Sexual Experience Induces the Expression of Gastrin-Releasing Peptide and Oxytocin Receptors in the Spinal Ejaculation Generator in Rats.

Male sexual function in mammals is controlled by the brain neural circuits and the spinal cord centers located in the lamina X of the lumbar spinal cord (L3-L4). Recently, we reported that hypothalamic oxytocin neurons project to the lumbar spinal cord to activate the neurons located in the dorsal lamina X of the lumbar spinal cord (dXL) via oxytocin receptors, thereby facilitating male sexual activity. Sexual experiences can influence male sexual activity in rats. However, how this experience affects the brain-spinal cord neural circuits underlying male sexual activity remains unknown. Focusing on dXL neurons that are innervated by hypothalamic oxytocinergic neurons controlling male sexual function, we examined whether sexual experience affects such neural circuits. We found that >50% of dXL neurons were activated in the first ejaculation group and ~30% in the control and intromission groups in sexually naïve males. In contrast, in sexually experienced males, ~50% of dXL neurons were activated in both the intromission and ejaculation groups, compared to ~30% in the control group. Furthermore, sexual experience induced expressions of gastrin-releasing peptide and oxytocin receptors in the lumbar spinal cord. This is the first demonstration of the effects of sexual experience on molecular expressions in the neural circuits controlling male sexual activity in the spinal cord.

The majority of dorsal spinal cord gastrin releasing peptide is synthesized locally whereas neuromedin B is highly expressed in pain- and itch-sensing somatosensory neurons.

BACKGROUND: Itch is one of the major somatosensory modalities. Some recent findings have proposed that gastrin releasing peptide (Grp) is expressed in a subset of dorsal root ganglion (DRG) neurons and functions as a selective neurotransmitter for transferring itch information to spinal cord interneurons. However, expression data from public databases and earlier literatures indicate that Grp mRNA is only detected in dorsal spinal cord (dSC) whereas its family member neuromedin B (Nmb) is highly expressed in DRG neurons. These contradictory results argue that a thorough characterization of the expression of Grp and Nmb is warranted. FINDINGS: Grp mRNA is highly expressed in dSC but is barely detectable in DRGs of juvenile and adult mice. Anti-bombesin serum specifically recognizes Grp but not Nmb. Grp is present in a small number of small-diameter DRG neurons and in abundance in layers I and II of the spinal cord. The reduction of dSC Grp after dorsal root rhizotomy is significantly different from those of DRG derived markers but similar to that of a spinal cord neuronal marker. Double fluorescent in situ of Nmb and other molecular markers indicate that Nmb is highly and selectively expressed in nociceptive and itch-sensitive DRG neurons. CONCLUSION: The majority of dSC Grp is synthesized locally in dorsal spinal cord neurons. On the other hand, Nmb is highly expressed in pain- and itch-sensing DRG neurons. Our findings provide direct anatomic evidence that Grp could function locally in the dorsal spinal cord in addition to its roles in DRG neurons and that Nmb has potential roles in nociceptive and itch-sensitive neurons. These results will improve our understanding about roles of Grp and Nmb in mediating itch sensation.

The Ewing tumor family of peripheral primitive neuroectodermal tumors expresses human gastrin-releasing peptide.

The Ewing tumor family of peripheral primitive neuroectodermal tumors (pPNETs) are characterized by chromosomal translocations leading to EWS-ETS gene fusions. These hybrid genes express chimeric proteins that are thought to act as aberrant transcription factors. We therefore used differential display-PCR to compare gene expression patterns in pPNET cell lines with those of other small round cell tumors (SRCTs) of childhood. This technique detected differential expression of sequences corresponding to human gastrin-releasing peptide (GRP) in pPNET cell lines but not in other SRCT cell lines. Subsequent Northern and reverse transcription-PCR analysis of SRCT cell lines confirmed GRP positivity in all pPNET lines tested. Of primary tumors tested by reverse transcription-PCR, GRP expression was found in 7 (44%) of 16 pPNETs but in no other primary SRCTs examined. Expression of the GRP receptor gene was demonstrable in 55% of pPNET cell lines and 25% of primary pPNET tumors but also in several other SRCTs. Radioimmunoassays and immunohistochemistry confirmed expression of bioactive GRP peptide in pPNET cell lines and primary tumors, respectively. Moreover, in vitro growth of a pPNET cell line was slowed by treatment with a GRP receptor antagonist and accelerated by a GRP receptor agonist. GRP is a known autocrine growth factor in small cell lung cancer and other neuroendocrine tumors. Its expression in pPNETs provides further evidence for a neuroectodermal histogenesis of these tumors and suggests that autocrine growth of this family of tumors may be at least partially regulated by GRP.

