0022-1554/92/$3.30 The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.

Original Article

Vol. 40, No. 4. pp. 487-493, 1992 Printed iff USA.

Binding of an Iodinated Substance P Analogue to Cultured Anterior Pituitary Prolactin- and Luteinizing Hormone-containing Cells'

PHILIP J. LARSEN,2 TORBEN SBRMARK, and SOREN E. MAU Itpstitrcte of Me&& Anatomy B (PJL), EEC Concerted Actions, and InStitute of Medical Physiology B (TS,SEM,), University of Copenhagen, Copenhagen, Denmark.

Received for publication February 19, 1991 and in revised form October 18, 1991; accepted October 29, 1991 (1A2244).

Several lines of anatomic, biochemical, and pharmacologi- cal evidence suggest that the neuropeptide substance P has a direct action on cells of the anterior pituitary lobe via a specific neurokinin-1 receptor. In the present study we confiied this association by combining Bolton-Hunter io- dinated substance Preceptor autoradiography with immu- nocytochemistry on cultured anterior pituitary cells. Radio- labeled substance P was bound to living cell cultures at O"C, and after a brief wash the cultures were fued and processed immunocytochemically for prolactin and luteinizing hor- mone. A large proportion of cultured anterior pituitary cells possessed substance P binding Sites. When receptor autora- diography was combined with immunocytochemistry, it

Introduction Substance P (SP) is a member of the tachykinin peptide family characterized by their common C-terminal amino acid sequence (Phe-X-Gly-Leu-Met-NH2) (Regoli et al., 1987). SP is known to ex- ert a wide variety of physiological actions in both the peripheral and the central nervous systems (Jessel, 1983).

The role of SP as a hypothalamic neuroendocrine regulator of anterior pituitary functions is well established (Aronin et al., 1986). In vivo as well as in vitro, SP has been shown to stimulate the re- lease of prolactin (PRL) from anterior pituitary cells (Eckstein et al., 1980; Chihara et al., 1978; Vijayan and McCann, 1978; Kat0 et al., 1976). In addition to its action on lactotrophs, SP is likely to influence gonadouophs. This is supported by the observation that SP inhibits LHRH-stimulated luteinizing hormone (LH) re- lease from cultured pituitary cells (KerdelhuE et al., 1979). The concept of a direct action is further supported by the recent find- ing of a moderate density of neurokinin-1 receptors on anterior pi-

' Supported by NOVO'S Foundation, Fonden til Lagevidenskabcn Frcmme, Dr. Med. Vet Axel Thomxn og hustrus Fond, P. Carl Petersens Fond, and Brdr. Hartmanns Fond.

Correspondence to: Philip Just Larsen, Institute of Medical Anatomy, Department B, The Panum Institute, Blegdamsvej 3, DK-2200 Copenha- gen N, Denmark.

was evident that both prolactin- and luteinizing hormone- immunoreactive cells were labeled with radiolabeled sub- stance F? However, a smal l proportion of the radioligand- labeled cells were not stained by the immunocytochemical procedure, suggesting that additional cell types possess sub- stance P receptors. The present study presents morphologi-

ing hormone-containing cells of the anterior pituitary lobe. Therefore, it is likely that substance P has a direct action on mammouophs and gonadotrophs. (JHisrochem Cpo- &em 40:487493, 1992) KEY WORDS: Substance P Anterior pituitary cell culture; Prolactin; Luteinizing hormone; Neuropeptide receptor; Rat.

cal evid" that substance P binds to prolactin- and luteiniz-

tuitary membranes as well as cultured anterior pituitary cells (Lar- sen et al., 1989a,b). When bound to anterior pituitary cells, SP is intemalized and induces activation of the polyphosphoinositide second messenger pathway (Mau et al., 1990).

There are many possible sources that might convey SP to an- terior pituitary cells. One is the portal vascular system (Mikkelsen et al., 1989; ?sur0 et al., 1983,1987). Another is that the moderate number of SP-immunoreactive nerve fibers present within the an- terior pituitary lobe itself could have a direct action on the endo- crine cells (Ju and Liu, 1989; Muelsen et al., 1989). Finally, it may be that SP produced within anterior pituitary cells influences part of the cells via a paracrine mode of action (Jonassen et al., 1987; Aronin et al., 1986; DePalatis et al., 1982; Morel et al., 1982).

Yet to be investigated is whether SP exerts its actions on PIU and LH-containing anterior pituitary cells directly via surface- localized receptors. The aim of this study was to investigate the possibility that among the heterogeneous cell populations in the anterior pituitary gland SP binding sites are present on PRZ, and LH-containing cells. To demonstrate this, we employed a newly de- veloped technique which sequentially visualizes cell-associated receptors and intracellular immunoreactive material.

