Peptides, Vol. 11, pp. 729-736. © Pergamon Press plc, 1990. Printed in the U.S.A.
0196-9781/90 $3.00 + .00
Subsets of Pituitary Intermediate Lobe Cells Bind CRH and Secrete ACTH/CLIP in a Reverse Hemolytic Plaque Assay G W E N V. C H I L D S
Department o f Anatomy and Neurosciences, University o f Texas Medical Branch 200 University Blvd., Room 731, Galveston, TX 77551
R e c e i v e d 4 April 1990
CHILDS, G. V. Subsets of pituitary intermediate lobe cells bind CRH and secrete ACTH/CLIP in a reverse hemolyticplaque assay. PEPTIDES 11(4) 729-736, 1990.--Dissociated neurointermediate lobe cells (75%) bound [biotinyl-Ser~]corticotropin releasing hormone (bio-CRH) and secreted adrenocorticotropin [ACTH(25-39)] in a reverse hemolytic plaque assay (RHPA). The bio-CRH was rapidly internalized (< 1 min) in vesicles and scattered vacuoles. Plaque percentages increased from 26 ± 4% to 53 + 8% after CRH stimulation. Areas increased from 1688 ± 791 ~m 2 to 3773 _+788 Ixm2. After AVP treatment for 1 hr, plaque percentages and areas were augmented further. The heterogeneity in secretion may indicate the presence of quiescent IL cells or cells secreting an opiocortin peptide not detected in the plaque assay. Corticotropin releasing hormone
Neurointermediate lobe cells
THE intermediate lobe (IL) of the rat pituitary synthesizes proopiomelanocortin (POMC) and cleaves this glycoprotein into bioactive peptides including a-melanocyte stimulating hormone (a-MSH), [3-endorphin, [3-1ipotropin and adrenocorticotropin (ACTH) (12,26). ACTH is acetylated and amidated and contains the sequence of o~-MSH in its N-terminal (amino acids 1-13). It contains corticotropin-like intermediate lobe peptide (CLIP) in the C-terminal (amino acids 17-39). It can be induced to respond to glucocorticoids (2,3). Early studies reported that a relatively low percentage of biologically active ACTH comes from the intermediate lobe of rats (13, 18, 25). It is considered to be the precursor for c~-MSH and CLIP (12, 13, 18, 25, 26). Synthesis of POMC and secretion of opiocortin peptides from the intermediate lobe are stimulated by corticotropin releasing hormone (CRH) (19,21) and inhibited by dopamine (4,15). Recently, Saland et al. (22) reported striking changes in the amount of immunolabeling for a - M S H following stimulation by CRH or serotonin, or inhibition by dopamine. The CRH-treated lobes showed a heterogeneous labeling pattern as if some cells had secreted more product than others. Recent studies by Chronwall et al. (8) also demonstrated heterogeneity in expression of POMC mRNA/IL cell following 6 hr of haloperidol treatment (a dopamine antagonist). In unstimulated ILs, the labeling pattern was heterogeneous. Some cells exhibited a lower rate of synthetic activity. More recently, Chronwall et al. (9) have correlated an increase in the proportion of dark cells rich in rough endoplasmic reticulum with an increase in the labeling for POMC mRNA. These workers concluded that
Adrenocorticotropin
Plaque assay
IL cells may normally be in different stages of the secretory cycle and that the light to dark cell transition may result from stimulation of protein synthesis. DeSouza and his colleagues have assayed CRH receptors in the IL by RRA (10,11). Gregoriadis and DeSouza recently characterized the binding affinity and molecular weight of the CRH binding protein. They reported that it was identical to that assayed in the anterior lobe. Autoradiographs showed that CRH binding sites were diffusely distributed in the intermediate lobe (11,14). In 1986, our laboratory reported electron microscopic cytochemical studies of the binding and internalization of a biotinylated analog of CRH by anterior lobe corticotropes (6). The analog had biotin attached to the N-terminal serine during synthesis and retained all biological activity (6). When bio-CRH and avidin fluorescein protocols were applied to a population of living dissociated neurointermediate lobe (NIL) cells, only a subpopulation of cells was labeled (less than 50%). Some of this might reflect the fact that NIL cells are diluted by epithelial cells, endothelial cells and the glial cells from the pars nervosa. Alternatively, this could reflect rapid uptake and degradation of the CRH by the IL cells. Therefore, further studies of binding and processing of CRH by IL ceils were initiated. At the same time, a reverse hemolytic plaque assay for ACTH had been developed with antisera to the C-terminal amino acids 25-39 (5) and applied to dissociated anterior lobe cells. The heterogeneous responses in intermediate lobe cells reported by Saland et al. (22) and Chronwall et al. (8,9) prompted a study of secretion from individual intermediate lobe cells with this assay.
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CHILDS
FIG. 1. Dark field view showing label for biotinylated CRH (3-min exposure) in intermediate lobe cells, 1 hr after dispersion and plating. Label glows bright on the cells or cellular processes. Examples of label are noted by +. Magnification x 546.
These studies demonstrated that a subset of IL cells internalized and processed CRH more rapidly than did cells of the anterior lobe. Furthermore, a subset of IL cells secretes ACTH/CLIP in the RHPA. However, the numbers of secreting cells increase after CRH stimulation. This report also describes the effect of arginine vasopressin (AVP) and angiotensin II on secretion from individual IL cells. METHOD Male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Houston, TX) were used to supply the pituitaries. They had been acclimated for 10 days on a 14-hr day, 10-hr night cycle and given food and water ad lib. They were anesthetized with sodium pentobarbitol and then killed in a separate room by decapitation. The pituitaries were removed rapidly. The neurointermediate lobes (NIL) were separated from the anterior lobes and dissociated according to the technique of Wilfinger et al. (28). The cells were plated 3,000-5,000/well on coverslips in 24-well trays for 1-2 days. They were grown in Eagles Minimum Essential Medium with 10% fetal bovine serum, 0.6% HEPES, and 0.3% bovine serum albumin (BSA) as described previously (5,27). Parallel sets of NIL cells (500,000/tube) were left in suspension for electron microscopic analysis of CRH processing (6). Parallel cultures were washed with MEM and exposed to either
biotinylated CRH for cytochemical labeling (6,27) or the RHPA (19). The biotinylated CRH was diluted in MEM containing 0.6% HEPES, 0.3% BSA, 10 4 M ascorbic acid, and aprotinin (100 KIU). It was added to the cells in dilutions of 0.5-1 nM. [These are saturating concentrations. Higher amounts produce no increase in labeling (6,27).] Exposure times ranged from 1-30 rain. For the light microscopic studies, the cells were fixed in 2% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4) at the end of the treatment period. Fixation continued for 30 rain after which the ceils were washed 4 × in phosphate buffer (0.1 M pH 7.4) containing 4.5% sucrose. The biotinylated CRH was then detected with the avidin-biotin-peroxidase complex (ABC) technique (Vector Laboratories, Burlingame, CA) and nickel intensified diaminobenzidine substrate (black) (DAB) as described previously (6,27). Some cultures were then immunolabeled for ACTH with the contrasting amber DAB substrate (6). Antisera to ACTH(1739) was produced in this laboratory and used at 1:20,000-1:30,000 dilutions. Controls testing specificity of reactions for bio-CRH or ACTH were repeated for NIL cultures as reported previously (6,27). In addition, as described in more recent studies (7,23), anti-ACYH(17-39) or ACTH(25-39) was tested on various opiocortin peptide antigens blotted on Immobilon nylon filter paper. Among the antigens tested (16K fragment, 13-endorphin, [3lipotropin, and ACTH), only ACTH showed a reaction. This has
FACING PAGE FIG. 2. After 1 min of exposure, the label for biotinylated CRH is on plasma membranes in patches (arrowhead). Label is also inside the cells, in structures that resemble a lysosome (a). (b) Illustrates an overview of another cell showing labeling in vesicles (v) or receptasomes after 1 min of exposure. Nuclei (N) and mitochondria (M) remain unlabeled. Magnification × 86,625 (a); 68,750 (b).
