Secretion Of Immunoreactive Beta-endorphin Research Articles

Temperature effect on the action of corticotropin-releasing hormone (CRH) on secretion of immunoreactive beta-endorphin (IRβEP) in TM3 and AtT-20 cell lines

Testicular function is sensitive to chemical and thermal stresses. To investigate the effects of small temperature changes on CRH-stimulated βEP release, we employed TM3 cells, a mouse prepubertal Leydig cell line that secretes irβEP. To monitor βEP secretion from these cells we used the reverse hemolytic plaque assay. After 3.5 hr incubation of cells with hormone, the EC 50 of the CRH dose-response curve at 34°C and 37°C were 0.1 nM and 1 nM, respectively. For comparison, we also investigated the effect of temperature on CRH-stimulated βEP release from a non-testicular cell line, AtT-20, a mouse anterior pituitary cell line. Using radioimmunoassay to measure irβEP levels in the media of AtT-20 cells, the EC 50 S for the CRH dose-response curve at 34°C and 37°C were 0.2 nM and 2 nM, respectively, at 1 h. After 3.5 h this temperature dependent difference in EC 50 was still observed. These results suggest that CRH receptors or post-receptor actions in Leydig cells and anterior pituitary corticotropes are sensitive to small temperature changes.

Effects of Protein Kinase C Down‐Regulation on Secretory Events and Proopiomelanocortin Gene Expression in Anterior Pituitary Tumor (AtT‐20) Cells

To elucidate the role of the diacylglycerol-protein kinase C (PKC) pathway in beta-endorphin synthesis and secretion in anterior pituitary corticotrope tumor cells (AtT-20), a procedure for down-regulating PKC activity in the cells was developed. Treatment of AtT-20 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) led to an increase in [3H]phorbol 12,13-dibutyrate binding to PKC in the membrane fraction of these cells 30 s after its addition to the culture medium. Thereafter, a decrease in both [3H]phorbol 12,13-dibutyrate binding and PKC-specific phosphotransferase activity occurred in a time- and dose-dependent manner in both the cytosolic and membrane fractions. For example, treatment of the cells with 100 nM TPA for 24 h resulted in an almost complete depletion of PKC activity. Immunoreactive beta-endorphin secretion was found to be stimulated two- to fourfold in the control cells after incubation with corticotropin-releasing factor (10(-7) M), forskolin (10(-6) M), or TPA (10(-7) M) for 4 h. In cells rendered PKC deficient, TPA-stimulated immunoreactive beta-endorphin release was abolished, forskolin-stimulated release was unaffected, and corticotropin-releasing factor-stimulated release was depressed. Treatment of control cells with any one of the three stimulatory agents led to an increase in proopiomelanocortin mRNA levels, and these responses were also depressed after TPA pretreatment. The results suggest that physiological processes thought to be entirely cyclic AMP dependent, such as corticotropin-releasing factor-elicited secretion, may be partially dependent on PKC-mediated biochemical events.

The role of IL-1 in the corticotropin-releasing factor and arginine- vasopressin-induced secretion of immunoreactive beta-endorphin by human peripheral blood mononuclear cells.

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) stimulate the secretion of beta-endorphin by human PBMC. It is shown here that peripheral blood B cells are responsible for the production of beta-endorphin after culture with CRF and AVP. The presence of CD14+ monocytes is, however, a prerequisite for the enhancing activity of CRF and AVP. The data presented here show that rIL-1 beta can replace CRF and AVP, whereas a mAb directed against IL-1 abrogates the response to CRF and AVP. These results indicate that IL-1 mediates the effect of CRF and AVP on beta-endorphin production by human PBMC.