Contribution of gastrin-releasing peptide and its receptor to villus development in the murine and human gastrointestinal tract.

Recent studies have shown that aberrantly expressed gastrin-releasing peptide (GRP) and its receptor (GRP-R) critically regulate tumor cell differentiation in colon cancers developing in humans and mice. This finding suggested that the ability of GRP/GRP-R to promote a well-differentiated phenotype in colon cancer might reflect a re-capitulation of a normal role in regulating intestinal organogenesis. To determine if this was the case, we compared and contrasted intestinal development in GRPR-/- mice with their wild type littermates. GRP/GRP-R co-expression in wild type mice was only observed in villous enterocytes between N-1 and N-12. During this time frame villous growth was completely attenuated in GRPR-/- mice. The contribution of GRP/GRP-R to villous growth was due to their act in increasing enterocyte proliferation prior to N-8 but increasing enterocyte size thereafter. From N-12 onwards, small intestinal villous growth in GRPR-/- mice resumed such that no difference in this structure could be detected at adulthood between mice of either genotype. We next studied GRP/GRP-R expression in human abortuses. These proteins were co-expressed by villous enterocytes only between weeks 14 and 20 post-conception, a time frame analogous to when they are expressed in the murine intestine. Thus, this study shows for the first time that GRP/GRP-R play a transient and non-critical role in intestinal development, yet provides a rationale for their re-appearance in colon cancer.

Calbindin-D(28K) cells selectively contact intra-SCN neurons.

Calbindin-D(28K)-immunoreactive cells are tightly packed within a discrete region of the caudal aspect of the suprachiasmatic nuclei of hamsters. These cells receive direct retinal input and are Fos-positive in response to a light pulse. Knowledge of their afferent and efferent connections is necessary to understand suprachiasmatic nucleus organization. The first aim of the present study is to identify interconnections between calbindin and other peptidergic cells of the suprachiasmatic nuclei, using epi- and confocal microscopy and intra-suprachiasmatic nucleus tract tracing. The results indicate that essentially all calbindin cells receive numerous appositions from vasoactive intestinal polypeptide (VIP), neuropeptide Y and serotonin fibers and that most receive appositions from gastrin releasing peptide (GRP) and cholecystokinin (CCK) fibers. Reciprocal connections are seen from VIP, GRP and CCK cells but surprisingly, not from dorsomedial vasopressin cells. Injection of biotinylated dextran amine into the suprachiasmatic nucleus indicates that the ventrolateral suprachiasmatic nucleus projects to the entire nucleus, while the dorsal and medial regions of the suprachiasmatic nucleus project densely to most of the nucleus, except to the calbindin region. Analysis of colocalization of the peptides in the calbindin cell region shows that 91% of the substance P cells, 42% of the GRP cells and 60% of the VIP cells in the calbindin subnucleus coexpress calbindin-D(28K). Our results reveal a highly specialized topographical organization of connections among suprachiasmatic nucleus cells.

Tissue-specific regulation of gastrin-releasing peptide synthesis, storage and secretion by oestrogen and progesterone.

