Characterization of stanniocalcin 1 binding and signaling in gill cells of Japanese eels.

Abstract

Stanniocalcin 1 (STC1) is a hypocalcemic hormone that is known to play an important role in calcium metabolism in teleost fish. An increase in blood Ca(2) (+) levels stimulates its synthesis and release. The biological action of STC1 inhibits gill Ca(2) (+) transport (GCAT), but we as yet have no clear understanding of how STC1 inhibits GCAT. In the present study, we characterized the binding, signaling, and action of STC1 on gill cells. Treatment of gill cell cultures with the extracts of corpuscles of Stannius or recombinant STC1 proteins (STC1-V5) led to an increase in cytosolic cAMP levels. Using in situ ligand-binding assays, we demonstrated that STC1-V5 binds to both lamellar and inter-lamellar regions of gill sections. The binding sites were significantly increased in gill sections obtained from fish adapted to high-Ca(2) (+) (2 mM) freshwater (FW) as compared with those from fish adapted to low-Ca(2) (+) (0.2 mM) FW. Receptor-binding assays illustrated specific binding of STC1-alkaline phosphatase to plasma membrane (Kd of 0.36 nM), mitochondria (Kd of 0.41 nM), and nuclear (Kd of 0.71 nM) preparations from gill cells. STC1 binding capacity was significantly greater in the plasma membrane preparations of gills obtained from fish adapted to high-Ca(2) (+) FW. Using isolated pavement cells and mitochondria-rich cells in cAMP assays, we obtained results indicating that both cell types responded to STC1. To illustrate the biological action of STC1, we conducted Ca(2) (+) imaging experiments to demonstrate the effects of STC1 on thapsigargin-induced elevation of cytosolic Ca(2) (+). Our results indicated that STC1 exerted its inhibitory action via a cAMP pathway to lower intracellular Ca(2) (+) levels. Intriguingly, we were able to block the action of STC1 using an inhibitor, NS-398, of cyclooxygenase-2 (COX-2), which is known to stimulate the activity of sarcoplasmic and endoplasmic reticulum Ca(2) (+)-ATPase (SERCA). A follow-up experiment in which gill cells were incubated with STC1 revealed a downregulation of the epithelial Ca(2) (+) channel (ecacl) but an upregulation of cox-2 expression. The ECaCl is a gatekeeper for Ca(2) (+) entry, whereas COX-2 mediates an activation of SERCA. Taking these results together, the present study is, to our knowledge, the first to provide evidence of STC1 binding and signaling as well as the first to decipher the mechanism of the effect of STC1 on fish gills.