Calcitonin from ultimobranchial glands of dogfish and chickens.

Acid extracts of thyroid glands from a small shark Squalus suckleyi and domestic fowl Gallus domestica contained no detectable calcitonin activity, while very potent hypocalcemic responses were obtained in rats with similar extracts from the ultimobranchial glands of these two species. The calcitonin concentration was 4 to 40 times that present in hog thyroid, which, as in most other mammals, contains ultimobranchial tissue. The evidence suggests that calcitonin is a fundamental calcium-regulating hormone present in all higher vertebrates and that it is an ultimobranchial rather than a thyroid hormone. It also indicates an important and hitherto unrecognized function for the ultimobranchial glands.

Stimulation by ATP of alkaline phosphatase in placental plasma membranes.

1. ATP stimulated the p-nitrophenyl phosphatase activity of placental plasma membranes, with an increase in activity of approximately 100% at 5 mM ATP. The stimulation was not dependent on the presence of Mg-2-+. 2. The K-m for p-nitrophenyl phosphate was not changed by the presence of 5 mM ATP. 3. ATP hydrolysis by the plasma membrane preparation under the same assay conditions as for alkaline phosphatase was not influenced by the presence of 5 mM p-nitrophenyl phosphate. 4. Extraction of the plasma membrane preparation with n-butanol abolished the stimulatory effect of ATP, as well as Ca-2-+-activated ATPase activity.

Calcium uptake by placental plasma membrane vesicles.

The placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca2+. This is 24-fold higher than the external Ca2+ concentration. This process is dependent on ATP hydrolysis by the placental Ca2+-ATPase. The Pi/Ca ratio is dependent on the external Ca2+ concentration, and reaches the value of 2 at 10 mM Ca2+. Phosphate (5 mM) can double Ca2+ uptake when measured in the presence of 5 mM Ca2+. Mg2+ increased Ca2+ uptake only at low Ca2+ concentrations, and had no significant effect at 5 mM Ca2+.

Salmon stanniocalcin and bovine parathyroid hormone have dissimilar actions on mammalian bone.

Stanniocalcin (STC), a calcium-regulating glycoprotein hormone isolated from the corpuscles of Stannius of salmon, was tested for effects on bone and calcium metabolism in mammalian species (rats and mice). STC generally failed to alter serum calcium of parathyroidectomized rats at concentrations equimolar with effective concentrations of parathyroid hormone (PTH). STC did not increase cAMP in ROS 17/2.8 or UMR-108 osteosarcoma cells, OK kidney cells, fetal rat limb bones, or neonatal mouse calvariae, and similarly failed to increase urinary cAMP in rats. STC did not consistently stimulate resorption in any of the rodent bone culture systems, although variable resorptive responses were elicited in fetal mouse calvariae. The results indicate that this fish hormone has limited, if any, PTH-like activity on calcium metabolism in mammalian systems.

Development of a primary culture system for rainbow trout corpuscles of stannius and characterization of secreted teleocalcin.

A primary culture system has been established for rainbow trout corpuscles of Stannius (CS). A RIA has also been developed to monitor and characterize the teleocalcin secreted by these cultures. The primary cultured CS cells actively synthesized and secreted teleocalcin for up to 39 days when maintained in amphibian and mammalian culture media. Numerous teleocalcin cells were also evident after immunocytochemical staining of the CS cultures. When the cultures were labeled with L-[35S]methionine, de novo synthesized and secreted teleocalcin was judged to be a glycosylated protein on the basis of Concanavalin-A-Sepharose binding and was similar in size to the intracellular form of the hormone. This culture system may prove to be ideal for identifying those factors that regulate teleocalcin secretion.

Immunological studies on teleocalcin and salmon corpuscles of Stannius.

This report describes the characterization of an antiserum to salmon teleocalcin, a glycoprotein hormone that has recently been isolated from sockeye salmon, Oncorhynchus nerka, corpuscles of Stannius (CS). Immunodiffusion studies showed that teleocalcin was immunologically identical in four species of Pacific salmon. Histological staining of adjacent sections of sockeye salmon CS by periodic acid-Schiff reagent and immunocytochemistry revealed that the teleocalcin-immunoreactive cells were also periodic acid-Schiff positive. In bioassays using rainbow trout (Salmo gairdneri), the biological effects of teleocalcin were completely abolished by pretreatment with the antiserum before injection. The antiserum was ineffective by itself, however, in neutralizing endogenous teleocalcin. Immunoprecipitation studies of in vitro translation products from salmon CS poly(A)+ RNA identified a 26K protein, which corresponds to the size of the teleocalcin monomer as predicted by cDNA sequencing studies. Similar banding patterns were obtained with purified teleocalcin and crude CS extracts by Western blot analysis. However, there was one extra band (32K) in the CS extracts that was not present in the purified hormone preparations. This may represent the pro-form of the hormone in salmon.

