0013-7227/92/1305-3057$03.00/0
Endocrinology Copyright 0 1992 by The Endocrine Society
Cellular Protein
Vol.
Printed
Localization in the Rat*
PETER E. LOBIE, JUANITA WILLIAM R. BAUMBACH,
of the Growth
GARCIA-ARAGGN, BOSCO J. WATERS
Hormone
130, No. 5
in U.S.A.
Binding
S. WANG,
AND MICHAEL
Department of Physiology and Pharmacology (P.E.L., J.G.-A., M.J. W.), The University of Queensland, Brisbane, Queensland 4072, Australia; and the Immunology Group (B.S. W.) and Molecular and Cellular Biology Group (W.R.B.), American Cyanamid Company, Princeton, New Jersey 08540
ABSTRACT. In the rat a GH-binding protein (GHBP) exists that is derived from the GH receptor gene by an alternative messenger RNA splicing mechanism such that the transmembrane and intracellular domains of the GH receptor are replaced by a hydrophilic carboxy terminus. Previous immunohistochemical studies detailing the localization of the GH receptor binding protein (BP) have used monoclonal antibodies that recognize extracellular region-specific epitopes common to both the GH receptor and GHBP. In this study we have used a monoclonal antibody (MAb 4.3) specific for the carboxy terminus of the rat GHBP to map its somatic distribution in the rat and have compared this distribution with that of a MAb recognizing both the BP and the GH receptor. A variety of tissues including the skeletal and muscular systems, the gastrointestinal tract and derivatives, the male and female reproductive systems, skin, central and peripheral nervous systems, and the 18 day gestation fetus were investigated. The distribution of GHBP immunoreactivity (MAb 4.3) was widespread and identical to that previously- reported for the extracellular region of the GH receptor (MAbs 263 and 43). Immunoreactivity was both cytoplasmic and nuclear, indicating a possible role for the GHBP in intracellular function. GHBP
G
H BINDING activity was first detected in the serum of pregnant mice by Peeters and Friesen (1). A specific GH-binding protein (GHBP) has since been reported in rabbit, human, and rat serum (2-6). The GHBP has been shown by several methods to contain the extracellular portion of the GH receptor (3, 79), and the GHBP in the rabbit and human has been proposed to originate by specific proteolysis of the GH receptor (7, 10). However, in rodents the GHBP is produced by alternate messenger RNA splicing of the eighth exon and contains a unique hydrophilic carboxy terminus (8, 9). The extracellular hormone binding domain is therefore common to both the GH receptor and the GHBP (7-9). Previous immunohistochemical studies mapping the Received October 281991. Address requests for reprints to: Peter E. Lobie, Department of Physiology and Pharmacology, The University of Queensland, Queensland 4072, Australia. * Supported by the National Health and Medical Research Council of Australia.
immunoreactivity was predominantly associated with epithelial/ endothelial cell subtypes and with mesenchymal elements such as muscle, chondrocytes, and osteoblasts, as previously described for the GH receptor extracellular region. We also report here the distribution of the GH receptor/ GHBP in the kidney, cardiovascular, and respiratory systems. The most prominent immunoreactivity (MAbs 4.3 and 263) was associated with the distal convoluted tubules and collecting ducts of the kidney, with the epithelium and smooth muscle of the broncho-alveolar tree (including type I and II pneumocytes), with the Purkinje and myocardial fibers of the heart and with the endothelium and smooth muscle of blood vessels. Thus we have identified sites of direct GH action in the cardiovascular, renal, and respiratory systems. In conclusion, the extensive cellular distribution of the GHBP in the rat indicates physiological function(s) other than the bindina of GH in nlasma. Since GHBP mRNA has also been reported in a number of tissues, it may be that the GHBP is synthesized locally to mediate intracellular transport of GH and/ or transcriptional regulation by GH in a variety of target tissues. (Endocrinology 130:3057-3065,1992)
somatic distribution of the GH receptor/binding protein (BP) at the cellular level have used monoclonal antibodies recognizing epitopes shared by both the GH receptor and the GHBP (11-15). As such, no distinction between the location of the GH receptor and the GHBP was possible. The finding of different mRNA transcripts for both GH receptor and GHBP in various rat tissues (1618) and the possibility that the GHBP may be involved in intracellular signal transduction (19) has prompted us to examine the cellular distribution of the GHBP in the rat. We have used a GHBP specific monoclonal antibody (MAb 4.3) (20) to localize the expression of GHBP by immunohistochemical techniques in the rat. The observed widespread distribution of the GHBP indicates a physiological function other than the modulation of free GH concentrations during pulsatile secretion (21). Materials Monoclonul
antibodies
and Methods
to the GHBP and GH receptor/BP
MAb 4.3 was raisedagainst a synthetic peptide corresponding to the predicted 17amino acid hydrophilic carboxy terminus 3057
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of the rat GHBP mRNA (CGPKFNSQHPHQEIDNHI). It specifically recognizes recombinant rat GHBP produced in bacteria and immunoprecipitates GH binding activity from rat and mouse serum (20). MAb 4.3 does not recognize the rat GH receptor as determined by Western blot analysis (20). MAb 263 was raised using as immunogen purified microsomal rat and rabbit GH receptor and reacts specifically against GH receptor in immunoblots (22, 23). Under certain conditions it precipitates rat and rabbit GH receptor, but it can also compete for hormone binding to subtypes of the GH receptor. MAb 263 also precipitates GHBP from serum and cytosol (3, 24). MAbs 7 and 50.8 were used as control antibodies (25). MAb 7 recognizes a species-specific epitope on the rabbit GH receptor. MAb 50.8 is a non-cross-reactive murine MAb raised against Brucella abortus. Production of recombinant human GH receptor
rat GHBP
and truncated
(l-239)
The recombinant rat GHBP wasproduced asdescribedpreviously (20). Briefly, a hybrid complementary DNA clone encoding the entire rat GHBP, including 3’ untranslated sequence,was mutagenized to create a NdeI restriction site at nucleotide 49. The resulting plasmid (pRat 7-6m) contains an 840basepair NdeI fragment capableof encodinga protein that beginsat met 18 of the wild type rat GHBP. The 840 bp NdeI fragment from pRat 7-6 m was ligated into the unique NdeI site of the bacterial expressionvector (PET 3a) in which transcription of the insertedgeneis driven by a phageT7 promotor. The Escherichiu coli strain JM 109 (DE 3) containing this construct was grown in M9 mediumcontaining 100 pM ampicillin to an Ahg5of 1.0 and inducedby adding IPTG to 0.4 mM. The cellswere harvested, frozen, resuspendedin H20, homogenized, and sonicatedto disrupt genomicDNA. The recombinant product (in the form of inclusion bodies) was pelleted, washedtwice in H20, solubilized in Hz0 at pH 12, and ultrafiltered in 10 mM Na borate buffer (pH 9) with hollow fiber filters [lo and 100 dissociationconstant (Kd) cut off, Amicon Corp., Lexington, MA]. Chromatography wasperformed in the samebuffer on diethylaminoethyl-SepharoseCL-GB, elution at 250 mM NaCl. This material was concentrated and dialyzed againstNH40H, pH 9 before lyophilization. Final purity of rat GHBP sampleswas 87-96% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A solubletruncated (l-239) version of the human GH receptor (23) was kindly supplied by Genentech Inc (South San Francisco,CA). Both recombinant proteins wereusedin preabsorption studies to demonstrate the immunohistochemical specificity of MAb 4.3 for the GHBP. Tissue preparation
for immunohistochemistry
Adult male and female Wistar rats (fed ad libitum) were anesthetizedby an ip injection of pentobarbitone and perfused transcardially with cold PBS followed by Bouin’s solution [0.9% picric acid, 9% (vol/vol) formaldehyde, 5% acetic acid]. Various rat tissuesincluding the gastrointestinal tract, skin, male and female reproductive systems,kidney, cardiovascular and respiratory systems,skeletal tissues,and the central nervoussystemwere dissectedand postfixed in Bouin’s solution for 4 h at 4 C. Eighteen-day-old embryoswereobtained from timed matingsand immersionfixed in Bouin’s solution. Tissueswere
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Endo. 1992 Vol 130. No 5
embeddedin paraffin by standardhistologicalprocedures.Semiserial 5-pm sections were cut and collected onto gelatin/ chromic potassiumsulfate-coatedslides. Zmmunohistochemistry
Sections were deparaffinized and subjectedto immunohistochemical staining (26) according to the following procedure: 1) elimination of endogenousperoxidase activity with 0.5% HzOz in PBS for 15 min at 20 C; 2) elimination of nonspecific protein binding by incubation at 20 C with 10% normal goat serumfor 1 h; 3) incubation overnight at 4 C with mouseantiGH receptor/GHBP (MAb 4.3, MAb 7, and MAb 263 at 12.550 pg/ml) or Brucella abortus MAb at 25 pg/ml in PBS-l% BSA; 4) incubation with goat or sheepantimousebiotinylated IgG (Amersham,Arlington Heights, IL) for 2 h at 25 C (diluted 1:150 in PBS-l% BSA); 5) incubation with avidin (streptavidin)-biotin horse radish peroxidase complex for 1 h at 25 C (diluted 1:150 in PBS-l% BSA and (b) incubation with 0.05 mg/ml diaminobenzidinein PBS containing 0.01% H,O, for 5 min. Between each step sectionswere washedthree times in PBS and oncein PBS-l% BSA. All incubationswereperformed in a humidified chamber. Sections were left uncounterstained or were counterstained in Mayer’s hematoxylin, dehydrated, and mounted. Controls were performed by 1) omissionof the primary antibody or 2) replacing the anti-GH receptor mouse IgG by unrelatedprimary antibodies(Bruce& abortus and MAb 7 [specific for rabbit GH receptor/BP)] of the sameisotype (IgGKl) and at the sameor greater concentration. Zmmunohistochemical
specificity of MAb 4.3
For demonstration of the immunohistochemicalspecificity of MAb 4.3, two experiments were performed. MAb 4.3 was preincubated for 24 h at 4 C with a 10 M excessof either the recombinant rat GHBP or the recombinant truncated human GH receptor (l-239) before application to the sections. MAb 263 was also similarly preincubated with a 10 M excessof the rat GHBP or truncated human GH receptor. Immunohistochemistry wasthen performed exactly as describedabove. Results General Localization of antigen was evidenced by the deposition of dark brown diaminobenzidine reaction product in test sections. The specificity of GHBP (MAb 4.3) and GH receptor/BP (MAb 263) immunoreactivity in this study was verified by 1) absence of immunoreaction when control MAbs 7 and 50.8 were used as primary antibodies; 2) abolition of MAb 4.3 immunoreactivity by preincubation of the MAb with recombinant rat GHBP but not recombinant human GH receptor (l-239) before application to sections (Fig. 1); 3) abolition of MAb 263 immunoreactivity by preincubation of the MAb with both recombinant human GH receptor (l-239) not containing the BP carboxy terminal sequence and the recombinant rat GHBP (Fig. 1). MAb 4.3 therefore specifically recognized the GHBP and not the GH receptor in verification of previous Western blot analyses (20). The
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FIG. 1. Immunohistochemical specificity of MAb 4.3 and MM 263 for the GHBP and GH receptor/BP, respectively. Magnification bar is 100 pm. A, MAb 4.3 immunoreactivity (IR) in the developing skeletal muscle of an l&day gestation embryo. B, Lack of MAb 4.3 IR in an adjacent section to (A) after preincubation (24 h at 4 Cl of the MAb with 10 M excess of recombinant rat GHBP before application to the section. C, Retention of MAb 4.3 IR in an adjacent section to (A) after preincubation (24 h at 4 C) of the MAb with 10 M excess of soluble recombinant truncated human GH receptor before application to the section. D, MAb 263 IR in the developing skeletal muscle of an 18 day gestation embryo. E, Lack of MAb 263 IR in an adjacent section to (D) after preincubation (24 h at 4 C) of the MAb with 10 M excess of recombinant rat GHBP before application to the section. F, Lack of MAb 263 IR in an adjacent section to (E) after preincubation (24 h at 4 C) of the MAh with 10 M excess of soluble recombinant truncated human GH receptor before application to the section.