Release from Inflamed Tissue of a Substance with Properties Similar to Corticotropin-Releasing Factor

In response to stressors involving tissue injury, pituitary corticotroph secretion of immunoreactive beta-endorphin (iB-END) could be either due to release of hypothalamic factors such as corticotropin-releasing factor (CRF) or to release of a tissue factor from the periphery. In the present experiments, we investigated whether inflamed tissue releases a factor which evokes pituitary secretion of iB-END. In an initial experiment, rats with an inflamed hindpaw due to carrageenan injection had significantly greater levels of circulating iB-END as compared to rats with saline-injected paws. Removal of afferent input, by hindlimb denervation, failed to block the carrageenan-induced increase in iB-END levels. Subcutaneous perfusates were then collected from inflamed and control hindlimbs and applied to rat anterior pituitary cell cultures. Pituitary release of iB-END due to administration of perfusate from inflamed paws was significantly greater than iB-END release due to perfusate from saline-injected paws or to basal release. The releasing activity in the perfusates was blocked in calcium-free medium and was not due to a direct action of carrageenan, bradykinin, substance P or calcitonin gene-related peptide. The results indicate that inflamed tissue releases a CRF-like factor which stimulates iB-END release both in the denervated rat and cultured pituitary cells.

Thymosin fraction 5 stimulates secretion of immunoreactive beta-endorphin in mouse corticotropic tumor cells.

In addition to reconstituting immune competence, the thymus gland preparation, thymosin fraction 5 (TSN-5), has recently been shown to stimulate secretion of hormones from the hypothalamic-pituitary adrenal axis in vivo and from pituitary corticotropes in vitro. The purpose of the present study was to investigate the effects of TSN-5 on secretion of immunoreactive beta-endorphin (i beta-E) by mouse corticotropic tumor cells. The release of i beta-E by AtT-20 pituitary tumor cells was increased in a dose-dependent manner by concentrations of 30-600 micrograms/ml of TSN-5, whereas concentrations greater than 1,000 micrograms/ml were increasingly less effective in stimulating secretion. TSN-5 (600 micrograms/ml) significantly stimulated i beta-E release within 7 min; maximal secretory responses (up to 275% of control release) occurred by 4 hr. The secretory response of AtT-20 cells to 600 micrograms/ml TSN-5 (37.9 +/- 2.0 vs. 16.1 +/- 1.0 ng i beta-E/ml/4 hr, mean +/- SE) was similar in magnitude to release evoked by 0.1 microM corticotropin-releasing factor (CRF). Combining TSN-5 and CRF treatments increased secretion of i beta-E to nearly 600% of control levels, an effect greater than an additive influence of the two independent treatments. Whereas CRF treatment reduced the levels of i beta-E in AtT-20 cell extracts after 24-hr treatment by 45% (231.8 +/- 24.7 vs. 417.2 +/- 17.8 ng i beta-E/mg protein, CRF vs. vehicle treatments, respectively), TSN-5 did not significantly alter cellular hormone content. Neither TSN-alpha 1 nor TSN-beta 4, two of the component peptides of TSN-5, affected basal or CRF-stimulated release of i beta-E, indicating that an unidentified constituent(s) is corticotropic.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenoceptor activation mediates stress-induced secretion of beta-endorphin-related peptides from intermediate but not anterior pituitary.

Recently, we reported that adrenaline can stimulate the secretion of immunoreactive beta-endorphin in the rat. This response is mediated by beta-adrenoceptors and requires circulating adrenaline concentrations which are known to occur during stress. We therefore studied whether catecholamines are implicated in the stress-induced secretion of immunoreactive beta-endorphin from the pituitary gland. We report here that in rat the beta-adrenoceptor antagonist (-)propranolol reduces or abolishes the rapid increase of immunoreactive beta-endorphin levels during some stress stimuli (footshock, passive avoidance, restraint) but not during others (ether, formalin, laparotomy). The propranolol-sensitive response is largely prevented by extirpation of the neurointermediate lobe of the pituitary gland but is unaffected by dexamethasone, which inhibits peptide secretion from the corticotroph cells of the anterior lobe. These results suggest that catecholamines activate the release of immunoreactive beta-endorphin from the intermediate lobe but not from the anterior lobe of the pituitary gland during certain stress conditions.