In the ovine endometrium, dramatic increases in gastrin-releasing peptide (GRP) mRNA and immunoreactivity are observed during the luteal regression phase of the oestrous cycle (24-fold) and during pregnancy (at least 150-fold). This study sought to determine whether oestrogen and/or progesterone were responsible for the temporal regulation of GRP observed in the uterus. Ovariectomized sheep were divided into four groups (n=4), as follows: 1, untreated; 2, given subcutaneous and intravaginal progesterone implants; 3, given subcutaneous oestrogen implants; and 4, treated with both oestrogen and progesterone. After 10 days, the animals were sacrificed and plasma, pituitary and endometrium were obtained. A fifth group of sheep with intact ovaries was included. Analysis of endometrial GRP-immunoreactivity (GRP-ir) revealed a twofold drop for groups treated with oestrogen, progesterone or both hormones. A dramatic reduction in endometrial GRP mRNA was o! bserved in the group treated with both hormones. GRP-ir was measured in whole pituitaries and found to vary greatly (1.7-53.7 pmol/g tissue) within all groups of ovariectomized animals. There were no significant differences between any of the five groups. A significant reduction in circulating GRP-ir was observed after 10 days of treatment with either oestrogen or progesterone. These studies demonstrate that, in sheep, the synthesis, storage and secretion of GRP are differentially affected by oestrogen and progesterone. Regulation appears to be tissue specific since GRP content in the pituitary is unchanged by oestrogen or progesterone whereas GRP expression in the endometrium is inhibited. Changes in GRP mRNA expression did not correlate with changes in endometrial expression of mRNA for oestrogen receptor alpha, oestrogen receptor beta and the progesterone receptor. This study is the first reported demonstration that expression of the GRP gene can be influenced by the presence of ovarian steroids, with the conclusion that oestrogen and/or progesterone act as negative regulators of endometrial GRP expression.

Dexamethasone regulation of gastrin-releasing peptide receptor in human lung cells.

We investigated the effects of the glucocorticoid, dexamethasone (Dex), on expression of the gastrin-releasing peptide (GRP) receptor by human small cell lung carcinoma (SCLC) SHP77 cells. After 12h of 10nM Dex exposure, a six-fold increase in the peak of GRP receptor mRNA compared with untreated controls (10.5+/-4 versus 1.65+/-0.15 attomols/microg total RNA, respectively, P<0.05) occurred. GRP receptor mRNA levels fell to less than 0.5 attomols/microg total RNA after 24h; in Dex-treated cells, these levels rose to 1.2 compared with 0.12 attomols/microg total RNA in the absence of Dex after 7 days. A significant increase (P<0.05) in the GRP receptor-specific binding was also found. Stimulation of SHP77 cell proliferation (25-35% in the presence of 10-100 nM Dex; P<0.0001) was observed after 4-8 days of exposure; this stimulation was inhibited by GRP receptor antagonists. SHP77 cell content and concentration of bombesin-like peptides (BLP) in conditioned medium (approximately 4 nM) was unchanged by Dex. Stimulation of human SCLC SHP77 cell proliferation by Dex may, in part, occur via effects on the GRP autocrine system in these cells.

Inhibition of proliferation of human small cell lung cancer cells expressing an autocrine system for gastrin releasing peptide by antisense oligodeoxynucleotides to gastrin releasing peptide receptor.

The objectives of this study were to investigate the effect of antisense (AS) oligodeoxynucleotides (ODNs) directed against gastrin releasing peptide (GRP) receptor mRNA on proliferation of human small cell lung cancer (SCLC) NCI-H345 cells which express the autocrine system for GRP. The methods used were to expose human SCLC cell lines to antisense ODNs or sense ODNs and to measure their proliferation by spectrophotometric assay or viable cell counts. Our results demonstrated that the single or combined AS ODNs against GRP receptor inhibited proliferation of human SCLC NCI-H345 cells significantly by 37% (P<0.01), but did not inhibit proliferation of either human bronchial epithelial BEAS 2B cells or human SCLC NCI-N417 cells, neither of which express the GRP autocrine system. The sense controls did not significantly inhibit proliferation compared with no treatment controls. Specificity was also demonstrated by the observation that cells exposed to AS ODNs had a decrease in GRP receptor expression as measured by specific binding of 34% (P<0.01), and when all three AS ODNs were used, binding was decreased by 60% (P<0.03). Furthermore, AS ODNs decreased by 75% the maximum percentage of cells responding to GRP in an intracellular calcium release assay. Our conclusions are that antisense ODNs directed against a GRP receptor which is involved in an autocrine loop in human SCLC cells inhibited proliferation of these cells by their impact on reducing GRP receptor expression. Further development of means of increasing AS ODN specificity and effectiveness in human SCLC cell is warranted.

Correlation of expression of bombesin-like peptides and receptors with growth inhibition by an anti-bombesin antibody in small-cell lung cancer cell lines.

