Development of a human stanniocalcin radioimmunoassay: serum and tissue hormone levels and pharmacokinetics in the rat.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fish and recently identified in humans and other mammals. In fish STC is produced by one gland, circulates freely in the blood and plays an integral role in mineral homeostasis. In mammals, STC is produced in a number of different tissues and serves a variety of different functions. In kidney, STC regulates phosphate reabsorption by proximal tubule cells, whereas in ovary it appears to be involved in steroid hormone synthesis. However there is no information on circulating levels of STC in mammals or the regulation of its secretion. In this report we have developed a radioimmunoassay (RIA) for human STC. The RIA was validated for measuring tissue hormone levels. However human and other mammalian sera were completely devoid of immunoreactive STC (irSTC). To explore the possibility that mammalian STC might have a short half-life pharmacokinetic analysis was carried out in rats. STC pharmacokinetics were best described by a two compartment model where the distribution phase (t1/2(alpha)) equaled 1 min and the elimination phase (t1/2(beta)) was 60 min. However the STC in the elimination phase no longer crossreacted in the RIA indicating it had undergone substantial chemical modification, which could explain our inability to detect irSTC in mammalian sera. When we compared the pharmacokinetics of human and fish STC in mammalian and fish models the human hormone was always eliminated faster, indicating that human STC has unique structural properties. There also appears to be a unique clearance mechanism for STC in mammals. Hence there are major differences in the delivery and biology of mammalian STC. Unlike fishes, mammalian STC does not normally circulate in the blood and functions instead as a local mediator of cell function. Future studies will no doubt show that this has had important ramifications on function as well.

Expression, purification, and bioassay of human stanniocalcin from baculovirus-infected insect cells and recombinant CHO cells.

Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia. Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated. In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors. Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors. Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps. The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers. The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars. In an in vivo bioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent. The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation. Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined.

Development of a dose-response bioassay for stanniocalcin in fish.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fish and recently identified in mammals. In fish, STC is released into the bloodstream in classical endocrine fashion and has well established regulatory effects on calcium and phosphate homeostasis. However, there are no suitable dose-response bioassays for STC and consequently no methods for assigning units of potency to preparations of the hormone. All the available in vitro bioassays are too complex from a technical standpoint to readily accommodate the large number of samples required in dose-response bioassays. Most in vivo bioassays are hampered by the fact that fish have natural rhythms governing plasma STC levels which tend to make them variably sensitive to the injected hormone. In this report we have developed a new in vivo bioassay for STC using rainbow trout. The key feature of the bioassay involves suppressing plasma STC levels to the extent that fish are always receptive to injected hormone. This has been accomplished by phosphate-loading the animals, which lowers their plasma calcium levels, removes the stimulus for STC secretion and brings about a reduction in resting plasma hormone levels. The net effect is an animal that is always responsive to injected STC. With this bioassay we have been able to obtain sensitive and reproducible, dose-related effects of salmon STC on gill calcium transport.

Salmon calcitonin inhibits whole body Ca2+ uptake in young rainbow trout.

Gill Ca2+ transport (GCAT) in fish is regulated by a number of different hormones. Stanniocalcin (STC) from the corpuscles of Stannius (CS) is an inhibitor of GCAT, whereas pituitary-derived prolactin and cortisol stimulate GCAT. Other than this, however, little is known about the effects of other hormones on this important transport process. The role of calcitonin (CT) in calcium homeostasis in fish is still controversial. Whereas many studies have shown significant effects of CT on plasma calcium levels, an equal number of studies have failed to find any correlations between plasma calcium and CT levels in fish. Previous in vitro studies have shown that salmon CT has potent inhibitory effects on GCAT in isolated, perfused fish gill preparations, a finding that has never been corroborated in vivo. Therefore, in this report we examined the effects of salmon CT on whole body 45Ca uptake (as a measure of GCAT) in young rainbow trout. In support of the in vitro findings, we found that CT had significant inhibitory effects on GCAT. In parallel studies, we found that CT had no effects on STC secretion and only modest, stimulatory effects on STC mRNA levels in cultured trout CS cells. These finding suggest that both CT and STC function as negative regulators of GCAT in fish.