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CHILDS
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3 FIG. 3. This field illustrates the types of structures that are labeled during the first 5 min of exposure. These include small vesicles (v), or membranes of secretory vesicles or vacuoles (sv) or lysosomes (L). Some contain dense cores. Label is also on the plasma membrane (arrows). Magnification x 65,312.
confirmed many tests of this antisera over the past 18 years. Extensive tests of specificity of the same antisera in the plaque assay are described in (5). NIL cultures were washed after 1 day in MEM and then exposed to the reverse hemolytic plaque assay for ACTH as described in previous reports (5). The antisera to ACTH was diluted 1:100-1:200 as in the previous studies of anterior lobe cells (5). The complement was diluted 1:20-1:50. The RHPA was then run for 4 hr in the presence of 0 - 5 0 nM CRH. Only plaques
containing a single IL cell were analyzed in the area measurements. The bio-CRH-labeled cells or plaques were counted and expressed as a percentage of the total cell population counted. A minimum of 100 cells/coverslip were analyzed. Each experimental group contained 3-4 coverslips and each experiment was run at least 2 times. Plaque areas were measured with the Bioquant image analysis system (R & M Biometrics, Nashville, TN) as described in previous studies (5). The plaques were projected on
INTERMEDIATE LOBE CELLS BIND CRH
733
FIG. 4. This field shows a higher magnification of label for bio-CRH on membranes of secretory vesicles (arrows), or small vesicles (V) (15-min exposure to Bio-CRH). Magnification × 82,293.
the video screen and drawn with a cursor. Areas were calculated automatically by the computer software. As described previously (5), one can comfortably quantify a set of 12-14 coverslips in one period (to avoid extensive time lapse between coverslips). This will allow counts of 100 cells and measurements of 20 plaque areas/coverslip × 2 coverslips/group. The experimental groups were repeated 5-21 times to obtain adequate sampling. The data from all experiments (cell percentages or areas) were then averaged to provide the final data points. ANOVA followed by Duncan's multiple range test at the 5% level determined significant differences between groups. For the electron microscopic studies, the cells were left in suspension and exposed to bio-CRH for 1-30 min. They were fixed by adding an equal volume of 4% glutaraldehyde diluted in phosphate buffer (pH 7.4; 0.05 M). The protocol for fixation and labeling with ABC was identical to that described in the previous EM study of anterior lobe corticotropes (6). The cell pellets were embedded in Araldite 6005, sectioned and then the ultrathin sections were examined with a Zeiss 10 electron microscope after counterstaining with lead citrate and uranyl acetate. The EM studies were run 3 times. Electron micrographs of labeled intermediate lobe cells were evaluated as described in previous studies (6). The site(s) of the
label were identified in 50 cell profiles/experimental group. This analysis detected changes in the distribution of label with different times of exposure to the bio-CRH. RESULTS
Binding, by' Biotinylated CRH (bio-CRH) The ABC protocol detected label for biotinylated CRH on 5 8 _+ 14% of NIL cells after 1 min of exposure, and 75_+ 11% of NIL cells after 3 min (values _+ sd, n = 3 cultures). This was not a significant increase. The percentage of labeled cells did not increase with higher concentrations of bio-CRH (>1 nM). Percentages were 61 -+6% after 10 min of exposure. The label for bio-CRH was in patches or on processes (Fig. 1). Dual labeling for bio-CRH and ACTH showed that, after 3 min of exposure, 65_+ 20% of bio-CRH-labeled NIL cells also exhibited label for ACTH. Interestingly, 34-+ 19% exhibited label only for bio-CRH as if the CRH had stimulated secretion of all detectable ACTH stores. Less than 2% of labeled NIL cells showed label for ACTH and not bio-CRH. Controls demonstrated the specificity of labeling. The percentage of cells labeled for 0.5 nM bio-CRH was not affected by the
734
CHILDS
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FIG. 5. Analysis of changes in labeling in each type of organelle with time of exposure to biotinylated CRH. The percentage of labeled sites on the membrane (mem) and in small vesicles (ves) decreases after 1 min. There is an increase in the labeling of receptasomes (rec), lysosomes (lys), and secretory granules (gran) or vesicles. Structures associated with the Golgi complex (gol) are seldom labeled.
addition of 100 nM AVP, somatostatin, or angiotensin II. Labeling was reduced uniformly in the presence of 1000 x unlabeled CRH (exposure time 3 rain). A 100-fold excess of CRH resulted in labeling of 41-+ 14% of the cells. A 1000-fold excess of CRH blocked binding by bio-CRH so that only 3 -+ 1% of the cells showed label. The reduction was significant, F(2,18) = 37.94. The excess CRH stimulated secretion of stores so that most IL cells did not show label for ACTH. The EM studies showed rapid internalization of the bio-CRH. In 1 rain, the label was distributed in patches on the plasma membrane (Fig. 2a and b). Evidence for internalization was also seen as early as 1 min. During the first 5 min, label was found in small vesicles, structures that resembled receptasomes, and on membranes of secretory vesicles or dense core granules (Fig. 3). This labeling pattern persisted at later times of exposure. In addition, the labeling over membranes of secretory vacuoles or granules became more prominent (Fig. 4). Label was rarely found in the Golgi complex or vacuoles in that region. Figure 5 summarizes the changes in distribution of label in the different organelles with time of exposure to the bio-CRH.