The murine anti-bombesin monoclonal antibody, 2A11, has been demonstrated to inhibit growth of some small-cell lung cancer (SCLC) cells in nude mice xenografts and in a clinical trial. To determine if the expression of bombesin-like peptides (BLP) and their receptors (GRP-R and NMB-R) correlate with an in vitro response to 2A11, we measured these parameters in seven SCLC cell lines. Gastrin releasing peptide (GRP) mRNA was detected in three of seven cell lines (NCI-H69, NCI-H345, NCI-H510) and neuromedin B (NMB) mRNA was detected in all seven lines using an RNase protection assay (RPA). Immunoreactive BLP was detected in the cell pellets of all lines (range 0.11-59.90 pmol/mg protein) by a solid phase GRP radioimmunoassay (RIA) using 125I-labeled 2A11. RPA detected GRP-receptor mRNA in two cell lines (NCI-H69 and NCI-H345) and NMB-receptor in three lines (NCI-H345, NCI-H510, and NCI-H660). Reverse transcriptase-PCR confirmed the presence of receptor mRNA in these lines and detected NMB-receptor in an additional three lines (NCI-H69, NCI-H82, and NCI-H187). Calcium mobilization in response to BLP stimulation was detected in the six cell lines expressing either GRP-R or NMB-R mRNA but not in NCI-N417, which had no detectable BLP-receptor. 2A11 (5 microg/ml) inhibited colony formation by 26-61% after 2 weeks in all cell lines except NCI-N417. Thus, growth inhibition by 2A11 requires the presence of at least one BLP-receptor. These findings may be useful in selecting patients with SCLC for treatment with 2A11.

Growth stimulation of normal melanocytes and nevocellular nevus cells by gastrin releasing peptide (GRP).

In order to know the possible effects of gastrin releasing peptide (GRP) on nevus cells and melanocytes, we studied the effect of GRP on the proliferation of cultured human nevus cells and normal melanocytes. MTS assay showed that GRP stimulated the growth of viable melanocytes at 1000 ng/ml. GRP also stimulated the growth of nevus cells in a dose dependent manner and maximum stimulation was obtained at 100 ng/ml of GRP. GRP was less effective for growth stimulation of normal melanocytes than nevus cells. The cytoplasm of nevus cells were positively stained by polyclonal anti-GRP antibody. We also detected the expression of GRP and GRP receptor mRNAs in these cells by RT-PCR. These results suggest that GRP acts as an autocrine growth factor for nevus cells and normal melanocytes.

Bombesin-like peptide and receptors in lung injury models: diverse gene expression, similar function.

We previously demonstrated that bombesin-like peptide (BLP) mediates lung injury in premature infants with bronchopulmonary dysplasia (BPD). We now investigate gene expression and function of BLP (gastrin-releasing peptide, GRP) and BLP-receptors (GRP-R and BRS-3) in lung from two baboon BPD models. In the "interrupted gestation model," only GRP mRNA was up-regulated. In the "hyperoxic model," GRP-R mRNA was up-regulated. In lung explants from O2-treated animals, all BPD animals responded to 1nM bombesin, whereas non-BPD animals did not; the opposite effect was observed with a BLP blocking antibody. Cumulatively, these observations suggest that novel BLPs and/or BLP receptors are likely to be implicated in the pathogenesis of BPD.

Receptors and ligands for autocrine growth pathways are up-regulated when pancreatic cancer cells are adapted to serum-free culture.

Overexpression of autocrine growth factors and their receptors has been reported in many human cancers. The study of autocrine-regulated pathways using in vitro culture systems can be hindered by the presence of fetal bovine serum in culture medium. A human pancreatic cancer cell line (HPAF) was slowly weaned from its dependence on fetal bovine serum and subsequently maintained in serum-free conditions. Growth factor secretion studies showed that production of autocrine growth factors such as transforming growth factor alpha, gastrin-releasing peptide, and insulin-like growth factor I from weaned cells increased three times compared with nonweaned cells (p < 0.01). The epidermal growth factor and gastrin-releasing peptide receptor densities were also increased in weaned cells (2 times and 2.5 times, respectively, p < 0.05). The proliferation of weaned cells cultured continuously in the same medium was significantly greater than of nonweaned cells (p < 0.05). Collectively, these data indicate that weaned pancreatic cancer cells can proliferate in the absence of serum by up-regulating autocrine pathways.

