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Keywords:

  • LHRH;
  • LHRH binding sites;
  • parietal cells;
  • suckling;
  • rat

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Luteinizing-hormone releasing hormone (LHRH) is a hypothalamic and milk-borne hormone that inhibits the cell proliferation of gastric epithelium in developing rats, although the mechanism of such action is unknown. We investigated the presence of binding sites for LHRH in the stomach of suckling rats after the injection of the hormone. Immunofluorescence at the confocal microscopy level revealed that LHRH binds to gastric cells, being particularly abundant over the gland. Different fluorescent lectins were used to identify gastric cell types and determine which were labeled by the hormone. Colocalization studies in these double-labeling experiments showed that LHRH staining colocalizes with parietal cells, suggesting the presence of binding sites in these cells. The three-dimensional (3-D) reconstruction of isolated parietal cells revealed the localization of the signal, which appears to be in the membrane of the canalicular region. These results suggest that there are binding sites for LHRH in the gastric epithelium, specifically in parietal cells, and they might play a role in the control of cell proliferation during suckling. Anat Rec 264:43–50, 2001. © 2001 Wiley-Liss, Inc.

The luteinizing-hormone releasing hormone (LHRH) is a gonadotropin-releasing hormone (GnRH) produced in the hypothalamus that plays a central role in the regulation of pituitary gonadotropes. Luteinizing (LH) and follicle-stimulating (FSH) hormones are released when GnRH binds to specific receptors at cell surface. Extrapituitary receptors were identified in the thymus (Marchetti et al., 1989), in testes (Kaiser et al., 1992), and in the ovaries (Kaiser et al., 1992; Kogo et al., 1999), suggesting that LHRH or GnRH may have other physiological functions. In tumors, inhibitory roles on cell proliferation were reported (Szende et al., 1989, 1990; Brower et al., 1992; Jungwirth et al., 1998; Mizutami et al., 1998) showing that the hormone interferes in cell cycle control and may induce apoptosis. Consequently, LHRH has become a valuable tool in cancer hormone-therapy.

In addition to these different functions, LHRH was isolated in human (Baram et al., 1977) and rat milk (Smith-White and Ojeda, 1984) and its physiological relevance has been discussed since then (Koldovský, 1989). Smith-White and Ojeda (1984) demonstrated that the milk-borne LHRH is a biologically active molecule in the stomach content and also that the number of ovarian binding sites is regulated by the feeding condition. Recently, by investigating the cell proliferation in the hyperproliferative gastric epithelium of suckling rats, we showed that both LHRH and its antagonist exert inhibitory responses. (Gama and Alvares, 1996, 1999). It is not known whether this inhibition is promoted directly by LHRH on gastric cells or by indirect effects on the stimulatory action of other growth factors. Although epidermal growth factor (EGF) and GnRH were shown to interact in pituitary cells (Leblanc et al., 1997), in the gastrointestinal tract, the activity of milk-borne factors remains unclear. Goldfeder and Alvares (1995) demonstrated that LHRH inhibits gastric cell proliferation in vitro, suggesting an isolated response that might be controlled by local binding sites.

A major drawback in the study of LHRH receptors in the different organs has been the diversity of techniques used to identify them. Since the 1970s, the internalization of labeled probes at light and electron microscope levels (Hopkins and Gregory, 1977; Hazum et al., 1980; Pelletier et al., 1982; Jennes, 1990) has been used, but antibodies to the receptor have only recently been tested (Karande et al., 1995; Rajeshwari and Karande, 1999). In this study, we have used an indirect procedure to determine the presence of LHRH binding sites in the gastric mucosa either by treating suckling rats with the hormone and checking for its presence in the tissue or by incubating cells with a fluorescent molecule and after its internalization. Observations were carried out at confocal laser scanning, and the colocalization in double-labeling experiments was analyzed by Silicon Graphics.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Animals

Wistar rats 18 days old were used from the Histology and Embryology Department Animal Colony according to the Procedures of the Animal Ethic Committee. Pregnant females were kept in isolated cages until the time of birth. The delivery day was set as day 0, and litters were culled to eight pups around the third day. Animals were kept under 12/12 hr light–dark schedule. Water was offered ad libitum, and it was weekly supplemented with a 0.9% multivitamin complex (Vitagold, São Paulo, Brazil).