Secretion of ACTH (CLIP) Previous LM and EM studies had demonstrated heterogeneity in expression of POMC mRNA (8,9) or a - M S H storage after stimulation (22). Therefore, the RHPA was used to detect any heterogeneity in areas of plaques formed by IL cells. In untreated cultures, 26 + 24% ( - sd) of the NIL cells formed plaques. In the presence of 1 nM CRH the percentage was 36 -+ 9, which was not different from untreated cells. In the presence of 10 nM CRH values increased to 53 -+ 7, which was significantly different from all other values, F ( 3 , 4 4 ) = 12.69. No further increase was seen with higher concentrations of CRH. When plaque areas were measured, they averaged 1 6 8 8+- 791 Fxm2 in unstimulated cultures. The range of areas was from 892-2852 p.m 2, which confirms studies by Saland et al. (22) who demonstrated heterogeneity in expression of MSH secretory function. In the presence of 0.5 nM CRH average areas increased to 2718-+332 ixm2 (range 1798-4883 txm2). A further increase to 3773 ± 6 3 3 was seen following exposure to l nM (range 1892-
6551 p.m2). These increases were significantly different from basal areas, F ( 4 , 8 5 ) = 8 . 3 7 . Figure 6 illustrates ACTH/CLIP secreting plaques in NIL cultures. In a separate group of experiments, 10 nM arginine vasopressin (AVP) or angiotensin 1I (A-II) were added with 0-5 nM CRH. No increases in plaque percentages were evident (over those caused by CRH alone). However, when NIL cultures were exposed to A-II or AVP for 1 hr before the plaque assays (run with 0.5 nM CRH) there was a 1.5-2.2-fold increase in plaque percentages above basal values. Furthermore, AVP pretreatment stimulated 1.8-fold increase in percentages over that caused by 0.5 nM CRH alone, F(11,120) = 7.66. Unfortunately, this set of experiments was done with a secondary set of anti-ACTH(25-39) sera that was not as sensitive in the RHPA. (The primary supply was exhausted.) Therefore, whereas plaque areas were comparable, the number of plaques formed were 50-70% of those formed with the best antisera used in the first set of experiments. In this second set of experiments, basal plaque areas were 1918_+ 774. After pretreatment for 1 hr with AVP or A-II, plaques formed in the presence of 0.5 nM CRH were 3647___570 i_tm2 (after A-If) or 4455_+ 1271 Ixm 2 (after AVP). Both values were different from basal levels. AVP-treated values were higher than levels seen with 0.5 nM CRH alone, F(11,48) = 3.15. DISCUSSION
The ability of neurointermediate lobe cells to respond to CRH is well documented. CRH stimulates increases in POMC mRNA levels (17) and opiocortin peptide secretion (1, 16, 19, 21,22, 26). High affinity receptors for CRH have been located in the intermediate lobe by autoradiography on sections (10, I l, 14). These studies agree that NIL cells bind CRH. After 3 min of exposure, 75% of NIL cells exhibited binding for bio-CRH. The NIL cultures contained endothelial cells, epithelial cells (lining the lobe) and pars nervosa glial cells. Therefore, it is not surprising to find that less than 100% of this dispersed NIL population is labeled for CRH. Dual labels for ACTH helped to define the target cell population further. These showed, however, that some NIL cells bound CRH, but did not store ACTH. Perhaps these unlabeled
INTERMEDIATE LOBE CELLS BIND CRH
735
FIG. 6. Reverse hemolytic plaque assay for ACTH/CLIP in NIL cultures showing variation in plaque areas. The lysed red blood cells cover the secreting IL cells in some cases. Large and small arrowheads point to examples of large and small plaques. Magnification x 157.
cells have lost their ACTH stores as a result of the stimulation by bio-CRH. The EM study showed striking contrasts between anterior and intermediate lobe corticotropes in the processing of bio-CRH or its metabolites. We had demonstrated previously that anterior lobe cells internalize the bio-CRH in 3-5 rain and transport it to the Golgi complex after which it (or its metabolites) is in lysosomes (multivesicular bodies), and a subpopulation of secretory granules (6). In contrast, intermediate lobe cells internalize the bio-CRH in small vesicles scattered throughout the cytoplasm during the first minute. The Golgi complex is never labeled. Instead, the vesicle membranes appear to coalesce with those of nearby secretory granules or vacuoles. Investigators have learned that the intermediate lobe cells express heterogeneity in cellular mRNA, cytology and storage levels (8, 9, 22). The plaque assays agree with Saland et al. (22) that NIL cells express heterogeneity in secretion. Saland et al. had shown this by a quantitative analysis of stored opiocortin in individual IL cells after stimulation. In the RHPA, only 26% of NIL cells appear to be secreting ACTH/CLIP basally. Furthermore, their plaque areas varied from less than 900 p~m2 to over 2800 txm 2. This suggests that some NIL cells are secreting different amounts of ACTH/CLIP, or at higher rates. The difficulties with interpretation of the present data from the RHPA revolve mainly around the scarcity of good antisera for other opiocortin molecules that will react in a plaque assay.
Previous studies have demonstrated a 1: 1 ratio of bioactive ACTH to C-terminal activity in anterior lobes (13, 18, 25). However, such is not the case in IL cells and it is likely that the assay detects secretion of CLIP (1). The nonsecretory IL cells could be actively releasing other types of opiocortin molecules. Added to this is the fact that the scarce supply of antisera to ACTH limited the scope of the RHPA experiments. This prevented comparable experiments with AVP and A-II and further tests of cultures stimulated with serotonin, dopamine antagonists, or stimulators of adenylate cyclase. In agreement with Chronwall et al. (8,9), stimulation of NIL cells increases the secretory potential of the population. Over 50% of NIL cells become secretory after 10 nM CRH or 0.5 nM CRH + a 1-hr pretreatment in 10 nM AVP. Plaque areas increase to a range of 1855-6000 p,m 2, indicating that the stimulated population also expresses heterogeneity in the rate of secretion or amount secreted. Perhaps the heterogeneity reflects division of labor in the population. There may be cells that have high CRH receptor levels, and ACTH/CLIP stores in a readily releasable pool. Other cells may be more quiescent and require further stimulation to express synthetic and secretory activity. A similar hypothesis was proposed by Chronwall et al. (8,9) after they correlated the appearance of dark and light IL cells with high and low expression of cellular POMC mRNA. Highly stimulated intermediate lobes contained more dark cells and became more homogeneous in their
736
CHILDS
expression o f P O M C m R N A . Unstimulated ILs were more heterogeneous. The dark cells were filled with rough endoplasmic reticulum which presumably bore the m R N A - p o l y r i b o s o m e complexes. Finally, it appears that A V P will potentiate the action o f 0.5 nM C R H when added 1 hr before the assay. Although limited in scope by the supply o f antisera, these studies with A V P and A-II
suggest that CRH may work with other neurotransmitters to stimulate IL function as it does in the anterior lobe. ACKNOWLEDGEMENTS The author wishes to thank Ms. Geda Unabia and JoAnn Burke tbr excellent technical assistance and Ms. Betty Williams for skilled typing. This study was funded by NSF #DCB 8718242.
