the vasopressin this peptide (CPP) in the brain and peripheral
was detectedin adrenal tissuesof the Brattlebororat. CPP-immunoreactivity medullarycells and in Leydig and Sertoli cells of the testis. In addition, CPP positivecells were found in the supraopticand paraventricular hypothalamicnuclei of colchicinetreatedand adrenalectomized rats. These resultssuggest that Brattlebororats may make a small amount of normal vasopressinprecursor. INTRODUCTION Vasopressinis synthesizedin magnocellularhypothalamicneurons and axonallytransportedto the posteriorlobe of the pituitary. There it is released into the bloodstream to act as an antidiuretic hormone. The homozygous Brattleboro rat suffers from diabetes insipidus. Because it is deficient in vasopressin, it cannot concentrate its urine and must drink very large volumes of water. Despite the fact that the brain and posterior pituitary of the Brattleboro rat have been reported to have little or no vasopressin (1,2), vasopressin has been shown to be present in its ovaries (31, adrenals (41, and sympathetic ganglia (5). Vasopressin is synthesized along with its neurophysin and a 39 amino acid glycoprotein, as part of a 20,000 MWprecursor (2). Schmale and Richter have recently cloned a vasopressin gene from one homozygous Brattleboro rat (61, and have found that a nucleotide within the second exon of the gene is deleted. This deletion would result in a frame shift so that the sequence of the entire C terminus of the vasopressin precursor would be altered. This portion of the precursor normally gives rise to the glycopeptide mentioned above (C-terminal propressophysin, CPP) (7) found in the brains and posterior pituitaries of normal (8,9,10) but not heretofore detected in Brattleboro (11) rats. Below we show that CPP and vasopressin are present in the brain and peripheral tissues of Brattleboro rats. This finding indicates that there may be a second gene encoding vasopressin in these animals and, presumably, in normal animals as well. MATERIALSANDMETHODS
Brattlebororats were bred at the NIH by di/di and di/+ matings. Homozygous rats were selected on the basis of their water intake. One group of di/di animals was untreated; a second group was administered colchicine intraventricularly (75 pg); a third underwent adrenalectomy. Rats were anaesthetized with Nembutal (4Omg/kg) and perfused through the ascending aorta
79
with a solution of 4% paraformaldehyde and 1.5% picric acid in 0.1 M phosphate The tissues were removed and postfixed in the same fixative buffer (pH 7.2). for 90 minutes at 4OC, then washed overnight in phosphate buffered saline (PBS) containing 5% sucrose. Twelve pm thick sections were cut in a cryostat, thawed onto gelatin coated slides, then incubated in 1 :I000 dilutions of one of the following antibodies (the references contain descriptions of the specificities of the antibodies used): polyclonal anti-vasopressin (121, polyclonal anti-CPP (II). The antibody solutions contained 0.6% Triton X in PBS and 10% normal goat serum. After 3 days incubation at 4OC the sections were washed and incubated with a 1:200 dilution of affinity purified FITC conjugated anti rabbit IgG (Boehringer-Mannheim) for one hour at room temperature . After rinsing, the sections were mounted in glycerol/PBS ( 1: 1) and examined under a Zeiss fluorescence microscope. For staining in the central nervous system the Avidin-Biotin (ABC) (16) method was used as described previously (17 1. Briefly, after the fixation procedure described above, 50 urn thick sections were cut with a Vibratome. The staining was carried out on floating sections; after an overnight incubation in a solution of the primary antibody at 4’C the tiSSUeS were placed in a solution of biotinylated secondary antibody (1:400) for one hour then in a solution of avidin-peroxidase complex (1:400). The peroxidase was then developed using DAB as substrate. RESULTSAND DISCUSSION In nontreated di/di rats CPP immunopositive nerve fibers were observed in the posterior lobe of the pituitary gland (Fig. IA) and in the thyroid gland (not shown). Stained cell bodies were found in the adrenal medulla (Fig. IB) and in the testis (Fig. IC) (Leydig and Sertoli cells). No staining of cell bodies or fibers was observed in the brain (Fig. 2A). Intraventricular colchicine treatment (120 ug; two days survival) or adrenalectomy (one week survival) resulted in the appearance of CPP in magnocellular (and a few parvocellular) cells in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei (Fig. 2B-F). Processes of these cells could also be seen (Fig. 2D,E,F). The staining was eliminated by absorbing the antibody with 72 pg/ml of CPP but was not affected by absorption with 100 vg/ml of neurophysin, vasopressin, or oxytocin (data not shown). Immunostaining with polyclonal antiserum against vasopressin revealed cells with a similar distribution to those seen when anti CPP was used (data not shown). Adrenalectomy has recently been reported to increase vasopressin staining in normal rats (12,13,14). Our results indicate that adrenalectomy also raises the level of the vasopressin associated glycopeptide above the threshold of detectability in Brattleboro rats. Colchicine treatment, which blocks axonal transport, also results in an increase in CPP in some brain regions. These results indicate that Brattleboro rats can make low levels of vasopressin precursor, but normally produce insufficient vasopressin to inhibit formation of urine significantly. Their vasopressin synthesis can, however, be induced by certain stimuli (e.g., adrenalectomy). Immunostaining revealed the presence of vasopressin in cells of the the sublingual gland and adrenal medulla (also reported by Nussey et al. (4)), the testis (Fig. lB,C). The staining observed in the peripheral tissues
80
examined positive
was less in di/di
intense animals.
than that
in normal S-D rats,
and fewer
cells
were
The presence of CPP in cells of the Brattleboro rat suggests one of the 1) The defect in the animals may not be the one reported by following: Schmale and Richter (6) (which would result in the replacement of CPP by an altogether different peptide incapable of being detected by an antibody raised against the CPP> . 2) Brattleboro rats may have a vasopressin gene with a single base deletion but may splice the message in such a way that the exon encoding the glycopeptide is in frame and intact (151, or 3) Brattleboro rats (and normal rats) may have more than one vasopressin gene. Recent preliminary findings of more than two rat genes which hybridize to a neurophysin probe (T. Bonner, personal cotmnunication) seem to support this hypothesis.
FIGURE LEGENDS Fig.
1.
A: Posterior pituitary of a di/di rat stained for CPP. Several immunopositive varicose nerve fibers can be seen. 11: Several adrenal medullary cells are stained by anti-CPP in the di/di rats. C: In the testis of the Brattleboro rats many interstitial (Leydig) cells are positive for CPP and some Sertoli cells within the canaliculi are positive as well. Magnification: A and C 300x; B 580x
Fig.
2.
A, B and C show coronal sections of the paraventricular hypothalamic nucleus of the Brattleboro rat. A: Untreated animal; no staining can be observed. B: Colchicine treated animal; magnocellular neurons with the same distribution as vasopressin-positive cells are stained by anti-CPP. C_: Adrenalectomized animal; cells in the same area as shown in B are stained. The staining of the cells is less intense than shown in B. A highly stained cell .body is shown. 2: CPP-positive neurons in the supraoptic nucleus of an adrenalectomized rat. Several stained cell bodies in the ventral region of the nucleus can be seen. Also some fibers entering the nucleus from dorsal direction are visible. E: high power of an iuxuunopositive neuron in the SON. 1: high power photomicrograph of PVN neurons in a colchicine treated rat. Abbreviations: III.third ventricle, ot - optic tract, son - supraoptic nucleus. Magnification: 130x (A,B,C,D); 500x (E) and 330x (F).
81
Figure 1 82
*
?* >
s
Figure 2 83
REFERENCES 1.
Sokol HW, ZimmermanEA, Sawyer WH, RobinsonAG (1976)The hypothalamic neurohypophysial system of the rat: localizationand quantitationof neurophysinby light microscopicinnnunocytochemistry in normal rats and in Brattlebororats. Endocrinology98, 1176-1188.
2.