Induction of gastrin releasing peptide by all-trans retinoic acid in small cell lung cancer cells.

All-trans retinoic acid (RA) has been shown to inhibit cell proliferation while increasing neuroendocrine differentiation in small cell lung cancer (SCLC) cells. RA and related compounds are rapidly becoming integrated into clinical trials to prevent lung cancers and other aerodigestive neoplasms. We found that expression of gastrin releasing peptide (GRP), which can promote lung tumorigenesis in model systems, was increased by RA in SCLC cells which have functional retinoid signaling. In SCLC cells that possess functional GRP receptors, ectopic expression of RARc increased GRP expression and augmented cloning efficiency, demonstrating that these maneuvers result in biologically active GRP. SCLC cells with defects in RA pathway signaling did not efficiently induce GRP upon RA exposure. In these cells, transfection of RARs rendered the cells competent to induce GRP upon RA exposure. These data show that activation of intact retinoid signaling by RA can induce GRP, a growth factor that can act as a tumor promoter. Our findings suggest the possibility that retinoids may increase, rather than decrease, lung cancer risks in some individuals.

[Morphofunctional characteristics of gastrin-releasing peptide synthesizing system of the hypothalamus in normal conditions and in experimental diabetes in rats]. / Morfofunktsional'naia kharakteristika gastrin-rilizing peptid-sinteziruiushchei sistemy gipotalamusa v norme i pri éksperimental'nom diabete u krys.

5 weeks development of streptozoticin-induced diabetes mellitus in the rats is accompanied with the increase of gastrin-releasing peptide (GRP) immunopositive neurons amount in parvocellular neurosecretory subdivisions of immunopositive fibers increased in these structures:the content of GRP increased in median eminence as well. In supraoptic nucleus and posterior magnocellular subdivision of paraventricular nucleus the amount of GRP-immunopositive neurons increased by the second week of diabetes development with its decrease by the fifth week. Thus, the increase of GRP synthesis in hypothalamic neurosecretory structures in diabetes mellitus may be considered as a compensatory reaction directed on the activation of the central mechanisms of feeding restriction and stimulation of insulin synthesis in the pancreas.

Aberrant activation of androgen receptor in a new neuropeptide-autocrine model of androgen-insensitive prostate cancer.

Treatment of advanced prostate cancer with androgen deprivation therapy inevitably renders the tumors castration-resistant and incurable. Under these conditions, neuroendocrine differentiation of prostate cancer (CaP) cells is often detected and neuropeptides released by these cells may facilitate the development of androgen independence. Exemplified by gastrin-releasing peptide (GRP), these neuropeptides transmit their signals through G protein-coupled receptors, which are often overexpressed in prostate cancer, and aberrantly activate androgen receptor (AR) in the absence of androgen. We developed an autocrine neuropeptide model by overexpressing GRP in LNCaP cells and the resultant cell line, LNCaP-GRP, exhibited androgen-independent growth with enhanced motility in vitro. When orthotopically implanted in castrated nude mice, LNCaP-GRP produced aggressive tumors, which express GRP, prostate-specific antigen, and nuclear-localized AR. Chromatin immunoprecipitation studies of LNCaP-GRP clones suggest that GRP activates and recruits AR to the cognate promoter in the absence of androgen. A Src family kinase (SFK) inhibitor, AZD0530, inhibits androgen-independent growth and migration of the GRP-expressing cell lines, and blocks the nuclear translocation of AR, indicating the involvement of SFK in the aberrant activation of AR and demonstrating the potential use of SFK inhibitor in the treatment of castration-resistant CaP. In vivo studies have shown that AZD0530 profoundly inhibits tumor metastasis in severe combined immunodeficient mice implanted with GRP-autocrine LNCaP cells. This xenograft model shows autocrine, neuropeptide- and Src kinase-mediated progression of androgen-independent CaP postcastration, and is potentially useful for testing novel therapeutic agents.

Transient upregulation of GRP and its receptor critically regulate colon cancer cell motility during remodeling.