Rats were injected i.p. with LHRH (1 μg/g body weight) (Relisorm, Serono, Mexico) and anesthetized with Rompun (Bayer, São Paulo, Brazil) and Ketalar (Parke-Davis, São Paulo, Brazil) (1:1, 0.5 ml/100 g body weight) immediately and at 1 and 2 hr after treatment.

Immunohistochemistry

Gastric samples were fixed in Bouin's solution and processed for embedding in paraffin. Five-micron sections were obtained, cleared of paraffin, rehydrated in 0.05 M PBS (pH 7.4), containing 1% bovine serum albumin (BSA) and 0.03% Triton X-100, and incubated in 10% goat serum. Then, sections were incubated with rabbit anti-LHRH (Peninsula Labs., Belmont, CA; Chemicon Intl., Temecula, CA) at 1:50 and 4°C for overnight, washed in PBS/BSA/Triton, and incubated in the secondary goat fluorescein isothiocyanate (FITC) -antibody (1:50) (Sigma-Genosys, TX) for 1 hr at room temperature (RT) in a dark chamber. After washing, the slides were mounted in 60% glycerol in PBS. For double-labeling experiments, the fluorescent lectins, Dolicus biflorum (DBA), Ulex europeus (UEA) agglutinins-TRITC and Concanavalin-A–TRITC (Sigma-Genosys, TX) (10 μg/ml) were added to the secondary antibody solution, incubated for 1 hr at room temperature and mounted as above. In control sections, the primary antibody was substituted by goat serum.

Parietal Cell Isolation

To test whether parietal cells were labeled by LHRH, the gastric mucosa of 18-day-old rats was scraped and processed after some modifications introduced to the method of Berglindh and Obrink (1976). Briefly, samples in 0.01 M PBS were centrifuged at 3,000 rpm and incubated with collagenase IV (Gibco, Rockville, MD) for 90 min at 37°C with agitation. After centrifugation at 2,000 rpm, the pellet was washed with 0.01 M PBS, centrifuged again, and treated with 0.12% EDTA for 30 min at 37°C. The mixture was centrifuged, and Leibovitz culture medium (Gibco, Rockville, MD) was added to the pellet, so that cells could be maintained for some hours. Cell suspension was plated onto coverslips and maintained in HotBox chambers for 3 hr under a 5% CO2 and 95% O2 tension at room temperature. Some coverslips were fixed in Bouin's solution and stained with hematoxylin-eosin, and others were incubated with 10 nM LHRH-FITC solution (1 hr in the dark) that was prepared according to Calbiochem FITC conjugation kit (Calbiochem, La Jolla, CA). DBA-TRITC and ethidium bromide (1:104) were also added to the culture medium for 60 and 30 min, respectively, for double labeling experiments. The coverslips were mounted onto slides containing 60% glycerol in PBS, rapidly frozen in dry ice and kept at −20°C. Controls for LHRH labeling were done in 5-μm sections of ovaries and pituitaries treated as above with the fluorescent molecule.

Confocal Microscopy

Gastric sections and isolated parietal cells were first studied in a Zeiss Axioscope 2 fluorescence microscope and then in a Zeiss LSM 510 confocal microscope by using two excitation line lasers at 488 nm (Argon) and 543 nm (Helium-Neonium). The data from the channels were collected simultaneously with fourfold averaging at a resolution of 512 × 512 pixels. The microscope was an inverted Zeiss Axioplan, and images were collected with 63× oil immersion objective lens, N.A. 1.4. In double-labeling experiments, channels were also analyzed separately. For quantitative analysis of colocalization, Z-series were generated by collecting stacks of 20–40 optical slices by using a step size around 0.35 μm in the z-direction. Stacks were exported to Silicon Graphics and processed by Bitplane Colocalization Software. To localize LHRH labeling in the cell, three-dimensional (3-D) surface reconstructions were used and stacks were also exported and analyzed in Silicon Graphics by Imaris 2.7 and 3.0 softwares, from Bitplane.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

To test whether polyclonal antibodies recognize the commercial LHRH injected in the animals we used the dot blotting assay, in which we incubated trans-blot membranes with 2 μg of LHRH and checked the reactivity of the two polyclonal antibodies (Peninsula Labs., Belmont, MA; Chemicon Intl., Temecula, CA) at different concentrations. We observed that both polyclonal antibodies label LHRH (data not shown) up to a dilution of 1:400, suggesting that they could be used in our immunohistochemical experiments.