REFERENCES 1. AI-Noaemi, M. C.; Edwardson, J. A.; Hyghes, D. Corticotropinreleasing factor (CRF) stimulates release of peptides from the intermediate lobe of the rat pituitary gland. J. Physiol. (Lond.) 332:85-86; 1982. 2. Antakly, T.; Sasaki, A.; Liotta, A. S.; Polkovits, M.; Krieger, D. T. Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe. Science 229:227-229; 1985. 3. Antakly, T.; Mercille, S.; Cote, J. P. Tissue-specific dopaminergic regulation of the glucocorticoid receptor in the rat pituitary. Endocrinology 120:1558-1562; 1987. 4. Bjorklund, A. Monoamine-containing nerve fibers in the pituitary neurointermediate lobe of the pig and rat. Z. Zellforsch. Mikrosk. Anat. 89:573-579; 1968. 5. Chitds, G. V.; Burke, J. A. Use of the reverse hemolytic plaque assay to study the regulation of anterior lobe adrenocorticotropin (ACTH) secretion by ACTH-releasing factor, arginine vasopressin, angiotensin II and glucocorticoids. Endocrinology 120:439-444; 1987. 6. Childs, G. V.; Morell, J. L.; Niendorf, A.; Aguilera, G. Cytochemical studies of corticotropin-releasing factor (CRF) receptors in anterior lobe corticotropes: Binding, glucocorticoid regulation, and endocytosis of [Biotinyl-ser~] CRF. Endocrinology 119:2129-2142; 1986. 7. Childs, G. V.; Yamauchi, K.; Unabia, G. Localization and quantifications of hormones, ligands, and mRNA with affinity gold probes. Am. J. Anat. 185:223-235; 1989. 8. Chronwall, B. M.; Millington, W. R.; Sue, W.; Griffin, T.; Unnerstall, J. R.; O'Donohue, T. L. Histological evaluation of the dopaminergic regulation of proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary, involving in situ hybridization and [3HI thymidine uptake measurement. Endocrinology 120:12011211; 1987. 9. Chronwall, B. M.; Hook, G. R.; Millington, W. R. Dopaminergic regulation of the biosynthetic activity of individual melanotropes in the rat pituitary intermediate lobe: A morphometric analysis by light and electron microscopy and in situ hybridization. Endocrinology 123:1992-2002; 1988. 10. De Souza, E. B.; Kuhar, M. J. Corticotropin-releasing factor receptors in the pituitary gland and central nervous system: Methods and overview. Methods Enzymol. 124:560-590; 1986. 11. De Souza, E. B.; Perrin, M. H.; Rivier, J.; Vale, W. W.; Kuhar, M. J. Corticotropin-releasing factor receptors in rat pituitary gland: Autoradiographic localization. Brain Res. 296:202-207; 1984. 12. Eipper, B. A.; Mains, R. E. Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides. Endocr. Rev. 1:1-27: 1980. 13. Greer, M. A.; Allen, C. F.; Panton, P.; Allen, J. P. Evidence that the pars intermedia and the pars nervosa of the pituitary do not secrete functionally significant quantities of ACTH. Endocrinology 96:718724; 1975. 14. Grigoriadis, D. E.; De Souza, E. B. Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: Biochemical
15.
16.
17. 18.
19.
20.
2 I.
22.
23.
24.
25. 26. 27.
28.
characterization and autoradiographic localization. Peptides 10:179188; 1989. Kraicer, J.; Morris, A. R. In vitro release of ACTH from dispersed rat pars intermedia cells. II. Effect of neurotransminer substances. Neuroendocrinology 21 : 175-192; 1976. Kraicer, J.; Gajewski, T. C.; Moore, B. C. Release of pro-opiomelanocortin-derived peptides from the pars intermedia and pars distalis of the rat pituitary: Effect of corticotrophin-releasing factor and somatostatin. Neuroendocrinology 41:363-373; 1985. Lundblad, J. R.; Roberts, J. L. Regulation of proopiomelanocortin gene expression in pituitary. Endocr. Rev. 9:135-158; 1988. Moriarty, C. M.; Moriarty, G. C. Bioactive and immunoactive ACTH in the rat pituitary: Influence of stress and adrenalectomy. Endocrinology 96:1419-1425; 1975. Proulx-Ferland, L.; Labrie, F.; Dumont, D.; Cote, J.; Coy, D. H.; Svieraf, J. Corticotropin releasing factor stimulated secretion of melanocyte-stimulating hormone from the rat pituitary. Science 217: 62-63; 1982. Rosa, P. A.; Policastro, P.; Herbert, E. A cellular basis for the differences in regulation of synthesis and secretion of ACTH/endorphin peptides in anterior and intermediate lobes of the pituitary. J. Exp. Biol. 89:215-237; 1980. Sakly, M.; Schmitt, G.; Koch, B. CRF enhances release of both MSH and ACTH from anterior and intermediate pituitary. Neuroendocrinol. Lett. 4:289-293; 1982. Saland, L. C.; Guitierrez, L.; Kraner, J.; Samora, A. Corticotropinreleasing factor (CRF) and neurotransmitters modulate melanotropic peptide release from rat neurointermediate pituitary in vitro. Neuropeptides 12:59~6; 1988. Sasaki, F.; Wu, P.; Rougeau, D.; Unabia, G.; Childs, G. V. Cytochemical studies of responses of corticotropes and thyrotropes to cold and novel environment stress. Endocrinology 127:(1), in press; 1990. Schacter, B. S.; Johnson, L. K.: Baxter, J. D.; Roberts, J. L. Differential regulation by glucocorticoids of proopiomelanocortin mRNA levels in the anterior and intermediate lobes of the rat pituitary. Endocrinology 119:1442-1444; 1982. Scott, A. P.; Lowry, P. J.; Ratcliffe, J. G.; Ress, L. H.; Landon, B. A. Corticotrophin-like peptides in the rat pituitary. J. Endocrinol. 61:335-367; 1974. Smith, A. L.; Funder, J. W. Proopiomelanocortin processing in the pituitary central nervous system and peripheral tissues. Endocr. Rev. 9:159-179: 1988. Westlund, K. N.; Wynn, P. C.; Chmielowiec, S.; Collins, T. J.; Childs, G. V. Characterization of a potent biotin-conjugated CRF analog and the response of anterior pituitary corticotropes. Peptide 5:627-638; 1984. Wilfinger, W. W.; Larson, W. J.; Downs, T. R.; Wilbur, D. L. An in vitro model for studies of intercellular communication in rat anterior pituitary cells. Tissue Cell 16:483-494; 1984.
