Kathi James

Evidence for calcium-sensing receptor mediated stanniocalcin secretion in fish.

The secretion of parathyroid hormone (PTH) and calcitonin (CT) in mammals are both tightly regulated by the prevailing levels of extracellular ionic calcium (Ca(2+)). And, it is now widely recognized that both of these Ca(2+) effects are mediated exclusively through a seven transmembrane calcium sensing receptor or CaR. As in the case of PTH and CT, the secretion of stanniocalcin (STC) in fish is tightly regulated by the levels of extracellular Ca(2+). Fish STC functions as an anti-hypercalcemic hormone such that a rise in extracellular Ca(2+) above the physiological set-point of approximately 1.2 mM provokes an immediate secretory response. Whether or not Ca(2+)-regulated STC secretion in fishes is mediated by similar type of receptor has never been addressed. Here, we have found that Ca(2+)-stimulated STC secretion in salmon is mimicked by CaR mimetics, pharmacological agents that increase the sensitivity of the CaR to calcium. NPS 467, a small organic molecule that acts as a positive allosteric modulator of the CaR and alters calciotropic hormone secretion in mammals, was examined for effects on serum levels of STC in trout. The IP administration of NPS R-467 had time and dose-dependent stimulatory effects on STC secretion that were indistinguishable from those of Ca(2+) loading. The effects of NPS 467 were stereospecific and had no effects on serum CT. NPS 467 induced STC release was also manifested by a downstream physiological response; the inhibition of gill calcium transport. A cDNA clone was amplified from a fish corpuscle of Stannius cDNA library with high homology to the human CaR. RT-PCR revealed that this transcript was also present in gill, kidney, pancreas, brain, muscle and spleen. These findings suggest that Ca(2+)-stimulated STC secretion in fishes is mediated by a calcium ion-sensing receptor similar to that in mammals.

The respiratory effects of stanniocalcin-1 (STC-1) on intact mitochondria and cells : STC-1 uncouples oxidative phosphorylation and its actions are modulated by nucleotide triphosphates

Stanniocalcin-1 (STC-1) is one of only a handful of hormones that are targeted to mitochondria. High affinity receptors for STC-1 are present on cytoplasmic membranes and both the outer and inner mitochondrial membranes of nephron cells and hepatocytes. In both cell types, STC-1 is also present within the mitochondrial matrix and receptors presumably enable its sequestration. Furthermore, studies in bovine heart sub-mitochondrial particles have shown that STC-1 has concentration-dependent stimulatory effects on electron transport chain activity. The aim of the present study was to determine if the same effects could be demonstrated in intact, respiring mitochondria. At the same time, we also sought to demonstrate the functionality, if any, of an ATP binding cassette that has only recently been identified within the N-terminus of STC-1 by Prosite analysis. Intact, respiring mitochondria were isolated from rat muscle and liver and exposed to increasing concentrations of recombinant human STC-1 (STC-1). Following a 1h exposure to 500 nM STC-1, mitochondria from both organs displayed significant increases in respiration rate as compared to controls. Moreover, STC-1 uncoupled oxidative phosphorylation as ADP:O ratios were significantly reduced in mitochondria from both tissues. The resulting uncoupling was correlated with enhanced mitochondrial (45)Ca uptake in the presence of hormone. Respiratory studies were also conducted on a mouse inner medullary collecting cell line, where STC-1 had time and concentration-dependent stimulatory effects within the physiological range. In the presence of nucleotide triphosphates such as ATP and GTP (5mM) the respiratory effects of STC-1 were attenuated or abolished. Receptor binding studies revealed that this was due to a four-fold decrease in binding affinity (KD) between ligand and receptor. The results suggest that STC-1 stimulates mitochondrial electron transport chain activity and calcium transport, and that these effects are negatively modulated by nucleotide triphosphates.

Stanniocalcin-1 secretion and receptor regulation in kidney cells

Kidney collecting duct principal cells are the main source of stanniocalcin-1 (STC-1) production and secretion. From there, the hormone targets thick ascending limb and distal convoluted tubule cells, as well as collecting duct cells. More specifically, STC-1 targets their mitochondria to exert putative antiapoptotic effects. Two distal tubule cell lines serve as models of STC-1 production and/or mechanism of action. Madin-Darby canine kidney-1 (MDCK-1) cells mimic collecting duct cells in their synthesis of STC-1 ligand and receptor, whereas inner medullary collecting duct-3 (IMCD-3) cells respond to additions of STC-1 by increasing their respiration rate. In the present study, MDCK cell STC-1 secretion was examined under normal and hypertonic conditions, vectorally, and in response to hormones and signal transduction pathway activators/inhibitors. STC-1 receptor regulation was monitored in both cell lines in response to changing ligand concentration. The results showed that NaCl-induced hypertonicity had concentration-dependent stimulatory effects on STC-1 secretion, as did the PKC activator TPA. Calcium and ionomycin were inhibitory, whereas calcium receptor agonists had no effect. Angiotensin II, aldosterone, atrial natriuretic factor, antidiuretic hormone, and forskolin also had no effects. Moreover, STC-1 secretion exhibited no vectoral preference. STC-1 receptors were insensitive to homologous downregulation in both cell lines. In contrast, they were upregulated when STC-1 secretion was inhibited by calcium. The findings suggest that hypertonicity-induced STC-1 secretion is regulated through PKC activation and that high intracellular calcium levels are a potent inhibitor of release. More intriguingly, the results suggest that the receptor may not accompany STC-1 in its passage to the mitochondria.