Localization of LHRH Binding Sites in the Gastric Mucosa

We have evidence that one of the possible mechanisms by which LHRH inhibits cell proliferation in the developing gastric mucosa might be by the presence of binding sites in the stomach (Goldfeder and Alvares, 1995; Gama and Alvares, 1996, 1999). To localize them, we attempted to detect the injected LHRH molecules in the gastric mucosa by immunohistochemistry. Different periods of treatment were used, and we compared controls (Fig. 1A), represented by immediate sacrifice, to 1- and 2-hr treatments (Fig. 1B). We observed that after 1 hr, LHRH labeling clearly appeared in some cells in gastric glands, whereas no labeling could be noted in the control group. After 2 hr of treatment, LHRH signal was localized similar to the 1-hr-treated group (images not shown). To localize which cells were labeled by LHRH, we used fluorescent lectins that are known to bind to specific cell populations in the gastrointestinal tract (Katsuyama and Spicer, 1978; Falk et al., 1994; Li et al., 1998). Concanavalin-A did not bind to chief cells, as expected, and such a result might be associated with the different composition of zymogen granules at 18 days (Katsuyama and Spicer, 1978). UEA clearly established the limit between the gastric pit and gland (Fig. 1C) by labeling mucosal superficial cells. None of these cells was double labeled by LHRH. DBA defined the parietal acid-producing cells that were also positive to LHRH antibodies (Fig. 1D).

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Figure 1. Luteinizing-hormone releasing hormone (LHRH) antibodies localize binding sites in paraffin sections of the gastric mucosa by confocal microscopy (A,B). Views of untreated rat (A) or treated with LHRH for 1 hr (B) and stained for LHRH. C:Ulex europeus (UEA) -lectin (red recolored to blue) labels the surface mucosal cells in the stomach, establishing the limit between the gastric pit and gland. D: Double-labeling of treated gastric glands with Dolicus biflorum (DBA) -lectin (red) that stains parietal cells and LHRH (green), resulting in an orange interference color. The four figures represent single optical sections obtained with a pinhole approximately 100 μm. Scale bars = 20 μm in A–D.

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Colocalization Studies

Our observation that LHRH binding sites might be in parietal cells prompted us to test the degree of colocalization in double-labeling experiments. We examined different optical stacks by confocal microscopy and we verified that the labelings from LHRH and DBA overlapped, resulting in the orange interference color (Fig. 1D). By image analysis with Silicon Graphics, the overlapping between signals was quantified, statistically analyzed, and transformed both into graphics (Fig. 2A) and dark speckles over the original field (Fig. 2B,C). After studying different images, the ratio of colocalization between LHRH and DBA signals, that measures how similar they are, ranged from 85 to 100%. All this calculation was performed by Bitplane Colocalization. Correlation values from 74 to 94% were also obtained, indicating that some background was present and interfered in the adjustment of the images. This additional statistical information showed that the degree of colocalization was high, but background was not avoided when we used our polyclonal antibodies.

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Figure 2. Colocalization of luteinizing-hormone releasing hormone (LHRH) and Dolicus biflorum (DBA) -lectin signals in confocal optical sections obtained from the gastric mucosa of treated rats. A: The two-dimensional histogram is mapping all the possible colocalization events in the gastric section (B), because it provides for each pair of intensities the information of how often it occurs. Perfect colocalization results in a diagonal line (r2 = 1) in the histogram and r2 = 0.95 was measured in A. C: View of the gastric section in B with black speckles over colocalization sites that appear to be inside the cell. Colocalization analysis was performed by Bitplane Colocalization software. Scale bar = 20 μm in B (applies to B,C).

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Because LHRH binding sites were shown in parietal cells, we attempted to isolate them and determine how they are labeled by free LHRH-FITC. The efficiency of cell isolation was confirmed when coverslips were fixed and stained with hematoxylin-eosin, and the morphology was checked by light microscopy.