0196-9781/90 $3.00 + .00
Subsets of Pituitary Intermediate Lobe Cells Bind CRH and Secrete ACTH/CLIP in a Reverse Hemolytic Plaque Assay G W E N V. C H I L D S
Department o f Anatomy and Neurosciences, University o f Texas Medical Branch 200 University Blvd., Room 731, Galveston, TX 77551
R e c e i v e d 4 April 1990
CHILDS, G. V. Subsets of pituitary intermediate lobe cells bind CRH and secrete ACTH/CLIP in a reverse hemolyticplaque assay. PEPTIDES 11(4) 729-736, 1990.--Dissociated neurointermediate lobe cells (75%) bound [biotinyl-Ser~]corticotropin releasing hormone (bio-CRH) and secreted adrenocorticotropin [ACTH(25-39)] in a reverse hemolytic plaque assay (RHPA). The bio-CRH was rapidly internalized (< 1 min) in vesicles and scattered vacuoles. Plaque percentages increased from 26 ± 4% to 53 + 8% after CRH stimulation. Areas increased from 1688 ± 791 ~m 2 to 3773 _+788 Ixm2. After AVP treatment for 1 hr, plaque percentages and areas were augmented further. The heterogeneity in secretion may indicate the presence of quiescent IL cells or cells secreting an opiocortin peptide not detected in the plaque assay. Corticotropin releasing hormone
Neurointermediate lobe cells
THE intermediate lobe (IL) of the rat pituitary synthesizes proopiomelanocortin (POMC) and cleaves this glycoprotein into bioactive peptides including a-melanocyte stimulating hormone (a-MSH), [3-endorphin, [3-1ipotropin and adrenocorticotropin (ACTH) (12,26). ACTH is acetylated and amidated and contains the sequence of o~-MSH in its N-terminal (amino acids 1-13). It contains corticotropin-like intermediate lobe peptide (CLIP) in the C-terminal (amino acids 17-39). It can be induced to respond to glucocorticoids (2,3). Early studies reported that a relatively low percentage of biologically active ACTH comes from the intermediate lobe of rats (13, 18, 25). It is considered to be the precursor for c~-MSH and CLIP (12, 13, 18, 25, 26). Synthesis of POMC and secretion of opiocortin peptides from the intermediate lobe are stimulated by corticotropin releasing hormone (CRH) (19,21) and inhibited by dopamine (4,15). Recently, Saland et al. (22) reported striking changes in the amount of immunolabeling for a - M S H following stimulation by CRH or serotonin, or inhibition by dopamine. The CRH-treated lobes showed a heterogeneous labeling pattern as if some cells had secreted more product than others. Recent studies by Chronwall et al. (8) also demonstrated heterogeneity in expression of POMC mRNA/IL cell following 6 hr of haloperidol treatment (a dopamine antagonist). In unstimulated ILs, the labeling pattern was heterogeneous. Some cells exhibited a lower rate of synthetic activity. More recently, Chronwall et al. (9) have correlated an increase in the proportion of dark cells rich in rough endoplasmic reticulum with an increase in the labeling for POMC mRNA. These workers concluded that
Adrenocorticotropin
Plaque assay
IL cells may normally be in different stages of the secretory cycle and that the light to dark cell transition may result from stimulation of protein synthesis. DeSouza and his colleagues have assayed CRH receptors in the IL by RRA (10,11). Gregoriadis and DeSouza recently characterized the binding affinity and molecular weight of the CRH binding protein. They reported that it was identical to that assayed in the anterior lobe. Autoradiographs showed that CRH binding sites were diffusely distributed in the intermediate lobe (11,14). In 1986, our laboratory reported electron microscopic cytochemical studies of the binding and internalization of a biotinylated analog of CRH by anterior lobe corticotropes (6). The analog had biotin attached to the N-terminal serine during synthesis and retained all biological activity (6). When bio-CRH and avidin fluorescein protocols were applied to a population of living dissociated neurointermediate lobe (NIL) cells, only a subpopulation of cells was labeled (less than 50%). Some of this might reflect the fact that NIL cells are diluted by epithelial cells, endothelial cells and the glial cells from the pars nervosa. Alternatively, this could reflect rapid uptake and degradation of the CRH by the IL cells. Therefore, further studies of binding and processing of CRH by IL ceils were initiated. At the same time, a reverse hemolytic plaque assay for ACTH had been developed with antisera to the C-terminal amino acids 25-39 (5) and applied to dissociated anterior lobe cells. The heterogeneous responses in intermediate lobe cells reported by Saland et al. (22) and Chronwall et al. (8,9) prompted a study of secretion from individual intermediate lobe cells with this assay.
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FIG. 1. Dark field view showing label for biotinylated CRH (3-min exposure) in intermediate lobe cells, 1 hr after dispersion and plating. Label glows bright on the cells or cellular processes. Examples of label are noted by +. Magnification x 546.
These studies demonstrated that a subset of IL cells internalized and processed CRH more rapidly than did cells of the anterior lobe. Furthermore, a subset of IL cells secretes ACTH/CLIP in the RHPA. However, the numbers of secreting cells increase after CRH stimulation. This report also describes the effect of arginine vasopressin (AVP) and angiotensin II on secretion from individual IL cells. METHOD Male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Houston, TX) were used to supply the pituitaries. They had been acclimated for 10 days on a 14-hr day, 10-hr night cycle and given food and water ad lib. They were anesthetized with sodium pentobarbitol and then killed in a separate room by decapitation. The pituitaries were removed rapidly. The neurointermediate lobes (NIL) were separated from the anterior lobes and dissociated according to the technique of Wilfinger et al. (28). The cells were plated 3,000-5,000/well on coverslips in 24-well trays for 1-2 days. They were grown in Eagles Minimum Essential Medium with 10% fetal bovine serum, 0.6% HEPES, and 0.3% bovine serum albumin (BSA) as described previously (5,27). Parallel sets of NIL cells (500,000/tube) were left in suspension for electron microscopic analysis of CRH processing (6). Parallel cultures were washed with MEM and exposed to either
biotinylated CRH for cytochemical labeling (6,27) or the RHPA (19). The biotinylated CRH was diluted in MEM containing 0.6% HEPES, 0.3% BSA, 10 4 M ascorbic acid, and aprotinin (100 KIU). It was added to the cells in dilutions of 0.5-1 nM. [These are saturating concentrations. Higher amounts produce no increase in labeling (6,27).] Exposure times ranged from 1-30 rain. For the light microscopic studies, the cells were fixed in 2% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4) at the end of the treatment period. Fixation continued for 30 rain after which the ceils were washed 4 × in phosphate buffer (0.1 M pH 7.4) containing 4.5% sucrose. The biotinylated CRH was then detected with the avidin-biotin-peroxidase complex (ABC) technique (Vector Laboratories, Burlingame, CA) and nickel intensified diaminobenzidine substrate (black) (DAB) as described previously (6,27). Some cultures were then immunolabeled for ACTH with the contrasting amber DAB substrate (6). Antisera to ACTH(1739) was produced in this laboratory and used at 1:20,000-1:30,000 dilutions. Controls testing specificity of reactions for bio-CRH or ACTH were repeated for NIL cultures as reported previously (6,27). In addition, as described in more recent studies (7,23), anti-ACYH(17-39) or ACTH(25-39) was tested on various opiocortin peptide antigens blotted on Immobilon nylon filter paper. Among the antigens tested (16K fragment, 13-endorphin, [3lipotropin, and ACTH), only ACTH showed a reaction. This has
FACING PAGE FIG. 2. After 1 min of exposure, the label for biotinylated CRH is on plasma membranes in patches (arrowhead). Label is also inside the cells, in structures that resemble a lysosome (a). (b) Illustrates an overview of another cell showing labeling in vesicles (v) or receptasomes after 1 min of exposure. Nuclei (N) and mitochondria (M) remain unlabeled. Magnification × 86,625 (a); 68,750 (b).