Traditionally, peptide receptor autoradiography has been per- formed on unfixed or lightly fixed tissue sections. This procedure is inconveniently combined with a concomitant immunocytochem-

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488 LARSEN, SERMARK, MAU

ical demonstration of the uansmitter content of the receptor-bearing cell types. Therefore, previous attempts to immunocytochemically characterize radioligand-labeled cells have involved in vivo autora- diography or neighboring tissue sections. To overcome some of the obvious disadvantages connected with these methods, we have de- veloped a technique using primary cell cultures to which radiola- beled peptides are bound while still viable, followed by fixation and immunocytochemical staining of the hormone content.

Materials and Methods Chemicals. Carrier-free Bolton-Hunter iodinated substance P ([ I2II]-

BH-SP) was purchased from Amersham (Buckinghamshire, UK). Substance P, leupeptin, chymostatin, and bacitracin were obtained from Cambridge Research Biochemicals (Cambridge, UK). Cell culture media were from Gibco (Paisley, UK). All other chemicals used were obtained from Sigma Chemi- cal (St Louis, MO). [I2'I]-BH-SP was dissolved in 0.1 M acetic acid and stored at - 20°C under nitrogen. Under these conditions the tracer was stable for at least 2 months, as evaluated from HPLC analysis, which in addition confirmed that the tracer was carrier free.

Preparation of Dispersed Anterior Pituitary Cell Cultures. Adult male Wistar rats were decapitated, the pituitaries removed, and the neurointer- mediate lobe separated from the anterior lobe. The anterior lobes were cut into about 10 smaller fragments and isolated pituitary cells were prepared by trypsinizing the organs according to the method of Gillies and Lowry (1978), as modified by Larsen et al. (1989b). The dispersed cell sample was re-suspended in Dulbecco's modified Eagle's medium (DMEM) contain- ing 10% fetal calf serum, 15 pg benzylpenicillinlml, and 25 pg streptomy- cinlml to a final concentration of 1 x lo6 cellslml. The dispersed anterior pituitary cells were placed in 8- or 16-well Tissue-Tek Chamber slides (Nunc; Roskilde, Denmark) with 5 x 10' to 1 x lo6 cells per well and kept at 37'C in a CO2lair incubator until the cells were adherent to the well, which usually took place after 3 to 5 days.

Substance P Receptor Binding Study. The binding experiments were performed essentially as described earlier (Larsen et al., 1989b), with mi- nor variations. To abolish internalization of the ligand and minimize the action of enzymatic degradation, pre-incubation and incubation with the radioactive ligand were performed at O'C. Primary anterior pituitary cell cultures were washed twice with Earl's balanced salt solution (EBSS) con- taining 0.2 % bovine serum albumin (BSA), followed by pre-incubation for 20 min in 0.5 ml EBSS containing 0.2% BSA and 3 mM MnC12 per well (Buffer A). Thereafter, the cell cultures were incubated in 0.5 ml of Buffer A to which 40 pg bacitracin, 40 wg chymostatin, 4 pg leupeptin,

and aprotinin (100 kallikrein-inactivating unitslml) were added. At time zero, [ 12'I]-BH-SP (spec. act. 2000 Cilmmol) was added, giving a final con- centration of 0.5 nM of the radioactive tracer. Nonspecific binding was es- timated from incubations with the presence of 1.5 LM non-iodinated sub- stance P. After 20 min incubation, the wells were washed with chilled EBSS-BSA four times for 30 sec, followed by addition of 0.5 ml4% parafor- maldehyde in 0.1 M phosphate buffer (pH 7.4) fixative for 1 hr.

Lmmunocytochemistry. After fixation, the wells were washed twice with PBS, pH 7.4. Immunocytochemical staining of fixed primary anterior pi- tuitary cell cultures to which ['251]-BH-SP was bound was performed no longer than 3 days after the binding experiment. The wells were washed twice for 10 min with PBS and then incubated for 30 min in 5% pre-immune swine serum in PBS. Thereafter, the cell cultures were incubated at 4°C for 24 hr in either rabbit anti-prolactin (PRL, 1:lOOO) antiserum, rabbit anti-luteinizing hormone (LH, 1:lOOO) antiserum, or a mixture of both dis- solved in PBS + 1% BSA. The PRL and LH antisera were supplied by the National Pituitary and Hormone Distribution, NIDDK (Bethesda, MD) and have previously been used successfully in several immunohistochemi- cal studies (Nikitovitch-Winer et al., 1987).