The optical stacks obtained above were from paraffin sections, thus, the isolation procedure allowed us to reconstruct the whole cell (Fig. 3A), and have a 3-D view. Figure 3B shows the DBA-labeling, representing a reconstruction of the whole cell, in which we observe a dense and irregular surface. In Figure 3C, a complex convoluted structure was labeled by LHRH-FITC, suggesting a surface that follows the shape of the cell, does not cover the nucleus and seems to be folded. In Figure 3D, the nucleus is visualized by ethidium bromide staining, and the LHRH-FITC signal can be observed around it, forming the complex network mentioned in Figure 3C. When the double labeling with DBA-TRITC and LHRH-FITC was studied in different optical stacks and in the 3-D reconstruction (Fig. 3A), we observed that, whereas DBA bound to a more external structure that resembles the plasma membrane, LHRH binding sites seemed to be more internalized. By making DBA labeling transparent in Figure 3E, we confirmed this observation, because when both signals were in the same structure only LHRH-FITC could be noted. These cells were also studied for colocalization and ratios around 85% were obtained, reassuring that both signals are localized in the membrane. These results support the idea that DBA lectin that binds specifically to N-acetyl-D-galactosaminyl carbohydrate residue completely labels the plasma membrane, including intracytoplasmic canaliculi. This structure represents invaginations of the plasma membrane within the parietal cell and may be functionally active, i.e., covered by microvilli, or inactive and folded into tubulovesicles. According to our observations, presented in Figure 3, LHRH binding sites seem to be restricted to this canalicular system.

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Figure 3. Treatment of isolated parietal cells with luteinizing-hormone releasing hormone–fluorescein isothiocyanate (LHRH-FITC) and Dolicus biflorum (DBA) -lectin characterizes the distribution of labeling in the membrane system of the intracellular canaliculus (A–D). A: View of parietal cell labeled by DBA (red) and LHRH (green) shown as a volume reconstruction with shadow projection (Easy 3D, Imaris 3.0 Software). Note that the nucleus is not seen, because the whole cell is visualized. DBA signal covers the cell, and LHRH appears more internalized. (B–D) three-dimensional iso-surface reconstructions of parietal cells by using Imaris 2.7 Software with electronic zoom. B: DBA labels the dense irregular surface consistent with A, suggesting the structure of the plasma membrane. C,D: LHRH labels a convoluted irregular network that may characterize the canalicular region (C) around the nucleus stained by ethidium bromide (D). E: Parietal cells in a paraffin section reconstructed as mentioned above, in which the DBA signal was transformed into a transparency, allowing the visualization of internal LHRH-labeled structure. Note that LHRH does not extend through the whole cell, confirming the intracellular canalicular region. F: Paraffin section of the pituitary incubated with LHRH-FITC and used as a control, showing dotted signals around the cells. Scale bars = 2 μm in A, 20 μm in F.

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The control of LHRH-FITC reactions was performed in pituitary and ovary sections, that are target organs for LHRH, and we observed positive signals with a dotted pattern at the periphery of the cells (Fig. 3F) in the pituitary. In the ovaries, labeling was determined in the granulosa cells of preantral follicles and in the interstitial cells (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

LHRH is a decapeptide synthesized by hypothalamic neurons, which integrates neural and endocrine systems and regulates reproductive function. It stimulates gonadotropes of the anterior pituitary to release LH and FSH by means of specific receptors (Conn, 1986). The extrapituitary roles for LHRH in normal and malignant tissues have been discussed (Szende et al., 1991, 1994; Imai et al. 1992; Chatzaki et al., 1996; Imai and Tamaya, 2000), and throughout the suckling period, active LHRH is supplied to the pup (Smith-White and Ojeda, 1984) and may be one of the milk-borne factors that control cell proliferation in the stomach (Gama and Alvares, 1996, 1999). In the gastrointestinal tract, Szende et al. (1991) localized LHRH binding sites in pancreatic tumors and characterized them as nuclear and membrane receptors of high and low affinities, respectively. We have some evidence of binding sites in the stomach, because Goldfeder and Alvares (1995) demonstrated LHRH inhibitory action on the gastric epithelium maintained in vitro, i.e., isolated from other factors that might interfere with the hormone response.