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3 FIG. 3. This field illustrates the types of structures that are labeled during the first 5 min of exposure. These include small vesicles (v), or membranes of secretory vesicles or vacuoles (sv) or lysosomes (L). Some contain dense cores. Label is also on the plasma membrane (arrows). Magnification x 65,312.
confirmed many tests of this antisera over the past 18 years. Extensive tests of specificity of the same antisera in the plaque assay are described in (5). NIL cultures were washed after 1 day in MEM and then exposed to the reverse hemolytic plaque assay for ACTH as described in previous reports (5). The antisera to ACTH was diluted 1:100-1:200 as in the previous studies of anterior lobe cells (5). The complement was diluted 1:20-1:50. The RHPA was then run for 4 hr in the presence of 0 - 5 0 nM CRH. Only plaques
containing a single IL cell were analyzed in the area measurements. The bio-CRH-labeled cells or plaques were counted and expressed as a percentage of the total cell population counted. A minimum of 100 cells/coverslip were analyzed. Each experimental group contained 3-4 coverslips and each experiment was run at least 2 times. Plaque areas were measured with the Bioquant image analysis system (R & M Biometrics, Nashville, TN) as described in previous studies (5). The plaques were projected on
INTERMEDIATE LOBE CELLS BIND CRH
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FIG. 4. This field shows a higher magnification of label for bio-CRH on membranes of secretory vesicles (arrows), or small vesicles (V) (15-min exposure to Bio-CRH). Magnification × 82,293.
the video screen and drawn with a cursor. Areas were calculated automatically by the computer software. As described previously (5), one can comfortably quantify a set of 12-14 coverslips in one period (to avoid extensive time lapse between coverslips). This will allow counts of 100 cells and measurements of 20 plaque areas/coverslip × 2 coverslips/group. The experimental groups were repeated 5-21 times to obtain adequate sampling. The data from all experiments (cell percentages or areas) were then averaged to provide the final data points. ANOVA followed by Duncan's multiple range test at the 5% level determined significant differences between groups. For the electron microscopic studies, the cells were left in suspension and exposed to bio-CRH for 1-30 min. They were fixed by adding an equal volume of 4% glutaraldehyde diluted in phosphate buffer (pH 7.4; 0.05 M). The protocol for fixation and labeling with ABC was identical to that described in the previous EM study of anterior lobe corticotropes (6). The cell pellets were embedded in Araldite 6005, sectioned and then the ultrathin sections were examined with a Zeiss 10 electron microscope after counterstaining with lead citrate and uranyl acetate. The EM studies were run 3 times. Electron micrographs of labeled intermediate lobe cells were evaluated as described in previous studies (6). The site(s) of the
label were identified in 50 cell profiles/experimental group. This analysis detected changes in the distribution of label with different times of exposure to the bio-CRH. RESULTS
Binding, by' Biotinylated CRH (bio-CRH) The ABC protocol detected label for biotinylated CRH on 5 8 _+ 14% of NIL cells after 1 min of exposure, and 75_+ 11% of NIL cells after 3 min (values _+ sd, n = 3 cultures). This was not a significant increase. The percentage of labeled cells did not increase with higher concentrations of bio-CRH (>1 nM). Percentages were 61 -+6% after 10 min of exposure. The label for bio-CRH was in patches or on processes (Fig. 1). Dual labeling for bio-CRH and ACTH showed that, after 3 min of exposure, 65_+ 20% of bio-CRH-labeled NIL cells also exhibited label for ACTH. Interestingly, 34-+ 19% exhibited label only for bio-CRH as if the CRH had stimulated secretion of all detectable ACTH stores. Less than 2% of labeled NIL cells showed label for ACTH and not bio-CRH. Controls demonstrated the specificity of labeling. The percentage of cells labeled for 0.5 nM bio-CRH was not affected by the
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FIG. 5. Analysis of changes in labeling in each type of organelle with time of exposure to biotinylated CRH. The percentage of labeled sites on the membrane (mem) and in small vesicles (ves) decreases after 1 min. There is an increase in the labeling of receptasomes (rec), lysosomes (lys), and secretory granules (gran) or vesicles. Structures associated with the Golgi complex (gol) are seldom labeled.
addition of 100 nM AVP, somatostatin, or angiotensin II. Labeling was reduced uniformly in the presence of 1000 x unlabeled CRH (exposure time 3 rain). A 100-fold excess of CRH resulted in labeling of 41-+ 14% of the cells. A 1000-fold excess of CRH blocked binding by bio-CRH so that only 3 -+ 1% of the cells showed label. The reduction was significant, F(2,18) = 37.94. The excess CRH stimulated secretion of stores so that most IL cells did not show label for ACTH. The EM studies showed rapid internalization of the bio-CRH. In 1 rain, the label was distributed in patches on the plasma membrane (Fig. 2a and b). Evidence for internalization was also seen as early as 1 min. During the first 5 min, label was found in small vesicles, structures that resembled receptasomes, and on membranes of secretory vesicles or dense core granules (Fig. 3). This labeling pattern persisted at later times of exposure. In addition, the labeling over membranes of secretory vacuoles or granules became more prominent (Fig. 4). Label was rarely found in the Golgi complex or vacuoles in that region. Figure 5 summarizes the changes in distribution of label in the different organelles with time of exposure to the bio-CRH.