GHBP displayed both cytoplasmic and nuclear immunoreactivity (Fig. 2, A-C) in most tissue locations in the rat. Although difficult to quantify, it appeared that the immunoreactivity observed with MAb 4.3 was less intense in cytoplasm and more intense in the nucleus than with MAb 263. Tissue distribution Skeletal and muscular systems. Chondrocytes
(including hypertrophic chrondrocytes) of the epiphyseal growth plates of the humerus, femur, and tibia were immunoreactive with both MAbs 4.3 and 263. Articular and fibrocartilagenous chondrocytes (Fig. 2, D-F) were also immunoreactive with both MAbs. Osteoblasts and osteoprogenitor cells of the periosteum and osteoblasts lining the bony trabeculae (Fig. 21) displayed immunoreactivity. Osteocytes were generally immunonegative with both MAbs, but occasional osteocytes close to the medullary cavity were immunopositive. GH receptor/BP (MAb 263) and GHBP (MAb 4.3) immunoreactivity was also observed on osteoclasts (Fig. 21) (identified by morphology and their proximity to Howship’s lacunae). Megakaryocytes (Fig. 21) and hemopoietic elements were reactive with both MAbs 4.3 and 263. Skeletal muscle from different locations was also immunoreactive with both MAbs 4.3 and 263. Gastrointestinal system. All components of the gastrointestinal tract were investigated for GHBP expression in
the same manner as previously described for the GH receptor/BP (12). GHBP (MAb 4.3) immunoreactivity was observed in identical cellular locations as was MAb 4.3 or 263 reactivity in that study, and confirmed with MAb 263 in this study. The most intense immunoreactivity was associated with epithelial cells of the esophageal mucosa, chief (zymogen) (Fig. 3, A-C) and enteroendocrine cells of the stomach, surface epithelial cells of the stomach, cecum and colon, peripheral pancreatic acinar groups, cells of the islets of Langerhans, and crypt and villous columnar cells of the small intestine. MAb 4.3 immunoreactivity was also ubiquitously associated with smooth muscular elements of the gastrointestinal tract. Central and peripheral nervous systems. GHBP
immunoreactivity was observed in all cell types (neuronal, glial, and ependymal) of the embryonic and neonatal central nervous system identical to the distribution observed by MAb 263 (27). Neurons of peripheral nerve ganglia (sympathetic, parasympathetic, and dorsal root ganglion) were also immunoreactive (MAbs 4.3 and 263) as were their satellite cells and Schwann cells of the axons. Immunoreactivity in adult rat brain was weak, and an ontogenic decline in GH receptor mRNA postnatally has been observed (Lobie, P. E., et al, in preparation and Ref. 27).
Male reproductive system. The distribution
of the GHBP
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3060
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(;HBP
Fr>,l~ . I i’dl \,,,ilI.,.
L FIG. 2. Immunohistochemical localization of the (;H receptor/BP (MAh 2631 and the (;HHI’ lPvlAh 1.31 in the rat. Magnification har for micrographs A-F and H-I, and 200 pm for C. A, 5’lAh 263 immunnreactj~it\ (IR) in the adult 112 week) rat. liver. Note the promment proportion of hepatocyte nuclei (arrtxl’.s 1 and general cytciplasmic IH. H. R~lAk) J.:i IH in t hr adult rat liler. Again note that a proportion section to 1.4) and (I%. Note are immunoreactive (nrrr3wr I as well as the cyt~lplasm. C’. bl.4h 7 IR in the adult MI lix,er, in an adjacent lack of irIlrnnni)reac.tic)n In both nuclear and cytoplasmic compartments. D. MAh Xi IR in chondrvcytrs of the articular cart ilaye of the
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is 100 pm IR in a of nuclei complete proxm~nl
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(MAb 4.3) was identical to that previously described for the GH receptor/BP (MAbs 43 and 263) (13) and for MAb 263 in this study. The most prominent immunoreactivity was associated with the epithelium of the ductus epididymis, vas deferens, coagulating gland, seminal vesicle, and prostate (secretory phase), the ductular epithelium of the bulbourethral gland, and to a lesser degree with Sertoli cells and Leydig cells. Smooth muscle components of the ductus epididymis, vas deferens, seminal vesicles, and prostate were also strongly immunoreactive. Female reproductive system. The
distribution of the GHBP (MAb 4.3) in the female reproductive system was again identical to that previously described for the GH receptor/BP (13) and to MAb 263 in the present study. The most prominent immunoreactivity was associated with oocytes, the germinal epithelium of the ovary, the endothelial lining of the ovarian and uterine vasculature, the epithelial lining of the fimbriae, oviduct (Fig. 2G), and uterus, some endometrial glands, and with cells of the corpus luteum.
Kidney. GH receptor/BP
immunoreactivity has not previously been described in the kidney. All components of the nephron (proximal convoluted tubules, Loop of Henle, distal convoluted tubules, and the collecting ducts/tubules) were immunoreactive with MAbs 4.3 and 263 (Fig. 2, J-L). In both cases the most prominent immunoreactivity was associated with the distal convoluted tubule and the collecting ducts. The cells of the juxtaglomerular apparatus were immunoreactive while cells of the glomerulus were weakly immunoreactive or were immunonegative. Renal stromal elements were also weakly immunoreactive. The urothelium of the renal pelvis and its associated smooth muscle were also immunoreactive with both MAbs 4.3 and 263. Respiratory and cardiovascular systems. GH receptor/BP
immunoreactivity has not previously been described for in the respiratory and cardiovascular systems. Both MAb 263 (GH receptor/BP) and MAb 4.3 (GHBP) were immunoreactive in identical locations. The respiratory epithelium of the entire respiratory tract (nasal cavity, trachea, extrapulmonary and intrapulmonary bronchi, bronchioles, and alveolar ducts) was intensely immunoreactive (Fig. 3, D-F). The olfactory epithelium and type
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I and II pneumocytes were moderately to strongly immunoreactive. Mesenchymal elements such as cartilage and blood vessels were immunoreactive as described above. Purkinje fibers of the heart were intensely immunoreactive. Smooth muscle fibers of the atria, myocardial fibers (Fig. 3, G-I), and cells lining the epicardium and endocardium were strongly immunoreactive. Stromal elements were either weakly immunoreactive or immunonegative. Endothelial cells of blood vessels (both arterial and venous) and smooth muscle cells of the tunica media were strongly immunoreactive (Fig. 3, JL). Again stromal elements were either weakly immunoreactive or were immunonegative. Skin. The distribution
of the GHBP in skin was identical to that previously described for the GH receptor/BP (14) and to MAb 263 in this study. The most prominent immunoreactivity with both MAbs was associated with cells of the stratum basale and spinosum (Fig. 2H) and all layers of the lower third of the hair follicle including the hair matrix cells of the dermal papillae. The outer epithelial root sheath of the upper two thirds of the hair follicle was also strongly immunoreactive, as were cells of the sebaceous glands. Fibroblasts and adipocytes of the dermis were immunoreactive but to a lesser degree. 18 Day gestation embryo. The distribution
of the GHBP in the 18-day gestation rat embryo was identical to that previously described for the GH receptor/BP (26) and for MAb 263 in this study. The most prominent immunoreactivity was observed in skeletal (Fig. 1) and smooth muscle, chondroprogenitor cells, epithelial lining cells (Fig. lH), neuronal ganglia and ependymal cells, and cells of the adrenal cortex. Discussion
In this study we have exploited a MAb raised against the carboxy terminus of the rat GHBP to demonstrate a widespread distribution of the GHBP in the rat. The distribution of MAb 4.3 immunoreactivity (GHBP) is identical to that previously observed in some tissues (1 l15, 28) with MAbs 43 and 263, which recognize epitopes shared by both the GH receptor and GHBP. It is therefore possible to ascribe some, if not all, of the immunoreactivity observed in these tissues to the presence of GHBP and not the full length receptor. However, the
humerus of a 12-week-old rat. Note the heterogeneous IR of chondrocytes as they progress towards the center of the epiphysis (arrow). E, MAb 4.3 IR in the chondrocytes of the articular cartilage of the proximal humerus of a 12-week-old rat. Again note heterogeneous IR of chondrocytes. F, Lack of MAb 7 IR in chondrocytes of an adjacent section to Fig. 1, D and E. G, MAb 4.3 IR in the oviduct of an adult female rat. Note the prominent IR in the oviductal epithelium (arrow) and in smooth muscle of the wall (S). L, Lumen. H, MAb 4.3 IR in the skin of an 18-day gestation rat embryo. Note prominent IR in cells of the epidermis (E), developing hair follicle (H), and in dermal fibroblasts (arrow) as well as endothelial cells. I, MAb 4.3 IR in cells of the femoral epiphysis of an adult rat. OB, Osteoblast; OC, osteocyte; M, megakaryocyte. J, MAb 263 IR in the renal medulla of the adult rat. Note the prominent IR associated with all renal tubular subtypes. K, MAb 4.3 IR in an adjacent section to that of (J). Again note the prominent IR associated with all renal tubular subtypes. L, Lack of MAb 50.8 IR in the renal medulla in an adjacent section of Fig. 2, J and K.