Although abundant information on the pituitary GnRH receptor is available, understanding of binding to some extrapituitary sites is still lacking. The methods to study these receptors have been the most diverse, from internalization of labeled probes at light and electron microscope levels (Hopkins and Gregory, 1977; Hazum et al., 1980; Pelletier et al., 1982; Jennes, 1990) to isolation and characterization of the cDNA encoding for the sequence (Kaiser et al., 1992). We used an indirect procedure to determine the presence of LHRH binding sites in the gastric mucosa either by treating 18-day-old rats with the hormone and checking for its presence in the tissue, or by incubating cells with a fluorescent molecule and after its incorporation. We observed positive cells in the epithelium, and we used lectins to identify specific cell types. Lectins are considered sensitive tools for defining cell populations in the gastrointestinal tract (Falk et al., 1994; Li et al., 1998), and we noted that from those that we tested at 18 days, only UEA and DBA stained specific cells. DBA-labeled parietal cells and LHRH colocalized with it in the different analyses, suggesting that binding sites for LHRH are localized in these cells. Gastrin receptor (Nakamura et al., 1987), TGFα and EGFR (Beauchamp et al., 1989; Hormi and Lehy, 1994; Hormi et al., 1995) and other gastric peptides were also identified in parietal cells, and appear to be functionally involved in acid secretion regulation.

Parietal cells produce HCl inside the intracytoplasmic canaliculi that are invaginations of the apical surface membrane within the cell. H+/K+ ATPase pump is located in this membrane system that is rearranged according to feeding condition, in a way that when the stomach is empty, acid secretion is low and most of the canalicular membrane is enclosed inside the cell as tubulovesicles. These dynamic changes are promoted by cytoskeletal components and lasp-1 and ezrin seem to regulate actin activity in such specific membrane trafficking events in the cell (Chew et al., 2000). DBA-lectin binds specifically to N-acetyl-D-galactosaminyl carbohydrate residues, and labeled the plasma membrane in different levels, i.e., outside and inside the cell, probably including intracellular canalicular region. We used 3-D surface reconstructions (Fig. 3) to evaluate how the signals colocalized and we observed that DBA labeling covered a larger area compared with LHRH sites, which appeared to be internalized, suggesting that LHRH signal might not be on the surface of the cell. By reconstructing only LHRH-positive structure, we demonstrated that it was an elaborated and convoluted network inside the cell and around the nucleus, which resembled the canaliculus. Thus, both DBA and LHRH were colocalized in the intracytoplasmic membranes of the canaliculus, whereas the surface of the cell was labeled by DBA only.