Russell JT, BrownsteinMJ, Gainer H (1980)Biosynthesisof vasopressin, oxytocinand neurophysins: Isolationand characterization of two common precursors(propressophysin and prooxyphysin).Endocrinology107, 18801891.
3.
Lim ATW, Lolait SJ, Barlow JW, AutelitanoDJ, Toh BH, Boublik J, Abraham J, JohnstonCI, Funder JW (1984) Immunoreactiveargininevasopressinin the Brattlebororat ovary. Nature 310, 61-64.
4.
Nussey SS, Ang VTY, Jenkins JS, ChowdryHS, Bisset GW (1984)Brattleboro rat adrenalcontainsvasopressin. Nature 310, 64-69.
5.
Hanley MR, Benton HP, LightmanSL, Todd K, Bones EA, Fretten P, Palmer S, Kirk CJ, Michell RH (1984)A vasopressin-like peptide in the mammalian sympatheticnervous system. Nature 309, 258-261.
6.
SchmaleH, RichterD (1984)Single base deletionin the vasopressingene is the cause of diabetesinsipidusin Brattlebororats. Nature 308, 705709.
7.
SeidahNG, BenjanettS, ChretienM (1981)The completesequenceof a novel human pituitaryglycopepidehomologousto pig posteriorpituitary glycopeptide. Biochemicaland BiophysicalResearchCommunications100, 901-907.
8.
Watson SJ, Seidah NG, ChretienM (1982)The carboxy terminusof the in rat precursorto vasopressinand neurophysin: imnunocytochemistry brain. Science217, 853-855.
9.
Lu CL, Cantin M, Seidah NG, ChretienM (1982)Distributionpattern in the human pituitaryand hypothalamusof a new neuropeptide: the C-terminal glycoprotein-fragment of human pro-pressophysin (CPP). Histochemistry 75, 319-326.
10. Watson SJ, KhachaturianH, Seidah NC, ChretienM, ZimmermanEA, Nilaver G, WimersmaGreidanusTB (1983)Immunocytochemistry of the C-terminal peptideof propressophysin(CPP): relationshipto vasopressin,oxytocin, and neurophysin. Neuropeptides3, 321-336. 11.
Lu CL, CantinM, Seidah NG, ChretienMJ (1982)Immunohistochemical localizationof the human pituitaryglycopeptide(HPGP)-like immunoreactivity in the hypothalamusand pituitaryof normal and homozygousdiabetesinsipidus(Brattleboro)rats. J. Histochemistryand Cytochemistry30, 999-1003.
84
12.
Kiss JZ, Mezey E, Skirboll L (1984) Corticotropin-releasing factorimmunoreactive neurons of the paraventricular nucleus become vasopressin positive after adrenalectomy. Proceedings of the National Academy of Sciences USA 81, 1854-1858.
13.
Sawchenko PI, Swanson LW, Vale WW (1984) Co-expression of CRF- and vasopressin-immunoreacativityin parvocellular secretory neurons of the adrenalectomized rat. Proceedings of the National Academy of Sciences USA 81, 1883-1887.
14.
Tramu G, Croix C, Pillez A (1983) Ability of the CRF immunoreactive neurons of the paraventricular nucleus to produce a vasopressin-like material. Neuroendocrinology 37, 467-469.
15. Bonner T, Brownstein MJ (1984) Vasopressin, tissue-specific defects and the Brattleboro rat. Nature 310, 17. 16. Hsu SM, Ree HJ (1980) An improved imnunoperoxidase technique for the detection of small amounts of antigens. American Journal of Clinical Pathology 74, 32-40. 17.
Mezey E, Kiss JZ, Skirboll LR, Goldstein M, Axelrod J (1984) Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline. Nature 310, 140-141.