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GI IBP
I
A, ’
Flc;. 3. Immunohistochemical locahzation of the GH receptor/BP (MAb 263) and the GHBP thIAb 4.31 in the rat. Magnification bar is 100 pm for micrographs A-I and 200 pm for .J-I,. A, MAb 263 immunoreactivity tIR1 in the gastric gland of an adult (12 week1 rat fundus. Note the prominent IR associated with the chief tzgmogenxl cells (cl and the heterogenous IR associated with parietal cells (PJ. B, MAh 4.3 IR in the gastric gland of an adult rat fundus. Note the prominent IR associated with the chief tzymogenic) cells (cl and the heterogeneous IR associated
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presence of both 3.9 and 1.2 kilobase Kb GH receptor/ BP mRNA (16, 18) in many of the tissues studied here suggests coexistence of both the full length GH receptor and the GHBP. Indeed, in all rat tissues expressing 1.2 kb mRNA (16, 18), immunoreactivity for GHBP was found to be present. We have previously reviewed literature regarding the roles of GH in reproductive (13) and gastrointestinal tract function (12), in skin (14) and in cartilage and bone (11). The cellular location of GH receptor/BP immunoreactivity in the kidney reported here is concordant with reports of GH receptor and GHBP mRNA in the rat and rabbit kidney (16,X3,29) and of GHBP expression in rat kidney (30) and rabbit kidney (31). The hypertensive effect of GH seen in acromegaly (32) and in the hypophysectomized rat (33) may be a result of direct action on salt resorption at the proximal tubule (34, 35) or of inappropriate stimulation of renal renin secretion (36, 37) leading to increased aldosterone secretion through elevated plasma angiotensin. The former mechanism is concordant with immunoreactivity in the proximal tubule, the latter with immunoreactivity in the juxtaglomerular apparatus. GH also increases renal resorption of phosphate (38) and hydroxylation of 25-hydroxycholecalciferol (39, 40). Moreover, at least 10% of circulating insulin-like growth factor 1 derives from the kidney (41), renal IGF-1 mRNA increases in response to GH (42), both GH and IGF-1 induce renal hypertrophy (43) and IGF-1 can influence renal plasma flow and GFR (44). Our finding of GH receptor/BP immunoreactivity in vascular smooth muscle indicates a role for GH in smooth muscle function. Indeed GH treatment increases type 1 collagen and decreases elastin in rat aorta (45) and hypophysectomized rats subjected to endothelial damage display markedly delayed intimal muscle hyperplasia (46). Moreover, Ledet (47) reported that GH antisera blocked the proliferative effect of diabetic serum on aortic smooth muscle proliferation. GHBP immunoreactivity in cardiac muscle is corroborated by the finding of 1.2 kb GHBP mRNA in rat heart (16, 18) in addition to the full length GH receptor transcript (29). It is not known whether the cardiac hypertrophy seen in acromegaly (48) is a result of increased pressure load and other mechanisms, or a more direct effect through the
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GH receptor/BP present in this tissue (see Results). There is, however, evidence that the hypertrophic effect is independent of systemic hypertension (48), implying a local effect via the cardiac GH receptor/BP. GH receptor mRNA is present in rat (29) and rabbit (49) lung. The finding of strong alveolar epithelial GHBP and GH receptor BP immunoreactivity identifies cellular location but does not define function as there are no functional correlates with this localization. Although the GHBP influences the half-life of plasma GH (21), the function of the GHBP at the cellular level is unknown. Since its distribution includes cells without ready access to serum proteins (neurons, oocytes, see Results) it appears that the GHBP possesses function(s) at the cellular/tissue level independent of GH binding in plasma. Such an interpretation is favored by the existence of GHBP mRNA in extrahepatic tissues (16-18) and their independent regulation (17). We have recently shown by radioligand, immunohistochemical, and immunogold electron microscopic techniques that the GH receptor/BP in the rabbit is associated with the chromatin fraction of the nucleus (19). Affinity labeling of the chromatin fraction with [ lz51]GH revealed a hormone binding subunit of M, 68,000. This corresponds to the approximate mol wt of the glycosylated GHBP. In that study we proposed that the GHBP may be involved in intracellular signal transduction of GH. This model was consistent with the recent characterization of a cis acting GH response element 5’ of a rat liver protease inhibitor gene (50). That the nucleus is immunoreactive with MAb 4.3 in the present study demonstrates that the alternatively spliced rat GHBP is associated with the nucleus. Although GH exerts its anabolic effects by stimulating mRNA transcription from the nucleus (51,52) it has not yet been demonstrated to exert effects on isolated nuclei. The direct action of GH-like polypeptides and/or associated molecules within the nucleus would presumably involve mechanism(s) that are distinct from conventional signal transduction mechanisms involving plasma membrane-bound receptors. Such parallel pathways may be analogous to the contrast between the direct activation of nuclear protein kinase C (53) by PRL and its influence on plasma membrane phospholipid breakdown. A number of other polypeptide hormones have been
with parietal cells (P). C, Lack of MAb 50.8 IR in the gastric glands of an adult rat fundus in an adjacent section to Fig. 3, A and B. D, MAb 263 IR in the intrapulmonary bronchiole and pulmonary venule (V) of the adult rat lung. Note the prominent IR associated with the epithelial and smooth muscle components of both structures. E, MAb 4.3 IR in the intrapulmonary bronchiole (B) and pulmonary venule (V) of the adult rat lung. Note the prominent IR associated with the epithelial and smooth muscle components as in Fig. 3D. F, Lack of MAb 7 IR in an adjacent section to Fig. 3, D and E. G, MAb 263 IR in the endocardium and myocardium of the adult rat left ventricle. Note the prominent IR associated with the myocardial fibers (M) and the endothelium (E) of the ventricular cavity. H, MAb 4.3 IR in the endocardium and myocardium of the adult rat left ventricle. Note the prominent IR associated with the myocardial fibers (M) and the endothelium as in Fig. 3G. I, Lack of MAb 50.8 IR in the endocardium and myocardium in a section adjacent to Fig. 3, G and H. J, MAb 263 IR in the thoracic aorta of the adult rat. Note the prominent IR associated with the endothelium of the tunica intima (E) and the smooth muscle (S) but not the elastin fibers (arrow) of the tunica media. L, Lumen. K, MAb 4.3 IR in the thoracic aorta of the adult rat. Note the prominent IR associated with the endothelium (E) and smooth muscle (S) as in Fig. 35. L, Lumen. L, Lack of MAb 7 IR in the thoracic aorta of the adult rat in an adjacent section of Fig. 3, J and K.
