Endogenous galanin modulates the gonadotropin and prolactin proestrous surges in the rat.

Galanin (GAL) has recently emerged as an important neuroendocrine regulator which participates in the control of several pituitary and hypothalamic hormones. Our earlier observation that GAL stimulates LHRH release from nerve terminals of the median eminence as well as basal LH and LHRH-induced LH secretion from pituitary cells in vitro prompted us to evaluate whether endogenous GAL plays a role in regulation of the physiologically occurring preovulatory surges of gonadotropins and PRL. Proestrous female rats were passively immunized against GAL using a high affinity sheep antirat GAL serum (FMS-FJL 17-5). Animals were implanted during diestrus with indwelling atrial cannulae. On the expected day of proestrus, rats received 1 ml of either normal sheep serum or GAL antiserum (GAL-AS), iv, 1 h before blood sampling started. Blood samples (0.5 ml) were collected at hourly intervals from 1400-2300 h, and plasma levels of LH, FSH, and PRL measured by RIA. At several time intervals after GAL-AS administration, the maximum binding ability of the rat plasma was evaluated using standard saturation assays. High neutralizing levels of immunoglobulins were present throughout the experimental period. GAL passive immunization blunted the LH preovulatory surge by 30%. Although maximum LH levels were unaffected by the treatment, the area under the secretory curve and LH levels at 1700, 1900, and 2000 h were significantly reduced. Conversely, FSH secretion was not significantly altered for either maximum FSH levels or area under the curve. However, FSH levels were significantly diminished in GAL-AS-treated rats at 1700 h. GAL passive immunization selectively reduced the plateau phase of the preovulatory surge of PRL. No significant differences were observed in the initiation of the surge or maximum PRL levels, whereas PRL levels were significantly reduced from 1700 to 2200 h. In addition, the area under the PRL curve was diminished in GAL-AS-treated animals by 40%. In conclusion, our results clearly demonstrate that endogenous GAL is involved in control of the preovulatory surges of LH and PRL without altering the FSH surge. In addition, they provide, for the first time, evidence of an important role for endogenous GAL in the regulation of physiological events leading to ovulation.

Estradiol induces galanin gene expression in the pituitary of the mouse in an estrogen receptor alpha-dependent manner.

Estradiol imprinting plays an important role in the regulation of galanin (GAL) gene expression in the rat. In the anterior pituitary gland, GAL gene expression is greatly induced by estrogen. The relative involvement that the two estrogen receptor subtypes, alpha and beta, have in regulating this induction is not known. We have utilized ER alpha-knock-out (ER alphaKO) mice to discriminate the roles of ER alpha and ER beta in the regulation of GAL gene expression in the anterior pituitary gland. Our goals were to measure the effects of estradiol on GAL gene expression by solution hybridization ribonuclease protection assay in wild-type mice and to determine the roles of ER alpha and, indirectly, ER beta by measuring the same response in the ER alphaKO mice. Estradiol treatment for one week elevated GAL gene expression 30-40 fold in the wild-type mouse pituitary. Evaluation of estrogen effects on GAL gene expression in the anterior pituitary of ER alphaKO animals revealed that ER alpha is essential, because no response to estrogen was observed in these animals. Since ER beta mRNA was identified in the anterior pituitary by RT-PCR, but estrogen had no effects on GAL gene expression in the ER alphaKO mice, the beta subtype of ER does not appear to participate in estrogen-evoked GAL gene expression in the mouse anterior pituitary.

Expression of functional estrogen receptors and galanin messenger ribonucleic acid in immortalized luteinizing hormone-releasing hormone neurons: estrogenic control of galanin gene expression.

The activity of estradiol on the LHRH neuronal network is crucial in the regulation of reproduction. In vivo, estradiol induces galanin (GAL) gene expression in LHRH neurons and GAL/LHRH colocalization is sexually dimorphic and neonatally determined by steroid exposure. The effects of estradiol on LHRH neurons, however, are considered to be indirect because estrogen receptors (ER) have not been detected in LHRH neurons in vivo. Using immortalized mouse LHRH neurons (GT1-7 cells), we demonstrated by RT-PCR and Southern blotting that GT1-7 cells express ER messenger RNA (mRNA). Sequencing of the amplification products indicated that GT1-7 ER is of the alpha-subtype (ER alpha). Additionally, estrogen receptors in GT1-7 cells were characterized by competitive radioligand receptor binding and IC50 values for 17beta-estradiol and ICI-182,780 were found to be 0.24 and 4.1 nM, respectively. The ability of endogenous GT1-7 cell ER to regulate transcription was determined in transient transfection studies using a construct that consisted of a luciferase reporter gene that is driven by tandem estrogen response elements (ERE) and a minimal herpes simplex virus thymidine kinase promoter. 17Beta-estradiol was found to enhance luciferase activity by 2.5-fold at physiological concentrations with an ED50 value of 47 pM. This induction was completely inhibited by ICI-182,780 which had an IC50 value of 4.8 nM. Raloxifene, tamoxifen, 4-hydroxytamoxifen, and droloxifene also fully blocked estrogen-mediated luciferase induction with IC50 values of 58.4, 89.2, 33.2, and 49.8 nM, respectively. In addition, GAL mRNA was detected and identified by RT-PCR followed by Southern blotting using a rat GAL complementary DNA (cDNA) probe. The ability of 17beta-estradiol to modulate expression of the endogenous GAL gene in immortalized LHRH neurons was also determined. Quantitative RT-PCR demonstrated that physiological concentrations of estrogen increase GAL gene expression by 2-fold with an ED50 value of 23 pM. ICI-182,780, raloxifene, and droloxifene completely blocked this induction. In summary, our data demonstrate the presence of ER alpha and GAL mRNA in GT1-7 cells. The ER in GT1-7 cells is biologically active because 17beta-estradiol enhances both endogenous GAL gene expression and an ERE-driven reporter gene. These results suggest that estrogenic control of GAL gene expression in immortalized LHRH neurons may be transduced by ER. Thus, hypothalamic-derived LHRH neurons appear to have the capacity to be directly regulated by estrogen.

Development and characterization of a specific and sensitive radioimmunoassay for rat galanin: measurement in brain tissue, hypophyseal portal and peripheral serum.

Galanin (GAL), a 29 amino acid peptide, is extensively distributed in both brain and intestine having been described as a putative neuroendocrine modulator. Up to this date, available radioimmunoassays for measuring GAL have the problem of nonparallel responses when rat biological samples are used. Due to the recent availability of synthetic rat GAL (rGAL), we pursued the development of a specific RIA for rGAL. Our RIA system presents a high specificity since porcine GAL (pGAL), which differs from rGAL in three carboxy-terminus substitutions, displays negligible crossreactivity. In addition, in the RIA a sensitivity of at least 1 pg/tube can be obtained. All biological samples tested in this study (serum samples from both peripheral and portal blood and arcuate-nucleus median eminence extracts) displayed perfect parallelism to synthetic rGAL standard. The RIA is suitable for measuring minute amounts of rGAL directly in serum samples from both portal and peripheral circulation. Interestingly, the IC50 for portal serum was approximately 10-fold lower than that for peripheral serum. This indicates that rGAL may be present in higher concentrations in portal than in peripheral blood. In addition, our data suggest that the pituitary gland contributes approximately 30% to the total levels of rGAL measured in peripheral serum. Together, this data reinforces the concept that GAL is involved in mechanisms controlling and/or regulating neuroendocrine functions.