Accepted 23/10/85
85
DEMONSTRATIONOF THE VASOPBESSINASSOCIATEDGLYCOPEPTIDEIN THE BRAIN AND PERIPHERALTISSUESOF THE BRATTLEBORORAT iva Mezey+, N.G. Seidah*,M. Chretien*,and M.J. Brownstein +Laboratoryof Cell Biology,NIMH, Bethesda,MD 20892, U.S.A. and *ClinicalResearch Instituteof Montreal,Montreal,Canada H2W 1RT (reprintrequeststo EM) ABSTRACT An antibodythat recognizes precursor
was used to visualize
the C-terminal portion of the vasopressin this peptide (CPP) in the brain and peripheral
was detectedin adrenal tissuesof the Brattlebororat. CPP-immunoreactivity medullarycells and in Leydig and Sertoli cells of the testis. In addition, CPP positivecells were found in the supraopticand paraventricular hypothalamicnuclei of colchicinetreatedand adrenalectomized rats. These resultssuggest that Brattlebororats may make a small amount of normal vasopressinprecursor. INTRODUCTION Vasopressinis synthesizedin magnocellularhypothalamicneurons and axonallytransportedto the posteriorlobe of the pituitary. There it is released into the bloodstream to act as an antidiuretic hormone. The homozygous Brattleboro rat suffers from diabetes insipidus. Because it is deficient in vasopressin, it cannot concentrate its urine and must drink very large volumes of water. Despite the fact that the brain and posterior pituitary of the Brattleboro rat have been reported to have little or no vasopressin (1,2), vasopressin has been shown to be present in its ovaries (31, adrenals (41, and sympathetic ganglia (5). Vasopressin is synthesized along with its neurophysin and a 39 amino acid glycoprotein, as part of a 20,000 MWprecursor (2). Schmale and Richter have recently cloned a vasopressin gene from one homozygous Brattleboro rat (61, and have found that a nucleotide within the second exon of the gene is deleted. This deletion would result in a frame shift so that the sequence of the entire C terminus of the vasopressin precursor would be altered. This portion of the precursor normally gives rise to the glycopeptide mentioned above (C-terminal propressophysin, CPP) (7) found in the brains and posterior pituitaries of normal (8,9,10) but not heretofore detected in Brattleboro (11) rats. Below we show that CPP and vasopressin are present in the brain and peripheral tissues of Brattleboro rats. This finding indicates that there may be a second gene encoding vasopressin in these animals and, presumably, in normal animals as well. MATERIALSANDMETHODS
Brattlebororats were bred at the NIH by di/di and di/+ matings. Homozygous rats were selected on the basis of their water intake. One group of di/di animals was untreated; a second group was administered colchicine intraventricularly (75 pg); a third underwent adrenalectomy. Rats were anaesthetized with Nembutal (4Omg/kg) and perfused through the ascending aorta
79
with a solution of 4% paraformaldehyde and 1.5% picric acid in 0.1 M phosphate The tissues were removed and postfixed in the same fixative buffer (pH 7.2). for 90 minutes at 4OC, then washed overnight in phosphate buffered saline (PBS) containing 5% sucrose. Twelve pm thick sections were cut in a cryostat, thawed onto gelatin coated slides, then incubated in 1 :I000 dilutions of one of the following antibodies (the references contain descriptions of the specificities of the antibodies used): polyclonal anti-vasopressin (121, polyclonal anti-CPP (II). The antibody solutions contained 0.6% Triton X in PBS and 10% normal goat serum. After 3 days incubation at 4OC the sections were washed and incubated with a 1:200 dilution of affinity purified FITC conjugated anti rabbit IgG (Boehringer-Mannheim) for one hour at room temperature . After rinsing, the sections were mounted in glycerol/PBS ( 1: 1) and examined under a Zeiss fluorescence microscope. For staining in the central nervous system the Avidin-Biotin (ABC) (16) method was used as described previously (17 1. Briefly, after the fixation procedure described above, 50 urn thick sections were cut with a Vibratome. The staining was carried out on floating sections; after an overnight incubation in a solution of the primary antibody at 4’C the tiSSUeS were placed in a solution of biotinylated secondary antibody (1:400) for one hour then in a solution of avidin-peroxidase