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shown to elicit actions at the nucleus concordant with nuclear localization of their receptors (54). If GH acts directly at the nucleus in association with cytosol-derived GHBP, more than one signal transduction mechanism therefore exists for GH. GH mutants that uncouple receptor binding from selected biological activities (55) may prove useful in distinguishing these mechanisms. In conclusion, we have demonstrated that the alternatively spliced GHBP of the rat has an extensive somatic distribution suggestive of a role other than modulating the availability of plasma GH to tissues. On the basis of its extensive intracytoplasmic and nuclear localization we have suggested possible involvement of GHBP in the intracellular signal transduction of GH. The proposed intracellular role of the GHBP would be independent of the modulatory role of hepatic derived plasma GHBP on GH secretory pulses and GH half-life. References 1. Peeters S, Friesen HG 1977 A growth hormone binding protein in the serum of pregnant mice. Endocrinology 101:1164-1179 2. Ymer SI, Herington AC 1985 Evidence for the specific binding of growth hormone to a receptor like protein in rabbit serum. Mol Cell Endocrinol41:153-1613 3. Barnard R, Waters MJ 1986 Serum and liver cytosolic growth hormone binding proteins are antigenically identical with liver membrane “receptor” types 1 and 2. Biochem J 237885-892 4. Herington AC, Ymer S, Stevenson J 1986 Identification and characterization of specific binding proteins in normal human sera. J Clin Invest 77:1817-1823 5. Baumann G, Stolar MW, Amburn K, Barsano CP, DeVries BC 1986 A specific growth hormone binding protein in human plasma: initial characterization. J Clin Endocrinol Metab 62:134-1416 6. Amit T, Barkey RJ, Bick T, Hertz P, Youdim MBH, Hochberg Z 1990 Identification of growth hormone binding protein in rat serum. Mol Cell Endocrinol 70:197-202 7. Spencer SA, Hammonds RG, Henzel WJ, Rodriguez H, Waters MJ, Wood WI 1988 Rabbit liver growth hormone receptor and serum binding protein: purification, characterization and sequence. J Biol Chem 263:7862-7867 8. Baumbach WR, Horner DL, Logan JS 1989 The growth hormone binding protein in rat serum is an alternatively spliced form of the growth hormone receptor. Genes Dev 3:1199-1205 9. Smith WC, Kuniyoshi J, Talamantes F 1989 Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol3:984-990 10. Trivedi B, Daughaday WH 1988 Release of growth hormone binding protein from IM-9 lymphocytes by endopeptidase is dependent on sulphydryl group inactivation. Endocrinology 123:2201-2206 11. Barnard R, Haynes KM, Werther GA, Waters MJ 1988 The ontogeny of growth hormone receptors in rabbit tibia. Endocrinology 122:2562-2569 12. Lobie PE, Breipohl W, Waters MJ 1990 Growth hormone receptor expression in the rat gastrointestinal tract. Endocrinology 126:299306 13. Lobie PE, Breipohl W, Garcia-Aragon J, Waters MJ 1990 Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems. Endocrinology 126:22142221 14. Lobie PE, Breipohl W, Lincoln DT, Garcia-Aragon J, Waters MJ 1990 Localization of the growth hormone receptor/binding protein in skin. J Endocrinol 126467-472 15. Lincoln DT, Waters MJ, Breipohl W, Sinowatz F, Lobie PE 1990 Growth hormone receptor expression in the proliferating rat mammary gland. Acta Histochem [Suppl] XL (Jena) 47-49 16. Carlsson B, Billig H, Dymo L, Isaksson OGP 1990 Expression of the growth hormone binding protein messenger RNA in the liver
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22. 23.
24. 25. 26.
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29. 30. 31. 32. 33. 34.
35. 36. 37. 38. 39.
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and extrahepatic tissues in the rat: coexpression with the growth hormone receptor. Mol Cell Endocrinol73:Rl-R6 Frick GP, Leonard JL, Goodman HM 1990 Effect of hypophysectomy on GH receptor gene expression in rat tissues. Endocrinoloav-126:3076-3082 Tiong TS, Herington AC 1991 Tissue distribution, characterization and regulation of mRNA for GH receptor and serum bindine protein-in the rat. Endocrinology 129:1628-1634 Lobie PE, Barnard R, Waters MJ 1991 The nuclear growth hormone receptor/binding protein. Antigenic and physiochemical characterization. J Biol Chem 266:22645-22652 Sadeghi H, Wang BS, Lumanglas AL, Logan JS, Baumbach WR 1991 Identification of the origin of the growth hormone binding protein in rat serum. Mol Endocrinol4:1799-1805 Baumann G, Amburn KD, Buchanan TA 1987 The effect of circulating growth hormone binding protein on metabolic clearance, distribution and degradation of human growth hormone. J Clin Endocrinol Metab 64:657-660 Barnard R, Bundesen PG, Rylatt DB, Waters MJ 1985 Evidence from the use of monoclonal antibody probes for structural heterogeneity of the growth hormone receptor. Biochem J 231:549-568 Leuna DW. Spencer SA. Cachianes G. Hammonds RG. Collins C. Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537-540 Barnard R, Quirk P, Waters MJ 1989 Characterization of the growth hormone binding protein of human serum using a panel of monoclonal antibodies. J Endocrinol 123:327-332 Barnard R, Bundesen PG, Rylatt DB, Waters MJ 1984 Monoclonal antibodies to the growth hormone receptor: production and characterization. Endocrinology 115:1805-1813 Hsu S-M, Raine L, Fanger G 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibodv (PAP) urocedures. J Histochem Cytochem 29:577-580 Lobie PE, Lincoln DT, Breipohl W, Waters MJ, Growth hormone receptor localization in the central nervous system. (Program of the 71st Annual Meeting of the Endocrine Society, 1989, Seattle, WA), p 71: (Abstract 771) Garcia-Aragon J, Lobie PE, Muscat GEC, Gobius KS, Norstedt G, Waters MJ, Prenatal expression of the growth hormone (GH) receptor/binding protein in the rat: a role for GH in embryonic and foetal development? Development, in press Mathews LS, Enberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905-9&O Bonifacino JS. Roauin LP. Paladini AC 1983 Formation of complexes between %-human or bovine somatotropins and bind& proteins in vivo in rat liver and kidney. Biochem-J 214:121-132 Herinaton AC, Stevenson JL. Ymer SI 1989 Bindine nroteins for growth hormone and prolactin in rabbit kidney c;tbsol. Am J Physiol255:E293-298 Karlberg BE, Ottosson AM 1982 Acromegaly and hypertension: role of the renin-angiotensin-aldosterone system. Acta Endocrinol (Copenh) 100:581-587 Simon CD, Honeyman TW, Fray JC 1984 Renin-angiotensin system in hypophysectomized rats. I. Control of blood pressure. Am J Physiol246:E84-E88 Ogihara T, Hata T, Maruyama A, Mikami H, Nakamaru M, Okada Y, Kumahara Y 1979 Blood pressure response to an angiotensin II antagonist in patients with acromegaly. J Clin Endocrinol Metab 48159-163 Biglieri EG, Watlington CO, Forsham PH 1961 Sodium retention with human growth hormone. J Clin Endocrinol Metab 21:361369 Honeyman TW, Goodman HM, Fray JC 1983 The effects of growth hormone on blood pressure and renin secretion in hypophysectomized rats. Endocrinology 112:1613-1617 Ho KY, Weissberger AJ 1990 The antinatriuretic action of biosynthetic human growth hormone in man involves activation of the renin-angiotensin system. Metabolism 39:133-137 Daughaday WH 1985 The anterior pituitary. In: Wilson JD, Foster DW (eds) Textbook of Endocrinology. Saunders, Philadelphia, pp 568-613 Wongsurawat N, Armbrecht HJ, Zensser TV, Forte LR, Davis BB
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40. 41. 42. 43. 44. 45. 46. 47.
LOCALIZATION
1985 Effects of hypophysectomy and growth hormone treatment on renal hydroxylation of 25-hydroxycholecalciferol in rats. J Endocrinol 101:333-338 Gray RW, Garthwaite TL 1985 Activation of renal 1,25-dihydroxyvitamin D, synthesis by phosphate deprivation: evidence for a role for growth hormone. Endocrinology 116:189-193 McConaghey P, Denhel J 1972 Preliminary studies of sulfation factor production by rat kidney. J Endocrinol 52:587-588 Murphy W, Bell GI, Duckworth ML, Friesen HG 1987 Identification, characterization and regulation of a rat cDNA which encodes insulin like growth factor-l. Endocrinology 121:684-691 Conti FG, Elliott SJ, Striker LJ, Striker GE 1989 Binding of IGFl by glomerular endothelial and epithelial cells. Biochem Biophys Res Commun 163:952-957 Hirshbere R. KODDk JD 1989 Evidence that insulin like arowth factor-l i&eases’ ;enal plasma flow and glomerular filtration rate in fasted rats. J Clin Invest 83:326-330 Bruel A, Oxlund H 1991 Biosynthetic growth hormone changes the collagen and elastin contents and biomechanical properties of the rat aorta. Acta Endocrinol (Copenh) 125:49-57 - Tie11 ML. Stemerman MB. Snaet T 1978 The influence of the pituitary on arterial intimal proliferation in the rat. Circulation Res 43:644-649 Ledet T 1977 Growth hormone antiserum suppresses the growth effect of diabetic serum. Diabetes 26:798-903
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48. Fraser R, Davies DL, Cone11 JMC 1989 Hormones and hypertension. Clin Endocrinol (Oxf) 31:701-746 49. Tiong TS, Freed KS, Herington AC 1989 Identification and tissue distribution of messenger RNA for the growth hormone receptor in the rabbit. Biochem Biophys Res Commun 158141-148 50. Yoon J-B. Bern, SA. Seelie S. Towle AC 1990 An inducible nuclear factor binds to a growth-hormone regulated gene. J Biol Chem 265:19947-19954 51. Norstedt G, Enberg B, Moller C, Mathews LS 1990 Growth hormone regulation of gene expression. Acta Paediatr Stand [Suppl] 36679-83 52. Doglio A, Dani C, Grimaldi P, Ailhaud G 1989 Growth hormone stimulates c-{OS gene expression by means of protein kinase C without increasing inositol lipid turnover. Proc Nat1 Acad Sci USA 86:1148-1152 53. Russell DH 1989 New aspects of prolactin and immunity: a lymphocyte derived prolactin like product and nuclear protein kinase C activation. Trends Pharmacol Sci 10:40-44 54. Gabriel B, Baldin V, Roman AM, Bose Bierne I, Noaillac-Depeyre J, Prats H, Teissie J, Bouche G, Amalric F 1991 Localization of peptide growth factors in the nucleus. Methods Enzymol 198:480493 55. Chen WY, Wight DC, Wagner TE, Kopchick JJ 1990 Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice. Proc Nat1 Acad Sci USA 87:5061-5065
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Endocrinology Copyright 0 1992 by The Endocrine Society
Cellular Protein
Vol.