After incubation with the primary antiserum, the cell cultures were washed three times for 10 min in PBS with 0.25 % BSA, followed by incu- bation in biotinylated swine anti-rabbit IgG (Dakopatts; Copenhagen, Den- mark) diluted 1:400 in PBS containing 1% BSA at room temperature for 60 min. The cell cultures were then washed three times for 10 min in PBS with 0.25% BSA and finally incubated at room temperature for 60 min in streptavidin-HRP complex (Dakopatts). Thereafter, they were washed in PBS with 0.25% BSA for 10 min and finally incubated in 0.05 M Tris- HCI buffer (pH 7.4) for a further 10 min. The cell cultures were reacted for peroxidase activity by incubation in the latter buffer containing 0.025% 3,3'-diaminobenzidine (DAB) and 0.003% H202 for 20 min. After wash- ing twice for 10 min in distilled water, the top section of the Tissue-Tek Chamber slide was removed and the tissue slides were air-dried and cov- ered with Ilford K-2 emulsion diluted 1:1 in distilled water as described by Hosli et al. (1975). The emulsion-coated slides were exposed in the dark for 4-7 days and finally developed with Kodak D19 developer. After de- veloping the emulsion, the slides went through a series of ethanols and were coverslipped.

Results

Receptor Autoradiography Binding of [ lZ5I]-BH-SP to anterior pituitary cells in primary cul- ture resulted in uptake and breakdown of the ligand at 37°C but

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Figure 1. Low-power photomicrograph showing [1251]-BH-SP-labeled anterior pituitary cell culture concomitantly immunostained for PRL and LH. The upper part is close to the center of the culture chamber, while the lower part is close to the periphery. Note the higher density of grains accumulated over cells near the center of the chamber. Bar a 250 pm.

Figure 2. High-power photomicrograph of rectangle in Figure 1. A cluster of [Iz51)-BH-SP binding cells is densely labeled, making it impossible to evaluate concur- rent immunocytochemical staining for either PRL or LH (curved open arrow). In addition, single immunopositive (straight open arrow) and immunonegative (solid arrow) areas possessing [1251J-BH-SP binding sites are seen. Bar = 50 pm.

Figure 3. High-power photomicrograph of anterior pituitary cells immunoreactive for either PRL or LH. All immunopositive cells are labeled with [1251]-BH-SP, although density of accumulated grains overlying individual cells varies considerably. Bar = 50 wm.

Figure 4. Medium-power photomicrograph showing PRULH-immunoreactive cells (open curved arrow) and non-immunoreactive cells (solid arrow) to which p i ) . BH-SP was bound in the presence of 1 pM unlabeled substance P (i.e., nonspecific binding). Bar = 100 wm.

Figure 5. High-power photomicrograph showing anterior pituitary cells immunoreactive neither to PRL nor to LH, possessing [1251]-BHSP binding sites. Bar = 50 pm.

Figure 6. High-power photomicrograph showing PRULH-immunoreactive anterior pituitary cells to which [12511-BH-SP was bound in the presence of 1 unla- beled substance P (i.e., nonspecific binding). The nuclei of individual cells are usually clearly discerned from the heavily immunostained cytoplasm. Bar = 50 wm.

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SUBSTANCE P RECEFIOR O N ANTERIOR PITUITARY CELLS 489

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490 M E N , SERMARK, MAU

not at 0°C (Larsen et al., 1989b). After incubation of primary an- terior pituitary cultures at 0°C with [12SI]-BH-SP, many cells showed binding of this radioligand when subjected to autoradiog- raphy. Cells were considered positively labeled when they were over- laid with a grain density five times that of the background. The majority of labeled cells were large and rounded (Figure l), while a minor population appeared multiform, probably representing flattened cells more extensively attached to the chamber slide. Some cells, however, remained free of [ 12SI]-BH-SP (Figure 1).

Developed grains were unevenly distributed throughout the area of the culture chamber. Close to the periphery of the chamber a moderate number of grains were associated with cell bodies, while the highest number of grains was observed within the center of the chamber. As shown in Figure 2, it was impossible to evaluate the immunocytochemical staining properties of such heavily labeled cells after 7 days of exposure. Therefore, the evaluation of com- bined radioligand labeling and immunohistochemical staining was performed on cells after 4 days of exposure. The radioligand label- ing was clearly associated with the surface of cultured cells, although it was impossible at the light microscopic level to exclude intracel- lularly localized radioligand. Most heavily labeled was the central part of cells, but the cell outgrowths characteristic of adherent cul- tured cells were also labeled (Figure 6). The binding of [12>I]-BH- SP was reversible, as demonstrated in cultures in which 1.5 pM non- radioactive SP was present during the incubation (Figures 4,7D, and 7E).