Secretion of ACTH (CLIP) Previous LM and EM studies had demonstrated heterogeneity in expression of POMC mRNA (8,9) or a - M S H storage after stimulation (22). Therefore, the RHPA was used to detect any heterogeneity in areas of plaques formed by IL cells. In untreated cultures, 26 + 24% ( - sd) of the NIL cells formed plaques. In the presence of 1 nM CRH the percentage was 36 -+ 9, which was not different from untreated cells. In the presence of 10 nM CRH values increased to 53 -+ 7, which was significantly different from all other values, F ( 3 , 4 4 ) = 12.69. No further increase was seen with higher concentrations of CRH. When plaque areas were measured, they averaged 1 6 8 8+- 791 Fxm2 in unstimulated cultures. The range of areas was from 892-2852 p.m 2, which confirms studies by Saland et al. (22) who demonstrated heterogeneity in expression of MSH secretory function. In the presence of 0.5 nM CRH average areas increased to 2718-+332 ixm2 (range 1798-4883 txm2). A further increase to 3773 ± 6 3 3 was seen following exposure to l nM (range 1892-
6551 p.m2). These increases were significantly different from basal areas, F ( 4 , 8 5 ) = 8 . 3 7 . Figure 6 illustrates ACTH/CLIP secreting plaques in NIL cultures. In a separate group of experiments, 10 nM arginine vasopressin (AVP) or angiotensin 1I (A-II) were added with 0-5 nM CRH. No increases in plaque percentages were evident (over those caused by CRH alone). However, when NIL cultures were exposed to A-II or AVP for 1 hr before the plaque assays (run with 0.5 nM CRH) there was a 1.5-2.2-fold increase in plaque percentages above basal values. Furthermore, AVP pretreatment stimulated 1.8-fold increase in percentages over that caused by 0.5 nM CRH alone, F(11,120) = 7.66. Unfortunately, this set of experiments was done with a secondary set of anti-ACTH(25-39) sera that was not as sensitive in the RHPA. (The primary supply was exhausted.) Therefore, whereas plaque areas were comparable, the number of plaques formed were 50-70% of those formed with the best antisera used in the first set of experiments. In this second set of experiments, basal plaque areas were 1918_+ 774. After pretreatment for 1 hr with AVP or A-II, plaques formed in the presence of 0.5 nM CRH were 3647___570 i_tm2 (after A-If) or 4455_+ 1271 Ixm 2 (after AVP). Both values were different from basal levels. AVP-treated values were higher than levels seen with 0.5 nM CRH alone, F(11,48) = 3.15. DISCUSSION
The ability of neurointermediate lobe cells to respond to CRH is well documented. CRH stimulates increases in POMC mRNA levels (17) and opiocortin peptide secretion (1, 16, 19, 21,22, 26). High affinity receptors for CRH have been located in the intermediate lobe by autoradiography on sections (10, I l, 14). These studies agree that NIL cells bind CRH. After 3 min of exposure, 75% of NIL cells exhibited binding for bio-CRH. The NIL cultures contained endothelial cells, epithelial cells (lining the lobe) and pars nervosa glial cells. Therefore, it is not surprising to find that less than 100% of this dispersed NIL population is labeled for CRH. Dual labels for ACTH helped to define the target cell population further. These showed, however, that some NIL cells bound CRH, but did not store ACTH. Perhaps these unlabeled
INTERMEDIATE LOBE CELLS BIND CRH
735
FIG. 6. Reverse hemolytic plaque assay for ACTH/CLIP in NIL cultures showing variation in plaque areas. The lysed red blood cells cover the secreting IL cells in some cases. Large and small arrowheads point to examples of large and small plaques. Magnification x 157.
cells have lost their ACTH stores as a result of the stimulation by bio-CRH. The EM study showed striking contrasts between anterior and intermediate lobe corticotropes in the processing of bio-CRH or its metabolites. We had demonstrated previously that anterior lobe cells internalize the bio-CRH in 3-5 rain and transport it to the Golgi complex after which it (or its metabolites) is in lysosomes (multivesicular bodies), and a subpopulation of secretory granules (6). In contrast, intermediate lobe cells internalize the bio-CRH in small vesicles scattered throughout the cytoplasm during the first minute. The Golgi complex is never labeled. Instead, the vesicle membranes appear to coalesce with those of nearby secretory granules or vacuoles. Investigators have learned that the intermediate lobe cells express heterogeneity in cellular mRNA, cytology and storage levels (8, 9, 22). The plaque assays agree with Saland et al. (22) that NIL cells express heterogeneity in secretion. Saland et al. had shown this by a quantitative analysis of stored opiocortin in individual IL cells after stimulation. In the RHPA, only 26% of NIL cells appear to be secreting ACTH/CLIP basally. Furthermore, their plaque areas varied from less than 900 p~m2 to over 2800 txm 2. This suggests that some NIL cells are secreting different amounts of ACTH/CLIP, or at higher rates. The difficulties with interpretation of the present data from the RHPA revolve mainly around the scarcity of good antisera for other opiocortin molecules that will react in a plaque assay.
Previous studies have demonstrated a 1: 1 ratio of bioactive ACTH to C-terminal activity in anterior lobes (13, 18, 25). However, such is not the case in IL cells and it is likely that the assay detects secretion of CLIP (1). The nonsecretory IL cells could be actively releasing other types of opiocortin molecules. Added to this is the fact that the scarce supply of antisera to ACTH limited the scope of the RHPA experiments. This prevented comparable experiments with AVP and A-II and further tests of cultures stimulated with serotonin, dopamine antagonists, or stimulators of adenylate cyclase. In agreement with Chronwall et al. (8,9), stimulation of NIL cells increases the secretory potential of the population. Over 50% of NIL cells become secretory after 10 nM CRH or 0.5 nM CRH + a 1-hr pretreatment in 10 nM AVP. Plaque areas increase to a range of 1855-6000 p,m 2, indicating that the stimulated population also expresses heterogeneity in the rate of secretion or amount secreted. Perhaps the heterogeneity reflects division of labor in the population. There may be cells that have high CRH receptor levels, and ACTH/CLIP stores in a readily releasable pool. Other cells may be more quiescent and require further stimulation to express synthetic and secretory activity. A similar hypothesis was proposed by Chronwall et al. (8,9) after they correlated the appearance of dark and light IL cells with high and low expression of cellular POMC mRNA. Highly stimulated intermediate lobes contained more dark cells and became more homogeneous in their
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expression o f P O M C m R N A . Unstimulated ILs were more heterogeneous. The dark cells were filled with rough endoplasmic reticulum which presumably bore the m R N A - p o l y r i b o s o m e complexes. Finally, it appears that A V P will potentiate the action o f 0.5 nM C R H when added 1 hr before the assay. Although limited in scope by the supply o f antisera, these studies with A V P and A-II
suggest that CRH may work with other neurotransmitters to stimulate IL function as it does in the anterior lobe. ACKNOWLEDGEMENTS The author wishes to thank Ms. Geda Unabia and JoAnn Burke tbr excellent technical assistance and Ms. Betty Williams for skilled typing. This study was funded by NSF #DCB 8718242.