Printed
Localization in the Rat*
PETER E. LOBIE, JUANITA WILLIAM R. BAUMBACH,
of the Growth
GARCIA-ARAGGN, BOSCO J. WATERS
Hormone
130, No. 5
in U.S.A.
Binding
S. WANG,
AND MICHAEL
Department of Physiology and Pharmacology (P.E.L., J.G.-A., M.J. W.), The University of Queensland, Brisbane, Queensland 4072, Australia; and the Immunology Group (B.S. W.) and Molecular and Cellular Biology Group (W.R.B.), American Cyanamid Company, Princeton, New Jersey 08540
ABSTRACT. In the rat a GH-binding protein (GHBP) exists that is derived from the GH receptor gene by an alternative messenger RNA splicing mechanism such that the transmembrane and intracellular domains of the GH receptor are replaced by a hydrophilic carboxy terminus. Previous immunohistochemical studies detailing the localization of the GH receptor binding protein (BP) have used monoclonal antibodies that recognize extracellular region-specific epitopes common to both the GH receptor and GHBP. In this study we have used a monoclonal antibody (MAb 4.3) specific for the carboxy terminus of the rat GHBP to map its somatic distribution in the rat and have compared this distribution with that of a MAb recognizing both the BP and the GH receptor. A variety of tissues including the skeletal and muscular systems, the gastrointestinal tract and derivatives, the male and female reproductive systems, skin, central and peripheral nervous systems, and the 18 day gestation fetus were investigated. The distribution of GHBP immunoreactivity (MAb 4.3) was widespread and identical to that previously- reported for the extracellular region of the GH receptor (MAbs 263 and 43). Immunoreactivity was both cytoplasmic and nuclear, indicating a possible role for the GHBP in intracellular function. GHBP
G
H BINDING activity was first detected in the serum of pregnant mice by Peeters and Friesen (1). A specific GH-binding protein (GHBP) has since been reported in rabbit, human, and rat serum (2-6). The GHBP has been shown by several methods to contain the extracellular portion of the GH receptor (3, 79), and the GHBP in the rabbit and human has been proposed to originate by specific proteolysis of the GH receptor (7, 10). However, in rodents the GHBP is produced by alternate messenger RNA splicing of the eighth exon and contains a unique hydrophilic carboxy terminus (8, 9). The extracellular hormone binding domain is therefore common to both the GH receptor and the GHBP (7-9). Previous immunohistochemical studies mapping the Received October 281991. Address requests for reprints to: Peter E. Lobie, Department of Physiology and Pharmacology, The University of Queensland, Queensland 4072, Australia. * Supported by the National Health and Medical Research Council of Australia.
immunoreactivity was predominantly associated with epithelial/ endothelial cell subtypes and with mesenchymal elements such as muscle, chondrocytes, and osteoblasts, as previously described for the GH receptor extracellular region. We also report here the distribution of the GH receptor/ GHBP in the kidney, cardiovascular, and respiratory systems. The most prominent immunoreactivity (MAbs 4.3 and 263) was associated with the distal convoluted tubules and collecting ducts of the kidney, with the epithelium and smooth muscle of the broncho-alveolar tree (including type I and II pneumocytes), with the Purkinje and myocardial fibers of the heart and with the endothelium and smooth muscle of blood vessels. Thus we have identified sites of direct GH action in the cardiovascular, renal, and respiratory systems. In conclusion, the extensive cellular distribution of the GHBP in the rat indicates physiological function(s) other than the bindina of GH in nlasma. Since GHBP mRNA has also been reported in a number of tissues, it may be that the GHBP is synthesized locally to mediate intracellular transport of GH and/ or transcriptional regulation by GH in a variety of target tissues. (Endocrinology 130:3057-3065,1992)
somatic distribution of the GH receptor/binding protein (BP) at the cellular level have used monoclonal antibodies recognizing epitopes shared by both the GH receptor and the GHBP (11-15). As such, no distinction between the location of the GH receptor and the GHBP was possible. The finding of different mRNA transcripts for both GH receptor and GHBP in various rat tissues (1618) and the possibility that the GHBP may be involved in intracellular signal transduction (19) has prompted us to examine the cellular distribution of the GHBP in the rat. We have used a GHBP specific monoclonal antibody (MAb 4.3) (20) to localize the expression of GHBP by immunohistochemical techniques in the rat. The observed widespread distribution of the GHBP indicates a physiological function other than the modulation of free GH concentrations during pulsatile secretion (21). Materials Monoclonul
antibodies
and Methods
to the GHBP and GH receptor/BP
MAb 4.3 was raisedagainst a synthetic peptide corresponding to the predicted 17amino acid hydrophilic carboxy terminus 3057
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of the rat GHBP mRNA (CGPKFNSQHPHQEIDNHI). It specifically recognizes recombinant rat GHBP produced in bacteria and immunoprecipitates GH binding activity from rat and mouse serum (20). MAb 4.3 does not recognize the rat GH receptor as determined by Western blot analysis (20). MAb 263 was raised using as immunogen purified microsomal rat and rabbit GH receptor and reacts specifically against GH receptor in immunoblots (22, 23). Under certain conditions it precipitates rat and rabbit GH receptor, but it can also compete for hormone binding to subtypes of the GH receptor. MAb 263 also precipitates GHBP from serum and cytosol (3, 24). MAbs 7 and 50.8 were used as control antibodies (25). MAb 7 recognizes a species-specific epitope on the rabbit GH receptor. MAb 50.8 is a non-cross-reactive murine MAb raised against Brucella abortus. Production of recombinant human GH receptor
rat GHBP
and truncated
(l-239)
The recombinant rat GHBP wasproduced asdescribedpreviously (20). Briefly, a hybrid complementary DNA clone encoding the entire rat GHBP, including 3’ untranslated sequence,was mutagenized to create a NdeI restriction site at nucleotide 49. The resulting plasmid (pRat 7-6m) contains an 840basepair NdeI fragment capableof encodinga protein that beginsat met 18 of the wild type rat GHBP. The 840 bp NdeI fragment from pRat 7-6 m was ligated into the unique NdeI site of the bacterial expressionvector (PET 3a) in which transcription of the insertedgeneis driven by a phageT7 promotor. The Escherichiu coli strain JM 109 (DE 3) containing this construct was grown in M9 mediumcontaining 100 pM ampicillin to an Ahg5of 1.0 and inducedby adding IPTG to 0.4 mM. The cellswere harvested, frozen, resuspendedin H20, homogenized, and sonicatedto disrupt genomicDNA. The recombinant product (in the form of inclusion bodies) was pelleted, washedtwice in H20, solubilized in Hz0 at pH 12, and ultrafiltered in 10 mM Na borate buffer (pH 9) with hollow fiber filters [lo and 100 dissociationconstant (Kd) cut off, Amicon Corp., Lexington, MA]. Chromatography wasperformed in the samebuffer on diethylaminoethyl-SepharoseCL-GB, elution at 250 mM NaCl. This material was concentrated and dialyzed againstNH40H, pH 9 before lyophilization. Final purity of rat GHBP sampleswas 87-96% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A solubletruncated (l-239) version of the human GH receptor (23) was kindly supplied by Genentech Inc (South San Francisco,CA). Both recombinant proteins wereusedin preabsorption studies to demonstrate the immunohistochemical specificity of MAb 4.3 for the GHBP. Tissue preparation
for immunohistochemistry
Adult male and female Wistar rats (fed ad libitum) were anesthetizedby an ip injection of pentobarbitone and perfused transcardially with cold PBS followed by Bouin’s solution [0.9% picric acid, 9% (vol/vol) formaldehyde, 5% acetic acid]. Various rat tissuesincluding the gastrointestinal tract, skin, male and female reproductive systems,kidney, cardiovascular and respiratory systems,skeletal tissues,and the central nervoussystemwere dissectedand postfixed in Bouin’s solution for 4 h at 4 C. Eighteen-day-old embryoswereobtained from timed matingsand immersionfixed in Bouin’s solution. Tissueswere
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Endo. 1992 Vol 130. No 5
embeddedin paraffin by standardhistologicalprocedures.Semiserial 5-pm sections were cut and collected onto gelatin/ chromic potassiumsulfate-coatedslides. Zmmunohistochemistry
Sections were deparaffinized and subjectedto immunohistochemical staining (26) according to the following procedure: 1) elimination of endogenousperoxidase activity with 0.5% HzOz in PBS for 15 min at 20 C; 2) elimination of nonspecific protein binding by incubation at 20 C with 10% normal goat serumfor 1 h; 3) incubation overnight at 4 C with mouseantiGH receptor/GHBP (MAb 4.3, MAb 7, and MAb 263 at 12.550 pg/ml) or Brucella abortus MAb at 25 pg/ml in PBS-l% BSA; 4) incubation with goat or sheepantimousebiotinylated IgG (Amersham,Arlington Heights, IL) for 2 h at 25 C (diluted 1:150 in PBS-l% BSA); 5) incubation with avidin (streptavidin)-biotin horse radish peroxidase complex for 1 h at 25 C (diluted 1:150 in PBS-l% BSA and (b) incubation with 0.05 mg/ml diaminobenzidinein PBS containing 0.01% H,O, for 5 min. Between each step sectionswere washedthree times in PBS and oncein PBS-l% BSA. All incubationswereperformed in a humidified chamber. Sections were left uncounterstained or were counterstained in Mayer’s hematoxylin, dehydrated, and mounted. Controls were performed by 1) omissionof the primary antibody or 2) replacing the anti-GH receptor mouse IgG by unrelatedprimary antibodies(Bruce& abortus and MAb 7 [specific for rabbit GH receptor/BP)] of the sameisotype (IgGKl) and at the sameor greater concentration. Zmmunohistochemical
specificity of MAb 4.3
For demonstration of the immunohistochemicalspecificity of MAb 4.3, two experiments were performed. MAb 4.3 was preincubated for 24 h at 4 C with a 10 M excessof either the recombinant rat GHBP or the recombinant truncated human GH receptor (l-239) before application to the sections. MAb 263 was also similarly preincubated with a 10 M excessof the rat GHBP or truncated human GH receptor. Immunohistochemistry wasthen performed exactly as describedabove. Results General Localization of antigen was evidenced by the deposition of dark brown diaminobenzidine reaction product in test sections. The specificity of GHBP (MAb 4.3) and GH receptor/BP (MAb 263) immunoreactivity in this study was verified by 1) absence of immunoreaction when control MAbs 7 and 50.8 were used as primary antibodies; 2) abolition of MAb 4.3 immunoreactivity by preincubation of the MAb with recombinant rat GHBP but not recombinant human GH receptor (l-239) before application to sections (Fig. 1); 3) abolition of MAb 263 immunoreactivity by preincubation of the MAb with both recombinant human GH receptor (l-239) not containing the BP carboxy terminal sequence and the recombinant rat GHBP (Fig. 1). MAb 4.3 therefore specifically recognized the GHBP and not the GH receptor in verification of previous Western blot analyses (20). The
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3059
FIG. 1. Immunohistochemical specificity of MAb 4.3 and MM 263 for the GHBP and GH receptor/BP, respectively. Magnification bar is 100 pm. A, MAb 4.3 immunoreactivity (IR) in the developing skeletal muscle of an l&day gestation embryo. B, Lack of MAb 4.3 IR in an adjacent section to (A) after preincubation (24 h at 4 Cl of the MAb with 10 M excess of recombinant rat GHBP before application to the section. C, Retention of MAb 4.3 IR in an adjacent section to (A) after preincubation (24 h at 4 C) of the MAb with 10 M excess of soluble recombinant truncated human GH receptor before application to the section. D, MAb 263 IR in the developing skeletal muscle of an 18 day gestation embryo. E, Lack of MAb 263 IR in an adjacent section to (D) after preincubation (24 h at 4 C) of the MAb with 10 M excess of recombinant rat GHBP before application to the section. F, Lack of MAb 263 IR in an adjacent section to (E) after preincubation (24 h at 4 C) of the MAh with 10 M excess of soluble recombinant truncated human GH receptor before application to the section.