complex (1:400). The peroxidase was then developed using DAB as substrate. RESULTSAND DISCUSSION In nontreated di/di rats CPP immunopositive nerve fibers were observed in the posterior lobe of the pituitary gland (Fig. IA) and in the thyroid gland (not shown). Stained cell bodies were found in the adrenal medulla (Fig. IB) and in the testis (Fig. IC) (Leydig and Sertoli cells). No staining of cell bodies or fibers was observed in the brain (Fig. 2A). Intraventricular colchicine treatment (120 ug; two days survival) or adrenalectomy (one week survival) resulted in the appearance of CPP in magnocellular (and a few parvocellular) cells in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei (Fig. 2B-F). Processes of these cells could also be seen (Fig. 2D,E,F). The staining was eliminated by absorbing the antibody with 72 pg/ml of CPP but was not affected by absorption with 100 vg/ml of neurophysin, vasopressin, or oxytocin (data not shown). Immunostaining with polyclonal antiserum against vasopressin revealed cells with a similar distribution to those seen when anti CPP was used (data not shown). Adrenalectomy has recently been reported to increase vasopressin staining in normal rats (12,13,14). Our results indicate that adrenalectomy also raises the level of the vasopressin associated glycopeptide above the threshold of detectability in Brattleboro rats. Colchicine treatment, which blocks axonal transport, also results in an increase in CPP in some brain regions. These results indicate that Brattleboro rats can make low levels of vasopressin precursor, but normally produce insufficient vasopressin to inhibit formation of urine significantly. Their vasopressin synthesis can, however, be induced by certain stimuli (e.g., adrenalectomy). Immunostaining revealed the presence of vasopressin in cells of the the sublingual gland and adrenal medulla (also reported by Nussey et al. (4)), the testis (Fig. lB,C). The staining observed in the peripheral tissues
80
examined positive
was less in di/di
intense animals.
than that
in normal S-D rats,
and fewer
cells
were
The presence of CPP in cells of the Brattleboro rat suggests one of the 1) The defect in the animals may not be the one reported by following: Schmale and Richter (6) (which would result in the replacement of CPP by an altogether different peptide incapable of being detected by an antibody raised against the CPP> . 2) Brattleboro rats may have a vasopressin gene with a single base deletion but may splice the message in such a way that the exon encoding the glycopeptide is in frame and intact (151, or 3) Brattleboro rats (and normal rats) may have more than one vasopressin gene. Recent preliminary findings of more than two rat genes which hybridize to a neurophysin probe (T. Bonner, personal cotmnunication) seem to support this hypothesis.
FIGURE LEGENDS Fig.
1.
A: Posterior pituitary of a di/di rat stained for CPP. Several immunopositive varicose nerve fibers can be seen. 11: Several adrenal medullary cells are stained by anti-CPP in the di/di rats. C: In the testis of the Brattleboro rats many interstitial (Leydig) cells are positive for CPP and some Sertoli cells within the canaliculi are positive as well. Magnification: A and C 300x; B 580x
Fig.
2.
A, B and C show coronal sections of the paraventricular hypothalamic nucleus of the Brattleboro rat. A: Untreated animal; no staining can be observed. B: Colchicine treated animal; magnocellular neurons with the same distribution as vasopressin-positive cells are stained by anti-CPP. C_: Adrenalectomized animal; cells in the same area as shown in B are stained. The staining of the cells is less intense than shown in B. A highly stained cell .body is shown. 2: CPP-positive neurons in the supraoptic nucleus of an adrenalectomized rat. Several stained cell bodies in the ventral region of the nucleus can be seen. Also some fibers entering the nucleus from dorsal direction are visible. E: high power of an iuxuunopositive neuron in the SON. 1: high power photomicrograph of PVN neurons in a colchicine treated rat. Abbreviations: III.third ventricle, ot - optic tract, son - supraoptic nucleus. Magnification: 130x (A,B,C,D); 500x (E) and 330x (F).
81
Figure 1 82
*
?* >
s
Figure 2 83
REFERENCES 1.