REFERENCES 1. AI-Noaemi, M. C.; Edwardson, J. A.; Hyghes, D. Corticotropinreleasing factor (CRF) stimulates release of peptides from the intermediate lobe of the rat pituitary gland. J. Physiol. (Lond.) 332:85-86; 1982. 2. Antakly, T.; Sasaki, A.; Liotta, A. S.; Polkovits, M.; Krieger, D. T. Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe. Science 229:227-229; 1985. 3. Antakly, T.; Mercille, S.; Cote, J. P. Tissue-specific dopaminergic regulation of the glucocorticoid receptor in the rat pituitary. Endocrinology 120:1558-1562; 1987. 4. Bjorklund, A. Monoamine-containing nerve fibers in the pituitary neurointermediate lobe of the pig and rat. Z. Zellforsch. Mikrosk. Anat. 89:573-579; 1968. 5. Chitds, G. V.; Burke, J. A. Use of the reverse hemolytic plaque assay to study the regulation of anterior lobe adrenocorticotropin (ACTH) secretion by ACTH-releasing factor, arginine vasopressin, angiotensin II and glucocorticoids. Endocrinology 120:439-444; 1987. 6. Childs, G. V.; Morell, J. L.; Niendorf, A.; Aguilera, G. Cytochemical studies of corticotropin-releasing factor (CRF) receptors in anterior lobe corticotropes: Binding, glucocorticoid regulation, and endocytosis of [Biotinyl-ser~] CRF. Endocrinology 119:2129-2142; 1986. 7. Childs, G. V.; Yamauchi, K.; Unabia, G. Localization and quantifications of hormones, ligands, and mRNA with affinity gold probes. Am. J. Anat. 185:223-235; 1989. 8. Chronwall, B. M.; Millington, W. R.; Sue, W.; Griffin, T.; Unnerstall, J. R.; O'Donohue, T. L. Histological evaluation of the dopaminergic regulation of proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary, involving in situ hybridization and [3HI thymidine uptake measurement. Endocrinology 120:12011211; 1987. 9. Chronwall, B. M.; Hook, G. R.; Millington, W. R. Dopaminergic regulation of the biosynthetic activity of individual melanotropes in the rat pituitary intermediate lobe: A morphometric analysis by light and electron microscopy and in situ hybridization. Endocrinology 123:1992-2002; 1988. 10. De Souza, E. B.; Kuhar, M. J. Corticotropin-releasing factor receptors in the pituitary gland and central nervous system: Methods and overview. Methods Enzymol. 124:560-590; 1986. 11. De Souza, E. B.; Perrin, M. H.; Rivier, J.; Vale, W. W.; Kuhar, M. J. Corticotropin-releasing factor receptors in rat pituitary gland: Autoradiographic localization. Brain Res. 296:202-207; 1984. 12. Eipper, B. A.; Mains, R. E. Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides. Endocr. Rev. 1:1-27: 1980. 13. Greer, M. A.; Allen, C. F.; Panton, P.; Allen, J. P. Evidence that the pars intermedia and the pars nervosa of the pituitary do not secrete functionally significant quantities of ACTH. Endocrinology 96:718724; 1975. 14. Grigoriadis, D. E.; De Souza, E. B. Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: Biochemical
15.
16.
17. 18.
19.
20.
2 I.
22.
23.
24.
25. 26. 27.
28.
characterization and autoradiographic localization. Peptides 10:179188; 1989. Kraicer, J.; Morris, A. R. In vitro release of ACTH from dispersed rat pars intermedia cells. II. Effect of neurotransminer substances. Neuroendocrinology 21 : 175-192; 1976. Kraicer, J.; Gajewski, T. C.; Moore, B. C. Release of pro-opiomelanocortin-derived peptides from the pars intermedia and pars distalis of the rat pituitary: Effect of corticotrophin-releasing factor and somatostatin. Neuroendocrinology 41:363-373; 1985. Lundblad, J. R.; Roberts, J. L. Regulation of proopiomelanocortin gene expression in pituitary. Endocr. Rev. 9:135-158; 1988. Moriarty, C. M.; Moriarty, G. C. Bioactive and immunoactive ACTH in the rat pituitary: Influence of stress and adrenalectomy. Endocrinology 96:1419-1425; 1975. Proulx-Ferland, L.; Labrie, F.; Dumont, D.; Cote, J.; Coy, D. H.; Svieraf, J. Corticotropin releasing factor stimulated secretion of melanocyte-stimulating hormone from the rat pituitary. Science 217: 62-63; 1982. Rosa, P. A.; Policastro, P.; Herbert, E. A cellular basis for the differences in regulation of synthesis and secretion of ACTH/endorphin peptides in anterior and intermediate lobes of the pituitary. J. Exp. Biol. 89:215-237; 1980. Sakly, M.; Schmitt, G.; Koch, B. CRF enhances release of both MSH and ACTH from anterior and intermediate pituitary. Neuroendocrinol. Lett. 4:289-293; 1982. Saland, L. C.; Guitierrez, L.; Kraner, J.; Samora, A. Corticotropinreleasing factor (CRF) and neurotransmitters modulate melanotropic peptide release from rat neurointermediate pituitary in vitro. Neuropeptides 12:59~6; 1988. Sasaki, F.; Wu, P.; Rougeau, D.; Unabia, G.; Childs, G. V. Cytochemical studies of responses of corticotropes and thyrotropes to cold and novel environment stress. Endocrinology 127:(1), in press; 1990. Schacter, B. S.; Johnson, L. K.: Baxter, J. D.; Roberts, J. L. Differential regulation by glucocorticoids of proopiomelanocortin mRNA levels in the anterior and intermediate lobes of the rat pituitary. Endocrinology 119:1442-1444; 1982. Scott, A. P.; Lowry, P. J.; Ratcliffe, J. G.; Ress, L. H.; Landon, B. A. Corticotrophin-like peptides in the rat pituitary. J. Endocrinol. 61:335-367; 1974. Smith, A. L.; Funder, J. W. Proopiomelanocortin processing in the pituitary central nervous system and peripheral tissues. Endocr. Rev. 9:159-179: 1988. Westlund, K. N.; Wynn, P. C.; Chmielowiec, S.; Collins, T. J.; Childs, G. V. Characterization of a potent biotin-conjugated CRF analog and the response of anterior pituitary corticotropes. Peptide 5:627-638; 1984. Wilfinger, W. W.; Larson, W. J.; Downs, T. R.; Wilbur, D. L. An in vitro model for studies of intercellular communication in rat anterior pituitary cells. Tissue Cell 16:483-494; 1984.