GHBP displayed both cytoplasmic and nuclear immunoreactivity (Fig. 2, A-C) in most tissue locations in the rat. Although difficult to quantify, it appeared that the immunoreactivity observed with MAb 4.3 was less intense in cytoplasm and more intense in the nucleus than with MAb 263. Tissue distribution Skeletal and muscular systems. Chondrocytes
(including hypertrophic chrondrocytes) of the epiphyseal growth plates of the humerus, femur, and tibia were immunoreactive with both MAbs 4.3 and 263. Articular and fibrocartilagenous chondrocytes (Fig. 2, D-F) were also immunoreactive with both MAbs. Osteoblasts and osteoprogenitor cells of the periosteum and osteoblasts lining the bony trabeculae (Fig. 21) displayed immunoreactivity. Osteocytes were generally immunonegative with both MAbs, but occasional osteocytes close to the medullary cavity were immunopositive. GH receptor/BP (MAb 263) and GHBP (MAb 4.3) immunoreactivity was also observed on osteoclasts (Fig. 21) (identified by morphology and their proximity to Howship’s lacunae). Megakaryocytes (Fig. 21) and hemopoietic elements were reactive with both MAbs 4.3 and 263. Skeletal muscle from different locations was also immunoreactive with both MAbs 4.3 and 263. Gastrointestinal system. All components of the gastrointestinal tract were investigated for GHBP expression in
the same manner as previously described for the GH receptor/BP (12). GHBP (MAb 4.3) immunoreactivity was observed in identical cellular locations as was MAb 4.3 or 263 reactivity in that study, and confirmed with MAb 263 in this study. The most intense immunoreactivity was associated with epithelial cells of the esophageal mucosa, chief (zymogen) (Fig. 3, A-C) and enteroendocrine cells of the stomach, surface epithelial cells of the stomach, cecum and colon, peripheral pancreatic acinar groups, cells of the islets of Langerhans, and crypt and villous columnar cells of the small intestine. MAb 4.3 immunoreactivity was also ubiquitously associated with smooth muscular elements of the gastrointestinal tract. Central and peripheral nervous systems. GHBP
immunoreactivity was observed in all cell types (neuronal, glial, and ependymal) of the embryonic and neonatal central nervous system identical to the distribution observed by MAb 263 (27). Neurons of peripheral nerve ganglia (sympathetic, parasympathetic, and dorsal root ganglion) were also immunoreactive (MAbs 4.3 and 263) as were their satellite cells and Schwann cells of the axons. Immunoreactivity in adult rat brain was weak, and an ontogenic decline in GH receptor mRNA postnatally has been observed (Lobie, P. E., et al, in preparation and Ref. 27).
Male reproductive system. The distribution
of the GHBP
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3060
CELLULAR
LOCALIZATION
OF RAT
(;HBP
Fr>,l~ . I i’dl \,,,ilI.,.
L FIG. 2. Immunohistochemical localization of the (;H receptor/BP (MAh 2631 and the (;HHI’ lPvlAh 1.31 in the rat. Magnification har for micrographs A-F and H-I, and 200 pm for C. A, 5’lAh 263 immunnreactj~it\ (IR) in the adult 112 week) rat. liver. Note the promment proportion of hepatocyte nuclei (arrtxl’.s 1 and general cytciplasmic IH. H. R~lAk) J.:i IH in t hr adult rat liler. Again note that a proportion section to 1.4) and (I%. Note are immunoreactive (nrrr3wr I as well as the cyt~lplasm. C’. bl.4h 7 IR in the adult MI lix,er, in an adjacent lack of irIlrnnni)reac.tic)n In both nuclear and cytoplasmic compartments. D. MAh Xi IR in chondrvcytrs of the articular cart ilaye of the
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is 100 pm IR in a of nuclei complete proxm~nl
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LOCALIZATION
(MAb 4.3) was identical to that previously described for the GH receptor/BP (MAbs 43 and 263) (13) and for MAb 263 in this study. The most prominent immunoreactivity was associated with the epithelium of the ductus epididymis, vas deferens, coagulating gland, seminal vesicle, and prostate (secretory phase), the ductular epithelium of the bulbourethral gland, and to a lesser degree with Sertoli cells and Leydig cells. Smooth muscle components of the ductus epididymis, vas deferens, seminal vesicles, and prostate were also strongly immunoreactive. Female reproductive system. The
distribution of the GHBP (MAb 4.3) in the female reproductive system was again identical to that previously described for the GH receptor/BP (13) and to MAb 263 in the present study. The most prominent immunoreactivity was associated with oocytes, the germinal epithelium of the ovary, the endothelial lining of the ovarian and uterine vasculature, the epithelial lining of the fimbriae, oviduct (Fig. 2G), and uterus, some endometrial glands, and with cells of the corpus luteum.
Kidney. GH receptor/BP
immunoreactivity has not previously been described in the kidney. All components of the nephron (proximal convoluted tubules, Loop of Henle, distal convoluted tubules, and the collecting ducts/tubules) were immunoreactive with MAbs 4.3 and 263 (Fig. 2, J-L). In both cases the most prominent immunoreactivity was associated with the distal convoluted tubule and the collecting ducts. The cells of the juxtaglomerular apparatus were immunoreactive while cells of the glomerulus were weakly immunoreactive or were immunonegative. Renal stromal elements were also weakly immunoreactive. The urothelium of the renal pelvis and its associated smooth muscle were also immunoreactive with both MAbs 4.3 and 263. Respiratory and cardiovascular systems. GH receptor/BP
immunoreactivity has not previously been described for in the respiratory and cardiovascular systems. Both MAb 263 (GH receptor/BP) and MAb 4.3 (GHBP) were immunoreactive in identical locations. The respiratory epithelium of the entire respiratory tract (nasal cavity, trachea, extrapulmonary and intrapulmonary bronchi, bronchioles, and alveolar ducts) was intensely immunoreactive (Fig. 3, D-F). The olfactory epithelium and type
OF RAT
GHBP
3061
I and II pneumocytes were moderately to strongly immunoreactive. Mesenchymal elements such as cartilage and blood vessels were immunoreactive as described above. Purkinje fibers of the heart were intensely immunoreactive. Smooth muscle fibers of the atria, myocardial fibers (Fig. 3, G-I), and cells lining the epicardium and endocardium were strongly immunoreactive. Stromal elements were either weakly immunoreactive or immunonegative. Endothelial cells of blood vessels (both arterial and venous) and smooth muscle cells of the tunica media were strongly immunoreactive (Fig. 3, JL). Again stromal elements were either weakly immunoreactive or were immunonegative. Skin. The distribution
of the GHBP in skin was identical to that previously described for the GH receptor/BP (14) and to MAb 263 in this study. The most prominent immunoreactivity with both MAbs was associated with cells of the stratum basale and spinosum (Fig. 2H) and all layers of the lower third of the hair follicle including the hair matrix cells of the dermal papillae. The outer epithelial root sheath of the upper two thirds of the hair follicle was also strongly immunoreactive, as were cells of the sebaceous glands. Fibroblasts and adipocytes of the dermis were immunoreactive but to a lesser degree. 18 Day gestation embryo. The distribution
of the GHBP in the 18-day gestation rat embryo was identical to that previously described for the GH receptor/BP (26) and for MAb 263 in this study. The most prominent immunoreactivity was observed in skeletal (Fig. 1) and smooth muscle, chondroprogenitor cells, epithelial lining cells (Fig. lH), neuronal ganglia and ependymal cells, and cells of the adrenal cortex. Discussion
In this study we have exploited a MAb raised against the carboxy terminus of the rat GHBP to demonstrate a widespread distribution of the GHBP in the rat. The distribution of MAb 4.3 immunoreactivity (GHBP) is identical to that previously observed in some tissues (1 l15, 28) with MAbs 43 and 263, which recognize epitopes shared by both the GH receptor and GHBP. It is therefore possible to ascribe some, if not all, of the immunoreactivity observed in these tissues to the presence of GHBP and not the full length receptor. However, the
humerus of a 12-week-old rat. Note the heterogeneous IR of chondrocytes as they progress towards the center of the epiphysis (arrow). E, MAb 4.3 IR in the chondrocytes of the articular cartilage of the proximal humerus of a 12-week-old rat. Again note heterogeneous IR of chondrocytes. F, Lack of MAb 7 IR in chondrocytes of an adjacent section to Fig. 1, D and E. G, MAb 4.3 IR in the oviduct of an adult female rat. Note the prominent IR in the oviductal epithelium (arrow) and in smooth muscle of the wall (S). L, Lumen. H, MAb 4.3 IR in the skin of an 18-day gestation rat embryo. Note prominent IR in cells of the epidermis (E), developing hair follicle (H), and in dermal fibroblasts (arrow) as well as endothelial cells. I, MAb 4.3 IR in cells of the femoral epiphysis of an adult rat. OB, Osteoblast; OC, osteocyte; M, megakaryocyte. J, MAb 263 IR in the renal medulla of the adult rat. Note the prominent IR associated with all renal tubular subtypes. K, MAb 4.3 IR in an adjacent section to that of (J). Again note the prominent IR associated with all renal tubular subtypes. L, Lack of MAb 50.8 IR in the renal medulla in an adjacent section of Fig. 2, J and K.
