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.

Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein.

The mechanisms which control the production of erythropoietin (Epo) remain enigmatic. Recent data suggest that the half-time of Epo messenger RNA (mRNA) is increased by hypoxia in Hep 3B cells, a human hepatoma line. The post-transcriptional regulation of other rapidly degraded mRNAs is mediated by sequence-specific mRNA binding proteins. In order to determine if Epo mRNA specific binding proteins exist, we probed cytosolic lysates from Hep 3B cells and mouse tissues with radiolabeled Epo RNA. A cytosolic protein that binds specifically to Epo RNA was identified in the Epo-producing, hepatoblastoma Hep 3B cell line by gel mobility shift assay. This protein was identified in both normoxic and hypoxic cells and bound specifically to a 120-base fragment of the 3'-untranslated region (3'-UTR) of Epo mRNA. Binding was completed with unlabeled Epo RNA, but not with granulocyte-macrophage colony-stimulating factor RNA. Ultraviolet light cross-linked Epo RNA-protein complexes migrated as two bands of 70 and 135-140 kD on sodium dodecyl sulfate-polyacrylamide gels. Binding activity was markedly increased in brain and spleen lysates from mice subjected to 24 h of hypoxia. Therefore, the post-transcriptional regulation of Epo expression in response to hypoxia may in part be due to the interaction of Epo RNA with its specific binding protein.

Analysis of regional responses to endothelin in hindquarters vascular bed of cats.

Regional responses to endothelin, a peptide derived from endothelial cells in culture, were investigated in the hindquarters vascular bed of cats, when flow varied naturally and when flow was maintained constant with a pump. Intravenous injections of endothelin at doses of 0.03 and 0.1 nmol/kg caused dose-dependent decreases in systemic arterial pressure and increases in distal aortic blood flow. Injection of endothelin at a dose of 0.3 nmol/kg iv caused a biphasic response characterized by an initial decrease in arterial pressure and an increase in blood flow, which was followed by a secondary rise in pressure and a fall in blood flow. When blood flow to hindquarters was maintained constant with a pump, intra-arterial injection of 0.03 nmol endothelin caused a decrease in perfusion pressure, whereas 0.1-1 nmol doses elicited biphasic responses characterized by an initial decrease followed by a secondary increase in perfusion pressure. When compared with other vasoactive peptides, the pressor activity of endothelin was less than angiotensin II by an order of magnitude but was threefold greater than that of neuropeptide Y in the hindquarters vascular bed. The pressor component of the response to endothelin and the response to the calcium agonist BAY K 8644 were decreased in a reversible manner by nisoldipine, a dihydropyridine calcium entry blocking agent. The results of these studies indicate that porcine-human endothelin has both vasodilator and vasoconstrictor activity in the hindquarters vascular bed of cats. The predominant response at a low concentration is vasodilation, whereas at higher concentrations a vasoconstrictor response that was dependent in part on the influx of extracellular calcium could be demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of lactogenic activity in the serum of the squirrel monkey (Saimiri boliviensis) using the Nb2 lymphoma bioassay.

Determination of squirrel monkey prolactin by immunoassay has been hampered by the lack of antiserum specific to prolactin from this species. As an alternate method, we have investigated whether the Nb2 lymphoma bioassay could be adapted for routine measurement of the lactogenic activity of samples of squirrel monkey serum. The growth of the Nb2 cells is absolutely dependent on the presence of lactogens in the culture medium. The cells were maintained in Fisher's medium supplemented with 10% horse serum, 10% fetal calf serum (FCS), and 10-4M ß-mercaptoethanol. For each assay, the cells were plated at an initial density of 1 × 105 cells/ml in 22-mm 12-well dishes in the above medium, but devoid of FCS. Serum samples were heated to 56°C for 20 minutes to abolish the unusually high cytolytic complement activity of squirrel monkey serum and were incubated for 72 hours with Nb2 cells at serial dilutions from 1/40 to 1/2,560. Growth curves were generated with pooled samples of squirrel monkey serum, and the level of lactogenic activity was estimated using a calibration growth curve generated with known concentrations of purified rhesus monkey prolactin standard. We have found that the Nb2 lymphoma bioassay provides a sensitive and adaptable means for determination of lactogenic activity in the serum of the squirrel monkey.