Sokol HW, ZimmermanEA, Sawyer WH, RobinsonAG (1976)The hypothalamic neurohypophysial system of the rat: localizationand quantitationof neurophysinby light microscopicinnnunocytochemistry in normal rats and in Brattlebororats. Endocrinology98, 1176-1188.
2.
Russell JT, BrownsteinMJ, Gainer H (1980)Biosynthesisof vasopressin, oxytocinand neurophysins: Isolationand characterization of two common precursors(propressophysin and prooxyphysin).Endocrinology107, 18801891.
3.
Lim ATW, Lolait SJ, Barlow JW, AutelitanoDJ, Toh BH, Boublik J, Abraham J, JohnstonCI, Funder JW (1984) Immunoreactiveargininevasopressinin the Brattlebororat ovary. Nature 310, 61-64.
4.
Nussey SS, Ang VTY, Jenkins JS, ChowdryHS, Bisset GW (1984)Brattleboro rat adrenalcontainsvasopressin. Nature 310, 64-69.
5.
Hanley MR, Benton HP, LightmanSL, Todd K, Bones EA, Fretten P, Palmer S, Kirk CJ, Michell RH (1984)A vasopressin-like peptide in the mammalian sympatheticnervous system. Nature 309, 258-261.
6.
SchmaleH, RichterD (1984)Single base deletionin the vasopressingene is the cause of diabetesinsipidusin Brattlebororats. Nature 308, 705709.
7.
SeidahNG, BenjanettS, ChretienM (1981)The completesequenceof a novel human pituitaryglycopepidehomologousto pig posteriorpituitary glycopeptide. Biochemicaland BiophysicalResearchCommunications100, 901-907.
8.
Watson SJ, Seidah NG, ChretienM (1982)The carboxy terminusof the in rat precursorto vasopressinand neurophysin: imnunocytochemistry brain. Science217, 853-855.
9.
Lu CL, Cantin M, Seidah NG, ChretienM (1982)Distributionpattern in the human pituitaryand hypothalamusof a new neuropeptide: the C-terminal glycoprotein-fragment of human pro-pressophysin (CPP). Histochemistry 75, 319-326.
10. Watson SJ, KhachaturianH, Seidah NC, ChretienM, ZimmermanEA, Nilaver G, WimersmaGreidanusTB (1983)Immunocytochemistry of the C-terminal peptideof propressophysin(CPP): relationshipto vasopressin,oxytocin, and neurophysin. Neuropeptides3, 321-336. 11.
Lu CL, CantinM, Seidah NG, ChretienMJ (1982)Immunohistochemical localizationof the human pituitaryglycopeptide(HPGP)-like immunoreactivity in the hypothalamusand pituitaryof normal and homozygousdiabetesinsipidus(Brattleboro)rats. J. Histochemistryand Cytochemistry30, 999-1003.
84
12.
Kiss JZ, Mezey E, Skirboll L (1984) Corticotropin-releasing factorimmunoreactive neurons of the paraventricular nucleus become vasopressin positive after adrenalectomy. Proceedings of the National Academy of Sciences USA 81, 1854-1858.
13.
Sawchenko PI, Swanson LW, Vale WW (1984) Co-expression of CRF- and vasopressin-immunoreacativityin parvocellular secretory neurons of the adrenalectomized rat. Proceedings of the National Academy of Sciences USA 81, 1883-1887.
14.
Tramu G, Croix C, Pillez A (1983) Ability of the CRF immunoreactive neurons of the paraventricular nucleus to produce a vasopressin-like material. Neuroendocrinology 37, 467-469.
15. Bonner T, Brownstein MJ (1984) Vasopressin, tissue-specific defects and the Brattleboro rat. Nature 310, 17. 16. Hsu SM, Ree HJ (1980) An improved imnunoperoxidase technique for the detection of small amounts of antigens. American Journal of Clinical Pathology 74, 32-40. 17.
Mezey E, Kiss JZ, Skirboll LR, Goldstein M, Axelrod J (1984) Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline. Nature 310, 140-141.
Accepted 23/10/85
85