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CELLULAR
LOCALIZATION
OF RAT
GI IBP
I
A, ’
Flc;. 3. Immunohistochemical locahzation of the GH receptor/BP (MAb 263) and the GHBP thIAb 4.31 in the rat. Magnification bar is 100 pm for micrographs A-I and 200 pm for .J-I,. A, MAb 263 immunoreactivity tIR1 in the gastric gland of an adult (12 week1 rat fundus. Note the prominent IR associated with the chief tzgmogenxl cells (cl and the heterogenous IR associated with parietal cells (PJ. B, MAh 4.3 IR in the gastric gland of an adult rat fundus. Note the prominent IR associated with the chief tzymogenic) cells (cl and the heterogeneous IR associated
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presence of both 3.9 and 1.2 kilobase Kb GH receptor/ BP mRNA (16, 18) in many of the tissues studied here suggests coexistence of both the full length GH receptor and the GHBP. Indeed, in all rat tissues expressing 1.2 kb mRNA (16, 18), immunoreactivity for GHBP was found to be present. We have previously reviewed literature regarding the roles of GH in reproductive (13) and gastrointestinal tract function (12), in skin (14) and in cartilage and bone (11). The cellular location of GH receptor/BP immunoreactivity in the kidney reported here is concordant with reports of GH receptor and GHBP mRNA in the rat and rabbit kidney (16,X3,29) and of GHBP expression in rat kidney (30) and rabbit kidney (31). The hypertensive effect of GH seen in acromegaly (32) and in the hypophysectomized rat (33) may be a result of direct action on salt resorption at the proximal tubule (34, 35) or of inappropriate stimulation of renal renin secretion (36, 37) leading to increased aldosterone secretion through elevated plasma angiotensin. The former mechanism is concordant with immunoreactivity in the proximal tubule, the latter with immunoreactivity in the juxtaglomerular apparatus. GH also increases renal resorption of phosphate (38) and hydroxylation of 25-hydroxycholecalciferol (39, 40). Moreover, at least 10% of circulating insulin-like growth factor 1 derives from the kidney (41), renal IGF-1 mRNA increases in response to GH (42), both GH and IGF-1 induce renal hypertrophy (43) and IGF-1 can influence renal plasma flow and GFR (44). Our finding of GH receptor/BP immunoreactivity in vascular smooth muscle indicates a role for GH in smooth muscle function. Indeed GH treatment increases type 1 collagen and decreases elastin in rat aorta (45) and hypophysectomized rats subjected to endothelial damage display markedly delayed intimal muscle hyperplasia (46). Moreover, Ledet (47) reported that GH antisera blocked the proliferative effect of diabetic serum on aortic smooth muscle proliferation. GHBP immunoreactivity in cardiac muscle is corroborated by the finding of 1.2 kb GHBP mRNA in rat heart (16, 18) in addition to the full length GH receptor transcript (29). It is not known whether the cardiac hypertrophy seen in acromegaly (48) is a result of increased pressure load and other mechanisms, or a more direct effect through the
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GH receptor/BP present in this tissue (see Results). There is, however, evidence that the hypertrophic effect is independent of systemic hypertension (48), implying a local effect via the cardiac GH receptor/BP. GH receptor mRNA is present in rat (29) and rabbit (49) lung. The finding of strong alveolar epithelial GHBP and GH receptor BP immunoreactivity identifies cellular location but does not define function as there are no functional correlates with this localization. Although the GHBP influences the half-life of plasma GH (21), the function of the GHBP at the cellular level is unknown. Since its distribution includes cells without ready access to serum proteins (neurons, oocytes, see Results) it appears that the GHBP possesses function(s) at the cellular/tissue level independent of GH binding in plasma. Such an interpretation is favored by the existence of GHBP mRNA in extrahepatic tissues (16-18) and their independent regulation (17). We have recently shown by radioligand, immunohistochemical, and immunogold electron microscopic techniques that the GH receptor/BP in the rabbit is associated with the chromatin fraction of the nucleus (19). Affinity labeling of the chromatin fraction with [ lz51]GH revealed a hormone binding subunit of M, 68,000. This corresponds to the approximate mol wt of the glycosylated GHBP. In that study we proposed that the GHBP may be involved in intracellular signal transduction of GH. This model was consistent with the recent characterization of a cis acting GH response element 5’ of a rat liver protease inhibitor gene (50). That the nucleus is immunoreactive with MAb 4.3 in the present study demonstrates that the alternatively spliced rat GHBP is associated with the nucleus. Although GH exerts its anabolic effects by stimulating mRNA transcription from the nucleus (51,52) it has not yet been demonstrated to exert effects on isolated nuclei. The direct action of GH-like polypeptides and/or associated molecules within the nucleus would presumably involve mechanism(s) that are distinct from conventional signal transduction mechanisms involving plasma membrane-bound receptors. Such parallel pathways may be analogous to the contrast between the direct activation of nuclear protein kinase C (53) by PRL and its influence on plasma membrane phospholipid breakdown. A number of other polypeptide hormones have been
with parietal cells (P). C, Lack of MAb 50.8 IR in the gastric glands of an adult rat fundus in an adjacent section to Fig. 3, A and B. D, MAb 263 IR in the intrapulmonary bronchiole and pulmonary venule (V) of the adult rat lung. Note the prominent IR associated with the epithelial and smooth muscle components of both structures. E, MAb 4.3 IR in the intrapulmonary bronchiole (B) and pulmonary venule (V) of the adult rat lung. Note the prominent IR associated with the epithelial and smooth muscle components as in Fig. 3D. F, Lack of MAb 7 IR in an adjacent section to Fig. 3, D and E. G, MAb 263 IR in the endocardium and myocardium of the adult rat left ventricle. Note the prominent IR associated with the myocardial fibers (M) and the endothelium (E) of the ventricular cavity. H, MAb 4.3 IR in the endocardium and myocardium of the adult rat left ventricle. Note the prominent IR associated with the myocardial fibers (M) and the endothelium as in Fig. 3G. I, Lack of MAb 50.8 IR in the endocardium and myocardium in a section adjacent to Fig. 3, G and H. J, MAb 263 IR in the thoracic aorta of the adult rat. Note the prominent IR associated with the endothelium of the tunica intima (E) and the smooth muscle (S) but not the elastin fibers (arrow) of the tunica media. L, Lumen. K, MAb 4.3 IR in the thoracic aorta of the adult rat. Note the prominent IR associated with the endothelium (E) and smooth muscle (S) as in Fig. 35. L, Lumen. L, Lack of MAb 7 IR in the thoracic aorta of the adult rat in an adjacent section of Fig. 3, J and K.