Immunocytochemistry Among the cells of primary anterior pituitary cell cultures, a high number of PRLIR cells was present, whereas the number of LH-IR cells was low. In a separate series of experiments the PRL and LH antisera were mixed, and using this procedure a very high number of immunoreactive cells was revealed. Despite the nature of the immunocytochemical reaction, a number of non-immunoreac- tive cells were always present, probably representing other cell types in the heterogeneous anterior pituitary cell population. The immunoreactive material was granular, and usually the nuclear area was seen as non-immunoreactive c e n d y located ovoid spot (Figures 3,6, and 7E). In the combined experiments, this phenomenon served as a useful guideline to determine whether a cell possessing [lZ5I]- BH-SP binding sites was at the same time an immunopositive cell. Cell outgrowths, when present, were less immunoreactive than the central part of the cells. Control cell cultures were incubated with pre-immune serum or primary antisera pre-absorbed with the pep- tides against which they had been raised. Pre-immune serum was unable to visualize immunoreactive material, and only peptides against which the antisera were raised could abolish the immuno- histochemical staining.

Combined Results When immunocytochemistry was combined with binding of [ 12SI]- BH-SP to anterior pituitary cell cultures, it was revealed that PRL- IR as well as LH-IR cells were labeled with the radioligand (Figures 7A-7C). The immunoreactive material was clearly distinguishable in most labeled cells but, as mentioned above, the density of grains

was, in some cases exposed for 7 days, so high that evaluation of the immunocytochemical reaction was impossible. However, a careful examination of cell cultures exposed for 4 days revealed that all PRLIR and LH-IR cells possessed SP receptors. In some cases non- immunoreactive cells were labeled with the radioligand (Figure 7F). When the intensity of the immunocytochemical staining was com- pared to cultures in which [lZSI]-BH-SP was not bound, no ap- parent difference in staining intensity could be observed.

Discussion The present study demonstrates that binding sites for SP are local- ized on a large proportion of cultured anterior pituitary cells. Bind- ing of ['*'I]-BH-SP appeared to be specific, because it was in- hibited to a great extent by addition of 1.5 DM unlabeled SP to the incubation medium. The presence of SP binding sites on an- terior pituitary cells is consistent with previously performed bio- chemical receptor binding experiments (Larsen et al., 1989a,b; Ker- delhuE et al., 1985). The combination with immunocytochemistry has made it possible to confine the binding of a radioligand to biochemically defined cells. The number of SP binding sites within the anterior pituitary gland is fairly low, and the receptor ligand complex is internalized and recycled (Larsen et al., 1989b). There- fore, it is likely that the synthesis rate of SP receptors in this organ is very low, and this could explain why a recent attempt to demon- strate mRNA coding for the SP receptor in anterior pituitary tissue extracts found specific mRNA content to be below the detection limit (Hershey et al., 1991).

The observed variation in grain density between the center and the outer perimeter of the culture chamber is likely to be caused by variations in thickness of the emulsion layer covering the cul- tures. First, the surface of a tissue culture is uneven, raising the possibility that the emulsion layer is thinner immediately above the cell body. Second, the tissue chamber slides employed have their removable top glued to the plastic slide with a silicone membrane. This membrane is highly hydrophobic, leaving the emulsion most likely to attach to the central area of the culture chamber. Because of the previously mentioned variability of radioligand labeling in a single culture chamber, a quantitative estimate of the receptor density was not given. After 7 days of exposure, a high grain den- sity was obtained in the central area of the culture chamber, ob- scuring the immunocytochemical staining. Therefore, it was not possible to assess with certainty the proportion of immunoreactive cells labeled with [ 12'I]-BH-SP. However, after only 4 days of ex- posure the grain density was sufficiently low to enable evaluation of most immunocytochemically stained cells labeled with the radi- oligand, revealing that all PRL and LH-containing cells possess SP receptors.