GnRH or LHRH pituitary receptors were also identified in dissociated gonadotropes incubated with molecules labeled with ferritin (Hopkins and Gregory, 1977), fluorescein (Hazum et al., 1980), and radioactive ligands (Pelletier et al., 1982; Marian and Conn, 1983; Jennes, 1990). These studies showed that the hormone binds to surface receptors, the complexes are internalized into endocytic vesicles, and within hours labeling is seen in the Golgi apparatus and in lysosomes. We did not attempt to trace the LHRH-FITC conjugate, although at electron microscope level, we observed LHRH binding sites also close to and in the nucleus (data not shown). The meaning of nuclear receptors is puzzling, because they were reported in tumors and might be involved in the cell cycle control, an effect that is not exerted in pituitary cells. By treating pancreatic (Szende et al., 1989, 1990), breast (Brower et al., 1992), and uterine (Mizutami et al., 1998) tumors with LHRH or GnRH, cell proliferation rates decreased and cell death was enhanced. Imai et al. (1998a,b) observed that apoptosis is triggered by increased Fas-ligand levels, in uterine and ovarian tumor cells stimulated with GnRH, suggesting that the antiproliferative action of the hormone is exerted in that way. Previously, we demonstrated that LHRH and its antagonist inhibit the hyperproliferation in the gastric epithelium of suckling rats and that apoptotic figures can also be seen (Gama and Alvares, 1999). As mentioned before, the exact role of the hormone in this regulation has not yet been elucidated, but it seems likely that, during postnatal development, milk-borne LHRH takes part in the complex maturation of the gastrointestinal tract. Therefore, we assume that the presence of LHRH binding sites in the gastric epithelium might be connected to the antiproliferative effects, suggesting a direct action of LHRH in the stomach of pups. Future work is required to determine the relevance of LHRH binding sites in gastric tumors, to evaluate whether the identified binding sites are functional receptors or not, and to understand LHRH major role throughout postnatal development.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Cruz Alberto Rigonati for technical assistance, and Roberto M. Cabado is acknowledged for his assistance with confocal microscopy and Silicon Graphics.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  • Baram T, Koch Y, Hazum E, Fridkin M. 1977. Gonadotropin-releasing hormone in milk. Science 198: 300302.
  • Beauchamp RD, Barnard JA, McCutchen CM, Cherner JA, Coffey RJ Jr. 1989. Localization of transforming growth factor α and its receptor in gastric mucosal cells. J Clin Invest 84: 10171023.
  • Berglindh T, Obrink KJ. 1976. A method for preparing isolated glands from the rabbit gastric mucosa. Acta Physiol Scand 96: 150159.
  • Brower ST, Schally AV, Redding TW, Hollander VP. 1992. Differential effects of LHRH and somatostatin analogs on human breast cancer. J Surg Res 52: 614.
  • Chatzaki E, Bax CMR, Eidne KA, Anderson L, Grudzinkas JG, Gallagher CJ. 1996. The expression of gonadotropin-releasing hormone and its receptor in endometrial cancer, and its relevance as an autocrine growth factor. Cancer Res 56: 20592065.
  • Chew CS, Parente JA, Chen X, Chapponier C, Cameron RS. 2000. The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia. J Cell Sci 113: 20352045.
  • Conn PM. 1986. The molecular basis of gonadotropin-releasing hormone action. Endocr Rev 7: 310.
  • Falk P, Roth KA, Gordon JI. 1994. Lectins are sensitive tools for defining the differentiation programs of mouse gut epithelial cell lineages. Am J Physiol 266: G987G1003.
  • Gama P, Alvares EP. 1996. LHRH and somatostatin effects on the cell proliferation of the gastric epithelium of suckling and weaning rats. Regul Pept 63: 7378.
  • Gama P, Alvares EP. 1999. LHRH antagonist inhibits gastric cell proliferation in suckling rats. Regul Pept 84: 97100.
  • Goldfeder EM, Alvares EP. 1995. Effects of somatostatin and LHRH on cell proliferation of fetal stomach in vitro. Cell Prolif 28: 194.
  • Hazum E, Cuatrecasas P, Marian J, Conn PM. 1980. Receptor-mediated internalization of fluorescent gonadotropin-releasing hormone by pituitary gonadotropes. Proc Natl Acad Sci USA 77: 66926695.
  • Hopkins CR, Gregory H. 1977. Topographical localization of the receptors for luteinizing -hormone releasing hormone on the surface of dissociated pituitary cells. J Cell Biol 75: 528540.
  • Hormi K, Lehy T. 1994. Developmental expression of transforming growth factor α and epidermal growth factor receptor proteins in the human pancreas and digestive tract. Cell Tissue Res 278: 439450.
  • Hormi K, Onolfo JP, Gres L, Lebraud V, Lehy T. 1995. Developmental expression of transforming growth factor α in the upper digestive tract and pancreas of the rat. Regul Pept 55: 6777.