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shown to elicit actions at the nucleus concordant with nuclear localization of their receptors (54). If GH acts directly at the nucleus in association with cytosol-derived GHBP, more than one signal transduction mechanism therefore exists for GH. GH mutants that uncouple receptor binding from selected biological activities (55) may prove useful in distinguishing these mechanisms. In conclusion, we have demonstrated that the alternatively spliced GHBP of the rat has an extensive somatic distribution suggestive of a role other than modulating the availability of plasma GH to tissues. On the basis of its extensive intracytoplasmic and nuclear localization we have suggested possible involvement of GHBP in the intracellular signal transduction of GH. The proposed intracellular role of the GHBP would be independent of the modulatory role of hepatic derived plasma GHBP on GH secretory pulses and GH half-life. References 1. Peeters S, Friesen HG 1977 A growth hormone binding protein in the serum of pregnant mice. Endocrinology 101:1164-1179 2. Ymer SI, Herington AC 1985 Evidence for the specific binding of growth hormone to a receptor like protein in rabbit serum. Mol Cell Endocrinol41:153-1613 3. Barnard R, Waters MJ 1986 Serum and liver cytosolic growth hormone binding proteins are antigenically identical with liver membrane “receptor” types 1 and 2. Biochem J 237885-892 4. Herington AC, Ymer S, Stevenson J 1986 Identification and characterization of specific binding proteins in normal human sera. J Clin Invest 77:1817-1823 5. Baumann G, Stolar MW, Amburn K, Barsano CP, DeVries BC 1986 A specific growth hormone binding protein in human plasma: initial characterization. J Clin Endocrinol Metab 62:134-1416 6. Amit T, Barkey RJ, Bick T, Hertz P, Youdim MBH, Hochberg Z 1990 Identification of growth hormone binding protein in rat serum. Mol Cell Endocrinol 70:197-202 7. Spencer SA, Hammonds RG, Henzel WJ, Rodriguez H, Waters MJ, Wood WI 1988 Rabbit liver growth hormone receptor and serum binding protein: purification, characterization and sequence. J Biol Chem 263:7862-7867 8. Baumbach WR, Horner DL, Logan JS 1989 The growth hormone binding protein in rat serum is an alternatively spliced form of the growth hormone receptor. Genes Dev 3:1199-1205 9. Smith WC, Kuniyoshi J, Talamantes F 1989 Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol3:984-990 10. Trivedi B, Daughaday WH 1988 Release of growth hormone binding protein from IM-9 lymphocytes by endopeptidase is dependent on sulphydryl group inactivation. Endocrinology 123:2201-2206 11. Barnard R, Haynes KM, Werther GA, Waters MJ 1988 The ontogeny of growth hormone receptors in rabbit tibia. Endocrinology 122:2562-2569 12. Lobie PE, Breipohl W, Waters MJ 1990 Growth hormone receptor expression in the rat gastrointestinal tract. Endocrinology 126:299306 13. Lobie PE, Breipohl W, Garcia-Aragon J, Waters MJ 1990 Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems. Endocrinology 126:22142221 14. Lobie PE, Breipohl W, Lincoln DT, Garcia-Aragon J, Waters MJ 1990 Localization of the growth hormone receptor/binding protein in skin. J Endocrinol 126467-472 15. Lincoln DT, Waters MJ, Breipohl W, Sinowatz F, Lobie PE 1990 Growth hormone receptor expression in the proliferating rat mammary gland. Acta Histochem [Suppl] XL (Jena) 47-49 16. Carlsson B, Billig H, Dymo L, Isaksson OGP 1990 Expression of the growth hormone binding protein messenger RNA in the liver
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22. 23.
24. 25. 26.
27.
28.
29. 30. 31. 32. 33. 34.
35. 36. 37. 38. 39.
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and extrahepatic tissues in the rat: coexpression with the growth hormone receptor. Mol Cell Endocrinol73:Rl-R6 Frick GP, Leonard JL, Goodman HM 1990 Effect of hypophysectomy on GH receptor gene expression in rat tissues. Endocrinoloav-126:3076-3082 Tiong TS, Herington AC 1991 Tissue distribution, characterization and regulation of mRNA for GH receptor and serum bindine protein-in the rat. Endocrinology 129:1628-1634 Lobie PE, Barnard R, Waters MJ 1991 The nuclear growth hormone receptor/binding protein. Antigenic and physiochemical characterization. J Biol Chem 266:22645-22652 Sadeghi H, Wang BS, Lumanglas AL, Logan JS, Baumbach WR 1991 Identification of the origin of the growth hormone binding protein in rat serum. Mol Endocrinol4:1799-1805 Baumann G, Amburn KD, Buchanan TA 1987 The effect of circulating growth hormone binding protein on metabolic clearance, distribution and degradation of human growth hormone. J Clin Endocrinol Metab 64:657-660 Barnard R, Bundesen PG, Rylatt DB, Waters MJ 1985 Evidence from the use of monoclonal antibody probes for structural heterogeneity of the growth hormone receptor. Biochem J 231:549-568 Leuna DW. Spencer SA. Cachianes G. Hammonds RG. Collins C. Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537-540 Barnard R, Quirk P, Waters MJ 1989 Characterization of the growth hormone binding protein of human serum using a panel of monoclonal antibodies. J Endocrinol 123:327-332 Barnard R, Bundesen PG, Rylatt DB, Waters MJ 1984 Monoclonal antibodies to the growth hormone receptor: production and characterization. Endocrinology 115:1805-1813 Hsu S-M, Raine L, Fanger G 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibodv (PAP) urocedures. J Histochem Cytochem 29:577-580 Lobie PE, Lincoln DT, Breipohl W, Waters MJ, Growth hormone receptor localization in the central nervous system. (Program of the 71st Annual Meeting of the Endocrine Society, 1989, Seattle, WA), p 71: (Abstract 771) Garcia-Aragon J, Lobie PE, Muscat GEC, Gobius KS, Norstedt G, Waters MJ, Prenatal expression of the growth hormone (GH) receptor/binding protein in the rat: a role for GH in embryonic and foetal development? Development, in press Mathews LS, Enberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905-9&O Bonifacino JS. Roauin LP. Paladini AC 1983 Formation of complexes between %-human or bovine somatotropins and bind& proteins in vivo in rat liver and kidney. Biochem-J 214:121-132 Herinaton AC, Stevenson JL. Ymer SI 1989 Bindine nroteins for growth hormone and prolactin in rabbit kidney c;tbsol. Am J Physiol255:E293-298 Karlberg BE, Ottosson AM 1982 Acromegaly and hypertension: role of the renin-angiotensin-aldosterone system. Acta Endocrinol (Copenh) 100:581-587 Simon CD, Honeyman TW, Fray JC 1984 Renin-angiotensin system in hypophysectomized rats. I. Control of blood pressure. Am J Physiol246:E84-E88 Ogihara T, Hata T, Maruyama A, Mikami H, Nakamaru M, Okada Y, Kumahara Y 1979 Blood pressure response to an angiotensin II antagonist in patients with acromegaly. J Clin Endocrinol Metab 48159-163 Biglieri EG, Watlington CO, Forsham PH 1961 Sodium retention with human growth hormone. J Clin Endocrinol Metab 21:361369 Honeyman TW, Goodman HM, Fray JC 1983 The effects of growth hormone on blood pressure and renin secretion in hypophysectomized rats. Endocrinology 112:1613-1617 Ho KY, Weissberger AJ 1990 The antinatriuretic action of biosynthetic human growth hormone in man involves activation of the renin-angiotensin system. Metabolism 39:133-137 Daughaday WH 1985 The anterior pituitary. In: Wilson JD, Foster DW (eds) Textbook of Endocrinology. Saunders, Philadelphia, pp 568-613 Wongsurawat N, Armbrecht HJ, Zensser TV, Forte LR, Davis BB
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40. 41. 42. 43. 44. 45. 46. 47.
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1985 Effects of hypophysectomy and growth hormone treatment on renal hydroxylation of 25-hydroxycholecalciferol in rats. J Endocrinol 101:333-338 Gray RW, Garthwaite TL 1985 Activation of renal 1,25-dihydroxyvitamin D, synthesis by phosphate deprivation: evidence for a role for growth hormone. Endocrinology 116:189-193 McConaghey P, Denhel J 1972 Preliminary studies of sulfation factor production by rat kidney. J Endocrinol 52:587-588 Murphy W, Bell GI, Duckworth ML, Friesen HG 1987 Identification, characterization and regulation of a rat cDNA which encodes insulin like growth factor-l. Endocrinology 121:684-691 Conti FG, Elliott SJ, Striker LJ, Striker GE 1989 Binding of IGFl by glomerular endothelial and epithelial cells. Biochem Biophys Res Commun 163:952-957 Hirshbere R. KODDk JD 1989 Evidence that insulin like arowth factor-l i&eases’ ;enal plasma flow and glomerular filtration rate in fasted rats. J Clin Invest 83:326-330 Bruel A, Oxlund H 1991 Biosynthetic growth hormone changes the collagen and elastin contents and biomechanical properties of the rat aorta. Acta Endocrinol (Copenh) 125:49-57 - Tie11 ML. Stemerman MB. Snaet T 1978 The influence of the pituitary on arterial intimal proliferation in the rat. Circulation Res 43:644-649 Ledet T 1977 Growth hormone antiserum suppresses the growth effect of diabetic serum. Diabetes 26:798-903
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48. Fraser R, Davies DL, Cone11 JMC 1989 Hormones and hypertension. Clin Endocrinol (Oxf) 31:701-746 49. Tiong TS, Freed KS, Herington AC 1989 Identification and tissue distribution of messenger RNA for the growth hormone receptor in the rabbit. Biochem Biophys Res Commun 158141-148 50. Yoon J-B. Bern, SA. Seelie S. Towle AC 1990 An inducible nuclear factor binds to a growth-hormone regulated gene. J Biol Chem 265:19947-19954 51. Norstedt G, Enberg B, Moller C, Mathews LS 1990 Growth hormone regulation of gene expression. Acta Paediatr Stand [Suppl] 36679-83 52. Doglio A, Dani C, Grimaldi P, Ailhaud G 1989 Growth hormone stimulates c-{OS gene expression by means of protein kinase C without increasing inositol lipid turnover. Proc Nat1 Acad Sci USA 86:1148-1152 53. Russell DH 1989 New aspects of prolactin and immunity: a lymphocyte derived prolactin like product and nuclear protein kinase C activation. Trends Pharmacol Sci 10:40-44 54. Gabriel B, Baldin V, Roman AM, Bose Bierne I, Noaillac-Depeyre J, Prats H, Teissie J, Bouche G, Amalric F 1991 Localization of peptide growth factors in the nucleus. Methods Enzymol 198:480493 55. Chen WY, Wight DC, Wagner TE, Kopchick JJ 1990 Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice. Proc Nat1 Acad Sci USA 87:5061-5065
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