The use of primary cell cultures instead of post-mortem tissue sections makes it possible to bind radioligands to living cells with presumably unaffected surface receptors, although it is still possi- ble that the dispersion and culturing procedures may have affected the number of binding sites as well as the biochemical properties of the receptor in question. Our earlier binding experiments with [ lZSI]-BH-SP clearly show that the kinetic data obtained from an- terior pituitary cell membranes closely match those obtained from cultured anterior pituitary cells (Larsen et al., 1989a.b). The pres-

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SUBSTANCE P RECEmYlR ON ANTERIOR PITUITARY CELLS 491

Figure 7. (A) Medium-power photomicrograph showing [1251]-BH-SP-labeled anterior pituitary cells immunoreactive for either PRL or LH. The grains of the photo- graphic emulsion are closely associated with the immunocytochemically stained cells. In a number of immunoreactive cells the nucleus is clearly designated as a clear spot, helping to determine the specificity of the staining. (B,C) Photomicrographs showing [1251]-BH-SP labeled anterior pituitary cells immunoreactive for either PRL (E) or LH (C). When [1251]-BH-SP was bound in the presence of 1.5 pM unlabeled substance P, the number of grains associated with PRL- immunoreactive (D) or LH-immunoreactive (E) cells was very low, thus representing nonspecific labeling. (F) Two anterior pituitary cells immunoreactive neither for PRL nor for LH and possessing [1251]-BH-SP binding sites are seen. (0) PRULH-immunoreactive cells concomitantly labeled with [1251]-BH-SP (open arrow) are seen close to a non-immunoreactive cell possessing SP binding sites (solid arrow). Bars: A = 100 pm; B = 50 pm; C-G = 25 pm.

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492 LARSEN, SKRMARK, MAU

ent technique combines some of the advantages from both in vivo and in vitro techniques. In most in vivo techniques, one has to load an entire animal with radioligand, which is economically disad- vantageous. When using cell cultures, it is possible, by washing, to reduce nonspecific binding to low levels. This is almost impossi- ble with most in vivo techniques. Finally, it is possible to arrest enzymatic breakdown and internalization of the ligand, which other- wise could cause serious interpretational problems. As evaluated from the present study and earlier HPLC analysis of the incuba- tion medium after 30 min of incubation to O'C, the [ lZ51]-BH-SP ligand remains intact and is not susceptible to internalization (Larsen et al., 1989b). When using in vitro techniques involving tissue sec- tions, to obtain acceptable cell morphology one must use lightly fixed sections. Preliminary experiments clearly showed that prepa- ration of fresh tissue sections with paraformaldehyde fixative abol- ished the binding of [ 1251]-BH-SP.

Concomitant visualization of radioligand binding sites and in- tracellular neurotransmitters has been done previously using tech- niques different from the present (Szigethy and Beaudet, 1989; Akesson and Micevych, 1988; Morell and Pfaff, 1983). The most successful report of combined receptor autoradiography and im- munocytochemistry involved in vivo binding of radiolabeled steroids (Morell and Pfaff, 1983).

Not all cells labeled with [ 1251]-BH-SP were immunocytochem- ically stained, and this was true even when the PRL and LH an- tisera were mixed. The anterior pituitary gland consists of a heter- ogeneous population of cells identifiable according to their content of pituitary hormones produced (Herlant, 1964). Therefore, it is likely that cell types other than the two presently investigated may possess SP receptors.

From several reports, SP has been shown to stimulate the re- lease of PRL from anterior pituitary cells in vivo as well as in vitro (Eckstein et al., 1980; Chihara et al., 1978; Vijayan and McCann, 1978; Kat0 et al., 1976). The presence of [ lZ5I]-BH-SP binding sites on PRGcontaining cells provides evidence that SP exerts its prolactin- releasing action directly on mammotrophs. In addition to this, SP may influence LH-containing gonadotrophs, which is in accordance with the finding that SP inhibits LHRH-stimulated LH release from anterior pituitary cell cultures (Kerdelhut et al., 1979). Fur- thermore, the number of SP binding sites within the anterior pitu- itary lobe is inversely correlated with the number of LHRH bind- ing sites during the estrous cycle (KerdelhuC et al., 198s). However, the influence of SP on the release of other anterior pituitary hor- mones is more complex. It has been reported that corticotropin- releasing hormone and arginine-vasopressin-stimulated release of corticotropin from anterior pituitaries was inhibited by addition of SP to the incubation medium (Nicholson et al., 1984). Recently, another study could not confirm any effect of SP on corticotropin- releasing hormone-stimulated release of corticotropin from dis- persed anterior pituitary cell cultures (Chawdrey et al., 1990). There- fore, it is still speculative whether corticotrophs are in fact influenced by SP.

We have previously shown that SP induces receptor-mediated hydrolysis of polyphosphoinositides in the rat anterior pituitary lobe (Mau et al., 1990). Consistent with the findings of the present study, it seems likely that SP induces its effects on mammotrophs and gonadotrophs via the polyphosphoinositide pathway. Since many

cells in cultures of anterior pituitary glands, not merely mammo- trophs, can be labeled by incubation with [ 1251]-BH-SP, their mixed cultures cannot be used for experiments concerned with sec- ond messenger pathways leading only to PRL secretion.

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