- Top of page
- MATERIALS AND METHODS
- 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.
- Top of page
- MATERIALS AND METHODS
- 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.