Changes in plasma calcitonin and calcium during the migration of salmon.

The levels of immunoassayable calcitonin and of calcium were measured in the preblood of sockeye salmon during their migration from sea water to fresh water to spawn. Plasma calcitonin was higher in the females than in the males during all stages of migration as was plasma calcium except during spawning. In both sexes calcium decreased progressively throughout migration. In the male, calcitonin decreased as the fish arrived in fresh water but increased again at the spawning. These observations suggest that calcitonin may play an important role in the reproductive cycle of fish.

Studies on the regulation and characterization of plasma stanniocalcin in rainbow trout.

Stanniocalcin (STC) is a hormone that is synthesized and secreted by the corpuscles of stannius (CS), endocrine glands that are unique to the bony fishes. The hormone inhibits Ca2+ transport from the aquatic environment into the bloodstream by way of the gills. Previous in vitro studies by our laboratory have shown that STC secretion is positively regulated by Ca2+ in a time- and dose-dependent fashion. In this report, we have examined circulating levels of STC in free-swimming, cannulated rainbow trout and how hormone levels are affected by surgical stress and intra-arterial infusions of mono and divalent cations. In addition, the plasma hormone has been concentrated by immunoaffinity chromatography and characterized by Western blot analysis. The results suggest that the in vivo response of the CS is extremely rapid and Ca(2+)-specific and that STC circulates in multiple molecular weight forms.

The gill calcium transport cycle in rainbow trout is correlated with plasma levels of bioactive, not immunoreactive, stanniocalcin.

Stanniocalcin (STC) is an inhibitor of gill Ca2+ transport that is produced by the corpuscles of Stannius, endocrine glands in bony fish. In young rainbow trout (Oncorhynchus mykiss), there are cyclical changes in the rate of gill Ca2+ transport, with alternating phases of accelerated and reduced uptake every 14 days. Previous studies by our laboratory have established that the responsiveness of young trout to the inhibitory effects of exogenous STC is dependent on this cycle. Trout are highly responsive to STC at peaks of Ca2+ uptake and unresponsive at nadirs, which has led us to suggest that the gill Ca2+ transport cycle may be regulated by a reciprocal cycle in the levels of plasma STC. In this report, we have further characterized the gill Ca2+ transport cycle in salmonids and investigated the role of STC in its regulation. Our results showed that the cycle is synchronous and is likely a characteristic feature in all salmonids but that it varies in amplitude between species. Surprisingly, we observed no correlation between circulating levels of radioimmunoassayable STC and the rate of gill Ca2+ transport in trout. To address this apparent contradiction, trout fry were passively immunized with STC antiserum to determine if there were variable amounts of bioactive STC in the circulation, at times when trout were either more or less sensitive to exogenous STC. We observed that during the times when trout were responsive to STC treatment (i.e., cycle peaks), passive immunization had no effect on the rate of gill Ca2+ transport in fish from the same population, indicating that there were low levels of bioactive STC in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and cDNA sequence analysis of coho salmon stanniocalcin.

Stanniocalcin (STC) (formerly known as both teleocalcin and hypocalcin) is an anti-hypercalcemic, glycoprotein hormone that is produced by the corpuscles of Stannius (CS), endocrine glands that are confined to bony fishes. The hormone has a unique amino acid sequence and exists as a disulfide-linked homodimer in the native state. In previous studies, we have described the purification and characterization of two salmon STCs, and examined the regulation of hormone secretion in response to calcium using both in vitro and in vivo model systems. This report describes the molecular cloning and cDNA sequence analysis of a coho salmon STC messenger RNA (mRNA) from a salmon CS lambda gt10 cDNA library. The STC mRNA in salmon is approximately 2 kilobases in length and encodes a primary translation product of 256 amino acids. The first 33 residues comprise the prepro region of the hormone, whereas the remaining 223 residues make up the mature form of the hormone. One N-linked, glycosylation consensus sequence was identified in the protein coding region as well as an odd number of half cysteine residues, the latter of which would allow for interchain bonding or dimerization of monomeric subunits. In addition, three sites were identified in the mature protein core of STC (two dibasic, one tribasic) that may be acted upon by endopeptidases to produce truncated forms of the hormone. In support of this latter possibility, Western blot analysis revealed multiple molecular weight forms of sTC within salmon glands.

Chromogranin A is present in and released by fish endocrine tissue.

Chromogranin A (CgA) is a protein that is present in many mammalian endocrine cells and co-secreted with their resident hormones. We have demonstrated the presence of CgA by immunohistology in the ultimobranchial glands and corpuscles of Stannius of rainbow trout. CgA was also detected by radioimmunoassay in the medium of incubated coho salmon ultimobranchial glands. Our observations demonstrate the presence of CgA in endocrine glands of evolutionarily divergent species. These observations are consistent with the hypothesis that CgA participates in the secretory process of a wide variety of hormones.