Differential regulation of chromogranin B/secretogranin I and secretogranin II by forskolin in PC12 cells.

The factors which regulate the expression of the granin family of secretory proteins have yet to be completely described. The present study investigated the effects of forskolin (FSK), an activator of adenylate cyclase, on the regulation of chromogranin B/secretogranin I (CgB) and secretogranin II (SgII) mRNA levels in rat PC12 cells. PC12 cells were treated with 10 microM FSK for time points up to 48 h and were harvested for cAMP determination, RNA isolation and Northern blot analysis, or fixed in 4% paraformaldehyde for immunocytochemistry. Cellular cAMP levels peaked after two h of FSK treatment and remained elevated for 48 h. Chromogranin B mRNA increased with FSK treatment, reaching a maximum of 7-fold above control after 24 h, while the level of SgII mRNA decreased to a level of 65 +/- 10% of control after 48 h. The effects of FSK on CgB mRNA appear to be mediated by cAMP, as 8-bromo-cAMP (500 microM) resulted in a 2.8-fold increase in CgB mRNA, and H-89 (30 microM), a selective inhibitor of cAMP-dependent protein kinase, reduced the FSK-mediated response. The level of CgB was also increased in FSK-treated cells, as evidenced by immunofluorescent analysis which showed a more intense staining in PC12 cells treated with FSK for 48 h than in untreated cells. The intensity of SgII staining was diminished by FSK treatment, most likely a result of a decreased rate of synthesis as well as an increase in the release of SgII. This study demonstrated that the mRNA and protein levels of CgB and SgII are differentially regulated by cAMP in PC12 cells.

Characterization of prolactin and growth hormone immuno- and bioactivities in the pituitary gland and serum of the squirrel monkey (Saimiri boliviensis boliviensis).

The immunological and biological activities of growth hormone (GH) and prolactin (PRL) in the pituitary gland and serum of the squirrel monkey (Saimiri boliviensis boliviensis) have been studied. Proteins in pituitary homogenates were solubilized in 1% SDS, electrophoresed on 12% polyacrylamide gels, and transferred to nitrocellulose. Squirrel monkey GH and PRL were identified by immunoblotting with anti-human GH antibodies and a monoclonal antibody to ovine PRL, 6F11, respectively. Squirrel monkey GH appeared predominantly as two proteins of apparent molecular weight 22 and 20 kD, corresponding to native and variant forms of human GH. Squirrel monkey PRL appeared as two proteins of apparent molecular weight 24 and 26.5 kD, which co-migrated with native and glycosylated forms of ovine PRL. The cross-reactivity of neutralizing antibodies to human GH and PRL with squirrel monkey GH and PRL were examined using the Nb2 lymphoma bioassay. One of three monoclonal antibodies to human GH (2A1) neutralized squirrel monkey GH with an apparent affinity for squirrel monkey GH (IC50 = 70 ng IgG/ml) which was fourfold lower than for human GH (IC50 = 15 ng IgG/ml). Both polyclonal [AR38-5(1)] and monoclonal (9C3) antibodies to human PRL inhibited the activity of squirrel monkey PRL, athough their affinities for squirrel monkey PRL were four- and twentyfold lower than for human PRL. The activities of antibodies 2A1 and 9C3 on GH and PRL in squirrel monkey serum were also examined by the Nb2 bioassay. The anti-glucocorticoid RU486 was used in all incubations with squirrel monkey serum to eliminate the effect of high glucocorticoid levels on Nb2 cell growth. The mitogenic activity of squirrel monkey serum in the Nb2 assay was completely eliminated in the presence of 2A1 and 9C3. This study represents the first description of the biochemistry of GH and PRL in the squirrel monkey.

A monoclonal antibody which inhibits the biological activity of rat prolactin, but not prolactin from other species.

Monoclonal antibodies generated to ovine prolactin were screened for their ability to neutralize the biological activity of prolactin from several species. By Western blot analysis, antibody 6F11 cross-reacted strongly with prolactin in homogenates of anterior pituitary glands from squirrel monkey, sheep and rat. In addition, this antibody (1 micrograms IgG/ml) completely inhibited the lactogenic activity of serum or purified prolactin (0.5 ng/ml) from rat, but not prolactin from any other source, in the Nb2 lymphoma bioassay. 6F11 cross-reacted with purified ovine and rat prolactin by enzyme-linked immunosorbentassay (ELISA) and Western blot analysis with similar affinities, suggesting that the 6F11 epitope was common to these peptides. Another monoclonal antibody (17D9, 35 ng IgG/ml) showed the opposite selectivity, completely inhibiting the activity of 0.3 ng/ml ovine prolactin, but not 0.5 ng/ml rat prolactin, in the Nb2 assay. Thus, we have identified monoclonal antibodies which cross-react with both ovine and rat prolactin, but selectively neutralize the lactogenic activity of prolactin from only one species.

Presence of ACTH and its receptor on a B lymphocytic cell line: a possible autocrine function for a neuroendocrine hormone.

A mouse B lymphocytic cell line, designated BCL1, was found to produce immunoreactive ACTH and to secrete this molecule into culture supernates. The BCL1-derived ACTH induced Y-1 adrenal cells to undergo a steroidogeneic response and was eluted from gel filtration columns at a molecular weight similar to that expected for pituitary-derived ACTH. Furthermore, ACTH receptors were detected on the surface of BCL1 cells using indirect immunofluorescence and 125I-ACTH binding studies. Scatchard analysis demonstrated the presence of high and low affinity binding sites with dissociation constants of 4.5 x 10(-12) M and 2.8 x 10(-9) M, respectively. The production of both ACTH and its receptor by this B lymphocyte cell line suggests that an autocrine mechanism might be important for the maintenance of the transformed phenotype.