  • Imai A, Tamaya T. 2000. GnRH receptor and apoptotic signaling. Vitam Horm 59: 133.
  • Imai A, Tatsuro F, Tamaya T. 1992. Is extrapituitary action of gonadotrophin-releasing hormone biologically significant? Ann Clin Biochem 29: 477480.
  • Imai A, Takagi A, Horibe S, Takagi H, Tamaya T. 1998a. Evidence for tight coupling of gonadotropin-releasing hormone receptor to stimulated Fas ligand expression in reproductive tract tumors: possible mechanism for hormonal control of apoptotic cell death. J Clin Endocrinol Metab 83: 427431.
  • Imai A, Takagi A, Horibe S, Takagi H, Tamaya T. 1998b. Fas and Fas ligand system may mediate antiproliferative activity of gonadotropin-releasing hormone receptor in endometrial cancer cells. Int J Oncol 13: 97100.
  • Jennes L. 1990. Postnatal development of gonadotropin-releasing hormone receptors in the rat anterior pituitary. Endocrinology 126: 942947.
  • Jungwirth A, Schally AV, Halmos G, Groot K, Szepeshazi K, Pinski J, Armatis P. 1998. Inhibition of growth of caki-I human renal adenocarcinoma in vivo by luteinizing hormone-releasing hormone antagonist Cetrorelix, somatostatin analog RC-160, and bombesin antagonist RC-3940 II. Cancer 82: 909917.
  • Kaiser UB, Zhao D, Cardona GR, Chin WW. 1992. Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun 189: 16451652.
  • Karande AA, Rajeshwari K, Schol DJ, Hilgers JH. 1995. Establishment of immunological probes to study human gonadotropin-releasing hormone receptors. Mol Cell Endocrinol 114: 5156.
  • Katsuyama T, Spicer SS. 1978. Histochemical differentiation of complex carbohydrates with variants of concanavalin A-horseradish peroxidase method. J Histochem Cytochem 26: 233250.
  • Kogo H, Fujimoto T, Park MK, Mori T. 1999. Gonadotropin-releasing hormone receptor mRNA expression in the ovaries of neonatal and adult rats. Cell Tissue Organs 164: 1422.
  • Koldovský O. 1989. Search for role of milk-borne biologically active peptides for the suckling. J Nutr 119: 15431551.
  • Leblanc P, L'Heritier A, Kordon C. 1997. Cryptic gonadotropin-releasing hormone receptors of rat pituitary cells in culture are unmasked by epidermal growth factor. Endocrinology 138: 574579.
  • Li Q, Karam SM, Coerver KA, Matzuk MM, Gordon JI. 1998. Stimulation of activin receptor II signaling pathways inhibits differentiation of multiple gastric epithelial lineages. Mol Endocrinol 12: 181192.
  • Marchetti B, Guarcello V, Moralle MC, Bartoloni G, Raeti F, Palumbo G Jr, Farinella Z, Cordaro S, Scapagnini U. 1989. LHRH agonist restoration of age associated decline of thymus weight, thymic LHRH receptors, and thymocyte proliferative capacity. Endocrinology 125: 10371045.
  • Marian J, Conn PM. 1983. Subcellular localization of the receptor for gonadotropin-releasing hormone in pituitary and ovarian tissue. Endocrinology 112: 104112.
  • Mizutami T, Sugihara A, Nakamuro K, Terada N. 1998. Suppression of cell proliferation and induction of apoptosis in uterine leiomyoma by gonadotropin-releasing hormone agonist (leuprolide acetate). J Clin Endocrinol Metab 83: 12531255.
  • Nakamura M, Oda M, Kaneko K, Yoney Y, Tsukada N, Komatsu H, Tsugu M, Tsuchiya M. 1987. Autoradiographic demonstration of gastrin binding sites in the rat gastric mucosa. Peptides 8: 391398.
  • Pelletier G, Dube D, Guy J, Seguin C, Lefebvre FA. 1982. Binding and internalization of a luteinizing hormone releasing hormone agonist by rat gonadotrophic cells. Endocrinology 111: 10681076.
  • Rajeshwari K, Karande AA. 1999. Molecular mimicry by antiidiotypic monoclonal antibody to gonadotropin releasing hormone. Immunol Invest 28: 103114.
  • Smith-White S, Ojeda SR. 1984. Maternal modulation of infantile ovarian development and available LHRH receptors via rat milk LHRH. Endocrinology 115: 19731983.
  • Szende B, Zalatnai A, Schally AV. 1989. Programmed cell death (apoptosis) in pancreatic cancers of hamsters after treatment with analogs of both luteinizing hormone-releasing hormone and somatostatin. Proc Natl Acad Sci USA 86: 16431647.
  • Szende B, Srkalovic G, Groot K, Lapis K, Schally AV. 1990. Regression of nitrosamine-induced pancreatic cancers in hamsters treated with luteinizing hormone-releasing-hormone antagonists or agonists. Cancer Res 50: 37163721.
  • Szende B, Srkalovic G, Timar J, Mulchahey JJ, Neill JD, Lapis K, Csikos A, Szepeshazi K, Schally AV. 1991. Localization of receptors for luteinizing-releasing-hormone in pancreatic and mammary cancer cells. Proc Natl Acad Sci USA 88: 41534156.
  • Szende B, Csikos A, Szepeshazi K, Neill JD, Mulchahey JJ, Halmos G, Lapis K, Schally AV. 1994. The concentration of LH-RH receptors in the nuclei of pancreatic cancer cells. Receptor 4: 201207.