Gastrin-releasing peptide (GRP) is typically viewed as a growth factor in cancer. However, we have suggested that in colon cancer, GRP acts primarily as a morphogen when it and its receptor (GRP-R) are aberrantly upregulated. As such, GRP/GRP-R act(s) primarily to modulate processes contributing to the assumption or maintenance of tumor differentiation. One of the most important such processes is the ability of tumor cells to achieve directed motility in the context of tissue remodeling. Yet the cellular conditions affecting GRP/GRP-R expression, and the biochemical pathways involved in mediating its morphogenic properties, remain to be established. To study this, we evaluated the human colon cancer cell lines Caco-2 and HT-29 cells. We found that confluent cells do not express GRP/GRP-R. In contrast, disaggreation and plating at subconfluent densities results in rapid GRP/GRP-R upregulation followed by their progressive decrease as confluence is achieved. GRP/GRP-R coexpression correlated with that of focal adhesion kinase (FAK) phosphorylation of Tyr(397), Tyr(407), Tyr(861), and Tyr(925) but not Tyr(576) or Tyr(577). To more specifically evaluate the kinetics of GRP/GRP-R upregulation, we wounded confluent cell monolayers. At t = 0 h GRP/GRP-R were not expressed, yet cells immediately began migrating into the gap created by the wound. GRP/GRP-R were first detected at approximately 2 h, and maximal levels were observed at approximately 6 h postwounding. The GRP-specific antagonist [d-Phe(6)]-labeled bombesin methyl ester had no effect on cell motility before GRP-R expression. In contrast, this agent increasingly attenuated cell motility with increasing GRP-R expression such that from t = 6 h onward no further cell migration into the gap was observed. Overall, these findings indicate the existence of GRP-independent and -dependent phases of tumor cell remodeling with the latter mediating colon cancer cell motility during remodeling via FAK.

Characterization of gastrin-releasing peptide and its receptor aberrantly expressed by human colon cancer cell lines.

Gastrin-releasing peptide (GRP) is a mitogen and morphogen important in the development of human colon cancers. Although epithelial cells lining the colon do not normally express GRP or its receptor (GRP-R), most human tumors express GRP-R mRNA. Yet functional protein has only been detected in 24 to 40% of colon cancers. To elucidate the reason for the difference between the expression of GRP/GRP-R mRNA and protein, we studied nine human colon cancer cell lines. Quantitative polymerase chain reaction revealed that all colon cancer cell lines expressed similar amounts of mRNA for both GRP as well as GRP-R. Yet binding studies using (125)I-Tyr(4)-bombesin detected functional receptors on only five of the nine cell lines studied. Conformational fragment-length polymorphism analysis indicated that although mRNA for the ligand GRP was never mutated, mRNA for the GRP-R was always mutated. Sequencing revealed that the message for GRP-R contained between two and seven separate mutations at the nucleotide level. This resulted in 14 separate coding mutations, 2 of which were observed in more than one cell line. Each mutation was individually recreated by site-directed mutagenesis and studied in transiently transfected Chinese hamster ovary-K1 cells. Alteration of Pro(145) into a tyrosine, of Val(317) into a glutamic acid, and insertion of a 32-nucleotide segment resulting in a frameshift distal to Asp(137) all resulted in GRP receptors incapable of binding ligand. Thus, these data indicate that human colon cancers commonly express GRP and GRP-R mRNA but that receptor mutations account for the failure of functional protein to be generated.

Immunohistochemical detection of gastrin releasing peptide in patients with prostate cancer.

Gastrin releasing peptide (GRP) is a neuropeptide that has been suggested to play a role in the development of some malignancies. Our aim was: (1) to identify the expression of GRP in cancerous prostate glands, and (2) to correlate its expression to various pathological parameters and to the patient's clinical outcome. Using standard immunohistochemistry, we evaluated GRP expression in both biopsy and radical prostatectomy specimens from 30 patients with prostatic adenocarcinomas. GRP was expressed in 18 radical prostatectomy specimens (60%) and in 15 biopsies (50%). There was an association between positive immunoexpression of GRP, relapse ( P=0.029) and advanced tumor stages (i.e. pT3, pT4) ( P=0.049). In the respective biopsies, GRP immunostatus was similar to that observed in the subsequent radical prostatectomy specimens. GRP immunoexpression may be of some value as a diagnostic and prognostic marker. Patients whose pathology specimens demonstrate GRP immunopositivity should be closely monitored, since they appear to be at higher risk of disease progression and relapse.